Advances in Applied Microbiology, Volume 45

ADVANCES IN

Applied Microbiology VOLUME 45

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ADVANCES IN

Applied Microbiology Edited by SAUL L. NEIDLEMAN Oakland, California

ALLEN I. LASKIN Somerset, New Jersey

VOLUME 45

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CONTENTS

One Gene to Whole Pathway: The Role of Norsolorinic Acid in Aflatoxin Research J. W. BENNETT,P.-K. CHANG, AND D. BHATNAGAR I. 11. 111. IV. V. VI. VII.

....................... Introduction. . . . . . . . . . . . . . . . . . . . . . . . . ................... Discovery.. . . . . . . . . . . . . . . . . . . . , . . . . . Aflatoxin Biosynthetic Pathway. . . . . . . . . . . . * . . . . . .. . . . . . . . . Enzymolo. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Genetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Strain Degeneration. . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reverse Genetics and Norsolorinic Acid. . , . . . . . . . . . .

.

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

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

...

1 2 3 6 7 10 11 12 13

Formation of Flavor Compounds in Cheese

P. F. Fox AND J. M. WALLACE 11. Glycolysis . . . . . . . . . . 111. Lipolysis . . . . . , . . V. Catabolism o

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

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

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

...

...

... ... ...

...

.. ...

17 20 27 31 45 70 75

The Role of Microorganisms in Soy Sauce Production

DESMONDK. O’TOOLE I. 11. 111. IV. V.

Introduction. . . , . . .............................. Types and Compos of Soy Sauce . . . . . . . . . . . . . . . . . . Aroma and Flavor of S h o p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Soy SauceProduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of Water Activity on Microorganisms . . . . . . . . . . . . . . . . . . . . . .

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

VII. VIII. IX. X. XI.

...

...

...

... ... ...

Bacteria .......................................... ... Production and Metabolism of Amino and Organic Acids in Moromi . . . . Production and Fate of Other Substances. . . . . . . . . . . . . . . . . . . . . . . . . . . Possible Role of Metal Ions in Soy Sauce Production . . . . . . . . . . . . . . . . . Conclusion. . . . ... References . . . . . ...

87 89 100 101 119 122 125 126 130 133 140 143

vi

CONTENTS

Gene Transfer Among Bacteria in Natural Environments XIAOMING YIN AND

G. STOTZKY

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. 111. IV. V.

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

Transformation Conjugation. . . Transduction.. . Concluding Rem References , . . .

153 154 166 177 195 198

Breathing Manganese and Iron: Solid-state Respiration KENNETH H.

NEALSONAND BRENDALITTLE

Introduction. . . . .. ........................ Respiration: Organismal and Environmental. . . . . . . . . . . . . . . . . . . . . . . . . . Metal-Reducing Bacteria in Captivity . . . . . . . . ............ Reduction of Metals by Iron and Manganese Reducers Electron Transport In Metal Reducers . . . . . . . . . VI. Metal-Reducing Bacteria in Natural Environme VII. Summa. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. 11. 111. IV. V.

..

.

213 214 219 221 220 231 235 237

Enzymatic Deinking

PRATIMA BAJPAI I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. Enzymes Used in Deinking. . . . . . . . . . . . . . . . 111. Performance of Enzymes in Deinking . . . . . . .

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

IV. Effect of Enzyme on Pulp Yield and the Qualit V. Possible Mechanisms of Enzymatic Deinking . . . . . . . . . . . . . . . . . . . . . . . . VI. Factors Affecting Enzymatic Deinking. . . . . . , . . . . . . . . . . . . . . . . . . . . . . . .

.

VIII. Current Research Needs IX. Conclusions. . .

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

241 242 245 256 258 261 263 263 265 266

CONTENTS

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Microbial Production of Docosahexaenoic Acid (DHA, C22:6) AJAYSINGH AND OWEN P. WARD I. 11. 111. IV. V. VI. VII.

Introduction ... ........ Biosynthesis ated ......................... Potential Microorganisms for DHA Production. . . . . . . . . . . . . . . . . . . . . . . . Kinetics of Microbial Growth and DHA Production. . . . . . . . . . . . . . . . . . . . Factors Affecting Microbial DHA Production . . . . . . . . . . . . . . . . . . . . . . . . . Downstream Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epilogue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. .

271 2 78 283 289 2 94 304 305 307

INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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CONTENTS OF Pmvrous VOLUMES.. . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . .

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One Gene to Whole Pathway: The Role of Norsolorinic Acid in Aflatoxin Research J. W. BENNETT Department of Cell and Molecular Biology Tulane University New Orleans, Louisiana 70118

P.-K. CHANG AND D.

BHATNAGAR

Southern Regional Research Center USDA Agricultural Research Service New Orleans, Louisiana 70179

I. 11. 111. IV. V. VI. VII. VIII.

Introduction Discovery Aflatoxin Biosynthetic Pathway Enzymology Molecular Genetics Strain Degeneration Reverse Genetics and Norsoloririic Acid Conclusions References

I. Introduction

Mycotoxins are toxic secondary metabolites produced by filamentous fungi. Although the mycotoxin problem is probably as old as agriculture (outbreaks of ergot alkaloid poisoning have been reported since medieval times (Bove, 1970)), contemporary interest in these toxic metabolites dates to the early 1960s and the discovery of aflatoxins from Aspergillus flavus and Aspergillus parasiticus (Goldblatt, 1969). The 15 years following the discovery of aflatoxins resulted in the identification of over 100 fungal toxins, an era described by Maggon et al. (1977) as a “mycotoxin gold rush.” Mycotoxins cause enormous economic losses in agriculture. One major study estimated that 25% of the annual food crop worldwide is mycotoxin-contaminated (Jelinek, 1987). Most mycotoxins are associated with veterinary diseases, which may be either acute or chronic (Richard and Thurston, 1986). For general reviews of 3 ADVANCES IN APPLIED MICROBIOLOGY,VOLUME 4 5 Copyright 0 1997 by Academic Press All rights of reproduction in any form reserved. OO~~-Z~M $25 /W nn

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1. W.BENNETT, P.-K. CHANG, AND D. BHATNAGAR

mycotoxins, see Betina (1984), CAST (1989), and Bhatnagar et al, (1992);more recent aspects of the aflatoxin problem have been reviewed by Eaton and Groopman (1994). Aflatoxins are the most important and best-studied mycotoxins. They act as potent carcinogens in laboratory tests, and there is strong evidence that they can act as hepatocarcinogens in human populations (Eaton and Groopman, 1994). Because there are no good treatments against aflatoxin-induced diseases, the emphasis in aflatoxin research has been to find methods for prevention of aflatoxin contamination of food and feed (Lee et al., 1992).Good agronomic practices that reduce preharvest and postharvest mold growth, coupled with careful regulatory surveillance for aflatoxins in foods intended for human consumption, comprise the main methods in use. Since these methods are inherently limited, considerable basic research on aflatoxin production has been funded by various agricultural organizations. There is hope that an understanding of the fundamental mechanisms leading to aflatoxin biosynthesis may translate into practical methods for blocking the process (Keller et a]., 1992). The first known intermediate in the aflatoxin biosynthetic pathway is norsolorinic acid, a red-pigmented polyhydroxyanthraquinone. In this review, we discuss the important role this compound has played in our knowledge of aflatoxin biosynthesis, aflatoxin genetics, and aflatoxin molecular biology. II. Discovery

The decaketide 2-hexanoyl-1,3,6,8-tetrahydroxyanthraquinone was named norsolorinic acid after the lichen Solorina crocea, from which it was first characterized (Anderson et al., 1966).Soon thereafter, a norsolorinic acid-producing mutant fungus was isolated after ultraviolet light irradiation of an aflatoxigenic strain of A. parasiticus (Lee et al., 1971). This mutant, later designated nor-I, was easily identified in liquid and agar culture because of the orange-red color characteristic of norsolorinic acid accumulation. An 80% reduction in aflatoxin production, as well as a correlation between aflatoxin and norsolorinic acid production, was observed in this A. parasiticus mutant. In chemically defined media under continuous illumination, in zinc-deficient media, and in the presence of para-aminobenzoic acid, both norsolorinic acid and aflatoxin production were inhibited. Growth in enriched complex medium resulted in higher yields of both compounds (Bennett et al., 1971).This correlation was useful because the bright orange-red pig-

NORSOLORINIC ACID IN AFLATOXIN RESEARCH

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ment of norsolorinic acid was an easy visual screen for the putative presence of aflatoxin. Other mutants of A . flavus and A . parasiticus that had lost the ability to produce aflatoxin were isolated on the basis of altered fluorescence under long-wave ultraviolet light. Mutants selected for altered fluorescence production also demonstrated abnormal pigment production. Likewise, mutants selected for lowered norsolorinic acid production had lowered aflatoxin production (Bennett and Goldblatt, 1973), suggesting that aflatoxin and mycelial pigment production might result from common precursors. Norsolorinic acid-accumulating mutants of aflatoxigenic species were also induced by nitrosoguanidine treatment in A . parasiticus (Detroy et al., 1973) and in A. flavus (Papa, 1982). Like nor-2, all of these norsolorinic acid-accumulating mutants were “leaky,” that is, they still produced aflatoxin, although in reduced quantities.

I l l . Aflatoxin Biosynthetic Pathway The polyketide origin of the carbon skeleton of aflatoxin B1 was established by 14C radioisotope labeling studies by Biollaz et al. (1968a,b). During the 1970s, several other anthraquinones, in addition to norsolorinic acid, were isolated from blocked mutants and shown to be aflatoxin precursors: averufin by Donkersloot et al. (1972) and versicolorin A by Lee et al. (1975). The role of sterigmatocystin as a late intermediate in aflatoxin biosynthesis was also established (Hsieh et al., 1973). When 14C labeling was used to follow incorporation of norsolorinic acid into aflatoxin, only 2.2% of label was recovered in aflatoxin B1, as opposed to 49.4% for averufin, 45.5% for versicolorin A, and 65.5% for sterigmatocystin (Hsieh et al., 1976). The availability of blocked mutants, coupled with the extensive use of both 14Cand 13C isotope studies, led to a generally accepted “canonical” scheme for aflatoxin biosynthesis in which the steps were usually summarized as acetate + malonate + anthraquinones 3 sterigmatocystins + aflatoxins B1. This pathway has been reviewed many times (see, e.g., Steyn et al., 1980; Bennett and Christensen, 1983; Turner and Aldridge, 1983; Dutton, 1988; Bhatnagar et al., 1992). In this scheme, norsolorinic acid is the decaketide product of a polyketide synthase and the first stable precursor in aflatoxin biosynthesis. Theoretically, the product of a polyketide synthase should be anthrone, but this compound has never been isolated, presumably due to its rapid oxidation to norsolorinic acid, a more stable conformation (Vederas and

4

J. W. BENNETT, P.-K. CHANG, AND D. BHATNAGAR AFBl (312)

POLYKETIDE

I

1

I

&

NOR(370)

OMST (338)

AVN(372)

1

ST (324)

AVNN(370)

KQ?J

HO \

OHMe

1

0

0

AVF(368)

I

HO

HO

0

OH

VERA (338)

0

HO 0

0

VHA (400)

FIG.1. Proposed scheme for aflatoxin biosynthesis based on mutation analysis, precursor feeding studies, and isotopic labeling (mol. wt.]. NOR = norsolorinic acid; AVN = averantin; AVNN = averufanin; AVF = averufin; VHA = versiconol hemiacetal acetate; VERA = versicolorin A, ST = sterigmatocystin; OMST = o-methylstwigmatocystin; AFBl = aflatoxin Bl.

Nakashima, 1980). Recognizing that acetyl CoA is not always the starter unit for polyketide synthesis, Townsend et al. (1984, 1988) provided an alternative proposal in which the sidechain of norsolorinic acid arises intact from hexanoic acid. Brown and Salvo (1994) have shown that a n ascospore pigment of Aspergillus nidulans, ascoquinone A, has a strong resemblance to norsolorinic acid. After norsolorinic acid, the polyketide undergoes an estimated 1 2 to 1 7 enzymatic transformations, leading through a series of pathway intermediates, including averantin, averufanin, averufin, versiconol hemiacetal acetate, versiconol, versicolorins A and B, demethyl sterigmatocystin, and sterigmatocystin, to the final product, aflatoxin B1 (see Fig. 1). In this scheme, the intermediate following norsolorinic acid is usually given as averantin, another polyhydroxyanthraquinone with a

5

NORSOLOFUNIC ACID IN AFLATOXIN RESEARCH

0 orsoiorinic acid (NOR)

2

Reductase

\

NADPH

Dehydrogenase

\

\

Oxidation

Dehydrogenase (a)

0

0

Averufanin (AV")

Averantln (AVN)

0 Averufin (open form)

HO

0

Averufln (ketal form) WF)

FIG.2. Proposed pathways for conversion of norsolorinic acid to averufin. After Bhatnagar et al. (1992).

C-6 sidechain. The enzymatic step entails reduction of the carbonyl group in the sidechain of norsolorinic acid to a hydroxyl group. The reaction is variously labeled as a reductase or a dehydrogenase step. Averantin is then converted to averufin. Alternatively, norsolorinic acid is reduced to averufanin and then to averufin (McCormick et al., 1987). Another postulated scheme channels norsolorinic acid to averantin, averufanin, and then to averufin (Bhatnagar et a]., 1992). These alternative schemes are outlined in Fig. 2. Experimental evidence for a norsolorinic acid * averantin hydroxyaverantin 3 averufin conversion has been provided by Yabe et al. (1991). In order to better understand the mechanistic details of these proposed transformations, it is necessary to isolate the relevant enzymes.

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J. W. BENNETT, P.-K. CHANG, AND D. BHATNAGAR

IV. Enzymology

Compared to other cellular proteins, secondary metabolic enzymes are often present only in small amounts, rendering the purification of aflatoxin pathway enzymes technically difficult. Additionally, cell-free extracts containing these secondary metabolic enzymes seem especially sensitive to denaturing conditions. Consequently, most efforts to purify these enzymes result in crude protein homogenates instead of pure protein extracts (Dutton, 1988). The enzyme that catalyzes the conversion of norsolorinic acid to averantin is usually called norsolorinic acid dehydrogenase. Chuturgoon and Dutton (1991) developed an affinity chromatography method for the purification of this enzyme activity. Using reactive green 19-agarose and norsolorinic acid agarose affinity chromatography, the dehydrogenase was purified from the mycelia of 84-hour A . parasiticus. A molecular weight of 140 kDa was determined by gel filtration chromatography on a Sephacryl S-300 column. Additionally, the temperature and pH optima of the dehydrogenase were found to be 35°C and 8.5, respectively. Like other fungal dehydrogenases, a pH below 6.5 or above 9.0 resulted in a decline in enzymatic activity. Further studies of K, values for norsolorinic acid dehydrogenase, using norsolorinic acid and averantin as substrates with various nicotinamide cofactors, showed that norsolorinic acid dehydrogenase favored the reaction of norsolorinic acid to averantin somewhat more than the reverse reaction of averantin to norsolorinic acid. The K,,, for norsolorinic acid (3.45 mMj was lower than the K,,, for averantin (3.72 mM), indicating that norsolorinic acid dehydrogenase had a higher affinity for norsolorinic acid than averantin (Chuturgoon and Dutton, 1991). Yabe et al. (1991) studied the enzymatic conversion of norsolorinic acid to averufin in Emericella heterothallica, a non-aflatoxigenic species that produces the anthraquinones averantin, averufanin, and averufin. Cell-free experiments supported the reversibility of the norsolorinic acid dehydrogenase reaction. The presence of the reduced forms of the cofactors resulted in greater product formation in the forward conversion of norsolorinic acid to averantin, while the oxidized forms (NAD' or NADP+)were more efficient in the reverse reaction of averantin to norsolorinic acid. NADPH was preferred to NADH in the norsolorinic acid dehydrogenase reaction. Yabe et al. (1991) also reported that norsolorinic acid dehydrogenase activity was media-dependent. Activity was present when a complex culture medium containing 2% yeast extract and 20% sucrose was used, but when the sucrose was replaced with 20% peptone no norsolorinic acid dehydrogenase activity was detected. The presence of NAD under yeast extract-

NORSOLORINIC ACID IN AFLATOXIN RESEARCH

7

peptone culture conditions restored low dehydrogenase activity (Yabe et al., 1991). Bhatnagar and Cleveland (1990) also worked on this enzyme activity, purifying a protein that catalyzed the transformation of norsolorinic acid to averantin from 3-day-old cultures of A. parasiticus by classical biochemical techniques. The multistep protocol involved ammonium sulfate precipitation and several chromatography steps, concluding with an HPLC BioScale anion-exchange column. The purified protein catalyzed the transformation of norsolorinic acid to averantin in the present of NADPH and had a pH optimum of 7.5, a temperature optimum of 3OoC, and an estimated molecular weight of 4 3 kDa. Using this purified enzyme, polyclonal antibodies were prepared; the prepared antiserum neutralized norsolorinic acid reductase enzyme activity significantly more than did control antisera (Lee et al., 1995). Because traditional biochemical approaches to enzyme purification have been so technically demanding, workers have turned to molecular genetics. Once relevant genes are isolated, it is possible to predict amino acid sequences from nucleotide sequences and engineer expression systems for producing purified proteins. In recent years, the tools of molecular genetics have been applied to the enzymes surrounding norsolorinic acid and other steps in the aflatoxin pathway. There have been some surprising discoveries. V. Molecular Genetics

Prior to 1989, a major obstacle to the study of these genes involved the need for efficient transformation systems that were met first for A. flavus (Woloshuk et al., 1989) and then for A. parasiticus (Skory et al., 1990). These transformation systems were developed for A. flavus by complementation of the pyr-4 gene of Neurospora crassa involved in pyrimidine biosynthesis, while the system developed for A. parasiticus used the homologous pyrG gene and involved the isolation and characterization of uridine auxotrophic mutants of A. parasiticus (Skory et al., 1990).

Almost simultaneously, another transformation method for A. parasiticus was developed based on complementation of nitrate reductase mutants with the homologous niaD gene (Chang et al., 1992). This complementation system was used to clone the first aflatoxin pathway gene. Specifically, a genomic library was constructed by inserting wildtype A. parasiticus DNA into a cosmid vector containing the nitrate reductase gene niaD. As a selectable marker, an aflatoxin-producing

8

J. W. BENNETT, P.-K. CHANG, AND D. BHATNAGAR

cosmid was isolated that complemented an aflatoxin-deficient (norsolorinic acid-positive), nitrate-nonutilizing mutant strain. The gene was called nar-1 for “norsolorinic acid related,” but later renamed nor-1. Further investigations showed that a 1.7-kb Bgl 11-Sph-I DNA fragment was responsible for the renewal of aflatoxin production (Chang et al., 1992). Shortly thereafter, a similar strategy was used to isolate a gene that encodes an activity associated with the conversion of versicolorin A to sterigmatocystin (Skory et al., 1992). This latter gene was named ver-1. The appearance of nor-1 and ver-1 transcripts was followed in batch cultures. The two genes were expressed at the end of trophophase. The timing of expression suggested that they were controlled by a common regulatory factor and that the accumulation of these transcripts partially regulated aflatoxin biosynthesis (Skory et al., 1993). Sequence analysis of nor-1 genomic DNA predicted a region containing three introns that encoded a putative protein of 294 amino acids with a molecular mass of 29 kDa. The deduced amino acid sequence of nor-1 had a high sequence similarity to other nucleotide-binding dehydrogenases-reductases (Trail et ul., 1994). A related nor-1 gene has been isolated from A. f l a w s (Brown-Jencoet al., 1994). Gene disruption was used to confirm the role of nor-1 in aflatoxin biosynthesis in A. parasiticus. Disrupted strains, like the original blocked norsolorinic acid mutations, accumulated norsolorinic acid, as well as retaining the ability to produce low levels of aflatoxin (Trail et al., 1994), supporting earlier biochemical hypotheses concerning alternative pathways from norsolorinic acid to averufin (Bhatnagar et al., 1992; Yabe et a!., 1991). There has been a burst of research activity concerning the molecular genetics of A . parasiticus and A. flavus. In addition to Complementation, genes have been isolated after purification of their relevant proteins (e.g., Yu et d., 1993) and by differential expression of transcripts during idiophase (e.g., Feng et al., 1992). See Bennett et al. (1994) and Trail et al. (1995a) for reviews of early aflatoxin molecular biology. Although classical parasexual studies had hinted that some of the aflatoxin pathway genes in A. flavus were linked (Bennett and Papa 1988), the high degree of linkage that has been revealed by molecular techniques was unexpected. Aflatoxin pathway genes in both A . flavus and A . parasiticus are clustered (Yu et a!., 1995; Silva et al., 1996). This fortuitous clustering has allowed “chromosome walking,” i.e., the identification of DNA sequences related to the pathway through the use of overlapping clones (Yu et a]., 1995). This technique has been exploited most fully in the study of the cognate polyketide pathway to sterigmatocystin in Aspergillus nidulans. This mold has been studied as a genetic model for over 50 years (Martinelli and Kinghorn, 1994),making

NORSOLORINIC ACID IN AFLATOXIN RESEARCH

9

it more amenable to sophisticated genetic analysis than either A. flavus or A. parasiticus. Using standard molecular techniques, Brown et al. (1996a) have described a 25-gene cluster of approximately 60-kb DNA that contains most, if not all, of the enzyme activities required for sterigmatocystin biosynthesis. The A. nidulans homologue of nor-1 is called stcE. It encodes a putative reductase and shares 56% deduced amino acid identity with nor-1. Molecular studies have also clarified details concerning the starter chain. If, as proposed by Townsend et al. (1984, 1988), hexanoic acid initiates the polyketide, norsolorinic acid would be assembled from hexanoyl CoA (synthesized by a fatty acid synthase, FAS) and 7 malonate units, and not from 10 classical polyketide condensation reactions. The isolation and characterization of the A. parasiticus polyketide synthase gene p k s A on the gene cluster (Chang et al., 1995a; Feng and Leonard, 1995; Trail et al., 1995b) gives molecular support to Townsend’s postulate. Sequence analysis indicates that the p k s A gene encodes P-ketoreductase, acyltransferase, acyl carrier, and thioesterase domains, but no P-acyl ketoacyl reductase or enoyl reductase domains, suggesting that the pksA gene does not encode catalytic activities that are required for processing P-carbon (keto group). Moreover, a fatty acid synthase gene, fas-ZA, is necessary for norsolorinic acid production in A. parasiticus. The predicted amino acid sequence for fas-ZA shows a high degree of similarity with the P subunit of a fatty acid synthase from yeast (Mahanti e t al., 1996), suggesting that fas-ZA encodes a fatty acid starter unit. Subsequent characterization and disruption of the fas-ZA gene and another gene Ifas-2A) encoding a 6.5-kb transcript demonstrated that these genes were directly involved in early pathway functions (Chang et al., 1995b; Trail et al., 1994). Enoyl reductase domains were found in both genes. It appears that the two FAS genes are required for the production of the C6 sidechain of norsolorinic acid. Metabolite feeding studies with p k s A and fas disruptants provided evidence that the protein products of these genes may physically associate during normal pathway function (Watanabe et al., 1996; Linz et al., 1996). A unique fatty acid synthase dedicated to the synthesis of a hexanoate moiety also has been identified from A. nidulans (Brown et al., 1996b). A pair of fatty acid synthase genes, stcJ and stcK, are required for sterigmatocystin biosynthesis, but not for growth. Disrupted stcJ and stcKstrains are restored to sterigmatocystin biosynthesis by the addition of hexanoic acid (Brown et a]., 1996b).

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J. W. BENNETT, P.-K. CHANG, AND D. BHATNAGAR

In summary, these data indicate that the sterigmatocystin-aflatoxin pathway is indeed primed by specialized fatty acid synthases to generate a fatty acid (hexanoic acid), which in turn is converted by a polyketide synthase to norsolorinic acid, thus supporting Townsend’s early hypothesis. The ability of the polyketide synthase to use exogenous hexanoic acid also has important implications for engineering polyketide synthase diversity. VI. Strain Degeneration

Filamentous fungi are known for their morphological and metabolic variability. Traits such as virulence, sporulation, and secondary metabolite production may be unexpectedly lost, especially in cultures that have been maintained in the laboratory for long periods. Such strain degenerations are poorly understood and are particularly troublesome in industrial fermentations. Proposed theories for the cause of strain degeneration in filamentous fungi include chromosome instability, transposable elements, heterokaryosis, and cytoplasmic inheritance (Kale and Bennett, 1992; Li et a]., 1994). The norsolorinic acid mutant of A. parasiticus has been used to study experimentally induced strain degeneration. The loss of red pigment is easily identified in both liquid and solid culture. In an early study on the effects of zinc and barium on norsolorinic acid and aflatoxin production, unpigmented colonies were regularly detected. These unpigmented non-aflatoxigenic variants were associated with serial transfers of inocula that contained hyphae rather than conidia, but were not associated with either zinc deficiency or the presence of barium ions in the medium (Bennett, 1981). In addition to loss of norsolorinic acid and aflatoxin production, these strains sporulated poorly and gave an unusual “fan-like” growth on Petri plates. Similar degenerate forms were subsequently isolated from wild-type and genetically marked strains after 5-12 serial transfers of mycelial macerates. These strains were designated s e c for “secondary metabolite minus” (Kale eta]., 1994).The Kale et al. (1994) paper presented a color plate that showed the progression of variation from bright red “parental” colonies that accumulated large quantities of norsolorinic acid, to colonies observed after serial transfer in which there was no detectable production of this pigment. These variants were stable and did not revert back to norsolorinic acid or aflatoxin production even after several years of storage in the laboratory (Kale et al., 1994).

NORSOLORINIC ACID IN AFLATOXIN RESEARCH

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The reproducible isolation of sec- forms, and the pleiotropic nature of the changes observed, made them an attractive model system for studying strain degeneration. In another study, it was demonstrated that the s e c strains lacked the enzymatic ability to convert certain aflatoxin precursors to aflatoxin. Moreover, Northern blots probed with pathway genes demonstrated lack of message for both nor-1 and a regulatory gene associated with the aflatoxin pathway, while the polymerase chain reaction confirmed the presence of the structural gene for nor-2 (Kale et al., 1996). In other words, although these s e c forms retained genes involved in norsolorinic acid production, the genes were neither transcribed nor translated. In summary, the norsolorinic acid marker has been important in developing a model system for studying degeneration. These s e c strains display a pleiotropic phenotype that includes changes in conidiophore development and sporulation patterns, suggesting that the regulation of aflatoxin synthesis and condiogenesis may be interlinked. The norsolorinic acid marker may prove to be as useful for dissecting developmental pathway genes as it has been in studying polyketide pathway genes. VII. Reverse Genetics and Norsolorinic Acid

The most recent chapter in the history of norsolorinic acid concerns the “reverse genetics” approach to isolating genes. As described above, three different groups have studied the enzyme activity that transforms norsolorinic acid to averantin. Although Trail et a]. (1994) stated that “two different norsolorinic acid reductases associated with the conversion of norsolorinic acid have been purified,” it is not yet known whether the ability reported by Chuturgoon and Dutton is actually different from the one reported by Bhatnagar and Cleveland (1990). Nor is it clear how the Emericella enzyme studied by Yabe’s group is related to A. parasiticus enzyme(s). What is clear is that polyclonal antibodies prepared against the A. parasiticus enzyme exhibiting norsolorinic acid reductase activity purified by Bhatnagar’s group did not react with the 29-kDa nor-1 gene product isolated by complementation (Lee et al., 1995). A monoclonal antibody to norsolorinic reductase was therefore prepared and used to screen a cDNA library. A gene with two introns was isolated that exhibited 68% nucleotide identity to an aryl alcohol dehydrogenase gene known from Phanerochaete chrysosporium (Cary et a]., 1996). The gene was located in the aflatoxin cluster and was named norA, because monoclonal antibodies used to isolate the gene inhibited the conversion of norsolorinic acid to averantin and because

12

J. W. BENNETT, P.-K. CHANG, AND D. BHATNAGAR

the purified 43-kDa protein used to generate the monoclonal antibody demonstrated norsolorinic acid reductase activity. NorA shows high sequence similarity with an aryl alcohol dehydrogenase from A . nidulans designated stcV (Cary, personal communication). Gene disruption experiments have been difficult to perform with norA, presumably due to duplication of a 12-kb region of the gene cluster (Trail et al., 1995b), so it is not yet known whether disrupted strains will accumulate pathway intermediates. However, it is known that nor-2 and norA are under common regulation of the aflatoxin biosynthetic pathway regulatory gene aflB (Chang et al., 1995b; Cary et a]., 1996). These findings underline the importance of multiple approaches to molecular cloning. In this case, complementation and reverse genetics identified separate genes implicated in norsolorinic acid metabolism. The nomenclature of these genes is confusing, and we recommend that workers studying the aflatoxin pathway develop a standardized system in the near future. VII I. Conclusions

Norsolorinic acid is a C-20 anthraquinone that is the first stable intermediate in aflatoxin biosynthesis. Mutants of A . flavus and A. parasiticus that accumulate norsolorinic acid also produce low levels of aflatoxin, raising interesting questions about the early steps of the pathway. The development of transformation systems for the aflatoxigenic species A. flaws and A. parasiticus, and the rapid cloning, sequencing, and analysis of several genes involved in the pathway, have facilitated new insights into aflatoxin biosynthesis. A gene involved in norsolorinic acid (nor-1)was isolated by complementation. Disrupted strains accumulated norsolorinic acid, confirming the role of this compound as an intermediate. Additionally, disruption of a fatty acid synthase has confirmed the role of hexanoic acid as the starter unit initiating polyketide biosynthesis. Researchers initially had difficulty in purifying the enzymes of the aflatoxin biosynthetic pathway, a common problem for other secondary metabolites as well. Nevertheless, several of the enzymes have been successfully purified to homogeneity in the past few years. These include a norsolorinic acid reductase associated with the conversion of norsolorinic acid to averantin. When reverse genetics was used to isolate the gene corresponding to this enzyme activity, surprisingly, it was different from nor-1. The new gene is called norA. An understanding of the regulation and expression of these genes in aflatoxin-producing strains at the molecular level continues to expand

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our knowledge of fungal polyketide synthesis and may ultimately lead both to new ways of generating hybrid polyketides and to improved methods for controlling the production of aflatoxins. ACKNOWLEDGMENTS

We thank Nancy Keller and John Linz for sharing unpublished data, and Aaron Allen and Tarry1 Gallo for manuscript preparation. JWB and PKC acknowledge a cooperative agreement with the U.S. Department of Agriculture (532 243). REFERENCES Anderson, H. A., Thomson, R. H., and Wells, J. W. (1966). J. Chem. SOC.,pp. 1727-1729. Bennett, J. W. (1981).J. Gen. Microbiol. 124,429-432. Bennett, J. W., and Christensen, S. B. (1983).Adv. Appl. Microbiol. 29,53-92. Bennett, J. W., and Goldblatt, L. A. (1973). Sabouraudia 11,235-241. Bennett, J. W., and Papa, K. E. (1988). In “Advances in Plant Pathology” (D. S. Ingram and P. H. Williams, eds.), Vol. 6, pp. 263-280. Academic Press, London. Bennett, J. W., Lee, L. S., and Vinnett, C. (1971). J. Am. Oil Chem. SOC.48,368-370. Bennett, J. W., Chang, P.-K., and Bhatnagar, D. (1994). In “The Genus Aspergillus” (K. A. Powell, A. Renwick, and J. F. Peberdy, eds.), pp. 51-58. Plenum, New York. Betina, V., ed. (1984). “Mycotoxins: Production, Isolation, Separation, and Purification.” Elsevier, Amsterdam. Bhatnagar, D., and Cleveland, T.E. (1990). FASEB J. 4,A2164. Bhatnagar, D.,Ehrlich, K. C., and Cleveland, T. E. (1992). In “Handbook of Applied Mycology” (D. Bhatnagar, E. B. Lillehoj, and D. K. Arora, eds.), Vol. 5, pp. 255-286. Dekker, New York. Biollaz, M., Buchi, G., and Milne, G. (1968a). J. Am. Chem. SOC.90,5017-5020. Biollaz, M., Buchi, G., and Milne, G. (1968b). J. Am. Chem. SOC.90,5019-5020. Bove, F. J. (1970). “The Story of Ergot.” Barger, New York. Brown, D. W., and Salvo, J. J. (1994). Appl. Environ. Microbiol. 60,979-983. Brown, D.W., Yu, J.-H., Kelkar, H. S., Fernandes, M., Nesbitt, T. C., Keller, N. P., Adams, T. H., and Leonard, T. J. (1996a). Proc. Natl. Acad. Sci. U.S.A. 93,1418-1422. Brown, D. W., Adams, T. H., and Keller, N. P. (1996b). Proc. Nutl. Acad. Sci. U.S.A. 93, 14873-14877. Brown-Jenco, C. S.,Brewer, J. F., and Payne, G. A. (1994). Phytopathology 84,1098. Cary, J. W., Wright, M., Bhatnagar, D., Lee, R., and Chu, F. S. (1996). Appl. Environ. Microbiol. 62,360-366. CAST [Council for Agricultural Science and Technology] (1989). “Mycotoxins: Economic and Health Risks.” CAST, Ames, 10. Chang, P.-K., Skory, C. D., and Linz, J. E. (1992). Curr. Genet. 21,231-233. Chang, P.-K., Cary, J. W., Yu, J., Bhatnagar, D., and Cleveland, T. E. (1995a). Mol. Gen. Genet. 248,270-277. Chang, P.-K., Ehrlich, K. C., Yu, J., Bhatnagar, D., and Cleveland, T. E. (1995b). Appl. Environ. Microbiol. 61,2372-2377. Chuturgoon, A. A., and Dutton, M. E. (1991). Prep. Biochem. 21,125-140.

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Detroy, R. W., Freer, S., and Ciegler, A. (1973). Can. J. Microbiol. 19,1371-1378. Donkersloot, J. A., Matales, R. I., and Yang, S. S. (1972). Biochem. Biophys. Res. Comrnun. 47, 1051-1055. Dutton, M. F. (1988). Microbiol. Rev. 52,274-295. Eaton, D.L., and Groopman, J. D. (1994). In “Human Health, Veterinary, and Agricultural Significance.” Academic Press, San Diego. Feng, G. H., and Leonard, T. J. (1995). J. Bacteriol. 177, 6246-6254. Feng, G. H., Chu, F. C., and Leonard, T. J. (1992). Appl. Environ. Microbiol. 58,455-460. Goldblatt, L. A., ed. (1969). “Aflatoxin: Scientific Background, Control, and Implications.” Academic Press, New York. Hsieh, D. P. H., Lin, M. T., and Yao, R. C. (1973). Biochem. Biophys. Res. Commun. 52, 92 2-997. Hsieh, D. P. H., Lin, M. T., Yao, R. C., and Singh, R. (1976). J. Agric. Food Chern. 24, 1171-1174. Jelinek, C. F. (1987). Joint FAO/WHO/UNEP lnt. Conf. Mycotoxins, Znd, Bangkok, Thailand. Kale, S. P., and Bennett, J. W. (1992). In “Handbook of Applied Mycology” (D. Bhatnagar, E. B. Lillehoj, and D. K. Arora, eds.), Vol. 5, pp. 311-331. Dekker, New York. Kale, S. P., Bennett, J. W., and Bhatnagar, D. (1994). Mycol. Res. 98,645-652. Kale, S. P., Cary, J. W., Bhatnagar, D., and Bennett, J. W. (1996). Appl. Environ. Microbiol. 62, 3399-3409. Keller, N. P., Cleveland, T. E., and Bhatnagar, D. (1992). In “Handbook of Applied Mycology” (D. Bhatnagar, E. B. Lillehoj, and D. K. Arora, eds.), Vol. 5, pp. 287-310. Dekker, New York. Lee, L. S., Bennett, J. W., Goldblatt, L. A., and Lundin, R. E. (1971). J. Am. Oil Chem. Soc. 48,93-94. Lee, I,. S., Bennett, J. W., Cucullu, A. F., and Stanley, J. B. (1975). J. Agric. Food Chern. 23, 1132-1134. Lee, L.S., Bayman, P., and Bennett, J. W. (1992). In “Biotechnology of Filamentous Fungi” (D. R. Finkelstein and C. Ball, eds.). Butterworth-Heinemann, Boston. Lee, R. C., Cary, J. W., Bhatnagar, D., and Chu, F. S. (1995). Food Agric. Irnmunol. 7,Zl-32. Li, A., Begin, A., Kokurewicz, K., Bowen, C., and Horgen, P. A. (1994). Appl. Environ. Microbiol. 60,2384-2388. Linz, J. E., Townsend, C. A,, and Bhatnagar, D. (1996). In “Proc. Noble Foundation Symp.: The Biochemical and Metabolic Aspects of 3-Ketoacyl Synthases.” In press. Maggon, K. K., Gupta, S. K., and Venkitasubramanian, T. A. (1977). Bacteriol. Rev. 41, 822-855. Mahanti, N., Bhatnagar, D., Cary, J. W., Jourbran, J., and Linz, J. E. (1996). Appl. Environ. Microbiol. 62,191-195. Martinelli, S. D., and Kinghorn, J. R., eds. (1994). “Aspergillus: 50 Years On.” “Progress in Industrial Microbiology,” Vol. 29. Elsevier, Amsterdam. McCormick, S. P., Bhatnagar, D., and Lee, L. S. (1987). Appl. Environ. Microbiol. 53, 14-16. Papa, K. E. (1982). J. Gen. Microbiol.128,1345-1348. Richard, J. L., and Thurston, J. R., eds. (1986). “Diagnosis of Mycotoxicoses.” Martinus Nijhoff, Boston. Silva, J. C., Minto, R. E., Barry 111, C. E., Holland, K. A., and Townsend, C. A. (1996). J. Bid. Chem. 271, 13600-13608. Skory, C. D., Horng, J. S., Pestka, J. J., and Linz, J. E. (1990). Appl. Environ. Microbiol. 56,3315-3320.

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Skory, C. D., Chang, P.-K., Cary, J. W., and Linz, J. E. (1992). Appl. Environ. Microbiol. 58, 3 52 7-35 3 7.

Skory, C. D., Chang, P.-K., and Linz, J. E. (1993).Appl. Environ. Microbiol. 59, 1624-1646. Steyn, P. S., Vleggar, R., and Wessels, P. L. (1980). In “The Biosynthesis of Mycotoxins: A Study in Secondary Metabolism” (P. S. Steyn, ed.), pp. 105-155. Academic Press, New York. Townsend, C. A., Christensen, S. B., and Trautweink, K. (1984). J. Am. Chem. Soc. 106, 3868-3869.

Townsend, C. A., Brobst, S. W., Ramer, S. E., and Vederas, J. C. (1988).1. Am. Chem. Soc. 110, 318-319.

Trail, F., Chang, P.-K., Cary, J., and Linz, J. E. (1994). Appl. Environ. Microbiol. 60, 4078-4085.

Trail, F., Mahanti, N., and Linz, J. E. (1995a). Microbiology 141, 755-765. Trail, F.,Mahanti, N., Rarick, M., Mehugh, R., Liang, S.-H., Zhou, R., and Linz, J. E. (1995b).Appl. Environ. Microbiol. 61,2665-2673. Turner, W. B., and Aldridge, D. C. (1983). “Fungal Metabolites,” Vol. 2. Academic Press, London. Vederas, J. C., and Nakashima, T. T. (1980).J. Chem. Soc., Chem. Commun., pp. 183-185. Watanabe, C. M. H., Wilson, D., Linz, J. E., and Townsend, C. A. (1996). Chem. Biol. 3, 463-469.

Woloshuk, C. P., Seip, E. R., and Payne, G. A. (1989).Appl. Environ. Microbiol. 55, 86-90. Yabe, K., Nakamura, Y . ,Nakajima, H., Ando, Y.,and Hamasaki, T. (1991). Appl. Environ. Microbiol. 57, 1340-1 345. Yu, J., Cay, J. W., Bhatnagar, D., Cleveland, T. E., Keller, N. P., and Chu, F. S. (1993).Appl. En viron. Microbiol. 59, 35 64-3 5 71. Yu, J., Chang, P.-K., Cary, J. W., Wright, M., Bhatnagar, D., Cleveland, T. E., Payne, G. A., and Linz, J. E. (1995). Appl. Environ. Microbiol. 61, 2365-2371.

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Formation of Flavor Compounds in Cheese P. F. FOX AND J. M.

WALLACE

Department of Food Chemistry University College Cork, Ireland

I. Introduction A. Ripening Agents in Cheese 11. Glycolysis A. Metabolism of Lactose by Lactic Acid Bacteria B. Metabolism of Lactic Acid in Cheese C. Effect of Lactose Concentration on Cheese Quality D. Citrate Metabolism 111. Lipolysis A. Lipases in Cheese B. Catabolism of Free Fatty Acids IV. Proteolysis A. Contribution of Individual Proteolytic Agents B. Proteolysis in Cheese C. Free Amino Acids in Cheese V. Catabolism of Amino Acids A. Decarboxylation and Production of Amines B. Deamination/Formation of Ammonia and Neutral or Acidic Compounds C. Transamination, Strecker Degradation, and Production of Aldehydes D. Catabolism of Sulfur Amino Acids E. Catabolism of Phenylalanine, Tyrosine, and Tryptophan F. Other Important Flavor Compounds from Amino Acid Catabolism G. Reactions of Free Amino Acids with Other Compounds in Cheese H. Nonprotein Amino Acids VI. Chemistry of Cheese Off-Flavors References

I. Introduction

The quality of cheese is determined by its flavor (taste and aroma), rheological properties (commonly referred to as body and texture, including such parameters as hardness, cohesiveness, sliceability, meltability, and stretchability), and visual appearance (e.g., intensity and uniformness of color, presence or absence of eyes or slits, closeness, uniformity of mould growth, or smear, if relevant). The relative importance of these three quality attributes varies with the cheese variety, and they are interrelated, at least to some extent. The flavor impact of cheese 17 ADVANCES IN APPLIED MICROBIOLOGY. VOLUME 45 Copyright 0 1997 by Academic Press All rights of reproduction in any form reserved. 0065-2164197 $25.00

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is very strongly influenced by its rheological properties, but, unfortunately, these attributes are rarely studied in a single investigation. The flavor of cheese has been the subject of scientific investigation for nearly a century. Initially, it was believed that cheese flavor might be due to a single compound, but it soon became apparent that cheese flavor is due to the correct balance and concentration of a wide range of sapid and aromatic compounds that led to the “Component Balance Theory” (Mulder, 1952; Kosikowski and Mocquot, 1958). The development of gas chromatography (GC) in the 1950s and its interfacing with mass spectrometry (MS)permitted detailed and sensitive analysis of the volatile compounds in cheese believed to be responsible for its aroma and to be major contributors to its overall flavor. Nonvolatile compounds, for example, acids, amino acids, and small peptides, also make significant contributions to cheese flavor, especially its taste. Paper, thin-layer, and ion-exchange chromatography were initially used to study the nonvolatile flavor compounds in cheese, but have been superseded by the much more powerful technique of reverse-phase high-performance liquid chromatography (RP-HPLC).An extensive literature on various aspects of cheese flavor has accumulated and has been reviewed extensively: 57 reviews on various aspects of cheese flavor are cited by Imhof and Bosset (1994). Two general reviews have been compiled by Urbach (1993, 1995). Techniques for studying the volatile fraction of cheese have been reviewed by Imhof and Bosset (1994). More than 200 volatile compounds have been recovered from Cheddar cheese (Aishima and Nakai, 1987; Maarse and Visscher, 1989). The principal compounds are listed in Table I. Most of the water-soluble nonvolatile compounds are produced by glycolysis and especially proteolysis; methods for monitoring these have been reviewed by Fox et al. (1995b) and McSweeney and Fox (1997). About 200 peptides, some of which may have an impact on flavor, have been identified in Cheddar cheese (Singh et a]., 1995, 1997, unpublished data; Mooney and Fox, 1997). The water-soluble fraction of cheese contains considerable concentrations of free amino acids that probably contribute to the background flavor of cheese and serve as substrates for various flavor-generatingreactions. All cheese varieties that have been studied in sufficient detail contain essentially the same range of sapid compounds. Their flavors differ owing to the absolute and relative concentrations of these compounds. In spite of very considerable progress on identification and quantitation of volatile and nonvolatile flavor compounds in cheese, it is not yet possible to fully describe cheese flavor in chemical terms. It is much

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TABLE I VOLATILE FLAVOR COMPOUNDS THATHAVEBEENIDENTIFIEXI IN CHEDDAR CHEESE' Acetaldehyde Acetoin Acetone Acetophenone P-angelicalatone 1,2-Butanediol n-Butanol 2-Butanol Butanone n-Butyl acetate n-Butyl butyrate n-Butyric acid Carbon dioxide p-Cresol y-Decalactone &-Decalactone n-Decanoic acid Diacetyl Diethyl ether

Dimethyl sulfide Dimethyl disulfide Dimethyl trisulfide S-Dodecalatone Ethanol Ethyl acetate 2-Ethyl butanol Ethyl butyrate Ethyl hexanoate 2-Heptanone n-Hexanoic acid n-Hexanol 2-Hexanone Hexanethiol 2-Hexenal Isobutanol Isohexanal Methanethiol Methional

Methyl acetate 2-Methylbutanol 3-Methylbutanol 3-Methyl-2-butanone 3-Methylbutyric acid 2-Nonanone 6-Octalactone n-Octanoic acid 2-Octanol 2 &Pentanediol Z-Pentanol Pentan-2-one n-Propanol Propanal Propenal n-Propyl butyrate Tetrahydro furan Thiophene-2-aldehyde 2 -'hi decanone 2-Undecanone

'Adapted from Urbach (1993).

easier to chemically define flavor defects that are usually due to a single compound or a class of compounds. Cheese flavor is normally assessed by subjective sensory methods. A key task here is establishment or recognition of the flavor characteristic of a variety. No stable reference exists, and personal preferences can be very important. Another major task is the development of a vocabulary of terms with which to describe cheese flavor. Very interesting work on this subject has been published by McEwan et al. (1989) and Muir and Hunter (1992).For various reasons, there has been a longstanding desire to develop objective chemical and physical methods for describing and assessing cheese flavor. In this review we do not intend to review the literature on cheese flavor or the methodology used but to describe the biochemical pathways for the formation of known sapid and aromatic compounds in cheese, compounds believed to contribute to its flavor. Particular attention is focused on the catabolism of amino acids.

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A. RIPENINGAGENTS IN CHEESE Cheese curd is bland, and its characteristic taste and aroma, as well as texture, develop during ripening. The ripening of cheese involves a series of reactions catalyzed by: 1. Living microorganisms (primary and secondary starters, and nonstarter microorganisms). 2. Enzymes secreted by these microorganisms or released from their cells after death and lysis, and enzymes indigenous in milk or added to the milk or curd, especially the coagulant. 3. Chemical reactions, principally involving interactions between and modification of the products of biological and enzymatic reactions. The principal sapid compounds in cheese are produced through glycolysis, lipolysis, and proteolysis, which are now fairly well established. The secondary reactions involved in the evolution of cheese flavor are less well characterized, but several have been elucidated. II. Glycolysis

A. METABOLISM OF LACTOSEBY LACTICACIDBACTERIA Most (-98%) of the lactose in milk is removed in the whey as lactose or lactic acid, and that retained in the cheese curd is metabolized to lactate, partly during curd manufacture and partly during the early stages of ripening, normally by the starter. The pathway for lactose metabolism is characteristic of the type of starter used (see Cogan and Hill, 1993). Lactococcus lactis ssp. lactis and Lc. lactis ssp. cremoris metabolize lactose to L(+) lactic acid. The glucose moiety is metabolized via the Embden-Meyerhof (EM) pathway, while galactose is metabolized via the tagatose pathway. In cheeses where a thermophilic starter is used, lactose is absorbed by S. thermophilus (which grows first as the curd cools) and hydrolyzed by P-galactosidase; the glucose moiety is metabolized via the EM pathway to L-lactate. S. thermophilus is unable to metabolize galactose, which it secretes. When the curd has cooled sufficiently, Lactobacillus spp. grow. The galactose-positive lactobacilli convert galactose via the Leloir pathway to Glu-6-P, which is then metabolized to DL-laCtate via the EM pathway. However, many strains of Lb. delbruekii ssp. lactis and Lb. delbruekii ssp. bulgaricus are galactose-negative. Lactic acid makes a major contribution to the flavor of acid-coagulated cheeses and contributes significantly to the flavor of young ren-

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net-coagulated cheeses. As the sapid compound present at the highest concentration, it probably makes some contribution to the flavor of all cheeses. However, the principal effect of the lactic acid fermentation on cheese quality is indirect: Cheddar cheese with a pH > 5.4 (which usually also has a high moisture content) has a high probability of developing off-flavors, while low pH cheese is crumbly (see Fox et al., 1990). A high pH also favors survival or growth of pathogenic and food-poisoning microorganisms. Starters for some cheeses include Leuconostoc spp., which isomerize the galactose moiety of lactose via the Leloir pathway to Glu-6-P and then metabolize it, and the glucose moiety of lactose via the phosphoketolase pathway to lactic acid, ethanol, and CO,. The lactose in brine- or dry-surface-salted cheeses is usually completely metabolized by the starter before salting, but in Cheddar-type cheeses, which are salted by mixing milled curd with dry salt, the salt concentration may rapidly reach a level that is inhibitory to the starter and residual lactose is then metabolized by salt-tolerant nonstarter lactic acid bacteria (usually mesophilic lactobacilli, but perhaps also micrococci and pediococci), some of which may be heterofermentative, producing lactic acid, ethanol, and CO,. Ethanol may contribute directly to cheese flavor or may be esterified with various fatty acids. The principal esters produced are ethyl hexanoate and ethyl octanoate, which are mainly responsible for the fruity off-flavors sometimes encountered in cheese. The CO, may be sufficient to cause pinholes and cracks in the cheese, generally considered undesirable in Cheddar-type cheese. B. METABOLISM OF LACTICACIDIN CHEESE

Young cheese contains 1.0-1.5% lactic acid, the fate of which depends on the variety (see Fox et al., 1990). In Cheddar, Dutch, and similar varieties, L-lactate is isomerized to DL-lactateby nonstarter lactic acid bacteria (NSLAB; Thomas and Crow, 1983). Racemization of L-lactate is not significant from the flavor viewpoint, but calcium D-lactate,which is less soluble than Ca-L-lactate, may crystallize in cheese, causing undesirable white specks, especially on cut surfaces (see Fox et al., 1990). Lactate can be oxidized by NSLAB in Cheddar, and probably in similar cheeses, to acetate and COz (see Fox et al., 1996). This reaction depends on the availability of 0,, which is determined by the size of the cheese and the oxygen permeability of the packaging material (Thomas, 1987). Acetate, which can also be produced by starter bacteria

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from lactose (Thomas et al., 1985), or citrate (see below), or from amino acids by starter bacteria and lactobacilli (Nakae and Elliott, 1965), is usually present at fairly high concentrations in Cheddar and is considered to contribute to its flavor, although high concentrations may cause off-flavors (see Aston and Dulley, 1982). Thus, the oxidation of lactate to acetate may contribute to Cheddar flavor. The fermentation of lactose and lactate in Swiss-type cheeses has been described comprehensively by Turner et al. (1983). Typically, Emmental cheese contains about 0.4 and 1.2% D- and L-lactate, respectively, at 1 4 days, at which time the sugars have been metabolized completely. On transfer to a warm room, propionibacteria grow and preferentially metabolize L-lactate to propionate, acetate and CO,: SCH3CHOHCOOH + ZCH,CH,COOH Citric acid

Propionic acid

+ CHzCOOH + COZ + HZO Acetic acid

The COz generated is responsible for eye development, a characteristic feature of these varieties. Acetate, and especially propionate, contribute to the flavor of these cheeses. The metabolism of lactate is very extensive in surface mold-ripened varieties, for example, Camembert, Brie, and Carre de 1’Est (see Noomen, 1983; Lenoir, 1984; Karahadian and Lindsay, 1987). After manufacture, these cheeses contain 1.O% lactic acid, produced mainly or exclusively by the mesophilic starter. Secondary organisms quickly colonize and dominate the surface of these cheeses, principally Penicillium caseicolum, and to a lesser extent Geotrichum candidum and yeasts, and perhaps Brevibacterium linens. G. candidum and f? caseicolum rapidly metabolize lactate to C 0 2 and H,O, causing an increase in pH. Deacidification occurs initially at the surface, resulting in a pH gradient from the surface to the center and causing lactate to diffuse outward. When the lactate has been exhausted, E! caseicolum metabolizes proteins, producing NH3, which diffuses inward, further increasing the pH; the pH of mature Camembert may be 7.5 at the surface and 6.5 at the center. The concentration of calcium phosphate at the surface exceeds its solubility at the higher pH and precipitates as a layer of CaJPO,), on the surface, thereby causing a calcium phosphate gradient within the cheese. Reduction of the calcium phosphate concentration in the interior helps to soften the body of the cheese. The elevated pH favors the action of plasmin, which together with residual coagulant, is responsible for proteolysis in this cheese rather than proteinases secreted by the surface microorganisms, which diffuse into the cheese to only a very limited extent, although the products of their action on

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23

surface proteins may diffuse into the cheese. The combined action of increased pH, loss of calcium, and proteolysis are necessary for the characteristic softening of the body of Brie and Camembert. The metabolism of lactate and the concomitant increase in pH probably influence the flavor of these cheeses, which is rather mild and creamy, but their flavor is dominated by the products of other reactions. The ripening of surface smear-ripened cheeses, for example, Limburger, Munster, Tilsit, and Trappist, is characterized by a very complex surface microflora that includes Brevibacterium linens, Br. ammoniagenes, Arthrobacter spp., Coryniforms spp., G. candidum, and various yeasts. The surface of these cheeses is colonized first by yeasts, which catabolize lactic acid, causing an increase in pH. Deacidification is essential for the growth of Brevibacterium spp., which do not grow below pH 5.8, but probably has little direct effect on the flavor of smear-ripened cheeses, which is dominated by the catabolic activity of Brevibacterium spp. A common defect in many cheeses arises from the metabolism of lactate (or glucose) by Clostridium spp. to butyrate, H2, and CO, (see Fox et al., 1996). This reaction leads to late gas blowing and off-flavors in many cheese varieties unless precautions (e.g., good hygiene, addition of NaN03 or lysozyme, bactofugation, or microfiltration) are taken.

c. EFFECTOF LACTOSECONCENTRATION ON CHEESE QUALITY Although the concentration of lactose in milk decreases with advancing lactation, its concentration in bulked factory supplies from cows on a staggered calving pattern is essentially constant. However, when synchronized calving is practiced ( e g , New Zealand, Ireland, and Australia), substantial seasonal changes occur in the concentration of lactose in milk and, consequently, in fresh cheese curd. Variations in the concentration of lactose in cheese curd probably affect the final pH of the cheese, which affects cheese texture, enzyme activity, and perhaps the nonstarter microflora. Cheese flavor is likely to vary owing to variations in the concentration of lactic acid and catabolites therefrom, and due to variations in the metabolic activity of the cheese microflora. The concentration of lactose in cheese curd is affected by certain features of the manufacturing process. As far as we know, the concentration of lactose in cheese curd is not increased intentionally for any variety, but it is advertently increased in curd made from milk concentrated by ultrafiltration (UF),and the concentration of lactose may have to be reduced to an appropriate level by diafiltration. However, the concentration of lactose in the curd for several varieties, including

24

P. F. FOX AND J. M. WALLACE

1.5

1.o

0.5

0.0

0

4

8

12

16

20

24

28

32

36

Time, weeks FIG.1. Metabolism of lactose in Cheddar cheese curds during ripening: control (O), lactose-enriched cheeses: 1 ( A ) , 2 (A), whey-replaced cheese (01, and washed-curd cheese ( 0 ) .(Modified from Waldron, 1997.)

Gouda and Edam, is reduced by replacing part of the whey by warm water. This process, which was probably introduced as a simple method for cooking curds on farmsteads lacking jacketed cheese vats, effectively controls the pH of the cheese. Curds are washed in the washed-curd variant of Cheddar cheese and perhaps in the production of low-fat cheese (to increase moisture content). The effect of variations in the concentration of lactose in cheese curd on the quality of mature cheese has received very little attention. In an attempt to vary the concentration of lactose in Cheddar cheese curd, Huffman and Kristoffersen (1984) added lactose to the curd-whey after cutting the coagulum, but because there is a strong outflow of whey from the curd at that stage due to syneresis of the curd, the increase in lactose concentration within the curd was quite small. In a study by Waldron (1997), the lactose content of Cheddar cheese curd was reduced by removing 35-45% of the whey shortly after cutting the coagulum and replacing it with an equal volume of warm water

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

25

4.6 0

4

8

12

16

20

24

28

32

36

Time, weeks FIG. 2. Changes in the pH of lactose-modified Cheddar cheeses during ripening: control (0),lactose-enriched cheeses 1 ( A ) , 2 (A),whey-replaced cheese (01and , washedcurd cheese (). (Modified from Waldron, 1997.)

or by washing the curd after milling, or increased by adding lactose to the cheese milk up to a level of -8%. The concentration of lactose in the curd at milling ranged from 0.6 to 2.5%. Changes in the concentration of lactose in the cheese during ripening and the pH of the cheese are shown in Figs. 1 and 2. The lactose in the two types of washed-curd cheese was completely metabolized within -2 weeks, but it persisted in the high-lactose cheeses throughout ripening. Not surprisingly, the pH of the cheeses was inversely proportional to the concentration of lactose in the curd. The pH of high-lactose cheeses continued to decrease (to -4.8) throughout ripening, whereas in the washed-curd cheeses the pH increased once the lactose had been exhausted, as is common for many varieties of cheese, probably due to proteolysis and production of NH,. Flavor development was substantially faster in the high-lactose cheeses than in the washed-curd cheeses, although it was considered to be rather harsh, perhaps due to the low pH. The flavor of the low-lactose cheeses was clean and mild. The rate of growth and final number of NSLAB

26

P. F. FOX AND J. M. WALLACE

were not affected by the concentration of lactose, suggesting that NSLAB do not depend on lactose as a growth substrate. They probably utilize amino acids or are physically constrained within the curd, or colony size is restricted by local production of toxic substances. The types of NSLAB in the various cheeses may have varied, but this was not studied. The results of this study suggest that the concentration of lactose in cheese curd has a substantial effect on the quality of Cheddar and probably other cheeses. Replacing some of the whey with water or washing the cheese curd might be considered when a mild, clean flavor is desired. Normal variations in the lactose content of milk from mixedcalving herds are probably not significant but may have a substantial effect when synchronized calving is practiced. The effect may be very substantial when UF-concentrated milk is used due to low levels of curd syneresis. Diafiltration of the cheese milk or washing of the curd would appear to be desirable. Cheese made from milk with a high content of fat and casein may have a reduced lactose content, as may cheese produced from milk, the protein content of which is increased by adding UF retentate. The foregoing discussion indicates that the metabolism of lactose and lactate in cheese is well understood. In quantitative terms, these changes are among the principal metabolic events in most cheese varieties. However, compared to other biochemical changes during cheese ripening, conversion of lactose to lactate may have relatively little direct effect on the flavor of mature cheese, but since it determines its pH, it is of major significance in regulating the various biochemical reactions that occur during ripening. The isomerization of lactate probably has little impact on cheese flavor, but its conversion to propionate and/or acetate is probably significant, and, when it occurs, the metabolism of lactate to butyrate has a major negative effect on cheese quality.

D . CITRATE METABOLISM Bovine milk contains -1.8 g liter-' citrate (-8 mM), about 95% of which is soluble and lost in the whey. Citrate is not metabolized by Lc. lactis or Lc. cremoris, but it is metabolized by Lc. lactis ssp. lactis biovar diacetylactis and Leuconostoc spp. with the production of diacetyl and GO2 (for reviews, see Cogan, 1985; Cogan and Daly, 1987; Cogan and Hill, 1993). It is not metabolized by S . thermophilus or by thermophilic lactobacilli (Hickey et al., 1983), but several species of mesophilic lactobacilli metabolize citrate with the production of diacetyl and formate (Fryer, 1970).

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

27

Production of COz from citrate is responsible for the characteristic eyes in Dutch-type cheese, and for undesirable openness and floating curd in Cheddar and Cottage cheese, respectively. Diacetyl is very significant for the aroma-flavor of cottage cheese, Quarg, and many fermented milks, and also contributes to the flavor of Dutch-type cheeses. Cheddar contains 0.2 to 0.5% (wt/wt) citrate, which is metabolized at a rate dependent on the NSLAB microflora. Diacetyl occurs at a considerable concentration in Cheddar and is generally regarded as a significant contributor to its flavor. Acetate from citrate may also contribute to cheese flavor. Diacetyl can be converted to acetoin, 2,s-butylene glycol, and 2-butanone. The latter is considered to be a major component of Cheddar cheese flavor (see, e.g., Dimos et al., 1996).

I I I . Lipolysis The fat fraction of cheese has a major effect on cheese texture and is important for the perception and development of cheese flavor. Poor development of flavor and texture in low-fat cheeses is a considerable technological problem that limits the market for such products. In addition to serving as a direct (fatty acids) or indirect (methyl ketones, lactones, esters) source of flavor compounds in cheese, fat serves as a solvent for sapid compounds produced from other constituents. It has been reported that a natural milk-fat emulsion yields better cheese than milk fat emulsified in skim milk, suggesting that the fat-water interface plays an important role in the development of cheese flavor; perhaps enzymes in the natural fat globule membrane (see Andrews et al., 19921 are important in flavor development. The fat in cheese can undergo degradation via lipolysis (enzymatic) or oxidation (chemical]. The degree of lipid oxidation in cheese is limited, probably due to the low redox potential of cheese and the presence of natural antioxidants. Many highly flavored compounds are produced via lipid oxidation, but the possible contribution of lipid oxidation to cheese flavor/off-flavor has been largely ignored. Fatty acids released upon lipolysis contribute directly to cheese flavor, especially in hard Italian and mold-ripened varieties and, probably, to a lesser extent in other varieties, especially when extra-mature, provided they are properly balanced by products of proteolysis and other reactions. However, extensive lipolysis is considered undesirable in most cheese varieties: Cheddar, Gouda and Swiss-type cheeses containing even a moderate level of free fatty acids (FFAs) would be

28

P. F. FOX AND J. M. WALLACE TABLE I1 TYPICAL. CUNCENTRATIONS OF FREE FATTY Acros (FFAs) IN SOME CHEESE VARIETFES

Variety

Sapsago Edam Mozzarella Colby Camembert Port Salut Monterey Jack Cheddar

FFA (mg1kg-I)

FFA (mg/kg-’)

Variety

Gjetost Provolone Brick Limburger Goat’s milk Parmesan Romano Blue-mould (US) Roquefort

21 1 356 363 550 681 700 736 1028 ~~

~~~

1658 2118 2150 4187 4558 4993 6754 32230 32453

~~

Date from Woo et a1 (1984)and Woo and Lindsay (1984).

considered rancid. The total concentrations of FFAs in some major cheese varieties are summarized in Table 11. A. LIPASESIN CHEESE Lipases in cheese originate from milk, rennet paste (some varieties), starter, adjunct starter or nonstarter bacteria, or exogenous sources. Milk contains a well-characterized lipoprotein lipase (LPL) that liberates fatty acids from the sn-1 and 3 positions of mono-, di-, and triglycerides and the 1 position of glycerophospholipids (Olivecrona and Bengtsson-Olivecrona, 1991; Olivecrona et al., 1992). In milk fat, highly flavored short-chain acids that are esterified predominantly at the sn-3 position and hence even low LPL activity can have a significant effect on flavor. LPL is associated mainly with the casein micelles and is incorporated into cheese. LPL probably causes significant lipolysis in raw milk cheese but probably contributes little in pasteurized milk cheese. Good-quality rennet extracts are free of lipolytic activity, but the rennet paste used in the manufacture of some Italian varieties (e.g., esterase Romano, Provolone) contains a potent lipase-pregastric (PGE)-that catalyses the extensive lipolysis responsible for the “piccante” flavor characteristic of such varieties. The literature on PGE, also called lingual or oral lipase, was comprehensively reviewed by Nelson et al. (1977) and updated by Fox and Stepaniak (1993).

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

29

PGE is highly specific for short-chain acids esterified at the sn-3 position. Slight differences in specificity between calf, lamb, and kid PGEs permit the manufacture of Italian cheeses with slightly different flavor characteristics. Most other lipases are unsuitable for the manufacture of Italian cheeses because of incorrect specificity, but it has been claimed that certain fungal lipases may be acceptable alternatives (see Fox and Stepaniak, 1993). The use of PGE to accelerate the ripening of other cheese varieties was discussed by Fox (1988-89). L Q C ~ O C O Cspp. C ~ Sare weakly lipolytic, but in the absence of strongly lipolytic microorganisms they probably make a significant contribution to the low level of lipolysis in Cheddar and Dutch varieties. The intracellular esterase-lipase of two Lactococcus strains has been isolated and characterized (Holland and Coolbear, 1996; Chick et al., 1997). Little is known about the genetics of these enzymes. Isolation of lipase/esterasenegative variants of Lactococcus would permit the significance of these enzymes in cheese ripening to be assessed. Both mesophilic and thermophilic lactobacilli possess (mainly) intracellular esterolytic-lipolytic activity (Khalid et al., 1990; Gobbetti et al., 1996). The esterolytic-lipolytic activity in cell homogenates of a number of lactobacilli was characterized by El-Soda et al. (1986), and an intracellular lipase and an intracellular esterase from Lb. plantarum were purified and characterized by Gobbetti et al. (1997a,b). Micrococcus spp., which constitute part of the nonstarter microflora of cheese, especially the surface microflora, produce lipases that may contribute to lipolysis during ripening (Bhowmik and Marth, 1990a,b). The nonstarter microflora of cheese may also include Pediococcus spp. that are weakly esterolytic and lipolytic (Tzanetakis and LitopoulouTzanetaki, 1989; Bhowmik and Marth, 1989). Propionibacterium shermanii possesses a lipase(s) (Paulsen et al., 1980) that contributes to lipolysis in Swiss varieties. An intracellular lipase of I! shermanii was partially characterized by Oterholm et al. (1970). Brevibacterium linens, a major component of the surface of smear-ripened cheeses, possesses intracellular lipases and esterases (Smhaug and Ordal, 1974; Foissy, 1974). The intracellular esterase of Br. linens ATCC 91 74 has also been purified and characterized (Rattray and Fox, 1997a). Very extensive lipolysis occurs in mold-ripened cheese, particularly blue mold varieties in which up to 25% of the total FFAs may be liberated (see Gripon, 1987, 1993). However, the impact of FFAs on the flavor of blue mold-ripened cheeses is less than for hard Italian varieties, possibly due to neutralization as the pH increases during ripening and the dominant influence of methyl ketones on the flavor of blue

30

I? F. FOX AND 1. M. WALLACE

mold cheeses. Lipolysis in mold-ripened varieties is due primarily to the lipases of Penicillium roqueforti or I! camemberti, which secrete potent extracellular lipases that are well characterized (see Kinsella and Hwang, 1976; Gripon, 1987, 1993). Psychrotrophs, which dominate the microflora of refrigerated milk, are potentially an important source of potent lipases in cheese if their numbers exceed lo7 ml-I (Cousins et al., 1977). Many psychrotrophic lipases are heat stable and may cause rancidity in cheese over a long ripening time (Mottar, 1989; Smhaug and Stepaniak, 1997). Unlike psychrotrophic proteinases, which are largely lost in the whey, psychrotrophic lipases adsorb onto the fat globules and are concentrated in the cheese.

B. CATABOLISM OF FREEFATTY ACIDS The flavor and aroma of blue mold cheese are dominated by saturated alkan-2-ones (n-methyl ketones). A homologous series of alkan-Zones with odd-numbered carbon chains from C3 to CI7, and some with even-numbered carbon chains, occurs in these cheeses: heptan-2-one, nonan-&one, and undecan-2-one are dominant during most of the ripening period (Dartley and Kinsella, 1971). Some reduction of alkan-2ones to alkan-2-01s occurs during ripening, which has an undesirable effect on flavor. The metabolism of fatty acids in cheese by Penicillium spp. involves four main steps: (1)release of fatty acids by lipases, (2) oxidation to a-ketoacids, (3) decarboxylation to alkan-2-ones with one less carbon atom, and (4) reduction of alkan-Zones to the corresponding alkan-2-01; step four is reversible under aerobic conditions. Alkan-2-ones can also be formed by mold from the ketoacids naturally present at low concentrations in milk fat (-1%of total fatty acids) or by the oxidation of monounsaturated acids. The rate of alkan-Zone production in cheese is affected by temperature, pH, the physiological state of the mold, and the concentration of fatty acids (Adda et al., 1982). Both resting spores and mycelia are capable of producing alkan-Zones at a rate that does not depend directly on the concentrations of FFA precursors. Indeed, high concentrations of FFAs are toxic to I! roqueforti (Fan et al., 1976). Lactones are formed by the intramolecular esterification of hydroxyacids through the loss of water to form a ring structure. a- and 0-lactones are very reactive and unstable, but y- and S-lactones are stable and occur in cheese. Lactones possess a strong aroma, which, although not cheese-like, may contribute to overall cheese flavor. y- and S-lac-

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

31

tones may be formed spontaneously from the corresponding y- and 6-hydroxyacids following their release from triglycerides; their concentration in cheese should therefore correlate with the extent of lipolysis. Lactones may also be formed from ketoacids after reduction (Wong et al., 1975). The formation and significance of lactones in cheese have received relatively little attention.

IV. Proteolysis

Proteolysis is the most complex and perhaps the most important biochemical event during the maturation of most cheese varieties. The extent of proteolysis varies from very limited (e.g., Mozzarella] to very extensive (e.g., blue mold varieties), and the products range from large polypeptides, comparable in size to intact caseins, through a range of medium and small peptides, to free amino acids. Proteolysis is mainly responsible for softening of the texture of cheese during the early stages of ripening and influences the development of cheese flavor via the formation of amino acids and peptides that make a direct, although probably limited, contribution to flavor. Amino acids also serve as substrates for the formation of numerous flavor compounds. Proteolysis also affects the mouthfeel of cheese and the release of flavor compounds from cheese during mastication. Various methods have been developed for quantifying the extent and pattern of proteolysis in cheese. They are reviewed by Fox et al. (1995b) and McSweeney and Fox (1997). A. CONTRIBUTION OF INDIVIDUAL PROTEOLYTIC AGENTS Proteolytic enzymes in cheese originate from milk, coagulant, starter, secondary starter, and nonstarter microorganisms. Information on the relative importance of individual proteolytic agents has been obtained from studies on cheeses with controlled microflora, in which the coagulant, plasmin, starter, and nonstarter bacteria, in various combinations, have been excluded or inactivated. These studies, which have been summarized by Fox et (11.(19931, indicate that rennet and plasmin are mainly responsible for the initial hydrolysis of as1-and p-caseins, respectively, and for the production of most of the water- or pH 4.6-soluble N in cheese. Although the lactic acid bacteria (Lactococcus, Lactobacillus, Streptococcus) are weakly proteolytic, they possess a very comprehensive

32

P. F. FOX AND J. M. WALLACE

proteolytic system that has been studied extensively and reviewed (see Fox and McSweeney, 1996; Kunji et al., 1996). The proteolytic system of Lactococcus has been particularly well studied. They possess a cell wall-associated proteinase, several intracellular proteinases, at least two intracellular oligoendopeptidases (PepO, PepF), at least three aminopeptidases (PepN, PepA, PepC), a dipeptidylaminopeptidase (PepX), a tripeptidase, a general dipeptidase, and proline-specific dipeptidases. However, Lactococcus spp. and most Lactobacillus spp. lack a carboxypeptidase. They also possess a number of peptide and amino acid transport systems. This comprehensive proteolytic system is necessary to enable the lactic acid bacteria to grow to the requisite high numbers ( 1 0 ~ - 1 0cfu/ml) ~~ in milk, which contains only low levels of small peptides and free amino acids. The proteolytic system of Lactobacillus spp. has been studied less thoroughly than that of Lactococcus, but the two systems appear to be generally similar. The cell wall-associated proteinase contributes to the formation of small peptides in cheese, probably by hydrolyzing larger peptides produced from a,,-casein by chymosin or from p-casein by plasmin. The aminopeptidases, dipeptidases, and tripeptidases, which are intracellular, are responsible for the release of free amino acids after the starter cells have died and lysed. The proteolytic system of NSLAB in Cheddar, and probably in similar cheeses, appears to supplement the proteolytic action of the starter, producing generally similar peptides and amino acids (Lynch et al., 1997). The proteinases and peptidases of P roqueforti and l? comemberfi make a major contribution to proteolysis in mold-ripened cheeses (see Gripon, 1993). Br. linens secretes an extracellular proteinase and an aminopeptidase and possesses a number of intracellular peptidases, which may be released on cell lysis (see Rattray et al., 1995; Rattray and Fox, 1997b). These enzymes probably contribute to proteolysis, including the production of free amino acids, in the surface of smear-ripened cheeses. Propionibacterium shermanii, the characteristic secondary microorganism of Swiss-type cheeses, is weakly proteolytic but has high peptidase activity, especially proline-specific peptidases, and contributes significantly to proteolysis in Swiss cheese. The proteolytic specificity of chymosin, plasmin, and the cell wallassociated proteinase of several lactococcal strains on a,,-, as2-, p-, and K-caseins has been determined (see Fox et al., 1995a, 1996; Fox and McSweeney, 1996). The specificity of the extracellular proteinases of €? roqueforti, €? camemberti, and Br. linens on as1-and p-caseins has been determined (see Gripon, 1993; Rattray et al., 1996, 1997).

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

33

B. PROTEOLYSIS IN CHEESE The extent and type of proteolysis in a number of the principal cheese varieties has been characterized (see Fox and McSweeney, 1996). Proteolysis in Cheddar is very well characterized and can be summarized as shown in Figs. 3, 4, and 5. a,,-casein is completely hydrolyzed by chymosin, typically within about 3 months, at the Phe2,-Phez4 bond. The larger peptide, a,,-CN f24-199, is further hydrolyzed extensively by chymosin at the Leulol-Lys,oz bond and to a lesser extent at Phe3,Gly33, Leug8-Leu,,, and Leulog-Glullo, probably not in sequence. Some of the Lyslo3-Tyrlo4 and Lyslo5-Vallos bonds are also hydrolyzed by plasmin. The large C-terminal peptides, a,,-CN f24-199,102-199,110199, 99-199, 33-199, 104-199, and 106-199, are detectable in the water-insoluble fraction (Fig. 6). The complementary N-terminal peptides (e.g., a,,-CN f24-101, 24-98, 24-109) have not yet been identified in the water-insoluble fraction, but since these are highly phosphorylated, they may be in the water-soluble extract. The large peptides, which have not yet been characterized, are also hydrolyzed by lactococcal CEP and possibly by lactococcal PepO and several small peptides produced therefrom and are present in the UF permeate or retentate of the water-soluble extract (WSE) (Fig. 3). The peptide a,,-CN fl-23 is hydrolyzed rapidly by the lactococcal CEP at bonds Glng-Glyl0, Gln13-Glu14, Glu14-Val15, and Leul6-Asnl7, and probably at other sites, depending on the specificity of the CEP. The peptides aSl-CNh-9,1-13, and 1-14 accumulate and dominate the UF permeate or 70% ethanol-soluble fraction of the water-soluble extract of Cheddar. According to Exterkate and Alting (1995), these peptides do not affect the flavor of cheese. The complementary C-terminal peptides (i.e., a,,-CN f10-23, f14-23, f15-23, and f17-23) have been identified in the UF permeate, and some of them have been partially hydrolyzed by an aminopeptidase, probably PepN, releasing amino acids (Fig. 3). Peptides from the N-terminal region of a,,-CN f24-199 are also hydrolyzed by lactococcal CEP, or possibly PepO or PepF. The N-terminals of some of these peptides do not correspond to known CEP cleavage sites, suggesting the action of aminopeptidase, PepO, PepF, or NSLAB enzymes. In Cheddar and most other cheeses, p-casein is much more resistant to hydrolysis than a,,-casein: only about 50% is hydrolyzed within 6 months. It is hydrolyzed mainly by plasmin, at Lys28-Lys,g, LyslOsGlnlos,and Lyslo7-Glulo8,yielding yl-,f-, and f-caseins (P-CN f29-209, 106-209, and 108-209, respectively), which are clearly evident in the

W ip

1

-19

93 -lY 14-

7

DF Rctcotatc I* -35 2s-u

IS-35

116-124

U n

IU

-am

115-121

15-Y 14

110-

3 4

!’

Cleavage sites uf crll-euvelupc pruteinas uf starter L.Clueoccur rpp.

14 1 ,

IW57

I

I

Y3

Zb-31

1-9

-

16-31

1) IJ

2J-m

-

?

IBS 1.1

*(-?

y ? 4l-?

17-7 18 - ?

!’

DF Pernienle

?

Jl-?

Water-hsoluble Fraction .................

2, .......... 21 .- ~

_ _ 33 .........

,

,

”......._..... ..

7y

,

-

199

.- . ~ . -. . ~ . . .................. 1...... 199 lu.-.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . lu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - IY, IIO.-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - I99 121 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 I*y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I99 .......... 1% ?

FIG.3. ~ - C a s e i n - d e r i v e dpeptides isolated from the water-insoluble (----I, the diafiltrate (DF), retentate (-1, and the DF permeate (-1 of the water-soluble fraction of a mature Cheddar cheese. (Modified from Singh et ol., 1997, and Mooney, 1997.)

I

DF Permeate

7

191175176-1

197

lU2 ZOC-207

Cleavage sites of Plasmin

61-71

61-70

2s

DF retentate

-=

2 7 -

41

FIG.4. aS2-Casein-derivedpeptides isolated from the DF retentate (-), of mature Cheddar cheese. (Data from Singh et a]., 1997.)

and the DF permeate (-)

of the water-soluble fraction

93

51 51 51

w

93

Q,

92

n-n

7 3 n

I

.

[-)

Zm

In .

109 W

FIG.5. p-Casein-derived peptides isolated from the water-insoluble fiaction (----I, the DF retentate (-1, and the D F permeate of the water-soluble fraction of a mature Cheddar cheese. (Modified from Singh et al., 1997, and Mooney, 1997.)

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

37

FIG.6. Urea polyacrylamide gel electrophoretogram of sodium caseinate (C) and the water-insoluble nitrogen fraction of Cheddar cheese after manufacture (slot 2) and ripening for 1, 2, 3, 4, 5, 6 , 8, 10, 1 2 , 14, 16, 18, and 20 weeks (slots 3-14, respectively). Peptides isolated from the 20-week sample were identified as follows: 1. P-CN f106-149, 2. P-CN flO6-128, 3. P-CN flO6-209 (y),P-CN f29-209 (r'),5. P-CN flO8-209 (y),6. 0-CN, 7. P-CN f1-187/192 (P-I-CN),8. P-CN fI-*, 9. asI-CN f99-199,lO. CX~I-CN f80-*, 11. asl-CN, 12. as1-CN f102-199, 13. asl-CN f24-199 (USi-I-CN), 14. as1-CN 33-*, 15. asl-CN f121199, 16. asl-CN fl29-199, 17. CXSI-CNf60-*, 18. asl-CN f110-199, 19. USI-CN f70-156. * = peptide not identified completely. (Modified from Mooney, 1997.)

water-insoluble fraction of Cheddar and most other varieties (Fig. 7), and proteose peptone 8 fast (P-CN fl-ZS), 8 slow (P-CN f29-105/107), and 5 (P-CN fl-105/107), which are present in the UF retentate or 70% ethanol-insoluble fraction of the WSE. Most of the peptides that have been identified in the UF retentate (or 70% ethanol-insoluble fraction) of the WSE are produced from p-casein by the action of lactococcal CEP, probably on proteose peptones rather than on intact p-casein, since none of the identified peptides contain a plasmin cleavage site (Fig. 5). The N-terminal of many of the identified peptides corresponds to the carboxyl residue of a plasmin or CEP cleavage site, but several appear to be 1 , 2 , or 3 residues short, indicating

38

P. F. FOX AND J. M. WALLACE

FIG.7 . Urea-PAGE of the water-insoluble fraction of a selection of cheese varieties. Lane 1 = Na-caseinate; lane 2 = Cheddar 1; lane 3 = Cheddar 2; lane 4 = Cheddar 3; lane 5 = Emmental 1; lane 6 = Emmental 2; lane 7 = Emmental 3; lane 7 = Maasdam; lane 9 = Jarlsberg; lane 10 = Edam 1; lane 11 = Edam 2; lane 1 2 = Edam 3; lane 13 = Gouda 1; lane 14 = Gouda 2; lane 15 = Gouda 3. (Modified from McGoldrick, 1996.)

aminopeptidase activity. The C-terminal of several peptides does not correspond to reported CEP cleavage sites, suggesting carboxypeptidase activity (which has not been reported in Lactococcus spp.) or unknown CEP cleavage sites, or the action of other proteinases, for example, from NSLAB or oligopeptidases (PepO, PepF). Thus, the large water-insoluble peptides in Cheddar are the C-terminal segments of asl-casein produced mainly by chymosin or of p-casein (y-CNs)produced by plasmin. Para-K-casein (K-CN fl-105) is very resistant to proteolysis and is not hydrolyzed during ripening. The concentration of a,,-casein appears to decrease during ripening, but its fate is

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

39

unclear. No large peptide derived from a,,-casein has yet been reported, and only eight small as2-derived peptides have been identified in the WSE (Fig. 4). Perhaps a,,-CN, which represents only -10% of total casein, is hydrolyzed rather nonspecifically to several peptides, none of which are present at a high concentration, and hence have been overlooked. The small water-soluble peptides appear to be produced from chymosin- or plasmin-produced peptides by lactococcal CEP and perhaps endopeptidases. Some of the small peptides are hydrolyzed by aminopeptidases. Although NSLAB dominate the viable microflora in Cheddar for most of the ripening period, they appear principally to supplement the peptidolytic activity of the starter, especially in the production of amino acids (Lynch et al., 1997). The accelerated maturation of cheeses with added lactobacilli appears to be closely related to the concentration of , Ardo and free amino acids (Lloyd et al., 1980; Hickey et ~ l . 1983; Pettersson, 1988; Ardo et al., 1989; Puchades et al., 1989; Broome et al., 1990; Lee et al., 1990a,b;Lynch et al., 1997). Although most lactobacilli were found to increase the concentration of amino acids and the intensity of cheese flavor, the flavor was found to be atypical when cheese milk was inoculated with Lb. helveticus (Lloyd et al., 1980), Lb. brevis (Puchades et al., 1989),or Lb. casei MIL2A (Broome et al., 1990). Proteolysis in Cheddar- and Dutch-type cheeses, and in many other varieties, is generally similar (Fig. 7). The high-cooked varieties, for example, Emmental and Parmesan, differ, partly because chymosin is extensively or totally inactivated during cooking while plasmin activity is increased, and partly because the thermophilic starter lactobacilli are more peptidolytic than Loctococcus spp. In addition, Swiss-type cheeses contain Propionibacterium, which has very high proline-specific peptidase activity. Commercially mature Emmental undergoes relatively little primary proteolysis: only -50% of the aSl-casein is hydrolyzed at Phe,,-Phe,, (by chymosin) and/or Lys103-Tyr104 and Lyslo5-Vallo, (by plasmin); the Leulol-Lysloz bond is not hydrolyzed. Although primary proteolysis is relatively limited in Parmesan, very extensive peptidolysis occurs; most of the WSE occurs as very small peptides or free amino acids.

c. FREEAMINOACIDSIN CHEESE The final products of proteolysis are free amino acids, the concentrations of which have been used as indices of ripening for many years

40

P. F. FOX AND J. M. WALLACE

(Harper and Swanson, 1949; Kosikowski, 1951; Law, 1981; Hickey et al., 1983; Wood et al., 1985; Amantea et al., 1986; Puchades et al., 1989; Wilkinson, 1993; Resmini et al., 1993). Resmini et a]. (1993) reported that the concentration of free amino acids in Grana Padano cheese correlated with flavor intensity but not with ripening time. However, others reported that amino acids appeared to have no effect on cheese flavor (Dacre, 1953; Mabbit et al., 1955; Law, 1966; Law and Sharpe, 1977). As discussed in Section IV.B, ripening and flavor development appear to be accelerated when high levels of free amino acids are produced early in the ripening process. Although catabolic products of amino acid degradation are thought to be mainly responsible for Cheddar flavor, small peptides and free amino acids contribute to the flavor of most cheese varieties (Urbach, 1995), for example, “brothy,” “nutty,” and “sweet” tastes (Biede and Hammond, 1979). The flavor characteristics and flavor threshold of free amino acids are summarized in Table 111. High levels of proline contribute to sweetness in Swiss cheese (Reinbold, 1973; Langsrud and Reinbold, 1973; Lloyd et al., 1978, 1980) and to a sweet Swiss cheeselike flavor in experimental Cheddar (Lloyd et al., 1980). Arginine has been associated with an unpleasant bittersweet taste (Puchades et al., 1989).Free amino acids can also contribute to bitterness in cheese (Ney, 1971). About 20 to 30% of the total N in a mature Cheddar cheese is soluble in water, while about 2 to 5 % is soluble in 5% phosphotungstic acid (PTA). The levels of peptides and free amino acids soluble in 5% PTA in cheese are considered to be reliable indicators of the rate of flavor development (Aston et al., 1983; Pham and Nakai, 1984; Amantea et al., 1986; Ardo and Pettersson, 1988); however, the composition of the amino acid fraction and the relative proportions of individual amino acids are thought to be most important for the development of a typical characteristic flavor (Broome et al., 1990). The release of specific amino acids, particularly Glu, Met, and Leu, coincides with the development of Cheddar cheese flavor (Broome et a]., 1990; Marsili, 1985). Leu and Met are considered to be major contributors to cheesy flavor in the water-soluble extract of Cheddar (Kowalewska el al., 1985; Marsili, 1985; Aston and Creamer, 1986). The concentration of free amino acids in cheese at any stage of ripening is the net result of the liberation of amino acids from casein and their transformation to catabolic products. The concentration of individual amino acids in a number of cheese varieties are compiled in Table IV. Comparison of free amino acid profiles in different varieties is limited for a number of reasons. Cheeses are ripened at different rates,

41

FORMATION OF FLAVOR COMPOUNDS IN CHEESE TABLE I11

TASTEDESCFXPTORAND THRESHOLD VALUESOF AMINOACIDS'

Q" Amino acid

Gly Ser Thr His Asp Glu

(cal mol-'I

0

-300

Taste threshold (mg 100 ml-')

Concentration'in Cheddar (wg g-'1

Perceptiand

Taste Sweet

130

371

***

150

1210

**I

***

400

260

649

500

20

436

0

3

606

0

5

5075

750

50

1737

A% Ala Met

500

60

337

1300

30

869

LYs

1500

50

2330

Val Leu Pro Phe

1500

40

2022

Salt

Sour

Bitter

* ***

** ***

**

*** ***

***

**

** ***

1800

190

4610

2600

300

389

2500

90

2400

*** *** ***

607

***

Tyr

2300

Ile

2950

90

466

3li.p

3400

90

-

Umami

***

*** ***

"Adapted from O'Callaghan (1994). bHydrophobicity values of Bigelow and Channon (1976). Wilkinson (1992); 6-month-old Cheddar; total concentration = 24.1 mg g-' cheese. dAmino acids in Cheddar are deemed to be perceived if their concentration in a water extract (50 g cheese containing 37% moisture to 100 ml water) is greater than their threshold concentration.

even within a single variety. The age of the cheeses in Table IV vary, and in some cases are unknown. Different analytical techniques lead to variations in results. The rate of amino acid degradation differs between and, to a lesser extent, within varieties due to different manufacturing and ripening conditions and variations in cheese microflora. Without monitoring protein-bound as well as free amino acids, no conclusions as to the exact concentrations of amino acids released may be drawn since some of the free amino acids may have been degraded prior to analysis. The relative proportions of individual amino acids appear to be similar in many varieties. Leu, Glu, and Lys are the principal amino acids in Cheddar (Wood et al., 1985). Glu has been described as an important contributor to Cheddar flavor by many authors (Harper and Wang, 1981; Marsili, 1985; Nsar and Younis, 1986; Broome et a]., 1990).

42

P. F. FOX AND J. M. WALLACE TABLE IV

CONCENTRATION (mg kg-' CHEESE) OF FREEAMINOACIDS AND THEIR RELATIVI?.PROPORTIONS (% TOTAL FREE AMINO ACIDS) I N DWI%RENT CHEESE VARIETIES Chnese [Ripening time]

(:hedrlm (1)

Cheddar (2)

I8 niol

19 mol

mg/ kg

'&of FAA

CYS

44.7

029

Asp

1532 8

9.94

Amino acid

1.99

-

Thr

-

SW

41fi 3

2.7

GlU

:i144 1

20.39

Pro

336.2

2.18

GlY

30fi.n~

mg/ kg

Edam (3)

% of FAA

mg/

Gouda (4)

HnrrgArd (5) [.I4wkl

Emmental (6) [fi mol

mg/

mg/ kg

% of FAA

% of FAA

mg/

%of

kg

kg

FAA

-

-

0.65

20.5

0.9

91.7

1.4

1.4

1fifi.S

0.9

1.11

144.5

6.0

272.9

4.2

2.3

688.5

3.7

1.85

71.1

3.0

167 3

2.fi

2.7

548.9

2.9

6.21

351.7

1 2 3!

992.1

15.1

14.3

26no.s

14.3

i5y.n

54

345.6

5.3

4.8

2535.2

13.5

2.32

34.ti

1 2

108.G

1.6

2.7

430.8

23

-

~

k

nf FAA

Oh

-

Ala

3Sfi.2

2.31

-

3.67

68.6

24

iun.9

2.8

2.7

568.2

3.0

Val

1096.4

7.11

-

1 5 26

167.4

6.8

47fi.s

7.3

7.0

1561.5

8.4

Met

434.8

2.82

-

8.14

60.3

2.3

176.1

2.7

1.n

502.7

27

1Ie

-

8 46

48 1

2.4

200.5

3.1

2.3

1051 1

5.G

26.90

428.0

17.5

1056.9

16.2

14 3

17949

9.6

89.2

3.4

165.3

2.5

2.3

2859

1.5

7.56

291.9

11.6

645.9

9.9

56

1279.1

fi.8

49.7

1.7

143.6

2.2

3.8

RGR.2

4.6

11 00

245.5

9.1

fi71.4

10.3

12 LJ

22198

11.9

0 00

130.5

4.2

167.3

2.6

11 7

192

0.1

~

i.PI1

2774.1

'Tyr I'hn

464.1

3.01

-

1472.6

9.55

-

17.99

IIis

-

-

LYS

1127.2

7.31

Arg

1096.4

7.11

Total

~

~

~

~

258'~

05211

Asrr Gln

7.7 18616

'The concentrations of amino acids were either given by, or calculated from tho results of 1: Wood et a]. (19851; 2: Puchades ot a]. (1989); 3,4,6: Antila and Antila (1968);5: Ardo and Gripon (1995).

High levels of Asp and Met were also observed by these authors. Comparatively low levels of Glu (as a percentage of total FAAs) were found in Cheddar by Puchades et al. (1989), who found that Met represented >8% of total FAAs; in contrast, Wood et ~ l(1985) . found that Met was only 2.8% of FAAs. According to Wood et al. (19851, -10% of total FAAs in Cheddar is Asp, while Puchades et al. (1989) reported a value of only 0.65%.Large variations within single varieties have been reported for Emmental, Gruyere, Parmagiano Reggiano, Camembert, Stilton, Danablue, Cabrales, and Mahon (Table IV). There are, however, clear similarities within, and differences between, different cheese varieties. High concentrations of free amino acids in Parmesan are thought to be due to high numbers of lactobacilli and a long ripening time. High

43

FORMATION OF FLAVOR COMPOUNDS IN CHEESE TABLE IV continued Cheese (Ripening time1 Gruyere (7)

Amino acid

mgi kg

70of FAA

Gruyhrr (HI

Appenznller (9)

mgl kg

B of FAA

mgi kg

%of FAA

23

Grana (101 Padano [60samples 10-20 wkl

mgl kg

*A of FAA

Parmigino (111 Parinigino (121 Reggiano Reggiano [mean of 60 cheeses1 mgl

kg

-

%of FAA

mgl kg

“A of FAA

-

115.0

0.3

3.1

3241.9

4.3

2231.9

fi.3

2.4

3.2

4033.3

5.3

1031.3

3.2

0.76

4.4

4459.5

5.9

1580.5

5.0

20.42

18.0

14489.0

19.0

7580.2

21.4

3299.3

9.87

9.5

-

-

3649.6

10.3

74Y.O

2.2

2.4

2115.6

2.8

667.3

1.9

2.65

925.65

2.8

2.7

2260.2

3.0

1028.2

2.9

2381.5

7.3

3594.4

10.8

7.2

6011.9

7.9

2330.9

6.6

868.3

2.7

922.0

2.8

2.5

2351.5

3.1

970.7

2.7

6.1

1901.8

5.97

1YY6.3

6.0

6.1

5205.2

6.H

1762.5

5.0

1778.7

9.1

3765.6

12.5

4176.1

12.5

9.1

7290.4

9.6

2905.6

9.1

321.3

1.6

992.9

2.7

884.2

2.7

2.7

2054.7

2.7

1963.7

5.5

1366 7

7.0

2839.4

7.6

2548.7

7.6

5.1

4314.9

5.7

1753.0

4.9

CYS

-

-

ASP

328.9

1.7

834.8

2.6

774 6

Thr

7R1.2

4.0

1017.2

3.1

1317.1

Snr

446.1

2.2

979.4

3.0

253.2

GI”

2963.5

15.2

6415.9

19.7

6823.4

Pro

2817.3

14.4

3546.9

10.9

Gly

493.0

2.5

716.0

2.2

Ala

597.6

3.1

865.1

Val

1817.9

8.3

Met Ile

532.1

2.7

1189.2

Leu TYr Phe His

680.6

3.4

1276.8

3.2

1081.3

3.2

3.3

-

-

1034.5

2.9

LYS

2227.8

11.3

4038.7

13.7

4573.7

13.7

12.2

10091

13.3

2876.9

8.1

‘4%

21.3

0.1

158.5

0

0

0

-

791.4

1.0

977.1

2.8

Tulal

19342

32598

33419

76100

34459

The concentrations of amino acids were either given by, or calculated from the results of 7: Antila and Antila (1968); 8: Lavanchy and Buhlmann (1983);9: Lavanchy et 01. (1979); 10: Resmini et al. (1993); 11: Resmini et al. (1988); 12:Mariani et a1. (1993).

concentrations of free amino acids are also present in mold-ripened and Swiss cheeses. Of the cheeses tabulated, Edam and Gouda contain the lowest levels of free amino acids. However, an Edam-type cheese made from goat’s milk contained high levels of FAAs, as did another goat’s milk cheese, Rumelia (Baltajieva et al., 1985). Resmini et al. (1993) attempted to “fingerprint” cheeses using chemometric models based on free amino acid profiles. These models allowed Parmigiano Reggiano, Grana Padano, and Fontina to be distinguished from each other and from similar cheeses. In another comparative study, Engels and Visser (1994) found that the amino acid profiles for Cheddar, Edam, Gouda, Gruyere, Maasdam, Parmesan, and Proosdij cheeses were

44

€? F. FOX AND J. M. WALLACE

TABLE IV

continued Cheese [Kipening time] Danish 1131 Camembert

Camnm- 114) hrt

Stil- (15) ton

Stik- (16) ton IMarketl

170 days]

Amino acid

mgl kg

YO of FAA

rngl kg

% uf FAA

mgl

%of

mgf

kg

FAA

kg

Stil- (17) tnn

of FAA

mgl kg

%of FAA

Oh

Dana- (18) blue [Market] rngl

kg

"h of FAA

-

fi.4

0.1

-

-

1020

10.4

-

5.9

99.7

4.0

5390

28

626.77

2.2

340

3.5

1098.1

Thr

-

2.5

134.8

6.9

751.6

4.0

837 5

3.5

1811

1.8

1526.6

5.4

Ser

-

3.2

56.2

27

761.0

4.0

621.4

2.6

57u

58

896.6

3.2

Glu Pro

-

22.5

566.6

20.5

1750.0

8,2

4238.4

175

1090

11 1

in302

6.5

3.1

192.3

6.6

1446.3

7.6

1267.2

5.2

510

52

332.0

12

G~Y

-

1.2

36.9

1.3

216.4

1.1

650.6

2.7

140

1.4

485.2

1.7

Ale

-

1,8

93.8

3.3

876.9

4G

793.8

3.3

350

357

13194

47

VAI

-

5.8

169.1

5.7

1664.6

8.8

2687.4

in 7

fiiu

6.22

3353.~~ 11s

cys Asp

~

~

3.9

Met

-

3.6

65.6

3.1

603.6

3.2

929.7

3.8

410

4.2

1679.R

Ile

-

5.8

134.2

4.2

848.4

4.5

1716.3

7.1

310

3.2

2434.G

8.6

LCU

-

11.8

317.6

11.5

3015.9

15.9

2920.3

12.0

1220

12.5

3708.6

13.1

TLr

-

5.3

160.1

5.9

1495.6

7.9

1733.2

7.1

260

2.7

2340.9

17.3

PllB

-

6.4

241.8

8.7

1776.5

9.4

2386.2

9.8

610

6.2

2fi89.9

9.5

2.6

175.1

6.6

6337.7

3.4

463.7

1.9

490

5

996.0

3.5

93

209.1

6.5

1975.8

10.4

2456.7

10.1

1560

15.9

2760.9

9.7

36.2

1.5

616.9

3.3

279.2

1.2

130

1.3

919.3

3.2

His

4 s

*u Tulal

~

~

~

1.4

2790

18980

24275

YB00

5.9

28375

The concentrations of amino acids were either given by, or calculated from the results of 14: Antila and Antila (1968); 13,17:Ismail and Hansen (1972); 14: Antila and Antila (1968); 15,16,18:Madkor et a]. (1987); 17:Ismail and Hansen (1072); 17: Zarmpoutis (1995).

similar, with Glu, Leu, and Phe being the principal amino acids; Val, Pro and Lys were also quite abundant in these varieties. Maasdam, Cheddar, Gouda, and Edam contained similar concentrations of free amino acids in the UF permeate (MW < 500 Da), but higher concentrations were found in Gruyere, Proosdij, and Parmesan cheeses. The authors concluded that flavor and the concentration of free amino acids could not be correlated since Cheddar, Maasdam, Gouda, and Edam have very different flavors, although the concentration and relative proportions of free amino acids were generally similar. Although the concentration of free amino acids depends on the cheese variety and the dairy plant where the cheese is manufactured, they generally increase during ripening, with the exception of Arg, the

45

FORMATION OF FLAVOR COMPOUNDS IN CHEESE TABLE IV continued Cheese [Ripening time] Dana- (20) blue 18.3 mol

Dana- 1191 blue

-____ Amino acid

mg/ kg

%of FAA

mgl kg

Gorgon- (21) zola

Cashel (22) (Blue1

~

%of FAA

mg/

kg

~ % of FAA

Chetwynd (23) Gamonedo (24) (Blue) (Blue) 190 days1 _ _

-

kg

%of FAA

mg/ kg

%of FAA

mg/

mg/ kg

% of FAA

CYS

1160

9.48

-

-

1380

5.3

740

13.7

590

10.2

-

ASP

300

2.5

-

2.9

1020

3.9

140

2.6

190

3.3

3.7 6.4+gly

Thr

190

1.6

-

3.8

530

2.0

80

1.5

100

17

Ser

1020

8.3

-

3.5

1570

6.0

330

6. 1

390

68

4.2

Glu Pro

1730

14.2

-

16.1

3940

15.1

620

11.5

850

14.7

18.5

2.77

530

4.3

2320

1.2

160

3.0

160

160

1.3

-

10.3

G~Y

1.6

390

1.5

70

1.3

100

1.7

+Thr

Ala

340

2.R

-

5.8

1140

4.4

120

2.2

150

2.6

6.2

Val

610

5.0

-

6.1

2220

8.5

350

6.5

360

6.2

7.8

Met

500

4.1

-

37

760

3.0

180

33

200

3.5

2.5

Ile Leu

300

2.5

-

6.5

1300

5.0

2 70

3.1

220

3.8

6.1

1530

12.5

-

10.1

2910

11.2

690

12.8

650

11.3

11.5

5 r

520

4.3

3.9

650

3.3

290

5.4

270

4.7

7.4

Phe

680

5.6

6.0

1590

6.1

320

5.9

310

5.4

5.9

His

filO

5.0

1.5

800

3.1

240

4.4

270

4.7

3.5

LYS

1540

12.6

-

7.5

3050

11.7

650

12.0

960

16.6

4.5

510

4.2

-

1.5

280

1.1

260

4.8

-

Told

12.230

~

~

26070

5410

10

5770

The concentrations of amino acids were either given by, or calculated from the results of 19,21, 22, 23: Zarmpoutis (1995): 20: Isrnail and Hansen (1972); 24: Gonzalez de Llano et 01. (1991).

levels of which are reported to decrease during the later stages of ripening (Puchades et a]., 1989; Broome et a]., 1991; Wilkinson, 1993). V. Catabolism of Amino Acids

The general catabolism of free amino acids is summarized in Fig. 8 (Hemme et al., 1982). A. DECARBOXYLATION AND PRODUCTION OF AMINES

Amines, including biogenic mines, are produced in cheese by enzymatic decarboxylation of free amino acids (Joosten and Stadhouders,

P. F. FOX AND J. M.WALLACE

46

TABLE IV continued Cheese [Ripening time]

Cabrales (251 (BhlC)

[4 rnol

Amino

mgl

acid

kg

%of FAA

Cabrales @ti) (Blue) 14 mol

rngl kg

'% of FAA

Danbu (27) 18.3 mu] mgl kg

of FAA

'Yo

Maribo (281 (rindless) [8.4 mo] ing/ kg

of FAA

Oh

Maribo (291 (rind) In.4 mol

mgl kg

% of FAA

Havarti (30) [Smear]

mg/

kg

Oh of FAA

-

-

-

-

ASP

2800 0

ti.7

2880.2

5.0

1.9

1.1

1.7

1.7

Thr

4190.0

13.6

3039 fi

52

27

1.5

3.0

2.0

Ser

1230 0

4.0

790.5

1.4

22

1.0

2.6

2.3

Glu

4140.0

13.4

7604.9

13.1

21.0

11.3

19.2

21.2

4.7

3.1

4.1

7.ti

Gly

590.0

1.9

1041.n

1.8

1.R

1.7

1.7

1.8

Aln

1970 0

6.4

1893.6

3.3

2.8

5.6

2.8

2.n

Val

2040.0

6.8

4504.1

78

90

10.0

8.7

78

Mct

11R0.0

38

2696.3

9G

3.4

4.3

3.4

33

110

1310.0

4.2

4124.1

71

6.2

5.9

5.8

5.0

LOU

2640.0

8.6

7525.2

13.0

14.0

17.8

1ti.0

13 2

Cyr

PR,

TY~

540.0

18

333Y.8

5.8

1.7

1.7

1 6

33

I'hn

1730.0

5G

4406.0

7.6

7.9

8.3

R.0

7.6

llis

1140.0

3.7

1403.3

2.4

1.Y

2.0

13

1.5

1.ys

3270.0

10.6

71335.5

13.2

8.8

6.8

7.4

R.8

AX Total

870.0

2.R

281.9

0.5

0.1

0.4

0.2

0.1

30890

57897

The concentrations of amino acids were either given by, or calculated from the results of 25: Gonzalez de Llano et al. (1988);26: Tuckey and Sahasrabudhe (1958);27,28,29,30: Ismail and Hansen (1'372).

1987). Although amines are thought to be important contributors to cheese flavor, some may cause bitterness (Ney, 1971). Amines are potentially toxic to humans due to their ability to react with nitrogen oxides, forming carcinogenic nitrosamines, and their ability to induce symptoms of hypo- or hypertension in certain individuals (Rice et al., 1978). Concentrations of amines in Cheddar are generally too low to cause adverse effects (Joosten and van Boekel, 1988; Flynn, 1992). The principal amines in most cheeses are tyramine and histamine, produced by decarboxylation of Tyr and His (Fig. 9) (Fox et al., 1995a). The concentrations of tyramine and histamine in cheeses inoculated with lactobacilli were twice as high as in control cheeses, indicating that decarboxylases of lactobacilli play a major role in their production

47

FORMATION OF FLAVOR COMPOUNDS IN CHEESE TABLE IV continued Cheese [Ripening time] Lim- (31) burger 110 wkl

Brick (32) I10 wkl

Roma- (33) dour

Hovi (341 (Gervais)

Kreivi (35) (Tilsit)

Kesti (36) Wlsit mil Kummeln~satc)

~

Amino acid

mg/ kg

% of FAA

mg/ kg

% of FAA

mg/ kg

% of FAA

mg/ kg

Yn of FAA

mg/ kg

% of FAA

mg/ kg

% of FAA

CYS

450

3.5

-

-

-

-

-

ASP Thr

450

3.5

430

7.2

1ofi.n

2.0

7.5

3.4

96.5

1.2

114.6

600

4.7

in0

3.0

75.1

1.4

6.3

2.6

457.4

5.9

375.6

5.1

Ser

290

2.3

210

3.5

61.6

1.2

6.7

3.0

276.0

3.5

242.2

3.5

Glu

2580

20.1

600

10.1

519.0

9.7

53.2

23.8

117R.7

15.2

782.9

13.1

Pro

320

2.5

244.6

4.6

14.0

6.4

534.3

6.4

368.5

5.6

Gly

380

3.0

500

8.4

111.2

2.1

3.0

1.3

131.1

1.6

113.2

1.7

Ala

890

6.9

460

7.7

171.7

3.2

6.2

2.8

213.0

2.5

180.2

2.7

Val

lain

14.1

190

3.2

610.6

11.4

6.1

2.7

608.9

7.4

491.0

7.5

Met

1100

8.6

480

8.0

230.0

4.3

3.1

1.3

229.4

2.8

192.5

2.9

333.9

6.2

5.6

2.2

288.0

3.2

215.9

3.3

13.8

-

Ile

1.8

Leu

1900

14.8

lion

in4

1039.1

19.4

16.6

7.3

1161.7

15.6

919.8

TYr

R90

6.9

420

7.0

a3 o

1.6

7.1

3.2

327.2

4.0

257.8

3.9

Phe

100

0.8

520

87

468.1

8.8

13.0

5.7

796.1

10.0

658.2

10.0

His

260

2.0

223.6

4.2

5.9

2.6

575.1

7.2

148.9

2.3

LYs

370

29

726 8

13.6

24.0

10.7

974.8

11.8

748.0

11.5

34 n

0.6

11.9

5.3

161.8

2.1

235.2

3.7

-

Arg

Total

12818

4624

224.2

799n

6461

The concentrations of amino acids were either given by, or calculated from the results of 31, 32: Tuckey and Sahasrabudhe (1958); 33,34, 35, 36: Antila and Antila (1968).

(Broome et al., 1990). The concentration of tyramine in cheese often exceeds that of histamine, with lesser amounts of phenylethylamine (from Phe), tryptamine (from Trp), cadaverine (from Lys), and putricine (from ornithine) (Tokita and Hosono, 1968); the relative proportion of amines depends on the cheese variety and the nonstarter microflora. Lactobacilli appear to be responsible for the production of tyramine, histamine, and putricine in Gouda cheeses. In normal Gouda and Maasdam produced under hygienic conditions from pasteurized milk, concentrations of biogenic amines were low, indicating the inability of starter cells to produce them (Joosten and Stadhouders, 1987). A similar conclusion applies to Edam and Emmental (Antila ef al., 1984). Enterococci and coliforms in Gouda are capable of producing biogenic amines (Gripon et al., 1991);Br. linens is capable of producing tyramine, hista-

P. F. FOX AND 1. M. WALLACE

48

TABLE IV continued Cheese [Ripening time) Trap- (37) pisttype 135 days]

Ras (38) (Egyptian) [4 mol

Kashkaval (39) Kopanisti (40) (Egyptian) (Greek) [4 mol 146 days]

Yo of

1.6

1217.3

4.2

1642.4

4.9

1403.8

4.0

2.0

2146.3

64

10615

64

211.0

2.3

71322

21.2

59159

212

7.3

815.3

8.8

18978

57

1719.0

5.7

2.8

286.6

3.1

12778

3.8

1244.7

3.8

8.1

1022.8

11.1

1221.0

3.6

970 1

36

44

1280.4

14.0

30742

92

26543

92

54

11670

35

936 8

3.5

11 1

2077.0

6.2

1524.2

G.2 12.0

mgi kg

% of

rngl

I'AA

kg

18.1

0.5

182.5

7.7

56.3

1.3

103.0

2.9

109.4

4.6

143.1

4.2

99.9

28

41.7

Gl"

784.2

22.8

700.0

I'J 8

401.0

Pro

m8.n

5.5

167.0

4.7

Gly

59.2

1.7

83.3

2.4

Ald

204.0

5.1)

279.0

7.g

Val

290.6

8.5

307.0

8.7

Me1

32.3

09

2530

7.2

66.5

2.8

500.4

Ile

12'J 7

38

261.0

7.4

81.5

34

1026.6

LCU

400.8

% uf FAA

Cye

12.4

0.4

-

Asp

130.0

3.8

Tlir

44.4

Ser

mgl

Edam- (42) type (goat's)

rngi kg

% uf I'AA

mgi kg

kg

Amino acid

Rumelia ( 4 1 ) (goat's)

rngi kg

FAA

0.6

530.8

462.1

5.0

1.8

181.1

16.9

173.7 67.7 191.1 1043

-

~

% uf FAA

~

% vf

FAA

~

14.3

345.0

9.8

2600

11.0

2033.1

22.1

4014.7

12.0

3281.2

Tyr

57

n

17

75.3

2 1

130.9

55

25.1

0.3

521.0

1.n

38S.5

1.6

Phe

270 3

7 9

315.0

8'1

295 0

12.4

11as.9

12 0

1850.6

5.5

1475.7

5.5

His

74 fi

22

87.1

2.5

62.2

2.6

98.4

1.1

863.5

2.6

1202.5

2.6

LYS

480.6

140

1590

4.5

126.1

5.3

106.3

1.2

3921.0

11.6

4103.4

11.6

Arg

46 4

14

279.0

7.9

80.3

3.4

-

-

254.4

0.8

216.5

0.8

Total

3439

3530

2380

m n

33594

29450

l'hc concentrations of amino acids were either given by, or calculated from the results of 37:Ades and Cone (1969); 38:Omar (1984); 39: Omar and El-Zayat (1986); 40: Kaminarides et aJ. (1990); 41: Baltajieva et al. (1985);42:Ramos et aJ. (1987).

mine, monomethylamine (Fig. 10a), monoethylamine (Fig. lob), trimethylamine (Fig. lOc), dimethylamine (Fig. lod), cadaverine (Fig. loe), monopropylamine (Fig. 100, dipropylamine (Fig. log), tripropylamine (Fig. loh), triethylamine (Fig. l O i ) , and piperidine (Fig, loj) in model systems (Tokita and Hosono, 1968; Hosono and Tokita, 1969). Simple decarboxylation can explain the formation of most amines found in cheese, but there is no readily available explanation for the formation of secondary and tertiary amines and n-butylamine in cheese (Adda et a]., 1982). No relationship has been found between the concentration of free amino acids and the production of amines in cheese (Smith, 1981), probably due to differences in the rate of decarboxylation of individual

49

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

TABLE IV continued Cheese [Ripening time1 Turunmss (43) (Finnish) Amino acid

rngi kg

% of I'AA

Sir0 (44)

(Finnish) rngi kg

Loustari (45) (Port Salut)

Mahon (46) (traditional)

Mahon (47) (Industrial)

%of

rngi

FAA

kg

% of FAA

1.9

60

1.3 5,5+gly

rngi kg

% of FAA

% of FAA

-

-

-

ASP

53.3

0.9

70.3

1.3

113.2

Thr

349.2

7.3

354.6

6.5

185.6

8.7

5.7+gly

Ser

139.2

3.0

186.8

2.9

+Ser

tSer

5.5

3.8

Clu

740.5

159

51G.G

95

942.0

17.2

17.2

15.3

Pro

253.3

6.1

237.9

4.4

176.1

4.4

9.7

9.0

tily

63.4

14

110.7

1.U

73.1

1.4

+Thr

+Thr

CYS

-

rngi kg

-

Ala

110.8

2.0

181.1

3.3

176.2

2.8

3.4

3.2

Val

372.1

8.4

420.4

7.7

420.2

7.6

13.8

12.7

Met

125.6

2.8

197.9

3.6

157.7

3.1

0

2.9

Ile Leu 5 r Phe

123.8

2.4

lfi4 9

3.0

218.7

3.6

9.1

18.8

-

707.2

16.6

704 4

12.9

800.1

15.5

242.0

1.9

164.7

3.0

161.1

3.1

-

-

500.1

11.7

600.9

11.0

567.5

11.3

22.2

20.3

His

63.3

0.8

114.1

2.1

100.4

1.8

-

-

Lys

434.8

7.8

542.1

9.9

527.8

96

A%

95.5

2.4

352.6

6.5

51.4

1 5

0 74

1.0

Total

4733

5453

-

5076

The concentrations of amino acids were either given by, or calculated from the results of 43,44,45: Antila and Antila (1968);46,47:Polo et al. (1985).

amino acids or in the rate of deamination of resulting amines (Polo et a]., 1985). B. DEAMINATION-FORMATION OF AMMONIA AND NEUTRAL OR ACIDICCOMPOUNDS

Deamination of free amino acids leads to the production of ammonia and a-keto acids (Hemme et al., 1982). Bassett and Harper (1956) identified a number of a-keto acids, including pyruvic and p-hydroxyphenyl pyruvic acids, in cheese. However, more recent studies have not identified a-keto acids, perhaps due to their instability when measured

50

P. E FOX AND J. M. WALLACE Amino acids

Transamination

I

Oxidative deamination

I

\ Amines

Amino acids

I

a-keto acids

_i,,

Aldehydes

v

Phenols

Indole

I

Other sulphur compounds

Alcohols

Acids

FIG.8. Catabolism of free amino acids in cheese. (Modified from Hemme et al., 1982.)

H~N-CH-COOH Tryptophan

H,N- JH, T ptamine

FIG.9. Production of biogenic amines from tyyosine and tryptophan.

FORMATION OF FLAVOR COMPOUNDS IN CHEESE (a)

51

(C)

NH,

CH,

CH,

N

I

I

C'H,

C$

Monomethyl amine

Trimethylamine

(d)

(0 H,N

CH,

I

N -CH,

CH,

I

CH,

H

Dimethylamine

Monopropylamine

(g)

(9

cY 7' CH

I I

I

CH,

I I

N-

NH

/: ' CH,

CH,

CHI-CH,

CH,

I

CH, CH,

Dipropylamine

Triethylamine

ti)

0 HI

Pipiradine

FIG.10. Amines that have been identified in cheese (Tokita and Hosono, 1968; Hosono and Tokita, 1969).

by GLC (Urbach, personal communication). Ammonia is an important constituent in many cheeses, such as Camembert, Gruyere, and Comte (Fox et a]., 1995a). G. candidurn is capable of deamination, and Br. linens strains possess very active deaminases and produce large quantities of ammonia via the catabolism of serine, and to a lesser extent of glutamine, asparagine, and

52

P. F. FOX AND J. M. WALLACE

COOH

OH

I

I

Isovaleric acid FIG.

Isobutyricacid

Acetic acid

COOH

I

Propionic acid

11. Volatile fatty acids produced by amino acid catabolism.

RCOOOH II 0

RCH + C Q II 0

0

Strecker aldehyde

FIG.12. Strecker degradation pathway. (Modified from Urbach, 1995.)

threonine (Hemme et al., 1982). Ammonia can also be formed by oxidative deamination of amines, forming aldehydes (Hemme et al., 1982) or volatile fatty acids. Isovalerate (Fig. l l a ) , isobutyrate (Fig. I l b ) , acetate (Fig. l l c ) , and propionate (Fig. I l d ) are produced by oxidative deamination of isoleucine, valine, glycine-alanine-serine, and threonine, respectively.

53

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

0

0 II CH

x I

0 II CH I

CH,- CH

I

CH, CH3

Phenylacetaldehyde

Isobutanal

3-methylbutanal

0

II

CH

I

3-methylthiopropmal

Methional

FIG.13. Aldehydes produced by Strecker degradation of amino acids.

c.

TRANSAMINATION, STRECKER DEGRADATION, AND PRODUCTION OF ALDEHYDES

Production of aldehydes from free amino acids can result from decarboxylation, deamination, transamination (Fox et al., 1995a), or via Strecker degradation (Keeney and Day, 1957; Dunn and Lindsay, 1985; Urbach, 1995) (Fig. 12). Enzymatically catalyzed transamination of a free amino acid results in the formation of an intermediary imide that is subsequently degraded by decarboxylation or the Strecker reaction, resulting in the formation of an aldehyde (Keeney and Day, 1957; Polo et al., 1985). Phenylacetaldehyde (Fig. 13a), isobutanal (Fig. 13b), 3methylbutanal (Fig. 13c), and 3-methylthiopropanal (Fig. 13d) and methional (Fig. 13e) can be formed by this mechanism from Phe, Leu-Ile, Val, and Met, respectively (Adda et al., 1982). Transamination also leads to the production of other amino acids (Fox et ai.,1995a), such as Glu from Phe (as in Section V.E.1) (Lee and Desmazeaud, 1985) Interconversion of amino acids has been demonstrated in Tallegio and other Italian cheeses by using radiotracers (Cicchi et al., 1979), giving

54

P. F. FOX AND J. M. WALLACE

rise to various metabolites such as a-ketoglutaric and pyruvic acids that could participate in further interconversion reactions (Polo et al., 1985). Aldehydes are thought to contribute to the flavor of many cheese varieties. In Parmesan, where they accounted for 2% of the total volatiles, their production is assumed to be via Strecker degradation (Barbeiri ef al., 1994). The low pH of Parmesan during ripening is thought to be responsible for the decarboxylation of amino acids to amines that are subsequently oxidized to aldehydes when there is an increase in pH during the later stages of ripening (Belitz and Grosch, 1987). When the concentration of Strecker-derived compounds exceeds a certain threshold in Cheddar, unclean flavors develop (Dunn and Lindsay, 1985).Poor flavors were noted when the concentrations of two Strecker aldehydes (3-methylbutanal/2-methylbutanal or 2-methylpropanal) exceeded 200 pg/kg in Cheddar (Dunn and Lindsay, 1985). Isovaleraldehyde, 2-methylbutanal, isobutanal, and methional were formed by Strecker degradation of Leu-Ile, Val, Phe, and Met, respectively, in aqueous extracts of cheese and cultures of cheese microorganisms (Griffith and Hammond, 1989). Dunn and Lindsay (1985) found no correlation between the concentrations of Strecker-derived compounds and concentrations of individual free amino acids in Cheddar cheese. D. CATABOLISM OF SULFUR AMINOACIDS

The catabolic products of sulfur amino acids have been implicated as major contributors to Cheddar cheese flavor (Manning, 1974; Manning et al., 1976; Adda et al., 1982; Dunn and Lindsay, 1985; Kristoffersen, 1985; Barlow et al., 1989; Gripon et al., 1991; Alting et al., 1995) (Fig. 14). Certain smear surface-ripened cheeses exhibit flavors described as “cabbagey,” “garlic,” or “putrid” (Adda eta]., 1978; Hemme et al., 1982; Ferchichi et al., 1985; Manning and Nursten, 1985). These flavors are attributable to sulfur compounds derived from methionine and perhaps to a lesser extent from cysteine. Sulfur compounds in cheese (e.g., methanethiol (CH,SH), hydrogen sulfide (H2S), dimethylsulfide (CH3SCH3), dimethyldisulfide (CH3SSCH3), dimethyltrisulfide (CH3SSSCH3),and carbonyl sulfide (O=C=S)) are thought to interact with each other and with other compounds in cheese, generating typical cheese flavors (Kim and Olson, 1989). Sulfur compounds have been identified in many cheese varieties, but their importance in smear surface-ripened cheeses appears to be accentuated by their high concentrations at the surface (Ferchichi et al., 1985; Gripon et al., 1991).

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

a

Homoalanine

CH,SH Methanethiol

3

Homocy steine

a-keto-thiornethy1 butyrate dernethiolase

Sulphur derivatives FIG.14. Catabolism of methionine. (Modified from Hemme et al., 1982.)

55

P. F. FOX AND J. M. WALLACE

56

(b)

I

L-Methionine

a-Ketoglutarate

L-Cy steine

FIG.15. Chemical structures of methionine, a-ketoglutaric acid, and cysteine.

The principal microorganisms in the smear of smear-ripened cheeses are coryneform bacteria, particularly strains of Brevibacterium linens. Sharpe et al. (1976) were the first to report the ability of Br. linens to produce methanethiol enzymatically. Br. linens is also found on the surface of many other cheeses, such as Camembert (Ferchichi et a]., 1985; Hemme and Richard, 1986) or Gruyere (Law, 1981), the distinct flavors of which have been attributed to the formation of sulfur compounds, particularly methanethiol, at the surface (Manning and Price, 1982).

The enzyme demethiolase, involved in methanethiol production, is highly dependent on the availability of Met (Fig. 15a) (Ferchichi et al., 1987) and the ability of cells to transport it into the cytoplasm (Forss, 1979; Ferchichi et af., 1985). The production of methanethiol involves a-ketoglutarate (Fig. 15b), which is used by the bacteria as a carbon source, as an intermediate (Hemme and Richard, 1986). Uptake of Met into the cells is strongly inhibited by high concentrations of Cys (Fig. 15c) (Ferchichi et al., 1987), as is its conversion to methanethiol (Urbach, 1995). Some authors (Adda et a]., 1982) claim that the enzyme has a preference for free methionine, but others (Ferchichi et a!., 1986b) have demonstrated higher activity on Met in the dipeptides L-Ala-L-Met and L-Met-L-Ala than on free Met. Methionine and methanethiol may affect characteristics of the cheese other than flavor. The production of a red-orange pigment by Br. linens appears to depend on the concentrations of methanethiol and dissolved O2 (Ferchichi et a]., 1986a). Methionine inhibits the germination of

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

57

certain mold spores; the active compound is thought to be methanethiol (Beattie and Torney, 1986). Manning et al. (1976) correlated the concentration of methanethiol with the flavor of Cheddar cheese but pointed out that this finding may only apply to experimental cheeses manufactured in their laboratory. Other authors (see Aston and Dulley, 1982) have since found that the concentration of methanethiol is a poor indicator of flavor, particularly in Cheddar cheeses subjected to accelerated ripening (Aston et a]., 1983). In cheeses such as Cheddar, which lack a surface microflora, flavor is produced by starter and nonstarter bacteria and their enzymes (Urbach, 1995). Since coryneforms do not occur in Cheddar, the production of methanethiol was thought to be a chemical process (Urbach, 1995). Certain NSLAB can produce methanethiol enzymatically from methionine, but the enzymes involved are thought to be very unstable in ripening cheese and are therefore unlikely to influence the concentration of methanethiol during ripening (Law and Sharpe, 1978). In cheese varieties such as Cheddar, the role of microorganisms in amino acid catabolism may be indirect. Key chemicals, such as methanethiol, may be produced chemically in the cheese as a result of starter-induced conditions (low pH and Eh) rather than enzymatically (Law, 1981; Kim and Olson, 1989). However, Urbach (1995) proposed that the secondary flora, particularly in Cheddar and Emmental, is likely to be more important than chemical reactions for the formation of sulfur compounds. This hypothesis was supported by Alting et al. (1995), who isolated an enzyme (similar to cystathionine P-lyase) from L. lactis ssp. cremoris B78 that was capable of producing methanethiol from sulfur amino acids under cheese ripening conditions (pH 5.2-5.4 and 4% NaCl). Engels and Visser (1996) produced typical Gouda flavor by adding methionine to cell-free extracts of this strain. Fermentation of lactose during manufacture reduces the pH to -5.1 and the redox potential from about +300 mV to -130 mV or lower (Law and Sharpe, 1977; Adda et al., 1982). In Swiss cheese, in which the highly reductive propionic acid fermentation results in a further decrease in Eh, the concentration of active sulfhydryl groups is twice that in Cheddar (Kristoffersen, 1973). Law and Sharpe (1977) found low levels of sulfur compounds in starter-free Cheddar cheeses (chemically acidified with GDL) and attributed this to the high redox potential of the GDL cheeses. Manning (1979a), who artificially reduced the Eh of GDL-acidified cheese using dithiotreitol and glutathione, demonstrated the nonenzymatic formation of methanethiol. Although the reducing conditions were created artificially, Manning concluded that the reduc-

58

P. F. FOX AND J. M. WALLACE

ing conditions produced by starter bacteria would be adequate to cause the same effect. Ponce-Trevino et al. (1987, 1988), who manufactured chemically and microbially acidified cheese slurries, found that the production of H2S,carbonyl sulfide, methanethiol, and dimethyl sulfide was highest in cheese slurries with added starters. When starter cells were inactivated by antibiotics, no H2S or (CH3)2Swas produced. When the Eh of chemically acidified slurries was reduced by using NADH, O=C=S and CH3SH were produced but H2S and (CH3)2Swere not. Samples (1985) commented that, while a negative redox potential may be necessary for the production of sulfur compounds in cheese, it is not sufficient. He felt that glutathione, which is stored in the cells of certain lactococcal strains (Fernandes and Steele, 1993), plays an important role. A low Eh does, however, have a stabilizing effect on sulfur compounds (Law et al., 1976a,b). Although the exact pathway for the nonenzymatic formation of CH,SH has not been established, a mechanism proposed by Manning (1979a,b) is generally accepted by most investigators (Law, 1981; Adda et al., 1982; Hemme et al., 1982; Barlow et a]., 1989; Fox et al., 1995a). The proposed pathway involves the release of H2S from cysteine in the presence of a reducing agent, followed by the reaction of H2S with methionine, in which the C-S bond is cleaved, with the release of CH3SH. This proposed pathway is supported by the fact that H2S is released initially in the curd prior to the formation of CHoSH(Manning, 1979a,b; Adda et a]., 1982). Green and Manning (1982) showed that low concentrations of H2S in cheese during the early stages of ripening coincided with the production of low levels of CH3SH. This appears to indicate that H2S is essential for the formation of CH3SH. However, Cys is an inhibitor of CH3SH production and the release of H2S from Cys may simply remove the inhibitor for the enzymatic production of CH3SH (Urbach, 1995). The thiol-producing enzyme system is thought to be inactivated by high heat treatment of milk (Samples, 1985), and indigenous milk enzymes may be responsible for the production of CH3SH in cheese (Urbach, 1995). The production of both CH3SH and H,S was increased by the addition of reduced glutathione to cheese (Samples, 1985), and glutamyltranspeptidase, which acts by desulfurating glutathione, is thought to be partially responsible for the formation of H2S, and possibly of CH3SH. Other enzyme(s) are also involved (Samples, 1985; Urbach, 1995). SH groups are a limiting factor in the formation of H,S (Samples 1985). Since there are few cysteine-cystine residues in casein, it seems likely that at least some of the sulfur groups arise from the

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

59

action of y-glutamyltranspeptidase on glutathione, which is introduced into the cheese in starter cells (Fernandes and Steele, 1993; Wiederholt and Steele, 1994; Folkertsma and Fox, 1996). The formation of H2S is thought to be influenced by Eh and the concentration of glutathione. The presence of H2S in fresh curd indicates that some may also be formed during pasteurization of cheese milk (Law et al., 1976a). Concentrations of H2S in cheeses with added NSLAB showed that some lactobacilli are capable of desulfurating Cys and producing H2S, while Group D streptococci, staphylococci, micrococci, and pediococci are not (Sharpe and Franklin, 1962; Law, 1981). While the concentration of H2S in cheese increased during ripening, Aston et al. (1983) found no correlation between its concentration (or that of other sulfur compounds) and flavor development. Similar observations were reported by Lawrence (1963) and Manning (1978). On the other hand, Kristoffersen and Nelson (1955)found that cheeses with the highest concentration of H2Sreceived the highest flavor scores. In later studies (Kristoffersen and Gould, 1960; Kristoffersen, 1967), fluctuations in H2S were observed throughout ripening, and the authors suggested that the ratio of H2S to short-chain fatty acids was important for flavor development. Barlow et al. (1989) found a high correlation between flavor and the concentration of H2S, particularly when the value for H2S was combined with either the concentration of water-soluble nitrogen or lactic acid. The authors concluded that these parameters (H2S + WSN/lactate) were better predictors of the way in which young cheeses would mature than their flavor or composition at an early age. A notable feature of Parmesan cheese is that, although a number of sulfur compounds have been identified, this cheese is characterized by low concentrations of H2S and CH3SH (Barbeiri et al., 1994). This may be due to inactivation of indigenous milk enzymes by the relatively high cooking temperature during manufacture (Barbeiri et al., 1994). Dimethylsulfide (DMS), dimethyldisulfide (DMDS), and dimethyltrisulfide (DMTS) are thought to be important contributors to cheese flavor (Manning et al., 1976; Barbeiri et al., 1994). DMS is a component of Swiss cheese flavor and is a metabolite of propionibacteria (Langler et al., 1966; Adda e f al., 1982). DMS levels remained constant in Cheddar cheese for up to 6 months of ripening and decreased thereafter (Aston et al., 1983).Manning et al. (1976) proposed that the concentration of DMS is seasonal and originates in the cheese milk rather than from the catabolism of sulfur amino acids. Variability of the concentrations of DMS and H2S throughout ripening rendered these two compounds of little value as indices of ripening (Aston and Douglas, 1983). DMDS is formed as an end-product of Strecker degradation (Belitz and Grosch, 1987). Strecker degradation involves a reaction between an

60

P. F. FOX AND J. M. WALLACE

---

CH,

I

Streckex Degradation

S

I

CH,

I

CH,

S

I

FH2 CH,

I

I

S N - CH - O H

o= CH

Methionine

Methional FIG.16. Structure of methional.

amino acid and a diketone, resulting in the formation of an aldehyde with one carbon atom less than the original amino acid (Aston and Dulley, 1982; Keeney and Day, 1957; Urbach, 1995) (Fig. 12). 3-Methylpropanal is formed in the first step of this reaction. DMDS has been identified in Parmesan (Barbeiri et al., 1994), Cheddar (Aston et al., 1983; Barlow et al., 1989), and surface-ripened cheeses (Jollivet et al., 1992). In Cheddar it was found to correlate fairly well with flavor scores (Barlow et al., 1989). DMTS is another potent aroma compound and has been associated with the aroma of cooked cabbage, broccoli, or cauliflower. It has been identified in Parmesan (Barbeiri et a]., 1994) and Cheddar (McGugan, 1975; Wood, 1989). Although DMDS is formed by Strecker degradation of methionine, the principal product of this reaction is methional (P-methylmercaptopropionaldehyde) (Keeney and Day, 1957; Aston and Dulley, 1982) (Fig. 16). Methional contributes positively to cheese only when present at low concentrations; a sweet corn-like aroma is detectable at high levels (Barbeiri et al., 1994). Methional is present in the volatile fraction of many cheeses, including Cheddar (Wijensundera and Urbach, 1993). Dunn and Lindsay (1985) noted that methional plays a role in flavor suppression in bland curds and proposed that this compound may play a critical role in masking off-flavors in cheese. Methional can also be produced enzymatically by many strains of lactic acid bacteria in enriched media (Tracey and Britz, 1989). Several other sulfur compounds have been identified in cheese volatiles. Thioesters, produced by esterification of methanethiol and acetic or propionic acid, have a cheesy aroma (Dumont and Adda, 1978). S-methylthioacetate (Fig. 17a) can be produced under certain conditions by selected strains of Br. linens (Ferchichi et al., 1986a,b) and has

61

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

-N

II

S-Methylthioacdate

Benzothiazole

0

II

FH

2-methyl propanal

FIG.17. Chemical structures of some volatile compounds derived from methionine.

CH,SH

Methanethiol

+

+ Formaldehyde->

Bis(methy1thio)methane

FIG.18. Production of bis(methylthio)methae by condensation of methanethiol with formaldehyde.

been found in Limburger cheese (Parliment et al., 1982). The mechanism of thioester formation is unknown, but it is thought to be enzymatically controlled as the presence of micrococci was required for their formation in model systems (Adda et al., 1982). A reported component of Parmesan aroma is the methionine-related benzothiazole (Meinhart and Scheier, 1986; Barbeiri et a]., 1994) (Fig. 17b). No mechanism for its formation has been reported. A sulfur-containing aldehyde, 3-methylpropanal (Fig. 17c), presumably formed by transamination of methionine, followed by decarboxylation of an intermediate imide, has been identified in Cheddar (Adda et al., 1982) and Parmesan (Barbeiri et al., 1994) and may coiitribute to the aroma of these varieties. Methylthiopropionate, which has a cheesy aroma, has been found in Cheddar (Cuer et al., 1979). Bis(methy1thio)methane (Fig. 18),an important component in Camembert flavor, can be produced by the condensation of methanethiol with formaldehyde (Dumont et al., 1976) (Fig. 18). The concentration of carbonyl sulfide increases throughout ripening and is less variable than other sulfur compounds (Aston and Douglas,

P. F. FOX AND J. M. WALLACE

62

CH, I

Phenylmethanol

c=o

OH

I

Phenylethanol

I

I

CHZ

I

0

CH,

CHZ

I

COOH

CH,

Phenylpropanone

Methylphenyl hydroxyacetic acid

CH, I

OH-CH-CH2 -COOH

O=CH

Phenylacetaldehyde

Pheny lpyruvic acid

Phenylethanol acetic acid

FIG.19. Some phenylalanine-derived catabolites that have been identified in cheese.

1983; Aston et al., 1983). It has not been established whether the concentration of carbonyl sulfide correlates with cheese flavor.

E. CATABOLISM OF PHENYLALANINE, TYROSINE, AND TRYPTOPHAN 1, Phenylalanine

Phe is released at high concentrations, particularly during the early stages of cheese ripening. Initial cleavage of the Phe,,,-Metlo6 bond of K-casein by rennet and of Phe,,-Phe,, of aSl-casein during early ripening provides terminal Phe, which could be easily released by bacterial exopeptidases (Dunn and Lindsay, 1985). A number of flavor compounds arising from Phe have been identified in cheese (Adda et al., 1982; Dunn and Lindsay, 1985; Lee and Desmazeaud, 1985) and in model systems (Jollivet et al., 19921, that is, phenylmethanol (Fig. 19a), phenylethanol (Fig. 19b), phenylpropanone (Fig. I ~ c ) methylphenyl , hydroxyacetate (Fig. 19d), phenylacetaldehyde (Fig. 19e), phenylpyruvate (Fig. 19f), and phenylethanol acetate (Fig. 19g). L-G~u(0.93 moll and phenylpyruvate (1.0 mol) were pro-

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

Phenylalanine

+

2-0xyglutaric acid

+

63

Phenylpyruvic acid + Glutamic acid

FIG.20. Transamination reaction with the production of glutamic acid from phenylalanine.

duced when Phe acted as amino group donor and 2-oxyglutarate as acceptor in a transamination reaction (Lee and Desmazeaud, 1985) (Fig. 20). Br. linens rapidly catabolyzes Phe and other aromatic amino acids and can utilize them as nitrogen sources. The enzymes involved in the initial breakdown of Phe are aromatic amino acid amino transferase, L-amino acid oxidase, L-phenylalanine ammonia lyase, L-aromatic amino acid decarboxylase, and phenylalanine dehydrogenase. Many of these enzymes have been identified in Br. linens (Jollivet et al., 1992, 1994), but their activity appears to be strain-dependent (Lee and Richard, 1984). Removal of the amino group from Phe by Br. linens is thought to be by oxidative deamination. A general L-amino acid oxidase is considered to be responsible for the ability of coryneform bacteria to utilize amino acids for nitrogen (Coudert and Vandcastelle, 1975). Surface yeasts in Camembert are thought to possess at least a transaminase and a decarboxylase active on Phe (Roger et al., 1988). In Cheddar (and other varieties with only primary starters),Phe is thought to be degraded by Strecker degradation (Dunn and Lindsay, 1985). The typical aroma of Camembert has been attributed to phenylethanol (Fig. 19b) produced by surface yeasts (Adda et al., 1982) involving transamination, decarboxylation, and reduction reactions (Dunn and Lindsay, 1985). Yeasts have been implicated as the main producers of phenylethanol in mold/smear-ripened cheeses (Roger et al., 1988; Jollivet et al., 1992); however, Br. linens degrades phenylethanol further (Jollivet et al., 1992). Phenylacetaldehyde (Fig. 19e) is considered to be an important flavor compound in many cheeses (Dunn and Lindsay, 1985; Jollivet et d., 1992). In Cheddar, phenylethanol and phenylacetaldehyde can be pro-

64

P. F. FOX AND J. M. WALLACE

duced by Strecker degradation of Phe (Dunn and Lindsay, 1985). Concentrations of phenylethanol in good- and poor-quality Cheddar cheeses (-100 kg/kg) were comparable, but the concentration of phenylacetaldehyde ranged horn 4 0 to 400 pg/kg, the highest levels being present in poor-quality cheese (Dunn and Lindsay, 1985). Both phenylethanol and phenylacetaldehyde have rosy-floral aromas and have been associated with off-flavors produced by Brewer’s yeast (Saccharomyces cervisiae) in beer (Sentheshanmuganathan, 1960). Unclean rose-like flavors observed in some poor-quality Cheddar have therefore been attributed to the presence of these compounds (Dunn and Lindsay, 1985). High concentrations of phenylacetaldehyde also contribute astringent, bitter, and stinging sensations to Cheddar flavor. The concentrations of these compounds were highest and their effects greatest in young and mid-ripened cheeses. In mature Cheddar cheese with more intense flavors, the effects of phenylethanol and phenylacetaldehyde are diluted, and off-flavors are either combined with, or masked by, other aroma compounds (Dunn and Lindsay, 1985). An important component of Camembert flavor is hydroxyphenylacetic acid, thought to be formed via initial deamination of phenylalanine followed by hydroxylation (Simonart and Mayaudon, 1956). Phenylethanolacetate (Fig. 19g)from Phe catabolism has been identified in many cheeses (Jollivet et al., 1992).Although phenylpropanone (Fig. 19c) is present in cheese (Jollivet et al., 1992), the mechanism for its formation has not been established. 2. Tyrosine and Tryptophan

Tyrosine serves as a precursor for three compounds in cheese: tyramine, formed by decarboxylation, and p-cresol (Fig. 21a) and phenol (Fig. 21b) by atypical Strecker degradation (Elsden et a]., 1976). Phenol and indole, pyruvate, and ammonia can be produced by the action of C-C lyases on Tyr and Trp (Hemme et al., 1982). Tyrosol (Fig. 21c) and tryptophol (Fig. 21d), two classical Strecker-type alcohols, have been reported to impart slightly bitter flavors to beer (Dunn and Lindsay, 1985);however, they have not been identified in cheese. p-Cresol and phenol have strong flavor potencies. While phenol (100225 mg k g * ) appears to have no detrimental effect on Cheddar flavor, p-cresol has been implicated in putrid “utensil” aromas (Dunn and Lindsay, 1985) when concentrations exceed 100 mg kg’ in Cheddar. Lactobacilli in Gouda and Vacherin cheeses are thought to be responsible for producing p-cresol (Badings et a]., 1968; Dumont et al., 1974). The concentration of phenol is unusually high in Limburger (Parliment eta]., 1982).

65

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

YCOH

p-Cresol

Phenol

Tyrosol

H&H,

Tryptophol

FIG.21. Aroma compounds produced by the degradation of tyrosine and tryptophan.

F. OTHER IMPORTANT FLAVOR COMPOUNDS FROM AMINO ACIDCATABOLISM Other compounds, thought to be important in cheese flavor, produced by Strecker degradation include benzaldehyde (Fig. 22a) and acetophenone (Fig. 22b) from Phe, 2-acetylthiazole (Fig. 22c) from Cys, and methylglyoxal alkypyrollizines from Lys. Amines can be acetylated, yielding to N-isobutyl acetamides, which have been identified regularly in Camembert (Dumont and Adda, 1978). Aldehydes are usually reduced as soon as they are formed (Polo et al., 1985) with the concomitant production of alcohols: isobutanal (Leu-Ile) is reduced to isobutanol (Fig. 22d), 3-methylbutanal (Val) to 3-methylbutanol (Fig. 22e), phenylacetaldehyde (Phe) to phenylethanol (Fig. 22f), and 3-methylthiopropanal (Met) to 3-methylthiopropanol (Fig. Zag) (Adda et al., 1982). 2,5-Dimethylpyrazine is produced in significant quantities for flavor perception in model systems containing Br. linens. This compound is present in many cheeses and can give a nutty, toasted note to the flavor. It could be produced by degradation of Thr to amino-acetone, the condensation of two amino acetone molecules forming one molecule of 2,5-dimethylpyrazine (Jollivet et al., 1992). Isobutanal (Leu-Ile) is reduced to isobutanol (Fig. 22d), 3-methylbutanal (Val) to 3-methylbutanol (Fig. 22e), phenylacetaldehyde (Phe) to phenylethanol (Fig. 22f), and 3-methylthiopropanal (Met) to 3-methylthiopropanol (Fig. 22g) (Adda et al., 1982). G. REACTIONSOF FREEAMINOACIDSWITH OTHER COMPOUNDS IN CHEESE

Cheesy flavors have been noted when free amino acids react with other molecules in the cheese. Addition of calcium or magnesium to

66

P. F. FOX AND J. M. WALLACE

0

0

II

II

L-’c-cH3 sYc-cH3 Benzaldehyde

Acetylthiazole

Acetophenone

(0 OH

OH

I

I

CK

x

CH3

CH,

Isobutanol

3-methylbutanol

Phenylethanol

(g) OH 1

CH-CH,SH I

YK CH3

Frc. 22. Some minor amino acid catabolic products found in cheese.

cas-amino acids enhanced their sweetness (Biede and Hammond, 1979), suggesting that Ca (or Mg) amino acid complexes may contribute to the sweet flavor of Swiss cheeses. A number of mono- and dicarbonyls are produced from free fatty acids in cheese (Reps et a]., 1987), particularly by lactobacilli. Kowalewska et al. (1985) showed that much of the flavor in the nonvolatile water-soluble fraction was generated from amino acid-carbonyl reactions. Glyoxal, methylglyoxal, dihydroxyacetone, and ethanal were prominent among the carbonyls, and Val, Leu-Ile, Met, Cys, Phe, Pro, and Lys were the principal amino acids that react chemically with them (Griffith and Hammond, 1989). Although the exact pathways for carbonyl-amino acid reactions are not known, Griffith and Hammond (1989) proposed a pathway for the

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

+

67

CH3

I

c= 0 I

H

Cysteine

Methylglyoxal

T

COOH I II KC- CH-N =CH- C-CH3 + CO,

I

SH

I

KC- C K - W C H - C-CH, + CO,

c=0

I

SH

2- Acdylthiazole FIG.23. Production of 2-acetylthiazole by reaction of cysteine with methylglyoxal; chemical pathway proposed by Griffith and Hammond (1989).

reaction of Cys with methylglyoxal (Fig. 23). The reaction of Lys with glyoxal or dihydroxyacetone can give rise to 6-valerolactam (Fig. 24a) or 2-acetyl pyrroline (Fig. 24b). Proline can react with ethanal, methylglyoxal, or dihydroxyacetone to produce 2-acetyl-1-pyrroline (Fig. 24c), 2-methyl benzaldehyde (Fig. 24d), and isomers of 2,3-dihydropyrolizine, respectively (Griffith and Hammond, 1989). These compounds

68

H I

+

c= 0 I c=o I

H

Ly sine

0)

-(2)-

P. F. FOX AND J. M. WALLACE

+

‘i“. CHZ +

CHZ

I

H2N- CH

6 -valerolactam (2-peperodine)

I

CHZ

‘iHz

-

H

I

I

Glyoxal

- COOH

I

c=o

CCH,

I H

COOH

Proline

+

Ethanal-

2- acdyl-1-pyrrolhe

FIG.24. Compounds formed by the reaction of amino acids with carbonyls. (Modified from Griffith and Hammond, 1989.)

were identified in aqueous extracts of cheese (Kowaleska et al., 1985) and Lb. bulgaricus cultures (Kowalewska et al., 1985; Reps et al., 1987).

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

69

COOH ;,H

""\/ \/" + YC

R;3

CH,-C-C,

H

- CH,

+ Methylglyoxal

Proline

I I

-

2 Methyl b d d e h y d e

(f 1

I

I I

I

yH3

7H3 S

I

I I

S

I

I

I

I I I I I

CH, I 0 II yH2 CH,C&C-NH-CH COOH

-

CH,

I

li' y".

I

CH,- C- NH-CH-COOH

I

I I I I

N-Propionyl methionine

I

N-acetyl methionine

I I

I

N-Ropionyl leucine

I I

I

N-Propionylphenylalanine FIG24d-h.

70

P. F. FOX AND J. M. WALLACE

Roudot-Algaron et al. (1993) isolated N-propionylmethionine (Fig. 24e), N-acetylmethionine (Fig. 24f), N-propionylleucine (Fig. 24g), and Npropionylphenylalanine (Fig. 24h) from the water-soluble fraction of Gruyere de Comte cheese: the first compound was cheesy, while the others had bitter flavors. AMINOACIDS H. NONPROTEIN

A number of nonprotein amino acids have been found in most cheese varieties, the principal being y-aminobutyric acid, formed by decarboxylation of Glu (Kaminarides et al., 1990) (Fig. 25a) and ornithine by arginase activity on Arg (Fig. 25b) (Broome et al., 1991). y-Aminobutyric is present at high concentrations in mold-ripened cheeses (Ismail and Hansen, 1972; Gripon et al., 1991). In Kopanisti cheese, y-aminobutyric acid levels increased 120-fold during ripening with a concomitant decrease in the concentration of Glu (Kaminarides et al., 1990). a-Aminobutyric acid has also been identified in many cheese varieties: Cabrales (Gonzalo de Llano et al., 1987; Ramos et al., 1987), Stilton (Madkor et al., 1987), Kopanisti (Kaminarides et al., 1990), and Gamonedo (Gonzalez de Llano et a]., 1991). However, its concentration was generally lower than that of y-aminobutyric acid. Enzymatic hydrolysis of the guanidino group of Arg leads to the formation of ornithine or citrulline (Hemme et al., 1982). Decreases in the concentration of Arg, particularly in the later stages of ripening, have been reported by a number of workers (Puchades et a]., 1989; Broome et a]., 1991). Certain strains of L. cremoris are capable of degrading arginine to a limited extent (Broome et a]., 1990). Low correlations between nonprotein amino acids and ripening time have been reported (Resmini et a]., 1969; Gonzalez de Llano et a]., 1991). VI. Chemistry of Cheese Off-Flavors

In addition to the characteristic desirable flavor of cheese, cheese frequently suffers from specific flavor defects. While normal desirable flavor has been difficult to define in chemical terms, the specific causes of many of the principal defects have been established more or less definitively and are described below. Bitterness in cheese results from the accumulation of hydrophobic short peptides that can originate from both asl-and p-caseins. Certain sequences in the caseins are particularly hydrophobic and when excised by proteinases can lead to bitterness. The action of chymosin has

71

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

Glutamkacid

decarboxylation

*

y -amino butyric acid

(Kaminarides et al., 1990)

(b)

W-CH

Arginine

Arginase

- COOH

> Ornithine

+

Urea

(Broome ad.,1991)

FIG.25. Formation of nonprotein amino acids.

been implicated in the formation of bitter peptides in cheese (see Lemieux and Simard, 1991, 1992), and thus factors that affect the retention and activity of rennet in the curd may influence the development of bitterness. Lawrence et al. (1972) pointed out the importance of both starter strain and rennet type and suggested that the major role of rennet in the development of bitterness may be the production of long peptides that are subsequently degraded to small bitter peptides by starter proteinases. These authors found a higher concentration of free amino acids in cheeses made with nonbitter starters, suggesting greater

72

P. F. FOX AND J. M. WALLACE

peptidase activity in these strains. Certain strains of starter and Penicillium spp. are associated with the development of bitterness (see Adda et nl., 1982), and it would appear that bitterness in cheese results from the action of chymosin on casein with the release of bitter peptides. These peptides accumulate in bitter cheese due to the inability of “bitter” starters to hydrolyze them to nonbitter peptides due to a deficiency in peptidase activity (Stadhouders and Hup, 1975), or perhaps by degradation by bacterial enzymes of peptides that otherwise would be too large to be perceived as bitter (Lemieux and Simard, 1991). The development of bitter taste is common in low-fat cheeses (Banks et a)., 1992). In full-fat cheese, a certain proportion of bitter peptides, being hydrophobic, probably partition into the fat phase, where they are less likely to be perceived as being bitter. The literature concerning bitterness in dairy products has been reviewed by Lemieux and Simard (1991, 1992). The majority of studies in which bitter peptides were identified were conducted in model systems consisting of isolated casein incubated with various enzymes. Bitter peptides have been identified in hydrolyzates of a,,-, as2-, and P-caseins. Para-K-casein is a potential source of bitter peptides (Visser, 1981), although in the few studies in which the hydrolysis of para-lc-casein was investigated it was found to be resistant to proteolysis. Bitter a,,-casein-derived peptides identified in hydrolyzates include f22-43 and f45-50 (see Sullivan and Jago, 1972), f23-34, f9l-100, and f145-151 (Hill and van Leeuwen, 1974). LeBars and Gripon (1989) considered that three short peptides produced from the C-terminal region of a,,-casein by plasmin (f198-207, f182-207, and f189-207) may be bitter. Bitter peptides found in hydrolyzates of p-casein include f203-208, fl95-209, f201-209 (see Sullivan and Jago, 1972), and f53-79 (Clegg et al., 1974). A number of bitter peptides isolated hom cheese or casein hydrolyzates are summarized in Table V. A number of authors have described the isolation of bitter peptides from cheese (e.g., Harwalkar and Elliott, 1971; Visser, 1977), but there is some disagreement as to the source of these peptides (Visser, 1981). However, unlike peptides produced from a,,-casein, the initial peptides produced from p-casein by chymosin are probably bitter, and thus the limited hydrolysis of this protein may contribute to bitterness. The concentration of NaCl has a major effect on the hydrolysis of p-casein by chymosin in solution (Fox and Walley, 1971) and in cheese (Kelly, 1993), and thus may be a factor in the development of bitterness. The formation of p-casein f193-209 (the primary product of chymosin action on p-casein and potentially bitter) was inhibited by increasing NaCl

73

FORMATION O F FLAVOR COMPOUNDS IN CHEESE TABLE V BITTERPEPTIDES ISOLATED FROM CHEESE‘ Cheese

Origin

Cheddar

aS,-CN fl4-17 a,l-CN fl7-21 a,,-CN f26-32 CX,~-CN26-33 8-CN f46-67 P-CN f46-84

P-CN f193-209 Gouda ondh

P-CN f84-89 P-CN f193-207 P-CN fl93-208 P-CN fl93-209

Sequence

Hydrophobicity Q, cal residue-’

E.V.L.N N.E.N.L.L A.P.F.P.E.V.F A.P.F.P.E.V.F.G Q.D.K.I.H.P.F.A.Q.T.Q.S.L. V.Y.P.F.P.G.P.1.P Q.D.K.I.H.P.F.A.Q.T.Q.S.L. V.Y.P.F.P.G.P.I.P.N.S.L.P.Q.N. 1.P.P.L.T.Q.T.P.V.V.V Y.Q.Q.P.V.L.G.P.V.R.G.P.F.P. 1.I.V

1162.5 1074.0 1930.0 1688.8

V.P.P.F.L.Q Y.Q.Q.P.V.L.G.P.V.R.G.P.F.P.1 Y.Q.Q.P.V.L.G.P.V.R.GP.F.P.I.1 Y.Q.Q.P.V.L.G.P.V.R.G.P.F.P. 1.I.V

1983.3 1686.7 1766.9

1580.5

1508.5 1762.4

1762.4

Alpkase

aSl-CN fl98-199

L.W

2710.0

Butterkase

P-CN f61-69

P.F.P.G.P.1.P.N.S

1792.2

“Adapted from Lemieux and Simard (1992).

concentrations (Kelly, 1993). The primary action of plasmin on p-casein probably does not produce bitter peptides. The fate of as2-casein in cheese is unclear, but plasmin can release potentially bitter peptides from this protein in solution (LeBars and Gripon, 1989). Guigoz and Solms (1974) found the bitter dipeptide, asl-CN fl98-199 (Leu-Trp), in Alpkese cheese. Stepaniak and Fox (1995) demonstrated the production of this peptide from asl-CN fl65-199 by a lactococcal endopeptidase (PepO). In addition to peptides, a number of other compounds can contribute to bitterness in cheese, including amino acids, amines, amides, substituted amides, long-chain ketones, and some monoglycerides (Adda et

d., 1982).

Rancidity, which is occasionally encountered in cheese, is due to excessive or unbalanced lipolysis caused by lipases-esterases from starter or NSLAB, psychrotrophs in the cheese milk or indigenous milk

74

P. F. FOX AND J. M. WALLACE

lipoprotein lipase. As discussed in Section II.C, a high concentration of lactic acid, perhaps arising from the retention of an excessive level of lactose in the cheese curd, imparts an acidic, harsh taste to cheese. Graders frequently use the term “over-acid” to describe a flavor defect in overripe cheese. The precise cause of this defect is not known, but it is probably due to excessive or unbalanced proteolysis rather than to a high concentration of lactic acid, The principal compounds responsible for fruitiness in Cheddar are ethyl butyrate and ethyl hexanoate, formed by esterification of FFAs with ethanol. Production of ethanol appears to be the limiting factor, as FFAs are present in cheese at relatively high concentrations (Bills et al., 1965). Ethyl esters are present at low concentrations in nonfruity cheeses and thus the fruity defect occurs as a result of increased production of ethanol or its precursors. The origin of “unclean” and related flavors in Cheddar was investigated by Dunn and Lindsay (1985), who quantified a number of Strecker-type compounds, including phenylacetaldehyde, phenethanol, 3-methylbutanol, 2-methylpropanol, phenol, and p-cresol. Phenylacetaldehyde concentrations were elevated in cheeses with an “unclean rosy” off-flavor, and the addition of this compound to clean-flavored mild Cheddar reproduced this defect. At higher concentrations (>400 pg/kg), phenylacetaldehyde imparted astringent, bitter, and stinging flavors to cheese. Concentrations of phenethanol were similar in most of the cheeses studied (-100 pg/kg). p-Cresol was found to impart a putrid “utensil”-type flavor when present in high concentrations (>lo0 pg/kg-l). Dunn and Lindsay (1985) also discussed the potential of short-chain fatty acids to potentiate the flavor impact of p-cresol. Branched-chain Strecker-type aldehydes (3-methylbutanal, 2methylbutanal, and 2-methylpropanal) did not cause flavor defects when added at concentrations below 200 pg/kg to clean-flavored cheese. Phenol (100-225 pg/kg-l) appeared to have no detrimental effect on cheese flavor and indeed enhanced the sharpness of Cheddar flavor (Dunn and Lindsay, 1985). Dunn and Lindsay (1985) added quinine to cheese to simulate the bitterness often associated with the development of Strecker-type compounds. It enhanced the flavor impact of these compounds. ACKNOWLEDGMENTS

The authors would like to express their thanks to Ms. Anne Cahalane for her assistance in preparing the manuscript.

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Manning, D. J., Chapman, H. R., and Hosking, Z. D. (1976). The production of sulphur compounds in Cheddar cheese and their significance in flavour development. J. Dairy Res. 43,313-320. Mariani, P., Bonomi, A., Sabbioni, A,, Lucchelli, L., Blanco, P., Zanzucchi, G., and Florentini, L. (1993). Caratteristiche organolittiche e chimico-bromatologiche des formaggio Parmigiano-Reggiano prodottd con il latte delle razze bruna e frisono Italiana. Rev. Sci. Alinientazione 22, 91-102. Marsili, R. (1985). Monitoring chemical changes in Cheddar cheese during aging by high performance liquid chromatography and gas chromatography techniques. J. Dairy Sci. 68, 3155-3161.

McEwan, J. A. Moore, J. D., and Colwill, J. S. (1989). The sensory characteristics of Cheddar cheese and their relationship with acceptability. J. Soc. Dairy Technol. 4, 112-1 17.

McGoldrick, M. A. (1996). “Intervarietal Comparison of Proteolysis in Cheese.” MSc. Thesis, National University of Ireland, Cork. McGugan, W. A. (1975). Cheddar cheese flavour. A review of current progress. J. Agric. Food Chem. 23, 1047-1050. McSweeney, P. L. H., and Fox, P. F. (1997). Chemical methods for characterization of proteolysis in cheese during ripening. Le Lait 77,41-76. Meinhart, E., and Scheier, P. (1986). Study of the flavour compounds from Parmigiano Reggiano cheese Milchwissenschaft 41,689-691. Mooney, J. S. (1997). “Isolation and Identification of the Water-Insoluble Peptides in Cheddar Cheese.” M.Sc. Thesis, National University of Ireland, Cork. Mottar, J. F. (1989). Effect on the quality of dairy products. In “Enzymes of Psychrotrophs of Raw Foods” (R. C. McKellar, ed.), pp. 227-243. CRC, Boca Raton, FL. Muir, D. D., and Hunter, E. A. (1992). Sensory evaluation of Cheddar cheese: The relation of sensory properties to perception of maturity. J. SOC.Dairy Technol. 45,23-30. Mulder, H.(1952). Taste and flavour forming substances in cheese. Neth. MilkDairy J. 6 , 15 7-1 67.

Nakae, T., and Elliott, J. A. (1965). Volatile fatty acids produced by some lactic acid bacteria, I: Factors influencing production of volatile fatty acids from casein hydrolyzate. J. Dairy Sci. 48, 287-292. Nelson, J. H., Jensen, R. G., and Pitas, R. E. (1977). Pegastric esterase and other oral lipases-a review. J. Dairy Sci. 60, 327-362. Ney, K. H. (1971). Prediction of bitterness of peptides from their amino acid composition. Z. Lebensm. Forsch. 147,64-68. Noomen, A. (1983). The role of the surface flora in the softening of cheeses with a low initial pH. Neth. Milk Dairy]. 37,229-232. Nsar, M. M., and Younis, N. A. (1986). Effect of adding alanine, phenylalanine and proline on the properties of Romi cheese. Egypf I. Food Sci. 14,385-388. O’Callaghan, D. M. (1994). “Physicochemical, Functional and Sensory Properties of Milk Protein Hydrolysates.” Ph.D. Thesis, National University of Ireland, Cork. Olivecrona, T., and Bengtsson-Olivecrona, G. (1991). Indigenous enzymes in milk: Lipase. In “Food Enzymology” (P, F. Fox, ed.), Vol. 1, pp. 62-78. Elsevier, London

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Olivecrona, T.. Vilaro, S., and Bengtsson-Olivecrona, G. (1992). Indigenous enzymes in milk, 11: Lipases in milk. In “Advanced Dairy Chemistry” (P. F. Fox, ed.), Vol. 1, pp. 292-310. Elsevier, London. Omar, M. M. (1984). Microstructure, free amino acids and free fatty acids in Ras cheese. Food Chem. 15, 19-29. Omar, M. M., and El-Zayat, A. I. (1986). Ripening changes of Kashkaval cheese made from cows milk. Food Chem. 22, 83-94. Oterholm, A., Ordal, Z. J., and Witter, L. D. (1970). Purification and properties of glycerol ester hydrolase (lipase) from Propionibacterium shermanii. Appl. Microbiol. 20, 16-22.

Parliament, T. H., Kolor, M. G., and Rizzo, D. J. (1982). Volatile components of Limburger cheese. J. Agric. Food Chem. 30, 1006-1008. Paulsen, P. V., Kowalewska, J., Hammond, E. G., and Glatz, B. A. (1980). Role ofmicroflora in production of free fatty acids and flavor in Swiss cheese. J. Dairy Sci. 63,912-918. Pham, A.-M., and Nakai, S. (1984). Application of stepwise discriminant analysis to high performance liquid chromatography profiles of water extract for judging ripening of Cheddar cheese. 1. Dairy Sci. 67, 1390-1396. Polo, C., Ramos, M., and Sanchez, R. (1985). Free amino acids by high performance liquid chromatography and peptides by gel electrophoresis in Mahon cheese during ripening. Food Chem. 16, 85-96. Ponce-Trevino, R., Richter, R. L., and Dill, C. W. (1987). Influence of lactic acid bacteria on the production of volatile sulphur containing compounds in Cheddar cheese slurries. J. Dairy Sci. 70, 59 (Abstr.). Ponce-Trevino, R., Richter, R. L., and Dill, C. W. (1988). Observations on effects of oxidation-reduction potential on volatile sulphydryl production in Cheddar cheese slurries. J. Dairy Sci. 71, 278 (Abstr.). Puchades, R., Lemieux, L., and Simard, R. E. (1989). Evolution of free amino acids during ripening of Cheddar cheese containing added lactobacilli strains. I. Food Sci. 54, 885-888, 946.

Ramos, M., Caceres, I., Polo, C., Alonso, L., and Jaurez, M. (1987). Effect of freezing on soluble nitrogen fraction of Cabrales cheese. Food Chem. 24, 271-278. Rattray, F. P., and Fox, P. F. (1997a). Purification and characterization of an intracellular esterase from Brevibacterium h e n s ATCC 9174. Int. Dairy J. In press. Rattray, F. P., and Fox, P. F. (1997b). Purification and characterization of an intracellular aminopeptidase from Brevibacterium linens ATCC 9174. Le Lait 77, 169-180. Rattray, F. P., Bockelmann, W., and Fox, P. F. (1995). Purification and characterization of an extracellular proteinase from Brevibacterium linens ATCC 9174. Appl. Environ. Microbiol. 61, 3454-3456. Rattray, F. P., Fox, P. F., and Healy, A. (1996). Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine a,,-casein. Appl. Environ. Microbial. 62, 501-506. Rattray, F. P., Fox, P. F., and Healy, A. (1997). Specificity of an extracellular proteinase from Brevibacterium linens ATCC 91 74 on bovine p-casein. Appl. Environ. Microbiol. 63, 2468-2471.

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Reps, A., Hammond, E. G., and Glatz, B. A. (1987). Carbonyl compounds produced by growth of Lactobacillus bulgaricus. J. Dairy Sci. 70, 559-562. Rice, G. H., Stewart, F. H. C., Hillier, A. J., and Jago, J. R. (1978). Uptake of amino acids and peptides by Streptococcus lacfis.J. Dairy Res. 45,93-107. Roger, S.,Degas, C., and Gripon, J.-C. (1988). Production of phenylethyl alcohol and its esters during ripening of traditional Camembert. Food Chern. 28, 129-140. Roudot-Algaron, F., LeBars, D., Einhorn, J., Adda, J., and Gripon, J.4. (1993). Flavour constituents of aqueous fraction extracted from Comte cheese by liquid carbon dioxide. J. Food Sci. 58,1005-1009. Samples, D. R. (1985). “Some Factors Affecting the Production of Volatile Sulphydryl Compounds in Cheddar Cheese Slurries.” Ph.D. Thesis, Texas A&M University. Cited in Urbach, 1995. Sentheshanmuganathan, S . (1960). The mechanism of the formation of higher alcohols from amino acids by Saccharomyces cervisiae. Biochern. J. 74,568-576. Sharpe, M. E., and Franklin, J. G. (1962). Production of hydrogen sulphide by lactobacilli with special reference to strains isolated from Cheddar cheese. In “Proc. 8th Int. Congr. Microbiol. BJI,” Vol. 3, p. 46. Sharpe, M. E., Law, B. A., and Phillips, B. A. (1976). Coryneform bacteria producing methanethiol. J. Gen. Microbiol. 94,430-435. Simonart, P. and Mayaudon, J. (1956). Chromatographic analysis of cheese, 3. Aromatic Acids. Ned. Melk Z. 10, 261-267. Singh, T. K., Fox, P. E , and Healy, A. (1995). Water soluble peptides i n Cheddar cheese: Isolation and identification of peptides in the UF retentate of water-soluble fractions. J. Dairy Res. 62,629-640. Singh, ’T. K., Fox, P. F.,and Healy, A. (1997). Isolation and identification of further peptides in the diafiltration retentate of the water-soluble fraction of Cheddar cheese. J. Dairy Res. 64.In press. Smith, T.A. (1981). Amines in food. Food Chern. 6, 169-200. Ssrhaug, T., and Ordal, Z. J. (1974). Cell-bound lipase and esterase of Brevibacterium linens. Appl. Microbiol. 27,607-608. Ssrhaug, T., and Stepaniak, L. (1997). Psychrotrophs and their enzymes in milk and dairy products: Quality aspects. Trends Food Sci. Technol. 8,35-41. Stadhouders, J., and Hup, G. (1975). Factors affecting bitter flavour in Gouda cheese. Neth. Milk Dairy J. 29, 335-353. Stepaniak, L., and Fox, P. F. (1995). Characterization of the principal intracellular endopeptidase from Lactococcus lactis ssp. lactis MG 1363. Int. Dairy]. 5,699-713. Sullivan, J, J., and Jago, G. R. (1972). The structure of bitter peptides and their formation from caseins. Aust. J. Dairy Technol. 27,98-104. ’Thomas, T. D. (1987). Acetate production from lactate and citrate by non-starter bacteria in Cheddar cheese. N. Z. J. Dairy Sci. Technol. 22,25-38. Thomas, T. D., and Crow, V. L. (1983). Mechanism of D(-)-lactic acid formation in Cheddar cheese. N . Z. J. Dairy Sci. Technol. 18, 131-141. Thomas, T.D., McKay, L. L., and Morris, H. A. (1985).Lactate metabolism by pediococci isolated from cheese. Appl. Environ. Microbial. 49,908-91 3.

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The Role of Microorganisms in

Soy Sauce Production DESMONDK. O’TOOLE Department of Biology and Chemistry City University of Hong Kong Kowloon, Hong Kong

I. Introduction 11. Types and Composition of Soy Sauce A. Types of Shoyu B. Amino Acid Composition of Soy Sauce C. Organic Acids in Soy Sauce D. Sugars in Soy Sauce 111. Aroma and Flavor of Shoyu IV. Soy Sauce Production A. Composition of Raw Materials B. The Manufacture of Soy Sauce C. Koji D. Moromi E. Use of Continuous Fermentation Processes to Produce Soy Sauce V. Effect of Water Activity on Microorganisms VI. Yeasts A. Effect of Oxygen on Yeasts B. Alcohol Production by Zygosucchuromyces rouxii VII. Bacteria VIII. Production and Metabolism of Amino and Organic Acids in Moromi A. Role of Tetrugenococcus halophilus B. Interaction Between Organic Acids and Soy Sauce Organisms IX. Production and Fate of Other Substances X. Possible Role of Metal Ions in Soy Sauce Production A . Metal Ions i n Metabolism B. Investigation of Metal Ions i n Soy Sauce and Its Manufacture C. Adequacy of Minerals in Raw Materials XI. Conclusion References

I. Introduction

Soy sauce is a salty condiment commonly used in Eastern Asia that is made from soy beans with varying amounts of wheat or no wheat at all. It is known as shoyu in Japan, chiang-yu (or -yi) in China, kecup in Indonesia, kunjang in Korea, toyo in The Philippines, and see-ieu in Thailand (Djien, 1982; Beuchat, 1985; Fukushima, 1989). It provides 67 ADVANCES IN APPLIED MICROBTOLOGY, VOLUME 45 Copyright 0 1997 by Academic Press All rights of reproduction in any form reserved. 0065-2164/97$25.00

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flavor in an otherwise bland diet, and nutritionally it provides salt (NaCI) and predigested proteins in a diet that is traditionally protein poor. It has been made for centuries on a small scale in many towns and villages in Asia, but since 1950, particularly in Japan, the manufacturing process has been studied and modernized so that its manufacture is now concentrated in large factories using modern, controlled methods of production (Sasaki and Nunomura, 1993). In Japan, soy sauce fermentation is a major food manufacturing activity. More than 1.1 million kiloliters of soy sauce was produced in 1986 by 3000 producers, and the Kikkoman Company supplied 30% of the market (Fukushima, 1989).By 1990 there were 2871 manufacturers, five of which produced about 50% of the total production (Sasaki and Nunomura, 1993). While modern methods are used for most of the soy sauce produced in Japan, and factory production in other Asian countries is growing, soy sauce is still produced by methods involving no modern technological inputs (Roling et a!., 1994a).

BKIEFHISTORY Soy sauce “was derived from a Chinese food called chiang,” which was basically a mash (Fukushima, 1989). First recorded in a book published around 1200 B.c.,chiang was made by growing yellow aspergilli on millet (to produce the equivalent of modern-day koji),then the molded grain, animal, poultry, or fish flesh and salt were mixed with a “good liquor” in a bottle for 100 days. The use of a liquid similar to soy sauce from this type of product was first mentioned during the Han dynasty (25-220 A.D.). The first mention of the use of soy beans in this product was in 535 A.D., and the first record of the use of all the materials used in modern soy sauce production was published between 1271 and 1368 A.D. The method for making chiang-yuwas not described in detail until the sixteenth century (Wang and Fang, 1986). From China, the fundamental principles of soy sauce production spread to other Southeast Asian countries and to Japan about the seventh century (Fukushima, 1989). During the seventeenth and eighteenth centuries, large quantities of soy sauce were exported from Japan by the Dutch to other parts of Asia and to Europe. During the twentieth century, the manufacture of soy sauce has spread from Japan to the United States, where a plant was first opened in 1909, which was not successful. However, production continued, and in 1972 a more modern plant was opened by the Kikkoman Company. Soy sauce production, as well as the production of a number of other traditional Asian foods (e.g., miso and rice wine) is based on a two-step

MICROORGANISMS IN SOY SAUCE PRODUCTION

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microbial process that has some similarities to beer production (Fukushima, 1989; Yong and Wood, 1974). Fungi are allowed to grow on the pretreated raw material, soy bean and wheat, and the molded grain is then steeped in brine. The enzymes from the mold break down the constituents (proteins, starch, and cellulose) in the legume and grain, and subsequent bacterial and yeast growth produce the characteristic flavor of the soy sauce. Beer production depends on enzymes (amylases) produced by the sprouting of the barley grain to break down the cooked starch in the endosperm to mono- and disaccharides that are subsequently fermented by yeasts. Our scientific knowledge of soy sauce production and manufacture has increased rapidly over the last 45 years, and there have been several published reviews about soy sauce. Most of the important work has been done and published in Japan. Some idea of the quantity of Japanese work can be gleaned from two Japanese reviews entitled “Review of Research Reports on Soy Sauce and Miso in 1986” (Mori, 1987), and “Reports on Soy Sauce and Miso, Published in Japan in 1981” (Mori, 1982), in which 534 and 743 references, respectively, to soy sauce and miso are listed. Also, 539 of the 1092 papers in which soy sauce occurs in Food Science and Technology Absfracfsfrom 1969 to December 1996 were published in Japanese. An early English-language review of soy sauce is that of Yokotsuka (1960), while non-Japanese perspectives on the topic were presented by Yong and Wood (1974) and Wood (1982). Recent English reviews from Japan include those of Yokotsuka (1985, 1986) and a really comprehensive review from Fukushima (1989). However, no critical reviews in English seem to have approached soy sauce from a microbiological perspective, as other aspects of soy sauce such as the biochemical, chemical, and technological aspects have attracted more attention. The purpose of this review is to examine soy sauce in relation to the role of microorganisms in its production with a view to how they produce the soluble components and flavors in the sauce, and what factors can and do influence the quality of the final product.

II. Types and Composition of Soy Sauce

The spread of the manufacture of soy sauce to many parts of Southeast Asia and the subsequent growth of cottage-style production led to differences in production techniques from place to place that ultimately impinged on the composition and types of soy sauce. In some countries

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DESMOND K. O'TOOLE

TABLE I THEGENEKAL COMPOSITION" Product Koikuchi shoyu, Japan lisukuchi shoyu, Japan Soy sauce, Taiwan Soy sauce, Korea Soy sauce, Hong Kong Soy sauce, Philippines Soy sauce, Singapore Soy sauce, Malaysia Soy sauce, USA

OF SOME SOY SAUCES FROM VARIOUS PARTS OF THE WORLD

BB

NaCl

TN

23.6 22.2 25.6 21.9 28.5 23.3 30.1 23.9 22.8

17.0 18.0 15.6 17.3 26.2 24.7 24.1 18.3 16.5

1.70 1.18 2.05 1.50 1.54 0.76 1.97 1.17 1.65

RS(1S) Alcohol 5.07 4.00 5.95 2.10 4.22 1.06 4.81 8.50 3.70

2.50 2.00 0.86 0.39 0.00 0.01 0.00 0.03 2.07

Color

++

+ ++ ++ if+

++ +++ +++ ++

aBB = spccific gravity, dcgrees Baum6; NaCl = sodium chloride (g 100 ml-'I: TN = total nitrogen (g 100ml-I); KS(1S) = reducing sugar (invert sugar) (g 100 rn1-l): alcohol = ethanol (ml 100 nil-'). bAdaptcd from Table 2 of Yokotsuka (1985).

these interregional differences have been formalized into systems that define the various types of sauce. Although there are similarities in the composition of soy sauce, as, for example, there are similarities between cheeses, there are significant variations in the composition of the soy sauces produced. The Indonesians add sugar to the soy sauce, so that they have two basic kinds of soy sauce, kecap asin and kecap rnanis (the sugared sauce) (Roling et al., 1994a). The general composition of some soy sauces from different parts of the world is shown in Table I. A. TYPESOF SHOYU Japan has an organized system of classification to define types of soy sauce. Such systems probably exist outside Japan, and other countries do set standards for soy sauce (see, e.g., Chang et a]., 1987). In Chinese societies, dark and light soy sauces are marketed (Anon., 1984). The types of soy sauce recognized by the Japanese Government and their typical composition are listed in Table 11. However, the typical ethanol levels in Table I1 for tamari, saishikomi, and shiro shoyu differ significantly from values published by Sasaki and Nunomura (1993), who reported typical values for ethanol in tamari, saishikomi, and shim shoyu of 3.41, 2.59, and 3.85% (v/v), respectively. These differences may be due to better control of the process used in the production of

91

MICROORGANISMS IN SOY SAUCE PRODUCTION

TABLE I1 TYPICAL PERCENTAGE

COMPOSITIONS OF

FIVEVARIETIES OF SOY

RECOGNIZED BY THE JAPANESE

Sauce

(shoyul

Koikuchi Usukuchi Tamari Saishikomi Shiro

NaCl w/v

Total nitrogen w/v

16.9 18.9

1.57 1.19

19.0 18.6

2.55 2.39

19.0

0.5

Formol nitrogen w/v 0.94

0.80 1.05 1.11 0.24

SAUCE

GOVEFNMENT~ Reducing Alcohol sugar wlv vlv

Color

3.0 4.2

2.3

Deep brown

2.1

5.3 7.5

0.1 trace trace

Light brown Dark brown Dark brown Yellow-tan

20.2

‘Specific gravity is from 22.0 to 29.9°Be; pH is from 4.6 to 4.8. Adapted from Table 2 of Fukushima (1989).

the minor types of shoyu through the application of results obtained from research on koikuchi shoyu, or the direct addition of ethanol. The differences between these types of soy sauce are quite significant, and they come about primarily from the proportions of raw materials, soy beans, and wheat used to make the sauce. Koikuchi is made with about equal quantities of wheat and soy protein, whereas usukuchi is made with more wheat (Yokotsuka, 1985), but Fukushima (1989) says that the difference between these two sauces is due only to the manner in which the fermentation is conducted to reduce the development of color. Tamari is made using a soy:wheat ratio of about lO:l, while saishikomi is made using equal quantities of soy and wheat, with the variation that raw soy sauce is used and not freshly made brine. Shiro is made using a very high level of wheat in the soy-wheat mixture (Fukushima, 1989);hence the high level of reducing sugar in this shoyu. The most popular Japanese soy sauce is koikuchi shoyu, which made up 82.9% of the shoyu produced and checked by the Japan Agricultural Standard in 1990; tamari shoyu comprised only about 1.9% of production (Sasaki and Nunomura, 1993). Koikuchi shoyu has quite a high alcohol content (=3%), which has long been considered important for stabilizing the quality of and adding flavor to Japanese shoyu (Iwasaki ef a]., 1991a). As Table I shows, ethanol is not a significant constituent in soy sauces from other parts of Asia. Tamari-type soy sauce is the most widely produced soy sauce type in Southeast Asia (Fukushima, 19891, and it is also the main Chinese type of soy sauce (Sasaki and Nunomura, 1993), because the cereal-legume mixture for

92

DESMOND K. O'TOOLE

tamari production contains about 10% wheat, which is similar to the proportion used in Chinese soy sauce (Fukushima, 1989). However, high-wheat sauces (e.g., sauces made with wheat:soy bean ratios of 1:2 and 1:l) are also produced in China (Butler, personal communication, 1997). In addition, Fukushima (1989) says that the process now used in China involves a defatted soy bean meal-wheat bran mixture at a 6:4 ratio that is incubated for 24 h for mold growth and is then held for 3 weeks at a temperature in the moromi of 40-45OC, which is for a very much shorter period and at a much higher temperature than that used traditionally by the Japanese. On the other hand, Chiao (1986) does not mention such a shortened method of production in China. B. AMINOACIDCOMPOSITION OF SOY SALJCE The amino acid composition of the five types of Japanese shoyu and some Chinese and Korean soy sauces are shown in Table I11 (Ren et al., 1993a). The data for koikuchi shoyu are from ten samples, and the data for the Chinese and Korean soy sauces are from eight samples (data from one of the published Chinese samples have been dropped from the data herein because the raw materials for the sauce included monosodium glutamate). These data show that there is great variation in the amino acid content between the various Japanese types of shoyu, but the variation between various samples of koikuchi shoyu was not very great. The variation in amino acid contents between samples of Chinese sauce and between samples of Korean sauce was almost as great as the variation between the various types of Japanese shoyu, which may reflect variation in the raw materials used and in the processes used in Chinese and Korean sauces similar to those from Japan but which are not formally recognized in those countries as they are in Japan. The total nitrogen data for these samples reflect this, as the values for koikuchi, usukuchi, tamari,saishikomi, and shiro are 1.49, 1.02, 1.49, 1.45, and 0.51 mg . g-l, respectively, while the figures for Chinese and Korean sauces fall in the ranges 0.82-2.03 and 0.86-1.21 mg . g-l, respectively (Ren et al., 1993a). However, the above data from Ren et al. (1993a) differ significantly from those published by Sasaki and Nunomura (1993) (Table IV). Even after taking into account the different methods for expressing the concentrations, mg . g-' versus mg . ml-l, there is significant variation, particularly with the data from tamari shoyu. Fukushima (1989) says that the amino acid composition of the final product is almost the same as the original mixture of soy beans and wheat, with some exceptions. To assess whether soy sauce contains

MICROORGANISMS IN SOY SAUCE PRODUCTION

93

TABLE 111 THEAMINO AclD COMPOSITION (mg . g-') Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine Wine Methionine Isoleucine Leucine Tyrosine Phenylalanine

OF SHOYu AND CHINESE AND

Usukuchi

Tamari

Saishikomi

3.71-4.31

2.76

0.31

2.13

1.00-1.32

1.29

0.95

1.04

1.93-2.39

2.60

5.41

2.53

3.35-5.42

2.15

1.87

2.77-2.93

1.60

3.61-4.16 12.38-1 7.33

Koikuchi

Shim

KOREANSOY

SAUCES

Chinese

Korean

1.15

1.15-4.44

0.97-4.53

0.25

0.42-1.21

0.51-1.59

0.72

0.61-3.41

0.34-4.53

4.95

0.50

1.85-4.85

1.88-7.45

1.63

2.35

0.53

0.95-2.37

1.33-2.81

1.92

2.07

3.44

0.79

1.31-3.58

1.87-3.76

4.20

3.90

7.82

5.15

2.28-9.26

5.85-15.36

3.464.15

2.01

0.28

2.91

1.37

1.20-3.91

2.00-4.07

2.03-2.58

0.98

1.34

1.84

0.36

0.55-3.45

0.92-3.50

4.15-6.49

1.69

1.86

3.48

0.80

1.59-5.65

2.08-5.06

0-0.05

0.06

0.95

0.12

0.09

0-0.20

0-0.09

3.73-4.27

2.05

1.98

3.36

0.70

1.49-4.00

1.59-3.48

0.94-1.14

0.82

0

0.92

0.24

0.42-0.91

0.43-0.85

3.05-3.68

1.91

2.14

3.00

0.58

1.29-2.61

1.46-2.59

4.61-5.65

2.48

3.46

4.50

1.00

2.23-3.96

2.37-4.53

0.66-0.81

0.76

0.35

0.91

0.64

0.56-1.83

0.35-1.35

1.22-2.14

1.48

1.95

2.34

0.83

0.59-1.50

0.28-1.56

'Adapted from Table 2 of Ren et of. (1993a).

amino acids in the proportions found in the raw materials, the data of Sasaki and Nunomura (1993) and Ren et al. (1993a) for the amino acid compositions of koikuchi and tarnari shoyu and the calculated values for amino acids from published values of amino acids for soy bean and wheat (Table IV) were used to determine the ratios of each amino acid in soy sauce and in the raw materials used. When the values are compared (Tables V and VI), we can see that the equivalence of the amino acid content with the constituents in the raw materials might not be the case and the values obtained really depend on the analytical results for the amino acids one chooses to use. In koikuchi shoyu, the ratio of amino acid concentrations in the shoyu to the theoretical amino acid levels in the raw materials is significantly higher for nine of the amino acids based on the results of Sasaki and Nunomura (1993), but only six are higher based on the results of Ren et al. (1993a). For tarnari shoyu, the discrepancy between the raw materials and the shoyu seems not so great by the results of Sasaki and Nunomura (1993), but by the results of Ren et al. (1993a) there are significant discrepancies between the s h o p values and the theoretical values for the raw materials.

94

DESMOND K. O'TOOLE

TABLE IV THEAMINO ACIDCOMPOSI'I'ION 01' SHoW (mg. rn-'), SOYBEANS (g . kg-l SED), DEFATTED SOY BEAN(g kg-'1, OF HARD R E D WINTER COMMERCIAL MILL WHEATMix (g . kg-' DRYWEIGHT) Soy beans

Shoyu"

Wheatb

Dc-

KOComponent Lysine Histidine Arginine Aspartic acid 'l'hreonine Serine Glutamic acid Proline Glycine Almine Cystine Wine Methionine Isoleucine Leucine Tyrosine Phenylalanine Protein Milling yield, 70

Tamari

Whole

fatted

kuchi

beansC

flourd

4.09

6.44

26.5

1.67

1.80

10.5

4.34

5.41

2.58

9.15

3.12

~

Whole grain

Flour

28.8-29.3

4.0

2.68

7.97

11.2-11.5

3.06

3.17

4.96

30.1

32.4

5.59

4.27

11.33

48.6

-

7.32

5.12

12.92

4.91

16

18.1-1 8.9

4.52

6.20

4.40

6.71

21.3

24.5-25.2

6.78

3.66 5.86

11.88

18.18

77.7

43.62

45.02

36.82

4.07

6.08

22.8

-

13.43

14.03

12.21

2.50

3.76

17.4

18.9-22.9

5.72

4.27

9.74

7.05

7.71

17.7

-

4.92

3.54

8.67

ND

0.28

5.5

6.8-6.9

2.0

1.95

-

4.82

6.73

20

22.5-23.4

6.12

5.12

9.03

Bran

8.14

1.17

1.17

5.3

6.3-6.9

1.R6

1.83

2.48

4.24

4.80

18.9

22.0-23.9

5.19

4.76

6.73

6.57

6.08

32.3

34.2-35.2

9.58

8.78

11.86

1.23

2.28

13

12.8-1 5.8

2.66

2.81

3.72

4.43

5.92

20.6

22.0-22.7

5.99

5.73

7.08

-

-

I22

177

-

-

133

-

-

100

72.8

21

ONunomura and Sasdki (1993). 'Mattern (1991).Table 40, calculated to g kg-' dry weight from g kg protein dry wcight using protein values in tahle above. 'Considine and Considine (1982). ' k d d r o u p and Smith (1989).

Three conclusions can be drawn from these data. The first is that amino acid values published for the various soy sauces may be very unreliable. This could reflect the difficulty of determining the amino acids in a soy sauce matrix, or the inherent difficulty of determining certain amino acids, The second is that a detailed study of the raw ingredients of batches and of the resulting soy sauce would be necessary to determine any equivalence between the amino acids in the raw

95

MICROORGANISMS IN SOY SAUCE PRODUCTION TABLE V ACIDSIN KO~KUCHI SHOYU AND IN THE RAW MATERIALS A COMPARISON OF AMINO OF SOUBEANAND WHEAT

Concentration of amino acids

Ref. 2

%, raw materials

Ref. 1

Ref. 2

6.0

6.7

4.79

125

140

2.5

2.2

2.45

100

90

6.4

3.6

5.84

109

62

Yo I

Ref. l n mg . ml-'

Ref. 2 m g ' g-'

Ref. 1

Lysine

4.09

3.97

Histidine

1.67

1.32

Amino acid

Amino acid ratios, sh oyu :raw materials, YO

shop

Arginine

4.34

2.16

Aspartic acid

2.58

5.42

3.8

901

8.78

43

104

Threonine

3.12

2.77

4.6

4.7

3.69

124

127

Serine Glutamic acid Proline

4.4

4.17

6.5

7.0

5.20

124

135

11.88

13.0

17.4

21.9

26.09

67

84

4.07

4.3

6.0

7.2

7.89

76

91

Glycine

2.5

2.85

3.7

4.3

4.31

85

100

Alanine

7.05

4.77

10.3

8.0

4.05

256

198

Cystine

0

0.05

0

0.1

1.44

0

6

Valine

4.82

4.23

7.1

7.1

4.79

148

148 132

Methionine

1.17

1.08

1.7

1.8

1.36

126

Isoleucine

4.24

3.05

6.2

5.1

4.30

145

119

Leucine

6.57

4.54

9.6

7.6

7.61

127

100

Tyrosine

1.23

0.81

1.8

1.4

2.61

69

54

Phenylalanine

4.43

1.22

6.5

2.1

4.81

135

44

68.16

59.44

100

100

100

100

100

Total

'Ref. 1 is Sasaki and Nunomura (1993):Ref. 2 is Ren et al. (1993a).The data from the shoyu samples with around 6 g . 100 ml-' of amino acids were used for this calculation.

materials and the resulting soy sauce. Finally, it is almost certain that amino acids are metabolized by microorganisms (see below), and, if so, the levels of amino acids are probably in part the consequence of microbial activity and not a straight conversion of protein in the raw materials to amino acids. If so, the levels of amino acids in soy sauce will never mirror the levels found in the raw materials and may vary depending on particular strains of microorganisms present in the brine stage.

96

DESMOND K. O'TOOLE

TABLE V1 A COMPARISON OF AMINOAcrns IN TAMAItl SllOYU AND IN T I E RAW MATERIAI.S 01'SOYBEANAND WHEAT

Concentration of amino acids Ref. I

Ref. 2

%, raw materials

Yo,

~

Amino acid

Ref. '1 mg. nil-'

Ref. 2 rng

. g'

shoy

~

Amino acid ratios, shoyuxaw materials, YO Ref. 1

Ref. 2

Lysine

6.44

0.31

6.6

1.02

6.20

107

17

Hi sti dine

1.80

0.95

1.9

3.12

2.57

72

121

Arginine

5.41

5.41

5.6

17.77

7.12

78

250

Aspartic acid

9.15

1.87

9.4

5.98

11.37

83

53

Threonine

4.91

1.63

5 .O

5.35

3.90

129

137

Serine

6.71

2.07

6.9

6.80

5.25

131

130

Glutamic acid

18.18

3.90

18.7

12.81

20.60

91

62

Proline

6.08

0.28

6.2

9.20

6.09

102

151

G1y cin e

3.76

1.34

3.9

4.4

4.31

90

102

Alanine

7.71

1.86

7.9

6.11

4.31

184

142

Cystine

0.28

0.95

0.3

3.12

1.38

21

226

Valinti

6.73

1.98

6.9

6.5

4.92

141

132

Methionine

1.17

0

1.2

0

1.32

91

0

Isoleucine

4.80

2.14

4.9

7.03

4.60

107

155

Leucine

6.08

3.46

6.2

11.34

7.92

79

143

Tyrosine

2.28

0.35

2.3

1.15

3.10

76

37

Phenylalanine

5.92

1.95

6.1

6.4

5.04

121

127

Total

97.41

30.45

100

100

100

100

100

'Rel'. 1 is Sasaki and Nunomura (1993);Ref. 2 is Ren ef ul. (1993a)

c.

ORGANIC

ACIDSIN SOY SAUCE

A variety of organic acids is found in soy sauce. Ushijima et al. (1982) reported levels of pyroglutamic, lactic, acetic, formic, citric, succinic, pyruvic, and malic acids found in individual batches of koikuchi and usukuchi shoyu, as shown in Table VII. More recent average figures (Sasaki and Nunomura, 1993) for some of these acids found in the five basic types of Japanese s h o p are shown in Table VIII. Lactic acid in koikuchi shoyu varied from about 3 to 1 2 mg . ml-l, with a median

97

MICROORGANISMS IN SOY SAUCE PRODUCTION

TABLE VII CONCENTRATIONS OF ORGANIC ACIDSFOUND IN T W O TYPES OF JAPANESE SHOYU (mg . m1-I)' PyrogluSample

Koikuchi s h o p A B C

D E F G H

tamic

Lactic

Acetic

Formic

Citric

Succinic

Total

3.30 3.65 3.78 2.78 7.89 5.17 3.59 5.11

10.22 8.81 9.18 6.39 2.30 7.15 12.64 12.49

1.92 1.93 1.70 1.43 1.09 1.74 1.97 2.14

0.16 0.18 0.15 0.19 0.22 0.22 0.20 0.25

0.18 0.40 0.18 0.81 2.15 0.84 0.12

0.34 0.37 0.37 0.50 0.59 0.43 0.31 0.34

16.20 15.40 15.40 12.30 14.70 15.60 18.80 20.40

1.63 2.67 1.62 5.64 2.87 4.90

6.74 6.74 7.94 2.29 4.70 3.92

1.25 1.22 1.60 0.70 1.73 0.67

0.08 0.09 0.17 0.08 0.14 0.12

0.39 0.35 0.33 0.42 0.29 0.43

10.10 11.70 12.10 10.70 10.60 11.90

trace

Usukuchi shoyu I

7 K L M N

trace 0.39 0.45 1.52 0.87 1.86

aAdapted from Table 5 of IJshijima et al. (1982). Malic acid concentration was either 0 or trace, except for two samples of koikuchi shoyu in which it was 0.12 and 0.42 mg . mlP. Pyruvic acid concentration ranged from 0 to 0.04, but one sample of usukuchi shoyu contained 0.27 rng . ml-'.

concentration of about 7 mg . rnl-l, and acetic acid was about 1.5 to 2.0 mg . ml-l. When the data in Tables VII and VIII are compared, we can see that the more recent levels of pyroglutamic acid are lower for koikuchi shoyu and for usukuchi shoyu, which may reflect improvements in the control of microbial and enzymic processes. Acetic and lactic acid concentrations are comparable, but formic and citric acids have virtually disappeared from the modern shoyu. Succinic acid has also been reduced when the 1982 data are compared with the recent data. In contrast to the above data, Ren et al. (1993b) reported significantly different levels of some of these acids in Japanese shoyu even after taking into account the different bases on which the levels have been calculated (mg . 8-l versus mg . ml-l). Table IX shows that the levels of citric and succinic acids were much greater and levels of acetic much lower than those reported by Sasaki and Nunomura (1993).

98

DESMOND K. O'TOOLE

TABLE VIII ORGANIC ACIDS IN THE

FIVE'rYI'ES

OF JAPANESE SHOYU

(mg .

PyroShop

glutamic

Lactic

Acetic

Formic

Koikuchi Usukuchi Tamari Shiro Saishikomi

3.67

7.59 7.61 9.01

1.81 1.19

0.09 ND

2.47

0.14

1.31 1.55

ND

N D ~ ND ND ND

0.10

ND

1.79 4.79 0.82

ND 4.12

8.18

Citric

succinic __ 0.26 0.25 0.30 ND 0.28

"Adapted from Table 5 of Sasaki and Nunomura (19931. hNon~ detected.

TABLE IX ORGANIC ACIDS IN THE FIVE TYPES (IF JAPANESE SHOYri AND RANGES OF THE IN

ACIDSFOUND

CHINESE AND KOREAN SOYSAUCE (mg . g-')' ~

SOY SdUCC

Koikirchi shoyu lisukuchi s h o p Tumari shoyu Shiro shoyir Saishikoirii s h o p

Chinese Korean

Pyroglutarnic 2.75-5.24 2.97 5.71 2.18 9.19 i.o3-~.2ti 1.48-5.24

Lactic

Acetic

Formic.

Citric

succinic

10.78-10.49

0-0.69 0

1.21-1.59 0.84

0

4.81

0 0 0-1.23 0-13.20

0 1.43

3.08-7.61 1.14 20.34 0 33.6 1.19-5.nn 0.94-7.61

9.10-11.15 18.4 18.49 4.21 24.27 1.99-18.33 1.45-5.26

8.08 19.39

4.66 13.48 3.72-19.67 3.19-11.5n

04.09

04.m

UAdaptedfrom Table 2 of Ken et nl. (1983b).

Chinese and Korean soy sauce samples (Ren et al., 1993b) showed great variation in the levels of organic acids, in line with the figures reported for the Japanese shoyu samples (Table IX). The level of lactic acid in old baceman (Table X) is comparable to the shoyu levels, but the level of acetic acid is lower.

D.

SUGARS IN SOY SAUCE

There are two sources of the sugars found in soy sauce. The first is from the sugars that arise from the breakdown of carbohydrates in the

99

MICROORGANISMS IN SOY SAUCE PRODUCTION TABLE X SIMPLE ORGANIC COMPOUNDS FOUND I N DIFFERENT BRANDS OF INDONESIAN BACEMAN(mg . ml-l)a Manufacturer and baceman age Ikan Lele at 28 d a y s Manggis a t 5 d a y s Purwokerto Libra a t 1 7 days

Lactic

Acetic

Ethanol

Glycerol

1.98 0.04 7.66 4.88

0.35 0.04 0.86 1.22

0.95 0.37 0.001 0.03

1.4 0.64 0.84 0.84

“Adapted from Table 1 of Roling et al. (1994a)

TABLE XI SUGARS IN THE FIVETYPESOF JAPANESE SHOYU(mg

Shop

Koikuchi Usukuchi Tamari Shiro Saishikomi

Mannose

Fructose

Arabinose

0.23 0.08 1.02 0.13 0.65

m 16.62 trace trace trace

0.83 1.72 0.39 1.30 0.25

rn1-’la

Galactose

Xylose

Glucose

2.26 2.66 3.80 trace 2.61

0.51 1.46 0.23 11.45 0.34

9.42 19.97 12.55 105.58 13.19

“Adapted from Table 5 of Sasaki and Nunomura (1993). ND = None detected. Sucrose at 1.88 mg . ml-’ was measured in shiro shoyu, and ribose was negligible in all shoyus.

mororni stage; the second is from the direct addition of sucrose, as occurs in Indonesian kecap manis. As seen in Table I, reducing sugars in soy sauces from around the world vary from about 1% to as much as 8.5%. Sasaki and Nunomura (1993) reported the levels of eight sugars in shoyu: namely, the pentoses ribose, arabinose, and xylose; the hexoses mannose, fructose, galactose, and glucose; and the disaccharide sucrose. They found that sugar levels in shoyu varied with the type of shoyu and that the levels of some sugars were relatively significant. Glucose varied from 9.42 mg . ml-1 for koikuchi shoyu to 105.6 mg . ml-1 for shim shoyu. Galactose was also present in significant quantities in most shoyu at a level of about 3 mg . ml-I (Table XI). In one baceman sample, about 4.4 mg . ml-l of glucose, fructose, and galactose combined was measured, but generally very little of any sugars is found in kecap asin (Roling et a]., 1994a).

100

DESMOND K. O’TOOLE

Ill. Aroma and Flavor of Shoyu

The aroma and flavor of shoyu are important aspects. Much of the Japanese work has been directed to improving and maintaining good flavor and aroma on a consistent basis while improving efficiency and yield. About 300 compounds have been identified as contributing to the final flavor and aroma of shoyu (Yokotsuka, 1985). Yokotsuka (1985) identifies some of them as follows: 1. methionon (3-methylthio-1-propanol) 2. 3.

4. 5.

6.

7.

4-ethyl guaiacol and 4-ethyl phenol phenol esters between phenolic compounds, such as $-ethyl guaiacol, vanillic acid, and vanillin, and organic acids such as benzoic acid and acetic acid maltol (3-hydroxy-2-methyl-4-pyrone; Zapsalis and Beck, 19861, and 5-hydroxy-malt01 hydroxy-furanones: 4-hydraxy-2(or5)-ethyl-5-(or 2)-methyl-3(2H)-furanone(HEMF) 4-hydroxy-5 -methyl4 (2H)-furanone (HMMF) 4-hydroxy-2,5-dimethyl-3 (2H)-furanone (HDMF) cyclothen: 2-hydroxy-3-methyl-2-cyclopentene-l-one lactones: 4-butanolide 4-pentanolide 2-methyl-4-butanolide 4-hexanolide

This list, however, omits ethanol and glutamic acid, which are also considered significant for the organoleptic qualities of shoyu (Fukushima, 1989). Typical levels of significant compounds that contribute to flavor and aroma that are found in Japanese s h o p are shown in Table XII. The compound considered the character impact substance contributing to the flavor and aroma of shoyu is HEMF because it has an intense sweet aroma similar to shoyu, it occurs in high concentrations in shoyu, and it has a threshold detection limit in water of <4 x mg . liter1 (Sasaki and Nunomura, 1993). However, it does not occur in tamori shoyu, which is made with very little wheat (Fukushima, 1989). The optimum level in shoyu of another volatile aromatic compound, 4-ethyl guaiacol, is 1-3 mg . liter1 (Hamada et al., 1990), but Ishihara et al.

MICROORGANISMS IN SOY SAUCE PRODUCTION

101

TABLE XI1

THEMAJORFLAVOR COMPONENTS AND THEIR CONCENTRATION (mg . kg-’) FOLJNDIN S H O Y ~ P Concentration

Flavor component Ethanol Lactic acid Glycerol Acetic acid 4-Hydroxy-5-methyl-3( 2H)furanone (HMMF) 2,3-Butanediol Isovaleraldehyde 4-Hydroxy-2(or 5)-ethyl-5(or 2)methyl-3(2H)-furanone (HEMF) Methanol Acetolb Ethyl lactate 2,6-Dimethoxyphenol Ethyl acetate Isobutyraldehyde Methyl acetate Isobutyl alcohol

31,501 14,347 10,209 2,108 256.4 239.6 233.1 232.0 62.4 24.6 24.3 16.21 15.13 14.64 13.84 11.95

Flavor component Furfuryl alcohol Isoamyl alcohol Acetoin n-Butyl alcohol 4-Hydroxy-2 ,s-dimethyl3(2H)-furanone (HDMF) Acetaldehyde 2-Phenylethanol n-Propanol Acetone Methionol 2-Acetylpyrrole 4-Ethylguaiacol Ethyl formate 4-Butanolide Methional 4-Ethylphenol Dimethyl sulfide

Concentration 11.93 10.01 9.78 8.69 4.83 4.63 4.28 3.96 3.88 3.65 2.86 2.77 2.63 2.02 1.42 0.34 0.04

OSasaki and Nunomura (1993j.Reprinted with permission from Academic Press. bThisis a registered trade name, the substance may be 1-hydroxy-2-propanone(Budavari,1989).

(1996) report the preferred concentration of 4-ethyl guaiacol to be “0.8 ppm” and of 3-methylthio-1-propanol “3.9 ppm.”

IV. Soy Sauce Production

The traditional production methods for soy sauce rely heavily upon natural microflora that respond to the environmental changes imposed on the raw materials. Soy beans are boiled, mixed with cracked wheat or flour, left covered for a few days while natural mycoflora grow, and then the molded beans are steeped in brine for many days or months (Wang and Fang, 1986). This process ultimately produces soy sauce. Scientific studies have explained the major components in the process, so that the process is better understood and can be controlled to give a more consistent product.

102

DESMOND K. O'TOOLE TABLE XI11 CCIMPOSPI'ION or SOYBEANS,SOME SOY BEANPRODUCTS, WHEATGRAIN, WHEAT FLOUR, ANU WHEAT BIUN (g . kg-l) Soy beans

Component

Whole bean"

Defatted flourh

Flourb

Whole grain

52

115-140

-

37-177

153 19 18.5 717

140-180 9-24

119-22 9

-

30-68 38-96 619

703

650-740

141

21-26

12-21 3

352

24

431

86

72

343

4 70

Carbohydrate

187 51 316

62 384

Starch-amylose

4

Free sugar Cellulose Hemicellulose

=50 4.3

Water Protein Oil

Ash

Wheatr

12

345 206 45 352

Flour

4-14

10.8

Bran

76

OAugustin and Klein (19891. 'Haytowitz and Matthews (1989). CDecker and Hanners (1991). %onstituents of soy bmri carbohydrate from Snyder and Kwon (1987).

A. COMPOSITION OF RAWMATERIALS

The raw materials used are soy beans, wheat, salt, and water. Soy beans are the seeds of the plant Glycine max (L.) Merr., wheat is the common name of the seeds of Triticum aestivum L., T compacturn Host, and ?: durum Dest. (Mattern, 1991), and the salt is usually sea salt (Wood, 1982). The water must be of good quality, free of nitrates, which can lead to the production of N-nitrosamines (Sung et al., 1991), and low in Fe++and Mn++ (Noda, 1980). The proximate analysis of raw materials used in modern soy sauce production, namely, soy beans, defatted flour, and full-fat flour, and of wheat grains, flour, and bran, is shown in Table XIII. The moisture content of whole beans is between 10 and 13% depending on the length of time they have been stored (Weingartner, 1987), as is the water content of wheat. The proximate compositions of soy beans and wheat (Table XIII) are for indication only, so as to give some idea of the relative abundance of the different components, because these values vary depending on plant variety, analytical methods, and the growing conditions of the crop. Table XI11 shows that there is about 34% protein and 20% oil in the beans and, of

MICROORGANISMS IN SOY SAUCE PRODUCTION

103

course, that the protein level is higher after defatting. The oil component is not thought to contribute to the final composition or flavor of soy sauce, but, according to Yokotsuka, sauce made from whole beans is “more stable” and more resistant to “yeast invasion” (Yong and Wood, 1974). The carbohydrate content of soy bean is about 30% and comprises a soluble fraction and an insoluble fraction. The soluble fraction, about 10% of the bean, is a mixture of sucrose (5%),raffinose (l%),and stachyose (4%), and the insoluble fraction consists of structural carbohydrates such as pectin (30% of insoluble carbohydrate), hemicellulose (50%),and cellulose (20%), and the starch level is less than 1% (Snyder and Kwon, 1987). Soy hemicellulose consists mainly of arabinogalactan (Hisamatsu ef a]., 1995). The easily soluble fraction is a very small part of the whole bean and could easily be lost during rehydration of the beans before heating, and would almost certainly be lost during boiling and decantation of the water in the traditional process, so the reported levels in raw beans here may not be the same as in the cooked beans when inoculated to make koji. The insoluble carbohydrate is complex and is more resistant to degradation than starch, and requires a more complex enzyme mixture to degrade these structures to mono- and disaccharides (Coughlin and Ljungdahl, 1988; Singh and Hayashi, 1995). On the other hand, wheat contains at least twice the carbohydrate level of soy beans, but most of it is starch-amylose, an easily degradable carbohydrate. The protein content of wheat, at 153 g . k g l , is much less than the 343 g . kg-1 in soy beans. Of the amino acids in whole soy beans, wheat, and bran, about 19.2, 33, and 23%, respectively, consist of glutamic acid, but in absolute terms soy beans supply more glutamic acid than wheat (Table IV), which is not quite consistent with Yokotsuka’s reported claim that wheat is added “to serve as a rich source of glutamic acid” (Yong and Wood, 1974). B. THEMANUFACTURE OF SOY SAUCE

The soy sauce-making process can be stated simply as follows. Soy beans or defatted soy flakes are soaked and cooked, and wheat is either roasted and crushed or used as raw flour. The cooked beans and added wheat component are mixed in proportions that depend on the type of soy sauce being produced. Fungi are grown on the mixture of soy beans and crushed and roasted wheat for several days to produce what the Japanese call koji (Fukushima, 1989). The molded soy beans are then mixed with an 18-20% NaCl solution, producing a mixture called

104

DESMOND K. O’TOOLE

moromi in Japanese, and steeped for 6 to 9 months. Bacteria and yeasts grow in the moromi to develop the s h o p flavor, and then the raw shoyu (kiage in Japan) is separated from the residue, pasteurized, and bottled. In older systems of production the soy beans are boiled in water, but in modern systems the beans are cooked at a high temperature under steam pressure. A variation is the species of mold used to produce the koji, Aspergillus oryzae versus A. sojae. In other parts of Asia, different names are used and variations in the soy sauce-making process occur. In Indonesia the koji, called bungkil, is sun dried before steeping (to produce the baceman (moromi)stage) for 1 day or for as long as 5 months (Roling et al., 1994a). In Korea the equivalent of kojj is maeju, a cake of molded soy beans (Lee and Cho, 1971). The treatment of the molded beans in Korea is virtually the same as that published in China during the Yuan dynasty, 1271-1368 A.D. (Fukushima, 1989). The Indonesian soy is spiced and boiled before bottling, and Korean soy sauce is also boiled before bottling (Lee and Cho, 1971). In Hong Kong and China, before pasteurization and bottling, some manufacturers place the raw soy sauce in the sun for 1to 2 months to concentrate the sauce (Chiao, 1986). Each component and process contributes in various ways to the final product and its flavor. The severity and degree of heating of the soy beans affect the yield of nitrogenous compounds in the final soy sauce. The mold provides the enzymes that during the moromi stage break down the proteins and the wheat starch. In addition, the mold secretes such macerating enzymes as pectinase, cellulase, and hemicellulase that improve access of other enzymes to the protein and carbohydrate in the soy bean and wheat by breaking down cell walls (Sugiyama, 1984). The wheat supplies protein that can be degraded and carbohydrates that enhance the growth of yeasts in moromi (Roling et al., 1 9 9 4 ~ ) and that are converted into organic acids, such as lactic acid, and ethanol that are produced during fermentation. The soy beans supply the base protein that is hydrolyzed to peptides and amino acids, which give the nutritive value of the product. The bacteria produce lactic acid and lower the pH, which allows the growth of yeasts that produce the essential flavor constituents. The final pasteurization step stimulates the chemical development of essential flavoring agents. C. KO~I

The Japanese name koji is derived from the Chinese character for molded grain. In Chinese it is called shui, and in Korean it is ku (Lotong, 1985). Koji production in Japan is important for the production of soy

MICROORGANISMS IN SOY SAUCE PRODUCTION

105

sauce, rniso, and a range of alcoholic beverages. In soy sauce manufacture the grain is soy bean, and it usually includes wheat grain or flour, except for China, where wheat bran is now included (Fukushima, 1989). However, substitution of wheat bran for wheat flour results in an unsatisfactory soy sauce (Yong and Wood, 1974). Traditionally the koji has been produced by allowing the natural environmental inoculation of the prepared soy bean-wheat mixture, or prepared soy bean, and incubating aerobically in shallow trays (but with forced airflow in modern systems) for 2-3 days. Known strains of A . oryzae and A . sojae are now used, and Sugiyama (1984) stipulated that strains used for koji production must not produce mycotoxins, and should possess high levels of proteolytic, amylolytic, and macerating enzymes. 1. Seed Koji The use of pure fungal strains for koji production requires the production of seed koji (tune koji) to act as a starter culture for the process. Seed koji is a mass of spores, not mycelia, of the koji mold grown on a suitable carbohydrate base, usually rice or wheat bran. The carbohydrate base is sterilized, inoculated, and incubated and the mold grown until it has sporulated. The spore mass and carbohydrate base are inoculated at about 0.1 to 0.2% of the mass of raw material prepared for soy koji (Lotong, 1985). In traditional production, naturally high levels of fungal spores are relied on for the inoculation of the prepared materials for koji. 2. Materials and Conditions for Enzyme Production

In Japan, full-fat beans have been replaced with defatted beans because the nitrogen yield in the final soy sauce is greater and the soy oil harvested from the beans can be used for other purposes. Another significant effect on nitrogen yields in the sauce is the degree of heating used to cook the soy beans. The higher the temperature and the shorter the time of heating, the greater the yield of nitrogen from the original soy beans (Fukushima, 1989; Yokotsuka, 1985). Thus, the yield of nitrogen in the soy sauce obtained in the early part of the twentieth century by boiling the beans was about 60%, but today, when temperatures of 1 7 O O C for 15 sec are used to cook the beans (Fukushima, 1989), yields of 88% are obtained. When defatted soy flakes are used, there can be variation in the size of the flakes in batches produced by different techniques ( h i s h i , 1970).When batches contained 84% of the soy flake in the size range of >8 mesh (>2.5 mm) and 10% in the 8-20 mesh (2.5-0.9 mm) range, the moromi fermentation proceeded more rapidly than in a batch where 35% of the soy flakes were in the >8 mesh size

106

DESMOND K. O'TOOLE

First stirring Max. 38OC

40oC

30OC

$

Y

a L

pf. 20c

+-,

B

%"c:

Room humidity: saturated

b

lOOC

4-~

-Temp. of koji mixture --_-_ Temp. of koji room

0°C

0

9

54 Time course of incuhation (Hours) 18

27

36

45

63

72

FIG.1. Temperature changes in koji produced by a traditional small tray method. Adapted from Fukushinia (1989).

and 55% in the 8-20 mesh range (onishi, 1970), suggesting that the larger size promoted moromi fermentation. Yu et al. (1972) in Korea studied the substitution of corn gluten for the defatted soy bean and found that substitution of up to 15% of the total bean and wheat content with corn gluten produced good-quality soy sauce, as measured by the amylase and proteolytic activities in the koji. When more than 15% corn gluten was used, a soy sauce of poor taste and low nitrogen content resulted that was attributed to the low enzyme activity in high-corn-gluten koji. However, in a similar study, when corn substituted up to 60% of the wheat, or barley substituted u p to 70% of the wheat, good-quality soy sauce was produced from the koji (Lee et al., 1972). Substitution of the carbohydrate portion has a lesser effect than substitution of the soy protein. In the traditional small-tray method of making koji (Fukushima, 1989), the incubation temperature of the koji at the beginning is about 35OC. It quickly drops to about 25"C, after which the temperature rises, due to fungal growth, to about 38"C, at which point the mixture is stirred and the temperature drops to about 33OC (Fig. 1). During the first 24 hours, the humidity is controlled to about 1009'0, but during the rest of the time room humidity is lowered and the temperature drops below 29°C and is at 25°C after 72 h have elapsed. Solid matter is lost during the koji stage, as Takeuchi ef ~ l(1968), . in a study of koji production for

107

MICROORGANISMS IN SOY SAUCE PRODUCTION TABLE XIV THE PROTEIN DEGRADING ENZYMES IN KoJr AND THEIR ACTIVITY Enzyme Proteinases Alkaline Semialkaline Neutral I Neutral I1 Acid I Acid I1 Acid 111 Acid carboxypeptidases I I1 I11 IV

Optimum pH

Activity”

10.5 8.3 7

929 55

6

3

9 44 10 5

3 4 3-4 3 3 4

0.25 0.18 0.01-0.05 0.02-0.11

8.5

0.1 0.25 0.15 0.15 0.11 0.01 0.03

80

3.2 3

Leucine aminopeptidases

I I1 111

IV V v1 VII Ar ylaminadase

5-a 8

7 -

8.5

0

~

‘Unit against casein per g of koji. Adapted from Tables 1 0 , 1 1 , and 12 of Fukushima (1989)

tamari manufacture, reported a 9.6-21.4% loss of solid matter during 4 days of koji manufacture, a loss that increased with increased moisture content in the mixed koji raw materials. Yamamoto et al. [1972)reported that, when koji prepared for “white shoyu” was incubated at 27-29OC for up to 70 h, phosphatase activity reached a high point after about 40 h, after which there was very little increase, while acidic and alkaline proteases, and a- and P-amylases, reached maximum activities after 48 h, and neutral protease activity peaked after 60 h. They concluded that 48 h gave the most desirable result, because amylase activity was maximal at this point. Yong and Wood [1974) reported a similar pattern of enzyme production. The enzymes produced by fungi include pectinase, cellulase, and hemicellulase, and the starch-degrading enzyme amylase, as well as the protein-degrading enzymes such as proteinases and peptidases [Fukushima, 1989). The range of particular protein-degrading enzymes includes those shown in Table XIV.

108

DESMOND K. O'TOOLE

The proteinases, with peak activity in alkaline and also in neutral and acid conditions, are of the endo type, that is, they do not possess any amino- or carboxy-peptidase action and so hydrolyze protein to peptides only, not amino acids (Fukushima, 1989). The peptidases are of the exo type that liberate amino acids from the carboxy- or amino-terminals of the peptides and show maximum activity in slightly alkaline or acid conditions (Fukushima, 1989). The proteinases break down the protein, leading to an increase in total soluble nitrogen but with no concomitant increase in formol nitrogen, which is a measure of amino acid nitrogen. It is only with the inclusion of peptidases that formol nitrogen increases along with an increase in glutamic acid content. Proteinase production by A. oryzae NISL 1913 in chemostat cultures using a medium containing 2.6% soy flour as a nitrogen source was accelerated when soy bean oil was used as the carbon source. The specific protease production rate on a 1.8% oil mixture was 2.85 protease units . (mg dry weight of mycelium)-' . h-l, which was threefold greater than that with starch as the carbon source, but a-amylase production on the oil was only about a third of that when the organism was grown with starch as the carbon source. Soy oil could be replaced by C14 and Cl8 fatty acids (Fukushima et aJ., 1991). The starch from wheat is also important for the growth of the koji fungi. Roling et aJ. (1994~)found that the growth of fungi during the bungkiJ stage, which usually includes soy beans only, was faster and enhanced by the addition of 20% rice flour. The amount of fungal mass produced was not measured, but protease was increased about 30%, the leucine amino peptidase concentration doubled, and glucoamylase increased 136%. 3. Studies of Koji Fungi Studies of A. oryzae and A. sojae strains used for shoyu production have focused on comparing these two fungal species and improving their enzyme-producing abilities. A study of the enzymatic differences between 11 strains of A . oryzae and 20 strains of A . sojae showed that the activities of neutral, acid, and alkaline proteases, xylanase, pectin lyase, phosphatase, and aminopeptidase were not significantly different. However, acid carboxypeptidase activity and a-amylase activity were higher from A. oryzae when compared with A. sojae strains, while endopolygalacturonidase activity was much higher from A. sojae than from A. oryzae (Terada et QJ., 1980). The ratios between a-amylase activity and endopolygalacturonidase activity of 0.5-2 for A. sojae and 20-2000 for A. oryzae were suggested as a differentiation criterion for

MICROORGANISMS IN SOY SAUCE PRODUCTION

109

the species (Terada et al., 1980). Hayashi et a]. (1981) compared the performance of these two fungi in s h o p production. They found that the activities of protease, acetic carboxypeptidase, and a-amylase were lower and those of endopolyglucuronidase and glutaminase were higher in koji made with A. sojae. In the moromi stage, the proportions of NH3 nitrogen (N), glutamic acid N, and total N were higher, and viscosity and heat residue were lower with A. sojae. The resulting concentrations of citric and succinic acids in the shoyu were significantly higher ( p < 0.001) with A. sojae than with A. oryzae. Ishihara et al. (1996) compared the volatile components in commercial koikuchi s h o p s from different factories using either A. oryzae or A. sojae and found that the concentrations of 1- and 2-propanol, furfuryl and benzyl alcohols, ethyl-benzoate, and lactate, acetate, pyrazines, carbonyl compounds such as ethanal, maltol, and phenyl acetaldehyde, phenol, and others, were higher in the shoyu from factories using the latter fungus, but concentrations of &methyl- and 3-methyl-1-butanol, 2-phenyl ethanol, 2-methyl- and 3-methyl-butanoic acid, 3-methylthio-1-propanol, HEMF, 4-ethyl guaiacol, 4-ethyl phenol, and others were greater in shoyu from factories using the former fungus. These results have prompted factory managements to use A. sojae for koji production. Using an unusual system, Yasui et al. (1982) tested a range of koji fungal strains for glutaminase production and found that, when a strain showing 16% higher glutaminase activity than its parent strain was compared with its parent in the production of shoyu, the final glutamic acid concentration was 10% higher. In the early 1950s, A. sojae KS was irradiated with X-rays by Iguchi to produce strain X-816 of A. sojae (Sekine et al., 1970). Sekine et al. (1970) obtained seven strains with superior alkaline phosphatase activity (130-190%) and highly active protease, peptidase, cellulase, and amylase activities that were better at decomposing soybean protein. Yokoyama and Kadowaki (1983) UV-irradiated A. sojae strain H and obtained mutant strains with total protease activities 2.5 times that in wheat bran and soy sauce kojis. The mutant strains were diploidized and combined with natural mutants from M strains, and strains TH and D-15 were produced that possessed higher total protease activities than the M strains, and grew well. However, W irradiation may stimulate the production of toxic elements in otherwise safe fungi. Kalayanamitr et al. (1987)W-irradiated A. flavus var. colurnnaris Raper and Fennel (ATCC 44310) to obtain mutant strains with high protease and amylase activities, and light-colored conidia. Some selected mutant strains were found to be acutely toxic to weanling rats, even though they were negative for aflatoxin production. The investi-

110

DESMOND K. O’TOOLE

gators suggested that the toxic compound could be one of four substances: maltorhyzine, aspergillic acid, kojic acid, or cycoopiazonic acid. protoplasts Furuya et al. (19831 fused, with an efficiency of lo%, derived from two strains of A. oryzae, one with a high growth rate and the other producing high levels of protease. Ttyo strains derived from successful fusions showed high stability, fast growth, and abundant sporulation and produced 2. 3 times more protease than the parent fast-growth strain. The growth and development of microorganisms on defatted soybean and ground wheat koji prepared with A. sojae were studied by electron microscopy by Kitahara et al. (1980). Growth of the mold on the surface of the soybean was rapid up to 24 h, at which point formation of sporing bodies began, and spores were released within 40 h. However, very little fungal growth was seen on the wheat surface, but yeasts were seen growing on the wheat. Growth of Micrococcus species became noticeable after 16 h, as did multiplication of lactobacilli. These observations on the growth of the koji mold are at odds with the observation that 10-%0”/0of the dry matter in koji is lost in the koji stage (Takeuchi et al., 1968) and the observations below on the significant consumption of carbohydrate during the koji stage. I suggest that significant penetration of the wheat endosperm should have been seen. During koji production, carbohydrate is consumed by the fungus, thus leaving less carbohydrate available to provide flavor compounds for the final shoyu produced (Furuya et al., 1985). This carbohydrate consumption is positively correlated with a-amylase activity in koji culture. To overcome the depletion of carbohydrate before the moromi stage, Furuya et al. (1985) derived mutants that utilized 10-50% less carbohydrate during preparation of koji than the parent strain, with about 1/3, 1/20, and 1/150 of the a-amylase activity of the parent strain of A . oryzae. Significantly increased amounts of carbohydrate-derived compounds were found in the resulting shoyu made with these mutants. Enhanced glutaminase activity in koji is desirable to increase glutamic acid production in soy sauce, and reduced conidial production in the koji reduces contamination of the air with floating conidia (Ueki et al., 1994a). A mixed t a m koji of two koji fungi, A. oryzae strains K2 and HG, increased glutaminase activity of the mixture to 11.3 units g-l dry koji, which was higher than the 4.7 or 4.4 units. g-l dry koji produced by the K2 strain or HG strain, respectively, and conidia production was reduced tenfold (Ueki et a]., 1994a). The mixed tane koji was used in the manufacture of soy sauce, and the resulting mixed koji made with 3.6 tons of defatted soybean and of wheat grain showed 1

MICROORGANISMS IN SOY SAUCE PRODUCTION

111

high glutaminase activity (5.5 units . g1dry weight koji) when compared to strain K2 alone (1.8 units . g-l dry weight koji). In addition, the number of conidia in the mixed culture was 2.5 x lo7 g-l dry koji, which was lower than 1.3 x lo8 g-l dry koji produced by strain K2 alone. The glutamic acid content of the raw soy sauce was 1.25 times higher than the glutamic acid level found in normal soy sauce (Ueki et al., 1994b). Kim and Cho (1975) investigated soy sauce production in Korea using a soy-wheat koji prepared with A . sojae, using natto, a soy bean product prepared with Bacillus natto, and using a mixture of the two in varying proportions. The natto-brine mixture had protease activity twice as high as the koji alone, and this was reflected in the protease activities found in mixtures of the natto and koji. On comparing the organoleptic qualities of soy sauces fermented for 3 months, the kojinatto at a ratio of 6:4 had the best flavor, followed by koji alone.

D. MOROMI The name moromi is applied to the mixture obtained when koji is immersed in a liquid (Koseki et al., 1996), which could be water or a brine solution as used for shoyu production. For shoyu the koji is mixed with a brine solution to give a final salt concentration of about 3 MNaC1, which stops the fungal growth but does not stop the activity of the fungal enzymes that can continue the degradation processes. The degradation process for proteins is outlined in Fig. 2. Macerating enzymes, such as cellulase, hemicellulase, and pectinase, break down the cellwall components to make the proteins more susceptible to attack by the protein-degrading enzymes (Fukushima, 1989). These enzymes are essential to allow satisfactory filtration of the raw shoyu. At the same time, amylase and other starch-degrading enzymes attack the starch to break it down to glucose. Another important enzyme is glutaminase, which converts glutamine to glutamic acid. However, this enzyme is sensitive to an acidic pH and to salt, so that lower temperatures (15-20OC) are recommended for shoyu production during the first month (Fukushima, 1989) to slow acid production by the lactic acid bacteria to prevent loss of glutaminase. While the mold enzymes are doing their work, other microorganisms begin to grow. Traditionally, when added to the brine, the koji already contains a range of bacteria and yeasts, many of which are also killed by the high salt concentration, but in the traditional process in a wellestablished factory a wild microflora exists that adheres to the walls of tanks, often made of concrete in traditional plants. The walls of soy

112

DESMOND K. O’TOOLE Soy protein

Wheat protein

i I

Macerating enzymes: cellolase, hemicellulase, pectinase

Released protein Alkaline proteinases Neutral proteinases

Polypeptides

I

Acid carbox ypeptidases Leucine arninopeptidases

Amino acids

Glutamine

I

Glutaminase

Glutamic acid

FIG.2. The breakdown process of soy and wheat proteins in moromi by koji enzymes. Redrawn from Fukushima (1989).

sauce concrete brewing tanks in Japan had populations of 1.0 to 4 . 5 x lo5 m-2 Zygosacchuromyces species and 7.0 x lo4 to 5.6 x lo5 m-2 Cundida species adhering to them (Noda et al., 1 9 8 2 ~ )They . become incorporated in the brine mixture and begin to grow, and a complex fermentation takes place. This natural inoculum then follows growth cycles dictated by environmental factors, the presence of particular ingredients, and the byproducts of the growth of other microorganisms. In the first stage, a lactic fermentation takes place due to the growth of halotolerant lactic acid bacteria. These organisms produce lactic acid from the glucose released from starch and lower the pH of the mix from about 6.6-7.0 to <5.0. When the low pH has been reached, the natural yeasts can then begin to grow (see, e.g., Yong and Wood, 1976), and these organisms are responsible for the most important flavor components. The second yeast stage has two parts. In the first part, yeasts grow and produce alcohol; in the second part, other yeast species sometimes grow and produce essential flavor components. The time required for the moromi stage is about 6 to 9 months. A most significant advance in soy sauce manufacturing to control the flavor of the final product has been introduction of greater control of the moromi process. Parameters controlled include temperature, aeration, and inoculation of starter microorganisms dominating in the rnororni.

MICROORGANISMS IN SOY SAUCE PRODUCTION

113

The above applies to Japanese s h o p , but it does not apply completely to Indonesian kecap (Roling et al., 1996). Satisfactory kecap is produced after a short fermentation time of 1month, and yeast fermentation is not important for flavor development. Roling et al. (1996) concluded that Japanese process technology and techniques were not suitable for kecap production.

Temperature of the Brine Stage Temperature control in the early stage of s h o p production has involved two divergent approaches. The first approach is to raise the temperature at the beginning of the brine stage to about 50-55"C, so that degradation of the soy bean and wheat starch is accelerated, making them available for microbial growth more quickly. This is usually done in a stirred tank with a salt content of 10-12% (Osaki et al., 1985; Hamada et al., 1989). Pure microbial cultures must then be added for fermentation to proceed. The second approach is based on the old observation that moromi batches commenced in late winter, when temperatures in the moromi were low, produced the best-flavored shoyu (Sasaki and Nunomura, 1993). Consequently, in this approach the brine at 0°C is mixed with the koji to keep the temperature in the early stage of the moromi below 15°C for several days (Sasaki and Nunomura, 1993). Otsuki et al. (1981a) investigated the effect of temperature in the moromi stage. They set up seven treatments and used the temperature regime as shown in Table XV over a period of 5 months. Their results showed that, the total amount of soluble N released after 1 month was equal to 60% of the final total in treatment (i)but 92% of the final total for treatment (vii), that is, the higher temperature favored enzyme activity, which solubilized N. Ethanol was only produced at 30°C in any of the treatments, and the concentration was highest in treatments (i), (ii), and (v),Citrate levels were negligible in treatments (i). (ii), and (iv), and low in treatment [iii), but noticeable in treatments (v), (vi), and (vii); therefore, the prolonged and early high temperature of 30°C promoted the retention of citrate in the moromi, but the lower temperatures, especially in the early period, promoted the disappearance of citrate, presumably through its metabolism by the microorganisms. In a further report of the experiment, Otsuki et al. (1981b) showed that amylase activity decreased rapidly at 30°C, protease activity was stable at 20°C but decreased at 3OoC, and glutaminase activity increased at 20°C but decreased rapidly at 30°C but, irrespective of treatment temperature, glutamic acid concentration increased in months 1to 3 and then leveled off. Otsuki et al. (1982) showed later that glutaminase activity was lost 1.

114

DESMOND K. O'TOOLE TABLE XV

EXPERIMENTAL PLAN OF OTStJKI ETAL. (1981) FOR THE STUDY OF EFFECT OF TEMPERATURE ON MOROMI FERMENTATION Temperature, "C, in moromi at various times Treatment

Beginning

lil

5

1 month 10

Beginning

1 month

2 months

5 months

5 5 10 10

20 30 20 30 30 30

30 30 30 30 30 30

30 30 30 30 30 30

(ii) (iii) (iv) (v) (vi) (vii)

20

30

1.5 months 20

2.5 months 30

5 months 30

rapidly when the temperature was 30°C and that in the moromi glutamine conversion to glutamic acid ceased after about 1.5 months and excess glutamine was converted to pyroglutamic acid. Leucine-amino peptidase and acid carboxypeptidase activities increased at temperatures above 20aC but showed a steady decline at 30°C. From the same laboratory in further experiments (Hirakuri et al., 1982), conducted using a slightly different temperature regime (Table XVI), it was shown over a 6-month period that a lower temperature in the early part of the moromi stage favored the production of glutamic acid and reduced the production of pyroglutamic acid. Conversely, the higher temperature in the early part of the moromi stage favored the production of pyroglutamic acid over the production of glutamic acid. The temperature regime did not affect the concentration of acetic acid (130-150 mg . TN-') (TN = total nitrogen), whereas, contrarily, at the lower early temperature lactic acid production was favored. In treatment (i) the lactic acid content was 800 mg . TN-l after 5 months, but after the same time in treatment (iv) the content was 400 mg . TN-l. This shows that the lower temperature favored the growth of the added lactic acid culture (Hirakuri et al., 1982). The concentrations of 8-phenyl ethanol and +ethyl guaiacol were both higher at 20°C. On the other hand, temperature is not so important for kecap production. Roling et al. (1994b) investigated the effect of constant temperatures of 24, 30, and 37°C for 4 2 days on the composition of kecap and found little significant difference between the three temperatures. How-

115

MICROORGANISMS IN SOY SAUCE PRODUCTION TABLE XVI

EXPERIMENTAL PLAN FOR THE STUDY OF TEMPERATURE EFFECTS ON kfOROA41 FERMENTATION AND RESULTSFROM HlRAKURI ETAL. (1982)

Concentration at 5 months, mg . TN-'

Time, months 1

2

Treatment

(i) (ii] (iii] (ivl

3

4

5

6

Glutamate

Temperature, "C

Pyroglutamate Lactate

5

10

10

20

20 30

30 20

20 20

20 20

820 808

166 193

20 30

30 30

20 20

20 20

20 20

20 20

721 704

250 309

800

400

ever, the temperature did affect the rate of growth of bacteria. Halotolerant lactic acid bacteria grew much more quickly at 37OC, and numbers peaked within a week at about 3 x TO7 CFU . ml-1 and then declined within 35 days to lo3 CFU. m F , but at 24 and 3OoC the numbers reached about 5 x lo8 CFU . ml-1 within 2 weeks and declined very little over the rest of the 42 days. It seems that the effect of temperature is complicated by interactions with the carbohydrate added to the soy beans. 2. Aeration of the Brine Stage

Traditionally, moromi has been given brief bouts of occasional aeration to mix the contents and to promote microbial growth (Fukushima, 1989), but no element of time is mentioned in more recent reports (Sasaki and Nunomura, 1993). However, in Thailand, where Taiwanbased technology is being used, the moromi is aerated for several hours three times a week for the first month. Aeration is required by the yeasts, which are strict aerobes and need oxygen for growth. In addition, aeration in the early stage retards the growth of salt-tolerant lactobacilli and wild yeasts (Sasaki and Nunomura, 1993), and the stirring causes interaction between enzymes and substrates. A high NaCl concentration in water reduces the solubility of O2in the water and consequently also in the moromi. As the amount of NaCl in a water solution increased from 10 to 18%, the amount of dissolved O2 in the solution fell from 60 to 42% of the concentration in pure water, and the absorption rate coefficient of O2 also decreased with increasing NaCl concentration (Miyauchi et al., 1981). Similarly, Matsumoto et al. (1982) found the concentration of dissolved O2 in water was three times

116

DESMOND K. O’TOOLE

that in an 18% salt solution. In unaerated moromi, the amount of dissolved O2 was zero, but in aerated moromi the level of dissolved O2 after 1 month was 60% of that in 02-saturated pure water: the level dropped to zero 15 min after stopping the flow of O2 (Miyauchi et al., 1981), showing that the O z was being consumed either by microorganisms or in chemical reactions in the moromi. The latter possibility cannot be excluded from consideration as a contributor to flavor, good or bad, in the final product. Moromi contains a significant quantity of carbohydrate, but baceman contains no added carbohydrate apart from that present in the soy beans. During baceman production it is stirred daily for 3 weeks and thereafter about three times a week (Roling et a]., 1994a). Roling et al. (1994b) compared aeration or lack of aeration on baceman fermentation. They sparged air (30 liter . h-l) through the baceman or directed Nzover the baceman (anaerobic conditions) during fermentation. They found that under aerobic conditions staphylococcal and coryneform bacteria were the dominant bacteria up to 2 1 days. Yeasts reached only about lo3 ml-l, but under anaerobic conditions the staphylococcal and coryneform bacteria were present at the beginning, and by day 7 halotolerant lactic acid bacteria were the dominant and only bacteria present. In summary, control of temperature and aeration affects the manufacture of good-quality shoyu and influences the final content of some important components. 3.

Microorganisms in the Brine Stage

Apart from temperature, the presence or absence of 02,and the presence or absence of carbohydrate, a dominant factor influencing the growth of yeasts and bacteria in moromi, is the high level of NaCl that lowers the water activity of the mixture. The water activity is about 0.88 in a 3 M solution of NaCl (Yokotsuka, 1985), which restricts growth to those organisms able to grow at such a low water activity and prevents growth of many potential spoilage organisms. The growth of yeasts in the high concentration of NaCl is eventually reduced by byproducts of yeast growth such as guaiacol, vanillin, and ethanol (Yokotsuka, 1985). Another important factor is the presence of utilizable carbohydrate. The soy protein seems only important for provision of amino acids and peptides. In the natural situation, where a natural inoculum from the walls of the vessel starts the fermentation, the microbial growth pattern is as shown in Fig. 3. The organisms present come from the environment of the factory at which the soy sauce is produced. No doubt, unless the vessels are sterilized, the microorganisms adhere to the walls of the

MICROORGANISMS IN SOY SAUCE PRODUCTION

:b

.... i

i

n

1

2

3

4

:.

i

m

5

6

117

n

7

Months FIG.3. The changes in microflora in moromi during 7-month incubation: 1, wild yeasts; 2, Micrococcus species; 3 , Bacillus species; 4, Tetragenococcus species; 5, Z. rouxii; 6, Candida species. Adapted from Tamagawa et al., 1975 (Yokotsuka, 1985).

moromi vessel and are present when the freshly prepared koji and brine are added. However, bacteria and yeasts may also grow in the koji stage, and bacteria have been known to reach levels of 108to lo9 per gram. In modern Japanese koji production systems, the contaminating bacteria are mostly species of Micrococcus (Yokotsuka, 1985). These organisms are then present in the morami at the beginning hut generally do not grow. The microorganisms that grow in the moromi and produce the desirable flavors are lactic acid bacteria and yeasts. The predominant desirable lactic acid bacterium is the halotolerant Tetrugenococcus halophilus, formerly known as Pediococcus halophilus (Anon., 1994; Collins et al., 1990). The general properties of this organism that make it suitable for growth in the high salt concentration in moromi are shown in Table XVII. In the next stage, Zygosaccharomyces rouxii grows and produces alcohol followed by Candida versatilis and C. etchellsii, which grow and produce essential flavor components. The characteristics of these organisms that make them suitable for growth in moromi are shown in Table XVII. Realization of their importance has led to the use of cultures of these organisms as starter cultures in the production of shoyu (Noda

118

DESMOND K. O'TOOLE TABLE XVII

THECHARACTERISTICS OF YEASTSAND BACTERIA WHICHAREIJsm As SI'AKI'EKS IN hfOf1OMI FERMENTATION [SASAKI AND NUNOMURA, 1993) Growth in medium with

Z . rouxii Limits

C. versatilis

C. etchellsii

Tet. halophilus

Opt.

Limits

25 25

-

-

4 0

2042

25-30

4-5.5 4-5

3-7 3-7

5 4-5

-

-

5.5-9.0

7.0

5-24

5-10

Opt.

Limits

Opt.

Limits

25-30 25

<35 <35

25-30 25

4 0

3-7 3-7

Opt.

'18111 perat itre fur

growth. "C NaCi 0%

NaCl 18% PH

NdCl 0% NaCl 18%

<35 <42

3-7 4.5-6.0 3.5-5.5 4-5

NaC1, YO wlv

<24-26

-

<26

-

<26

-

Water activity

0.787-0.81

-

<0.787

-

0.787

-

0.9'+0.808 0 . 9 S 0 . 9 4

et al., 1982b). In Japan, when the moromi reaches pH 5.2-5.3, a culture of Z. rouxii is added to give a concentration of 5 x lo5 to loGcells per g of moromi (Ishihara et al., 1996). A different situation occurs during the production of baceman. The lack of excess carbohydrate does not stop bacterial growth but retards yeast growth, which eventually reaches levels comparable with those levels found in carbohydrate-augmented baceman (Roling et a]., 1994a,c). T halophilus also occurs in baceman, but equal numbers of staphylococcal and coryneform bacteria also grow in it (Roling et a]., 1994a). Yeasts in baceman form films on the top of some batches that can cause off-flavors in the kecap.

E. USE OF CONTINUOUS FERMENTATION PROCESSES TO PRODUCE SOYSAUCE Production of soy sauce on a large scale ties up large fermentation vessels for a long time. To speed up manufacturing, continuous processes and bioreactors have been studied. The first continuous method involved an enzymic process to hydrolyze the raw materials followed by lactic fermentation (Anon., 1982).The next step was the introduction

MICROORGANISMS IN SOY SAUCE PRODUCTION

119

of whole cells of T halophilus, Z. rouxii, and C. versatilis immobilized in calcium alginate gel to ferment the moromi (Osaki et al., 1985). Following enzymic hydrolysis and passage through a column bioreactor containing immobilized I: halophilus cells for lactic fermentation, the production stream was split in two and put through either a Z. rouxii or a C. versatilis reactor. Each stream was then heated, filtered, and combined. The production time was equivalent to 2 weeks. Sauce was produced continuously for 80 days through a 280-liter reactor, but it had an aroma pattern different from traditional sauce. However, the gel was unstable to heat and not resistant to a high-salt solution, so Horitsu et al. (1990) introduced a ceramic bead carrier for the whole cells and could produce satisfactory soy sauce within 8 days. Hamada et al. (1991) used a system based on “Chitopearl” to immobilize glutaminase and on alginate gel to immobilize the microorganisms. The koji mixture was hydrolyzed in 8.5% NaCl at 45OC for 3 days, filtered, and then put through the glutaminase reactor followed by the microbial reactors. Although production took about 2 weeks and the system ran continuously for 100 days, the product still did not completely match the traditional product (Hamada et al., 1991). Koseko et al. (1993), using a Microbe Immobilized Ceramic reactor, successfully fermented nonsterilized moromi mash with the microorganisms named above and were able to produce sauce with good taste and flavor, With a residence time of 30 h for Z. rouxii, 2% ethanol and 38 mg . liter-’ 2-phenyl ethanol was produced, and with a residence time of 48 h for C. versatilis the same level of ethanol and 3.7 mg . liter-l 4-ethyl guaiacol were produced. This is not an exhaustive look at the work done in this area. Other relevant work includes that of Iwasaki and Ueno (1990) and Iwasaki et al. (1991a,b, 1992). So far this apparently attractive approach to the problem of reducing the production time and thereby increasing productivity has not yet become a commercial reality. V. Effect of Water Activity on Microorganisms

Water activity