Advances in Food Research, Volume 15

ADVANCES IN FOOD RESEARCH VOLUME 15

CONTRIBUTORS TO THIS VOLUME

ADELABDEL-KADER GEORGED. ARMERDING B. BORENSTEIN R. H. BUNNELL R. J. FORTLAGE SAMUEL A. GOLDRLITH D. E. HATHWAY E. C. MAXIE ROGERJ. ROMANI N. F. SOMMER

ADVANCES I N FOOD RESEARCH VOLUME 15

Edited b y E. M. . \ I & ~ K

C. 0. CH~CHESTER

Uiiiwemity of Californiu

University of Calipoinia Davis, Califorilia

Davis, California

G. k'.

S.11<\1-.4K1.

ITniversity o f California Davis, California

Editoyial B o a d S. LEPKOVSKY E. C. BATE-SMITH EDWARD SELTZER H. COOK W. M. URBAIN M. A. JOSLYN J. F. VICKERY

w.

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CONTRIBUTORS TO VOLUME 15

ADEL ABDEL-KADER, U n i v e w i t y of Califomia, Davis, California*

GEORGED. ARMERDING, Mojonnie?. Bros. Co., Oakland, California+

B. BORENSTEIN, Hoff mann-La Roche Inc., Nutley, N e w Jersey R. H. BUNNELL, Ho.ffmann-La Roche Znc., Nutley, N e w Jersey R. J. FORTLAGE, Department of Po?nology, University of Califomia, Davis, Califomia

SAMUEL A. GOLDBLITH,Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massac hzt set t s D. E. HATHWAY, Tunstall Labomtot y, “Shell” Research L i m i t e d , Sittirigboume, Kent, Englaml E. C . MAXIE, University of Califo?.nia, Davis, California

ROGERJ. ROMANI,Llepartment of Pomology, University of Califor)iia, Davis, C‘alifomia N. F. SOMMER, Department of Pomoloyy, U n i v e w i t y of Califomia, Da 23 is, Calif o m ia * Present address : Department of Horticulture, College of Agriculture, Cairo University, Cairo, Egypt. * Present address: 3477 St. Mary’s Road, Lafayette, California.

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CONTENTS

(:OSTRIRIITOHS

TO VOLl'hlE

15 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V

Metabolic Fate in Animals of Hindered Phenolic Antioxidants in Relation to Their Safety Evaluation and Antioxidant Function

D. E. HATHWAY I. Introduction . . . . . ............................... 11. Hindered Phenolic ts ............... 111. Metabolic F a t e of Hindered Phe in Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Antioxidant Action . . .............................. V. Aspects of the Physiological Chemistry of Hindered Phenolic Antioxidants . . . . . . . . . . . ........ VI. Evaluation of the Safety of Food Antioxidants ........ References . . ...............................

1 6 9 25 32 39

50

Radiobiological Parameters in the Irradiation of Fruits and Vegetables

ROGERJ. ROMANI I . Introduction

..............................................

11. Radiation Units and Dosimetry

............................ ........

111. Radiation Mechanisms . . . . . . I\'. Chemical and Biochemical Events in Irradiated F r u i t getables . . . . . . . . . . . V. Metabolic Aspects ................................ ................................ VI. Radiation Sources V I I . F u t u r e Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Food Irradiation-Physiology

E. C.

57 58 63

82 87 92 93

of Fruits as Related to Feasibility of the Technology

RIAXIE AND

ADF.1, ABDEL-KADER

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. F r u i t Respiration in Relation to Radiation EffectsClimacteric Class of F r u i t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. F r u i t Respiration in Relation to Radiation EffectsNonclimacteric F r u i t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

105 107 114

...

CONTENTS

v111

IV. EfTect of I r r a d i a t i o n on T e x t u r e of F r u i t s . . . . . . . . . . . . . . . . . . V. Effect of Radiation on Chemical Components of F r ~ i i t s. . . . . . . VI. Effects of Radiation on Organoleptic A t t r i b u t e s of F r e s h F r u i t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1'11. Wholesomeness of I r r a d i a t e d F r u i t s . . . . . . . . . . . . . . . . . . . . . . . . VIII. Czone Produced hy ionizing Rildiation ailti I t s R-latiorr t:) F r u i t Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Feasibility of I r r a d i a t i o n as a Commercial Technology X. Concluding R e m a r k s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . ................................

116 123 129 1:G 1:il

ionizing Radiation for Control of Postharvest Diseases of Fruit and Vegetables

h'. F. SOMMER A N D R. J. FORTLAGE I. Introduction . . . . . . . . . . . . . . . . . . . . . 11. Fungicidal and F u n g i s t a t i c Effects 111. IV. V. VI. VII.

......................

Techniques f o r P o s t h a r v e s t Disease R a T h e N a t u r e a n d Causes of P o s t h a r v e s t Diseases . . . . . . . . . . . . . . Disease-Control Investigations . . . ................. Protective P a c k a g i n g . . . . . . . . . . . . . . . . . . . . . . . .... Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................................... References . . . . . .

147

166

170 182 183 184

Carotenoids: Properties, Occurrence, and Utilization in Foods

B. BORENSTEIN ASD R. H. BUISPZELL I. Inti,oduction . . . . . . . . . . . . . . . . . . . .

...................... ........................... ......

1'35 1'36

111. Occurrence a n d Stability of N a t u r a l Carotenoids in Food . . . . IV. Added Carotenoids in Food Processina . . ............... V. Additional Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . ...............

21.5 244 262

11. General P r o p e r t i e s

264

Basic Principles of Microwoves and Recent Developments

SAMUEL A . GOLDBLITII I . Introduction . . . . . . ........ ......................... I I . Radio-Frequency E n e r g y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. H o w Does R F E n e r g y H e a t Foodstuffs? . . . . . . . . . . . . . . . . . . . . . 11.. T h e P o w e r E q u a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... I-. P e n e t r a t i o n of Microwaves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1. Electrical Power a n d T h e r m a l E n e r g y . . . . . . . . . . . . . . . . . . . . . . V I I . Dielectric Loss F a c t o r s . . . . . . . . . . ................. V I I I . T y p e s of Microwave Process Devices . . . . . . . . . . . . . . . . . . . . . . . . 1X. Efficiency of Microwave Ah-ol.ption into Foods . . . . . . . . . . . .

.>--

--I1

278 279 "0

281 281 282 282 2Ni

CONTENTS

ix

X . Choice of Frequency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X I . Possible U s e s of R F in Food Processing . . . . . . . . . . . . . . . . . . . . . X I 1. S u m m a r y a n d Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ................................................

287 288 295 295

Evaporation Methods as Applied to the Food Industry GEORGE .

I. I1 . 111. I V.

V.

V I. VII. VIII. IX. X. XI. XI1 . XI11 . X I V. XV. XVI. XVII. X V I I I. XIX .

XX . XXI. XXII. X X I I I. X X I V. X X V. XXVI. X X V I I. X X V I I I.

ARlIERDING

. . . . . . . . . . . . . . 305 E a r l y Methods of E v a p o r a t i o n . . . . . . . . . . 310 Metallurgy a n d E . . . . . . . . . . . . . . 315 Producing the Vacuum . . . . . . . . . . . . . . . . . . . . 318 The Heating Surface . . . . . . . . . . . . . . . . . . . 321 Condensers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T h e V a c u u m Pail 322 The Calandria P a n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32-f Entrainment Separators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 Tubular Evaporators ................................. 328 330 Forced-Circulation E v a p o r a t o r s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................... 331 Falling-Film Evaporators . . . . . . . . . . . Heat-Pump Evaporators . . . . . . . . . . . . . . . . . . . . . . . 333 Indirect Heat-Pump Evaporat ................... 334 C e n t r i f u g a l T h i n - F i l m Evapoi.a t o r s . . . . . . . . . . . . . . . . . . . . . . . . 336 The Vacreator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 . . . . . . . . . . . . . . . . . . . 341 Plate-Type Evaporators . . . . Expanding-Flow Evaporators . . . . . . . . . . . . . . 341 F r u i t - S p r e a d Cookers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 Concentration by F r e e z i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 A u t o m a t i c Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 Control E q u i p m e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 Sonic a n d Ultrasonic Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 T h e Carver-Greenfield Process .................... 349 Essence Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 P r e - e v a p o r a t i n g Conditioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353 Electrodialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 Reverse Osmosis . . .................................. 356 Evaluation of E v a p o r a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

SUBJECT ISDEX

....................................................

359

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METABOLIC FATE I N ANIMALS OF HINDERED PHENOLIC ANTIOXIDANTS IN RELATION TO THEIR SAFETY EVALUATION AND ANTIOXIDANT FUNCTION

I. Inti~oduction ............................

..............

A . Metabolic Considerations in Relation to Food Additives . . . . . . . . . . . . ................... H. Autoxidation a n d Antioxidant ......... 11. Hindered Phenolic Antioxidants . . . . . . . . . . . . . . . . . . . . . . A. S t r u c t u r a l Formulas . . . . . . . . . . . . ................... B. Specifications f o r Identity a n d P u r i t y of Food Additives . . . . . . 111. Metabolic F a t e of Hindered Phenolic Antioxidants in Animals . . A. Compounds Exhibiting F a i l u r e of Alimentary Absorption . . . . H. Compounds t h a t a r e Rapidly Absorbed, Metabolized, a n d Completely Excreted . . . . . . . . . . . . . . . . . . . . . . . . C. Compounds with Intermediate Rates o and Elimination f r o m Body Tissues . ................ IV. Antioxidant Action ......................... sni to Antioxidant Action i ) ~ Vif/’O . . V. Aspects of the Physiological Chemistry of Hindered Phenolic ..................... Antioxidants . . . . . . . . . . . o Micellar Forniation A . Alimentary Absorption in 8 . Secretion into Bile . . . . . . . . ......................... C:. T r a n s p o r t f r o m Mother t o E .......................... D. Stimulation of Hepatic-Drug-Metabolizing Enzymes .......... VI. Evaluation of t h e S a f e t y of Food Antioxidants ......... A. Toxicological Inforniation, with Spec BHA, and BHT . . . . . . . . . . . . . . . . . . ............... B. Relationship of Metabolic F a t e a n d Related Aspects of Physiological Chemistry to Evaluation of t h Use of Food Additives . . . . . . . . . . . . . . . . . . . . ........... C. A Possible Role f o r Synthetic Antioxidants i Biochemistry . . . . . . ......................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..............

2

11

17 25 25 32 32 31 34 36 39 30

46 49

50

2

D. E. HATHLVAY

I.

INTRODUCTION

A. METAHOLICC‘OKSIDEKATIOKS I K RELA-\? I O N TO SAFFTY E V A L U A T I O N O F F O O D ADDITIVKS Metabolic studies a r e important to evaluation of the safety of a food additive ; thuq, ‘ ‘ a substance might be considered safe a- ii result of im accurate knowledge of its metabolism in man” (Jlinist r y of Agriculture, Fisheries a n d Food, 1 9 6 5 ) . Of value, therefore, i n reaching a conclusion about toxicity, is information > i b ( ~ ~ i t the alimentary absorption of a n additive, the eliminative route, tissue concentrations a n d r a t e of elimination of the original substance. a n d its metabolic products in animals. Metabolic studies in animals may be designed t o elucidate the metabolic p i t h \ \ a y i i 11(1 identify excreted or st o red metabolites . SLI ch info i*iiiat i on i s eqsentia! t o accounting f o r t h e adnii?iistered dose in tern?> vf t h e metabolic products ( N a t l . Acad. of Sci., 1 9 6 0 ) . Measurement of the toxicity of key metabolites will determine whether the -Libhtance h a s been transformed iii viva into h a r m f u l o r hamiless p 1’0(1 11c t 9 . hlctabol i c a n d b i och em ic a 1 investigations can son1e t i ni es oiter ;L conclusive proof of safety. Thus, if’ a substance produces n o toxic symptonis d u r i n g its toxicological testing, the f a t e of a dose might be traced so as t o provide convincing evidence of its hxrmlcssness (Ministry of Agr., Fisheries a n d Food, 1 9 6 3 ) . Such e\planation might depend on : (1) uniform failure of alimentary absorption among species ; ( 2 ) rapid metabolism and complete etioii of t h e initial substance and its metabolic products ; o r ( 3 ) conversion of the additive into a normal physiological rnetiibolite or metabolites. Knowledge about t h e metabolism of a food ; i d d i t i \ e would therefore be decisi\ e in assessing the s a f e t y oi ci I)iwpobetl use. F ~ o t antioxidants l a r e used t o preserve the niitritional 1 a l ~ eo f the diet of animals a n d man. TIThere species differences o c c ~ ~ r , either i t i t h e metabolic pathways of foreign compounds o r in their toxicity, extrapolation t o m a n from the results of animal e\pei.inients is difficult ( v i c l c , iwtcr cilia, Beyer, 1 9 6 0 ) . I t is therefore important t o understand t h e r a t e s of metabolimi of foreign o r ganic compounds in various experimental animals a s cc)~:paii (1 n i t h those in m a n (Erodie, 1 9 6 2 ) . Side efl’ects in nian cannot easily be predicted from animal studies, because of species tlifi e,,cnces (Zbinden, 1 9 6 3 ) . Thus, the fact t h a t phenothiazines do :lot callhe l i l e r damage in dogs ~ L I Ldo alter intrabiliary pressui‘e, 1r2Iy tJ\plain the development of jaundice in men exposed t o theye

METABOLISM OF HINDERED PHENOLIC ANTIOXIDANTS

3

drugs (Popper e t ul., 1 9 5 7 ) . It m a y therefore be prudent t o assess t h e effect of a new food additive on m a n whenever evidence can be adduced f r o m existing sources (Ministry of Agr., Fisheries a n d Food, 1965). I t is generally recognized t h a t there can be no absolute assurance t h a t use of a chemical compound in food processing, pacltaging, o r storage will not prove h a r m f u l in a n y degree. E f f o r t should accordingly be directed t o ensure t h a t t h e hazard associated with a particular use of a food additive o r antioxidant is very small i n relation t o t h e benefit t o be derived. I n this consideration, metabolic studies in m a n of dose levels near those to be encountered in the proposed use might be useful, a f t e r prior laboratory-aninial studies have indicated profitable lines of approach (see Natl. Acad. of Sci., 1 9 6 5 ) . This chapter h a s been written in view of t h e importance of metabolic a n d physiological chemical considerations t o evaluation of t h e safety of antioxidants f o r food a n d food-wrapping materials. In particular, Sections 111, V, a n d VI deal with important aspects of this subject.

B. AIJTOXIDATION A N D ANTIOXIDANT ACTION The meaning of antioxidant action within the connotation of food preservation a n d protection requires explanation. Advances have been made in knowledge of olefinic peroxidation (Uri, 1961a) since it became generally accepted t h a t t h e propagation sequence

r R.

RO.0.

+ 0,

t RH -ROOOH

RO. 0. f

Re

1

is a fundamental mechanism in many deteriorative reactions. Termination m a y be brought about by interaction of t h e chainbearing radicals ( R . a n d R O . 0 . ) o r by reaction with a n inhibitor ( A H ) (Bolland, 1 9 4 9 ) . The amount of inhibitor required t o suppress Ol-absorption is very small, usually as little as O . O l % , because of t h e large over-all kinetic chain lengths. T h e amounts of products formed from the inhibitor a r e accordingly very small. Sufficient evidence h a s accumulated concerning the dominant role of alkylperoxy-radicals ( R O . 0 . ) in autoxidation (Bolland, 1949 ; Uri, 1961a). Since t h e concentration of alkyl radicals ( R . ) is generally low, a s a result of rapid reaction with 02,it may be assumed t h a t t h e inhibitor operates mainly by reac-

4

D. E. HATHWAY

tion with R O . 0 . . This has been investigated by preparation of alkyl radicals, followed by isolation of products (e.g., Bickel et al., 1953 ; Campbell and Coppinger, 1952 ; Kharasch and Joshi, 1957).

1:o. .\.

+ A\ll

+ 1:011

+1~0*0*

---j

+ .I.

I{O*O.I

(3) (4)

Hence, 2ijo.oir

+ M I + iio.oiz + I W H + I r 2 0

The over-all equation shows that only catalytic quantities of Co naphthenate a r e necessary for the reaction. Reaction processes 3 and 4 exemplify that hydrogen abstraction is the most important mechanism for breaking the reaction chain and inhibiting peroxidation (Uri, 1961b). This chemical knowledge provides a chemical basis for the fact that animals, which contain oxygen-labile polyunsaturated lipids, require lipid antioxidants ; the biological essential of a lipid antioxidant is mainly fulfilled by vitamin E. The chemical basis of vitamin E function is its reactions with free-radical intermediates of lipid peroxidation and with lipoperoxides. Section VI,C deals with the possible role of antioxidants in nutritional biochemistry. In this context the word peroxidation does not have the same meaning a s it does in enzymology. To the enzymologist, peroxidation refers to the special case of oxidation when the H-acceptor or electron-acceptor is H20, o r an alkyl peroxide. Thus, horseradish peroxidase catalyzes the oxidation of ascorbate by peroxide t o dehydroascorbate and water ; i.e., the product is not a peroxide. The heme-catalyzed peroxidation of unsaturated lipids by H,O, possibly follows the same pattern, but in this case the product is a peroxide. Lipid antioxidants belong to many classes of organic compound, of which phenols a r e of primary importance. This chapter deals only with synthetic hindered phenolic antioxidants. Each phenolic group belonging to the individual antioxidants, except BHA, is hindered by the steric effects of tert-butyl substituents in each ortho-position. Antioxidant action might here be illustrated by reference to BHT, which, for example, reacts with alkyl or aralkyl hydroperoxide in the presence of cobalt naphthenate

METABOLISM O F HINDERED PHENOLIC ANTIOXIDANTS

5

catalyst to afford 2,6-di-te~t-butyl-4-methyl-4-a1kyl(or aralkyl) peroxy-2,5-cyclohexadien-l-one :

+$-y C (CH,)

Co naphthenate 2R-4. (CH,),C

I"~'C,$,C(CHJ~

CH,

+ RO,H

H,C

O,R

BHT

Section IV deals with certain aspects of antioxidant action. 11.

HINDERED PHENOLIC ANTIOXIDANTS

A. STRUCTURAL FORMULAS Since the biochemical approach is fundamental to the subject matter, no attempt is made within the limits of the present chapter to include a n account of the methods used for the synthesis of hindered phenolic antioxidants. Reference to syntheses a r e made, however, in Table I, which gives the formulas, physical constants, and reactions of seven hindered phenolic antioxidants. With the metabolic considerations in prospect, the compounds in Table I a r e arranged in the same order as in Section 111. Antioxidants exhibiting a failure of alimentary absorption (Ionox 330 and Ionox 312) precede those that are rapidly metabolized and completely excreted (BHA and Ionox loo), which a r e followed in turn by others with intermediate rates of absorption, metabolism, and elimination (Ionox 220, Ionox 201, and B H T ) . FOR IDENTITY AND PURITY B. SPECIFICATIONS O F FOOD ADDITIVES

Of the compounds discussed in this chapter, only BHA and BHT enjoy approval as food additives ; specifications have been compiled for their identity and purity (Food and Agricultural Organization, 1962). BHA is a mixture of isomers (Table I ) , and, by definition, the commercial antioxidant contains not less than 98.5% of the empirical formula, C, ,H,,,O,, whereas commercial BHT contains 99% of authentic antioxidant. Besides color reactions (Table I ) , identification tests also include solubility determinations. Stringent limits have been set for arsenic and lead impurities, and, as a further criterion of purity, a freezing-point determination is

D. E. HATHWAY

TABLE I FORMULAS AND PHYSICAL A N D CHEMICAL PROPERTIES OF INDIVIDUAL ANTITOXIDANTS Physical constants and reactions

Reference to the svnthesis

Ionox 330 PI. 17:s 200"

and approx. Red with c a r boll tetrach:oride-SbCl, reagent. Further c h a r acterization by infrar e d spectroscopy. 245OC.

OH

c(cH3)3

Rocklin and Van Winkle, 1962

0H

2,4, E-Tr:-(3', 5'-di-rc2rt -butyl-4'Lpdroxybenzp 1)niesitylene

Ionox 312

..-

C (CU5) 1

OH

h ' ,J

bH

m.p. 1G3"C. R e d w i t h carbon ietrachloride53C1, reagent.

Jafflvor

ot-7

METABOLISM OF HINDERED PHENOLIC ANTIOXIDANTS

7

TAHLIC I (('ontinrted) Formula .

~

_~__

~~~

Reference to the synthesis

Physical constants and r e a c t i o n s ~~~~

BHA ( M i x t u r e a of 2 - and 3-tcJ.t-butyl4 - hydroxyanisole)

,T, : OCH,

I

OCH,

C( CHJ3

2%, li

&

''~(CH,),

?,-L

OH

OH ( 1 85%)

( " 15%)

2 - and 3- tt~~~t-Butyl-4-methoxyphenol

Rosenwald, 1949a; m . p . 54-58°C; b.p. 264270"C/133 mm Hg. BHA, Rosenwald, 1949b; Young and r e a c t s w i t h FeC1,-2,2'diDvridvl R o d g e r s , 1955. _ . . r e a g e n t t o give a r e d c o l o r . T h e 2-[som e r , which m e l t e d a t 62.564.0°C, gave blue-brown with NH, -AgNO,, o r a n g e with P a u l y ' s r e a g e n t , and blue-gray with Gibb's reagent. T h e 3 - i s o m e r , which m e l t e d at 64.5", gave blue-black with NH,-AgNO, o r a n g e - r e d with P a u l y ' s r e a g e n t , and blue-purple with Gibb's reagent.

Ionox 100 m . p . 1 4 1 " C ; b . p . 162"C/ 2 . 6 m m Hg. R e d with carbon ietrachlorideSbC1, r e a g e n t ; m a g e n t a with Gibb's reagent.

OH

Wright ct a / . , 196Sb.

CH,OH 2 , 6 - D i - tc~-t-butyl-4-hydroxymethylphenol Ionox 220 (CH,),C

,C(CH,),

/ >OH $

HO
/

/

m.p. 155-156"C;b.p. 2 1 7 O Filbey and Coffield, 1957 C/1 m m Hg. Vermilion with c a r b o n t e t r a c h l o r i d e SbC1, r e a g e n t ; blue w i t h Gibb's r e a g e n t .

'CC(CH,),

(CH,),C

D i - ( 3 , 5 -di- to-t - buty 1- 4 - hy droxyphenyl) m e t h a n e Ionox 201 (CH,),C

C(CH3),

Gibb's r e a g e n t .

L(CH,',C

m.p. 138". R e d with c a r bon tetrachloride-SbC1,

'C(CHJ,

Di(3, 5-di- I<,i-t-butyl- 4-hydroxybenzy1)ether

M o r r i s and Sullivan, 1963.

8

D. E. HATHWAY

Physical constants and r e a c t i o n s

Formula ~~~

.-

~.

~

~

~

~~~~~~~~~~~

Reference to the synthesis ~~

~

~

BHT (3, 3 - D i - f e r f-butyl-.i -hydroxytuluene) OH

(CH,),C

-. ’\-C(CH,), Li

,&’ CH3

2,6-Di- terf-butyl-0-cresol

71”C.I1.u. 265°C. Red w i t h carl;on t e t r a rloride-SbCI, r e a g e n t . magenta w i t h Gibli’s r e agent. B H T d o e s not r e a c t with FeC1,-2, 2’-dipyridyl r e a g e n t . Further c h a r a c t e r i z a t i o n by inf r a r e d spectroscopy.

11i.u.

Cowie. 1953: Stillskn, 1947

“ P e r c e n t a g e composition d e t e r m i n e d by i n f r a r e d s p e c t r o s c o p y ifor d e t a i l s , s e e Section 11, 8).

specified f o r BHT, which is a chemical entity. Eminently suitable infrared methods are stipulated f o r estimation of these two a n tioxidants. Thus, in the case of BHT, t h e net absorption spectrum of a test specimen w a s obtained by subtraction of t h e recording f o r t h e solvent f r o m t h a t of a standard solution in carbon disulfide under s t a n d a r d instrumental conditions, between 11 a n d 14 p. T h e corrected absorption a t a selected wavelength (12.85 p ) was then referred t o a calibration curve prepared f r o m standard solutions of pure BHT. A similar exercise is also made f o r a test specimen of BHA, but in t h i s case t h e isomer ratio h a s also t o be determined. F r o m t h e optical-density values a t 10.75 and 10.95 p, t h e optical-density ratio can be used to calculate the percentage of the 3-isomer in the sample by reference t o a second calibration curve, in which optical-density ratios a r e plotted against t h e corresponding concentrations of 3-te~t-butyl-4hydroxyanisole in standard mixtures of t h e pure isomers. The specifications (Food a n d Agriculture Organization, 1962) also include colorimetric procedures f o r estimating the two antioxidants ; t h e colors generated by either FeC1,-2,2’-dipyridyl o r Gibb’s reagent can be used f o r t h e assay of BHA. Brief reference ought also t o be made t o Ionox 330, which exhibits failure of alimentary absorption in various animal species (Section II1,A) a n d a lack of toxicological effects in r a t s (Section V1,A). The approval of Ionox 330 f o r incorporation into food-wrapping materials a n d its efficacy a s a n antioxidant in polyunsaturated lipids a t physiological temperatures suggest the possibility of wider commercial application. I n step with specifications f o r other antioxidants, Ionox 330 might be identified by

METABOLISM O F HINDERED PHENOLIC ANTIOXIDANTS

9

the color-reaction (Table I ) and solubility data, and its purity could be defined in terms of the double melting point and characteristic infrared spectrum. Infrared and cryoscopic measurements are available for assay purposes; small quantities of impurities (which are more volatile than Ionox 330) can be estimated by gas chromatography. 111.

METABOLIC FATE OF HINDERED PHENOLIC ANTIOXIDANTS I N ANIMALS

A. COMPOUNDSEXHIBITING FAILURE OF ALIMENTARY ABSORPTION

Unchanged Ionox 330 was quantitatively eliminated by the fecal route in dogs, rats, and man after oral administration, and virtually no I 4 C was measured in the urine and expired gases of rats dosed orally with ["C] Ionox 330 (Wright et aZ., 1965a). The very low concentrations of I4C that were measured in the combined carcasses and remaining viscera after removal of the alimentary canal of these animals 72 h r after dosage was ascribed to slight contamination of the fur, which had not been removed in this experiment. ( A small degree of contamination of the fur is bound to occur in all metabolic experiments with rodents.) Dogs and rats did not show a sex difference in this pattern of elimination. Quantitative elimination of ['"C] Ionox 330 and the absence of I4C in the carcass and viscera of rats 72 h r after dosage showed that the antioxidant did not accumulate in the body. No metabolites were formed in consequence of the ingestion of Ionox 330. Rats eliminated three-quarters or more of a dose (285.7 mg kg body wt) of Ionox 330 in 24 hr, and the remainder during 24-48 hr. Dogs eliminated the whole dose (90 mg/kg body w t ) within 48 hr, and a variable proportion within 24 hr. These rates of elimination, which did not seem to be affected by the size of dose (300 mg/kg body wt to rats, 90 mg/kg body wt to dogs, and 1.25 mg,/kg body wt. to man) or by the presence of triglyceride f a t or phospholipid in the diet, a r e consistent with the passage of unabsorbed material through the alimentary canal. Tissue radioactivity was therefore measured in male and female rats that had been killed 8, 16, and 24 h r after the ingestion of [14C] Ionox 330. The absence of radioactivity in the skinned carcass and remaining viscera after removal of the alimentary canal at times before total elimination indicated that the remaining [ "C]

10

D. E. HATHWAY

Ionox 330 w a s still in the alimentary canal. Hence i t is unlikely t h a t a big proportion of t h e antioxidant t h a t w a s eliminated in the feces originates in the bile. Since r a t s have no gall bladder, the bile cannot be concentrated, a n d a small degree of absorption might have escaped detection, especially if entero-hepatic circulation were rapid. The absence of l 4 C in the 24-hi- bile of r a t s with M a r y fistLilae t h a t had been dosed orally with ["C] I o n o s 330 strongly suggests t h a t ["C] Ionox 330 is not absorbed f r o m t h e gastrointestinal tract. Since bile was not available t o assist alimentary absorption in the experiment with r a t s with biliary fistulae, the 24-hr bile was collected from pigs with biliary fistulae, a n d f r e s h bile was supplied by stomach tube a f t e r treatment of the animals with ["C] Ionox 330. I n comparison with t h e experiment with r a t s with biliary fistulae, the pigs with biliary fistulae were allowed t o recover post-operatively a n d maintained in a physiological condition throughout t h e experiment. The absence of I 4 C in the bile of these pigs confirms t h a t ["C] Ionox 330 is not absorbed f r o m t h e gastrointestinal t r a c t . Because of the structural similarity of Ionox 312 a n d Ionos 330, tissue radioactivity was measured in male a n d female r a t s t h a t had been killed 8, 16, 24, a n d 48 hi- a f t e r the ingestion of I'("[ Ionox 312. Virtually no I ' C ~ v a smeasured in the urine a n d expired gases of these rats, a n d the absence of radioactivity in t h e skinned carcass a n d remaining viscera a f t e r removal of t h e alimentary canal a t times before total elimination could have occurred indicated t h a t t h e remaining ["C] Ionox 312 was still in the alimentary canal. This strongly suggests t h e absence of alimentary absorption. The absence of "C in the 24-hr bile of r a t s with biliary fistulae t h a t had previously been dosed orally with ["C] Ionox 312 confirms t h a t [IIC] Ionox 312 is not absorbed f r o m the gastrointestinal tract. Despite t h e high lipid solubility of Ionox 330 a n d I o n o s 312 a n d the un-ionized state of the molecules a t the p H of gastric a n d intestinal mucosal fluids, these compounds Ivere not absorbed from the gut. 1o:'ox 3310 and Io;qc\- 316> xvcre precipit::tetl f r o m lipid solution in the g u t as t h e lipid is absorbed. Foreign organic substances a r e absorbed when their properties a r e siniilai. to those of n a t u r a l physiological metabolites, f o r which t h e r e ai e absorption mechanisms. Because of their size, shape, a n d colloidal properties, Ionox 330 a n d Ionox 312 a r e unlikely to be absorbed by a n y to the mechsnisms available (see K r i g h t c t u l . , lSC-.Ja). Section V,A deals ~ v i t hcertaiii aspects of alimeiltary absoiytion.

11

METABOLISM O F HINDERED PHENOLIC ANTIOXIDANTS

Ionox 330 is not metabolized by animals, because of its nonabsorption a n d because of its stability t o t h e soluble enzymes of the alimentary canal a n d intestinal flora ( W r i g h t et nl., 1965a).

B. COMPOrJNCS T H A T ARE RAPIDLYABSORBED, METABOLIZED, AND COhlPLETELY EXCRETED Mammalian metabolism of BHA was first investigated by Dacre et ul. (1956) (see also Dacre, 1958, 1 9 6 0 ) . Those workers found t h a t rabbits dosed orally with 1 g of the antioxidant excreted 46% as glucuronides, 9% as ether sulfates, a n d 6% as f r e e phenols in the 24-hr urine. A f t e r a 0.5-g oral dose the recovery of glucuronides w a s 60% a n d of ether sulfates was 1256, a n d a f t e r a 0.25-g oral dose t h e recovery of glucuronides was 84% a n d of ether sulfates lvas 18%. Excretion of glucuronides in the urine was lowered by repeated dosage (0.5 g o r 1 g ) of BHA, a n d f o u r o r five daily dcses of 1 g to a rabbit had cumulative a n d lethal effects. I n experiments in which t h e elimination w a s not quantitative, the f a t e of t h e yemainder of t h e dose was unknown. F r o m the urine of treated animals, Dacre e t al. (1956) isolated t h e glucuronides of the t w o BHA isomers a s barium ( 2 - t c t t butyl-4-methoxyphenylgl~1copyranosid) uronate a n d barium (3te~t-butyl-4-methoxyphenylglucopyranosid) uronate. Dacre et crl. (1956) found t h a t when a rabblt \vaa maintained f o r some weeks on a low-sulfur diet i t showed no increase in ether sulfate excretion a f t e r administration of BHA, whereas administration of' 1 g of NalSO, by stomach tube together i ~ i t h t h e KHA resulted in a n excretion of 20% of t h e dose as ether sulfate. The proportion of administered BHA which was excreted as ether sulfate could therefore be varied experimentally. In study of the nietabolism of B H A in rabbits (Dacre crl. 1956), the dose was very large in comparison with human intake, which is probably less than 0.1 mg kg. Since variations in dose may alter tbe ii -opo:.tions of metabolites excreted w h e x more t h a n cne nieta5olic p)ath\vay is available ( c j . pheucl; Bray c t c i l . , 1952), it might be n;guecl t h a t the metabolic pattern ()Light therefore to be studied with a range of doses including t h e Icwest possible so t h a t a n y unsuspected changes in nietalxlic pattern might k:e detected. TheFe considerations prompted reinLestigntion of the metabolism cf CIIA, and another animcil > pecies XI a s chosen (Astill ( t n l , 1 9 6 0 ) . I n the r a t , t h e m e t a h l i s m crf lev; doses c,f E H A resembled fairly closely t h a t described (Dwcre c t crl., 19.56) f o r the rabbit c j t

12

D. E. HATHWAY

for doses of 0.13-0.55 g/kg. At all the doses studied in the rat (Astill et al., 1960), BHA and its component isomers were absorbed and readily metabolized. The main eliminative route was via the kidneys ; fecal excretion in repeated-dose studies accounted for only 10.2% of the dose. For single doses of 0.002, 0.01, 0.025, 0.05, 0.10, and 0.40 g of BHA/'kg, the 5-day recoveries were high (82-100% of dose). Excretion of unchanged BHA was 11% of a dose of 10 mg/kg, and 3-7% of a dose of 400 mg/kg, but the slightly higher proportion of unconjugated BHA excreted a t the lower dose does not appear to be related to any tissue storage. It was concluded that at these low doses, BHA was largely absorbed and largely excreted; these doses approximate more closely to suspected human-use doses. Astill et al. (1960) were unable to isolate the glucuronide (11) (see Scheme 1) of 2-tert-butyl-4-hydroxyanisole ( I ) (cf. Dacre et al., 1956), but crude methyl 0-triacetate, purified by column chromatography, was converted into 3-tert-butyl-4-methoxyphenyl-p-glucuronamide, identical to a synthetic specimen. The Q-sulfate (111) of the 2-isomer was isolated as its Safranine bluish salt (Dodgson et al., 1955), which was converted by cation

+

OH

Y SCHEME I

OH

a

METABOLIC PATHWAYS OF BHA IN THE RAT

METABOLISM O F HINDERED PHENOLIC ANTIOXIDANTS

13

exchange into potassium 3-tert-butyl-4-methoxyphenyl sulfate; both salts were identical to synthetic specimens. The glucuronide (VI) of 3-tert-butyl-4-hydroxyanisole (V) was isolated as barium (2-tert-butyl-4-methoxyphenyl glucopyranosid) uronate from the glucuronide gum; no O-sulfate was isolated from the 3-isomer. The only other metabolite of BHA in rat urine was 2 (or 3) -tert-butyl-4-hydroxyphenyl sulfate (IV) , which gave SO.,- and tert-butylquinol on acid hydrolysis. A scheme for the metabolic pathways of BHA is shown in Scheme 1. 3-tert-Butyl4-methoxyphenyl sulfate is the principal metabolite of 2-tertbutyl-4-hydroxyanisole, which was appreciably converted into the O-glucuronide when the sulfate pool was depleted. When dogs were dosed with 350 mg of BHA/kg, the recovery in the excreta during 4 days was almost quantitative (Astill et al., 1962); 62% of unchanged BHA was eliminated by the fecal route, because of nonabsorption (Astill et al., 1962). Sulfate conjugation of the absorbed material in dogs occurred to the extent of 23% of dose. Separation of the O-sulfates as their phenazinium salts, followed by ion exchange and mild hydrolysis of the potassium salts, afforded 3-tert-butyl-4-hydroxypheny! sulfate and a sulfate of a nuclear hydroxylation product of BHA. Excretion of unchanged BHA in the urine is in roughly the same proportion of the dose in dogs as in rabbits and rats. Only 5% of the dose was excreted as glucuronides, and glucuronide conjugation involved the 4-hydroxyl of BHA. The value of comparative metabolic studies is illustrated by the different metabolic pathway for BHA in dogs than in rabbits and rats. When human volunteers ingested BHA (0.4-0.7 mg k g ) , measurement of glucuronides in the urine established that the BHA absorbed was rapidly metabolized. Maximum excretion of glucuronides occurred within 17 hours of dosage, and excretion was complete within 48 hours of dosage. Subject to the errors of the analytical methods employed, 27-77% of the dose was excreted in the urine of men, as glucuronides. Free BHA was not detected in human urine, and there was little evidence for sulfate conjugation. This work (Astill et al., 1962) shows t h a t a major metabolic pathway for BHA is common to rabbits, rats, and man. The possible storage of BHA in mammalian fatty tissues has also been investigated. When chickens were fed for 8 weeks and pigs for 4 months with 1000 ppm of BHA in their diet, no unchanged antioxidant was found in the dorsal and perinephric fats at the end of these feeding trials (Franqois and Pihet, 1960).

14

D. E. HATHWAY

Similarly, when groups of dogs were maintained f o r one year on diets containing B H A at 0.3, 3.0,30, a n d 100 m g k g a s a 50% solution in propyleneglycol, t h e r e was no storage of B H A in t h e body f a t , brain, liver, o r kidney (Hodge et al., 1964). Johnson e t al. (1958) found t h a t ingestion by rats of B H A in amounts equivalent t o 100 a n d 500 times t h e concentration permitted in human food caused a n increase in t h e stability of the perirenal f a t , whereas administration of B H A at the normal level of use did not produce a n y significant increase in stability. I n comparison with the amounts of antioxidant ingested by the rats, extraction of t h e f a t depots afTorded very small quantities of material with t h e antioxidant activity a n d color reactions of BHA. Whether B H A was directly responsible f o r the observed increase in stability of t h e f a t was not proved. B H A m a y have stabilized the biological antioxidants a n d caused them to accumulate in the f a t t y tissues. The foregoing studies on the metabolic f a t e of B H A were made with isotopically unlabeled antioxidant, a n d i t is f a i r comment t h a t t h e design of experiments a n d t h e resulting d a t a were entirely dependent on t h e scope a n d limitations of t h e analytical methods employed. I n t h i s section, reference is therefore made t o experimental detail. When very small doses (97 pg) of [U- :HI 3-tert-butyl-4hydroxyanisole were administered intraperitonea!ly t o r a t s , more t h a n 91% of t h e .:H w a s excreted in t h e urine d u r i n g 4 days (Golder et nl. 1962). The r a t e of excretion of bH when rats were dosed with [U- 'HI 3-te?f-butyl-4-hydroxyanisolew a s similar t o t h a t of the metabolic products (Astill ct al., 1960) when unlabeled BHA a n d its component isomers \ v e x administered at dose levels r a n g i n g f r o m 4 t o 200 times as great. A t a very low dose, B H A is probably excreted almost completely in a short time, a n d these results were considered to confirm the safety of E H A as a food antioxidant. Parenteral administration of B H A a n d B H T t o rats by Golder ~t crl. (1962) deserves comment. Thus, t h e best method of making a n intravenous injection of a lipid-soluble foreign compound is by slow infusion of a f a t t y emulsion o r colloidal solution which has previously been equilibrated with plasma itL vit7.o. Highly polar organic solvents are not ideal f o r t h i s purpose, since they may cause considerable loss of material through penetration of t h e venous wall n e a r the point of administration. Intraperitoneal administration of d lipid-soluble compound causes precipitation

METABOLISM O F HINDERED PHENOLIC ANTIOXIDANTS

15

in the peritoneal cavity, with possible immobilization th e r e of a variable proportion of the dose. This explanation may be relevant to the very variable proportion of a single intraperitoneal dose of [ I ' CI BH T which was excreted in t h e urine (Golder ct a l . 1962) ; elimination of I4C in th e feces was not. however, recorded. The metabolisni of Ionox 100 in the dog a n d r a t was investigated by W r i gh t et al. (1965b). A single dose (250 m g k g ) of "'C] Ionox 100 (2,6-di-te~t-butyl-4-hydroxy [14C]methylphenol) administered orally to rats was excreted almost quantitatively in 11 days. Very little (0.29 A: 0.02%) of th e dose w a s present in the carcass a nd remaining viscera a f t e r removal of th e gut. There were two main eliminative routes, 15.6-70.8% of "C being excreted in the urine, an d 75.2-27.0% in t h e feces d u r in g 11 days. During 96 h r , 12.5-66.9% of the "C w a s excreted in th e urine and 60.8-24.395 in the feces. The big variation in the elimination pattern of individual animals suggested t h a t the same p rima r y metabolic product ( s ) is ( a r e ) eliminated primarily by t h e fecal and ur i na r y routes. Approximately th e same proportion of a single dose as t h a t eliminated fro m r a t s d ur in g 96 hours w a s eliminated from dogs. Dogs arid rats do not show a species difference in this pattern of elimination. In dogs, excretion of "C in th e feces was t he main eliminative route. A fte r oral administration of ["C] Ionox 100, the r a t e of elimination a n d recovery of radioactivity from animals did not seem to be affected by th e size of t he dose (250 m g ' k g an d 20 m g j k g were given to ra ts , a n d 17.62 m g k g t o dogs) o r b y th e presence of triglyceride f a t in th e diet. Ionox 100 was completely metabolized in dogs a n d r a t s ; unchanged Ionox 100 was absent from th e urine a n d feces, a n d from the carcass when elimination was complete. F r om the urine of r a t s treated with [14C] Ionox 100, th e re were ['"C] oic acid a n d (3,5-diisolated 3,5-di-te?f-butyl-4-hydroxybenz tert-butyl-4-hydroxyhenz [ I .IC] oyl-p-D-glucopyranosid) uronic acid, which was similarly prepared fro m the urine of rabbits dosed with 3,5-di-te?*t-butyl-4-hydroxybenzoic acid. Th e crude reaction product from successive methylation an d acetylation w a s purified by column chromatography to give methyl (3,5-di-tert-butyl-4-hydroxybenz [ "C] oy1 tri-0-acetyl-p-D-glucopyranosid)uronate, identical with material similarly obtained fro m th e metabolic product of 3,5-ditert-butyl-4-hydroxybenzoic acid in rabbits. 3,5-Di-tert-butyl-4hydroxybenz ["C] oic acid accounts quantitatively f o r th e 14C in extra c t s of the feces. In rats, 3,5-di-tert-butyl-4-hydroxybenzoic acid accounts f o r 50-85% of a dose of Ionox 100, a n d (3,5-di-tert-butyl-

16

D. E. HATHWAY

4-hydroxybenzoyl-p-~-glucopyranosid)uronic acid f o r 47-10 c/o ; in dogs, t h e unconjugated acid accounts f o r 85% a n d t h e ester glucuronide f o r 10-12%. 3,5-Di-tert-butyl-4-hydroxyhippuricacid, which was synthesized, is not formed in vivo. Other metabolites detected in small quantity in th e feces and urine of animals dosed with Ionox 100 have not been identified. I n t hi s work, 3,5-di-tert-butyl-4-hydroxybenzoic acid (111) , Scheme 2, a nd the related ester glucuronide ( I V ) were shown to be m a j or metabolites of th e structurally related antioxidants B H T ( I ) a nd Ionox 100 (11) in rats. Th e elimination of Ionox 100 metabolites fro m rats is faster th a n t h a t of B H T a n d its metabolites. Unlike BHT, unchanged Ionox 100 could not be detected in the bodies of these animals.

SCHEME 2. SCHEME FOR THE METABOLISM IN RATS

OF BHT

(11 AND IONOX 100

(El

After publication of our results on t h e metabolism of Ionox 100 ( W r i ght et al., 1965b), o u r attention w a s d r a wn to work by Akagi a nd Aoki (1962a) in which i t wa s claimed t h a t 3,5di-te?*t-butyl-4-hydroxybenzaldehyde,3,5-di-tert-butyl-4-hydroxybenzoic acid, di- (3,5-di-tert-butyl-4-hydroxybenzyl),a n unidentified glucuronide (not [3,5-di-tert-butyl-4-hydroxybenzoyl-/3-~glucopyranosid] uronic acid, an d unchanged Ionox 100 had been isolated from t h e urine of rabbits dosed with Ionox 100 (1.6 g / k g body w e i ght ) . Identification of the free acid agrees with findings of JVright et al. (1965b) f o r rats, an d it is rath er surprising t h a t the corresponding ester glucuronide was not also identified, but it would have been hydrolyzed in th e exhaustive extraction with ether if several changes of solvent were not used a n d if the e th e r extracts

METABOLISM O F HINDERED PHENOLIC ANTIOXIDANTS

17

were not dried immediately a f t e r collection. However, excretion of t he lipid-soluble compounds Ionox 100, 3,5-di-tert-butyl-4hydroxybenzaldehyde, an d di- (3,5-di-te~t-butyl-4-hydroxybenzyl) in t he urine is not feasible. It seems possible that, at the high level of dose, some Ionox 100 might conceivably have been eliminated in t he urine a s a conjugate, which could have been hydrolyzed under the conditions described f o r the extraction, a n d undergone subsequent transformation in th e work-up. Scrutiny of the experimental procedure reveals, however, t h a t the researchers claimed t o have separated 3,5-di-tert-butyl-4-hydroxybenzaldehyde a nd Ionox 100 by extraction with aq. 10% KOH, in which neither compound is soluble, so g reat caution should accordingly be exercised in interpreting these results. Moreover, regardless of wh at happens with rabbits, rats rapidly metabolized Ionox 100 into 3,5-di-tert-butyl-4-hydroxybenzoic acid. Thus, a f t e r ingestion of ["C] Ionox 100, 90% of th e I'C was secreted into th e 20-hr bile of one male a n d one female rat with biliary fistulae (Dr. Wright, unpublished observation), and this radioactivity could also be accounted f o r as th e f r e e acid a n d its corresponding ester glucuronide.

c:.

COMPOUNDS W'ITH INTERMEDIATE RATESO F ABSORPTION, METABOLISM, AND ELIMINATION FROM BODYTISSUES

When male an d female rats were dosed orally with ["C] Ionox 220 (di- (3,5-di-tert-butyl-4-hydroxyphenyl) ["C] methane) ( 10 m g / k g ) , a nd p airs were killed 4, 8, 16, a n d 24 days a f t e r dosage, th e main eliminative route was fecal; 89.4-97.5% of th e original label was excreted in th e feces during 24 days (Wr ig h t et al., 1966a). Ra t s do not show a sex difference in th e p a tte r n of elimination of Ionox 220 an d its metabolites. Initial elimination in th e feces, however, was comparatively rapid, 86.58-94.62% (8 animals) of t he l*C in 4 days. Retention of I-'C in th e body tissues of these animals a n d secretion of "C into th e bile of animals with biliary fistula showed t h a t there was appreciable alimentary absorption a f t e r oral dosage. After removal of th e g u t fro m r a t s killed 4 days a f t e r dosage, the carcass a nd remaining viscera of animals contained 8.2% of I4C, a level t h a t fell t o 4.0% in animals killed 24 days a f t e r dosage. A f t e r a n oral dose of [I4Cc] Ionox 220, more l-'C w a s stored in t he f a t t y tissues, including th e intraintestinal a n d subcutaneous f a t s a nd pelts, t h a n in th e soft organs. Retention of "C in th e f a t t y tissues was g reater in female rats th a n in males. Ob-

18

D. E. HATHWAY

seruntions on th e secretion of 1 4 C into bile suggested t h a t a low degree of alimentary absorption had taken place a n d t h a t diffusion of ["C] products fro m plasma to bile w a s slow. A t least 15% of the I4C excreted in th e feces d u rin g 24 days originated in the bile, since 11% of 14C was secreted in the 30-hr bile a n d 4.1': of "C was eliminated f r o m the organs a n d tissues d u r in g 4-24 days. When allowance is made f o r the retention of I4C for 24 days after dosage, i t follows t h a t approximately 20% of a single dose of [' 'C] Ionox 220 was absorbed in ra ts . The small amount of radioactivity (<1% of dose) in the urine of r a t s dosed with ["C] Ionox 220 was due to 3,5-di-tert-butyl4-hydroxybenz ["C] oic acid an d 3,5-di-tert-butyl-4-hydroxybenz [I oyl-p-D-glucopyranosid) uronic acid. These are th e metabolites t h a t were excreted in the urine of animals dosed with Ionox 100 (Secticn 111,B). Most of th e I4C in t h e total lipid fraction of the feces of treated r a t s was accounted f o r by unchanged [I4C] Ionox 220, but there were also small proportions of 3',5'-di-tertbutyl-4'-hydroxyphenyl- (2,6-di-tei?-butyl-p- benzoquinone) [I4C] methide ( ["C] quinone methide) (11) (see Scheme 3 ) , 3,5-ditcrt-butyl-4-hydroxybenz [ l 'C] oic acid (111), a n d a polar unidentified [ l 'C] component contaminated with traces of 3,5-difcrf-butyl-4-hydroxybenz[14C]oic acid. Unlabeled 2,6-di-tertbutyl-p-benzoquinone ( I V ) was also detected a n d identified in the fecal lipids by its chromatogrqphic a n d spectroscopic properties. I n t he body f a t of rats dosed with [ ' 'C] Ionox 220, 97% of th e I 4 C n.as present a s unchanged ["C] antioxidant, a n d the remainder was due to a small proportion of 3 ,5 -d i-t~ t- b u ty l- 4 hydi oxybenz [ I'C] oic acid an d traces of the L ' 'C] quinone methide a nd of the unidentified [ ' 'C] component. n'hen a biliary fistula was established in r a t s t h a t had pre~ i o u s l ybeen dosed with ["C] Ionox 220, the radioactivity t h a t was collected in the 24-hr bile was due to variable proportions of (3,5-di-tert-butyl-4-hydroxybenz [14C] oyl-p-D-glucopyranosid) uronic acid ( V ) , the f r e e [I4C] acid, and unchanged ["C] Ionox 220. Rigger amounts of th e free ["C] acid a n d [I4Cc] ester glucuronide t han those excreted by th e kidneys were secreted into bile. A simple scheme f o r th e metabolism of Ionox 220 in vivo is shown in Scheme 3 . On account of th e relationship of th e metabolic f a t e of t his antioxidant t o its suspected antioxidant action in vitro, t h e metabolic pathway suggested in Scheme 3 requires

METABOLISM O F HINDERED PHENOLIC ANTIOXIDANTS

19

slight amendment; th is is discussed in Section IV. T h e metabolic pathway of Ionox 220 differs f r o m those of other diphenylmethanes. Unsubstituted diphenylmethane is excreted largely as th e 4-hydroxy derivative (Stroud, 1940), a n d p-bromodiphenylmethane is not oxidized in vivo (Klingenberg, 1 8 9 1 ) . 0 OH

0

I

II

m

Y

SCHEME 3. SCHEME FOR THE METABOLISM OF IONOX 220 IN RATS

One-fifth of a single oral dose of Ionox 220 wa s absorbed in rats, and not all of th is proportion of t h e dose w a s metabolized, since some unchanged antioxidant accumulated in t h e f a t t y tissues in vivo a n d was secreted unchanged into t h e bile. Among th e metabolic products of Ionox 220, 3,5-di-te)f-butyl-4-hydroxybenzoic acid accounted f o r ju st over 5% of th e dose, (3,5-di-tertbutyl-4-hydroxybenzoyl-p-~-glucopyranosid) uronic acid f o r up to 0.4% of the dose. th e quinone inethide f o r ju s t over 5% of t h e dose, a nd t he unidentified polar constituent f o r u p to 3% of the dose. Thus, 13-1492 of a single oral dose was metabolized. The r a t e of metabolism of foreign compounds is lowered by factors t h a t diminish th e effective concentration of foreign compounds at t he active sites of th e liver microsomal drug-metabolizing enzymes. Ionox 220 seems to have been precipitated in th e gastrointestinal tract, an d dissolution of th e solid s ta te take place slowly, with th e result t h a t its r a t e of metabolism by liver microsomal enzymes is limited by th e r a t e of alimentary absorption a nd concentration in portal blood. The effective concentration of Ionox 220 in peripheral circulation is lowered by localization in t he f a t t y tissues, which f o r a long period maintain a low plasma concentration of antioxidant t h a t is metabolized a n d excreted very slowly. Other physiological an d biochemical implications which result fr om ingestion of Ionox 220 were discussed by Wr ig h t et al. (1966a).

20

D. E. HATHWAY

When rats equipped with biliary fistulae \yere treated orally with [ " C ] Ionox 201 (di- (3,5-di-te~~t-butyl-4-hydroxybenz-".'C]yl) ether), measurements of radioactivity in the bile, exhaled air, and urine, and in the carcass and viscera remaining after removal of the gut, showed that up to 32% of the I-'C had been absorbed (Wright e t al. 196613). When rats were dosed orally with ["C] Ionox 201 (34 mg kg body weight), the pattern of elimination was essentially similar to that for oral dosage with [l4CC] Ionox 220. Thus, in a n experiment in which pairs were killed 1, 4, 8, 16, and 24 days after dosage, the main eliminative route was fecal, and 86.8-97.2% of the original label was excreted in 24 days. Up to 5.6% of the l l C was excreted via the kidneys during this period, and 0.8% in the exhaled a i r during 48 h r . Initial elimination of the I 4 C in the feces was slow, 20.2% in 24 hr, but 90.0% in 4 days (8 animals). Rats did not show a sex difference in this pattern of elimination of Ionox 201 and its metabolites (Wright et nl., 1966b). The carcass and viscera remaining after removal of the g u t and contents of animals killed 24 h r after dosage contained 9.2% of 14C, a concentration that fell to 5.5% in animals killed 4 days after dosage, but the fact t h a t similar levels-5.0 (3.4-5.9) %were also found in the carcasses of animals killed 8, 16, and 24 days after dosage is indicative of storage. After oral dosage of [14C] Ionox 201, more was stored in the fatty tissues than in the organs. Retention of I4C in fatty tissues was greater in female rats than in males. At least 25% of the I4C excreted in the feces during 24 days originated in the bile. Secretion of I-'C into the bile was maximum approximately 6 h r after dosage. After 16 hours, the concentration of "C in the bile fell very slowly. These observations indicate a low degree of alimentary absorption and slow diffusion of I4C-labeled products from plasma to bile. About 65.0% of I'C in the feces of animals dosed with [14C] Ionox 201 was due to unchanged antioxidant, 30.0% t o 3,5-ditert-butyl-4-hydroxybenzoic acid, 3.5% to unidentified polar conand stituent (s), 1.4% to 3,5-di-tert-butyl-4-hydroxybenzaldehyde, 0.1% to 3,3',5,5'-tetra-tert-butyl-4,4'-stilbenequinone.A variable proportion of I-'C in the urine was due to 3,5-di-tert-butyl-4hydroxybenzoic acid (40-60% ) , and the remainder (60-40% to the ester glucuronide, when the animals were treated with different dose levels of antioxidant. In eight animals dosed with 6.78 m g of [14C] Ionox 201, one-third of I 4 C in the bile was due to the

METABOLISM O F HINDERED PHENOLIC ANTIOXIDANTS

21

free acid, 45% to the ester glucuronide, 20% to a n unidentified constituent, and 2% to unchanged antioxidant, and in two animals dosed with 13.56 mg there was a smaller proportion of free acid and a larger proportion of ester glucuronide. About 80% of 14C in the body f a t was due to unchanged antioxidant, 19% to the free acid, and 1% to 3,5-di-tert-butyl-4-hydroxybenzaldehyde. In summary, up t o one-third of a single oral dose of Ionox 201 was absorbed in rats, and not all of the absorbed dose was metabolized, since some unchanged antioxidant was concentrated in the fatty tissues in vivo. A proportion of the dose was transformed into (metabolic) products in the intestinal tract. Of a single dose of Ionox 201, 36.2% is metabolized : 3,5-di-te~t-butyl-4hydroxybenzoic acid accounts for 30.2%, 3,5-di-tert-butyl-4-hydroxybenzoyl-p-D-glucopyranosiduronic acid for 1.4%, 3,5-di-tertbutyl-4-hydroxybenzaldehyde for 1.3%, 3,3’,5,5’-tetra-tert-butyl-4, 4’-stilbenequinone for 0.1 %, and unidentified polar metabolite ( s ) for 3.2%. (CHihC HO (CH3)3C

/ \

C(CH3h

\

CHZ.O.CHZ

t1

QCH,)s

OH

1

SCHEME 4. SCHEME FOR THE METABOLISM OF IONOX 201 IN RATS

A scheme for the metabolism of Ionox 201 ( I ) in vivo is tentatively suggested in Scheme 4. There a r e two metabolic pathways : on one of them lies 3,5-di-tert-butyl-4-hydroxybenzaldehyde (11), the benzoic acid (111), and the ester glucuronide ( I V ) , and on the other lies 3,3’,5,5’-tetra-tert-butyl-4,4’-stilbenequinone ( V ) . It may be relevant that, since compounds 11, 111, and

22

D. E. HATHWAY

V can also be produced by mild oxidation i j i ? * i t t o , their formation iii vivo need not necessarily be enzymic. However, conjugation of 111 i?i v i m t o t h e ester glucuronide ( I V ) is undoubtedly a n enzymic step. As will be discussed (Section I V ) , t h e metabolism of Ionox 201 i ~ i?'ire is c’ Jely related to its antioxidant action iii z’iti.0, a n d i t follows t h a t t h e presence of 3,5-di-te~t-butyl4-hydroxybenzaldehyde (11) a n d 3,3’,5,5’-tetra-f~? t-butyl-4,4’stilbenequinone ( V ) in t h e feces, a n d of I1 in the f a t t y tissues, probably arose by radical processes in t h e two biological situations. Since compounds I1 a n d V were found also in the feces of germ-free animals, they were not formed by the intestinal flora. Until recently, the metabolic f a t e of B H T in animals had been investigated with unlabeled antioxidant, and, a s with BHA, the design of experiments a n d resulting d a t a were entirely dependent on the scope a n d limitations of t h e analytical methods used. Thus, Dacre (1961) found t h a t rabbits dosed orally with B H T (0.8 g / k g body w t ) excreted 54.1 I7.2% of t h e dose in the urine d u r i n g 4 d a y s ; none of this was unchanged antioxidant. Dacre (1961) says t h a t the f a t e of the remainder of t h e dose is unknown. Golder ef al. (1962) a n d Ladomery ef al. (1963) measured the elimination of ’ H a n d 14C in the urine of rats dosed intraperitoneally with [U-’H] B H T a n d [I4Cc] B H T (0.5 m g l k g body w t ) . Those workers found t h a t t h e amount of radioactivity in t h e urine corresponded t o about 33% of the dose d u r i n g 4 days, a n d they did not measure radioactivity in the feces. Those experiments suggested t h a t there might be a n appreciable retention of BHT in t h e body tissues a f t e r a single dose, a n d some evidence f o r this had also arisen f r o m investigations of t h e possible storage of B H T in animal tissues. Thus, supplementation of the diet of chronically fed r a t s with 10% of a 1% solution of BHT in coconut oil increased the stability of the perirenal f a t of male a n d female rats (Johnson ct al., 1958). As with BHA, extraction of these f a t depots failed t o establish whether B H T w a s directly responsible f o r the observed increased stability of f a t . When chickens were fed f o r 8 weeks a n d pigs f o r 4 months with 1000 ppm of BHT in cheir diet, BHT was stored in t h e f a t of the chicken at a concentration of 60 ppm, but none was detected in the body f a t of pigs. The concentrations f o r chickens agree ivith values (52-56 ppm of B H T ) found in the body f a t of chickens fed chronically with 500 ppm of B H T in their diet (van S t r a t u m and Vos, 1 9 6 5 ) . Daniel a n d Gage (1965) recently found t h a t 80-9096 of a single

METABOLISM O F HINDERED PHENOLIC ANTIOXIDANTS

23

oral dose of [14C]BHT ( 5 mg/kg) in rats was excreted during

4 days. Over-all excretion was slightly faster in female rats than in males. I n females, about 40% of the dose was excreted via the kidneys, and in males about 25%. When a single dose of [l4CC] BHT was administered t o rats fed - diet containing 0.5% of BHT, excretion was slightly more rapid, and approximately 40% of the I4C was excreted in the urine of male and female rats. This may be related to development of a tolerance observed in rats chronically fed diets containing BHT. Two and four days after oral administration of [14C] BHT ( 5 mg/kg) to rats, respectively 14.8 and 3.8% of the I4C was retained in the body tissues. The bulk of this radioactivity was in the small intestine, even a t 4 days, when excretion of 14C in the feces was low. This evidence, together with the somewhat higher concentration of I4C in liver than in the other organs and the considerable secretion of I J C into bile, provides strong evidence that a large proportion of the BHT metabolites are secreted into bile and reabsorbed from the intestine. Since the low concentrations of 14C in the other tissues decrease a t about the same rate as does the amount of 14C in the gut, the kinetics of the excretion of BHT metabolites seems to be explicable in terms of enterohepatic circulation, not of tissue retention. Daniel and Gage (1965) also determined, by chemical analysis, the concentration of BHT in the liver and body f a t of rats chronically fed a diet containing 0.5% of BHT. The concentration of BHT in f a t depots rapidly attained an equilibrium concentration, which was maintained throughout the experimental period: 30 ppm for males and 45 ppm for females. During a ’I-week exposure, there was no evidence for progressive accumulation of BHT in the fatty tissues. After the ingestion of BHT had ended, the Concentration of antioxidant in the fatty tissues fell logarithmically, giving a half-life period for BHT of 7-10 days. Throughout the experimental period the concentration of BHT in the fatty tissues and liver was very low. Daniel and Gage (1965) found that 0.064% of a single dose of BHT was present in 1 g of f a t 2 days after dosage, and since the animals received a daily dose of 100 mg of BHT, the concentration in f a t would be expected t o exceed 100 ppm at that time. I n an unpublished report that later appeared in summary form, Tye e t al. (1965) gave results rather similar to those of Daniel and Gage (1965) for the excretion and retention of I4C in rats after ingestion of [“C] BHT. Repeated oral dosage did not in-

24

D. E. HATHWAY

fluence the rapid elimination of I4C from the body tissues. For female rats, the main eliminative route was via the kidneys, and for males most of the I4C was eliminated in the feces. Greater absorption of ['C] B H T in females led to higher tissue concentrations of 14C than in males. There was no evidence for progressive accumulation of ['*C] B H T in the body. Rats dosed subcutaneously with ["C] BHT excreted 91% of the 14C during 4 days when the dose was 0.0003 mmoles/kg body wt, but only 37% of the 14C when the dose was increased to 0.025 mmoles/kg body wt. It might be unsound to make deductions from this observation, since unknown proportions of these doses may have been localized near the point of administration. Dacre (1961) found t h a t glucuronides account f o r 36% of a single oral dose of BHT in rabbits and that 16% of the dose could be attributed to ester-type glucuronide. Two metabolites were isolated from the urine as crystalline methylated-acetylated derivatives. The ester glucuronide was (3,5-di-tert-butyl-4hydroxybenzoyl-p-D-glucopyranosid) uronic acid, which was chromatographically indistinguishable from the glucuronide similarly isolated from the urine of rabbits treated with 3,5-di-tertbutyl-4-hydroxybenzoic acid. Methyl (3,5-di-tert-butyl-4-hydroxybenzoyl tri-0-acetyl-p-D-glucopyranosid) uronate prepared from the ester glucuronide in the urine of rabbits dosed with BHT was identical in every respect to the compound prepared from the authentic ester glucuronide recovered from the urine of rabbits dosed with the free acid. Besides the ester glucuronide, the urine of rabbits dosed with BHT contained a glucuronide, which was -pp-diformulated as p- (3-tert-butyl-2-hydroxy-5-methylphenyl) methylethyl-p-D-glucpyranosiduronic acid, and which was isolated as the corresponding methyl tri-0-acetyl uronate. The aglycone, which has not been synthesized, was nevertheless proas p- (3-tert-butyl-2-hydroxy-5-methyl\-isionally identified phenyl ) -pp-dimethylethanol. On the assumption t h a t this is correct, BHT is oxidized in the rabbit either by conversion of the methyl substituent into a carboxyl group or by w-oxidation of one of the te~t-butylsubstituents to the primary alcohol stage. There was precedent for w-oxidation of a tert-butyl substituent, since Robinson and Williams (1955) had found that up to 90% of a dose of tert-butylbenzene was oxidized in rabbits to 2,2-dimethyl8-phenylethanol, and excreted as the glucuronide. I n addition t o the two glucuronides, the urine of rabbits dosed with BHT also contained free 3,5-di-te?*t-butyl-4-hydroxybenzoic acid (Dacre,

METABOLISM O F HINDERED PHENOLIC ANTIOXIDANTS

25

1961). When rats were dosed with BHT, however, Dacre’s (1961) ether glucuronide was not found among the metabolites in the urine, in which 3,5-di-tert-butyl-4-hydroxybenzoicacid and the ester glucuronide were present (Wright et al., 1965b). 3,5-Di-tertbutyl-4-hydroxybenzoic acid was not conjugated with glycine in dogs, rabbits, or rats (Wright e t al., 196513). The metabolism of BHT in rats is postulated in Scheme 2. At the time of Dacre’s (1961) investigation of the metabolites of BHT in the urine of rabbits, a similar study was made in Japan, also with rabbits (Akagi and Aoki, 1962b), and, in addition, those workers isolated unchanged BHT from the feces. From the urine, isolation of the following compounds was claimed : Ionox 100, 3,5-di-tert-butyl-4-hydroxybenzaldehyde,3,5-di-te~tbutyl-4-hydroxybenzoic acid, di- (3,5-di-tert-butyl-4-hydroxybenzyl), and an ether glucuronide as the corresponding methyl 0-triacetyl glucopyranosiduronate with the same [a] value and melting point as Dacre’s ether glucuronide. The identification of the free acid in the urine agrees with Dacre’s (1961) findings, and the corresponding ester glucuronide, which was also present, would have been hydrolyzed in the 60-hr ether extraction if several changes of solvent had not been used, and if the ether extracts were not dried immediately after collection. No attempt was made to hydrolyze the ether glucuronide, and the evidence for its chemical structure is due entirely t o Dacre (1961). A possible explanation for the alleged presence of three lipid-soluble constituents in the urine is that a small quantity of Ionox 100 might have been excreted via the kidneys as the 0-sulfate, which could conceivably have been hydrolyzed under the extraction conditions and have undergone subsequent transformation in the work-up. However, the researchers claimed to have separated 3,5-di-tert-butyl-4-hydroxybenzaldehyde and Ionox 100 by extraction with aq. 10% KOH, in which the two compounds are insoluble, so great caution should be exercised in interpreting their data. IV.

ANTIOXIDANT ACTION

RELATIONSHIP O F METABOLISM TO ANTIOXIDANT ACTIONin V i t ~ o Section 1,B made reference to the fact that the phenolic group in the BHA isomers is not hindered in the same way as are the phenolic groups belonging to the other antioxidants discussed in

26

D. E. HATHWAY

this chapter. It is perhaps not surprising that a similarity between metabolism in vivo and antioxidant action in vitl'o, which characterizes some of the other antioxidants, does not hold for RHA. When the molar disappearance of BHA was compared with the molar appearance of peroxide oxygen during the aging of cereals, approximately 5 moles of peroxide oxygen were found to be present for every mole of BHA oxidized (Anderson et al., 1963). Since the amount of peroxide oxygen (present in each cereal) in relation to the BHA destroyed was approximately proportional to the amount of lipid present, the rate of disappearance of BHA was related to the concentration of lipoperoxides in the cereals, and was therefore independent of the total amounts of peroxides or the amount of lipid present. The reaction of BHA with peroxide radicals is more complicated than a simple equimolar reaction, which would be expected to give a dimeric oxidation product corresponding t o the destruction of two oxidation chains. In that work, no 2,2'-dihydroxy-3,3'-di-te~t-butyl-5,5'-dimethoxydiphenyl, which is the isolable oxidation product of BHA (Rosenwald and Chenicek, 1951), was formed. The results of Anderson e t al. do not conflict with the supposition that BHA is destroyed by reactions with peroxide radicals to form complexes of antioxidant with peroxide radicals and possibly a hydroperoxide o r alkylperoxide, in a manner analogous to that proposed by Boozer et al. (1955). BHA is readily transformed iii vivo into hydrophilic conjugates that are rapidly excreted via the kidneys, and the metabolic reactions involved are not related to the lipoperoxide-induced oxidations that have been described. In contrast to the antioxidant behavior of BHT, exemplified in Section I,B (see also Scheme 9 ) , 3,5-di-tert-butyl-4-hydroxybenzaldehyde is formed from Ionox 100 by an attack of the alkylperoxy- or alkoxy-radical on the p-hydroxymethyl group (Scheme 5 ) . This implies that using Ionox 100 as an oxidation inhibitor will afford hydroperoxide only in the inhibitor step. Since Ionox 100 lvas oxidized it& vivo into 3,5-di-tert-butyl-4-hydroxybenzojc acid OH

OH

+ 2 RO*OH SCHEME 5. ANTIOXIDANT BEHAVIOR OF lONOX 100

METABOLISM OF HINDERED PHENOLIC ANTIOXIDANTS

27

(111) (Scheme 2 ) , the p-hydroxymethyl substituent of Ionox 100 is therefore attacked both in mammalian metabolism and in the lipoperoxide-induced oxidations, which would be expected to take place in the protection of 02-labile polyunsaturated lipids by this antioxidant in vitro. The metabolism of Ionox 220 in vivo seems to be closely related to its expected antioxidant action in vitro. Thus, Wright et al. (1966a) have demonstrated that in vitro oxidation of Ionox 220 ( a ) (Scheme 6 ) to Coppinger’s (1957)

id-

~

_p--.--i

SCHEME 6. ANTIOXIDANT ACTION OF IONOX 2 2 0

stable phenoxyradical (c ) proceeds via 3’,5’-di-tert-butyl-4’-hydroxyphenyl- (2,6-di-te~t-butyl-p-benzoquinone) methide ( b ) (the quinone methide). Further, Kharasch and Joshi (1957) have shown that oxidation of 2 molar proportions of Coppinger’s (1957) stable phenoxy radical (c) by molecular oxygen in alkali affords 1-molar proportions of each of the products: the quinone methide ( d ) , and 2,6-di-tert( b ) , 3,5-di-tert-butyl-4-hydroxybenzaldehyde butyl-p-benzoquinone (e) . Since these compounds were also formed by autoxidation of Ionox 220 on a thin-layer plate of SiO,-gel in the presence of sunlight, the chain reaction suggested in Scheme 6 appears to typify the antioxidant action of Ionox 220 in vitro. Since the metabolism of Ionox 220 (u) (Scheme 7) in vivo affords the quinone methide ( b ), 3,5-di-tert-butyl-4-hydroxybenzoic acid ( d ) , the ester glucuronide ( f ) , and 2,6-di-tertbutyl-p-benzoquinone ( e ) , the preferred scheme for the metabolism of Ionox 220 in vivo is that shown in Scheme 7 ( c f . Scheme 3 ) . Coppinger’s (1957) stable phenoxy radical ( c) has not, how-

28

D. E. HATHWAY

LL

0

METABOLISM O F HINDERED PHENOLIC ANTIOXIDANTS

29

ever, been isolated in metabolism investigations. The striking similarity between Schemes 7 and 6 reflects the close relation between the in vivo metabolism of Ionox 220 and its autoxidation in vitro. Since compounds b-e (Scheme 7) can also be produced by gentle oxidations in vityo, their formation in vivo need not necessarily be enzymic, and may result from antioxidant action in the feces and body fats (Wright et al., 1966a). The conjugation of compound d, which results in the ester glucuronide ( f ) , is undoubtedly enzymic. The metabolism of Ionox 201 in vivo appears to be closely related to its antioxidant action in vitro. The antioxidant action of Ionox 201, which involves a radical mechanism, was simulated by reaction with a single-electron oxidizing agent, lead dioxide (Wright et al., 1966b). Produced besides compounds 11, 111, and V (Scheme 4) were the following compounds : di- (3,5-di-te~t-butyl4-hydroxybenzyl) (VI) (Scheme 8 ) , 3,3’,5,5’-tetra-tert-butyl-4, 4’-diphenoquinone (VII) , 2,6-di-tert-butyl-p-benzoquinone(VIII) , the quinone methide (IX) , and Coppinger’s stable phenoxy radical (X) All of these compounds, 11-X, except f o r the ester glucuronide ( I V ) (Scheme 4 ) , can be derived from two radicals (Cosgrave and Waters, 1951), a and b (Scheme 8 ) , themselves generated in the oxidation reaction. It follows that the presence of 3,5-di-tc~t-butyl-4-hydroxybenzaldehyde (11) and 3,3’,5,5’-tetratert-butyl-4,4‘-stilbenequinone(V) i n the feces and of I1 i n the fatty tissues probably arose by radical processes in the two biological situations (see Section II1,C). The oxidation of BHT ( I ) (Scheme 9) has been investigated with various oxidants. Thus, small quantities of the corresponding stilbene-4,4’-quinone (11) and the related 4,4‘-dihydroxydibenzyl (111) were produced by protracted reaction of I with perbenzoic acid (Cosgrave and Waters, 1951; Yohe e t al., 1953), and these compounds were also obtained by reaction of BHT with lead dioxide or alkaline ferricyanide (Cook, 1953; Cook et al., 1955). As mentioned in Section I,B, treatment of BHT with alkyl or aralkylhydroperoxide and cobalt naphthenate gave 2,6-di-tert-butyl-4methyl-4-alkyl (or aralkyl) peroxy-2,5-cyclohexadien-l-one(IV) (Campbell and Coppinger, 1952; Bickel et al., 1953). I n general, reaction of BHT with molecular oxygen in the presence or absence of alkali gave very low yields of products. Thus, a small quantity of 3,5-di-tert-butyl-4-hydroxybenzaldehyde ( V ) was formed in lubricating oil at 110°C in the presence of copper (Wasson and Smith, 1953), and a small amount of 2,6-di-tert-butyl-p-benzoqui-

.

m

P

0

w

METABOLISM OF HINDERED PHENOLIC ANTIOXIDANTS

31

none ( V I ) was isolated from a heated reaction mixture in the presence of a copper-iron catalyst (Metro, 1955). It follows that the primary reaction product of BHT with 0 2 is unstable, and that this undergoes further reactions under the influence of heat or in the presence of alkali. When a solution of BHT in alcoholic potash was shaken with O2 until one molar equivalent was absorbed,

P

m

AcOH,Nal y1

t 0 (CHhC414

,b H3CAOH

C(CH3h

I'

m

SCHEME 9 OXIDATION REACTIONS OF BHT /N VITRO

there was recovered in good yield a hydroperoxide that liberated iodine from acidified iodide reagent (Kharasch and Joshi, 1957). The hydroperoxide (VII) and the iodide reaction product (VIII) have been elucidated by their spectroscopic properties and chemical transformation products (Kharasch and Joshi, 1957). When the molar disappearance of BHT was compared with the molar appearance of peroxide oxygen during the aging of cereals, the rate of disappearance of BHT was found to be related to the

32

D. E. HATHWAY

concentration of lipoperoxides in the cereals, and was independent of the total amounts of peroxides or the amounts of lipid present. (111) nor 3,3‘,5,5‘Neither di- (3,5-di-te?t-butyl-4-hydroxybenzyl) tetra-tel-t-butyl-4,4’-stilbenequinone(11) was formed. These results (Anderson et al., 1963) do not conflict with the supposition that BHT is destroyed by reactions with peroxide radicals to form a hydroperoxide ( V I I ) or alkylperoxide ( I V ) and complexes of the antioxidant with peroxide radicals, in a manner analogous to that proposed by Boozer et a?. (1955) and Campbell and Coppinger (1952). Since B H T was oxidized i r ~vivo into 3,5-di-tert-butyl-4-hydroxybenzoic acid (111) (Scheme 2 ) , it follows that the methyl substituent in 4-position of the BHT molecule is attacked in mammalian metabolism ; and in the lipoperoxide-induced oxidations, which would be expected to occur in the protection of 0,labile polyunsaturated lipids by BHT in vitro, the 4-position is attacked by the alkylperoxy radical to afford 2,6-di-tert-butyl-4methyl-4-alkylperoxy-2,5-cyclohexadien-l-one( I V ) (Scheme 9 ) . The w-oxidation of a tert-butyl substituent, which results from the metabolism of BHT in rabbits (Dacre, 1961), is without counterpart in the oxidation and autoxidation reactions of BHT in vitro. Under certain circumstances, it is possible that alkyl- and hydroperoxides might be formed in the antioxidant action of some structurally related hindered phenolic antioxidants, such a s Ionox 330, but these products have not been described. V.

ASPECTS OF THE PHYSIOLOGICAL CHEMISTRY OF HINDERED PHENOLIC ANTIOXIDANTS

A. ALIMENTARY ABSORPTION I N RELATIONTO

MICELLARFORMATION Transport of foreign organic compounds in animals depends on the availability of existing physiological mechanisms. Relevant to the intestinal absorption of lipid-soluble hindered phenolic antioxidants is the absorption of lipids, particularly sterols. Experiments on the absorption of sterols suggest that they a r e absorbed from a molecular dispersion in the lumen and then transferred to the lipoproteins of the mucosal cells. Besides fats dispersed in micellar form by bile salts and lower glycerides, intestinal fluid also contains finely dispersed cholesterol, either in micellar form or as a lipoprotein. Cholesterol forms mixed micelles

METABOLISM O F HINDERED PHENOLIC ANTIOXIDANTS

33

with sodium taurodeoxycholate and lecithin, either individually or collectively, and bile entering the intestine already contains cholesterol dispersed in bile salt-phospholipid micelles. Lysolecithin, formed from bile lecithins by the action of pancreatic lecithinase A, is more effective than lecithin in solubilizing cholesterol prior to absorption (Barton and Glover, 1965), and is also superior to bile salts and to a n optimum combination of taurodeoxycholate and lecithin in solubilizing lipids. Lysolecithin forms micelles at lower concentrations ( <0.1 mmole/liter) than bile salts (ca. 1.0 mmole/liter) , and, consequently, dilution is less likely to cause breakdown of lysolecithin-cholesterolmicelles (Barton and Glover, 1965). On this basis, Glover (private communication, 1965) examined the capacities of Ionox 330, Ionox 100, and BHT to enter mixed phospholipid-cholesterol micelles (Table 11), and found that, in comparison with BHT and Ionox 100, Ionox 330 was poorly dispersed in the free micelles. The chance of its being taken up by the more highly organized lipid in the membrane of the mucosal cells is even less likely, and this is consistent with the nonabsorption of this antioxidant from the gastrointestinal tract (Wright et al., 1965a). Further, micellar solutions containing Ionox 330 a r e not as translucent a s those containing B H T and Ionox 100, suggesting that the micellar solutions of Ionox 330 a r e more susceptible to breakdown. On the evidence that BHT is taken up by phospholipid-cholesterol micelles more effectively than Ionox 330 (Table 11), the former antioxidant would be expected to be reasonably well absorbed. In agreement with which, Daniel and Gage (1965) found (.‘.\I> \ r . i n OF

TABIJC I1 ITISIIEIEDI’IIEYOI,IC AXTIOSII).IKTSTO ENTER ~ I I X E II’imwinmw) C1inmsmitoL ~ I I C E L L E S ~

Rlicellar solutiotis IAccitliin 1,ccithiti Lwithin 1,ysolecithiir J,ysolecithin Lysolccithin

: Cholesterol : Ionox 3:)O Ionox 100

: Cholcstcrol : : Cholestcrol : : Cliolcsterol : : Cholesterol : : Cholesterol :

BHT Ionox 330 Ionox 100

BHT

r .

4G.0 : 9 . 5 : 1 : 3.i : 1 : 1.4 : 1 : 14.5 : 1 : 1.3 : 1 6 . 5 : 1.8 : 1

21.0 5 5 51.0 9.0

1 hr niiccllar solutions were made t o contain a constant amount of the phospholipid

:it, 2 irirnol(./litc~rj)liosphate huffer, pH 6 8 (0 14.11).

34

D. E. HATHWAY

t h a t 53% of a single dose was secreted into th e 40-hr bile of a male r a t a nd 17% into t h a t of a female, a n d when t h e correspondin g ur i na r y excretions were taken into consideration, minimal figures f o r absorption were 68% of th e dose f o r th e male r a t a n d 56% f o r t he female. The in T i t m studies (Table IT) also suggested t h a t t he r e would be a poorer absorption of Ionox 100 t h a n of BHT, whereas 90% of a n oral dose was secreted into the 20-hr bile of one male a nd one female rat with biliary fistulae (Wright, unpublished observation). I t seems t h a t th e formation of stable niiscelles is a prerequisite of intestinal absorption, an d t h a t th e absorption of hindered phenolic antioxidants corresponds roughly with their capacity to expand phospholipid-cholesterol micelles. B. SECRETION INTO BILE

3,5-Ili-tof-butyl-4-hydroxybenzoic acid a n d its corresponding ester glucuronide, which are majo r metabolites of Ionox 100, Ionox 220, Ionox 201, an d BHT, a r e secreted into bile, a n d the concentrations in bile a r e related to the r a t e of absorption a n d subsequent metabolism of each of the fo ur antioxidants. I n addition to 3,5-di-tert-butyl-4-hydroxybenzoicacid a n d its ester glucuronide, other substances, including th e unchanged antioxidants themselves, are secreted into th e bile of rats dosed with Ionox 220 o r Ionox 201. The presence of these antioxidants in th e bile could be controlled by th e n atu re of th e lipid complex in bile, which m a y be considered as a mixed micelle, containing phospholipids, bile acids, f r e e f a t t y acids, an d cholesterol (Desai et al., 1965). It is known t h a t other unionized compounds, such as neut r a l steroid metabolites an d the xanthophylls, are also excreted via t he liver. B H A isomers a r e transformed i l l ciro into conjugates with massi\-e renal clearance, an d ingestion of BH A is not followed by biliary secretion of BIIA metabolites. The secretion of foreign organic compounds into body fluids is important, but, as f a r as we are aware, t h e milk of lactating animals f e d fodders containing hindered phenolic antioxidant (s) h a s not been examined f o r th e possible presence of unchanged antioxidant (s) a nd its (th eir) metabolic products.

c. TRANSPORT FROM MOTHERTO EMBRYO Lipid-soluble foreign organic compounds, which a r e absorbed and not rapidly metabolized an d excreted, a r e tr a n s f e r r e d in

METABOLISM O F HINDERED PHENOLIC ANTIOXIDANTS

35

small proportion from the maternal blood to the developing enibryo (i??tcr alia, Hathway et al., 1966) ; this applies to some hindered phenolic antioxidants. Thus, in the egg lipids of laying hens with a dietary intake of 500 ppm of BHT, a fairly constant concentration of unchanged antioxidant (20 ppm) was found during 2-16 weeks after dosage (van Stratum and Vos, 1965). The lowest dietary level of BHT that led to detectable amounts of antioxidants in egg lipids was 100 ppm, and in this diet the lipid component contained twenty times the concentration of BHT recommended for the stabilization of f a t s and oils. Hence, if lipids stabilized with the recommended concentration of BHT were incorporated into the rations of laying hens, detectable amounts of antioxidants are unlikely to be found in the eggs. In the eggs of laying hens chronically fed diets containing 200 ppm of BHT, 2 ppm of antioxidant was found after one week (Frawley et al., 1965a), and that concentration did not increase during a very short feeding trial. The accumulation of hindered phenolic antioxidants in the eggs of laying birds may explain the high concentrations of vitamin A and carotenoids observed in the eggs of hens with a dietary intake of 0.125% BHT (Shellenberger e t al., 1957). After 4 months’ egg production, laying birds that had been continuously fed diets containing this high concentration of BHT were artificially inseminated, and eggs were collected for a 10-day period. Dietary BHT, some of which we know would certainly have been transferred to these eggs, had no harmful effect on their hatchability. The early viability of the resulting chicks was excellent, and their growth was normal (Shellenberger et al., 1957). The accumulation of small quantities of BHT in hens’ eggs therefore does not appear to be harmful either to the developing embryo or to the egg-consuming human population. The transfer of lipid-soluble insecticides from the pregnant doe to developing embryos (Hathway et al., 1966), and t h a t of BHT from the laying hen to eggs, strongly suggests the passage of BHT from pregnant animals to fetuses. No direct measurement has been made of fetal uptake, but early investigations indicated that BHT could be harmful if transferred to rat fetuses in large enough quantity. Thus, a t the 1.55% level, BHT caused loss in weight in pregnant females and fetal deaths (Ames et al., 1956), but it is fair comment that at that dose the rats were receiving daily approximately one-half of the single dose LD,,, of BHT (Deichniann e t al., 1955). More recently, Brown et al. (1959)

36

D. E. HATHWAY

studied the effect on rats of BHT at concentrations of up to 0.5% in a diet containing 10 or 20% of added fat, amounts of f a t t h a t a r e not unusual in the human diet but a r e abnormal in the diet of the laboratory rat. Three of thirty rat litters contained young born without eyes, when the parents had been fed on diets containing BHT for 5 months; anophthalmia was not observed in any of the stock or other experimental animals. This suggests that the fetal deaths and embryotoxic effect may have been caused by diet a r y BHT transferred to the embryos at a critical stage in morphogenesis. This supposition has not been confirmed by subsequent work, however. When mice were fed diets containing 0.1 or 0.5% BHT and 10 or 20% lard for 18 months (Johnson, 1965), none of the 7765 mice born showed evidence of anophthalmia, although 12 of the 144 mothers had been selected from a n established anophthalmic strain. Clegg (1965) employed three dose schedules : single massive doses ( 1 g’kg body weight) on a specified day after gestation; repeated daily doses (750 mg/kg) from the time of mating throughout pregnancy; and daily doses (250-500 mg/kg for mice, and 500 and 750 mg/kg for rats) during a 7-10-week period before mating and continuing throughout gestation until the animals were killed. No significant embryotoxic effect was detected by histopathological examination of the skeletal and soft tissues of the fully developed fetuses when either BHT or BHA were administered according to these schedules. In a multigeneration study, Frawley et al. (1965b) chronically fed weanling rats with diets containing 300, 1000, or 3000 ppm BHT and 20% lard, and found no reproductive or embryotoxic effects in two litters from each of the first two generations. I n the more recent work, no embryotoxic effect was associated with the transfer of BHT from maternal blood to the developing embryos, under the experimental conditions and with the strains of mice and rats that were used. Some characteristic fetal abnormalities resulted when pregnant rats were dosed with vitamin A (Clegg, 1965), and this might provide a clue to the results previously obtained with BHT by Brown et ul. (1959), who could not exclude the possibility of a teratogenic effect. D. STIMULATION

OF

HEPATIC-DRUG-METABOLIZING ENZYMES

Living organisms a r e able to adapt themselves to different physiological situations, and in many cases the basis of adaptation is a change in the enzymic equipment of the tissues. One type

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37

of mechanism that brings about such a change and seems to operate when animals a r e exposed to a foreign organic compound, involves variation in the rate of enzyme synthesis-repression of the formation of some enzymes and induction of the formation of others. As a result, there is a significant variation of the actual enzymic activities of the tissues. In the livers of rats dosed chronically with BHT, Gilbert and Golberg (1965) measured the activities of typical drug-metabolizing enzymes associated with liver-cell microsomes (broken-down smooth endoplasmic reticulum) : hexobarbitone oxidase, aminopyrine demethylase, and nitro-anisole demethylase. They correlated enhanced enzymic activity with increase in relative liver weight and with BHT storage in the fatty tissues. The fate of BHT in the body would be expected to be regulated by the activity of the hepatic-drug-metabolizing enzymes, which process lipid-soluble foreign compounds. Thus, when rats were treated with 500 m g BHT/kg/day, the concentration of BHT in fatty tissues rose to 230 ppni in females and 162 ppm in males in 2 days, at which time the liver weight had increased significantly, with corresponding elation of enzymic activities. Thereafter, the enzymic activities and liver weight continued to rise, but in both sexes the concentration of BHT in fatty tissues fell to 10 ppm. Gaunt e t al. (1965a) found that the onset, degree, and duration of liver enlargement induced by either BHA or BHT was followed by increased output of urinary ascorbic acid. With BHA, increased excretion of ascorbic acid in the urine was rapid but transient, whereas with BHT it was slower in onset but more prolonged. stimulation of hepatic-drug-metabolizing enzymes, increase in excretion of ascorbic acid in the urine, and increase in relative liver weight brought about by BHT, were unaffected by 14 days of starvation. All these changes were entirely reversible during 14 days of recovery on a normal diet (Gaunt, e t al., 1965b). Hence BHT caused variation in the rate of liver-cell enzyme synthesisrepression of the formation of some enzymes and induction of the formation of others. Changes in liver enzyme equipment were related to relative liver weight, but a better standard of reference would have been either the concentration of microsomal protein/unit weight of liver o r the concentration of liver microsomal DNA, since other foreign organic compounds a r e known, f o r example, to cause fatty infiltration of the liver, and some fatty change has been instanced f o r BHT (Feuer e t al., 1965). Such increases in liver weight and

38

D. E. HATHWAY

niicrosomal protein as have been discussed do not necessarily indicate net synthesis of enzyme protein, but they might be expected if such variation occurred. Increases in liver enzyme equipment similar to those cited for BHT were caused by chlordan, and they could be blocked by administration of the amino-acid antagonist DL-ethionine ( H a r t e t al., 1963). It is also well-established that foreign compounds, which stimulate liver-cell microsoma1 enzymes in the way described by Golberg and co-workers, also accelerate the metabolism of D-glUCOSe, D-galaCtOSe to D-glucuronic acid, L-gulonic acid, and L-ascorbic acid through the glucuronic acid pathway (Conney, 1962). Increased output of ascorbic acid in the urine, described by Golberg and co-workers, reflects such stimulation (Gaunt et al., 1965a). The claim that “BHT is not hepatotoxic” requires substantiation, and it would have been relevant to have determined whether this antioxidant induced a nonspecific stimulation of liver-cell microsomal enzymes, as in the case of chlordan, phenobarbital, and most other drugs, or a specific stimulation, as in the case of the polycyclic hydrocarbons. In this connection, Gillette ( 1963) had found t h a t high doses of phenobarbital plus 3,4-benzpyrene increased the activities of liver-cell microsomal enzymes much more than the same doses of either agent administered separately. Conversely, there were no additive effects with regard to enzyme activity when 3,4-benzpyrene and 3-methylcholanthrene were administered simultaneously (Gillette, 1963) or when phenobarbital and y-chlordan were given together ( H a r t and Fouts, 1965). I t is also of interest that the metabolism of such endogenous substances as testosterone and a4-androstene-3,l7-dioneis accelerated by the presence of a foreign compound that causes nonspecific stimulation of liver-cell microsomal enzymes but is not influenced by polycyclic hydrocarbons (Conney, 1962 ; Conney and Klutch, 1963). As has been stated, a nonspecific stimulation of hepatic microsonial enzymes is indicative of proliferation of liver-cell smooth endoplasmic reticulum, and this can now be photographed in the intact cells with improved electron-microscopic techniques. This represents another approach to the subject (Fouts and Rogers, 1965). Feurer et al. (1965) approached this problem in a different way, and thereby broke new ground. They selected three hepatotoxinscarbon tetrachloride, coumarin, and ethionine-that did not stimulate the liver-cell microsomal enzymes, and they compared the re-

METABOLISM OF HINDERED PHENOLIC ANTIOXIDANTS

39

sponse of the liver of rats exposed for one week to each of these substances with the response produced by BHT or BHA. At high doses, carbon tetrachloride, coumarin, and ethionine caused a decrease in activity of glucose-6-phosphatase and a corresponding increase in activity of glucose-6-phosphate dehydrogenase. Similar changes in the activities of the two enzymes were induced by 500 mg of BHT/kg body weight, but not by 200 mg/kg, whereas BHA did not cause this variation in enzymic activity. BHA, BHT, and coumarin produced a n increase in relative liver weight, with the histological alterations consisting principally of fatty change. Carbon tetrachloride caused pronounced fatty infiltration, which was less apparent with ethionine and still less with BHT and coumarin ; BHA caused no fatty change in the liver. When the animals were allowed to recover on a normal diet, Feuer et al. (1965) distinguished between the quicker reversibility of liver changes due to BHT and the more prolonged effects induced by the hepatotoxins. Thus, decreased glucose-6-phosphatase activity caused by the hepatotoxins was still apparent after 14 days of recovery and returned to normal only on the twenty-eighth day. Fatty infiltration brought about by dosing with carbon tetrachloride or coumarin persisted a t a reduced level throughout twenty-eight days of recovery, whereas the effect produced by BHT disappeared comparatively rapidly. Thus, BHT brings about various changes in liver enzyme equipment, and the reversibility of these changes on the recovery of the animal is significant. Exposure to BHT induces changes in liver enzyme activities some of which are brought about by nonspecific stimulatory agents and others by hepatotoxins. I n the present state of knowledge, this division of enzyme responses is incompletely understood. VI.

EVALUATION OF THE SAFETY OF F O O D ANTIOXIDANTS

A. TOXICOLOGICAL INFORMATION ON IONOX 330, BHA, AND BHT 1 . lono.?. 330

a. A c x t e Toxicity. When Ionox 330 was administered orally, the LD,,, value for rats (expressed as mg’kg body weight) was more than 5000 (Stevenson et al., 1965). b. S h o i f - T e r m S t u d y . When Ionox 330 was fed to groups of rats for 90 days a t various dietary levels, weight gain was slightly less

40

D. E. HATHWAY

in animals with the highest concentration of Ionox 330 (31600 ppm) in their diet. This growth suppression seems to be due to dietary intake and was not interpreted as a toxic effect by Stevenson e t al. (1965). 2. BHA ( 1 . Acute To.ricity. When BHA was administered orally, the LD,,, values (expressed as mg/kg body weight) were 2000 (mice) and more than 5000 (rats) (Bunnell et al., 1955; Lehman e t al., 1951). b. Short-Term Studies. I n a half-year feeding study with groups of rats on various dietary levels of BHA, animals on the 3% level did not eat enough to gain weight and had to be put onto the 2% diet for a time and then returned to the 3% regimen. Even a t the 2% level, food consumption was below optimum. No effects due to the food additive were revealed by histopathological examination (Wilder and Kraybill, 1948). Combination of BHA with other food additives a t fifty times the levels normal in bread had no adverse effects when fed (in bread) to groups of rats for 32 weeks (daily doses of BHA ranged from 3.3 to 7.0 mg/kg body weight) (Graham et al., 1954 ; Graham and Grice, 1955). When rats were fed with BHA at 500 and 600 m g kg body weight f o r 82 and 68 days, respectively, no behavioral changes were observed, but a reduction was found in growth rate and in the activities of blood catalase and peroxidase. BHA caused increased relative liver weight in rats (Karplyuk, 1959). Daily oral administration of BHA (1 g ) to rabbits for 5-6 days caused a tenfold increase in the excretion of N a + and a 20% increase in the excretion of K + in the urine. A decrease in extracellular fluid volume approximately restored the concentration of plasma Na 4 . Serum K fell after 5 days of treatment, and in muscle cells, K + was being replaced with N a + ; changes in heart muscle followed those in skeletal muscle but were not as severe. BHA may have a direct action on the kidneys; there were some changes in the adrenal cortex, and increased excretion of aldosterone occurred together with loss of Na- and K- (Denz and Llaurado, 1957). When BHA was fed to groups of dogs f o r one year at various dietary levels (0.3, 3.0, 30, and 100 mg/kg body weight day) in propylene glycol, there were no ill effects; renal function, hematology and histopathology of the main tissues, and organ weights were normal (Hodge et aL., 1964). 7

METABOLISM O F HINDERED PHENOLIC ANTIOXIDANTS

41

c. Long-TeFm Studies. BHA fed to groups of rats for 22 months at various dietary levels caused no ill effects. Weight gains were

comparable in all groups, reproduction was normal, and young rats fed on the same diet grew normally. The litters were strictly comparable, for all animals of all groups, with regard to number, size, weight, weight gain, and mortality. No changes due t o this antioxidant were revealed by histopathological examination (Wilder and Kraybill, 1948). Similar tests were made with a n additional group of rats fed f o r 21 months with a diet containing 0.12% BHA and supplemented with 2% lard. Histopathological examination, again, revealed no changes, and animals fed BHA were identical to the controls (Wilder and Kraybill, 1948). Brown et al. (1959) fed BHA to rats for 2 years at various dietary levels, and in some of the animals on the highest (0.5%) level there was a slight reduction in mature weight and increased relative liver weight. There were no effects on: reproductive cycle ; histology of kidneys, liver, skin, and spleen ; relative weights of heart, kidneys, and spleen; or mortality. The toxicity of BHA was unaffected by the dietary load of fat. Wilder et al. (1960) fed BHA for 15 months a t various levels to groups of Cocker Spaniel pups, and found that throughout this period the general health and weight gains of the dogs were good. Hemoglobin and blood-cell counts were not affected significantly a t the dietary levels of antioxidant used. Histopathological examination showed no changes other than normal variation except that three of the animals that had received the highest dose (250 mg/kg body weight/day) suffered liver damage. Subacute toxicity data for BHA are satisfactory with regard to the numbers of species tested. Tests on BHA have taken into consideration the possibility t h a t the toxicity of antioxidants may be changed a t the temperatures used in food processing. 3. BHT a. Acute Toxicity. When BHT was administered orally, the LD,, values (expressed a s mg/kg body weight) were 1700-1970 (Deichmann et al., 1955), 2450 (Karplyuk, 1960) ( r a t ) , and 2000 (mouse) (Karplyuk, 1960) ; and the LD,,,, values were: 3500 ( r a t ) , 2500 (mouse) (Karplyuk, 1960), 940-2100 ( c a t ) , 2100-3200 (rabbit), and 10700 (guinea pig) (Deichmann e t al., 1955). b. Skort-Term Stu,dies. Weanling rats of each sex were chronically fed diets containing 10 and 20% lard supplements with 0.0001, 0.1000, o r 0.5000% RHT added, and the 0.5% BHT caused

42

D. E. HATHWAY

increased concentrations of serum cholesterol and phospholipid within 5 weeks. Female rats fed for 8 months on a diet containing 10% lard and 0.1% BHT showed increased serum cholesterol levels (Day e t al. 1959). Brown e t al. (1959) studied the effect on rats of BHT (and BHA) at levels up to 0.5% in a diet containing 10 or 20% added fat. BHT compared unfavorably with BHA in the extent to which it diminished initial growth rate and increased relative liver weight. Loss of hair from the heads of rats was claimed with diets containing 0.1% BHT or more; this was enhanced by increasing lard concentrations in the diet. Three of thirty rat litters contained young born without eyes when the parents had been fed 5 months on diets containing BHT, and it is relevant that anophthalmia was not observed in any of the stock or other experimental animals. Deichmann e t al. (1955) had previously reported that rats chronically fed diets containing BHT found 0.5 or 1.0% of the antioxidant unpalatable. The animals could be conditioned to ingest such foods if these concentrations were attained gradually. Paired feeding experiments with groups of 5 or 10 rats for 25 days demonstrated that diets containing 0.8 or 1.0% BHT depress food intake, and that a concentration of 1.0% of BHT in the diet retards weight gain. In another exploration of the safety evaluation of BHT, Karplyuk (1959; 1960) fed rats for two or three months with EHT, BHA, or propyl gallate, equivalent to 0.3% of the diet, dissolved in Iard. Judged to be the most toxic of these antioxidants was BHT. This antioxidant alone affected phospholipid synthesis in liver, and the hydrolysis and transport of neutral fat from the liver. Only BHT decreased the activities of three plasma enzymes-catalase, cholinesterase, and peroxidase-whereas BHA reduced catalase and peroxidase activities, and propyl gallate reduced cholinesterase and peroxidase activities. Sporn and Schobesch (1961) also concluded that BHT had a toxic effect. They also considered that the low margin of safety of BHT made it undesirable a s a food additive. Those workers used groups of 16 white rats fed diets containing 9.6, 16.6, and 19.9% of casein, and the test groups also received 0.2% of BHT in their diet. In each case, BHT improved growth and protein efficiency. Liver nitrogen content was greatly reduced in BHT-treated animals unless the concentration of BHT in the diet was only 0.02%. It was also alleged that after an unspecified period of fasting, the recovery of liver protein was impaired in rats chronically fed

METABOLISM O F HINDERED PHENOLIC ANTIOXIDANTS

43

0.2% BHT. Liver lipids increased at the higher dietary level of BHT, but not at 0.02% BHT. Further, the increased ascorbic acid content of the adrenal medullae of rats fed 0.2% BHT was considered indicative of stress imposed by BHT. The present author, however, claims that when ascorbic acid is expressed in terms of adrenal concentration (pgig of tissue), it becomes clear that no significant increase in concentration has occurred. Finally, under short-term studies with rats, mention ought also to be made of work of Gaunt et al. (1965a) in which groups of 48 rats were chronically administered diets containing 0.1% BHT or BHA. Measurements of body growth, food consumption, and organ weights, together with detailed histological examination, showed no differences from the control group. During 16 weeks, increases occurred in relative liver weight and in relative adrenal weight, without histopathological evidence of damage. Histochemical observations on glucose-6-phosphatase and glucose-6-phosphate dehydrogenase again showed no differences from the control group. The findings of Golberg and co-workers concerning changes in liver enzyme equipment caused by BHT have been discussed (Section V.D) ; reference might be made here to their reversibility during recovery of the animals. In rabbits, acute effects on electrolyte excretion similar to those described for large doses of BHA (Section V1,A) were also obtained after administration of 500-700 m g of BHT/kg body weight (i.e., about 2% in the diet). At lower doses these effects were not encountered (Denz and Llaurado, 1957). Deichmann e t al. (1955) induced severe diarrhea in a group of four dogs fed doses of 1.4-1.7 g of BHT/kg body weight every 2-4 days for. 4 weeks. No gross histopathological changes were revealed post-mortem. Dogs fed 0.17-0.94 g of BHT kg body weight 5 days a week for 12 months showed no symptoms of intoxication and no histopathological changes post-mortem. As has been stated (Section V,C), fertility, hatchability of eggs, and health of chicks did not differ from those of a control group when 10 laying hens were fed 0.125% B H T for 34 weeks (Shellenberger e t al., 1957). c . Lorig-Temi Studies. Deichmann et al. (1955) found that, when groups of 15 male and 15 female rats were given diets containing 1% lard and 0.2, 0.5, or 0.8% BHT for 2 years, they showed no symptoms of intoxication and no histopathological changes post-mortem. In a n experiment designed t o take into account the possibility that the toxicity of the antioxidant (0.5%

44

D. E. HATHWAY

BHT) may be changed under conditions used in food preparation (150°C for 30 m i n ) , Deichmann et al. (1955) showed that there were no effects on weight gain or on the hematopoietic system; histopathological studies were negative. Ingestion of 1 % BHT by male and female animals caused subnormal weight gain and increased relative liver and brain weights, but histopathological examinations were negative for this group as well. A t these levels of dose, BHT did not affect the hematopoietic system. I n all of these experiments a number of rats died, but deaths were not interpreted as being related to the concentration of antioxidant ingested (Deichmann et al., 1955), When rats were chronically fed diets containing 0.5% BHT, Brown et al. (1959) found no adverse effects on reproductive cycle, structure of kidneys, liver, skin, and spleen, or relative weights of heart, kidneys, or spleen. When rats were fed a diet containing 0.1% BHT and 10% hydrogenated coconut oil for 2 years, mortality was not increased. Karplyuk (1960) administered 0.2% BHT ( o r BHA) in lard at a dose level of 4 mg, kg body weight day for 6 months, followed by 8 mg’kg body weight day for a n additional 6 months. A t the end of the first 5 months, the rats were mated. The 1-month-old young were separated and put on a regimen of 8 mg of BHT/kg body weight day. After another 6 months, a third generation was produced by the second, and treated with the same BHT regimen for 6 months. All three generations were examined for weight gain. The hematopoietic system, serum albumin, blood cholesterol, nonprotein nitrogen, blood enzymes (catalase, cholinesterase, and peroxidase) , Takata-Ara reaction, and Weltmann coagulation test were investigated. Relative liver weight, dry weight, and cholesterol, fat, glycogen, and phosphorus contents of livers were determined. Histopathological examinations were made of the brain, liver, and several other organs; and the numbers of young, their weights, appearance, and age when eyeopening occurred were recorded, Rats treated with BHT (and BHA) were normal in all these respects. In a later paper (Karplyuk, 1962), milk and meat were Ivithdrawn for 14-day periods from the diets of animals committed to the long-term feeding regimen with RHT (Karplyuk, 1960) during the seventh and eleventh months of test; body weights of test and control animals followed similar patterns. I,a st 1y , under 1on g- t e r m st u d i e s with r at s, the mu 1ti gen e r at i on

METABOLISM O F HINDERED PHENOLIC ANTIOXIDANTS

45

study of Frawley et al. (196513) should be mentioned (see also Section V,C). A t 100 days, sixteen females at each dietary level (300, 1000, or 3000 ppm BHT) were mated with animals belonging to the same dietary level. Examined in mothers were fertility, parturition, and lactation ; examined in the young were survival, viability, and growth. Ten days after weaning, the females were mated again, and similar observations were recorded for mothers and young. The following clinical tests were made on the first generation : full blood count, cell indices, reticulocyte count, prothrombin time, serum total cholesterol, cholesterol esters, GOT, GPT, alkaline phosphatase, fasting blood sugar, blood urea nitrogen, icterus index, urinary reducing substances, albumin, and microscopic elements. All clinical tests on the first generation were repeated on the second generation, together with the following additional clinical tests : serum lipids, phospholipids, mucoproteins, a- and p-lipoproteins, cholinesterase, erythrocyte and cerebral acetylcholinesterase, adrenal ascorbic acid, cholesterol, and cholesterol esters. Complete histopathological examinations were made after the second litter was weaned. In the first and second generations, thirty organs and tissues were examined histologically. After the second litter was weaned, these offspring were fed the same diet as their parents until they were 100 days old, when they were mated according to the accepted practice. The same clinical and pathological observations were recorded for these animals and the two litters of young. At the level of 3000 ppm BHT, growth gain of parents and young was depressed 10-20%. After 28 weeks, there was a 20% increase in serum cholesterol over controls, and after 42 weeks a 10-2096 increase in relative liver weight. The remaining observations at the 3000-ppm level, and all observations at the 300- and 1000-ppm levels, were strictly comparable with those of controls. Throughout the trial, reproduction was normal and no teratogenic effects were noticed. The only long-term study with a nonrodent species was made by Karplyuk (1960). He fed pairs of dogs for one year with f a t containing 4 % BHT (or BHA) at a dose level of 4 m g of antioxidant/kg body weight/day. No toxic effects were found; results cited refer particularly to liver dry weight, glycogen, lipid phosphorus, and cholesterol. Subacute toxicity data f o r BHT a r e satisfactory with regard to the numbers of species tested, especially in the short-term studies.

46

D. E. HATHWAY

Tests on BHT have taken into consideration the possibility that the toxicity of antioxidants may be changed a t the temperatures used in food processing. B. RELATIONSHIP O F METABOLIC FATEA N D RELATED ASPECTSOF PHYSIOLOGICAL CHEMISTRY TO EVALUATION O F THE SAFETY-IN-USE O F FOOD ADDITIVES

If a substance produces no toxic symptoms in toxicological testing, the fate of a dose may be traced to provide convincing evidence of its harmlessness (see Section 1,A). Commensurate with this supposition, investigations with Ionox 330 have yielded very promising results. Thus, no toxic symptoms were detected in a short-term study with rats (Stevenson et al., 1965), and, in a n investigation of the fate of Ionox 330 (Wright et al., 1965a), nonabsorption was demonstrated in pigs and rats; unchanged antioxidant was quantitatively recovered from the 48-hour feces of rats, dogs, and man. F o r a full safety evaluation, subacute toxicological testing would have to include work with a nonrodent species and a long-term study that might incorporate some carcinogenicity testing. Since no symptoms would be expected, a feeding test with humans could be conducted. BHA has for long enjoyed the reputation of being a safe food additive (see World Health Organization, 1962a), and extensive toxicological testing has not exposed significant toxic symptoms. The data on toxicity to animals a r e satisfactory with regard to numbers of animals and species, but the duration of long-term tests with nonrodent species might be open to criticism. The experimental observations allow for the effect of heat on the antioxidant and for the possibility that other additives in the diet may potentiate the toxicity of BHA. Metabolism studies have shown that single doses of BHA a r e absorbed, metabolized, and excreted in a short period (Section III,B) . These results and the absence of toxic symptoms during toxicological testing a r e considered to vindicate the safety of BHA as a food additive (see Section 1,A). Further, on account of the conjugation of BHA isomers in the liver, some accompanying variation would be expected in the rate of liver enzyme synthesis-repression of some enzymes and induction of the formation of others. In fact, enhanced activities of microsomal drug-metabolizing enzymes rapidly returned to normal when the animals were allowed to recover. At high doses, BHA caused neither changes in the activities of other smooth endoplasmic reticular enzymes, glucose-6-phosphatase and glucose-6-phos-

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47

phate dehydrogenase, nor fatty changes in the liver (Section V,D) . Any changes in the liver due to BHA were therefore caused by hyperfunction. Some liver damage, previously reported in dogs after long-term testing with the antioxidant a t high dose levels, would not have been predictable on the basis of the recent liverenzyme studies with rats. I n common with other food antioxidants, BRA has not been tested for carcinogenicity. No direct feeding tests on humans seem to have been reported, although no symptoms would be expected. BHT formerly enjoyed conditional acceptance as a food additive (inte? alia, World Health Organization, 1962b), the estimate of acceptable intake for humans being 0-0.5 mg/kg body weight/ day. This antioxidant has also been used widely for protecting lipid-containing food supplements for f a r m animals. More recently, the use of BHT a s a food additive in the diet of humans has been censured (Ministry of Agr., Fisheries, and Food, 1963). BHT and other antioxidants were shown to behave similarly when concentrations of up to 0.8% were added to animal diets containing 1% lard, but, when the diet contained 20% lard (Brown et al., 1959), BHT decreased growth rate, increased liver weight, and caused loss of hair. One-tenth of the rats were born without eyes, and BHT was also considered to inhibit important blood enzymes. The case against the use of BHT is based on these results and on the slower elimination of this antioxidant and its metabolic products than of BHA and its metabolites (Ministry of Agr., Fisheries and Food, 1963). Brown e t al. (1959) did not test BHA when the diet contained 20% lard ( c f . World Health Organization, 1962a ; Ministry of Agr., Fisheries and Food, 1963), and this subtracts from the weight of evidence on which decisions concerning BHT were based. Recent experimentation has shown, however, that a lower dietary concentration of BHT (0.1%) in a diet containing 20% lard has no adverse effect on the growth rate of rats through two generations (Frawley et aZ., 1965b), which puts controlled use of BHT in a less unfavorable perspective. Similarly, the relative liver weight was not increased a t a low dietary concentration of 0.1% BHT. In work that has been discussed (Section V,D), higher dose levels of BHT induced proliferation of liver (microsomal) enzyme protein, associated with increased relative liver weight and increased excretion of ascorbic acid in the urine. Since these changes in the liver were reversible when the animals

48

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were allowed to recover (Gaunt e t aZ., 1965b; Gilbert and Golberg, 1965), they seemed to be manifestations of liver hyperfunction rather than of irreversible pathological damage. Nevertheless, significant variation in the activities of other smooth endoplasmic reticular enzymes produced a t high dose levels of BHT, which were also caused by selected hepatotoxins, were unaffected by comparable doses of B H A ; such changes in enzymic activity attributable t o BHT were also readily reversible. Consistent with earlier contentions, which were held t o be serious, high doses of BHT caused some degree of fatty change in liver cells that, although reversible when the animals were allowed to recover, were not produced by similar treatment with BHA. Recent work has thus established that doses of BHT f a r exceeding the input commensurate with commercial protection of the diet have no adverse effects on the liver, even though high doses of BHA cause changes in the liver (apparently reversible). Work by Clegg (1965), Frawley et al. (1965b), and Johnson (1965) has not confirmed the association of embryopathic effects with chronic intake of BHT that might be inferred from the earlier work of Brown e t al. (1959). No cases of anophthalmia were observed among any of the stock or other experimental rats of the Australian workers. This therefore seems to be a case where an experimental biological finding was unsubstantiated by the results of three other investigations. How many negative biological results are necessary to negate a positive finding is a matt e r for subjective assessment. On the other hand, if such a clearcut effect (Brown et al., 1959) were due to BHT, it is probable that this would be reproducible. I n this connection, a two-generation feeding test (Frawley e t al., 1965b) did not reveal any adverse effect of BHT on the reproduction and fertility of rats, and the subsequent growth rate and health of the young were normal. The reservation based on the inhibition of blood enzymes by BHT appears to be unjustified. Thus, Frawley et aZ. (1965b) found that the activities of brain, erythrocyte, and plasma cholinesterases were unaffected in rats chronically dosed with 3000 ppm of BHT. Indeed, it is difficult to understand why there should be any disturbance, since BHT would not be expected to possess anticholinesterase properties. There is considerable retention of radioactivity after administration of a single dose of [14C] BHT to rats, and BHT shares this property with the related antioxidants Ionox 220 and Ionox 201 (Section II1,C). Delay in the metabolism is due to the opera-

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49

tion of three factors: 1) an induction period in which the liver drug-metabolizing enzymes become activated ; 2) entero-hepatic circulation ; and 3 ) the withdrawal of this lipid-soluble food additive into the fatty tissues. Much of the time-lag in the over-all metabolism of BHT can be attributed to the difficulty in oxidizing BHT to Ionox 100, since an oral dose of the latter antioxidant was excreted almost quantitatively in 11 days (Wright e t al., 1965b). Tissue retention of BHT diminishes with increasing activity of the drug-metabolizing enzymes. Where enzyme activation is high, as in chronically fed animals, Daniel and Gage (1965) found a half-life of 7-10 days for the antioxidant. In conclusion, a t the 0.1% level of chronic ingestion, BHT causes no demonstrable effect on either weight gain or relative liver weight, and the comparatively slow rate of metabolism presents, pc?rse, no toxicological hazard. A no-effect level of 100 ppm BHT in a high-fat diet has been demonstrated for two generations of rats and their offspring. FAO/WHO experts (World Health Organization, publication during 1966 ; Dr. L. Golberg, personal communication) have recently agreed on a n unconditional acceptable daily intake for man of 0-0.5 mg BHT/kg body weight, and a conditional acceptable daily intake of 0.5-2.0 mg/kg body weight. When BHT and BHA are used together, the total conditional acceptable daily intake agreed on is 0.5-2.0 mg/kg body weight. Much has therefore been done to meet the reservations brought against the use of BHT in the diet of humans (Ministry of Agr., Fisheries and Food, 1963), but specific investigation of possible carcinogenicity is missing and there appears to be no information about the effect on man. However, long-term studies would have indicated any carcinogenic potential of BHT.

c. A

ROLE FOR SYNTHETIC ANTIOXIDANTS IN NUTRITIONAL BIOCHEMISTRY

POSSIBLE

There is a possibility that hindered phenolic antioxidants with intermediate rates of absorption, metabolism, and elimination from body tissues (see Section III,C) simulate some of the biological properties of vitamin E in vivo. Ionox 220, Ionox 201, and BHT a r e poorly absorbed from the intestine and give rise in the liver to initial high concentrations that are not maintained. Unchanged antioxidants accumulate in the fatty tissues. Vitamin E shares these distributive properties, and, since it is poorly absorbed, the more that is ingested the

50

D. E. HATHWAY

greater is the absorption. lonox 220, Ionox 201, and vitamin I3 are transformed into p-benzoquinones in vivo. It has not been established whether the ingestion of BHT, Ionox 220, or Ionox 201 prevents testicular dystrophy in male rats and browning of the uterus in females that have been reared on a vitamin-E-deficient diet, but BHT and diphenyl-p-phenylenediamine were effective agents for preventing encephalomalacia in vitamin-E-deficient chicks (Bunnell et al., 1955). As has been stated (Section V,C), the eggs of laying hens chronically fed diets containing BHT accumulate a low concentration of unchanged antioxidant in the lipid fraction (van Stratum and Vos, 1965), and the sparing action of this synthetic antioxidant may explain the high concentrations of vitamin A and carotenoids observed in the eggs of hens with a dietary intake of BHT (Shellenberger e t al., 1957). Further, fatty livers induced either by carbon tetrachloride or ethanol have been modified significantly by administration of synthetic antioxidants (di Luzio and Costales, 1965). These results suggest that there is a common factor operating in both hepatotoxic actions. This may be the breakdown of hepatic lysosome membranes, which are known t o be protected in vivo by vitamin E and may be similarly protected by synthetic antioxidants. Administration of synthetic antioxidants might therefore be used to prevent liver injury. Thus, the hindered phenolic antioxidants which accumulate in the fatty tissues have some biological properties in vivo in common with vitamin E. Hence, these compounds or their structurally related analogs may be useful in various biological situations, and in this connection a reduction in the severity of atherosclerosis in male rats has been correlated with intake of BHT (Brown e t al., 1959). ACKNOWLEDGMENTS The author thanks Professor R. A. Morton, F.R.S., f o r help and advice, Dr. A. S. Wright f o r invaluable help with the metabolism studies, and Dr. L. Golberg for preprints of t h e British Industrial Biological Research Association’s work on BHT.

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tivity by phenobarbital, chlordane, benzpyrene or methylcholanthrene in rats. J . Pharmacol. Exptl. T h e r a p . 147, 112. Francois, A.-C., and Pihet, A. 1960. Influence de l’ingestion d’antioxygenes s u r la composition de certains tissus e t s u r l a stabilitk des graisses de rCseive du porc e t du poulet. Ann. Zootech. 9, 195. Frawley, J. R., Kay, J. M., and Calandra, J. C. 1965a. The residue of butylated hydroxytoluene ( B H T ) and metabolites i n tissue and eggs of chickens fed diets containing radioactive BHT. Food Cosmet. Tozicol. 3, 471. Frawley, J. R., Kohn, F. E., Kay, J. H., and Calandra, J. C. 1965b. Progress report on multigeneration reproduction studies in r a t s fed butylated hydroxytoluene ( B H T ) . Food Cosmct. Toxicol. 3, 477. Gaunt, I. F., Feuer, G., Fairweather, F. A., and Gilbert, D. 1965a. Liver response tests. IV. Application to short-term feeding studies and butylated hydroxytoluene ( B H T ) and butylated hydroxyanisole ( B H A ) . Food Cosmet. Toxicol. 3, 433. Gaunt, I. F., Gilbert, D., and Martin, D. 196513. Liver response tests. V. Effect of dietary restriction in a short-term feeding study with butylated hydroxytoluene ( B H T ) . Food Cosmet. Torcicol. 3, 445. Gilbert, D., and Golberg, L. 1965. Liver response tests. 111. Liver enlargement and stimulation of microsomal processing enzyme activity. Food Cosmet. Toxzcol. 3, 417. Gillette, J. R. 1963. Factors t h a t affect the stimulation of t h e microsomal d r u g enzymes induced by foreign compounds. A d v a n c e s in E n z y m e R e g u latio?t 1, 215. Golder, W. S., Ryan, A. J., and Wright, S . E. 1962. The urinary excretion of tritiated butylated hydroxytoluene in the rat. J . P h a r m . and Pharmacol. 14, 268. Graham, W. D., and Grice, H. C. 1955. Chronic toxicity of bread additives to rats. 11. J . P h a r m . and Pharmacol. 7 , 126. Graham, W. D., Teed, H., and Grice, H. C. 1954. Chronic toxicity of bread additives to rats. J . P h a r m . and Pharmacol. 6, 534. Hart, L. G., and Fouts, J. R. 1965. Studies of t h e possible mechanisms by which chlordane stimulates hepatic microsomal d r u g metabolism in the rat. Biochem. Pharmacol. 14, 263. H a r t , L. G., Shultice, R. W., and Fouts, J. R. 1963. Stimulatory effects of chlordane on hepatic microsomal d r u g metabolism in t h e rat. Toxicol. A p p l . Pharmacol. 5 , 371. Hathway, D. E., Moss, J. A., Rose, J. A,, and Williams, D. J. M. 1966. Transport of dieldrin from mother to blastocyst and from mother t o fetus in pregnant rabbits. Bzochem. J . (in p r e s s ) . Hodge, H. C., Fassett, D. W., Maynard, E. A., Downs, W. L., and Coye, R. D., Jr. 1964. Chronic feeding studies of butylated hydroxyanisole in dogs. Toxicol. A p p l . Pharmacol. 6, 512. Jaffe, G . S., Rocklin, A. I,., and Van Winkle, J. L. 1964. Polynuclear phenolic antioxidants. B r i t i s h P a t e n t 946,603. Johnson, A. R. 1965. A re-examination of t h e possible teratogenic effects of butylated hydroxytoluene ( B H T ) and its effect on the reproductive capacity of t h e mouse. Food Cosmet. Toxicol. 3, 371.

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Johnson, A. R., O’Halloran, M. W., and Hewgill, F. R. 1958. Phenolic antioxidants and the stability of perirenal rat f a t . J . A m . Oil Chemists’ Soc. 35, 496. Karplyuk, I. A. 1959. Toxicological characteristics of phenolic antioxidants of edible fats. T’oprosy I’itaniyu 1 8 ( 4 ) , 24. Karplyuk, I. A. 1960. Elementary r a w fats containing phenolic antioxidants, a n evaluation from the point of view of hygiene. Voprosy Pitaniya 19 ( l ) , 67. Karplyuk, I. A. 1962. Effect of phenolic antioxidants of nutritive f a t s o n the animal organism. T r u d y 2-01 ( v t o r ) N a u c h n o - K o f . p . V o p r o s y Probl. Zh. v Pitawiya L c n i ) i g r . p. 318. Kharasch, M. S., and Joshi, B. S. 1957. Reactions of hindered phenols. 11. Base-catalyzed oxidations of hindered phenols. J . Org. C h e m . 22, 1439. Klingenberg, K. 1891. Studien iiber die Oxydationen aromatischer Substanzen im thierischer Organismus. J . ber. F o r t s c h r . Tierchem. 21, 57. Ladomery, L. G., Ryan, A. J., and Wright, S. E. 1963. The u r i n a r y excretion of “C-labelled butylated hydroxytoluene by the r a t . J . P h a m z . and P h a m r a c o l . 15, 771. Lehnian, A. J., Fitzhugh, 0. G., Nelson, A. A., and Woodward, G. 1951. Pharmacological evaluation of antioxidants. A d v a n c e s in Food RPscnrch 3, 197. di Luzio, N. R., and Costales, F. 1965. Inhibition of the ethanol and carbon tetrachloride induced f a t t y liver by antioxidants. Exptl. Mol. Pathol. 4, 141. Metro, S. J. 1955. 2,6-Di-t-butylbenzoquinone. d . Am. C h e m . Soc. 77, 2901. Ministry of Agr., Fisheries and Food, 1963. “Food Standards Committee Report on Antioxidants in Food.” H e r Majesty’s Stationery Office, London. Ministry of Agr., Fisheries and Food, 1965. “Memorandum on Procedure f o r Submissions on Food Additives and on Methods of Toxicity Testing.” London: H e r Majesty’s Stationery Office. Morris, R. C., and Sullivan, W. J. 1963. Novel antioxidant 3,5-dialkyl-4hydroxybenzyl ethers and their preparation. British P a t e n t 932,818. Natl. Acad. of Sci., 1960. “Principles and Procedures f o r Evaluating t h e Safety of Food Additives.” N a t l . R e s e a w h Counc. Publ. No. 750, Washington, D.C. Natl. Acad. of Sci., 1965. “Some Considerations in the Use of Human Subjects in Safety Evaluation of Pesticides and Food Chemicals.” N a t l . Research Counc. Publ. No. 1270, Washington, D.C. Popper, H., Dubin, A., Bruce, C., Kent, G . , and Kushner, D. 1957. The effect of chloropromazine upon experimental hepatic injury. J . Lab. Clin. Med. 49, 767. Robinson, D., and Williams, R. T. 1955. Studies in detoxication. G 1 . The metabolism of alkylbenzenes, tert.-butylbenzene. Biochem. J . 59, 159. Rocklin, A. L., and Van Winkle, J. L. 1962. Polynuclear polyphenols. U.S. P a t e n t 3,026,264 ( t o Shell Oil Co.). Rosenwald, R. H. 1949a. Separation of alkyl-alkoxyphenol mixtures. U.S. P a t e n t 2,459,540 ( t o Universal Oil Products Co.). Rosenwald, R. H. 1949b. Alkylation of phenols. U.S. P a t e n t 2,470,902 ( t o Universal Oil Products Co.).

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Rosenwald, R. H., and Chenicek, J. A. 1951. Alkylhydroxyanisoles as antioxidants. J . Ani. Oil Chemists’ Soc. 28, 185. Shellenberger, T. E., P a r r i s h , D. B., and Sanford, P. E. 1957. Effects of antioxidants, D P P D and BIIT, on health, production and reproduction of laying hens. l’oidtry Sci. 36, 1313. Sporn, A.! and Schobesch, 0. 1961. Research on the toxicity of butylhydroxytoluene ( B H T ) . Illicrobiol. E p i d c m i o l . 9, 113. Stevenson, D. E., Chambers, P. L., and Hunter, C. G. 19G5. Toxicological studies with 2,4,6-tri- (3’,5’-di-icrt.-butyl-4’-hydroxybenzyl) niesitylenc i n the r a t . Food Cosmet. Toxicol. 3, 281. Stillson, C;. H. 1947. Alkylation of phenols. U.S. P a t e n t 2,428,745 (to Gulf Research and Development Co.). Stroud, S. W. 1940. Metabolism of the p a r e n t compounds of some of the simpler synthetic oestrogenic hydrocarbons. J. Endociinol. 2, 55. Tye, R., Engel, J. D., Rapien, I., and Moore, J. 1965. Summary of toxicological d a t a . Disposition of butylated hydroxytoluene in t h e rat. Food Cosnie’t. Toxicol. 3, 547. Uri, N. 1961a. Physico-chemical aspects of autoxidation. 172 : “Autoxidation and Antioxidants.” Vol. 1, p. 55. W. 0. Lundberg, ed. Interscience Publishers, New York. Uri, N. 1961b. Mechanism of antioxidation. 112: “Autoxidation and Antioxidants.” Vol. 1, p. 133. W. 0. Lundberg, ed. Interscience Publishers, New York. van S t r a t u m , P. G. C., and Vos, H. J. 1965. The t r a n s f e r of dietary butylated hydroxytoluene ( B H T ) into the body and egg f a t of laying hens. Food Cosmct. Toxicol. 3, 475. Wasson. J . I., and Smith? W. 111. 1953. Effect of alkyl substitution on antioxidant properties of phenols. I d . E w g . C h e m . Ind. E d . 45, 197. Wilder, 0. H. M., and Kraybill, H. R. 1948. S u m m a r y of toxicity studies on butylated hydroxyanisole. University of Chicago. American M e a t Institute Foundation, Chicago. Wilder, 0. H. M., Ostby, P. C., and Gregory, B. R. 1960. Effect of feeding butylated hydroxyanisole to dogs. J . Agr. Food Chern. 8, 504. World Health Organisation. 1962a. Evaluation of the toxicity of a number of antimicrobials and antioxidants. Sixth report of the joint F A O / W H O expert committee on food additives. World Health O r g . Tech. R e p t . Scr. No. 228, p. 41. World Health Organization, Geneva. nisation. 196213. Evaluation of the toxicity of a number s and antioxidants. Sixth report of the joint F A O / W H O expert committee on food additives. World Herrlth O r g . T e c h . R c p t . Scr. No. 228, p. 45. World Health Organization, Geneva. Wright, A . S., and Crowne, R. S . 1965. The f a t e of 2,4,6-tri-(3’,5’-di-tc?-t.buty1-4’-hydroxybenzyl) phenol (Ionox 312) i n the rat. Unpublished work. .lVi,iglit, A. S., Crowne, 12. S., and Hathway, D. E. 1965a. The f a t e of 2,4,6-tri- (3’,5’-di-tc~t.-butyl-4’-hydroxybenzyl) mesitylene (Ionox 330) in the dog and r a t . Bioclicm. J. 95, 98. Wright, A. S., Akintonwa, D. A. A., Crowne, R. S., and Hathway, D. E. 1965b. The metabolism of 2,6-di-tert.-butyl-4-hydroxymethylphenol(Ionox 100) in the dog and rat. Biochem. J . 95, 303.

56

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Wright, A . S., Crowne, R. S., and Hathway, D. E. 1966a. The f a t e of Di- (3,5,-di-tert.-butyl-4-hydroxyphenyl) methane (Ionox 220) in the r a t . Biochem. J . 99, 146. W i i g h t , A. S., Crowne, R. S., and Hathway, D. E . 196613. The f a t e of di (3,5-di-tcit.-butyl-4-hydroxybenzyl) ether (Ionox 201) in the r a t . Riocliem. ,I. (in p r e s s ) . Tohe, G. R., Hill, D. R., Dunbar, J. E., and Scheidt, F. 1LI. 1953. Coal oxidations. Comparative studies on phenols. J . Am. Chem. SOC.75, 2688. l o u n g , de W. S., and Rodgers, G . F. 1955. tcrt.-Butylhydroquinone and t c T t . butyl-4-methoxyphenol. U.S. P a t e n t 2,722,556 ( t o Eastman Kodak Co.) . Zhinden, G. 1963. Experimental and clinical aspects of d r u g toxicity. A d v a n c e s i i i Phaiwzcrcol. 2, 1.

RADIOBIOLOGICAL PARAMETERS IN THE IRRADIATION OF FRUITS AND VEGETABLES B Y ROGERJ. ROMANI D e p a r t w m i t of Pomology, U n i v w s i t y of California, Davis, Califomiri

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Radiation Units and Dosimetry A. Units . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

57

.........

............................................. 111. Radiation Mechanisms . . . . . . . A. Temporal Aspects . . . . . . . . . . . . . . . . . . .............. B. Quantitative and Spatial Aspects .................... IV. Chemical and Biochemical Events i n Irradiated F r u i t s and Vegetables ........................ .................. A. Immediate Chemical Changes . . . . . . . ............... B. Protracted Chemical Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Cell Wall Constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Metabolic Aspects . . . . . . . . . . . . ............................. A. Intracellular Studies . . . . . . . .................. B. Involvement of Phytohormo ............. C. Repair . . . . . . . . . . . . . . . . . . . VI. Radiation Sources . . . . . . . . . . . A. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Irradiators Especially Designed f o r the LowDose Food Irradiation Program . . . . . . . . . . . . . . . . . C. Types of Research Facilities in General Food Irradiation . . . . . . . . . . . . . . . . . . . . ............. D. Commercial Units . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. F u t u r e Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . References ... .. .. .. .. .. .. .. .. . ........................

I.

58 59 59 63 63 68

77 77 79 80 82 82 85 86 87 87

87 89 91 92 93

INTRODUCTION

The validity and persistent challenge of the basic concepts in food irradiation remain as tributes to pioneering research in that field. Application of these concepts to fresh fruits and vegetables, however, has yielded little beyond a n admixture of future promises and continuing enigmas. The experimentation has been in large part exploratory and empirical leading to Dupaigne’s (1964) 57

58

ROGER J . ROMAN1

exhortation t h a t progress now lies in understanding the fundamentals. Toward this end one can profitably d r a w upon related studies in radiation biology. I n t r i g u i n g subjects of radiobiological research, i.e., t h e sites of cellular radiation injury, t h e metabolic p a t t e r n s in i n j u r y development, t h e cellular capacities f o r repair, have vital counterp a r t s in fresh-food irradiation. This review comprises a n a t t e m p t t o discuss radiation mechanisms a n d c u r r e n t t r e n d s in radiation a n d radiobiological research a s they relate to the i r r a diation of foods, especially f r e s h f r u i t s a n d vegetables. The challenge of radiation research w a s well p u t by Andrews (1961), who reflected upon the mysterious aspect whereby a radiation event t h a t is complete in lo---' second manifests itself clays o r years later. Equally mysterious a r e t h e forces which allow f r u i t cells t o continue their temporal progress through ripening a n d senescence a f t e r exposure t o kilorad doses ( a s many a s 5 >, 10" ionizing events per cell). Time becomes a vitnl pnrnnieter in analyzing radiation mechanisms a n d in interpreting radiation effects : time f o r completicn of the radiochemical event, time f o r development of t h e biochemical lesion a n d its manifeatation, a n d time f o r radiation repair o r f o r eventual cell death. Equally vital parameters a r e t h e quantitative, energetic, a n d spatial aspects of radiation events, all of which bear upon t h e ultimate f a t e of the irradiated specimen. These aspects of radiation mechanisms will be discussed following a brief review of units a n d dosimetry, the essential tools of a n y radiation research. Chemical, biochemical, a n d metabolic events observed in irradiated f r u i t s a n d vegetables will be viewed f r o m a radiobiological context. T h e final section is devoted to a discussion o i radiation facilities. II.

RADIATION UNITS AND DOSIMETRY

E s s e n t i d l y all recent t e s t s i n radiobiology devote considerable space to discuhsing radiation uiiits, dose, a n d dosimetry. And justifizbly so, f o r t h e very fact t h a t units of energy, space, a n d time can be assigned t o a radiation event facilitates t h e formation of conceptual images which become t h e foundations of fruitful re 9 e a r ch . The importance of adequate dosimetry t o a food research pro;)rani is attested by t h e extensive investigations conducted under the U.S. Army Quartermaster Corps program (Taimuty, 1959).

IRRADIATION O F F R U I T S A N D VEGETABLES

59

Uniform dosimetric methods among several cooperating laboratories a r e essential t o progress in a broadly based radiation prog r a m . I n t h i s connection, t h e U.S.-A.E.C. low-dose food irradiation program h a s held two dosimetry sessions under the auspices of the Brookhaven National Laboratory. Inadequate dosimetry, Zimmer (1964) warned, leads t o findings of questionable value.

A. UNITS The classical unit of radiation, “roentgen,)’ is clearly defined as t h e quantity of ionizing irradiation t h a t will produce sufficient ions in 1 cc of d r y a i r t o c a r r y 1 esii of electricity. The roentgenequivalent-physical, o r “rep,” was meant t o represent a n amount of radiation t h a t w-ould release 1 roentgen of energy in one g r a m of tissue. Equivalence is difficult t o establish, however, a n d the r e p h a s been assigned a r a n g e of values f r o m 84 t o 9 3 e r g s per gram. To resolve this ambiguity, the “rad” h a s been proposed a n d defined a s t h a t unit of radiation which i m p a r t s 100 e r g s of energy per g r a m of t h e irradiated substance. This is a most functional measure since i t relates t o t h e absorbed o r effective energy. F o r most purposes t h e following conversion will apply: r a d = r e p X 1.06. The International Commission on Radiological Units ( 1962, 1963) clearly defines the quantitative units f o r radiation, a n d suggests the following usage : rad -solely f o r absorbed doses roentgen - solely f o r exposure curie - solely f o r activity Other t e r m s useful in t h e measurement of radiation a n d in understanding the function of chemical dosimeters a r e : ionic yield ( u n i t effect/ion p a i r ) a n d “G”-va!ue (unit effect/100 ev of e n e r g y ) . Since “W,” the amount of energy required to form a n ion pair, is generally accepted a s being 34 ev, a “G”-value of I is approximately equal to a n ionic yield of 3. The foregoing a n d othpr commonly used terms a n d conversion factors a r e listed in ‘I‘able I. B. DOSIMETRY Various princip!es of dosimetry a s well a s the use of ion chambers, calorimeteix, chemical dosimeters, a n d other means of radiation detection a n d calibration a r e discussed in a book edited by

60

ROGER J . ROMAN1

Hine a nd Brownell (1956). Whyte (1959) offers a thorough development of radiation dosimetry in a manner particularly suited t o biologists. Articles of interest a r e also found in "Selected Topics in Radiation Dosimetry," a recent International Atomic E n e rg y Agency report (1961). F o r a succinct description of dosimetry systems used in food irradiation, see a paper by Ahnstrom a n d Eh r e nbe r g (1960). Dosimetry systems suitable f o r large radiation facilities an d industrial uses a r e thoroughly discussed by Jefferson (1964).

Trmr,I: I I

JV

I,ET I
VIP<,

TERM<, ~ N I
C Y I ' -

rnergy for foi illation of an ioii pair jca. 3.1 c\ ) lincnr enrrgy trniisf(~r(ev/p) = relativr biological rficic~nc.y =

=

= 1. t i x 10-12 crga I curie = 3 . 67 X 10'" disiritegr:itions per s w Akvogadro's110. (Xj = G.025 x 1023 molccules/iiiole

1 cv

The Fricke or ferrous sulfate aqueous dosimeter (F e z - + Fe" + ) is widely used in food irradiation studies (Taimuty, 1 9 5 9 ). F e rric molecules formed in th e exposed ferrous sulfate solution a r e measured spectrophotometrically at 305 mp. Determination of the extinction coefficient may be hindered by a difficulty in obtaining chemically pure FeCiT. Schuler an d Allen (1956) resolved the problem by adding readily determined equivalents of ceric sulfate to a solution of excess ferrous ions, resulting in a stoichiometric conversion t o ferric. Th e extinction coefficient (21 at 305 mp) of the resultant cerous m u st be subtracted. It is well t o remember th a t the temperature of th e solution during O.D. measurement is important since th e extinction coefficient will increase 0.8% per 1 C. rise in temperature (Lazo e t al., 1954). A commonly used value f o r the extinction coefficient of fe rric is 2177. When t he Fricke system is to serve as a s ta n d a r d f o r calibration of sources a nd secondary dosimeters (Romani e t al., 1963b), i t is

IRRADIATION O F F R U I T S A N D VEGETABLES

61

well to take careful precautions, for even minor impurities have been reported to affect the “G”-value (Sutton, 1962 ; Branislav et al., 1964)., i.e., the quantitative response of the dosimeter to radiation. I n this regard, Lazo et al. (1954) and Schuler and Allen (1956) made careful studies of the “G”-values obtained with the Ferric dosimeter. H a r t (1957, 1963) elaborated on the chemical events which lead to oxidation of the iron molecule. The “G”value has been found t o be independent of dose rate up to 109 rad,‘sec. (Thomas and Hart, 1962) and to be reliable for all except pulsed high-energy sources. It seems reasonable to assume that, with the use of doubly distilled water, good reagents, and glassware cleaned with nitric acid, considerable confidence can be placed in the generally accepted “G”-value of 15.6. A limitation of the Fricke dosimeter in food irradiation studies is its upper threshold of approximately 40,000 rads (Weiss, 1952), resulting from the expenditure of dissolved oxygen. This threshold may be raised by bubbling O2 through the original solution. Alternative possibilities for raising the upper dose limits a r e either purging the ferrous solution with nitrogen, resulting immediately in the reduced “G”-value normally obtained above 40,000 rads, or adding 0.01N cupric sulfate, which drastically lowers the “G”-value to about 0.66 (Jarrett, 1964). The ceric dosimeter ( Ce4+ --f Ce“+ ) has a “G” value considerably lower than the Fricke (Taimuty, 1959; Weiss, 1952) and is usable over a much wider dose range. Unfortunately,, the system is reported to be generally unstable and troublesome (Nicksic and Wright, 1955). Instability was discerned in the extinction coefficient at low ceric concentrations, and in the “G”-value at low doses (Romani e t al., 1963b). The oxalic acid dosimeter introduced by Draganic (1959) is not directly applicable to low-dose food irradiation ; however, its extremely high and extensive dose range (1.6 to 160 megarads) may make it serviceable in other radiation processes or in measuring cumulative doses. Various plastic dosimeters a r e described by Artandi (1959). Use of the thermoluminescent properties of lithium fluoride f o r reliable dosimetry over the range of 1 to los rad seems promising and is thoroughly discussed by Karzmark et al. (1964). Routine dosimetry in fruit packages o r within fruit may be achieved with cobalt-60 glass-chip dosimeters of the type described by Kreidl and Blair (1956a,b) and Caldwell and Frainey (1958). The chips (Bausch and Lomb, melt F-061) a r e inexpensive, small (approx. 1 x 3 >< 6 cm.), and easily readable in a Bausch and

62

ROGER J . ROMAN1

Lonib “Spectronic 20.” By ;I choice of wavelengths (350-430 n i p ) and associated extinction coefficients t h e chips a r e useful in th e range of 6 x 1 0 ’ to 2 x loG rads. Dose distribution patterns within a n irradiation chamber o r food package a r e generally determined with the use of “phantom racks” t o position dosimeters in a geometric a rra y . Such a device is seen in Fig. 1 alongside th e upper portions of th e Mark I1 Cobalt60 food irradiator (see Section VI). Brynjolfsson (1963) and

FIG.1. A view of the Mark I1 food irradiator designed a t the Brookhaven National Laboratory and installed a t the University of California, Davis. A11 three irradiation chambers, shown in the “up” position, a r e lowered t o the Cobalt-60 plaques f o r exposure. A t the right is the lid of one side-chamber I\ it11 attached dosimeter rack and vials (Romani e t al., 1962).

IRRADIATION O F FRUITS AND VEGETABLES

63

Thaarup (1963) report the use of polyvinylchloride ( P V C ) films to discern dose distribution patterns, and Hansen et al. (1963) describe dose distribution measurements in food cans. In summary, accurate measurements of dose rate and dose distribution a r e necessary to successful radiation experiments. With appropriate precautions the Fricke ferrous sulfate dosimeter can be a very reliable standard for food irradiation laboratories. Ill.

RADIATION MECHANISMS

A. TEMPORAL ASPECTS Table 11, taken from three recent publications (Bacq and Alexander, 1961 ; Mole, 1962 ; and Dorfman, 1963) and modified t o include elements of fruit irradiation, depicts the time scales of various sequential events in a n irradiated food. TABLI: I1 TE\IIWR.\I, SEQCENCE Time intervd

O F E V E N T S I N I I l R 4 D I A T E D FOODS"

]<:vent Impact and transfer of energy to the recipient molecule Fastest molecular dissoriation 1)ielectrir relaxation in water Track reactions in water lieactions of freely dif'fusing radicals Chemical changes: splitting of molecules, beginriirig of biochemical events (ascorbate oxidation, sulfhydryl oxidation, ethylene formation, breakdown of membranes, disruption of protein and riucleic avid metabolism, etc.)

R C 1 ~ 0 I 1 1 I St o

hours

1)issipation o f long-lived radicals L)ef;nition of biochemical events, development of physiological disorders

Fruits Put Ji oyciis Changcis in rcspiration Blocking of cell I m s o f turgidity division Changcs i n intracellular protrins I k a t h or repair Gross 1)hysiologiral efl'ects: I'roliferation o f delay i n chlorophyll destruction, survivors delay of textural ch:tnges, physiological breakdown and/or spoihge

64

ROGER J . ROMAN1

1. Pli Usicctl Eveiits

Those events t r a n s p i r i n g in less t h a n lo-‘ second fall predominately in the domain of radiation physics. They include t h e point of impact upon t h e t a r g e t molecule, release of secondary electrons ( t r u e f o r either X-ray, gamma, o r electron i r r a d i a t i o n ) , a n d the formation of ionized species a n d triplet states. A n d r e w (1961), H a r t (1963), Bacq a n d Alexander (1961), a n d Pottinger (1962) offer succinct a n d lucid introductions t o these concepts. Much of the research in radiation physics h a s been carrried out with gaseous phases, and, a s p u t metaphorically by Willard (1963), may relate more directly t o “food f o r thought r a t h e r t h a n food f o r the body.” Nonetheless, these initial, fleeting, t r a n sitional states embody all t h e benefits, o r determents, which follow. Gray (1961) described t h e phased techniques used t o study the n a t u r e a n d decay r a t e s of intermediate ionized species. As much as 1.5 Mev of energy m a y be pulsed into a system in 1-2 microsecond, followed by millisecond flash spectral analysis. Though the use of such high-energy pulsed techniques f o r processing foods may be precluded by t h e dangers of induced radioactivity, they could be vital analytical a n d experimental tools. Dosimetry at these high dose r a t e s would require special consideration since chemical dosimeters exhibit reduced “G”-values at dose r a t e s above 10’’ r a d s second ( H a r t , 1963). 2. C h e m i c a l Everits Many of the events discussed in t h e preceding dosimetry section t r a n s p i r e in approximately lo--’ second. To represent the temporal sequence, Andrews (1961) utilized t h e following dimensions : velocity of 1 Mev electron, 2.8 x 10“’ cm second; second; time t o pass through 1 atomic diameter, av. molecular vibration time, second.

Thus, in second t h e energy is distributed in t h e molecule leading t o dissociation in lo-? t o 10P4 second. I n this interval t h e chemical species a r e formed. Chemical events in t h e irradiated system a r e generally classified as direct, occurring i n t h e solute molecule, o r i n d i r e c t , via t h e formation of solvent radicals which may either recombine o r attack t h e solute phase. The radiation chemistry of aqueous syst e m s h a s been studied extensively, with several (often complex)

IRRADIATION O F FRUITS AND VEGETABLES

65

reaction sequences having been postulated f o r th e formation of different radicals, hydrated electrons, an d other reactive species. A thorough discussion of these principles is offered by Allen (1961). Many of t he i n d i w c t reactions shown to occur in irradiated aqueous solutions have been assumed to occur also i.ii vivo. Since frui t s a nd vegetables a r e highly hydrated, th e assumption would seem t o be especially valid. However, it is difficult t o ascertain a distinct separation of important p a r t s of th e cell, cell wall, organelles, etc., an d th e contiguous an d more aqueous cytoplasm. Bacq a nd Alexander (1961) pointed out t h a t bound oxygen a n d bound w a t e r should be considered p a r t of t h e “parent” molecule insofar as t he radiochemical event is concerned, t h u s favoring d i w e t effects. a. E x c i t e d States. The temporal sequence of radical formation and attack of th e solute, o r recombination a n d decay, ma y be bypassed with the formation of excited states. These result when th e energy imparted t o molecules, though insufficient t o cause ionization, does render them unstable a n d chemically more reactive. H a r t (1963), quoting work of F ra n c k a n d Platzman (1954), suggested t h a t excited states may be much more prevalent tha n w a s formerly surmised. Reid (1963) emphasizes the electron-capturing potential of biological molecules which yields excited states t h a t m ay be th e ultimate vectors of radiation damage. An excited molecule can t r a n s f e r its energy in ways diverse from t he stoichiometric patterns in ideal solutions, i.e., 34 ev = 1 ionization I1 radical = 1 chemical event. 11. Long-Lived Radicals. A f u r t h e r deviation f r o m stoichiometry and t he normal time scale of events may result f r o m t h e formation of long-lived radicals. Eh ren b erg ( 1961) reviewed recent evidence f o r long-lived radicals in biological systems, a n d Cook ( 1964), using especially designed electron-spin resonance techniques, clearly demonstrated t h e presence of fre e radicals with finite lifetimes in seeds having as high as 70% moisture. The formation a nd lifetime of th e radicals is affected not only by th e degree of hydration b u t by th e presence of various chemicals such as nitric oxide (Cook, 1964), oxygen (E h re n b e rg e t al., 1962; Snipes a n d Gordy, 1963), chemical blocking agents (Pihl a n d Sanner., 1963), p-mercaptothylamine (Smaller a n d Avery, 1959), and undoubtedly other radioprotective o r radiosensitizing materials. Based on t he above evidence it seems reasonable to assume t h a t

66

ROGER J. ROMAN1

some long-lived radicals a r e formed in irradiated fresh food, and, when present, would prolong the chemical events f o r seconds o r minutes (Miiller a n d Zimmer, 1961 ; Zimmer, 1960). Ho\vever, it is highly unlikely t h a t they would exist long enough t o be a health concern in the consumption of irradiated f r u i t s a n d vegetables . 3 . Bioclic?iiicnl Evetits

Although events a t t h e chemical a n d molecular levels m a y have finite lifetimes, t h e duration of ensuing biochemical phenomena m a y depend on t h e site a n d degree of injury, t h e metabolic r a t e a n d stage of development of t h e irradiated tissues, a n d the lifetime of t h e organism. An index of i n j u r y most generally used in radiobiological studies h a s been the cessation of cell division, often referred t o as reproductive death, which may manifest itself in t h e irradiated generation o r in one of several generations thcrea f t e r . Recently, emphasis on repair mechanisms h a s elaborated t h e time vector f o r t h e repair processes; the significance of “repair,” biochemical, a n d intracellular events t o f r e s h food irradiation is discussed under “Metabolic Aspects.”

4. Biological Evciits It is now clear t h a t time with regard t o t h e life of a f r u i t o r vegetable, i.e., its stage of m a t u r i t y o r ripeness when irradiated, is of crucial importance in determining its radiation response (Hendel a n d B u r r , 1961; Koval’skaya ef nl., 1963; Maxie cf al., 1964; Mullins a n d B u r r , 1961; Romani et al., 1961; Romani, 1964; a n d De Zeeuw, 1 9 6 1 ) . These reports generally agree t h a t with advancing maturity, o r ripeness, there is a reduced radiation response amounting t o a n a p p a r e n t increase in radiation tolerance. Vidal (1963) believes t h a t t h e contrary m a y be t r u e in some instances. Respiratory trends during a n d immediately following exposure have been utilized to illustrate the time course of radiation stress in f r u i t (Romani, 1964). As shown in Fig. 2, t h e magnitude of t h e radiation response is related directly t o dose a n d t o f r u i t age ( m a t u r i t y ) . The lower response of more m a t u r e f r u i t t o ionizing radiation, and, in a pragmatic sense i t s greater resistance, is likely t h e result of a n age-limited capacity t o respond t o i n j u r y r a t h e r t h a n resistance pc?’ sc. The final time factor t o consider, Our preliminary report of transient E S R signals i n ii radiated f r u i t (Romani, 19G4b) has been confirmed (Romani, 1966).

IRRADIATION O F FRUITS A N D VEGETABLES

67

and a most vital one to technological development, is the period of postirradiation storage and handling. These aspects a r e covered in accompanying reviews by Sommer and Fortlage., and Maxie and Abdel-Kader.

FIG.2. Respiration r a t e of Eureka lemons during (solid lines) and immediately following (dashed lines) the absorption of 200 K r a d of Cobalt-60 gamma. Curves A to D represent f r u i t of increasing m a t u r i t y : A, dark-green f r u i t ; B, yellow to green yellow; C, yellow; and D, deep yellow, without luster (Romani, 1964).

5. Dose Rates

It is well to distinguish between two aspects of dose rate (Mole, 1962) : dose rate a t the site of injury and dose rate in the recipient tissue. From the brief mathematical representation of molecular events shown above (Andrews, 1961), one can surmise that, regardless of the number of ionizing events per unit time (rate of dose absorption by the tissue), the rate of energy dissipation a t the molecular site will be the same f o r all radiations of a given energy and species. Thus, radiochemical events are generally regarded as being dose-rate-independent. Dose rates with respect to the tissue, however, may be slow, fast, or intermittent, with accompanying divergencies in biological effect. Of particular significance to control of fruit pathogens is Beraha’s (1964) report of a significant dose rate effect on the control of Penicilliurn italicurn and BotrzJtis cinerea. It is not clear what factors bring about a dose-rate dependence, although the presence of repair mechanisms (Section V,C) may be of consequence.

68

ROGER J . ROMAN1

Except in ;L recent rep o rt by Kahan e t al. (1965) a n d the observations of Massey e t al. (1961), dose rate effects, as such, have not been studied with f r u i t o r vegetable tissues. Massey a n d colvorkers concluded t h a t the respiration of irradiated lettuce is dose-rate-dependent a n d not related to total dose. T h is is in cont r a s t t o results obtained by Romani (1964) in which th e respiration r a t e s of several f r u i t s increased linearly with dose, a t least up t o threshold values. Assuming th e response of lettuce leaves and f r u i t t o be qualitatively th e same, the discrepancies could be attributed to a marked difference in dose rate. T h e highest dose r a t e s in t h e lettuce experiments were 16.2 Kra d /h r, whereas the dose rates in th e f r u i t experiments were 250-300 K ra d /h r. One could invoke th e presence of rep air reactions t h a t establish a steady st a t e of radiation damage an d correction t o explain th e constant respiration rates obtained by Massey et al. (1961). A t much higher dose rates th e relatively slower repair mechanisms would be ineffective until cessation of th e exposure.

B.

QUANTITATIVE AND SPATIAL

ASPECTS

1. Radiochemical Y i e l d s

“G”-values an d “ionic yields” a r e used t o denote radiocheniical efficiency in relatively p u re an d dilute systems. Luse (1964) justifiably questioned th e feasibility of applying similar quantitative expressions t o t h e complex, “impure,” an d relatively concentrated systems composing living tissaes. Nonetheless, th e re is some merit in exploring these concepts since they ma y suggest interpretations of events in radiopasteurized food. I n addition, th e re is the possibility t h a t the relatively massive doses employed in food irradiation m a y result in a sufficient number of direct chemical events t o establish a “quasi” quantitative relationship with th e observed biological phenomena. Table I11 gives calculated “G”-values f o r 4 chemical changes 01‘ products observed in irradiated f r u i t o r in th e atmosphere about them. Each of these is particularly important. Ozone is biologically effective an d a known product of radiation in a i r (Kertesz a nd Parsons, 1963). Radiation will induce carbon dioxide production (respiration) above th e normal r a t e ; however, Ha nna n (1956) wonders whether th e CO, ma y not also result f r o m direct radiation scission. Ascorbate levels a r e readily a ffected by radiation, an d sulfhydryl groups are recognized f o r their radiosensitivity an d importance in many enzyme reactions.

69

IRRADIATION O F FRUITS AND VEGETABLES

“G”-values a r e readily arrived at by using the units and conversion factors given in Table I. Dividing the ev deposited per unit volume of tissue by the number of molecules of gas produced o r molecules chemically altered in the same unit volume, X 100, yields the number of chemical events per 100 ev, or “G”-value. Barring a n extremely radiosensitive species or the presence of chain reactions, the “G”-values, even in relatively pure chemical systems, a r e likely to be less than 1 (Hine and Brownell, 1956,

C’heniical cvcnt

_ _ _ ~ _ _

Ozonr product ioii

( ( 0 2

pro-

Dose

10 Iirad 120 I
300 1ir:td

duc%loll

Ascwhtt: oxidittioil

400 Iii~atl

Loss of -411

400 l*;I.a,d

groups

-

Total production

“G”-value (molcculrq atTectcd/100 cv)

1.G ppm in 330 nila 7 ppm in 20 1 1 8 . 5 ppni in ‘LO I

5.4 8.9 2.8

~~

110 nil 1 2 0 2 (grccii lemons) 18 ml CO2 (mature lemons)

~

15.7 2.7

7 mg/100 1111 of cstrac.tctl orange j uirc

1.0

2 . 8 fi moles/100 i d of rxtracted orange juiccc

0.0ti

Data froni Kertesz and Parsor~s(1963), assuming 0.0012!)3 g air per cc and U C ~ U C O U S dosimetry. Based on the increase in respiration rate of lcinoiis during first Iiour of irradkttioii (Itommi et al., 1963%)and assuming unit dcnsity of fruit. c Calculated from data of Romani et al. (1963:~) with thr :issumptioil that tlic offec~tive volume in s i f u of 1 1111 juice was 1 cc.

p. 364). As discussed by Gordon (1957), the probability of having selective radiochemical events in such a complex system as living tissue is indeed highly remote. Thus, it would seem untenable t h a t such a large “G”-value for Con,as given in Table 111, would result from direct radiochemical scission. Respiration must be looked upon as the principa1, if not sole, contributing mechanism. This may be demonstrated more directly by irradiating a killed portion of tissue (Romani, 1964), which results in no measurable production of CO,. Kertesz and Parsons (1963) give data for ozone yields in closed systems over rather long exposure periods. Calculations made

70

ROGER J. ROMAN1

from these data do not tak e into account th e r a te s of concomitant formation a nd decomposition of ozone by radiation (Harteck c t nl., 1965) resulting in “G”-values (Table 111), which first rise an d then fall with increasing dose. This characteristic raises the possibility of minimizing ozone concentrations in t h e atmosphere about a food product by a n appropriate ra te of air exchange d u r i n g irradiation. High “G”-values f o r ascorbic acid reduction ma y result f r o m the action of ascorbate ions a s scavengers of oxidative radicals Eorrned dur i ng irradiation. Much lower “G”-values found f o r -SH groups, on t he other hand, likely reflect their scarcity a n d low probability of “hit” in such low-protein systems as citrus. I t must be emphasized th at these are mere conjectures to illustrate t he possible relation between total energy input a n d observed phenomena, a s expressed b y “G”-values. I n most instances this quantitative expression of results will have only limited meaning, or none, unless buttressed by d a ta on th e quantit y a nd radiosensitivity of th e compound in its biological milieu. 2 . LET a n d RBE

I n biological systems such a s f r u i t tissues o r their respective pathogens, t he efficacy of radiation will depend not only upon total energy input but also on th e locus of the ionizations a n d th e concentration of energy at each locus. Th e te r m LET, o r “linear energy transfer,” denotes the amount of energy dissipated per given length of ionization track, i.e., Kev per micron. Recalling t h a t “W” is the energy required to fo rm a n ion p a ir (approximately 34 e v ) , the following relationship will be obtained : I,E:T,’W

T

X(J.of ionizations/unit length of ionization track

Rates of energy dissipation by various f o r ms of radiation have been summarized by Gunckel an d Sp arro w (1961). Typical values a r e shown in Table IV. I n general, lower-density ionizations a r e more effective when th e end result is dependent upon small a n d well-dispersed chemical changes, such as in the inactivation of enzymes, a nd high-density events are more effective where large, localized effects a r e operative, such as chromosome breakage. T he comparable effectiveness of various f o r ms of radiation is denoted by t he term “relative biological efficiency” (RBE) . Lamerton (1962) summarized the complex interrelationship between LET a nd RBI? an d how it may be influenced by dose rate, absence

71

IRRADIATION O F FRUITS AND VEGETABLES

or presence of repair, temperature, oxygen, nitric oxide, and other factors. Augenstein et al. (1964)., Pottinger (1962), and Luse (1964), in discussing radiobiological mechanisms, pointed to the unknowns which complicate attempts to relate physical parameters of radiation to the ultimate biological effect. Nonetheless, considerations of this nature a r e useful in evaluating the effectiveness of different forms of radiation a s illustrated by Conger et al. (1958). I n irradiation of fruit and other foods a choice of radiation forms or conditioning parameters may be experimentally useful and could lead to the very pragmatic goal of increasing RBE with respect to the pathogen while decreasing RBE with respect to the host. I.ET

TABLE I V IONDENSITIES CHARACTERISTIC OF DIFFERENT FORMS A N D ENERGIES O F R A D I A T I O N ( A F T E R G R A Y1955) ,

AND

Linear energy transfer (I<ev/micron)

Ions per micron (mean linear ion density)

Ionizing partirles

Ilndint inn

Energy

High rnergy p and -

20-30 Mev

0 28

8 5

Electron

1 llev" 200 Kev 1 5Kev

0 49 2 6 15

15 80 460

Electron Elertron Electron

12 Mev 400 Kcv

9.5 35.8

290 1110

Proton Proton

3700

OL

s-I

:1>

Ncut 1.1 111'

Alplin

120

" 1;ol c-)nipnrison, the higher of the two energy gnn:nia rays from

=

particles

1 33 l k v .

3 . Density of Ionization E v e n t s

L E T refers to the energy dissipated (ionizing events) per given length of ionization track. The ultimate biological effectiveness of these events is dependent on the probability of their hitting vital molecules. Pollard (1963) offers a lucid illustration of the relationship between the size of a molecule or sensitive area, radiation dose, and the probability of experiencing a n ionization event. The expression log n/n, = VI/2.3

72

ROGER J. ROMAN1

holds where n/n, = the fraction escaping a n ionization, V = the volume of the molecule or site, and I = the number of primary events per given volume. A t higher gamma and X-irradiation doses the distribution of events may be considered to be uniform throughout a tissue. I is thus readily calculated (see units, Table 11) from the dose in rads. The relationship is shown graphically in Fig. 3. Table V was prepared to illustrate how such calculations can be applied to sensitive sites and radiation doses of particular interest in food irradiation. It can readily be seen that a target with the volume of a ce!l or even that of a mito-

B

A 96 %

2 010

FIG.3. Schematic representation of probability t h a t a n ionizing event will occur within a molecular structure. Cubes represent volumes of roughly 1 pa, spheres represent large sensitive molecules (0.004 p 3 ) , dotted lines t h e zone of migration of radicals, and each dot a n ionization event. I n A t h e dose is 10 rads, and i n B it is 400 rads. The percent figures a r e t h e numerical estimates of the probability of escape for t h e molecule i n question ( a f t e r Pollard, 1963). TABLE: V BIOLOGICAL SITE A K D I~VENTS PER 100 KRADDOSE

1 ~ E L . i T I O N S H I PB E T W E E N SIZE OF

Site

Approximate dimensions

h71~MBEROF

IOSIZIS~;

Approximate volume

Peach cell

1 :3 Y 101'

11itoc~liondrioIior 1 X 2 p section o f p w c h cell v::tll

1 g:tlsctosr molcculr

7 x 10'

7 x 10-7 01' 1 ioiiizntiori per 1 -1 million molrculrs

a

From Reeve, 1959.

* From summation of interatomic bond distances

IRRADIATION O F FRUITS AND VEGETABLES

73

chondrion will receive thousands of ionizing events. On th e other hand, a single 6-carbon unit would have less t h a n one chance in a million of being ionized by direct hit. The case f o r quantitative considerations at t h e cellular level is clearly presented by Setlow an d Pollard (1962), who made a theoretical analysis of events within a bacterial cell receiving 1000 r. G r a nt in g a number of assumptions, th e y estimated t h a t 370 protein molecules were damaged by direct h i t a n d 230 by agents formed in water out of a total of 4.7 million protein molecules. If t h e dose had been at food-pasteurizing levels (ca. 200,000 r ) , perhaps 100,000 molecules might have been altered. While still a small fraction of th e total molecules present, th e possibility of measuring direct radio-chemical events is obviously enhanced a t r;i.dio-pasteurization doses.

4. Target Theoiy and Associated Calculatiom A simplified an d clear discussion of th e “ ta rg e t theory” is given by Epstein (1963). General discussions of th is theory a n d associated quantitative derivations are found in most te x ts on radiobiology. Zimmer (1961) offers a vigorous a n d thorough development of t he subject matter. I t is well t o state t h a t all authors w a r n against oversimplification a nd mal-application of th e concepts. Yet under ideal conditions, where t he molecular species o r th e cellular site in question is inactivated by 1 o r a known number of ionizing events, it is possible to estimate targ et size o r sensitive volume f r o m doseinactivation data. A schematic example is shown in Fig. 4. Without recourse t o mathematical derivation one can sense t h a t the slope of a dose-response curve relates t o th e sensitive volume, i.e., a smaller t a r get size requires higher dose o r g re a te r density of radiation events f o r a given percent inactivation. T h is is essentially the converse of “probability of hit,” discussed in th e preceding section. I n analogous fashion, radiation of high L E T ma y be used to estimate t he t arg et a r e a (Epstein, 1963). A t high L E T , th e ionization density is so g reat t h a t each ionization track passing through the site will cause a n ionization event within it. Knowing th e number of incident particles per em.”, one can estimate th e a r e a of t he target. Thus, by a combined use of low- a n d high-LET irradiation, estimates of targ et volume a n d shape ma y be developed. Vital phenomena a r e reflected in the shoulders o r lag phase of

74

ROGER J. ROMAN1

inactivation curves such as those depicted in Fig. 4. A l a g in radiation effect m a y result f r o m t h e presence of more t h a n one vital site per t a r g e t (cell, spore, etc.), a need f o r cumulative events t o achieve site inactivation, o r concomitant radiation repair. I n instances of multiple sites a quantitative estimate is obtained by extrapolating t h e inactivation curve to t h e “Y” axis, giving t h e “extrapolation number.” Thus, f o r curve ( b ) the extrapolation number 3 m a y represent t h e number of sites which m u s t undergo radiation damage in a cell o r spore prior t o its reproductive death. --

~

~~

0

~

~~

1

GGh

J

01

COCiL

1 ~~~~

~~

--

23SE

FIG,4. Schematic radiation inactivation c u r v e s r e p r e s e n t a t i v e of single cells ( A & B ) a n d multicelled colonies ( C & U ) . C u r v e B s u g g e s t s t h e presence of a p p r o x i m a t e l y 3 ( e x t r a p o l a t i o n no.) inactivation sites p e r cell, curve C \vould r e s u l t f r o m t h e presence of ea. 100 viable cells p e r colony, a n d c u r v e I ) s u g g e s t s t h e presence of 2 inactivation sites p e r cell in t h e colony. T h e intei~ventioiiof r a d i a t i o n r e p a i r could also account f o r t h e presence of a shoulder :I< in cui’ves I:. C, 8 II ( L i t t b r a n d a n d Revesz, 1 9 6 4 ; Elkind a n d S u t t o n , 1 9 6 0 ) .

Of more direct interest to food irradiation is t h e use of similar analysis t o estimate t h e number of viable cells o r spores in irradiated colonies (Elkind a n d Sutton, 1960). As shown schematically i n F i g . 4 (curves c a n d d ) , t h e percent survival would r e f e r t o t h e number of colonies containing one o r more s u n - i l i n g cells a f t e r a given dose. The extrapolation number is now a quantitative estimate of reproductive units (cells, spores) within the original colony. This is equivalent t o cell inactivation except t h a t now the biological unit of interest is t h e colony instead of the cell, a n d t h e vital radiation t a r g e t is t h e viable cell instead of

IRRADIATION O F FRUITS A N D VEGETABLES

75

the intracellular site. Mathematical derivations of these data, as given by Elkind and Sutton (1960), a r e complex; however, one can readily deduce that the dose required to inactivate all or a given percentage of the pathogens on or in a food is directly dependent on the number of viable pathogens originally present. In practice, a working correlation must be established between radiation dose and the desired end effect. F o r instance, Schmidt and Nank (1960) and Schmidt et al. (1962) define their chosen “end point” ( D value) as the minimum dose resulting in complete destruction (sterilization) of inoculum in 90% of the food cans. Sommer et al. ( 1964b) describe qualitatively similar end-point analyses especially useful for assessing the radiation control of fruit pathogens. 5 . Spatial Chamcteristics of Di.fferent Forms of Radiation

At the tissue level, different forms of radiation will markedly affect the distribution of energy because of their varying penetrating power and LET. This is shown schematically in Fig. 5. While electrons a r e the ultimate effective species for both betaand gamma- or X-irradiation, the penetration of beta rays (electrons) is limited by the charged nature of the particle and is proportional to its energy. On the other hand, hard (high-energy) X-rays or gamma-, such as from Co-60, have much greater penetration. Particulate forms of radiation (protons, deuterons, alpha particles) offer a n interesting advantage by permitting the distribution of high-LET radiation at controllable depths within a t i s u e (D’Angio and Lawrence, 1963). The area of high relative dose is referred to as the Bragg Peak. Cellular and molecular

D E P T H IN TISSUE

FIG.

(cm)

5. Depth-dose characteristics of different forms of radiation : A , 3.-MeV

electrons; I:, 6-Mev electrons; C, 52-Mev protons; and D , Co-60 gamma.

76

ROGER J. ROMAN1

effects of densely ionizing radiations are discussed by Tobias and Monney (1964). Though beta a n d g a m m a sources have been used in experimental food irradiation, t h e choice h a s often been dictated by source availability o r size of package a n d need f o r penetrating power. T h u s f a r , little h a s been done in t h i s field t o exploit t h e penetration characteristics of different radiations t o experimental advantage. It is somewhat surprising, f o r instance, t h a t t h e respiration response of pears, a measure of t h e degree of stress, h a s been remarkably similar a f t e r exposure t o either 5.6-Mev electron (Maxie a n d Nelson, 1959) o r Co-60 g a m m a r a y s (Romani et al., 1 9 6 1 ) . Equivalence in effect of gamma- a n d beta-irradiation of pears was also noted b y Hansen a n d Grunewald (1961). A t the indicated energy t h e 2 1 5 % dose penetration of t h e electrons used by Maxie a n d Nelson would have been approximately 1.8 em, whereas g a m m a rays would have distributed energy uniformly throughout the fruit. Given this disparity in dose distribution, can t h e equivalent response be attributed t o g r e a t e r sensitivity of t h e surface tissues? Or do t h e skin a n d immediate subsurface cells contribute the m a j o r portion of the normal respiration? AIthough these questions m a y be of no direct concern t o the radiation technologist, they do relate t o the normal radiation physiology of t h e host food a n d t o its postirradiation behavior. I n recently completed preliminary experiments made possible by D r . John Lawrence, Dr. Graeme Welch, a n d Mr. Douglas Pounds of t h e Donner a n d Lawrence Radiation Laboratories, we observed t h e respiratory response of f r u i t exposed t o t h e following approximate doses of different f o r m s of radiation :

( a ) 300 K r a d of low-penetrating (1-2 nim) I-Mev electrons. ( b ) 300 K r a d of penetrating (cn. 2 e m ) 7-Mev electrons. ( c ) A 42-Krad dose of 7-Mev electrons equivalent in total energy imparted t o t h e f r u i t t o t h e 1-Mev treatment. ( c l ) 300 K r a d of gamma rays f r o m CO-60. ( e ) 48-Mev protons with the R r a g g Peak adjusted t o fall j u s t below t h e skin surface (approx. 2 m m ) . ( f ) Similar protons with the E r a g g Peak a t a depth of 1.8 cm. Thus, on a per-cell basis, ( a ) a n d ( I ) ) received the same dose, but case ( h ) had proportionately severalfold more cells exposed ( i n different a r e a s of t h e f r u i t ) , because of the much greater

IRRADIATION O F FRUITS AND VEGETABLES

77

depth of penetration. In case (c ) the same total volume of fruit was exposed as in ( b ) , but with one-seventh the total energy deposited per unit vol., or per cell, with a n equivalent total energy per fruit as in case ( a ) . Some of the resulting respiration rates, expressed as a percent of the control, a r e shown in Table VI. From the electron data one can tentatively surmise that the surface layers of cells contribute a major portion of the respiratory activity. The proton data a r e not a t variance with this interpretation, as it may first appear, since at 2 mm depth ( f ) , the high LET Bragg Peak may have caused such extensive damage as to obviate a postirradiation respiration. In case ( e ) , the plateau region of the Bragg curve passing through the surface cells had a n LET nearly similar to the beta- and gamma-radiation, causing a qualitatively similar respiration response. IV.

CHEMICAL A N D BIOCHEMICAL EVENTS IN IRRADIATED FRUITS A N D VEGETABLES

A. IMMEDIATECHEMICALCHANGES Given the complexity of biological systems and the limited resolution, on a molecular basis, of most chemical analytical techniques (Setlow and Pollard, 1962), it is not surprising that few well-defined, direct radiochemical changes have been noted in irradiated fruits and vegetables. Ascorbic acid, a n exception, is affected immediately and quite noticeably by radiation, likely because of its redox function. The number and diversity in reported values for radiation-induced ascorbic acid losses have been discussed (Romani et al., 1963a). So as to gain some concept of the time vector involved, ascorbic acid levels have been measured at close time intervals immediately following exposure of the fruit (Romani et al., 1963a). While most of the ascorbate oxidation occurred during the exposure period ( 1 h r and 20 min), there was a continuing decrease in ascorbic acid levels for about one hour after irradiation to a n absorbed dose of 400 Krad. Since metabolic processes continue at abnormal rates for hours and days after exposure, there is a strong implication that the ascorbic changes found during and immediately after irradiation a r e either of radiochemical origin or very near in time and locus to the original radiochemical events. The postirradiation changes could represent, in part, the dissipation of long-lived radicals (Section IIl,A,l,b).

Days after esposure

a

Control pears (mg COzikglhr)

48-Mev protons with Bragg Peak a t approximately

Accelerated electrons 1 >lev

7 >lev

7 L'Irv

Coso Gamma

Ihplic.ate samples of four or more fruit each. Respiration rate expressed as percent iiicre:tsc over control.

1 . 8 ('111

2 mm

IRRADIATION O F FRUITS A N D VEGETABLES

79

A pattern of change somewhat similar to that of ascorbic acid was noted for sulfhydryls in irradiated citrus (Romani et al., 1963a). I n lemons the quantitative determination of -SH groups was affected markedly by the release of nitrogenous compounds after irradiation. Such a structural change in the tissues, which can affect both the extraction and quantitative expression of a given constituent, may explain some of the wide divergence in analyses of radiation effects in fruits and vegetables. Radiagroups have also been noted in tion-induced changes in -SH tomato products (Villareal e t al., 1961) and in potatoes (Korableva, 1959) (see also Section B , l ) .

B. PROTRACTED CHEMICALCHANGES A preponderance of the chemical analyses of irradiated fruits and vegetables have been carried out at varying postirradiation times. Because of the pronounced effect of ionizing radiation on the progress of fruit maturation and senescence, it is often questionable whether the observed change in constituents is due to radiation events pe?* se or is a consequence of some more fundamental radiation stress. Some examples are the transient changes in the sugar content of potatoes (Cloutier et al., 1959), increases in inorganic phosphorous, loss of nucleic acids, and minor changes in fatty acids of irradiated potatoes (Schwimmer et al., 1958), and a decrease in the acidity of apples and pears (Fernandes and Clarke, 1962; Clarke, 1961). Grebinskii e t al. (1962) discussed the effects of radiation on the minor transformation of various storage substances in sprouting seeds. Frumkin et al. (1961) reported little or no change in the coloration of anthocyanin-containing fruits with doses above 1.5 megarad; however, Markakis et al. (1959) did show some pigment destruction in irradiated strawberries. Minor changes in the reducing and nonreducing sugars of mangoes were reported by Mathur and Lewis (1961), and slight increases in the protein content of pears were noted by Clarke and Fernandes (1961). A loss of astringency in irradiated kaki (persimmons), possibly associated with solubilization of pectins, was reported by Kitagawa e t al. (1964). Other chemical changes in irradiated fruits and vegetables have been reported and reviewed by Salunkhe (1961) and in the accompanying review by Maxie and Abdel-Kader. The question of protracted versus direct chemical events is particularly relevant as i t applies to the production of ethylene,

80

ROGER J . ROMAN1

because of t he efficacy of th e g as in promoting physiological activities in f r u its (Biale, 1960a,b). Young (1965) suggested t h a t ethylene is not a direct product of radiochemical events but represents, instead, a physiological response to more basic radiation injuries. His suggestion was premised on the observation t h a t ethylene production was more directly dependent on physiological s t a t e of th e f r u i t at time of radiation t h a n on radiation dose. This observation is at variance with reports of Maxie a n d co-workers (1965), a discrepancy which ma y be due, in part, to the different f r u i t s used an d to differences in dose r a te s (Section III,A,5). Young varied dose rate s while keeping th e exposure time constant, whereas Maxie e t al. employed a constant dose r a t e with varying exposure times.

C. CELL WALL CONSTITUENTS As shown by Reeve (1959), ripening f r u i t undergo changes in cell wall thickness, in texture, an d in t h e pectin fractions. Since large polymers would have a n increasing probability of being “hit” by ionization tracks, an d since one scission in a polymer chain can have marked effects on viscosity a n d other physical attributes, i t is perhaps not surprising t h a t the most obvious and detrimental effects of ionizing radiations in f r u i t s a n d vegetables are t he change in texture an d loss of firmness. Whether one ascribes the effect to altered permeability, as implied by work of Skou (1963), Nor-Arevyan (1963) a n d Hluchovsky a n d S r b (1963), or to events i n t h e middle lamella leading t o a decrease in intercellular cohesion, as suggested by extensive studies of Kertesz a n d co-workers at Cornell (1963, 1964), cell wall constituents a r e undoubtedly involved. A recent paper by Kertesz c t nl. (1964) describes changes in pectins a nd cellulose fractions extracted f r o m irradiated apples, carrots, a nd beets. Similar descriptive studies have been reported by McArdle an d Nehemias (1956), Massey e t al. (1964), a n d Somogyi a nd Romani (1964). These, with associated determinations of t he viscosity an d electrophoretic changes in irradiated pectin solutions (Skinner an d Kertesz, 1960), a n d a recent study of homogenate viscosities (Jo n as an d Romani, 1965), point to a n increase in pectin solubility an d loss of viscosity via scissions in the polymer chains. There remains a d earth of definitive information on t h e structu r e a nd radiation chemistry of pectins a n d other natural polymers, though progress h a s been made to wa rd elucidating some

IRRADIATION O F FRUITS AND VEGETABLES

81

of the radiochemical events. Tomada and Tsuda (1961) found that the extent of cross-linking in gelatin was dependent on both dose rate and solute concentration. Cross-linking was increased by radiation in the presence of N,, whereas H bonds were broken in the presence of 0,. Analogous studies with aerobic and anaerobic pectin solutions have not shown such a n effect from 0, (Somogyi and Romani, 1964). Ricketts and Rowe (1954) employed branched and straightchain dextrans to differentiate between molecular degradation and cross-linking effects of radiation. Degradative processes were found t o predominate in irradiated 1% solutions of dextran. Deshpande et al. (1964) reported the breakdown of pectin to be linear with radiation dose. They were plagued with erratic results, however, when the irradiated pectin was used as a substrate for enzymes. This difficulty may have been due to the instability of irradiated pectins, a s implied in the description of “aftereffects” by Glegg and Kertesz (1956). Following a thorough analysis of several by-products of pectin irradiation, Luck and Dell (1963) suggested that radiation scission occurs at the 1-4 glycosidic bond. A similar bond breakage is indicated by recent findings in our laboratory which include such radiation effects as decreases in equivalent wt., increases in titratable methoxyl groups, and decreases in the suitability of irradiated pectin as a substrate for pectin polygalacturonase and pectin transeleminase. Random hydrolytic scission of the pectin chain has also been indicated in earlier work of Skinner and Kertesz (1960). In a discussion of experimental results based on the presence of 3.7 X lo2 units per gram of cellulose, Charlesby (1955) estimated that 1 megarad fractured 0.16% of the monomer units in the main chain. Cowling (1963) pointed out that the enzymatic degradation of cellulose is dependent upon its accessibility to the enzyme, a concept that may be relevant to other cell wall polymers. As a working hypothesis, radiation could be thought of as causing both a scission of polymer chains and formation of additional cross-links among remaining polymer units. This latter possibility has been shown for cellulose (Tomada and Tsuda, 1961) and is implied by the transient increase in viscosity of pectin fractions from irradiated fruits (Kertesz e t al., 1964), and in the gelation of irradiated pectin solutions (Wahba e t al., 1963). The presence of these divergent and concurrent events would explain the immediate softening effects of radiation (via

82

ROGER J . ROMAN1

scission) a n d the stabilization against f u r t h e r change with time (reduced accessibility t o enzymes) which result in irradiated fruit, often reported as a delay of “ripening.” As a final comment, t h e widely held view t h a t pectins a r e t h e structurally determinative cell wall material m a y be open t o question. Sterling ( 1962) emphasized t h a t secondary linkages o r bridge structures within t h e cell wall contribute importantly t o its architecture and, by implication, its functional properties. This view is supported by Bell (1962), who emphasizes t h e importance of bindings between components of t h e cell mall, especially between polysaccharides a n d protein. V.

METABOLIC ASPECTS

Interactions of living m a t t e r with radiation, t h e purview of radiobiological research, a r e especially relevant t o f r u i t and vegetable irradiation. This is most obvious in t h e case of pathogens, where reproductive death, o r a t least reproductive inhibition, is t h e ultimate objective. B u t although cell division a n d proliferation a r e not of direct concern in t h e irradiated f r e s h food, i t s tissues must still continue t o live their normal storage life span a n d must evolve along established patterns of senescence so as to result in a properly ripened a n d marketable item. I n instances where radiation m a y extend a “shelf-life’’ normally terminated by decay, additional adjustments m a y be required in t h e physiological processes of t h e f r u i t or vegetable. These important a n d functional aspects of postirradiation a n d postharvest physiology a r e covered in accompanying reviews by Maxie a n d Abdel-Kader a n d by Sommer a n d Fortlage. W h a t follows below is a comment a r y on the altogether too sparse information about the cellulai. responses of f r u i t s a n d vegetables to radiation exposure. The scarcity of such studies is particularly regrettable since the outward manifestations of phenomena such as senescence a n d radiation i n j u r y have their origins a n d development in t h e cell.

A. INTRACELLULAR STUDIES Giinckel a n d Sparrow (1961), in their extensive review of plant radiobiology, were compelled to d r a w upon investigations i n t h e micro b i a1 a n d m 21,m ma 1i a n fie1ds t o i I1 u st r a te import a n t current trends in radiation biochemistry. The reader is referred t o the above review a s well a s to general discussions on the intracellular effects of radiation in radiobiology texts (Bacq a n d Alexander,

IRRADIATION O F FRUITS A N D VEGETABLES

83

1961 ; E r r e r a Forssberg, 1961 ; Hollaender, 1954 ; Kuzin, 1962 ; Lea, 1947; Pottinger, 1962; Setlow and Pollard, 1962). I n addition, several papers offer brief but lucid discussions of specific cellular radiobiological events. Among these is one by Dendy (1964) which discusses the development of radiation damage in the cell, the importance of cellular organization, the effects of membrane changes, and the enzyme release hypothesis. Kuzin (1963) also emphasizes events at the cellular level. Puck (1961) elaborates on vital concepts of radiation and aging, albeit in mammalian tissues, and Van Lancker (1962) offers views on the “Cytochemical Injury of X-radiation.” Thus far, the most obvious metabolic response in fruits and vegetables has been the increase in respiration (Smock and Sparrow, 1957; Massey et al., 1961; Maxie and Nelson, 1959; Romani et al., 1961). Respiration rates during and immediately after exposure (Massey et al., 1961; Romani and Bowers, 1963) afford some measure of the immediate cellular response to radiation damage. These experiments a r e discussed above (Section III,A,5). On the a p 7 * i 0 7 i assumption t h a t gross tissue respiration results from mitochondrial activity, the fate of these organelles in irradiated tissue becomes particularly significant. By analogy with findings of Schwarz e t al. (1961)) who analyzed r a t liver mitochondria following doses of 1 Krad or less, one would suspect that the mitochondrial lipids in fruit would reflect extensive radiation damage as a result of the much higher doses. However, no change was noted in the fatty acid composition of mitochondria from irradiated apples and pears (Breidenbach, 1963). Minor changes in the lipid fraction were noted with the ripening of both control and irradiated tissues, affirming the sensitivity of the assay. One is led to suspect that the reported effects o f rsc!iat;on were physiological adjustments to stress rather than the direct results of radiation. Since the radiation-induced increase in respiration is sustained f o r several days, damage to the mitochondria must not be so extensive a s to preclude their metabolic function. This would apply to doses up to 250-300 Krad, which appear to be well below the threshold for massive damage to fruit cells. Doses of 1000 Krad or higher invariably produce a sharp fall in respiration and a loss of tissue integrity (Romani, 1964), and more drastic intracellular effects may be expected. Mitochondria have been examined from fruit exposed to these two levels of radiation. Some of the data a r e summarized in Table VII. The oxidative capacity of the

84

ROGER J . ROMAN1

mitochondria, even following a I-Mrad exposure, is surprisingly high. * Electron microscopy and bound enzyme reactions (Romani and Miller, 1965) suggest maintenance of the particulate structure at these high doses. I n studies utilizing density gradients to discern possible mitochondrial fragmentation, Miller et al. (1964) found little effect of radiation but a diminution in density of the intracellular particles with aging of the tissues. This effect of aging, however, was enhanced by an earlier exposure to 1000 Krad.

0 0 2

Time :Lft(Ir irmdi:ition

('ontrol

250 1ir:d

1 huur 3 d:lys (i d:qs

45.3 26.2 27.0

35. 3 43. 7 43.0

~~~~

1000 l
.__ __

30.8 40.0 fruit rotted

XOTE Cytoplasniic particles isolated with standard differential centrifugation techniques and washed once. Reaction mixture was 0 . 5 6 1 in sucrose and included the following, in niicroniolar quantities: PO,, 25; ADI-', 5; ATP, 0.1; DPN, 0.1; Cyto C . , 0.02; E'AIX, 0 . 1; NgCls, 10; CoA, 0.013; Succinnte, 20. Final volume was 3 ml per reaction vessel including 5-7 m g particulate protein.

I n contrast to the lack of significant qualitative effects, a decrease in overall particulate yield was noted following irradiation of the fruit tissues (Romani and van Kooy, 1962). This apparent loss of intracellular structures is followed by a partial recovery after lower doses (250-300 K r a d ) , but not after higher doses (600-1000 Krad) . The capacity for recovery of particulate material is a function of fruit age and is not evident if radiation takes place when the tissues have already reached their climacteric peak. There a r e interesting corollaries in recent reports of Bain and Mercer (1964), Cocking and Gregory (1963), and Romani e t al. (1965), who provide evidence for a considerable amount of intracellular structural change with ripening of fruit. Interrelationships between the stresses of radiation and aging on the

* I n recent studies (Romani, R. J., and Yu, I. K., A r c h . Biochem. & Biophys., 1966, i ) t p r e s s ) mitochondrial respiratory control was also found t o be resistant t o 250 Krad of in viwo irradiation and t o recover following 500 o r 750 K r a d doses.

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capacity of cellular repair, and indirectly radiation tolerance, are the subjects of our further investigations. A final comment regards the intracellular milieu at the time of irradiation. J u s t as some stages of nuclear division are more sensitive than others to radiation damage (Evans, 1963 ; Borstel and St. Amand., 1963), conditions within the cytoplasm may also alter its sensitivity (Bacq and Alexander, 1964). Radiation sensitization of bacteria, for instance, resulting in a dose reduction factor as high a s 3, has been achieved with chemical pretreatments (Emmerson and Howard-Flanders, 1964).

B. INVOLVEMENT OF PHYTOHORMONES One of the early and prominent studies in plant radiobiology was that of Skoog (1935), on the effects of radiation on auxin responses and auxin destruction. Subsequently, Gordon (1957) reviewed evidence supporting the acute sensitivity of indoleacetic acid (IAA) and the IAA-synthesizing systems to ionizing radiation. Cervigni and Belli (1962) offered further evidence for the radiation sensitivity of auxin in solution, and King and Galston ( 1960) suggested the radiation destruction of endogenous auxin. The auxin effect is cited as one of the most sensitive of cellular responses to radiation (Kelly,, 1961), although this sensitivity may not be universal in all plants or present at all times in the same plant (Curtis, 1960 ; Sparrow, 1961). Some instances of hormone-induced reversals of radiation effects were noted following relatively low doses of 4 to 50 Krad. These include the report of Mathur (1961) on the reversal of radiation-induced dormancy in potatoes by gibberellic acid, further experiments by Mathur (1963) on the reversal of decay susceptibility by the methyl-ester of indolyl-3-acetic acid, and the report by Teas et al. (1962) on the reversal of low-dose (25 Krad) ripening inhibition of bananas by 1 ppm ethylene or 1000 ppm 2,4-dichlorophenoxyaceticacid. Morris e t al. ( 1964) demonstrated low-dose inhibition of geotropic responses in asparagus which may have involved auxin transport or destruction. Effects of auxin and other hormones a r e not likely to be very dramatic where growth or other marked physiological changes are not involved. However, given the interrelationship between sulfhydryls and auxin activity (Marrk and Arrigoni, 1957) and the known sensitivity of -SH groups to ionizing radiation, some interactions may be expected. Related to radiation effects o r radiation susceptibility may be suggestions of Glasziou and Inglis

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(1958), Glasziou e t al. (1960), Xorden and Thimann (1963), and Sacher (1957) that auxin levels control pectin binding, cell wall permeability, and the progress of senescence, and Albersheim’s (1963) finding of ozone effects on auxin-induced pectin transeliminase inhibition. Re-evaluation of possible radiation effects \ria plant hormones is encouraged by Pollard’s (1964) recent report of inhibition at the DNA-RNA translation level by ionizing radiation, a finding in support of Gordon’s (1957) earlier suggestion that the radiosensitivity of the auxin system could result from effects on enzyme synthesis.

C. REPAIR Recent advances in radiobiology have elaborated the presence of repair mechanisms that may stem or reverse radiation damage. Initial studies were reviewed by Wolff (1960), and many interesting articles on the repair of genetic radiation damage a r e contained in a volume edited by Sobels (1963). The presence of repair is illustrated by split-dose experiments, such as those of Elkind and Sutton (1960), Littbrand and Revesz (1964), and Elkind et al. (1964), where the time interval between exposure allows for metabolically dependent correction of radiation lesions. Splitdose phenomena indicative of repair have also been reported by Hall and Lajtha (1963) with Vicia f a b a , and Beatty and Beatty (1963) with pollen. I n the latter experiments, protein synthesis was implicated as the underlying factor in repair. Das and Alfert (1961) showed that DNA synthesis in onion root may actually be enhanced by radiation. Of microbiological interest a r e the effects of recovery on partial synchronization of radiation survivors and subsequent sensitivity of a colony as discussed by Sinclair and Morton (1964) and Kallman (1963). Littbrand and Revesz (1964), studying the effects of oxygen on recovery, concluded that it may proceed only in air, implying the requirement for metabolically directed energy as proposed by Sommer et al. (1964a) in studies with irradiated Rliixopus stolonif el’ sporangiospores. Similar conclusions were reached by Patrick and Haynes (1964), who worked with yeast. Our own observations (Romani et al., 1964; Romani and Miller, 1965) on respiration and mitochondria1 yields suggest the presence of repair mechanisms in f r u i t (see footnote, p. 84). Radiation repair can have direct and important implications in fresh-food irradiation, where one would seek to maximize repair in the commodity, thus increasing its radiation tolerance,

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while minimizing repair in pathogens so as to reduce the dose required for effective control. Understanding the repair mechanism could well suggest means of effecting these differential controls. VI.

RADIATION SOURCES

In contrast to the foregoing-on the functional aspects of radiobiology-a discussion of radiation sources must remain essentially descriptive. Nonetheless, sound radiation research often depends on the availability of adequate radiation facilities. A. GENERAL Conceptual designs as well as descriptions of food irradiation sources in actual operation have been previously discussed (Hannan, 1956; Brownell et al., 1957; Desrosier and Roenstock, 1960; Pomerantz e t al., 1957; Pomerantz and Siu, 1957a). An excellent description of radiation sources is found in a recent book edited by Charlesby ( 1964) which contains several papers describing the more common, as well as some unique, sources of radiation. Included in the volume a r e discussions of nuclear reactors, by Bopp and Parkinson ; isotopic sources, by Kuhl and Ballantine ; linear accelerators, by Miller ; resonant transformers, by Westondrop; and Van de Graff accelerators, by Burrell. The characteristics of each particular radiation source a r e discussed, with specific reference to outputs, advantages, and disadvantages, characteristics of radiation, etc. Elements of radiation safety, particularly as they apply to the larger radiation sources, a r e discussed by Cooper and Siu (1957) and Saxon (1964). Economic considerations have been reviewed by Huber and Klein (1960), Kraybill and Brunton (1960), and Pomerantz and Siu (1957b), who compare machine and elemental sources. Methods for estimating costs of gamma-radiation processing have been outlined by Kukacka and Manowitz (1965).

B. IRRADIATORS ESPECIALLY DESIGNEDFOR T H E LOW-DOSE FOOD IRRADIATION PROGRAM Many of the radiation sources available in the past have been designed for maximum versatility or to serve given purposes in radiation physics, chemistry, engineering, etc. Unfortunately, all too often the specific needs f o r food irradiation or f o r a given

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food commodity have been overlooked. As emphasized i n th e preceding portions of th is review an d in t h e accompanying review by Maxie a nd Abdel-Kader, physiological processes in th e food commodity a n d th e milieu in which it is placed a r e of ma jo r importance. II'hether control of parameters such as temperature, dwell time (dose r a t e ) , an d atmosphere is essential d u r in g exposure, remains problematical. However, the design of th e research irradiator should facilitate answering these questions. With this in mind, some of the features most essential t o a research irradiator a r e outlined in Table VIII.

A very significant contribution to th e c u r r e n t low-dose program ha s been t he construction of radiation facilities by the AEC'$ tha t fulfill most of the research requirements. These units, a s well a s modified versions, a r e discussed by Manowitz e t al. (1964) and Kuhl a nd ILillantine (1964). The first of these to be used f o r f r u i t irradiation was installed at th e University of California, Davis (Romani c>t aZ., 1962). Others a r e now located a t MIT a n d at the University of Washington (primarily f o r research \vith sea foods), Jvhile comparable units at the UniL ersity of Florida and the University of Hawaii will be used principally f o r research with f r u i t products. F i gur e 1 illustrates th e general features of these research irradiators. The essential components a r e the stainless-steel tank, with parallel an d vertical plaques of Co-60 positioned a t th e bottom, a nd 3 water-tight irradiation chambers which a r e lowered, \ i a the elevator system, between an d on either side of the plaques. Following t h e Brookhaven prototype, additional expel iniental food i n atliat o i s have been constructed by t h e Piocess'g E q u i p m e n t Corporation, Lodi, N e w .Jersey.

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The center chamber has proven especially serviceable, because of the good dose distribution ( 2 8 % ) within a large volume, and facility for temperature and atmospheric control (Romani et al., 1963b). For dose uniformity the contents of the outer chambers are rotated 180 at half-dose. Two obvious limitations of these facilities a r e the lack of variable dose rates other than what can be achieved with lead shielding o r positioning the source above the gamma field, and the fixed size of the irradiation chamber. However, with 30,000 curies (approximately ‘4 maximum capacity) the throughput is ca. 30 megarad pounds per hour at a dose rate of approximately 300 Krad/hour. This is adequate for most food research, with the possible exception of the final phases, where quasi-commercial conditions and pilot shipments of fruits or vegetables may be considered. The research versatility of such a gamma source may be greatly increased if utilized a s a “pool,” with additional physical arrangements of CO-60 source rods and access tubes for the exposure of smaller amounts of materials at various dose rates. After the research phase, transitional-type irradiators may be required for introduction of a process into commercial channels. One such unit is the Marine Products Development Irradiator (MPDI) at Gloucester, Massachusetts (Miller and Herbert, 1964), and the truck-mounted irradiator now under construction for the fruit program. This latter unit is somewhat larger than the transportable Canadian unit (Anon., 1964) and incorporates additional desirable features. Other design concepts as well as larger commercial radiation facilities now in use a r e discussed in the review articles cited a t the beginning of this section.

c. TYPES O F

RESEARCHFACILITIES I N GENERALUSE FOR FOOD IRRADIATION In general, isotopic sources a r e of four characteristic types : open-air field sources, shielded chambers, water-attenuated pools, and self-contained lead-shielded units. Field and greenhouse sources have been used primarily for the exposure of whole plants to rather low, chronic doses, although pasteurization doses can be obtained near the source tube. Examples of such facilities a r e those a t the Brookhaven National Laboratory (Sparrow, 1960 ; Woodwell, 1963), one near Rome (D’Amato et al., 1962), one in Japan (Kawara, 1963), and one in Florida (Teas, 1958) which has received considerable use in the irradiation of fruit. More commonly used in biological research is the radiation

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chamber, where the gamma source is raised from a storage well into a room that is concrete walled or otherwise protected. The Phoenix facility at Michigan State has been used extensively in food-irradiation research (Nehemias e t al., 1954), and the installation at Cornell (Kertesz, personal communication) was designed for use with fruit and other plant products. These and similar “chamber” facilities incorporate the advantages of accommodating many samples of varied sizes which may be positioned in concentric circles about the source and thus be exposed to different dose rates. Such a n arrangement is illustrated by Matthee and Marais (1963). While the samples to be irradiated are easily accessible for remote monitoring systems, there exists the disadvantage of variable dose rates, especially within larger packages, such a s a box of fruit. One of the simplest installations is the well-type unit, with the radiation sources placed in the bottom under sufficient water for safe attenuation. The size and strength of such a facility is limited principally by the volume and depth of the pool, and the utility is dependent upon the s0phisticatic.n of the mechanical contrivances. Lead-shielded self-contained sources are available commercially from several companies. Others have been custom-designed f o r specific requirements, such as one by Kuhl et al. (1964) for the irradiation of circulating blood, suggesting the possibility of similar application for any moving stream. Riegert and Spinks (1961) described a n interesting unit in which the emitting isotope is rolled out of its lead container onto a horizontal track to expose samples positioned along the way. A4source at Wantage (Hannan and Thornley, 1957) utilizes radially arranged tubes which emerge from behind a shield. Pellets of Co-60 are moved along the tubes to the exposure position or retracted to the safe position for introduction of the food sample. A unit at Riso, Denmark, described by Brynjolfsson (1960) is unique in that the GO-60 is positioned in tubes arranged in a V. Food samples pass at right angles to the source tubes, with the exposure of large samples ( a t lower dose rates) achieved by positioning the co-60 at increasing distances from the vertex of the V. The foregoing discussion has been limited t o isotopic sources principally because they have been most readily adaptable to the several food research programs. However, it may be well to note that some of the more promising early research with fruit irradi-

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ation (Maxie and Nelson, 1959) was accomplished with linear accelerators and that the current research programs at the University of Michigan and Michigan State University include a comparative study of electron and gamma sources for the irradiation of fish and fruit (L. Kempe and B. Schweigert, personal communication). As discussed by Jefferson (1964, p. 303) one cannot generalize at this early date regarding the comparative merits of either machine or isotopic sources of radiation. However, with specific reference to fresh fruit or vegetable irradiation one can point to the obvious need for uniform treatment of the food sample. The size, shape, and easily bruised nature of most fresh foods favors the use of uniformly penetrating radiation such as gamma- or X-rays. Penetrating radiation can be obtained from a machine source utilizing the bremsstrahlung phenomenon (Whyte, 1959). As described by Kraybill and Brunton (1960), machine sources a r e generally one of 5 basic types: cascade generator, resonant transformer, high-frequency capacitors, Van de Graff generators, or linear accelerators. F o r a description of machine sources used in food irradiation, see articles by Thaarup (1963) and Hansen e t al. (1963) as well a s the general references cited above.

D. COMMERCIALUNITS In some commercial-scale irradiators the sources a r e contained in lead shielding and then raised to the appropriate position when in operation. A plant at Wantage, England, makes use of multiple passes of the product about the source unit, resulting in efficiencies as high as 40% (Tunstall, 1960). Another unique design has been incorporated in the Canadian mobile irradiator (Anon., 1964) where the packages revolve in a Ferris-wheel-like arrangement about the point source. Other and varied designs a r e discussed in the general references cited above. The many designs available with isotope sources a r e not generally seen with the “machine)’ counterparts. The great limitation of the electron sources-the lack of penetration at the lower permissible beam energies-may be especially serious in commercial units, where large packages would likely be involved. However, where applicable, surface treatments would reduce the extent of undesirable effects, such as changes in texture, experienced with penetrating radiation. Continued engineering achievements or a raising of Mev tolerance levels could well favor the machine sources. )

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VII.

FUTURE RESEARCH NEEDS

In most instances of fresh-food irradiation the dose thresholds f o r desired and detrimental effects unfortunately overlap. Thus,

while the promise of radiation pasteurization persists, it must be extracted from a maze of little-understood radiobiological phenomena. What is tiow required is the development of knowledge to facilitate the control and beneficial manipulation of primary radiation efTects and associated biological stresses. Experimentation toward this end must include a familiarity not only with the food and its noxious pathogens, but also with the intrinsic radiation responses of each. The objective in this review has been to illustrate the relevance of various radiobiological parameters and to substantiate the view that a working knowledge of radiation itself may be requisite for future progress in fresh-food irradiation. As to specific research needs, adverse textural changes appear to be the single most apparent detrimental effect of radiation in fresh fruits and vegetables. This has been well recognized by Kertesz and co-workers at Cornell. Their many excellent contributions to date on the radiation of aqueous model systems or intact tissues must be supplemented with experiments that explore the intricacies of the cell wall. Cellular reactions in general underlie radiation tolerance. This is well illustrated by the limited response, or apparent increased tolerance, of more senescent cells. The relationship between physiological state, metabolic activity, and radiation response must be explored. To this must be added considerations of radiation repair a n d recovery, factors of singular importance in achieving control of the pathogen and preservation of the host. All, or most all, of these problems a r e radiobiological in nature. M'ith full recognition of radiation parameters, the food scientist can thus contribute significantly to the radiobiological sciences while advancing the quest for a food irradiation technology. ACKNOWLEDGMENTS Appreciation is expressed t o the several colleagues, and especially to U I s. D. Brown, M. Goldman, and B. Schweigert, who have offered valuable suggestions f o r improvement of the manuscript. Mrs. Nancy Deghan arid Niss Lily Lim were most helpful in preparation of the manuscript. Original research reported in the paper received support f r o m the U.S.-A.E.C., Contract A T (11-1)-34, Project Number 112.

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REFERENCES Ahnstrom, G., and Ehrenberg, L. 1960. Dosimetry of Radiations Used f o r Food Preservation. Riso Report No. 16 (Proc. 1st Nordic Meeting on Food Preservation by Ionizing Radiations) pp. 15-16. Danish Atomic Energy Comm. Albersheim. P. 1963. Auxin induced product inhibition of pectin transeliminase a s shown by ozonolysis. Plant Physiol. 38, 426-429. Allen, A. 0. 1961. “The Radiation Chemistry of Water and Aqueous Solutions.” Van Nostrand, New Jersey. Andrews, H. L. 1961. “Radiation Biophysics.” Prentice-Hall, Inc., Englewood Cliffs, New Jersey. Anonymous. 1964. A mobile cobalt-60 source. Food P r o c e s s i z g a n d P a c k a g i n g (G. Brit.) 33, 149-150. Artandi, C. 1959. Plastic dosimetry. Nucleoxics 17 ( 2 ) , 62-63. Augenstein, L., Mason, R., and Rosenberg, B. (eds.) 1964. “Physical Processes in Radiation Biology.” Academic Press, New York. Bacq, Z. &I., and Alexander, P. 1961. “Fundamentals of Radiobiology.” Perganion Press, New York, London. Bacq, Z. M., and Alexander, P. 1964. Importance f o r radio-protection of the reaction of cells to sulfhydryl and disulfide compounds. Nature 203, 162164. Bain, J. &I., and Mercer, F. V. 1964. Organization resistance and the respiration climacteric. A u s t r a l i a n J . Biol. Sci. 1 7 ( l ) , 78-85. Beatty, A. V., and Beatty, J. W. 1963. Radiation recovery enhanced through inhil)itors of protein synthesis and amino acids. P ~ o c Natl. . Acad. Sci. U.S. 431. Bell, L. G. E. 1962. Polysaccharide and cell membranes. J . Theoret. Biol. 3, 132-133. Reraha, L. 1964. Influence of gamma-radiation dose r a t e on decay of citrus, pears, peaches, and on Penicillium italicurn and Botrytis cinerea i ) z vitro. P h ? J t o p O f h O ~ O g54 ~ ( 7 ) , 755-759. Biale, J . B. 1960a. Respiration of fruits. 1 x “Encyclopedia of P l a n t Physiology,” Vol. XII, pp. 536-592, Springer Verlag, Berlin. Biale, J . B. 1960b. The post-harvest biochemistry of tropical and subtropical fruits. Advaiiccs i~ Food Research 10, 293-354. Borstel, R. C. von, and St. Amand, W. 1963. Stage sensitivity to X-radiation during meiosis and mitosis in the egg of the wasp H u b r o b m e o i l . I n “Repair from Genetic Radiation Damage.” pp. 87-97. ( F . H. Sobels, ed.) The RIacniillan Co., New York. Branislav, R., Karapandiit, M., and Gal, 0. 1964. G-value measurements with differential calorimeter. Nuclcoiiics 22, 52-54. Breidenbach, W. R. 19F3. The effects of ionizing radiation on the lipid composition of the cytoplasmic particulates of f r u i t tissue. M.S. thesis. Univ. of Calif., Davis, Calif. Brownell, L. E., Neheniias, J. V., and Purohit, S. N. 1957. Gamma-irradiation facilities designed t o process commercial quantities of food products. I?L “Atomic Energy in L4griculture.” pp. 367-389 (C. L. Colmar, ed.), AAAS, Washington, D.C.

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Brynjolfsson, A. 1960. The Co-60 irradiation facility a t the Danish Research Establishment, Riso. Riso Report No. 1 6 (Proc. 1st Nordic Meeting on Food Preservation by Ionizing Radiations) pp. 5-7. Danish Atomic Energy Conini. Hiyijolfsson, A. 1963. Three-dimensional dose distribution in samples i r r a diated by electron beams. Radiation Research (Proc. of a n Intern. Conf., Natick, Mass. J a n . 1963) pp. 116-129, Dept. of Conim., P B 181506. Caldwell, E. F., and Frainey, J. 1958. Use of cobalt glass dosimetry in electron beam radiation of foods. Food Rcseurch 23, 599-602. Cervigni, T., and Belli, bl. L. 1962. Modifications de l’acide ;i-indolacetique p a r les radiations ionisantes. (Proc. Symposium o n Biol. EfFects of Ionizing Radiation a t the Molecular Level) ( I A E A , ed.) , pp. 193-202, Vienna, 1962. Charlesby, A. 1955. The degradation of cellulose by ionizing radiation. J . Poly?ner S c i . 15, 263-270. Charlesby, A. (ed.) 1964. “Radiation Sources.” The 3lacniillan Co., New Tork. Clarke, I. D. 1961. Some effects of gamma radiation on the chemical and physiological changes in f r u i t s . 111 “Recent Advances in Botany.” Univ. of Toronto Press, Toronto, Canada. pp. 1176-1180. Clarke, I. D., and Fernandes, S. J. G. 1961. Effects of gamma-radiation on the protein content of apples and pears. Iiiterii. J . i l p p l . R a d i a t i o n a n d Isotopes 11, 186-189. Cloutier, J. A. R., Cox, C. E., Manson, J. &I., Clay, 31. G., and Johnson, L. E. 1959. Effect of storage on carbohydrate content of two varieties of potatoes grown i n Canada and treated with gamma radiation. Food R e s c a ~ c l ~ 24, 659-664. Cocking, E. C., and Gregory, D. W. 1963. Organized protoplasmic units of the plant cell. I. Their occurrence, origin and structure. J . Exptl. B o t u n y . 14, 504-51 1. Conger, A. D., Randolph, M. L., S h e p p a d , C. W.> and Luippold, H. J . 1958. Quantitative relation of R B E in T r a d e s c a x t i a and average L E T of gamma-rays, X-rays, and 1.3, 2.5 and 14.1 &lev f a s t neutrons. Radiation R e s e a r c h 9, 525-547. Cook, R. F. 1964. The effects of w a t e r and a protective agent on gamma-ray induced f r e e radicals in mustard seeds. I n t e m . J . R a d i a t i o n Biol. 7 ( 5 ) , 497-504. Cooper, R. D., and Siu, R. G. H. 1957. Radiological safety. R a d i a t i o n l’rcscrv. of Foods. U.S. A r m y QM. pp. 399-409. Cowling, E. B. 1963. S t r u c t u r a l features of cellulose t h a t influence its susceptibility to enzymatic hydrolysis. I n “Advances in Enzymic Hydrolysis of Cellulose and Related Materials.” ( E . T. Reese, ed.) pp. 1-32, Pergamori Press, New York. Curtis, H. J. 1960. Discussion following paper by J. W. King and A. W. Galston. I n “Radiobiology.” (Proc. 3rd Australasian Conf. on Radiobiology) , ( P . L. T. Ilbery, ed.) p. 244, Butterworth’s, London. U’Amato, F., Scarascia, G. T., Belliazzi, U., Bassani, A., Cambi, S., Cevolotto, P., Giacalone, P., and Tagliati, S. 1962. T h e gamma radiation field of the “Comitato Nazionale per L’Energia Nucleare,” Rome. R a d i a t i o n Botany 1 ( 3 ) , 243-246.

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D’Angio, G. J., and Lawrence, J. H. 1963. Medical research with high energy heavy particles. Nucleonics 21, 56-61. Das, N. K., and Alfert, &.I.1961. Accelerated DNA synthesis in onion root meristem during X-irradiation. PTOC.Natl. Acarl. Sci. U.S. 4 7 ( l ) , 1-6. Dendy, P. P. 1964. The role of radiation in cell biology. S ci . I ’ Y o ~ J . .52, 191204. Deshpande, S. N., Cherry, J. N., Draudt, H. N., and Desrosier, N. W. 1964. Degradative processes in pectinic acid exposed to high energy radiations and actions of pectin enzymes. Abstr. 24th Ann. Meeting, Inst. Food Technologists, Wash. D.C., p. 67. Desrosier, N. W., and Roenstock, H. &I. 1960. “Radiation Technology in Food, Agriculture, and Biology.” Avi Publ. Co., Westport, Connecticut. De Zeeuw, D. 1961. Experiments on the preservation of fresh f r u i t by irradiation. Food I r r a d i a t i o n 1( 3 ) , A5-A7. Dorfman, L. M. 1963. Pulse radiolysis: F a s t reaction studies in radiation chemistry. In “Radiation Research.” (Proc. Intern. Conf., Natick, Mass., J a n . 1963) pp. 59-73, Dept. of Comm., P B 181506. Draganic, I. 1959. Action des rayonnements ionisants s u r les solutions aqueuses d’acide oxalique. J. Chim. Phys. 56, 9-15. Dupaigne, P. 1964. Application des radiations ionisantes aux produits fruitiers. Fruits ( P a r i s ) 19, 31-42. Ehrenberg, A. 1961. Research on free radicals in enzyme chemistry and in radiation biology. I,/ “ F r e e Radicals in Biological Systems.” (M. S.Blois, Jr., H. W. Brown, R. M. Lemmon, R. 0. Lindblom, 111. Weissbluth, eds.) pp. 337-350, Academic Press, New York. Ehrenberg, A., Ehrenberg, L., and Loefroth, G. 1962. Radiation-induced p a r a magnetic centers in plant seeds a t different oxygen concentrations. A b h a x d l . dczct. A k a d . ll’iss. ( B e d i n ) No. 1, 229-232. ( U S . Atomic Comm. N u c l e a r S c i . Abstr. l i , 40570) (1963). Elkind, &I. M., and Sutton, H. 1960. Radiation response of mammalian cells grown in culture. I. Repair of X-ray damage in surviving Chinese hamster cells. Radiation Resczarch 13, 556-593. Elkind, M. M., Whitmore, G. F., and Alescio, T. 1964. Actinomycin-D: Suppression of recovery in X-irradiated mammalian cells. Science 143 (2), 1454-1457. Eninierson, P. T.. and Howard-Flanders, P. 1964. Sensitization of anoxic bacteria to X-rays by di-butyl nitroxide and analogues. N a t u r e 204, 1005-1006. Epstein, H. T. 1963. “Elementary Biophysics.” Chapt. 9. Addison-Wesley Publ. Co., Reading, 3Iass. E r r e r a , M., and Forssberg, A. 1961. “Mechanisms in Radiobiology.” Academic Press, New York, London. Evans, H. J. 1963. Possible reasons f o r variations in chromosome radiosensitivity during mitotic and meiotic cycles. 112 “Repair f r o m Genetic Radiation Damage.” ( F . H. Sobels, ed.), pp. 31-44. The Macmillan Co., New York. Fernandes, S. J. G., and Clarke, I. D. 1962. Effects of ionizing radiation on the acid metabolism of apples (Cox Orange Pippin). J . Sci. Food A g r . 13, 23-28.

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Tomada, Y., and Tsuda, M. 1961. Some aspects of cross-linking a n d degradation of gelatin molecules i n aqueous solution irradiated by Coo’ gamma rays. J . P o l y m e r Sci. 54, 321-328. Tunstall, J. 1960. Britons open pilot plant t o t r y radiation processes. Nucleonics 18, 100-104. Van Lancker, J. L. 1962. Cytochemical injury of X-radiation. Federation Proc. 21, 1118-1123. Vidal, P. 1963. Preservation of soft f r u i t by radiopasteurization. Food Irrarliation 4 (1-2), A2-A9. Villareal, F., Luh, B. S., and Romani, R. J. 1961. High-velocity electron i r r a diation of tomato paste. Food Technol. 15, 220-223. Wahba, I. J., Tallman, D. F., a n d Massey, L. M., Jr. 1963. Radiation-induced gelation of dilute aqueous pectin solutions. Science 139,1297-1298. Weiss, J. 1952. Chemical dosimetry using ferrous and ceric sulfate. Nucleoiiics 10, 28-31. Whyte, G. N. 1939. “Principles of Radiation Dosimetry.” p. 124. J. Wiley & Sons, New York. Willard, J. E. 1963. Radiation chemistry of gases. I n “Radiation Research.” (Proc. Intern. Conf., Natick, Mass.) pp. 77-84. U.S. Dept. of Comm., P B 181506. Wolff, S. 1960. Chromosome aberrations. I n “Radiation Protection and Recovery.” (P. Alexander, ed.) pp. 157-174. Pergamon Press, New York. Woodwell, G. 111. 1963. Design of the Brookhaven experiment on the effects of ionizing radiation on a terrestrial ecosystem. R a d i a t i o n Botany 3 ( 2 ) , 125-133. Young, R. E. 1965. The effect of ionizing radiation on respiration and ethylene production of avocado fruit. N a t u r e (in press). Zimmer, K. G. 1960. The development and prospects of quantitative radiobiology. I n “Immediate a n d Low Level Effects of Ionizing Radiations.” ( A . A. Buzzati-Traverso, ed.) pp. 1-10. Taylor and Francis Ltd., London. Zimmer, K. G. 1961. “Quantitative Radiation Biology.” (Translation from German by H. B. Griffith.) Oliver and Boyd, Edinburgh and London. Zimmer, K. G. 1964. Reviewing “Dosimetrie ionisierender Strahlung” by K. K. Aglinzew. R a d i a t i o n B o t a n y 4, 344.

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F O O D IRRADIATION-PHYSIOLOGY OF FRUITS AS RELATED T O FEASIBILITY OF THE TECHNOLOGY* By E.

c. M A X I E

AND /lDEL

ABDEL-KADER

Univcysifu o f C a l i f o m i a , Davis, C a l i f o n i i a

I. Introduction . ................... 11. F r u i t Respirati Climacteric Class o f F r u i t s ................................... 111. F r u i t Respiration in Relation to Radiation EffectsNonclimacteric F r u i t s . . . . . . . . . ............... IV. Effect of Irradiation on Texture of F r u i t s . . . . . . . . . . . . . . . . . . . . . . V. Effect of Radiation on Chemical Components of F r u i t s . . . . . . . . . . A. Nutritionally Significant Compounds B. Other Chemical Constituents . . . . . . . VI. Effects of Radiation on Organoleptic Attributes of F r e s h F r u i t s . . VII. Wholesomeness of Irradiated F r u i t s . . . . . . . . . A. Induced Radioactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Wholesomeness of Irradiated F r u i t s . . . . . . . . . . . . . . . . . . . . . . . . VIII. Ozone Produced by Ionizing Radiation and its Relation t o F r u i t Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Feasibility of Irradiation as a Commercial Technology with Fresh F r u i t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Concluding Remarks . . . . . . . . ................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I.

105 107 114 116 123

129 133 134

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INTRODUCTION

The concept of using ionizing radiation to extend the shelflife of fresh fruits is not new. The basis for its potential application dates from a paper by Prescott (1904) published shortly after the discoveries of the X-ray by Roentgen (1898) and of natural radioactivity by Becquerel (1896). Attempts t o develop a practical technology f o r perishable commodities was given impetus by a paper by Brasch and Huber (1947). Hannan (1955, 1956) and Desrosier and Rosenstock (1960) have reviewed some of the technical problems involved in the irradiation of foods, including fruits. Dupaigne (1958, 1964) has written popular reviews on fruit irradiation.

* Preparation of this review w a s supported by t h e U.S. Atomic E n e r g y Commission, Contract UCD 34P80. 105

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Postharvest deterioration of f r u i t s an d vegetables can be classified into t hr e e categories : 1) biochemical a n d physiologicalendogenous changes associated with ripening a n d senescence ; 2 ) pathological-derived f r o m attacks by rot organisms, usudehydration a n d mechanially f ungi ; a nd 3 ) physical-primarily cal injury. Considering th e effects of radiation on living systems, one might expect some beneficial effect in categories 1 a n d 2. With category 3 , only adverse effects seem probable. Two approaches may be made in applying radiation t o f r u i t s : 1) complete sterilization of all ro t organisms by employing high doses ; or 2) substerilization doses t h a t inhibit ro t o r physiological processes only temporarily. Complete sterilization of fre s h f r u its an d vegetables is not feasible, f o r i t damages quality attributes such as appearance, aroma, flavor, an d texture. Work on sterilization has been summarized by Han n an (1956) a n d b y th e U.S. A r m y Quartermaster Corps (1957). Th e present review emphasizes t h e low-dose aspects of f r u i t irradiation. There is, perhaps, no area in applied biology where claims f o r th e effectiveness of a protective process have been as extravagant as f o r t h e irradiation of fruits. Stories in th e popular press have implied t h a t irradiated fresh f r u i t s m ay be kept at room temperat u r e f o r as long as two y ears with no loss in quality. This is a biological absurdity, f o r it ignores th e living n a tu re of th e products a nd t h e endogenous degradative processes in them. Maxie a nd Sommer (1964) listed most of the following points t h a t m us t be established before irradiation can become a practical technology f o r an y perishable commodity : 1) There mu st be a distinct benefit, i.e., less decay in th e product, or reduced losses due to physiological disorders. 2 ) There must be no majo r loss in th e nutritional quality of th e product. 3 ) Radiation-induced changes in texture must not niake the commodity excessively susceptible to impact a n d vibration inju r i e s dur i ng shipment by rail o r truck-or adversely affect its palatability. Radiation-induced flavors an d odors must not be objectionable and, preferably, should not make th e product atypical of th e species a nd/ or variety. 5 ) T he appearance of th e irradiated product must be attractive and, preferably, typical of th e species and,’or variety. 6 ) There m u st be no radiation-induced substances harmful to humans, a nd a legal requirement in th e United States t h a t mu s t

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be met is clearance of the irradiated product for human consumption by the Federal Food and Drug Administration. 7) The postirradiation susceptibility of the product to infections by decay organisms must not be greatly enhanced. 8 ) Methods f o r irradiation treatment must not entail extensive additional handling of the commodity. 9) The refrigeration requirement for removal of respiratory heat from the irradiated commodity must not be excessive. 10) The cost of the technology must not exceed the benefits to be gained; certainly, it must not result in a major increase in the price of the commodity t o the consumer. No product has met all the above requirements. Satisfying some of them requires detailed studies involving the physiology of the commodity. To date, no review has appeared concerning the radiation physiology of fruits. Therefore, the objectives of this review a r e to: (1) review the physiology of fruits as it may affect their response to irradiation; (2) indicate methods for valid research on the irradiation of fruits and other perishable products; ( 3 ) evaluate the effects of irradiation on the quality attributes of f r u i t s ; and (4) evaluate the prospects for a commercial technology. Several excellent reviews have been written on the biochemistry and physiology of fruits. Biale (1950,, 1960a) wrote on fruit metabolism in general and (1960b) published a specialized review on tropical and subtropical fruits. Biale and Young (1962) reviewed the biochemistry of fruit maturation. Hulme (1958) covered biochemical and physiological studies with apples and pears. Porritt (1951) and Burg (1962) dealt with ethylene in relation to fruit ripening. Miller (1946, 1958) treated the physiology of citrus fruits in storage. Knowledge of the general biochemical and physiological systems in fruits is a prerequisite to planning, performing, and interpreting the data of meaningful experiments involving any stimulus or stress condition. Unfortunately, such systems have received little consideration in many papers concerned with fruit irradiation. 11.

FRUIT RESPIRATION I N RELATION T O RADIATION EFFECTSCLIMACTERIC CLASS O F FRUITS

Fruits may be classified into two categories with regard to their respiratory behavior during ripening (Biale, 1960a,b). The first, known as the climacteric class, exhibits a rate of CO,

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evolution and oxygen consumption that slowly declines to a minimum just prior to the onset of ripening. As ripening begins, the respiratory rate increases dramatically. Simultaneously, green colors fade, yellow and red colors appear, and flesh firmness declines, at least in part from changes in pectic substances. Chemical components comprising the soluble solids in the cell sap increase, the rate of production of ethylene and other volatile materials increases, and the fruit takes on the appearance of ripeness. The respiratory rate reaches a peak within a few days. Most fruits of the climacteric class a r e considered “eating ripe” at or near this peak. The declining rate of respiration following the climacteric peak signifies final degradation of the fruits, leading to death. The preclimacteric minimum, in the sequence just described, is a pivotal event with regard to the response of the climacteric class of fruits to stimuli. Fruits harvested a t a stage of maturation just prior to the onset of the climacteric rise may be held in cold storage longer than the same variety harvested after onset of the rise (Maxie and Baker, 1954). Fruits subjected to controlled atmospheres (low-oxygen and high-carbon dioxide concentrations in the ambient a i r ) seem to respond much more favorably if harvested near the minimum point of the preclimacteric respiratory pattern than if harvested after ripening has begun, findings of Smock (1949) and Smock and Van Doren (1941) imply. Ethylene gas, in concentrations of 0.1-10 parts per million or higher, hastens the ripening process if applied to fruits in the preclimacteric stage (Biale, 1960a) but has little effect if applied after onset of the climacteric rise (Gane, 1937; Hansen and Hartman, 1937). Dinitrophenol applied to the tissues markedly stimulated the respiration of preclimacteric avocados but had much less effect on climacteric fruits (Millerd et al., 1953). Considering these studies, one would predict that the stage of a f r u i t in the climacteric sequence might have a marked effect on the fruit’s response to radiation. Mikaelson and Roer (1960) noted that irradiation lengthened the storage life of preclimacteric apples. Romani et a l . (1961) reported that the respiratory rate of Bartlett pears ivas affected much less if the fruits were irradiated a s they approached the climacteric peak than if irradiated in the preclimacteric state. They made only one treatment, however. Maxie et al. (1966) used doses of 300 kilorad on Bartlett pears a t several stages of the climacteric sequence. Fruits irradiated before the onset of the climacteric showed a marked increase in

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respiration rate, as shown in Fig. 1. Despite the accelerated respiration, the irradiated fruits did not ripen normally. Fruits irradiated a f t e r t h e climacteric was well advanced showed only a small increase in respiratory rate, and ripening was not affected. The climacteric is associated with ripening of fruits, and it is incorrect to apply this term to a n increase in respiratory rate unless the biochemical and physiological changes associated with ripening occur simultaneously. The events described above, relative to the climacteric, a r e concerned with stages of ripeness, not 60

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FIG. 1. Effect of gamma irradiation at various stages of the climacteric on the respiratory rate of Bartlett pears. (Redrawn from Maxie, e t al., 1966.)

maturity. Maturity is a measure of the capacity of fruits to ripen t o acceptable quality if harvested, and is determined by events occurring with the fruit attached to the plant. A fruit does not “mature” after harvest; a n immature fruit may ripen but will be of low quality. In some fruits, such a strawberries, “mature” and “full ripe” are synonymous. In these cases fruits harvested less than ripe a r e immature. In t h e climacteric class of fruits, ripening is intimately related to ethylene production by the fruit. Two theories exist for the role of ethylene in the ripening of these fruits: One holds that the production of ethylene by the fruit triggers the onset of the climacteric and ripening (Hansen, 1942; Kidd and West, 1945; Burg and Burg, 1962) ; the other maintains that ethylene is only a by-product of ripening (Biale et al., 1954). Regardless of the

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mechanism of ethylene action in fruit ripening, i t is to be expected that the irradiation of fruits will have a marked effect on the production and/or action of this compound. Smock and Sparrow (1957) showed that doses of 20,000 and 40,000 roentgens of gamma rays decreased ethylene production in post-climacteric Rhode Island Greening apples. Maxie et aZ. (1966) showed t h a t doses of 100-400 Krad induced a n immediate burst of ethylene from preclimacteric pears. The effect was temporary, particularly at the higher doses. As shown in Fig. 2, fruits subjected to 300 110,

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FIG.2. Effect of gamma irradiation on ethylene production by Bartlett pears. (Redrawn from Maxie, e t al., 1966.)

and 400 Krad exhibited a much lower rate of ethylene production (after the initial surge described above) than did fruits subjected t o 200 Krad or less. Fruits subjected to 100 and 200 Krad ripened normally, and the onset of ethylene production associated with ripening began at the same time as in unirradiated fruits. The peak in the rate of ethylene evolved, however, was much higher f o r the latter two irradiated lots. The origin of radiation-induced ethylene in fruits is not known. Two possibilities a r e worthy of consideration. Firstly, physical injury is known to induce ethylene production in some fruits (Burg, 1962), and the textural changes that gamma irradiation

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O F FRUITS

induces in fruits indicate that the fruits a r e physically injured. Secondly, fruits contain many C2-Co alcohols and esters. Bombardment of alcohols with helium nuclei (Henry, 1957) and gamma rays (Maxie et al., 1966) produces traces of ethylene. Similarly, fumaric and linolenic acids produce C,H I when subjected to doses of gamma rays as low as 100 Krad (Maxie and Rae, 1965). Pears irradiated a t the climacteric peak showed a decreased rate of ethylene production, but changes in the fruits associated with senescence were not affected (Maxie e t al., 1966). Since gamma radiation can slow ethylene production in fruits t h a t produce large quantities and since irradiation with 300 and 400 Krad inhibited the ripening of pears, one might conclude that the ripening is retarded by reducing the capacity of the fruits to produce ethylene. That is not the case, however. When pears were subjected to inhibitory levels of gamma radiation and then exposed t o ethylene gas, the fruits subsequently produced the gas a t a rate which would stimulate ripening in unirradiated fruits, as shown in Fig. 3. Nevertheless, the ethylene-treated irradiated

2

4

6

8

10

12

14

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TIME IN DAYS

FIG.3. Effect of gamma irradiation and ethylene treatments on ethylene production by Bartlett pears. Applications were in the order listed in dual treatments. (Redrawn from Maxie e t al., 1966).

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fruit still failed to ripen normally. Pears subjected to 300 and 400 Krad of gamma rays and then held 8 days in 1000 p.p.m. of ethylene, remained green, failed to soften, and were insipid in flavor. Thus, gamma rays have a dual action on preclimacteric pears : they not only reduce ethylene production but also reduce sensitivity to the ripening action of ethylene. One must not conclude that the behavior described above for Bartlett pears is typical of all climacteric types of fruit. Maxie and Sommer (1963) showed that peaches and nectarines-both climacteric types-irradiated in the preclimacteric state were stimulated to ripen by doses a s high as 600 Krad. Figure 4 shows

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8

Late

that gamma irradiation stimulates onset of the climacteric in peaches. This action is apparently caused by radiation-stimulated ethylene productions, as shown in Fig. 5 . The respiratory and ethylene response t o gamma irradiation was accompanied by changes in the color (greens, yellows, and reds), aroma, and flavor normally associated with ripening. It thus appears that, unlike Bartlett pears, peaches and nectarines irradiated a t the same doses as pears lose neither their sensitivity to ethylene nor their capacity to produce the gas. Evidence is still insufficient for drawing any firm conclusions

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O F FRUITS

regarding the relationship between ethylene and the radiation physiology of the climacteric class of fruits. It may develop that the response obtained with a particular fruit will depend on the dose, with some doses stimulating ethylene production and ripening, and other doses inhibiting both processes. Inhibition of ripening by radiation has been reported for bananas (Hannan, 1956; Teas et al., 1962) ; tomatoes (Hannan, 1956; Burns and Desrosier, 1957; Salunkhe et al., 1959a; Salunkhe

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1961; Truelsen, 1963 ; Abdel-Kader e t al., 1965) ; plums (Hannan., 1956) ; and papayas (Maxie and Sommer, 1963). I n every case where irradiation has delayed ripening significantly, t h e fruits subsequently either failed to ripen or exhibited an anomalous ripening and were of poor quality except with bananas subjected to low doses (Teas e t al., 1962; Amezquita, 1965). It appears that with many fruits the degree of ripeness may be critical with regard t o the feasibility of irradiation as a protective technology. The position of the f r u i t in t h e climacteric sequence is an excellent physiological guide t o the response

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E. C. MAXIE A K D ADEL ABDEL-KADER

that may be expected. If the fruits a r e preclimacteric, the effects of irradiation will likely be more adverse than if the fruits a r e beyond the onset of the climacteric rise in respiration. With respect to ripening, inhibition can be expected only in preclimacteric fruits. Therefore, with fruits of the climacteric class, the stage of t h e climacteric must be known at the time of irradiation if the data obtained a r e to be meaningful. Ill.

FRUIT RESPIRATION IN RELATION TO RADIATION EFFECTSNONCLIMACTERIC FRUITS

Fruits of the nonclimacteric class exhibit a slowly declining rate of respiration after harvest. Biale (1960a,b) listed several fruits in this class, but indicated that some of them a r e classified only tentatively. Some characteristics other than respiratory drift distinguish fruits of the nonclimacteric class from climacteric f r u i t s ; they a r e often fully ripe at harvest, show no rapid chemical or physiological changes associated with ripening either on or off the tree, possess no reserve of starch, produce but little ethylene, show a respiratory drift curve from ethylene treatment that differs in pattern from that of untreated fruits, and show a respiratory response proportional to ethylene concentration at low concentrations. Considering the inherent differences between climacteric and nonclimacteric fruits, one would expect some clearly defined differences between fruits of the two classes in respiratory response to irradiation. Maxie and Sommer (1963) showed that the respiratory rate of lemon and orange fruits increased immediately after treatment with a wide range of doses of gamma rays. They noted that irradiated green lemons “degreened” more rapidly than did unirradiated fruits, and showed that doses of 50, 100, and 200 Krad induced in green lemons a respiratory response resembling the climacteric (Fig. 6 ) , which is atypical of unirradiated citrus. Within 30 minutes after irradiation at all doses, ethylene could be detected in the internal atmosphere of the irradiated lemons (Fig. 7 ) . Measurements with intact fruits showed that t h e r a t e of ethylene production in irradiated fruits increased to a peak at about the time of the maximum respiratory activity, then declined, giving a curve f o r ethylene production not unlike t h a t f o r fruits of t h e climacteric class. The green color of the irradiated fruits had disappeared by the time of the decline in rates of respiration and ethylene evolution. This, too, is a re-

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sponse typical of clirnacteric fruits. However, lemon fruits do not ripen after harvest (though they change color). Therefore, one must not conclude that irradiation induces a climacteric in green lemon fruits. Oranges and grapefruit show a n increased respiratory rate foilowing irradiation. They also evolve measurable quantities of ethylene, but there is no peak comparable to that shown by lemons (Maxie et al., 1965a). Since these two fruits are harvested after the green color disappears, no color change I20

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FIG.7. Effect of gamma irradiation on r a t e of ethylene production by E u r e k a lemons. (Redrawn from Maxie e t al., 1965a.)

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E. C. MAXIE AND ADEL ABDEL-KADER

comparable to that in lemons was observed. When immature, green oranges were irradiated, ethylene production was stimulated, and the green color partially disappeared; but the effects were much less pronounced than with lemons. The irradiated oranges never developed the full, ripe color t h a t lemons did. It appears that the difference between lemons and oranges in response to gamma irradiation is more of a species variation than a difference in degree of maturation at harvest. Information is insufficient to indicate a clear difference between climacteric and nonclimacteric fruits in response to irradiation. As more evidence accumulates, however., response to ionizing radiation may prove of value in classifying fruit into either the climacteric or nonclimacteric category. IV.

EFFECT OF IRRADIATION ON TEXTURE OF FRUITS

At the very outset it must be emphasized that evaluation of the effects of irradiation on the texture of fruits requires objective data, obtained by reliable instrumentation or by trained taste panels following simulated or actual transit tests. In fruits held in stationary tests, textural changes due to irradiation may not be apparent unless the doses a r e well in excess of the practical tolerance of the product. The effects of irradiation on the texture of fruits must be considered from two points of view : the direct (and immediate) effect on the firmness of the tissues, and a delayed, secondary effect on ripening of the fruits. The effect of irradiation on ripening is inhibitory in apples (Massey et aZ., 1964), bananas (Teas et al., 1962; Amezquita, 1965) ; papayas (Maxie and Sommer, 1 9 6 3 ) ; pears (Hannan, 1956; Maxie and Sommer, 1 9 6 3 ) ; and tomatoes (Hannan, 1956; Burns and Desrosier, 1957; Truelsen, 1963; Abdel-Kader e t al., 1964). Irradiation seems to stimulate ripening in nectarines and peaches (Maxie and Sommer, 1963). There a r e two important aspects to the effects of irradiation on the texture of fruit tissues: (1) the susceptibility of irradiated fruits to mechanical i n j u r y ; and (2) the contribution of texture to the palatability of fruits when they a r e subsequently ripened. In the manipulations involved in harvesting, packing, storing, shipping, and retailing fresh fruits, four types of mechanical injury must be considered (Sommer and Luvisi, 1960) : cuts,

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compression, impact, and vibration. Cuts result from contact of the fruits with sharp edges. It is not likely that irradiation would increase the sensitivity of fruits t o this type of wound. Compression wounds result from pressure on the fruits. It is probable that gamma irradiation would increase the susceptibility of fruit to this type of injury. Maxie and Sommer (1963) showed that lemons and oranges lose resistance to compression following irradiation. Impact damage results from sharp jolts when individual fruits or packages of fruits a r e dropped, or when carrier vehicles accelerate or decelerate. Symptoms of this injury are not often immediately visible, but may appear later as bruises in the flesh of the fruit. Vibration damage is caused by the abrasive scuffing of individual fruits against one another or against container surfaces during movement of the carrier vehicle. Such injury is normally confined to the surface tissues of the fruit and is often visible immediately after it occurs. Before any firm conclusions can be drawn concerning the feasibility of irradiation a s a technology with any fruit, data must be obtained on postirradiation susceptibility of the product to compression, impact, and vibration damage. It is likely that irradiated fruits will behave very differently after a shipment of several hundred miles from their behavior when held stationary, even under the same condition of temperature and relative humidity. Mechanical injury in fruits is important f o r two reasons: it is unsightly and may detract from the marketability of the product, and any open wounds could provide access to infection by pathogens. Unfortunately, little attention has been given to the susceptibility of irradiated fruits to mechanical injury. One might expect that vibration injury might be enhanced in fruits subjected to either electrons or gamma rays. It is likely that electron irradiation would affect vibration injury primarily because penetration is limited to tissues at or near the surface of the fruit. Fruits subjected to gamma-rays, in contrast, might show compression and impact injury as well a s vibration damage because of the deeper penetration, giving more extensive textural changes deep in the fruit tissues. I t is difficult to evaluate the effects of radiation on fruit tissues in terms of a practical technology for shelf-life extension. The reasons a r e : (1) most experiments have been stationary, with the irradiated fruits not subjected to mechanical forces that might divulge and/or intensify injury; (2) many papers

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present only observations and opinions of fruit response to irradiation, with no objective measurements reported of textural change in response to treatment; ( 3 ) the physiological status of the fruit-such a s maturity-at the time of treatment is often not determined; ( 4 ) species and varieties of fruits often vary considerably in responses to stress, but this has not been investigated to any extent; (5) climatic conditions and cultural practices often have a striking effect on the response of fruits to stress. This, too, is largely unexplored with respect to irradiation effects. Hannan (1956) used sterilization doses of electrons ( 2 M.e.v.) on a number of fruit tissues, and reported no major adverse effects on fruit texture. His criteria for judging the seriousness of the irradiation effect, however, were not clearly stated. I t is now clear that sterilizing doses a r e not feasible for most fruits (Maxie and Sommer, 1964). Therefore, only doses in the range of 0-500 Krad are considered here. When many fruits ripen, one of the most striking changes that occur is a conversion of insoluble protopectin to soluble pectin (Biale, 1960a; Joslyn, 1962; Kertesz, 1951). This change is a logical one to study with respect to the softening associated with irradiation. Kertesz and his associates at Cornell University, Geneva, New York, have contributed several outstanding papers on the subject, which a r e cited below. Glegg et al. (1956) showed that the threshold dose for softening was 34,700 r in Gravenstein apples, 166,000 r in Chantenay carrots, and 316,000 r in Detroit Dark Red beets. Boyle e t al. (1957) later showed a wide variation in threshold values among seven varieties of apples, from 4.2 x lo3 to 107 x 103r. The dose required for 50% softening of the flesh varied from 123 x lo3 to 134 x 103r. Massey et al. (1964) showed immediate softening in some varieties of apples at doses above 10 Krad, but after long-term storage the irradiated fruit was firmer. Maxie et al. (1966) showed a similar postirradiation delay in softening in Bartlett pears subjected to 300 and 400 Krad. Kertesz et al. (1956) noted that pectin in solution was degraded at doses of 8300 r. Skinner and Kertesz (1960) showed that gamma radiation caused random hydrolytic breakage of the polygalacturonide macromolecule, yielding fragments of lower molecular weight. Kertesz et al. (1964) showed t h a t in apple fruits the threshold dose was the same for the degradation of cellulose and pectin as for softening of the tissues. Somogyi and

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Romani (1964) dosed pears and peaches with 300, 600, and 900 Krad, and showed a good correlation between tissue softening, decreases in protopectin, and increases in pectin and pectate. They postulated that increased pectin methylesterase activity may contribute to the initial pectin degradation in irradiated fruits. Their enzyme assays, however, were made on cherry fruits, for which no textural or pectin degradation data were presented. It is improbable that the striking softening of fruits during irradiation is primarily an enzymatic phenomenon. The reasons are: (1) the rate of change and the magnitude and distribution of the effect in the tissues a r e not compatible with enzymatic capabilities ; ( 2 ) the effect is temperature-insensitive ; and ( 3 ) there is no measurable aftereffect immediately following treatment. If radiation stimulated enzymatic reactions capable of causing textural changes a t the rate found in fruits during irradiation, one would expect the reaction to continue f o r some time after the treatment ends. It should be noted t h a t both cellulose and pectin show a nonenzymatic aftereffect in degradation if irradiated dry (Glegg and Kertesz, 1956). The aftereffect in cellulose is initiated by oxygen and can be terminated by the presence of moisture (Glegg, 1957). To date, no significant aftereffect has been shown in fresh fruits. I n very starchy fruits, such as green bananas, it is possible that the mass of starch grains in the flesh may contribute to the firmness of the tissues as measured by mechanical devices. Kertesz et al. (1959) studied the effects of radiation on the starch and starch fractions. They found 60 Krad to be the threshold for a major change in starch, and t h a t the number of reducing groups increased markedly in amylopectin and amylose a t doses above 100 Krad. Glegg and Kertesz (1956, 1957), Glegg (1957), and Kertesz e t al. (1964) have shown cellulose t o be degraded by ionizing radiation. Kertesz et al. (1956) showed t h a t cellulose degradation occurred in apples at the same dose that caused softening of the tissues. It is well established that ionizing radiation degrades at least three of the major polymers-cellulose, pectin, and starch --that could contribute to the texture of fruits. It is possible that a role in the loss of texture. The each polymer-if present-has major role, however, is likely played by the pectins, for starch is lacking in many fruits, and in others is present in only low amounts by the time the fruits a r e harvested. Thus, it is unlikely t h a t starch degradation by radiation contributes much to

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losses in texture in an y appreciable number of f r u its . Similarly, cellulose is not a m ajo r fraction in most fru its . Degradation of cellulose embedded in th e cell walls of f r u i t s by radiation would probably contribute to a loss of texture, b u t it seems unlikely t h a t t his compound is th e p rimary structural component being degraded (Kertesz et al., 1964). T h a t pectin degradation in f r u i t s by ionizing radiation involves radiochemical as well a s physical phenomena is shown by the proctective action of anoxia. Maxie a n d Sommer (1963) showed t h a t Barlett pears irradiated in anoxia ripened in ii near-normal fashion following doses t h a t produced anomalous ripening in f r u its irradiated in air. Somogyi a n d Romani (1964) showed t h a t anoxia protected against textural changes in nectarines, peaches, an d pears irradiated t o 300 a n d 900 Krad. Glegg (1957) showed t h a t exclusion of oxygen prevented t h e postirradiation degradation of cellulose. Bussel (1965) showed t h a t t h e internal atmosphere of Bartlett pears could be completely freed of oxygen in about 40 minutes when th e f r u i t s n e r e held in pure nitrogen flowing at a rate of 200 ml. per minute. Similarly, f r u i t s whose internal atmosphere wa s completely depleted of oxygen would come t o a n equilibrium of 19-20% oxygen within 40 minutes of retu rn in g to a i r flowing a t a r a t e of 200 ml per minute. One hour was as effective as 24 hours of ;mosia in reducing th e amount of radiation in ju r y to th e f r u it. With beets, Kertesz e t a l . (1964) did not find a close relationship between softening an d radiation-induced changes in pectins a nd cellulose. They suggested t h a t other factors, such as cell turgidity, may be involved in th e response of th is tissue to irradiation. Despite t h e speculative n atu re of th e ir suggestion, i t is worthy of f u r t h e r research. I t is impossible a t th is time to specify tolerable doses of radiation for the various f r u i t species based on existing litei-attire. Salunkhe (1961), a f t e r noting t h a t irradiation degrades complex components of foods, such as proteins, f a ts , a n d carbohydrates (starch, sugar, insulin, cellulose, an d pectins), stated ; “HOWe i e r , \\hen the product w a s irradiated in th e vicinity of 2-3 10’ r i d s no unacceptable softening was found.” This finding is in conflict n-ith subsequent work by Cooper a n d Salunkhe (1963) n-ith st r a w be r ries; Maxie e t nl. (1964b) with grapes ; Truelsen (1960, 1963) with plums an d raspberries ; Beraha (1964) a n d Maxie a nd Sommer (1963) with p ears; Phillips (1959) with apples ; a nd Maxie an d Sonimer (1963) with peaches, nectarines,

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O F FRUITS

and grapes, where the conclusion was reached that doses a s low as 200-300 Krad damaged fruit texture to a n unacceptable degree. These conclusions, furthermore, were reached with fruit in stationary cold storage, and it is possible that, when irradiated fruits are actually shipped, the maximum permissible doses will have t o be revised downward. Johnson e t al. (1965) made a detailed study of the effects of gamma irradiation on the Shasta and Lassen varieties of strawberries, using both a taste panel and the Allo-Kramer shear press. They concluded that these fruits could tolerate 200 Krad without harming their marketability. The strawberry exhibits an unusual behavior in cold storage, as shown in Fig. 8, by becoming progressively firmer with time. At all doses above 50 Krad, there is an immediate loss in flesh firmness following irradiation, and the irradiated fruits remain softer in relation to the control fruits a t all levels for the entire storage period. However, the firmness in all fruits increases until irradiated fruits even a t 400 Krad a r e firmer than unirradiated fruits were a t harvest. The increasing firmness of strawberry fruits has not been explained. Water losses are not great enough t o account

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E. C. MAXIE AND ADEL ABDEL-KADER

f o r the increasing firmness in cold storage. One might speculate that reabsorption of calcium from the cell sap followed by calcium bonding in the pectins may contribute to the increasing firmness. Maxie rt al. (1964a) showed in irradiated lemon fruits a severe breakdown of the segment walls which apparently reflects dissolution of the cementing substances between the cells. Maxie and Sonimer (1964) concluded that textural changes xvoultl likely be the factor limiting the application of irradiation as a practical technology for fruits. Although they felt that a minimum dose of 175 Krad will be required for effective inhibition of postharvest fungi, they concluded that 225 Krad will be the maximum dose that most fruits can tolerate without unacceptable susceptibility to transit injury. Whether commercial irradiators can be designed to accommodate such a narrow maximum-minimum dose range is problematical. Climate and cultural practices (fertilizers, irrigation, pruning, etc.) exert a strong influence on the texture and other quality attributes of fresh fruits (Villiers et al., 1963). Truelsen (1963) noted that stran.berries harvested during rainy weather did not resist radiation injury as well a s fruits harvested during sunny weather. Maxie ct nl. ( 1 9 6 4 ~ )found strawberries grown during cool weather to be injured by doses t h a t gave no injury later in the summer. There a r e two things of importance in evaluating the work reported in the papers by Truelsen and Maxie e t al.: (1) the level of field infection with fungi is probably higher during inclement weather; and ( 2 ) fruits grown in cool or wet ueather seem less resistant to mechanical injury than a r e fruits grown under more nearly ideal conditions. Very little has been clone in evaluating the effects of cliniate and cultural conditions on the response of fruits to irradiation. These parameters need thorough study with any fruit th2.t seems to hold promise for a feasible irradiation technology. Since irradiation induces physiological stress in living cells, one would predict that the treatment might intensify symptoms of other stress conditions-chilling injury, high temperatures, etc. Abdel-Kader P t al. (1965) have shown that irradiation intensifies chilling symptoms in tomatoes, and, conversely, that incipiently chilled tomatoes show irradiation injury more readiIy than u n c h i 1led fruits . I t is likely that gamma irradiation may cause increased water loss from some fruits. Abdel-Kader ct nl. (1964) showed a marked

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increase in w a t e r loss f r o m tomatoes. Maxie e f al. (1964a) showed a n increased dehydration in irradiated lemons. V.

EFFECT OF RADIATION ON CHEMICAL COMPONENTS OF FRUITS

A. NUTRITIONALLY SIGNIFICANT COMPOCNDS 1. E n e r g y - Y i e l d i n g Value If ionizing radiation is t o be a feasible technology with fresh fruits, there m u s t be no significant loss in t h e nutritive value of the irradiated product. Read e t al. (1957) estimated t h a t changes in t h e chemical constituents of foods f r o m irradiation a t sterilizing doses affect only 0.003% of all t h e compounds present. If t h a t is so, irradiation a t doses t h a t f r u i t s can tolerate should not affect their energy values by a measurable amount. W a t t a n d Merrill (1950) have tabulated t h e macro- a n d micronutrient composition of foods, including fruits. Their d a t a shonthat, on a fresh-weight basis, f r u i t s contain between 4 a n d 30% carbohydrate. Read (1959) noted t h a t irradiation, even at sterilization doses, h a s little effect on t h e macronutrients a n d the energy-yielding value of foods. Johnson a n d Metta (1956) a n d Friedemann (1956) noted no loss in t h e energy value of carbohgdrates a n d f a t s in peas a n d Lima beans at doses of 3 X 10" rep. Cahall e t al. (1957) a n d Worth e f al. (1957) found t h a t the protein, f a t , a n d carbohydrate were fully a s available t o rats in nine foods irradiated t o 5.58 Megarad a s in unirradiated foods. Johnson (1960) reported t h a t doses as high as 10 Megarad did not alter the energy value of foods. The effects of ionizing radiation on cellulose, pectins, a n d starch were descriked earlier with reference t o the role of these compounds in t h e textural quality of irradiated fruits. T h e most noteworthy effect on the macromolecules w a s a hydrolysis t o simpler compounds having t h e same general structure. Therefore, if irradiation exerts a n effect on the digestibility of these polymers, one would expect them t o be more digestible, r a t h e r than less so, a f t e r irradiation. It might be assumed t h a t t h e higher respiration r a t e s induced in f r u i t s by irradiation might cause some loss of energy-yielding compounds. However, most f r u i t s have a low respiration r a t e in terms of actual carbon loss. Maxie e t nl. ( 1 9 6 4 ~ )showed t h a t strawberries subjected t o 200 K r a d of gamma r a y s a t 5 ' C evolved only 34 m g of CQ, kg. hr., compared t o 23.4 m g f o r tin-

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E. C. MAXIE A N D ADEL ABDEL-KADER

irradiated fruit. This represents only 3 mg of actual dry matter lost per kg/hr as a result of irradiation. Thus, one can safely conclude that irradiation at doses that fruit can tolerate will not reduce the caloric value of the product by a significant amount. 2. Vitamins Fruits are important sources of vitamin A (or its precursors) and vitamin C, but a r e relatively low in thiamine, riboflavin, and niacin (Watt and Merrill, 1950). Destruction of vitamin A or C in fruits by irradiation would be undesirable. In evaluating the effect of ionizing radiation on the destruction of vitamins in fresh fruits, it is desirable that the analyses be made after the fruit has been subjected to a n actual or simulated storage and marketing sequence. Unfortunately, few studies of this nature have been made with fresh fruits. Vitamin C is one of the more radiosensitive vitamins (Proctor and Goldblith, 1949). Clarke (1959) found t h a t gamma irradiation of strawberries a t doses of 0.3 and 0.4 Megarad resulted in respective losses of ascorbic acid of 62 and 81%. Salunkhe e t nl. (1959a) reported that gamma irradiation of 0.93 x lo5 and 4.65 X lo5 rad at rates of 0.093 X loG and 0.93 x 10" rad per hour markedly reduced the amount of ascorbic acid in strawberries, with fruits treated at the faster rate showing the greater rate of loss. Their unirradiated fruit contained only 4 mg. of the vitamin per 100 g. of fresh weight of fruit, a much lower level than is normal in this fruit (Watt and Merrill, 1950; Maxie and Sommer, 1963). Zeeuw (1961) found no effect of electrons on the ascorbic acid content of the Senga variety of strawberries at doses as high as 500 Krad. Maxie and Sommer (1963) found that the ascorbic acid content of strawberry fruits subjected to 200 Krad varied from 84 to 97% of the content of unirradiated fruits after a simulated marketing period of 12 days at 5°C. It was concluded that even the lowest value did not represent a nutritionally significant amount, since the irradiated fruits still contained 60 mg. of the vitamin per 100 g . of fruit. The importance of cold storage to losses of ascorbic acid in lemons was shown by Maxie and Sommer (1963). Twenty-four hours after irradiation with 100, 200, 300, and 400 Krad, the respective amounts of ascorbic acid as percent of levels in unirradiated fruits were 102, 100, 95, and 93. After 40 days a t 15°C: the respective values were 94, 29, 10, and 5%. However, in every

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case the irradiated f r u i t s showed severe in ju ry in th e f o r m of cavities along th e segment walls following cold storage, a n d it was concluded t h a t gamma-irradiation could not be used with lemons destined f o r long-term storage. With Washington Navel oranges, Maxie a n d Sommer (1963) found no digerenee in the ascorbic acid levels of f r u i t s subjected to 0 a nd 200 K r a d even a f t e r 95 days at 0'C. I n contrast to results with lemons, th ere was no evidence of in ju ry i n t h e irradiated f r ui t . With Krummel peaches, Maxie et al. ( 1 9 6 4 ~ ) showed respective losses of ascorbic acid of 23 an d 35% in comparison to unirradiated f r uits d u rin g 10 days a t 5-C in f r u i t s subjected to 150 and 300 Krad. They also found t h a t Bing cherries subjected to 200 a nd 400 Krad lost 3 an d 10.5% more ascorbic acid d u rin g 24 hours a t 5 C th an did unirradiated fru it. A t 10 days of storage a t 5 C., respective losses were 0, 3, a n d 2% in f r u i t s subjected t o 0, 200, and 400 Krad. Abdel-Kader ct (11. (1965) studied t h e effects on ascorbic acid in tomato f r ui ts of degree of ripeness, dose, a n d time of storage at 20 c'. T he results a r e shown in Tables I a n d 11. I n general, the amount of ascorbic acid in fru its within each ripeness class declined with increasing dose an d time. F r u i t s irradiated at th e pink and full-ripe stages of ripeness contained more ascorbic acid exen a f t e r 400 K r a d t h a n was found in subsequently ripened unirradiated f r u i t s harvested in th e mature-green stage. I t should be noted t h a t mature-green f r u i t s show a n anomalous ripening when subjected to 400 an d 600 Krad. Considerably more work mu st be done before a n y firm conclusions can be d rawn concerning the effects of radiation on th e ascorbic acid content of fruits. Niacin (Proctor, 1954 ; Proctor a n d Goldblith, 1948 ; Goldblith a n d Proctor, 1949) and riboflavin (Proctor a nd Goldblith, 1949) have a protective action f o r ascorbic acid against radiation. However, most f r u i t s contain only tr a c e amounts of niacin an d riboflavin ( W a t t a n d Merrill, 1 9 5 0 ), s o this mechanism is of doubtful significance in explaining differences in t he radiosensitivity of ascorbic acid among the various f r ui t s . Carotene i n i r u i t s h a s not received much attention as regards its sensitivity to ionizing radiation. I n solution, carotene is moderately sensitive to radiation (Chalmers e t al., 1945 ; Goldblith a n d Proctor, 1949; Knapp an d Tappel, 1961; Lukton a n d MacKinney, 1956). I n plant tissues, carotene seems more resistant

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E. C. MAXIE AND ADEL ABDEL-KADER

to destruction by radiation, probably because of the protection against radiation-induced free radicals by other compounds in the tissues (Lukton and MacKinney, 1956). These workers reported that carotene losses were negligible in irradiated whole tomatoe3 even at doses of several million rep. Using a dose of 1.86 Megarads, Franceschini et al. (1959) showed that carotenoid destruction in canned samples varied from 5 to 95% in green

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beans, 25 t o 60% in broccoli, 3 to 20% in sweet potatoes, and 0 to 56% in carrots, depending on packing conditions. Calloway and Thomas (1961) used canned samples and showed that doses of 2.79 and 5.58 Megarad caused negligible losses of carotene in carrots stored six months at 72”F, whereas peaches lost approximately 50% of their carotene. Burns and Desrosier (1967) showed that the development of red color, phytofluene, gamma-carotene, and lycopene was inhibited in tomatoes subjected to 4.5-Mev. cathode rays at doses of 0.5 Megarep. The content of beta-carotene was not affected by irradiation, and in-

FOOD IRRADIATION-PHYSIOLOGY

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12‘7

creased a s the f r u i t ripened, as was found in unirradiated fruits. Insufficient data a r e available to permit a firm conclusion on the significance of losses of carotene in fruits subjected to low doses (less than 0.5 Megarad). However, it seems likely that, with most fresh fruits., adverse textural effects and anomalous ripening will limit irradiation to doses well below those required f o r a nutritionally significant loss in carotene.

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Vitamins other than A and C have received little attention in irradiated fruits, particularly a t doses of less than 500 Krad. Ziporin e t al. (1957) studied frozen peaches subjected to 2.8 and 5.6 Megarad of gamma rays. Thiamine was destroyed almost completely. Niacin, which is usually radioresistant, was reduced 48 and 56% by the two doses. This was interpreted as a n effect of the high amounts of ascorbic acid used t o prevent browning i n frozen peaches. As noted earlier, ascorbic acid seems to increase the sensitivity of niacin to radiation (Proctor, 1954; Proctor and Goldblith, 1948, 1949). Gerber et al. (1958) reported that thiamine in fruits and vegetables was not affected by radiation.

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Considering the low amounts of niacin, thiamine, and riboflavin in fruits, and based on what meager data a r e available concerning the effects of low doses of radiation on these vitamins in vizw, it appears unlikely that their destruction will limit the applicability of radiation to fresh fruits.

B. OTHER CHEMICALCONSTITUENTS Fruits contain a multitude of acids, alcohols, amino acids, organic acids, esters, fats, etc. Unfortunately, little attention has been directed toward determining the effects of low doses of radiation on these compounds in vivo. Fernandes and Clarke (1962) reported t h a t irradiated apples seemed to taste sweeter than unirradiated fruit. It was found t h a t the amount of malic acid was much less in irradiated apples than in control fruit. Citric acid increased in all samples with time in storage, with irradiated apples having slightly higher levels. Quinic acid was not affected. Hulme (1959) noted that succinic acid accumulated in irradiated fruit, and suggested that this phenomenon might be involved in radiation injury to the fruits. Maxie e t al. (1964a) showed a marked decrease in total acidity (expressed as percent citric acid) in irradiated lemon fruits. The biochemical or physiological explanation for the decrease in organic acids in irradiated fruit is not known a t this time. Considering the marked increase in rate of respiration in fruits following irradiation, one might predict that the major loss in acids is by way of the tricarboxylic-acid cycle. Romani (1964) presented data indicating that the acid loss is not a direct radio-decarboxylation of acids. Auxins in fruits have not received much attention in relation t o irradiation efl’ects. Mathur (1963) studied the effects of the methyl ester of indole-3-acetic acid on radiation-induced susceptibility to decay of potatoes and tomato fruit. He noted slower respiration and transpiration in irradiated fruits treated with the auxins, and suggested t h a t radiation may act by inactivating the endogenous auxin. That radiation can destroy auxins was shoxvn by Skoog (1934, 1935) and Gordon and Weber (1950). Maxie e t al. (1964a) and Maxie and Sommer (1963) showed that gamma irradiation induced a condition similar t o “Black Button” in lemons. “Black Button” can be controlled by dipping lemons in a solution of 500 ppm of 2,4-dichlorophenoxyacetic acid (Stewart, 1948). Maxie e t al. (1964a) and Maxie and Sommer (1963) showed that this auxin, as well as kinetin (6-fur-

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furylaniino purin e), applied either before o r a f t e r irradiation, was ineffective in preventing radiation in ju r y to th e lemon buttons. This finding indicates t h a t g amm a radiation inactivates o r inhibits some Fysteni concerned with auxin metabolism in the lemon buttons. There is a need fo r detailed studies of the effects of low doses of irradiation (0-0.5 Megarad) on th e various chemical constituents of f r u its. These effects a r e important because of their direct effect on th e quality attributes of irradiated f r u its . They are also important in a secondary m an n e r since they m a y alter the metabolism of th e fruits, thereby producing delayed changes affecting dessert an d storage qualities. T h e U.S. A r m y Quartermaster Corps (1957) an d Brownell (1961) have presented discussions of radiochemical phenomena an d their possible effects on foods, many of which a r e undoubtedly important in th e response of f r u i t s to radiation. VI.

EFFECTS OF RADIATION ON ORGANOLEPTIC ATTRIBUTES O F FRESH FRUITS

One of the areas in f r u i t irradiation t h a t is most difficult to evaluate is t he effect of radiation on the quality attributes of the commodity. There a r e several reasons why th is is s o : (1) th e failure of many investigators t o keep in mind t h a t f r u i t s are alive a nd t h a t m an y quality attributes a r e expressions of dynamic systems which cannot persist if th e f r u i t s a r e killed ; ( 2 ) ignorance of, o r disregard of, wha t constitutes acceptable quality; ( 3 ) failure to subject th e irradiated product to actual or simulated marketing conditions; ( 4 ) a n assumption t h a t freedom from decay, quality, an d shelf life a r e synonymous; (5) lack of objective evaluation of irradiated commodities by appropriately trained taste panels with a n application of adequate statistics ; ( 6 ) unrealistic experimental conditions, such as a irtight packages an d o r doses f a r in excess of th e practical tolerance of the commodity; an d (7 ) failure t o recognize th e significance of product matu rity and,‘or degree of ripeness at th e time of treatment on the subsequent expression of quality attributes. I t should be emphasized t h a t “marketable quality” involves more t ha n physical survival of th e fruits. Unless irradiated f r u i t s a r e acceptable in appearance, aroma, flavor, a n d texture, they cannot be classed as “marketable.” Salunkhe ( 1961) summarized taste-panel evaluations of a number of irradiated products. H e

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used a n “adjusted preference score” in which the panel ra tin g (11 th e commodity on a hedonic scale of 1 (dislike extremely) to 9 (like extremely) lvas multiplied by the percent of surviving fr ui t s. This technique combines two attributes which a r e not necessarily related an d will more often th a n not bias th e results of the experiment in fav o r of irradiated f r u its , such as cherrie.-: and strawberries. lvhere the incidence of decay in th e unirradiated samples is high. Th e efYect of bias is demonstrated in the evaluation of Shasta strawberries a f t e r 1 5 days a t 40 F and 2 days a t room temperature. Panel evaluations f o r taste indicated th a t the irradiated berries were unacceptable. T h e “adjusted preference scores,” however, showed t h a t berries subjected t o 200 a nd 300 K rad were practically as good a s they were at t h e s t a r t of the experiment. Strawberries seem t o hold more promise a s subjects of irradiation t h a n most fru its. Heeney et al. (1964) found n o detectable diff‘erence between f r u i t s subjected to 110, 220, a n d 330 Kra d a f t e r two weeks a t 40 F . Truelsen (1963) showed no significant difference in taste-panel scores f o r berries subjected t o 0, 100. 200, 300, a n d 400 K r a d f o r t h e “first fe w days” a f t e r tre a tment a t 7 C. After 8 days, the irradiated berries were judged better t ha n the control fru its. After 14 days all berries were judged unacceptable. Johnson e t al. (1965) used Lassen and Shasta strawberries in a simulated marketing sequence representative of t he American t r a d e (10 days at 4 1 °F) a f t e r irradiation a t doses of 0, 100, 200, an d 300 Krad. The ta s te panel could detect differences between irradiated an d unirradiated f r u i t s in appearance, aroma, an d texture b u t not in flavor. T h e panel did not consider objectionable th e differences noted in t h e irradiated berries. Salunkhe e t al. (1959b) irradiated f o u r varieties of strawberries in sealed cans an d showed th a t th e taste-panel preference declined a s th e dose increased, but they did not indicate how long the f r u i t was stored prior t o evaluation. They concluded t h a t strawberries were “too sensitive f o r gamma radiation,” but did not indicate the basis f o r their conclusion. Maxie c~t al. (196410) showed t h a t every quality a ttrib u te of irradiated E mp ero r an d Tokay grapes declined with increasing doses of gamma rays an d with increasing time in storage at 32 F. Salunkhe (1961) reported no off-flavors following three months a t 4 0 - F in Thompson Seedless grapes given doses as high as 500 Krad. H e noted, however, t h a t f r u i t s subjected to 300, 300. and 500 Krad were unmarketable a f t e r one month, be-

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cause of a deep-brown color. It is strange that fruits injured enough by irradiation to show severe discoloration after one month in storage would not have off-flavors after a n additional two months. I n determining how a treatment affects quality attributes such a s appearance, aroma, flavor, and texture, it is imperative that fruits from the different treatments be a t the same stage of ripeness a t the time of sampling. U’ith fruits whose ripening is affected by irradiation, this is difficult to accomplish. Peaches and nectarines apparently a r e stimulated to ripen by gamma radiation (Maxie and Sommer, 1963). Thus, the ripening effect must be compensated for in unirradiated fruits. Pears and tomatoes pose the reverse problem when irradiated at doses of 300-400 Krad o r more. Bartlett pears given 300 Krad or more of gamma rays (Maxie et al., 1966) cannot be ripened, and a t doses of 600 Krad or more a r e severely injured (Maxie and Sommer, 1963). Considering these effects it is difficult to understand data of Salunkhe e t al. (1959b) in which taste preference scores of 5 or more, on a scale of 1 (dislike extremely) to 9 (like extremely), were assigned to pears subjected to doses of 100-750 Krad. Data presented by Salunkhe et a l . (1959b) raise a n important point in taste-panel evaluation of quality in fresh fruits. A small “expert panel” (8-12 people) is of value only in detecting differences in appearance, aroma, flavor, texture, etc., in samples subjected to some sort of treatment. The panelists must first be selected for a n ability to detect small differences in quality attributes of the product being evaluated. Secondly, the panelists should be given control samples of acceptable quality to use as a reference when judging treated samples. It should be noted that data taken with a n “expert panel” cannot be projected to indicate consumer acceptance. Much additional research is needed on the effects of irradiation on the quality of fresh fruit. It appears likely, however, that anomalous ripening and adverse textural changes will in most cases limit the irradiation of fruits to doses lower than a r e required for severe adverse effects on aroma and flavor. The appearance of irradiated fruits is so intimately associated with changes in texture and ripening that this attribute will likely show a n adverse response at about the threshold dose for adverse eflects on these two phenomena. Pigmentation is a n important attribute in the appearance of

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fresh fruits. In evaluating the effects of ionizing radiation on pigments, one must recognize immediate and direct effects on pigments p e se, ~ as opposed to delayed and indirect effects on fruit ripening. Markakis et al. (1959) showed that 0.465 Megarad of cathode rays destroyed 55% of the anthocyanins in strawberry juice. The dose employed is above the tolerance of fresh strawberry fruit, and further, there is no assurance that anthocyanins in intact fruit would be equally susceptible to destruction. Truelsen (1960) found no effect of gamma rays a t doses of 200 Krad on red color in strawberries. At doses of 350 Krad and higher the color of the fruit was noticeably lighter. Maxie e t al. ( 1 9 6 4 ~ ) noted no change in the external color of strawberries given a dose of 200 Krad. Johnson e t al. (1965) noted that strawberries subjected to 300 Krad had less luster than unirradiated fruit. It was not determined whether this response was due to a loss in color or a change in the physical nature of the fruit surface. They noted t h a t the red color from the peripheral tissues of the berries seemed to diffuse into the white tissues near the center of the berry. There is a possibility, however, that the apparent increase in red pigments in the center of the fruit was a result of increased synthesis of pigments rather than diffusion from adjacent tissues, for Maxie e t al. ( 1 9 6 4 ~ ) showed that red pigments increased markedly in irradiated peaches and nectarines. This phenomenon was associated with faster ripening of irradiated fruit. However, unirradiated fruit never developed as much red color as did irradiated fruit, indicating that the higher pigmentation was a radiation-induced response. Cook et al. (1957) showed that the color of Jonathan apples was not affected by doses of gamma rays which controlled blue mold. Several workers have studied the effect of radiation on the development of red color in green tomatoes. Burns and Desrosier (1957) reported that 0.5-1.5 x loG rep inhibited red development and t h a t the irradiated fruit contained less phytofluene and lycopene. The doses used were probably above the practical tolerance for tomatoes. Salunkhe et al. (1959a) reported that about 1.86 x 10: rad was the threshold at which the ripening of tomatoes became inhibited, and that Jycopene development was retarded. They concluded that lycopene was more susceptible to degradation by radiation than were carotenoids and chlorophyll. The validity of this conclusion is questionable, considering the radiation inhibition of ripening in the tomato fruit. Until

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irradiated fruit of a degree of ripeness comparable to that of unirradiated samples a r e analyzed, one must consider Salunkhe’s data as showing only differences in ripeness. Salunkhe et al. (1959a) reported that the intensity of red color in “cherry drink” was reduced by 0.93-3.72 x loG rad. Such doses a r e far above the practical tolerance of fresh fruits to radiation. Maxie et al. ( 1 9 6 4 ~ )noted that Bing cherries subjected to 300 and 350 Krad of gamma rays and then shipped by rail from Stockton, California, to Pittsburgh, Pennsylvania, showed some skin discoloration. It could not be determined whether this was a direct effect of irradiation on the color of the fruit or a n indirect effect of greater susceptibility of irradiated fruit to transit injury. Fruits subjected to less than 300 Krad were not affected. Much more research is needed on the effects of irradiation on chlorophyll in fresh fruits. To date, no objective measurements have been made of the chlorophyll content of irradiated fruits. Maxie and Sommer (1963) noted that irradiated peaches and nectarines seemed to lose their green color more rapidly than did unirradiated fruits. Similarly, Maxie and Sommer (1963) showed that lemon fruits degreened within 8 days of treatment with 25-100 Krad of gamma rays. It was concluded that the disappearance of green color in nectarines, peaches, and lemons was an indirect effect of irradiation. I n all three fruits, production of ethylene by the fruits was stimulated, resulting in faster ripening of nectarines and peaches, and faster degreening of lemons. Considering the data currently available, it seems unlikely that irradiation with doses that fresh fruits can tolerate will have a direct and significant adverse effect on the color of the commodity. Serious delayed effects may develop, however, as a result of inhibited o r enhanced ripening in response to irradiation. VII.

WHOLESOMENESS OF IRRADIATED FRUITS

A. INDUCEDRADIOACTIVITY The possibility of induced radioactivity was of concern in early attempts to develop a n irradiation technology for foods. To review the considerable work done on this problem is beyond the scope of this paper. However, from the data accumulated on sterilization levels for both plant and animal products, it is clear that induced radioactivity from gamma irradiation will not be a

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problem at doses that fresh fruit can tolerate (Stanford Research Institute, 1961).

B. TYHOLESOMENESS O F IRRADIATED FRUITS There is no evidence to date that doses of 500 Krad or less mill induce in fresh fruits chemical changes that will pose any health hazard to man o r animal (Kraybill, 1955, 1957, 1959a,b, 1960a; Kraybill and Huber, 1957; Kraybill e t ul., 1956; Read, 1959, 1960 ; Read e t al., 1957). It should be noted t h a t before any irradiated product is placed on the American market, clearance must be obtained from the Federal Food and Drug Administration. For clearance, the FDA requires animal feeding studies varying from 90 days to 215 years, thus reducing to a minimum the probability that any foodstuff cleared for human consumption will be hazardous. There a r e no fully verified results in the literature to date to indicate that wholesomeness will be a problem in the irradiation of fresh fruits. VIII.

OZONE PRODUCED BY IONIZING RADIATION AND ITS RELATION TO FRUIT BEHAVIOR

In the presence of a i r or oxygen, gamma rays or electrons may produce high levels of ozone (Kertesz and Parsons, 1963; Maxie and Sommer, 1963; Less and Swallow, 1 9 6 4 ; Shah and Maxie, 1 9 6 6 ) . Ozone is a powerful oxidizing agent which reacts rapidly with compounds of the type that make up fresh fruits (American Chemical Society, Advances in Chemistry Series 21, 1959). It is thus probable that many of the responses of fresh fruits to ionizing radiation are expressions of ozone action rather than direct radiochemical events. Fruits offer a unique system for studying the relationship between direct radiation phenomena and the indirect effects of ozone. Lemons, pears, peaches, etc., may contain as much as 10-20 ml of gas in their internal atmosphere. The oxygen concentration in this gas is around 18-20% a t cold-storage and rooni temperatures (Eaks and Ludi, 1960; Trout e t aZ., 1 9 4 2 ) . Therefore, penetrating radiations, such as gamma rays, undoubtedly produce ozone within the tissues of the fruit. Shah a n d Masie (19GC) have estimated that ozone produced inside fruits may amount to as much as 58 ppm. I t is likely that ozone produced in the internal atmosphere of f r u i t s would react immediately with water vapor and other components of the fruit.

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Ozone may elicit toxic responses in plant tissues at concentrations of 1 p a r t ozone in 8,000,000 p a r t s of a i r (Homan, 1937). Thus, it is expected t h a t much of th e i n ju r y occurring in f r u i t tissues dur i ng irradiation with g amm a r a y s m a y result f r o m ozone a nd its reaction products. The internal atmosphere of f r u i t s can be altered very quickly. a nd t h e tissues can tolerate anoxia f o r several hours without ill effect. Tissue slices may be taken f r o m f r u i t s f o r direct exposure to ozone a n d f o r metabolic studies. It is probable th a t studies on anoxia as a means of protecting f r u i t s f r o m irradiation will be largely of academic interest. T h e pathogens t h a t attack f r u its, an d adverse endogenous physiological processes in f r u i t s such as ripening, would probably be protected by anoxia t o about th e same degree as th e f r u i t s themselves. Thus, irradiation of f r u i t s in anoxia o r other protective conditions is not likely to be a practical procedure. IX. FEASIBILITY OF IRRADIATION AS A COMMERCIAL TECHNOLOGY WITH FRESH FRUITS

It is unfortunate t h a t highly optimistic press releases have appeared concerning th e general applicability of irradiation to fresh f r ui t s a n d vegetables. These releases a n d t h e “data” to support them often originate with persons without experience in postharvest pathology, physiology, an d marketing. P rio r to 1961, facilities were lacking f o r adequate evaluation of t h e irradiation process on a laboratory scale. Except f o r work done in 1963-1964 by th e U.S. Department of Agriculture a t Fresno, California,“ there have been no sizable test shipments of irradiated fruits. Such shipments would be essential to t h e development of a n y technology in which fresh f r u i t s are subjected t o stress. Maxie a nd Sommer (1964) concluded t h a t irradiation will not be a n all-inclusive technology with fres h f r u i t s a n d vegetables. Its role, they believe may be confined to a limited number of species o r perhaps varieties within species. Even more specifically, irra diation m a y be limited to certain physiological stages of a species or variety. I t now a ppears t h a t f r u i t s an d th e pathogens t h a t attack them differ very slightly in tolerance to radiation injury. This ’ We a r e a w a r e of t h e U.S.D.A. work. U n f o r t u n a t e l y , t h e i r m a n u s c r i p t h a d n o t reached LIS by t h e deadline f o r t h i s review.

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conclusion led Maxie an d Sommer (1964) to decide t h a t a minimum-maximum dose ran g e of 175-225 K r a d will apply f o r most fruits. Admittedly, this conclusion is subject to modification with additional research. When additional evidence is developed, however, i t is probable t h a t t h e range of practical doses will be more restrictive, r a t h e r t h a n less. Maxie a nd Sommer (1964) have suggested t h a t a practical irradiation technology is most likely with f r u i t s t h a t have a relatively short postharvest life, f o r the following reasons : ( 1 ) these f r u i t s a r e th e most susceptible to decay a n d th u s suffer t h e greatest losses in mark etin g ; (2) they a r e fully ripe o r radiation-induced anomalies in ripening nearly so at harvest-so a r e not a f a c t o r ; ( 3 ) symptoms of radiation in ju ry ma y develop slowly in some f r u i t s an d will be noted only if they a r e held f o r long periods in cold sto rag e; an d (4 ) irradiation will almost certainly a dd some cost to the marketing of a n y treated fruit. The longer a f r u i t can be held in ordinary cold storage without severe losses, th e less likely is acceptance by t h e tra d e of a n y process t h a t would give additional storage life but with added costs. With most irradiated fruits, appearance, aroma, energy content, flavor, nutritional adequacy (vitamins), a n d wholesomeness will not be limiting factors a t doses th e f r u i t will tolerate. The factors t h a t seem most limiting a r e : (1) a loss in texture, Tvith a n associated increase in susceptibility to mechanical in ju ry ; ( 2 ) anomalous ripening; an d ( 3 ) failure t o achieve a satisfactory fungicidal effect a t doses th e f r u i t will tolerate. The first two of these m a y exert adverse effects on t h e quality attributes noted above, a n d m us t be considered as important causes of lower quality. Maxie a nd Sommer (1964) have emphasized t h a t the number of days of freedom f r o m decay is not synonymous with the shelf life of f resh fru its. The irradiated product mu s t be typical of t he species an d variety an d at least of comparable quality. They believe t h a t the principal benefit t h a t might be derived from ir r a di a t i ng most perishable commodities is a reduction in th e amount of decayed f r u i t d u rin g a normal marketing sequence. If all other features of good handling, such as prompt cooling, rigorous temperature control at th e best storage temperature, etc., a r e adhered to, this could mean 3-4 additional days f o r marketing. Doubling o r tripling th e storage an d shelf life of f r u i t s by irradiation seems unlikely. T he economic feasibility of irradiation a s a technology is not

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a matter for this review. It will be a critical consideration, however, if attempts a r e made to commercialize the technology with any fruit. There are certain areas for which the irradiation of fresh fruits holds promise. If the work of Teas et al. (1962) and Amezquita (1965) on the inhibition of ripening in bananas by 25-50 Krad can be confirmed, the perplexing and costly problem of overripe and “turning” f r u i t may at last be solved. The nature of the banana industry in Latin America, where large acreages with year-round productivity a r e confined to a single locale, W O L I ~make ~ irradiation seem economically advantageous. Disinfestation of fruit flies in papaya and seed weevils in mango seems certain to be a practical application (Balock and Christenson, 1956; Balock et al., 1963) provided that the U.S.D.A. Quarantine Service will allow admission of sterilized insects. There is no reason to doubt that the doses required for disinfestation of papayas will be less harmful to fruit quality than the fumigation procedure now in use. Strawberries in Californiawith large acreages, a long season, and high percentage of decay a promising subject for in current marketing procedures-seem irradiation. Irradiation may prove beneficial in hastening the drying rate of prunes and other dried fruits (Markakis, 1964; Maxie et al., 1965b). Faster drying could mean better flavor in irradiated fruits. The decrease in firmness in irradiated dried fruits may prove to be a desirable characteristic in the marketing of these products (Markakis, 1964). There are certain fruits where irradiation is not likely to find a n application. For example, most apples may be stored with good success in ordinary cold storage or under controlled atmospheres for as long as is desirable in most cases. This review was written with the American fruit industry in mind. This industry has highly developed facilities and techniques f o r handling perishable commodities. We would be remiss if we did not indicate the possibility that irradiation may prove feasible in the emerging nations, where facilities and techniques a r e limited. I n our opinion, this potential application of irradiation should be explored. X.

CONCLUDING REMARKS

At this writing, fruit irradiation must be considered still in a n early stage of development. From research in this area, a feasible technology may develop for one or more fruits. It is by

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no nieaiia a n assured technology f o r a n y specific fru it. There a r e many fascinating responses of fru its to irradiation, but th e investigator must avoid “chasing rainbows” with regard t o applicability on a commercial scale. Stresses f r o m irradiation should prove of value in elucidating biochemical a n d physiological phenomena in f r u i t ripening an d senescence. This f a c t alone is justification f o r research in th is area. Indeed, such use of irradiation m a y prove t o be its principal value. I n our opinion, irradiation can be used to advantage in studying a n y biochemical o r physiological event in fresh fru its . I t is of particular value in studying ripening, f o r i t delays t h e senescence of tissues in some cases while hastening i t in others. Studies of irradiation effects on the interrelationships of r a t e of respiration an d the production of ethylene an d other volatiles will be helpful in understanding these processes. Much research is needed on radiological degradation an d o r induced synthesis of th e compound< associated with aro m a an d flavor. V’e have cited th e series of elegant papers by Professor Kertesz and his colleagues on irradiation effects on the texture of tissues, and commend them f o r their work. However, much more work is needed in this area, particularly on methods of modifying th e injury. Such studies may reveal the means f o r a feasible irradiation technology with fresh f r u i t s ; certainly, they will contribute to our knowledge of cell stru ctu re a s it m a p afl’ect responses t o stress conditions. The relationship of direct radiation i n ju ry a n d th e secondary effects of ozone produced within the tissues via ionization of a i r needs much attention. Again, th e d ata obtained will be of broad interest in biochemistry an d physiology. Fruits a r e admirably adapted t o such studies. The reciprocal influences of irradiation a n d other stress conditions need much more attention, a s do the effects of climatic and cultural factors concerned with f r u i t growth a n d development on t he response of f r u i t s to irradiation. One th in g is paramount in all irradiation research designed to extend th e shelf life of fresh f r u i t s : i t must be done by a person knowledgable in postharvest pathology, physiology, an d biochemistry.

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Goldblith, S. A., and Proctor, B. E. 1949. Effect of high voltage X-rays and cathode r a y s on vitamins (riboflavin and carotene). Arucleonics 5 ( 2 ) , 50-58. Gordon, S. A., and Weber, R. P. 1950. The effect of X-radiation on indoleacetic acid and auxin levels in the plant. Am. J . B o t a n y 37, 678. Hannan, R. S. 1955. Scientific and technological problems involved i n using ionizing radiations f o r the preservation of food. G t . Brit.,D e p t . Sci. I n d . Research, Food I n v e s t . Special R e p t . N o . 61, 192 pp. Hannan, R. S. 1956. “Research on the Science and Technology of Food Preservation by Ionizing Radiation,” pp. 1-192. Chem. Publ. Co., New York. Hansen, E. 1942. Quantitative study of ethylene production in relation t o respiration of pears. Botan. Gaz. 103, 543-558. Hansen, E., and Hartman, H. 1937. Effect of ethylene and certain metabolic gases upon respiration and ripening of pears before and a f t e r cold storage. P l a n t Physiol. 12, 441-454. Heeney, H. B., Rutherford, W. M., and MacQueen, K. F. 1964. Some effects of gamma radiation on the storage life of fresh strawberries. Can. J . P l a n t S c i . 41, 188-194. Henry, M. C. 1957. Action of ionizing radiations on alcohols, organic acids and esters, aldehydes and ketones. I n : “Radiation Preservation of Foods.” pp. 113-118. U.S. Army Quartermaster Corps P.B. 151493. Homan, C. 1937. Effects of ionized a i r and ozone on plants. P l a n t Physiol. 12, 957-978. Hulme, A. C. 1958. Some aspects of the biochemistry of apple and pear fruits. A d v a n c e s in Food R e s e a r c h 8 , 297-413. Hulme, A. C. 1959. Unpublished data. (Cited by Fernandes, S. J. G . , and Clarke, I. D., 1962. J . Sci. Food A g r . 13, 23-28.) .Johnson, B. C. 1960. Comments on the wholesomeness of irradiation-processed foods. Food I r r a d i a t i o n 1 ( 2 ) , A4-A5. Johnson, B. C., and Metta, V. C. 1956. Effect of irradiation sterilization on nutritive value of protein and energy of food. F e d e r a t i o n Proc. 15, 907-909. Johnson, C. F., Maxie, E. C., and Elbert, E. M. 1965. Physical and sensory tests on fresh strawberries subjected to gamma irradiation. Food T e c k no!. 19, 119-123. ,Joslyn, M. A. 1962. The chemistry of protopectin: A critical review of historical d a t a and recent developments. A d v a n c e s in Food R e s e a r c h 11, 1-107. Kertesz, %. I. 1951. “The Pectic Substances.” 628 pp. Interscience Publishers, New York. Kertesz, %. I., and Parsons, G . F. 1963. Ozone formation in a i r exposed to cobalt-60 gamma radiation. Science 142, 1289-1290. Kertesz, %. I., Morgan, B. H., Tuttle, L. W., and Lavin, M. 1956. Effect of ionizing radiations on pectin. R a d i a t i o n R e s e a r c h 6, 372-381. Kertesz, %. I., Schulz, E. R., Fox, G., and Gibson, M. 1959. Effects of ionizing radiations on plant tissues. IV. Some effects on starch and starch fractions. Food R e s e a ~ c k24, 609-617. Kertesz, %. I., Glegg, R. E., Boyle, F. P., Parsons, G. F., and Massey, L. M., Jr. 1964. Effect of ionizing radiations on plant tissues. 111. Softening and

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changes in pectins and cellulose of apples, carrots a n d beets. J . Food Sci. 29, 40-48. Kidd, F., and West, C. 1945. Respiratory activity and duration of life of apples gathered a t different stages of development and subsequently maintained at a constant temperature. Plant Physiol. 20, 467-507. Iinapp, F. W., and Tappel, A. L. 1961. Comparison of t h e radiosensitivities of the fat-soluble vitamins by gamma irradiation. J . A g r . Food C h c m . 9, 430-433. Kraybill, H. F. 1955. Nutritive effects on foods sterilized by ionizing radiations. Nutritioii Rev. 13, 193-195. Kraybill, H. F. 1957. Radiation preservation of food. Public Health R c p t s . ( U S . ) 72 ( 8 ) , 675-680. Kraybill, H. F . 1959a. Safety in the operation of radiation sources and use of irradiated foods. 1)itern. J . A p p l . Radiation Isotopes 6, 233-254. Kraybill, H. F. 195913. Nutritional and biochemical aspects of foods preserved by ionizing radiation. J . Home Econ. 51, 695-700. Kraybill, H. F. 1960a. Are irradiated foods h a r m f u l ? Nuclcoitics 18 ( l ) , 112-1 17. Kraybill, H. F. 1960b. Evaluation of the wholesomeness of irradiated foods. Food Irradiation l ( 2 ) . A2-A3. Kraybill, H. F., and Huber, T. E . 1957. The wholesomeness of irradiated food and its military implications. J I i l i t a r y AIcd. 120 ( 6 ) , 417-422. Kraybill, H. F., Read, &I. S., and Friedemann, T. E. 1956. Wholesomeness of gamma irradiation treated food fed to r a t s . Federation Proc. 15, 933-937. Less, L. N., and Swallow, A. J. 1964. Estimating the hazard due to radiolytic products f r o m air. Nzccleotrics 22, 58-61. Lukton, A., and MacKinney, G. 1956. Effect of ionizing radiations on carotenoid stability. Food Tcchnol. 10, 630-632. ?IIarkakis, P. 1964. (Personal communication.) Xarkakis, P., Livingston, G. E., and Fagerson, I. S. 1959. Effect of cathode r a y irradiation on the anthocyanin pigments of strawberries. Food Rescarch 24, 520-528. Afassey, L. M. J r . , Parsons, G. F., and Smock, R. ill. 1964. Some effects of gamma radiation on the keeping quality of apples. J . A g r . F o o d Chcm. 12, 368-274. X a t h u r , 1'. €3. 1963. Reversal of gamma-ray-induced susceptibility to decay of potato tubers and tomato f r u i t by methyl ester of indolyl-3-acetic acid. N a t u r r 199, 1007-1008. Masie, E. C., and Baker, C. E . 1954. A i r filtration studies in a commercial type apple storage. Proc. A m . Soc. Hoyt. Sci. 61, 235-247. AIasie, E. C., and Rae, H. L. 1965. Unpublished data. Maxie, E. C., and Sommer, N. F. 1963. Radiation technology in conjunction with post-harvest procedures a s a means of extending the shelf-life of 30, 1963) Contract f r u i t s a n d vegetables. Ann. Rept. (Feb. 1, 1962-Jan. No. A T (11-1)-34, Project Agreement No. 80, U.S. Atomic Energy Comm., Div. Tech. Inform. Jfasie, E . C., and Sommer, N. F. 1964. S t a t u s of gamma irradiation as a technology f o r perishable commodities. Proc. F r u i t and Veg. Handling Conf., Univ. of California, Davis. March, 1964, 96-100.

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Maxie, E . C., Eaks, I. L., and Sommer, N. F. 1964a. Some physiological effects of gamma irradiation on lemon fruits. Rudiutiox B o t u n y 4, 405-411. Maxie, E. C., Nelson, K. E., and Johnson, C. F. 196413. Effect of gamma i r radiation on table grapes. I’roc. Am. SOC.Hort. Sci. 81, 263-268. Maxie, E. C., Sommer, Ii. F., and Brown, D. S. 1964c. Radiation technology in conjunction with post-harvest procedures a s a means of extending the shelf-life of f r u i t s and vegetables. U.S. Atomic Energy Comm. Research and Develop. Rept. No. UCD-34P80-2. Maxie, E . C., Eaks, I. L., Sonimer, N. F., Rae, Henry L., and El-Batal, Salah. 19658. Effect of gamma radiation on r a t e of ethylene and carbon dioxide evolution by lemon fruit. P l a n t Physiol. 40, 407-409. Jlaxie, E. C., Somnier, N. F., and Brown, D. C. 196513. Radiation technology in conjunction with post-harvest procedures a s a means of extending the shelf-life of f r u i t s and vegetables. U S . A t o m i c E n e r g y Comm. Research a n d Llevelop. R e p t . N o . UCD-34P80-3. Jlaxie, E. C., Sommer, N. F., Muller, C., and Rae, H. L. 1966. Effect of gamma radiation on the ripening of Bartlett pears. P l a n t Physiol. 41, 437-442. hlikaelsen, K., and Roer, L. 1960. Radiation experiments with potatoes and other plant products in Norway. R e p t . DuTiish Atomic E n c r g y Comm., R ~ S16, O 65-66. Niller, E. V. 1946. Physiology of citrus f r u i t s in storage. E o f a n . Rev. 7, 303423. Miller, E . V. 1958. The physiology of citrus f r u i t s i n storage. 11. Botan. R t c . 24, 43-59. Millerd, A, Bonner, J., and Biale, J. B. 1953. The cliiiiacteric rise in f r u i t respiration a s controlled by phosphorylative uncoupling. P l a n t Physiol. 28, 521-531. Phillips, W. R. 1959. Irradiation of apples. 1959 R e p t . C ~ HComm. . Fruit V e g . pp. 16-17. Porritt, S. W. 1951. The role of ethylene in f r u i t storage. Sci. A g r . 31, 99-112. Prescott, S. C . 1904. The effect of radium r a y s on the colon bacillus, t h e diptheria bacillus and yeast. Science 20, 246-248. Proctor, B. E. 1954. Food preservation with the use of irradiation. I n d . R e f r i g . 127 (Z), 17-20. Proctor, B. E., and Goldblith, S. A. 1948. Effect of high voltage X-rays on vitamins (Niacin). Xucleonics 3 ( 2 ) , 32-43. Proctor, B. E., and Goldblith, S. A. 1949. Effect of soft X-rays on vitamins (niacin, riboflavin and ascorbic acid). Nucleoxics 5 ( 3 ) , 56-62. Read, M. S. 1959. The effects of ionizing radiations on the nutritive value of foods. Proc. Intern. Conf. on t h e Preservation of Foods by Ionizing Radiations. July 27-30, 1959. Massachusetts Inst. Technol., Cambridge, Massachusetts, pp. 138-152. Read, M. S. 1960. A summary of t h e wholesomeness of gamma irradiated foods. Federation Proe. 19, 1055-1059. Read, M. S., Kraybill, H. F., Linder, R. O., and Huber, T. E. 1957. Wholesomeness of irradiated foods. I n : “Radiation Preservation of Foods.” pp. 295-303. U.S. A r m y Q u a r t e r m a s t e r Corps, R e p t . N o . P.B. 151493. Roentgen, W. C . 1898. I. Ueber eine neue A r t von Strahlen. Ann. Physik u. C h e m . 64, 1-11.

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Romani, R. J. 1961. Radiation physiology of fruit-respiration during and immediately following kilorad doses of ionizing radiation. Raditr f i o n C o f a i i ] ] 4, 299-307. Romani, R. J . , Van Kooy, J., and Robinson, B. J. 1961. Gamma irradiation of fruit-preliminary physiological studies. Food Ir)arliatioa 2 ( 2 ) , All-A13. Salunkhe, D. K. 1961. Gamma radiation effect on f r u i t s and vegetables. Econ. Cottitiy 15 (l), 28-56. Salunkhe, I). K., Gerber, R. K., and Pollard, L. H. 1959a. Physiological and chemical effects of gamma radiation on certain fruits, vegetables and their products. Proc. Am. Soc. H o r t . Sci. 74, 423-429. Salunkhe, D. K., Pollard, L. H., and Gerber, R. K. 1959b. Effects of gamma radiation dose, r a t e and temperature on the taste preference and storage life of certain f r u i t s , vegetables and their products. Proc. Am. Soc. IJort. Sci. 74, 414-422. Shah, J., and Maxie, E. C. 1966. Gamma r a y radiosnythesis of ozone from air. I n t e m . J . Appl. R a d i a t i o n Isotopes 17, 155-159. Skinner, E. R., and Kertesz, Z. I. 1960. T h e effect of gamma radiation on the structure of pectin; a n electrophoretic study. J . FO!U?)7(’rSci. 47 (149), 99109. Skoog, F. 1934. The effect of X-rays on growth substance and plant growth. Science 79, 256. Skoog, F. 1935. The effect of X-irradiation on auxin and plant growth. J . Cclliilar C o m p . Physiol. 7, 227-270. Smock, R. M. 1949. Controlled atmosphere storage of apples. C,’or)i(~lI E . r t c 7 ~ . Bntll. 759. Smock, R. M., and Sparrow, A . H. 1957. A study of the effect of gamma radiation o n apples. I’roc. Am. SOC.Hort. Sci. 70, 67-69. Smock, R. M., and Van Doren, A. 1941. Controlled atmosphere storage of apples. Coriicll I;)tiv. A g r . Expt. S t a . Bull. 762. Sonimer, N. F., and Luvisi, D. 1960. Choosing the right package f o r fresh f r u i t . P a c k a g e E ) i g . 5, 37-43, 46. Soniogyi, L. P., and Romani, R. J. 1964. Irradiation-induced textural changes in f r u i t s and its relation t o pectin metabolism. J. Food S c i . 29, 366-371. Stewart, W. S. 1948. The effects of 2,4-D and 2,4,5-T on citrus f r u i t storage. C i t r u s Leaves 28 (ll), 5, 24-27. Stanford Research Inst. 1961. Radiation preservation of selected f r u i t s and vegetables. SRIA-30, J a n u a r y 1961. Teas, H. J., Quintana, D. C., and Campos, J . 0. 1962. Inhibition of hanana f r u i t ripening by gamma radiation. 2)id Zntern. C o ~ g r .Radiation Rc.scar.ch, Harrognte, E x g l a n d , Abstr. 179. Trout, S. A., Hale, E. G., Robertson, R. N., Hackney, M. V., and Sykes, S. M. 1942. Studies in t h e metabolism of apples. A z c s t r u l i a ~J~. E x p t l . Biol. Aled. S c i . 20, 219-231. Truelaen, T. A. 1960. Irradiation of fresh f r u i t s a n d vegetables with pasteurizing doses. R c p t . Danish. Atomic E n e r g y Comm., RisO 16, 73-75. Truelsen, T. A . 1963. Radiation pasteurization of fresh f r u i t s and vegetables. Food Tcehwol. 15, 336-339. U.S. Army, Quartermaster Corps. 1957. Radiation preservation of food. Rep o r t P.B. 151493, 461 pp.

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Villiers, C;. D. B. de, Brown, D. S., and Tompkins, R. G. 1963. The effect of weather and climate upon the keeping quality of f r u i t . I. Zv: World Meteorological Organization, Technical Note No. 53, pp. 1-112. W a t t , B. K., and Merrill, A . L. 1960. Composition of foods. U.S. Dcpt. Ayr. Ha>idbooli 8, 147 pp. Worth, W. S., Read, M. S., and Kraybill, H. F. 1957. Deterniination of metabolizable energy of frozen irradiated food a s fed t o r a t s of the second generation in long-term toxicity studies. U.S. A r m y Medical Nutrition Laboratory Rept. No. 210, Denver, Colorado. Zeeuw, D. de. 1961. Experiments on the preservation of f r e s h f r u i t by irradiation. Food Irradiation 1 ( 3 ) , A5-A7. Ziporin, Z. Z., Kraybill, H. F., and Thach, H. J. 1957. Vitamin content of foods exposed to ionizing radiations. J . N u t r i t i o n 63, 201-209.

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IONIZING RADIATION FOR CONTROL OF POSTHARVEST DISEASES OF FRUITS AND VEGETABLES

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Fungicidal and Fungistatic Efiects of Radiation . . . . . . . . . . . . . . A. Radiobiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R. Factors Affecting Dose Requirer.ients . . . . . . . . . . . . . . . . . . . . . . . C. Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Techniques f o r Postharvest Disease Radiation Studies . . . . . . . . . . . . A. I i i Vztro Studies ................................... B. 1~ Viwo Studies . . . . . . . .................... IV. The N a t u r e and Causes of ases . . . . . . . . . . . . . . . . V. Disease-Control Investigations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Irradiation-Induced Susceptibility to Infection . . . . . . . . . . . . . . . . VI. Protective Packa ......................... VII. Research Needs ........................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

147 149 149 151 158 159 160 166 166 170 170 180 182 183 184

INTRODUCTION

The bactericidal and fungicidal properties of ionizing radiation have been studied from a time soon after Roentgen’s (1898) discovery of X-rays, in 1895, and Becquerel’s (1896) discovery of radioactivity. Notable early reports include those of E r r e r a (1896) on the effects of X-rays on P h y c o m ~ c e s ;Dauphin (1904) on the influence of radium on the growth and development of certain lower fungi; and Minck (1896) and Rieder (1898) on the bactericidal properties of X-rays. Many of the earliest researchers reported no damage to the irradiated organism, but Rieder reported decided damage to bacteria. The early investigations on the biological effects of ionizing radiation have been reviewed in detail by Duggar (1936) for bacteria and by Smith (1936) for fungi. The possibility of utilizing ionizing radiation for disease control was pointed up by the successful use of X-rays to cure 147

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dermatophytoses (Jungling, 1919 ; Levy, 1913 ; Melchoir, 1916 ; Sardemann, 1914). Similarly, Heyderdahl (1926) cured actinomycosis with gamma rays from radium. These demonstrations that parasites could be selectively treated in host tissue suggested the possible use of ionizing radiation to eliminate certain plant parasites in seed-borne diseases. Pichler and Wober (1922) reported that X-rays killed Ustilugo t ~ i t i c i(Pers.) Rostr. in wheat seeds, and C. m t d a (Jens.) Rostr. in barley seeds. I n addition, those researchers reported elimination of the wart disease fungus, Svnchyt?*iumendobioticum (Schilb.) Percival, from potato tubers. From later studies, however, Tascher (1933) concluded that radiation doses sufficient to kill fungi in seeds would injure embryos, resulting in abnormal germination and seedling growth. More recently, Lo (1964) reached similar conclusions. Studies have been conducted on the effect of X-radiation on the crown gall disease and its causal organism, Ag~obacteiiuni tzimefacieizs (E. F. Sm. and Town.) Conn. (Levin and Levine, 1917; Rivera, 1929 ; Waggoner and Dimond, 1952a). The possible application of ionizing radiation to disease control is evidently severely limited by the susceptibility of host plant meristems to damage by relatively low doses. Treatments sufficient t o inactivate pathogens would generally be expected to result in abnormal or inhibited growth (Dimond, 1951 ; Hellmers, 1959 ; Schwinghamer, 1957 ; Waggoner, 1956 ; Waggoner and Dimond, 1952b). Thus, deeply penetrating ionizing radiation does not appear promising for therapy of plants or plant parts destined for later growth. Consequently, such treatments a r e evidently ruled out for seed grains and propagules such its bulbs, tubers, and cuttings. Much more promising subjects for irradiation a r e harvested plant parts destined for consumption. Although the commodities a r e alive, cell division has often virtually ceased. In other cases, cell division may occur after harvest but abnormal o r reduced growth from irradiation may not be objectionable. Where the effects on growth can be ignored, relatively high doses can frequently be tolerated by the host. The present capabilities for source construction and design suggest that radiation could economically be used to treat food if benefits were sufficient. An area of particular interest in this regard is the possible use of ionizing radiation as a fungicidal treatment for the control of postharvest diseases of fruits and vegetables (Droge, 1963). Here, radiation treatments appear to have important potential advantages. Not the least among them

RADIATION FOR DISEASE CONTROL

I45

is the complete absence of chemical residues. Furthermore, radiation provides a fungicidal treatment uniquely different from chemical applications. The extreme penetration, particularly of gamma rays, permits the host to be considered essentially transparent to the rays. Therefore, organisms deep within the host tissue niay be treated as readily a s if they were on the surface. Because of this feature, pathogenic organisms may be treated within host tissue to provide a therapeutic effect in a manner ordinarily not possible with chemical fungicides. With radiation as with chemicals, control of postharvest diseases requires a thorough understanding of the diseases to be controlled. Particularly important are the time, place, and manner of infection; the influence of temperature and other environmental conditions on disease development ; the propensity for spread by contact ; and the physiological length of the postharvest life of the fruit o r vegetable. Extended storage following radiation may affect host injury responses (Maxie and Abdel-Kader, 1965) as well as change the relative importance of various diseases. The effectiveness of radiation treatments may therefore be determined in large measure by factors relating to disease etiology. Equally important a r e those facets of radiation biology which affect the fungicidal and fungistatic consequences of radiation. Therefore, these topics are considered at this time to lay a foundation for discussions of recent investigations in the area of irradiation of fresh fruits and vegetables. 11.

FUNGICIDAL AND FUNGISTATIC EFFECTS OF RADIATION

A. RADIOBIOLOGY Ionizing radiation is a form of energy which, if absorbed, acts upon living cells to produce injuries. The absorption of energy ionizes and electronically excites molecules in a way that produces molecular changes. The action is termed “direct” if the damage occurs in the molecule in which the energy has been absorbed, and is called “indirect” if it results from highIy reactive free radicals formed in water and reacting with cell constituents. The injury may directly damage genetic material, producing mutations which may or may not prove lethal. Molecular change may cause so-called biochemical lesions which a r e developed or intensified by metabolic processes of the cell. Biochemical lesions may affect vital genetic syntheses to result in a mutation which, if sufficiently severe, may result in cell death. On the other hand,

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biochemical lesions may affect nongenetic processes and result in an altered physiology which may also prove lethal to the cell. The fundamentals of radiation biology are treated adequately in a number of excellent reports. Particular attention is called to those of Bacq and Alexander (1961a), E r r e r a and Forssberg (19611, Harris (1961), Jenkinson (1963), Kimball (1957), Lea (1955), Romani (1965), Platzman (1952), Setlow e t al. (1961), and Spear (1953). Fungi were given special attention in the reports of Beck and Roschenthaler (1960), Pomper and Atwood (1955), and Tatum (1950). Certain aspects of radiation effects a r e of particular, and sometimes unique, importance to studies of the use of ionizing radiation in the pathology of harvested fruits and vegetables. These a r e the considerations which receive primary attention here. Ionizing radiation may, in pathogens, cause morphological abnormalities as well as genetic mutations and altered physiology (Atwood and Pittenger, 1955a ; Brace, 1950 ; Burns, 1955 ; Johnson, 1932). The morphological effects on fungi a r e especially striking immediately after germination. When irradiated spores are plated on a medium conducive to prompt and rapid germination, the germ tubes produced often have diameters much larger than normal (Berk, 1952a, 1953; Brace, 1950; Dimond and Dugger, 1940 ; Luyet, 1932). Frequently grotesque swellings occur at various places in the mycelium. Commonly, the germ tube may grow only a short distance and then round up at the end t o produce a monster germinant resembling a “dumbbell.” Occurring just as commonly, however, is extensive germ tube development, including branching. The amount of germ tube development is evidently inversely related to dose. Characteristically, cross walls a r e almost totally lacking in species that normally form regular and prominent walls in the germ tubes. Extensive growth may occur, but a point invariably seems to be reached at which all further development stops and death follows. The absorption of energy to cause lethal damage may be separated from death by many hours. If irradiated spores a r e stored in a n environment not conducive to germination, the time between energy absorption and death may be extended to a number of days. During that time respiration, the production of certain enzymes, and other metabolic functions a r e known to proceed at a normal or accelerated rate (Sommer et aZ., 1963b, 1965a). The stimulation of metabolism or germination and growth (Buchwald and Wheldon, 1939 ; Vasudeva e t aZ., 1959) following lethal or sublethal irradia-

RADIATION FOR DISEASE CONTROL

151

tion must presumably require a n explanation based upon cell injury . In considering control, the relation of radiation dose to the ability of fungus spores to germinate is of little importance (Uber and Goddard, 1934). Much greater significance must be attached t o loss of the ability t o form a colony capable of indeterminate growth. In fungi the capacity for indefinite growth is lost a t a much lower dose than the ability to germinate (Beraha et al., 1959a,b). In experiments with Rhixopzts stolonifer, a dose of 500 Krad reduced the surviving fraction (i.e., ability t o form a colony) to less than 1% but hardly affected the ability to germinate (though many germ tubes produced were abnormal). Reducing germination to nea.r 1% required a dose of 1500 Krad. An even more resistant process than germination is the ability of the spore to swell prior t o emergence of the germ tube (Sommer ef al., 1963a). When colonies are irradiated at a dose insufficient to inactivate permanently, growth is halted temporarily (Beraha et al., 1959a,b; Nelson et al., 1959), to be resumed after a delay which can amount to several days. The basis for the delay is not well understood. It is likely, however, that most of the mycelium has been irreversibly injured. Only certain portions of hypha may, with time, recover. Essentially normal growth then occurs from localized areas.

B. FACTORS AFFECTINGDOSE REQUIREMENTS 1. Genetic

The use of radiation in postharvest pathology obviously depends on the radiation sensitivity of the fungus compared with the ability of the host t o withstand the treatment with little or no obvious injury or deleterious side effects. The apparent sensitivity of the fungus is determined by a number of factors. One factor is the inherent resistance of a fungus to inactivation by ionizing radiation. The resistance varies widely between species, is evidently of a complex nature, and is genetically controlled. The genetics of fungi a r e reviewed by Fincham and Day (1963), while radiation genetics have been discussed by Wolff (1961). I n fungi it has been demonstrated, particularly with yeast, t h a t diploid cells a r e more radiation-resistant than haploid cells of the same species (Fig. 1 ) (Beam, 1955; Latarjet and Ephrussi, 1949 ; Mortimer, 1954 ; Mortimer, 1961 ; Zirkle and Tobias, 1953).

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N. F. SOMMER A N D R. J. FORTLAGE

It a ppe a r s t h a t th e greater radiation resistance of th e diploid condition is a direct result of th e presence of a second set of chromosomes. Th e nuclear redundancy of th e diploid condition presumably provides protection against recessive lethal mutations. Da t a f r o m higher plants suggest t h a t the f u r t h e r chromosome duplication of polyploids does not im p ar t added radiation resistance (Sparrow an d Miksche, 1961). Similarly, in fungi, Mortimer (1961) found t h a t hexaploid yeast cells were less resistant th a n triploids.

I

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FIG. 1. Survival of haploid and diploid yeast cells a f t e r X-radiation. I. IIaploid. 11. Diploid. Redrawn from L a t a r j e t and E p h r u s s i (1949).

T he nuclei of fungus spores a r e usually haploid, but, commonly, t w o or more nuclei m ay be present within a single-celled spore. Here, multinuclearity imp arts a resistance analogous t o the efiect of ploidy. I n experiments with hrewospo?.a spores, Norman (1951 ) found uninucleate microconidia to be less radioresistant th a n conidia containing fro m one t o several nuclei but averaging about two (Fig. 2 ) . Similarly, Atwood an d Pittenger (195513) found multinucleate Sezwospo?*n ascopores more resistant t h a n microconidia. The results suggest t h a t th e spore can survive if one ilucleus escapes inactivation. I n the case of multicellular spores the presence of the several cells in a spore would presumably impa r t resistance: as long as lethal in ju r y was escaped by a n y one constituent cell, th e spore would retain th e ability to f o r m a colony.

153

RADIATION FOR DISEASE CONTROL

Although the relation between nuclear conditions and radiation resistance is unmistakably important, damage to the cytoplasm is also important (Kimball, 1957). Furthermore, nuclear damage may not be all genetic damage.

I . /

0

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20

30

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DOSE IN QUANTA CM* x FIG.2 . Survival of uninucleate microconidia and multinucleate (av. 2.27) conidia of .Vcztrospoi.a o‘assa. Redrawn from Norman, (1951).

2. P o p u l a t i o n S i z e The size or amount of fungus “tissue” present affects the apparent sensitivity of a species. With filamentous fungi, direct determination of population size of mycelium is difF.cult in vitro and may be impossible in vivo. Problems of technique in studies involving mycelium are discussed in a later section. Obviously, however, the minimum dose required to inactivate with a high degree of probability all “cells” in a population is related to the number ol‘ cells in the population (Couey and Bramlage, 1965). In studying t h e inactivation of mycelium, so-called “lethal doses” depend upon the size of the colonies. Presumably “lethal dose” values would always differ if the amount of mycelium was not the same. For example, a number of “lethal doses” have been reported for B o t r v t i s cinerea. Skou (1960) reported the “lethal dose” to be greater than 470 Krad. Beraha e t al. (1960) reported t h a t the approximate doses lethal to young mycelium in vitro varied from 0.95 to 2.03 Krad. In the host, a comparable effect required 2.74 to 4.56 Krad. Saravacos e t al. (1962) reported a

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N. F. SOMMER AND R. J . FORTLAGE

“lethal dose” of 500 Krad for the fungus in culture. Kljajic (1960) reported that doses of 600-1,000 Krad were needed for a “lethal” effect. Sommer e t al. (1964a,b) studied the doses required to inactivate every “cell” in widely varying sizes of populations of B. cineyea conidia and young mycelial “cells.” The young mycelium was obtained by germinating conidia until the resulting germ tubes had three to five cross walls. Figure 3 shows the doses which a r e capable of inactivating every “cell” within 80% of the populations when populations of several different sizes a r e compared. Careful consideration of population sizes is of great importance in interpreting the results of radiation treatments for the control of postharvest diseases in fruits and vegetables. If colonies are large, even radiation-sensitive fungi may require relatively large doses. For example, among Pviirius fruit decay fungi, Moiiilinia f i ircticola conidia a r e relatively radiation-sensitive whereas Rki:opus stolonife?. spores a r e much more resistant (Fig. 3 ) . Yet, because of population differences, &I. fructicola may appear the 600/

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POPULATIONS FIG.3. Approximate doses required to inactivate every cell in 80‘r (colony inactivation dose 80) of various sized populations of postharvest disease f u n g i . Data a r e f o r spores in vitro. 1) Trichoderma viridc. 2 ) P h o ~ o p s i s citn’. 3 ) Penicillium i t a l i c u m . 4) Pcnicillium e x p a m u m . 5 ) Pcxicilliltm digifatzLm. 6 ) Gcotr.idiunz canrlitlum. 7 ) Moiiilinia f r u c t i c o l a . 8) B o t r y t i s ciiicrea. 9 ) Dip l o d i a izatalc nsis. 10) R h i x o p u s stolonifer. ll) Alternaria citri. 1 2 ) Clado.iporicim h c r b a m m . Redrawn from Sommer c t al., (1964a,b).

RADIATION FOR DISEASE CONTROL

15.5

more resistant when fruit is irradiated at a given dose. The reason is that the R. stolonifer population may consist of only a few spores contaminating harvest or handling wounds whereas M . f m c t i c o l a may be present in well-established colonies resulting from infections occurring in the orchard before harvest. 3. Fungicidal or Fungistatic Effect Required

The degree of the fungicidal effect sought greatly influences the size of the radiation dose required. A relatively small dose frequently suffices if complete inactivation of lesions is not required. At doses insufficient to inactivate lesions, growth is halted temporarily, with t h e length of the delay related to the size of the dose (Beraha et al., 1959a,b; Nelson et al., 1959). I n the first place, fungus “cells” given sublethal doses a r e evidently injured sufficiently for a time lag t o result before growth is resumed. Secondly, most of the constituent mycelium of 8 colony may be inactivated, so that resumption of colony growth is dependent on the activity of localized areas of a few hyphae. Such a temporary halt in lesion growth may provide a good and sufficient control in commodities that normally have only a short physiological life, such a s strawberry or sweet cherry fruits. With longer-lived hosts a highel. proportion of fungal lesions will likely require inactivation.

4. Dose-Modifying Factors Experimental conditions before, during, or after irradiation may influence the results of experiments designed to determine the effectiveness of radiation a s a fungicidal treatment. Some factors may magnify the damaging qualities of the irradiation, whereas others may alter the ability of the living cell to withstand injury. a. Clxy.c/en E f f e c t . Perhaps the most striking dose-modifying variable is the presence or absence of oxygen. Oxygen materially enhances the effectiveness of a given radiation dose (Bridges and Horne, 1959 ; Laser, 1954 ; Stapleton and Hollaender, 1952). The increased lethality is evidently the consequence of the formation of toxic peroxides in the presence of oxygen. I n tests with several postharvest disease fungi the dose required to reduce survivors to the 1% level was only about 60% as great in the presence of oxygen as in anoxia (Sommer et nl., 1 9 6 4 ~ ) . Fungi or bacteria parasitizing fruit or vegetable tissue are

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~ i ~ ~ i ain l l ya n aerobic environment, but, under certain conditions, low oxygen tensions may develop. F o r example, if t h e f r u i t s t o be irradiated a r e sealed in plastic bags or other containers, the respiration o i f r u i t and pathogen may modify t h e atmosphere by depleting oxygen while carbon dioxide accumulates. Similarly, Sungi prepared f o r irradiation iti v i t w may consume oxygen t o t h e extent t h a t near-anaerobic conditions may result unless guarded against. A related factor is the presence of ozone produced by t h e i r radiation of oxygen. Evidently concentrations toxic t o host and parasite can accumulate, particularly if the irradiation is performed in a closed container. Rapid a i r movement through the irradiation chamber would presumably prevent accumulation of a n ozone atmosphere around the f r u i t or vegetable host. A more difficult measure, however, would be t h e prevention of ozone accumulation within host tissues, since these tissues m a y contain as high as 20% a i r space (Maxie a n d Abdel-Kader, 1965). b. P?-otection. A radiation dose is commonly less effective i t i civo t h a n in v i t m . The lower lethality is believed t o be t h e consequence of t h e chemical protection afforded by host constituents. Many chemicals a r e known t o exert a protecting effect against radiation damage (Bacq and Alexander, 1961b ; Hollaender, 1960 ; Riley, 1955). I n some cases t h e protective effect results f r o m t h e compound’s removal of oxygen or f r o m t h e promotion of oxygen removal by the cell by oxidation. Other compounds, however, a r e able t o exert a protection greater t h a n found by removal of oxygen a n d a r e believed t o specifically protect radio-sensitive sites or promote recovery (Stapleton, 1960). c. R a t e of Application of Radiation. The r a t e of application of a given dose or fractionation of the dose into two or more portions separated by time influences t h e biological effect achieved. Sometimes t h e greater biological effects of rapidly applied doses have been suggested t o result from fewer opportunities for repair (Whiting, 1960). Beraha e t al. (1959d) have studied t h e effects of t h e rate of application of the gamma-radiation dose in connection with certain postharvest diseases of fruits. Within t h e range of 137-182 Krad, a high flux of 7 Krad/min. controlled Pythium deba?*yanz~nr Hesse inoculated in potato tubers better t h a n t h e same dose applied at 3 K r a d min. I n later studies (Beraha, 1964), Pexicilliu?~?, italicurn Weh. inoculated in oranges was controlled f o r 12 days by 137 K r a d applied at 40 K r a d l m i n or 182 K r a d applied at 20

1,5:

RADIATION FOR DISEASE CONTROL

Krad min (Fig. 4 ) . Similar advantages of a high dose r a t e were reported f o r Moriiliiiiu f ructicola (Wint.) Honey in peaches, a n d Rotqjfis cinema Pers. ex Fr. in pears. Absorption of the dose by th e host ma y reduce th e dose a t critical sites, modifying th e expected radiation effect. However, this is a problem of dosimetry and dose distribution ra th e r t h a n a dose-modifying effect. 5 0 40 Kradlmin 3 Krad/rnin

% 4

a u

i i

a 3

z 0 L2 W

lL

z-I 0

56812 5 6 8 12 5 6 8 12 5 6 8 12 5 6 8 12 DAYS AFTER IRRADIATION 1-0-1 /--0.9-I I-1.25-1 1--1.57-1 1-1.82GAMMA

I

DOSE ( X lo5 R o d )

FIG.4. Effect of the r a t e of application on t h e effect of radiation doses. Average infection g r a d e of blue mold (Penicillium italicurn) on oranges a t 75°F. following gamma irradiation with different doses delivered a t 40 K r a d / min. and 3 Kradlmin. Redrawn from Reraha, (1964).

d. RecoveTy. I n v i f r o studies have demonstrated th a t, under certain conditions, the postirradiation environment can influence th e dose effect, evidently b y permitting th e operation of recovery mechanisms. Inactivation as a response to irradiation h a s been reported t o be reduced by holding bacteria in suspension before plating (Roberts and Aldous, 1949) ; incubation at suboptimal (Latarjet, 1943, 1934; Stapleton et ul., 1953) o r supraoptimal (Stein a n d Meutzner, 1950; Anderson, 1951; Buzzell, 1956) temperature ; o r starvation (Alper an d Gillies, 1958). Alper a n d Gillies (1958) have suggested t h a t a common fe a tu re of these procedures is their ability to slow metabolism a n d growth a f t e r irradiation. Repair of genetic radiation damage h a s been discussed by Sobels (1963). Experiments with sporangiospores of t h e fungus R h i x o p m

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stdotlifer (Ehr. ex Fr.) Lind. have shown that a portion of the potentially lethal irradiation injuries can be restored to the nonlethal condition if germination is delayed for a time. The recovery involves metabolism, with the required energy supplied either by oxidative respiration or, if a glucose substrate is present, by anaerobic fermentation. When the germination of spores was inhibited either by a lack of a medium suitable for germination or by anaerobiosis, the number of repaired spores was greatest a t a temperature near optimum for growth (Sonimer et al., 1963a, 1964c, 196Sa,b ; Sommer and Creasy, 1964). The significance of the recovery mechanism as a factor niodifying the dose required for disease control has not been completely evaluated. Certainly recovery could influence the results of it/ vitt.0 studies of sensitivity to radiation. Whether the recovery mechanism is likely to operate in v i m is less certain. The number of recovered spores is greatest when growth has been prevented or slowed. The condition most likely to halt fungus growth in the host is cold storage, but the low temperature would be expected to limit metabolic recovery drastically. Modification of the atmosphere to provide a n elevated carbon dioxide level or a depressed oxygen content, or both, is sometimes employed in fruit storage or transit. Here, growth would be slowed while other conditions might permit metabolic repair. However, modified atmospheres are used most often in combination with low temperatures, which would likely negate the possibility f o r extensive repair. e. Preiwacliatio?z Conditions. Preirradiation conditions evidently may influence the dose effect as well as the environment during or after irradiation (Stapleton, 1960). The reasons f o r the different sensitivities have not been established. It has been suggested that, under certain preirradiation conditions, protective chemicals may be produced; that the number of nuclei per cell may be increased; or t h a t after growth on a rich medium, nietnbolic reserves may enhance the potentialities for repair. C. MUTATIONS Irradiation treatments result in many mutants among surviving pathogens (Berk, 195213; Catcheside, 1948 ; Diller e t al., 1946 ; Hollaender and Emmons, 1946 ; Stapleton and Martin, 1949). Mutants a r e usually less vigorous than the original species. Of particular concern in connection with fruit and vegetable irradiation treatments a r e mutants which may exhibit increased radio-

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resistance or become more vigorous pathogens as a result of mutation. Variation to radioresistance h as been observed in a number of studies (Clark an d Frad y , 1959; Hill an d Simson, 1961; Witkin, 1947). Greenberg (1964) h a s studied th e genetics of radiation resistance in Esciicrichia coli (Migula) Cast. & Chaf. There a p pears little doubt t h a t radioresistant stra in s of phytopathogenic fungi could appear a f t e r a radiation technology is adopted. If their original level of pathogenicity should be maintained, such mut a nt s might become troublesome. Th e extent of t h e potential problem cannot yet be adequately evaluated. Most mut a nts appear to be less pathogenic th a n their parents (Beraha et al.,1 9 6 4 ). Even a m u tan t with a n in vit7.o growth rate of about th ree times normal did not have g re a te r pathogenicity (Buddenhagen, 1958). On th e other hand, F lo r (1958) an d Schwinghamer ( 1959) observed mutation to wider virulence in t he flax r us t organism, M e l a v i p s o m Zini (Ehrenb.) Lev. Similarly, Day (1957) observed mutation t o virulence in C l a d o s p o ~ i u n i fulvzrm Cooke tested on tomato plants resistant to th e original isolate. The occasional occurrence of increased pathogenicity f r o m niutation ha s usually been demonstrated in plants having a resistance introduced by a breeding program. Commonly, such plants a r e resistant t o certain fungus strain s b u t not others. Furthermore, th e parasitism exhibited by ru st fungi is a highly advanced type which depends upon a delicate physiological balance between host and pathogen (Gaumann, 1950). I n nature, new fu n g u s races may appear as a result of mutation o r recombination, making breeding f o r resistance a continuing program. By contrast, f r u i t and vegetable ro t disease organisms exhibit a primitive type of parasitism (Gaumann, 1950) in which t h e physiological balance between host an d parasite is relatively less important. Although there appears to be no reason to believe t h a t increased pathogenicity could not appear among postharvest disease mutants, th e increased pathogenicity is likely t o be much less dramatic t h a n in th e case of rusts. Ill.

TECHNIQUES FOR POSTHARVEST DISEASE RADIATION STUDIES

I t appears logical t h a t studies of th e fungicidal effect of radiation must be based on critical investigations of th e radiation biology of t he fungi involved. Attempts to determine t h e effec-

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tiveness of radiation by merely irradiating commercially h a r\ ested commodities a r e usually unsuccessful. A t best, only qualified conclusions can be drawn. Often th e results a r e misleading.

A. Z I I V i t r o STUDIES 1. Eapei*ii?iciital M c t h o d

A number of different approaches have been used in studying the dose response of fungi t o irradiation. Since all approaches have various limitations o r defects, the method mu s t be selected with care. F o r in v i t w studies, the methods usually used with yeasts o r bacteria frequently require modification because of the filamentous habit of growth of most postharvest decay species. Techniques used in studying chemical control of decay must be modified t o take into account t h e g reat penetration of th e ra y s along \vith t he frequent inability to achieve 100% inactivation a t doses t h a t t he host can withstand. Some studies have been made in which fungus populations have been represented by all o r p a r t s of mycelial colonies o r by uniform loops of a spore suspension (Beraha et ul., 1960; Nelson et nl., 1959; Saravacos e t ul., 1962; Skou, 1 9 6 0 ). A common defect of these methods is the use of only one fu n g u s population of a size which m a y be poorly defined. F o r example, colonies of different species might be grown on a g a r media in P e tri dishes. Uniformsized pieces of a g a r bearing fu n g u s mycelium a r e then removed and irradiated with various doses, an d each fra g me n t is then placed on media in a P e t r i dish o r tube slant. T h e dose at which nearly all, or a certain fraction, fails t o grow f u r t h e r is determined. This method is easy an d rapid, but th e relative sensiii:+ ties between species or conditions can usually only be approsimated. Since population sizes influence th e probability of inactivation, accurate determinations a r e needed. However, here there is usually no way of determining sizes except by measurements, such as diameters. Since th e density of th e mycelial ma t l a r i e s with the species, th e same size of block ma y not provide comparable mycelia. Th e same defect m a y occur when th e same species is grown under different conditions of medium o r environment. Furthermore, th e medium constituents would presumably influence t h e dose response by exerting a protective action during irradiation. The degree of protection might v a ry with th e medium used. The medium constituents may change with age o r as a

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consequence of fungus growth. If the medium is present during irradiation, the risk is added that changes in constituents might affect the physiology of the fungus after irradiation. The greatest obstacle of all, however, appears to be the absence of any convenient method of estimating population sizes accurately. Whole colonies in viti.0 can be irradiated with a cellophane technique, however. The medium in the Petri dish is covered with sterile cellophane approximately 1 mil thick. Commercial cellophane may contain undesirable waterproofing additives which can be removed by soaking overnight in acetone followed by thorough washing in running t a p water. The fungus inoculum is placed on the cellophane near the center of the dish. After the desired growth, the cellophane bearing the colony may be removed for irradiation. The irradiated cellophane has not appeared to affect the fungus dose response, but for the most critical studies the fungus colony may be aseptically scraped from the cellophane before irradiation. After irradiation, the fungus colony, with or without cellophane, is placed on fresh medium in a Petri dish. To ensure that all surviving fungus cells will be in contact with the medium, the irradiated colony is then covered with a thin layer of medium cooled to ca. 45°C before i t is poured. An obvious disadvantage of the cellophane technique is the inability to directly quantitate the fungus “tissue” using the same colonies as those irradiated. An indirect quantiation is possible, however, by relating the d r y weight, protein, or nucleic acid content of similar colonies as discussed in a later section. The conditions of medium o r environment during growth must be standardized. If a fungus sporulates abundantly, spores a r e the most convenient fungus structure to use for dose-response studies. The spores a r e discrete individuals which can usually be easily quantitated by counting procedures. The ease of obtaining reliable quantitative data is another reason that spores a r e the structures of preference when experimental requirements permit their use. A number of limitations must be considered, however, when spores a r e used. I n some cases, sufficient numbers cannot be obtained. I n other cases the spores a r e large and of indefinite multicellularity, causing difficulties in quantitating on a cellular basis. I n still other cases, spores of more than one type may be present in a culture, making it difficult or impossible to obtain uniform spore suspensions. Furthermore, the objects to be inactivated in fruit a r e more likely to be mycelia than spores.

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2 . E J 1.1w s s i i i g

Ex p e r i me ?i ta 1 Data

(1. Ilosc I:cJspo?isc Crrwcs. Studies of the radiation sensitil ity of bacteria and yeast species have generally involved comparisons of the shape an d slope of the curve established by plotting th c (lose against the log percent survival. F u n g u s spores can be studied in a similar fashion (Fig . 5 ) . Exponential survival can be explained either on th e basis of a single hit to kill o r a population distributed exponentially with respect to resistance to radiation, with the first possibility appearing the more plausible (Lea, 1955) . Most often, however, fungus curves are sigmoidal.

DOSE (Krad)

5. A p p r o x i m a t e dose response curves f o r spores of p o s t h a r v e s t disease f u n g i . 1) Triclioderma viradc. 2 ) P h o m o p s i s citri. 3 ) Penicillium italiczcm. 4) Pc?iicillium r x p a a s u m . 5 ) Penicillium cligitatum. 6 ) G e o t r i c h u m candidurn. 7 ) .IZ
A common reason f o r sigmoidal inactivation in fu n g u s spores is a multinucleate o r multicellular spore condition. P la tin g e r r o r s due t o clumping would also result in a sigmoidal curve (Snyder, 1947). Atwood an d Norman (1949) an d Kimball (1953) have discussed t h e interpretation of multi-hit survival curves. Since th e survival of discrete individuals is determined, th is technique is not adapted to studies with mycelium, where cells ma y be ill defined. Furthermore, th e technique appears to be limited to ~ T ~ i t r ostudies.

L

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163

0 . Eutl-Poiirt Am1usi.s. To determine th e doses required to inactivate a large percentage of each size of f u n g u s population, a n “end-point’’ technique appears ideally adapted. Studies of the control of f r u i t disease organisms by irradiation frequently involve the probability of inactivating fungus populations of different sizes in host lesions by different doses. Such a technique h a s the advantage of being adapted to spores, mycelium, o r sclerotia. Certain types of studies can be made i?i vivo as well as i?z vit?o. The “end-point” technique should permit infection levels in “orchard-run” f r u i t to be estimated an d compared in te r ms of end-point results in experiments involving known populations. The proportion of infections inactivated by various doses would be related t o population sizes behaving similarly in controlled experiments, a n d th e field infections could be expressed in te rms of “spore equivalents” o r some other index of population size, a s discussed later. I n studies involving th e inactivation of bacterial spores in canned foods, Schmidt et al. (1962) an d Schmidt a n d N a n k (1960) used a n end-point technique with results expressed as D values. Survival in different experiments were compared by the D values, calculated a s follows :

Radiation dose (megarad) D=---~ Log M - Log s where JI = t h e total inoculum; t h a t is, t h e spores per container times the number of containers, an d S Z= th e number of containers not sterile, assuming one survivor per container. D values could presumably be calculated in experiments in which known numbers of fu n g u s spores constitute th e inoculum. The technique is applicable to certain in vitro o r in vivo experiments. However, D value calculations depend on th e ability t o determine cell numbers in th e inoculum, a procedure usually difficult or impossible with mycelia o r sclerotia. I n a variation of the end-point technique, Sommer et nl. ( 1964a,b) experimentally determined the doses required to inactivate every fungus cell, i?i vivo an d in vitro, in populations of inocula varying in number fro m ca. 10 to 10‘. cells o r spores. Twenty replicates were used f o r each population size a n d f o r each dose. F o r each population size, doses were selected so that, ideally, t he lowest dose would inactivate no populations, intermediate doses would halt some replicates, a n d t h e highest dose would halt almost all.

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With each inoculum size, the inactivation of populations was plotted to establish preliminary inactivation curves. Doses inactivating 50, 80, and 90% (or any other level) could be approximated by interpolation (Fig. 6 ) . The doses inactivating 80% (and sometimes 50 and 9 0 % ) of the populations were then plotted against the exponent of the population (Fig. 3 ) . With the populations and doses used, the points were experimentally determined to lie sufficiently near a straight line to permit ready interpolation between widely separated points.

DOSE ( K r o d ) FIG. 6 . Plot of doses required to inactivate every cell in various sized populations of conidia of Monilinia fructicola when each population and every dose was provided with 20 replicates. Doses inactivating 80% of the populations (C.I.D.,o) were determined by interpolation and constitute line 7 in Figure 2. D a t a f r o m Somnier ct al., (1964a).

F o r i y b vit?*o end-point studies, portions of each suspension, representing the desired population, were pipetted into test tubes containing potato-dextrose agar slants immediately after irradiation. The incubation provided was at least two weeks a t room temperature. For in vivo studies with spores, a portion of a suspension coiltaining the desired number of individuals was injected into each fruit with a hypodermic syringe. Fruits containing locules, such as the tomato, can be readily inoculated by inserting the needle through the locule wall. With other commodities, a hole about 5 mm deep and 5 mm in diameter was made with a cork borer. One-tenth or one-hundredth ml of a spore suspension of a con-

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centration that delivers the required number of spores was placed in the hole, which was immediately covered with cellulose acetate tape. To ensure isolation and to prevent contamination, each fruit was placed in a paper cup of appropriate size, which was tightly covered with cellulose acetate film. The atmosphere surrounding certain fruits was tested by gas chromatography to detect the possible development of conditions of low oxygen o r high carbon dioxide. Young mycelial cells of certain species have been obtained by placing the desired number of spores in a suitable liquid medium and incubating on a culture shaker until germ tubes of about four to six cells were present (Sommer e t al., 1964a,b). The germinated spores were collected on sterile filter paper, resusnended in water in the presence of a surface-tension-reducing agent, and agitated briefly in a Waring Blendor t o disperse clumps of germinated spores. Average numbers of cells per germ tube and absence of tube breakage from blending were verified microscopically. Suspensions in appropriate aliquots were dispensed by pipette. With mature mycelia and sclerotia, cellular units cannot be readily counted to provide a cellular basis f o r population sizes. In the Plrycomycetes the mycelium is nearly devoid of the deliminating cross walls which permit discrimination of regular units of hypha for a “cell” count. Yet, careful studies of the radiation biology of pathogens must include the all-important mycelium as well as spores. The technique used should not only permit determination of the size of fungus populations but enable the preparation of comparable populations whenever required. F or i~ vitro studies, fungus colonies can be grown on cellophane. Colonies to be irradiated can be related to dry weights of colonies of similar size. The sensitivity to irradiation of several colony sizes could be determined by the end-point technique. The use of dry weight, however, poses some problems arising from the heterogeneity of the mycelium. Young mycelium near the edge of the advancing colony is metabolically active and is filled with protoplasm, while the hyphal walls a r e very thin. In older mycelium, in contrast, the walls may be much thicker and the protoplasm may contain large vacuoles. In some cases, individual hyphae may become devoid of protoplasm. Therefore, on a dryweight basis young mycelium might be expected to be much more resistant than old mycelium, and more resistant than spores if the latter have thick walls. In studies with B. cinerea, young

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mycelium was more resistant t h a n either conidia o r old mycelium when d r y weights were compared (Sommer e t al., 1965a). On a cellular basis, however, young mycelium (germ tubes) a n d conidia were nearly equal in sensitivity (Sommer, 1964a,b). Since lethal radiation in ju ry involves th e protoplast, protein analyses probably provide a more fundamental basis f o r comparison t ha n d r y weights. However, if th e lethality is a consequence of injuries to t h e genetic ap p ar a tu s within th e nucleus, protein content would presumably provide a valid comparison only if nuclear numbers were uniform within t h e protoplasm. Analyses of deoxyribonucleic acid would presumably provide th e best basis f o r comparison.

B. I N VIVO STUDIES F u r i u rivo experiments, mycelial inoculum evidently cannot be used conveniently, with th e limited exception t h a t germinated spores provide young mycelium in th e for m of germ tubes. However, it a ppe a r s t h a t fu n g u s colonies growing in f r u i t could be sized indirectly by comparing the radiation dose required f o r inactivation of the lesion with th e spore population size similarly affected by t he same dose. The in vivo mycelial colony sizes would then be expressed as “spore equivalents.” Reasonably uniform colonies would be required. A high degree of uniformity could presumably be achieved by giving careful attention t o th e number of spores constituting the initial inoculum, th e incubation temperatu r e , a nd time a f t e r inoculation. The end-point technique is particularly well adapted to studies of colony inactivation. It appears to be equally usuable where only lesion delay, not inactivation, is measured. IV.

THE NATURE AND CAUSES OF POSTHARVEST DISEASES

F r e s h f r u i t s an d vegetables a r e living plant p a r t s subject t o infection by parasites at all stages of their growth, maturation, and senescence. Defense mechanisms protect living fru its a n d vegetables f r om disease. Only organisms capable of avoiding or overcoming host defenses can be successful pathogens. However, a f t e r death occurs, a much wider range of fungi a n d bacteria a r e capable of colonizing th e tissue. Most causal agents of postharvest diseases a r e facultative parasites. Some species commonly exist as saprophytes, becoming parasitic only under certain conditions. With some important ex-

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ceptions t he agents of postharvest diseases a r e filamentous fungi f r o m t he Phycowzycetes, Ascom ycetes, Basidiomycetes, a n d Deuteromycetes. Probably the most important exception to t h e n e a r monopoly of postharvest diseases by filamentous f u n g i is t h e bacterium Erzuinia carotovora (Jones) Holland, th e cause of s o ft r o t in a wide r a nge of hosts, primarily vegetables ( S mith a n d Friedman, 1953). Species of bacteria an d fungi which m a y be in contact with fr ui t s a nd vegetables probably number in th e thousands. Yet, very few are capable of invading living tissue. Even a list of th e relatively f e w species known to be sometimes pathogenic is deceptively long. Th e most important fu n g i a n d bacteria which are pathologically associated with f r u i t s an d vegetables are well known a nd have been carefully studied (Anderson, 1956; Klotz, 1961 ; Walker, 1952 ; Wiant an d Bratley, 1948). Most postharvest pathogens are of minor importance, because of their ra rity , slow growth, o r control by refrigeration. As a case in point, California st r a w be r r y f r u i t s are usually attacked b y only about five o r six species of fungi. Only two of these, Botrytis cinerea Pers. ex Fr. an d Rhixopus StoZozife?. (Ehrenb. ex F r . ) Lind., commonly develop extensively a f t e r harvest. Of these two, R. stolonifer is held in check by temperatures below about 10°C. (Brooks a n d Cooley, 1921 ; Muller, 1 9 5 6 ). Therefore, modern refrigeration methods, which also extend th e physiological life of th e fruit, effectively control this fungus. B. cinerea growth, in contrast, is slowed b u t not entirely stopped by lowering th e temperature t o about 0°C. Refrigeration is consequently only partially effective in controlling the g r a y mold disease caused by B. cinerea. As a ma tte r of fact, if' the fungus h as already colonized th e f r u i t extensively, as is common with strawberries, rot development ma y be rapid despite refrigeration. Hence, studies of s tra w b e rry irradiation are, in large measure, studies of t h e radiation biology of B. cinprcn; other species a r e usually of minor importance o r nonpathogenic. The reason some microorganisms can attack th e living tissue of f r ui t s a nd vegetables while others cannot, is a complex problem which extensive investigations have not yet, in most cases, explained. I n some cases th e living tissue simply ma y not meet all the requirements of a good medium f o r th e growth of certain fungi. T h a t this explanation is not generally applicable is easily demonstrated by th e fact t h a t media made fro m cooked o r dried fr ui t s o r vegetables a r e frequently nearly ideal f o r a wide r a n g e

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of saprophytic organisms t h a t a r e entirely incapable of attacking th e living host tissue. I n some cases th e lack of pathogenicity m a y be t he consequence of a failure to synthesize necessary enzymes a nd toxins. However, much of th e host-pathogen specificity depends upon host resistance. The basis f o r the resistance ma y be mechanical, physiological, o r biochemical. Resistance by exclusion of organisms is provided by the epidermis of t he f r u i t o r vegetable. Overlying th e epidermal cells is a ba r r i e r layer of relatively inert cutin, which is difficult to penetr a t e . Additionally, th e cuticle b arrier provides a surface unfavorable f o r spore germination an d growth because of the lack of w a t e r a nd nutrients. However, small openings (stomata o r lenticels) through th e epidermis permit the entrance of some fungi which could not otherwise penetrate the cuticle. Many pathogens a r e strictly wound parasites which gain entrance only a f t e r t h e epidermis has been cut o r broken, usually during harvest or handling. Air- o r water-borne spores ma y be lodged in the wound, or objects t h a t produce the wound ma y be contaminated by spores of t he pathogen. Wounds a r e th u s a favorite means of entrance even by species whose germinating spores are capable of penetrating t he epidermis. Often, however, even wounds ma y serve as infection courts f o r only limited periods. F o r example, f r u i t tisdue immediately adjacent to wounds ma y rapidly become desiccated, no longer providing a good infection site. JITounds m a y heal, a s in th e suberization of potato tubers. I n some husts, such a s sweet potato roots, wounds m a y be walled off h\- the differentiation of a cork periderm. (>rind Most organisms placed directly within a susceptible a r e unable t o parasitize th e tissue. Although fu n g u s spores ma y germinate, growth is only limited. In some cases, host mettibolic products a r e toxic to certain fungi, resulting in a physiological or biochemical resistance. Toxic products may result f r o m hostparasite interaction. F o r a fuller coverage of disease resihtance and susceptibility, some excellent discussions a r e available (Allen, 1959 ; Barnett, 1959 ; Butler an d Jones, 1949 ; Cruickshank, 1963 ; F a r k a s a nd KirBly, 1962; Giiumann, 1950; Horsfall a n d Dimond, 1957 ; Tomiyama, 1963). F o r the majority of postharvest fu n g u s pathogens, th e sequence of events leading to infection and f r u i t o r vegetable ro t is essentially a s follows: The first step in th e infection process is spore germination. Spores on the host surface swell by a n uptake of water, which requires energy f o r a t least p a r t of the 71

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process. The protrusion and growth of a germ tube follows. I n species capable of direct penetration, the tip of the germ tube may form a n appressorium from which a very fine infection peg grows to penetrate the host cuticle and epidermis. Entrance may similarly be achieved through stomata, lenticels, o r wounds. Once within the tissue, mycelia branch repeatedly. Bacteria that cause postharvest diseases have no means of penetrating the plant epidermis and must usually depend upon mechanical or insect wounds. Sometimes, however, entrance through natural openings may be a n important factor. Once inside the host, postharvest disease pathogens commonly kill cells in advance of actual contact. Fungus hyphae kill and degrade host tissues by the production of toxins and enzymes (Barnum, 1924; Braun and Pringle, 1959; Cole, 1956; Cole and Wood, 1961a,b; Ludwig, 1960 ; McCalla and Haskins, 1964 ; Norkrans, 1963 ; Tomiyama, 1963; Wood, 1959). The cycle is completed when the fungus produces spores on the surface of the rotting fruit and these spores a r e released and disseminated, usually by air, insects, or water (Butler and Jones, 1949 ; Gaumann, 1950). Once a fruit or vegetable is intensively rotted, other fruits in contact with it may be invaded by pathogens capable of contact infection. Contact infections occur by mycelium growing from the rotted into healthy fruits or vegetables, with the healthy epidermis presumably being penetrated without any need for wounds. Some fungus pathogens have a well developed capacity to grow thus from f r u i t to fruit, producing a “nest” of decaying fruits held together by the intertwining mycelium. Thus, one infected fruit in a container may lead t o eventual loss of the entire contents. Environmental conditions play a n important role in postharvest diseases. Spore germination requires very high humidity or free water. Disease spread could be much reduced by lowering the humidity, but the drying environment would be objectionable because of weight loss and shrivel of the fruit or vegetable. Furthermore, the humidity may be much higher at the commodity surface than in the a i r of the storage room. If it is in a fresh Ivound, the spore may be bathed in liquid regardless of the humidity of the air. Temperature, the second major environmental factor, influences both spore germination and growth. If fiuits a r e cooled to a temperature unfavorable to fungus growth, rot stops. For example, if f r u i t temperatures a r e reduced t o O’C, all rotting ceases except t h a t caused by a few

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cold-tolerant fungus species (Brooks and Cooley, 1921 ; Muller, 1956 ; McClure, 1958; Smith and McClure, 1960). Particularly dificult to control a r e those postharvest diseases resulting from infection that may occur in the field o r orchard. Since fungus lesions a r e present in fruits a t harvest, protective chemical fungicides a r e of no avail. A good example is the gray mold disease of strawberries. Field infections by B. ciiierea normally occur during blossoming, when senescing or dying floral parts a r e invaded by fungus spores. The fungus spreads from the floral parts to the developing fruit receptacle. There the fungus may remain relatively quiescent until the fruit starts to ripen (Powelson, 1960). Pickers attempt to reject rotting fruits a t harvest, but some fungus lesions a r e difficult t o see, so rotting berries become randomly distributed among healthy fruits in the container. Then large losses may result during transit, because of the ability of the fungus to grow at low temperatures and because of vigorous contact spread and “nesting.” Protective chemical fungicidal sprays in the field have been relatively ineffective. Postharvest treatment with chemicals may not affect the fungus, because it is growing within the fruit tissue . V.

DISEASE-CONTROL INVESTIGATIONS

A. GENERAL Although a major motive for irradiating fruits and vegetables would be postharvest disease control, not a single disease appears to have been studied in depth. Similarly, little study has been made of the radiation biology of postharvest pathogens in relation to the diseases they cause. There a r e many reports of the reduction or delay of decay, but, all too frequently, no mention is made of the disease o r organism involved. The reader is often uninformed as t o the diseases being controlled, the circumstances leading to infection, whether infection occurred before or after harvest or before o r after irradiation, the extent of the infections at the time of irradiation, or the extent of control sought. In some studies the more serious diseases must have been entirely missing, for the organisms reported were relatively innocuous. In most cases the experiments were based upon natural infections, which can yield meaningful results only if the nature and incidence of the diseases a r e representative of usual conditions. If

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the various important diseases a r e not present in t h e frequency required t o provide a good test, experiments of th is type can yield results which lead to overly optimistic o r pessimistic conclusions as t o the fungicidal value of radiation. In relation to disease control, the most important contribution of studies of this type is the determination of the maximum doses tolerated by the host species o r variety. Such a dose will presumably be the one generally used f o r disease control. It a p p e a rs abundantly clear t ha t the optimum dose desired f o r pathogen control will almost invariably be higher t h a n th e host can tolerate. Thus, the radiation dose used will be determined by th e host, not by the pathogen. Different host species a n d varieties within a species differ in radiation tolerance. Additionally, th e tolerance m a y be influenced by the stage of ripeness at th e time of tre a tment. Since adverse effects m a y appear days o r weeks a f t e r i r radiation, the dose tolerated may be influenced by the presence or absence of extended storage. Generally, f r u i t s destined f o r storage a r e limited to a lower radiation dose (Maxie a n d AbdelKader, 1965). Many f r u i t an d vegetable species have been subjected t o irradiation treatments, primarily t o test th e effect on the host ra th e r th a n t o study th e relation of the treat me n t to disease control. I n some studies, the objectives of radiation were not to control disease but t o control specific host responses such as sprouting or ripening. Commodities which have received th e most attention or which appear to be th e most likely candidates f o r irradiation will be briefly considered at this juncture. F o r other discussions of t he s t a t us of investigations of ionizing radiation f o r f r u i t a n d vegetable postharvest disease control, consult surveys by Clarke (1959), Dupaigne (1964), Salunkhe (1961), a n d Willison (1963). Attention is also called to reports by DeZeeuw (1961), Heeney ~t a l . (1964), Maxie e t al. (1 9 6 5 ), Rubin e t al. (1959), Saravacos and Macris (1963), Tamburino (1959), Truelsen (1963), a n d Workman c.t a l . (1960). 1. Bewies

The st r a w berry appears to be the f r u i t most likely t o benefit in t he near f u tu re fro m irradiation as a fungicidal treatment. This is particularly tru e in California, where benefits are likely to be maximum because of special circumstances : relatively long producing periods in a single location ( 6 t o 8 months) ; th e high m a r ke t value of th e f r u i t ; th e presence of a destructive disease

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which cannot be adequately controlled by other pre- or postharvest treatments ; long distances to market (up to 3,000 miles by rail or 6,000 miles by air) ; and important benefits to be derived from only a few days' delay in disease development. Probably the most important disease, by f a r , is gray mold caused by Botiytis cinerea Pers. ex F r . Because small lesions a r e already present a t harvest (Powelson, 1960), decay may proceed without the delay that would be caused by the time necessary for infection and colonization. Under refrigerated transit conditions the growth of the fungus is only slowed, not stopped, by the low temperatures. Furthermore, the fungus can spread vigorously from fruit to fruit by contact. Spread by conidia is relatively unimportant in harvested strawberries, because of the time required for infections to develop at low temperatures and because of the short postharvest life of the host. Nelson e t al. (1959) reported that strawberries could not be sterilized at noninjurious doses. However, i~ vivo and i?i vitr.0 studies determined that the growth of mycelial colonies of B. cixei-ea could be halted by a dose of 200 Krep (Fig. 7 ) . At 5'C, a normal rate of growth resumed after 10 to 14 days. Contact infection and nesting were prevented for a similar period.

-"

I0 1 4 - 7 2 - 2 6

-Yo

DAYS AFTER RADIATION

FIG.7. The effect of ionizing radiation (electrons) on the subsequent growth of B o t t y t i s cimel-a in pure culture a t 3-4"C. Redrawn from Nelson, e t 111. (1959).

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The other major disease of strawberry fruits is “leak,” usually caused by Rhixopus stolonifer (Ehr. ex F r . ) Lind. although other mucoraceous species may sometimes be associated with the disease. R. stolonifer commonly infects wounds inflicted during harvesting and handling. Like B. cinerea, R. stolonif er may also infect by contact from fruit to fruit, producing an extremely rapid growth throughout the contents of a fruit container. F o r rapid development, however, temperatures must be warm. At temperatures near the optimum for fungus growth, i.e. 25-27”C, all strawberries within a container may be reduced to a watery residue within one or two days. Unlike B. cinerea, R. stolonifer does not grow at temperatures below about 10°C. Modern handling practices provide for quick cooling and maintenance a t ca. 5°C or less from the grower to the housewife’s refrigerator. Under these conditions the disease cannot occur. The possibility of using radiation to extend the marketing period in the absence of refrigeration appears extremely unlikely for strawberries. At elevated temperatures the physiological life of the fruit is extremely short, while the irradiation induced delay in fungus growth is minimal (Maxie et al., 1964). Moreover, even in the unlikely event that R. stolonifer could be completely controlled by radiation, the possibility of postirradiation infections must be considered because of the explosive nature of the growth of this pathogen at near-optimal temperatures. Prevention of postirradiation infections would likely require a sophisticated (and costly) packaging program. A third fungus species, Cladospo?.ium ke?.Dal-um Lk. ex Fr., is common in strawberries. It is not only capable of growing a t refrigerated temperatures but is radiation-resistant as well (Figs. 3, 5 ) . Fortunately, its growth is slow and it is evidently only weakly pathogenic. Fruit rots caused by Phzjtophthom sp. and Rhizoctoxia sp. seldom develop vigorously after harvest. Skou (1964a) has shown t h a t Aureobasidium pulluluns (de By.) Arnaud is radiation-resistant and might be a limiting factor with irradiated fruits and vegetables. An evaluation of this possibility requires further information regarding its pathogenicity in irradiated strawberries and other fruits. 2 . Stone Fruits

I n America, cherry, peach, nectarine, plum, and apricot fruits a r e destroyed by two major an d several minor postharvest diseases (Rose e t al., 1937). Brown rot, incited by Monilinia

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fructicola (Wint.) Honey an d closely related species, a n d K k i z o pus stolonife,. ( E h r . ex F r . ) Lind., sometimes associated with other mucoraceous species, result in widespread losses. I n extended cold storage, g r a y mold caused by B. c i n e ~ e a and blue mold caused by Penicillium e x p a m u m Lk. ex Thom. ma y cause important damage. Gray mold h as been discussed in connection with strawberries, an d blue mold is discussed with pome f r u i t diseases. Cludosporium her?mmm Lk. ex Fr. is of particular importance in sweet cherries, an d this or a closely related species of Cludosporizini may produce a serious rot of other stone f r u i t s if they a r e held in cold storage f o r extended periods. The brown rot disease is destructive both before a n d a f t e r harvest. Particularly d u rin g periods of wet weather, f r u i t r o t in orchards m a y be extensive even if a comprehensive protective spr a y program is followed. Because of t h e spread in t h e orchard, small lesions may be present at harvest. Since th e mycelium of lesions is internal, postharvest sp r a y s o r dips a r e usually entirely ineffective in eradicating t h e fungus. Chemical tre a tments a r e sometimes applied to reduce th e occurrence of infections a f t e r harvest, however. Although small lesions ma y be present a t harvest, the contamination of harvest a n d handling wounds by spores constitutes a n ever-present a n d important means of infection. Growth of M . f m c t i c o l a m ay be extremely slow a t, or halted by, temperatures below 5°C. Th e brown r o t disease cannot develop under refrigeration. Ho.vever, stone f r u i t s are frequently harvested while still firm in order t o limit tr a n s it a n d handling injuries a nd to provide added time f o r marketing. When these f r u i t s are removed fro m refrigeration to permit final ripening, th e brown rot disease is then free to develop. If, as a ppe ars likely, th e maximum permissible radiation dose f o r stone f r u i t s is 200 to 250 Krad (Maxie a n d Abdel-Kader, 1965), i t would appear unlikely t h a t a large proportion of large, Lvell established colonies could he inactivated. This conclusion is borne out by a failure t o inactivate a high proportion of infection sites when inoculated f r u i t was permitted t o incubate at room temperatures fo r 48 hours before irradiation or when massive inoculum was used (Sommer e t aZ., 1964a). At best, only a delay in the f u r t h e r development of well established lesions could be expected. On th e other hand, irradiation soon a f t e r harvest should inactivate a large proportion of very small lesions. An even higher proportion of contaminated harvest a n d

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handling wounds should be inactivated. I t appears almost certain that it will be highly important to accomplish cooling and irradiation as quickly as possible after harvest. Rhizopus rot may be, under certain conditions, the most destructive postharvest disease of peaches ( Wiant and Bratley, 1948) and may attack other stone fruits a s well. Particularly seriously attacked are fruits that a r e shipped without adequate refrigeration or fruits in which cooling has been delayed. Disease development seldom occurs at 10°C. or below (Brooks and Cooley, 1921). If fruits a r e shipped ripe and a r e refrigerated from grower to home refrigerator, Rkixopus rot should not be a problem. If fruits a r e harvested and marketed only partially ripe, as is common with peaches and nectarines, the disease may develop during the final ripening period. I n general, however, it has been noted that the incidence of Rhixopus rot is less if fruits a r e promptly cooled and held under refrigeration f o r a few days (McClure, 1958; Pierson et al., 1958). Smith and McClure (1960) reported that, under certain conditions, merely holding inoculated fruit at O'C. for 5 days might reduce the incidence of Rhixopas rot by nearly 50%. The reason f o r this cold-induced reduction in disease development is not known. The development of Rhixopus rot in market areas may be associated with poor temperature management, particularly delayed cooling or elevated transit temperatures. Presumably, however, much of the Rhixopus rot may be of local origin. In markets, the incidence of Rhixopus rot of peaches and nectarines is often limited to very ripe fruit held without refrigeration. Almost omnipresent with these ripe fruits a r e vinegar flies, D?,osophila melunoyaster Meig. One wonders if the flies a r e not inoculating the fruit with spores of R. stolonifer during oviposit. Inoculation and spread of R. stolonif e r by spore-contaminated U . melanoguste?, has been demonstrated in ripe canning tomatoes (Butler and Bracker, 1963). Alternatively, extensive handling and resulting injuries, particularly at retail markets, provide ample opportunity for inoculation and disease development if fruits a r e not refrigerated. Whether Rhixopus rot can be controlled by irradiation is still uncertain. It is true that R. stolonifer is relatively radiationresistant (Figs. 3 and 5 ) . However, if the irradiation is performed promptly, the fungus population to be inactivated may consist of only a few spores contaminating harvest wounds. In such case a modest dose should inactivate the fungus in a high

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proportion of inoculation sites. However, radiation would not alleviate and might actually increase the possibilities for inoculation and infection in market areas. Among stone fruits, C. he?-bamm causes a n important rot of sweet cherries grown along the Pacific Coast (Rose et al., 1937). Infection and fungus growth occur in the orchard, particularly in fruits that have cracked or in doubles in which one of the fruits has aborted. The fungus normally grows relatively slowly. The rot develops gradually in infected fruits, and a spread to other fruits during transit and marketing appears to be only a minor problem even though growth can occur at low temperatures. The radiation resistance of this fungus, its slow growth, and the fact that well established infections exist at harvest suggest that the benefits from irradiation as a fungicidal treatment to control this disease would be minimal. 3 . Citrus Fruits

Postharvest losses in Citrus fruits a r e severe from Penicilliunt itulicum Whemer (Citrus blue mold) and Penicillium d i g i t a t u ~ Sacc. (Green mold), or mixtures of the two (Klotz, 1961). Infections generally result from contamination of harvest and handling wounds. According to Klotz (1961), the postharvest development of blue mold is directly proportional to the concentration of spores in the air and on the surfaces of eyuipmerit. Considerable spread by fruit-to-fruit contact occurs in the case of the blue mold disease. In addition to inducing decay, both species sporulate profusely and the colored spores may be deposited on other fruits, rendering them unsightly in appearance. Present control measures include the avoidance of injuries ; placing in packages such volatile materials as biphenylor ammonia-emitting chemicals or nitrogen trichloride-forming chemicals which tend to prevent in-transit spread and sporulation; or fumigation in cars or storage by nitrogen trichloride or ammonia (Eckert and Kolbezen, 1963a,b, 1964; Eckert ct al., 1963; Harvey and Pentzer, 1953; Smith, 1962). Citrus brown rot, caused by Phytophthora spp., occurs in the orchard when motile spores a r e splashed from the soil to lowerhanging fruits. Small or incipient infections may be present when fruits a r e picked. Sound fruits may be infected by washing in contaminated water. When Citrus brown rot is a problem it is controlled in the packing house by immersing all fruits for 2-4 minutes in water or fungicide solutions at 46 to 49°C. (Klotz, 1961).

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Stem-end fruit rots a r e caused by A l t e m m i a citri Ellis and Pierce ; Diapor.tlie citri (Faw.) Wolf ; Pleospom l r e r b a m ~ n(Pers.) Rab. ; Bot)yosphao,ia ribis Dug. ; and Diplodia .iiatalensis PoleEvans. Extensive disease development is often preceded by low vitality caused by poor growing conditions or extremes of temperature before or after harvest. Other fruit diseases which may sometimes be destructive include gray mold, caused by B. ciwerea; Trichoderma rot, caused by T r i c i i o d e r m lignorurn (Tode) H a r z ; cottony rot, caused by Sclerotirhia sclerotiorzm (Lib.) Mass. ; and sour rot, caused by Geotrichum candidum Lk. ex Pers. (Klotz, 1961). Possible use of ionizing radiation to control Citrus fruit rots was investigated by Beraha et al. (1959a,c), who gave particular attention to the Penicillium blue and green molds. Under carefully controlled conditions, a radiation dose of 150-200 Krep protected inoculated fruits against rotting by P. digitatum for about 12 days at 75°F. and 17 days at 55°F. Results were equal or better when P. italicurn was the organism involved. No detailed studies seem to have been made of other postharvest Citrus diseases. However, Sommer et al. (196413) studied the sensitivity of the more important Citrus f r u i t decay fungi in vivo and in uitT.0 and reported dose-response and end-point studies, mainly of spores, under various conditions. An increased incidence of Alternaria stem-end rot following irradiation was noted by Beraha et al. (1959a,c). It was found that A. citri could be isolated from the calyces of irradiated or unirradiated fruits, but decay developed only in the former. Investigations of Maxie et al. (1965) confirmed this greater incidence of Alternaria rot, apparently associated with irradiationinduced death of calyx tissue and possibly with destruction of auxin (Gordon and Weber, 1955; Skoog, 1934, 1935). Since A. citr.i is very radiation-resistant (Sommer e t al., 1964b), incipient lesions known to sometimes be in calyx tissue (Bartholomew, 1923) may escape inactivation. If the calyx tissue remains alive and healthy, no rot occurs. If the calyx tissue dies, however, the fungus grows into the fruit proper. The usual means of delaying calyx senescence, i.e., plant growth regulators, were ineffectual in prolonging life following irradiation (Maxie e t al., 1965).

4. Pome Fruits Apples and pears held in modern cold storage suffer large losses from several diseases, both parasitic and nonparasitic. Of

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greatest importance among parasitic diseases of stored apples is the blue mold rot, caused by Penicillium expmrsum Lk. ex Thom. and possibly certain other species of Penicillium. The fungus is capable of saprophytic growth on a wide range of decaying matter. Since the fungus typically sporulates profusely, the spores can be considered almost omnipresent. Contamination of harvest and handling wounds is a major means of gaining entry into the fruit. Infection may also occur through natural openings, the lenticels (Anderson, 1956). Although P. expansum can grow at temperatures as low as fruit can endure without danger of freezing, fungus development is slow near 0°C. Consequently, if fruit is cooled to the storage temperature without delay, several months may be required before the disease becomes readily apparent. If cooling is delayed, however, the spores in contaminated wounds may germinate and form small colonies before growth is slowed by the cold. After such a start, the rot lesions appear much earlier in the storage period (Ramsey and Smith, 1953). The most serious disease of pears in storage is usually gray mold, caused by Botrytis cinerea (see Strawberries for a more complete discussion). Apples a r e attacked less seriously. An abundant source of inoculum is generally present since B. ci?lerea grows saprophytically on many rotting materials in orchards and around packing houses. Infection is frequently found in stems, which the fungus colonizes before rotting the fruit proper. Prompt cooling reduces the seriousness of the disease. The fungus can grow only very slowly a t 0”C, but losses may become serious in long-term storage. The seriousness of the disease is intensified by the “nesting” which results from contact infections, with the fungus growing from one rotting fruit to infect adjacent fruits. Contact spread is frequently prevented by wrapping individual fruits in tissue paper containing copper compounds (Ramsey and Smith, 1953). A serious storage disease, Bull’s Eye rot, caused by GLoeospoi.ium perennuns Zeller & Childs, is sometimes prevalent in the Pacific Northwest and certain other parts of the world. In humid regions, younger branches a r e infected, producing a canker disease in the orchard (Perennial Canker). F r u i t infections may occur at any time between petal fall and harvest. Rot does not become extensive, however, until later in storage (Sprague, 1958). Rots of similar appearance may be caused by Gloeosporium albunz Osterw., G. f ructigenuin Berk., and certain other fungi.

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Scald is the name given to physiological diseases of apples and pears which develop in storage. They have in common the irregular browning of the surface and immediate underlying fruit tissue. The cause of the development of scald diseases is not well understood. Beraha et al. (1957) reported t h a t Penicillizcm expamzcrri inoculated into Jonathan apples and incubated for 24 or 96 hours prior to irradiation was suppressed for 10 days at 70-75°F. by 200 Krep. In later work, Beraha et al. (1961) reported that 50 Krep did not reduce P. erpaiiswm rot whereas 100 Krep reduced day-old infections and 200 Xrep was required to check decay in 4-day old infections. The sensitivity of P. ezpansunz as determined in vitT.0 and in hosts other than apples suggests t h a t radiation could effectively control the blue mold disease. Such a conclusion is based on the assumption that disease lesions a r e not already present in the fruit when harvested and that the fruit can withstand a dose of about 200 Krad. Similarly, gray mold should be readily controlled if the source of infection is primarily the contamination of harvest and handling wounds. The effect of radiation on Bull’s Eye rot appears not to have been studied. A reduction in the incidence of apple scald has been reported (Massey et al., 1964; Phillips et al., 1960; Phillips and MacQueen, 1961). Similarly, another physiological disease, called brown core or core flush, was reduced by irradiation in the same studies. In other studies, core flush was variable or was increased by irradiation (Anon., 1961). Although pathogenic or nonpathogenic storage diseases of pome fruit might be controlled by radiation, apples and pears appear to be unlikely candidates for such treatment at this time. Interest in irradiation would appear to be limited by the availability of reasonably satisfactory and cheap chemical treatments, on the one hand, and the possible development of delayed irradiation injury in storage, on the other. 5. Bulb, Tuber, and Root Crops

The prevention of sprouting or growth by radiation treatments has been suggested as a means of maintaining quality during storage. Comparatively little work has been done with storage diseases of these crops. However, Beraha et al. (1959d) investigated some potato storage rots. Gamma-rays at 17.7 to 477.4 Krad did not prevent decay in Red Pontiac tubers previously inoculated with the soft rot bacterium, Erzoinia ca?*otoz.om

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(Jones) Holland. Higher doses caused extensive discoloration and softening. Against the late blight tuber rot, caused by Phgtophthora i w f e s t a m (Mont.) de By., 45.64 Krads prevented tuber decay without injuring the tubers, but did not control natural infections of F u s a ~ i i c msp. in the same tubers. In tubers inoculated with Pgtliiunz debaiyaiizim Hesse, doses of 137 Krad gave almost complete control under certain conditions, but a slight softening of the tubers occurred. The susceptibility of irradiated potato tubers to storage rots is discussed by Brownell e t al. (1957), Duncan et al. (1959), Hooker and Duncan (19591, and Waggoner (1955). 6. Tomatoes

Irradiation treatments have been reported to extend the shelf life of tomatoes by delaying ripening (Maxie and Abdel-Kader, 1965). No detailed studies seem to have been made on the use of radiation as a fungicidal treatment for postharvest diseases such as Alternaria rot (Altemaria tenuis Nees ex Corda) or Rhizopus rot (Rhixopus stolonifer) .

B. IRRADIATION-INDUCED SUSCEPTIBILITY TO INFECTIOK Attention has frequently been called to a n increase in susceptibility to infection and decay following irradiation. In some reports the changes followed high doses which must have resulted in near death or extreme injury to host tissue. Such massive, injury-inducing doses would seemingly impair host resistance in a manner analogous to that resulting from excessive heat or cold. From the standpoint of food irradiation, it is of primary importance to determine any increased susceptibility in hosts resulting from irradiation at near the highest dose that does not impair quality factors. Susceptibility could be increased by a reduction in the physiological and biochemical resistance of the host tissue. The result would be more rapid growth of a parasite in host tissue. Furthermore, some fungi that a r e normally weakly pathogenic might vigorously colonize tissue of lowered resistance. In addition, opportunity for infection may be greater in irradiated hosts. Radiation-induced tissue softening might, under some conditions, render the fruit or vegetable more injury-prone during transit or handling. Particularly if the injury resulted in rupture of the epidermis, natural inoculation by contamination of the wound by the pathogen would likely result.

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Skou (1964b) suggested a n alternative possibility in which changes in cell wall pectins and increased permeability of host tissues would “provide the saprophytes with the same growth possibilities which the parasites obtain independently on untreated material.” It is evidently assumed t h a t the presence of exudates from host tissue would permit colonization of organisms on the surface, which would facilitate eventual penetration of the host epidermis. Cell wall changes would presumably further facilitate penetration by fungi. It would appear, however, that these manifestations of host injury a r e accompanied by a seriously altered physiology which would also reduce the physiological and biochemical resistance of the host. An example of radiation-induced susceptibility of a host to a weak pathogen is seen in the previously mentioned Alternuria stem-end disease of Citrus. Incipient infections in the caIyx evidently do not develop extensively while the host tissue remains healthy. If, on the other hand, the fruits have been subjected to adverse conditions, rotting may be extensive. Irradiation at 150-200 Krad is followed by senescence of the calyx accompanied by development of A . citri, which is now capable of growing from the calyx into the main body of the fruit. The fungus, which is radiation-resistant (Figs. 3 and 5 ) , presumably escaped inactivation. As might be expected, if spores of A. citri a r e placed in knife wound of healthy Citrus fruits, few rot lesions develop. If, however, the fruits have been subjected to extended storage or have undergone poor growing conditions, fungus colonization and rot occur. Similarly, irradiated f r u i t a r e made susceptible to artificial inoculations. Alternaria rot of tomato fruits, caused by Alternaria tenuis, seldom occurs in fruits of high vitality. The disease may appear, however, after storage at low temperature if incipient chilling injury has occurred (i.e., 0 4 ° C f o r several weeks). Also, after gamma irradiation of 300-400 Krad, the fruits seem more susceptible to this weak pathogen. The relatively low doses of gamma irradiation used t o inhibit the sprouting of potatoes have been reported to increase susceptibility to storage rot organisms. At least a portion of the increased susceptibility is evidently due to greater opportunity for pathogens to gain entrance via wounds. Irradiation has been reported to slow suberization and wound healing and thereby render wounds highly susceptible to infection f o r a longer period (Henriksen, 1960; Isleib, 1957; Sawyer and Dallyn, 1955, 1961).

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It has thus been amply demonstrated that irradiation may render fruits or vegetables more susceptible to subsequent infection. The extent of the problem is yet unclear, for many observations of increased disease incidence followed excessively high irradiation doses. More information is needed on the increased susceptibility following doses of 250 Krad or less. The diseases should preferably be studied individually, with carefully controlled inoculations. VI.

PROTECTIVE PACKAGING

The use of packaging to protect fruits or vegetables from postirradiation infection has been suggested by examples of increased postirradiation susceptibility and by the fact t h a t irradiation does not leave a protective residue. Used most commonly have been bags or plastic films. Obviously, if rot organisms were completely eliminated by irradiation, a barrier would prevent any subsequent infection or rot. Seldom, however, will the highest permissible dose even approach this ideal effect. If postirradiation rot results primarily from field infections that are not completely inactivated, a s in Alternaria rot of Cit?*us,a protective film will be of little benefit. Similarly, the postirradiation gray mold rot of strawberries occurs primarily from lesions, established in the field before harvest, that a r e slowed by irradiation but not halted completely. Spores reaching susceptible infection sites may establish new infections, but the life of the strawberry, even under the best of conditions, is so short t h a t new infections originating from single spores a r e unlikely to be of consequence. Furthermore, protective packaging will not protect from contact infections and “nesting” unless fruits are packaged or wrapped individually. On the other hand, if rotting following irradiation occurs from postirradiation infections-not irradiation escapes-protective packaging might be helpful. Any packaging material used must be sufficiently permeable to permit ready passage of oxygen and carbon dioxide. Otherwise, suboxidation or carbon dioxide injury to the fruit or vegetable may result from a respiration-induced atmosphere modified by reduction of oxygen or a n accumulation of carbon dioxide or both. Furthermore, to be effective the barrier likely must be sealed. The use of ventilation holes in films to increase the gas exchange almost certainly destroys the protective effect. The area thus exposed is small, but the movement of bags and

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temperature changes would cause the air t o be "pumped" in and out of the bag, thereby permitting the entrance of air-borne contamination. Furthermore, if insects or other small animals a r e present, the openings permit them to enter and bring contaminating organisms with them (Cooper and Salunkhe, 1963; Salunkhe ct al., 1959; Sommer and Luvisi, 1960). VII.

RESEARCH NEEDS

It is readily apparent at this time that ionizing radiation will not have universal or even, perhaps, widespread application for the control of postharvest diseases of fruits and vegetables. In many cases a n adequate control can be achieved by chemical fungicides or by temperature management. In other cases the sensitivity of the host to the damaging effects of radiation (offflavors, softening, tissue death) will not permit the application of adequate doses. Where diseases exist that cannot be adequately controlled by other means, however, radiation may permit reduction of important losses. Furthermore, the great penetration of some rays provides a therapeutic effect not ordinarily possible with chemical fungicides. Pathogens in established lesions within host tissues can be controlled by this fungicidal treatment, whereas chemicals a r e usually only protective in nature. Consequently, those individual postharvest diseases which a r e most difficult to control by chemicals should receive particular attention. Various problems of radiation biology should receive more attention in relation to the control of postharvest diseases. A few of these a r e the following: the relation of high vs. low rates of application of a given dose ; comparisons of highly penetrating gamma-rays vs. the limited penetrating electrons with regard to the level of control and to adverse host responses; the radiation resistance and pathogenicity of survivors; the effect of atmosphere modification on postirradiation disease expression ; the effect of irradiation on subsequent fruit transit injury and postirradiation infection ; careful evaluation of the need for protective packaging; and the sensitization of pathogens by heat. Probably the greatest need is for investigators interested in both radiation and pathology. The postharvest pathologist must be competent in mycology ; be thoroughly acquainted with postharvest diseases ; and be knowledgeable about problems involved in the storage. transport, and marketing of fruits and

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vegetables. He must also be familiar with plant pathological techniques and be prepared to develop new methods as needed. Finally, an intense interest in radiobiology must be developed. If such a person is included in all research teams, faster progress can be expected.

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Poniper, S., and Atwood, K. C. 1955. Radiation studies on fungi. 1~ “Radiation Biology” ( A . Hollaender, ed.), Vol. 11, pp. 431-453. McGraw-Hill Book Co., New York. Powelson, R. L. 1960. Initiation of strawberry f r u i t rot caused by 1;otryti.s cinerea. P h y t o p a t h o l o g y 50, 491-494. Ranisey, G. B., and Smith, M. A. 1953. N a r k e t diseases caused by fungi. U.S. Dept. Agr. Yearbook. pp. 809-816. Kieder, H. 1898. Wirkung der Rontgen Strahlen auf Bakterien. Mii ticlt. 7,iotl. Wochsch. 45,101-104. Riley, H. P. 1955. The protective effect of various chemical compounds against damage to chromosomes by gamma radiation. Am. J . Botany 42, 765-769. Rivera, V. 1929. Influenza del trattamento di tubi emanazione sopra lo sviluppo di alcuni microorgaismi vegetali. Boll. w g i n stnz. p ( i f o / . wegetalc K o m a , 11s. 9, 241-247. Roberts, R. B., and Aldous, E. 1949. Recovery from ultraviolet irradiation in Escherichia coli. d . B‘acfw-iol. 57, 363-375. Roentgen, W. C. 1898. I. Ueber ein neue A r t von Strahlen. Anx. I J h y s i k r o t d Chcniia 64, 1-11. Koniani, R. J. 1965. Radiobiological parameters in the irradiation of f r u i t s and vegetables. A d v a n c e s i)r Foot1 R c s c a r c h ( T h i s volume). ltose, D. H., Fisher, D. F., Brooks, C., and Bratley, C. 0. 1937. Market diseases of f r u i t s and vegetables: Peaches, plums, cherries and other stone fruits. U.S. Dept. Agr. M i x . Publ. 228. liubin, B. A . , Metlitskii, L. V., Sal’kova, E. G., Mukhin, E. N., Korableva, N. P.. and Morozova, N. P. 1959. The use of ionizing irradiations for the regulation of the dormancy of potato tubers in storage. Biolihim. I’lodov. i OvoslwheY Acatl. ,Vauk S.S.S.R. I ? i s t . Biolihiw. iui. A . N . Baklici 3, 5101. R e v . A p p l . Mycol. 39,188-189. 1960. Salunkhe, D. K. 1961. Gamma radiation effects on f r u i t s and vegetables. h’co)/. B o t a n y 15, 28-56. Salunkhe, D. K., Pollard, L. H., Gerber, R. K., Wilcox, E. B., and Sinion, &I. 1959. Packaging effects on the flavor and shelf-life of g a m m a irradiated fresh f r u i t s and vegetables. Package Eng. 4, 39-52. Saravacos, G., and illacris, B. 1963. Pasteurization of Greek table grapes by gamma radiation. N.R.C. “Democritos” Radiation Technol. Dept., Athens, Greece. Saravacos, G. D., Hatzipetrou, L. P., and Georgiadou, E. 1962. Lethal doses of g a m m a radiation of some f r u i t spoilage microorganisms. Food Irrrrdiuti072 3 (1-2), A6-A9. Sardeniann, E. 1914. Ueber die Behandlung der Aktinomykose mit Rontgenstrahlen. ,!?runs’ Beitr. klin. Chi,.. 90, 157-167. Sawyer, R. L., and Dallyn, S. L. 1955. The effect of gamma irradiation on storage life of potatoes. Am. P o t n t o d . 32, 141-143. S a u y e r , R. L., and Dallyn, S. L. 1961. Effect of irradiation on storage quality of potatoes. A m . Potato J . 38, 227-235. Schmidt, C. F., and Nank, W. K. 1960. Radiation sterilization of food. I. Procedures f o r the evaluation of the radiation resistance of sporcs of Clostridiuin botulinum in food products. Food R e s c a r c h 25, 321-327. Schmidt, C. F., Nank, W. K., and Lechowich, R. V. 1962. Radiation sterilization of food. 11. Some aspects of the growth, sporulation, and radiation

RADIATION FOR DISEASE CONTROL

191

resistance of spores of Clostridium b o t u l i m c m , type E. J . Food Sci. 27, 77-84. Schwinghamer, E. A. 1957. Effect of ionizing radiation on r u s t reaction in plants. Science 125, 23-24. Schwinghamer, E . A. 1959. The relation between radiation dose and t h e frequency of mutations f o r pathogenicity in Melampsora lini. Phytopathology 49, 260-269. Setlow, R., e t al. 1961. “Fundamental Aspects of Radiosensitivity.” Brookhaven Natl. Lab., Upton, N.Y. 308 pp. Skoog, F. 1934. The effect of X-rays on growth substance and plant growth. Science 79, 256. Skoog, F. 1935. The effect of X-irradiation on auxin and plant growth. J . Cellular Comp. Physiol. 7, 227-270. Skou, J. P. 1960. Microbiological studies in connection with irradiation of carrots. Danish Atomic E n e r g y Comm., Riso, Den., Riso R e p t . N o . 16, 79-83. common Skou, J. P. 1964a. Aureobasidium pullulans (de By.) Arnaud-a and a very radio-resistant fungus on f r e s h f r u i t s and vegetables. In “Radiation Preservation of Foodstuffs.” (Kinnell, P. 0. and Vera Runnstrom-Reio, eds.) Second Scand. Meet. Food Preserv. Radiation. Skou, J. P. 1964b. Radiation induced damage t o plant tissues as a cause of the intensified attacks by microorganisms following irradiation. I n “Radiation Preservation of Foodstuffs.” (P. 0. Kinnell and Vera Runnstrom-Reio, eds.) Second Scand. Meet. Food Preserv. Radiation. Smith, E. C. 1936. The effects of radiation on fungi. In “Biological Effects of Radiation.” (B. M. Duggar, ed.) Vol. 2, pp. 889-918. McGraw-Hill Book Co., New York. Smith, W. L., Jr. 1962. Chemical treatments to reduce postharvest spoilage of f r u i t s and vegetables. Botan. Rev. 28, 411-445. Smith, W. L., Jr., and Friedman, B. A. 1953. The diseases bacteria cause. U.S. Dept. Agr. Yearbook, pp. 817-821. Smith, W. L., Jr., and McClure, T. T. 1960. Rhizopus r o t of peaches as affected by postharvest temperature and moisture. Phytopathology 50, 558-562. Snyder, T. L. 1947. The relative e r r o r s of bacteriological plate counting methods. J . Bacteriol. 54, 641-654. Sobels, F. H. 1963. Repair from genetic radiation damage. 454 pp. Macmillan Co., New York. Sommer, N. F., and Luvisi, D. 1960. Choosing the right package f o r fresh fruit. Package Eug. 5, 37-43, 116. Sommer, N. F., and Creasy, M. T. 1964. Recovery of Rhizopus stolonifer sporangiospores a f t e r potentially lethal gamma irradiation. Radiation Research 22,074. Sommer, N. F., Creasy, M., Romani, R. J., and Maxie, E. C. 1963a. Recovery of gamma irradiated Rhixopus stolonifer sporangiospores during autoinhibition of germination. J . Cellular Cowip. Physiol. 61, 93-98. Sommer, N. F., Creasy, M., Maxie, E. C., and Romani, R. J. 196313. Production of pectolytic enzymes by Rhizopus stoloxifer sporangiospores a f t e r “lethal” gamma i i ~ a d i a t i o n A. p p l . Jlicrobiol. 11, 463-466.

192

h-.F. S O M M E R A N D R. J . FORTLAGE

Somnier, N. F., illaxie, E. C., and Fortlage, It. J . 196da. Quantitative t1os.eresponse of Prioiics f r u i t decay fungi to gamma irradiation. Rutlintioii Botccicy 4, 309-316. Sonimer, N. F., hlaxie, E. C., Fortlage, R. J., and Eckert, J. W. 1964b. Sensitivity of Citrus f r u i t decay fungi to gamma irradiation. Rudiutiotc Botcrwy 4, 317-322. Soninier, N. F., Creasy, AT., Roniani, R. J., and Naxie, E. C. 1 9 6 4 ~ An . oxygendependent postirradiation restoration of Rlrizopits s t o l o v i f e i sporangiospores. Radiation R e s c u i d i 22, 21-28. Sommer, N. F., Fortlage, R. J . , and Buckley, P. 111. 1965a. Unpublished data. Dept. of Pomology, Univ. of California, Davis. Soninier, N. F . , Gortz, J. H., and Maxie, E. C. 1965b. Prevention of repair in irradiated Rhizopits stolouiic‘r sporangiospores by inhibitoi.~of protein synthesis. R a d i u t i o n Rescarch 21, 390-397. Sparrow, A. H., and Miksche, J. P. 1961. Correlation of nucleai, volume and D N A content with higher plant tolerance to chronic radiation. Scicticc 131, 282-283. Spear, F. G. 1953. “Radiations and living cells.” 222 pp. Chapman and Hall, London. Sprague, R. 1958. A world-wide review of the control of hull’s eye rots. Proc. 54th Ann. Meet. Wash. S t a t e Hort. Assoc., Yakima, Wash. pp. 195198. Stapleton, G. E. 1960. Protection and recovery of bacteria and fungi. I / / “Iiadiation Protection and Recovery.” (A. Hollaender, ed.). Pp. 87-116. Perganion Press, N.Y. Stapleton, G. E., and Hollaender, A. 1952. Mechanism of lethal and mutagenic action of ionizing radiations on Aspergillus tcrreus. 11. Use of modifying agents and conditions. J . Cellular Comp. PhysioE. 39, 101-113. Stapleton, G. E., and Martin, F. L. 1949. Comparative lethal and mutagenic effects of ionizing radiations on Aspergillus tcrrezts. A m . J . Botutr!g 36, 816. Stapleton, C;. E., Billen, D., and Hollaender, A. 1953. Recovery of X-irradiated bacteria at suboptimal temperatures. J . Cellular C o m p . I’h:/sioZ. 41, 34.5.357. Stein, W., and Meutzner, I. 1950. Reaktivierung von UV-inaktivierteni E u L tcTiccm coli durch Warme. Natur?L.isse)zschaftcii37, 167-168. Tamburino, S. M. 1959. Sulla conservazione di arance mediante irradiazione con raggi gamma dcl Co““. Tccxica A g r . ( I t a l y ) 11, 631-635. Hort. A h s f ) . . 31, 3185. 1961. Tascher, W. R. 1933. Experiments 011 the control of seed-borne diseases hg X-rays. .J. A g r . R c s e a r c h 46, 909-815. Tatuni, E. L. 1950. Effects of radiation on fungi. J . CclluZar Conip. I’h!j.siol. 35, (Sup. 1 ) 119-131. Toniiyama, K. 1963. Physiology and biochemistry of disease resistance of plants. Ann. Rcv. I’hytopathol. 1, 295-324. Truelsen, T. A. 1963. Radiation pasteurization of fresh f r u i t s and vegetables. Food Tcchrrol. 17, 336-339. Uber, F. M., and Goddard, D. R. 1934. Influence of death criteria on the X - r a y survival curves of the fungus, Ncurospora. J . Gcw. I ’ h ~ s i o l . 17, 5 1 1-590. -77

RADIATION FOR DISEASE CONTROL

193

Vasudeva, It. S., Bajaj, B. S., Chatrath, M. S., and Ganju, P. J. 1959. Stimulating effect of ionizing radiations on certain microorganisms. I,rdiair I’hytoputhol. 12, 19-24. Rev. A p p l . AIIycol.39, 277. 1960. Waggoner, P. E. 1955. Radiation and resistance of tubers to rot. A I U .P o t a f o .I. 32, 448-450. Waggoner, P. E. 1956. Altering disease resistance with ionizing radiation. I’hytopatholoyy 46, 125-127. Waggoner, P. E., and Dimond, A. E . 1952a. Crown gall supprcssion by ionizing radiation. A m . J . Botany 39, 679-684. Waggoner, P. E., and Dimond, A. E. 195213. Examination of the possibility of therapy of plant disease with ionizing radiation. P h y t o p u t h o l o y y 42, 5!)9--602. Walker, J. C. 1952. “Diseases of Vegetable Crops.” 529 pp. McGraw-Hill Book Co. Whiting, A. R. 1960. Protection and recovery of the cell f r o m radiation damage. Z?L “Radiation Protection and Recovery” (A. Hollaender, ed.). Pp. 117-156. Pergamon Press, N.Y. Wiant, J. S., and Bratley, C. 0. 1948. Spoilage of fresh f r u i t s and vegetables in rail shipments unloaded at New York City. U S . D e p t . A g v . Circ. 773, 62 PP. Willison, R. S. 1963. Ionizing radiation f o r t h e control of plant pathogens; a review. Cay?. Pla?it Disease Suvvey 43, 39-53. Witkin, E. 1947. Genetics of resistance to radiation in Escherichia coli. Gc?cc’tics 32, 221-248. Wolff, S. 1961. Radiation genetics. I I L “Mechanisms in Radiobiology.” Vol. I. Pp. 419-475. (ill. E r r e r a and A. Forssberg, ed.) Academic Press, New York. Wood, R. K. S. 1959. Pathogen factors in the physiology of diseases-pectic enzymes, 112 “ P l a n t Pathology, Problems a n d Progress, 1908-1958.” (C. S. Holton e t al., ed.). Univ. of Wisconsin Press, Madison. 588 pp. Workman, M., Patterson, M. E., Ellis, N. K., and Heiligman, F. 1960. The utilization of ionizing radiation to increase the storage life of white potatoes. Food Technol. 14, 395-400. Zirkle, R. E., and Tobias, C . A. 1953. Effects of ploidy and linear energy t r a n s f e r on radiobiological survival curves. Arch. Biochcm. B i o p h y s . 47, 282-306.

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CAROTENOIDS: PROPERTIES, OCCURRENCE, A N D UTILIZATION IN FOODS BY B. BORENSTEIN A N D R. H. BUNNELL Hoffmanii-La Roche I I I C . , N u t l c y , N e w J e r s e y

I. Introduction . ...... .................... 11. General Prope ......................... A. Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Nomenclature ..... .......................... C. Cis-Trans Isomerizat D. Physical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Occurrence of Carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Function in N a t u r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Provitamin A Activity . . . . . ............................ I. Synthesis of Carotenoids . . . . . . . . ....... J. Toxicology of Commercial Sy .............. 111. Occurrence and Stability of N a t u r a l Carotenoids in Foods . . . . . . . . A. Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Stability . . . . . . . . . . . . . IV. Added Carotenoids in Food Processing . . . . . . ........ A. N a t u r a l Carotenoid Colorants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Food Applications of Synthetic Carotenoids . . . . . . . . . . . . . . . . . . . . C. Synthetic Carotenoid Hues in Food Applications . D. Stability of Added Carotenoids in Foods . . . . . . . . E. Indirect Coloration of Foods . . . . . . . . . . . . . . . . . . F. Market Forms of Synthetic Carotenoids . . . . . . . . . . . . . . . . . . . . . . V. Additional Research Needs . . . . . . . . . . . . . . . . . ..... A. Biosynthesis and Function of Carotenoids B. Commercial Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Stability of Carotenoids in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Coloring D r y Mixes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Coloring Clear Aqueous Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . ..............

I.

195

197 202 205 205 208 209 210 212 215

244 246 248

260 262 262 263

263 263 263 264

INTRODUCTION

Nature relies on a variety of compounds for pigmentation of living organisms : carotenoids, anthocyanins, porphyrins, and chlorophylls. The carotenoids are the most widespread, occurring 195

196

B. BORENSTEIN AND R. H. BUNNELL

in both the animal and plant kingdoms, and appear to have the most varied functions. A wide variety of foods-yellow vegetables, tomatoes, apricots, peaches, orange juice, egg yolk, chicken, butter, lobsters, trout-owe their color mainly to carotenoids, as do a variety of food colors from natural sources-paprika, annatto, saffron. This review discusses the properties of carotenoids, their occurrence and stability in food, and the utilization of synthetic carotenoids in coloring food products. This review does not provide a complete bibliography of the thousands of articles written on carotenoids, but does offer a useful bibliography for those interested in further study. II.

GENERAL PROPERTIES

A. DEFINITION

According to the definition proposed by Karrer, carotenoids a r e yellow t o red pigments of aliphatic or aliphatic-alicyclic structure composed of isoprene groups, usually 8, linked so that the two methyl groups nearest the center of the molecule a r e in positions 1 : 6 and all other lateral methyl groups a r e in positions 1 : 5, with a series of conjugated C-C double bonds constituting the chromophoric system of the carotenoids. The basic structure is demonstrated by the formula for beta-carotene, a symmetrical hydrocarbon with 40 carbon atoms, as shown in Fig. 1. Of the approximately 100 known carotenoids, all can be related structurally to the parent compound, lycopene, the familiar red pigment of the tomato. A wide range of pigments can be derived from the parent carotenoid by chemical changes such as double-bond migration, introduction of hydroxyl, keto, o r methoxyl groups, partial hydrogenation, cyclization, oxidative degradation, or isomerization.

FIG. 1. Structural formula of beta-carotene.

Figure 2 shows the structural formulas of a few commonly found carotenoids. The history and vast literature concerning isolation, separation, chemical properties, and structure proof of this large family of compounds were thoroughly reviewed by Zechmeister and Cholnoky (1943) and Karrer and Jucker (1950). The

CAROTENOIDS-PROPERTIES

A N D FOOD U S E S

197

distribution of carotenoids in n atu re wa s also discussed in th e above texts a nd by Gcodwin (1954a ; 1955). More recently isolated carotenoids a r e not systematically discussed i n th is chapter. Table I is a brief summary of th e historical development of a fe w of the more interesting carotenoids.

B. NOMENCLATURE The ma j or carotenoid subgroups a r e carotenes a n d xanthophylls. The former include a!] the hydrocarbon carotenoids, a n d th e latter all the hydroxy, epoxy, an d OXY derivatives of th e carotenes. Xanthophylls a r e also frequently esterified, as, f o r example, physalien, which is th e dipalmitoyl ester of zeaxanthin. Many of the carotenoids were named by their discoverer f o r some special pioperty or f o r their source, e.g., carotene ( f r o m c a rro ts ), cryptoxanthin (hidden p ig m en t), an d zeaxanthin (fro m Z e a ,nays). A different classification system subdivides th e carotenoids into acyclic, monocyclic, an d bicyclic derivatives. T h e respective parent compounds of each category a r e lycopene, gamma-carotene, a n d beta-carotene. I n addition, th e prefix “neo” is used t o designate carotenoid stereoisomers containing a t least one cis configuration in the double-bond chain, th e prefix “pro” t o designate some polycis carotenoids, an d th e prefix “apo” to designate a carotenoid which ha s been derived f r o m another carotenoid by loss of a structu r a l element by degradation. A detailed discussion of t h e chemical nomenclature of the carotenoids was presented by Goodwin (1954a).

C. C i s - T r a m ISOMERIZATION The number of possible carotenoids is increased by c i s - t m i s isomerization. Zechmeister (1944 ; 1962) reviewed this subject extensively. Theoretically, each double bond in the beta-carotene chain, including th e two in th e beta-ionone rings, can exist in two configurations. Therefore, 1,056 cis-tmns beta-carotene isomers a r e theoretically possible. I n actuality, th e methyl groups along th e chain cause steric hindrance, which limits rearrangement (Zechmeister, 1944). According to Pauling (1939) th e cis-configuration in the open chain of carotenoids can be assumed only b y those double bonds which a r e joined by two CH groups:

> CII-CII=C--CII I

<

198

B. BORENSTEIN AND R. H. BUNNELL

OH

I

Y OH

Lutein

OH

":e-:' ' 7 '

Isozeaxanthin

/\

J

"

b/

7

%-/%P\V\ I

OH

I

l e mxanthin

iYi-

pq-

HO

FIG.2. S t r u c t u r a l formulas of carotenoids.

-2..'

,

.

CAROTENOIDS-PROPERTIES

A N D FOOD USES

199

ed COOH

Torularhodin

Astaxanthm

HO

0

FIG.2 ( c o n k ) . Carotenoids

Zechmeister (1944) showed the positions of the stereochemically effective double bonds of four important carotenoids (see Fig. 3 ) . He gave the following formula for calculating the number of possible isomers. For unsymmetrical chains with n effective double n bonds, the formula is N = 2”. For symmetrical chains-when is an odd number, Ay = 2(”-1J/?( 2 ( r z - l 1 / 2 + 1 ) [I1 and when

?i

is even, =

2(71/2-11

(2d2

+ 1)

I‘“

Beta carotene, for example, has 20 cis-tram isomers. The special curve of carotenoids is modified by isomerization from the trans to the cis form, which is characterized generally by the appearance of a “cis peak” in the near ultraviolet (e.g., 340 mp) and a corresponding lowering and slight shifting toward the ultraviolet of the main absorption peaks. The magnitude of the cis peak also varies among the different cis isomers. The spectral characteristics of three stereoisomers of beta-carotene, for example, are shown in Fig. 4, taken from Stitt e t al. (1951). It will

TABLE I HISTORICAL DEVELOPMENT

-

Yellow carotenoids

Dnte

p-Cm:)tcnc

T)ihydmp-c:troteIie .

Xa.nthophg.11 -

Zeaxatitkiiri

Cryptoxanthiri

Physn.Iir rL

Wac henro derl 1831

Bixin

Berzeliusl 1937

0

1910

1930

z

Karrer3 1929-1931 Ka.rrer-Inholrcn5 1950 Islcr et aZ.8 1953

c3

E

W illstatter2 Wuest' 101s

Karrer3 KHr.l.t.1'3

€Ieisuschka2 1,917 Kuhn3 1028-103 1

1907

1930-1933

Karrers 1929-1 Ir33

Yamamntd 1932

Ku hn3

1931-1941

1950

E

Thudichum' 1869 Willstatterz 1906

w m

1850 1870

1890

0 0

-.___-

BollrssixI~nult' 1825

1830

f.3

Kulrii' 1'320 ICrihn3

1933

l9sn-lsxn

Isler et al.5 1956

Isler et a1.j 1055

Inhofkl~ 1850

Isler et a.E.5 1955 R.ed carotmoidx

Weedorib 1951

Z !

* 2 ?d

x d

z

5P

r

Pouchct' 1 S7G

Kuhn rt al.4 1 !I33

Zrchmeister et al.' 193.1 Zrchmeister et nl.2 1935 Kuhn el ~ 1 . ~Zcchmeister et al.3* 1938 1934-1 935 Kuhn et nL3 103s

]!I50

1 960

Karrcrs 1!I50 1slc.r et ~ l 1056

.

Hnxo' 1950 ~ Zcdinieister et a2.* 1956 Zrrhmeistcr el nL5 1056

Lederer' 19.73

Zochmcister rt aL3* 1927- 1935 Knrrcr et a1.3* 1!)27-19:L5

Karrer et al.' 1913 Knrrer et ~ l . ~ * 1OK

Knrrcr et aL3* 105G

Weeden et al. Structure Proof 1960 Weeden Synthesis 1965

v1

+ Weeden et al. Structure I'roof 1960 Wecdcn Synthesis 1965

5 Total synthesis. * Proposed structure. First mention. Constitution. llolecular formula. ' Isolation. Commercial method. Adapted from Islcr cl al. (1958);the above listings therefore do not necessarily represent references cited in this chapter.

2

3

8U 2 M

v1

202

B. BORENSTEIN AND R. H. BUNNELL

All-tJvlls -a-carotene

All-tr.nns

- p-carotene

All- tynns-7-carotene

All-tvniic -1ycopene F I G . 3. Stereochemically effective double bonds (marked by asterisk) of f o u r carotenoids. From Zechmeister (1944).

be noted t h a t the cis peak is higher f o r neo beta-carotene-B. The highest cis peak of any beta-carotene isomer, however, occurs with 340 mp 1010). the central 15,15’ mono cis beta-carotene (Elcml% The majority of the carotenoids, however, occur in nature as the all-trans form, with mono-cis forms occurring occasionally and poly-cis forms rarely.

D. PHYSICAL PROPERTIES Carotenoids crystallize in a variety of forms, the color of the crystals varying from deep red through violet to almost black.

CAROTENOIDS-PROPERTIES

AND FOOD USES

203

200

50

0 32C

350

400 Wovelengih

45c (mi)

500

FIG. 4 . Absorption spectra of three stereoisomers of beta-carotene. B = neo-p-carotene-B ; U = neo-p-carotene-U ; T = all-fruris-p-carotene. a , b , c , d respectively indicate the location of mercury a r c lines 334.1, 404.7, 435.8, and 491.6 mp. From S t i t t c t al. (1951).

Their melting points are usually fairly high and tend to increase with increasing molecular weight and functional groups. F o r example, beta-ap0-8'-carotenal, beta-carotene, and canthaxanthin have respective melting points of 136"-140°C, 176"-182'C, and 2O8"-21O0C. Because of their system of conjugated double bonds, the crystalline materials are very sensitive to oxidative decomposition when exposed to air. The crystals must therefore be stored a t low temperatures in sealed containers under vacuum or inert gas. When suspended or dissolved in vegetable oil, however, their stability is adequate for practical use in food coloring. The use of food-grade antioxidants gives a further stability improvement. The spectral absorption curves of the carotenoids, particularly in the visible region 400-500 mp are widely used for purposes of identification and assay. The extinction coefficients are quite high, and, in general, the greater the number of double bonds in conjugation, the higher the extinction and the more intense the color. values of several of the carotenoids are shown in The E,,.,,L1$h Table 11.

204

B. BORENSTEIN AND R. H. BUNNELL

In work with carotenoids, a knowledge of their solubility characteristics is quite useful. The carotenoids a r e insoluble in water, slightly soluble in vegetable oils, moderately soluble in aliphatic and aromatic hydrocarbons, and very soluble in chlorinated hydrocarbons (e.g., chloroform). The solubilities of beta-carotene, canthaxanthin, and beta-apo-8’-carotenal a r e given in Table 111. In

~

,;1c< I < 181 ~

2500 10 1 0 2640 2,550 2200 3450 3150 1800

spite of their relatively low solubility in vegetable oils, their high tinctorial power overcomes this apparent handicap. I n conditions of practical use in oil-based foods, the levels of carotenoid used are well below the solubility limits. The solubility in vegetable oils can be dramatically increased by heating, and, although crystallization occurs on cooling, this property can be p u t to practical use both in preparirlg market forms and in coloring certain foods.

CAROTENOIDS-PROPERTIES

A N D FOOD USES

205

E. OCCURRENCE O F CAROTENOIDS In nature, the carotenoids occur in solution in f a t depots, in colloidal dispersion in lipoid media, or combined with protein in the aqueous phase. An example of the latter is the work of Nishimura and Takamatsu (1957), who isolated a carotene-protein complex from parsley and spinach leaves. Carotenoids a r e found in flowers, leaves, roots, fruit, algae, bacteria, fish, mammalian ovaries, muscle, etc. Caroteaoids may be extracted by maceration of tissue, denaturation of the protein, and extraction with organic solvents. The various carotenoids can then be separated by column chromatography, and assayed spectrophotometrically. Zechmeister and Cholnoky (1943) authored a classic work on the chromatography of carotenoids. F.

BIOSYNTHESIS

It should be noted that carotenoids a r e synthesized in their entirety only by higher plants and by Protista; all animal carotenoids are ultimately derived from these two sources, although they may be slightly altered by oxidative metabolism for accumulation in various animal tissues (Goodwin, 1963). Only recently has the biosynthesis of carotenoids been partially clarified. Reviews have been written by Mackinney and Chichester (1960), Goodwin (1961), Porter and Anderson (1962), Davies (1962), and Grob (1963). Grob suggested t h a t biosynthesis takes place in three major steps : 1) Preparation of active isoprene (isopentenyl pyrophosphate) 2) Chain construction by head-to-tail linkage of active isoprene forming geranyl-geranyl-pyrophosphate, then reductive condensation to lycopersene, the parent substance 3 ) Dehydrogenation to the colored pigments

Porter and Anderson (1962) proposed the synthetic pathway from geranyl-geranyl-pyrophosphate to lycopene and beta-carotene in tomatoes (shown in Fig. 5 ) . Their mechanism is similar to that of Grob (1963) except t h a t lycopersene is not believed to be a n intermediate. Davies e t al. (1961) did not find lycopersene in carotenogenic bacteria or in higher plants. Lycopersene was not found in carrots or tomatoes in radioactive tracer studies by Anderson and Porter (1961 ; 1962). The use of radioactive tracers has facilitated the study of carotenoid biosynthesis. C1-'-tagged carbon dioxide, acetic acid, and mevalonic acid have been studied in carotenoid biosynthesis by

206

B. BORENSTEIN A N D R. H. BUNNELL

ge r anyl p y r o p h u s p h ate

(;er.myl

TPN

CH,

(31,

H,C

C

C H

H

H

tl C C

II C H

H C H

C H

C

C C C H H i t

H,C

CH,

H&

C

2'

c

'

CH,

CH,

C H

C. H

C - ~ CC~ C

C

H

H

C

C H

C

( C C C H H H I I

C

7

CIi

H

H

C

C

C-C C

H

C

H

H H C ~ C - ~ C - LC ~c~ H H H H H

H H I t

C

H

c 11, C C C I I H H

C

CH,

CH'

C

C li,

CH,

Cll,

~

C

c H,

CH,

CH,

H,CC

CH,

H H C ~ - C - ~ CC H H H

C

C

H

C H

C H

C

C H

C C C H H H

(

C C i I I H H

C

H

C C - C H H

H,( ( C C C H H H

H C H

11

( H

C

C

Cit,

H

,CH

c',

C C H t I I C H ' H,C

CH

('H,

C H,

CH,

C

c

c

CH'

H'

H, i

c

lr

,tt I,?

FIG.5. A proposed path\\ ay f o r t h e hio5ynthesis of lycopene and @-carotene. From P o r t e r and Anderson ( 1 9 5 2 ) .

CAROTENOIDS-I'ROPERTIES

AND FOOD VSES

207

molds, tomatoes, carrots, an d maize. The lack of significant randomization of 2-C1'-acetate in beta carotene indicates t h a t t h e tricarboxylic acid cycle h as no direct role in carotenoid biosynthesis (Goodwin, 1961). Mevalonic acid is incorporated into carotenoids 10-20 times as effectively as acetate (Braithwaite a n d Goodwin, 1957; Goodwin, 1961). Acetate is apparently a precursor of mevalonic acid (Goodwin, 1961). Mevalonic acid-2-C1-' was incorporated into phytoene, phytoAuene, zeta-carotene, beta-carotene, gamma-carotene, a n d lycopene in tomatoes (Purcell e t al., 1959). Shneour a n d Zabin (1959) found t h a t cell-free homogenates of tomato also converted mevalonic acid-2-C'-' into lycopene. Yields were highest in th e uresence of ATP, pyridine nucleotides, glutathione, manganese ion, a nd oxygen. Ripening tomatoes converted tagged mevalonic acid into phytoene, phytofluene, zeta-carotene, neurosporene, lycopene, gamma-carotene, an d beta-carotene (Anderson e t al., 1960b). The concentration of mevalonic acid in plants is low, a n d it h a s been determined definitively only in carrots-2 to 4 pmoles per 100 g on a wet basis (Modi an d Patwa, 1961). A mechanism f o r the formation of isopentenyl pyrophosphate fro m mevalonic acid is shown in Fig. 6. Isopentenyl pyrophosphate is apparently a universal intermediate in terpenoid synthesis. Anderson a n d Po rter (1961) demonstrated th e incorporation of C14 of terpinol pyrophosphates into phytoene by isolated plastids of carrots, an d into phytoene, phytofluene, zeta-carotene, CH,OH

H,

c/"~2:

CH,O%

MnZ+

I

y z

n y H2 ATP c om

Mevalonic acid

ADP

,,C?H

H,C

CH,COOH

/

I ~ I G G. . Formation of isopentenyl pyrophosphate. From Goodwin (1961).

208

B. BORENSTEIN A N D R. H. BUNNELL

neurosporene, lycopene, gamma-carotene, a n d beta-carotene by plastids of tomatoes (Anderson an d Porter, 1962). Relatively little is known about th e biosynthetic relationship of the various carotenoids, about rin g closure in th e formation of cyclic carotenoids, o r about t h e introduction of oxygen functions. Por t e r a nd Anderson (1962) gave evidence f o r th e conversion of lycopene t o beta-carotene in tomatoes fro m inheritance studies. Goodwin (1961) was not certain of lycopene intermediates a n d did not agree t h a t lycopene is th e precursor of beta-carotene. Purcell (1964) suggested t h a t tomato carotenoids a r e formed on stroma within chromoplasts an d t h a t the carotenoids do not become p a r t of a metabolic pool. Yamamoto et al. (1962) demonst r a t e d t he incorporation of molecular 1802 into xanthophylls, a n d of the oxygen of water into epoxides. Torularhodin isolated from R h o d o t o m l a rubra grown in a n enriched atmosphere of IRO2contai ns 1 atom of “0 in th e carboxyl group (Simpson e t al., 1964).

G. FUNCTION IN NATURE Carotenoids play a number of vital roles in th e economy of plants ; f o r example, they are intimately concerned with photosynthesis, t he fundamental reaction on this planet (Stanier, 1960 ; Goodwin, 1961). F o r th is reason, th e absorption spectra of carotenoids m a y be their most important characteristic. T h e nutritional value of carotenoids to the animal kingdom is of great importance, but the fact t h a t light absorbed by t h e carotenoids is utilized f o r photosynthesis mu st be considered their prime function. T he chief absorption bands of most of th e carotenoids lie between 400 an d 500 mp. There is close agreement among worker s on the positions of th e band maxima in a given solvent. It is clear t h a t carotenoids p artak e in photosynthesis a n d photosensitization in higher plants (Goodwin, 1961). T h e role of accessory pigments in photosynthesis h as also been reviewed by Anderson et al. (1960a). They do not initiate photosynthesis, but apparently t r an sfer energy to chlorophylls with a n efficiency of 30-10095, depending on th e organism. E n e r g y tra n s fe r has been demonstrated in th ree classes of algae-Chlorophyceae (green), Phaeoyhqceae (b ro wn ), an d Bacillariophyceae (diatoms) (DLwsens, 1951 ; Blinks, 1954)-and in photosynthetic bacteria (Goedheer, 1959). Work with photosynthetic bacteria-Rhodopseudomonas spheroides a nd R. rubrum (Griffiths et al., 1955; Cohen-Bazire a n d Stanier, 1958) -suggested t h a t carotenoids protect against photo-

CAROTENOIDS-PROPERTIES

AND FOOD USES

209

sensitization. This h as not been substantiated by work with carotenoid-deficient m u tan ts of higher plants. Zsolt e t al. (1963) suggested t h a t carotenoid epoxides a r e oxygen carriers in ripening plant tissues. The widespread occurrence an d accumulation of carotenoids in reproductive structures of plants an d animals suggests a role in reproduction, but little evidence is available to substantiate this idea.

H. PROVITAMIN A ACTIVITY Since only six o r seven of th e more th a n 100 carotenoids have provitamin A activity, it is not correct to consider vitamin A activity a general property of carotenoids. Nevertheless, herbivorous animals derive almost all their vitamin A fro m carotenoids (Moore, 1957). Carnivores obtain their vitamin A by eating th e herbivores. Man obtains his vitamin A by consuming fru its , vegetables, t he milk of animals, an d th e liver of animals a n d fish. I n all cases, carotenoids a r e t h e original source of man's vitamin A. The National Research Council (1958) estimated t h a t twothi r ds of t he vitamin A value of th e normal American diet is obtained a s provitamin A carotenoids. Beta-carotene is the most important carotenoid f r o m a nutritional viewpoint, both because of its prevalence in n a tu re a n d because it has t he highest provitamin A activity of all carotenoids. The beta-ionone r i n g is essential to provitamin A activity, a n d beta-carotene contains two such rings. It should be noted t h a t beta-carotene, even a t high input levels, cannot cause hypervitaminosis (Bagdon et al., 1960; Abrahamson a n d Abrahamson, 1962), although yellow pigmentation of th e skin (xanthodermis) can occur on consumption of fro m 4 t o 8 pounds of r a w carrots daily (Lord Cohen of Birkenhead, 1958). Cis isomers of betacarotene have somewhat lower provitamin A activity th a n the alltrans isomer. Other carotenoids with vitamin A activity include beta-apo-8'carotenal, alpha-carotene, crypotoxanthin, echinenone, a n d torularhodin. Anhydrolutein can be converted to vitamin A? by chicks (Budowski et al., 1963). T h e biochemistry of t h e conversion of carotenoids t o vitamin A is not discussed here. Glover a n d Redf e a r n (1954), Glover (1960), an d Anonymous (1963) reviewed the conversion of beta-carotene to vitamin A. Marusich e t al. (1960a) described the determination of beta-ap0-8'-carotenal vitam i n A activity by th e curative rat-growth assay. Rubin a n d De

210

B. BORENSTEIN

AND R.

H. BUNNELL

Ritter (1954) reviewed the carotenoid requirements of different species based on the differences of availability of carotenoids in different foods. The metabolism of the carotenoids was thoroughly reviewed by Deuel (1957).

1. SYNTHESIS O F CAROTENOIDS The synthesis of carotenoids is a choice example of the geometric rate at which science progresses. Karrer and Jucker (1950), in their definitive text, reported t h a t no total synthesis of a natural carotenoid had been achieved although Karrer and Solmssen (1935) and Karrer and Jucker (1947a) had converted several natural carotenoids into others. In 1950, two teams (Karrer and Eugster, 1950 ; Inhoffen et al., 1950) synthesized beta-carotene independently, and Karrer et al. (1950) synthesized lycopene By 1957, methyl bixin (Ahmad and Weedon, 1953), diniethyl crocetin (Inhoffen et al., 1953), zeaxanthin, isozeaxanthin, physalien, cryptoxanthiq, canthaxanthin (Isler et al., 1956b,c,e, 1957), and neurosporin (Eugster et al., 1956) had been synthesized. In 1963, Isler and Schudel (1963), in reviewing the synthesis of carotenoids, reported that 62 totally synthetic all-trans carotenoids had been prepared. They also reported on the commercial synthesis of beta-carotene, canthaxanthin, beta-apo-8’-carotenal, and beta-apo-8’-carotenoic acid ethyl ester. Capsanthin and capsorubin were synthesized in 1965 by Weedon and Warren. 1. Commercial Synthesis

The chemistry involved in the commercial synthesis of betacarotene and canthaxanthin is closely related to the synthesis of vitamin A now employed by a number of chemical manufacturers (Isler et al., 1955; 1956a,d; Isler and Zeller, 1957). In this procedure, citral, obtained from lemon grass oil, is the starting material for beta-ionone, which is converted into C, $ aldehyde. C , , aldehyde is transformed to C I G aldehyde, and then to C1, aldehyde, two moles of which a r e condensed with acetylene dimaynesium bromide to yield, on further treatment, beta-carotene (see Fig. 7 ) . Beta-ionone can also be synthesized from acetone (see Fig. 8) (Kimel et al., 1957, 1958). An interesting synthesis f o r beta-carotene was proposed by Surmatis and Ofner (1961) using C I j aldehyde building a C,, aldehyde + C,,, Wittig compound scheme.

+

CAROTEIVOIDS-PROPERTIES

A N D FOOD U S E S

Citral

+

211

cycllzation

,3-I"KJ"
glycidester synthesis, /alkali treatment

a;AHo 1 t

?-C,,-Aldehvde

J-C,,-Aldehyde

'Q

I

O H C Y

1

chain lengthening with vinyl e t h e r

EL 2 2i -.v (pACH0 CHO

O

H

C

v

chain lengthening with propenyl e t h e r

~

t

,J-C,,-Aldehvde

o

H

C

y

y

A

&

1

g r i g n a r d reaction with acetylene

8-C4,-Dioi splitting-off w a t e r with r e a r r a n g e m e n t

I S , 15'-Dehydro8-carotene

1 p a r t i a l hydrogenation,

1isornerization

FIG.7. Commercial synthesis of @-carotene.

The commercial synthesis of beta-apo-8'-carotenal is shown in Fig. 9 (Isler and Schudel, 1963), and that of beta-apo#-carotenoic acid esters in Fig. 10 (Isler and Schudel, 1963). Several syntheses of canthaxanthin have been proposed (Isler and Schudel, 1963).

212

B. BORENSTEIN AND R. H. BUNNELL

Acetone

Ethynylation

Hydrogenation

Ketonization

Oy’

--

t

Py

, . Y

I

Ethynylation

’

‘

, Cyclization \

13-Ionone

FIG.8. Synthesis of p-ionone from acetone.

2 . Microbial Szjnthesis

The microbiological synthesis of beta-carotene has received attention in recent years (Grob and Butler, 1954; Goodwin, 1958; Lotspeich et al., 1959). Phzjconzyces blakesleeanus and Mucor hiemalis have been used to prepare C14-labeled beta-carotene. The Northern Regional Research Laboratory has been interested in developing a commercial microbiological synthesis (Hesseltine and Anderson, 1957; Anderson et al., 1958; Ciegler et al., 1962, 1963). Lilly et al. (1960) obtained 4 m g beta-carotene per gram dry mycelium Phycomyces blakesleeanus. Ciegler et al. (1962) reported yields of 17.5 m g beta-carotene per gram dry fermentation solids using Blakeslea trispora in shake flask culture with a grain-based medium containing kerosene, nonionic detergent, and beta-ionone to increase yields. Lower-cost syntheses of carotenoids will continue t o be of interest because of the relatively high cost of these compounds.

J. TOXICOLOGY O F COMMERCIAL

S Y N T H E T I C CAROTENOIDS

The toxicology of the commercially available synthetic carotenoids has been studied intensively, but relatively little has been published.

CAROTENOIDS-PROPERTIES

AND FOOD USES

O-C,,-Aldehyde

F

C,-acetal

C

H

O +

I g r i g n a r d reaction

Oxyacetal (CJ I

acid t r e a t m e n t

CHO

Dehydro- apo - 12’carotenal (C,J

I

chain lengthening with vinyl e t h e r

I

chain lengthening with propenyl e t h e r

p a r t i a l hydrogenation, isomerization

p - Apo - 8’carotenal

(c3J FIG. 9. Commercial synthesis of p-apo-8’-carotenal.

213

214

B. BORENSTEIN AND R. H. BUNNELL

1 partial ~

3-Apo-8 acid e s t e-cnrotenoic r

hydrogenation lsomerization

XpJLLpw 4

7

COOR

I

A\

FIG. 10. Commercial synthesis of p-apo-8’-carotenoic acid ester.

1. Beta-Carotene

The chronic toxicity of beta-carotene in rats and dogs has been reported (Zbinden and Studer, 1958; Bagdon et al., 1960). Administration of 0.1% beta-carotene by weight of diet to four generations of rats, f o r periods including the entire life span of some groups, produced no toxic manifestations. Acute oral toxicity in dogs was greater than 8000 mg per kg. Doses of 100 mg betacarotene per k g body weight 5 days a week for 13 weeks evidenced no influence on the growth of dogs or any toxicity. Greenberg e t al. (1959) administered 100,000 units beta-carotene per day to 15 human subjects for 3 months. Serum betacarotene increased from 128 pg% to 308 pg%, but there were no clinical signs of vitamin A toxicity. 2 . Beta-Apo-S’-Carotenal

A 2-year chronic-toxicity study with rats-first generatioriplus a second two-year study with the F, generation plus a oneyear study with the F, generation showed no toxic effect on feeding beta-apo-8’-carotenaI a t 0.1 % except that degenerative changes in the epithelium of the seminiferous tubules were slightly more frequent ( P = 0.17) in the treated animals (Scharer and Studer, 1961). This was apparently due t o the fact that high-level feeding of beta-ap0-8’-carotenal depresses vitamin E tissue stores (Marusich, 1962; Brubacher e t al., 1965). Bagdon et al. (1962)

CAROTENOIDS-PROPERTIES

AND FOOD USES

215

fed dogs a daily dose of 1000 mg beta-apo-S’-carotenal for 14 weeks with no toxic manifestations. Beta-apo-8’-carotenoic acid was excreted in the urine. 3. Canthaxanthin

The chronic toxicity of canthaxanthin in rats has been studied in a 3-generation test similar to that of beta-apo-8’-carotenal above (Scharer, 1960 ; Scharer and Studer, 1961). No toxic indications were noted. Four grams per day for 15 weeks had no toxic effects on dogs (Bagdon e t al., 1962).

4. Beta-Apo-t?’-Carotenoic Acid E s t e r s The chronic toxicity of beta-apo-8’-carotenoic acid methyl ester has been studied in rats. As with beta-apo-€Y-carotenaI, the only toxic findings were testicular damage. Experiments feeding 1% methyl and ethyl esters plus added d,Z-alpha-tocopheryl acetate to rats for 1 year left fertility completely unimpaired (Scharer, 1963). This substantiated the hypothesis that vitamin E deficiency caused by the large amounts of vitamin A-in the form of betaapo-8‘-carotenal, beta-apo-8‘-carotenoic acid methyl or ethyl ester-was the cause of the testicular damage noted in the rats on a marginal vitamin E intake. It has been demonstrated that high-level feeding of beta-apo-S’-carotenoic acid ethyl ester depresses vitamin E tissue stores (Brubacher e t al., 1965). Ill.

OCCURRENCE A N D STABILITY OF NATURAL CAROTENOIDS I N FOOD

This section reviews the literature on the total carotenoid content and variety of carotenoids present in foods commonly used in the American diet. In some cases, space is given to a food because of its interesting carotenoid chemistry rather than its importance to the diet. A. OCCURRENCE There have been a vast number of publications concerning the distribution and varieties of carotenoids in foodstuffs, and t o review them in any detail would be impossible in a n article of this size. F o r more detailed information the reader is again referred to more extensive monographs in this field (Goodwin, 1954a; Moore, 1957). Table IV summarizes the provitamin A value of foods. The carotenoid pattern in foods may vary from relatively simple mix-

216

B. BORENSTEIN AND R. H. BUNNELL

tures to extremely complex ones. The simplest mixtures a r e usually found in foods of animal origin, because of the limited ability of the animal to absorb and deposit a great variety of carotenoids. At the other extreme is the analytically formidable a r r a y of carotenoids encountered in citrus fruits, which have only recently succumbed to sophisticated modern analytical methods. These extremes of complexity, however, a r e not easily discernible to the TABLE IV PROVITAWN A VALTJE OF FOODV

r.u./100 g* C:trrots, mature Carrots, yourig

>hit Parsley Spinach L)audelion leaves Spinach beet I E ~ V P R Turnil) leaves Crws

Kale Co1l:Lrds 3lustard greens Stvert potato IVatercrcss 1 k d iVP

20,000 10,000 19,000 14,000 13,000 13,000 11,000 10,000 8000 8000 8000 8000 6000 5000 4000

1.1-./100 gb Broccoli Apricots Lettuce Toinatoe Asparagus Bean, French Cabbage Peach Brussels sprouts Bean, runner Watermelon Bannn:i Tellow maize G ooscberry Orange juicc

3500 2000 2000 1200 1000 1000 500b 600 700 (is0 550

400 350 :100 200

Adapted from 11oore (1957). Roundcd figure. c The valuc is greatly aflectcd hy suniplirig, accordiiig t o whr tlicr tlir out( I’ g i . c c ~ or~ iriiivr nliite leaves are takcn. a

naked eye, so that a mixture of a few carotenoids may give the same yellow-to-orange hue as a mixture of 20-30 different carotenoids of complex structure. The determination of beta-carotene in fruits and vegetables has been of particular interest since it is used as a measure of the provitamin A content of foods. Column chromatography has been the method used most frequently (Strachan et al., 1951 ; AOAC, 1955). The validity of the AOAC method depends on the assumption that beta-carotene is the major hydrocarbon carotenoid present, since alpha-carotene, beta-carotene, and cryptoxanthin a r e retained on the column and a r e not eluted separately. Results of this procedure a r e usually labeled carotene, total carotene, or

CAROTENOIDS-PROPERTIES

217

AND FOOD USES

beta-carotene in the literature. I n this review, the term “total carotene” is used in reporting column-chromatography values in the literature that do not separate individual carotenes, and all assays are reported as ppm on a fresh-weight basis. Bickoff (1957) has an excellent review of the determination of total carotene as well as beta-carotene stereoisomers in foods and feeds. The difference in results between total carotene values obtained by column chromatography and beta-carotene values obtained by partition chromatography of butternut squash was discussed by Lewis and Merrow (1962) (see Table V ) . TAEI,I5 V CAROTENOIDS (FREWU I ~ I C .I)N BUTTERNUT SQUASHE~ D C I ~ I ~STOR\(,E~~ G Weeks of St orage 0 1 2 3 4

-

a

Partition chroinntogi aphy

AOAC carotene

a-Carotene

&Carotene

Xanthophy11\

2.09 3.71 3.28 3.90 4.24 4.5‘3

0.37 0.45 0.37 0.68 0.86 0.51

0.44 0.64 0.86 0.82 0.79 0.80

0.88 0.98 1.29 1.50 1.44 2.14

Adapted from Lewis and 3lerrow (1962). Mean of 8 replications expressed as mg p-carotene in 100 g.

NOTE:

Curl (1953) pioneered the use of countercurrent distribution plus column chromatography t o separate the complex carotenoid mixtures of fruits. Dozens of carotenoids have been identified. These methods, unfortunately, have not been used widely in vegetables and other foods to identify the distribution of trace carotenoids. 1. Fruits

In ripening fruit the decrease in chlorophylls is frequently accompanied by a n increasing concentration of cartenoids and by an increase in the ratio of carotenes to xanthophylls. Alpha-, gamma-carotene, and lycopene are the common fruit carotenes. The xanthophylls of fruit a r e often esterified. Oxygen is required for maximum carotenoid production, and the temperature range is critical. Light, however, is not required during maturation for carotenoid synthesis. The total carotene content of 54 fruits and vegetables grown in Israel has been summarized (Halevy et al.,

218

B. BORENSTEIN AND R. H. BUNNELL

1957). Carotenoids in fruits, juices, and concentrates were reviewed by Bauernfeind (1958). Table VI summarizes recent data on the total carotenoid content of fruits. The structural formulas of carotenoids commonly found in fruits are shown in Fig. 11. TABLE VI Twr \ L

<: \ R O T E S O I D

Fruit

Total carotenoids (ppm wet basis)

Applex" Xpric ot,s Black figsb Blackberriesb Hlueherriesb Cherries" Cling peachesb Cranberl.iesb Grapesb

0.9-5.4 35 8.5 5.9 2.7 5-1 1 27 5.8 1.8

Galler and Nackinncy (1'365). (19G4b).

* Curl

C O N T E N T OF

FKIIT,

Fruit

.Japanese prrsinimolis" Lemonc Navel orange pulp" PearsY Pomegranatesb Prunesd StrawberryPb Tangerine pulp* Tornat oes c

d

Total citrotcnoids (p p n i \VP t basis)

54 2.3 23 0.3-1.2 0.16 21 0 . ti-1. .5 27 t5 1

Curl (196%). Curl (1963j.

a. Orange. Intensive investigations of carotenoids of the orange have been recently reviewed by Mackinney (1961). Major workers in this field a r e Zechmeister and Tuzson (1931, 1936, 1937), Karrer and Jucker (1944, 1947b), Natarajan and Mackinney (1952), Curl and Bailey (1954, 1955, 1956a, 1961), and Curl (1953, 1965a). Natarajan and Mackinney (1952) reported that the cartenoids of Valencia orange juice were 66% xanthophyll esters, 25% unesterified xanthophylls, and 9% epiphasic carotenoids (carotenes). The greater portion of the xanthophylls a r e epoxides (Curl and Bailey, 1956a). The cryptoxanthin, lutein, zeaxanthin, hydroxy alpha-carotene, and xanthophyll 5,6-epoxides are mainly esterified, and the isomeric xanthophyll (5,8-epoxides) furanoxides are mainly unesterified (Curl and Bailey, 1955). Table VII summarizes the carotenoids found in orange pulp and orange peel by Curl and Bailey (1956a). Beta-ap0-8'-carotenal was isolated from orange peels by Winterstein et al. (1960) and from peel and pulp by Thommen (1962). Curl (1965a) reported beta-citraurin to be present in orange peel. A pink sport of the Shamouti orange contains lycopene (Monselise and Halevy, 1961). Color is a n important characteristic of citrus juices because it

CAROTENOIDS-PROPERTIES

219

AND FOOD USES

is one of the criteria of present commercial standards. The total color is affected by variety, maturity, processing methods, etc. Taylor and Witte (1938) determined total carotenoids of orange juice using trans beta-carotene as a primary standard, and found wide variation in market oranges arriving in the New York area (Table VIII) . These differences were due not only to variety but to district of origin. Florida oranges were generally lower in carotenoids than California oranges. To the food processor the total carotenoid content of oranges is a satisfactory measure of total color, and variations of carotenoid type a r e not significant except in the case of isomerization of 5,6-epoxides to 5,g-epoxides.

~~

Orange ( ‘onstituent

I’liytoene I’liytofluene a-Carotene fi-Carotent. %( ta-Cnrot.enc 0 H-a-C:troteneli li c (’ryptoxantliin cposidcl ike Cryptoxanthin Cryptofiavirililie Cryptocliroiiiclike 1,u t eiri Z:c:tsarit h i r i

PUlP

(%)

4.00 13

0.5 1.1 5.4 1.5

. .. 5.3 0.5

. ..

2.9 4.5

Fresh orange peel (%)

3.18 6.1 0.1 0.3 3.5 0.3 0.4 1.2 1.2 0.8 1.2 0.8

Constituent Capsanthinlike Antheraxanthin 1Iut:ttoxanthins Violaxanthin Luteoxanthiiis Aurosanthin Valcnciasanthin Sincnsiasanthin Trollixanthinlike Valenciachrome Sjric,isi:tchromeliIcc. Trolliclirornclike

Fresh orange Orange pulp (%) peel (70)

5.8 6.2 7.4 17 12 2.8 2.0 2.9 1.0 t . .

3.0

0.3 6.3 1.5 44 16 ‘2.3 2.2 3.5 0.5 0.7 0.2 0.8

Apllroaiiiinte va1ur.s. (Assuniirig a l l constituents to liavc same spwific extinction (wefficient). Curl aud U:~ilc>-(1‘356~).

A stability study of canned orange juice (Curl and Bailey, 1956b) demonstrated that the 5,6-epoxides, which a r e prevalent in fresh juice, isomerized completely to 5,8-epoxides during storage. This loss of one double bond from the conjugated double-bond system causes both a shift in the wavelength absorption maximum and a decrease in the molar absorbance. These changes partly account for an apparent loss of 20-30% total carotenoids in canned orange juice during one year of storage a t 70°F. Frozen 6-fold-concentrated Fiorida Vslencia orange juice contains 39 ppm total carotenoids, and the distribution of the carote-

220

B. BORENSTEIN A4ND R. H. BUNNELL

Anther axanthin

Mutatoxanthin

Auroxanthin

FIG.11. Structural formulas of common f r u i t carotenoids.

CAROTENOIDS-PROPERTIES

A N D FOOD USES

B-Carotene

7 -Carotene

5 -Carotene

Lycopene

Cryptoxanthin

FIG. 11 ( c o n t . ) .

221

222

B. BORENSTEIN AND R. H. BUNNELL

noids is similar to that in single-strength juice (Curl and Bailey, 1959). Dehydration of this concentrate to 3% moisture caused little change in carotenoid distribution. Conditioning of the powder a t 77°F f o r 78 days to reduce moisture further caused a large decrease in the diepoxide diols because of isomerization of 5,6epoxides to 5,8-epoxides. On subsequent storage the remaining 5,6-epoxides isomerized. Stability of the provitamin A sources, cryptoxanthin and beta-and alpha-carotene, was excellent in the entire process. TAB1,C VIII (? ~ K O T E N O I D CONTENT O F F R E ~XI I I\ R K E

I'

ORINGB~"

SO.

Origin

Tinic,

of samples -

Chlif omia Florida Califorrii:~-\\~a~liirrgtori b'lorida Florida Californi:t-\~ashi~igtori ~~aliforriia-\~ashington ~aliforiii:t-\~ashington California-Washington (:aliforni:t-Washingtori California-Washington (:alifornia-~ashirigtori

b

14 34 G8 32 I (i

->

1 -1 18

8 8 10 S

0.65-2.74 0.18-1.05 0.32-2.97 0 . 05-0. so 0 . 10-0.70 0.23-0.34 0.3-1.77 0.32-1 . i 4 0.56-1.31 0.70-1.75 0.43-1.9!) 0.!)2-2.!)7

1.65 0.5T 1.07 0 . 34 0.32 -

0.90 0.93 0.90 1.28 1.05 1.83

Taj lor :tiid \Vitte (1938). \lilligranis Iwr liter.

0. Taszgeyine. Tangerines also exhibit a complex spectrum of carotenoids, and generally have a higher color intensity than the orange. Curl and Bailey (1957a), using their two-pronged attack of countercurrent distribution and chromatography, unraveled the problem of the carotenoids of the tangerine, as they had done so ably for the orange. They found that the redder color of the tangerine than of the orange was due to higher concentrations of cryptoxanthin in the pulp and peel a s well as of beta-carotene in the pulp, and a hydroxy canthaxanthin-like substance in the peel. Minor amounts of other carotenoids not found in the orange were also reported. Measuring the total carotenoid content of the pulp and peel in a n Evelyn colorimeter and calculating as betacarotene gave values of 186 ppm for the peel and 26.5 ppm for

CAROTENOIDS-PROPERTIES

AND FOOD USES

223

the pulp. Thus, the peel of the tangerine has about twice the carotenoid content of the peel of a typical Valencia orange, and the pulp about the same content as the Valencia pulp. The carotenoid constituents of the pulp and peel as taken from Curl and Bailey (1957a) a r e shown in Table IX. More recent work of Curl (1962b, 1965a) has characterized five carbonyl carotenoids from tangerine peel-reticulataxanthin, tangeraxanthin, beta-citraurin, beta-apo-8’-carotenal, and apo-l2‘-violaxanthal. These carotenoids a r e obtained principally from the peel, and betacitraurin makes a particularly important contribution to the redder color of the tangerine. c. GTapefruit. Khan and Mackinney (1953) studied the pigments of white, pink, and red grapefruit, and reported 11 carotenoid bands on chromatography. In the pink strains, beta-carotene and lycopene predominated, with smaller amounts of zeta-carotene and phytofluene and traces of alpha- and gamma-carotene. Lycopene and beta-carotene account for 80% or more of the color of the pulp of Ruby Red Grapefruit (Curl and Bailey, 1957b). Most of the xanthophylls found in oranges and tangerines a r e present as minor constituents. Lime et al. (1954) have shown the relationship of visual color and the lycopene and carotene content during the maturation of Ruby Red. As the fruit reaches optimum maturity, lycopene decreases and carotene increases (Fig. 12). Analytical methods for beta-carotene and lycopene in Ruby Red Grapefruit have been compared (Lime et al., 1957). d . Peach. Mackinney e t al. (1942) reported that beta-carotene was 10% of the total carotenoids of peaches. Curl (1959) found cling peaches to contain primarily violaxanthin, cryptoxanthin, beta-carotene, and persicaxanthin, and identified 25 other carotenoids, including neoxanthin. Neoxanthin has been identified tentatively as 3,3’5‘-trihydroxy 5’6’-dihydro 5,6-epoxy betacarotene (Curl, 1965b). McCarty and Lesley (1954) found that peaches contain eight pigments, with five of them identified as alpha-carotene, beta-carotene, cryptoxanthin, lutein, and zeaxanthin. Strachan et al. (1951) studied the total carotene content of 7 British Columbia tree fruits. Table X summarizes their data on 9 varieties of peaches. Varietal differences a r e great. e . Apricot. Apricots a r e excellent sources of beta-carotene (see Table X ) . They contain little, if any, xanthophylls, as opposed to peaches. Beta-carotene, gamma-carotene, and lycopene a r e the main carotenoids (Goodwin, 1954a). Curl (1960b) found that the major carotenoid distribution was ca. 60% beta-carotene,

TABLE I S CAROTENOID CONSTITUENTS OBTAINED FROM TANGERINE PULP Approximate yo of total cnrtotrnoidb Fraction

Constiturri t

l’ulp

.4ND PEEL”

.4ppro\irn:it~ yoof total (*:trotenold-

1I-

Pcrl

17rartion

Constiturnt

Pulp

PCC~l ~~

Hydrocarbon

Diol Phytoene Phytofluene a-Carotene Phytofluenelikc p-Carotene Zeta-Carotene Gam,ma-Cnrotrnelike Ly cop? ne

N @

5,s

4.2

7.2

3.5

0.3 0.1 4.1

0.2 0.1 0.4 “0 0.02

6.!J

0.1 0.1

Lutcin Zcnsnnthiti H~dros~-C:ttitli:tsnntliiitli kv

a

Curl and Railrp (195ia).

1.0 0.!l 33

-

<).i 2.2

O.s

-

(i.2

2.S

Ihrther diol Violaxaiithiti J.utrosanthin.: Aurosnnt~liins

0.ti 1.4

14

21

3.5

!I. I

0.1

1.9

0.2 0.2 1.0 0.9 0.3

0. 1 1.1

1’01 yo1

24 0.4 3.4 0.2 0.1

3.3 3.5 2.7

llotioc~thc~r diol

niorloi

II y droxy-a-Carotenelihc Cryptosanthin eposidc Cryptosanthin Hydroxy-a-Carote ne Furanoxidelike Cryptoflavinlike Rubisanthinlike Cryptochromelike

2.!) 3.3 0.1

’ ’

V:ilcrtc.i:is:inthiii SinsPnsictsanthin Trollisnnthinlilie Trollein Trollichromclike

2.6 -

0.7

AND FOOD USES

CAROTENOIDS-PROPERTIES

r

- -

A = Lycopene

B = Carotene C = Index of

A

I 1

I-

fading

/

1.251

;;

_. .

Qm

~ 1

1

Q

1

m m

1

m

? o .

1

#

o o

,

--

!

-

z z ,

-

1

/

N

,

(\i

1 ~

~

I -

~.

Good

I

of

Dale ~~~~~

F e l l e n 1

. . .

-

Good

3~~

,

-

,

N

cum

m

p

I

sampling

1

Z Q E, m, E, r n,

Falr

I

I

Poor

FIG. 12. Carotene content of Ruby Red g r a p e f r u i t dnring growth and maturity. From Lime e t ul. (1954).

5% lycopene, 5% gamma-carotene, 4% cryptoxanthin, and 2%' lutein and that the carotenes included cis and polycis isomers. f. Miscellaneous Fruits. The total carotenoid content of miscellaneous fruits is shown in Table VI. I n the elderberry the principal pigments are beta-carotene, cryptoxanthin, lutein, flavoxanthin, and neoxanthin (Goodwin, 1956). Papaya contains cryptoxanthin and violaxanthin, and pineapple contains beta-carotene and lutein. Major components of Passion fruit a r e beta-carotene and ununesterified and esterified xanthophylls (Pruthi and Lal, 1958). The carotenoid distribution of cranberries, figs, grapes, blackberries, and blueberries is shown in Table XI (Curl, 1946b). Galler and Mackinney (1965) discussed the carotenoid distribution of apples, pears, strawberries, and cherries. Xanthophylls, mainly esterified diepoxy diols and polyols, predominate in apples, pears, and strawberries, while carotenes predominate in cherries. The carotenoids of the pulp of the Meyer lemon a r e mainly cryptoxanthin (Curl, 1962a). The peel includes a number of xanthophylls, primarily monoepoxide monols and diepoxide diols. Italian prunes contain violaxanthin, beta-carotene, and lutein as the primary carotenoids (Curl, 1963).

226

B. BORENSTEIN AND R. H. BUNNELL

The carotenoids of the Japanese persimmon have been investigated intensively (Brossard and Mackinney, 1963 ; Curl, 1960a). Forty varieties contained 20-1 15 ppm total carotenoids (Brossard and Mackinney, 1963). The major carotenoid was cryptoxanthin (30-35% ), with a n extremely variable lycopene content (0-3076 ) . Curl ( 1 9 6 0 4 isolated 26 carotenoids from the Hachiya variety. The major carotenoids were cryptoxanthin (38% ) and zeaxanthin (18%). ( ’ I ROI ’ ENE

T.-IRT,l: X APRICOT\\ \ I ) 1’E

CONrEUT O F

\CHI,\“

V:tric.ty .

~~

.ipricots All varieties Rlenheini (Koy:tl) 1,:nglish 3 f o o r p r k Iialcden Idie Perfection Iteliablc Rose Tilton Wenatxhee lloorpark Varicty No. 27-5 I’eaches All varieties 1l;lberta IG shc r J. H. Hale ltochester Spotlight Superior Valiant Vedette Veteran a

23 2 1 4 4 1

1

4 3

a

1.94 2.23 3.49 1.46 1.85 2.30 2.45 2.20 1.94 1 .2 1 2.15

0.70-4.15 I ,5552,so 2.40-4.15 0.80-2.05 -

0.79 0 .4 2 0. i 2 0 .4 4 0. Ti 0 .7 8 0 .8 8 1.20 1.01 0 . 78

0.31-1.4ci 0.38-0.48

2.10-2.50

1 .75-2,6.5 I , 50-2.35 0.70-2.05 -

-

0 . 3 1-0.60 0.46-1.08 -

1. 03-1 . 40

0.85-1.25 0 34-1.4fi

Strachan el a1 (1951). mg/100 g.

2 . Vegetables

u. Currot. Garden varieties of carrots averaged 54 ppm total carotene, mainly alpha- and beta-, with zeta-carotene prevalent and lycopene present in some varieties (Harper and Zscheile,

CAROTENOIDS-PROPERTIES

('oristituc'rit

(hailberry

Hydrocarbons (I) Pliytoene Phytofluc~ne alpha-Carotenc Unidentified beta-Carotene zeta-Carotene Mutatochronicd

4.7 0.9 0.14 5.3 0. G O.G

hlouols (11) H3.droxy-alphe-carotcne-lilie Hydros3-alpha-carotene Cryptoxanthin

0.4 1.1 1.8

1)iols (111-4) Lutein Zeasanthiii

31.3 2.8

1Ionoepoxitle diols (IIIB) Carbonyld Lutein-5,6-epoxide Antheraxanthin (cis) Lutein-5,s-epoxide Mutat oxinthins

0.18 10.5 4.6 0.7 0.33

1)ieposide diols (IIIC) Carbonyld Violasanthin Luteoxanthins

0.37 20.7 1.8

r ~ ~ i y o(i s1 ~ ) Valenrinsanthind Sinrnsiasanthind Neoxanthin Sinensi:it~antliiii-lili(~

1.1 0.25 6.9 1.8

AND FOOD USES

Fig

Grape

2.4

+

0.7 0.24 -

9.9

0. 2 0.2 0.4 32.3

227

Blackberry Blueberry (11.0)c

1.5 0.2 1.1 -

9.5

-

-

-

-

-

-

(2.1)c

(2.1)c

(3.2).

(2,L')C

28.8 1.1

22.0 1.1

44.3 2.3

39.2 2.1

(2.0)C

(6.0)C

(1.9)"

(13.7)c

(22.0)c

(19.4)"

(14.1)"

(11.4)c

-

40.0 2.5

15.7 1.6

0.4 1.0

7.1 1.0

(12.2)C

Curl (1064b).

* Percentages based upon total absorbance of each constituent :it its principal sprctrxl absorption maximum. c Percentage of fraction froin countercurrent distribution; fmctions not chromatographed. d Tentative identification. Spectral absorption curve of cntire fraction very simi1:tr to that of neoxanthin.

228

B. BORENSTEIN AND R. H. BUNNELL

1945). Various workers have reported 5 4 6 % alpha-carotene content (by weight of carotenes) in carrots (Goodwin, 1954a). Gamma-, delta-, zeta-carotene, and lycopene are also present in small amounts. The xanthophylls fraction is usually only 5-1095 of the total carotenoid content (Booth, 1945), but yellow varieties contain 75-93% xanthophylls, and wild carrots contain 95% xanthophylls. The phloem has a higher concentration of carotenoids than the xylem, as is obvious from the color (Booth, 1951a). Carrots stored at 6°C continue to synthesize carotenoids for 30 days or more, with increases of up to 20% (Booth, 1915b). A comparison of carotenoids in frozen and canned carrots in a 4-year study (Weckel e t al., 1962) indicates a higher carotenoid concentration in the canned products : 852-1352 ppm dry weight, vs. 534-831. This is apparently due to a leaching of the carrot soluble solids into the canning liquid. Carotenoid content of the canned products is shown in Table XII. Canning caused losses in provitamin A activity of 7-12%, because of cis-trans isomerization of both alpha- and beta-carotene. Carotene oxidation and off-flavor development have been correlated in dehydrated carrots (Falconer et al., 1964). Off-flavor can be avoided by packaging in a n inert atmosphere. Dehydrated carrots stored under high vacuum have good carotenoid stability (Mackinney et al., 1958). Lantz (1949) reported excellent stability of total carotene in carrots stored 17 weeks a t 42°F. Cooking under different conditions had little effect on total carotene. A process for commercial extraction of carotenes from carrots has been described (Barnett et al., 1958). b. Corn. White, yellow, and red varieties of corn are used widely as foods. A substantial fraction of the nonsweet varieties used as food are processed and consumed as corn flour, meal, and grits. Kuhn and Grundmann (1933, 1934) isolated zeaxanthin, cryptoxanthin, and beta-carotene from dried corn. Quackenbush ct al. (1961) separated 11 carotenoid fractions in 5 corn strains, and compared theoretical provitamin A activity with bioassay results. In these 5 strains, lutein and zeaxanthin predominated (see Table XIII) . Total carotenoids (excluding colorless polyenes) ranged from 18 to 55 ppm. Hungarian-grown dentidormis Koern of Zea maize L. was studied a t two stages of maturity (Zsolt et al., 1963). Carotenes, with beta-carotene predominating, were approximately % of the total carotenoids. The xanthophylls, with zeaxanthin predominating, were the remaining :<. The total carotenoids ranged from 10 ppm, in white corn, to 24 ppm, in red corn.

TABLE XI1 AVERAGECONTENT OF TOTAL CAROTENOID A N D CAROTENE COMPONENTS O F PROCESSED (CANNED) C A R R O T S GROWNO N M U C K A N D h l I N E R A L SOILS (1957-60)" Percentage

N

%

Year

Total carotenoids (ppm dry Wright)

Neo-alpha B

All-trans alpha

Keo-alpha 1.

Keo-beta B

All-trans bvta

Neo-beta 1'

Loss in provitamin A activity duc t o isomerization

~~

1957

879 f 243

2 1

30 9

2.7

6.0

42.0

3.8

T.3

1958

92i 5 l i 9

6 4

21 8

1.6

13.1

41.5

4.5

11.7

1959

852 f 149

5 4

24 2

2.4

8.5

41.!)

47

9. 7

1960

1352 & 335

2 3

2G G

2.5

10 7

46.4

4.2

8.3

Weckel et al. (1962).

230

B. BORENSTEIN AND R. H. BUNNELL

Quackenbush (1963) reported that 11%-moisture corn lost 75% of initial carotenes in 3 years a t 25”C, and 50% a t 7’C. Losses of total carotenoids were most rapid during the early part of the storage period. The xanthophylls were more stable than the carotenes.

Frwtitrn 1

1

, 4 1

(i

-,

Chief component Ptiytocrie Phytofluene @-Carotene P-Zeacnroteiic. Zc%a-Caroteiicl Zrinosanthin Cryptosanthiii ISsters Luteiii Zeasanthin Polyosy pigrnrnts l’otal polyenes

Corn 1 (Os420) 21.1 4.8

2 i 0.4 0.9 2.3 1.4 0.8 11.7 6.0 0.8 5 2 . $1

Corn :3 l).C.b 15.5 3 .8 1.8 0.5 0.3 1.1 3. 1 1.1 11.9 11.3 1.1 51.5

19.3 3.7 2.0 1.2 1. 0 2.5 2.1 1.1 14.1 6.8 1.8 S . ( j

Corn 4 Oh45

25.t; 0 .3 4.4 4.0 4.2 2.2 a.2 4.0 “0.2 5.4 1.;3 02.S

10.7 1.3 0.6 0 .1 u.2 0.6 1.0 0 .5 10.5 3 ,9 0.8 30.2

” Quackenbubh et al. (1961). Seed from the double cross (A23 X \V8:t) X (Kys X 38-11) g r o ~ nundrr open1)ollinntion conditions.

Golden Cross Bantam sweet corn contained ca. 1 ppm total car1940). otene by chemical assay and bioassay (Zimmerman et d., Sweet golden corn varieties contained 1.2-5.3 ppm total carotene (Scott and Belkengren, 1944). Evertender C and Royal Gold sweet corn contained 8-9 ppm total carotenoids, including 0.91.3 ppm beta-carotene and 2.3-3.1 ppm cryptoxanthin (Tichenor c t aL., 1965). Beta-carotene in these varieties was stable in refrigerated and frozen storage. Diol and polyol carotenoids composed ca. 50% of the total carotenoids of both varieties. A recently discovered carotenoid in corn is zeinoxanthin (Petzold and Quackenbush, 1960). It has no provitamin A activity and appears to be a n all-tmns monohydroxy alpha-carotene. Considerable work has been done to breed corn with high carotenoid contents (Brunson and Quackenbush, 1962; Blessin et al., 1963). Blessin et al. (1963) concluded that breeding high-xanthophyll corn is feasible and desirable.

CAROTENOIDS-PROPERTIES

AND FOOD USES

231

c. Tomato. Von Euler et al. (1931) found that green tomatoes ripened normally a t 20°C, but became yellow rather than red at 30°C. Lycopene is by f a r the most predominant carotenoid of tomatoes, with beta-carotene, gamma-carotene, and xanthophylls (see Table XIV) (Went et al., 1942). Curl (1961) determined the xanthophyll distribution of tomatoes : 15% were monols, 49% diols, 4 monoepoxide diols, 22 diepoxide diols, and 11 polyols. The outer pericarp of tomatoes has the highest total carotenoid concentration, and the locular contents have the highest carotene content (McCollum, 1955). The effect of ripeness on carotenoid content of different parts of the tomato is shown in Table XV. Kargl et al. (1960) discussed a strain of tomatoes high in delta-carotene. d. Potato. Potatoes (Solanum tuberosum) , Katahdin variety, contain 3 ppm carotenoids, dry weight (Brunstetter and Wiseman, 1947). The same workers identified lutein, 0.50-0.77 ppm; beta-carotene, 0.3 ppm ; and xi carotene (aurochrome) , 0.11 ppm ; and isolated several unidentified carotenoids. Six British varieties all contain beta-carotene, beta-carotene-5,6-monoepoxide, cryptoxanthin 5,6-diepoxide, lutein, cis-violaxanthin, cis-antheraxanthin5,6-monoepoxide, cis-neoxanthin, and an unknown (Pendlington et al., 1965). During growth, the epoxides are especially abundant. At maturity the nonepoxides are present in equal ratio; during storage, nonepoxides, primarily lutein, increase, and epoxides, primarily cis-violaxanthin, decrease. e . Sweet Potato. The carotenoids of sweet potatoes (Zpotnoea bata tns Poir) have received considerable attention. Matlack (1937) demonstrated that beta-carotene was the principal pigment. Ezell and Wilcox (1946, 1948) reported that flesh color was a reliable indicator of the provitamin A value of sweet potatoes. The total carotene content, 0-151 ppm, depends greatly on variety (Miller and Covington, 1942) and varies more than 400% within a variety (Ezell and Wilcox, 1946). Total carotene and total carotenoids in 2 varieties were determined by Ezell and Wilcox (1958) (Table XVI). Cordner e t al. (1959) suggested that breeding could increase total carotene from 50 mg/100 g to 80 mg/100 g dry weight. Storage causes increases in proyitamin A activity in most mrieties (MacLeod et nl., 1935), but storage temperature is a significant variable in carotenoid fate (Ezell and Wilcox, 1952). They rcicommended a temperature of 60°F for maximum total carotene production during storage. The effects of variety, cur-

CAROTENOID CONTENT (MC/100 G

DRY

FRUIT)

OF

TABLE XIV TOMATOES GROWN I'NDER

VARIOUS E N V I R O N M E N T A L

CONDITIONS"

Jhvironmcntal conditions*

A Temp. ("C) in day: 2G 5 Temp. ("C) in night. 20 0

N

Total pigment (as lycopcne) Xanthophyll Lycoxanthinc, Lycopenc Neolycopene A Neolycopene €3 1-nidentified carotenr 7-carotcne nro-7-carotc rif' 1-nidrntifit d Isomcr of p - c : ~otcaric, +carotene Neo-P-carotenc

+

386 4.6 21.2 270 17.7 1.5 1.7

A 26 5 20 0 286 :3 8 12.1

IS0 l(i .5

A 26 5 20 0 302 3 .8

11.4 188 I!) 5

1.2 0.5 2.1

-

-

0 .1

ti. 2

:i1

0.2 0 . (i 2.5

1.4

0 .7

0 .7

B

B

c

I)

I5

F

20 0 20 0

20 0 20 0

20 0 20 0

26 5 20 0

26 5 26 6

20 0 26 5

219 3.4 9.8 138 12.1 1.8 1.0 1.1 0.5 1.7 4.7 1.7

365 5.8 16.5 220 21.7 0.4 0.4 0.2 0.5 1 .:3 0.6

485 7.1 20.3 308 14.8 2.7 1.5 2.0 0.5 2.8

615 8. 7

512 5.2 "(i S

708 7.8 :30 7 412 "j 5 1.5 1.2 1.4 0.3

8.1)

3.5

2(i. 6

:375 20.6 3.2 2.5 1.5 0.3 1 . li 1 . (i 0.5

,

:33 I I (i.3

6.7 -

:3 c, 2.2 1 :I 7 . (i

1.4

1 . !)

7.S 2 :i

233

AND FOOD USES

CAROTENOIDS-PROPERTIES

TABLL XV CAKOTESOIDS OF THE REGIONS OF THE STORESDALE TOMATO VARIETY AT DIFFERENT STAGES OF RIPENEW Number of days ripened 4

8

12

16

20

4.40

2.96 4.80

7.84 4.40 4.16

8.64 4.40 4.00

8.64 4.00

8.40 4.40

6.56

6.32

0.23 0.22 0 60

0.23 0.12 0.71

0.18 0.15 0.60

0.18 0.18 0.57

0.26 0.30 0.55

Ilegion of fruit Total c:trotenoiti.; (ing/100 g) Outer peric:trp Inner perirarli Locul:ir cviiteiits Caroteric (mgj100 g ) Outer prricarp I n n r r Iwricnrli 1,ocul:u contriit.;

:i 4

47 30

37 6

7

47 22 12

23 11

12

ing, storage, and planting and harvest time on sweet potatoes grown in Southern United States were intensively studied (Georgia Expt. Sta., 1953). f . Squash. Seven varieties of summer squashes (Cucurbita p e p 0 L. and Czrcurbita moschata L.) contained from 0.6 ppm total carotene, f o r Early White Bush Scallop, to 62 ppm, for Golden Cusham (Holmes e t a l , 1945). Flesh color is a crude guide to carotenoid content. Lewis and Merrow (1962) demonstrated that Butternut squash has approximately equal quantities of alphaand t:et:i-c:timtene and considerably higher quantities of mono-

0r:ingc 1,it tlc Sic’iii

Trllow

.Ji,rscs\-

1951 1952 1051 1952

a

l h l l :uid \\ilcos (1958).

* 1’:ii.t~ p r r niillio~i,frrsli txisis

00 27 1.9 0.8

08 34 7.1 1.7

234

B. BORENSTEIN AND R. H. BUNNELL

and dihydroxy carotenoids. Distribution of carotenoids in Butternut squash and their increase during storage are shown in Table V. The total carotene content of 6 varieties of winter squash was determined a t harvest and a t 5-week storage intervals (Hopp e t ul., 1960). Total carotene ranged from 4 to 29 ppm at harvest. After 10 weeks of storage, total carotene ranged from 6 to 36 ppm on a n adjusted fresh-weight basis. g . P e p p e r s . Red bell peppers have a complex carotenoid mixture, including capsanthin and capsorubin, and a t least 6 other pigments containing cyclopentane rings (Curl, 1 9 6 2 4 . Total carotenoids concentration is 127-248 ppm. Green bell peppers contain 9-11 ppm total carotenoids. Major carotenoids are lutein, beta-carotene, violaxanthin, neoxanthin, and luteoxanthin (Curl, 1964a). TABLJ: xvrr TOTAL CAROTENE CONTENTIN V E G E T ~ B L E S

Asp;Lragusb Bret tops6 Broccolic

Brussel sprouts" Cabbage hcadsc Cnbbagr outer ]cawsc Collardsd Lettuce"

5.0 22.0 6-46 1.2-5.8

trace 0 . 3 39. 3ti. 8-G8

!

Okrac Olives< Parslcyc Pumpkina Raped Spinsc;1ic Turnip grvviisd l-anic

c

d

Goodwill (1954a). Ilzc~lland \\ilcos (1!)62).

IL. Miscellaneous V e g e t a b l e s . The total carotene content of 16 vegetables is shown in Table XVII. Fresh broccoli contains 6-46 ppm total carotene. The total carotene content of asparagus is 5.8-7.0 ppm (Zimmerman e t a:., 1941; McConnell e t al., 1945). Lima beans, fresh or frozen, contain ca. 2.3 ppm total carotene, % of which is beta-carotene (Zimmerman e t al., 1941), and frozen baby Lima beans contain 1.2 ppm total carotene (Cook e t aZ., 1961). Snap beans contain 5.5 ppm (McConnell e t al., 1945), of which 2/3 is beta-carotene (Zimmerman e t al., 1941). Spinach contains 53-58 ppm total carotene plus 57-74 ppm lutein plus violaxanthin and unidentified carotenoids (Tan and Francis, 1962). The predominant hydrocarbon carotenoid of spinach is beta-carotene (Karrer and Schlientz, 1934).

CAROTENOIDS-PROPERTIES

235

A N D FOOD U S E S

The carotenoids of the rutabaga root are noteworthy for the unusually high proportion of poly cis isomers (Joyce, 1954). Six chromatographic bands of apparently different poly cis lycopenes were obtained in addition to all trans lycopene, zeta-carotene, and beta-carotene. Pumpkin seeds contain both carotenes and xanthophylls. Soy beans, cow peas, Lima beans, Thomas Laxton peas, and French (snap) beans contain 2-7 ppm total carotene. The total carotene content of 34 early and late varieties or strains of peas was found t o be 3.4-7.1 ppm (Heinze e t al., 1947). Varietal differences were highly significant. 3 . So?.yhzim

The grain of common varieties of sorghum contains 1.5 ppm total carotenoids; some yellow strains contain 8-9 ppm (Blessin et al., 1958). Major carotenoids were identified as lutein, zeaxanthin, and beta-carotene. Yellow sorghum carotenoids had 3553% losses during 41-day weathering periods (Blessin e t al., 1962). Blessin (1962) compared the total carotenes, xanthophylls, and xanthophyll esters of corn and sorghum (see Table XVIII) .

~~

Nonsaponified Sulnple 1Iigli-s:intlio~)hyl1 cor11 Hybrid yellow corn ('om glutert (60% protein) 1ic.d sorghunl Sorghuni gluten (607.) protein) a

Carotenes Xanthophylls

Saponified Carotenes

Xanthophylls

Xanthophyll esters

3.0 3.0

35.9 20.1

2.6 1.6

36.3 20.5

0.4

16.9 0.3

301.6 1.8

13.2 0. 2

205.3 1.9

3.7

0. 8

6.5

0.G

6.7

0.3

0.4 0. 1

Blessiii (1962).

4. Wheat Zechmeister and Cholnoky (1940) found less than 0.01 ppm beta-carotene in Hungarian wheat flour and reported that the major carotenoid present was 1-2.5 ppm lutein. Chromatography be-

236

B. BORENSTEIN AND R. H. BUNNELL

fore and after rigorous saponification indicated the presence of esterified xanthophylls. The major xanthophylls found by Irvine and Anderson (1949) were lutein and taraxanthin. The carotenoids of four types of wheat a r e shown in Table XIX, taken from Parker and Harris (1964). The total carotenoid content of whole wheat was 1.8-2.3 ppm, and the embryo contained the highest concentration (4-11 ppm).

Class of Wheat

Pgk

%

Hard red spring Carotene Santhophyll Santhophyll cstri Total

0.09 0.84 0. ti4 1.57

5.7 53.5 40.8

0.11 0.77 1. 12 2.00

5.5 :M.S 56. 0

liard red winter Carotene SaIlthophyll

xnrlthopllyll < st (31. Total

(1.72 5.78 0. ti!) 7 . l!)

0.80 5.98 I 06 ,

10.0 80.3 !). ti

0.04 0.42

0 .4 7 0.93

0.m

3.4 :iG.4 0 . 5:< I i O . 2

0.2j 1.33 0 .72 2 , 30

0.83 0.60 0 . !I5

0 , u3 0.32

1.13

10.2

!I,:() 0.21 I 1 . 0-l

s7.s

0.50 2 !I:( 0.70

12.2 7 0 .s

0.10 1.31

17.0

0 . SI 2.22

10.0 55.0 35.0

1 .8 0

0.21 0.7‘3 1.04 2,O-i

0.02

L!l

0 .1 8 0.99

2.1 34.8 63.2

10.2

7G.2 I :<.5

7.84

4 I:(

4.3 4 5 .1 50.5

0 . ss

10.3 38.7 %51.0

10.8

57,s :{I. 4

4 .5

0.15

i.ri

59.0

1.67

s-I.s

4(i. 5

0.1.5 I 97

71;

The principal pigment of Cnntharellus ci?rneDariwus, a n edible pink mushroom, is canthaxanthin (Haxo, 1950). Beta-carotene has also been identified in this mushroom. The most important commercial U.S. mushroom, Aga?>icuscampestris L., has little if any carotenoids.

CAROTENOIDS-PROPERTIES

AND FOOD USES

237

6 . Vegetable Oils

Of the vegetable oils that are widely consumed, palm oil has by f a r the highest concentration of carotenoids, usually 0.05-0.2% in the unbleached crude oil. Beta- and alpha-carotene a r e the major carotenoids present (Hunter and Krakenberger, 1946), usually in a 60 : 40 ratio. Alkali refining has little effect on the carotenoids of vegetable oils, but bleaching and hydrogenation degrade them almost completely. I n Africa, palm oil is a significant source of provitamin A. Several methods have been proposed for commercial extraction of carotenoids from palm oil, including extraction with propane (Larner, 1947), extraction of palm oil soap (Tabor et al., 1948), and conversion of triglycerides to methyl esters followed by distillation (Eckey, 1949). 7. Milk

Early studies indicated that milk from grazing cattle contained a higher content of carotenoids than milk produced from winter

feed (Gillam et al., 1933). Carotenes predominated, 0.5-6.0 ppm by weight of butterfat, over xanthophylls, 0.1-0.7 ppm. The carotenoid content of cow milk showed little change between the second and fortieth weeks of lactation (Chanda, 1952). Total carotenes were 4.3-6.0 ppm, 65-85% of the total carotenoids. New Zealand butterfat contains 9-14 ppm total cartenoids, depending on the season of the year (McGillivray, 1956). Reinart and Brown (1953) summarized variation in the total color of butt e r from Sweden and parts of the U.S. and Canada, a s shown in Fig. 13. Total carotenoids range from 2 to 13 ppm. Goat milk contains no carotenoids (Chanda, 1952). Fluorescent light causes destruction of 20-40% of beta-carotene in fluid milk in 12 h r of exposure (Sinha, 1963). 8. Food Animals

The enormous variation in carotenoid content in the fat, muscle, and organs of food animals makes i t impossible to give more than generalizations on occurrence and concentrations. Goodwin (1954b) classified mammals into three groups according t o ability to metabolize carotenoids. I n Group A, the tissues accumulate carotenoids indiscriminately ( m a n ) . In Group B, mainly carotenes accumulate (cattle and horses). In Group C, carotenoids do not accumulate at all (sheep, pigs, goats). Deuel (1957) suggested Group D (birds), which accumulate xanthophylls.

238

B. BORENSTEIN AND R. H. BUNNELL

..

color

L

Jon

I

Feb

I

I

I

I

Mar

Apr

May

June

I

July

of

butter

I

1

I

I

Aug

Sepi

Oct

Nov

Dec

FIG.13. Variations in the natural pigments in butterfat according to different investigations, expressed as micrograms of carotene per one g r a m of butterfat. F r o m Reinart and Brown ( 1 9 5 3 ) .

It is obvious from this classification system t h a t pork, mutton, and lard have little or no carotenoids. Sheep liver contains a trace of total carotene, approximately 1-3 ppm (Peirce, 1945). Palmer and Eckles (1914) established the fact that the chief carotenoid in cattle tissue is beta-carotene. Approximately 2 ppm of betaplus alpha-carotene were crystallized from cow f a t by Zechmeister and Tuzson (1934). No xanthophylls were present. Palmer (1922) reported xanthophylls in chicken f a t and skin and egg yolks. The livers of hens and turkeys contain xanthophylls (Guilbert and Hinshaw, 1934). Egg yolks contain 3-89 ppm carotenoids (Osadca and De Ritter, 1965). The primary pigments a r e lutein, zeaxanthin, and cryptoxanthin. ,9. Ma?.ize Life

Astaxanthin, 3,3’-dihydroxy-4,4’-diketo-beta-carotene, is widely distributed in the crustaceans (Goodwin, 1954a). A protein com-

CAROTENOIDS-PROPERTIES

AND FOOD USES

239

plex of astaxanthin is the characteristic pigment of the lobster carapace. It is well established that astacene, tetraketo-betacarotene, which is reported in the earlier literature as a crustacean pigment, is an extraction artifact of astaxanthin (Kuhn e t al., 1939). Daphnia contain canthaxanthin, echinenone, and betacarotene (Thommen and Wackernagel, 1964). Astaxanthin, lutein, and taraxanthin are the major carotenoids of the many red fishes (Goodwin, 1950). Thommen and Gloor (1965) reported recently that the seatrout (Salmo t ~ u t t a )contained considerable quantities of canthaxanthin as well as astaxanthin and beta-carotene. Astaxanthin and beta-carotene have been found in several salmon species (Deuel, 1957). The major storage sites of the carotenoids in fish are the skin, muscle, and ovaries. The xanthophylls of the skin and muscle are esterified, whereas those of the ovaries are not. B. STABILITY The stability of food carotenoids during and after food processing has received considerable attention. Specific examples have been cited above. The common unit operations of food processing in general have only minor effects on the carotenoids. The naturally occurring carotenoid-protein complexes apparently are more stable than the carotenoids per se. Blanching, retorting, and freezing generally cause little o r no degradation. Frozen foods and heat-sterilized foods, with few exceptions, exhibit excellent carotenoid stability throughout their normal shelf life. Powdered dehydrated fruits and vegetables have poor carotenoid stability unless stored in a n inert atmosphere. 1. Canning

Fellers (1940), in a review, indicated that canning and subsequent storage at ordinary temperatures did not materially affect the total carotene content of processed foods. Booher and March (1941), from a r a t bioassay, reported a n increase in vitamin A value of green beans, collard, endive, kale, iceberg lettuce, peppers, frozen spinach, and turnip greens on canning. Total carotene is relatively stable during the canning of sweet potatoes (Arthur and McLemore, 1957). Canned tomato juice retained over 95% of original total carotene during 2 years at 50-80°F (Sheft et al., 1949). Cis-trans isomerization occurs in carrots on canning (Table XII). Total carotene in spinach is stable on canning, but lutein undergoes substantial losses when spinach is proc-

240

B. BORENSTEIN AND R. H. BUNNELL

essed a t 240"F, and minimal losses a t 270-280°F (Tan and Francis, 1962). Isomerization of 5,6-epoxides to 5,8-epoxides in canned orange juice has already been discussed. Peaches lost 50% of their initial total carotenoids on canning (Mitchell e t al., 1948). Since the carotenoid distribution of peaches is similar to that of orange juice (Curl, 1959), and the peach pH is low, one may conjecture that isomerization of 5,6-epoxides is a factor in the high losses in the canning of peaches. Glass packing of peas, spinach, carrots, and tomato juice, with subsequent exposure t o diffused light, had little effect on total carotene (Fellers and Buck, 1941). Both glass-packed and canned asparagus, snap beans, peas, and corn had excellent carotene stability (McConnell e t al., 1945). Gstirner and Saad (1959) reported losses of 33% carotene in 44 days in light-exposed glasspacked spinach puree, but in those experiments sealed test tubes were only '/3 filled with product, resulting in unrealistically large headspaces compared with commercial glass packing. 2. B l a n c h i n g and F r e e z i n g

Blanching had little effect on the total carotene content of asparagus, snap beans, peas, and corn (McConnell et al., 1945). Blanching and freezing had little effect on asparagus and Lima beans carotenoids (Zimmerman e t al., 1941). Frozen corn had little change in beta-carotene, cryptoxanthin, or total carotenoids during 9 months of storage at 0 ° F (Tichenor e t al., 1965). Frozen broccoli had no loss in total carotene during storage a t O'F for 61 weeks (Martin e t al., 1960). 3 . Dehydratiow

Powdered dehydrated carrots lost 21% carotenoids in one month a t 40°C stored in air, 3s. 0% loss stored in vacuum (Mackinney e t al., 1958). Commercially dehydrated sweet potatoes lost 10% of their total carotene in 18 weeks at 40"-50"F (Mallette e t al., 1946). Concentration of tomato solids in the manufacture of tomato purke causes lycopene degradation, but this is minimized by deaeration and high-temperature short-time heat treatment (Monselise and Berk, 1955). The degradation of lycopene in centrifuged washed tomato pulp under different heating conditions has been studied (Cole and Kapur, 1957). Heating for 3 h r a t 100°C in daylight under a current of oxygen caused the greatest degradation : 33% loss.

CAROTENOIDS-PROPERTIES

A N D FOOD USES

241

4. Storage o f Unprocessed Foods The storage of leafy green vegetables at 32"-70"F causes carotenoid losses (Ezell and Wilcox, 1962). Wilting cannot be used as a measure of rate of carotenoid destruction. Four days at 32°F caused 0-13% losses in carotene in turnip, kale, collards, and rape. A t 50"F, losses were 17-41%. As already discussed under individual vegetables, the storage of roots and squash actually causes increases in carotenoid content due to continued synthesis. 5 . G a m m a Radiation

The effects of gamma radiation on carotenoids have been studied in a variety of systems. Lukton and Mackinney (1956) found films of beta-carotene and lycopene in the solid state to be surprisingly stable: 2% loss at 2 million rep. Solutions of betacarotene and lycopene were unstable in petroleum ether, methyl stearate, methyl oleate, and methyl linoleate. Stability was better in stearate than in oleate and linoleate. They concluded from this work that destruction is caused by secondary reactions and depends upon the extent to which free radicals or peroxides, formed in the surrounding medium, a r e available f o r reaction with carotenoids. The same workers studied the effects of gamma radiation on tomato purkes, whole tomatoes, carrot purkes, and prawns. Carotenoid stability was excellent at doses up to 12 X lo6 rep in the vegetable products (see Table X X ) , but the astaxanthin content of the prawns decreased as much as 60% at 4 X loG rep. Tissue damage occurred in whole tomatoes at doses as low a s 1.0 x lo6 rep. Irradiation of green tomatoes retarded the synthesis of lycopene, and at high levels prevented it (Salunkhe et al., 1959). The red color of tomatoes faded at doses of from 5 X lo5 to 1 X lo6 rads. Doses up t o 3.72 x loc rads had no apparent effect on the carotenoids of canned apricot nectar, peach nectar, or peach halves (Salunkhe et al., 1959). Franceschini et al. (1959) studied the effects of gamma radiation on the carotenoids of carrots, sweet potatoes, green beans, and broccoli. Green bean carotenoids were unstable when irradiated at 1.86 megarads after freezing, but reasonably stable when irradiated at room temperature. The other vegetables in this study did not exhibit this freezing-radiation interrelationship. The carotenoids of sweet potatoes showed relatively little de-

242

B. BORENSTEIN AND R. H. B U N N E L L

T.4BJ.E S X

s:LI1IpIc

rep X

loG

11011f’

5. 95 Ilollt’

12 0 nonr 20.0

C‘oohed tomato purCcs

none 1 2 4

()i~:tngc-typetomato pur& emulsified with methyl liiiolcriatc

none 1 2 3

4 5

.4ir

Kitrogm

Air

Nitrogen

i ( j .ti ,.-

80.8 80.4 83.0 79. 1 99.0 82.0

63.6 55.4 100.8 ‘30.0 100.0 80.5

72.3 63.3 102.1 103.1 100.5 S3.0

is.5

82.2 81.1 83.0 74.5

75.0 80.0 79.0 76.0 69.0 66.0 65.0 65.0 65.0 G4.0

\\ hole red tomatoes

:ifter irradiation, (d:iys) 0

4

7

11

none 1 2 nontl 1 2 nonc 1 2 none 1 2 nnrw 1

2 4 none 1 2

62.5 63.0 63.0 61.5 65.5 G6.0

57.0 50 . 0 59.0 56.3 GO. 5 59.0 1”” 128 12s !J8 94

89 80

Air

CAROTENOIDS-PROPERTIES

243

AND FOOD USES

TABLE XX-(Continued)

DOSC

Bcta-carotene

Lyropeiie

P d g

PRIR

-

Astaxan thin

OD485 0.303 0.218 0.239 0.164

Boilrd prawns

0.356 0.261 0.180 0.165

none 1 2 4

1,uhtori and Rlitckinney (1'356).

struction on irradiation a t 1.86 megarads. However, visual color changes were greater than pigment changes in storage and were highly dependent on storage conditions. These data a r e summarized in Table XXI. Carotenoid destruction of broccoli was 25-50% a t 1.86 megarads. Carotenoid destruction of carrots was moderate at 1.86 megarads except when the carrots were irradiated in a n air atmosphere. Packing in nitrogen improved retention of carotenoid pigments of irradiated sweet corn (Tichenor et al., 1965). Stability data after irradiation and storage are shown in Table XXII. Retention of beta-carotene, cryptoxanthin, and other carotenoids is good a t 1.0 megarad but falls off a t 3 and 5 megarads. Lai e t al. (1959) gamma-irradiated both a hard red spring wheat and a hard red winter wheat. Total carotenoids per 100 g of flour decreased from 13.7 mg to 10.5 at 1.0 X loF rep. These values for carotenoids in flour are higher than those reported by other workers. 6 . Home Cooking

Cooking had little effect on carotenoids in Lima beans (Cook et al., 1961). Retention of total carotene was almost 100% when fresh o r frozen broccoli was cooked (Martin et al., 1960). Microwave cooking had no effect on total carotene in peas (Eheart and Gott, 1964). Losses were small or zero in carrots and broccoli that were microwave-cooked (Thomas et al., 1949; Chapman et al., 1960). Cooking carrots in distilled, tap, or salt water a t at-

244

B. BORENSTEIN AND R. H. BUNNELL

Irrndiated a t room ten?perst urc Vacuum Kitroger1

1rradi:itr.d

Air

iI’(JZt’1l

Vacuum n‘itrogcii

__ ;\ir

Green bearis Caroteneb Xanthophyllb Broccoli Carotene Xanthophyll

95 155

15;

Sweet potatoes Total carotenoids p-carotene Xanthophyll Reflectancec Huec Chroma

351) ;325 1.72 19.7 34.5 27.2

Carrots Total carotenoids p-carotene a-carotene Xsnthophyll Reflectance Hue Chroma

574 352 109 6.3 12.0 41.3 30.4

a

(a2

78 129

103 174

111 “13

92 1i!)

393 354 1.75 18.9 34.3 2ti.8

374

396 363 2.04

11;

3i1 338 1.67 17.0 29.G 23.0

496 326 84 5.35 12.5 40.8 30.2

344 222 58 6.43 12.9 37.6 28.7

hl

339 1.65 18.2 29.9 24.S

20.4 34.0 27.0

585 377 115 5.38 12.9 40.9 31.4

;B:$ 2.U:3 18.1 33.0 25.2

11.5 2,5!J

_3I

4.ljl 14.5 42.2

32.5

3 18 208 s:3 3.93 15.3 39.5 ;10.4

Franceschini et al. (1959). Pignient contents in p g / g dry weight. The reflectance, hue, and chroma were respcctivc~lyrepresented by ISd, tnn-’ a / b , and

+

b2)l’z.

>:itch datum was obtninrd from mi average of 10 cans, i.c., 2 at, c:tch of five storage

periods.

mospheric pressure or in a pressure cooker had no effect on total carotene (Lantz, 1949). IV.

ADDED CAROTENOIDS IN

FOOD PROCESSING

Carotenoid addition to foods predates the commercial synthesis of beta-carotene. Carrot extracts, palm oil extracts, annatto extracts, and oleoresin paprika have been used for generations

TABLE SSll CAROTENOID COKTENT (pg/g) OF IRRADIATEDA N D UNIRRADIATED SWEET Coma

Pack Nitrogen

Atmospheric

a

Gammairradiation treatment (megarads)

Storage tcmpcrature (OF)

Beta-carotene*

Cryptoxanthinb

Other carotenoidsb

4

92

197

4

02

1%

4

!)3

197

None

0

.8

1.0

1.0

2.9

3.4

3.5

4.6

4.7

4.9

None 1.0 3.0 5.0

35 35 35 35

1.0 .G .4

1.3 .7 .4

9

.%

1.1 .8 .(i .2

2.8 2.8 1.7 1.0

2.5 2.7 1.7 1.0

3.0 2.9 1.6 1.0

4.1 4.0 2.3 1.5

4.8 4.0 2.5 1.4

5.6 4.4 2.6 l.G

.o

1.1

3.4

2.8

3.0

4.7

4.0

4.8

1.0 .2 .3 .2

1.0 .3 .3 .2

3.0 1.9 1.3

2.5 2.8 .i .7 1.2 .!)

4.7 2.3 1.8 1.4

4.9

5.6 .7 1.2 1.1

None

0

None 1.0 3.0 5.0

35 35 35 35

.I

-

. I

.9

.7 .4 .2

Tichenor et al. (19G5). Values of carotenoid content are given for 4, 92, and 197 days of storage.

.7

.7

.7

.G l.G 1.0

246

B. BORENSTEIN AND R. H. BUNNELL

to color cheese, butter, soups, sausage products, etc. The advent of pure synthetic carotenoids has increased interest in coloring foods with these compounds because of the obvious advantages of working with well-controlled, reproducible color sources. Carotenoids a r e added to foodstuffs for both nutrition enrichment and color improvement. The major carotenoids, natural and synthetic, used to color foods a r e listed in Table XXIII. TABLI: s s I r r NATI-RAL .\SD R Y s n i m i r C~ROTENOIDS T T .\< FOOD ~ ~ C o~~ o a i s ~ .

___-.

.

(’nrotmoi d

IJisiii cu-carotcne @-carotene P-apo-8’-caroten:tl Canthasnnthin P-apo-S’-carotenoic acid ethyl cst,er Capsanthin Capsorubin

1.1-.x lOG/g Provitamin A activity Xonc 0.88 1.66 1.20 Xonc 0.4 None None

A. NATURALCAROTENOIDCOLORANTS The natural carotenoid extracts of major importance are annatto and oleoresin of paprika. Carrot extracts and palm oil extracts, both composed primarily of alpha- and beta-carotene, a r e also available. Bixin (2) is the primary pigment of annatto (Karrer and Jucker, 1950 ; Diemair e t al., 1952).

Commercial extracts a r e either dilute oil solutions 01- alkaline aqueous solutions. McKeown and Mark (1962) believed that pigments other than bixin in annatto preparations a r e not derived from the Bixa seeds but a r e formed during extraction. Table XXIV shows typical bixin and total pigment concentrations of commercial annatto preparations. Major uses of annatto a r e in coloring butter and cheese.

CAROTENOIDS-PROPERTIES

A N D FOOD U S E S

247

The carotenoids of paprika have received extensive attention. The major carotenoids were identified by Zechmeister and Cholnoky (1934) as esters of capsanthin, capsorubin, cryptoxanthin, and zeaxanthin. Beta-carotene was identified as the major carotene (Zechmeister and Cholnoky, 1943).

Bixin (% of total pigments)

Total pigments

c I>

I.:

F

Run

(niloo R )

1 2

0.608 0.608

0.266 0.277

4i.7

1

2

0 , 33% 0 332

0 . lo:% 0.103

31.0

1 2

0.570 0.575

0.337 0,340

59.1

1 2

1.45 1.45

0.468 0.462

32.1

1 2

0.248 0.252

0.129 0.128

51.6

1 2

0.685 0 . G80

0.297 0.288

4%.5

McKeown and Mark (1962).

Commercial paprika preparations are usually color-standardized oleoresins. Bunnell (1957) chromatographed commercial oleoresin of paprika, a 50,000-color-units product (see Table XXV) . The correct structures of capsorubin and capsanthin have only recently been determined (Barber e t al., 1960) although cap-

____

TABLE XXV CAROTENOID COMPOSITION OF OLEORESIN PAPRIKA~ ~~

Carotenes Zeaxanthin and rryptoxanthin esters Capsanthin esters Capsorubin esters a

Bunnell (1957).

10.6% 22.6% 43.5% 18.7%

248

B. BORENSTEIN AND R. H. BUNNELL

santhin was first crystallized in 1927 and capsorubin in 1934 (Zechmeister and Cholnoky, 1934). The major uses of paprika a r e in coloring cheese, dressings, and sausage products.

APPLICATIONS OF S Y N T H E T I C CAROTENOIDS B. FOOD 1. Beta-Carotene

The commercial syntheses of carotenoids were discussed in Section 1. Beta-carotene is probably the most widely used synthetic carotenoid. The major uses in the United States a r e to color and fortify margarine, shortening, fruit drinks, popcorn, and baked goods. Many of these applications a r e reported in the literature. Bauernfeind et al. (1958) discussed the coloring of fat-base foods such as butter, margarine, shortening, and processed cheese. The use of beta-carotene in citrus beverages, primary cheese, egg yolk products, ice cream, and cake mixes was studied by Bunnell e t al. (1958). Stability of beta-carotene in fruit drinks is shown in Table XXVI (Bauernfeind et al., 1962). Analytical methods have been developed for distinguishing added carotenoids in fruit drinks (Bauernfeind et al., 1962). The coloring of popcorn was discussed by Borenstein (1962). Beta-carotene, ethyl bixin, Yellow AB, and Yellow OB were compared for use in fat-based foods by Geminder and MacDonough (1957). The use of synthetic beta-carotene to standardize butter color throughout the year has been studied successfully (Riel and Johns, 1957). Beta-carotene has been used commercially to color frozen French-fried potatoes. Margarine is frequently fortified and colored simultaneously by using the combination of beta-carotene plus vitamin A palmitate to achieve the U.S. Standard of Identity requirement of 15,000 units of vitamin A per pound. The concentration of beta-carotene added to produce a pleasant, butterlike color ranges from 4,000 to 6,000 units (2.5-4.0 mg) per pound, the remaining quantity of vitamin A being supplied by synthetic vitamin A palmitate. Whipped margarine requires higher levels of colorants to achieve butterlike hues; normally used in this application is 5-7 mg of beta-carotene per pound. Stability of beta-carotene in margarine is excellent (Marusich et al., 1957 ; Melnick et al., 1953 ; Deuel and Greenberg, 1953)) (see Table XXVII) . An interest by food processors in standardized egg yolks of darker-color for use in bakery products, macaroni, and mayonnaise has prompted investigation of the addition of beta-carotene

STABILITY

OF

TABLE XXVI ADDED@-CAROTENEI N COMMERCIALLY PROCESSED JUICES,

JUICE

DRINKS, AND JUICE CONCENTRATES" 8-C,zrot,erw

Yo retention Size of can Product

N Ip

W

Juice Orange juice Juice drinks Juice blend Orange drink I. Enamel-lined can Plain tin can 11. Enamel-lined can Plain tin can 111. Enamel-lined can Plain tin can Enamel-lined can Enamel-lined can IV. Enamel-lined can Orangeade Pineapple drink Pineapple-orange drink Juice blend Concentrates Orangeade base Orangeade base Orangr juice Orangr drinka

(02)

Type of processing

8-carotene product

Initial assay mg/8 oz

6 months a t 75°F

12 months a t 75°F

46

Heat

Beadlets

1.04

83

-

29

Heat

Beadlets

1.28

90

100

10

Heat

Beadlets

1.12 1.10 1.12 0.96 1.26 1.01 0.89 0.80 1.23 2.2 0 0.31 0.63 I .:!I2

90 94 96 108 89 104 86 100 100 125 84 100

i.87 i .i 3 !).I)!) 0.9!)

93 104 85

12

Heat

46

Heat Heat Heat Heat Heat

Beadlets Beadlets Beadlets Beadlets Beadlets Emulsion Emulsion Beadlets Beadlets Beadlets Emulsion

Heat Heat Heat, Frozen

Beadlets Beadlets Beadlets Headlet s

6

46 46 46 6 G :3 2

6.4

-

92

-

95 117 85 10G 85 91 100 90 129 -

90 -.

S:i

210

R. BORENSTEIN AND R. H. RUNNELL

After 2 months' s t o m p

Tnitinl assay provitamin A 1 I:i riufwturer (unitsflb) I05 9i

Ii I,

5:wo 5800

!IS !Ki

6100

100

S ,?

!I2 10" ~

-

~

!lI

!I1

1 0I 10:i

I'

(1 A v.

!I1 105

!)I

101

!)!I s4

!J4

5750

!Ji

ti000

!I5

53t50 5030

!)-i !li

01 !I0 !I2 !J5

5500 ti500

!)S

!I7 10" lo:( 107 !)ti 97

9!J

98

94

8!)

t o Irozen a n d dried yolk products. The color of yolk products h a s been expressed in t e r m s of carotene concentration by t h e Technical Committee of t h e National Egg Products Association (see Table X X V I I I ) . A color assay method in which the absorbance of a n acetone ext r a c t of yolk products is determined at 455 mp. a n d then converted t o equivalent beta-carotene concentration is used by the AOAC (1960). The stability of added beta-carotene in frozen a n d dried egg yolk is shown in Table XXIX, taken from

CAROTENOIDS-PROPERTIES

AND FOOD USES

261

Bauernfeind et aZ. (1958). Under usual storage conditions, retention was 90-95%. Sweet-dough pastries have been commercially colored with betacarotene. Three m g per lb produces a n attractive yellow. Cupcakes with 3-5 m g beta-carotene per l b have been marketed. I n these applications, beta-carotene oil suspensions a r e dissolved in t h e oil o r shortening before t h e dough is mixed.

15 40 70 90 120 150

Poultry p a r t s have been colored experimentally by immersing them in cracked-ice chill t a n k s containing emulsified beta-carotene. The immersion time required depends on the beta-carotene concentration. A n attractive, uniformly yellow skin is produced. Chicken p a r t s colored in this manner can t h e n be frozen, a n d t h e color is stable f o r 12 months at -10 C. T h i s application h a s potential f o r frozen poultry dinners a n d other consumer convenience foods. 2 . Bet a-A po-8 '-Ca1'0t e nal

~:eta-apo-$'-carotenal is used t o color f r u i t drinks, dressings, a n d gum candies. The utilization, stability, a n d analysis of betaapo-8'-carotenal in carbonated beverages, p r i m a r y a n d processed cheese, cake mixes, a n d F r e n c h dressing was discussed by Bauernfeind a n d Bunnell (1962). Food uses were also reported by P a r m a n a n d Borenstein (1964). I n m a n y applications, beta-apo8'-carotenal produces hues similar t o t h a t of oleoresin of paprika. This is particularly t r u e in sausages a n d French dressing. I n tinctorial potency, 20% beta-apo-8'-carotenaI oil suspension is equivalent t o 340,000 C.V. paprika (Borenstein a n d Dowell, 1964).

TABLb: XXIX D-('AROTENE STABILITY I N I'm YOLK

Form of carotene Frozen egg yolk 1 . None (NEPA No. 2) 2. @-rarotcnrsuspension,n I d I323 3 . @-rarotmcsuspension,a*b I,ot 0001~

Container Polyethylene bag inside sanitary metal can

Appearancc I-niforni light y e l l o ~ I'niform orangc 17niforin or:ingv

:< .I I2 7 1:< 5

:I, 0 12 7 I:< 5

N

~-

u1

4 0 10 -1 I 1 :<

__

~

N

Dried egg yolk 4. None (NEPA No. 2) 5 . @-carotenesuspensiona 6. @-carotenesuspcnsiona>" a

Polyethylene bag in cardboard containcr

@-caroteneaddition converts NEPA KO. 2 yolk t o N E P A No. 6.

* Laboratory preparation with especially small carotene crystals. 3 months storage. 15 months storage.

Initial

45"l;'.

6.2 I-niforni light yellow Cniforni yc~llow-orang(% 2 3 . 4 I'niform ycllow-orangs 24.0

4.8 2'2. G 21.9

'75'FL 81i"P'" 4,s 20.4 20.6

4.5 21.4 21.7

%OFr

4.8 21.7 21.2

CAROTENOIDS-PROPERTIES

AND FOOD USES

253

The use of beta-apo-8’-carotenaI in combination with betacarotene expands the color range of these compounds. For ex3 mg beta-carotene per Ib of ample, 5 mg beta-apo-8’-carotenaI processed cheese has been preferred by some cheese makers to cheese colored with either carotenoid separately.

+

3 . Canthaxanthin

Canthaxanthin, which is not yet approved in the U.S., is used in Europe to color tomato products and is fed to poultry to color their shanks and skin. Three to six mg per pound simulates the color of cured meat pigments in sausage products (Borenstein and Smith, 1964). Beta-apo-8’-carotenoic acid ethyl ester is used in Europe to pigment egg yolks.

c. SYNTHETIC CAROTENOID HUESI N

FOOD APPLICATIONS

The hues obtainable with carotenoids a r e dependent both on the market form used and the type of food involved. I n general, beta-carotene produces yellow to orange colors in oil solution. One to 10 mg per liter produces a yellow hue; 30-50 m g per liter approaches the orange range. Usual use rates of beta-carotene in foods a r e 2-20 ppm. Beta-ap0-8’-carotenal is more orange than beta-carotene in oil solution, and becomes red as concentration increases. The solubility of carotenoids in triglycerides is low, as shown in Table 111. It is frequently necessary to heat oils to achieve the desired concentration (see Section I1 for a discussion of physical properties). The solubility of canthaxanthin in triglycerides is so low that practical applications in fat-based foods have not yet been achieved, despite its very high tinctorial potency. In certain food uses the low solubility of canthaxanthin may be advantageous, e.g., in coIoring formulated tomato-based foods-spaghetti sauce, tomato soup, pizza sauce-it is desirable that the incorporated vegetable oil blend unobtrusively. A water-dispersible form of canthaxanthin has been developed which simulates the natural color of lycopene-rich tomatoes very well. When added to tomato products, the canthaxanthin remains in the aqueous phase, producing an attractive red hue, while the oil phase has a slight yellow hue which is not readily observed. It is interesting to note the relative tinctorial potency of carotenoids in oil. Bunnell (1958), using the Dubosq colorirneter, reported the data shown in Table XXX. In aqueous foods it is necessary to use water-soluble or waterdispersible forms of carotenoids. The only commercially available

254

B. BORENSTEIN AND R. H. BUNNELL

water-soluble carotenoid is bixin, which is a carboxylic acid. Bixin is soluble in alkaline aqueous solutions a n d is available in this f or m. The other carotenoids can be solubilized by preparation of oil-in-water emulsions in which th e carotenoid is in t h e oil phase. Such products are commercially available in variety a n d are discussed later in th is article. W a t e r dispersions of th e carotenoids frequently produce colors which differ fro m the equivalent concentration in vegetable oil. Beta-carotene dispersions are yellow-orange t o orange r a th e r th a n yellow. Beta-apo-8’-carotenaI produces peach-orange hues at low concentration an d tomato-like colors a t 40 ppm. Canthaxanthin is reddish at all concentrations in aqueous dispersion a n d resembles tomato juice o r whole blood a t 30-40 ppm.

llethyl bixin Ethyl hixin Bixiii Lycopene Cant haxanthiri Isozeaxanthin Beta-apo-S’-caroteiiaI

1.31 0 . !)‘I

53 2.85

:j

4.76 0.30 3 .3 0

Water-dispersible carotenoids do not produce the sparkling clarity of t r uly water-soluble compounds, a n d hence a r e limited in applications where clarity is necessary. The opacity resulting from higher use levels of water-dispersible carotenoids in aqueous foods simulates the insoluble f o r m of th e natural pigments in f r u i t tissue a nd therefore produces x more natural appearance th a n water-soluble azo colors in f r u i t drinks. The carotenoids, therefore, are t he preferred colorants f o r orange drinks. Food emulsions such as Fren ch dressihg can be colored with carotenoids in either oil- or water-dispersible forms. Emulsion colors a r e generally lighter t h a n th e colors produced by the same concentration of carotenoid dispersed in water. Stability of beta-apo8’-carotenal in Fren ch dressing is shown in Table X X X I. The stability of beta-ap0-8‘-carotenal in French dressing exposed t o sunlight is superior to t h a t of oleoresin of paprika, as shown in Table XXXII (Jah n s, 1965).

CAROTENOIDS-PROPERTIES

AND FOOD USES

255

TABLE S X X I

STABILITY OF BEPA-APo-S-C'W O T E N A L (per ccnt rctention)

IS

I < ' I ~ X C I1 I)RESSIS(;

\ v I T H O U T L4NTI OXI DANT S

Initial assay (ppm)

-

3 wc(llis' su~iliglit" 3 weeks 113°F

10 (1

88

58

3 iiionths 11T

-

100

Plus 2 montils at ItT.

The carotenoids are among the highest-tinetorial-potency colorants available, and are generally used a t concentrations of 120 ppm by weight of food. The FDC azo dyes, in comparison, are used a t concentrations of 40-300 ppm. For example, 60 ppm FDC Yellow #6 is a commonly used concentration in coloring orange soda. This color can be closely simulated by 10 ppm betacarotene or a combination of 5 ppm beta-carotene plus 1 ppm beta-apo-8'-carotenal. It is not always possible to match the color of azo dyes with carotenoids; however, combinations of carotenoids can be made which expand the number of hues achievable.

B L E A C H I N G OF

TABLE xxxrr FRENCH DRESSING I N DIRECT SUNLIGHT" Rcta-npo-S'-CarotenaI

1 day 2 days 3 days

None Slight Moderatc

Olcoresin paprika

Moderate Severc Complctc

___a J:diiis (1965).

._

D. STABILITY O F ADDEDCAROTENOIDS I N FOODS Stability data for added carotenoids in specific foods reported by Burinell and Bauernfeind (1962) and Bunnell et al. (1958)

are given elsewhere in Section IV. 1. EfSect of p H

Carotenoids are not pH-sensitive in the normal food range of 2-7 except for those which have a carboxyl group such as bixin, which changes hue and solubility with pH. Beta-carotene, betaapo-8'-carotenal, and canthaxanthin do not change hue with pH, and are stable in foods a t p H 2-7. Beta-ap0-8'-carotenal is unstable at high p H because of its aldehyde group.

256

I3. BORENSTEIN AND R. H. BUNNELL

2 . OzidatioiL All carotenoids have a conjugated carbon-carbon double-bond chain, which is responsible for their colorant properties. The large number of double bonds is similarly responsible for the sensitivity of carotenoids to oxidation. All carotenoids have this property t o some degree. Lycopene is perhaps the most unstable carotenoid. Synthetic lycopene crystals are unstable even in nitrogen-gassed ampoules. The formation of beta-ionone rings-lycopene compared to beta-carotene-markedly increases oxidative stability. Canthaxanthin is somewhat more stable than beta-carotene, and betaapo-8'-carotenal somewhat less stable, but these generalizations must be used very cautiously in food applications. Oxidation of carotenoids is accelerated by light. It is advisable, therefore, to minimize the oxygen content of clear-glass-packed food products colored with carotenoids. This can be accomplished by several methods, e.g., hot packing, vacuum sealing, oxygen scavenging with ascorbic acid. The addition of antioxidants, e.g., BHA, BHT, is helpful if the carotenoid is in the oil phase of the food products. Oxidative degradation of beta-carotene and betaapo-8'-carotenal causes a loss of color and a loss of vitamin potency. The rupture of any double bond in the chain causes a complete loss of color and vitamin A activity. Stability of waterdispersible canthaxanthin in carbonated soda is shown in Table XXXIII (Manz, 1965). Canthaxanthin can be used to color carbonated soda without oxygen scavenging. The stability of betaTABLI: S X X I l I C INTH I X A N T H I S I > CAI~BONATEII SOD1

7 '0 \ I y/hot tlc

12.0

l r i i t i d determination

I Xiect sunlight-ivoiii

tenl1)

1 month '2 months ti months 12 months

I )ark room-roo111 6 months 1'2 months

retention

11.2 10.0 !I. 0 8.4

!IS 8:1 75

11.4 !I. 5

95 79

i0

tcinp.

CAROTENOIDS-PROPERTIES

A N D FOOD USES

257

TABLE XXXIV S T A B I L I T Y 01.' C A R O T E N O I D S I N CARFJON.4TED BEVER-LOES L v I T H .4ND W I T H O L T

ASCORBIC ACID

~-

Per ccnt retention 1niti:il

1 month direct sunlight

(mgiqt)

____

2 months

86°F

3 months RT

I

.2l,o-carotenal, no ascorbic acid Apo-cm.otena1 100 mg ascorbic acid 8-carotene, no ascorbic arid /3-raroi,c~ne 100 mg ascorbic acid

+

+

3.51

0

83

78

3.G9

16

94

90

5.87

68

07

S6

5.62

78

100

95

carotene and beta-apo-t3'-carotenal is improved by the addition of ascorbic acid, as shown in Table XXXIV. 3 . Heat

Carotenoids are stable to heat in systems with a minimal oxygen content. Oil solutions can be heated under nitrogen to 150°C with only small losses. When beta-carotene solutions a r e heated above 60"C, cis-trans isomerization occurs, resulting in a mixture of stereoisomers consisting principally of all-trans beta-carotene, neo-beta-carotene-B, and neo-beta-carotene-U, with minor amounts of other isomers (Deuel, 1951). The absorption spectra of these three isomers are shown in Fig. 4. Cis-trans isomerization also occurs slowly a t room temperature, but has relatively little effect on tinctorial potency or hue. It should be noted that cis-trans isomers of beta-carotene also occur in foods, e.g., leafy raw vegetables contain 76% trans-beta-carotene, 10% neo-beta-carotene-U, and 10 neo-beta-carotene-B (Fraps, 1947). At temperatures above 200°C, e.g., popping corn, the stability of carotenoids is poor. In a 90-second corn popping cycle, 8010095, of initial beta-carotene content may be destroyed. It is desirable to incorporate 0.01-0.02% antioxidants by weight of popping oil to produce satisfactorily colored popcorn in commercial machines operating a t 200-250°C. The resulting popcorn color is stable up t o 1 year a t room temperature in translucent packages. The degradation of beta-carotene a t popping temperatures causes color loss, but the shape of the spectral curve does not change or shift significantly (Borenstein, 1962). Day and Erd-

258

H. BORENSTEIN A N D R . €I. BUNNELL

man (1963) reported the degradation products shown in Tablc X X X V a f t e r heating a 1% solution of beta-carotene in benzene at 188 C for 72 hours.

The s a n e compounds were also obtained by prolonged heating in a x'acuum at 240°C (Mader, 1964). "hen a nna t to is used i n corn popping, th e spectral curve shifts to t he ultraviolet, an d greenish hues are produced, r a th e r th a n yellow. Similar results are obtained by heating a n n a tto in oil at 125": (McKeown an d Mark, 1962; Fig. 1 4 ) . High-temperature

h a v e l P r g t h (rnpi!

F I G . 14. Absorption spectra of a solution of bixin in corn oil heated at 125°C f o r t h e times indicated (diluted 500 times with chloroform, 10 mm cell). F r o m McKeown a n d M a r k (1962).

CAKOTE'NCIDS-PROPERTIES

AND FOOD USES

259

extyaction, 125"C, of annatto seeds causes degradation of bixin ester of unsymmetrical dito a yellow pigment-monomethyl methyltetradecahexaene-dioic acid plus m-xylene ( McKeown, 1963). Methylbixin produces the corresponding dimethylester. Carotenoids are used to color heat-sterilized canned foods. Water-dispersible beta-carotene and beta-apo-8'-carotenal have good stability in commercially canned soups in retorting cycles of 90 minutes at 240°F. Stability of beta-carotene in commercially processed fruit products is shown in Table XXVI. Stability of beta-carotene in baked goods is dependent on the market form of carotene used and the composition of the baked goods. Retention in white bread is only 5 0 4 0 % . Cakes baked at 350" and 400°F retain 90-95% of the initial values of beta-carotene applied in a 10% water-dispersible beadlet form (see Table XXXVI) (Borenstein and Jahns, 1965). TAB1,T; XXSVI S T t n I L I 1 ' Y 01'

360°F 350°F 350°F 400°F 400°F 400°F ~

~~~~

10'7,

f l - C & R O r E V h I ~ E h D L G r 5IN

c \KE

Addition rate (my/lb of cakr)

Per cent retention

2 3 I0 2 3 10

94

92 !J4

95 90 93

~

Rorenstein and Jnhns (1965).

4. Flavor Added carotenoids in foodstuffs rarely cause flavor problems. At high use rates under conditions causing carotenoid degradation, haylike odors and flavors may develop. 5 . Enzyme

Lipoxidase from soybeans and other legumes will rapidly bleach oil solutions of carotenes, xanthophylls, and bixin (Sumner and Sumner, 1940). This is not often a problem in food applications.

E. INDIRECT COLORATION OF FOODS Carotenoids have been added t o the feeds of animals, poultry, and fish t o color the eventual food products prepared from them. This has been intensively studied since the advent of synthetic

260

B. BORENSTEIN AND R. H. BUNNELL

carotenoids (Bunnell and Bauernfeind, 1962), and is briefly discussed in this section. The pigmentation of broilers, fryers, and market eggs was discussed by Bunnell and Bauernfeind (1958). The coloration of yolks has received particular attention because modern poultry raising practices tend to lighten yolk color. The primary yolk pigments are lutein, zeaxanthin, and cryptoxanthin, but their ratios vary with the feed source (Osadca and De Ritter, 1965). The pigmenting characteristics of 8 synthetic carotenoids for egg yolks were reported by Marusich et a2. (1960b). The use of betaapo-8’-carotenal and canthaxanthin was reviewed by Bauernfeind (1962). A stabilized beadlet form of beta-apo-S‘-carotenal at 4.36 grams per ton of feed produced adequate color for table eggs laid by three layer breeds (Bunnell et al., 1962). De Groote (1964) compared with yolk-pigmenting values of alga meal, paprika, and synthetic carotenoids, and evaluated combinations on the basis of economics, Deethardt et al. (1965) studied the quality of sponge cakes prepared from egg yolks whose color was produced by different feed additives. I n general, cakes made with dark yolks produced by natural xanthophylls had stronger flavor, were less tender, and were coarser in texture than dark cakes colored with yolks containing canthaxanthin or beta-apo-S‘-carotenal. Skin and shank were effectively pigmented by 1-6 g canthaxanthin per ton of feed (Marusich and Bauernfeind, 1962). Eight to 24 g canthaxanthin per ton of broiler finisher ration containing yellow corn and alfalfa meal increased pigmentation effectively (Camp et al., 1963).

F. MARKETFORMS O F SYNTHETIC

CAROTENOIDS

Market forms of synthetic carotenoids are of two general types: oil- and water-dispersible. The pure crystals are rarely used, be-

cause of their poor solubility and stability. The major oil-dispersible forms of commerce are suspensions of micronized crystals in vegetable oil. Such suspensions are very stable because of the low oxygen solubility in the oil phase and the relatively small surface exposure of crystals compared to solutions (Hartmann and Barnett, 1949). These suspensions do not require antioxidants for per s e stability, but may contain antioxidants to stabilize the carotenoids in the end use applications, e.g., popcorn. Stability data are shown in Table XXXVII. Bauernfeind et al. (1962) discussed the vitamin A potency of market forms of synthetic beta-carotene. One gram of all-trans

CAROTENOIDS-PROPERTIES

261

AND FOOD USES

beta-carotene contains 1,666,667 units of vitamin A based on the results of 12 collaborative laboratories. Beta-carotene market forms contain cis stereoisomers, which have been reported (Fraps, 1947) to lower vitamin A activity. The biological potency of water-dispersible market forms of beta-carotene containing both trans and cis isomers is equal to that of all-trans beta-carotene, apparently due to better absorption of emulsified beta-carotene than of oil solutions of beta-carotene. TABI,I< XXXVII STABILITY OF i3-APO-8’-CAROTEN I L O I L

SUSPEN\IOU

Prr cent IC-n~)o-8’-carotenal No Antioxidant

1iiiti:tl 3 weeks a t 113°F 6 neeks at 113°F 6 weeks a t 98°F 3 Iiiontlis at 98°F 3 iiioriths at 75°F I:! Inonths a t 75°F

HHT

+ BHA Added

A

B

A

B

26.0 23.6 26.0 25.7 26.6 23.2 26.6

29.0 2i.!) 28.0 28.0 28.8 29.0 %.ti

26.0 25.6 26.0 25.6 27.4 27.4 27.0

30.7 31.2 29.7 31.2 31.0 31.6 30.1

The water-dispersible carotenoid market forms are available a s liquids and as dry products. I n both types the carotenoids are dissolved or suspended in a n oil phase which is emulsified into a n aqueous phase. The emulsions are stabilized with colloids such a s gelatin or vegetable gums (Bauernfeind and Bunnell, 1958). One method for preparation of a dry water-dispersible product is to heat and dissolve crystalline beta-carotene in vegetable oil containing food-approved antioxidants. Antioxidants can affect the stability of beta-carotene in the market form as well a s in the final food product. As soon as the carotene is dissolved, the hot oil solution is emulsified in a gellable colloid plasticizer composition, such as an aqueous gelatin-sucrose solution. The key to successful preparation of this product is complete emulsification of the hot carotene solution before crystallization has started. After emulsification is complete, the emulsion is sprayed and dried. The oil phase of this product is a supersaturated solution of beta-carotene and remains so on extended storage as a beadlet or when the beadlet is dispersed in aqueous systems (Bauernfeind and Bunnell, 1958). The mechanism by which crystallization is inhibited in this product is not known, but the high surface energy

262

B. BORENSTEIN AND R. H . BUNNELL

of fine emulsions may be involved, or the viscosity of the interior phase may be so high (because of the large surface area) t h a t crystallization is prevented as in glass. There is no generally accepted theory for organic compound crystal nucleation (Walton, 1965). A process has been described (Mueller and Tamm, 1963) for producing emulsions of carotenoids with particularly small oil droplets. In this process the carotenoid plus antioxidants is dissolved in a low-boiling water-immiscible solvent such as chloroform, which is then emulsified into a n aqueous phase. The chloroform is then evaporated, resulting in near-colloidal-size oil droplets. High-potency products of this type a r e now available commercial 1y . The colors, physical stability, and efficacy of water-dispersible carotenoids in specific food uses depend on the food and on the oil globule size of the carotenoid. Colloidal- o r near-colloidalsize oil globules a r e physically stable in aqueous foods, i.e., they are unaffected by the specific gravity of the food. Macro-type oil globules > 2 p. in diameter may cream, depending on the relative specific gravities of the oil and aqueous phases. This can be avoided by adjusting the specific gravity of the oil phase with brominated vegetable oil to match that of the aqueous phase. The macro-type oil globule is unsatisfactory for coloring maraschinotype cherries. I t is necessary to use a colloidal dispersed carotenoid to diffuse through the cell walls of cherries and thus color the product uniformly. A combination of FDC Red # 2 plus betaapo-8'-carotenal produces a n attractive, natural red cherry color in this application. The color is superior to the purplish-red of Red # 2 , when used alone, but is distinctly different from the typical maraschino cherry color produced by Red +t4. A stable dry composition of carotenoids complexed with protein is described by Wingerd and Saperstein (1964). In this method a n emulsion is prepared with skim milk used as the exterior phase, and the emulsion is spray-dried. The disadvantage of this type of product is that the concentration of active ingredient is generally low, i.e., below 1.0%.

V.

ADDITIONAL RESEARCH NEEDS

A. BIOSYNTHESIS A N D FUNCTION O F CAROTENOIDS The intensive work under way in this field promises to unravel the many unsolved problems related to the biochemical mechanisms of synthesis and function. The importance of isopentenyl

CAROTENOIDS-PROPERTIES

AND FOOD USES

263

pyrophosphate in carotenoid synthesis has been well established. Less is known about chain construction from C, to C40, and the precursor interrelationships between the carotenoids have not been clarified. The mechanism of ring closure and the introduction of oxygen function is not understood. The role of accessory pigments in photosynthetic plant tissue needs clarification. The mechanism and significance of energy transfer from carotenoid t o chlorophyll is not known. Other purposes of carotenoids in photosynthetic plant tissue a r e not known. The role of carotenoids in nonphotosynthetic plant tissue is not understood. Almost nothing is known about the possible role of carotenoids in reproduction.

B. COMMERCIALSYNTHESIS Less expensive routes to synthetic carotenoids will continue to be of interest for obvious reasons.

c.

STABILITY O F CAROTENOIDS I N

FOODS

Relatively little is known about the state of combination of the carotenoids in foods o r of the mechanism of carotenoid degradation during food processing. I n most foods, carotenoid degradation is not a serious problem from a color or nutritional point of view. D. COLORINGDRY MIXES An unsolved problem is the coloring of dry mixes and the resulting reconstituted food products. Water-dispersible dry carotenoids in beadlet form are stable in d r y mixes and disperse readily when the dry mixes a r e added to water, e.g., gelatin dessert, cake mixes. Dry mixes colored with beadlets have a spotted or mottled appearance, which is unattractive in the sophisticated U.S. market place. When liquid carotenoid forms a r e used to color dry mixes uniformly, the stability of the carotenoid is deceased. The stability problem can be minimized by spraying the dry mix with corn syrup or other oxygen barriers. This procedure has the disadvantage of increasing product costs. Dry mixes containing f a t s or oils can be colored by dissolving the carotenoid in the f a t phase. In general, at least 5% f a t is necessary to achieve good stability.

E. COLORINGCLEAR AQUEOUSFOODS Methodology that maintains absolute clarity when aqueous foods are colored with fat-soluble carotenoids would be desirable.

264

B. BORENSTEIN AND R. H. BUNNELL

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K a r r e r , P., and Solmssen, U. 1935. Ueberiiihrung von Rhodoxanthin in Zeaxanthin. Helv. Ch im. A c t u 18, 477. Karrer, P., Eugster, C. H., and Tobler, E. 1950. Synthesen von Carotinoidfarbstoffen. 111. Totalsynthese des Lycopins. Helv. Ch h. A c t u 33, 1349. Khan, M., and Mackinney, G. 1953. Carotenoids in grapefruit, C i t r u s p,artrtlisi. PZmt Physiol. 28, 550. Kimel, W., Surmatis, J. D., Weber, J., Chase, G. O., Sax, K. W., and Ofner, A . 1957. Synthesis of pseudoionone homologs and related compounds. J . O r g . Chcnt. 22, 1611. Kimel, W., Sax, N. W., Kaiser, S., Eichman, G. G., Chase, G. O., and Ofner, A . 1958. Total synthesis of pseudoionone and a n isomeric ketone. .I. O r g . Chem. 23, 153. Kuhn, R., and Grundmann, C. 1933. Ueber Kryptoxanthin, ein Xanthophyll der Formel C,,,H,,O. Bet,. 66, 1746. Kuhn, R., and Grundmann, C. 1934. Kryptoxanthin a u s gelben Mais. I:(,),. 67, 593. Kuhn, R., Stene, J., and Sorensen, N. A. 1939. Ueber die Verbreitung des Astaxanthins im Tier- und Pflanzenreich. Ber. 72, 1688. Lai, S., Finney, K. F., and Milner, M. 1959. Treatment of wheat with ionizing radiations. IV. Oxidative, physical, and biochemical changes. Cereal Cheni. 36, 401. Lantz, E. M. 1949. Carotene and ascorbic acid in carrots during growth, storage and cooking. N e w Mexico Agr. E x p t . Sta. Bull. 350. Larner, H. B. 1947. Concentration of vitamin A carotenoids. U.S. P a t e n t 2,432,021. Lewis, E. P., a n d Merrow, S. B. 1962. Influence on t h e estimation of $-carotene by other carotenoids in butternut squashes at harvest and during storage. J . A g r . Food C h e m . 10,53. Lilly, V. G., Barnett, H. L., and Krause, R. F. 1960. The production of carotene by Phyconzyces b~ulicslccunzis.West Virginia Uxiv. A g v . E x p t . S f a . Bull.

441T. Lime, B. J., Stephens, T. S., and Griffiths, F. P. 1964. Processing characteristics of colored Texas grapefruit. I. Color and m a t u r i t y studies of Ruby Red grapefruit. Food Technol. 8, 566. Lime, B. *J., Griffiths, F. P., O’Connor, R. T., Heinzelman, D. C., and McCall, E. R. 1957. Spectrophotometric methods f o r determining pigmentationRuby Red grapefruit. J . Agr. Food C h e w . beta-carotene and lycopene-in 5 , 941. Lord Cohen of Birkenhead. 1958. Observations on carotenemia. A x ? ! . I n t e r , / . M c d . 48, 219. Lotspeich, F. J., Krause, R. F., Lilly, V. G., and Barnett, H. L. 1959. The degradation of labeled p-carotene. J. X i o l . C h e m . 234, 3109. Lukton. A , , and Mackinney, G. 1956. Effect of ionizing radiations on carotenoid stability. Food TcchnoZ. 10, 630. Mackinney, G. 1961. Coloring matters. 171: “The Orange : I t s Biochemistry and Physiology.” (W. B. Sinclair, ed.) p. 302. University of Calif. Mackinney, G., and Chichester, C. 0. 1960. Comparative biochemistry and photoreactive systems. In : “Biochemistry of Carotenoids.” p. 205. A c a demic Press, New York.

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illackinney, G., Aronoff, S., and Bornstein, B. T. 1942. Some assays of p ~ o vitamin A carotenoids. I n d . Eng. C h e m . Anal. E d . 14, 391. JIackinney, G., Lukton, A,, and Greenbaum, L. 1958. Carotenoid stability i n stored dehydrated carrots. Food Technol. 12, 164. AlacLeod, F. L., Armstrong, M. R., Heap, M. E., and Tolbert, L. A. 1935. The vitamin A content of five varieties of sweetpotatoes. J . A g r . Research 50, 181. hlader, I. 1964. Beta-carotene : thermal degradation. Science 144, 533. I. Dawson, C. R., Nelson, W. L., and Gortner, W. A. 194G. Rlallette, &F., Commercially dehydrated vegetables. Oxidative enzymes, vitamin content, and other factors. I n d . E n g . C h e m . 38, 437. Manz, U. 1965. Unpublished work. F. Hoffmann-La Roche & Co. Ltd., Basel. Martin, M. E., Sweeney, J. P., Gilpin, G. L., and Chapman, V. J. 1960. Factors affecting the ascorbic acid and carotene content of broccoli. J . A g r . Food C h e m . 8, 387. RIarusich, W. L. 1962. Unpublished work. Hoffniann-La Roche Inc., Nutley. Marusich, W. L., and Bauernfeind, J. C. 1962. Canthaxanthin a s a broiler pigmenter. (Abstract) P o u l t r y Sci. 41, 1664. Marusich, W., De Ritter, E., and Bauernfeind, J. C. 1957. Provitamin A activity and stability of p-carotene in margarine. J . Am. Oil Chcmisfs’ SOC.34, 217. RIaiusich, W., De Ritter, E., Vreeland, J., and K r u k a r , R. 1960a. Vitamin A activity of beta-apo-S’-carotenal. J . A g r . Food C h e m . 8, 390. Marusich, W., De Ritter, E., and Bauernfeind, J. C . 1960b. Evaluation of carotenoid pigments f o r coloring egg yolks. P o u l t r y Sei. 39, 1338. Matlack, M. B. 1937. The carotenoid pigments of the sweetpotato (Ipon~oeu batatns, Poir). J . Wash. A c a d . Sci. 27, 493. McCarty, C. D., and Lesley, J. W. 1954. The carotenoids, amygdalin content and titratable acidity of white and yellow-fleshed peaches within a nearly isogenic line. Proc. Am. Soc. Hort. Sci. 64, 289. JIcCollum, J. P. 1955. Distribution of carotenoids in the tomato. Food R e s e a ~ ~ l z 20, 55. McConnell, J. E. W., Esselen, W. B., Jr., and Guggenberg, N. 1945. Effect of storage conditions and type of container on stability of carotene in canned vegetables. F r u i t Prods. J . 24, 133. McGillivray, W. A. 1956. The vitamin content of New Zealand butterfat. New Zeala?itl J . Sci. Tccliinol. Sect. A 38, 466. McKeown, G. G. 1963. Composition of oil-soluble annatto food colors. 11. Thermal degradation of bixin. J . Assoc. O f i c . A g r . C h e m i s t s 46, 790. McKeown, G. G., and Mark, E. 1962. The composition of oil-soluble annatto food colors. J . Assoc. O fi c . A y r . Chcmists 45, 761. BIelnick, D., Luckmann, F. H., and Vahlteich, H. W. 1953. The retention of preformed vitamin A and carotene in margarine based upon physicochemical assays. Food Research 18, 504. JIiller, J. C., and Covington, H. M. 1942. Some factors affecting the carotene content of sweet potatoes. Proc. Am. Soc. H o r t . Sci. 40, 519. Nitchell, J. H., Van Blaricom, L. O., and Roderick, D. B. 1948. The effect of canning and freezing on the carotenoids and ascorbic content of peaches. S. Carolina A g r . Expt. Sta. Bull. No. 372.

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AND FOOD USES

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Modi, V. V., and Patwa, D. K. 1961. Occurrence of mevalonic acid in carrots. N a t u r e 191, 1202. Monselise, J. J., and Berk, Z. 1955. Oxidative destruction of lycopene during the manufacture of tomato purke. J . S e i . Food A g r . A b s t r . 6, i, 315; and Bull. Research Council Israel 4, 188 (1954). Monselise, S. P., and Halevy, A. H. 1961. Detection of lycopene in pink orange f r u i t . Science 133, 1478. Moore, T. 1957. “Vitamin A.” Elsevier Publishing Co., New York. Mueller, P., and Tamm, R. 1963. Process of making a carotenoid preparation. U.S. Patent 3,110,598. N a t a r a j a n , C. P., and Mackinney, G. 1952. Carotenoid pigments of orange juice. J. Sci. I n d . Research ( I n d i a ) 11B, 416. National Research Council. 1958. Recommended dietary allowances. N a t l . A c a d . Sci., N a t l . Research Council. Publ. No. 589 Nishimura, M., and Takamatsu, K. 1957. A carotene-protein complex isolated from green leaves. N a t u r e 180, 699. Osadca, M., and De Ritter, E. 1965. Unpublished work. Hoffmann-La Roche Inc. Palmer, L. S. 1922. “Carotenoids and Related Pigments.” Chemical Catalog Co., New York. F’almer, L. S., and Eckles, C. H. 1914. Carotin-the principal natural yellow pigment of milk f a t : i t s relation to plant carotin and the carotin of the body f a t , corpus luteum, and blood serum. 111. The yellow lipochrome of blood serum. J . Biol. C h e m . 17, 223. Parker, H. K., and Harris, M. C. 1964. Wheat pigments and flour color. I n : “Wheat Chemistry and Technology.” (I. Hlynka, ed.). p. 435. American Association of Cereal Chemists, St. Paul, Minnesota. Parman, G . K., and Borenstein, B. 1964. Apo-carotenal: a potent color. Food E n g . 36 ( 5 ) , 77. Pauling, L. 1939. Recent work on t h e configuration and electronic structure of molecules with some applications to natural products. F o r t s c h r . C h e m . org. N a t u r s t o f f e 3, 203. F’eirce, A. W. 1945. The effect of intake of carotene on the general health and on the concentration of carotene and of vitamin A in t h e blood and liver of sheep. A u s t r a l i a n J. E x p t l . Biol. M e d . Sci. 23, 295. Pendlington, S., Dupont, M. S., and Trussell, F. J. 1965. The carotenoid pigments of S o l a n u m tuberosum. Biochem. J . 94, 25P. Petzold, E. N., and Quackenbush, F. W. 1960. Zeinoxanthin, a crystalline carotenol from corn gluten. A r c h . Biochem. B i o p h y s . 86, 163. Porter, J. W., and Anderson, D. G. 1962. The biosynthesis of carotenes. A r c h . Biochem. B i o p h y s . 97,520. Pruthi, 3. S., and Lal, G. 1958. Carotenoids i n passion f r u i t juice. Food Research 23, 505. Purcell, A. E. 1964. Biosynthetic relationships between carotenes. A r c h . Biochem. Biophys. 105, 606. Purcell, A. E., Thompson, G. A., J r . , and Bonner, J. 1959. The incorporation of mevalonic acid into tomato carotenoids. J . Biol. C h e m . 234, 1081. Quackenbush, F. W. 1963. Corn carotenoids: effects of temperature and moisture on losses during storage. Cereal Chem. 40, 266.

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€3. BORENSTEIN A N D R. H. BUNNELL

Quackenbush, F. IV., Firch, J . G., Rxbourn, W. J., RlcQuistan, &I., Petzold, P;. N., and Kargl, T. E . 19G1. Analysis of carotenoids in corn grain. J . A g v . Food Chcwi. 9 , 132. Reinart, A., and Brown, R. W. 19%. Standardizing the colour of butter. I n t c ~ D~ a. i r y Co,cyr. Z’roc., 13th Congr. The Hague 111. p. 1233. Eiel, R. R., and Johns, C. K. 1957. The use of synthetic p-carotene f o r coloring butter. J . D a i r y Sci. 40 ( X L ) , 192. Rubin, S. H., and De Ritter, E. 1954. Vitamin A requirements of animal species. V i t a m i n s a)rd Hormo,rcs 12, 101. Salunkhe, D. K., Gerber, R. K., and Pollald, L. H. 1959. Physiological and chemical effects of gamma radiation on certain f r u i t s , vegetables, and their products. Proc. Am. Soc. H o r t . Sci. 71, 423. Scharer, K. 1960. Unpublished work. F. Hoffmann-La Roche B Co. Ltd., Basel. S c h l r e r , K. 1963. Unpublished work. F. Hoffmann-La Roche & Co. Ltd., Basel. S c h l r e r , K., and Studer, F. 1961. Unpublished work. F. Hoffmann-La Roche & Co. Ltd., Basel. Scott, G. C., and Belkengren, R. 0. 1944. Importance of breeding peas and corn f o r nutritional quality. Food R e s c a r c h 9, 371. Sheft, R. B., Griswold, R. M., Tarlowsky, E., and Halliday, E. G. 1949. Effect of time and temperature of storage on vitamin content of commercially canned f r u i t s and f r u i t juices. Zxtl. E x g . C h e m . 41, 144. Shneour, E. A., and Zabin, I. 1959. The biosynthesis of lycopene in tomato homogenates. J . Biol. C h e m . 231, 770. ‘impson. K. L., Nakayama, T. 0. RI., and Chichester, C. 0. 1964. The biosynthetic origin of the carboxyl oxygen atoms of the carotenoid pigment, torularhodin. Biochem. J . 92, 508. Sinha, S. P. 1963. The effect of fluorescent light on the vitamin A and pcarotene content of milk. Inter12. Z. Vitaminforsch. 33, 262. Stanier, R. 1960. Carotenoid pigments : problems of synthesis and function. In: “The Harvey Lectures, 1958-1959.” p. 219. Academic Press, New York. S t i t t , F., Bickoff, E. M., Bailey, G . F., Thompson, C. R., and Friedlander, S. 1951. Spectrophotometric determination of beta-carotene stereoisomers in alfalfa. J . Assoc. O f i c . A g r . C h e m i s t s 31, 460. Strachan, C. C., Moyls, A. W., Atkinson, F. E., and Britton, J. E., 1951. Chemical composition and nutritive value of British Columbia tree f r u i t s . Caw. DPpt. A g r . P ~ h l .No. 862. Sunincr, J. B., and Sumner, R. J. 1940. The coupled oxidation of carotene and f a t by carotene oxidase. .J. Ciol. Chcm. 134, 531. Surmatis, J. D., and Ofner, A. 1961. A new synthesis of trans-p-carotene and decapreno-p-carotene. d. 0 t . y . Chcrtz. 26, 1171. Tabor, J. M., Seibert, H. F., and Frohring, P. R. 1948. Method f o r extracting pigments. U.S. P a t e n t 2,440,029. T a n , C. T., and Francis, F. J . 1962. Effect of processing temperature on pigments and color of spinach. J . Food S c i . 27, 232. Taylor, A. L., and Witte, P. J. 1938. Carotene in oranges. I n d . E n g . Chcwz. 30, 110. Thomas, M. H., Brenner, S., Eaton, A,, and Craig, V. 1949. Effect of electronic cooking on nutritive value of foods. J . A m . Dictet. Assoc. 25, 39.

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AND FOOD USES

275

Thommen, H. 1962. Ueber das Vorkomnien von Beta-Apo-8’-Carotenal in1 S a f t und in der Schale von frischen Orangen. Na t u rwi sse n sc h a f t c n 49, 517. Thornmen, H., and Gloor, U. 1965. Zum Vorkommen von Keto-Carotinoiden in der Forellr. Natzirzc zssc’nschaften 52, 161. Thommen, H., and Wackernagel, H. 1964. Zum Vorkommen von Keto-Carotinoiden in Crustaceen. Naturwissenscha f t e n 51, 87. Tichenor, D. A., Dudley, C. M., and Wells, C. E. 1965. Carotenoid content of frozen and irradiated sweet corn. Food Technol. 19, 406. von Euler, H., K a r r e r , P., Krauss, E. v., and Walker, 0. 1931. Zur Biocheniie der Tomatenfarbstoffe. Helv.Chim. Aeta 14, 154. Walton, A . G. 1965. Nucleation of crystals from solution. Science 148, 601. Weckrl, K. G., Santos, B., Hernan, E., Laferriere, L., and Gabelman, W. H. 1962. Carotene components of frozen and processed carrots. Food Technol. 16 ( 8 ) , 91. Weedon, B. C. L., and W a r i e n , C. K. 1965. Carotenoid compounds. U.S. P a t e n t 3,180,892. Went, F. W., LeRosen, A. L., and Zechmeister, L. 1942. Effect of external factors on tomato pigments as studied by chromatographic methods. Plant Ph ysiol. 17, 91. Wingerd, W. H., and Saperstein, S. 1964. Carotenoid pigment and protein coniplex and method of producing the same. U.S. Patent 3,125,451. Winterstein, A., Studer, A., and Ruegg, R. 1960. ATeuere Ergebnisse der Carotinoidforschung. Bcr. 93, 2951. Yamamoto, H. Y., Chichester, C. O., and Nakayama, T. 0. M. 1962. Biosynthetic origin of oxygen in the leaf xanthophylls. Arc h . Btoehem. B i o p h y s . 96, 645. Zbinden, G., and Studer, A. 1958. Tierexperimentelle Untersuchungen uber die chronische Vertraglichkeit von p-Carotin, Lycopin, 7,7’-Dihydro-p-Carotin und Bixin. 2. Lebcnsm.-Untmwch. u. Forsch. 108, 113. Zechnieister, L. 1944. Cis-trans isomerization and stereochemistry of carotenoids and diphenylnolyenes. Chcm. Rev. 34, 267. Zechmrister, L. 1962. “Cis-Trans Isomeric Carotenoids, Vitamin A and Arylpolyenes.” Academic Press, New York. Zechmeister, L., and Cholnoky, L. 1934. Untersuchungen uber den PaprikaFarbstoff. VII. Adsorptionsanalyse des Pigments. Ann. 509, 269. Zechmc,ister, L., and Cholnoky, L. 1940. Carotenoids of Hungarian wheat flour. J . B d . Chem. 135, 31. Zechmrister, L., and Cholnoky, L. 1943. “Principles and Practices of Chromatography.” Translated by Bacharach, A . L., and Robinson, F. John Wiley & Sons, New York. Zechmeister, L., and Tuzson, P. 1931. Ueber d a s Pigment der Orangenschale. NtrturwissenschaftP?z 19, 307. Zechmeister, L., and Tuzson, P. 1934. Zur Kenntnis der tierischen Fettfarbstoffe. Ber. 67, 154. Zechmeister, L., and Tuzson, P. 1936. Ueber das Polyen-Pigment der Orange. ( I . Mitteilung) Ber. €9, 1878. Zechmeister, L., and Tuzqon, P. 1937. Ueber das Polyen-Pigment der Orange. (11. Mitteilung) : Citraurin. R c ) . 70, 1966.

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Zimmerman, W. I., Tressler, D. K., and Maynard, L. A. 1940. Determination of carotene in fresh and frozen vegetables. I. Carotene content of green snap beans and sweet corn. Food Research 5 , 93. Zinimerman, W. I., Tressler, D. K., and Maynard, L. A. 1941. Determination of carotene in fresh and frozen vegetables by a n improved method. 11. Carotene content of asparagus and green lima beans. Food Research 6, 57. Zsolt, J., Schneider, G., and Matkovics, B. 1963. Carotenoid changes in different maize varieties during ripening. Caz. J . Biochem. and Physiol. 11, 481.

BASIC PRINCIPLES OF MICROWAVES AND RECENT DEVELOPMENTS* BY SAMUEL A. GOLDBLITH D e p a r t m e n t of N u t r i t i o n and Food Science, Massachusetts I n s t i t u t e of Technology, Cambridge, M a s s a c h u s e t t s

I. Introduction .......................... ............. 11. Radio-Frequency Energy . . . . 111. How Does R F Energy H e a t F IV. The Power Equation ..................... ......... V. Penetration of Microwaves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Electrical Power and Thermal Energy VII. Dielectric Loss Factors . . . . . . . . . . . . . . VIII. Types of Microwave Process Devices 1X. Eficiency of Microwave Absorption i n X. Choice of Frequency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

277

Blanching of Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Defrosting of Frozen Foods . . . . . . . . . . . . . . . Microwaves i n Freeze-Drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................... Microwave Cooking . . . . . . . . . . . . . Microwaves f o r Sterilization . . . . . . . . . . . . . . . . . . . . Use of R F Energy f o r Baking . . . . . . . . . . . . . . . . . . . . . . . . . L. Use of R F Energy f o r Processing Potato Chip

290

...........................

296

F. G. H. I. J. I(.

..........

References

................

I.

INTRODUCTION

That high-frequency electrical fields may affect biological systems has been known since d’Arsonva1 (1893; d’Arsonval and

* Contribution No. 889 from the Department of Nutrition and Food Science, Massachusetts Institute of Technology. 277

278

SAMUEL A. GOLDBLITH

Charrin, 1896), who showed that laboratory animals exhibited marked rises in temperature and that bacterial toxins underwent fundamental changes when exposed to high-frequency fields. A good deal of work done over the next fifty years showed, by and large, that the action of high-frequency or radio-frequency ( R F ) fields was due to the heat produced in the material being exposed. It should be stated, however, that some workers have felt that the lethal changes induced in bacteria or the degree of heating produced in mice by exposure to R F energy was due to some intrinsic properties of the radiations other than heat, and that this effect was frequency-dependent. The early work has been reviewed by Webber et al. (1946) and also by Ark and P a r r y (1940), the latter pointing out that, although d’Arsonval’s work led to the development of diathermy, it was not until work of others in the mid-1920’s and early 1930’s that the destructive effects on bacteria (and, later, insects) was followed up. During World War I1 and shortly thereafter, a number of people began to experiment with dielectric heating and, later, microwave heating for food processing. RADIO-FREQUENCY ENERGY

11.

The general area of the electromagnetic spectrum covered by R F heating is shown in Fig. 1. The dielectric region of the spect r u m is generally of longer-wave length radiations than the mi915 MC 2450MC

\

\

/ /

\

\

/

/ /

f

Visible r e d T blue

W

I G a m m a rays and

~

‘ ~ / ~ ~ ~ t

x-rays

Frequency, cycles/second

FIG.1. Electromagnetic spectrum (courtesy Cryodry Corp.).

I

MICROWAVES I N FOOD PROCESSING

279

crowave region, which has seen a great deal of development in the past twenty years. The frequencies which have been allocated for industrial, scientific, and medical uses (ISM frequencies) a r e presented in Table I. In general, in the microwave region of the spectrum, two frequencies a r e of importance today. These a r e 915 and 2450 megacycles, and it is with these two frequencies that much of the more recent work over the past five years has been carried out.

Frequency (cycles/sec)

13,580 27,130 40,680 915 2450 5800 'L2,125 a

X lo3 X lo3 x 103 X loG x 106 X lo6 X lo6

iVltve length (cm)

-" '1'120, 1100. i35. 32.8 12.24 5.17 1.36

From FCC Rules arid Itegullttiorls, Part 18, Jan. 1964.

Ill.

HOW DOES RF ENERGY HEAT FOODSTUFFS?

All matter is made up of electrically charged particles, both positive and negative. In normal, undistributed matter, there a r e equal numbers of positively and negatively charged particles. Thus, most materials a r e electrically neutral. If the material is nonconducting, i.e., a dielectric, and is placed in a n electromagnetic field, the charged asymetric molecules, of which the dielectric (such as a foodstuff) is composed, a r e driven first one way and then another. Each of the asymmetric molecules attempts to align itself with the rapidly changing alternating-current field. I n this field, the molecules act as miniature dipoles, and, while oscillating around their axes in a n attempt to go to the proper positive and negative poles, intermolecular friction is created and is manifested as a heating effect. A t the microwave frequencies, e.g. 915 megacycles, the molecules oscillate back and forth 915 million times per second. Figure 2 illustrates the attempts of the molecules to align themselves to rapidly changing fields, thus causing heat.

280

SAMUEL A. GOLDBLITH

644G 1 -

I

i -

=

e

:

=

=

in ‘I

-

IV. THE POWER EQUATION The amount of power which can be generated in a nonconductor placed in a n electromagnetic field is given in Eq. 1. 2’

=

E’ X

Y

X

tr”

X 55 (il X

111

where P is the power dissipated, in watts/cm3; E is the electric field strength (volts/cm) ; 1 is the frequency (cycles/sec) ; and € , I f is the dielectric loss factor. E,“, the dielectric loss f a c t o r , is the over-all measure of the ability o f the material to respond when placed in a n electromagnetic field. It is related t o the dielectric coristant ( E , ’ ) , which is the ratio of the capacity current through the material t o the capacity current which would flow if the same field intensity were applied to freeIf one p u t s a conducting material (such a s copper) directl!! in contact n i t h the terminals of a n electric-field generator, forces a r e exerted by the electric field on the free negatively-charged particles, and t h e electrons accelerate and pass through the conductors as electric current. In moving through the conductor, the electrons bounce against t h e molecules of the conductor, creating motion and friction, thereby raising the temperature of the conductor. In the case of the conductor, the violence of the field is trivial in comparison with the motion of the free electrons. d

MICROWAVES I N FOOD PROCESSING

28 1

space, and to the dissipation f a c t o r (tan 8, or the loss tangent) as in Eq. 2. tr

c7

= e,'

x

tan 6

I21

The increase in temperature of a material per unit time, AT, is given in Eq. 3 : AT

=

P/'Cp

[31

where: P is the power from Eq. 1; C is the specific heat of the is the density of the material. material (cal/cms) ; and Since 1 watt-minute = 14.4 calories (1 kw can raise the temperature of 1 kg of water 14.4"C/min), then A T = 14.4PjCp ("C/min)

or

Examination of Eq. 1 shows that the power absorbed in a foodstuff is dependent upon E , +",and v. E is limited by the voltage breakdown of the a i r ; E,." is a characteristic of the dielectric properties of the particular foodstuff and varies with temperature. Thus, one can increase the power absorbed by a product (if all else is constant) only by increasing the frequency ( v ) . V.

PENETRATION OF MICROWAVES

The penetration of R F energy is usually described by the half power depth (HPD) or that thickness of material which reduces the power of the surface to one half. HPD is given in Eq. 6 . IIPD

= __

0.693 ______

dz-t a n 6 . 3 3 . G 1 . lo-'"

[(;I v

Thus, in terms of the two frequencies under commercial study today, the depth a t 915 megacycles is much greater than a t 2450 megacycles. VI.

ELECTRICAL POWER AND THERMAL ENERGY

Because the observed effects of microwave power are expressed as thermal energy, it is of interest to develop the relationship be-

282

SAMUEL A. GOLDBLITH

tween the two: since 1 kilowatt can raise the temperature of 1 kilogram of water 14.4'Cl min, then I (h\.i-hg mini

x

2 2 (lh hg)

x

14 4'C'

x

9 .5

x

1

(Iirii

Ili'Ti

or 57 Btu,min kw or 3420 Btu/hr/kw. VII.

DIELECTRIC LOSS FACTORS

Data on dielectric loss factors in foodstuffs and their components are relatively sparse, and basically from these relatively few sources : Von Hippel (1954) Morse and Revercomb (1947) Kan (1961) Bengtsson e t al. (1963) Dunlap and Makower (1945) Harper et al. (1962)

Hasted et al. (1947) Owen et al. (1961) Ede and Haddow (1951). Takashima and Schwann (1965) Schwann (1956)

Some data have been obtained on fats a t a relatively low frequency (Gouw and Vlugter, 1964) and on long chain fatty acids and their methyl esters in the microwave region (Buchanan, 1954). I t is obvious that many more data a r e needed on foodstuffs, particularly a t the permitted frequencies. Systematic studies of the dielectric properties of various foods at different temperatures and moisture contents a r e also needed. The value of such studies has been shown by Smyth in his excellent text (Smyth, 1954). VIII.

TYPES OF MICROWAVE PROCESS DEVICES

At the lower frequencies (the dielectric region) the time required to generate sufficient power into materials is relatively long (power being a direct function of frequency, Eq. 1 ) . This was pointed out particularly by Bengtsson (1963). With the subsequent development of tubes of higher power in the microwave region, much emphasis has been given in recent years to use of the microwave region. At the lower-frequency dielectric region of the spectrum, the 7var.e length of the radiation is relatively longer ; hence, flat-plate electrodes may be used to expose the samples with relatively little loss in power a t the f o u r edges. A t higher frequencies of the microwave region of the spectrum, the container must be closed in order to avoid the lower-wave length radiation from escaping into

MICROWAVES IN FOOD PROCESSING

283

the room. This situation in continuous processing shows the need for smaller openings at the frequency of 2450 megacycles than at 915 megacycles. It also shows the need for the energy-trapping devices described later in this review. The first type of microwave process device to be developed was the batch-type oven, a s shown in Fig. 3. The radiation is emitted from a power tube (magnetron) into a cavity, bounces back and forth, and is absorbed into the food material. Originally, this type of oven operated a t 3000 megacycles; now it uses the 2450megacycle band. Much of the work prior to 1960 was done with such a unit and was limited by a power level of 1 t o 2 kw. This type of unit is being manufactured and produced by the Raytheon Co., Litton Industries, the Philips Co., and the Eimac Division of Varian Associates.

p, /

1

//

/ / ,/

/

/

/ /

FIG.3. Batch oven.

The second general type of microwave process device utilizes five 1-2-kw parabolic radiators (Fig. 4 ) radiating onto foodstuffs placed on a belt made of a low-loss dielectric material. It was developed by the Philips Co., Eindhoven, Holland, and is similar to the type used by Bengtsson (1963). I n it the foodstuff is exposed t o the microwave energy for about a 5-em distance on the belt, and then the food equilibrates until it reaches the next parabolic radiator. A total of five 2-kw radiators a r e used. This unit may have particular use in defrosting frozen meals where foods of different dielectric loss characteristics are present and where equilibration time would be of advantage. Litton Industries, in 1963-1964, developed a somewhat similar multi-modular system utilizing five 2-kw-power module units feed-

SAMUEL A. GOLDBLITH

284

Transport channel

Water

load

FIG.4. Parabolic devices.

ing power along the conveyor line. This system also operates a t 2450 megacycles (Fig. 5). I n 1962 the Cryodry Corporation, San Ramon, California, developed a continuous-type microwave oven which feeds microwave power from one single large power tube (25 kw) into a tunnel through a series of slit openings (Fig. 6 ) . At the end of the belt a r e energy-trapping devices which serve t x o functions : (1) They prevent excess radiation from leaking _ Energy , trap

2 kw

kw

Energy /

FIG.5. Litton device. 10-kw Microwave tunnel using series of 2-kw power units.

MICROWAVES I N FOOD PROCESSING

285

out of the ends of the tunnel, instead being absorbed in the water loads. (2) Being thus absorbed when there is no load in the tunnel, there is no energy to go back into the power tube and damage it. The forced-air system with heating attachment removes the water vapors driven from the interior to the surface of the product. Large power tube

Forced

air system.

cj

devtces

FIG.6. Cryodry unit (continuous-type microwave oven with one large power tube).

One of the latest developments relates to a folded-waveguide, or nienntler, system, wherein the energy is piped into one end and the product passes through the waveguide (Fig. 7 ) . Thus, more energy is available a t the beginning of the transit through the Water

Iood

Wove Wave

FIG.7. Meander system.

guide

286

SAMUEL A. GOLDBLITH

meander guide, where the product is wetter. As the product passes along, more energy is absorbed, and finally, near the end of the waveguide, where the product is driest, the energy available is also least. This system is particularly useful f o r textile and paper drying, lamination of plywoods, etc. Still another and relatively recent development is the Amplitron tube. Allaire and Sample (1964), Allaire (1965a,b), and Brown (1965) announced the availability of a new tube, the Aniplitron, which can produce up to 425 kw of 3000-megacycle microwaves. Allaire (1965a,b) suggested the use of and presented a design concept for this tube as a heat exchanger. It remains to be seen whether or not the tube will have a marked influence on the state of the a r t of microwave technology. Further information will be needed on tube life and control, as well as demonstration that power as high as 400 kw can be taken out of a tube, delivered into a waveguide, and coupled into a product with reasonable efficiency. IX.

EFFICIENCY

OF M I C R O W A V E ABSORPTION I N T O FOODS

It would seem advisable to approach the problem of efficiency of microwave absorption into foods on a thermodynamic basis, since the use of microwaves in the food industry seems to be based in large measure on the following: ( 1 ) Microwave processing offers a means of rapidly providing uniform heat energy throughout a product without suffering the limitations of normal heating, i.e., conduction and convection heating with surface crust formation. ( 2 ) The sole effect of microwaves in foods is that of heat. ( 3 ) The amount of heat that can be absorbed is a function of frequency and of the dielectric-loss characteristics of the foodstuffs. Thus, assuming that one is finish-drying 2 lb of vegetables per hour and reducing the moisture content from a% to b % , and the inlet temperature of the vegetables into the dryer is 150"F, then the total amount of heat needed to remove ( a - b)/100 lb of watei is the sum of a, b, and c, as follows: a ) the amount of heat needed to bring the water in the vepetables up t o 212°F o r 113 of n-ater in

x

\pecific heat of w i t c r X A 7'

01'

7X

(I

100 111) h ~ X) 1 . 0 Btu, "F X (212" - 150°F)

= __

13tu/hl'

MICROWAVES IN FOOD PROCESSING

28'7

b) the latent heat of the water being evaporated, or Zlh'hr X ( a - b),/l00 X O i O ti (Btu'lb)

=

-Rtu,'hi

c) the amount of heat needed to bring the solids in the vegetables up t o 212°F) or %Ih:hr X [ Z - Z(q'lO0) ]

x

Cp iolids iBtu "Fi (1Ti

Or

Z ( I t ) liriX [Z -(oZ/IOO)][O 373(Btu/"F)(212 - 130°Fi] = --Rtii

111.

+ +

Total Btu needed is the sum of a b c. Inasmuch as 1 kw z 3420 Btu'hr (from Section VI, above), one can calculate microwave power needed vs. that power actually used and thus obtain the efficiency of the process. X.

CHOICE OF FREQUENCY

One of the most difficult things to answer relates to suggesting an optimum frequency. Obviously, several factors bear on this : (1) recognition of the various factors of the power equation (eq. 1) ; (2) depth of material to be processed (eq. 6) and the inverse relationship of frequency; ( 3 ) the fact that certain foodstuffs a r e of such a shape and configuration as to need a large entrance and exit cavity through which to pass; (4) the fact t h a t power requirements for the particular throughput of a conventional plant could be easily as large as 50, 100, or 200 k w ; and ( 5 ) the fact that foodstuffs a r e heterogeneous in nature, with the several major components differing in dielectric loss characteristics. The power equation states t h a t the power capacity of a system is R direct function of the frequency and dielectric loss of a foodstuff. Thus, the higher the frequency, the more energy one can put into a system. On the other hand, the HPD is inversely proportional to frequency. Foodstuffs that a r e too thick may be unable to be handled at the higher frequency, whereas certain thin materials may be ideally suited to the higher frequency (2450 megacycles). Consequently, in terms of the important basic equations, it is difficult t o predict which frequency may be optimum. Each situation and particular use must be analyzed with its own parameters and requirements, not the least of which is an adequate knowledge of the dielectric properties of the foodstuffs. With regard to power requirements (bearing in mind that 1 kw represents 3420 B t u ) , the throughput of a product through a nom-

288

SAMUEL A. GOLDBLITH

inal-sized plant may well be in excess of multiples of 50-kw units. From a n engineering point of view, the concept of multiple 2.5kw-powered modules may be difficult to manage. This remains to be seen when plants of several hundred kilowatts a r e built (to be expected in the not too distant future). XI.

POSSIBLE USES OF RF IN FOOD PROCESSING

Among the processes and effects studied over the past forty years have been the following : A. IMPROVEMENT O F STORAGE QUALITYO F COTTONSEED Lyman et al. (1948) demonstrated that the enzymes responsible for the free-fatty acid formation and rapid removal of moisture were destroyed by exposure to R F energy at 14 megacycles per second.

B. DESTRUCTION O F FOOT AND MOUTH DISEASE V I R U S Zarotschenzef? (1944) raised the temperature of meat rapidly throughout its mass t o the temperature necessary for destruction of the virus, and then cooled the meat in order t o avoid a cooked flavor.

c. DEHYDRATION O F VEGETABLES Rushton et al. (1945) experimented with 13-megacycle R F heating of vegetable blocks in order to reduce the respective nioisture contents of cabbage and potato from 9 and 15% to 5 and 7 % . The higher moisture contents a r e needed t o ensure proper compression of the shreds into blocks; the lower moisture levels a r e needed to ensure proper drying. Conventional hot-air drying was found to be too slow and costly. It was found possible to finishdry the blocks in either a i r or vacuum (pressure 12-13 ern H g ) . However, a number of practical difficulties were encountered, such as control of block temperature and heat damage. Yet, in airfinish-drying by R F energy, the time required was about one-fifth of that in a cross-air blow dryer; vacuum-drying increased the drying rate by a further 50%. More recent work by Jeppson (1964) on the finish-drying of vegetables demonstrates a synergistic effect between hot a i r and microwaves. Further work by Huxsoll and Morgan (1966) is in progress on the use of microwaves in “puffing” foods.

MICROWAVES I N FOOD PROCESSING

D. DESTRUCTION OF

INSECTS I N

289

GRAIN

Destruction of insects in grain through the temperature reached in bulk or in packages or containers of grain or flour was studied by Smith (1944), Anon. (1945)) and Webber e t al. (1946). One problem observed with certain cereals was arcing or voltage breakdown before the necessary temperature was reached.

E.

PRODUCTION O F MOLD-FREEBREAD

Cathcart (1946a)) Cathcart et al. (1947), Bartholomew et aZ. (1948)) and Godkin and Cathcart (1949) have shown that R F may be used for producing mold-free bread. In this early work, frequencies of 13-27 megacycles were used, and destruction could be obtained on a small scale with uniform products such as bread, but problems of arcing occurred with some breadstuffs. Uneven heating occurred with fruitcake, because of the varied materials composing it and the different dielectric loss factors of these materials. A t that time the process did not appear to be practical on a commercial basis. More recent work has been done by Jeppson (1964) and Olsen (1965) on the destruction of molds on bread by continuous microwave systems, and the process appears to be effective.

F. BLANCHING O F FOODS Early work on the blanching of foods was done by Moyer (1945), Moyer and Stotz (1945), Moyer and Holgate (1947)) Proctor and Goldblith (1948), Samuels and Wiegand (1948), Anon. (19511, and Hard and Ross (1956). Electronic blanching has a n advantage over the conventional hot-water method in that it does not leach out nutrients and coloring compounds such as chlorophylls and carotenoids. Copson ( 1954) studied the inactivation of enzymes, by microwaves, in orange juice concentrate. G. DEFROSTING O F FROZEN FOODS One of the early uses of R F energy in the food field was for the defrosting of frozen foods (Cathcart, 1946b ; Eikelberg, 1950 ; Morse and Revercomb, 1947; Satchel1 and Doty, 1951). More recently, extensive experiments on this use have been carried on in Europe by Jason and Sanders (1962a,b), Bengtsson et al. (1963), and Bengtsson (1963). While defrosting is a possible use for highfrequency R F energy, it should be recognized that the difference in the loss factor between ice and water is very great, and the

290

SAMUEL A. GOLDBLITH

differences between various components of a frozen food are large enough to cause uneven heating and even “runaway” heating where there is a “hot spot” of R F energy. The latter occurs when a portion of the ice melts rapidly (especially so in comparison with the unmelted ice) and boils while much of the meal is still frozen. Decareau (1964) reviewed some of the advantages of this method for different classes of foods, and compared (1965) 915 megacycles and 2450 megacycles for this use. Walter (1965) also reviewed the use of dielectric heating for thawing. Heisig and Kobe (1958) used R F energy for selective melting of frozen sol u t ion s.

H. MICROWAVES I N FREEZE-DRYING C‘opson and Decareau (1957), Harper and Tappel (1957), Jackson et al. (1957), and Copson (1958a,b) were among the first to suggest R F a s energy source for the sublimation of foods in order to speed up the drying process. Harper and Chichester (1960a,b) probably initiated the early significant efforts in this field, and delved as f a r as anyone into this possible use of R F energy in lyophilization. Further work has been done o r reported on in this field by Decareau (1961), Harper et al. (P962), Decareau (1962), Decareau (1963), Simatos (1963), Meryman (1964) and Hoover et al. (1966a,b). Burke and Decareau (1964), besides Meryman (19641, offer excellent reviews of the possibilities of microwaves in this field as well as considering the limitations. The limitations appear to be control of the energy input in order t o prevent melting of the product, the differences in the dielectric loss factors of the frozen core vs. the dry layer, and cost of development of the theory into practical commercial utilization, which could prove to be considerable. Even if a successful system of using microwaves in freeze-dehydration could be developed, it is questioned whether o r not there is economic justification. I. MICROWAVE COOKING

A great deal of early work on microwave cooking or dielectric cooking was devoted primarily to studies on nutrient retention (Proctor and Goldblith, 1948; Thomas et al., 1949; Causey and Fenton, 1951a,b; Fenton, 1957; Campbell e t al., 1958). These studies generally showed that retention of nutrients was greater with electronic heating because of relatively uniform heat distribution in the foodstuffs plus reduced leaching-out of foodstuffs.

MICROWAVES I N FOOD PROCESSING

291

Later, applications of dielectric thawing of precooked frozen meals for hospitals and other institutions were made by Bollman (1948), Stevens and Fenton (1951), Copson et al. (1955), Bechtel (1959), Decareau (1959), Gordon and Noble (1959), Pollak and Fain (1960), Schmidt (1960), and Smith (1963). Special meals prepared in control kitchens and then frozen may be delivered to hospital floors and thawed electronically, thus minimizing the need of multidietetic kitchens. Lacey e t al. (1965) have suggested the additional benefit of reduction of bacterial count in foods reheated by microwaves and served to patients in a n “ultraclean” unit. Later studies by Pircon et al. (1953), Jeppson (1964), and Decareau (1965) have been directed toward utilizing R F energy for heating and cooking on a n industrial scale, such a s the cooking of chicken. Earlier work by Essary (1959) showed advantages in reduction of dark bone in frozen chicken. The advantages of this method over conventional cooking are the lower losses during cooking and the greater retention of flavors. This is covered in a recent publication (Anon., 1966a,b). Van Zante (1966) recently suggested a novel means of evaluating the uniformity of heat distribution in a n electronic oven using egg whites. J. MICROWAVESFOR STERILIZATION Early work in this field showed that R F energy could reduce the bacterial count of products exposed to it. Fleming (1944) demonstrated the destruction of bacteria in weak electrolytes. Brown et al. (1947) were unable to repeat Fleming’s work, however. In later and more definitive work, Brown and Morrison (1954) were convinced that there was no significant destruction of bacteria other than that brought about by thermal effects (the frequencies studied were 60 cycles ; 190 kilocycles ; and 26, 65, and 60 megacycles). This work is considered t o be a most definitive and critical study. Similar results were obtained by Proctor and Goldblith (1953), i.e., the effect of the R F energy (3000 megacycles) was due t o heat and not to the radiation p e r se. Other early work in this field was done by Brown et al. (1947), who were able to pasteurize beer with 28-megacycle R F energy. Titus (1946) attempted to pasteurize beer by R F energy (25 megacycles) with relatively short bursts (5 or 10 seconds). He found but little effect of the energy on the temperature of the beer, and thus no effect on the yeasts. Yang et al. (1947) sterilized wine successfully with R F energy of 26-34 megacycles.

292

SAMUEL A. GOLDBLITH

Throughout all of the work on R F sterilization, the question has been raised frequently as to whether there is any effect of R F energy p e r se (other than that of heat) and whether there is a n optimum frequency. While, in general, it is believed that there a r e no effects p e r s e and that there is no optimum frequency, there have been some studies which indicate possible lethal effects due to R F energy p e r s e (Gaden and McMahon, 1947). A recent article (Robe, 1966) cites work by two R F experts of Tacoma, Washington-Julius Mann and George F. Russell-who claim lethal effects of R F energy p e r s e (frequency not specified). A possible optimum frequency has been indicated by Bach (1965). However, at this writing the two ideas mentioned above (a specific effect pel. s e and an optimum wave length) a r e not generally accepted. The question of optimum frequency for biological effect has not been limited entirely to microbiological activity. Nelson (1965) has stated that a n optimum resonant frequency has been found for the irradiation of cholinesterase (near 13 megacycles). Because of this reported work and because methods a r e needed to inactivate the catheptic enzymes in meats prior to preservation by ionizing energy, this company has been engaged in a study to ascertain whether there is any effect p e r se of R F energy and to determine the optimum frequency on catheptic enzymes isolated from meat. To date, none has been found around 13 megacycles. Takashima (1966) studied the effects of 60 megacycles R F energy on solutions of DNA and of alcohol dehydrogenase and found no specific effects due to the radiation p e r se. The studies by Jeppson (1964), as well as by others such as Jackson (1947), indicate distinct possibilities for the use of microwaves as a rapid means of providing sufficient heat throughout a container, thereby achieving a sterilization temperature with a relatively short come-up time and with uniform heating. Obviously, to achieve this on a commercial basis would require development, but possibilities a r e suggested with either glass (with which Jeppson did his work) or with the new plastic heat-stable containers. A t 915 megacycles one should be able to achieve sufficient penetration f o r relatively large container sizes.

K. U S E O F R F ENERGY FOR BAKING The use of R F energy in baking has been discussed previously for reducing the mold content of the baked product and for thawing frozen baked goods. Proctor and Goldblith (1948) demon-

MICROWAVES I N FOOD PROCESSING

293

strated the use of microwaves a t 3000 megacycles as a means of baking gingerbread and devil’s food mixes (products which can be served without a crust), and showed excellent retention of thiamine but not of riboflavin in these products. Holland (1963) reviewed the use of R F heating for the finish baking of biscuits or crackers, the R F energy being applied in the latter stage of the baking process, when the coloring and drying of the biscuit takes place. The result is a more rapid removal of moisture from the biscuit by means of increased heat transfer to the center of the biscuit, and thus a greater throughput through the oven. The process has been fairly well adopted in a number of biscuit factories in England and in a t least one in the United States. Jeppson (1964) suggested a similar application utilizing 915megacycle microwaves. Some o€ the users in the United States and in England are listed by Anon. (1965). One of the recent United States installations is described by Heppner e t al. (1965).

L. USE O F R F ENERGY FOR PROCESSING POTATO CHIPS One of the early commercial uses prophesied for microwave energy was the completion of continuous tunnel systems using microwave energy for the finish frying of potato chips-and this was done in 1964. By frying to about 8% moisture content and then finishing off the dehydration in a microwave oven at 212°F (instead of the 320°F temperature of the f r y e r ) , it is possible to use most varieties of potatoes, regardless of temperature of storage, without causing excessive browning from the Maillard reaction. Excess reducing sugars occur in potatoes stored at lower temperatures (under 50°F). Moreover, there would be further potential savings with the use of potatoes stored at lower temperatures, because losses a r e relatively much greater (due to sprouting) at higher storage temperatures. This work has been described in a series of articles by Davis et al. (1965a,b,c) and discussed by Decareau (1965). Economic justification has been claimed by Davis e t al. (1965a,b,c). Blau e t al. (1965) presented economic and product justification for the use of microwaves at 2450 megacycles in the manufacture of potato chips using the Litton multiplemodule system. O’Meara (1966) presented the economic savings obtained by using 915-megacycle microwaves for the finish frying of potato chips. Blau’s paper describes a full-scale system, with operating costs. This would require -60 kw in order to achieve a production rate of 1500 Ib of chips per hour and a reduction of

294

SAMUEL A. GOLDBLITH

moisture from -5.5 t o 1.5%, with the inefficiencies of absorption and coupling of R F energy (35% estimated). Some of the calculations from Blau et al. (1965) are presented in Table 11. Since one of the earliest commercial uses for microwaves was in the processing of potato chips, O'Meara's (1966) economic justifications (Figs. 8, 9 ) are developed in terms of in-

Blnu et al. (19ti5).

75

r

INCREASED C H I P Y I E L D ( L B S K W T INTO STORAGE t

FIG. 8. Profits due to increased chip yield with microwave drying. Basis: Cryodry model IV-50-CP machine (nominal capacity 1500 lb chips per h o u r ) ; microwave drying cost: $25 per hour (includes lease, utilities, and maintenance); additional chips valued at 25 and 35 cents per pound. From O'RIeara cost calculations.

MICROWAVES I N FOOD PROCESSING

0

295

I-0

0

/

/

0

/

FIG.9. Profits due to r a w potato price differential with microwave drying. Basis : Cryodry model IV-50-CP machine (nominal capacity 1500 lb chips per h o u r ) ; microwave drying cost: $25 per hour (includes lease, utilities, and maintenance). Price differential includes transportation savings. From O'Meara cost calculations.

creased chip yield and in savings in r a w potatoes due to the price differential made possible by removing the need for careful storage of chipping potatoes.

M. GENERAL The categories above represent most of the important uses that have been suggested for R F energy in the processing of foods. A recent review on applications of R F energy in the food industry was published by Garrick (1966). In all instances these uses appear t o depend on the ability of the R F energy to introduce heat into the product. XII.

SUMMARY AND CONCLUSIONS

It is now some fifteen years since the first review on this subject (Proctor and Goldblith, 1951). I t becomes fairly obvious, after a critical review of the literature plus personal experience in the field, that great progress has been made in the uses and sources of R F energy. The bibliography contained herein is but a n indication of the quantity of work done and the types of ac-

296

SAMUEL A. GOLDBLITH

complishment made. Undoubtedly, there are many more references in this field that might have been cited, and many of these may be quite pertinent. Nevertheless, it is felt that the scope of work in the years intervening since the earlier review is represented by the references cited in this paper. The development of continuous types of tunnels and waveguides has rekindled the interest of the food industry in using R F energy f o r food processing. However, there still remains a paucity of knowledge on the basic dielectric properties of foodstuffs under a variety of conditions. Such data are necessary and relevant to the problem of judging the optimum frequency for various types of foodstuffs and for different processing operations. These data a r e necessary not only for indicating the limitations of microwaves but also for indicating other possible uses of this new method of processing.

ACKNOWLEDGMENT The author is particularly indebted to the Cryodrg Corporation f o r permission to use several illustrations in this paper.

REFERENCES Allaire, R. P. 1965,. Industrial microwave heating. Elretyo7iic I’iogy. 9 ( 4 ) , 14-20. Allaire, R. I?. 196513. Potential applications f o r the microwave heat exchanger. Food Techml. 19 ( 8 ) , 40-42. Allaire, R. P., and Sample, J. G. 1964. Super power microwaves f o r advanced systems. Cornell Hotel Restaurant Admin. Q u a r t . 5 ( l ) , 71-75. Anon. 1945. Electronic sterilization. A m . M i l l e r 73 (4),43, 48. Anon. 1951. Continuous electronics blanching proved feasible in pilot plant. Food Eng. 23 (5), 81. Anon. 1965. High frequency heating gains. Food Eizg. 37 ( l l ) , 62-63. Anon. 1966a. Microwave cooking cuts labor cost in half. Broiler Znd. Anon. 1966b. Microwave oven with steam atmosphere produces higher quality, more profitable precooked chicken. Food PYOC.and MarkctirTg 27 (4), 92-100. Ark, P. A., and P a r r y , W. 1940. Application of high-frequency electrostatic fields in agriculture. Q u a r t . Rev. Biol. 15 ( 2 ) , 172-191. Bach, S. A. 1965. Biological sensitivity to radio frequency and microwave energy. F e d e r a t i o n Proc., Suppl. 14, Pt. I11 24( 1 ) , S22-S26. Bartholomew, J. W., H a r r i s , R. G., and Sussex, F. 1948. Electronic preservation of Boston brown bread. Food Technol. 2 ( 2 ) , 91-94. Bechtel, J. 1959. Electronic oven speeds service of tasty hospital food. J . A m . Dietet. Assoc. 35 ( 3 ) , 257-258. Bengtsson, N. 1963. Electronic defrosting of meat and fish at 35 and 2450 mc.-a laboratory comparison. Food Ttchnol. 17 ( l o ) , 97-100.

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Bengtsson, N. E., Melin, J., Remi, K., and Soderlind, S. 1963. Measurements of the dielectric properties of frozen and defrosted meat and fish in the frequency range 10-200 MHz. J. Sci.Food A g r . 14 ( 8 ) , 592-604. Besser, E. D., and Piret, E. L. 1955. Controlled temperature dielectric drying. Cheni. E n g . Progr. 51, 405. Blau, R., Powell, M., and Gerling, J. E. 1965. Results of 2450 megacycle microwave treatments in potato chip finishing. Proc. 28th Ann. Conf. and Exhibit of the Potato Chip Inst. Intern., New York. Bollman, M. C. (1948). Application of electronic cooking to large scale feeding. J . Am. Dietet. Assoc. 24 (12), 1041-1048. Brown, W. C. 1965. Electronic components f o r microwave power engineering. EIectronic P r o g r . 9 ( 4 ) , 9-13. Brown, G. H., and Morrison, W. C. 1954. An exploration of t h e effects of strong radio-frequency fields on micro-organisms i n aqueous solution. Food Technol. 8 ( 8 ) , 361-366. Brown, G. H., Hoyler, C. N., and Bierwirth, R. A. 1947. Theory and Application of Radio-Frequency Heating. 370 pp. D. Van Nostrand Co., New York. Buchanan, T. J. 1954. The dielectric properties of some long-chain f a t t y acids and their methyl esters in the microwave region. J . C h e m . P h y s . 22 (4), 575-584. Burke, R. F., and Decareau, R. V. 1964. Recent advances in t h e freezedrying of food products. A d v a n c e s in Food R e s e a r c h 13, 1-88. Campbell. C. L., Proctor, B. E., and Lin, T. Y. 1958. Microwave vs. conventional cooking. J . Am. Dietet. Assoc. 31, 365-370. Cathcart, W. H. 1946a. High frequency heating produces mold-free bread. Food Znd. 18, 864-865. Cathcart, W. H. 194613. Frozen foods defrosted by electronic heat. Food Znd. 18, 1524-1525. Cathcart, W. H., Parker, J. J., and Beattie, H. G. 1947. The treatment of packaged bread with high frequency heat. Food Technol. 1, 174. Causey, K., and Fenton, F. 1951a. Effect of reheating on palatability, nutritive value and bacterial count of frozen cooked foods. I. Vegetables. J . Am. Dictet. Assoc. 27, 390. Causey, K., and Fenton, F. 1951b. Effect of reheating on palatability, nutritive value and bacterial count of frozen cooked foods. 11. Meat dishes. J . A m . Dietet. Assoe. 27, 491. Copson, D. A. 1954. Microwave irradiation of orange juice concentrate f o r enzyme inactivation. Food Tcchnol. 8 ( 9 ) , 397-399. Copson, D. A. 1958a. Methods and a p p a r a t u s f o r radio-frequency freeze drying. U.S. Patent 2,859,534. Copson, D. A. 1958b. Microwave sublimation of foods. Food TeeIznoZ. 12 (6), 270-272. Copson, D. A., and Decareau, R. V. 1957. Microwave energy in freezedrying procedures. Food R e s e a r c h 22, 402-403. Copson, D. A., Neumann, B. R., and Brody, A. L. 1955. High frequency cooking, browning methods in microwave cooking. J . A g r . Food C h e m . 3, 424-427. d’Arsonval, A. 1893. Influence de la frCquence s u r les effets physiologiques des courants alternatifs. C o m p t . rend. A c a d . Sci. ( P a r i s ) 116, 630-632.

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d’Arsonva1, A,, and Charrin, M. 1896. Action des diverses modalites s u r les toxines bacteriennes. Compt. vend. Soc. Biol. 48, 96-99. Davis, C. O., Smith, O., and Olander, J. 1965a. Xicrowave processing n i potato chips. P t . I. P o t a t o C h i p p w 2 5 ( 2 ) , 38-58. Davis, C. O., Smith, O., and Olander, J. 1965b. Microwave processing of potato chips. Pt. 11. P o t a t o C h i p p e r 25 ( 3 ) , 72-92. Davis, C. O., Smith, O., and Olander, J. 1 9 6 5 ~ .Microwave processing of potato chips. Pt. 111. P o f a t o C h i p p e r 25 ( 4 ) , 78-94. Decareau, R. V. 1959. The microwave oven in hospital food service. Hospifcil M a ii a y e ni e ii t. Decareau, R. V. 1961. How micyowaves speed freeze drying. Food E t i g . 33 ( 8 ) ,3-1. Decareau, R. V. 1962. Limitations and opportunities f o r high frequency energy in the freeze-drying process. I n : Freeze Drying of Foods, Proc. of a Conf. pp. 147-162. F. R. Fisher, ed. N.A.S.-N.R.C., Washington, D.C. Decareau, R. V. 1963. Microwave freeze-drying. In : Freeze-Drying of Foodstuffs. 295 pp. S. Cotson and D. B. Smith, eds. Columbine Press, Manchest e r and London. Decareau, R. V. 1964. Microwave defiosting and heating. C o l - ) i ~ / IHotel ( r l i i ; Rcstazcl-a?zt Admiii. Qzcal-t. 5 (l), 76-78. Decareau, R. V. 1965. F o r microwave heating tune to 915 nic o r 2450 n i c . Food E u g . 37 (‘i), 54-56. Dougherty, T. J . 1965. Electrical properties of ice. I. Dielectric relaxation in pure ice. d . C h o n . Phys. 4 3 ( 9 ) , 3247-3252. Dunlap, W. C., J r . , and Makower, B. 1945. Radiofrequency dielectric properties of dehydrated carrots-application to moisture determination by electrical methods. J . Phys. Chem. 49, 601-622. Ede, A. J . , and Haddow, R. R. 1951. The electrical properties of foods a t high frequencies. Food il.1aiiu.i’. 26, 156. Eikelberg, E. W. 1950. Electronic heating f o r frozen foods. Quick Fro;( Foods. 1 2 ( 1 2 ) , 48-49. E s s a r y , E. 0. 1969. Influence of microwave h e a t on bone discoloration. Poicltry Sci. 38, 527-529. Fenton. R. 1957. Research on electronic cooking. J . Hoiiic E c o x . -19 ( 9 ) , 709-712, Fleming, H. 1944. Effect of high-frequency fields on microorganisnis. Elcctrowie E n g . 6 3 ( 1 ) , 18-21. Gaden, E. L.. and McMahon, E. K. 1947. The lethal effects of a high frequency electrical field on Eschcrichic~ coli. S.M. thesis. 24 pp. and appendix. Columbia Univ., Dept. Chem. Eng., New York. Garrick P. 1966. Applications of high frequency energy in t h e food industi,y Food T r a d e Rev. 57-61. Godkin, W. J., and Cathcart, W. H. 1949. Effectiveness of h e a t in controlling insects infesting the sui,face of bakery products. Food Tccliiiol. 3 ( 8 ) . 254-257. Gordon, J., and Xoble, J. 1959. Comparison of electronic vs. conventional cooking of vegetables. J . A m . D i e t e t . Assoc. 35 ( 3 ) , 241-244. Gouw, T. H., and Vlugter, J. C. 1964. Physical properties of f a t t y acid methyl esters. V. Dielectric constant. J . Am. Oil Chemists’ Soe. 41 (10 j . 6 7 ;i-6 78. Hard. 31. McG., and Ross, E. 1956. Dielectric scalding of spinach, peas, and snap beans f o r freezing preservation. Food T e c h x o l . 10 ( 6 ) , 241-2-14.

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Harper, J., and Chichester, C. 0. 1960a. Freeze-drying applications of dielecMilitary Industry tric heating. I n : Freeze-Dehydration of Foods-A Meeting, Chicago, Food and Container Inst., Sept. 11-14. Harper, J. C . , and Chichester, C. 0. 1960b. Microwave spectra and physical characteristics of f r u i t and animal products relative to freeze-dehydration. Final Report, Contract No. DA 19-120-qm-1349. Quartermaster Food and Container Inst. f o r t h e Armed Forces, Chicago. Harper, J. C., and Tappel, A. L. 1957. Freeze-drying of food products. A d i ~ t r i t c ciii . ~ Food R e s e a r c h 7, 171-234. Harper, J . C., Chichester, C. O., and Roberts, T. E. 1962. Freeze-drying of food--dielectric heating applied to dehydrated food production. A g r . E72g. 43 ( 2 ) , 78-81. Hasted, J. B., Ritson, D. N., and Collie, C. H. 1948. Dielectric properties of aqueous ionic solutions. P a r t s I and 11. J . Chem. Phvs. 16 (1), 1-11. Heisig, C. G., and Kobe, K. A. 1958. Selective melting of frozen solutions with R.F. power. Iizcl. Eng. Chem. 5 0 ( 1 0 ) , 1517-1524. Heppner, W., Rabinsky, R., Thornbury, T., and Robe, K. 1965. Crisper crackers without scorching. Food Processing/Marketillg p. 18G-187. Holland, J. IN. 1963. High frequency baking. Ann. Meeting of the Biscuit and Cracker Inst. 29 pp. Hoover, ill. W., Markantonatos, A., and P a r k e r , W. N. 196Ga. Dielectric heating in experimental acceleration of freeze drying of foods. Food Teehnol. 20 ( 6 ) , 1OY-107. Hoover, $1. W., Markantonatos, A,, and Parker, W. N. 196613. Engineering aspects of csing U H F dielectric heating to accelerate the freeze drying of foods. Food Techiiol. 20 ( 6 ) , 107-110. Huxsoll, C. C., and Morgan, A. I., Jr. 1966. Use of microwaves in t h e food industry. Annual Meeting, Institute of Food Technologists. Jackson, J. M. 1947. Electronic sterilization of canned foods. Food E x g . 19 ( 5 ) , 124-126. Jackson, S., Rickter, S. L., and Chichester, C. 0. 19.57. Freeze-drying of f r u i t . Food Tcchnol. 11 ( 9 ) , 468-470. Jason, A. C., and Sanders, H. R. 1962a. Dielectric thawing of fish. I. Experiments with frozen herrings. Food Tech7tol. 16 ( 6 ) , 101-106. Jason, A. C., and Sanders, H. R. 196213. Dielectric thawing of fish. 11. Experiments with frozen white fish. Food TechrLol. 16 ( 6 ) , 107-112. Jeppson, M. R. 1964. Techniques of continuous microwave food processing. Coriiell Hotel a n d R e s t a i i ~ a i i Admix. t Q u a v t . 5 ( l ) , 60-65. Iian, B. 1961. Report No. A338, 196 on Contract DA 19-129-qm-E46 to U.S. Army Natick Laboratories f o r t h e period of 16 April 1961 to 15 J u n e 1961. liinn, T. P. 1947. Basic theory and limitations of high frequency heating equipment. Food T e c h z o l . 1, 161-173. Lacey, B. A . , Winner, H. I., McLellan, N. E., and Bagshau-e, K . D. 1965. Effects of microwave cookery on the bacterial counts of food. J . A p p l . Bnctcriol. 28 ( 2 ) , 331-33.5. Lyman, C,. PI.,Burda, E. J., and Olschner, P. Q. 19-18. The effect of dielectric heating on storage quality of cottonseed. J . Am. Oil Chemists’ Soc. 25 ( 7 ) , 246-249.

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Rleryman, H. T. 1964. Induction and dielectric heating f o r freeze-drying. I n : Aspects Thkoriques e t Industriels de la Lyophilisation. 653 pp. Louis Rey, ed. Herniann, Paris. Morse, P. W., and Revercomb, H. E. 1947. U.H.F. heating of frozen foods. Electronics 20 ( l o ) , 85-89. Moyer, J. C. 1945. Electronics in the service of food technology. Farm Research 11(4), Farm Research reprint no. 59 of the N.Y. S t a t e Expt. Sta., Geneva, N.Y. Bloyer, J. C., and Holgate, K. C. 1947. Cooling a f t e r water and electronic blanching. Food Ind. 19, 1370-1372. Moyer, J. C., and Stotz, E. 1945. Electronic blanching of vegetables. Science 102, 68-69. Nelson, S. 1965. Effects of radio-frequency irradiation on t h e enzymes of beef muscle tissue. Tech. Report FD-33 of Melpar, Inc., under Contract No. DA 19-129-AMC-262(N) with the U.S. Army Material Command. Dec. Olsen, C. M. 1965. Microwaves inhibit bread mold. Food E n g . 3 7 ( 7 ) , 51-53. O’Meara, J. P. 1966. Progress report on microwave drying. Proc. Ann. Meeting of the Potato Chip Inst. Intern., Las Vegas, Nevada, J a n . 30Feb. 3, 1966. Owen, B. B., Miller, R. C., Milner, C. E., and Cogan, H. L. 1961. The dielectric constant of w a t e r as a function of temperature and pressure. J . Phys. Chem. 65, 2065-2070. Pircbn, L. J., Loquercio, P., and Doty, D. M. 1953. High frequency cookinghigh frequency heating as a unit operation in meat processing. J . A g r . a n d Food Chem. 1( 1 3 ) , 844-847. Pollak, G. A., and Fain, L. C. 1960. Comparative heating efficiency of a microwave and conventional electric oven. Food Technol. 14,454-457. Proctor, B. E., and Goldblith, S. A. 1948. Radar energy f o r rapid food cooking and blanching and its effect on vitamin content. Food Technol. 2, 95-104. Proctor, B. E., and Goldblith, S. A. 1953. Electromagnetic radiation fundamentals and their applications in food technology. Adwanees i)z Food Robe, K. 1966. Improve flavor of pasteurized products. Food PToccssiiig a d Jlarl;cti?ry. 2 7 ( 3 ) , 84-86.

Rushton, E., Stanley, E. C., and Scott, A. W. 1945. Compressed dehydrated vegetable blocks-the application of high frequency heating. Chcm. & Znd. ( h ? r d o ? i ) 1945 ( 3 5 ) , 274-276. Saniuels, C. E., and Wiegand, E . H. 1948. Radio frequency blanching of cut corn and freestone peaches. Fruit Prods. J . 28 ( 2 ) , 43-44. Satchell, F. E., and Doty, D. M. 1951. High frequency dielectric heating f o r defrosting frozen pork bellies. Am. W c u t I n s t . Bull. No. 12. Chicago. Schwan, H. P. 1965. Electrical properties of bound water. 111: Forms of W a t e r in Biologic Systems. A V H .N.Y. Acad. S c i . 125, 344-354. Schmidt, W. 1961. The heating of food in a microwave cooker. Phillips Tech. R e v . 22(3), 89-102. Simatos, D. 19G3. Constante dielectrique et teneur en eau des produits lyophilisbs. Proc. XI11 Intern. C o n f . on Refrig. 6 ( C - 4 ) , 6 pp. Smith, C. 1944. How t o prevent insect contamination. I x t c r n . Coi?iectio?ier 5 1 ( 9 ) , 30, 48, 52.

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Smith, L. L. W. 1963. Progress abroad in microwave cooking. Cornell Hotel aiid R e s t a u r a n t Admin. Q u a r t . 4 ( 1 ) , 75-78. Smyth, C. P. Dielectric Behavior and S t r u c t u r e . McGraw-Hill Book Co., Inc. New York, 1954. 441 pp. Stevens, H. G., and Fenton, F. 1951. Dielectric vs. stewpar, cookery. J . A m . Dil,tcJf.Assoc. 2 7 ( 1 ) , 32. Sugiura, Y.,Koga, S., and Akabori, H. 1964. Dielectric behavior of yeast cells in suspension. J . Gen. A p p l . Microbiol. 10(2), 172-183. Takashima, S., and Schwann, H. P. 1965. Dielectric dispersion of crystalline powders of amino acids, peptides, and proteins. J . Phjjs. C h c m . 69, 41764182. ‘Yakashima, S. 1966. Studies on the effect of Radio-frequency waves on biological macromolecules. IEEE T r a n s . o n Bio-Medical E n g i n e e r i n g , BME-13 ( I ) , 28-31. Titus, A. C. 1946. Attempted pasteurization of beer by dielectric h e a t treatment. Am. B r e w e r 79 ( 2 ) , 23-24, 65-66, ‘Thomas, &.I. H., B r m n e r , S. B., Eaton, A., and Craig, V. 1949. Effect of electric cooking on nutritive value of foods. J . A m . Dietet. Assoc. 25, 39--.14. Van Zante, H. J . 1966. Determination of cooking power distribution in electronic ranges. J . H o m e Econ. 58 ( 4 ) , 292-295. Walter, L. 1965. Dielectric thawing f o r frozen foods. Caxner and P a c k e r 36-37. Webber, 13. H., Wagner, R. P., and Pearson, A. G. 1946. High frequency electric fields as lethal agents f o r insects. J . Econ. Entomol. 3 9 ( 4 ) , 487-498. Von Hippel, A. R. 1954. Dielectric Materials and Applications. 438 pp. The Technology Press, Cambridge, Mass. Yang, H. Y., Johnson, J. H., and Wiegand, E . H. 1947. Electric pasteurization of wine. Fruit Prods. J . 2 6 ( 1 0 ) , 295-299. Zarotschenzeff, M. T. 1944. Will dielectric heating pave the way f o r South American m e a t ? M e a t J . 1-4.

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EVAPORATION METHODS AS APPLIED TO THE FOOD INDUSTRY By GEORGED. ARMERDING M o jo7iuier. Bms. Co., Onklnxtl, Cnlit’or~icr

I. E a r l y Methods of Evaporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 A. The Solar Pond . . . . . . . . . .................. 305 . . . . . _ . . . . . _ . . _ _ . . . . .306 . B. Shijoka . . . . . . . . . _. . . . . . . . . . . . . . . . . . . . . . . 306 C. The Open Kettle . . . . . . . . . . . . . . . . . . . D. The Jacketed Kettle . . . . . . . . . . . . . . . . . . . . . 307 ........................ E . Vacuum Cooking . . . 309 ....................... 310 310 312 313 314 C. H e a t Conductivity . . . . . . . . . . . . . . . . . 314 D. Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314 ............................. E. Plastics . . . . . . . . . . . . . . F. Glass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 315 318 321 322 324 327 IX. Tubular Evaporators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 X. Forced-Circulation Evaporators . , . . . . . . . . . . . . . . . . . . . . . . . . . . 330 XI. Falling-Film Evaporators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 XII. Heat-Pump Evaporators . . . . . . . . . . . . . . . . . . . . . . . . . 333 X I I I . Indirect Heat-Pump Evaporators . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 XIV. Centrifugal Thin-Film Evaporators . . . . . . . . . . . . . . . . . . . . . . . . . 336 XV. The Vacreator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 XVI. Plate-Type Evaporators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 XVII. Expanding-Flow Evaporators ......................... 342 XVIII. Fruit-Spread Cookers . . . . . . . ......................... 342 XIX. Concentration by Freezing . . . . . . . . . . . . . . . . . . . 343 XX. Automatic Cleaning , . . . . . . . . . , . . . . . . . . . . . . . . . , . . . . . . . . . 344 XXI. Control Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 XXII. Sonic and Ultrasonic Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 XXIII. The Carver-Greenfield Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 XXIV. Essence Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 A. Stripping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351 B. Noncondensable Gases . . . . . . . . . . . . . . . . . . . . . . . 352 XXV. Pre-evaporation Conditioning . . . . . . . . . . . . . . . . . . . 353 303

.

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XXVI. Electrodialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XXVII. Reverse Osmosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XXVIII. Evaluation of Evaporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

355 356 356

357

The removal of water from food products before marketing may be referred t o as dehydration, drying, or evaporation. The term “evaporation” is usually used when the resultant product is still in a liquid or semisolid state. With certain products, a combination of terms may be involved. For example, in the production of milk-solids-not-fat, evaporation may be used to remove the bulk of the water, followed by a drying process completely separated from the evaporation unit. There a r e many reasons for the evaporation, o r removal, of water from food products. While the most important of these may be economic, there a r e certain food products that are not acceptable to the consumer in the original form. Maple sap, as it comes from the tree, would certainly find no place in the markets of today. Other food products are concentrated to improve keeping quality o r to change the natural qualities to those desirable for consumer acceptance. Equipment to accomplish these results has been modified over and over again, with the objective of doing the job economically, scientifically, and quickly. Some of the most primitive methods of evaporation are still in use today and may be with us for years to come. A t the same time there a r e influences a t work bringing about changes so rapidly that even the best informed technicians cannot keep abreast of each new development. As we trace these changes from the primitive to the highly developed complex units of today we may observe a trend which will point the way to even greater improvement in the years ahead. Manufacturers and designers of evaporating equipment have been striving t o build units that will retain all the original food value while removing the water. At the same time the evaporator must be efficient, sanitary, easy to clean, of high capacity, built for long life, simple to operate, and reasonably low in cost. It must be versatile, with the ability to produce new products as they are developed by food technologists. Design changes alone could never bring about the desired results. The materials or metals used, the auxiliary and control equipment, plus new manufacturing techniques-all have a part. The general advance of science in many

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fields has contributed, in a large measure, to the success so f a r obtained. Even as we think that we have reached the ultimate, new demands and new research a r e making obsolete the masterpieces of yesterday. I.

EARLY METHODS OF EVAPORATION

A. THE SOLARPOND From the earliest records of man we learn that the solar pond was the chief source of salt. The unlimited supply of ocean water plus the evaporating characteristics of warm a i r currents made use of the solar pond extremely practical, and it is still a n economical means of salt production even in these days of automation. The largest solar ponds in the world a r e located in the San Francisco Bay area (Calif. Bureau of Mines, 1957). The Leslie Salt Company, the largest producer in the area, employs approximately 30,000 acres of tide land for the production of over a million tons of salt annually. The land used is relatively cheap, with a n assessed valuation of about $150.00 per acre (normally 42% of the actual value). With a series of ten ponds involved, the sea water is admitted at high tide and brings with it approximately 0.22 pound of salt per gallon of water (10% salometer). Through the dry summer months, evaporation proceeds at a n annual rate of 34 to 49 inches of water. A contributing factor is the strong prevailing wind of low relative humidity. When the brine reaches the last pond the volume has been reduced in volume t o ?/loth of the original sea water admitted to the system. The evaporation rate decreases with increasing concentration, and at saturation is only 30% of the volume of evaporation of fresh water. The maximum yield equals 40 tons to the acre. Harvest begins late in the year, and is carried on around the clock until the heavy rains, in December. A feature of the solar pond system is the loss of undesirable salts, which drop out in the first evaporating ponds so that, by the time the brine reaches the saturation point in the last pond (25.6" Baume), it contains 99.6% pure sodium chloride and is fit for many commercial uses (Salt Institute, 1962). Further refining and other methods of evaporation a r e needed f o r the production of table salt, which is 99.9% pure. As long as land values and taxes a r e low, the solar pond must remain as a n inexpensive and practical means of obtaining salt from sea water. When land values rise, as they did in the Long

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Beach (California) area in 1946, th e solar pond will be forced out of service. I n addition to the salt recovered, th e process also yields a n end product known a s bittern, which in itself is a valuable source of zdditional chemicals f r o m th e sea.

B. SHIJOKA A unique system used f o r the production of salt f r o m sea w a te r is known in J a p a n as Shijoka (Anon., 1961). This evaporator is made up with a series of racks, 4 to 6 meters high, covered with bamboo branches which h a n g over th e wooden frames. A distri but i ng pipe along th e to p allows th e brine to spill down over th e branches. Air moving through th e fr a me s evaporates the water , a nd concentration proceeds to a point of saturation, with the salt dropping out into ponds below. Sand beds a r e also used, but these require constant turning, a n d th e labor required v i g h t well prove too costly f o r production of a n item as low in value as salt.

C. THE OPEN KETTLE Xext t o t h e solar pond, th e open kettle is probably th e oldest means of evaporation. Primitive man hollowed out a log to evapor a t e brine f o r salt production. The American Indian used the same method t o produce maple syrup. Th e open kettle still occupies a n important place in th e production of such items as maple syrup, j a m s a n d jellies, ketchup, an d concentrated soups. Simple as t h e kettle may seem, it h as nevertheless undergone many improvements d u rin g th e last century. T h e single shallow pan in use f o r centuries f o r the manufacture of maple s y r u p has given place t o a twin unit provided with meandering channels. This construction adds heating surface and makes possible continuous production even while concentrating fro m a low of 1'2% suga r in t he r a w s a p t o a finished product testing 86" B rix (86% s u g a r ) . A float valve controls the input a n d maintains a constant depth of product in th e first unit, which is called th e s a p pan (U.S. Dept. Agr., 1958). Th e evaporation continues as th e sap flows through th e channels, an d then, at about 55-60" Brix, i t is siphoned into the finishing pan. Anywhere fro m 5 to 10 gallons is finished at a time, with th e boiling continuing until the temperature is 7°F above th e exact boiling point of water. The entire process requires 114 to 2 hours. This period is essential to produc-

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tion of the flavor and color which a r e characteristic of maple syrup. The fuel f o r evaporation of maple sap has been almost exclusively the hardwood obtained from nearby forests in the area of production. With increased labor costs the industry is now turning t o oil-fired kettles and, more recently, to the steam evaporator. There a r e advantages as well as disadvantages to use of the newer heat sources. It has been determined t h a t one gallon of fuel oil will evaporate approximately 8.4 gallons of water, so t h a t about five gallons of oil is required to produce one gallon of maple syrup (Vermont Agr. Expt. Sta., 1962). When high-pressure steam is used as a heat source, a boiler is required and the evaporating pan must be equipped with either a coil or jacket. The necessity for a licensed engineer plus water conditioning problems have made use of the steam boiler rather objectionable. The evaporator requires about 10 boiler horsepower to produce one gallon of syrup per hour (Vermont Agr. Expt. Sta., 1962; p. 1 9 ) . In some instances, oil has been used for the initial boiling in the sap pan, with a steam coil provided in the finishing pan. Vacuum evaporators have been used for the production of maple syrup, but the product is not as salable as syrup produced in a n open kettle. With maple syrup, proper color and flavor both depend on long boiling with high heat. When the open kettle is used for jams or jellies, the original color and flavor must be preserved. To do so, batches must be kept small and cooking time must be minimal. Consumer acceptance often dictates the method of evaporation.

D. THE JACKETED KETTLE The use of a steam jacket or a heating coil marked the first step in accurate control of the temperature of the heating medium. Open-fire heating invariably provided excess heat, which affected both the color and flavor of the finished product. The controlled use of steam for heat also provided a means for measuring evaporation rates. Overlooking a slight loss of heat through radiation, one pound of steam normally evaporates one pound of water. For practical purposes a ratio can be used of 1.1 pounds of steam to one pound of water. By collecting the condensate through a trap the operator can determine how fast evaporation is being accomplished. By this means it was also learned that evaporation slows down as the product increases in density, but increasing the steam pressure can maintain the evaporation rate up to a point.

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The problem of burn-on is undoubtedly the main drawback to open-kettle cooking. This has been controlled in five ways: 1) 2) 3) 4) 5)

Reduce steam pressure in coils or jacket Submerge all heating surfaces with product Provide agitation for the product Clean thoroughly between batches Limit cooking time by processing only small batches.

When high-pressure steam is used it is important that sufficient distance be allowed between the reducing valve and the heating surface. The super-heat of the steam is dispelled in energy. A “desuperheater” may also be installed, using either cold water o r condensate, to bring the temperature of the steam down to the desired level. Burn-on is usually most severe a t the surface of the cooking level. If all of the heating surface is submerged the problem will be greatly reduced. For this reason, some manufacturers of kettles have divided the heating area into zones. As each zone is submerged, steam is admitted. As concentration reduces the level of the liquid, the steam for the exposed heating surfaces is shut off. Agitation is not always desirable, nor is it always necessary. Light products boil violently and provide ample movement to prevent burning. With products such as strawberry jam or preserves, however, agitator will damage the product. Still other products demand thorough mixing while cooking since heat transfer is low. Cheese kettles may be equipped with double agitators. Almost any type of agitator is available, including rotary coils, propeller-type agitators, scraper agitators, and paddles. Some processors prefer to roll the kettle for agitation, t o prevent product damage. To prevent entrainment of air, the agitator should be kept completely submerged. As evaporation progresses in the open kettle a certain amount of precipitation is bound to occur on the heating surface. This may be made up of salts or sugar, or even protein. It is important t o remove this precipitate between batches, to avoid further burn-on or prevent a slow-down in the cooking process. Even when operation is continuous, precipitates, with few exceptions, will accumulate on the heating surfaces. Fast cooking with small batches will also reduce the possibility of excessive burn-on. When certain products such as ketchup are produced in jacketed kettles it is possible to add the various ingredients at different stages of the cooking process. This precludes using a closed o r

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vacuum-type cooker. However, in order to retain color, the ketchup must be cooked quickly-never over 45 minutes, with one-half hour being better (Campbell, 1950). Ordinary table syrups are usually produced in atmospheric kettles to control both color and flavor. Most concentrated soups a r e also processed in this type of equipment.

E. VACUUMCOOKING Any evaporation carried out a t pressures below atmospheric is termed in the trade %acuum cooking.” Whoever originated the process probably observed that a t higher altitudes water would boil under 212°F and evaporation take place just as well as, or perhaps even better than, at the higher temperatures and lower altitudes. To accomplish the same results at sea level it would be necessary to provide a closed vessel and reduce the pressure inside by mechanical means. The first recorded work of producing a vacuum was that of Evangelista Torricelli, early in the 17th century (Encyclopedia Americana, 1963). However, positivedisplacement pumps were in use even before the Christian era and may have been employed to reduce pressure in vessels before the work of Torricelli. These pumps were constantly improved until the invention, in 1840, of the direct-acting reciprocating steam pump by Henry Rossiter Worthington (Encyclopedia Americana, 1963; Vol. 23 p. 1). With the availability of a good pump to produce a vacuum, low-temperature evaporation came into being. Vacuum evaporation took hold commercially early in the 19th century. The sugar industry led the way, but shortly thereafter vacuum evaporators found a place in the dairy industry. Gail Borden began producing sweetened condensed milk in about 1850, and somewhere between 1880 and 1885 the evaporated-milk industry was born. Other food processors were slow to follow. It was not until almost the middle of the present century that suitable equipment was found for concentrating delicate products such as orange juice. This report details the advances in evaporating equipment and methods that have brought u s to the highly efficient and well constructed units of the present day. The thermodynamics of vacuum concentration have been ably described in much of the literature now available. This phase of the subject is not a part of this report, which instead centers on advances in the design and construction of equipment to meet present-day demands.

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F. T H E VACUUMEVAPORATOR There a r e four major essential parts to any piece of equipment t h a t is to be used for removing water from a food product by low-temperature evaporation. 1) A vessel of material and design suitable to withstand the external pressures created by a vacuum within the vessel and with sufficient space to do a satisfactory job. It may be spherical, egg-shaped, erect, horizontal, cylindrical, or conical ; it may be made of wrought-iron, cast iron, copper, brass, lead or tin, stainless steel, earthenware, glass, or porcelain (Hausbrand, 1908). 2) A vacuum-producing unit such as a reciprocating pump, a centrifugal pump, positive-displacement pump, a water or steam jet, and/or multiples of such jets with inter-condensers. 3) Heating surface to transmit the heat from a n outside source to the product being concentrated. Such surface may be in the form of a coil or series of coils, a jacketed area, or a system of tubes f o r use with steam, hot liquids, or direct fire. 4 ) A condenser or sink to dispose of the vapors released from the product. This may be either a wet or dry type, spray or shell construction, con-current o r counter-current, superimposed, or off-set. Two other features have recently been developed : 1) A system for the recovery of volatile esters or essence. Such a system must precede the evaporation unit and may be designed to fraction off a varying amount of the product, depending on the ruggedness or stability of the essence. 2) Equipment to clean the unit without dismantling. This may be built into the unit itself; removable, and perhaps manually controlled, partially automatic, or completely automatic. The development of the various essential features as listed has taken years of engineering and practical experience at tremendous cost to both the equipment manufacturer and the processor. A detailed study of these developments should point the way to even greater advances in evaporating technique.

II. METALLURGY A N D EVAPORATING EQUIPMENT

The steady advance in the development of metals and fabrication technique has had a profound effect on improvements in evaporating equipment and its uses. Early equipment was in-

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sanitary in construction, subject to corrosion, a n d unsuitable f o r variety of products now being produced i n vacuum equipment. Many of the first vacuum cookers were made of mild steel and cast iron, in many cases bolted together with gasketed flanged unions. The porous gasket material used wa s a source of contamination. T he crevices were difficult t o clean. Oxidation a n d corrosion often destroyed the desirable qualities of th e food product being processed, an d in m a n y cases produced off-flavors as well. To overcome these difficulties, the iron a n d steel were replaced with copper a n d bronze. Th e problem of suitable gasket material remained, but the oxidation difficulty wa s avoided by using tin as a coating f o r all contact surfaces. There still remained th e task of cleaning, which required many man-hours of h a r d work and often left much to be desired. The development of stainless steel opened th e way f o r tremendous improvement not only in evaporator construction but in sanitation methods. Th is was not a simple development, f o r i t brought with i t a number of special problems in welding, th e tensile s t r e ngth of the metals used, an d h e a t tra n s fe r. Once the food industry recognized th e potential, however, no effort was spared to Overcome the difficulties involved. JVelded construction of processing equipment was hampered since suitable welding rods had not been developed. E a r l y fabrication of stainless-steel evaporators involved th e use of so-called silver solder. The constant contraction a n d expansion of the metals f r om temperature changes caused fatigue cracks at the .joints, requiring frequent an d costly repairs. I n ma n y instances th e repairs proved unsatisfactory, an d expensive equipment had to be junked. FVelding rods of many types have since been developed, as well as welding equipment of special design which was not previously available. Metallurgists an d engineers have worked together t o overcome these problems, with excellent results. The t e r m “stainless steel” covers a wide ra n g e of products with different properties an d characteristics. These a r e classed under three broad general headings : Austenitic, Ferritic, a n d Martensitic steels. Since the Austenitic steels are those generally used f o r food processing equipment, we will not concern ourselves with th e other two categories. T he Austenitic steels a r e iron-chromium-nickel steels in which nickel exceeds about 756, an d chromium exceeds about 17%, with o r without moderate additions of other elements (U.S. Steel Corp., 1968 ; p. 7 ) . These steels a r e normally nonmagnetic. Included in R

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this group a r e all steels with AISI Type numbers in the 200 and 300 series. Throughout the food industry this metal is commonly known as 18-8 Stainless and is further identified as type 304, 308, or 316. Other types of stainless steel, substituted briefly during the war years were quickly discarded when the 18-8 series became readily available. Since types 304 and 308 a r e less costly than type 316, these steels are largely used in evaporator construction. The three types analyze as in the following tabulation (U.S. Steel Corp., 1962; p. 8 ) .

Types 304 and 308 stainless steels a r e generally corrosionresistant to most of the acids in food products but in a few instances a r e subject to attack by such acids as the malic acid found in apple juice. It is generally recommended that all evaporators for f r u i t juices and those subject to vinegar fumes be fabricated of stainless steel type 316. Dairy products containing lactic acid may be safely processed in stainless-steel equipment of either type 304 or 308. It should be remembered that some cleaning materials a r e harmful t o stainless steel, but this is dealt with later in this chapter. A. WELDINGTECHNIQUES Welding techniques generally fall into two major classes, known a s a r c welding and fusion welding. Fusion welding is objectionable for evaporator construction since it normally requires overlapping of the sheets, which creates problems which a r e not encountered when sheets are butt-welded. The a r t of welding has been refined to a point where little more could be desired, but careless workmanship will often create field problems. Electrodes for welding should have the composition equivalent of the parent metal. When sheets of metal a r e joined, many manufacturers a r e now using submerged-arc welding, in which the process is shielded with a gas such as argon or helium or a mixture of both. The results are highly satisfactory for food processing equipment.

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B.

SURFACE

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FINISHOF METALS

The polished finishes of stainless steel a r e identified by number. The unpolished surface texture, known a s a standard (or pickle) finish, may be acceptable for exterior use but is not satisfactory for surfaces that contact the product. A polished finish is considered a necessity. The lowest acceptable type of polish, indicated as no. 4 finish, is obtained with a 120/150 grit-cutting compound. Brushing with finer abrasives such a s powdered pumice or silica flour mixed with oil will produce a satin or smooth matte (dull) finish, indicated a s no. 6. F o r most food products this is considered satisfactory, but certain health officers insist on polishing to obtain a smoother surface, or mirror finish. The no. 7 finish will still have a few fine scratches even though a 240-grit compound is used for polishing. A scratch-free mirror finish, identified a s no. 8, is obtained by using a 320/400-grit cutting compound followed by color buffing compounds. Sanitarians will contend that smoother surfaces are more easily cleaned with clean-in-place (CIP) systems and for that reason are more desirable. As against this there a r e several objections to the higher polish or finish. Polishing to a no. 8 finish increases costs of equipment a s much as one-third above the cost of equipment with a no. 6 finish, even though such polishing is limited t o product-contact surfaces. Operators have also observed that with highly polished surface it is dificult to obtain full filming on heating or cooling surfaces. Instead of spreading smoothly over the surface the liquids have a tendency to flow in little rivulets, giving incomplete coverage of the surface. This is objectionable for efficiency reasons, for it becomes impossible to obtain maximum heat transfer and, in addition, it encourages “burn-on” a t the fringe of the uncovered areas. There is a definite relationship between surface tension and filming, which is afected by the condition of the heating surface. The advantage on the cleaning or sanitation side becomes a disadvantage on the operating side. As opposed to this, submerged surfaces may have a much higher heattransfer rate with the highly polished finish when good agitation is provided. A slow-moving film of product in contact with the surface means low conductivity. A rapidly moving film of product on a polished surface will bring about higher conductivity and a greater coefficient of heat transfer. Similarly, the slow-moving product will foul the surface much sooner than the fast-moving product. The film which adheres to the surface becomes thinner

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as the velocity of the product is increased. Artificial turbulence

created by a n agitator increases velocity and overcomes to a marked degree the problem of surface tension.

C. HEATCONDUCTIVITY The conductivity of heat is much higher through copper than through stainless steel. The ratio is between nine and ten to one (Farrell, 1942 ; p. 6 4 ) . Theoretically, therefore, it would require nine to ten times as much stainless-steel surface as copper surface for heat transfer to do the same amount of work. Actually, this is not true-for several reasons: the greater strength of stainless steel permits the use of much thinner material (in some instances manufacturers have reduced the gauge of the metal used to a point where, for economical reasons, they have gone beyond a reasonable safety factor) ; the absence or reduction of surface tension provides greater velocity of the product across the heating surface and almost completely offsets losses from poor conductivity ; and there is less fouling of the heating surface. The actual advantage or disadvantage of the one metal above the other, as pertaining to heat transfer, has never been reduced to a simple equation. More research is needed to determine the relationship of one metal to the other when all of the above factors a r e taken into consideration. Clad metals with a combination of iron and stainless steel or copper and stainless steel have been used, but increased manufacturing problems discourage the use of such combinations. Improved techniques will need to be developed before such clad metals will find general acceptance for processing equipment.

D. MAINTENANCE Stainless steel is f a r superior in maintenance factor and life expectancy to other materials used in evaporator construction. Oxidation and corrosion a r e practically unknown if the steel is properly selected to suit the product being processed. Stainless steel is also resistant to the abrasive qualities of sugar crystals which form during evaporating processes, a s well a s to materials such a s the tartrates inherent in Concord grape juice.

E. PLASTICS The rapid development of plastics and bonding materials may, once again, open the way to the use of base metals, properly coated, in evaporating equipment. Certain component parts are already being manufactured of plastic materials, and this use will

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undoubtedly increase. The materials used a r e inert, and costs a r e very favorable. Single-service equipment liners may speed up cleaning time. Many rubber-lined evaporators a r e now in service in chemical plants, but their use in food plants is limited.

F. GLASS The use of glass has been restricted because of the extreme danger of breakage. The production of a n unbreakable shatterproof glass will open the way for new design in evaporating equipment and overcome some of the objections to in-place cleaning. The possibility of transparent equipment is certainly intriguing since it may reveal a number of processing facts which a r e now concealed and remain unkown.

Ill.

PRODUCING THE VACUUM

The eficiency of early vacuum evaporation equipment depended largely on the vacuum pump. Since steam was the source of heat in the evaporator it was only natural t h a t early vacuum pumps were steam driven. The exhaust steam from the pump in many applications was used a s a source of heat in the coils or jacket of the evaporator. The so called “wet” vacuum pump did double or even triple duty by removing the water from the condenser, producing a vacuum, and providing heat. Early milk evaporators used the “wet” vacuum pump almost exclusively. There were exceptions where recovering the distillate separately was desirable or when barometric condensers were used. The punips were of heavy construction, with reciprocating pistons operating a t about 100 strokes per minute. The valve area was large, limiting the service to about 27 inches Hg on the vacuum gauge. The dry vacuum pumps used were also of the piston type but were operated at much higher speed-up to about three times that of the wet vacuum pump. This eliminated most of the pulsations but left much to be desired. Equipped with a flywheel or governor, they could be either steam-driven or connected by belt t o a gasoline engine or motor. Although both the wet and dry vacuum pumps were of rugged construction, limitations in speed and capacity soon forced the development and use of other types of vacuumproducing equipment. The centrifugal pump was invented in 1689 by Denis Papin, a Frenchman (Osbourne, 1944). It has largely supplanted the old-

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fashioned reciprocating pump for vacuum equipment. As the name implies, the pump depends on centrifugal force in its operation. The casing remains static, while the impeller, revolving at high speed, tends to force a i r o r liquid away from the center inlet, forcing it through a n outlet a t the periphery of the impeller. There are a number of manufacturers of rotary pumps of this type. Two of the most commonly used a r e the Nash Pump and the Beach-Russ. In the Nash pump (Nash, 1950) the impeller consists of a series of curved vanes revolving in a n elliptical casing. The principle of operation requires water in the pump a t all times. The water rotates with the impeller. A t the narrow part of the casing the water fills the space between the vanes but retracts from these spaces a s the impeller revolves into the long end of the casing. This produces a vacuum and, in turn, a pressure as the rotation continues. The water seal is maintained constantly and provides a means for the dispersal of heat produced in compression of the air. The inlet and the outlet a r e on opposite sides of the pump casing. A pump of this type will eficiently maintain a vacuum up to 27 inches Hg in single stage, and higher in two stages. The maximum vacuum is dependent on the barometer reading and will vary as in the following tabulation.

The vacuum produced is nonpulsating, a feature which is very desirable in the operation of vacuum evaporating equipment. The Beach-Russ (Beach-IZuss Co., 1958) is a rotary-piston pump with a minimum of moving parts: the rotor and its slide valve, which a r e always completely oil-sealed in the pump cylinder. The rotor is set eccentrically in the cylinder, with the elliptical cylinder bore made to such close tolerances that the end of the slide valve in the rotor maintains uniform minimum clearance from the cylinder wall through its rotation. Thus, a s the rotor turns, the slide valve acts as a piston, forcing all the air o r gas out of the cylinder

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through the discharge valves. Simultaneously, a constant vacuum is created behind the slide valve piston as new air or gas is drawn in through the manifold heads and intake ports in the hollow slide valve. The air space on the discharge side of the pump is steadily reduced, forcing the a i r through the exhaust port into the double spring-loaded disc valves. These valves, submerged in oil, make it possible for the pump to discharge against atmospheric pressure for a small part of the revolution, and prevent back-leakage of air into the pump. Many operations still employ vacuums in the range from slightly under atmospheric pressure to 28 inches Hg. Today, however, a r e common. The pump sustained vacuums of from 5 to 10 described above will produce such vacuums, but where lower pressures a r e necessary, two-stage pumps a r e available with the ability to provide pressures as low a s .0004 to .0002 mm H g absolute. The ability of vacuum pumps to produce practically any desired pressure within the evaporator leaves little room for further demands in this direction. Another method for the evacuation of equipment is the jet ejector. The most common type is the steam-jet ejector, in either single-stage or multiple-stage for low-pressure evaporation. The ejector was first conceived in the early part of this century by a European and a n Englishman, Le Blanc and Parsons, who a r e credited with the original development (Graham, 1963). Ejectors were introduced into the United States in about 1915. The ejector is composed of four stationary parts: steam chest, nozzle, suction chamber, and diffuser. I n operation, high-pressure steam flows through the steam chest and into a nozzle, where the steam’s pressure energy is converted to velocity (usable kinetic energy). During this conversion the steam accelerates, expands, and decreases in pressure. As the steam leaves the nozzle it has accelerated to its maximum velocity, which is supersonic, and expanded to the minimum pressure. This high-velocity low-pressure jet of steam now enters the suction chamber, where the gas or vapor to be evacuated is drawn around it, entrained, and carried into the diffuser. The design characteristics (Graham, 1963) as claimed by one leading manufacturer of jet ejectors are a s follows: a ) Stability in operation up to the guaranty point, but with a reasonable overload factor

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b ) Economical steam consumption and economical water consumption if inter and after condensers a r e used d ) Low first cost c) Operation that is devoid of mechanical trouble e) Ease of replacing any worn, corroded, or broken part f ) Durability through proper selection of materials and ample thickness of all parts. The first ejectors featured multiple jets, but it has since been found that a single jet gives better performance. The small orifice in the mutiple jet had a tendency to clog from scaling with boiler compounds or cleaning solutions. The large single jet has overcome this objection in some measure, but the difficulty still exists. A single-stage ejector may produce a vacuum as high as 50-100 mm H g Abs. with reasonable steam pressure, but for high vacuum a n ejector with two or more stages is desirable. The two-stage ejector will produce a higher vacuum with less steam. There a r e a number of difTerent desigm, from the single stage up to seven stages, depending on the vacuum desired. A condenser is recommended betweea the stages. The temperature of the water used in the condenser affects the capacity of the unit. The colder the water, the greater the capacity. Similarly, the steam pressure mill also affect the efficiency of the ejector. The higher the pressure, the greater the benefits. Water-jet ejectors a r e also available. With water pressures of 40 psig or higher, a vacuum in the range of 4.0 inches to 1.0 inch H g Abs. is possible with a single-stage unit. IV.

THE HEATING SURFACE

The type and design of the heating surface and the total area of such surface required a r e dependent on a long series of variables. Since many of these variables a r e still unknown and have not yet been identified, it is exceedingly difficult to determine beforehand the size or design of equipment for any particular job. To complicate the problem further, many evaporators must be utilized for more than one product and at varying capacities. The determination of the amount of heating surface required is dependent on the coefficient of heat transfer, sometimes referred to as U value or I< value (Spencer 1929). The unit of heat transfer, the U value, also called the specific thermal conductivity, is the number of thermal units transferred per hour from the heat-

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ing medium through the heating surface to the product f o r each degree difference of temperature, Fahrenheit, between the heating medium and the product for each square foot of heating surface. When expressed in caiories (the amount of heat required to increase the temperature of one kilo of water by one degree centigrade), the coefficient of transmission of heat is the figure which gives the number of calories which pass in one hour from a warmer to a colder liquid through one square meter of the partition when the difference in temperature between the warmer and colder fluids is one degree centigrade. The coefficient is represented by K (Hausbrand, 1908). The efficiency of the heating surface, whether in terms of U or K , will be determined by any one or all of the following variables, which a r e vital to the function of the evaporating equipment : 1) Kind and type of metal or material used to construct the heating chamber 2) Thickness o r gauge of the metal or heating surface 3 ) Temperature of steam or liquid used as a heating medium 4) Quality of the steam 5 ) The uniformity of steam pressure or steam flow 6 ) Proper distribution of the heating medium 7 ) Absolute pressure within the vessel 8) Velocity of steam o r heating liquid over the heating surface 9 ) Presence of a i r or other noncondensable gases, in the heating chamber 10) Presence of moisture or condensed vapor in the heating chamber. 11) The cleanliness of the heating surface-"burn-on" 12) The character and thickness of any scale or deposit on either side of the heating surface 13) Rate of product in-feed 14) Temperature of in-bound product 15) Foaming characteristics of the product 16) Viscosity of the product 17) Pectin content of product 18) Starch content of product 19) Gum content of product 20) Presence of tartrates 21) Total solids of inbound materials 22) Hydrostatic head above heating surface

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23) Boiling-point rise 24) Turbulence 25) Escape area for vapors 26) Temperature of condensing water 27) Even flow and distribution of condensing water 28) Barometric pressure 29) Ambient temperature 30) Heat loss through evaporator walls 31) Skill of the operator 3 2 ) Accuracy of instrumentation 33) Efficiency of all auxiliary equipment 34) Design of auxiliary equipment 35) Location and control of product inlet 36) Design and location of condensate removal equipment 37) Efficient removal of noncondensable gases from heating chamber. Other variables could undoubtedly be added to the above list. Some operators claim that there a r e at least fifty. Designers of evaporating equipment have improved design and operating technique to off set many of these so-called variables. Unfortunately, much of the valuable information obtained from actual plant operation has never been published, and much additional information is classed as trade secret and never released. Since such factual information is not generally available, pilot-plant operations have been recommended to provide the answers. Many of the actual operating conditions cannot be duplicated in a pilot model, which leaves some of the problems to be resolved in the field, often a t great cost and inconvenience. It is a known fact that duplicate pieces of equipment fail to produce the same results in different areas of the country and with different operators. In the experience of the writer, operation of the equipment will vary with the time of day and even with wind velocity when portions of the unit are exposed. Knowledge obtained from experience and keen observation of contributing factors has had a profound effect on evaporator design, so that present-day units a r e giving highly satisfactory performance. The introduction of new raw materials and new products creates new problems. Our wealth of experience, when properly applied, should help anticipate such problems and guard against difficulties created by faulty design, construction o r operation.

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V.

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CONDENSERS

When evaporation is accomplished at atmospheric pressure, the vapors produced, being lighter than air, rise and a r e dispelled into the atmosphere. When the evaporation is carried on in a vessel at lower than atmospheric pressure, a means for condensing these vapors is needed, unless they can be evacuated in some other way. Since the volume of the vapor increases with decreased pressure, the cost of moving these vapors without condensing is prohibitive ; hence the need for a condenser. The condenser must be a closed vessel into which the vapors a r e drawn. The condensation is accomplished by withdrawal of heat with either cold water or a i r or a refrigerant. Since the former is cheaper, practically all condensers use water as a means of condensing the vapors. The water may be used in two ways: either the cooling water is injected directly into the vapor to be condensed, or the vapor is conducted over surfaces cooled by water or a i r or a refrigerant. These condensers a r e identified as either j e t condensers, sometimes referred to as barometric condensers, or surface condensers. J e t condensers may be of either the wet or dry types. Where large quantities of vapor, having no commercial value, a r e to be condensed, the wet or barometric condenser is used. This is considered practical with vacuum up to 26 inches Hg. The condensed vapor, the water and the noncondensables a r e all drawn off through the same pump. With a vacuum above 26 inches Hg, a barometric condenser is used in combination with a n a i r ejector or a dry vacuum pump. The condensed vapor and the water flow down through a tailpipe to a hot well, and no pump is necessary if the bottom of the condenser is 34 feet or more above the water sur€ace in the hot well. The a i r ejector, o r dry vacuum pump, in turn, removes the noncondensable gases. I n place of a hot well, a gooseneck at the bottom of the tailpipe with a flapper valve may be used provided the flapper valve is at least 34 feet below the bottom of the condenser. When such height is not available, a suitable pump may be used at any convenient level. The surface condenser is made up of a number of tubes, either horizontal or vertical. I n some cases the vapor is condensed on the outside of the tube, with the water or a refrigerant on the inside, but others a r e just the reverse. Where the vapor is condensed on the inside of the tube, the pressure drop encountered may be objectionable.

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Jet condensers are made in a number of styles, but the basic principle has not changed. The spray type depends on the amount of surface of water exposed. The baffle type (Byer type) depends on a series of cascades through which the vapor must pass to reach the ejector or vacuum pump. A minimum of three cascades is required, but for higher efficiency four or five passes a r e generally provided. Basically, the outgoing water from the condenser must be lower in temperature than the vapor in the evaporator. F o r best use of the water, the closer this temperature is maintained, the better. Where control of the water in-feed is manual, the temperature difference may be as much as 1 0 ° F or even more. Automatic controls can narrow this difference to under 5"F, and sometimes even as low as 2 ° F with satisfactory results. The vacuum should always be somewhat higher (i.e., the pressure somewhat lower) in the condenser than in the evaporator. A recent development in evaporator design employs a n entirely new approach to the vapor-condensing problem. Instead of attempting to compress the high-volume water vapor for reuse, a transfer of heat is made from the water vapor to another vapor (NH,) which has the characteristic of low specific volume, and then this low-volume vapor is compressed. The use of water is at a minimum, and the heat thus absorbed can be salvaged again in the heating section of the evaporator. This type of low-temperature equipment is discussed in detail later in this chapter. VI.

THE VACUUM PAN

As stated earlier, the primary purpose of a vacuum cooker is to remove water from a product a t a lower temperature than that required at atmospheric pressure. To evaporate one pound of water in a n atmospheric kettle, starting with a temperature of 32°F requires the expenditure of 1146.6 BTU. To remove the same amount of water under vacuum at 140°F requires the expenditure of 1123.3 BTU, a saving of 2.03% in heat units (Mojonnier and Troy, 1920). Further, the amount of heating surface required is considerably lower when evaporation is accomplished at reduced pressure. For example, at 14.72 psi absolute and a boiling temperature of 212'F, the pounds of water evaporated per hour per square foot of surface a r e approximately 8.2 whereas at 2.034 psi absolute (25.85 inches H g in a column), the boiling temperature is reduced to 126.15"F and the pounds of evaporation

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per hour per square foot of heating surface a r e increased to 30.2 (Hunziker, 1920). The earliest vacuum cookers were commonly called vacuum pans. The “pan” consisted of a cylinder with a dished bottom and a domed top from which the vapor was carried off to the condenser through a large gooseneck. The pan was constructed of either copper or iron, possibly 5-6 f t in diameter. Sometimes the height was the same as the diameter, while other manufacturers increased the height for supposedly better operation. Claims were made that the taller equipment was less likely to splash over into the condenser, but, against this, it was contended that the narrow, tall cylinder increased velocity, with greater carry-over into the condenser. The lower part of the vacuum pan was dished f o r strength, and jacketed to provide the necessary heating surface. Distribution of the steam in the jacket was poor, with hot spots causing scorching or burning of the product. Heat losses by radiation from the outer surface of the jacket raised steam requirements s o that i t was not uncommon to use 1.1-1.2 Ib of steam for every pound of water removed. To overcome such radiation losses the jacketed cooker gave way to a pan equipped with a coil installed in the lower part of the cooking chamber. Steam was admitted at one end of the coil, usually the top, and the condensate removed through a t r a p at the other end of the coil. This type of construction brought with it a new series of problems. The coils, spaced near the wall of the vessel, were very difficult to clean. The condensate forming in the coil insulated the heating surface, cutting down on the heat transfer, and causing burn-on, which added to the cleaning problem. The next logical step wa.s to divide the coil into separate units, each with its individual inlet and outlet. As the product was drawn into the pan the steam would be admitted to the coils as they were covered. This overcame much of the burn-on problem. Various types of coil construction were used, such as round, elliptical, or even box-shaped. These, in turn, were mounted in series, with a large coil surrounding several smaller coils. The banks of coils were superimposed above each other three or four high, and sometimes augmented with a jacket on the pan itself to provide additional heating surface. The cleaning and condensate draining problems remained unsolved. Further design difficulty was experienced from expansion and contraction of the heating coils, causing metal fatigue with subsequent cracking. Heating

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surface in the largest unit of this type was limited to about 300 ft. The vacuum pans described above were usually operated by what is commonly known as the “batch” method. Product was drawn into the pan, keeping pace with the evaporation, until the capacity of the unit had been reached for a finished batch. Samples would be drawn from time to time, by means of a striking cup, to test for density. When the proper concentration was reached for the full batch, the steam was shut off, the vacuum released through a vacuum breaker, the product, drawn off with a pump or by gravity, and the whole process repeated. A complete cycle might require a s much as 3 4 h r hours or even longer. No attempt was made t o wash the equipment between batches. After three o r four such batches had been processed, the coating of product on the coils was generally so severe t h a t further use of the equipment was not practical. Stickage and entrainment losses were excessive, but, since no better equipment was available, such losses were tolerated. Heat damage to the product was another serious drawback to such equipment, but, even with all of these objections, many of the primitive “pans” a r e still in operation as this is being written. VII. THE CALANDRIA PAN

The term “calandria,” unique to evaporating equipment, simply means a tube bundle or a tube chest with the product flowing through the tubes and the heating medium around the tubes. The tubes a r e relatively short and seldom longer than six feet. In the center of the vertical tube bundle, a large downtake pipe was installed, usually larger than necessary, but this was considered good practice. The pattern of the vertical tubes surrounding the down-pipe was dictated by manufacturing convenience. The entire tube nest, or calandria, was installed in the lower part of the vacuum pan. The dished space below would permit circulation of the product. The outer wall of the calandria was also the outer wall of the pan. A t least one manufacturer departed from this construction to provide a basket-type calandria which was entirely separate from the wall of the pan. With this unit, circulation was up through the heating tubes and down through the space allowed between the basket and the side wall as well as through a downpipe. There was no definite path for the liquid to follow, resulting in wildness and varying turbulence.

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The heating tubes varied in diameter from 2 inches up, but most manufacturers considered 2 or 254 inches the proper size. For t o 1/3 t h e best operation, a product level was recommended at tube height, but since no level control was provided, such levels were practically impossible to maintain. The vapor space above the tube nest varied in diameter a s well a s in height. Some evaporators were made with a diameter the same as the calandria for the full height. Others were provided with a flared rim a t the top of the calandria to provide a larger diameter for the vapors, while still others were constructed using a n inverted cone with the vapor body tapering down in diameter to the size o f the gooseneck which conveyed the vapors to the condenser. One such model is called the Garrigue evaporator (Webre and Robinson, 1926; p. 447). The vapor body was extended to a diameter of 8 ft. The heating tubes were in the center of the calandria, and 2 inches in diameter, with 4-inch tubes in the outer pattern for down flow. Another modified calandria pan is called the Manistee evaporator (Webre and Robinson, 1926; p. 452). It provides mechanical agitation. The calandria has a large central down-pipe. A propellor, 75% of the diameter of the down-pipe, is mounted slightly below the bottom o f the calandria and driven by a shaft extending through the top of the evaporator. The agitator revolves at 40 rpm. The heating tubes a r e 5 f t long and either 2 o r 2% inches in diameter, with circulation up through the small tubes and down through the large center pipe. The level of the liquid is carried well above the tube nest in this unit, and the vapor chamber is flared out for greater diameter. The vapor space is constructed as a n inverted cone. An occasional unit was built as large as 30 f t in diameter, and even larger sizes have been reported. The problems with the calandria pan were numerous. The tube sheets provided areas where the product would remain for indefinite periods, resulting in severe burn-on. Condensate forming in the steam chest would accumulate in varying depths on the lower tube sheet, insulating this heating surface a s well as a portion of the tubes. The removal o f noncondensable gases was on a hitand-miss basis, depending on a petcock which was allowed to bleed off steam into the atmosphere a t all times. The entry of the steam into the tube-nest chamber created a problem of even distribution, which some manufacturers tried to overcome with baffles or multiple inlets. Another more serious problem was

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GEORGE D. ARMERDING

that of cleaning. Access was provided into the vapor chamber through a manhole, usually not more than 16 inches in diameter. A similar opening was provided for access to the space below the calandria. To assist the cleaning of this lower portion of the evaporator, the size was increased, but this also increased the amount of product necessary for operation of the evaporator. The job of cleaning was a menial one and dreaded by all employees. As a result, strong cleaning solutions with one t o two pounds of caustic soda to each cubic foot of water were allowed to remain in the unit overnight and sometimes over a week-end, with final clean-up just prior to starting the equipment. Treatment with the caustic soda was occasionally followed by a n acid rinse. The chief objection to the calandria type of pan was undoubtedly the large amount of product resident in the equipment in order to obtain circulation. Heat damage was severe, primarily because of the depth of product and the increased hydrostatic head. Spencer (1929) has shown that with a head of 2 f t of 50.6'F Brix syrup with a vacuum of 26 inches Hg, water boils at 125.5"F. Down 2 f t the water boils at 141.8"F plus 4 degrees for the boiling-point rise of the syrup, making a total of 145.8"F to produce boiling, or a loss of degrees equal to 20.3"F. This higher temperature is not apparent to the operator but may be very destructive to the product itself and at the same time materially reduce the efficiency of the unit. To overcome the problem of the large volume of product below the calandria, the down-pipe was extended several inches, thus increasing the turbulence in that area and preventing dead pockets of product. The calandria type of pan made possible the reuse of heat from the vapors produced, by operating a series of pans at reduced temperatures with the vapors from the first unit entering the calandria of the second unit, and so on. The vacuum would be 5-7 inches H g in the first effect, 14-17 inches H g in the second effect, and 26-28 inches H g in the third effect. Such multipleeffect units have been operated in up to a series of five or six in certain industries, with the cost of equipment being the limiting factor. The first multiple-effect evaporator was invented by Norberto Rilleux, a free Negro, in New Orleans in 1840. He contended that a pound of steam in the first effect would evaporate a pound of water. The vapor produced would evaporate a pound of water in the second effect, and so on. This phenomenon is termed Rilleux's rule, in honor of its inventor (Van Hook 1944).

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At least one manufacturer (Manzani, Italy) constructed a calandria pan with one unit superimposed above the tomato products. The upper, or top, calandria uses natural, or thermo-siphon, circulation. The product then flows by gravity to the lower calandria, where forced circulation is employed. One common entrainment separator and condenser is used for both stages, since pressure is constant throughout the unit. Horizontal-tube evaporators were also constructed, with the steam on the inside of the tubes and the product on the outside, just the reverse of the ordinary calandria type of pan. Such equipment was limited for use with products t h a t do not have scaleforming tendencies or that do not require mechanical cleaning. VIII.

ENTRAINMENT SEPARATORS

The carryover of liquid particles from the vapor chamber to the condenser is called entrainment. The loss of product may be excessive unless some method is devised to control the carryover of such liquid particles. Numerous safeguards have been built into present-day evaporators to control carryover a t its source. It is important to introduce the product into the evaporator in such a way that there is no conflict between the escaping vapors and such liquid. The tangential method of introducing product has proven very effective. Centrifugal force and gravitation overcome the tendency f o r the product to escape. With foamy products, additional precautions must be taken, and f o r this reason numerous separators have been developed to control losses. An intermediate, or external, separator is sometimes provided between the vapor chamber and the condenser. The flow of the vapor is directed against the side of the separator, and, again, centrifugal force and gravitation combine to reduce losses. Internal separators a r e mounted above the vapor chamber and are constructed in many different patterns. The vapor velocity is reduced, with a corresponding reduction in product loss. Claims made by manufacturers a r e inconclusive, for there a r e many contributing influences which might well offset such claims. Use of an entrainment separator increases the clean-up problem, adds materially to the cost of the equipment, and in many cases causes a pressure differential between the condenser and the vapor chamber, with a corresponding increase in operating temperatures. The prevention of entrainment by removal of the cause of such losses is much more satisfactory than any method of re-

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GEORGE D. ARMERDING

covery. There is a tendency on the part of the operator to become careless, depending on the equipment, to recover product that may carry-over. Once the product starts up into the condenser, it becomes very difficult to prevent it from going all the way. I n certain evaporators, such as the centrifugal thin-film type, it is possible to provide a mechanical unit which is claimed to be highly efficient in preventing carry-over. This unit is offered in several designs. In one type the vapors are passed into the unit in spiraling streams through a stepped series of rotating concentric rings which deflect the vapor radially inward while any entrained droplets a r e thrown outward and returned to the thermal section. The combination of rotary velocity, centrifugal action, and change of direction results in a very compact and effective separator. Another type has vertical rotating blades which pass any entrained droplets to stationary fins, which return them to the thermal section. High centrifugal action, coupled with impingement, makes this an efficient separator suited to products producing a foam (Rodney-Hunt Machine Co., 1963). IX.

TUBULAR EVAPORATORS

A transition from the calandria pan to the tubular type of construction was inevitable. The problems referred to in the discussion of the calandria pan demanded a solution. Paul Kestner, a n eminent French engineer,, first patented the tubular evaporator in 1899. Other engineers, applying themselves to the job, developed almost as many designs a s there were manufacturers to construct them-or even more. Nor is the end in sight. New designs are offered even prior to the installation of existing designs. No attempt is made here to give an exhaustive description of all the designs that were ever offered. Such a treatise could not be included in the confines of this chapter. However, certain weI1defined variations are given careful consideration. Tubular evaporators may be grouped into three general classifications : thermo-siphon, forced circulation, and falling film. The thermo-siphon tubular evaporator depends on the energy developed from heat for circulation of the product through the tubes. The direction of flow is from the bottom of the tube t o the top. The vapor-separating chamber may be mounted directly above the tube nest or off to one side. Product is drawn into the unit either at the base of the tube nest or into the vapor-separating chamber. A level must be maintained high enough in the tubes

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that there will be sufficient liquid in the thermal tubes t o induce circulation. The height in the tubes varies with the pressure within the unit, the temperature difference (AT) between t h e steam and the product, the tube size, and the specific gravity of the product. It can readily be seen that the amount of product must be great enough to keep the evaporator cycle constant. This amount compares favorably with similar-capacity units of the calandria type. The improvement in the quality of the product in the tubular evaporator was immediately apparent, and steps were then taken to reduce still further the amount of product resident in the equipment. Vapor chambers were built in a cone shape, thus reducing the capacity of the lower portion and still providing adequate space for vapor removal. The space beneath the tube bundle was kept to a minimum, and the transfer pipe from the vapor chamber to the tube chest was reduced in diameter until further reduction would restrict flow. The transition from the calandria pan to the tubular evaporator came about so quickly that very little research, if any, was done by the manufacturers in their own laboratories. The many different designs produced were field-tested in production plants, often a t great expense to the equipment manufacturer as well as to the processor. Tube sizes varied in diameter from 1 to 3 inches, and in length from a short 6 feet 8 inches t o a long 20- or 24-foot size, and sometimes even longer than that in experimental equipment. Tube bundles were set vertically alongside the vapor separating chamber. Qthers were tilted a t a n angle, while still others were built within the vapor chamber itself. Many of the design features conformed to economic requirements instead of operational demands. Since tube manufacturers standarized on 20-foot lengths, this dictated the lengths used in the thermal chambers-such as 20 f t , 10 ft, and 6 f t 8 inches. A long tube provided more surface for less money, and in the interest of economy very little thought was given to a problem such as expansion and contraction. As a result, many tubes bowed and buckled and often ruptured or completely withdrew from the tube sheet. Supporting members were then introduced to keep the tubes in place. This, in turn, led t o difficulty with steam distribution and condensate removal. Vapors condensing in the top of the tube bundle would produce a coating of condensate on the lower portion of the tubes, where heat was vitally needed for thermo-siphon action. Removal of the condensate from the steam chest depended on gravity flow, generally through a trap, and many traps would not function until a good

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GEORGE D. ARMERDING

head of condensate would accumulate. I n some units i t was found possible to improve capacity by simply lowering the condensate t r a p or pump. Other problems developed in removing noncondensable gases from the steam chest. Vent lines were installed between the steam chest and the condenser. The noncondensables were drawn by vacuum through the condenser into the ejector and exhausted. No one seemed to know, for sure, what size such a vent line should be or whether it should connect to the top or the bottom of the tube chest. To overcome this difficulty, the pipe was generally oversized, provided with a valve,, and connected to both the top and bottom of the tube chest. Seldom did the operator adjust such valves, and, as a result, steam losses were excessive or evacuation of noncondensables was not complete. A number of the undesirable features of the calandria pan were still present in the tubular evaporator. The problem of cleaning remained. When burn-on occurred it was found necessary to clean the tubes with a wire brush. This, in turn, called for clearance above the evaporator sufficient to use a brush with a handle 4-6 f t longer than the tubes. Openings were provided for this purpose in the roof of the building housing the equipment. A further objection to the thermo-siphon tubular evaporator was the inability to obtain proper circulation with heavy or viscous products. Increased steam pressure was used to overcome this difficulty, but the cleaning problem became more acute. X.

FORCED-CIRCULATION EVAPORATORS

The foiced-circulatiorr. evaporator followed the design of the thermo-siphon system but with a pump added between the vapor separator and the thermal-tube bundle. The purpose of the pump was to provide enough circulation to prevent burn-on and to speed up heat transfer, especially with heavy products. Capacity with lighter free-flowing products could also be increased. The circulating pump brought about a new series of problems and added to the cost of the equipment. .Operation of the pump itself required power, increasing production costs. A t the same time, changes in design became necessary to ensure continuous uninterrupted performance of the equipment. It was found that a cone-shaped vapor chamber with a tangential inlet produced a swirl of the product, causing a vortex to form directly above the inlet of the pump. This vortex carried down into the pump, resulting in cavitation and severe vibration. To overcome this diffi-

EVAPORATION A S APPLIED TO T H E FOOD INDUSTRY

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culty, the level of the product was raised or swirl breakers were installed. Construction changes replaced the cone with a half-cone design. Most of these problems were solved in production plants, again at great cost to equipment manufacturers and processors. The advantages of the forced-circulation system over the thermo-siphon system a r e chiefly the ability to produce a higherdensity concentrate and to do this without serious burn-on. The higher velocity of the product through the tubes increased the rate of heat transfer, reducing the amount of heating surface necessary f o r a given capacity. Heat damage and stickage were reduced, allowing longer periods of operation without shutting down for clean-up. Density control of the finished product could be maintained on a constant basis. The increased capacity reduced the residence time, improving quality further. One manufacturer (Chicago Bridge & Iron Company, Oak Brook, Illinois) listed certain advantages of the forced-circulation evaporator as follows : 1) Can achieve high final concentration 2) Controlled operating conditions such as level of product and retention time 3 ) Retards scale 4 ) Easy to clean; lends itself to in-place cleaning 5) Lower investment 6 ) Economy because of greater number of effects 7 ) Stability of operation and capacity over longer periods. XI.

FALLING-FILM EVAPORATORS

The development of the falling-film evaporator has undoubtedly been the greatest single step forward in evaporator design and construction since the introduction of vacuum cooking. In this unit, a thin film of product flowing down the inside surface of tubes is evaporated by a heating medium, such as steam, on the outside of the tubes. This type of equipment has made possible low-temperature evaporation, either in single pass or by recirculation, with a short retention time within the unit and the absence of all hydrostatic pressure differences in the product. One of the unique characteristics of this equipment, as pointed out by Farrell (1963), is the importance to maximum efficiency of uniform distribution of the product on the inside of the tubes. This has been a major problem with manufacturers of the equip-

332

GEORGE D. ARMERDING

ment and has resulted in a variety of designs-with inconclusive results. Let us assume that a unit with a capacity of 20,000 lb of in-feed per hour utilizes 100 tubes to do the work. Each tube should receive 200 pounds of product. J u s t how can this be accomplished? Of the number of methods of distribution designed, some have given fairly satisfactory results, but it cannot be claimed that any particular design has overcome the problem completely. The various methods used a r e briefly described as follows : A perforated plate placed on top of the tube nest had holes drilled above each tube, and a constant head of product above the plate assured that a stream of product entered each individual tube. There was no assurance that the liquid entering the tube would be distributed evenly on the heating surface. I n addition, coagulation of product soon cut off flow t o some of the tubes, with subsequent fouling and burn-on. One manufacturer provided a series of weirs, or collars, inserted in the tubes. The liquid was allowed to overflow the weirs, assuring distribution on the entire circumference of the tube. The product on the tube sheet was subjected t o constant heat, and soon accumulated a layer of coagulated or burned material. To offset this objection, perforations or slots were provided in the weirs t o reduce the level of the product and keep it in motion. Another means of even distribution utilized a cone head above the tube nest. The product enters the top of the cone through a swirled insert, breaking up the stream into a spray, with fairly satisfactory results. Still other designs employ a series of baffles to accomplish even distribution. The success or failure of the entire unit depends, in a large measure, on satisfactory distribution of the product to the tubes. Further improvements in distribution systems will undoubtedly be developed. The success of any given system can be determined by inspecting the tubes after the operation is complete. An absence of precipitation or burn-on is substantial proof that distribution is satisfactory. I n the many units t h a t a r e cleaned, automatically, such subsequent inspection is of doubtful value. There a r e many advantages to the falling-film evaporator, and it lacks many of the disadvantages of evaporating equipment enumerated previously. One manufacturer ( Goslin-Birmingham Mfg. Co.) claims that in any discussion of the advantages of the falling-film evaporator not the least important consideration is the liquor velocity obtainable with a given flow of liquor to each

EVAPORATION AS APPLIED TO THE FOOD INDUSTRY

333

individual tube. Let us consider, for example, a Il/,-inch tube 20 f t and 0 inches long; in a forced-circulation evaporator, it would be necessary to pump a quantity of liquid equivalent to 40 gallons per minute to obtain a velocity of 8 f t per second, whereas in a falling-film evaporator, pumping about 3 gallons per minute per tube would give velocities up to 20 feet per second a t the lower end of the tube. Another important advantage of the falling-film evaporator is the small amount of product resident in the equipment during operation. Since most such evaporators a r e constructed in multiplc effects or stages, the entire concentration can be accomplished in a single pass or with a minimum of recirculation in the final stage. There a r e certain disadvantages to be weighed in light of the performance of the equipment. Falling-film evaporators have a high cost compared with conventional equipment. The operation is dependent on accurate control equipment. Some operators have found it necessary to s t a r t out with water in order to get the equipment in “balance,” and then follow with product. This may take as little as 10 minutes or less, but may require as much as 40 minutes or more.

XII.

HEAT-PUMP EVAPORATORS

A comparatively recent development of the tubular falling-film evaporator involves a heat pump to compress the product vapor, thus raising the temperature and reusing this energy as a heat head for further evaporation. With use of the pump, the same heat is cycled over and over from the condensing vapor to the evaporating fluid. No steam or other external heat supply is required, and a heat sink is needed only to remove from the system the small amount of s u r p l u s heat which is generated by compression. Because of the high volume of vapors a t low pressure, as in a vacuum vessel, a second type of heat-pump evaporator has been developed which first transfers the latent heat from the high-volume product vapor to a low-volume secondary vapor, and then compresses this secondary vapor to a temperature which is higher than the distillation o r evaporating temperature. The amount of energy required to compress vapor in a heatpump evaporator is minute compared with the heat energy, which in a steam system is first used to supply heat f o r vaporizing the water, and then expensively discarded in a heat sink o r condenser.

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GEORGE D. ARMERDING

Various types of turbo-compressors a r e available, operating in a range of 4 or 5 to 1, either singly or in series. A similar development involves the use of a steam recompression j e t which boosts the temperature of the product vapor so that it can be reused to furnish heat for further vaporization. I n this system, high-pressure steam enters a steam-vapor compressor, which pressure is converted to velocity by passing through a nozzle. The high-velocity steam, in passing through a Venturishaped throat, entrains a part of the product vapor, and the mixed low-pressure motive steam and product vapor pass into the shell of the heat exchanger. The latent heat flows from the mixed vapor, condensing on the outside of the product tubes, to produce vaporizing at a lower temperature inside the tubes. Since this recompressor consumes only a part of the product vapor, the excess is available for use in additional effects. When a total of three effects are used, each 1 lb of high-pressure steam will motivate the flow of latent heat from about 5 lb of condensing vapor to the evaporating product. The usual practical maximum is about 6 Ib of total evaporation from each pound of high-pressure steam used. I n a single-effect system, slightly over 1 lb of steam is required f o r each pound of water evaporated, and this energy is then expensively discarded to a heat sink. XIII.

INDIRECT HEAT-PUMP EVAPORATORS

Despite the outstanding economy of the direct vaporcompression evaporator, the practical usefulness of the system is limited to processes in which a relatively high evaporation temperature is not objectionable. Water vapor has a high specific volume. A t 212"F, the volume is 27 cu f t per Ib of vapor, and the specific volume increases in a geometric ratio as the temperature is lowered. A t 85"F, for example, the volume is 543.3 cu f t per lb. Both the initial cost of the vapor compressor and the operating cost of the vapor compression system increase in geometric ratio as evaporation temperatures a r e lowered, and the economy is questionable a t evaporation temperatures of much less than 212°F. With a n indirect heat-pump system, the latent heat of the product vapor is first transferred to a secondary low-volume vapor such as NH3 before compression, and this secondary vapor is then compressed. This procedure removes the handicap of the high specific volume of water vapor. For example, 1 lb of water vapor a t 85'F has a volume of 543.3 cu f t , with latent heat of 1,044.7

EVAPORATION AS APPLIED TO T H E FOOD INDUSTRY

335

Btu. If this latent heat is transferred to NH,, boiling at 80"F, the volume of the NH, vapor which is produced will be 5.095 cu f t and the weight of this vapor will be 2.095 lb. This 4 cu f t of vapor will have the same latent heat as the 543.3 cu f t of water vapor (Cross, 1960). It would obviously be economically unsound to compress over 500 cu f t of vapor for each 1,000 Btu of heat to be salvaged, but the compression of 4 cu f t of vapor f o r the same purpose is practical and economical, and can be accomplished in well developed and highly efficient NH, compressors. I n any evaporator, the magnitude of the heat head which is required per unit of evaporation is in indirect proportion to the area of heat-exchange surface. I n a heat-pump evaporator, the amount of power which is required for compression is in direct proportion to the heat head. Thus, each increment of increased area of exchange surface lowers both the initial cost of the heatpump equipment and the amount of power required for compression. However, the cost of extra surface is usually more than the savings in cost of the heat-pump equipment, and, in general, the lower the power which is required, the higher the cost of the equipment per unit of evaporation. The optimum balance between capital costs and power requirements is influenced by many variables. If the system is to be online 24 hours per day, 365 days of the year, then the overall economy favors generous areas of exchange surface, with consequent higher capital costs but lower operating costs. If duty is occasional or seasonable, then the design is usually slanted toward minimum amounts of surface, with lower capital costs but higher operating costs. If low-cost heat-exchange material such a s mild steel is permissible, then more generous areas of exchange surface may be incorporated than if high-priced fabrication materials such as type-304 or type-316 stainless steel a r e mandatory. The main advantage of present indirect heat-pump evaporators is the ability to process such heat-sensitive products as orange juice a t relatively low temperature. Boiling temperatures as low as 70°F are not uncommon. It will readily be seen that the product will not be damaged. Undoubtedly this development in evaporation has accounted for the phenomenal growth of the concentsated-orange-juice industry. There a r e a number of other advantages derived from low-temperature processing in a n indirect heat-pump evaporator. When water is in short supply it must be pointed out that the amount required with this equipment is nominal, and, with reuse of the condensate produced, no additional supply is necessary. Similarly, there is no sewage disposal

+

33G

GEORGE D. ARMERDING

problem from high water use. Since the system is usually in perfect balance, utilizing a11 the energy without waste, the cost of operation is low. Manufacturer’s claims (Mojonnier Bros. Co.) indicate that 75 kwh or less is required to distill 1,000 gallons of water. The chief objection to the indirect heat-pump evaporator is the high initial cost. Because of the low AT, large surface area is required. Where heat-sensitive products a r e concentrated, the additional expense is justified. On the other hand, where continuous operation over long periods is required, the low cost of operation may well offset the high original investment. Because no percipitation or scaling develops, the equipment may be kept in service longer without apparent decrease in operating efficiency. XIV.

CENTRIFUGAL THIN-FILM EVAPORATORS

The equipment described so f a r is limited in its performance to free-flowing materials or products having low viscosity. Since many applications of evaporating equipment have to do with heavy viscous materials, a special type of concentrating unit has been developed for this purpose. These evaporators, generally known as centrifugal thin-film evaporators, are manufactured in several designs. As the name implies, the evaporation process is dependent on a mechanical device, known as a rotor, which is used to keep the product in motion and assist in a n even distribution of heat as the material is in transit. All of these units are tubular in design and equipped with blades mounted on a rotating shaft. They a r e constructed either vertical o r horizontal, with a straight-side or cone-shaped cylinder. One type allows a clearance between the thermal wall and the blade; another uses a contact scraper, while some of the horizontal cone-shaped units have a blade or series of blades that may be adjusted for wall clearance. The function of a centrifugal thin-film evaporator is to concentrate heavy or viscous products rapidly in a thin film, usually in a single pass, and for the most part in a vacuum or partial vacuum. The vertically arranged rigid-blade evaporator utilizes fixed blades with a predetermined clearance a t the thermal wall (approximately % 2 inch) to provide a scrubbing action on the liquid film. The blade clearance is determined by the viscosity, surface tension, thermal conductivity, and throughput rate of the material. A rolling fillet is formed on the leading edge of the blade, with the fillet size dependent on the physical properties of

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the material. The turbulent action and mixing imparted to the film, and the turnover of exposed area on the fillet surface, result in high heat and mass transfer. The liquid film on the wall continues downward in a spiraling manner subject to the turbulent action oS the rotor blades (Rodney Hunt Machine Co., 1963; p. 13). The in-feed is located just above the top of the heating jacket. The material to be concentrated spirals down the side wall by gravity and is unloaded through a special type of discharge head located j u s t below the thermal wall. Vapors pass upward in the space between the blades and a r e drawn into a condenser, which may be any one of a number of types. The transit time is never of long duration and is determined by the amount of concentration desired. A second type of centrifugal thin-film evaporator employs a hinged blade or blades and is sometimes called the plowing-blade system. The rotor may be a single scraper or made of a number of scrapers or a combination of both, as may be required to do any particular job. The blades glide on the liquid film on the thermal wall, providing a wiping action. A slight lifting wave builds up in front of each blade, but this is not a fillet, as with the fixed-clearance blade, since the lifting wave is essentially independent of the thickness of the wall film. Both blade types operate a t moderate top speed and a r e swung outward by centrifugal force into contact with the liquid film. The combination of viscous drag, shear effect, and instant release of blade pressure results in thorough agitation and film mixing. Reductions of 20 to 1 can be obtained, being equivalent to 95% evaporation. On products of certain characteristics, reductions of 50 to 1 or higher can be obtained continuously in a single pass. The units a r e suitable for high vacuum ranging from 0.25 to 4.0 mm H g absolute pressure. One manufacturer (Blaw-Knox) offers a choice of operating temperatures. A third type is mounted in a horizontal rather than a vertical position, is conical shaped, and is available in a number of styles. The product is fed into the top of the unit a t the small end of the cone and is drawn out a t the bottom of the large end of the cone. This is termed “forward taper.” When fed a t the large end and withdrawn at the small end, this is termed “reverse taper.” Vapors may be removed a t the top or the bottom or may even be condensed within the unit itself. The blades may be rigid, not touching the sides of the cone, o r they may be of the floating type, contacting the sides as in the vertical models. A number of varia-

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GEORGE D. ARMERDING

tions have been built into the centrifugal thin-film evaporators, each manufacturer claiming superior performance with his particular design. The blade clearance may be fixed a t the factory so that changes cannot be made except by removing the rotor and modifying the blades (Rodney-Hunt). Other blades are hinged and a r e self-regulating as to clearance (Sambay) , while still others may vary the clearance by shifting the position of the rotor within the cone. The blades may be made of rayon, carbon, Teflon, stainless steel, or other inert materials. Normal blade life is 1-2 years, depending on materials used. The cylinder or cones must be of sturdy construction to withstand high vacuum on the product side and high pressures up to 600-800 lb on the steam side. Heating is not limited to steam, but may be supplied by water, Dowtherm, Hot Aroclor, oil, or even a n electric element. The jacket may be equipped with spiral inserts to maintain even distribution of the heating element and to increase velocity. A major problem encountered with this type of equipment is providing a satisfactory seal for the rotating shaft, which is driven from an outside source of power. External roller bearings with single- or double-faced seals a t both ends of the shaft a r e used, or the shaft may be sealed with a water-cooled stuffing box. A t least one manufacturer (Pfaudler) uses an internal bearing at the base of the vertical cylinder. There a r e objections t o all of these features from a sanitation standpoint. I n addition, the bearings score easily and are subject to distortion due to variation of temperature within the unit. The power requirements depend on the speed of the rotor, the viscosity of the product, size of the unit, blade clearance, diameter of the chamber, and temperature of the product. The peripheral speed of the rotor in the rigid-blade type with medium-viscosity products is 20-50 ftlsec, irrespective of size. For thin or light materials the blade speed is higher. With nonrigid blades the speed ranges from 10 to 15 ft/sec, and power requirements are less. No satisfactory formula has been devised to calculate total power needs. Recommendations depend on the experience of the manufacturer. The feed rate of the centrifugal thin-film evaporator is generally near the upper limits specified by the manufacturer. The rated capacity may vary from 10 lb/hr/sq f t of heating surface t o a s much as 300 lb/hr,’sq f t with total capacities from a low of 60 lb/hr in a unit with only 1 sq f t of heating surface (Labora-

EVAPORATION AS APPLIED TO T H E FOOD INDUSTRY

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tory Model) to a high of 5,000 Ib h r in a unit with 140 sq f t of heating surface (Kontro). It is very important t h a t sufficient product be admitted to the unit to avoid “starving” any portion of the heating surface. Again, excess product feed would flood the entire body, leaving no space for vapors and rendering the unit ineffective. The retention time within the unit is variable. A number of factors a r e involved such as feed rate, viscosity, and blade clearance. A change in one of these factors can affect the others, and often does. The residence time may be less than 1 min o r 3-4 min or even longer. Virtually any degree up to 99% evaporation can be accomplished in a single pass with assured “wetting” and agitation of the thin film throughout the length of the heat-transfer surface so as to prevent d r y spots and, in turn, “burn-on” (Gudheim, 1964). The manufacturers claim numerous distinct advantages for the centrifugal thin-film evaporators, as follows : 1) Light liquids to very heavy viscous materials a r e processed in a single positive pass within a relatively short period. 2 ) The equipment can be operated on a continuous, automatic basis. 3 ) Heat-transfer surface is large in relation to quantity of material in process. 4) Higher heat treatment is possible because of the short exposure time. 5) Higher heat allows use of smaller compact units. 6 ) Rotary blades develop high turbulence and prevent fouling of heating surface. 7) Overall heat-transfer coefficients of 50-550 Btu ’hr ’ft’ :F a r e obtained.

Examples of concentration of food products from feed material to end product a r e as in the following table. (‘oncentration,

yo

Food product

Fred

E n d product

Apricot p r c e coffee Gelatin Sorbitol Tomato paste

12 20

24

i0

40 35 99

20

40

1T

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It should be noted that many of these products can be concentrated to a much higher percent solids, depending upon process requirements (Rodney-Hunt, 1963). Since the cost of the centrifugal evaporator is many times that of a n ordinary tubular evaporator, thin-film processors should not be considered instead of normal evaporating equipment unless evaporation cannot be obtained in any other way. One manufacturer (Luwa Corporation) (Reay, 1963) reported, claims a number of advantages f o r the vertical cylinder compared with the horizontal conical type. These can be summarized as follows : 1) The vertical position guarantees the maximum possible uniformity of film flow in the axial direction. The horizontal machine permits back-mixing of the film, resulting in unfavorable distribution of residence time. 2) From the point of view of design and fabrication, the vertical position of the rotor is very important. It is very difficult t o manufacture a horizontally supported rotor that is stiff enough to ensure the required small wall clearance under varying temperature conditions for anything but relatively small machines. This renders it difficult to make reliable horizontal machines in economically large sizes. 3 ) The vertical machine operates in a countercurrent manner a s regards liquid and vapor flow through the machine. The resulting fractionation effect can be very helpful in distillation processes. The horizontal machine, on the other hand, usually requires with-current operation, so that no fractionation is possible. Furthermore, with-current operation in a horizontally disposed machine presents the designer with a great problem a s regards entrainment and carry-over of droplets in the vapor. 4 ) The vertically disposed machine takes up much less floor space than the horizontal type. XV.

THE VACREATOR

The vacreator, a special type of evaporator designed especially for the dairy industry, is used for concentration of fluid milk in combination with pasteurization. It is made up of a series of three vacuum chambers plus the condenser. Capacity is limited, and this equipment has not yet been offered for volume production. The operation is on a recirculation basis, with the product flowing from a supply tank into a heat exchanger, where the

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temperature is raised to approximately 205°F (Hunziker, 1949). The transit time from the heat exchanger through the vacuum chambers equals about 10 seconds. The temperature drops to 100-110°F as the product passes from one vacuum to another. This unit is particularly well adapted to small ice cream plants for the manufacture of ice cream mix. XVI.

PLATE-TYPE EVAPORATORS

The use of plate-type heat exchangers to replace tube bundles is a rather recent departure for evaporator construction. Many plate designs, or patterns, have been tried to overcome the problems of pressure, turbulence, scaling, burn-on, etc. The gasket problems have been fairly well resolved. One great advantage is the flexibility of the plate-type unit with regard to capacity. The capacity of the unit can easily be adjusted by increasing or reducing the number of plates. There a r e limits, however, since such variations must remain within the confines of the vapor chamber, condenser, and other related equipment. The compact nature of plate equipment is very desirable, as is the possibility of inspection of the heating surface. A reasonable amount of success has been experienced with products such as orange juice, but other products, such as grape juice, have not been processed successfully. Precipitates on the heating surface can very easily upset performance. Further work is necessary before this type of heating unit will find general acceptance with evaporator users. The advantages to be gained seemed to weigh heavily in favor of continued research. XVII.

EXPANDING-FLOW EVAPORATORS

One of the newer designs in evaporators features a compact unit having a cone shape. The product is introduced through a hollow central hub and is distributed into passages between nested stainless-steel cones. It boils as it flows upward and outward on the heating surface of the cones. The space for the expanding yapor increases as vapor travels toward the outer periphery of the cones. The steam is countercurrent to the flow of the product, entering the conical annular spaces at the periphery. The condensed product falls from the outer edge of the cone and is drawn off tangentially to the shell t o provide a cyclone effect.

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The manufacturer (De Laval) claims a number of advantages for the unit, such as the compact design. The heating element and the expansion vessel a r e a single unit. Concentration is obtained in a single pass, with only about 1 min of process time for a double-effect unit. A similar unit offered by the same manufacturer is called the Centri-Therm evaporator. It differs from the expanding-flow evaporator in that the cones are inverted, with the apex up, and, in addition, the cones rotate at high speed. The product is introduced into the underside of the top of the cone assembly through a spray arrangement. It flows rapidly downward and outward by centrifugal force, spreading in a thin film with a heat exposure time of about 1 see. The steam enters from the outside edge of the cone and flows upward and inward to the apex. A paring device is used t o remove the condensate, just as a similar method is used to remove the concentrate. XVIII.

FRUIT-SPREAD COOKERS

There is still a variance of opinion among manufacturers of jellies, jams, and preserves regarding the merits of vacuum processing. A number of packers still use open kettles with small batches. Those who use vacuum cooking claim several advantages : 1) Low-temperature cooking with less browning 2) Faster cooking 3 ) Better evacuation of air, resulting in faster sugar absorption by the fruit.

The advantage of the low temperature o r short cooking time is offset to a certain extent since packing is done a t high temperatures. Present cooling methods after packaging are still ineffective in removing the heat fast enough to prevent a certain amount of damage. Vacuum-processed fruit appears to have better color and longer shelf life, according to claims made by processors. To offset some of the problems of vacuum cooking, such as time required to evacuate the air and subsequent unloading time, the trend has been to large-diameter cookers with a shallow bottom. The depth of product is kept a t a minimum, and unloading is speeded up by using air pressure after releasing the vacuum. The shallow-dished cooker prevents excessive rolling of the preserves, minimizing damage to fruits such as strawberries or peaches. This seems to be a desirable result.

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Essence recovery is difficult with vacuum equipment since certain flavors a r e lost through the vacuum ejector or pump and cannot be reclaimed because of the risk of contamination in such auxiliary equipment. On the other hand, essence recovery is equally difficult with atmospheric cookers, primarily because the batch method of cooking does not lend itself to satisfactory operation of distillation columns. It is apparent that there is a real need for a continuous processor that could function well with a n essence recovery system, but such equipment is not presently available. The trend of the industry toward larger processing plants may offer opportunity to develop continuous equipment for the manufacture of high-quality preserves. The use of fresh-frozen fruits and berries has changed processing from a seasonal to a yeararound production schedule, with opportunity for reasonable amortization of the type of equipment evidently needed.

XIX.

CONCENTRATION BY FREEZING

A t present, at least two methods make limited use of concentration by freezing. The Seahl method is based on freezing and deaerating t o a slush state and then centrifuging to remove concentrated liquids. This process is repeated for higher concentration. The Noyes process applies the same principles but omits the centrifuge, using draining or siphoning to remove the concentrate. Desalting of sea water by refrigeration methods has been under study by the Office of Saline Water since 1956. (Rinne, 1963). Separation of a pure water-solid phase from a salt solution may best be brought about by direct methods of cooling; that is, the latent heat of fusion is removed by evaporating a portion of precooled sea water under reduced pressure or by vaporizing a refrigerant such as n-butane in direct contact with sea water. In either case the absence of a heat-transfer barrier permits the use of relatively low driving forces in both the freezer and the melter. The condensation of water vapor or refrigerant vapor by direct contact with the washed ice, producing fresh water, assures continuous recovery of the latent heat of fusion. Hydraulic forces have been applied successfully to move a porous bed of ice upward in a column against a downward flow of water. Experimental work is still in progress to determine the

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feasibility of centrifugal separation. Freezing continues to show promise of becoming a n economical process with sea-water desalting, and may become equally promising in the food industry. XX.

AUTOMATIC CLEANING

The development of automatic cleaning, otherwise known as C I P (clean-in-place), has proved a real boon to the operation of evaporating equipment. The drugery of manual cleaning is no longer a problem if no severe burn-on is encountered. The production of satisfactory reagents necessary for cleaning, together with improvements in design and construction, brought about this very practical departure from old and laborious cleaning methods. The assurance of a satisfactory cleaning and sanitizing job is dependent on a number of factors, all of which a r e important. The failure or success of CIP depends largely on the design of the equipment. Every square inch of surface exposed to the product must be exposed to the cleaning procedure. In addition, there must be no areas where either product or cleaning agents could lodge. All surfaces must be properly drained. Flat or level surfaces within the equipment a r e permissible only if such surfaces are subject to sufficient scrubbing action of the cleaning agents to assure a complete cleaning job. I n addition, the equipment must be constructed of materials not affected by either the caustic or acids used during the cleaning process. Types of stainless steel now being used for evaporator construction apparently meet the necessary requirements. Cleaning apparatus that forms a permanent part of the equipment, such as spray balls, jets, or piping, must be designed and located so as to avoid fouling by either the product in transit or the cleaning solution itself. Such devices must be properly placed to assure adequate and complete circulation of the cleaning solutions to all areas of the equipment. When cleaning devices are removable and not in place during operation of the equipment, fouling or interference by the product is avoided. Since, in many cases, design and method of operation do not permit the removal of cleaning apparatus, the problem remains and demands a satisfactory solution. The time required for the cleaning cycle and the cost of cleaning materials are also important factors in the success of a C I P system. Since a n operator must be available and since cleaning is done a t about the same temperature as that required for

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processing, it will readily be seen t h a t the cost of the cleaning time per hour is equal to t h a t of processing time per hour. In addition, there must be added the cost of the cleaning compounds and water requirements. The down time (time required for cleaning) may also be important during high production periods, when every hour counts. The development of CIP methods has improved to a point where the entire job is almost completely automated. The operator may still be required to add caustic or acid, and may be involved in a few of the programed procedures, but since his presence is necessary in any case, such tasks have not proven objectionable. Most of the automatic cleaning equipment is designed so as to give sufficient warning to the operator whether his attention is necessary. This may be a light signal or a n annunciator, or both. The length of time required for a complete cleaning cycle may vary from 90 min to 240 min or even more. The condition of the equipment, reflecting scaling, burn-on, or simple neglect, will have a profound influence on the cleaning time required. The strength or effectiveness of the cleaning agents used will also increase or decrease the elapsed time f o r the job. The programing of the cleaning cycle may require as many as 40-50 separate steps, depending on the complexity of the apparatus and the functions of each unit. One manufacturer (Ladish Co.) has enumerated the various steps in the procedure as follows :

Pre-qinse: a ) Rinse tank automatically filled b) Temperature control set initially at approx. 100°F c) Rinse through lines and discharge to drain until clear d) Return to rinse tank when clear ; circulate 5 min. rlllcali-wash: a ) Solution tank automatically filled b) Alkali wash powder added to solution tank by operator c ) Initial time period set to approx. 30 min d ) Temperature control set initially at approx. 165’F e) Circulate at adjusted temperature and time, discharging back t o solution tank f ) Cycle ends with all solution returned into solution tank.

Rinse: a ) Rinse tank full from last prerinse return b) Rinse at 165°F for 2 min, max. Discharge to drain.

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A c i d wash: a ) Solution t a n k automatically filled b ) Acid cleaner added t o solution t a n k by operator c ) Initial period set at approx. 20 min d ) Temperature control set initially at approx. 165°F e ) Circulate a t adjusted temperature an d time, charging back t o solution tank.

Riiise: a ) Rinse t a n k full fro m last prerinse re tu r n b ) Rinse a t same temperature a s prerinse f o r 5 min a n d discharge t o drain c ) Circulate rinse t a p water temperature f o r 10 min, re tu rn t o rinse tank.

Snuitize: a ) I nse r t card f o r sanitizing program f o r best results b ) Sanitize chemical powder added to rinse t a n k by operator c ) Circulate cold f o r period advised by sanitizer manufacturer. which is programed on card d ) Discharge to drain. Since each operation may require a start a n d stop, th e automated system must function accordingly. T he cost of cleaning materials will depend on th e amount and type used as well a s th e efficiency of th e C I P system. A n operator who used 100 Ib of caustic an d 3 gallons of acid t o clean a quadruple-effect evaporator found t h a t he could reduce his caustic requirements to one-half by installing a diffusing nozzle a t the inlet of t he first effect. Th e better distribution of th e caustic solution improved th e effectiveness of th e cleaning. It is apparent t h a t f u r t h e r study is necessary f o r improvement of processing equipment design to assure easy access f o r cleaning, more efficient cleaning apparatus, relative stren g th of reagents, a n d teniperatu r e s required. Corrosion of equipment is a factor t o be considered in C I P and automated systems. Corrosion of stainless steel a n d related metals consists of a degradation of the metals a n d alloys which causes them t o retu rn to one o r more of t h e fo rms found in their natural ores. When this degradation is rapid enough to be noticeable without precise laboratory analysis, the process is called corrosion. When the process is slower, i t is considered to be under control or not corroding.

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Although stainless steels a r e very resistant to corrosion, under certain conditions they corrode readily and rapidly. This corrosion usually takes the form of pitting. Corrosion may result from direct chemical attack or from one of several electrolytic cells which may exist. Acids and chlorine readily attack stainless steel. Phosphoric acid and nitric acid do very little harm providing they cover the entire surface. When they a r e present in droplets or a r e present along with soil particles, they act as electrolytes f o r one of the cell-type reactions. Chlorine and most of the chlorides react directly with the stainless steel especially in the presence of moisture. Corrosion by chlorine (and other halogens) is much m w e severe when the environment is acidic. Electrolytic or galvanic-cell corrosion takes place when dissimilar metals a r e in contact or connected by a metallic bridge and submerged in a n electrolyte. The electrolyte can be any liquid capable of carrying a n electric current-even water with a very small amount of impurities. The more noble metal (according to the Galvanic Series of Metals) becomes the cathode, and the less noble is the anode. The current flows from the cathode through the metal to the anode and returns through the electrolyte. Positively charged metal ions a r e carried out into the electrolyte to the cathode, where they a r e met by electrons which move through the metal in a direction opposite to the current. The removal of metal ions corrodes the anode, causing a pit to form. Austenitic stainless steels a r e peculiar in that two areas of the same piece may act as dissimilar metals. An oxide film which forms on the surface when clean, dry stainless steel is exposed t o the a i r makes the surface passive. If the film is disrupted, the active metal is exposed and this becomes the anode, and when it is flooded by a n electrolyte, a cell forms and pitting may occur. Other cells, known as oxygen cells and concentration cells, may exist when soil or some other material on the surface excludes oxygen or causes a difference in concentration of ions between the stainless surface and the main body of electrolyte. Here, the area of least oxygen becomes anodic, and a pit may begin beneath the soil particle. This is why proper cleaning and the avoidance of dead ends or areas of stagnated flow are so very important in automated C I P systems. The corrosion of metals may limit the application of C I P or the types of cleaners which may be used. F o r example, parts of the

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equipment which have chromium plating to give hardness necessary f o r a certain function cannot be cleaned with acid cleaners, for the acid removes the chromium. Often, a bearing in the product zone is necessary. If a dissimilar metal is used, as is invariably the case, corrosion may occur more rapidly. With automatic cleaning, inspection is often neglected, allowing corrosion to proceed t o a point of breakdown. XXI.

CONTROL EQUIPMENT

The use of control equipment usually involves temperature, pressure, time, mass, and movement. Manufacturers of control equipment a r e now supplying various components to handle all of these factors in a very satisfactory manner. Many of the control problems can be solved in several ways. Combinations such as a pressure control with a pneumatic or a i r valve can sustain steam pressures within a range limited to ounces. Time controls a r e available with limitations in seconds. Density controls, with charts or without, function satisfactorily within a narrow range. Float controls that use either float balls or probes a r e now in use, confining limits to within a fraction of a n inch. The limitation in the use of controls is a matter of cost involved. A completely automated system is possible, but not always advisable. As noted in the control of the cleaning operations, there a r e certain jobs which can be performed by the person in charge more economically than without such personal attention. With present technical knowledge there is no function of the evaporator that cannot be controlled automatically. The use of controls is recommended wherever practical, since uniformity of operation is essential with an evaporator. Variations outside a very narrow range invariably upset the operation, causing malfunction and product spoilage. XXII.

SONIC A N D ULTRASONIC CLEANING

Sonic and ultrasonic cleaning actions can be produced by mitting sound waves of certain frequencies into a tank of containing a chemical cleaning agent. The transmitted waves create a special form of mechanical agitation. The features of these cleaning methods a r e (Boka, 1964) :

transliquid sound major

1) Among the most thorough cleaning processes devised

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2 ) Highly effective, often producing results not available by any other process 3 ) Extremely rapid action, with cleaning time often reduced to a matter of seconds.

Working at great speed and force, the sound waves produce a swirling sea of submicroscopic bubbles in the liquid cleaner, rapidly forming and collapsing. This phenomenon is known as cavitation. The rapid implosion or inward collapse of thousands of these bubbles gives a scrubbing action of a speed and vigor that is impossible to achieve with conventional means. Furthermore, because cavitation can be produced throughout the entire tank or body of liquid, it can produce a scrubbing action even in blind holes and inside tubing, crevices, and other inaccessible places. Assemblies are often cleaned by simply immersing them in the ultrasonic tank. Although several factors a r e involved, the degree of cavitational energy is controlled principally by the choice of frequency and power input levels. The lower the frequency, the more violent the cavitation. The higher the frequency, the smaller the bubble becomes, resulting in less violent cavitation and a more diffused cleaning effect. Although cleaning in the sonic range (within the range in which humans can hear the sound) is quite effective, it is also quite annoying. The high-pitched whine is very distressing to the operating personnel. The ultrasonic cleaning method has two basic elements, mechanical action and chemical action. In essence, the ultrasonic agitation accelerates and enhances the detergency of the chemical cleaner. XXIII.

THE CARVER-GREENFIELD PROCESS

This Carver-Greenfield process is a patented process used primarily for inedible food products, but it may be developed for future use with edible materials. Basically, it involves the addition of oil o r tallow as a means of reducing moisture content to a n absolute minimum. I n a normal evaporating process the removal of water is limited to a point at which the resultant product refuses to flow or where good heat transfer can no longer be obtained. A t this point, or even earlier in the process, a vegetable or animal oil in liquid form is added to produce a slurry t h a t can be pumped through the evaporating equipment. Heat

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transfer is again possible, and any remaining moisture is removed. The oil used to produce the slurry is recovered in a dual process involving a centrifuge f o r the preliminary treatment and a n expeller for the final separation. The oil thus separated may be reused, although a portion is lost and remains with the finished product. While the moisture may be reduced to less than 296, the retained f a t or oil may be as much as 6-8%. This process is now being used successfully in rendering plants and appears to have real possibilities for further development. XXIV.

ESSENCE RECOVERY

The highly volatile constituents of fruits which a r e responsible for their typical aromatic flavor a r e present in fruit juices in only minute traces. It has been estimated that the essential essence of apple flavor, for example, is present in the fresh juice to the extent of only 15 parts per million. Since these essences are, in general, more volatile than water, they a r e lost either partly or completely when fruit juices a r e concentrated by evaporation if the vapor is discarded. I n the concentrated orange juice industry it has been demonstrated t h a t the aromatic constituents of the flavor a r e so robust that the sense of taste may be saturated when these components a r e present to the extent of less than 10% of their concentration in freshly extracted orange juice. The unusual characteristic has made it possible to concentrate fresh juice five to one, removing substantially all of the flavor in the process, and to restore a sufficient amount of flavor to the concentrate by blending one part of fresh unconcentrated juice with two parts of the deflavored concentrate. The resulting blend may be diluted by mixing three volumes of water with one volume of the blend, and the reconstituted juice, now containing less than 1 0 % of the original aromatic flavor constituents, usually conveys the illusion of a fullflavor fresh juice. When the “cut-back’’ process described above has been applied to the production of concentrates other than citrus juices, the results have been disappointing. The volatile aromatic flavor constituents of many fruit juices either a r e present in smaller proportions or have less impact upon taste perception. The illusion of a full, robust flavor cannot be conveyed by blending fresh unconcentrated juice with concentrate in any commercially practical proportions. Grape juice, strawberry juice, pineapple juice, and

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many other juices, when reconstituted from concentrate, may require up to 100% of the aromatic constituents of fresh juice in order to be indistinguishable from the unconcentrated fresh juices. If full-flavor concentrates a r e to be prepared from these juices it is necessary first to strip the essence from the fresh juice, then to concentrate the stripped juice, and finally to blend the two concentrates. The concentrates of the fresh volatile flavor components a r e commonly known as essences. These essences a r e aqueous solutions of organic compounds and are of a highly complex composition. The constituents of apple essence, for example, have been identified as butyl alcohol, 2-methybutanol, acetaldehyde, hexyl alcohol, methanol, caproaldehyde, propyl alcohol, acetone, and furfurol, in descending order of their percentages in the essence. Esters may be important constituents of some fruit flavors, but the dominant constituents a r e non-esters. Ethyl alcohol is usually present in varying percentages. It is not thought to be an essential constituent, but rather a n unavoidable adulterant, and its separation from the essential components is difficult or impractical. A process for recovering the aroma of apple juice in concentrated or essence form was developed in 1944 at the Eastern Regional Research Laboratories. Since that time, publications by that Laboratory, supplemented by publications of the Western Regional Research Laboratories and others, have discussed the application of this process to the juices of other fruits, and to the preparation of full-flavor concentrated fruit juices. Existing installations of essence recovery and concentrating equipment usually follow the processes and the equipment designs which are discussed in those publications. The essential elements of the system developed by those investigators a r e outlined as follows :

A. STRIPPING The system developed by the Regional Laboratories commences with the raw juice, such as apple juice, just a s it comes from the presses without any previous treatment other than passing through a simple strainer to remove extraneous material. The juice is pumped through a preheater, where the temperature is raised from room or storage temperature to 220°F. It is further heated in a vaporizer and flashed into a n atmospheric vapor chamber, where about 10% of the original volume is released. This vapor passes through a fractionating column and thence into

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a sanitary surface condenser, with the condensate flowing back over the plates or ceramic rings. A t the base of the fractionating column, a reboiler raises the temperature of the condensate t o release any entrained essence. The cycling continues with the essence drawn off at the desired rate by a metering pump from the reservoir at the base of the sanitary surface condenser. An overflow is provided in the base of the fractionating column, where the spent condensate is discharged to the sewer. The apple juice, reduced now to about 90% of the original volume, is drawn into a flash chamber under vacuum, where the temperature is reduced to about 120"F, thus releasing approximately another 10% of t h e volume in the form of vapor, which is condensed into a countercurrent wet condenser and discharged to the sewer. The stripped juice is then cooled and is ready for depectinizing, clarification, and further evaporation. The essence-bearing liquid which has been drawn from the sanitary surface condenser, together with any noncondensable gases., is pumped through a combination essence and vent-gas cooler and then through a final stripping column into a n essence storage tank, ready to be added back to the concentrated apple juice. The final essence may be 100-150-fold or even higher, at the discretion of the processor, and is held a t about 35°F.

B. NONCONDENSABLE GASES All fresh juice contains varying quantities of air, CO-, o r other gases which a r e noncondensable at the temperatures and pressures used in the apparatus. The volume of these gases may vary from a trace in carefully prepared juice from fresh unfermented fruit, to the atmospheric pressure saturation volume of CQ, in juice which is slightly fermented or which has been expressed from slightly fermented fruit. These gases a r e vented with an essence concentration which is in equilibrium with the essence at its highest concentration. Early laboratory research indicated that, in the production of apple essence, the loss of essence in vent gases was not excessive under the following conditions : a ) Fresh juice, with a minimum of air and other noncondensable gases, is used f o r stripping b ) Essence of 150-fold or less is produced c ) The gases a r e vented at atmospheric pressure d ) The gases a r e scrubbed by passing upward through a small

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packed column, countercurrent to chilled essence not exceeding 150-fold in concentration. The above process has proven very satisfactory with a number of fruit juices other than apple, including grape, pineapple, and various berries.

XXV.

PRE-EVAPORATION CONDITIONING

The composition of the product t o be evaporated will determine the type of equipment to be used as well as the procedure during the evaporation process. Preconditioning of the product to conform to certain standards may simplify the process and greatly assist in operation of the evaporator. Other methods of product treatment may do exactly the opposite, increasing the problems. The method used (with tomato juice, for example) is highly important in determining the type and capacity of the evaporating equipment. Since i t is possible to modify the raw product, and by so doing change entirely the evaporation characteristics, more information is needed in order to determine:

1) The type of preliminary treatment 2 ) The design of the equipment 3) The operating procedure. The tomato industry employs two methods for juice production, known as the cold-break and the hot-break. The cold-break is, simply, a delayed heating procedure, while the hot-break method provides instant heating a t the time of crushing. With the latter, the temperature of the tomato pulp is raised, within a few seconds, to a point where the enzymes a r e inactivated and practically all of the pectin retained. This is desirable for a heavybodied tomato juice, but when juice so treated is processed through a n evaporator the capacity of the equipment is greatly reduced and the degree of evaporation is limited. Special equipment must be provided to handle this product. With the coldbreak method the viscosity is low,, evaporation rates a r e high, and greater concentration is possible. Kopelman and Mannheim (1964) showed that when tomato juice was centrifuged to remove all suspended solids i t was possible to increase evaporation rates and continue evaporation t o a much higher concentration than with juices not centrifuged. For

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these determinations, they used two types of equipment: one mas a n atmospheric open-pan type of evaporator; the other was a thinfilm short-time evaporator with agitator. In concentrating serum only, the evaporation rate increased in a ratio of 1.7 t o 1. On the other hand, at 20% solids the U value was 470 f o r regular juice and 1,200 for the serum, or a ratio of 2.5 to 1. The researchers concluded that the heat-transfer coefficients mere superior f o r the serum methods. This is reflected in reduced evaporation time, i.e., in a n increase in unit capacity. These findings can be expected to favor improved product quality. With many types of fruit juices the evaporation rate and the amount of concentration a r e both greatly restricted unless some pretreatment is given the juice. When apple juice is concentrated, treatment with a n enzyme such as pectinole (Smock and Seubert, 1950) is necessary to hydrolyze colloidally suspended material into more or less soluble substances. Similar treatment with diastase, or a commercial product called Rhozyme, may be necessary, particularly with immature fruit, where the starch content is high. Without such treatment, evaporation may be limited to as low as 45" Brix to avoid gelation. When treated, the same juice may easily be concentrated to 70" Brix or higher. Naturally, such treatment changes the nutritional value of the product. Very little work has been done to determine the extent of such changes. It is apparent, however, that pretreatment of many fruit juices, such as peach, plum, and apricot, may bring into being many new products not now available. Further studies a r e undoubtedly needed to determine the effect of such pretreatment on the appearance, nutritional value, and salability of such products. The standardizing or pretreating of products ahead of the evaporation process could have a profound effect on equipment design and use. In practically every operation the finished product must meet certain predetermined standards, whereas the raw product often shows large variations in moisture content, viscosity, and other characteristics. To offset these variations, the equipment must be operated at different capacities, at fluctuating steam temperatures and pressures, with product recycling and other objectionable procedures to obtain the desired end product. Pretreatment may eliminate many, if not all, of these difficulties. Single-pass evaporation, with its many advantages, can be obtained more easily when the condition of the inbound product remains constant. Foaming of the product within the evaporator is not always the

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result of a i r leaks. The entrainment of gases such as CO, or nitrogen may cause excessive foaming. Treatment of the inbound product with a n antifoam compound such as Antifoam “A” (Dow Corning) at the rate of 10 to 30 p.p.m. will help control this difficulty. There a r e certain limitations on the use of such antifoam compounds as prescribed by the Food and Drug Administration. Products used for human food a r e within these restrictions.

XXVI.

ELECTRODIALYSIS

A process for separating substances by diffusion through a semipermeable membrane is termed dialysis. The driving force is a difTerence in concentration developed when a solvent and solution a r e separated by a semipermeable membrane. Electrodialysis is similar to dialysis except t h a t it works on the basis of electrical charges on the material to be separated. A process such as this could conceivably be used to remove dissolved salts from a solution and thus eliminate the expensive process of evaporation, which is dependent on heat transfer to do the job. Use of electrodialysis has been very limited in the food industry. An installation at Appleton, Wisconsin (Wyeth Laboratories), is producing desalted whey f o r infant food (Stribley, 1963). Concentrated whey is pumped through spacers to be directed over surfaces of membrane in a tortuous path to ensure maximum use of the membrane area. Sodium and other cations migrate toward the cathode, with the negatively charged cation membrane permitting their passage. Chloride and other anions move toward the anode; they can pass freely through positively charged anion membranes. Whey is constantly circulated on one side of the membranes. An acidified brine is passed over the other side. Salts from the whey enrich the brine, which is then discarded. After the production cycle of six or more hours, the electrodialysis stack is shut down for in-place cleaning and sterilization and regeneration of the membranes to permit them to work at top efficiency. The possibilities for this method have yet to be explored. Research under the direction of the United States Department of the Interior for the distillation of sea water may provide suficient information to launch further investigation in the food industry. J u s t how f a r this will go toward replacing present evaporating processes is strictly a matter of conjecture.

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XXVII.

REVERSE OSMOSIS

When pure water and a salt solution are on opposite sides of a semipermeable membrane (ideally, permeable t o water but immeable to salt), the fresh water flows through the membrane and dilutes the salt solution. This is the well-known process of osmosis. If pressure is applied to the salt solution in excess of its osmotic pressure, fresh water flows through the membrane in the direction opposite t o normal osmotic flow. Thus, the term “reverse osmosis.’’ This process has long been recognized as one that potentially could be operated at a high thermodynamic efficiency (Sieveka, 1963). Membranes have been developed with flow rates suitable for pilot-plant operation, but research is continuing to develop a membrane suitable for economic desalinization of sea and brackish water. This might well open up the way for further research in handling food products. A combination of processes such as centrifuging, filtering, protein precipitation, and reverse osmosis must be explored to determine the total possibilities in this area. These systems might well replace evaporation by heat treatment. Sanitation and total cost will be among the important factors. XXVIII.

EVALUATION OF EVAPORATION

The development of evaporation technique from the solar pond of prehistoric days to present-day highly automated equipment has been a n arduous, expensive task, with a mixture of failure and success. The objective of one hundred years ago was vastly different from that of today. Engineers and scientists could not forsee all of the present-day problems created by new products, new demands for sanitation, and the business economy. Nor can we, with any degree of surety, foresee what the next century may demand. As we review progress we see a trend of reasoning which has brought into existence present evaporating equipment and methods. These may be catalogued as follows: 1) 2) 3) 4)

Improvement of product Sanitation Flexibility of operation Economics.

Some of the steps that have been taken have embraced all of the factors involved, while others have been confined to simply one

EVAPORATION AS APPLIED TO THE FOOD INDUSTRY

357

facet of’ the operation. Until very recently, all phases of the development were tied in with heat processes of one form or another, with the emphasis on more efficient use of the Btu’s involved, improvement of the product,, and lowered cost of operation. There are undoubtedly other methods for the removal of soluble solids from a liquid which have not yet been dreamed of. The challenge to discover and develop such methods should inspire the present generation with courage to depart from long-established practices and launch out into the realm of the unknown, just as the astronauts are probing space for the answers to other scientific problems.

REFERENCES Anon. 1961. S a l t Industry in J a p a n . Japan Monopoly Corp. Tokyo, p. 3. Reach-Russ. 1958. Bulletin No. 90 158, Beach-Russ Co., New York 7 . p. 4. Berkeley, F. D. 1958. Hydrocarbon processor and Petroleum Refiner. Dec. Gulf Publ. Co., Houston, Texas. p. 99. Boka, V. P., Jr. 1964. Milk Dealer. July. Olsen Publ. Co., Milwaukee, Wis. Calif. Bur. of Mines. 1957. S a l t In California. Bull. No. 175, Calif. Div. of Mines, F e r r y Bldg., S a n Francisco, Calif. Campbell, Clyde H . 1950. Campbell’s Book, 3 r d ed. p. 54. Johnson Press, Pontiac, Ill. Cross, J. A. 1960. H e a t Pump Evaporation Theory and Applications. Unpublished Paper, Mojonnier Bros, Co., Chicago, Ill. Encyclopedia Americana. 1963. Vol. 27, p. 627. Americana Corp., New York 22, N.Y. Farrell, A. W. 1942. Dairy Engineering. p. 64. John Wiley & Sons, New York, N.Y. Farrell, A. W. 1963. Engineering f o r Dairy & Food Products. p. 402. John Wiley & Sons, New York, N.Y. Graham, 1963. Bull. No. 70B, p. 3. Graham Mfg. Co., G r e a t Neck, N.Y. Gudheim, A. R. 1964. Unpublished Paper. Kontro Co., Petersham, Mass. Hausbrand, E. 1907. Evaporating & Cooling Apparatus. Scott, Greenwood & Sons, England. p. 56. Hunziker, 0. F. 1920. Condensed Milk & Milk Powder. 3rd ed. Published by Author. LaGrange, Ill. p. 87. Kopelman, J. and Mannheim, H. C. 1964. Evaluation of two methods of tomato juice concentration. Food Technol. 18 ( 6 ) , 117. Mitten, H. L. 1964. Design Factors Which Affect the Automation of Processing Equipment. Unpublished Paper. Creamery Package Mfg. Co., Chicago, Ill. Nash, 1950. Nash Engineering Bulletin No. 1 V-CT-373C. p. 9. Nash Engineering Co., So Norwalk, Conn. Osborne, A. 1944. Modern Marine Engineer’s Manual. Vol. 1, Sec. 14, p. 51. Cornell Maritime Press, New York.

358

GEORGE D. ARMERDING

Reay, W. H. 1963. Recent Advances in Thin Film Evaporation. Reprint from Industrial Chemist, June, 1963, Harrison and Sons, Ltd., S t Martin’s Lane, London, England. Rinne, W. W. 1963. Saline Water Conversion Report. U.P. Dept. of Interior, Washington, D.C. p. 115. Rodney H u n t Machine Co. 1963. Bull. No. 123, Rodney H u n t Machine Co., Orange, Mass. p. 11. S a l t Institute, 1962. Processing F o r Market. Bull. Salt Institute, Chicago, Ill. Sieveka, E. H. 1963. Branch of Membrane Processes Report. p. 102, U.S.Dept. of Interior, Office of Saline Water, Washington, D.C. Smock, R. M. and Neubert, A. M. 1950. Apples and Apple Products. p. 335, Interscience Publ., New York, N.Y. Stribley, R. C . 1963. Electrodialysis: F i r s t Food Use. Food Processitiy J a n u a r y 1963, p. 49. Food Processing Publ. Co., Chicago, Ill. U.S. Dept Agriculture, 1958. Maple Syrup Producers Manual. Agr. Handbook No. 134. p. 17. U.S. Govt. Printing Office, Washington, D.C. U.S. Steel Corp. 1962. Fabrication of Stainless Steel. 3rd. ed. U.S. Steel Corp. Pittsburgh 30, Pa. Webre, A. L., and Robinson, C. S. 1926. Modern Library of Chemical Engineering. p. 447. Reinhold Publ. Co., New York, N.Y. Van Hook, A. 1949. Sugar. p. 35. Ronald Press, New York, N.Y. Vermont Agr. Expt. S t a . 1962. M i x . Publ. No. 20. p. 7.

SUBJECT INDEX A Acetic acid, 205 Acetone, 210 Algae, carotenoids in, 208 Alimentary absorption, 9 micellar formation in, 32 Alkylperoxy radicals, 3 Alpha radiation, 71 .4Iteriiuria citri, 177 Aminopyrene demethylase, 37 Annatto, 246, 258 Anoxia, 120 Anthocyanins, 132 Antifoam “A,” 355 Antioxidants, 3, 5, 25, 35, 50, 257 /3-Apo-8‘-carotenal, 211, 246, 251 Apricots, 223 Ascomycetes, 167 Ascorbate oxidation, 68 Ascorbic acid, 77, 124 Astaxanthin, 241 Aureobasidium pullulaizs (de By), 173 Automatic cleaning, see Cleaning Autoxidation, 3 Auxins, 85, 128 Azo dyes, 255

Blackberries, 225

Blakeslea trispora, 212 Blanching, 240, 289 Blueberries, 225 Blue mold, w e Penicillium italicurn Botryosphucria ribis, 177 B o t r y t i s cinerea, 167, 172 Bragg Peak, 75 Bread, 289 Brine, 305, 355 Brown core, 179 Brown rot, 173 Bulbs, 179 2-tert-Butyl-4-methoxypheno1,5, 11, 22, 40 3-tcrt-Butyl-d-methoxyphenol, 5, 11, 22, 40

C

Cake, 260 Calandria pans, 324 Canning, 239 Canthaxanthin, 210, 246, 253 Capsanthin, 246 Capsorubin, 246 Carbon dioxide, in irradiated fruits, 68 Carbon tetrachloride, 38 B a-Carotene, 246 Bacteria, 291 p-Carotene, 126,205, 209, 241 carotenoids in, 208 ?-Carotene, 126, 206 Baking, 292 [-Carotene, 206 Basidiomycetes, 167 Carotenoids, 125, 132, 202 Beach-Russ pump, 316 biosynthesis, 204, 210, 262 Berries, 171, 225 color, 218 Beta radiation, 71 commercial synthesis, 210, 263 BHA, see 2-tert-Butyl-4-methoxyphefunction, 208, 262 no1 microbial synthesis, 210 BHT, see 3,5-Di-tert-butyl-4-hydroxy- nomenclature, 196 toluene occurrence i n animals, 260 Bile, 34 in f r u i t , 217, 225 Biscuits, 292 i n vegetables, 226, 234 Bixin, 246 properties, 202, 231, 239, 254 359

360

SUBJECT INDEX

in storage, 241 Diathermy, 277 toxicology, 212 2.B-Di-terf-but) 1-4-h yclroxymeth yl Carrot, 226 phenol, 7 C a i ver-Greenfield process, 349 3,5-Di-tc~)f-butyl-4-hydroxytoluene. 5, Cakitation, 348 11, 22,31,41 Cellulose, 81,118 Di (3,5-di-fert-butyl-4-hydroxybenzyl) Cell walls, 80 ether, 7 Chickens, 260 Di- (3,5-di-fart-butyl-4-hydroxyChlordan, 38 phenyl), 7 Chlorophyll, 133 Dielectric constant, 280 Cholesterol, 33 Dielectric heating, 278 Cholinesterase, 292 Dielectric loss factor, 280 Citral, 210 Diplodia ? i a t a l e m i s , 177 3-Citraurin, 223 Diseases, postharvest, see Postharvest Citrus brown rot, see P h y t o p h t h o r a diseases Citrus fruits, 176,218 Dissipation factor, 281 juice, 218,350 Dosage, of pathogens, 151,155 Clarlosporum e r b a rzini, 173,176 Dosimetry, 58 Cleaning Drosophila ~ ~ e l a n o g a s t r175 i, automatic, 344 Dyes, azo, 255 sonic, ultrasonic, 348 Cimiate, effect on irradiated f r u i t s , 122 E Color, 241 of fruit, 126 Eggs, 35,248 synthetic, 253 Elderberry, 225 Colorants, 246 Electrical fields, 277 Condensers, 321 Electrodialysis, 355 Hyer type, 322 Electromagnetic spectrum, 278 Control equipment, in evaporators, 348 Embryos, 35 Cooking, 241 End-point analysis, 163 microwave, 290 Energy dissipation, 71 vacuum, 309 Energy, thermal, 281 Core flush, see Brown core Entrainment separators, 327 C o r n , 228, 235 Enzymes, 259,292 Corrosion, 346 in liver, 36,50 Cottonseed, 288 Erwinia c n r o f o v o r a , 167,179 Couniarin, 38 Essence recovery, 342,350 Cranberries, 225 Ethionine, 38 Crustaceans, 238 Ethylene, 7'3 Cryoday unit, 285 Evaporation, in food industry, 303 ff Curie, 59 Evaporators, Centri-Therni, 342 expanding flow, 342 D falling film, 331 Defrosting, of food, 289 forced circulation, 330 Dehydration, 240 heat pump, 333 by microwaves, 288 plate type, 341 Desalination, 305,343,355 thin film, 336 Deuteromycetes, 167 tubular, 328 Dinporthe cityi, 177 vacreators, 340

361

SUBJECT INDEX

F Figs, 225 Fish, 238 Flavor, 259 Foaming, 354 Food additives, 2, 46 Food, frozen, see Frozen food Food irradiation, 105 ff., see also Irradiation Food storage, 288 Freeze-drying, 290 Freezing, of food, 240, 343 Frequency, of microwaves, 287, 292 Frozen food, 289 Fruits, irradiated, 57 ff., 105 ff., 147 ff., 173 climacteric, 107 culture of, 122 effect of climate, 122 of ozone, 135 ethylene production in, 109 injury of, 110,116 nonclimacteric, 114 pathogens, 135 respiration, 107 ripeness, 108, 131 F r u i t spread cookers, 342 Fungi, 149, 289 germination, 150 irradiation, 153 mutations, 158 FusariuwL, 180 G

Gamma radiation, 71, 110, 241 Genetics, effect of radiation on, 152 Geotrichum candidum, 177 Geranyl-geranyl-pyrophosphate,205 Gloeosporium, 177 Glucose-6-phosphate, 39 Glucuronides, 11 Grain, 289 Grapefruit, 223 Grapes, 225 Green mold, see Penicillium digitatum “G”-Value, 59

H Hepatoxins, 38 Hexobarbitone oxidase, 37

Humidity, effect on disease spread, 169 Hydroxy a-carotene, 218

I Indole acetic acid, 85, 128 Insects, 289 Ionic yield, 59 Ionizing radiation, 119, 147 ff. Ionox antioxidants, 6, 9, 17, 21, 28, 30, 39 Irradiation, see also Radiation effect on auxins, 128 on chemical compounds, 123 on genetics, 152 on metabolism, 82 on pigments, 126, 132 on taste, 129 on texture, 116 on vitamins, 124 of foods, 105 ff. of f r u i t , 116 of fungi, 149 ff. in vitro, 159 in vivo, 166 Isoprene, 205 Isomerization of carotenoids, 197,239

J J a m , 342 Jelly, 342 J e t ejectors, 316

L Lemon, 225 Lethal radiation doses, 153 Linear energy transfer, 60, 70 Litton device, 284 Liver-cell microsomal enzymes, 36 ff., 50 “Lossiness,” 280 Lutein, 218, 239 Lycopene, 126, 132, 218, 223, 241 Lycopersene, 205

M Maillard reaction, 293 Maple syrup, 306 Margarine, 248 Meander system, 285 Mevalonic acid, 205 Micelles, effect on aIimentary adsorption, 32

362

SUBJECT INDEX

JIicrowave(s), 277 ff. absorption into food, 286 baking, 292 cooking, 290 frequencies, 279, 287 heating, 278 process devices, 282 sterilization, 291 Microsomes of liver, 3; Milk, 237 condensed, 309 evaporated, 309 Mitochondria, 83 Mold, 289 Moniliii in fi‘rlic f icola, 173 Mucor hiemalis, 212 Mushroom, 236 bIutations of fungi, 159 Mycelium, 153, 160

N Nash pump, 316 Seurosporene, 206 Niacin, 127 Nitroanisole demethylase, 35 Noyes process, 343

0 Oil-suspensions, 261 Oleorosin, 246 Optimum frequency. of microwaves, 292 Oranges, 218, 350 Organic acids, 128 Oscillation of molecules, 280 Oxygen, effect on carotenoids, 257 on f r u i t s , 155 Ozone, effect on f r u i t s . 68, 134, 156

P Packaging, 182 Paprika, 246 Pathogens, 135 effect of radiation on, 150 postharvest, 168 Peaches, 223 Pectin, 80, 118 P r nic il l i u m rligitutum, 176 expa’tisurn, 178 italicurn, 176

Peppers, 231 Persimmon, 226 Phospholipids, 33 P h y c o m y c e s blukeslceaniis, 212 Phycomycetes, 167 Phytoene, 206 Phytofluene, 126, 132, 206 Phytohormones, 85 P h u t o p h t h o r a , 176, 180 Pigmentation, 132 Pleospora h c t , b u ? x m , 177 Pome f r u i t s , 177 Postharvest diseases, 147 ff. cause, 166 control of, 170 ff. i n berries, 171 i n f r u i t s , 173, 176 in tomatoes, 180 in vegetables, 179 Potato chips, 293 Potatoes, 231 Preserves, 342 Provitamin A , 209 value in foods, 216 Prunes, 225 P y t h i u m d e b u v u a n u m , 180

R Radiation, 71, 241, see ctlso Irradiation application rates, 156 dose rates, 67 mechanisms, 63 sources, 87 units, 58 Radicals, 65 Radiochemical yields, 68 R B E (Relative-biological efficiency). 60, 70 Respiration, 78, 83 Reverse osmosis, 356 R h i z o p u s stolonifer, 167, 173 Riboflavin, 128 Roots, 179 S Salt, 305, 343 Scald, 179 Sclerotinia sclerotium, 177 Sea water, 343 Shijoka, 306

363

SUBJECT INDEX

Solar ponds, 305 Sorghum, 235 Spore count, 161 Squash, 233 Starch,118 Steam recompression, 334 Strawberries, 121 Stripping, 351 Sulfhydril groups, 68, 79 Sweet potato, 231

T Tangerines, 222 “ T a r g e t theory,” 73 Taste panels, 131 Temperature, effect of on carotenoids, 231, 257 i n food dehydration, 335 i n irradiated fungi, 169 Texture, 80 Thermal energy, see Energy Thiamine, 127 Tomatoes, 231 Toxicity, i n food, 39 Trichoderma lignorum, 177 5,4,6-Tri- (3’,5’-di-tert-butyl-4’hydroxybenzyl) mesitylene, 6

2,4,6-Tri- (3’,5’-di-tert-butyl-4’hydroxybenzyl) phenol, G Tubers, 179 Turgidity, 120

v Vacuum cooking, 309 Vacuum evaporator, 310 Vacuum pumps, 315 Vegetables, 57 ff.,147 ff. carotenoids in, 216 dehydrated, 288 cils in, 237 Vitamin A , 209, 248 in canning, 239 Vitamin C, 124 Vitamin E, 49, 215

W Wheat, 235

x Xanthophylls, 217 X-Rays, 71

Z Zeaxanthin, 218

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