Advances in Food and Nutrition Research, Volume 52 (Advances in Food & Nutrition Research)

ADVANCES IN

FOOD AND NUTRITION RESEARCH VOLUME 52

ADVISORY BOARD KEN BUCKLE University of New South Wales, Australia

MARY ELLEN CAMIRE University of Maine, USA

BRUCE CHASSY University of Illinois, USA

DENNIS GORDON North Dakota State University, USA

ROBERT HUTKINS University of Nebraska, USA

RONALD JACKSON Quebec, Canada

DARYL B. LUND University of Wisconsin, USA

CONNIE WEAVER Purdue University, USA

RONALD WROLSTAD Oregon State University, USA

SERIES EDITORS GEORGE F. STEWART

(1948–1982)

EMIL M. MRAK (1948–1987) C. O. CHICHESTER (1959–1988) BERNARD S. SCHWEIGERT (1984–1988) JOHN E. KINSELLA STEVE L. TAYLOR

(1989–1993) (1995– )

ADVANCES IN

FOOD AND NUTRITION RESEARCH VOLUME 52

Edited by

STEVE L. TAYLOR Department of Food Science and Technology University of Nebraska Lincoln, Nebraska USA

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CONTENTS

Contributors to Volume 52. . . . . . . . . . . . . . . . . . . . . . .

ix

Sweet Potato: A Review of Its Past, Present, and Future Role in Human Nutrition Adelia C. Bovell-Benjamin I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . II. Sweet Potato in Human Diets . . . . . . . . . . . . . . . III. Biochemical and Nutritional Composition of the Sweet Potato . . . . . . . . . . . . . . . . . . . . . . . . IV. Sweet Potato Utilization as Value-Added Products in Human Food Systems . . . . . . . . . . . . . . . . . . . V. Sweet Potato Starch Utilization in Human Food Systems VI. Advances in SPF Production and Utilization for Human Food Systems . . . . . . . . . . . . . . . . . . . . . . . VII. Other Potential Sweet Potato Products . . . . . . . . . . VIII. Potential of Sweet Potato in the Ugandan Food System . IX. Sweet Potato Processing and Utilization EVorts in Kenya X. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . XI. Recommendations . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .

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I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Canine distemper virus . . . . . . . . . . . . . . . . . . . . . . III. Rous-Associated Virus-7 . . . . . . . . . . . . . . . . . . . . .

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Infectobesity: Obesity of Infectious Origin Magdalena Pasarica and Nikhil V. Dhurandhar

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IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV. XVI.

Chlamydia pneumoniae . . . . . . . . . . . . Scrapie Agent . . . . . . . . . . . . . . . . Borna disease virus . . . . . . . . . . . . . . Gut Microbiota . . . . . . . . . . . . . . . Adenoviruses . . . . . . . . . . . . . . . . . SMAM-1 . . . . . . . . . . . . . . . . . . . Adenovirus Type 36 . . . . . . . . . . . . . Adenovirus Type 5 . . . . . . . . . . . . . . Adenovirus Type 37 . . . . . . . . . . . . . Adipogenic Potential of Other Adenoviruses Role of Pathogens in Human Obesity . . . . Infection, Inflammation, and Obesity . . . . Conclusions . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

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77 78 81 83 84 85 87 90 91 91 92 92 93 94 94

Refrigerated Fruit Juices: Quality and Safety Issues Maria Jose Esteve and Ana Frı´gola I. Introduction . . . II. Review . . . . . . Acknowledgments References . . . .

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142 145 156 208 220 221

Tetrodotoxin Poisoning Deng-Fwu Hwang and Tamao Noguchi I. II. III. IV. V.

Introduction . . . . . . TTX Poisoning . . . . . Causative Agent: TTX . Highlight of Viewpoint. Summary . . . . . . . . References . . . . . . .

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Marine Biotechnology for Production of Food Ingredients Rosalee S. Rasmussen and Michael T. Morrissey I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Sources of Marine-Derived Food Ingredients . . . . . . . . . .

238 244

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III. Marine-Derived Food Ingredients . . . . . . . . . . . . . . . . IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

257 283 284

Fruits of the Actinidia Genus Ichiro Nishiyama I. II. III. IV. V. VI.

Introduction . . . . . . . . . . Actinidia Species and Cultivars Fruit Components . . . . . . . Allergenic Properties . . . . . . Health Benefits . . . . . . . . . Perspectives. . . . . . . . . . . Acknowledgments . . . . . . . References . . . . . . . . . . .

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294 295 299 317 317 318 319 319

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

325

CONTRIBUTORS TO VOLUME 52

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Adelia C. Bovell-Benjamin, Department of Food and Nutritional Sciences, Tuskegee/NASA Center for Food and Environmental Systems for Human Exploration of Space (CFESH), Tuskegee University, Tuskegee, Alabama 36088 (1) Nikhil V. Dhurandhar, Department of Infections and Obesity, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana 70808 (61) Maria Jose Esteve, Department of Food Chemistry and Nutrition, University of Valencia, Avda. Vicent Andres Estelles, s/n. 46100, Burjassot, Spain (103) Ana Frı´gola, Department of Food Chemistry and Nutrition, University of Valencia, Avda. Vicent Andres Estelles, s/n. 46100, Burjassot, Spain (103) Deng-Fwu Hwang, Department of Food Science, National Taiwan Ocean University, Taiwan, Republic of China (141) Michael T. Morrissey, Seafood Laboratory, Department of Food Science and Technology, Oregon State University, Astoria, Oregon 97103 (237) Ichiro Nishiyama, Department of Food and Nutrition, Komazawa Women’s Junior College, Inagi, Tokyo 206-8511, Japan (293) Tamao Noguchi, Department of Food Science, National Taiwan Ocean University, Taiwan, Republic of China; Tokyo Healthcare University, Setagaya, Tokyo, Japan (141) Magdalena Pasarica, Department of Infections and Obesity, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana 70808 (61) Rosalee S. Rasmussen, Seafood Laboratory, Department of Food Science and Technology, Oregon State University, Astoria, Oregon 97103 (237)

SWEET POTATO: A REVIEW OF ITS PAST, PRESENT, AND FUTURE ROLE IN HUMAN NUTRITION ADELIA C. BOVELL-BENJAMIN Department of Food and Nutritional Sciences, Tuskegee/NASA Center for Food and Environmental Systems for Human Exploration of Space (CFESH) Tuskegee University, Tuskegee, Alabama 36088

I. Introduction II. Sweet Potato in Human Diets III. Biochemical and Nutritional Composition of the Sweet Potato A. Protein: Sweet Potato Leaves B. Protein: Sweet Potato Roots C. b-Carotene D. Mineral Contents E. Dietary Fiber F. Anthocyanins IV. Sweet Potato Utilization as Value-Added Products in Human Food Systems V. Sweet Potato Starch Utilization in Human Food Systems A. Bulk Ingredients from Three Cv. of Sweet Potatoes: Composition and Properties (Bovell-Benjamin et al., 2004) B. EVects of Processing Technology on SPS Yield and Quality (Jianjun, 2004) C. Physicochemical and Viscometric Properties of an SPS Syrup (Miller et al., 2003) D. EVects of pH and Concentration Times on Selected Functional Properties of a Sweet Potato Syrup (Yousif-Ibrahim et al., 2003) E. Influence of a-Amylase on the Physical Properties and Consumer Acceptability of SPS Syrup (Bovell-Benjamin et al., 2005) F. Sensory and Consumer Evaluation of SPS Syrup (Miller et al., 2003) VI. Advances in SPF Production and Utilization for Human Food Systems A. Breadmaking Properties of SPF (Greene et al., 2003) B. Macroscopic and Sensory Evaluation of Bread Supplemented with SPF (Greene and Bovell-Benjamin, 2004) C. Development and Storage Stability of Breads Supplemented with SPF and Dough Enhancers (Hathorn et al., 2005)

ADVANCES IN FOOD AND NUTRITION RESEARCH VOL 52 # 2007 Elsevier Inc. All rights reserved

ISSN: 1043-4526 DOI: 10.1016/S1043-4526(06)52001-7

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VII.

VIII. IX. X. XI.

D. Sensory Characterization of a Ready-to-Eat Sweet Potato Breakfast Cereal by Descriptive Analysis (Dansby and Bovell-Benjamin, 2003b) E. Physical Properties and Sixth Graders’ Acceptance of an Extruded Ready-to-Eat Sweet Potato Breakfast Cereal (Dansby and Bovell-Benjamin, 2003c) F. Preparation of SPF and Its Fermentation to Ethanol (Reddy and Basappa, 1997) G. Genetic Variation in Color of SPF Related to Its Use in Wheat-Based Composite Flour Products (Collado et al., 1997) H. Quality Evaluation of SPF Processed in DiVerent Agroecological Sites Using Small-Scale Processing Technologies (Owori and Hagenimana, 2000) I. SPF-Like Products Other Potential Sweet Potato Products A. Consumer Acceptance of Vegetarian Sweet Potato Products Intended for Space Missions (Wilson et al., 1998) B. Sweet Potato ‘‘Kunuzaki’’ (Tewe et al., 2003) C. Sweet Potato Beverage D. Selected Patents Regarding Sweet Potato Products E. Preparation, Evaluation, and Analysis of a French-Fry-Type Product from Sweet Potatoes F. Textural Measurements and Product Quality of Restructured Sweet Potato French Fries (Walter et al., 2002) Potential of Sweet Potato in the Ugandan Food System Sweet Potato Processing and Utilization EVorts in Kenya Conclusions Recommendations Acknowledgments References

The overall objective of this chapter is to review the past, present, and future role of the sweet potato (Ipomoea batatas [L.] Lam) in human nutrition. Specifically, the chapter describes the role of the sweet potato in human diets; outlines the biochemical and nutritional composition of the sweet potato with emphasis on its b-carotene and anthocyanin contents; highlights sweet potato utilization, and its potential as value-added products in human food systems; and demonstrates the potential of the sweet potato in the African context. Early records have indicated that the sweet potato is a staple food source for many indigenous populations in Central and South Americas, Ryukyu Island, Africa, the Caribbean, the Maori people, Hawaiians, and Papua New Guineans. Protein contents of sweet potato leaves and roots range from 4.0% to 27.0% and 1.0% to 9.0%, respectively. The sweet potato could be considered as an excellent novel source of natural health-promoting compounds, such as b-carotene and anthocyanins, for the functional food market.

SWEET POTATO: A REVIEW

3

Also, the high concentration of anthocyanin and b-carotene in sweet potato, combined with the high stability of the color extract make it a promising and healthier alternative to synthetic coloring agents in food systems. Starch and flour processing from sweet potato can create new economic and employment activities for farmers and rural households, and can add nutritional value to food systems. Repositioning sweet potato production and its potential for value-added products will contribute substantially to utilizing its benefits and many uses in human food systems. Multidisciplinary, integrated research and development activities aimed at improving production, storage, postharvest and processing technologies, and quality of the sweet potato and its potential value-added products are critical issues, which should be addressed globally. I. INTRODUCTION Currently, in some developed countries, overnutrition rather than undernutrition presents a major public health challenge. However, from a global perspective, undernutrition, food insecurity issues, droughts, and limited agricultural technologies are major problems. In developing countries, many farmers are highly dependent on root and tuber crops, as contributing, if not principal, sources of food, nutrition, and cash income (Scott et al., 2000). From this standpoint, there is need to critically reevaluate versatile, locally available, hardy root and tuber crops with wide ecological adaptability for their usefulness in human nutrition. The sweet potato (Ipomoea batatas [L.] Lam) is one such crop because it is high yielding and drought tolerant, with wide adaptability to various climates and farming systems (Diop, 1998; Jiang et al., 2004). Furthermore, a single sweet potato plant may produce 40–50 roots ranging in length from a few to 30 cm, and weighing between 100 and 1000 g, and the roots, leaves, and shoots are all edible (CIAD et al., 1996). It has been emphasized that the sweet potato roots and leaves (greens or tips) can support more people per unit hectare than any other food (Woolfe, 1992). The leaves of the sweet potato are dark green and are expected to have nutritive values comparable to common dark green leafy vegetables (Ishida et al., 2000). Regular consumption (100 g or half-cup daily) of yellow- or orange-fleshed sweet potato roots having about 3 mg/100 g b-carotene on a fresh weight basis provide the recommended daily amount of vitamin A for children less than 5 years old (Tsou and Hong, 1992). Jalal et al. (1998) reported that incorporation of orange-fleshed sweet potato into meals eaten by 3- to 6-year olds improved vitamin A status. Weight for weight, some orange-fleshed sweet potato cultivars (cv.) contains 20–30 times more b-carotene than Golden Rice

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(Ye et al., 2000). Also, sweet potato is a typical food security crop because it can be harvested little by little over several months. It is because of these unique features and nutritional value of the sweet potato that the National Aeronautics and Space Administration (NASA) has selected it as a candidate crop to be grown and incorporated into the menus for astronauts on space missions. The sweet potato has immense potential and has a major role to play in human nutrition, food security, and poverty alleviation in developing countries. Sweet potato is a creeping dicotyledonus plant with the following scientific classification (Kingdom: Plantae; Division: Magnoliophyta; Class: Magnoliopsida; Order: Solanales; Family: Convolvulaceae; Genus: Ipomoea, and Species: batatas). The sweet potato, its leaves and roots are shown in Figure 1. Common names for the root include: sweet potato (English); batata, boniato, camote (Spanish); kumar (Peru); kumara (Polynesian); and cilera abana, ‘‘protector of the children’’ (eastern Africa); kara-imo, ‘‘Chinese potato’’ (southern Kyushu, Japan); Ubhatata (South Africa); and satsuma-imo, ‘‘Japanese potato’’ (most of the other parts of Japan). The sweet potato is said to have originated in the New World; however, the exact origin has not been well defined. Austin (1988) proposed the origin of the sweet potato as being between the Yucata´n Peninsula of Mexico and the Orinoco River in Venezuela. In 1514, nine sweet potato cv. were identified in Honduras. Currently, hundreds of cv. are grown throughout the world and are unique to countries or smaller regions within countries. All cv. are more or less sweet flavored. In developed countries, limited numbers of sweet potato cv. are grown. In the United States, producers tend to grow only one or two major cv. for regional and national markets, but may grow several cv. in small amounts for local markets. The two cv., which account for most of the current US acreage, are ‘‘Jewel’’ and ‘‘Beauregard.’’ There is a need to diVerentiate between the sweet potato and yam because of some confusion that exists regarding them in the United States. Sweet potatoes and yams are both angiosperms (flowering plants), however, they are botanically diVerent. Sweet potatoes, often called ‘‘yams’’ in the United States, are dicotyledons (having two embryonic seed leaves) and are from the Convolvulaceae or morning glory family. On the other hand, yams are monocotyledons (one embryonic seed leaf ) and are from the Dioscoreaceae or yam family. Also, the edible storage organ of the sweet potato is a true root, and for the yam it is a tuber. A tuber is a thickened part of the stem or rhizome (Kays et al., 1992). The root system of the yam is a rhizome, which is a thickened stem that grows horizontally underground. The appearance and shape of the sweet potato and yam are also diVerent. For example, the sweet potato is usually smaller, short, and blocky with tapered ends, while yams are usually long and cylindrical with some toes. b-Carotene content is usually high in orange-fleshed sweet potatoes

SWEET POTATO: A REVIEW

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FIG. 1 Sweet potato leaves and roots. (A) Sweet potato leaves. (B) Orange-fleshed sweet potato roots. (C) White-fleshed sweet potato roots (http//encyclopedia.laborlawtalk.com).

but low in yams. The growing season is 90–150 and 180–360 days for the sweet potato and yam, respectively (http://www.loc.gov/rr/scitech/mysteries/sweetpotato.html). This chapter focuses on the sweet potato and not yams. The overall objective of this chapter is to review the past, present, and future role of the sweet potato in human nutrition. Specifically, the paper will:
Describe the role of the sweet potato in human diets
Outline the biochemical and nutritional composition of the sweet potato with emphasis on its b-carotene and anthocyanin contents

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Highlight sweet potato utilization and its potential for incorporation into value-added products for use in human food systems
Use case studies to demonstrate the potential of the sweet potato in the African context II. SWEET POTATO IN HUMAN DIETS The records left by early Europeans indicated that sweet potato provided a staple food source for many indigenous populations in southern Central America and South America (Sauer, 1950). Piperno and Holst (1998) reported that starch grain analysis revealed sweet potato as a staple food source for inhabitants in coastal Peruvian sites. The sweet potato or kumara was one of the most important crops in the diets of the Maori people, early New Zealand settlers of Polynesian descent, and was already an important staple food in Hawaii in 1778 (Huang et al., 1999). Commercial cultivation of sweet potato began in Hawaii in 1849, and since then many cv. have been introduced (Valenzuela et al., 1994). The sweet potato remains an important staple food in the diets of the Maori people, Papua New Guineans, and Hawaiians (Cambie and Ferguson, 2003; Huang et al., 1999; Sawer, 2001). Sweet potato, which is the commonest traditional root crop in Papua New Guinea, is consumed daily by 66% and 33% of the rural and urban population, respectively (Sawer, 2001). The Satamu sweet potato provides the largest part of the energy intake and contributes to self-suYciency in Okinawa in the Ryukyu Island (Sho, 2001). Sweet potato is also an important staple in countries such as the Solomon Islands, Tonga, and New Caledonia (Hijmans et al., 2002). In parts of West, Central, and East Africa, sweet potato is an important source of calories and is consumed by people of all age groups (Hagenimana et al., 1998a). For example, in Nigeria, the traditional utilization of sweet potatoes includes: (1) boiled and eaten with stew; (2) boiled and pounded with either boiled or fermented cassava as ‘‘foofoo’’ or boiled or pounded yam; (3) dried and milled for sweetening of gruel or ogi porridge; and (4) sliced into chips, dried, and fried in vegetable oil or boiled with beans or vegetables. Koreans value sweet potato leaves as a very nutritious and tasty vegetable. The leaves are usually cooked together with other ingredients in various Korean dishes or can be dried and stored for later use as a boiled or fried vegetable (www.agnet.org/library/article/ pt2001034.html). The sweet potato is also an important starch source in China, Vietnam, Korea and Taiwan, and the Philippines (Collado et al., 1999; Marter and Timmins, 1992). Although sweet potato may contribute essential nutrients, it is usually consumed for its sensory properties, as a substitute or supplement to corn, rice, or wheat or as the main ingredient of traditional, but infrequently consumed dishes in many developing countries (Shewry, 2003; Van Den and del Rosario, 1984).

SWEET POTATO: A REVIEW

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In 1999, sweet potato accounted for approximately 20% of the total world production of root and tuber crops (FAO, 1999). Asia is the largest sweet potato-producing region in the world, with an annual production of 125 million tonnes. China produces roughly 65% of the world’s sweet potato, making it the leading supplier of sweet potatoes in the world (Hijmans et al., 2002). Latin America and North America produce about 1.9 million and 600,000 tonnes annually, respectively. In the United States, North Carolina and Louisiana each grow 40% of the total sweet potatoes, while California, Alabama, and Texas grow the bulk of the remainder (Burden, 2005). The only European country that produces considerable quantities of sweet potato is Portugal, at 23,000 tonnes annually (FAO, 1999). There are considerable concentrations of sweet potato in the Caribbean region; Indonesia; New Guinea, Papua New Guinea; and Vietnam (Hijmans et al., 2002). Production of sweet potato in Africa amounts to 6% of world production (Karyeija et al., 1998). The largest sweet potato growers in Africa are: Uganda, the third largest producer in the world (2.2 million metric tonnes); Rwanda (1.1 million metric tonnes); Kenya and Burundi (0.7 million metric tonnes each); Tanzania (0.4 million metric tonnes); and Ethiopia (0.2 million tonnes) (Diop, 1998; Karyeija et al., 1998; http://www.harvestplus.org/sweetpotato.html). The other African countries with annual sweet potato production (103) exceeding 100,000 tonnes are shown in Figure 2. However, over the last four decades, global sweet potato production has remained static, and demand for the crop is greatly decreased, possibly because of diversified eating habits and little knowledge about its nutritional and functional properties among other things (Kays, 2005; Yamakawa and Yoshimoto, 2002).

III. BIOCHEMICAL AND NUTRITIONAL COMPOSITION OF THE SWEET POTATO The sweet potato has immense potential to help prevent and reduce food insecurity and mal-, under-, and overnutrition in developing and developed countries because of its nutritional composition and unique agronomic features. However, paucity of information regarding the nutritional composition of the sweet potato greatly limits its exploitation. Improved awareness of the nutritional quality, utilization, and future economic importance of the crop has important implications for human food systems, nationally and internationally (Scott et al., 2000). The sweet potato contains many nutrients including protein, carbohydrates, minerals (calcium, iron, and potassium), carotenoids, dietary fiber, vitamins (especially C, folate, and B6), very little fat, and sodium. As described in the following sections, the nutrient composition of the sweet potato varies greatly according to genetic and environmental factors.

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Egypt 160

Guinea 143

Uganda 2235 Ethiopia 155 Cameroon 180

Rwanda Kenya 1100 630 Zaire 407 Burundi Tanzania Madagascar 451 674 560 Angola 200

FIG. 2 The 12 African countries with an annual sweet potato production (103) exceeding 100,000 tonnes. Note the main production area concentrated around Lake Victoria (in black). Modified from Karyeija et al. (2000) and FAO (1995).

A. PROTEIN: SWEET POTATO LEAVES

In Africa and Japan, the leaves of the sweet potato are eaten, and the protein content has been reported to be as high as 27% protein on dry weight basis (dwb; Diop, 1998). Tewe et al. (2003) reported that the protein content of sweet potato leaves was 18.4%, while fiber content was between 3.3% and 6.0%. Ishida et al. (2000) studied two kinds of sweet potatoes and reported that the leaves contained high amounts of protein (3.8 and 3.7 g/100 g), total dietary fiber (5.9 and 6.9 g/100 g), and ash (1.9 and 1.5 g/100 g). Ishiguro et al. (2004) described a newly developed sweet potato cv. (Suioh) for utilization as vegetable greens. The nutritional composition of the greens is shown in Table I. The total polyphenol content and radical-scavenging properties of the Suioh were reported to be much higher than that of spinach, broccoli, cabbage, and lettuce (Ishiguro et al., 2004). Also, sweet

SWEET POTATO: A REVIEW

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TABLE I NUTRITIONAL CONTENT OF SUIOH SWEET POTATO GREENS

Nutrient

Percentage (dwb)

Iron (mg/100 g) Calcium (g/100 g) Vitamin E (mg/100 g) Total carotenoid (mg/100 g)

2.6–26.4 1.2 15.8 34.7

Source: Ishiguro et al. (2004).

potato tea made from Suioh greens was more acceptable to sensory judges than other teas (Ishiguro et al., 2004). Pace et al. (1996) described the nutritional content of sweet potato greens as 4.0–6.0% protein, 8.0–12.0% carbohydrate, 60-mg/100 g calcium, and 80-mg/100 g phosphorus. Tewe et al. (2003) also reported that in some parts in Nigeria, sweet potato leaves were acceptable as soup ingredients in terms of flavor, appearance, palatability, softness, and acceptability. Islam (2006) reported that sweet potato leaves contain at least 15 biologically active anthocyanins, which are beneficial to human health and may also be useful as natural food colorants. B. PROTEIN: SWEET POTATO ROOTS

The crude protein content of sweet potato (Kjeldahl nitrogen  6.25) generally ranges from 1.3% to >10% dwb (Bradbury et al., 1985; Purcell et al., 1978). However, substantial variation has been shown to exist. Ishida et al. (2000) reported 2.1% and 1.3% protein for Koganesengan and Beniazuma sweet potato cv., respectively. Diop (1998) reported 1.0–2.4% protein in sweet potato while Bovell-Benjamin et al. (2001) and Dansby and Bovell-Benjamin (2003a) reported protein contents ranging from 1.2  0.05% to 1.8% (fresh weight) for hydroponically grown sweet potatoes. Oboh et al. (1989) analyzed 49 varieties of sweet potato sold in Nigerian markets and reported protein contents between 1.4% and 9.4%. The protein contents of sweet potato roots from 16 cv. grown in Sri Lanka ranged from 3.0% to 7.2% on dwb (Ravindran et al., 1995). Cambie and Ferguson (2003) reported 1.7% protein content for sweet potato while Gichuhi et al. (2004) reported 4.5%, 4.7%, and 9.0% protein (dwb) for cv. J6/66, Beauregard (commercial), and TU-82-155. Bovell-Benjamin et al. (2004) observed a wide variation in the protein content of three cv. of sweet potato with TU-82-155 containing almost twice as much protein (8.7  0.1%) on dwb as J6/66 (4.4  0.03%) and Beauregard (4.7  0.5%). Sporamins A and B, the major storage proteins in sweet potato, which account for more than 80% of the total protein, are also of importance (Gichuhi et al., 2004; Scott and Symes, 1996). It has been reported that sporamins A and B,

10

A. C. BOVELL-BENJAMIN

which are proteinase inhibitors, may have some anticarcinogenic properties (Maeshinia et al., 1985). Although the biological significance is still unclear, Hou and Lin (1997) reported that sporamins have antioxidant activity, acting as dehydroascorbate reductase and monodehydroascorbate reductase, which are associated with intermolecular thiol/disulfide exchange. Although regarded as a low-protein food in the United States, sweet potato serves as an important protein source to a large segment of the world’s population (Walter et al., 1984; Woolfe, 1992). For example, the highlanders of Papua, New Guinea rely on sweet potatoes as a major source of protein and for 60–90% of their energy requirements (Clark and Moyer, 1988).

C. b-CAROTENE

Globally, sweet potato has a significant role to play in the fight against vitamin A deficiency (VAD). VAD is of public health significance in developing countries, causing temporary and permanent eye impairments and increased mortality, especially among children, pregnant, and lactating women. It has been shown that more than 230 million of the world’s children have inadequate vitamin A intake, with 13 million of them being aVected by night blindness (Schweigert et al., 2003; Stephenson et al., 2000; Underwood and Arthur, 1996). Children in South Asia, eastern, western, central, and southern Africa have the highest prevalence of VAD (Mason et al., 2001). VAD is caused by habitual inadequate intake of bioavailable carotenoids (provitamin A) or vitamin A to meet physiological needs (Van Jaarsveld et al., 2005). Plant foods do not contain vitamin A, however, they contain precursors or provitamin A (b-carotene and other carotenoids), which the human body converts to vitamin A. One approach to controlling VAD is improving dietary quality and quantity through diversification. Dietary diversification includes the production of b-carotene-rich crops such as orange-fleshed sweet potato for human consumption (Van Jaarsveld et al., 2005). More than eight decades ago, Steenbock (1919) reported that sweet potato eliminated the symptoms of VAD in rats. Later, Ezell and Wilcox (1948) stressed the importance of sweet potato as a source of b-carotene. Haskell et al. (2004) concluded that daily consumption of cooked, pureed green leafy vegetables or sweet potatoes impacts positively on vitamin A stores in populations at risk for VAD. In Kenya, orange-fleshed sweet potatoes have been recognized as the least expensive year-round source of provitamin A (Low et al., 1997). Hagenimana et al. (1999) reported that consumption of orange-fleshed sweet potato increased the dietary vitamin A intake in Kenyan children and women. Another study indicated that consumption of orange-fleshed sweet potato could contribute substantially

SWEET POTATO: A REVIEW

11

to reducing VAD in sub-Saharan Africa (Low et al., 2001). The consumption of diets containing primarily orange-fleshed sweet potatoes as a source of b-carotene significantly increased serum retinol (vitamin A status) levels of Indonesian children with marginal VAD (Jalal et al., 1998). Van Jaarsveld et al. (2005) also concluded that increased consumption of orange-fleshed sweet potato could be a feasible food-based strategy for controlling VAD in children in developing countries. Sweet potato varieties exist in many colors of skin and flesh, ranging from almost pure white through cream, yellow, orange, or pink, to a very to deep purple, although white and yellow-orange flesh are the most common (Onwueme, 1978). For example, white- to cream-colored flesh sweet potatoes are common in the South Pacific, Africa, the Caribbean, and most other developing countries. In contrast, sweet potatoes commonly consumed in the United States and other developed countries, normally have yellow to orange flesh (Bradbury and Holloway, 1988). The commercial sweet potato varieties in Hawaii have white, cream, yellow to orange, and purple color flesh. The flesh colors and b-carotene content in sweet potato roots vary widely because they are aVected by genetic variety, maturity, growing conditions, postharvest storage, season, and which part of the vegetable is consumed (Hart and Scott, 1995; Hulshof et al., 1999). The intensity of the yellow or orange flesh color of the sweet potato is directly correlated to the carotenoid content (Ameny and Wilson, 1997). Sweet potato is an excellent source of carotenoid because its major carotenoid is all trans-b-carotene, which exhibits highest provitamin A activity among the carotenoids. Carotenoids, which can be red, yellow, or orange, are a diverse group of structurally related isoprenoids biosynthesized mainly by plants that have the capacity to trap lipid peroxyl radicals and singlet oxygen species (Ben-Amotz and Fishler, 1998). Provitamin A carotenoids are those which can be cleaved to yield retinaldehyde. b-Carotene, the primary carotenoid in sweet potato, is cleaved in the intestinal mucosa by carotene dioxygenase, yielding retinaldehyde, which is reduced to retinol (vitamin A). The total amount of vitamin A in foods is expressed as microgram retinol equivalents (Bender, 2002). Nutritionally, 6 mg of dietary b-carotene is equivalent to 1 mg of retinol (Bender, 2002). Provitamin A from orange-fleshed sweet potato appears to be more bioavailable than that from dark green leafy vegetables (de Pee et al., 1998; Jalal et al., 1998). Ben-Amotz and Fishler (1998) reported relatively high b-carotene content in sweet potato (Table II). Hagenimana et al. (1998a) reported varying b-carotene for orange, cream, and white flesh sweet potato roots, respectively, while Holland et al. (1991) reported b-carotene values for orange-fleshed sweet potatoes (Table II). Huang et al. (1999) reported b-carotene contents for 18 commercial varieties of sweet potato roots grown in Hawaii.

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TABLE II b-CAROTENE CONTENTS OF SWEET POTATO REPORTED BY DIFFERENT RESEARCHERS

References

b-Carotene content (mg/100 g) unless otherwise indicated

Holland et al., 1991

1800–16,000

Simonne et al., 1993 Bhaskarachary et al., 1995 Hulshof et al., 1999 Ben-Amotz and Fishler, 1998 Hagenimana et al., 1998a

Dansby and Bovell-Benjamin, 2003a

100–19,000 1000 5.0  1.0 to 58.0  70.0 79.8 2130  109 to 7983  339 158  39 to 1071.3  101 19.6 6700–13,100 <0.1–0.6 7.9  0.2 4.5  0.3 1566  306

Namutebi et al., 2004 Bendech et al., 2005

6800–12,500 1911–2348

Huang et al., 1999 Sungpuag et al., 1999

Sweet potato type Orange-fleshed sweet potatoes on fresh weight basis dwb Yellow-fleshed variety ‘‘kiran’’ Indonesian sweet potato varieties dwb Orange flesh, cream flesh, white flesh Orange flesh, yellow, white, and purple flesh Raw, yellow-flesh cooked, yellow-flesh Raw, orange-flesh, hydroponic, fresh weight Orange-flesh Orange-flesh grown in Burkino Faso

The b-carotene content of raw orange-fleshed hydroponically grown sweet potato was reported by Dansby and Bovell-Benjamin (2003a). Simonne et al. (1993) reported the b-carotene content in blanched sweet potato roots from diVerent cv. (Table II). Bendech et al. (2005) reported mean b-carotene contents for orange-fleshed sweet potatoes grown in Burkino Faso as shown in Table II. Namutebi et al. (2004) and Sungpuag et al. (1999) detailed high b-carotene concentrations for orange-fleshed, and raw and cooked yellow sweet potato varieties (Table II). Traditional Indian sweet potato cv. are white fleshed and contain no carotenes, however, a new yellow-fleshed variety, ‘‘kiran’’ showed significant amounts of b-carotene, and mean b-carotene content for six samples was 1087  0.14 mg/100 g (Bhaskarachary et al., 1995). Cancer is not a rare disease in most developing countries. For example, in women, cancer cumulative mortality from cancer in developing countries is actually higher than in developed countries (Parkin et al., 2005). Several epidemiological studies have shown associations between carotenoids such as b-carotene, and decreased risk for cancer, heart disease, and age-related macula degeneration (Kohlmeier and Hastings, 1995; Niizu and Rodriguez-Amaya, 2005; Olson, 1996; Russell, 1998; van Poppel and Goldbohm, 1995). Using a case-control study, Pandey and Shukla (2002) evaluated the possible role of diet

SWEET POTATO: A REVIEW

13

in gallbladder carcinogenesis. The authors reported that a significant reduction in odds ratio (OR 0.33; 95% CI 0.13–0.83) was seen with the consumption of sweet potato, radish, and green chili. On the basis of its b-carotene content, there is a potential role for sweet potato in cancer prevention and risk reduction. D. MINERAL CONTENTS

It has been argued that the mineral content of agricultural products varies with geographic location. Makki et al. (1986) reported that in two Egyptian sweet potato cv., the mineral in highest concentration was calcium followed by magnesium, iron, copper, zinc, and manganese. However, older data reported by Ekpenyong (1984) from FAO (1972) cited phosphorous as the mineral in highest concentration for sweet potatoes. The data indicated 56-, 36-, 0.9-, 2.0-, and 387-mg/100 g for phosphorus, calcium, iron, zinc, and manganese, respectively. Olaofe and Sanni (1988) reported potassium (3617 mg/100 g) as the most abundant mineral in sweet potato roots followed by magnesium (580 mg/100 g) and calcium (112 mg/100 g). Manganese, iron, copper, and zinc were present in low amounts of 8.8, 14.0, 1–5.0, and 3.0 mg/100 g, respectively. A more extensive discussion of mineral occurrence in sweet potato is contained in Woolfe (1992). E. DIETARY FIBER

The importance of dietary fiber in noncommunicable disease prevention has been extensively discussed elsewhere (Guillon and Champ, 2000; Schneeman, 1998). Dietary fiber contains all the polysaccharides and lignin of the diet that are not digested by human enzymes (Brody, 1994). Dietary fibers include: resistant starches; cellulose; hemicellulose; b-glucans; pectins, nonstarch polysaccharides and lignin (Brody, 1994). For populations that consume sweet potato as a staple food, its dietary fiber contribution could be critical. Huang et al. (1999) reported that the total dietary fiber content of orangefleshed sweet potato cv. ranged from 2.0 to 3.2 g/100 g fresh weight, which is higher than the 0.7 g/100 g listed in the United States Department of Agriculture (USDA) database. Four purple-fleshed sweet potatoes had dietary fibers ranging from 2.3 to 3.9 g/100 g, while yellow/white-fleshed sweet potato cv. had values between 2.3 and 3.3 g/100 g (Huang et al., 1999). Crude dietary fiber ranged from 3.8% to 5.9% in 49 varieties of sweet potatoes analyzed by Oboh et al. (1989). Two Egyptian sweet potato cv., ‘‘Abees’’ and ‘‘Giza 69’’ studied by Makki et al. (1986) had crude dietary fiber contents of 5.6% and 5.7%, respectively, dwb. Total dietary fiber content of two kinds of sweet potato roots were reported as 3.4 and 2.3 g/100 g by Ishida et al. (2000).

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A. C. BOVELL-BENJAMIN

However, in agreement with Tsou and Hong (1992), it should be noted that crude dietary fiber which consists of cellulose and lignin does not adequately express the role of sweet potato as a dietary fiber provider in human nutrition. Research is being conducted to provide consumers with a sweet potato, which is low in cellulose and lignin but high in other components of dietary fiber (Tsou and Hong, 1992).

F. ANTHOCYANINS

Until the mid-1800s, the only external sources of colorants used in food systems were natural (Gilbert, 2005). Over the next 50 years, synthetic organic dyes became the most common type of food coloring (Gilbert, 2005). However, during the last decade, the demand for food colorants from natural sources has increased because of legislative and consumer pressure to reduce the use of synthetic additives in foods (Giusti and Wrolstad, 2003). Evidence of this trend is reflected in food industry’s replacement of synthetic dyes such as FD&C red 40 and the banned FD&C red 2 with natural plant colorants (Fabre et al., 1993). Additionally, most natural colorants are bioactive, and they add status to food products marketed as ‘‘natural’’ and ‘‘organic.’’ Anthocyanins are a large group of water-soluble pigments responsible for the attractive orange, red, purple, and blue colors of fruits and vegetables (Plata et al., 2003). Anthocyanins extracted from plants are commonly used in soft drinks, jams, confectioneries, and bakery products as a natural colorant (Plata et al., 2003). Deep purple sweet potato flour (SPF) and paste are also used as coloring materials for bread, snacks, and noodles (KNAES, 1995). Cevallos-Casals and Cisneros-Zevallos (2002) reported Peruvian, purple sweet potato colorant as having much higher color retention and more stability than commercial red grape colorant. Additionally, there is an increasing demand for carotenoids, such as b-carotene, as food colorants because of their natural origin, lack of toxicity, and flexibility of providing both lipo- and hydrosoluble colorants (Ben-Amotz and Fishler, 1998; Cinar, 2005). Consumption of the sweet potato has always been associated with good health and enhanced human nutrition. However, limited research has addressed the role of sweet potato in human nutrition and health. In human life, cell damage from oxygen free radicals (OFRs) is ubiquitous. OFRs are known to have carcinogenic potential (Dreher and Junod, 1996). Dietary or natural antioxidants such as carotenoids are said to be protective against the eVects of OFRs. Antioxidants exert their eVect on OFRs by neutralizing them (Scheibmeir et al., 2005). It has been reported that white, yellow, orange, and purple-fleshed sweet potato cv. have antioxidative and radical-scavenging activities, with the purple

SWEET POTATO: A REVIEW

15

Ayamurasaki

FIG. 3 Ayamurasaki sweet potato with high-antioxidative and radical scavenging activities (Cevallos-Casals and Cisneros-Zevallos, 2002; Furuta et al., 1998).

ones such as cv. Ayamurasaki (Figure 3) having the highest activity (CevallosCasals and Cisneros-Zevallos, 2002; Furuta et al., 1998). Antioxidant activity of purple sweet potato was observed to be 3.2 times higher than that of a blueberry variety (Cevallos-Casals and Cisneros-Zevallos, 2002). Interestingly, the antioxidant activity in sweet potato skin was found to be almost three times higher than in the rest of the tissue. Animal studies have shown that when purple sweet potato juice from cv. Ayamurasaki was fed to rats it reduced liver injury induced by carbon tetrachloride (Suda et al., 1998). Persons consuming cv. Ayamurasaki juice daily for 44 days eVectively decreased the blood serum levels of l-guinidine triphosphate (GTP), glutamin-oxaloactive transaminase (GOT), and glutamin-pyruvic transaminase (GPT) (indicators of liver injury) (Suda et al., 1998). Suda et al. (2003) reported that purple-fleshed sweet potato is a good source of anthocyanin. Some foods contain mutagens, which are associated with carcinogenesis. Purple-fleshed sweet potato roots were reported to have high antimutagenic activity (Yoshimoto et al., 1999). Various studies have reported the relationship between consumption of anthocyanin-rich foods and improved health. Health benefits associated with anthocyanin extracts include chemopreventive activities such as antimutagenicity and antioxidative potential (Table III). It has also been shown that a glycolipid (ganglioside) found in sweet potato juice reduces the multiplication of cultured human cells in cancers of the womb neck and melanoma (Shimozono et al., 1996). Sweet potato juice has been associated with antihypertensive and antidiabetic properties (Kusano and Abe, 2000; Matsui et al., 2001; Suda et al., 1998). Further, the anthocyanins, chlorogenic acid, and other polyphenolic compounds in sweet potato have the capability to inhibit carcinogens generated during food processing and cooking.

16

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TABLE III POSSIBLE HEALTH BENEFITS ASSOCIATED WITH ANTHOCYANINS

Possible health benefit

References

Chemopreventive activities (e.g., antimutagenicity) Vasoprotective and antiinflammatory properties Enhancement of sight acuteness Antidiabetic, controlling diabetes, and antineoplastic agents Hepatoprotective Vasotonic agents Radiation-protective agents Antioxidant capacity

Karaivanova et al., 1990; Morazzoni and Magistretti, 1986 Lietti et al., 1976

Antihypertension Fibrocystic disease of the breast

Politzer, 1997; Timberlake and Henry, 1988 Kamei et al., 1995; Matsui et al., 2002, 2004; Scharrer and Ober, 1981 Mitcheva et al., 1993 Colantuoni et al., 1991 Akhmadieva et al., 1993; Minkova et al., 1990 Islam et al., 2002, 2003; Prior et al., 1998; Rice-Evans and Miller, 1996; Tamura and Yamagami, 1994; Wang et al., 1997 Suda et al., 1998; Yoshimoto, 2001 Leonardi, 1993

Sweet potato also contains the coumarins scopoletin (3, R —H), aesculetin (3, R —OH), and umbelliferone (3, R —OMe) compounds, which have anticoagulation properties and are postulated to inhibit HIV replication (Weiss and Finkelmann, 2000). The sweet potato could be considered as an excellent novel source of natural health-promoting compounds, such as anthocyanins, for the functional food market. Ultimately, this could increase utilization and demand for the crop by consumers, and the food industry. In sum, the commonly consumed white to cream-colored-flesh sweet potatoes in the South Pacific, Africa, the Caribbean, and most other developing countries contain less b-carotene than the orange-fleshed sweet potato. Orange-fleshed sweet potato is one of the most promising plant sources of b-carotene (Hagenimana and Low, 2000). However, it should be noted that although orange-fleshed sweet potato roots contain abundant amounts of b-carotene, the content decreases over time with processing (van Hal, 2000). Sungpuag et al. (1999) also demonstrated that boiling yellow-fleshed sweet potato resulted in further loss (43%) of b-carotene. Increased consumption of fresh orange-fleshed sweet potato roots and sweet potato-based processed foods in developing countries could contribute substantially toward improving vitamin A nutrition and enhancing food security. Sweet potato contains b-carotene and other dietary carotenoids, which have anticancer properties. The high concentration of anthocyanin and b-carotene in sweet potato combined with the high stability of the color extract make it a promising and healthier alternative to synthetic coloring agents in food systems.

SWEET POTATO: A REVIEW

17

IV. SWEET POTATO UTILIZATION AS VALUE-ADDED PRODUCTS IN HUMAN FOOD SYSTEMS Over the last 40 years, utilization of the sweet potato has shifted from being a ‘‘subsistence,’’ ‘‘food security,’’ or ‘‘famine relief’’ crop in some developing countries. Although traditional uses are still important in many countries, new uses are emerging, especially in China and parts of sub-Saharan Africa. Average annual per capita consumption of fresh roots for 1994–1996 was estimated at: 73, 18, 9, 7, 5, and 2 kg in Oceania, Asia, Africa, Japan, Latin America, and United States, respectively (FAO, 1997). In contrast to potato, per capita sweet potato consumption in Canada, Europe, and Australia is extremely limited and often confined to immigrant populations. In the developing world, sweet potato consumption varies by countries, regions, season of year, and income. For example, in Africa annual per capita sweet potato consumption in Rwanda is estimated at 160 kg; Burundi, 102 kg; and Uganda 85 kg. In northeast Uganda (one of the poorest parts of that country), sweet potato becomes a seasonal staple during the dry season when most other foodstuVs are in short supply. Currently, US per capita consumption is roughly 9.5 kg down from a recorded high of 30.6 kg in 1949. Several years ago, Carver (1918) recognized and demonstrated the importance of processing the sweet potato into value-added products by encouraging economically deprived farmers in the southern United States to grow and process it. Approximately 100 new products were processed from the sweet potato in Carver’s era, including flour, starch, sugar bread, mock coconut, tapioca, and vinegar (Carver, 1918). Additionally, SPF has been fermented to make products such as soy sauce and alcohol, or if immediately cooked, could be further processed into wine, vinegar, and ‘‘Nata de coco.’’ ‘‘Nata de coco’’ is a popular dessert or ‘‘on-the-go’’ food in the Philippines, and adjacent Asian countries, and is becoming very popular in Japan. It is a chewy, translucent, ready-to-eat mixture of coconut, sweet potato, and fruit, which resembles the American canned ‘‘fruit cocktail.’’ Today, long after Carver’s era, most of the global sweet potato production is marketed as cleaned, but otherwise unprocessed roots (Burden, 2005). Unprocessed sweet potato roots have a short shelf life compared to carrots, white potatoes, and so on, and they are diYcult to store. In developing countries, where limited transport infrastructure exists, processing the sweet potato into value-added products provides an alternative to the diYculties associated with storage and transport of the raw roots (Dansby and Bovell-Benjamin, 2003a). Furthermore, vegetable industry data suggest that 15% of all vegetables marketed in the United States are now in more consumer-friendly, convenient forms (Burden, 2005). If the sweet potato is to gain increased market visibility

18

A. C. BOVELL-BENJAMIN

and play a more important role in human nutrition, some of the total production must be converted into more consumer-friendly, convenient forms such as: (1) ‘‘minimally processed’’(ready-to-use—peeled and packaged or peeled, cut, and packaged); (2) ‘‘intermediate processed’’ (bulk ingredients such as flour and starch for use in other products, and so on); and (3) ‘‘preprocessed’’ (readyto-eat products). The following sections discuss some potentially feasible value-added products from the sweet potato. V. SWEET POTATO STARCH UTILIZATION IN HUMAN FOOD SYSTEMS In developing countries, sweet potato oVers much potential for income generation through small-scale processing because they are eYcient producers of carbohydrate. Carbohydrates could be converted into stable intermediate bulk ingredients (starch and flour) that are suitable for diverse markets in the food industry (Wheatley et al., 1996). Bulk ingredients from the sweet potato can be incorporated into the menus for astronauts and human food systems by further processing them into convenient, ready-to-use products. For example, sweet potato starch (SPS) can be enzymatically processed into diVerent products such as glucose syrup, which have a wide range of applications in the food and pharmaceutical industries (Sarikaya et al., 2000). Starch is very versatile with a number of uses in most major industries, but in developing countries, it is used predominantly to make processed foods. Worldwide, the biggest user of starch is the sweetener industry. In the food industry, starch is used to impart ‘‘functional’’ properties to processed foods such as thickening, binding, and filling. Starch is also used in canned soups, instant desserts, ice creams, noodles, processed meats, sauces, and bakery products. It can be processed into sweeteners and syrups, and in the manufacture of monosodium glutamate, a taste enhancer (Fuglie and Oates, 2002; Zhang and Oates, 1999). About 5% of total SPS production is used for making distilled spirit ‘‘shochu’’ in Japan as well as lactic acid, butanol, acetone, vinegar, and yeast (Woolfe, 1992). SPS has been successfully used to thicken chocolate pudding at Tuskegee University (HoVman and BovellBenjamin, 2001). Major users of starch in nonfood industries include the textile, paper, plywood, and adhesive industries and pharmaceuticals. Starch is a naturally occurring biopolymer in which glucose is polymerized into amylose, an essentially linear polysaccharide, and amylopectin, a highly branched polysaccharide. Starch occurs in plant tissues in the form of discreet granules whose size, shape, and form are unique to each botanical species (Woolfe, 1992). Sweet potato roots contain approximately 80–90%

SWEET POTATO: A REVIEW

TCAM-SB

0000

30 kv 10 µm

19

×2.000

FIG. 4 Scanning electron micrographs of sweet potato starch granules (Miller et al., 2003).

carbohydrate, mainly starch, making it a good raw material for the starch industry (Garcia and Walter, 1998; Lu and Sheng, 1990; Miller et al., 2003). Brabet et al. (1998) evaluated 106 sweet potato clones and reported an average total starch content of 61.5% dwb. Miller et al. (2003) reported 81% starch content on dwb in SPS. According to Woolfe (1992), sweet potato starches occur in oval-, round-, or polygonal-shaped granules, and mean granule size ranges from 12.3 to 21.5 mm. Bovell-Benjamin et al. (2004) isolated SPS and reported granule sizes in the range of 9.0–34.2, 2.6–15.8, and 5.3–21.8 mm for J6/66, Beauregard, and TU-82-155 cv., respectively (Figure 4). These findings were consistent with those of Mandamba et al. (1975) and Chen et al. (2003) who reported the same phenomenon for the granule size of three Chinese sweet potato cv. Kays (1992) also reported a range from 1 to 30 mm for SPS granules. Starch processing from sweet potato can create new economic and employment activities for farmers and rural households and can add nutritional value to food systems. Conversion of sweet potato to starch should be especially attractive to developing countries because it can help to lessen the need for imported materials, which reduces import bills (Garcia and Walter, 1998). For example, sweet potato accounted for 26% of starch production in Asia, and the global SPS production of 4.15 million metric tonnes in the early 1990s came from Asia (Ostertag, 1993). SPS is widely used in a variety of food and industrial applications in Asia (Tian et al., 1991). An industry based on SPS extraction has been developed in several regions in China (Li et al., 1991) and may account for more than two million tonnes annually, but the statistics are not quite precise (Wheatley and Bofu, 2000). The starch is utilized primarily

20

A. C. BOVELL-BENJAMIN

for the production of traditional noodles, although some factories use it for production of derived products such as maltose. For noodles, starch quality is an important factor, and starch from sweet potato is preferred over starch from other crops such as corn and cassava (Fuglie and Oates, 1990). Vietnam exports about 840 tonnes of sweet potato annually to China for production of maltose and glucose syrups, which are used in the manufacture of baby foods in some developed countries. Recently, SPS has emerged as a commercially important value-added product in the Philippines with the institution of three starch plants in the last 9–10 years (Collado et al., 1999). CIAD et al. (1996) assessed the potential demand for SPS in both food and nonfood industries in China, and industry estimated starch demand at over 200,000 million tonnes versus 100,000 million tonnes of maize starch actually supplied to the industrial sector. This situation reflects a great potential for SPS in human food systems, although some uncertainties have been reported. Researchers have reported that starches from diVerent sweet potato cv. exhibit a variety of nutritive, physical and sensory properties, such as higher starch content and more extractable starch (Madhusudhan et al., 1996). Maximizing starch yield from the sweet potato requires varieties with high dry matter and starch contents; however, these genetic characteristics can be manipulated. Ideally, for most food products SPS should have a smooth texture, which is consistently soft and flexible at low temperature and must be able to retain its thickening power at high temperature (Garcia and Walter, 1998). In SPS production, quality of the fresh root and the amount of extractable starch are important because of their influence on final product quality and eVects on process eYciency. The key issue in SPS production is product quality, especially the chemical (ash, protein, fiber, and so on) and functional properties of the starch itself. Wheatley and Bofu (2000) reported that SPS has low viscosity values on pasting when compared with potato starch, and it may not be suYciently white in color. However, Miller et al. (2003) reported L* color values of SPS, which are comparable to those of cornstarch. Excessive moisture content, as high as 15%, has been reported for SPS, but Miller et al. (2003) and Bovell-Benjamin et al. (2004) reported a 4.4  0.2%, 5.0  0.2%, and 6.0  0.3% moisture contents for starch extracted from three sweet potato cv. (J6/66, TU-82-155, and Beauregard, respectively). Garcia and Walter (1998) extracted starch from seven sweet potato cv. and reported moisture levels extending from 9.8% to 15.3%. The limited chemical properties (impurities) reported could be addressed through process improvements such as more eVective separation, additional purification steps, and more eYcient drying. For example, in China, Timmins and Marter (1992) added ‘‘sour liquid’’ from a traditional fermentation process (liquid fermentate from peas or

SWEET POTATO: A REVIEW

21

beans) at the separation stage to remove impurities, which produce oV-colors in the SPS (Figure 5). The process of starch isolation from the sweet potato using a modified technology with more eYcient drying is shown in Figure 5. The functional properties of SPS can be manipulated by processing (milling, sifting) as well as by cv. selection and chemical or enzymatic modifications. The seasonal nature of the sweet potato harvest limits the processing period,

A Freshly harvested sweet potato roots Water Washing Washed roots Water Grinding SP slurry (mash) Water

Extraction

Sieving SP starch slurry Sweet potato residue (pig feed)

Aqueous solution (drain)

Sour liquid solution (1st liquid)

Sour liquid solution (2nd liquid)

(Mix) Sedimentation (1) Wet starch (Mix) Sedimentation (2) Wet starch

Sour liquid Purification Water

(Mix) Sedimentation (3) Wet starch

Dewatering Moist starch

Aqueous solution (drain) Drying (sun)

Dry sweet potato starch

FIG. 5 (continued )

Final preparation

22

A. C. BOVELL-BENJAMIN B

Water

Sweet potato roots

Wash, peel, weigh

Water, dirt

Homogenize (2:1 water to sweet potato ratio)

Centrifuge (3000 rpm, 5 minutes)

Decant

Repeat last two steps Moist starch

Dehydrate (708C, 12 hours)

Grind

Mill

Sweet potato starch

FIG. 5 Procedures for sweet potato starch production. (A) Sweet potato starch production by the sour-liquid method (Timmins and Marter, 1992). (B) Sweet potato starch isolation (Miller et al., 2003).

and such constraints help to make large-scale investment in production of SPS uneconomic. However, small- and medium-scale enterprises may not suVer from this drawback. The summaries of the following studies demonstrate the potential usefulness of SPS in human food systems in space and on Earth.

SWEET POTATO: A REVIEW

23

A. BULK INGREDIENTS FROM THREE CV. OF SWEET POTATOES: COMPOSITION AND PROPERTIES (BOVELL-BENJAMIN ET AL., 2004)

The study processed three sweet potato cv. into starch and evaluated the suitability of the starch for use as a bulk ingredient on space flights and on Earth. Starch was isolated from TU-82-155, Beauregard, and J6/66 cv. as shown in Figure 5B, and X-ray diVraction (XRD) was used to determine starch crystallinity level. The protein, ash, moisture, carbohydrate, and fat contents of starch from the three diVerent cv. were similar (Table IV). Figure 6 shows the XRD patterns of the three sweet potato starches, as well as that for TU-82-155 hydroponically grown sweet potato. The Beauregard, J6/66, and the hydroponic sweet potato starches had similar XRD patterns. However, the field grown TU-82-155 exhibited only one XRD peak, indicating that the sample was almost entirely amorphous. According to Morris et al. (2005), the strong ˚ spacings are consistent with the presence of crystallites character3.8- to 5.8-A ized by a hexagonal array of sixfold amylase double helices, whose central channel is occupied by another double helix. This structure, which is typical of cereal starch crystallites, was also recognized in SPS. These results suggest that it is possible to relate the functional properties of the SPS to the XRD patterns, and hence predict their potential use in human food systems. B. EFFECTS OF PROCESSING TECHNOLOGY ON SPS YIELD AND QUALITY (JIANJUN, 2004)

The eVect of processing technologies that aVect the quality and extraction rate of SPS was examined by orthogonal design. An orthogonal test was made with nine trials which included three combinations of technologies on milling method, separating fineness, and precipitation method of starch extraction from ‘‘Xushu 18’’ sweet potato cv. Two sets of technology to enhance starch quality and extraction rate were identified. Recommendations to improve

TABLE IV PROXIMATE COMPOSITION STARCH PROCESSED FROM THREE SWEET POTATO CV.

Cultivar

Moisture (%)

Carbohydrate (%)

Protein (%)

Ash (%)

Fat (%)

J6/66 TU-82-155 Beauregard

4.4  0.2 5.0  0.2 6.0  0.3

93.6 92.8 92.1

1.3  0.0 1.2  0.0 1.1  0.1

0.6  0.01 0.7  0.04 0.6  0.01

0.1  0.1 0.3  0.2 0.2  0.1

Source: Bovell-Benjamin et al. (2004).

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FSST 100

50

0 BSS 100

Intensity (counts)

50

0 HSST 100

50

0 JSS 100

50

0 5

10

15

20 25 2-Theta (8)

30

35

40

FIG. 6 X-ray intensity curves for starch from three sweet potato cv. FSST, starch from TU-82-155 cv. (field grown); BSS, starch from Beauregard cv.; HSST, starch from TU-82155 cv. (hydroponic); JSS, starch from J6/66 cv. (Greene, 2003).

SWEET POTATO: A REVIEW

25

starch quality were the combination of saw tooth milling at 1000 rpm, separation using 120-size mesh, and precipitation with sour liquid. A combination of hammer mill in 4200 rpm, separation using 120-size mesh, and natural precipitation was recommended to improve extraction rate. C. PHYSICOCHEMICAL AND VISCOMETRIC PROPERTIES OF AN SPS SYRUP (MILLER ET AL., 2003)

This study processed syrup from SPS and determined its physicochemical [refractive index (RI) and color] and viscometric properties during storage at 21  3 and 4 C. SPS was isolated from field-grown Hillbilly sweet potato cv. (Figure 5B). The SPS was rehydrated, heated to 102 C, treated with a-amylase at 90 C for 5 hours, cooled, and further treated with glucoamylase at 62.5 C for 12 hours (Figure 7). The solutions were filtered, evaporated, and cooled. RI, color, and viscometric properties were measured. The RI of the sweet potato syrup was significantly higher (p < 0.01) than that reported in the literature (1.42  0.02 vs 0.66). The mean L*, a*, and b* values of the syrups were 68.8  0.6, 0.7  0.1, and 18.7  0.6, respectively.

Sweet potato starch and water L I Q

Heat to 1028C Thermostable bacterial α-amylase Incubate at 1028C

Dextrins (branched and linear) Cool adjust to pH 6.4

Glucoamylase S A C

300 µl, incubate at 62.58C, 12 hours Filtration and evaporation

High glucose syrup

FIG. 7 Overview of enzymatic hydrolysis of sweet potato starch into glucose syrup. LIQ ¼ Liquefaction, SAC ¼ Saccharification (Bovell-Benjamin et al., 2005).

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The mean viscosity of the sweet potato syrup was 1.98 Pas at room temperature and 0.95 Pas at 4 C. The viscosity for the sweet potato syrup gradually increased as storage time increased. With syrups stored at both temperatures, increased shear stress decreased their viscosities. However, when a commercial corn syrup was tested under the same conditions, there was no decrease in viscosity as shear stress increased. Overall, the SPS syrup had RI, color, and viscosity comparable to that of the commercial corn syrup. D. EFFECTS OF pH AND CONCENTRATION TIMES ON SELECTED FUNCTIONAL PROPERTIES OF A SWEET POTATO SYRUP (YOUSIF-IBRAHIM ET AL., 2003)

The overall objective of this study was to optimize previously developed sweet potato syrup. Specifically, the study determined the eVect of varying pH and time at the liquefaction and concentration stages, respectively, on the moisture, RI, color, and yield of an SPS (Figure 10). SPS was used to process three syrups in which the protocol remained constant, except for the pH and concentration times. At the liquefaction stage, the pH was adjusted to 6.4, 7.0, and 8.0 for Syrup A (SPSA), Syrup B (SPSB), and Syrup C (SPSC), respectively. At the concentration stage, the syrups were exposed to 35, 40, and 43 minutes of heat. The moisture content of the syrups concentrated for 35 minutes was significantly (p < 0.05) diVerent, with the SPSC having the highest moisture. Similarly, the moisture content for the SPSC heated for 40 minutes was highest, but lowest when heated for >43 minutes. The mean refractive indices were similar for the syrups, ranging from 1.4 to 1.5. As the concentration time increased, the L* value decreased for the syrups, with SPSA being lightest in color at all concentration times. At all concentration times, the total syrup yield from 60-g SPS was highest for SPSC. The syrup with pH 8.0, and 40 minutes, at the liquefaction and concentration stages, respectively, had the most desirable RI, color, and overall yield. E. INFLUENCE OF a-AMYLASE ON THE PHYSICAL PROPERTIES AND CONSUMER ACCEPTABILITY OF SPS SYRUP (BOVELL-BENJAMIN ET AL., 2005)

Technology was developed on a laboratory scale for the production of SPS syrup, and the eVect of varying levels of a-amylase on syrup quality, storage stability, and consumer acceptance were evaluated. Three levels of thermostable bacterial a-amylases (1.5, 3.0, and 4.5 ml) were used for conversion of SPS into glucose syrup. The 1.5-ml a-amylase-treated SPS was dropped

SWEET POTATO: A REVIEW

27

from the experiment because there was no hydrolysis. The enzymatic conversion of SPS into glucose was significantly higher (p < 0.05) for the 4.5-ml a-amylase-treated compared to the 1.5 and 3.0 ml levels. The RI was 1.5 and 1.4 for the 4.5 and 3.0 ml a-amylase-treated syrups, respectively, while moisture content (16.7 vs 12.5) and  Brix (65.0 vs 57.0) were higher for the 4.5 ml a-amylase-treated syrup. Syrups were stored at room (RSPSS) and refrigerated (RESPSS) temperatures. During storage, the L* color value for RESPSS was significantly higher ( p < 0.05) than that of RSPSS. Similarly, the  Brix was 64.1  1.1 and 66.0  1.7 for the RSPSS and RESPSS, respectively. The RSPSS was stable for 12 weeks. The sweet potato starch syrup (SPSS) and two commercial syrups (maple and ginger) were evaluated by 112 children between the ages of 12 and 13 years on a nine-point hedonic scale with plain waZe as a ‘‘carrier.’’ For the younger children, there were no significant diVerences in liking between the SPSS (6.1) and maple syrup (5.8). However, the mean preference for the ginger syrup was significantly less (p < 0.001). Overall, the SPSS had acceptable physical and consumer properties, indicating the need for further research to convert it into a commercially viable product.

F. SENSORY AND CONSUMER EVALUATION OF SPS SYRUP (MILLER ET AL., 2003)

This study evaluated selected sensory attributes of SPS using a modified magnitude estimation (ME) procedure, and determined consumer acceptance using a nine-point hedonic scale. A syrup made from hydroponic sweet potato starch (HSPS) was tested against two commercial control (ginger and corn) syrups. For the ME, 11 undergraduate students on Tuskegee University campus were trained as judges to do ME scaling. The judges were trained in ratio scaling by contrasting a reference line with relative lengths of experimental lines in random order in proportion to the fixed modulus of 15 (Giovanni and Pangborn, 1983). The three syrups were evaluated in triplicate with the modulus anchored at ‘‘30.’’ Five attributes, namely, mouthfeel, pourability, aroma, sweetness, and color were evaluated. The samples were evaluated relative to the modulus, for example, if a sample was twice as sweet as the modulus, it would be assigned the value of ‘‘60.’’ For the consumer testing, 43 untrained judges evaluated the syrups for color, sweetness, aroma, viscosity, and overall preference. The pourability of the HSPS and corn syrups was significantly ( p < 0.05) higher than the ginger syrup, which is a more desirable feature for consumers. The ME judges scored the HSPS as having a more desirable color than both commercial syrups. Ratings for the HSPS and ginger syrups were similar for the aroma attribute, but significantly higher for the corn syrup. The ginger

28

A. C. BOVELL-BENJAMIN

and corn syrups were equally sweet, but the HSPS was significantly ( p < 0.05) less sweet. Consumers liked the color HSPS and corn syrups moderately (7.0 score on the hedonic scale), and this is promising because a 7.0 indicates that it is highly likely the particular attribute will be acceptable to consumers. The viscosity of the HSPS was also liked slightly and equally well as the commercial ginger syrup. However, the sweetness and aroma attributes were disliked slightly. Further optimization of the HSPS syrup such as isomerization into fructose syrup could help to improve the sweetness and aroma attributes. VI. ADVANCES IN SPF PRODUCTION AND UTILIZATION FOR HUMAN FOOD SYSTEMS Peters and Wheatley (1997) conducted an in-depth diagnostic assessment of the feasibility of establishing a household-level SPF processing industry in East Java. Their results identified SPF as a promising product to increase rural income with potential markets in bakery, noodle, and various snacks. This finding is also relevant to other developing countries such as Kenya, Uganda, and Nigeria where the potential for small-scale SPF production was identified as an alternative for traditional flours (Hagenimana and Owori, 1996; Hagenimana et al., 1995; Omosa, 1994). SPF could be marketed as a low-cost alternative to imported wheat flour, thus reducing bakery costs and foreign exchange bills. For example, Peters and Wheatley (1997) postulated that for a 5% substitution rate, the potential savings from wheat import reductions for Indonesia would be US$40,000,000 based on 1995 import volume and value. SPF can also serve as a source of carbohydrate, b-carotene, minerals; can add natural sweetness, color and flavor to processed products; and can prevent food allergies in some instances (van Hal, 2000). Food allergies have become a public health issue in many countries including the United States (Maleki, 2001). Cereals containing gluten are ranked among the eight most common causes of food allergy (Taylor and Hefle, 2001). It is estimated that up to 5% of the population has serious allergies to some foods, including the gluten in wheat (Mannie, 1999). Celiac disease aVects the small intestines due to sensitivity to gluten present in certain cereals including wheat, wheat starch, rye, barley, triticale, and probably oats, and the only eVective treatment is strict adherence to a 100% gluten-free diet for life (Caperuto et al., 2000). SPF can serve as an alternative for individuals diagnosed with celiac disease or with allergies to the gluten in wheat. Perhaps, more importantly, van Hal (2000) estimated that SPF could contribute 0–100%, 20%, 20–40%, 17%, and 10% of the daily nutrient needs [based on the recommended daily allowances (RDA)] for b-carotene, thiamin, iron, vitamin C, and protein, respectively. SPF can also be used

SWEET POTATO: A REVIEW

29

directly or as a raw material for processing into other products. A variety of products such as doughnuts, biscuits, muYns, cookies, fried sweet potato cakes, extruded sweet potato chips, ice creams, porridge, brownies, pies, breakfast foods, and weaning foods have been made from SPF (Fuglie and Hermann, 2004; Greene, 2003). In India, dried sweet potatoes grounded into flour are used to supplement flours in bakery products, chapatis, and puddings, while in the Philippines, dried sweet potato chips are pounded into flour for use in gruel (Nair et al., 1987). The steps involved in processing sweet potato into flour are shown in Figure 8. The ratio of the weight of the flour to the weight of the fresh sweet potato roots expressed as a percentage is termed the yield or conversion rate. Limited information regarding flour yields from sweet potato is available, but it is known that flour yield is dependent on factors such as sweet potato cv., technologies used, and the dry matter of the final flour. Dawkins and Lu (1991) reported a 13–18% yield in flour prepared from steam blanched, microwave blanched, and unblanched sweet potatoes. A flour yield ranging from 17% to 38% for diVerent sweet potato cv. was also reported by Gakonyo (1993a). Two Philippine sweet potato cv. Georgia Red and Ilocos Sur were reported to yield 12 and 37 kg, respectively, of flour from 100-g fresh roots (Van Den, 1984). Dansby and Bovell-Benjamin (2003a) calculated the mass balance for flour made from hydroponic sweet potato and reported a yield of 15%. Average sweet potato root:flour conversion rates ranging from 23% to 27% were reported by Peters et al. (2005). Suismono (1995) reported 24% fresh root:flour conversion rate, while Martin (1984) reported a 17–38% range. Peters and Wheatley (1997) calculated the data from two on-farm trials, and the fresh

Fresh sweet potato roots

Grind

Mill

Weigh, wash

Dehydrate, 708C, 12 hours

Peel, wash

Shred

Sweet potato flour

FIG. 8 Production of sweet potato flour (Dansby and Bovell-Benjamin, 2003a).

30

A. C. BOVELL-BENJAMIN

root:flour conversion rates were approximately 20%. Overall, the reported literature indicates that fresh sweet potato root:flour conversion rate ranges from 13% to 38%. Variations in yield could be aVected by factors such as drying times, cv., processing methods, and calculations. It is important to understand the functional, physical, and chemical characteristics of SPF if its suitability for use in value-added products has to be determined. Previous researchers have noted a lack of information on SPF quality. For example, Hagenimana and Owori (1996) mentioned that SPF is very convenient for utilization by the food industry, but its quality and shelf life are not documented. The quality characteristics of SPF include high starch content, white color, low acidity, crude fiber, and ash contents (Antarlina, 1990). The moisture content of SPF is especially important because high moisture can speedup chemical or microbial deterioration (van Hal, 2000). According to Woolfe (1992) and Collado and Corke (1999), the moisture content of SPF ranges from 4.4% to 13.2%. Reddy and Basappa (1997) prepared SPF using diVerent peeling methods: hand, lye, mechanical abrasive, no peeling, and no peeling and cooking. Final SPF had moisture contents between 9.3% and 12.6%. The sensory, nutritional, and microbiological quality of SPF processed from nonsoaked and soaked slices using minimum amounts of water were compared by Owori and Hagenimana (2000). The moisture contents of SPF soaked for 0, 30, 60, and 90 minutes are shown in Table V. Fuglie and Hermann (2004) and TABLE V MOISTURE, CARBOHYDRATE, PROTEIN, AND b-CAROTENE CONTENTS OF SWEET POTATO FLOUR

References

Moisture (%)

Carbohydrate

Protein (%)

b-Carotene (mg/100 g)

SWEET POTATO: A REVIEW

31

Dansby and Bovell-Benjamin (2003a) also evaluated the moisture contents of SPF processed from conventionally and hydroponically grown sweet potatoes (Table V). The hydroponic SPF had lower mean initial moisture than those reported by Woolfe (1992) and Collado and Corke (1999). However, the final moisture content is dependent on the drying method and time. The bulk of SPF is carbohydrate between 85% and 95% dwb, but this varies according to cv. Several researchers have reported the total carbohydrate content of SPF as ranging from 86.1% to 94.8% dwb (Gurkin-Ulm, 1988; Maneepun et al., 1992; Woolfe, 1992). Dansby and Bovell-Benjamin (2003a) reported carbohydrate values for hydroponic SPF stored for 5 months at room and refrigerated temperatures (Table V). Yadav et al. (2005) determined the eVect of drying methods (native, drum dried, and hot air-dried) on the functional properties of SPF from red- and white-skinned sweet potato. Fuglie and Hermann (2004) documented carbohydrate content for a complex instant SPF as shown in Table V. The protein content of SPF is usually low, ranging from 1.0% to 14.2% (Collado et al., 1997; Woolfe, 1992). Owori and Hagenimana (2000), Dansby and Bovell-Benjamin (2003a), Yadav et al. (2005), and Fuglie and Hermann (2004) reported protein values for SPF (Table V). The b-carotene content of SPF processed from hydroponically grown sweet potato reported by Dansby and Bovell-Benjamin (2003a) was within the range of that reported by Woolfe (1992) for field-grown sweet potatoes (Table V). Total dietary fiber of SPF was reported to range between 11.0  0.1% and 17.6  0.3% by Dansby and Bovell-Benjamin (2003a) and Yadav et al. (2005), respectively. Dietary fiber content of SPF is between 0.4% and 13.8% (van Hal, 2000). Fuglie and Hermann (2004) reported 2% crude dietary fiber in SPF. SPF has higher amounts of b-carotene than wheat products and is an eVective way of increasing dietary carotenoid intake. Although SPF is identified as one of the most promising sweet potato products, its quality and storage stability need to be further researched. Dansby and Bovell-Benjamin (2003a) evaluated the proximate composition and color of processed hydroponic SPF during storage. The researchers concluded that the storage temperatures and time (4 or 21  4 C for 5 months) did not significantly aVect the proximate composition and color of the SPF. However, although no discoloration or browning of the SPF was observed, the flour stored at 21  4 C lost more of the orange/yellow color than that stored at refrigerated temperature. Orbase and Autos (1996) found that there was no discoloration in flour from four sweet potato cv. stored for 6 months in polyethylene, cotton, and polyethylene/cotton packaging. No deterioration was observed in flour stored for 5–7 months in polyethylene and polypropylene bags (Woolfe, 1992). Similarly, Gurkin-Ulm (1988) reported that the proximate composition and dietary fiber of SPF stored for 1.5 month were

32

A. C. BOVELL-BENJAMIN

not aVected by storage time, temperature, or atmosphere. In general, the SPF retains most of the nutritional composition of fresh sweet potato roots, and many advances have been made in the technology of SPF production, its storage stability and nutrition, making the flour a highly feasible, commercial value-added product. Many studies have reported the feasibility of using SPF as an alternative to wheat especially in bakery products. Woolfe (1992) reported that at SPF substitution levels above 20%, bread became unacceptable in terms of loaf volume, flavor, and texture. In Peru, commercial bakeries are producing widely accepted bread supplemented with 15% and 30% sweeet potato (Huaman, 1992). The technology has advanced, and substitution levels as high as 65% SPF have resulted in bread with acceptable loaf volumes, flavor and texture. For example, breads supplemented with 50%, 55%, 60%, and 65% SPF had acceptable texture and loaf volumes (Greene et al., 2003). Marklinder et al. (1996) considered barley sourdough bread that yielded volumes >450 ml as acceptable, and 990-ml loaf volume for standard wheat bread as acceptable. The studies reported below illustrate the advances in utilization of SPF. A. BREADMAKING PROPERTIES OF SPF (GREENE ET AL., 2003)

There is limited research regarding the processing of sweet potato bread. The objectives of this research were to: (1) determine the chemical properties (moisture, loaf volume, and texture) of bread supplemented with diVerent levels of SPF, and (2) evaluate the structural properties of bread supplemented with diVerent levels of SPF using scanning electron microscopy (SEM) and diVerential scanning calorimetry (DSC). Whole-wheat bread formulations were supplemented with 50%, 55%, 60%, and 65% SPF. The maximum percentage strain required to cut the breads into two pieces was used to indicate texture (firmness). The Jeol 5800 SEM and DSC 2010 were used to determine the morphological structure and enthalpies of the breads, respectively. The moisture contents of sweet potato bread ranged from 36.8  0.7% to 40.4  0.7%. The bread supplemented with 50% SPF had the highest loaf volume, which was not significantly diVerent from the other breads. Loaf volumes ranged from 825 ml (bread with 65% SPF) to 1450 ml (bread with 50% SPF). The loaf volume of the bread containing 65% SPF was acceptable and contrary to Woolfe’s (1992) findings. All the sweet potato breads were less firm than the control (a 100% whole-wheat bread). The SEM showed that the 50% SPF bread had the most gelatinization of starch granules when compared with the others. The sweet potato breads had high enthalpies, possibly because of the presence of larger granules in the SPS. The chemical and structural properties of the sweet potato breads were similar. Overall, the sweet potato breads had similar loaf volume, texture, morphology, and

SWEET POTATO: A REVIEW

33

enthalpies. The moisture content of the breads supplemented with 55% and 60% SPF were similar, but significantly diVerent from those with 50% and 65% SPF. The SEM revealed that starch gelatinization was less in the breads supplemented with 55%, 60%, and 65% SPF. The DSC results indicated a high enthalpy in the bread with 50% SPF, which showed the most gelatinization. It can be concluded that sweet potato bread is a feasible option for incorporation into the diets of astronauts and consumers on Earth based on its chemical and structural properties. B. MACROSCOPIC AND SENSORY EVALUATION OF BREAD SUPPLEMENTED WITH SPF (GREENE AND BOVELL-BENJAMIN, 2004)

The macroscopic and sensory properties of bread supplemented with 50%, 55%, 60%, and 65% SPF were evaluated. Trained judges evaluated 10 samples of freshly baked and 5 samples of day-old breads supplemented with 50%, 55%, 60%, 65% SPF, and two commercial controls (100% wholewheat bread and potato bread). The proximate analysis showed that increasing percentages of SPF increased the b-carotene contents of the breads, while decreasing their protein contents. Twelve perceived sensory attributes, which could be used to diVerentiate the appearance (color, cell size, uniformity), texture (chewy, soft, gritty, denseness), and flavor (fresh bread smell, strong wheaty smell, strong sweet potato smell, wheaty taste, aftertaste) of breads supplemented with SPF were generated. The judges’ perceptions for color and texture were in agreement with those from the instrumental measures used in the study. SPF could be used to substitute whole-wheat flour at 50%, 55%, 60%, and 65% levels in bread making. C. DEVELOPMENT AND STORAGE STABILITY OF BREADS SUPPLEMENTED WITH SPF AND DOUGH ENHANCERS (HATHORN ET AL., 2005)

Sweet potato and hard red spring wheat (HRSW) have been selected by the NASA to be grown in space. The objective of this study was to determine the storage stability of bread supplemented with SPF. Sweet potato and HRSW were processed into flour, and two dough enhancers, with and without SPS, were developed. Six breads, with two levels of SPF (50% and 65%) with and without dough enhancers were made. The breads were stored at room temperature (22  2 C) for 7 days. Samples were randomly withdrawn every 24 hours, and the proximate composition, color, loaf volume, texture, and mold and yeast growth measured. Moisture contents ranged from 30% to 41% in all breads. The color of the breads lightened as storage time increased.

34

A. C. BOVELL-BENJAMIN

FIG. 9 (A and B) Loaf volumes of breads supplemented with 50% and 65% sweet potato flour, respectively.

Loaf volumes were highest in breads containing 50% SPF and dough enhancers (Figure 9). Breads containing 65% SPF and no dough enhancer had the lowest loaf volume. Texture (firmness) was similar for all breads during the storage period. Yeast counts were low, but higher than mold counts throughout the storage. During storage, mold counts were <4 CFU/ml for all breads. On the seventh day, the breads deteriorated, and yeasts were too numerous to count. The findings indicate that sweet potato bread can be stored for 6 days at room temperature without deterioration in texture and adverse growth of yeasts and molds. D. SENSORY CHARACTERIZATION OF A READY-TO-EAT SWEET POTATO BREAKFAST CEREAL BY DESCRIPTIVE ANALYSIS (DANSBY AND BOVELL-BENJAMIN, 2003b)

A trained panel evaluated the sensory attributes of extruded, sweet potato ready-to-eat breakfast cereals (RTEBC) using sensory descriptive analysis. Three cereal formulations with varying amounts of processed hydroponic SPF and/or wheat bran [100% SPF, 75%/25% SPF/whole-wheat bran (SPFWWB), and 100% whole-wheat bran (WWB)] were developed. Ten panelists evaluated the sensory attributes of the three RTEBC, and a commercial cereal, Fiber OneÒ (the control). The intensity of 12 sensory attributes of the four RTEBC was rated. The appearance attributes evaluated were color, gloss, and sticklike; the flavor attributes were blandness, sweetness, sharp smell, cinnamon aftertaste; and the texture attributes were crunchiness, grittiness, hardness, chewiness, and dryness. Descriptive analysis revealed that the samples were significantly diVerent for all attributes. Fiber OneÒ had a significantly (p < 0.05) lighter brown color than the sweet potato RTEBC, but the 100%

SWEET POTATO: A REVIEW

35

SPF and SPFWWB were the crunchiest. The WWB was the sweetest cereal and significantly diVerent to all the others. For the sweetness attribute, the SPFWWB and the Fiber OneÒ were not significantly (p < 0.05) diVerent, but the 100% SPF cereal was sweeter and significantly diVerent from them. The sweet potato RTEBC was chewier than the control. The underlying principle of descriptive analysis is the development of a descriptive language to be used for scoring the product. In this study, a descriptive language for sweet potato RTEBC was developed, and 12 terms could be used to describe and diVerentiate the appearance, texture, and flavor of sweet potato RTEBC. The data collected could be used to facilitate future training of similar panels and to enhance sensory communication regarding sweet potato RTEBC. Stone and Sidel (1993) and Pal et al. (1995) noted that descriptive analysis could contribute directly or indirectly to other activities such as cost reduction, determination of consumer reaction, quality maintenance, and evaluation in product development. Therefore, the data collected in this could be useful for further optimization of the sweet potato RTEBC and help to increase utilization of SPF. Finally, the data can be used in the design of follow-up consumer studies of the sweet potato RTEBC with the relevant population groups. E. PHYSICAL PROPERTIES AND SIXTH GRADERS’ ACCEPTANCE OF AN EXTRUDED READY-TO-EAT SWEET POTATO BREAKFAST CEREAL (DANSBY AND BOVELL-BENJAMIN, 2003c)

This study was a continuation of the authors’ earlier investigations in a broader project to develop palatable, nutritious food ingredients and value-added products from the sweet potato. The nutritive, physical properties and sixth graders’ acceptance of a newly developed RTEBC were determined. Extruded RTEBC were made from 100% and 75% SPF, 100% WWB, and extrusion cooking. The proximate composition, bulk density, expansion ratio, color, morphology, water absorption index, and water solubility index of the RTEBC were determined. Seventy-three sixth grade students evaluated three sweet potato RTEBC on a nine-point hedonic scale, with the nine structural levels ranging from 9 ‘‘supergood’’ through 5 ‘‘maybe good or bad’’ to 1 ‘‘superbad.’’ The samples were: (1) 100% SPF cereal, (2) 75% SPF/25% WWB cereal, (3) 100% WWB cereal, and (4) Fiber OneÒ (a commercial ready-to-eat breakfast cereal, made from wheat and corn bran was the control). Wheat bran was chosen as a control because the main ingredient in Fiber OneÒ is wheat bran, and it is the commercial ready-to-eat breakfast cereal, which is similar in appearance to the sweet potato cereals. The mean moisture content was similar for both sweet potato RTBC, but significantly higher for the 100% WWB. The crude ash content was significant (p < 0.05) for all the RTEBC, being highest in the 100% WWB and lowest in the

36

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100% SPF. All the RTEBC had similar fat contents. As expected, carbohydrate and ascorbic acid contents were higher in the 100% SPF. The b-carotene content in the 100% SPF was significantly (p < 0.05) higher than that of the 100% WWB, which had negligible amounts (6495 mg/100 g compared with 280 mg/100 g). However, the dietary fiber in the 100% WWB was almost three times as high as that of the 100% SPF (29% vs 10%), not surprising because wheat is a richer source of dietary fiber than sweet potato. The vitamins thiamin and riboflavin contents were similar for the cereals. The bulk density and expansion ratio of extruded products describe the degree of puYng of the extrudates. The bulk densities and water absorption index were similar for the cereals. However, expansion ratio was highest in the 100% SPF cereal. The 100% WWB had the lightest color and most fibrous morphology. For the consumer testing, degree of liking (DOL) among the RTEBC diVered significantly (p < 0.05). Fiber OneÒ was liked significantly more than the other cereals. The 100% WWB was the least-liked cereal and was significantly diVerent from all other cereals. The extruded RTEBC containing 100% SPF and 75%/25% SPFWWB received high ratings 6.7  1.7 and 6.2  1.7, respectively. In hedonic ratings, scores 5 reflect liking of the product by the consumers. The 100% WWB was unacceptable to the sixth graders which was not surprising because sensory descriptive analysis done earlier confirmed that it was the blandest, most gritty, most chewy cereal. Extruded RTEBC containing 100% SPF and 75% SPF are promising products to be included in human diets because they were well liked and acceptable to sixth graders. However, further work is continuing to decrease the chewiness of the sweet potato RTEBC and evaluate its stability during long-term storage studies. F. PREPARATION OF SPF AND ITS FERMENTATION TO ETHANOL (REDDY AND BASAPPA, 1997)

The economic preparation of SPF and eYcient conversion to ethanol were studied. SPF was prepared using abrasive peeling, hand peeling, lye peeling, and drum drying. The SPF was treated with pectinase, and then with culture filtrate (a-amylase and glucoamylase) of Endomycopsis fibuligera. The inoculated samples were fermented for 3 days. A winelike product with ethanol up to 8.6% (w/v), desirable aroma and color was processed. G. GENETIC VARIATION IN COLOR OF SPF RELATED TO ITS USE IN WHEAT-BASED COMPOSITE FLOUR PRODUCTS (COLLADO ET AL., 1997)

SPFs vary widely in color depending on genotype, and when used in wheatbased composite flours they will impart characteristic colors, which may be favorable or unfavorable for particular food products. SPF was prepared from

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44 genotypes and analyzed for proximate composition and biochemical properties. The Hunter color L*, a*, b* values of the dry SPF and their modified Pekar slicks (PS) with water and alkali were measured. Polyphenol oxidase activity, a-amylase activity, and total sugar were significantly correlated to L* values of dry SPF and of their PS tests with water and alkali. The yellow pigment level was significantly correlated to the yellowness (b*) of the dry flour and of the PS test with water, but less correlated to b* of the PS test with alkaline. The results indicated a complex biochemical basis to SPF color, and no single biochemical factor examined was adequate to predict the color of a food product made from SPF. However, the PS color parameters were highly correlated with the color of dough sheets for white-salted and yellow-alkaline noodles made from wheat and sweet potato composite flour (75:25). Thus, the simple modified PS test could be used in screening of genotypes for color stability and suitability for a specific end-use. SPF genotypes conferred a wide range of colors on composite flour dough preparations. Some colors, particularly the range of greens and bright orange, may be useful in specialty product development. H. QUALITY EVALUATION OF SPF PROCESSED IN DIFFERENT AGROECOLOGICAL SITES USING SMALL-SCALE PROCESSING TECHNOLOGIES (OWORI AND HAGENIMANA, 2000)

Owori and Hagenimana (2000) developed appropriate processes for smallscale production of SPF with the desired degree of odor, color, and nutritional and microbiological quality. It was reported that slicing and soaking the sweet potato roots for 1.5 hours prior to sun drying reduced the odor of the flour. For the color, as the soaking time increased, browning in the SPF decreased. There was a decrease in the total sugars, reducing sugars, starch, and proteins in flour processed from sweet potatoes soaked for 90 minutes. There were no diVerences in the microbial quality of SPFs made from soaked and nonsoaked sliced roots. The researchers concluded that soaking sliced sweet potato roots for 90 minutes prior to sun drying could be a suitable method for small-scale production of flour. I. SPF-LIKE PRODUCTS

In Uganda, drying is the traditional way to preserve sweet potato. Women crush and sun dry chunks of the fresh root to prepare a coarse inginyo. For amukeke chips, the men slice up the roots into round, flat pieces, which the women then spread out to dry; both keep for 4–5 months (CIP, 1998). The inginyo or amukeke are ground into a coarse flour, which is rehydrated with water, boiled, mashed, and then eaten directly as a thick porridge known as atapa, starchy staple (CIP, 1998). The dried sweet potato is also boiled in

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sauces along with beans and vegetables. The recovery rate of fresh roots to amukeke dried chips is from 40% to 45%, and for every 45 kg of fresh sweet potato roots about 18 kg of dried chips (at 14% moisture) are produced. The nutritional composition of sweet potato chips has been reported as 5.2%, 0.1%, and 2.6% for crude protein, crude fiber, and ash, respectively (Tewe et al. 2003). Equally suitable as a human snack food, amukeke dried chips can be found in the marketplace, but command little consumer interest (CIP, 1998). In Mali, West Africa, sweet potatoes are peeled, cut into small pieces, and sun-dried. The dehydrated sweet potatoes can be stored for several months. They are usually rehydrated and added to a sauce with other condiments and eaten with a stiV cereal porridge or rice (Scheuring et al., 1996). Researchers have indicated that shade-dried sweet potato pieces still had high levels of b-carotene (Scheuring et al., 1996). Sweet potato is processed into two local products called Michembe (the roots are withered, cut into slices, and dried) and Matobolwa (the roots are boiled, sliced, and dried) in Tanzania. These products can last for 5–8 months (Gichuki et al., 2005). Other products that have been prepared in Tanzania include cakes, chapatis, donates, kaimati, and buns (Gichuki et al., 2005). VII. OTHER POTENTIAL SWEET POTATO PRODUCTS Potential sweet potato products (some with limited commercialization) include sweet potato bread pudding, casserole, tart, muYns, scalloped sweet potato, and refrigerated sweet potato pieces (Figure 10). Other value-added, commercial SPF products sold in supermarkets in the United States include sweet potato pancake mixes and sweet potato chips. Some east coast restaurants in the United States, especially in New York and Florida now feature sweet potato fries (Adam, 2005). An extensive sweet potato recipe list including dishes from China, Ghana, Guyana, India, Japan, and the United States is available elsewhere (Hill et al., 1992). The following section gives a more detailed description of selective potential sweet potato products. A. CONSUMER ACCEPTANCE OF VEGETARIAN SWEET POTATO PRODUCTS INTENDED FOR SPACE MISSIONS (WILSON ET AL., 1998)

The study determined consumer acceptability of products containing from 6% to 20% sweet potato on dwb. Vegetarian products made with sweet potato were developed for use in nutritious and palatable meals for future space explorers. Sensory (appearance/color, aroma, texture, flavor/taste, and overall acceptability) studies were conducted using panelists at NASA/Johnson Space Center, Houston, Texas. All the products were vegetarian with the exception of a

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FIG. 10 Potential sweet potato products (some with limited commercialization). (A) Bread pudding, (B) casserole, (C) bran muYns, (D) scalloped sweet potato, (E) refrigerated sweet potato pieces, and (F) tart.

sweet potato pie. A nine-point hedonic scale (9 ¼ like extremely, 5 ¼ neither like nor dislike, and 1 ¼ dislike extremely) was used to evaluate 10 products and similar commercially available products (controls). The products tested were sweet potato pancakes, waZes, tortillas, bread, pie, pound cake, pasta, vegetable patties, doughnuts, and pretzels. All the products were liked either

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moderately or slightly with the exception of the sweet potato vegetable patties, which were neither liked nor disliked. Because of their consumer acceptability, these products were recommended to NASA’s Advanced Life Support Program for inclusion in a vegetarian menu plan designed for lunar/Mars space missions. B. SWEET POTATO ‘‘KUNUZAKI’’ (TEWE ET AL., 2003)

In the northern part of Nigeria, sweet potato is processed into a local drink called kunuzaki. Fresh sweet potato is peeled, blended/grated or ground, sifted separated into boiled and unboiled portions, mixed, and left overnight to ferment and sugar added. C. SWEET POTATO BEVERAGE

Van Den (1992) reported two beverages from orange-fleshed sweet potato. One of the beverages, which were highly acceptable to consumers, was fruity and resembled fruit juice drinks. Sweet potato roots were processed into precooked powder, which was sieved and mixed with cocoa powder. The sweet potato–cocoa (85:15) hot drink was also acceptable to consumers. In the United States, Gladney (2005) reformulated and processed a sweet potato beverage. Selected chemical and physical properties of the beverage (color,  Brix, pH, and ascorbic acid) were evaluated. The beverages contained 19%, 22%, and 26% sweet potato puree and other fruit juices. The results indicated that the color was similar for all beverages; however, those with higher amounts of sweet potato were darker. In general, the  Brix was higher than those reported for a fruity beverage developed by Van Den (1992). D. SELECTED PATENTS REGARDING SWEET POTATO PRODUCTS

A technology with methods for producing cooked sweet potato products was patented in 2001 (Walter et al., 2001). The patent, docket, and serial numbers are: 6197363, 22696, and 9216518, respectively. The invention is a process for converting sweet potatoes into convenient and nutritional finished products for consumers and the food service industry. Similar to frozen Idaho potatoes and fries, the invention uses a method that combines cooked and pureed sweet potatoes with approximately 25% dry matter such as potato flakes or starch. Gelling agents such as alginate, methylcellulose and hydroxypropylmethyl cellulose, sucrose, calcium, and water are added. The final mixture can either be spread or injected into a rectangular mold or extruded and could be cut into bite-sized pieces, strips, wafers, and patties, frozen and packed for distribution. The resulting products have low-fat content and can be adjusted by food processors for desired fat levels.

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Products made using the invention also contain a high degree of b-carotene to meet or exceed RDA. US patent 5,204,137, which describes processes for products from sweet potato, was issued in 1993. The invention is concerned with the utilization of orange sweet potato roots, with the purpose of producing various flours, other valuable edible products, and industrial products from them. The invention is best described by reference to certain specific examples given in this patent. A partial listing of these examples includes orange sweet potato bread, orange sweet potato imitation corn bread, orange sweet potato cake dough, orange sweet potato muYns, orange sweet potato pancake mix, orange sweet potato pizza dough, orange sweet potato waZes, orange sweet potato dumplings, and so on (http://patft.uspto.gov). Patent 5,204,137 also reports earlier patents, which are described here. Dried, ground orange sweet potatoes were patented for use as an ingredient in coVee blends (US patent 100,587 issued in 1870; http://patft.uspto.gov); uncooked orange SPF was patented by Marshall (US patent 77,995; http://patft.uspto.gov) and Baylor (US patent 100,587; http://patft.uspto.gov). E. PREPARATION, EVALUATION, AND ANALYSIS OF A FRENCH-FRY-TYPE PRODUCT FROM SWEET POTATOES

The researchers prepared a French-fry-type product from Jewel and Centennial sweet potatoes. The sweet potato roots were washed, lye peeled, sliced into strips, and blanched in hot water containing 1% sodium acid pyrophosphate. The blanched strips were partially dried at 121 C. The dehydrated strips were frozen until fried at 175 C. Panelists evaluated the color, flavor, and texture of the products on a five-point scale. The flavor and texture results indicated that a good product could be prepared from both cv. of sweet potato. F. TEXTURAL MEASUREMENTS AND PRODUCT QUALITY OF RESTRUCTURED SWEET POTATO FRENCH FRIES (WALTER ET AL., 2002)

The study investigated: (1) the applicability of using alginate–calcium gelling system to produce a high-quality sweet potato French-fry-type product from sweet potato puree, (2) the physical and sensory properties of the product, and (3) the relationship between measured instrumental texture parameters and sensory properties of this product. Cooked, pureed sweet potatoes were mixed with potato flakes, sucrose, tetrasodium pyrophosphate, alginate, and calcium sulfate, formed and gelled, cut into strips, frozen, and fried. The sweet potato fries containing 0.35-g alginate/100 g and 0.5 g-CaSO4/100 g

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were most acceptable to consumers who evaluated them. The researchers concluded that restructured sweet potato fries with consistent texture and consumer acceptability can be made from sweet potato puree using the alginate–calcium gelling system. VIII. POTENTIAL OF SWEET POTATO IN THE UGANDAN FOOD SYSTEM Uganda is the biggest sweet potato producer in the Africa and third largest producer in the world (Scott and Maldonado, 1999). In the 1900s, sweet potato was already an important crop in the western part of Uganda (McMaster, 1962). Winter et al. (1992) noted that the crop was traditionally grown in appreciable quantities by the Arube tribe and Konjo of the Ruwenzori slopes. Sweet potato cultivation spread to northern Uganda during the early part of the nineteenth century (Drisberg, 1923). In the 1950s, sweet potatoes ranked as the most important crop after millet, bananas, and cassava (Hakiza et al., 2000). Today in Uganda, sweet potato is a major staple food and the fifth most important food crop, with regard to land area (after bananas, beans, maize, and finger millet), but is rated third in importance on a fresh weight basis (Ministry of Agriculture, Animal Industries and Fisheries, 1992). In districts such as Lira, Pallisa, and Soroti, sweet potato is ranked as one of the three most important crops grown. The crop is an important source of carbohydrates because of rapid population growth, and its ability to produce massive amount of energy in short periods of time (Odongo et al., 2004). Additionally, the importance of sweet potato increased over the last two decades because of the serious decline of cassava production subsequent to attack by the cassava mosaic disease, cassava green mite, and cassava mealy bug (Odongo et al., 2004). Also from the mid-1980s, the region has experienced increased food insecurity, and the people have relied on the sweet potato for their nutritive requirements. In Uganda, sweet potato production, which is mainly concentrated in the densely populated, mid-altitude regions, has increased from 231,000 ha in 1980 to 560,000 ha in 1999 with outputs of 1.2 and 1.9 metric tonnes, respectively (Hakiza et al., 2000). During the last 5 years, sweet potato production trends have increased in the northeastern region of Uganda (Owori and Hagenimana, 2000). Bashasha et al. (2001) ranked sweet potato as second to banana in the western and central regions, and after finger millet in the northern and eastern regions in terms of food preference. Smith et al. (1996) reported that sweet potato ranks first and third as a food crop and cash crop, respectively, in Soroti District, Uganda. However, the postharvest technologies and outlets have not kept pace with the increased production.

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Researchers, policymakers, and other stakeholders have become increasingly interested in the role of sweet potato in the Ugandan food system and its potential to improve nutrition, reduce food insecurity, and generate rural incomes (Bashasha et al., 2001). With this recognition, the government of Uganda has given high priority to research directed at enriching the nutrient contents of the sweet potato through the development of orange-fleshed cv. and diversifying sweet potato-based products and fortification. In Ugandan food systems, the sweet potato assumes five roles, of which four are unique to rural areas and one mainly to urban areas. According to Hall et al. (1998) and Mwesigwa (1995), these roles are: (1) a predominant staple providing the majority of calories for most of the year, although sweet potato production is seasonal; (2) it is a major complementary staple eaten throughout the year but with seasonal peaks; (3) a famine reserve staple typically consumed only in significant quantities during shortages of the dominant staple; (4) a source of cash income either produced strictly for sale or more commonly as a source of revenue from petty trading; and (5) a low-priced complementary staple for the poor and lower-income urban groups. As with other developing countries, the marketing of sweet potato in Uganda is influenced by its bulkiness, perishability, transportation issues, poor physical infrastructure such as roads, poor storage, and marketing information systems, limited availability of adapted sweet potato-processing technologies, and limited demand for traditionally processed products (Owori and Hagenimana, 2000). However, the adaptation of sweet potato processing technologies and value-added products can enhance consumption among consumers. Some researchers have concluded that it is not profitable for Ugandan farmers to engage in sweet potato processing, while others have reported otherwise. For example, in Soroti, the sweet potato is consumed year-round as boiled roots; traditionally processed food products, such as dried chips; flour primarily for domestic use; household food security; and on a more limited scale for sale in rural markets (Owori and Hagenimana, 2000). EVorts are being made to expand the SPF market, and there is a potential for SPF in Uganda’s baking industry. Collaborative research in Lira and Soroti districts has also demonstrated that there is a market for common snack products such as mandazi (doughnut), chapati, buns, and cakes, which have been supplemented with 30% SPF (Owori et al., 2000). Bashasha and Scott (2001) conducted a study in northeastern Uganda to assess the status and market potential of processed sweet potato, namely, inginyo and amukeke. Their results indicated that sweet potato was processed primarily for household food security. Respondents reported lack of time, high labor cost, and lack of market as the main bottlenecks, while peeling and slicing were the most labor-intensive activities. Owori and Hagenimana (2000) conducted research to improve the quality (odor, color, sensory, nutritional, and

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microbiological) of SPF processed from nonsoaked and soaked slices and recommended appropriate processing techniques for small-scale production. Soaking the sweet potatoes for 90 minutes decreased the odor intensity of the flour, and browning reduced with increased soaking time. The nutrient content of the SPF reduced slowly as soaking time increased, moisture content increased, ash, starch, protein, total and reducing sugars decreased. The research demonstrated that routine microbiological analysis of SPF was needed to control the production process, and soaking sliced sweet potato roots for 90 minutes before sun drying is the most suitable method for small-scale production. In another research process, Mudiope et al. (2000) conducted a survey in Kumi subcounty, Uganda to evaluate the role of sweet potato in the farming system by measuring the production, marketing, and processing constraints facing the crop. Production constraints such as labor shortage and lack of planting materials were the major limitations in the sweet potato farming system. Constraints in processing of sweet potatoes included lack of processing tools. Peters (1998) examined related aspects of sweet potato production, processing, utilization, and marketing to set postharvest research strategies for addressing food security and income generation in Uganda. The relevant findings indicated the need for more research focused on planting material, appropriate technology for processing, storage of fresh or processed roots, and the commercial potential for SPF as a substitute for wheat flour. Peters (1998) also indicated that SPF could be marketed as ‘‘atap’’ (a low-quality flour consumed by the general population as a staple). The outcomes of the above-mentioned studies stress the need for further research eVorts to address the production and processing aspects of the sweet potato to enable it to play a greater role in ensuring food security in Uganda. Over the last decade or more, sweet potato production has increased in Uganda while the decline in cassava production has allowed the crop to play a role in food security. However, the sweet potato has the potential to play a much more substantial role in the Ugandan food system. Further fundamental research is needed to upgrade the quality of sweet potato production, processing and product development, and identify ways to eVectively educate Ugandans regarding exploitation of the nutritional benefits of the sweet potato, and how it can play a role in eradicating VAD and food insecurity. IX. SWEET POTATO PROCESSING AND UTILIZATION EFFORTS IN KENYA In Kenya, East Africa, sweet potato is an important secondary and ‘‘food security’’ crop when maize supply is low and in times of drought (Gakonyo, 1993b; Mutuura et al., 1992). For example, Mutuura et al. (1992) reported

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that in western Kenya, 40–60% of households ate sweet potatoes more than four times weekly, while 65–95% consumed them once per week during the ‘‘hunger season’’ and when maize was plentiful, respectively. Alumira and Obara (1997) reported that sweet potato is not a main dietary staple in Nairobi. In their 1997 survey, the majority of participants (from middle- and low-income groups) indicated that they ate less sweet potato than 5 years ago because of changes to more ‘‘urban/modern-type’’ foods. Other reasons given for the decreased consumption include unavailability of sweet potato, unreliable sweet potato harvests, less land space available for production of sweet potato, and high cost for sweet potatoes. On the other hand, 37% of the participants indicated that eating habits have changed in favor of the sweet potato because they are cheap and easy to cook, they serve as an alternative when maize price is high, and they are preferred to bread at breakfast time because of the sugar content and low price. The production of sweet potato in Kenya has increased from 55,000 ha in 1988 to 65,000 ha in 1996 (FAO, 1997). The average annual per capita sweet potato consumption is approximately 24 kg (Scott and Ewell, 1992). In the western part of Kenya, including the Lake Victoria basin and Nyanza Province, sweet potato is grown widely and is mostly a woman-tended crop (Nungo et al., 2000; Oyunga-Ogubi et al., 2005). In the Nyanza Province, 82% of households grow sweet potato (Oyunga-Ogubi et al., 2005). VAD is widespread in Kenya, and most of the dietary vitamin A is supplied by carotenoids (GoK and UNICEF, 1995). Plant foods such as the sweet potato with concentrated provitamin A can contribute to decreasing food insecurity and VAD in Kenya. However, full exploitation of the sweet potato is limited by its bulkiness, perishability, high cost per unit sold, poor consumers’ perceptions, and the varieties cultivated (GTZ, 1998). The predominant sweet potato cv. grown and consumed in Kenya are white or pale-yellow flesh types, which are low in b-carotene (Hagenimana et al., 1998b). In contrast, orange-fleshed varieties, which are rich in b-carotene, are not so popular. The orange-fleshed sweet potato is an aVordable, rich, yearround source of b-carotene, therefore, eVorts are being made to adopt it in Kenya. If farmers and consumers switch from nonorange to orange-fleshed sweet potato cv., it is hypothesized that this could have a substantial impact on vitamin A status. Hagenimana et al. (1998b) concluded that the high carotenoid level of orange-fleshed sweet potato cv. would provide an excellent and appropriate mechanism for combating VAD. In an 18-month village-level study in western Kenya, Low et al. (1997) and Hagenimana et al. (1999) confirmed that potential exists to successfully substitute orange-fleshed sweet potatoes for the commonly used white-fleshed varieties. In Kenya, SPF was consumed in large quantities in the 1950s when some families used the flour to make porridge mixed with sorghum of finger millet

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flour (GTZ, 1998). However, with the introduction of maize, utilization of SPF declined as it became an inferior crop associated with poverty (GTZ, 1998). According to Nungo et al. (2000) and Gakonyo (1993a), processing and utilization of sweet potato in Kenya have been limited to washing, roasting, boiling, and mashing with other foods. Since 1995, the Allendu Women Group from Allendu location has been processing and marketing sweet potato products including chapati, mandazi, crisps, chips, and cakes. However, accurate data on the volume of sweet potato processed are unavailable. Oyunga-Ogubi et al. (2005) reported that improved provitamin A content was seen in population groups when SPF was incorporated into traditional products such as buns, chapatis, and mandazis in Rongo, Ndhiwa, and Kendu Bay districts in Nyanza Province. Nungo et al. (2000) prepared, tested, and evaluated several recipes made from sweet potato leaves and roots for color, taste, texture, and acceptability, and compared them to other recipes from cassava, rice, and carrots. The products were scored on a five-point scale where 5 ¼ very desirable; 3 ¼ moderately desirable; and 1 ¼ undesirable. Overall, 10 sweet potato products mshenye, doughnuts, relish, porridge, chips, ugali, cake, bread, mandazi, and chapati were selected as suitable and marketable products. Mshenye and relish were very acceptable to 90% of the consumers. Oyunga-Ogubi et al. (2005) investigated the adoption of an intervention technology for increasing vitamin A intake through the use to high-b-carotene sweet potatoes in the Siaya district, Kenya. They reported on the fresh roots, SPF, and novel products from traditional dishes (mandazis, chapati, ugali, porridge, chips, and crisps). Consumer tests showed high acceptability of the novel products. Mandazis and chips were preferred by males, while all products except ugali were highly acceptable to females. Children of diVerent ages and gender had variable preferences. Method of preparation of products was acceptable to women. However, on increased SPF processing, equipment was an issue. Men preferred most equipment as presented, but women and children suggested changes. For example, in flour production, cleaning and peeling were tedious, and the large amount of wash water resulted in increased workload. Also, women and children felt that the rotary slicer should be modified to allow a sitting position, the drying should fit under their normal practice, and there should be education on storage of chips and flour. The researchers also concluded that there is insignificant sweet potato processing in Siaya, but there is potential for adoption of this technology particularly by women. Hagenimana et al. (1999) identified orange-fleshed sweet potato varieties with high acceptability of appearance and taste that are appropriate for consumption by adults and young children in Kenya. In both Ndhiwa/Nyarongi and Rongo districts, consumer evaluation of the taste and appearance of cooked sweet potatoes indicated that cv. of orange-fleshed sweet potatoes

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were acceptable to community members. Pumpkin cv. was preferred for use in weaning foods. Local cv. ranked high in terms of taste and appearance in all districts. Also processed food products, such as mandazis made from new sweet potato cv. were acceptable to food processors and consumers. Mashed and flour products were preferred over grated products. The processing of modified food products did not require additional labor or production costs. A cost analysis indicated that substituting sweet potato for wheat flour in mandazis made the product more profitable for market vendors, and substituting sweet potato for other ingredients increased the b-carotene content of processed food products. Mandazis, chapatis, and buns with and without sweet potato contained approximately 100- and 800- to 3200-mg b-carotene/100 g, respectively (Hagenimana et al., 1999). As part of the promotion of orange-fleshed sweet potato, farmers in some parts of Kenya have received hands-on demonstrations to make various sweet potato products, including vegetable stew (relish), mashed food (mshenye/Irio), chapati, samosa, and biscuits (Gichuki et al., 2005). In sum, preliminary studies indicate that orange-fleshed sweet potatoes are available and acceptable to western Kenyans and are a good addition to the diets to reduce VAD. Processing of the orange-fleshed sweet potato into value-added products for yearround consumption and to increase provitamin A intake is limited. However, encouraging processing and expanding the use of the orange-fleshed sweet potato in the Kenyan food system will help to meet food requirements, improve nutritional status, enhance food security, and reduce poverty. X. CONCLUSIONS The sweet potato combines a number of advantages from nutritional to socioeconomic to environmental, which makes it a potentially good candidate for reducing the increasing food insecurity, VAD, and the under- and overnutrition that is occurring globally, especially in developing countries. However, consumers’ poor perception of the sweet potato in many developing countries, its static production, lack of storage technology for fresh sweet potato, lack of processing equipment, scarcity of data on the demand for fresh sweet potato or its products, inadequate processing and postharvest technology, and lack of knowledge regarding its nutritional benefits must be overcome. Repositioning sweet potato production and its potential for value-added products will contribute substantially to utilizing its benefits and many uses. Accurate estimates of the market demand for value-added sweet potato-based products have not been established, but advertisement and promotion will enhance their utilization.

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XI. RECOMMENDATIONS Multidisciplinary, integrated research and development activities aimed at improving production, storage, postharvest and processing technologies, and quality of the sweet potato and its potential value-added products are critical issues, which should be addressed globally. Additionally, widespread, eVective farmer and consumer education (awareness) regarding the exploitation of the nutritional, health and economical benefits of the sweet potato, and how it can play a role in eradicating VAD and food insecurity should be undertaken. Other important issues that need to be addressed urgently include: (1) research that will lead to the development of nutritious, consumer-acceptable valueadded products from the roots and leaves of the sweet potato; (2) determination of the critical physical and chemical factors modulating the acceptance of sweet potato by consumers; (3) more urgent collaborative eVorts by plant breeders, horticulturists, sensory scientists, and nutritionists to characterize and improve the starch and flour properties of sweet potato cv.; (4) although SPF is one of the most promising sweet potato products, its quality and storage stability need to be further researched; (5) providing farmers and consumers with acceptable recipes and samples of value-added products are also necessary approaches to increase production and consumption of the crop; and (6) low-cost, appropriate sweet potato processing technologies, and policies for their transfer to developing countries should be developed. Finally, governments, policy makers, and stakeholders should adopt food-based strategies that encourage and expand utilization of the orange-fleshed sweet potato cv. instead of the white-fleshed cv. Such strategies will assist in meeting food requirements, improve nutritional status, enhance food security, reduce poverty, and could eVectively improve vitamin A status of young children in particular, and populations in general. ACKNOWLEDGMENTS This chapter is, in part, an output from the sweet potato research work experiences of the author at Center for Food and Environmental Systems for Human Exploration of Space (CFESH), Tuskegee University funded by NASA; therefore, the author also wishes to acknowledge NASA, the team members of the Food Processing and Product Development Team in particular, and the rest of the CFESH team. REFERENCES Adam, K.L. 2005. ‘‘Sweetpotato: Organic Production’’. ATTR (http://www.attra.ncat.org). Akhmadieva, A.K., Zaichkina, S.I., Ruzieva, R.K., and Ganassi, E.E. 1993. The protective action of a natural preparation of anthocyanin (Pelargonidin-3-5-diglucoside). Radiobiologiia 33, 433–435.

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Matsui, T., Ebuchi, S., Fujise, T., Abesundara, K.J.M., Doi, S., Yamada, H., and Matsumoto, K. 2004. Strong hyperglycemic eVects of water-soluble fraction of Brazilian propolis and its bioactive constituent, 3,4,5-tri-O-caVeoylquinic acid. Biol. Pharm. Bull. 27, 1797–1803. McMaster, D.N. 1962. ‘‘A Subsistence Crop Geography of Uganda’’, pp. 72–76. Geographical Pub. Ltd., England. Miller, S.-A., Dean, D., Ganguli, S., Abdalla, M., and Bovell-Benjamin, A.C. 2003. Physicochemical and viscometric properties of a sweetpotato syrup. Paper presented at ARD 2003. Association of Research Directors, Inc., 13th Biennial Research Symposium, March 29–April 2, Atlanta, GA. Ministry of Agriculture, Animal Industries and Fisheries 1992. Kampala, Uganda. Minkova, M., Drenska, D., Pantev, T., and Ovcharov, R. 1990. Antiradiation properties of alpha tocopherol, anthocyans, and pyracetam administered combined as a pretreatment course. Acta Physiol. Pharmacol. Bulg. 16, 31–36. Mitcheva, M., Astroug, H., Drenska, D., Popov, A., and Kassarova, M. 1993. Biochemical and morphological studies on anthocyans and vitamin E on carbon tetrachloride induced liver injury. Cell. Mol. Biol. 39, 443–448. Morazzoni, P. and Magistretti, M.J. 1986. EVects of Vaccinium myrtillus anthocyanosides on prostacyclin-like activity in rat arterial tissue. Fitoterapia 57, 11–14. Morris, V.J., Ridout, M.J., and Parker, M.L. 2005. AFM of starch: Hydration and image contrast. Prog. Food Biopolymer. Res. 1, 28–42. Mudiope, J., Kindness, H., and Haigenemana, V. 2000. Socio-economic constraints to the production, processing, and marketing of sweetpotato in Kumi District, Uganda. African Potato Association Conference Proceedings 5, 449–453. Mutuura, J., Ewell, P., Abubaku, A., Mungu, T., Ajarga, S., Irunga, S., Owori, F., and Masbe, S. 1992. Sweet Potatoes in the food system of Kenya. Results of a socio-economic survey. In ‘‘Current Research for the Improvement of Potato and Sweetpotato in Kenya’’ (J. Kabira and P. Ewell, eds). International Potato Centre, Nairobi, Kenya. Mwesigwa, T.W. 1995. Sweetpotato consumption levels and patterns in urban household with particular reference to Kampala city. B.Sc. Thesis, available at Library, Department of Agriculture, Makerere University, Kampala, Uganda. Nair, G.M., Ravindran, C.S., Moorthy, S.N., and Ghosh, S.P. 1987. Sweetpotato research and development for small farmers. International Sweetpotato Symposium, Baybay, Philippines, May 20–26, (K.T. Mackay, M.K. Palomar, and R.T. Sanico, eds), p. 301. SAEMEO-SEARCA, Philippines. Namutebi, A., Natabirwa, H., Lemaga, B., Kapinga, R., Matovu, M, Tumwegamire, S., Nsumba, J., and Ocom, J. 2004. Long-term storage of sweetpotato by small-scale farmers through improved post harvest technologies. Uganda J. Agric. Sci. 9, 922–930. Niizu, P.Y. and Rodriguez-Amaya, D.B. 2005. New data on the carotenoid composition of raw salad vegetables. J. Food Compost. Anal. 18, 739–749. Nungo, R.A., Ndolo, P.J., and Hagenemana, V. 2000. Promoting sweeetpotato processing and utilization: Experience in Western Kenya. African Potato Association Conference Proceedings 5, 481–482. Oboh, S., Ologhobo, A., and Tewe, O. 1989. Some aspects of the biochemistry and nutritional value of the sweetpotato (Ipomoea batatas). Food Chem. 31, 9–18. Odongo, B., Mwanga, R.O.M., and Niringiye, N. 2004. Promoting improved sweet potato varieties and processing technologies in Lira, Pallisa and Soroti Districts. Final Technical Report, October 2004-12-09. NAARI, Kampala, Uganda. Olaofe, O. and Sanni, C.O. 1988. Mineral contents of agricultural products. Food Chem. 30, 73–77. Olson, J.A. 1996. Benefits and liabilities of vitamin A and carotenoids. J. Nutr. 126, 1208S–1212S. Omosa, M. 1994. Current and potential demand for fresh and processed sweetpotato products in Nairobi and Kisumu, Kenya. International Potato Center, Nairobi, Kenya.

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INFECTOBESITY: OBESITY OF INFECTIOUS ORIGIN MAGDALENA PASARICA AND NIKHIL V. DHURANDHAR Department of Infections and Obesity, Pennington Biomedical Research Center Louisiana State University System, Baton Rouge, Louisiana 70808

I. Introduction II. Canine distemper virus A. CDV General Information B. CDV-Induced Obesity C. Mechanisms of CDV-Induced Obesity III. Rous-Associated Virus-7 A. RAV-7 General Information B. RAV-7-Induced Obesity C. Specificity of RAV-7-Induced Obesity D. Mechanism of RAV-7-Induced Changes IV. Chlamydia pneumoniae A. CP General Information B. CP Association with Higher BMI C. Mechanism of CP EVects V. Scrapie Agent A. General Information B. Scrapie-Induced Obesity C. Mechanism of Scrapie-Induced Obesity VI. Borna disease virus A. General Information B. Borna disease virus-Induced Obesity C. Mechanism of BDV-Induced Obesity VII. Gut Microbiota A. General Information B. Microbiota-Induced Obesity C. Mechanism of Microbiota-Induced Obesity VIII. Adenoviruses IX. SMAM-1 A. SMAM-1 General Information B. SMAM-1-Induced Obesity C. Mechanism of SMAM-1-Induced Obesity X. Adenovirus Type 36 A. Ad-36 General Information B. Ad-36-Induced Adiposity C. Mechanism of Ad-36-Induced Obesity

ADVANCES IN FOOD AND NUTRITION RESEARCH VOL 52 # 2007 Elsevier Inc. All rights reserved

ISSN: 1043-4526 DOI: 10.1016/S1043-4526(06)52002-9

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M. PASARICA AND N. V. DHURANDHAR XI. Adenovirus Type 5 A. Ad-5 General Information B. Ad-5-Induced Obesity C. Mechanism of Ad-5-Induced Obesity XII. Adenovirus Type 37 XIII. Adipogenic Potential of Other Adenoviruses XIV. Role of Pathogens in Human Obesity XV. Infection, Inflammation, and Obesity XVI. Conclusions Acknowledgments References

The rapid increase in obesity and the associated health care costs have prompted a search for better approaches for its prevention and management. Such efforts may be facilitated by better understanding the etiology of obesity. Of the several etiological factors, infection, an unusual causative factor, has recently started receiving greater attention. In the last two decades, 10 adipogenic pathogens were reported, including human and nonhuman viruses, scrapie agents, bacteria, and gut microflora. Some of these pathogens are associated with human obesity, but their causative role in human obesity has not been established. This chapter presents information about the natural hosts, signs and symptoms, and pathogenesis of the adipogenic microorganisms. If relevant to humans, ‘‘Infectobesity’’ would be a relatively novel, yet extremely significant concept. A new perspective about the infectious etiology of obesity may stimulate additional research to assess the contribution of hitherto unknown pathogens to human obesity and possibly to prevent or treat obesity of infectious origins. I. INTRODUCTION The World Health Organization recently declared that we are experiencing an epidemic of obesity. In the past 20 years, the prevalence of obesity has increased by 30% in the United States and several developed as well as developing countries have reported increased prevalence (Astrup et al., 1998; Powledge, 2004; Rossner, 2005). In the best of hands, obesity treatment usually yields marginal and transient weight loss. More eVective and longer lasting strategies for prevention and treatment of obesity are urgently needed. Understanding contribution of various etiologic factors of obesity may lead to treatments directed specifically toward the cause, and consequently, its successful management. In addition to genetic, behavioral, endocrinal, and other causative factors of obesity, infectious etiology has been known for over two decades

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(Sclafani, 1984) but largely ignored for the purpose of obesity prevention and treatment strategies. To date, adipogenic eVects of 10 pathogens have been described, which include human and nonhuman viruses, scrapie agents, bacteria, and gut microflora. A comprehensive understanding of their adipogenic role may facilitate further investigation in determining certain infections as an etiologic factor in human obesity. This chapter describes various adipogenic pathogens reported since the first report about obesitypromoting eVects of Canine distemper virus (CDV) in 1982 (Lyons et al., 1982). A summary of the effects of these adipogenic pathogens on the host and the potential mechanism involved is provided in Tables I and II.

II. CANINE DISTEMPER VIRUS A. CDV GENERAL INFORMATION

The first obesity-promoting pathogen reported (Lyons et al., 1982), CDV of the genus Morbillivirus, subgroup of paramyxoviruses, is a lymphotropic and neurotropic negative-stranded RNA virus (Rozenblatt et al., 1985; Vandevelde and Zurbriggen, 1995; Yamanouchi, 1980). CDV causes a frequently fatal disease that aVects dogs and a wide range of mammals (Leisewitz et al., 2001; Lyons et al., 1982; Raine, 1976; Summers and Appel, 1994; Vandevelde and Kristensen, 1977). CDV and Measles virus (MV) produce similar systemic disorders and are antigenically related (Hall et al., 1980). Although an association of CVD with other human diseases was suggested (Summers and Appel, 1994), CDV is not considered a human pathogen. CDV invades the nervous system and replicates in neurons and glial cells of the white mater subgroup (Vandevelde and Zurbriggen, 1995). Mice or dogs experimentally infected with CDV showed viral inclusions, giant cell formation, reactive gliosis, and myelin degradation (Lyons et al., 1982; Raine, 1976; Vandevelde and Kristensen, 1977). Bernard et al. (1993) suggest that CDV infection spreads by humoral and intracellular space pathways or by axonal transport. CDV infection results in acute encephalitis and death of 40–50% of the animals, followed by either motor impairment or obesity syndrome in a late phase in the survivors (Bernard et al., 1988, 1993). B. CDV-INDUCED OBESITY

Lyons et al. (1982) first reported that CDV induces obesity in Swiss albino mice. Results were confirmed later by Bernard et al. (1988, 1991, 1993). CDV produced significant increase in body weight and fat cell number as well as

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TABLE I ADIPOGENIC PATHOGENS—AN OVERVIEW

Animal model used, age, and sex

Duration of onset of obesity

EVect of infection on body weight

EVect of infection on biochemical parameters

Association with human obesity

CDV

1982 (Lyons et al., 1982)

Swiss albino mice, male and female, 4–5 weeks old (Lyons et al., 1982)

Developed 6–10 weeks postinfection and reaches a plateau after 16–20 weeks (Lyons et al., 1982)

Mean weight of infected obese animals was 63.7 g versus 33.1 g in the uninfected group (Lyons et al., 1982)

Association with human multiple sclerosis (Summers and Appel, 1994)

RAV-7

1983 (Carter et al., 1983a)

10-day-old embryos— SC white leghorn chicken line (Carter et al., 1983a,b, 1984; Carter and Smith, 1983)

Stunting—14 days posthatching (Carter et al., 1983a) Obesity—3 weeks of age (Carter et al., 1983b)

Stunting " Visceral fat (Carter et al., 1983a,b)

BDV

1991 (Gosztonyi and Ludwig, 1995)

Weaning/adult Lewis rats (Gosztonyi and Ludwig, 1995; Herden et al., 2000)

2-month postinoculation (Gosztonyi and Ludwig, 1995)

Obesity with " visceral fat (Gosztonyi and Ludwig, 1995)

" Insulin (Bernard et al., 1988, 1999) No significant change in glucose (Bernard et al., 1988) # Leptin levels (Bernard et al., 1999) # MCH, NPY (GriVond et al., 2004) # Catecholamine (Lyons et al., 1982) # Neuropeptides (GriVond et al., 2004) Anemia Hyperlipidemia Immune suppression " Amylase " Insulin # Glucose # Thyroxine (Carter et al., 1983a,b, 1984) " Tryglyceride Moderate " blood glucose (Gosztonyi and Ludwig, 1995)

No

Transmission of BDV from animals to humans is possible (Ludwig and Bode, 2000)

M. PASARICA AND N. V. DHURANDHAR

Year first reported

1968 (Pattison and Jones, 1968)

6- to 9-week-old female mice SJL, C57NL, A2G, SAMP8, SAMR1, AKR, 22L (Kim et al., 1987, p. 91; Carp et al., 1998) 3-week-old white leghorn broilers (Dhurandhar et al., 1990, 1992)

12 weeks postinfection (Carp et al., 1984)

" Body weight due to fat accumulation (Carp et al., 1984, 1989)

" Blood glucose (Vorbrodt et al., 2001) # GLUT-4 density in certain brain areas (Vorbrodt et al., 2001)

Not reported

SMAM-1

1990 (Dhurandhar et al., 1990)

3 weeks postinoculation (Dhurandhar et al., 1990, 1992)

Stunting " Visceral fat (Dhurandhar et al., 1990, 1992)

# Cholesterol # Triglyceride (Dhurandhar et al., 1990, 1992)

2000 (Dhurandhar et al., 2000)

Chickens Mice Male rhesus and marmoset monkeys Rats (Dhurandhar et al., 2000, 2001, 2002; Pasarica et al., 2006)

4 weeks (Dhurandhar et al., 2000, 2001), 6- to 7-month postinoculation (Dhurandhar et al., 2002; Pasarica et al., 2006)

" Body weight " Visceral, epididymalinguinal, retroperitoneal fat (Dhurandhar et al., 2001; Pasarica et al., 2006)

2002 (Atkinson et al., 2002)

3-week-old white leghorn chickens (Atkinson et al., 2002)

4-weekpostinoculation (Atkinson et al., 2002)

No diVerence in body weight " Visceral fat (Atkinson et al., 2002)

# Cholesterol (Dhurandhar et al., 2000, 2001; Pasarica et al., 2006) # Triglyceride (Dhurandhar et al., 2000, 2001; Pasarica et al., 2006) # HOMA (" insulin sensitivity) (Pasarica et al., 2006) " Leptin (Pasarica et al., 2006) # Corticosterone and norepinephrine (Pasarica et al., 2006) # Triglycerides (Atkinson et al., 2002)

Yes. Seropositive obese subjects were heavier versus seronegative obese counterparts (Dhurandhar et al., 1997b) Yes. Greater prevalence of seropositivity in obese versus nonobese subjects. Seropositive subjects were heavier than seronegative counterparts (Atkinson et al., 2005)

Ad-36

Ad-37

Keratoconjunctivitis and genitourinary tract infections in humans (de Jong et al., 1981)

65

(continued)

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Scrapie

66

TABLE I (continued) Animal model used, age, and sex

Duration of onset of obesity

EVect of infection on body weight

EVect of infection on biochemical parameters

Association with human obesity

Ad-5

2005 (So et al., 2005)

3-week-old female outbread CD1 mice (So et al., 2005)

22- to 13-week postinoculation (So et al., 2005)

–

Chlamydia pneumoniae

2000 (Ekesbo et al., 2000)

Humans (Dart et al., 2002; Ekesbo et al., 2000)

Association with increased BMI (Dart et al., 2002; Ekesbo et al., 2000)

" Body weight " Whole body adiposity (So et al., 2005) Significantly higher BMI (Dart et al., 2002; Ekesbo et al., 2000)

Gut microbiota

2004 (Backhed et al., 2004)

8- to 10-week-old germfree male B6/NMRI mice (Backhed et al., 2004)

After 14 days colonization (Backhed et al., 2004)

Respiratory tract infection in humans (Limbourg et al., 1996) Association with increased BMI 27.3 versus 25.8 kg/m2 (Ekesbo et al., 2000) 26.6 versus 25.5 kg/m2 (Dart et al., 2002) –

No diVerence in body weight " Whole body fat " Epididymal fat # Lean mass (Backhed et al., 2004)

Association with CHD (Danesh et al., 1997; Kuo et al., 1993; Saikku et al., 1988)

Insulin resistance # Metabolic rate " Hepatic triglyceride content " Expression of de novo fatty acid synthesis pathway # Expression Fiaf (Backhed et al., 2004)

M. PASARICA AND N. V. DHURANDHAR

Year first reported

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TABLE II MECHANISM OF ACTION OF ADIPOGENIC PATHOGENS

Natural host

Organs/systems aVected

Mechanism of action

CDV

Dogs (Leisewitz et al., 2001; Summers and Appel, 1994)

Brain Pancreas Reproductive organs Kidneys Liver (Lyons et al., 1982)

RAV-7

Avian

BDV

Horses and sheep (Gosztonyi and Ludwig, 1995)

Scrapie

Sheep and goats (Hunter, 1972)

# Thymus weight # Bursa weight Fatty, yellow, enlarged livers Lymphoblastic infiltration of thyroid and pancreas (Carter et al., 1983a,b; Carter and Smith, 1983) Brain Hyperplasia islet of Langerhans (Gosztonyi and Ludwig, 1995) # Size—spleen, liver, kidney, brain, uterus " Adrenal weight (Carp et al., 1984; Kim et al., 1988)

Alters hypothalamic integrity (Bernard et al., 1993, 1999; Nagashima et al., 1992; Verlaeten et al., 2001) # Leptin long receptor and " leptin (Bernard et al., 1999) # MCH (Verlaeten et al., 2001) # Neuropeptides (GriVond et al., 2004) # Catecholamine (Lyons et al., 1982) " Cytokines production (Bencsik et al., 1996) Changes in lipid metabolism (Anderton et al., 1982, 1983a,b; Wild et al., 1986) # Thyroxine (Carter and Smith, 1983)

Inflammatory lesions and viral replication in hypothalamus (Herden et al., 2000) Brain function alteration (Kim et al., 1987; Vorbrodt et al., 2001) # GLUT-1 density, # glucose tolerance (Vorbrodt et al., 2001) " Adrenal weight disturbs hypothalamicpituitary-adrenal axis (Kim et al., 1988) (continued)

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TABLE II (continued) Natural host

Organs/systems aVected

Mechanism of action

SMAM-1

Avian (Ajinkya, 1985)

Impaired liver function (Dhurandhar et al., 1992) Impaired lipogenesis (Dhurandhar et al., 1992) Glucagons deficiency (Dhurandhar et al., 1992)

Ad-36

Human (Wigand et al., 1980)

Liver—" size, congested, fatty, infiltrated with intranuclear inclusion bodies Kidney—" size # Size of bursae, thymus, spleen (Dhurandhar et al., 1990, 1992) –

Ad-37

–

Ad-5

Human (de Jong et al., 1981) Human (Limbourg et al., 1996)

Chlamydia pneumoniae

Humans (Hogan et al., 2004)

Respiratory tract (Kuo et al., 1995)

Gut microbiota

Human gut (Xu and Gordon, 2003)

–

–

Increased replication, diVerentiation, and lipid accumulation in preadipocytes (Pasarica et al., 2005; Vangipuram et al., 2004) " Insulin sensitivity (Yu and Dhurandhar, 2005) # Leptin secretion (Vangipuram et al., 2007) and expression (Yu and Dhurandhar, 2005) in fat cells Increased Increased preadipocyte diVerentiation (So et al., 2005) Anti-inflammatory response determined by infection (So et al., 2005) Mechanism unknown. Preponderance of seropositivity in obese subjects is not an eVect of obesity (Dart et al., 2002) Increased processing of polysaccharides Increased calories Increased ineYcient metabolism (Backhed et al., 2004)

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moderate increase in fat cell size in mice surviving the infection (Lyons et al., 1982). Food intake was slightly but not significantly increased for the obese group (Bernard et al., 1988). Obese and lean animals had comparable titers of anti-CDV-neutralizing antibodies (Lyons et al., 1982). Obesity was observed in 26% of the animals after an intracerebral inoculation of CDV, while only 16% of animals become obese following an intraperitoneal inoculation. Body weights of the obese animals were comparable to those seen in genetic or hypothalamic models of obesity. When the body weight plateaued at 16–20 weeks postinoculation, mean weight of infected obese animals was 63.7 g versus 33.1 g in the uninfected group (Lyons et al., 1982). Obese animals had three times more lipid per cell and twice as many fat cells in the inguinal fat pads compared with the lean counterparts. A comparable change was observed in the gonadal and retroperitoneal fat pads (Lyons et al., 1982). Brains and reproductive organs were lighter and livers, kidneys, and pancreas were heavier in the obese animals, and this diVerence was statistically significant only for the females. There was no diVerence in the adrenal and pituitary gland weights. The increase in liver weight was mainly due to accumulation of triglyceride, while no change was detected in phospholipid composition. Obese mice had decreased lipogenesis in white adipose tissue with unaVected glycogenesis (Bernard et al., 1988). Prior to developing obesity, the CVD-infected group of mice showed increased insulin levels that attained statistical significance of 4–6 months postinfection, when they were six times greater than those in lean animal (Bernard et al., 1988, 1999). However, no significant diVerence in glucose levels was observed (Bernard et al., 1988). There was an initial increase, followed by a decrease in leptin long receptor and a dramatic increase in leptin levels accompanied by low levels of viral RNA in targeted CDV brain regions of infected animals (Bernard et al., 1999). Melanin-concentrating hormone precursor mRNA (ppMCH) was downregulated in the late stage of the acute phase CDV infection in mice (Verlaeten et al., 2001). In addition, GriVond et al. (2004) reported decreased expression of neuropeptide Y (NPY), melanin-concentrating hormone (MCH), and other neuropeptides in the obese animals. Catecholamine levels in the obese-infected mice were reduced significantly in forebrain, but not in the brain stem (Lyons et al., 1982). In substantia nigra, CDV infection downregulates tyrosine hydroxylase (TH), the ratelimiting enzyme of dopamine synthesis, which leads to motor impairment of infected animals (Bencsik et al., 1996).

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M. PASARICA AND N. V. DHURANDHAR C. MECHANISMS OF CDV-INDUCED OBESITY

As described below, several possible pathways have been proposed to explain the mechanism involved in CDV-induced obesity. 1. CDV replication in the rain The occurrence of obesity was correlated with the neurovirulence of the virus strain (Bernard et al., 1999). Prior vaccination with a vaccinia recombinant coding for CDV surface antigens partially protected against acute encephalitis and obesity (Sixt et al., 1998; Wild et al., 1993). Neuroadapted CDV strain inoculation by other routes (intranasal, footpad, and subcutaneous) does not produce obesity, suggesting that viral replication in the brain is a prerequisite for development of obesity, as suggested by Bernard et al. (1999). 2. Hit-and-run eVect Oldstone et al. (1982) reported a novel way by which viruses may cause disease, ‘‘a hit-and-run’’ model. Virus replicates in the cells that make growth hormone, thereby disrupting homeostasis and decreasing growth hormone synthesis but surprisingly without producing any cell death or inflammation. Bernard et al. (1999) speculated that the initial impact of CDV on hypothalamus may initiate changes that would continue to promote obesity in animals even months after the acute infection, suggesting a ‘‘hit-and-run’’ eVect. Viral products are expressed in high levels in the acute stage of infection and at lower levels in the late stage (1 year after infection). Survivors of acute encephalitis complete the elimination of virus-producing cells or suppress expression of viral antigens within a few weeks postinfection (Nagashima et al., 1992). CDV nucleoprotein transcripts and F gene mRNA levels decrease with time, reaching low levels in the hypothalami of the infected rats 1 year postinoculation (Bernard et al., 1999). F surface protein of CDV has an essential role in infectivity, allowing viral spread. Defective expression of F gene, along with viral proteins M and HA, may produce viral persistence in brain cells, probably by escaping the host immune system (Liebert et al., 1986). There are no signs of inflammation in the hypothalamus (Bernard et al., 1999). Infected cells appear intact, despite vital presence (Bernard et al., 1993). Bernard et al. (1999) suggested that the hypothalamus acts as a reservoir for CDV, and this noncytolytic infection impedes specific hypothalamic functions like weight maintenance (Bray and York, 1971; Olney, 1969; Weingarten et al., 1985).

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3. CDV alters hypothalamic integrity Hypothalamic nuclei, neuropeptides, and neurotransmitters integrate central and peripheral signals in order to regulate energy balance of the organism (Elmquist et al., 1999; Kalra et al., 1999; Sawchenko, 1998). Verlaeten et al. (2001) suggested that CDV alters hypothalamic integrity and subsequently modifies the homeostasis of the brain, which leads to obesity. Although CDV targets specific brain structures such as hypothalamic nuclei, thalamus, limbic system, substantia nigra, pars compacta, locus ceruleus, and raphe nuclei (Bernard et al., 1993), viral transcripts are only present in few structures, including hypothalamus (Bernard et al., 1999). Nagashima et al. (1992) reported that CDV produces a disorganization or destruction of hypothalamic nuclei, including the paraventricular nucleus. Furthermore, obese-infected animals lost pro-opiomelanocortin (POMC) and TH cell bodies (Nagashima et al., 1992). Loss of POMC bodies is shown to be associated with development of obesity (Scallet and Olney, 1986). These findings suggest a role for food intake disregulation in the CDV-infected animals. In agreement with these findings, Lyons et al. (1982) had reported that food intake of the infected group was greater, but did not attain statistical significance. Perhaps, the small additional energy intake over several weeks eventually contributed to biologically significant gain in body energy stores. 4. CDV downregulates long leptin receptor and increases leptin In the acute phase of infection, the expression of long leptin receptor was upregulated in all tissues targeted by CDV. With the exception of the hypothalamus, the same change was present in the late phase of the infection (Bernard et al., 1999). Long form of leptin receptor expression in the hypothalami was downregulated 70% and blood leptin levels were dramatically increased (53.4 mg/l vs 7.1 mg/l) in the late phase of infection when obesity occurred. Leptin, a protein mainly secreted by adipocytes, modulates food intake and energy metabolism (Paracchini et al., 2005). Downregulation of leptin receptor produces a state of leptin resistance. Consequentially, an increase in food intake and body weight may follow. 5. CDV downregulates melanin-concentrating hormone Melanin concentration hormone, synthesized in lateral hypothalamus and zona incerta (Bittencourt et al., 1992), is recognized to be a potent anorectic peptide that is involved in hypothalamic regulation of feeding behavior (Presse et al., 1996; Qu et al., 1996; Shimada et al., 1998). CDV causes an

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initial downregulation of ppMCH in infected mice and this change is maintained in the late stages of infection only in the mice that become obese (Verlaeten et al., 2001). Verlaeten et al. (2001) speculated that either a disturbance in cAMP second messenger pathway or a strong induction in TNF-a is responsible for ppMCH downregulation. Expression of ppMCH mRNA is reduced in obese-infected mice by about 70% when compared to lean infected or uninfected mice. MCH protein expression is also decreased even in the absence of neuronal damage or reduction in cell number in hypothalamus, which suggests the ‘‘hit-and-run’’ eVect described by Oldstone et al. (1982). Verlaeten et al. (2001) suggested that this downregulation is related to CDV-induced obesity, rather than CDV infection itself. 6. CDV decreases expression of neuropeptides In the acute phase of infection, CDV decreases the expression of neuropeptides, in particular NPY, MCH, hypocretin, vasopressin, and tachykinins, disturbing homeostatic equilibrium. In the late stage of infection, the lean animals recover, while some of the obese animals surprisingly still have disturbed equilibrium. Interestingly, POMC, the precursors of anorexigenic a-MSH, is not modified, whereas orexigenic peptides like NPY, MCH, and hypocretin are downregulated (GriVond et al., 2004). NPY is involved in the control of hunger, satiation, and satiety (Ramos et al., 2005); MCH participates in food intake and energy expenditure modulation (Collins and Kym, 2003; Kawano et al., 2002); and hypocretins contribute to appetite and satiety regulation, energy equilibrium, arousal and sleep wakefulness, and vigilance and defense behaviors (Burdakov, 2004; Mazza et al., 2005; Sakurai, 2003). Implications of these findings are still unclear (GriVond et al., 2004). 7. CDV decreases catecholamine levels Changes in adrenergic system play a key role in regulating energy balance, and are associated with obesity (Clement et al., 1997; Fisher et al., 1998; Lipworth, 1996). Catecholamine levels were two to three times lower in CDV-infected mice (Lyons et al., 1982). Lyons et al. (1982) did not find significant lesions in the brain. Therefore, at the time, they considered reduced catecholamine levels as the cause of the obesity observed in CDV mice. 8. Increase cytokine production Cytokines produced in the brain play an important role in virus–host interaction that may influence viral spread and gene expression and consequently the functioning of neural cells (D’Arcangelo et al., 1991; Merrill and Chen, 1991;

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Patterson and Nawa, 1993; Zalcman et al., 1994). TNF-a, IL-b, and IL-6 transcripts are selectively expressed in CDV-targeted brain structures, suggesting a possible participation of cytokine in CDV-induced changes. Cytokines are only expressed in brain regions permissive to CDV, suggesting that viral replication is necessary for cytokine expression (Bencsik et al., 1996). Upregulation of cytokine expression may alter neural cell function, which the author speculates could trigger the ensuing neurological disorders (Bencsik et al., 1996).

III. ROUS-ASSOCIATED VIRUS-7 Rous-associated virus-7 (RAV-7) was the second pathogen reported to cause obesity. In addition, it causes stunting and hyperlipidemia in chickens (Carter et al., 1983a). The following is a description of the adipogenic eVects of RAV-7. A. RAV-7 GENERAL INFORMATION

RAV-7, an avian leukosis virus (Carter and Smith, 1984), has an 8.2-kb RNA (Carter et al., 1983a). It is the most common naturally occurring avian retrovirus associated with neoplastic disease condition in domesticated poultry (Fadly, 1997). Avian leukosis viruses are RNA tumor viruses, a subfamily of the retroviridae (Vogt and Hu, 1977). All RNA tumor viruses carry three genes: gag (group-specific antigen), pol (directs the synthesis of the RNA-dependent DNA polymerase of the virion), and env (the gene for the envelope glycoprotein). The envelope glycoprotein of oncoviruses determines type-specific properties of the virion: host range, interaction with neutralizing antibodies, and viral interference. In the avian leukosis viruses, these specific properties are markers for virus classification into five subgroups: A, B, C, D, and F (Vogt and Hu, 1977). RAV-7 shares the least antigenic cross-reactivity within the subgroup C (DuV and Vogt, 1969). Avian retroviruses have also been classified into two groups based on the time required to produce tumors (Graf and Beug, 1978). However between these groups, there is another group that produces tumors like osteopetrosis after an intermediate latency time (Smith and Moscovici, 1969), immunosuppression (Smith and Van Eldik, 1978), stunting (Banes and Smith, 1977), and anemia (Paterson and Smith, 1978; Smith and Schmidt, 1982). Carter

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showed that RAV-7 is classified as an avian retrovirus that causes disease at an intermediate time.

B. RAV-7-INDUCED OBESITY

Carter et al. (1983a) showed that RAV-7 induces obesity in chickens, which is characterized by stunting and hyperlipidemia. Birds, when infected as 10-day-old embryos, developed fat deposition around crop and abdominal fat pads. Whereas inoculation of 1-day-old hatched chickens did not produce stunting or obesity (Carter et al., 1983a). DiVerence in food intake was observed only after 3 weeks, when stunting and obesity developed (Carter et al., 1983b). Stunting of growth was the most striking eVect of RAV-7. Failure to grow became apparent 14 days posthatching, and the infected animals had grown four times slower than the uninfected controls (3.9 g/day vs 16.3 g/day). Fifty days posthatching, mean weight of RAV-7-infected chickens was 194 g compared to 515 g of the control group. RAV-7 infection caused a marked reduction in thymus weight, whereas the spleen size remained unchanged (Carter et al., 1983a). Fifty days after hatching, bursa weight was 0.2 g versus 3.1 g in the uninfected controls. Three weeks after hatching, infected chickens developed fatty, yellow colored livers, hepatomegaly, anemia, and immune suppression. Infected livers represented 6.2% of the body weight versus 2.4% in the uninfected controls. The liver cells from the 30-day-old RAV-7-infected chickens showed marked histological changes, with disorganization and swollen mitochondria with less cristae. RAV-7-infected chickens showed hyperlipidemia. The most striking eVect was observed in several 30-day-old infected chickens that had serum triglyceride levels greater than 2000 mg/dl and interestingly one greater than 14,000 mg/dl. There was no evidence of neoplastic transformation in the infected chickens, but lymphoblastoid infiltration of the thyroid and pancreas was present as early as 7 days posthatching, and the infiltration involved more tissue with time. Hypoglycemia was present in the infected animals 20 and 40 days after hatching. Serum amylase levels were increased for the infected animals, and this diVerence was significant 40 days posthatching. Carter et al. (1983a) suggested that increased amylase levels and decreased serum glucose values accompanied by histological alteration of the pancreas indicated pancreatic damage. Insulin levels in RAV-7-infected chickens were above normal all through 43 days posthatching and ranged from 114% to 352% of normal, with the exception of 3 days. Twenty and 30 days posthatching, glucagon

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levels dropped to 34% and 44% of normal. This period also coincided with high mortality in RAV-7-infected chickens (Carter et al., 1983a). C. SPECIFICITY OF RAV-7-INDUCED OBESITY

Carter et al. (1983a,b) reported that the obesity-promoting eVects of RAV-7 are common for the subgroup C of avian leucosis viruses, but not for the other subgroups (A, B, D, or F). Chickens infected with subgroups A, B, D, E, and F showed osteopetrosis (predominant for MAV-1 and MAV-2), anemia, liver lesions, blood cysts, fibrosarcoma, or nephroblastoma (predominant for MAV-2), but did not develop adiposity (Carter and Smith, 1984). Subgroup C viruses tested (RAV-7, transformation-defective B77, transformation-defective Prague strain of Rous sarcoma virus, RAV-49) caused stunting, lymphoblastoid infiltration of the thyroid and pancreas, decreased thyroxine, and increased insulin and liver weight, with RAV-7 showing the greatest change. These results were obtained from 10-day-old embryos intravenously inoculated with diVerent types of viruses belonging to avian leukosis class. Subgroup C virus-infected chickens were significantly stunted 20, 30, or 40 days posthatching. The possibility that the envelope of avian leukosis virus is involved in the pathogenesis of disease was examined by comparing the changes induced by RAV-7, and other viruses in subgroups A, B, D, and F. Subgroup C viruses produced similar changes, which were not common to those from other subgroups. This suggested that the envelope may be involved in pathogenicity (Carter and Smith, 1984). RAV-7 shares the least antigenic cross-reactivity within the subgroup C viruses (DuV and Vogt, 1969), which may explain its greater pathogenicity compared to other viruses in the same subgroup. At 10 days posthatching, 25–75% of thyroid tissue from all subgroup C virus-infected chickens appeared to be lymphoblastoid and at 30 days posthatching, the eVect was more prominent, showing the presence of plasma cells. T4 levels in the serum were significantly lower for RAV-7- and tdB77-infected chickens at 10 days posthatching, while those for RAV-49infected chickens attained significantly lower levels after 20 days. T4 levels continued to decrease until 45 days posthatching, when all the viruses in subgroup C had significantly lower T4 levels. Infiltration of the pancreas varied at 10 days posthatching among the viruses, being the most severe for RAV-7 and RAV-49. However by 20–30 days posthatching, all the subgroup C viruses tested showed germinal center formation. Insulin levels were increased 10 days after hatching compared with the control group and by 45 days after hatching the diVerence was significant for all subgroup C viruses.

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Livers from all infected chickens showed mild lymphocytic infiltration 10 days posthatching with slight enlargement of hepatocytes after 30 days, which the authors attribute to fat accumulation. Chickens infected with all subgroup C viruses had significantly increased relative liver weights at all times compared with the controls. Serum triglyceride levels in chickens infected with all subgroup C viruses were increased. The greatest change was induced by RAV-7 with a mean of 1500 mg/dl after 45 days compared with a range of 68.2–90.5 mg/dl in uninfected chickens. Cholesterol levels decreased initially followed by a significant increase, 45 days posthatching. Marked hyperlipidemia was present only in RAV-7-infected chickens. Histological examination of thyroid, pancreas, and liver showed no similarity between subgroup C-induced changes and those by Rous-associated viruses of other subgroups (A, B, D, and F). Fat accumulation as adipose tissue and fatty liver was apparent only for RAV-7-infected chickens. Relative liver weights were higher for all infected animals. MAV-1- and RAV-1infected chickens showed significant increase in insulin levels, but the increases were smaller compared to those induced by RAV-7. All avian leucosis virus-infected chickens showed decreased T4 levels, with a significant diVerence in RPV and RAV-61 (subgroup F). Changes in T4 levels in these chickens were smaller compared to the changes induced by RAV-7, which also showed thyroiditis. D. MECHANISM OF RAV-7-INDUCED CHANGES

A decrease in thyroid hormone levels is the major metabolic change that may explain adiposity and other changes induced in RAV-7-infected chickens (DuV and Vogt, 1969). Chicken embryos infected with RAV-7 showed alteration in thyroid tissue 2 days posthatching, lymphoblastoid infiltration 7 days posthatching, and extensive infiltration, loss of normal thyroid structure, and significantly lower levels of T3 and T4 16 days posthatching. Chickens with hypothyroidism induced by Tapazole have near normal growth characteristics after daily administration of T4 (Singh et al., 1968). When RAV-7-infected chickens received supplementation with Synthroid, the disease symptoms alleviated showing a decrease in body weight, relative liver weight, and insulin levels. Although an increased mortality in the group fed low-fat diet was noted, fat content of the diet did not influence stunting or obesity (Carter et al., 1983b). Levels of T4 were decreased for all avian leukosis virus-infected chickens and correlated with stunting of animals (Carter and Smith, 1984). The possibility of autoimmune thyroiditis was considered due to the similarity in thyroid infiltration and change of T4 levels (Carter and Smith, 1984) present in an obese chicken line and RAV-7-infected chickens. The obese chicken

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line is a phenotypic expression of the genetically determined autoimmune thyroiditis (Wick et al., 1981). However, the possibility of autoimmune thyroiditis was excluded since no precipitating antithyroglobulin in the sera of RAV-7-infected chickens was observed. RAV-7-induced hyperlipidemia could be a result of hypothyroidism. Hyperlipidemia appears to be common in hypothyroid animals, where there is a decrease in lipid utilization or catabolism (Hoch, 1974). Similar etiology was suggested for increased insulin levels and localized lymphoblastoid infiltration of the pancreas (Carter and Smith, 1984) because it was observed that amelioration of thyroid function by Synthroid administration reduces insulin levels (Carter and Smith, 1983). Increased mortality that occurred 20 and 30 days posthatching may be the result of a severe drop in glucagon levels (Carter and Smith, 1983; Carter et al., 1983a), which determines severe hypoglycemia (Mikami and Ono, 1962).

IV. CHLAMYDIA PNEUMONIAE Chlamydia pneumoniae (CP) is the first bacteria reported to be associated with increased body mass index (BMI) in humans. Experimental inoculation of animals with CP to study the eVect on body weight has not been reported. A. CP GENERAL INFORMATION

Chlamydia is a eubacteria that causes widespread infection in humans (Hogan et al., 2004). CP respiratory infection occurs in almost all humans during lifetime (Hogan et al., 2004) and results in 10% of communityacquired pneumonia and 5% of bronchitis and sinusitis cases (Kuo et al., 1995). Association between CP and coronary heart disease (CHD) is equivocal. Some studies show an association between CP and CHD (Danesh et al., 1997; Kuo et al., 1993; Saikku et al., 1988), while others found none (Caligiuri et al., 2001; HoVmeister et al., 2000). HoVmeister et al. (2000) reported that indicators of atherogenic lipid profile, like decreased HDL-C, HDL-C to total cholesterol ratio, apoAI, and increased apoB, were associated with current infection with Helicobacter pylori, but not with CP or Cytomegalovirus (HoVmeister et al., 2001). B. CP ASSOCIATION WITH HIGHER BMI

Some studies reported an association between CP infection and increased BMI (Dart et al., 2002; Ekesbo et al., 2000), while others did not find any relation (Blanc et al., 2004; Falck et al., 2002; Muller et al., 2003).

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Ekesbo et al. (2000) reported that a group of subjects with combined serology for CP and H. pylori had significantly higher BMI compared to the control group (27.3 kg/m2 vs 25.8 kg/m2). Greater age, lower socioeconomic status, and greater fasting levels of insulin also characterize the antibody positive subjects. The authors suggested that ‘‘Obesity might be a marker not only for lower social class but also for greater than normal susceptibility to such infections’’ (Ekesbo et al., 2000). Similar correlation was found in another study where subjects positive for IgG to CP had significantly higher mean BMI compared to the seronegative subjects 26.6 kg/m2 versus 25.5 kg/m2, respectively (Dart et al., 2002). There was no association between CHD or coronary artery stenosis and infection with CP. Antibody positive subjects had increased BMI, smaller LDL particle, and greater insulin levels. However, after multivariate analysis, only BMI continued to be associated with seropositivity (Dart et al., 2002). C. MECHANISM OF CP EFFECTS

Dart et al. (2002) postulated that CP infection is causally related to increased BMI, although the mechanism is completely unknown. Obesity is associated with impaired immunity (Fried et al., 1998; Tanaka et al., 1993; Visser et al., 1999) and as suggested by Ekesbo et al. (2000) obesity might be the indicator for increased susceptibility to H. pylori and CP infection (Ekesbo et al., 2000). However, Dart et al. (2002) found no preponderance of antibodies to Chlamydia trachomatis and Chlamydia pssittaci in the same subject population. Lack of relationship of BMI with prevalence of antibodies to C. trachomatis and C. pssittaci indicated that increased BMI did not necessarily predispose the subjects to ‘‘catch’’ infection. Experiments with animal models designed to investigate the adipogenic potential of CP and human sero-epidemiological data are needed to further determine the role of CP in obesity. V. SCRAPIE AGENT Scrapie agents have been reported to cause obesity in mice, and the eVect is dependent on the strain of scrapie. Relevance of the findings to humans has not been established. A. GENERAL INFORMATION

Scrapie is a neurodegenerative disease with a long incubation period, known to occur in sheep and goats, however, mice and other small rodents can be infected with it (Hunter, 1972). Certain scrapie strains induce obesity in

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experimentally infected animals, although the main manifestation of scrapie infections is abnormal behavior and motor dysfunction in mice (Kim et al., 1987) and hamsters (Carp et al., 1990). Incubation period determined by the mouse gene Sinc and manifestation of disease varies with the scrapie and mouse strain (Dickinson and Meikle, 1971). B. SCRAPIE-INDUCED OBESITY

Weight increase in scrapie-infected animals was observed as a preclinical manifestation as early as 1968 by Pattison and Jones (1968) and was followed by Outram and Markovits in 1972 and 1981, respectively (Markovits et al., 1981; Outram, 1972). Mice infected with certain strains of scrapie determine a significant increase in body weight in the preclinical phase of the disease (Carp et al., 1984). Regardless of the mouse strain, the scrapie strain ME7 injected in the hypothalamus-induced obesity (Carp et al., 1984, 1998; Kim et al., 1987, 1988). This eVect was also observed for 22L scrapie strain injected in certain type of mice (Carp et al., 1984, 1998; Kim et al., 1987), but not for 139A (Carp et al., 1984, 1998; Kim et al., 1987, 1988) or 22A scrapie strains (Carp et al., 1984). Hypothalamic injection of ME7 injection induced obesity in SJL, C57NL, A2G, SAMP8, SAMR1, and AKR mice (Carp et al., 1984, 1998; Kim et al., 1987, 1988). Twelve weeks postinfection with ME7, SJL mice weighed significantly more than the control group and this diVerence reached maximum after 4 weeks. Infected animals showed 80% and 68% increase in body weight when injected in the hypothalamus or cortex, respectively, versus 41% in the control group. The weight increase was due to fat accumulation, and not due to edema (Carp et al., 1998; Outram, 1972). Surprisingly, spleen, liver, kidney, brain, and uterus weights were lower than those in the control (Carp et al., 1984; Kim et al., 1988). Adrenal gland was the only organ with significantly greater weight in the infected group. This increase was due to marked enlargement in zona fasciculate and slight enlargement in zona glomerulosa of the cortex, without any cytopathic changes (Kim et al., 1988). Weight gain in ME7-infected mice paralleled increase in food consumption (Kim et al., 1988). The weight gain appeared 67 days postinfection and continued throughout the preclinical phase of the disease (Kim et al., 1988). Unexpectedly, at the same time there was an increase in food consumption in scrapie strain 139A-infected SJL mice, which did not become obese. The author suggests a possibly separate mechanism for food consumption and weight gain (Kim et al., 1988). ME7 injected in the hypothalamus, but not in cortex, induces obesity in C57NL mice. ME7 produced the same pattern of vacuolation in nine

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diVerent brain regions, regardless of the injection site (Kim et al., 1987). The only diVerence was in the brain region predominantly aVected: ME7 aVected the forebrain region, 22L aVected the cerebellum, and 139A the white matter (Kim et al., 1988). 22L strain of scrapie injected in the cortex or hypothalamus of SJL mice induced obesity (Carter and Smith, 1984; Kim et al., 1987). However when injected in SAMP8, SAMR1, AKR (Carp et al., 1998), and CBA mice (Carp et al., 1984), the scrapie strain did not produce any diVerences in body weight. SJL-infected mice showed 73% and 70% increase in body weights when injected in hypothalamus or cortex, respectively, versus 36% increase in the control group. Furthermore, the weight diVerence became significant earlier, compared to when inoculated with 22L (10 weeks vs 12 weeks postinjection) (Kim et al., 1987). A significant decrease of GLUT-1 in thalamus, cerebellum, and hippocampus along with reduced glucose tolerance and hyperglycemia in ME7infected mice suggested disregulation in function of the aVected brain regions (Carp et al., 1989; Kim et al., 1988). C. MECHANISM OF SCRAPIE-INDUCED OBESITY

Involvement of the brain is strongly implicated in scrapie-induced obesity. Kim et al. (1987) speculated that scrapie-induced obesity is related to changes in CNS and neuroendocrine dysfunction and that the condition was also dependent on the route of inoculation. ME7 induced obesity in C57NL mice only when injected in hypothalamus, but not cortex, whereas hypothalamic and not cortical route of inoculation was required for increasing body weight in SJL. Similar weight gain and patterns of vacuolation were obtained whether ME7 was injected unilaterally or bilaterally in the hypothalamus of SJL mice, suggesting that scrapie can spread to the opposite hemisphere after replication at the site of injection (Kim et al., 1987). Role of brain function alterations was further supported by the findings of Vorbrodt et al. (2001), who reported a decrease in GLUT-1 density in certain brain regions, reduced glucose tolerance and hyperglycemia in ME7infected mice. Glucose is of critical importance for normal functioning of the nervous system and impaired glucose transport could untimely lead to impaired brain function. The authors recommend further studies to elucidate the regional diVerence in GLUT-1 expression and the eVect on brain function (Vorbrodt et al., 2001). In addition to the role of higher centers, a hypothalamic-pituitary-adrenal axis-mediated mechanism for scrapie-induced obesity has been suggested (Kim et al., 1988). Adrenals in the scrapie-induced mice were significantly larger than controls and adrenalectomy performed before ME7 injection

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prevented weight gain in mice, which support the hypothesis. Although one of the possible downstream eVects of impaired higher brain center functions is food intake dysregulation, increased food intake did not necessarily explain obesity in all models of scrapie strains. ME7- and 139A-infected SJL mice showed increase in food consumption, however only ME7 induced obesity (Kim et al., 1988). Kim et al. (1988) suggested that the pathways involved in increasing food consumption and weight gain may be separate in these models. Moreover, the duration of scrapie incubation does not seem to play a role in the development of obesity. Carp et al. (1984) showed that Sinc mouse gene, which determines the incubation period, is not involved in scrapieinduced obesity. 22L strain was injected into three mouse strains with the same s7s7 Sinc genotype, which resulted in obesity in SJL mice, but not the other two strains (Carp et al., 1984).

VI. BORNA DISEASE VIRUS A. GENERAL INFORMATION

Borna disease virus (BDV) is an enveloped, nonsegmented, negative-stranded RNA virus belonging to the Mononegavirales order (Kao et al., 1993; Rott and Becht, 1995; Stitz et al., 1995). Although it can produce a strong immune response and the disease can be fatal, BDV may persist in the nervous system with no major symptoms (Ludwig and Bode, 2000; Narayan et al., 1983; Solbrig et al., 1995). Experimental infection with BDV has been shown in chickens, mice, rats, rabbit, hamsters, and rhesus monkeys models (Ludwig et al., 1988; Richt et al., 1992). Rats infected with BDV showed behavior changes and learning deficits (Ludwig and Bode, 2000). BDV causes encephalopathy in a broad range of animals like horses, sheep, cattle, cats, and dogs (Ludwig and Bode, 2000). BDV incidence was reported worldwide: in UK (Thomas et al., 2005), Iran (Bahmani et al., 1996), Japan (Terayama et al., 2003), China (Yang et al., 2003), Germany (Bechter et al., 1987), and the United States (Kao et al., 1993). Ludwig and Bode (2000) postulated that a cross species transmission of BDV from animals to humans may be possible. BDV received considerable attention when de la Torre (1994) demonstrated that BDV can infect human brain, by detecting BDV-specific antigen in four autopsied human brains. Fifty BDV studies were reported in humans before the beginning of 2000, including case studies, serological, or viral prevalence studies. Chalmers et al. (2005) reported in their review that seroprevalence in humans varied from 0% to 48% and viral prevalence from 0% to 82%. Mental disorders like depression

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and schizophrenia are associated with BDV-specific antibodies prevalence (Chen et al., 1999; Ferszt et al., 1999; Richt et al., 1997) and BDV RNA in the peripheral blood (Chen et al., 1999). Chen et al. (1999) provided some evidence for a possible human to human transmission of BDV infection, by showing that psychiatric patient’s family members and the mental health workers had higher prevalence of BDV antibodies than control. However, Ferszt et al. (1999) postulated that the higher prevalence of BDV antibodies in depressive patients may be a nonspecific aspect of immunosuppression. Although the BDV pathogenesis is not yet clear, a pathogen once thought to only infect animals certainly appears to infect humans as well. B. BORNA DISEASE VIRUS-INDUCED OBESITY

Gosztonyi et al. (1991) described an obesity syndrome induced by BDV in rats. Induction of obesity was dependent on age and genetic characteristics of animals and virus strain (Gosztonyi and Ludwig, 1995; Gosztonyi et al., 1991). Weanling or adult rats developed acute encephalitis 1–4 months postBDV infection. Two moths later, surviving animals developed marked obesity. Virus-specific antigen and inflammatory infiltrate were present in brains of obese rats, and decreased progressively with time, reaching very low levels 18–24 months postinfection (Gosztonyi and Ludwig, 1995; Gosztonyi et al., 1991). The lymphomonocytic inflammatory infiltration was found in the hypothalami of all infected rats. Progressive neural degeneration located mainly in the dentate gyrus was accompanied by marked hydrocephalus. Spongy degeneration foci were present in the cerebellum, followed by reactive astrocytosis. Obese rats had massive visceral fat deposition with increased triglyceride and hyperplasia of Islets of Langerhans, with moderate increase in blood glucose (Gosztonyi and Ludwig, 1995). C. MECHANISM OF BDV-INDUCED OBESITY

Herden et al. (2000) compared the eVect of two diVerent BDV variants, one that induces obesity (BDV-ob) and one that induces the biphasic course of the disease (BDV-bi) in order to determine the mechanism of BDV-induced obesity. No antigenic variation was present between the two diVerent phenotypes. Four-week-old Lewis rats were inoculated intracerebrally with the infectious brain homogenate of BDV-ob or BDV-bi representing the control group. Fifty-six days postinfection animals infected with BDV-ob reached significantly higher body weight (p < 0.0001) compared with the control group. No neurological signs were observed in the obese group, whereas the control group developed the biphasic form of infection with ataxia, hyperactivity, and weight loss followed by apathy and somnolence. Both groups

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developed nonpurulent meningoencephalitis with mononuclear perivascular and parenchymal infiltration. These were generalized in the control group, but restricted to the ventromedial tuberal hypothalamus, septum, hypocampus, and amygdala in the obese group. Viral antigen expression presented a similar pattern of distribution. BDV-ob infection produced low levels of replication, in contrast with BDV-bi where replication was persistent for more than 210 days. Therefore, Herden et al. (2000) hypothesized that BDVinduced obesity may be due to inflammatory lesions and viral antigen expression in brain, especially in the hypothalamus, which is known to regulate body weight and food intake. VII. GUT MICROBIOTA A. GENERAL INFORMATION

Microbiota is a collection of microorganism, growing in the human gut. It is mostly composed of anaerobic bacteria, which are essential for processing dietary polysaccharides (Xu and Gordon, 2003). These bacteria act in a symbiotic relationship with the human gut, consuming as well as distributing energy by processing nutrients inaccessible to humans in other ways (Backhed et al., 2005). Moreover, it regulates human immune system (Braun-Fahrlander et al., 2002), fortifies the mucosal barrier, and stimulates angiogenesis (Hooper, 2001; Hooper et al., 2003; Stappenbeck et al., 2002). B. MICROBIOTA-INDUCED OBESITY

Backhed et al. (2004) reported that gut microbiota regulates fat storage. Fourteen days postconventionalization with normal microbiota, germfree mice developed more body fat and insulin resistance, with surprisingly lower food intake. These eVects were seen in male and females in two diVerent mouse strains (B6 and NMRI) (Backhed et al., 2004). Eight- to ten-week-old male germfree mice, which experience colonization for 14 days, showed 57% increase in total body fat with 61% increase in epididymal fat and 7% decrease in lean mass with no diVerence in body weight. Lipid profile did not change after colonization. A similar change was determined by colonization of germfree animals at birth or for diVerent amount of time; however, increasing colonization time did not amplify the eVect on adipose tissue (Backhed et al., 2004). Subsequently, germfree mice were colonized only with Bacteroides thetaiotaomicron, which is predominant in gut microbiota and has the ability to degrade plant polysaccharides (Hooper et al., 2001). B. thetaiotaomicron is

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associated with induction of host monosaccharide transporters (Xu and Gordon, 2003). After a 14 days colonization period, these mice developed 23% more body fat than their germfree counterparts showing a similar change to the complete colonization eVect, but to a smaller extent (Backhed et al., 2004). Ley et al. (2005) reported that obesity correlates with a shift in microbiota composition. Obese mice showed 50% decrease in Bacteroides and significant increase in Firmicutes (p < 0.05), independent on kinship or gender. OVspring shared cecal microbiota with their mothers, independent of their ob phenotype. The authors suggest that changes in microbiota of ob/ob animals ‘‘may represent an unheralded contributing factor to their fuel partitioning’’ (Ley et al., 2005). C. MECHANISM OF MICROBIOTA-INDUCED OBESITY

Colonization for 14 days produced a 27% decrease (p < 0.01) in metabolic rate, whereas high energy phosphate stores remained unchanged. Thus, obese colonized animals consume more oxygen without increasing high energy phosphate stores, which suggest the presence of ineYcient metabolism (Backhed et al., 2004). The microbiota determined a 2.3-fold increase in hepatic triglyceride content, with no change in cholesterol, but significantly increased expression of de novo fatty acid synthesis pathway genes (Backhed et al., 2004). Moreover, colonization decreased the expression of fasting-induced adipocyte factor (Fiaf) (Backhed et al., 2004), also called angiopoietin-like protein 4, an inhibitor of lipoprotein lipase (LPL) in vitro (Yoshida et al., 2002). This eVect is not dependent on mature lymphocytes or PPAR-g (Backhed et al., 2004). The authors suggest that the microbiota act via Fiaf to increase hepatic lipogenesis as well as increase adipose tissue LPL. Thus, the extra energy harvested from the gut by microbiota is converted to lipids in the liver and LPL helps deposition of this lipid in adipocytes and adiposity ensues (Backhed et al., 2004). VIII. ADENOVIRUSES Four adipogenic pathogens are adenoviruses. Adenoviruses were first isolated in 1953 by investigators attempting to establish cell lines from the adenoidal tissue of children after tonsillectomy (Shen and Shenk, 1995). Adenoviruses are widespread in nature, infecting birds, mammals, and humans. In humans, adenoviruses are frequently associated with acute upper respiratory tract infections, enteritis, or conjunctivitis. Presence of serum antibodies

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to adenovirus is common in the general population (Foy and Grayston, 1976). Adenoviral DNA is detected in adult human lymphocytes, and the number of positive cells increases with the age of the person (Horvath et al., 1986). There are six major subgroups among the 50 human adenoviruses maintained by the American Type Culture Collection (ATCC). Each subgroup has a number of specific serotypes. Adenoviruses are nonenveloped DNA viruses with icosahedral symmetry and a diameter of 65–80 nm (Pereira et al., 1963). All adenoviral genomes have the same general organization, that is the genes encoding specific functions are located at the same position on the viral chromosome (Shen and Shenk, 1995). The genome consists of a single linear, double-stranded DNA molecule consisting of five early transcription units (E1A, E1B, E2, E3, and E4), two delayed early units (IX and IVa2), and one major late unit which generates five families of mRNAs (L1–L5). Adenoviruses can be readily propagated in primary cell cultures or cell lines, replicate within the nucleus of the infected cells, and produce characteristic cytopathic changes in the cell. IX. SMAM-1 Avian adenovirus SMAM-1 causes adiposity in chickens and shows association with human obesity. It is the first virus to be linked with human obesity. Increased adiposity with hypolipidemia are the peculiar features of this syndrome. A. SMAM-1 GENERAL INFORMATION

Adenoviruses are responsible for approximately 8% of the viral infection worldwide (Rubin, 1993). Avian adenovirus SMAM-1 was discovered during a poultry epidemic in early 1980s (Ajinkya, 1985) and it is serologically related to avian adenovirus Chick Embryo Lethal Orphan virus (CELO). B. SMAM-1-INDUCED OBESITY

Three-week-old chickens experimentally inoculated with SMAM-1 developed excessive visceral fat compared to the uninfected controls 3 weeks postinoculation. Another group of chickens sharing the cage with the infected group (in-contact group) also developed significant visceral adiposity, suggesting a horizontal transmission of the virus in cage mates (Dhurandhar et al., 1990, 1992). Visceral fat was greater by 53% and 33% in the infected and in-contact group, respectively. Interestingly, SMAM-1-induced adiposity was accompanied by paradoxically lower serum cholesterol and triglyceride levels.

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Food intake of the chickens could not explain the adiposity (Dhurandhar et al., 1990, 1992). At baseline, body weights were similar for all groups; however, after 5 weeks the infected group weighted significantly less than the control (924.8
102.1 g vs 995.7
97 g). Furthermore, weight gain between the third and sixth week was 473.8
90.7 versus 571.8
63.5 in the infected and control groups, respectively. The in-contact group tended to have lower body weight after 6 weeks, but the diVerence was not significant (Dhurandhar et al., 1992). One week postinoculation, the infected and in-contact group had significantly lower cholesterol and triglyceride levels when compared to the control. Cholesterol values were 46.1
10.3 mg/dl versus 56.5
10.8 mg/dl and triglycerides 19.9
7.9 mg/dl versus 67.1
18.5 mg/dl in the infected and control groups, respectively. After 2 weeks, cholesterol as well as triglycerides remained lower for the infected and the in-contact groups, but only cholesterol showed statistical significance 3 weeks postinoculation (Dhurandhar et al., 1992). Livers were significantly heavier for the infected and in-contact groups. Livers and kidneys of the infected animals were enlarged and pale brown. Histopathology of the livers showed severe congestion, fatty infiltration, and presence of intranuclear inclusion bodies. The in-contact group showed similar but less severe changes. Bursae, thymus, and spleen were atrophied in the infected and in-contact group (Dhurandhar et al., 1992). Subsequently, Dhurandhar et al. (1997a) reported an association between SMAM-1 seropositivity and human obesity. This was the first virus reported to be associated with human obesity. Using an agar gel precipitation assay, Dhurandhar et al. (1997a) reported 20% prevalence in 52 obese subjects from India screened for the presence of antibodies against SMAM-1. Similar to the syndrome caused by SMAM-1 in chickens, the seropositive human subjects had significantly higher body weight and BMI and significantly lower blood lipid levels. These subjects were men and women from an outpatient treatment program in Bombay, India, with a mean age of 39 and a mean BMI of 31.6 kg/m2. The antibody positive subjects had significantly greater BMI (35.3
1.5 kg/m2) compared to antibody negative subjects (30.7
0.6 kg/m2, p < 0.001). A similar diVerence was seen in body weight. The antibody positive subjects weighted significantly more than the antibody negative ones (95.1
2.1 kg vs 80.1
0.6 kg, p < 0.02). The diVerences were valid even within gender. Serum cholesterol was 15% lower (p < 0.02) and triglyceride 60% lower (p <001) in the antibody positive subjects compared to antibody negative counterparts (Dhurandhar et al., 1997a). In addition, two of the positive serum samples cross-reacted with each other, suggesting presence of active viremia. The findings were surprising as avian adenoviruses have been

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thought to be serologically diVerent from human adenoviruses (Asch et al., 1979; McFerran et al., 1975), and therefore, unable to infect across species. C. MECHANISM OF SMAM-1-INDUCED OBESITY

Dhurandhar et al. (1992) suggested that SMAM-1 infection could impair normal liver function, causing fatty liver and decreasing cholesterol and triglyceride levels. Consequent to impaired hepatic lipogenesis, increased visceral fat accumulation may be due to either increased compensatory lipogenesis or reduced lipolysis in fat cells. Moreover, since chickens are very sensitive to glucagon, the authors expressed the possibility that glucagon deficiency could be responsible for greater accumulation of visceral fat due to reduced lipolysis (Dhurandhar et al., 1992). Both theories have not been tested. The authors postulated that high infectivity of SMAM-1 and the temporal association between the appearance of SMAM-1 in chickens in India and worldwide increase in obesity may suggest the role of SMAM-1 in human obesity (Dhurandhar et al., 1997a). However, further research is necessary to demonstrate such a role. X. ADENOVIRUS TYPE 36 Animals experimentally infected with Adenovirus type 36 (Ad-36) develop greater adiposity but a relative hypolipidemia. Sero-epidemiological studies show association of Ad-36 antibodies with human obesity. In vitro experiments in 3T3-L1 preadipocytes showed that Ad-36 promotes their proliferation, diVerentiation, and lipid accumulation. However, the exact mechanism of adipogenic action in vivo is yet unknown. Adipogenic eVects of Ad-36 are described below in detail. A. Ad-36 GENERAL INFORMATION

Ad-36 was first isolated in 1978 in Germany from the feces of a 6-year-old girl suVering from diabetes and enteritis (Wigand et al., 1980). Ad-36 infection was thought to occur rarely, but work indicates that 11–30% of the individuals screened in the United States are seropositive for Ad-36 antibodies (Atkinson et al., 2005). B. Ad-36-INDUCED ADIPOSITY

Ad-36 promotes adiposity in animals and shows association with human obesity.

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1. Animal models In separate experiments, chickens, mice, or marmosets (nonhuman primates) inoculated with human adenovirus Ad-36 developed a syndrome of increased adipose tissue and paradoxically low levels of serum cholesterol and triglycerides (Dhurandhar et al., 1992, 2000, 2001). Food intake of the infected and the uninfected controls was not significantly diVerent. Using a capillary electrophoresis assay (Kolesar et al., 2000), Ad-36 DNA was detected in the adipose tissue of the infected animals but not in the skeletal muscle. The amount of Ad-36 DNA present in the visceral fat correlated with the amount of total visceral fat in the chickens (r ¼ 0.41, p ¼ 0.025). Sections of the brain and hypothalamus of Ad-36-inoculated animals did not show any overt histopathologic changes. Dhurandhar et al. (2001) demonstrated the transmissibility of adenovirus infection and adiposity in a chicken model by parenteral route. Four groups of 3-week-old chickens (infected donors and recipients, and control donors and recipients) were used. The infected and the control donors were inoculated with Ad-36 or media. Thirty-six hours postinoculation, blood from the infected or a control group was transfused intravenously in the respective recipient groups. Infected donors and recipient animals developed significantly greater total body fat than the control groups, despite similar food intake. The visceral fat depots in the infected donors and receivers were heavier (142% and 80% greater, respectively) compared to the control. When obesity was defined as body fat 85th percentile of that in the control group, 64% of the infected donors and 72% of the infected recipients were considered obese versus only 18% in control donors and recipients together. Serum cholesterol was significantly lower in the infected and infected-recipient animals compared to the control group, showing that Ad-36 infection and subsequent obesity was transmissible. Subsequently, two more studies were conducted to demonstrate the adipositypromoting eVect in nonhuman primates (Dhurandhar et al., 2002). The first study used stored plasma samples from adult male rhesus monkeys, which were collected every 6 months over a period of 84 months at the University of Wisconsin Regional Primate Research Center in Madison, Wisconsin. The results showed spontaneous appearance of Ad-36 antibodies followed by weight gain in the year following seroconversion versus the year preceding it (1.8 kg vs 0.1 kg, respectively). Lipid profile was improved, showing a decrease in serum cholesterol by 35 mg/dl. In the second study, male marmosets, separated into two weight- and age-matched groups, were inoculated intranasally with either Ad-36 or medium. Twenty-eight weeks postinoculation, the infected animals showed a fourfold gain in body weight and an increase in visceral fat (7.3
2.5 g vs 4.4
0.9 g, p ¼ 0.089).

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Ad-36 inoculation induced adiposity in male Wistar rats despite similar food intake. Twenty-seven weeks postinoculation, Ad-36-infected rats attained greater body weight (628.3 þ 45.1 g vs 587.5 ¼ 30.6 g, p < 0.008). Their epididymal-inguinal, retroperitoneal, and visceral fat pads were 60%, 46%, and 86% greater, respectively (p < 0.0001). Paradoxically, Ad-36 group developed higher insulin sensitivity compared to the control (Yu and Dhurandhar, 2005). Leptin levels were greater and corticosterone and norepinephrine lower in the infected group, suggesting that Ad-36 may aVect the hypothalamo-pituitary-adrenal axis (Pasarica et al., 2006; Shin et al., 2004). 2. Association with human obesity Sera of 360 obese (BMI  30 kg/m2) and 142 nonobese (BMI < 30 kg/m2) subjects from Wisconsin, Florida, and New York were screened for the presence of Ad-36 antibodies using a constant-virus-decreasing-serum neutralization assay, a gold standard for detecting neutralizing antibodies. The eVect of Ad-36 seropositivity on the risk of obesity was highly significant (Atkinson et al., 2005), which was independent of age, sex, and the data collection site, Ad-36 antibodies were more prevalent in obese subjects (30%) than in nonobese subjects (11%). Seropositive obese as well as nonobese subjects had significantly greater BMI versus their respective seronegative counterparts (Atkinson et al., 2005). Prevalence of Ad-36 antibodies in human population may vary by the geographic locations. Only 5% of the subjects screened in Denmark had antibodies to Ad-36 (Raben et al., 2001). Ad-36 is the first human virus reported to show an association with human obesity. Due to ethical reasons, it is not possible to infect humans with a virus, which precludes unequivocal demonstration of causative role of Ad-36 in human obesity. Future experiments are likely to generate indirect and circumstantial evidence. C. MECHANISM OF Ad-36-INDUCED OBESITY

The adipogenic mechanism of Ad-36 is unclear, but several in vitro and in vivo studies provide some insight about the molecular eVect of Ad-36 on adipose tissue metabolism. Studies show that Ad-36 has similar eVects as thiozolinediones, a class of antidiabetic drugs. Ad-36 increases replication (Pasarica et al., 2005), diVerentiation (Vangipuram et al., 2004), lipid accumulation (Vangipuram et al., 2004), and insulin sensitivity (Yu and Dhurandhar, 2005) and reduces leptin secretion (Vangipuram et al., 2007) and expression (Yu and Dhurandhar, 2005) in fat cells. 3T3-L1 preadipocytes as well as human primary preadipocytes when infected with Ad-36 show increased levels of glycerol 3-phosphate dehydrogenase (GPDH), a diVerentiation specific

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enzyme (Vangipuram et al., 2004). These eVects were not observed with Ad-2, a nonadipogenic human adenovirus. These findings suggest considerable involvement of the adipose tissue in adipogenic eVect of Ad-36. Increased diVerentiation of 3T3-L1 preadipocytes (Vangipuram et al., 2004) and upregulated expressions of adipogenic regulator genes (like PPAR-g, CEBP b and a, and GPDH) in visceral fat of infected animals (Yu et al., 2004) suggest an eVect of Ad-36 on fat cell diVerentiation. Moreover, Ad-36 increases proliferation of preadipocytes, generating more cells capable of accumulating lipids, which may explain the adipogenic eVect in vivo (Pasarica et al., 2005). Greater number of preadipocytes and adipocytes due to Ad-36 infection may also contribute to the enhanced insulin sensitivity in Ad-36-infected rats (Yu et al., 2004). As expected, adipose tissue of Ad-36-infected rats showed enhanced expression of genes of insulin-signaling pathway and de novo lipogenesis such as GLUT-4, insulin receptor, fatty acid synthetase (FAS), and acetyl CoA carboxylase (ACC-1) (Yu et al., 2004). Thus, it appears that Ad-36 increases number of fat cells, enhances glucose uptake by adipocytes, and promotes its conversion to lipids via de novo lipogenesis in 3T3-L1 cells. XI. ADENOVIRUS TYPE 5 A. Ad-5 GENERAL INFORMATION

Adenovirus type 5 (Ad-5) sequence was completely described in 1992 (Chroboczek et al., 1992). Ad-5 has been extensively used for gene therapy (Kanerva and Hemminki, 2005) because these vectors are safe and eYcient and can accommodate large antigen-encoding structures (Wu et al., 2005). Ad-5 causes respiratory tract infections in humans (Limbourg et al., 1996). B. Ad-5-INDUCED OBESITY

So et al. (2005) showed that Ad-5 induces adiposity in mice. Three-week-old female CD1 mice were injected intraperitoneally with either Ad-5 or saline solution. Ad libitum access at food and water was provided. After 22–23 weeks, infected animals attained significantly greater body weight compared to the control group (14.8 g vs 13.5 g; p < 0.05) despite similar food intake. The infected group had a mean adiposity of 6.7%, compared with 2.4% in the control group (p < 0.05) representing a 279% increase, as measured by in vivo 1H magnetic resonance spectroscopy (1H MRS), which is a new rapid and noninvasive technique used to determine body composition. Compared to the control group in the upper quartile for body weight, 66.7% of the infected animals were heavier versus only 16.7% in the control group. In vivo 1H MRS

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showed that there was no diVerence in the intrahepatic lipid levels (So et al., 2005). Serum lipid levels were not reported in this study. C. MECHANISM OF Ad-5-INDUCED OBESITY

The infected group accumulated more body fat, which accounted for the increased body weight. The authors suggest that like Ad-36, increased preadipocyte diVerentiation by viral infection (Vangipuram et al., 2004) may be responsible for the increased body fat. Alternatively, the authors proposed the eVect of inflammation on lipid metabolism. PPAR-g is a pivotal transcription factor responsible for adipocyte diVerentiation and is also involved in modulating inflammatory response (Clark, 2002). The authors speculated that increased adiposity may be a side eVect of the anti-inflammatory response elicited by Ad-5 infection. XII. ADENOVIRUS TYPE 37 Human adenovirus type 37 (Ad-37) was discovered by de Jong et al. (1981). It causes keratoconjunctivitis and genitourinary tract infections in humans (de Jong et al., 1981). Ad-37 was shown to cause adiposity in chickens (Atkinson et al., 2002). Visceral fat pads were three times heavier for the Ad-37 group compared to the control (p < 0.0009). Interestingly, unlike Ad-36, it did not reduce serum cholesterol levels. Food intake of the animals could not explain the adiposity. No information is available about the possible mechanism involved. XIII. ADIPOGENIC POTENTIAL OF OTHER ADENOVIRUSES The adipogenic potential of Ad-36 is not shared by all adenoviruses. Avian adenovirus CELO or human adenoviruses type 2 or 31 did not promote adiposity in animal models (Atkinson et al., 2002; Dhurandhar et al., 2000). Unlike Ad-36, Ad-2 and Ad-31 did not show association with human obesity. For Ad-31 and Ad-2 the antibody prevalence between the obese and the nonobese subjects was similar (76% vs 81% for Ad-2 and 70% and 80% for Ad-31). BMIs and serum lipids were virtually identical for ABþ and AB subjects (Atkinson et al., 2005). Moreover, almost equal distribution of seropositivity among the obese and nonobese subjects for Ad-2 and Ad-31 suggested that the greater prevalence of Ad-36 antibodies in obese subjects is not a result of obesity. Of the 50 known serotypes of human adenovirus, Ad-36, Ad-37, and Ad-5 are adipogenic and Ad-2 and Ad-31 are not adipogenic. Adipogenic

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potential of the remaining serotypes is unknown. Further information about the mode of action and elucidating the mechanism involved may help in screening the remaining serotypes for their adipogenic eVects. XIV. ROLE OF PATHOGENS IN HUMAN OBESITY Although some of the adipogenic pathogens are conventionally considered to be nonhuman pathogens, possible use of a human host by these pathogens cannot be ruled out. Such was the case with BDV, which was originally considered to be a virus of horses and sheep but now shows clear evidence of human infections (Chalmers et al., 2005; de la Torre, 1994; Gosztonyi and Ludwig, 1995; Ludwig and Bode, 2000). Similarly, humans showed antibodies to SMAM-1, the adipogenic avian adenovirus (Dhurandhar et al., 1997a). Moreover, human adenovirus Ad-36 appears to have little selectivity for species and infects chickens, mice, nonhuman primates, rats, and hamsters (Dhurandhar et al., 1997a, 2000, 2001, 2002; Pasarica et al., 2006). Changing of host by pathogens has been well documented. For instance, CDV changed species to infect feline species and considerably reduced Serengeti’s lion population (Morell, 1996). Therefore, nonhuman adipogenic pathogens cannot be ruled out as nonpathogenic for humans. Although several pathogens have been unequivocally shown to cause obesity in animal models, determining their contribution to human obesity is of greatest significance, but equally challenging. Unlike the animal models, human cannot be experimentally infected with these pathogens due to ethical reasons. This limitation precludes any direct demonstration of a cause and eVect relationship of a pathogen and adiposity and the evidence will have to be indirect and circumstantial. Moreover, insidious onset of obesity makes it diYcult to link it to a particular episode of infection experienced in the past. If an adipogenic pathogen uses ‘‘hit-and-run’’ mechanism to induce obesity, the pathogen may not even be detectable in the body, by the time adiposity is noticed. Therefore, elucidating the mechanism of adipogenic action of a pathogen is important. In addition to treatment, prevention could be a longterm goal of researchers investigating Infectobesity. Vaccines against adipogenic pathogens could be expected to provide protection against obesity due to that specific pathogen. XV. INFECTION, INFLAMMATION, AND OBESITY The rapid increase in obesity, its comorbidities, and the associated health care costs have prompted a search for newer and better approaches for its prevention and management (WHO, 2000). Such eVorts may be facilitated by

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understanding the etiology of obesity. Of the nine etiologic factors (Sclafani, 1984), the viral etiology of obesity first reported in 1982 (Lyons et al., 1982) has been largely ignored. In the last two decades, 10 adipogenic pathogens were reported (Atkinson et al., 2002; Backhed et al., 2004, 2005; Carter et al., 1983a,b; Dart et al., 2002; Dhurandhar, 2004; Dhurandhar et al., 1990, 1992, 2000, 2001, 2002; Gosztonyi and Ludwig, 1995; Kim et al., 1987; Lyons et al., 1982; So et al., 2005). These astonishingly high number of reports include human and nonhuman viruses, scrapie agents, bacteria, and gut microflora. Some of these pathogens are associated with human obesity (Atkinson et al., 2005; Dart et al., 2002; Dhurandhar et al., 1997a), but their causative role has not been established. Although a causative role of certain infections in obesity is a relatively novel concept, adipose tissue involvement with modulators and mediators of immune response is well documented. For instance, Cousin et al. (1999) showed that preadipocytes function like macrophages and possess phagocytic and microbicidal activity. Leptin, an adipocyte-secreted hormone involved in body weight regulation, also enhances proliferation and activation of human circulating T lymphocytes and stimulates cytokine production (Martin-Romero et al., 2000). In addition, adipocytes themselves secrete various cytokines (Fried et al., 1998; Sewter et al., 1999) and, in turn, preadipocytes and adipocytes are subject to cytokine-directed modulations (Gregoire et al., 1992; Kras et al., 2000). With such an extensive interaction between the immune system and the adipose tissue, expansion of the latter in response to certain infections is conceivable. For instance, macrophage colony-stimulating factor (MCSF), which promotes the production of macrophages, is also secreted by adipocytes and when over expressed in vivo induces significant adipose tissue hyperplasia (Levine et al., 1998). It is unknown if the obesity-promoting pathogens stimulate MCSF production to increase the growth of adipose tissue. MCSF is an example of pro-inflammatory cytokine. Relationship of infections with inflammation is well known. Moreover, a recent body of evidence shows association of obesity with cytokines and markers of inflammation. Elevated levels of IL-6 (Roytblat et al., 2000) and C-reactive proteins (Visser et al., 1999) are observed in obese individuals. Interestingly, Duncan et al. (2000) showed that markers of inflammation can predict weight gain in middle-aged adults. It remains to be determined if inflammation is a cause or eVect of obesity. XVI. CONCLUSIONS In conclusion, Infectobesity or ‘‘Obesity of infectious origin’’ would be a relatively novel, yet extremely significant concept, if shown to be relevant to humans. An adequate understanding of such pathogens is needed for the

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better management of obesity. A new perspective about the infectious etiology of obesity may stimulate additional research to assess the contribution of hitherto unknown pathogens in human obesity and possibly to prevent or treat obesity of infectious origins. ACKNOWLEDGMENTS This work was partly funded by NIH grant 1R01 DK066164-01 to NVD. REFERENCES Ajinkya, S. 1985. ICAR: ‘‘Final Technical Report.’’ Red and Blue Cross Publishing, Bombay, India. Anderton, P., Wild, T.F., and Zwingelstein, G. 1982. Phospholipids in a measles virus persistent infection: Modification of fatty acid metabolism and fatty acid composition of released virus. J. Gen. Virol. 62(Pt. 2), 249–258. Anderton, P., Wild, T.F., and Zwingelstein, G. 1983a. Accumulation of radiolabelled fatty acids in the neutral lipid fraction of measles virus persistently infected BGM cells. Biochem. Biophys. Res. Commun. 112, 29–34. Anderton, P., Wild, T.F., and Zwingelstein, G. 1983b. Measles-virus-persistent infection in BGM cells. Modification of the incorporation of [3H]arachidonic acid and [14C]stearic acid into lipids. Biochem. J. 214, 665–670. Asch, B.B., McCormick, K.J., and Trentin, J.J. 1979. Unusual features of the oncogenicity of chicken embryo lethal orphan (CELO) virus in hamsters. Prog. Exp. Tumor. Res. 23, 56–88. Astrup, A., Lundsgaard, C., and Stock, M.J. 1998. Is obesity contagious? Int. J. Obes. Relat. Metab. Disord. 22, 375–376. Atkinson, R., Whigham, L., Kim, Y., Israel, B., and Dhurandhar, N. 2002. Evaluation of human adenoviruses as an etiology of obesity in chickens. Am. J. Clin. Nutr. 75, 380S. Atkinson, R.L., Dhurandhar, N.V., Allison, D.B., Bowen, R.L., Israel, B.A., Albu, J.B., and Augustus, A.S. 2005. Human adenovirus-36 is associated with increased body weight and paradoxical reduction of serum lipids. Int. J. Obes. Relat. Metab. Disord. 29, 281–286. Backhed, F., Ding, H., Wang, T., Hooper, L.V., Koh, G.Y., Nagy, A., Semenkovich, C.F., and Gordon, J.I. 2004. The gut microbiota as an environmental factor that regulates fat storage. Proc. Natl. Acad. Sci. USA 101, 15718–15723. Backhed, F., Ley, R.E., Sonnenburg, J.L., Peterson, D.A., and Gordon, J.I. 2005. Host-bacterial mutualism in the human intestine. Science 307, 1915–1920. Bahmani, M.K., Nowrouzian, I., Nakaya, T., Nakamura, Y., Hagiwara, K., Takahashi, H., Rad, M.A., and Ikuta, K. 1996. Varied prevalence of Borna disease virus infection in Arabic, thoroughbred and their cross-bred horses in Iran. Virus Res. 45, 1–13. Banes, A.J. and Smith, R.E. 1977. Biological characterization of avian osteopetrosis. Infect. Immun. 16, 876–884. Bechter, K., Herzog, S., Fleischer, B., Schuttler, R., and Rott, R. 1987. Findings with nuclear magnetic resonance tomography in psychiatric patients with and without serum antibodies to the virus of Borna disease. Nervenarzt 58, 617–624. Bencsik, A., Malcus, C., Akaoka, H., Giraudon, P., Belin, M.F., and Bernard, A. 1996. Selective induction of cytokines in mouse brain infected with canine distemper virus: Structural, cellular and temporal expression. J. Neuroimmunol. 65, 1–9.

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REFRIGERATED FRUIT JUICES: QUALITY AND SAFETY ISSUES MARIA JOSE ESTEVE AND ANA FRI´GOLA Department of Food Chemistry and Nutrition, University of Valencia Avda. Vicent Andres Estelles, s/n. 46100, Burjassot, Spain

I. Introduction II. Review A. Physicochemicals B. Nonenzymatic Browning C. Fatty Acids and Free Amino Acids D. Aroma and Flavor E. Vitamin C F. Carotenoids/Vitamin A G. Anthocyanins/Flavonoids H. Antioxidant Activity I. Color J. Pectinesterases K. Polyphenol Oxidase L. Yeast M. Lactobacillus brevis N. Lactobacillus plantarum O. E. coli P. Staphylococcus aureus Q. Salmonella enteritidis R. Neosartorya fischeri Acknowledgments References

Fruit juices are an important source of bioactive compounds, but techniques used for their processing and subsequent storage may cause alterations in their contents so they do not provide the benefits expected by the consumer. In recent years consumers have increasingly sought so-called ‘‘fresh’’ products (like fresh products), stored in refrigeration. This has led the food ADVANCES IN FOOD AND NUTRITION RESEARCH VOL 52 # 2007 Elsevier Inc. All rights reserved

ISSN: 1043-4526 DOI: 10.1016/S1043-4526(06)52003-0

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industry to develop alternative processing technologies to produce foods with a minimum of nutritional, physicochemical, or organoleptic changes induced by the technologies themselves. Attention has also focused on evaluating the microbiological or toxicological risks that may be involved in applying these processes, and their eVect on food safety, in order to obtain safe products that do not present health risks. This concept of minimal processing is currently becoming a reality with conventional technologies (mild pasteurization) and nonthermal technologies, some recently introduced (pasteurization by high hydrostatic pressure) and some perhaps with a more important role in the future (pulsed electric fields). Nevertheless, processing is not the only factor that aVects the quality of these products. It is also necessary to consider the conditions for refrigerated storage and to control time and temperature. I. INTRODUCTION The consumption of refrigerated juice in the United States is currently of the order of 4.4 billion liters (Jago, 2004). Orange juice is in highest demand, followed by apple, pineapple, and grapefruit. The preferred tropical juices are pineapple, passion fruit, and mango, followed by guava and soursop, which are generally used for mixtures and not in single-flavor beverages. The countries most receptive to consumption of tropical fruit juices are France and Spain, although demand is also high in the UK. The presence of Asian and Latin-American ethnic groups stimulates demand for these flavors. In the European Union (EU) the production of concentrated orange juice is low in comparison with Brazil and the United States. Spain and Italy are the main producers and have a reputation for high quality. Sales of juices and concentrates among the 15 members of the EU were 9.7 billion liters in 2004, and the trend has been increasing during the last decade. Germany is the main market for fruit juices and concentrates, with a consumption of 40.3 liters per person per year. It is followed by Finland, Austria, Spain, and Denmark. According to the Mexican–European Union Business Centre, consumer preferences currently show a tendency toward processed products that satisfy safety and hygiene regulations, and that are low in fats and contain no artificial preservatives (Legiscomex.com, 2006). Governments throughout the world advocate the inclusion of fruit juices in a healthy diet. A juice that is 100% derived from its parent fruit or fruits is almost universally regarded as a healthy and nutritious part of a human diet. The main emphasis in health-promoting dietary recommendations is increased consumption of fruit and vegetables. According to the CODEX General Standard for Fruit Juices and Nectars, an authentic fruit juice product

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must maintain the ‘‘essential physical, chemical, organoleptical, and nutritional characteristics of the fruit(s) from which it comes.’’ Juices are more convenient to consume and generally have a longer shelf life than fresh fruit (IFFP, 2005). Although fruits and vegetables are generally consumed fresh, many of them have to be processed and/or preserved, for financial or logistic reasons, to improve their digestibility, because of culinary needs, or to make them easier to consume for certain consumer groups (children, the elderly, the sick, or people with little time to prepare food, and so on). The consumer demands safe foods obtained by processing, with a preparation that takes up as little time as possible. These new habits have led to an increase in the consumption of prepared fruit and vegetable juices. A recent WHO/FAO joint report recommends consumption of about 400 g of fruit and vegetables a day as an invaluable aid to prevent chronic diseases, including cardiovascular diseases, cancer, type II diabetes, and obesity. According to WHO data, low fruit and vegetable intake causes some 2.7 million deaths each year and is one of the 10 risk factors contributing to mortality (WHO, 2003). In a study on food sources of nutrients in the diet of Spanish children, fruit juices and citrus fruit were shown to be the principal sources of vitamin C, accounting for 43% (Royo-Bordonada et al., 2003). Fruit juices contain a complex mixture of nutrients that are beneficial to the maintenance of good health, and they have intrinsic disease risk reduction properties. In addition to the major nutrients (e.g., vitamins, minerals) inherent in the fruit itself, juices also contain phytochemicals (often referred to as phytonutrients) derived from the fruit. The biological activity of phytochemicals has been studied in numerous in vitro and in vivo tests and in tests involving humans (Cassano et al., 2003; Duthie, 1999; Giovannucci et al., 1995; Granado et al., 1997; Hertog et al., 1994; Kurowska et al., 2000; Omaye and Zhang, 1998; Simon et al., 2001; Topuz et al., 2005; Tribble, 1998). Antioxidant activity is a common characteristic for all these bioactive compounds because of its ability to capture oxygen radicals (hydroxyl, peroxyl, superoxide, and simple oxygen), nitrogen radicals, and organic radicals (lipid hydroperoxides, and so on). Free radicals appear in tissues in situations of oxidative stress, polluted atmospheres, and so on. The accumulation of these species causes the appearance of oxidative damage in DNA, and also in proteins and lipids in cell membranes. All this damage leads to a consequent aging of tissues and the appearance of degenerative diseases (Halliwell, 1996). Lampe (1999) describes various mechanisms by which fruits and their constituents can have a protective eVect. Epidemiological studies show that consumption of fruit and vegetables has a great protective eVect against the risk of certain diseases connected with age,

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such as cancer, cardiovascular diseases, cataracts, macular degeneration, and diabetes, because these foods are rich in antioxidant vitamins such as vitamins C and E, phenolic compounds, and carotenes (Bazzano et al., 2002; Block et al., 2001; Burns et al., 2003; Epler et al., 1993; Forastier et al., 2000; Gardner et al., 2000; John et al., 2002; Lichtentha¨ler and Marx, 2005; Schieber et al., 2001; Simopoulos, 2001; Slattery et al., 2000). Some studies of supplementation with these antioxidant compounds, especially b-carotene, have produced contradictory results. Many epidemiological studies (ATBC, CARET, Women’s Health Study) with b-carotene supplements, alone or associated with vitamin E, show that they do not avoid cardiovascular diseases or some types of cancer, and they are not very recommendable for smokers (ATBC, 1994; Hennekens et al., 1996; Taylor, 1996). Nevertheless, epidemiological studies with fruits and vegetables or derivatives do show beneficial results for the health (Aviram et al., 2000; Kris-Etherton et al., 2002; Temple and Gladwin, 2003). Adapting to new trends and consumer demands is one of the primary objectives of orange juice producers. For some years, therefore, they have been producing juices with mild pasteurization, marketed in refrigerated conditions and with limited shelf life. Although conventional thermal processing ensures the safety and extends the shelf life of foods, it often leads to detrimental changes in the sensory qualities of the product (Bull et al., 2004). New products are being introduced, with juice mixtures that provide increased quality (nutritive value, color, and so on), this being the factor that most contributes to consumer acceptance and an increase in the value added to the product. As a result of the problem that has arisen because of the development of pathogens in some (unpasteurized) fresh orange juices (Ghenghesk et al., 2005; Parish, 1998a; Parish et al., 1997), the image and safety of these juices have been damaged, and the FDA is recommending manufacturers of these juices to increase safety measures by introducing the Hazard Analysis and Critical Control Points (HACCP) system in their processes and by applying a pasteurization or treatment that will ensure five decimal reductions of Escherichia coli (Food and Drug Administration, 1998). Product quality is determined by a number of parameters. Without going into detail about quality concepts, the most important attributes for a food product, in our view, are: organoleptic properties (taste, flavor, texture, appearance, and color); microbiological (absence of pathogens and microbial toxins) and toxicological safety; nutritive value; shelf life; convenience; and, last but not least, healthiness. Processing is generally done to achieve some desired results such as killing or inactivating microorganisms or inactivating enzymes and antinutritional factors. Such treatments also frequently result in undesired reactions, mostly of a chemical nature, especially during heat treatment (Van Boekel and Jongen, 1997).

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Many studies have been carried out on the quality and stability of pasteurized orange juices (Graumlich et al., 1986; Kaanane et al., 1988; Martı´n et al., 1995). But in some cases the juices are obtained from concentrates (Fellers and Carter, 1993), while in other cases (Graumlich et al., 1986; Martı´n et al., 1995) the pasteurization conditions applied are fairly intense (from 90
C, 15 s to 110
C, 15 s), anticipating storage at ambient temperature or else a very long shelf life in refrigeration. In these latter cases the impact of pasteurization on quality is clearly appreciable. In other cases, high storage temperatures have been studied in order to observe clearly the eVects of temperature on certain factors (browning, development of hydroxymethylfurfural, loss of vitamin C, and so on), and to deduce the kinetic models that define these changes (Manso et al., 2001a). And, finally, in other cases there are studies on the eVects of certain processes, processing conditions, canning, or storage, and on one or more specific quality parameters (Ayhan et al., 2001; Choi et al., 2002; Decio and Gherardi, 1992; Fan et al., 2002; Johnston and Bowling, 2002; Manso et al., 2001b; Nienaber and Shellhammer, 2001a; Parish, 1998b; Sa´nchez-Moreno et al., 2003; Trammell et al., 1986). Refrigerated juices that are not obtained from concentrates and have been subjected to mild pasteurization (75
C for 30 s) partly satisfy the requirements of the higher quality demanded by consumers. The shelf life of these juices ranges between 28 and 45 days in refrigeration and their quality approaches that of freshly squeezed juices. Newly developed food technologies usually focus on preservation while keeping food quality attributes. Therefore, the frequently used concept of ‘‘minimal processing’’ is not absolutely apt because really the principle of ‘‘as little as possible, but as much as necessary’’ is meant. Nonthermal methods allow processing of foods below the temperatures used during thermal pasteurization, so flavors, essential nutrients, and vitamins undergo minimal or no changes. Obstacles to commercialization include the lack of systematic inactivation kinetic data, the interpretation of nonlinear death kinetics, and the need to establish equivalent control measures for nonthermal treatments in comparison with traditional heat processes (Stewart et al., 2002). Foods can be nonthermally processed by irradiation, high hydrostatic pressure, antimicrobials, ultrasound, filtration, and electrical methods such as pulsed electric fields (PEFs), light pulses, and oscillating magnetic fields. As a result of technological developments, high-pressure and high-intensity PEF processing have received increased attention during the last decade (Butz and Tauscher, 2002). The main requirement that these new technologies must meet is to ensure product microbial safety while preserving sensory and nutritional characteristics so as to obtain products as similar as possible to fresh foods.

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Food preservation using high pressure is a promising technique in the food industry as it oVers numerous opportunities for developing new foods with extended shelf life, high nutritional value, and excellent organoleptic characteristics (Cheftel, 1995). High pressure is an alternative to thermal processing. Industrial applications are already known in Japan, the United States, France, and Spain. The European Commission includes products obtained by this technology in the Novel Foods group, subject to Novel Foods Legislation. After successful application and verification of this technology, the Food Standards Agency, UK, released a statement saying that the technology is not regarded as novel provided the foods are of fruit or vegetable origin, with a pH below 4.2, and germination of clostridia is prevented during shelf life (Houska et al., 2006). The first attempts to treat foods with electroimpulses were reported at the end of the 1920s in the United States. During recent years, research in this technology has been reinforced again. Various laboratory and pilot-scale treatment chambers have been designed and used for high-intensity PEF treatment of foods. Two industrial-scale systems are available, including treatment chambers and power supply equipment (Butz and Tauscher, 2002). Energy loss due to heating of foods is minimized, reducing detrimental changes in the sensory and physical properties of the foods. Microbial inactivation by PEFs has been explained by several theories. The possibility most studied is electrical breakdown and electroporation (Barbosa-Ca´novas et al., 1999; Grahl and Maerkl, 1996; Jeyamkondan et al., 1999). To study optimum treatment conditions for these new refrigerated juices, and also to guarantee the absence of microorganisms (whether pathogenic or otherwise), it is essential to know the characteristics (physicochemical and quality) of these juices and their possible storage variations in order to establish their shelf life. For all these reasons, in this chapter we propose to give details of studies that have been performed in recent years on refrigerated juices, first to ensure their safety, and second (but also importantly, as the many studies show) to ensure juices that are organoleptically as similar as possible to fresh juice and with the maximum content of bioactive compounds.

II. REVIEW A. PHYSICOCHEMICALS

Physical measurements are important because of their potential impact on sensory evaluation parameters such as mouthfeel. Viscosity, density, and amount of cloud are related to the quantity and consistency of the juice pulp.

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Pasteurization appears to have a major impact on the stability of juice cloud in orange juice concentrates because of the deactivation of pectinesterase enzymes (Chandler and Robertson, 1983). Rodrigo et al. (2003a) studied the physicochemical and quality characteristics of various refrigerated mixed orange and carrot juices, and their changes with storage time and temperature. Density, dry extract,
Brix values, acidity, turbidity, formol index, pectin methylesterase (PME), hydroxymethylfurfural, essential oils, ascorbic acid, and color varied with storage time and temperature. Some of the parameters could be used as indicators of quality loss or spoilage of the juices. Except for calcium, diacetyl index, and pulp, there were statistically significant diVerences (p < 0.05) between the juices, attributable to diVerences in the raw material and the processes used. However, the contents of ash, calcium, phosphorus, magnesium, potassium, total nitrogen, nitrates and nitrites, pH, conductivity, proline, diacetyl index, and particle size did not vary during storage, irrespective of temperature. Esteve et al. (2005) studied the physicochemical and quality characteristics of various minimally pasteurized refrigerated Spanish orange juices, and their changes with storage time and temperature. Except for pH, all the characteristics studied (essential oils, acidity, color, conductivity, density, diacetyl index, and viscosity) varied with storage time at 4 and 10
C, but density variation was not statistically significant at 4
C. In a study by Farnworth et al. (2001), Mexican orange juice bottled without pasteurization and frozen (–18
C); orange juice that was pasteurized, bottled, and frozen; and orange juice pasteurized and stored at 1
C in plastic bins were sampled monthly for 9 months. The viscosity and cloud of the orange juice stored in refrigeration for 9 months decreased significantly, whereas the density did not vary. The density of the unpasteurized orange juice was less than the density of the pasteurized juices (subsequently stored at –18 and 1
C). The cloud measurement indicated that pasteurization had a dramatic eVect on this physical property of the juice. Orange juices that were not pasteurized settled out over time, but no settling or clearing was observed for pasteurized orange juices. However, no significant changes were observed in the value of the density. The concentration of sugars (glucose, sucrose, and fructose) in the orange juice did not vary during storage, but the ºBrix increased with time. No significant diVerences in ºBrix were observed with type of treatment, although the pasteurized juices had a greater sugar content and higher ºBrix value. Sadler et al. (1992) reported that sucrose concentrations in Valencia orange juice decreased during storage at 4
C, apparently due to microbial contamination. The smallest decrease in sucrose was observed in unpasteurized orange juice. The malic acid content of the unpasteurized orange juice was significantly lower than of the pasteurized juices, and its concentration

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increased during storage. However, the authors did not find changes in the concentration of citric acid during storage or as a result of processing. Yeom et al. (2000a) compared PEF-treated orange juice (35 kV/cm, 59 ms) and heat-treated orange juice and observed that the PEF-treated juice had a smaller particle size than that of the juice pasteurized by heat. There were no diVerences between the PEF-treated juice and fresh juice. They did not observe diVerences between the
Brix and pH of the juices after treatment or during storage. Del Caro et al. (2004) did not see significant changes in the pH, acidity,
Brix, and dry matter of freshly squeezed juice of Shamouti oranges, Red Blush grapefruit, and Salustiana oranges during 15 days’ storage at 4
C. Polydera et al. (2003) treated orange juice with HHP (500 MPa at 35
C for 5 minutes). They found that the consistency index did not change significantly during storage of thermally treated orange juice, leading to an almost constant apparent viscosity. In the case of high pressurized orange juice, the consistency index increased with storage time. Higher apparent viscosity values were determined for high-pressurized orange juice compared with thermally treated orange juice immediately after processing and on each storage day. The same authors (Polydera et al., 2005a) found similar results when they treated the juice with HHP (600 MPa, 40
C, 4 minutes) and thermal pasteurization (80
C, 60 s). However, a small decrease in the consistency index, which also means a decrease in the corresponding apparent viscosity values, was observed during storage in diVerent conditions. This decrease was more pronounced in the case of high pressurized orange juice, while the consistency index of thermally treated juice did not change significantly with storage time. Bull et al. (2004) compared the quality and shelf life of high-pressureprocessed (600 MPa, 20
C, 60 s) Valencia and Navel orange juices with fresh juice and thermally pasteurized juice (65
C, 1 minute), and their subsequent storage at 4 and 10
C for 12 weeks. For both juice types, the pH,
Brix, viscosity, titratable acid content, and alcohol insoluble solids of the pressure or thermally treated juices were not significantly diVerent from fresh, untreated juices. The parameters did not change significantly over storage time. Clarification (cloud loss) occurred in all treatments, but no diVerence was found between treatments. The degree of clarification increased significantly over time across all treatments. Similar results were obtained by other authors (Goodner et al., 1999; Parish, 1998a), who required treatments of at least 700 or 500 MPa/60
C to obtain cloud stable juices. Fiore et al. (2005) compared diVerent juices purchased in the market and found that pH was slightly lower in sterilized (long shelf life) orange juices (2.66–3.20) than in pasteurized and refrigerated orange juices (3.12–3.34). Garde-Cerdan et al. (2007) studied the eVect of thermal and PEF treatments on various physicochemical properties of Parellada grape juice.

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No significant changes were noticed in the physicochemical properties measured such as reducing sugar content, total acidity, and pH. B. NONENZYMATIC BROWNING

The control of furanic aldehydes is important in the evaluation of nonenzymatic browning, adulterations, heating, incorrect storage, and sensory characteristics of food. Rodrigo et al. (2003a) and Esteve et al. (2005) observed an increase in 5-hydroxymethyl-furaldehyde (5-HMF) during storage (up to 6 weeks) of mixed orange and carrot juice and orange juice, respectively. The increase was greater at 10
C than at 4
C. Fan (2005a) investigated the formation of furan from sugars, ascorbic acid, and organic acids aVected by ionizing radiation and thermal treatments. The results showed that both thermal treatments and irradiation induced formation of furan from ascorbic acid, fructose, sucrose, or glucose. Little furan was produced from malic acid or citric acid. The pH and concentration of sugars and ascorbic acid solutions had strong influences on furan formation due to either irradiation or thermal treatment. The rate of irradiation-induced furan formation increased with decreasing pH from 8 to 3. Approximately 1600 times less furan was formed at pH 8 than at pH 3. At the same pHs, the amounts of furan formed from irradiation of ascorbic acid, fructose, and sucrose were always higher than that from glucose. As the pH decreased from 7 to 3, an increase in thermally induced furan was observed for sucrose and ascorbic acid solutions; for glucose solution, however, less furan was formed at pH 3 than at pH 7. The levels of sugars commonly found in fruits and fruit juices would, on irradiation, be high enough potentially to produce low parts per billion (ppb) levels of furan. The concentration of ascorbic acid at which a maximum of furan was produced on irradiation was about 0.5 mg/ml, a level commonly found in some foods. Five furan derivatives were tentatively identified in thermally treated ascorbic acid solution, while one furan derivative was tentatively found in both irradiated and thermally treated samples. The same author (Fan, 2005b) studied the formation of furan in freshly prepared apple and orange juices aVected by ionizing radiation and thermal treatments, using a newly developed solid-phase microextraction method coupled with gas chromatography–mass spectrometry (GC–MS). The results showed that furan levels increased linearly as the radiation dose increased from 0 to 5 kGy. Irradiation induced more furan in apple juice than in orange juice. During postirradiation storage at 4
C, furan levels increased in both apple and orange juices, particularly in the first 3 days. On the other hand, irradiation degraded deuterated furan (4-furan) spiked in water and fruit juices. The rate of degradation as a function of radiation dose was highest in water and lowest in orange juice. Submerging

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the juice samples in boiling water for 5 minutes induced higher amounts of furan in orange juice than in apple juice, but autoclaving (121
C, 25 minutes) resulted in more furan formation in apple juice than in orange juice. The results reported suggest that both ionizing radiation and thermal treatments induce furan formation in fruit juices. Yeom et al. (2000a) observed a linear increase in the browning index in PEF-treated orange juice (35 kV/cm, 59 ms) and pasteurized juice (94.6
C, 30 s) after a storage period of 28 days at 4
C. The browning index was lower in the PEF-treated juice than in the heat-treated juice during storage at 4
C, although no diVerences were observed when the juices were stored at 22
C. Roig et al. (1999) studied the occurrence of nonenzymatic browning during storage of freshly produced commercial citrus juice, aseptically filled in TetraBrik cartons. The rate of browning of the samples was directly related to temperature (room temperature and 5
C). Although formation of 5-HMF has been detected in degraded juice samples, its presence could not be used as an index of browning. 5-HMF has been found to be unreactive in the browning process in citrus juices and its contribution to browning in products of this type is insignificant if not negligible. Increasing the L-ascorbic acid (added as an antioxidant) concentration extends the nutritional value of the products but also increases the severity of browning. Bull et al. (2004) did not find significant diVerences in the browning index of high-pressure-processed (600 MPa, 20
C, 60 s) Valencia and Navel orange juices, fresh juice, and thermally pasteurized juice (65
C, 1 minute). The significant increase in the browning index seen over time was observed across all treatments. C. FATTY ACIDS AND FREE AMINO ACIDS

Although the formol index is not specific, it is used to estimate the total content of amino acids in a juice. Rodrigo et al. (2003a) and Esteve et al. (2005) determined the formol index of refrigerated (mild pasteurization, 77
C for 20 s) mixed orange and carrot juice and orange juice, respectively, and their evolution during storage for 6 weeks at 4 and 10
C. In both cases, the formol index decreased with storage time and temperature. This decrease might be due to consumption of amino acids by microorganisms responsible for the start of fermentation of the juices. Kaanane et al. (1988) observed that the formol index of pasteurized orange juices did not vary during storage. However, when Trifiro` et al. (1995) studied the eVect of storage time and temperature on the quality of fresh orange juices, they observed an increase in the formol index which they attributed to a proteolysis eVect, and they found that it was related to the origin of the juice and storage temperature. Villamiel et al. (1998) studied the influence of heat treatment (conventional and microwaves) on orange juice and reported that there were no diVerences

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in the content of amino acids between the untreated juices and the juice treated by microwaves, but they observed a decrease in some amino acids in the juice treated conventionally. Garde-Cerdan et al. (2007) studied common thermal and PEF treatments to assess their eVect on fatty acid and free amino acid contents of Parellada grape juice. These compounds are of great importance in winemaking as nutritive compounds for yeast growth. Neither thermal nor PEF treatments modified the total content of fatty acids and free amino acids in Parellada grape juice. However, the concentration of lauric acid diminished after PEF processing, and the concentration of some amino acids varied after both treatments. Lipids and nitrogen compounds play an important role in the fermentative steps of winemaking. Fatty acids and sterols have a great influence on the growth of fermentative yeast and thus on the development of alcoholic fermentation. Zulueta et al. (2007) evaluated the effect of HIPEF treatment on various physicochemical properties and fatty acid profile changes of the orange juice– milk beverage. After HIPEF treatment, nonsignificant changes in the contents of saturated fatty acids, monounsaturated fatty acids, or polyunsaturated fatty acids were observed, only a small reduction in fat content (p, 0.05) was found. D. AROMA AND FLAVOR

The flavor of orange juice is easily altered during processing and storage. Irreversible changes are produced in the flavor of the juice as a result of chemical reactions that are initiated or occur during thermal processing (Braddock, 1999). The changes in flavor are also associated with a number of deteriorative reactions that take place during storage, giving rise to the development of oVflavor. Nonenzymatic browning such as ascorbic acid degradation causes deterioration of flavor as well as loss of nutrients and darkening (Kaanane et al., 1988). Jordan et al. (2003) performed a comparative study between the aromatic profile of fresh orange juice versus deaerated and pasteurized juices. At the qualitative level, all the volatile components in the fresh orange juice were also found in the counterparts after deaeration and pasteurization processes. According to statistical analyses, significant losses in the concentration of volatile components occurred during the deaeration process, while there were no statistically significant diVerences between the concentrations of volatile components in the deaerated and pasteurized juices. The results show that during industrial processing of orange juice the biggest losses in the concentration of volatile components occur during deaeration. The pasteurization process does not significantly change the analytical composition of deaerated orange juice for any of the 42 quantitated volatile compounds (alcohols, aldehydes, esters, ketones, and terpenic hydrocarbons). In a study performed by Butz and Tauscher (2002), in high-pressureprocessed orange juice the changes in aroma and flavor and general quality

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after 21 days’ storage were imperceptible. However, Baxter et al. (2005) found that the odor and flavor of the HPP juice was acceptable to consumers after storage for 12 weeks at temperatures up to 10
C. Farnworth et al. (2001) found that in Mexican orange juice the concentrations of acetaldehyde and ethyl acetate were higher in unpasteurized juice. a-Pinene, b-myrcene, limonene, a-terpineol, 1-hexanol, 3-hexen-1-ol, and sabinene concentrations were higher in the unpasteurized juice than in the pasteurized juice. As storage time increased, PEF-treated orange juice showed a significantly higher content of flavor compounds than heat-pasteurized orange juice during storage at 4
C (Yeom et al., 2000a). Polydera et al. (2003, 2005a) found that high-pressure processing resulted in better retention of the flavor of untreated juice and superior sensory characteristics compared with thermal pasteurization. E. VITAMIN C

Vitamin retention studies to assess the eVects of food processing on the nutritive value of foods are of great importance to food technologists and consumers. Vitamin C is thermolabile and therefore in fruit and vegetables it provides an indication of the loss of other vitamins and acts as a valid criterion for other organoleptic or nutritional components such as natural pigments and aromatic substances. Its concentration decreases during storage, depending on storage conditions such as temperature, oxygen content, and light (Alwazeer et al., 2003; Blasco et al., 2004; Esteve et al., 1996; Polydera et al., 2003; Zerdin et al., 2003). Kabasakalis et al. (2000) studied the ascorbic acid content of commercial juices and its loss with storage time and temperature. The juices that they analyzed were divided into three groups: long-life commercial fruit juices without preservatives (100% orange; 100% grapefruit; 100% cocktail of orange, peach, grapefruit, pineapple, apple, mango, kiwi; 17% lemon; 50% cocktail of apple, orange, apricot, peach, grapefruit, pineapple); short-life commercial fruit juices (refrigerated) without preservatives (100% orange, 9% lemon); and fresh fruit juice (orange). A loss of ascorbic acid was observed in short-life 100% orange juice as the expiration date approached (42.7-mg ascorbic acid/100 ml at 26 days before expiration and 38.9-mg ascorbic acid/100 ml at 8 days before expiration). Loss of ascorbic acid in various commercial fruit juices stored in closed containers for a period of 4 months at room temperature ranged between 29% and 41%. When the containers were opened for consumption and then stored in the refrigerator for 31 days, commercial 100% orange juice lost 60–67% of its ascorbic acid, whereas under the same conditions ascorbic losses in fresh orange juice were much lower (7–13%).

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Farnworth et al. (2001) also obtained similar results. They studied Mexican orange juice bottled without pasteurization and frozen, orange juice that was pasteurized, bottled, and frozen, and orange juice pasteurized and stored at 1
C in plastic bins, sampled monthly for 9 months. The concentration of ascorbic acid was aVected by the method of production. The amount of ascorbic acid diminished as storage time increased. Vitamin C was evaluated by Gil-Izquierdo et al. (2002) in orange juices manufactured by diVerent techniques (squeezing, mild pasteurization, standard pasteurization, concentration, and freezing). They found that mild and standard pasteurization slightly increased the total vitamin C content and the contribution from the orange solids parts, whereas concentration and freezing did not produce significant changes. Rodrigo et al. (2003a) determined the concentration of ascorbic acid in various refrigerated mixed orange and carrot juices, and their changes with storage time (for 5 weeks) and temperature (4 and 10
C). They calculated the mean life of the juices on the basis of the vitamin C concentration, obtaining a period of 32 and 43 days at 10 and 4
C, respectively. This would ensure that if the storage temperature increased the juice would conserve its nutritive characteristics during its shelf life. Del Caro et al. (2004) stored squeezed juices of various species and cultivars (Red Blush grapefruit, Salustiana, and Shamouti oranges) for 15 days at 4
C and only in the Salustiana orange juice did they observe a significant decrease (13%) in the vitamin C concentration. Esteve et al. (1996) found a decrease of 5% in the concentration in freshly squeezed orange juice after 7 days’ storage at 4
C. Trifiro` et al. (1995) also reported a maximum decrease of 8% in the ascorbic acid concentration in fresh blood orange juice stored at 3
C, although the juice was pasteurized and stored for 30 days. Vikram et al. (2005) studied the status of vitamin C during thermal treatment of orange juice heated by diVerent methods (conventional heating, electromagnetic processing including infrared, ohmic heating, and microwave heating) and at diVerent treatment temperatures (50, 60, 75, and 90
C) and times (0–15 minutes). The degradation kinetics of vitamin C in terms of reaction rate constant, destruction kinetics, enthalpy, and entropy for the diVerent heating methods were discussed. The destruction of vitamin C was influenced by the heating method and the processing temperature. The degradation was highest during microwave heating, owing to the uncontrolled temperature generated during processing. Of the four methods studied, ohmic heating gave the best result, facilitating better vitamin retention at all temperatures. The activation energies for both vitamin and color were within the range of the literature values, 7.54–125.6 kJ/mol. The activation enthalpies agreed with the literature values of vitamin destruction in other food products. The z-values were also within the literature values of 20–30
C for vitamin destruction, except for microwave heating.

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Choi et al. (2002) studied the retention of ascorbic acid with storage in blood oranges and observed a linear reduction in concentration with time. Fan et al. (2002) also observed a linear degradation of ascorbic acid in orange juice with time, whether irradiated or not. Alwazeer et al. (2003) studied the eVect of both redox potential (Eh) and pasteurization of orange juice on its stability during storage at 15
C for 7 weeks. Gassing the juice with N2 or N2–H2 increased color retention and ascorbic acid stability. The study showed that the juice must be reduced just after heat treatment in order to stabilize color and ascorbic acid during storage. The eVects of storage temperature and time on the stability of PEF and thermally pasteurized orange juice were studied by Yeom et al. (2000a). PEFtreated orange juice retained a higher ascorbic acid content than that of heat-pasteurized orange juice during storage at 4
C. PEF-treated orange juice showed no diVerence in ascorbic acid concentration compared with heat-pasteurized orange juice during storage at 22
C. Polydera et al. (2003, 2005a,b) studied the eVect of HPP treatment (500 MPa, 35
C, 5 minutes or 600 MPa, 40
C, 4 minutes) and thermal pasteurization (80
C, 30–60 s) on orange juice and its subsequent storage (0–30
C, 1–3 months). In all cases, the ascorbic acid degradation rates were lower for high pressurized juice, leading to an extension of its shelf life compared with conventionally pasteurized juice. The shelf life of the HAPtreated juice (based on ascorbic acid retention) was greater than that of pasteurized juice. However, Bull et al. (2004) did not find significant diVerences between HAP-treated juice (600 MPa, 20
C, 60 s), pasteurized juice (65
C, 1 minute), and fresh juice. Nevertheless, they found a decrease in ascorbic acid concentration in all the juices with storage time, irrespective of the treatment applied and storage temperature (4 and 10
C). Fiore et al. (2005) did not find diVerences in vitamin C content (38.9–89.0 mg/100 ml) between sterilized (long shelf life) orange juices and pasteurized (refrigerated) orange juices. Esteve et al. (2005) studied nutritional characteristics of orange juices that can be found on the market and their evolution with time (1–6 weeks), and storage temperature in refrigeration (4 and 10
C). The ascorbic acid content of the juices decreased during storage, faster at 10 than at 4
C. The shelf life of the juices, based on 50% of the initial ascorbic acid concentration, was 42 days at 4
C and 35 days at 10
C. Torregrosa et al. (2006) compared the shelf life of a PEF-treated mixture of orange and carrot juice with a heat-treated juice (98
C, 21 s), kept in refrigerated storage at 2 and 10
C. The concentration of ascorbic acid remaining in the pasteurized orange–carrot juice was 83%, whereas in the PEF-treated juice it was 90%. The ascorbic acid degradation rate in the juice stored at 2
C was less than in the juice stored at 10
C, and in the pasteurized juice it was greater.

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F. CAROTENOIDS/VITAMIN A

Although they can easily be degraded, carotenoids can be retained during industrial processing if good technological practices are observed. Processing at lower temperatures and for the shortest possible treatment times is recommended. Retention of provitamin A is favored during storage at low temperatures, protected from the light, with the exclusion of oxygen and the presence of antioxidants. Rodriguez-Amaya (1993) reviewed the susceptibility or resistance to degradation of carotenoids during storage, performing a detailed analysis of the eVects of factors such as carotenoid structure, nature of the matrix, available oxygen, moisture content/water activity, light, temperature, antioxidants, pro-oxidants, fatty acids, sulfites, and sodium chloride in models and foods. In 1997, the same author made an exhaustive review of the influence of manipulation of foods on carotenoids, analyzing the retention of provitamin A carotenoids in prepared, processed, and stored foods (Rodriguez-Amaya, 1997). Lee and Coates (2003) studied changes in carotenoid pigments as a result of thermal pasteurization of Valencia orange juices. Total carotenoid pigment content loss was significant after thermal pasteurization at 90
C for 30 s. Thermal eVects on carotenoid pigment contents, especially violaxanthin (46.4%) and antheraxanthin (24.8%), were clearly observed. With the loss of violaxanthin and antheraxanthin, lutein became the major carotenoid, followed by zeaxanthin, in pasteurized Valencia orange juice. EVects of high-pressure treatment on orange juice carotenoids (b-carotene, a-carotene, zeaxanthin, lutein, and b-cryptoxanthin) associated with nutritional (vitamin A) values were investigated by De Ancos et al. (2002). Various high-pressure treatments (50–350 MPa) combined with diVerent temperatures (30 and 60
C) and treatment times (2.5, 5, and 15 minutes) were assayed. The juice was subsequently stored at 4
C. The authors found that high-pressure treatments at 350 MPa produced significant increases of 20–43% in the carotenoid content of fresh orange juice (from 3.99 to 4.78–5.70 mg/liter). In the treatment at 350 MPa/30
C/5 minutes, they observed an increase in the vitamin A value from 164 to 238 RE/liter (45%). During storage of the orange juice subjected to high pressures, it was better preserved and even increased its total content of carotenoids and vitamin A activity. The authors indicated, therefore, that high-pressure treatment might be an eYcient processing method for preserving orange juice as freshly squeezed for up to 30 days from the point of view of sensory (carotenoids) and nutritional (vitamin A) quality. However, when Bull et al. (2004) studied high-pressure-processed (600 MPa, 20
C, 60 s) Valencia and Navel orange juices, thermally pasteurized juice (65
C, 1 minute), and fresh juice, they did not find changes in the b-carotene concentration. They also observed no significant variations during storage at 4 and 10
C (12 weeks).

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Torregrosa et al. (2005) studied the eVect of pulse treatment on carotenoids in an orange–carrot mixture (80:20, v/v), using diVerent field intensities (25, 30, 35, and 40 kV/cm) and treatment times (30–340 ms). In parallel, a convectional heat treatment (98
C, 21 s) was applied to the juice, and the results were compared. Of all the carotenoids studied, only five decreased significantly: 9-cis-violaxanthin þ neoxanthin, antheraxanthin, a-cryptoxanthin, 9-cis-a-carotene, and 9-cis-b-carotene; the rest increased significantly, with the exception of lutein, mutatoxanthin, b-carotene, and x-carotene. The largest increase was in the concentration of 13-cis-b-carotene, followed by zeaxanthin and cis-b-cryptoxanthin. The decrease in violaxanthin þ neoxanthin and antheraxanthin after pasteurization coincided with an increase in mutatoxanthin. Vitamin A increased in the orange–carrot juice in comparison with the untreated juice. When the juice was treated with pulses the authors observed that the concentrations of the 9-cis-violaxanthin þ neoxanthin mixture, antheraxanthin, cis-b-cryptoxanthin, and 9-cis-acarotene increased with treatment time, and that the rate of formation of those carotenoids increased with treatment intensity. They concluded that PEF processing generally caused a significant increase in concentrations of the various carotenoids identified in the orange–carrot mixture as treatment time increased, whereas when conventional pasteurization was used to process the juice, the concentrations of most of the carotenoids decreased or else showed a nonsignificant increase. With PEF treatment of the orange–carrot mixture at 25 and 30 kV/cm, it was possible to obtain a vitamin A concentration higher than that found in the pasteurized juice. Corte´s et al. (2006a) studied the eVect of pasteurization and PEF treatment on carotenoids in orange juice. In their study, they found that PEF processing generally caused an increase in the concentrations of the carotenoids identified as treatment time increased. The decrease in the concentrations of carotenoids with provitamin A activity was very small, although it always decreased in comparison with untreated fresh juice. The concentration of total carotenoids decreased by 12.6% in the pasteurized orange juice in comparison with untreated fresh orange juice, as opposed to decreases of 9.6%, 6.3%, or 7.8% when fields of 25, 30, or 40 kV/cm were applied. The same authors (Corte´s et al., 2006b) subsequently compared the evolution and modification of various carotenoids and vitamin A in untreated orange juice, pasteurized orange juice (90
C, 20 s), and orange juice processed with high-intensity pulsed electric fields (HIPEF) (30 kV/cm, 100 ms) during 7 weeks of storage at 2 and 10
C. The concentration of total carotenoids in the untreated juice decreased by 12.6% when the juice was pasteurized, whereas the decrease was only 6.7% when the juice was treated with HIPEF. Vitamin A was greatest in the untreated orange juice, followed by HIPEF-treated

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orange juice (decrease of 7.52%), and, finally, pasteurized orange juice (decrease of 15.62%). The decrease in the concentrations of total carotenoids and vitamin A during storage in refrigeration was greater in the untreated orange juice and the pasteurized juice than in the HIPEF-treated juice. During storage at 10
C, auroxanthin formed in the untreated juice and the HIPEF-treated juice. This carotenoid is a degradation product of violaxanthin. The concentration of antheraxanthin decreased during storage and it was converted into mutatoxanthin, except in the untreated and pasteurized orange juices stored at 2
C. From the results obtained the authors concluded that nonthermal treatments had less eVect than conventional thermal treatments on concentrations of total carotenoids and vitamin A in refrigerated orange juice. With HIPEF treatment there was no significant decrease in the concentration of any carotenoid in comparison with the untreated juice. During storage in refrigeration, total carotenoids and vitamin A were maintained for longer in the juice treated with HIPEF than in the juice conserved using conventional pasteurization treatments. G. ANTHOCYANINS/FLAVONOIDS

Anthocyanins are included in the list of natural compounds known to work as powerful antioxidants. Blood orange anthocyanins are not very stable: during thermal treatment and storage they can degrade and form colorless or undesirable brown-colored compounds; the juice loses its bright red color and gains a brown color (Maccarone et al., 1985). Gil-Izquierdo et al. (2002) determined the eVect of individual orange juiceprocessing techniques at industrial scale (pasteurization, concentration, and freezing) on phenolic compounds, and the eVect of pasteurization on the pulp added to the final juice. In pulp, pasteurization led to degradation of several phenolic compounds, that is, caVeic acid derivatives, vicenin 2, and narirutin, with losses of 34.5%, 30.7%, and 28%, respectively. Flavonones were the major phenolic compounds in orange juice (narirutin, hesperidin, and didymin). Therefore, these flavonones were stable in the whole juice at pasteurization temperatures. Mild and standard pasteurization techniques did not show changes in the total content of phenolics in either the soluble fraction or the cloud fraction. No important changes were observed during the juice concentration process. In the case of the freezing technique, there was a dramatic decrease in phenolic compounds in comparison with the contents before this process (loss of 35%). Del Caro et al. (2004) stored squeezed juices of various species and cultivars (Red Blush grapefruit, Salustiana, and Shamouti oranges) for 15 days at 4
C. They found a decrease in the amount of single flavonoids (narirutin,

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hesperidin, and didymin), and therefore in the total flavonoid content in the orange juices. Grapefruit juice showed significant diVerences only for narirutin, hesperidin, and total content, whereas they did not observe variations in didymin, naringin, neohesperidin, and poncirin. Fiore et al. (2005) studied the anthocyanin contents in pasteurized pure orange juice with 40 days of shelf life, and a sterilized beverage containing from 25% to 35% of concentrated orange juice, with a long shelf life (1 year). In the refrigerated juices, the major compounds were cyanidin glucoside and cyanidin 3-(600 -malonylglucoside). The concentration of anthocyanins found in the refrigerated juices was similar to those found in fresh orange juices, indicating that the pasteurization treatment and storage conditions applied to this type of commercial sample do not damage anthocyanins. In the sterilized juice almost no anthocyanins were detected, which was partly due to the lower percentage of fruit juice present in the beverage and also to the severe degradation of the anthocyanins. Kirca and Cemeroglu (2003) showed that losses of anthocyanins were 14.4%, 21.5%, and 60.9% after heating for 120 minutes at 70, 80, and 90
C, respectively. They also confirmed that anthocyanin levels decreased very rapidly for samples stored at 37 and 20
C, whereas samples stored at 5
C showed a remarkably slower degradation. Choi et al. (2002) pasteurized orange juice at 90
C for 30 s and observed a decrease of 25% in the total anthocyanin contents after 7 weeks’ storage at 4.5
C. Vanamala et al. (2006) studied the variation in the bioactive flavonoid contents in 12 made-from-concentrate (MFC) orange juices, 14 pasteurized not-from-concentrate (NFC) orange juices, and 5 NFC grapefruit juices. The results obtained showed that the total flavonoid content of the MFC orange juices (53 mg/100 ml) was significantly higher than that of the NFC orange juices (36 mg/100 ml). Hesperidin was found to be the major flavonoid, followed by narirutin and didymin in orange juice. Naringin, narirutin, and poncirin were the major flavonoids in all brands of grapefruit juices. The concentration of didymin was considerably higher in the NFC orange juices than in the MFC orange juices. H. ANTIOXIDANT ACTIVITY

Fruits and vegetables contain many antioxidant compounds, especially ascorbic acid, phenolic compounds, thiols, carotenoids, and tocopherols. Polydera et al. (2005b) studied the total antioxidant activity of highpressure-processed fresh Navel orange juice (600 MPa, 40
C, 4 minutes) compared with thermally pasteurized fresh Navel orange juice (80
C, 60 s) as a function of storage in diVerent isothermal conditions (0–30
C). They also evaluated the contribution of ascorbic acid (among other antioxidant

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compounds of orange juice) to the total antioxidant activity. The reaction rate constant of nth order kinetics of the decolorization of ABTS radical cation solution, after addition of orange juice, was used as a measure of the total antioxidant activity. A mathematical description of this reaction rate constant as a function of storage temperature and time was established. Total antioxidant activity of both juices decreased during storage, mainly owing to ascorbic acid loss. High-pressure treatment led to a better retention of the antioxidant activity of orange juice compared with conventional pasteurization. Piga et al. (2002) evaluated the evolution of the ascorbic acid concentration and overall antioxidant properties of the water-soluble fraction of minimally processed mandarin juice during storage at 4
C for 4, 8, and 12 days. The evolution of the antioxidant properties as aVected by processing and storage conditions was not entirely related to ascorbic acid changes. The mandarin juices showed good retention of the original antioxidant activity at the end of storage. Gil-Izquierdo et al. (2002) found that mild pasteurization, standard pasteurization, concentration, and freezing of orange juices did not aVect the total antioxidant capacity of the juice, but they did aVect it in pulp, where it was reduced by 47%. In their study, they also found that ascorbic acid provided at least 77% of the antioxidant capacity, whereas the contribution of phenolic compounds was comparatively irrelevant. De Ancos et al. (2002) studied the eVect of high-pressure treatments (50–350 MPa) combined with diVerent temperatures (30 and 60
C) and times (2.5, 5, and 15 minutes) on the antioxidant capacity of orange juice, measured as free radical-scavenging capacity. During storage of the highpressure-treated juice at 4
C, a decrease in the free radical-scavenging capacity of the untreated and high-pressure-treated orange juices was observed. There were significant diVerences between the untreated sample (37.5% inhibition) and the orange juices treated at 350 MPa/30
C for diVerent treatment times (2.5, 5, and 15 minutes), with approximately 20% inhibition. They did not find a correlation between the carotenoid concentrations and free radical-scavenging capacity. Lo Scalzo et al. (2004) studied the eVect of thermal treatment on the antioxidant activity and antiradical activity of blood orange juice. The samples were processed in diVerent ways: blanching at 80
C for 6 minutes before squeezing; pasteurizing the juice at 80
C for 1 minute; and blanching the fruit, then squeezing it, and pasteurizing the juice. The results obtained showed that the inhibition of enzymatically mediated linolenic acid peroxidation was  increased by thermal treatments, while the scavenging eVects toward OH ,  generated by Fenton reaction, and DPPH , decreased. The authors indicated

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that the first point was sustained by the amounts of some phenolic substances with antioxidant action (anthocyanins and hydroxycinnamates). It was evident that the thermal treatments induced a decrease in the free radicalscavenging activity and were also responsible for the degradation of ascorbic acid in the blood orange juice. During storage of orange juice (Salustiana and Shamouti oranges) and Red Blush grapefruit juice at 4
C for 15 days, Del Caro et al. (2004) found that the antioxidant capacity increased significantly in the Red Blush grapefruit juice, decreased in Salustiana orange juice, and did not change in Shamouti orange juice. They found that the antioxidant capacity of the juices was significantly correlated with the ascorbic acid content rather than with the presence of flavonone glycosides. Fiore et al. (2005) studied two orange-based products: pasteurized pure juice with 40 days of shelf life (refrigerated), and a sterilized beverage containing a minimum 12% of fruit juice concentrate. All the assays gave clearly diVerent values for the two groups of juice, with the refrigerated juice having greater antioxidant power than the sterilized juice. The total concentration of anthocyanin was positively correlated with ABTS (2,20 -azino-bis (3-ethyl benzthiazoline-6-sulfonic acid)) values and, of course, with the cyanidin glucoside content. I. COLOR

The bright color of citrus juices is one of the important quality factors in citrus products. Detrimental changes in color, primarily caused by nonenzymatic browning, reduce consumer acceptance of citrus juices (Klim and Nagy, 1988). Storage can also cause an alteration in the color of juice because the action of heat, air, and light cause carotenoids to suVer oxidation, cis/trans changes, and changes in epoxide rings, with alteration of color. Rodrigo et al. (2003a) studied the color of various refrigerated mixtures of orange and carrot juice (mild pasteurization, 77
C, 20 s) and their stability over time (1–6 weeks). The juices were studied at an optimal storage temperature of 4
C and a suboptimal storage temperature of 10
C. The color variations at 4
C were minimal (nonsignificant diVerences). At 10
C, no variations were seen in a* (red to green color), but there was a reduction in the yellow component (b*, yellow to blue color), a decrease in hue which was translated into a color that was more red and less yellow. Lee and Coates (2003) studied the changes in color due to thermal pasteurization (90
C, 30 s) of Valencia orange juices. There was a perceptible color change after pasteurization of the juice, which led to the juice color becoming lighter and more saturated. Decreases in CIE a* value and increases in CIE L* (brightness), b*, h* (hue angle), and C* [chroma, (a*2 þ b*2)1/2] were the

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major color changes after pasteurization. Overall increases in reflected light might also influence the perception of color to a great extent in pasteurized orange juice. The total color diVerence (
E* ¼
L*2 þ
a*2 þ
b*2) compared with the fresh juice was 2.92  0.97 (p < 0.05). Fiore et al. (2005) studied sterilized (long shelf life) orange juices, and pasteurized and refrigerated orange juices purchased in the market. Measurement of the color density and polymeric color gave an immediate distinction between the two groups of samples. In the refrigerated orange juices, the color density was higher and the percentage of polymeric color was considerably lower than in the long shelf life orange juices. This clearly indicates that the color of sterilized orange juice is mainly due to the presence of chemical colorant added. Vikram et al. (2005) studied changes in visual color (as an index of carotenoids) during thermal treatment of orange juice heated by conventional, infrared, ohmic, and microwave heating at various treatment temperatures (50, 60, 75, and 90
C) and times (0–15 minutes). The degradation of visual color was expressed by the combined (a  b) values. The results obtained confirmed the influence of temperature on the degradation of color. The highest activation energy value corresponded to ohmic heating, followed by infrared, and it was lowest for microwave. Higher activation energy implies that a smaller temperature change is needed to degrade color more rapidly. The thermal resistance (Z) and activation energy values indicated that, with microwave heating, color degradation requires a higher temperature. Esteve et al. (2005) studied various refrigerated orange juices purchased in the market and evaluated color variation during storage at 4 and 10
C for 6 weeks. During storage at 4
C, there were slight decreases in L* and variations in a* and b* which were not significant in any of the three indices. With storage at 10
C there was a significant increase in L* compared with the initial value, and there was also a significant increase in a and significant reductions in b* in the juices. The color evaluations were always higher in the samples stored at 4
C than in those stored at 10
C. Ibarz et al. (2005) presented UV–visible irradiation as a possible process for destruction of the polymeric compounds (melanoidins) present in fruit derivative juices. In order to study the process, apple, peach, and lemon juices with diVerent soluble solid contents and diVerent browning degrees were irradiated using a lamp that emitted in the UV–visible irradiative spectrum. The data obtained showed an increase in the brightness of the juices with irradiation time. It was possible to describe the increase by means of a first order kinetics. The authors found that the colorimetric parameters a* and b* both decreased with irradiation time, indicating that the eVect that produced the irradiation was contrary to the browning process. An increase in soluble solids and in higher colored polymer contents led to a smaller percentage decrease in the colorimetric parameters.

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When they studied the eVect of PEF and heat treatment on the color of orange juice, and its subsequent storage at 4 and 10
C, Yeom et al. (2000a) observed that the PEF-treated orange juice had higher brightness (L), and higher hue angle values than the heat-pasteurized orange juice during storage at 4
C, whereas they saw no diVerences when the juice was stored at 22
C. Corte´s et al. (2006a) found that HIPEF-treated and pasteurized orange juices showed a greater tendency to yellow color and a lesser tendency to red compared with untreated orange juice, although this tendency was greater in the pasteurized juice. Polydera et al. (2003) found that color measurements of orange juice stored in laminated flexible pouches indicated that, although the color changed slightly with storage time (1–2 months), the change did not correlate with the type of processing (500 MPa at 35
C and thermal pasteurization at 80
C for 60 s) and storage temperature (0–15
C). The same authors (Polydera et al., 2005a) subsequently studied a high-pressure treatment of 600 MPa at 40
C for 4 minutes and postprocessing storage of fresh orange juice at 0–30
C compared with conventional thermal pasteurization (80
C, 60 s). HPP treatment led to lower rates of color change compared with thermal pasteurization at all the storage temperatures studied, except at 30
C (which is above the range of normal storage temperatures). An increase in storage temperature resulted in higher rates of browning of the orange juice. Similar results to those found in these studies were obtained by Bull et al. (2004) when they studied high-pressure processed (600 MPa, 20
C, 60 s) Valencia and Navel orange juices and compared them with thermally pasteurized juice (65
C, 1 minute) and fresh juice, and stored them at 4 and 10
C for 12 weeks. In comparison with untreated orange juice, HPP or thermal treatment had no eVect on the color of the juices. The results showed that there was an increase in the total color diVerence with time, regardless of the treatment. J. PECTINESTERASES

Collet et al. (2005) stated that the study of pectinesterase inactivation behavior is important because pectinesterase is responsible for juice cloud stability loss, is composed of several isoenzymes, and occurs naturally in orange. Freshly squeezed juice of Pera orange (Citrus sinensis) was pasteurized at temperatures of 82.5, 85.0, and 87.5
C. At least five runs with diVerent holding times were performed for each temperature. As the isothermal curves obtained showed deviations from the expected first-order kinetics, the data was statistically treated by applying a nonlinear regression, and the estimated best fit was a three-parameter-multicomponent-first-order model. At 82.5
C, the isothermal curves showed a nonzero asymptote of inactivation, indicating that at this temperature the most heat-resistant

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isoenzyme could not be totally inactivated. The 87.5
C isotherm showed the highest inactivation among the temperatures studied. These observations agree with the batch inactivation data found in the literature, but the holding time required for a satisfactory inactivation was significantly shorter than the time found in the literature, suggesting that the proposed model can be used to design continuous processes with more accuracy. Ingallinera et al. (2005) compared total pectinesterase activity of Sicilian blood oranges (Sanguinello, Moro, and Tarocco) with the blonde Navel cultivar, checking enzyme stability with various pasteurization time and temperature (70–85
C) conditions in order to optimize the heat treatment and increase the shelf life of the pasteurized juice. To do this they stored the juices at 4, 15, and 25
C for times ranging between 10 minutes and 50 days. Decimal reduction time and temperature (D and z) and the kinetic constant (k) were established to optimize and increase the shelf life of the pasteurized juice. Finally, a heat treatment (85
C  3 minutes) of both microbiological and enzymatic eYcacy was developed that does not compromise anthocyanin stability. Heat pasteurization of orange juice is designed to inactivate PME, which is more heat resistant than vegetative microorganisms (Chen and Wu, 1998). PME exhibits greater heat and pressure resistance than common orange juice spoilage microorganisms and can thus be used as a processing index for both HHP and thermal processes (Goodner et al., 1998; Parish, 1998c; Versteeg et al., 1980). Quoc et al. (2006) studied the development of a process that permitted quick inactivation of PME, which is present in cloudy or unclarified apple juice. This enzyme is responsible for opalescence instability. In order to achieve this objective, acidification of the apple juice to pH 2.0 was performed by electrodialysis, followed by mild heat treatment at temperatures of 40, 45, and 50
C for 0–60 minutes. Opalescence of the adjusted juice was more stable than for an untreated cloudy apple juice when stored at 4
C for 3 months. Bayindirli et al. (2006) studied the eVectiveness of treatment on pectinesterase activity in orange juice, comparing the application of high hydrostatic pressure with a mild heat treatment. The residual pectinesterase activity in the orange juice after treatment at 450 MPa and 50
C for 30 minutes was determined as approximately 7  1.6%. This compares with 12  0.2% after a treatment of 40
C and 450 MPa for 60 minutes. The inactivation was irreversible and the enzyme was not reactivated when stored at 4 and 25
C for 1 week. Guiavarc’h et al. (2005) studied combined thermal and high-pressure inactivation of PME in white grapefruit. The results showed that combined mild heat and high-pressure processing cannot be used for full inactivation of PME in grapefruit juice. However, by eliminating up to 80% of the PME

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activity (labile fraction), this treatment can probably contribute to a significant delay of the cloud loss defect observed in grapefruit juices while allowing pasteurization and good quality retention. Combining high-pressure and mild-temperature processing with other nonthermal approaches (e.g., use of PME-inhibitor) could be of interest for the creation of juices with extended shelf life. Lacroix et al. (2005) studied the eVect of dynamic high-pressure (DHP) homogenization, alone or in combination with prewarming, on PME activity and opalescence stability of orange juice. DHP without heating reduced PME activity by 20%. PME inactivation was further increased by adjusting the pH downward prior to treatment. The orange juice was stored for 16 days at 30
C in order to accelerate loss of opalescence compared with storage at 4
C. These results suggest that the opalescence stability of orange juice treated by DHP does not depend entirely on PME activity but also depends on particle size reduction and structural changes to pectin resulting from treatment. The freshness attributes of orange juice treated by warming were improved by DHP treatment. This treatment at pH 3.8 resulted in opalescence being maintained for 8 days, compared with 1 day for the control, 2 days for prewarmed but not pressure-treated, and about 10 days for pasteurized juice. In the study carried out by Bull et al. (2004), PME was not completely deactivated in the Valencia juice (pH 4.3) by HPP (600 MPa, 20
C, 60 s) or thermal treatment. In the Navel orange juice (pH 7.3), PME was reduced with thermal treatment (85
C for 25 s) and with HPP (45%). Polydera et al. (2004) studied the inactivation kinetics of endogenous PME in freshly squeezed orange juice under high hydrostatic pressure (100–800 MPa) combined with moderate temperature (30–60
C). PME inactivation followed first order kinetics with a residual PME activity (5–20%) at all pressure–temperature combinations used. Pressure and temperature were found to act synergistically, except in the high temperature–low pressure region, where an antagonistic eVect was found. Yeom et al. (2000b) studied the eVect of PEF treatment (35 kV/cm, 59 ms) and heat (94.6
C for 30 s) on the relative PME activity of orange juice during storage at 4 and 22
C. PEF treatment decreased 88% of PME activity, and the inactivated PME was not restored at 4 and 22
C for 112 days. Heat pasteurization inactivated 98% of PME activity. K. POLYPHENOL OXIDASE

The purpose of the study by Quoc et al. (2006) was to develop a process to enable quick inactivation of the polyphenol oxidase enzyme, which is present in cloudy or unclarified apple juice. This enzyme is responsible for enzymatic

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browning. In order to achieve this objective, the apple juice was acidified to pH 2.0 by electrodialysis (bipolar–anionic membranes), followed by mild heat treatment at temperatures of 40, 45, and 50
C for a duration of 0–60 minutes. It was shown that the application of mild heat treatment at 45
C for 5 minutes to the acidified juice was suYcient for quick inactivation of the enzyme. The authors also found that the organoleptic properties of the juice were preserved after treatment, and the adjusted juice (pH readjusted to its initial value) had a better color than untreated apple juice when the juice was stored at 4
C for 3 months. Bayindirli et al. (2006) studied the eVectiveness of treating polyphenol oxidase activity in apple juice by applying high hydrostatic pressure with mild heat treatment (350 MPa at 40
C). L. YEAST

Many organisms, particularly acid-loving or acid-tolerant bacteria and fungi (yeasts and molds), can use fruit as substrate and cause spoilage, producing oV-flavors and odors and product discoloration. If the contaminating microorganisms are pathogens, they could also cause human illness. Toxigenic fungi, on the other hand, under favorable conditions could produce mycotoxin in fruit products such as juice (Varma and Verma, 1987). Before pasteurization, fruit juices contain a microbial load representative of the organisms normally found on fruits during harvesting plus contaminants added postharvest (during transport, storage, and processing). Many reports of bacterial growth in fruit juices exist in the literature, but most of the ones describing human illness due to contaminated juice deal with unpasteurized juice (Besser et al., 1993; Krause et al., 2001). Some investigations regarding fungal contamination of pasteurized fruit juice are also available (Kurtzman et al., 2001; Mendoza et al., 1982). Most of these reports have shown yeasts to be the predominant fungi involved in juice spoilage (Parish and Higgins, 1989). Yeast spoilage of fruit juice can result in formation of haze, production of CO2 and oV-odors, and changes in color. Tchango et al. (1997) studied the resistance of Candida pelliculosa and Kloeckera apis, two spoilage yeasts, isolated from pasteurized tropical fruit juices and nectars produced in Cameroon, in pineapple juice, guava nectar, and passion fruit nectar, as it relates to the pasteurization process of this beverages. The results showed that 22% of the pasteurized fruit juice samples tested contained live fungi, due either to inadequate pasteurization or to postpasteurization contamination during cooling, bulk storage, and bottling. Juice processors and packers should therefore take care to eliminate yeasts from juices and pack these products under strict aseptic conditions in

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order to avoid losses due to yeast spoilage, which results in products of poor or unacceptable quality. Silva et al. (1999) investigated the influence of temperature (85–97
C), total soluble solids (5–60
Brix or wt.%), and pH (2.5–6.0) on D-values (decimal reduction time) of Alicyclobacillus acidoterrestris (type strain, NCIMB 13137) spores, and they fitted a model using response surface methodology. Within the factor ranges studied, temperature was the parameter that most aVected the D-value. Soluble solids came next, and pH value was last. In general, D-values measured in real fruit systems, such as orange, apple, and grape juices, black currant concentrates, cupuac¸u (exotic fruit) extract, and orange juice drink, were higher than those predicted by the malt extract broth model. Parish (1998c) obtained decimal reduction times (D-values) for Saccharomyces cerevisiae ascospores inoculated into pasteurized orange juice ranging from 4 to 76 s at pressures between 500 and 350 MPa. At the same pressures, D-values of S. cerevisiae vegetative cells ranged from 1 to 38 s, while for the native microflora in nonpasteurized Hamlin orange juice they ranged between 3 and 74 s. The corresponding z-values were 123, 106, and 103 MPa for ascospores, vegetative cells, and native microflora, respectively. Alwazeer et al. (2003) studied the eVect of both redox potential (Eh) and pasteurization of orange juice on growth recovery of S. cerevisiae during storage at 15
C for 7 weeks. Oxidizing conditions were the most eVective for thermal destruction of S. cerevisiae, while reducing conditions decreased recovery of heated cells of S. cerevisiae. Tahiri et al. (2006) evaluated the potential of DHP technology to inactivate S. cerevisiae. The inactivation eYcacy of DHP depended on the pressure applied and the number of passes. Garde-Cerdan et al. (2006) studied the eVect of thermal and PEF treatments on several physicochemical properties and a population of inoculated S. cerevisiae. Both technologies reduced the population of the spoilage microorganism inoculated in grape juice. No viable cells were observed after thermal processing of grape juice, whereas PEF treatment achieved four logarithmic reductions of the microbial viability. Tournas et al. (2006) studied 65 pasteurized fruit juice samples (apple, carrot, grapefruit, grape, and orange juices, apple cider, and soy milk) purchased from local supermarkets in the Washington, DC area and found that 22% of the pasteurized fruit juice samples tested contained live fungi due either to inadequate pasteurization or to postpasteurization contamination during cooling, bulk storage, and bottling. Some of the yeasts isolated from these products were capable of growing under refrigeration, completely spoiling the product before its expiration.

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Yeast species commonly isolated from fruit juices were Rhodotorula rubra, Candida lambica, Candida sake, and K. apis, with C. lambica being the organism most frequently encountered in these products. Small numbers of Penicillium and Fusarium spp. were isolated from 20%, whereas Geotrichum spp. were present in 40% of the grapefruit juice samples tested. All other products contained no molds. The fact that these organisms were present in very low quantities indicated that they were random contaminants not able to grow in the refrigerated juice. Various authors have studied the inactivation of pathogenic and nonpathogenic microorganisms, mesophile flora, molds, and yeast flora (Abram et al., 2003; McDonald et al., 2000; Rodrigo et al., 2001, 2003b; Spilimbergo et al., 2003). A number of authors have studied the evolution of quality and safety factors in orange juice after nonthermal treatment, in some cases making a comparison with the evolution after heat treatment (Ayhan et al., 2001; Jia et al., 1999; Linton et al., 1999; Yeom et al., 2000a,b; Zook et al., 1999). M. LACTOBACILLUS BREVIS

Elez-Martı´nez et al. (2005) studied the inactivation of spoilage microorganisms such as Lactobacillus brevis by HIPEF and pasteurization. The eVects of HIPEF parameters (electric field strength, treatment time, pulse polarity, frequency, and pulse width) and heat pasteurization (90
C/1 minute) were evaluated on samples of orange juice inoculated with L. brevis (108 CFU/ml). HIPEF processing of orange juice was more eVective in inactivating L. brevis than thermal processing. The extent of microbial inactivation depended on the processing parameters (p < 0.01). L. brevis destruction was greater when the electric field strength and treatment time increased, and also when the pulse frequency and pulse width decreased. L. brevis was inactivated to a maximum of 5.8-log reductions when inoculated orange juice was processed at 35 kV/cm for 1000 ms using a 4-ms pulse width in bipolar mode and 200 Hz at less than 32
C. Mechanical breakdown of cell walls was observed in L. brevis when orange juice was processed by HIPEF. N. LACTOBACILLUS PLANTARUM

Alwazeer et al. (2003) studied the eVect of both redox potential (Eh) and pasteurization of orange juice on growth recovery of microorganisms during storage at 15
C for 7 weeks. Three Eh conditions, þ360 (ungassed), þ240 (gassed with N2), and 180 mV (gassed with N2–H2) were applied to orange

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juice. Both thermal destruction and recovery of sublethally heat-injured cells of Lactobacillus plantarum were investigated. Oxidizing conditions were the most eVective for thermal destruction of L. plantarum. Tahiri et al. (2006) evaluated the eVect of DHP technology to inactivate pathogenic and spoilage microflora in orange juice. The inactivation eYcacy of DHP depended on the pressure applied and the number of passes. O. E. COLI

Exposure of E. coli to microwave treatments results in a reduction of the microbial population in apple juice. Can˜umir et al. (2002) determined the eVect of pasteurization at diVerent power levels (270–900 W) on the microbial quality of apple juice, using a domestic 2450 MHz microwave. The data obtained were compared with conventional pasteurization (83
C for 30 s). Apple juice pasteurization at 720–900 W for 60–90 s resulted in a 2- to 4-log population reduction. Using a linear model, the D-values ranged from 0.42  0.03 minutes at 900 W to 3.88  0.26 minutes at 270 W. The value for z was 652.5  2.16 W (58.5  0.4
C). These observations indicate that inactivation of E. coli is due to heat. Heinz et al. (2003) focused on improving the energy eYciency of PEFs treatment for pasteurization of apple juice inoculated with E. coli by investigating the relation between the reduction achieved in the survivor count and electric field strength and treatment temperature. To evaluate the thermal load of the product the pasteurization unit and cook value, key benchmarks for the thermal load, were used to compare PEF and conventional heat treatment. Tahiri et al. (2006) evaluated the potential of DHP technology to inactivate E. coli O157:H7 ATCC 35150 in orange juice. Complete inactivation and 5-log reduction of E. coli O157:H7 were achieved in orange juice at 200 MPa and 25
C after 5 and 3 passes, respectively. Bayindirli et al. (2006) found that high hydrostatic pressure with mild heat treatment (350 MPa at 40
C) caused inactivation of E. coli O157:H7 933 in apple, orange, apricot, and sour cherry juices. P. STAPHYLOCOCCUS AUREUS

Bayindirli et al. (2006) studied the eVect of high hydrostatic pressure with mild heat treatment on Staphylococcus aureus 485 in apple, orange, apricot, and sour cherry juices. The results showed that commercially practicable pressure processes can be used to inactivate even the most pressure-resistant microorganisms. The use of HPP (350 MPa) at 40
C could be considered

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for treating the fruit juices studied to improve microbial kill, with respect to the pressure-resistant strains of the pathogens studied. Q. SALMONELLA ENTERITIDIS

Korolczuk et al. (2006) used a pilot-scale continuous PEF treatment of liquid products to study the eVects of energy input (0–300 kJ/kg), electric field strength (25–70 kV/cm), square wave pulse width (0.05–3 ms), and initial product temperature (4–20
C) on Salmonella enteritidis. For energy input (Q), 0–100 kJ/kg, the decimal reduction number can be considered as linearly related to Q, with the decimal reduction energy (QD) varying between 44  3.2 kJ/kg for 0.05 ms, 37  2.5 kJ/kg for 0.1 ms, and 32  1.4 kJ/kg for 0.25–3 ms pulse width. For Q ¼ 0–300 kJ/kg, the relation between Q and log(N0/N) was of power law type, with the threshold energy level Q0 ¼ 9  2.6 kJ/kg and the power coeYcient 3.17  0.21. Bayindirli et al. (2006) studied the eVect of high hydrostatic pressure on S. enteritidis FDA in apple, orange, apricot, and sour cherry juices. They found that commercially practicable pressure processes (350 MPa at 40
C) can be used to inactivate S. enteritidis. R. NEOSARTORYA FISCHERI

Ascospores of heat-resistant molds can survive the heat pasteurization treatments normally applied to fruits and derivatives, and may spoil these products by germination and subsequent growth under reduced oxygen conditions. Neosartorya fischeri, Byssochlamys fulva, Byssochlamys nivea, Talaromyces flavus, and Eupenicillium are some of these fungi. These heatresistant molds are also known to produce various mycotoxins during their growth in fruit products (Rajashekhara et al., 2000). N. fischeri (anamorph Aspergillus fischeri) is one of the most frequently reported heat-resistant molds causing spoilage in fruit products (Nielsen, 1991). Salomao et al. (2007) studied the heat resistance of N. fischeri in three diVerent juices (apple, pineapple, and papaya). The optimum heat activation temperature and time for ascospores of N. fischeri (growth for 30 days at 30
C) was 85
C for 10 minutes. The z-values for apple, papaya, and pineapple juices were 5, 5.5, and 5.9
C, respectively. The sterilization F-values (4-log reduction) for apple, pineapple, and papaya juices were 56.3, 38.0, and 7.2 s, respectively. Considering the thermal treatments commercially applied to pineapple (96
C/30 s) and apple juices (95
C/30 s), the authors concluded that such treatments would not guarantee that less than 1 ascospore in each set of 103 packs survives. Only the treatment applied to papaya juice (100
C/30 s) would be suYcient because the F-value was less than 30 s.

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Silva, F.M., Gibbs, P., Vieira, M.C., and Silva, C.L.M. 1999. Thermal inactivation of Alicyclobacillus acidoterrestris spores under diVerent temperature, soluble solids and pH conditions for the design of fruit processes. Int. J. Food Microbiol. 51, 95–103. Simon, J.A., Hudes, E.S., and Tice, J.A. 2001. Relation of serum ascorbic acid to mortality among US adults. J. Am. Coll. Nutr. 20, 255–263. Simopoulos, A.P. 2001. The Mediterranean diets: What is so special about the diet of Greece? The scientific evidence. J. Nutr. 131, 3065S–3073S. Slattery, M.L., Benson, J., Curtin, K., Ma, K.N., SchaeVer, D., and Potter, J.D. 2000. Carotenoids and colon cancer. Am. J. Clin. Nutr. 71, 575–582. Spilimbergo, S., Dehghani, F., Bertucco, A., and Foster, N.R. 2003. Inactivation of bacteria and spores by pulsed electric field and high pressure CO2 at low temperature. Biotechnol. Bioeng. 82, 118–125. Stewart, C.M., Tompkin, R.B., and Cole, M.B. 2002. Food safety: New concepts for the new millennium. Innov. Food Sci. Emerg. Technol. 3, 105–112. Tahiri, I., Makhlouf, J., Paquin, P., and Fliss, I. 2006. Inactivation of food spoilage bacteria and Escherichia coli O157:H7 in phosphate buVer and orange juice using dynamic high pressure. Food Res. Int. 39, 98–105. Taylor, S. 1996. Beta-carotene, carotenoids, and disease prevention in humans. FASEB J. 10, 690–701. Tchango, J., Tailliez, R., Njine, P.E., and Hornez, J.P. 1997. Heat resistance of the spoilage yeasts Candida pelliculosa and Kloeckera apis and pasteurization values for some tropical fruit juices and nectars. Food Microbiol. 14, 93–99. Temple, N.J. and Gladwin, K.K. 2003. Fruit, vegetables, and the prevention of cancer. Res. Chall. Nutr. 19, 467–470. Topuz, A., Topakci, M., Canakci, M., Akinci, I., and Ozdemir, F. 2005. Physical and nutritional properties of four orange varieties. J. Food Eng. 66, 519–523. Torregrosa, F., Corte´s, C., Esteve, M.J., and Frı´gola, A. 2005. EVect of high-intensity pulsed electric fields processing and conventional heat treatment on orange-carrot juice carotenoids. J. Agric. Food Chem. 53, 9519–9525. Torregrosa, F., Esteve, M.J., Frigola, A., and Cortes, C. 2006. Ascorbic acid stability during refrigerated storage of orange-carrot juice treated by high pulsed electric field and comparison with pasteurized juice. J. Food Eng. 73, 339–345. Tournas, V.H., Heeres, J., and Burguess, L. 2006. Moulds and yeasts in fruit salads and fruit juices. Food Microbiol. 23, 684–688. Trammell, D.J., Dalsis, D.E., and Malone, C.T. 1986. EVect of oxygen on taste, ascorbic acid loss and browning for HTST-pasteurised, single-strength orange juice. J. Food Sci. 51, 1021–1023. Tribble, D.L. 1998. Further evidence of the cardiovascular benefits of diet enriched in carotenoids. Am. J. Clin. Nutr. 68, 521–522. Trifiro`, A., Gherardi, S., and Calza, M. 1995. EVetti della temperatura e del tempo di magazzinaggio sulla qualita` di succhi freschi di arance pigmentate. Industria delle Conserve 70, 243–251. Van Boekel, M.A.J.S. and Jongen, W.M.F. 1997. Product quality and food processing: How to quantify the healthiness of a product. Cancer Lett. 114, 65–69. Vanamala, J., Reddivari, L., Yoo, K.S., Pike, L.M., and Patil, B.S. 2006. Variation in the content of bioactive flavonoids in diVerent brands of orange and grapefruit juices. J. Compos. Anal. 19, 157–166. Varma, S.K. and Verma, R.A. 1987. Aflatoxin B1 production in orange (Citrus reticulate) juice by isolates of Aspergillus flavus Link. Mycopathologia 97, 101–104. Versteeg, C., Rombouts, F.M., Spaansen, C.H., and Pilnik, W. 1980. Thermostability and orange juice cloud destabilizing properties of multiple pectinesterases from orange. J. Food Sci. 45, 969–971. Vikram, V.B., Ramesh, M.N., and Prapulla, S.G. 2005. Thermal degradation kinetics of nutrients in orange juice heated by electromagnetic and conventional methods. J. Food Eng. 69, 31–40.

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TETRODOTOXIN POISONING DENG-FWU HWANG* AND TAMAO NOGUCHI*,{ *Department of Food Science, National Taiwan Ocean University Taiwan, Republic of China { Tokyo Healthcare University, Setagaya, Tokyo, Japan

I. Introduction II. TTX Poisoning A. Incidents in the World B. Symptoms and Signs C. Treatment D. Prevention III. Causative Agent: TTX A. Distribution of TTX-Bearing Organisms B. TTX Elaborator C. Mechanisms of TTX Infestation to Animals D. TTX Detection Method E. Chemistry of PuVer Toxin F. Pharmacology of TTX G. Therapeutic Application of TTX IV. Highlight of Viewpoint A. Proposed Programs of New Food Industry for PuVer B. Method of Species Identity for PuVer by Genome Techniques C. Method of Species Identity for PuVer by Protein Techniques D. TTX as Attractant and Hibernation Agent V. Summary References

Tetrodotoxin (TTX) is one of the most potent and oldest known neurotoxins. The poisoning cases due to ingestion of TTX-containing marine animals, especially for puVer, have frequently occurred in Asia since a long time ago. This chapter describes various topics on TTX poisoning including the tendency of poisoning incidents, typical case report, treatment and prevention, biology distribution, original source, infestation mechanism, detection methods, characteristics of chemistry and pharmacology, and therapeutic application.

ADVANCES IN FOOD AND NUTRITION RESEARCH VOL 52 # 2007 Elsevier Inc. All rights reserved

ISSN: 1043-4526 DOI: 10.1016/S1043-4526(06)52004-2

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Furthermore, the protocols for how to make puVer safe to eat and how to prevent puVer products made from toxic puVers have been suggested. Finally, the biological significance and neurophysiological role of TTX have been elucidated and TTX may act as an important drug like anesthetic in future. I. INTRODUCTION Tetrodotoxin (TTX), a puVer toxin named after its order name Tetraodontiformes by Professor Y. Tahara in 1880, is one of the most potent nonproteinaceous toxins known, responsible for numerous fish poisonings. This toxin is one of the oldest known natural toxins recorded as early as 2700 BC in Chinese literature, describing the toxicity of puVer, and around 2500 BC in Egyptian history. TTX poisoning cases due to ingestion of puVer have frequently occurred especially in Japan where these fish have been a traditional food since a long time ago. The preferred forms of this delicacy are slices of raw flesh (‘‘sashimi’’) and slightly cooked liver (‘‘kimo’’), Japanese people are aware of the puVer’s toxicity and have devised ways to reduce TTX in the liver. However, TTX poisoning incidents continue to occur in Japan yet. Since there is no cure for the poisoning patient, its mortality is very high. Judging from the statistics (Table I) provided by Japanese Ministry of Health and Welfare (MHW), the number of deaths due to puVer poisoning have steadily declined, from more than 10 cases with high mortality every year between 1960 and 1981 to less than 10 cases with low mortality every year since 1982 (MHW, 1997; Noguchi, 2003). This decline in incidence is probably not only due to complete performance of government regulations but also due to increase of cultured puVer (nontoxic) instead of decrease of wild puVer (toxic). TTX is a heterocyclic guanide compound whose chemical structure has been characterized (Figure 1). TTX has one guanidium moiety. As being seen in Figure 2, many derivatives of TTX have been found, although their toxicities vary widely. Since being first isolated in 1964 from puVer of the family Tetraodontidae (Tsuda et al., 1964), TTX has been found in diverse organisms, including marine goby and an octopus as well as some terrestrial amphibians such as newts and frogs (Table II). TTX has been found in several bacterial species, including Shewanella sp. and Vibrio spp., and is now believed to be of bacterial origin. Recently, several poisoning cases due to ingestion of, in addition to those of puVers, big and small gastropods have caused in Japan, Taiwan, and China. Especially small gastropods in China have caused many poisoning for a long time, resulting in many deaths and caused serious problems in public health.

TETRODOTOXIN POISONING

143

TABLE I PUFFER POISONING INCIDENTS IN JAPANa

Year

Number of incidents

Number of patients

Number of deaths

Mortality (%)

1965 1970 1975 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002

106 46 52 46 30 26 18 23 30 22 35 26 31 33 29 33 28 16 30 21 28 27 20 29 31 32

152 73 75 90 46 33 34 39 41 38 52 46 45 55 45 57 44 23 42 34 44 39 34 40 52 49

88 33 30 15 12 8 6 6 9 6 4 5 5 1 3 4 4 1 2 3 6 4 2 0 3 5

57.9 45.2 40.0 16.7 26.0 24.2 17.6 15.4 21.9 15.8 7.7 10.9 11.1 1.8 6.7 7.0 9.1 4.3 4.8 8.8 13.6 10.3 5.9 0 5.8 10.2

a

Data cited from MHW (1997) and Noguchi (2003).

HO

H

H H N

+

H2N

N H

OH

HO O

+

OHO

H2N

H

HO H

CH2OH

H

H Hemilactal form

H O

H H N N HO

OH OHO H

H

H

OH

H

CH2OH

H OH

10,7-Lactone form

FIG. 1 The tautomers of TTX.

144

D.-F. HWANG AND T. NOGUCHI (1) Hemilactal type OH HO R1 10 9 H + 2 O H H2N 4 R2 O N 3 5 4a H 8a 1N 7 R5 6 R H 4 8 H H R3 H

11-deoxyTTX 6-epi-11-deoxyTTX TTX-8-O -hemisuccinate

R1 H OH H H OH H

R2 OH H OH OH H OH

Chiriquitoxin 11-norTTX-6(S )-ol 11-norTTX-6(R )-ol 11-norTTX-6,6-diol 11-oxoTTX TTX-11-carboxylic acid

H H H H H H

OH OH OH OH OH OH

OH OH H OH OH OH

HO

R2

TTX 4-epi TTX 6-epi TTX

R3 OH OH CH2OH

R4 CH2OH CH2OH

R5 OH OH OH OH OH OOC(CH2)2COO−

OH CH3 CH3 CH2OH

OH OH OH

R S CH(OH)CH(NH+3)COO− H OH OH CH(OH)2

OH OH OH OH OH OH

COO−

(2) Lacton type O

H H N

+ H2N

R4 R3 O H

N HO R1

H

R6

H R5

H R1

R2

R3

R4

R5

TTX (lactone) 6-epi TTX (lactone)

H

H

OH

OH

H

H

OH

OH

OH CH2OH

R6 CH2OH

11-deoxyTTX (lactone)

H

H

OH

OH

OH

OH CH3

11-norTTX-6(S )-ol (lactone)

H

H

OH

OH

OH

H

11-norTTX-6(R )-ol (lactone)

H

H

OH

OH

H

OH

11-norTTX-6,6-diol (lactone)

H

H

OH

OH

OH

5-deoxyTTX

H

H

OH

H

OH

OH CH2OH

5,11-dideoxyTTX

H

H

OH

H

OH

CH3

6-epi-5,11-dideoxyTTX

H

OH

H

H

OH

CH3

1-hydroxy-5,11-dideoxyTTX

OH

H

OH

H

OH

CH3

5,6,11-trideoxyTTX

H

H

OH

H

H

CH3

4-epi-5,6,11-trideoxyTTX

H

OH

H

H

H

CH3

TETRODOTOXIN POISONING

145

(3) 4,9-Anhydro type OH H + H2N

O

H N N H

H O

H

R3

H H

4,9-Anhydro TTX 4,9-Anhydro-6-epi -TTX 4,9-Anhydro-11-deoxyTTX 4,9-Anhydro-TTX-8-O-hemisuccinate 4,9-Anhydro-TTX-11-O-hemisuccinate

−OOC + H2N

O

H N N H

R2

H R1

R1 OH CH2OH OH OH OH

R2 CH2OH OH CH3 CH2OH CH2OCO(CH2)2COO−

H H OH H OH H

HO H H

O

+ H2N

H

H N N

CH2OH

O

H

OH

O H O

H H

HO

CH3

H H

Tetrodonic acid (tetrodoic acid)

R3 OH OH OH OOC(CH2)2COO− OH

OH

4,9-Anhydro-5,11-dideoxyTTX

FIG. 2 The structure of three types of TTX analogues. (1) Hemilactal type, (2) Lacton type, and (3) 4,9-Anhydro type (Yotsu-Yamashita, 2001).

Accordingly, TTX in TTX-bearing organisms is found to come from concentration through several steps of the food web, starting with TTX production by bacteria. II. TTX POISONING A. INCIDENTS IN THE WORLD

1. Japan In Japan, many puVer poisoning cases occur every year, resulting in numerous deaths with approximately 8.3% mortality over the past 10 years (Table I). Japanese people know that puVer, especially its liver (‘‘kimo’’), is very toxic, but more than a few ‘‘kimo’’ fans dare to ingest the liver, believing that the toxin has been eliminated from it using their own ‘‘special’’ (traditional) detoxification method. Consequently most puVer poisoning cases are caused by ingestion of toxic livers. Since the Japanese MHW published a guideline of edible puVers in 1983, with updates in 1993 and 1995 (Table III) (MHW, 1983), these guidelines only prohibit puVer livers to be served in restaurants. As a result, many TTX poisoning still occur due to

146

D.-F. HWANG AND T. NOGUCHI

TABLE II DISTRIBUTION OF TTX IN ANIMALS

Animals 1 2

3

Platyhelminthes Turbellaria Nemertinea

Mollusca Gastropoda

4

Cephalopoda Annelida Polychaeta

5

Arthropoda

6

Chaetognatha

7

Echinodermata

8

Vertebrate Pisces Amphibia

9 10

Red Clacareous alga Dinoflagellate

Part Flatworms Planocera spp. Ribbon worms Lineus fuscoviridis Tubulanus punctatus Cerebratulus lacteus Cephalothrix linearis Charonia sauliae Babylonia japonica Tutufa lissostoma Zeuxis siquijorensis Niotha clathrata Natica lineata Rapana spp. Cymatium echo Pugilina ternotona Hapalochlaena maculosa Pseudopotamilla occelata Lepidonotus helotypus Halosydna brevisetosa Harmothoe imbricata Atergatis floridus Zosimus aeneus Carcinoscorpius rotundicauda Arrowworms Eukrohonia hamata Parasagitta spp. Flaccisagitta spp. Starfish Astropecten polyacanthus Astropecten latespinosus Astropecten scoparius Takifugu spp. Yongeichthys criniger Taricha spp. Notophthalmus spp. Cynops spp. Triturus spp. Ambystoma sp. Paramesotriton sp. Polypedates sp. Atelopus spp. Colostethus spp. Jania spp. Alexandrium tamarense

Whole body Whole body Whole body Whole body Whole body Whole body Digestive gland Digestive gland Digestive gland Digestive gland Digestive gland Whole body Digestive gland Digestive gland Digestive gland Postsalivary gland Whole body Whole body Whole body Whole body Whole body Whole body Egg Head Head Head Whole body Whole body Whole body Skin, liver, ovary Skin, viscera, gonad Skin, egg, ovary, muscle, blood Skin, egg, ovary Skin, egg, ovary, muscle, blood Skin, egg, ovary, muscle, blood Skin, egg, ovary, muscle Skin, egg, ovary, muscle Skin Skin Skin Whole body Whole body

TETRODOTOXIN POISONING

147

TABLE III EDIBLE PART OF PUFFER IN JAPAN

Edible part Family

Tetraodontidae

Diodontidae Ostracidae

Species

Muscle

Skin

Male gonad

‘‘Kusafugu’’ Takifugu niphobles ‘‘Komonfugu’’ Takifugu poecilonotus ‘‘Higanfugu’’ Takifugu pardalis ‘‘Shousaifugu’’ Takifugu snyderi ‘‘Nashifugu’’ Takifugu vermicularisa ‘‘Mafugu’’ Takifugu porphyreus ‘‘Mefugu’’ Takifugu obscurus ‘‘Akamefugu’’ Takifugu chrysops ‘‘Torafugu’’ Takifugu rubripes ‘‘Karasu’’ Takifugu chinensis ‘‘Shimafugu’’ Takifugu xanthopterus ‘‘Gomafugu’’ Takifugu stictonotus ‘‘Kanafugu’’ Lagocephalus inermis ‘‘Shirosabafugu’’ Lagocephalus wheeleri ‘‘Kurosabafugu’’ Lagocephalus gloveri ‘‘Yoritofugu’’ Sphoeroides pachygaster ‘‘Sansaifugu’’ Takifugu flavidus ‘‘Ishigakifugu’’ Chilomycterus reticulatus ‘‘Harisenbon’’ Diodon holocanthus ‘‘Hitozuraharisenbon’’ Diodon liturosus ‘‘Nezurnifugu’’ Diodon hystrix ‘‘Hakofugu’’ Ostraction immaculatum

s s s s s s s s s s s s s s s s s s s s s s

— — — — — — — — s s s — s s s s — s s s s —

— — — s s s s s s s s s s s s s — s s s s —

Applicable to muscle of the species caught in Shimabara Bay, Tachibana Bay, and the Inland Sea of Kagawa and Okayama, and to male gonad in Shimabara Bay and Tachibana Bay. s, Edible; —, nonedible. a

consumption of homemade puVer liver dishes from wild fish usually caught by a family member. Recently, 80% of puVer in fish market are cultured and rarely are they toxic. Accordingly, puVer is imaged from toxic to nontoxic. On the contrary, the poisoning events due to ingestion of other marine animals bearing TTX have occurred. The toxicity of puVers is related to their spawning with the highest toxicity levels, especially in the ovary, between March and June in Japan. The two typical examples of TTX poisoning cases in Japan are as follows: 1. Poisoning due to the liver of the finepatterned puVer (Takifugu poecilonotus) A 48-year-old man in Nagasaki City, Nagasaki Prefecture, ate more than four slices of slightly cooked liver (‘‘kimo’’) along with some flesh of wild

148

D.-F. HWANG AND T. NOGUCHI

T. poecilonotus in the evening in October 1996. The fish had been caught earlier in the day. After 30–60 minutes of ingestion of the puVer tissues, the man began to suVer from numbness in his hands and limbs, followed by cyanosis and respiratory failure during the next 60 minutes. Although he was immediately hospitalized, the patient died during the following hour. TTX toxicity of puVer is commonly determined by mouse bioassay. The mouse bioassay is performed by intraperitoneal injection. After injection with TTX-associated extracts, the mice show the characteristic signs and symptoms, like unique scratching of the shoulders/mouth by their hind limbs, weakness progressing to paralysis in hind limbs, uncoordinated movement, shallowness of breathing, convulsion, and jumping, followed by respiratory failure. Toxicity of an extract is expressed in terms of mouse units (MU), where 1 MU is defined as the amount of TTX required to kill a 20-g male of ddY or ICR strain in 30 minutes (Hwang and Jeng, 1991; MHW, 1991). Minimum detectable limit is about 0.2 mg of TTX (1 MU) per assay. In these cases, toxicity scores of the puVer livers and flesh were 715–4260 MU/g (equivalent to 0.14–0.85 mg of TTX per gram) and less than 5 MU/g (equivalent to 0.001 mg of TTX per gram), respectively. The cause of death was determined to be TTX in the wild T. poecilonotus liver. The victim died due to ingestion of a total toxicity score of more than 10,000 MU (about 2-mg TTX), equivalent to the LD50 of TTX for man (Noguchi and Akaeda, 1998). 2. Poisoning due to the digestive gland of a big gastropod trumpet shell (Charonia sauliae) In December 1979, a 41-year-old man ate a boiled digestive gland (
60 g) of ‘‘boshubora’’ or trumpet shell (C. sauliae) that he had caught in the Shimizu Sea. Thirty minutes after ingestion, he was immediately hospitalized and received artificial respiration. Although he remained unconscious for 2 days, he recovered fully in 5 days. The leftover digestive gland of the C. sauliae showed a toxicity score of 17,000 MU (equivalent to 3.4 mg of TTX) in the bioassay for TTX (Narita et al., 1981). Thus, the victim may have ingested about 10,000 MU (2 mg of TTX), the approximate LD50 of the toxin for man. TTX was also identified as the causative agent in samples from similar TTX poisoning cases in Wakayama in 1982 and in Miyazaki in 1987 using thin-layer chromatography (TLC), electrophoresis, and instrumental analyses with infrared (IR), proton nuclear magnetic resonance (1H NMR), and gas chromatography–mass spectrometry (GC–MS). 2. Taiwan and China In Taiwan and China, many food poisoning cases due to ingestion of wild puVer have occurred (Hwang, 2003a,b), while consumers here do not often eat puVer. According to data of TTX poisoning in Taiwan (Table IV), TTX

TETRODOTOXIN POISONING

149

TABLE IV FOOD-BORNE OUTBREAKS CAUSED BY TTX IN TAIWAN, 1994–2003

Time 1994 1995 1997 1997 1998 1999 2000

2001

January, April, May April, May June February, December February, March May, June, December January January, March, August February, April July March, April April

2002 2003

April February March March, September

Food

Patients/deaths

Place

PuVer

10/0

Gastropod Goby PuVer

26/0 2/0 4/1

Changhua, Taitung, Pingtung Pingtung Miaoli Changhua, Kaohsiung Kaohsiung, Tainan Taitung, Tuaoyuan, Taipei Taipei, Changhua Changhua, Keelung

Goby Dried dressed fish fillet PuVer PuVer Pried dressed fish fillet Gastropod PuVer Flake of dried mullet roe Gastropod Gastropod PuVer PuVer

5/0 5/0 18/1 14/0 7/2

Changhua, Tainan

1/0 13/1 1/0

Chaiyi Taichung, Keelung, Taipei, Lunlin Kaohsiung

5/0 1/0 6/3 6/0

Taipei Pingtung Taitung Kaohsiung

Total: 30 outbreaks, 124 patients, 8 deaths. Source: Hwang et al. (2003).

poisoning cases seem to occur by ingesting puVer roe as fake of ‘‘karasumi’’ dried mullet roe, toxic puVer muscle by mistake, and the dried dressed fish fillet produced from toxic puVers (Du et al., 1999; Hsieh et al., 2003; Hwang et al., 2002a). New faces of small gastropods as causative foods of TTX poisoning newly appear, such as Niotha clathrata, Zeuxis scalaris, Natica vitellus, Oliva miniacea, Oliva mustelina, Oliva hirasei, and Nassarius glans (Hwang et al., 1995, 2002b, 2003, 2005; Shiu et al., 2003). In China, food poisoning cases due to ingestion of puVer have occurred. Recently, the epidemic investigation on small gastropod poisoning incidents in Zhoushan has been shown in Figure 3. The toxic organism and causal agent were identified as Zeuxis samiplicutus and TTX (Shui et al., 2003; Sui et al., 2002).

150

D.-F. HWANG AND T. NOGUCHI 12

(6)*

10

200 50

* : Number of death

8

40

6

30

4

20

2

(1) (3) (1)

0

(1)

Patients (o)

Outbreaks ( )

(2)

10

(2) 0

1977 1979 1981 1983 1985 1987 1989 1991 1993 1995 1997 1999 2001 Year

FIG. 3 Yearly distribution of snail poisoning outbreaks and patients in Zhoushan City (Shui et al., 2003).

The two typical examples of TTX poisoning cases in Taiwan are as follows: 1. A food poisoning incident due to ingestion of an unknown fish A food poisoning incident following fish ingestion occurred in Chunghua Prefecture, western Taiwan on January 2, 2000. A total of five victims (four men, 58- to 64-year old, and one woman, 46-year old) were reported. Symptoms included paralysis, coma, nausea, vomiting, ataxia, aphasia, and diYculty of respiration. Among these victims, two men suVered from more serious symptoms and were treated with intravenous fluids, mechanical ventilation, and intensive treatment in the hospital. They were then discharged uneventfully after 1 week of management. Presently, all patients are normal and healthy. The fish, which might be puVer was reported by victims, was collected from the coastal area of Chunghua Prefecture. The small residue (11.02 g) of cooked fish liver was retained by the victims in this incident. The cooked fish liver retained by the victims was assayed for toxicity and mitochondrial DNA. Meanwhile, eight live specimens of puVer Takifugu niphobles were also assayed. The toxicity of cooked fish liver was 280 MU/g. All specimens of T. niphobles showed high toxicity (more than 850 MU/g) in the liver. The toxin from cooked fish liver and liver of T. niphobles was identified to be TTX. The cooked fish and fresh liver of T. niphobles showed the same

TETRODOTOXIN POISONING

151

sequence genotype and the same single restriction site for BsaI. Therefore, the species of cooked fish liver was suggested as T. niphobles (Hsieh et al., 2002). 2. Goby intoxication in a uremic patient The patient was a 52-year-old uremic woman. The uremia was of undefined etiology. Over the past 3 years she has received regular hemodialysis. One day in April, 1998, she and her husband, a healthy 55-year-old man, had fish soup. About 4 hours after the meal she developed a headache and a lingual and circumoral tingling sensation and numbness at the distal parts of all four limbs. She was dizzy and unsteady, had diYculty in swallowing, and became very weak. She was taken to the emergency service and was placed on machine-assisted ventilation as respiratory distress and cyanosis developed. Her husband remained asymptomatic throughout this time. The patient’s condition kept on deteriorating, developing eventually into a comatous-like state with no spontaneous or reflexive eye or limb movement within 30 minutes of intubation. On neurological examination, the papillary light reflex was absent and oculocephalic maneuver elicited no ocular movements. All four limbs were areflexic and Bafinski’s signs were absent. Brain computerized tomography (CT) and laboratory studies of arterial blood gas (under assisted ventilation), electrolytes, liver function, blood glucose, and cerebrospinal fluid (CSF) were unremarkable. An examination of renal function indicated chronic renal insuYciency with mild azotemia. An electric encephalography (EEG) recorded 18 hours after the onset of symptoms when the neurological condition was unchanged, showed posterior dominant alpha waves intermixing with trains of short duration, diVuse theta waves. At the same time brief noxious stimuli were replaced transiently by beta activities. The findings suggested that the profound neurological dysfunction might be peripheral in origin. The patient was given a course of hemodialysis according to the set schedule for uremia at 21 hours after onset of the symptoms. Her condition improved dramatically within an hour. She could open her eyes and she communicated and answered questions correctly by blinking. Pupillary reflex recovered and voluntary eye movements were limited only at the extreme lateral gaze. Muscle power was grade 3 and 4 in the proximal and distal parts of the four limbs. Tendon reflexed were still absent. She was taken oV mechanical ventilation the next day. Her clinical condition continued to improve and her symptoms subsided in a stepwise pattern, in response to each course of hemodialysis. She regained her initial strength by the time she was discharged on day 16. When analyzing the remains of the cooked fish (identified as Yongeichthys nebulosus), TTX was demonstrated by TLC, high-performance liquid chromatography (HPLC), and cellulose acetate membrane electrophoresis. Toxicity was assayed by using ICR (Institute of Cancer Research) strain adult male mice and the toxicity score was 25 MU/g in fish muscle. The synergistic eVect of uremia and TTX is obvious in this incident in which the patient and her

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husband ingested roughly an equal amount of TTX (about 0.2 mg, calculated from toxic score times the weight of ingested fish). The amount is about 10% of the estimated lethal dose in humans and caused no clinical evidence of poisoning in the healthy person. In this case, hemodialysis is suggested as an eVective method in the treatment of TTX intoxication (Lan et al., 1999). 3. Other countries Two Dutch sailors died within 17–20 minutes after ingestion of the liver of a South African puVer (Halstead, 1987; Noguchi and Ebesu, 2001). Ten human fatalities from the consumption of puVer have been reported in the United States (Ebesu et al., 2000). Three of them occurred between 1908 and 1990 (Lange, 1990; Redy and Hayes, 1989) and four in Hawaii between 1903 and 1925, all of the poisoning originated from the consumption of Arothron sp. (Helfrich, 1963). In 1986, a further poisoning incident occurred in Hawaii due to consumption of the liver of Diodon hystrix, aVecting one man (Sims and Ostman, 1986). Although the victim did not attend hospital until 24 hours of exposure, he recovered within 1 week. In Bangladesh on November 16, 1998, a food poisoning incident due to ingestion of roe of a puVer Takifugu oblongus occurred, aVecting eight people inclusive of five deaths (Mahmud et al., 1999b). Their symptoms were as follows: dyspnea, numbness of the lips, paralysis, and stomachache followed by vomiting. These appeared after 2 hours of ingestion. Two victims became unconscious within 5 hours of exposure. On the way to the hospital, two among eight patients died while the remaining six were admitted. Three of the six patients died in the hospital and three recovered and left the hospital. Poisonings due to ingestion of horseshoe crab (Carcinoscorpius rotundicauda) eggs have occasionally been reported in Thailand (Smith, 1933; Tiammeth, 1953; Trishnananda et al., 1966). The symptoms of the victims were mostly similar to these caused by TTX or paralytic shellfish poisons (PSPs). The responsible toxin was identified as PSP as the major toxin, with a minor unknown toxin (Fusetani et al., 1982, 1983). C. rotundicauda toxin was known to consist of mainly TTX, anhydroTTX, and only a small amount of saxitoxin (STX) and neosaxitoxin (neoSTX) (Kungsuwan et al., 1987). B. SYMPTOMS AND SIGNS

In human, the MLD50 of TTX is approximately estimated to be 10,000 MU, equivalent to 2 mg of TTX. Minimum dose for developing TTX poisoning symptoms in human is supposed to be near MLD50. TTX is heat-resistant and in general cooking process it is not decomposed.

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TABLE V THE MAIN DEGREE OF TETRODOTOXICATION AND ASSOCIATED SYMPTOMS

Degree

Characteristic symptoms

First

Neuromuscular symptoms (paresthesia of lips, tongue, and pharynx; taste disturbance; dizziness; headache; diaphoresis; pupillary constriction); gastrointestinal symptoms (salivation, hypersalivation, nausea, vomiting, hyperemesis, hematemesis, hypermotility, diarrhea, abdominal pain) Additional neuromuscular symptoms (advanced general paresthesia; paralysis of phalanges and extremities; pupillary dilatation, reflex changes) Increased neuromuscular symptoms (dysarthria; dysphagia, aphagia; lethargy; incoordination, ataxia; floating sensation; cranial nerve palsies; muscular fasciculations); cardiovascular/pulmonary symptoms (hypotension or hypertension; vasomotor blockade; cardiac arrhythmias including sinus bradycardia, asystole, tachycardia, and atrioventricular node conduction abnormalities; cyanosis; pallor; dyspnea); dermatologic symptoms (exfoliative dermatitis, petechiae, blistering) Respiratory failure, impaired mental faculties, extreme hypotension, seizures, loss of deep tendon and spinal reflexes

Second Third

Fourth

Tetrodotoxication is characterized by a few symptoms of the victims. The type, severity, and range of symptoms depend on the amount of toxin ingested, age, and health of the victim. The four main stages or degrees of tetrodotoxication based are shown in Table V. First degree is as follows: Depending on the amount of toxin ingested, symptoms usually appear within 10–45 minutes of exposure, though some cases have been reported to be a symptomatic until as much as 3–6 hours after exposure. Oral paresthesia is usually the initial symptom and gradually spreads to the extremities and trunk. Other early symptoms include taste disturbance, dizziness, headache, diaphoresis, and papillary symptoms of salivation, hypersalivation, nausea, vomiting, hyperemesis, hematemesis, hypermotility, diarrhea, and abdominal pain. These symptoms are characteristic of first degree of tetrodotoxication in a system devised by Fukuda and Tani (1941). Second degree is as follows: Symptoms are characterized by additional neuromuscular ones, such as advanced general paresthesia, paralysis of phalanges and extremities, papillary dilation, and loss of papillary and corneal reflexes. Respiratory distress may also begin. Third degree is as follows: Patient’s experience increased neuromuscular symptoms. Paresthesia of the larynx may lead to dysphagia and aphagia. Other neuromuscular symptoms include dysarthria, lethargy, muscular incoordination, and ataxia: sensations of

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floating due to numbness; cranial nerve palsies; and muscular fasciculations. Cardiovascular and pulmonary symptoms of hypotension or, more rarely, hypertension; vasomotor blockade; cardiac arrhythmias including sinus bradycardia, asystole, tachycardia, and atrioventricular node conduction abnormalities; cyanosis, pallor, and dyspnea may also occur during this stage of intoxication. Dermatologic symptoms of exfoliative dermatitis, petechiac, and blistering are also often observed. Fourth degree is as follows: Symptoms involve respiratory failure, extreme hypotension, seizures, and loss of deep tendon and spinal reflexes. Although some patients may exhibit impaired mental faculties and may even become comatose, most patients remain fully conscious in 6–24 hours, if the patient survives past 24 hours the prognosis for recovery is good. Otherwise, death is caused by progressive ascending paralysis involving the respiratory muscles. In addition to the symptoms just described, several unusual symptoms have been reported in a few cases. These include hypertension (Deng et al., 1991; Yang, 1967) and cranial diabetes insipidus (Tambyah et al., 1994). In the patients who experienced dramatic increases in blood pressure in response to TTX, Deng et al. (1991) noted they all had preexisting hypertension and sensitivity to sympathetic stimulation. The case involving cranial diabetes insipidus is described below. C. TREATMENT

Although monoclonal anti-TTX antibody to detect TTX has recently been developed, there are no known antidotes or antitoxins to TTX. So treatment of symptoms is supportive. Diagnosis is based on the clinical symptoms and history of consumption of toxic organisms. To reduce exposure to unabsorbed TTX, emetics may be administered if vomiting has not already occurred. In addition, gastric lavage, especially with 2% sodium bicarbonate, followed by activated charcoal is recommended (Sims and Ostman, 1986). Fluid and electrolyte replacement therapy may be used to reduce resulting fluid loss. Atropine may also be given to counteract hypotension and bradycardia (Sims and Ostman, 1986). In cases of respiratory diYculty or failure, oxygen and other ventilatory support, including endotracheal intubation, is often necessary. Lan et al. (1999) suggested that hemodialysis might be an eVective method in the treatments of TTX intoxication. Several researchers report that administration of the anticholinesterases edrophonium and neostigmine enhance the recovery of motor power and markedly reduce paresthesia and numbness (Chew et al., 1983, 1984; Sorokin, 1973; Tan, 1980; Torda et al., 1973). Although these reports are contrary to those of Kao (1966), Chew et al. (1983, 1984) suggest that TTX causes a

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competitive reversible block at the motor end-plate as well as at the motor axon and muscle membrane. The eVectiveness of the anticholinesterases can thus be explained by their action of increasing the quantal release of acetylcholine at the neuromuscular junction, thereby reversing the TTX blockage (Chew et al., 1983). D. PREVENTION

PuVer poisoning occurs when people always intentionally consume toxic specimen or its tissues. In many cases, the consumers cannot diVerentiate the toxic puVer species, the strong toxic parts of puVer, and other TTXbearing animals (toxic gobies, gastropods, crabs, horseshoe crabs, and so on). Hence, TTX-associated poisoning incidents do not minimize or prevent. In Japan, puVer fish is eaten primarily between October and March. Production of the traditional ‘‘fugu-kimo’’ is in the speciality stores of ‘‘puVer fish.’’ Livers separated from the whole body of wild puVer fish are used to squeeze TTX by repeatedly many presses with both hands after being run with a lot of water over night and then boiled with dilute saline solution, resulting in being served as ‘‘dish of puVer fish liver’’ with special vinegar. However, a little bit of TTX is generally left in the prepared liver, which is no problem to cause a food poisoning. Food poisonings due to ingestion of the liver generally do not occur in the speciality stores of ‘‘puVer fish’’ since according to the secret manual handed down there for a long time to eliminate TTX, the liver had been treated. Most of puVer fish poisonings occur in the family. Many patients die due to their incomplete treatment to release TTX from the toxic livers. A practical TTX elimination method is dependent on each speciality store of ‘‘puVer fish.’’ The method is secret, never opened. To prevent the gourmets from ingestion, it is the best way to advise them not to take the liver of toxic puVer. Recently, because harvest of wild puVer is decreasing, cultured puVer is flourishingly reared in many districts of Japan, which is supplied to meet up the big demand of consumer. Hopefully, these puVer fish reared in net in Japan are found to be nontoxic. In near future, nontoxic liver of these puVers generally may be served to the gourmets, with safety. On the other hand, to ensure the safety of consumers in some other Asian countries where the puVer consumption has been increasing, a comprehensive toxicological study should be carried out to identify the toxic and nontoxic puVer fish. On the basis of the results, consumer awareness should be created through several media in order to reduce the poisoning cases. In Japan, as described before, the death number decreases to less than one order although food poisoning cases occur every year. In Taiwan, puVer food poisoning occasionally occur due to mistaking toxic species and dried dressed fish fillet produced from toxic puVer fish

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(Lin et al., 2002a). Hwang et al. (1994a) indicated that the toxic puVer fillet can be treated with 3% NaCl to salt out more than 80% toxin at 4 C for 24 hours. The processed fillets become nontoxic and can be used as the material of dried dressed fish fillet. Another possible way to ultilize toxic puVer meat is first to avoid the toxin contamination from liver and ovary. Then the puVer meat is washed by 0.3% NaCl water, mixed with other nontoxic fish meat to form minced fish meat, and then produced into minced products such as fish ball, fish sausage, ‘‘kamaboko,’’ ‘‘chikuwa,’’ and ‘‘tempura.’’ This process includes the salting out to eliminate the toxin from puVer meat and mixing other nontoxic fish meat to minimize the toxin in the final products. However, any final puVer products should not contain more than 10 MU/g.

III. CAUSATIVE AGENT: TTX A. DISTRIBUTION OF TTX-BEARING ORGANISMS

1. PuVer It is well known that puVer generally contains TTX. TTX intoxication in humans most often results from ingestion of the liver of certain toxic species. Takahashi and Inoko (1889a,b) first attempted to study the chemical and physical properties of TTX partially purified from puVer. Later, TTX was successfully purified from the ovaries of puVer Spheroides rubripes. The structure of TTX was, however, approved in 1964 at the Fourth International Symposium on the ‘‘Chemistry of Natural Products’’ held in Japan, with the molecular formula of C11H17N3O8. Subsequently, the adopted term TTX attracted the attention of toxicologists, chemists, and pharmacologists, particularly for human intoxication. Records of puVer poisoning have been described in the ancient literature from various parts of the world, particularly in Japan and China (Halstead, 1965; Kainuma and Baba, 1984). In Japan, the oldest record of puVer poisoning is found in Nara and Heian eras (800 AD). PuVer frequently appeared in ‘‘senryu,’’ ‘‘haiku,’’ and poetry in the Edo era (1603–1868). These literatures indicate that puVer was a popular food item in those days. In China, puVers were eaten and caused human intoxication since the ancient time, nearly 2000 years ago. Tani (1945) extensively studied the toxicity of Japanese puVer. Of 21 species assayed, 14 were toxic, such as Takifugu porphyreus (purple puVer), Takifugu rubripes (tiger puVer), Takifugu pardalis (panther puVer), Takifugu obscurus (‘‘mefugu’’), Takifugu flavidus (towny puVer), Takifugu snyderi (vermiculated puVer), T. poecilonotus (finepatterned puVer), Takifugu chrysops (purple puVer),

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T. niphobles (grass puVer), Takifugu xanthopterus (striped puVer), Takifugu stictonotus (spotty back puVer), Takifugu pseudommus (‘‘radamashi’’), Lagocephalus inermis (smooth-backed blowfish), and Canthigaster ribulata (scribbled toby). In addition to these species, Takifugu vermicularis (pear puVer), Tetraodon alboreticulatus (‘‘shiroamifugu’’), Takifugu chinensis (eye spot puVer), Lagocephalus sceleratus (slack-skinned puVer), Chelonodon patoca (milkspotted blaasop), and Takifugu exascurus (‘‘mushifugu’’) were identified as TTX-bearing puVer. The toxicity of 23 species of Taiwanese puVers was later studied by Hwang et al. (1992a). Only two puVers, Lagocephalus gloveri (brown-backed toadfish) and Lagocephalus wheeleri (brown-backed toadfish), are nontoxic in all tissues and may be used as materials for producing dried dressed fish fillets. But Lagocephalus lunaris (green toadfish), T. oblongus (oblong toadfish), and T. niphobles are the most toxic in all tissues and occasionally cause food poisoning incidents in Taiwan (Table VI). Later, Lin et al. (2002b) reported that the imported puVer Tetraodon ocellatus contained moderate amounts (100–999 MU/g) of toxin in skin and viscera and another one Tetraodon nigroviridis contained weak amounts (10–99 MU/g) of toxin in skin. The toxin from both species was composed of TTX and anhydroTTX. In Japan, the toxicity of puVer is related to their spawning season, with the highest toxicity levels occurring between March and June, though the pattern varies to some degree among species. The toxin is concentrated in the ovaries, liver, and often the skin, making the female puVer more toxic than the males, especially during spawning season. The parts of the puVer considered less

TABLE VI THE TOXICITY OF TAIWANESE PUFFERS

Fish species

Ovary

Testicle

Liver

Gall

Skin

Intestine

Muscle

Lagocephalus gloveri Lagocephalus wheeleri Lagocephalus inermis Lagocephalus lunaris Takifugu xanthopteus Takifugu oblongus Takifugu flavidus Takifugu alboplumbeus Takifugu niphobles

— — ⋆⋆ ⋆⋆⋆ ⋆⋆⋆ ⋆⋆⋆ ⋆⋆⋆ ⋆⋆⋆ ⋆⋆⋆

— — ⋆ ⋆ — ⋆⋆ ⋆⋆ — ⋆⋆

— — ⋆⋆ ⋆⋆⋆ ⋆⋆⋆ ⋆⋆⋆ ⋆⋆⋆ ⋆⋆ ⋆⋆⋆

— — ⋆⋆ ⋆⋆ ⋆⋆ ⋆⋆⋆ ⋆⋆⋆ ⋆⋆⋆ ⋆⋆⋆

— — ⋆ ⋆⋆ ⋆ ⋆⋆ ⋆⋆ ⋆ ⋆⋆

— — ⋆⋆ ⋆⋆ ⋆⋆ ⋆⋆ ⋆ ⋆⋆ ⋆⋆

— — — ⋆⋆ — ⋆⋆ ⋆ ⋆ ⋆

⋆⋆⋆, much higher toxicity, death after ingesting less than 10 g; ⋆⋆, high toxicity; ⋆, low toxicity; —, nontoxicity. Source: Hwang et al. (1992a).

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toxic are the musculature, fins, and testes. Although muscle tissue may be toxic in at least three puVer species, T. pardalis, L. lunaris, and C. patoca, no fatal cases of tetrodotoxication have yet been reported due to eating the meat of puVer as ‘‘sashimi,’’ or sliced raw fish, in Japan. On the other hand, the dishes known as ‘‘chiri,’’ fillets partially cooked in a stew of skins, livers, intestines, or testis, and ‘‘kimo,’’ partially cooked livers, have been associated with much fatal intoxication. Japanese toxic puVer, especially the species from northern parts generally contains an elevated level of TTX in the liver and gonad, while C. patoca inhabiting Okinawa and south of Amami-Oshima Islands, the subtropical part of the country, contains the highest toxicity in the skin (Khora et al., 1991) and comparatively high toxicity in ovary, muscle, and testis (Mahmud et al., 2001). Hashimoto (1979) described that the established toxicological knowledge on puVer of the Ryukyus and Amami-Oshima Islands indicating the possible environmental eVect on their toxicity. Recently, each toxin of Thai brackish water puVer T. nigroviridis and Tetraodon steindachneri has been characterized as same TTX. Toxin distribution pattern was similar to that of C. patoca. TTX as the toxic principle was also detected in the marine puVers from Taiwan and Bangladesh (Hwang et al., 1988; Mahmud et al., 1999a,b, 2001). Seasonal individual and local variations of toxicity and toxin composition in puVer are occasionally observed. For example, panther puVer (T. pardalis) collected from the northern Pacific Coast of Japan exhibited much higher toxicity in the liver (mean 935 MU/g) compared to that form the southwestern part (337 MU/g). PuVers Arothron mappa, Arothron manilensis, C. patoca, Arothron nigropunctatus, Arothron hispidus, Arothron Stellatus, and Arothron reticularis collected from the Philippine water showed a remarkable individual diVerence in toxicity (Sato et al., 2000). With a few exceptions, considerable amounts of STXs were detected in these species as well as in the ovary of Japanese marine puVer Arothron firmamentum (Nakashima et al., 2004), although puVers inhabiting Japan contained mainly TTX. The toxic principle of the Thai freshwater Tetraodon fangi was reported as TTX (Laobhripatr et al., 1990), but later the major toxin was identified as STX (Sato et al., 1997). It is suggested that either TTX or PSP is overwhelmingly dominant in puVer. Besides TTX, some researchers reported the presence of TTX derivatives in puVer. Nakamura and Yasumoto (1985) first isolated and identified tetrodonic acid (TDA), 4-epiTTX and anhydroTTX from T. pardalis and T. poecilonotus. Subsequently, 11-norTTX-6(R)-ol, 6-epiTTX, and 11-deoxyTTX from T. niphobles and 11-norTTX-6(S)-ol from A. nigropunctatus were isolated and characterized (Endo et al., 1988; Yotsu et al., 1992a).

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2. Newts Unexpectedly, TTX was detected in the eggs of a newt (Taricha torosa) by Mosher et al. (1965), which was the first report of the occurrence of this toxin in the animals other than puVer. Later, TTX was detected also in several species of newts from various countries (Wakely et al., 1966). In Japan, there are three species of newts—Cynops pyrrhogaster, Cynops ensicauda, and Tylototiton andersoni. Among them, C. ensicauda from Okinawa and C. pyrrhogaster from main Japan were reported with special emphasis on their toxin profiles. TTX and its analogues 4-epiTTX, 6-epiTTX, 4,9-anhydro-6-epiTTX, 11-deoxyTTX, 11-deoxy-4-epiTTX, and 4,9-anhydroTTX were detected from C. ensicauda (Yasumoto et al., 1988). C. pyrrhogaster toxin was found to consist of TTX, 6-epiTTX, and 11-deoxyTTX (Yotsu et al., 1990a,b). However, T. andersoni was shown as a nontoxic species. Yotsu et al. (1990a,b) also reported the occurrence of 6-epiTTX and 11-deoxyTTX in a few species of newts Taricha granulosa, Taricha oregon, Triturus vulgaris, Notophthalmus viridescens, and Ambystoma tigrinum from the United States, in Paramesotriton hongkongensis from China, and Triturus alpestris from Italy. Toxin concentrations, measured by a fluorometric HPLC analyzer, were higher in T. granulosa (882 MU/g) and N. viridescens (673 MU/g) among them. A total of 382 specimens of the Japanese C. pyrrhogaster were collected from western Japan and assayed for toxicity and toxin profiles (Tsuruda et al., 2001). Most of them showed toxicity scores ranging from 5 to 370 MU/g. Among the parts, the skin and muscle showed higher toxicity score (56 MU/g) than the liver, stomach, intestine, and gonad whose scores ranged from less than 2 to 33 MU/g. Little seasonal, but large gender and regional variations of toxicity were recognized (Table VII). C. pyrrhogaster toxin consists of TTX and 6-epiTTX as the main components, and of 4-epiTTX, 4,9-anhydro-6-epiTTX, and 4,9anhydroTTX as the minor ones. The marked interspecies diVerences in toxin profile may reflect their metabolic pathway of accumulated TTX. 3. Gobies Goby toxin was isolated as crystalline state from the goby Yongeichthys criniger inhabiting the Amami-Oshima and the Ryukyu Islands and was identified from IR, 1H NMR, and elemental analysis as TTX (Noguchi and Hashimoto, 1973). There was a rumor in these areas that farmers used to treat the dry goby as a rodent killer in the fields. In the subsequent study, goby fish was found to be toxic (Noguchi et al., 1971). TTX concentration was generally high in the skin (with fins), followed by the viscera and muscle. In a few toxic specimens, the highest toxicity was in the mature testis. A rather wide local variation of toxicity was sharply observed (Table VIII).

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TABLE VII LOCAL AND SEXUAL VARIATION OF TOXICITY IN A JAPANESE NEWT C. PYRRHOGASTER

Toxicity (MU/g) Place of collection

Date of collection

Sexa

Range

Mean  S.D.b

Kishiku

April 1997

Nagasaki

May 1998

Togitsu

September 1997

Ohseto

June 1997

Isahaya

July 1997

Ohmura

May 1997

Nakabaru

June 1997

Kiyama

June 1997

Gounoura

May 1997

Houjou

May 1999

? / ? / ? / ? / ? / ? / ? / ? / ? / ? /

6–11 <5–35 12–160 12–250 <5–68 <5–240 64–140 5–37 67–210 27–370 <5–42 6–33 9–17 24–59 9–10 <5 <5–29 <5–13 15–150 32–120

82 15  13 72  47 96  63 24  28 120  103 83  33 21  12 127  52 163  138 12  17 18  12 12  3 50  15 10  1 00 93 46 77  49 68  37

Five specimens were assayed for each sex. Mean  S.D. was calculated on the assumption that toxicity of less than 5 MU/g was zero. Source: Tsuruda et al. (2001). a b

Several fatal poisoning incidents and frequent deaths of duck, both due to ingesting the goby occurred in Taiwan (Lin et al., 1996; Yang, 1967). An acute food poisoning due to ingestion of the gobies Y. nebulosus and Sillago japonica occurred in southern Taiwan, aVecting two male persons (Lin et al., 1999). TTX was identified as the causative agent of this food poisoning. The highest toxicity scores were 7650 and 1460 MU per specimen of Y. nebulosus and S. japonica, respectively. The toxicity of 12 species of Taiwanese gobies was examined and the specimens of three species of gobies, Y. nebulosus, Prachaeturichtys palynena, and Radigobius caninus were found to be toxic. The highest toxicity of Y. nebulosus was 4998 MU per specimen (Lin et al., 2000). 4. Frogs The discovery of TTX in newts and goby fish tempted scientists to search for new TTX-bearing animals. In 1975, TTX unexpectedly detected from the skin of Costa Rican frogs (average weight 1.3–1.8 g) Atelopus varius varius,

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TABLE VIII LOCAL VARIATION IN TOXICITY OF THE GOBY COLLECTED FROM JAPAN AND TAIWAN

Place of collection Island/Country

Site

Amami

Tekebu Ikuma Shinokawa Nakamagawa Naguragawa Hukidougawa

Iriomote Ishigaki Taiwan

Number of specimens tested 35 19 20 11 5 15 24

Toxicity (MU/g) Range (MU/g)a

Mean  S.D.

<20–160 <20–150 <20–40 <20–20 <20 – <20–120

64.9  5.5 38.9  9.7 8.0  3.0 5.5  2.8 – 66.7b 30.0  6.2

The value <20 MU/g is calculated as 0 MU/g. Fifteen specimens were combined and assayed. Source: Noguchi et al. (1971). a b

Atelopus varius ambulatorius, and Atelopus chiriquiensis (Kim et al., 1975). Toxicity in the skin ranged from 100 to 120 MU per frog. A. chiriquiensis toxin consisted of a mixture of TTX (
30%) and chiriquitoxin whose structure replaces –C(12)-CH2OH in TTX with –CH(OH)CH(NH3þ)COO–. Pavelka et al. (1977) found TTX and TTX-like compound in the eggs remained in a bound form. In 1992, TTX and its derivatives 4-epiTTX and 4,9-anhydroTTX were detected in the toad Atelopus oxyrhynchus from Venezuela (Yotsu et al., 1992b). This species is able to retain a high level of TTX and its analogues in long captive position under artificial environment. Besides its occurrence in the frogs of the family Bufonidae, TTX and TTX-like substance were detected in the Panamanian frog Colostethus inguinalis (Family: Dendrobatidae) (Daly et al., 1994) and Brazilian frog Brachycephalus ephippium (Family: Brachycephalidae) (Sebben et al., 1986), respectively. The skin extract from C. inguinalis showed 0.05–6 MU TTX-equivalents per individual frogs in the mouse assay, showing a remarkable variation in toxin concentration. As described above, TTX-bearing frogs/toads had been confined to certain species of genus Atelopus (Family: Bufonidae), Brachycephalus (Family: Brachycephalidae) and Colosthetus (Family: Dendrobatidae) from South and Central America (Daly et al., 1994; Kim et al., 1975; Mebs and Schmidt, 1989; Mebs et al., 1995; Pavelka et al., 1977). TTX was detected also in the skin of rhacophoridid frog Polypedates sp. from a subtropical country (Tanu et al., 2001). In the mouse assay, the skin toxicity ranged from 31 to 923 MU/g. Bigger specimens generally showed higher toxicity in comparison with that of smaller one and a wide local variation of toxin concentration was observed. In the skin extract, TTX was found by HPLC, electrospray ionization-time of flight/mass spectrometry

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(ESI-TOF/MS), and 1H NMR. In ESI-TOF/MS analysis, protonated molecular ion peak (M þ H)þ of the toxin appeared at m/z ¼ 320.1103, suggesting the molecular weight to be 319.1025, the same as that of authentic TTX (C11H17N3O8 ¼ 319.10116). The skins of previously described Atelopus frogs from northern Panama and Costa Rica contain TTX and another chemically distinct but related toxin (chiriquitoxin). Kim et al. (2003) reported that Atelopus zeteki, a bright yellow-colored frog with variable black markings and only in a single local region (Valle de Anton) in Panama, was found to contain a new toxin (zetekitoxin). The molecular weight was determined by high-resolution electron spray ionization mass spectra (HRESIMS) (50% MeOH) [M þ H]þ, m/z, 553.1369. The observed fragmentation [–SO3 þ H]þ, m/z, 473.1725 suggested the presence of O–SO3H or N–SO3H group. NMR experiments TOCSY, HSQC, 13C–1H, and 15N–1H HMBC revealed the STX backbone structure. The N7 substitution is CH2O–CO–NHOH. The amino group of this carbamate was determined by the fragmentation pattern to be hydroxylated. Other substitution moieties are suggested by rotating frame overhause eVect spectroscopy (ROESY) and nuclear overhauser enhancement spectroscopy (NOESY). The C11 substitution is OSO3H and other moieties of C6 and C11 are suggested to form fused bicyclic amide ring. Zetekitoxin is a new STX derivative which has a higher molecular weight than that of STX (Kim et al., 2003). 5. Horseshoe crab In the spawning season, female specimens of the horseshoe crab possess a large amount of eggs which Thai people are fond of eating, and which result in sporadic outbreak of poisoning (Banner and Stephens, 1966; Kanchanapongkul and Krittaya, 1995; Trishnananda et al., 1966). There are two species of horseshoe crab C. rotundicauda and Tachypleus gigas inhabiting Thailand. The causative species was identified as C. rotundicauda in all the poisoning incidents. Sometimes, T. gigas is mistaken for C. rotundicauda, resulting in poisoning incident (Banner and Stephens, 1966). Both are very similar to each other. The morphological diVerences between the toxic species (C. rotundicauda) and nontoxic one (T. gigas) are as follows: total length is approximately 300 mm in C. rotundicauda and 450 mm in T. gigas. The surface of the tail is smooth in the former while equipped with many prickles in the latter. Cross section of the tail is round in the former while triangle in the latter. The symptoms of horseshoe crab poisoning are similar to those of paralytic shellfish poisoning or TTX intoxication. The causative agent of poisoning was characterized as PSP (Fusentani et al., 1982, 1983). In 1987,

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however, the toxin of the same species from Thailand was shown to consist of TTX as the major toxin, with the highest score of 16 MU/g egg (Kungsuwan et al., 1987). Tanu and Noguchi (1999) reported the occurrence of TTX in the same species from Bangladesh. Most of 39 specimens collected were toxic. Among the tissues, the egg showed the highest toxicity, ranging from 2.0 to 7.4 MU/g (Table IX). Toxicity scores of the testis and viscera were less than 5 MU/g. The eggs showed a higher toxicity than the testis. However, the toxicity level in all tissues of Bangladeshi specimens was lower than the quarantine limit for human (10 MU as TTX per gram edible part). As a matter of fact, there has been no oYcial record of horseshoe crab, and also no outbreak of poisoning. It is assumed that TTX is involved in a defense mechanism to protect the horseshoe crab eggs from predators (Jeon et al., 1984; Sheumack et al., 1984). Horseshoe crab mainly feeds on mollusks, arthropods, and detritus (Chatterji et al., 1992). Some bacteria inhabiting the marine sediments might be the primary origin of TTX for this crab. 6. Xanthid crabs The occurrence of TTX in xanthid crabs Atergatis floridus, Atergatopsis germaini, Zosimus aeneus, Lophozozymus pictor, Demania reynaudi, and Eriphia sebana expands the diversity of TTX-bearing animals. The toxin composition of xanthid crabs A. floridus and Z. aeneus widely diVered depending on the habitat. These two species had been known to possess only PSP (Daigo et al., 1985; Koyama et al., 1981; Yasumoto et al., 1981). Noguchi et al. (1969) isolated crabtoxin from Z. aeneus in pure state and characterized as STX from its IR, specific rotation, specific toxicity, and TLC. Noguchi et al. (1983) first detected TTX as the major toxin (90%) in A. floridus collected from Miura Peninsula near Tokyo. In 1986, TTX was again reported as the major toxin in A. floridus collected from Kojima of Ishigaki Island in Okinawa, Oita, and Kumamoto Prefecture, with relatively low toxicity levels (38–80 MU/g) (Noguchi et al., 1986a,b). In early 1990s, two novel TTX analogues 11-oxoTTX and 11-norTTX-6(R)-ol were detected in A. floridus collected from the former Okinawa (Arakawa et al., 1994). In Z. aeneus specimens from the Philippines, TTX was identified as a minor toxin, with PSP as the main (Arakawa, 1988). Out of 32 specimens, 29 were toxic, with the toxicity scores of 14–850 MU/g. In this connection, Z. aeneus was examined for resistibility against PSP and TTX. The MLD of TTX in this species was estimated to be 1000–2000 MU/20 g body weight, in a strong contrast to the MLD (<1 MU/20 g) in nontoxic species (Koyama et al., 1983).

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D.-F. HWANG AND T. NOGUCHI

TABLE IX INDIVIDUAL TOXICITY OF HORSESHOE CRAB C. ROTUNDICAUDA FROM BANGLADESH

Date of collection November 1998

December 1998

October 1999

November 1999

Specimens number

Sex

Body weight (g)

1 2 3 4 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7

? ? / / ? / / / / / / / / ? ? ? / / / / / / / / / / ? ? ? ? ? / /

121 172 275 260 190 255 232 249 243 237 223 241 254 180 122 158 237 246 275 210 190 221 186 240 250 265 187 190 210 168 180 230 220

Toxicity (MU/g) Egg

Testis

Viscera

— — 7.0 5.3 — 6.7 6.6 7.4 6.2 5.4 5.8 5.2 6.2 — — — 2.6 2.7 3.2 3.7 3.8 2.8 3.8 <2 4.0 3.3 — — — — — 3.8 4.4

2.6 3.9 — — 4.8 — — — — — — — — 3.8 4.2 <2 — — — — — — — — — — <2 <2 3.6 4.2 2.6 — —

2 2.5 3 2.2 2.8 2.8 3.9 3.2 2.9 3.3 2.7 3.5 3.8 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2

Source: Tanu and Noguchi (1999).

The presence of TTX in Z. aeneus irrespective of the habitat, in addition to its resistibility against TTX, suggests that this toxin is essential for this species to survive. In Taiwan, xanthid crabs A. floridus, A. germaini, D. reynaudi, Z. aeneus, Xanthias lividus, and L. pictor were reported as TTX as well as PSP-bearing

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TABLE X TOXICITY AND TOXIN COMPOSITION OF SIX TOXIC CRAB SPECIES COLLECTED FROM TAIWAN

Species

Toxin composition

Zosimus aeneus

TTX (82) GTX1–4 (18) TTX (89) GTX1, 3 (11) GTX (50) hySTX (40) STX (7) TTX (3) TTX (85) GTX1–4 (15) TTX (88) GTX2–4, neoSTX (12) TTX (83) GTX1–4 (17)

Lophozozymus pictor Atergatopsis germaini

Atergatis floridus Demania reynaudi

Xanthias lividus

Approximate toxicity (MU/g) 7 5 40

Collection place Lanyu, Wanlitung Hsiaoliuchiu Keelung Keelung

8

Hsiaoliuchiu

3

Keelung

6

Lanyu, Hsiaoliuchiu

Data in parentheses represent the percentage. Source: Hwang and Tsai (1999) and Tsai et al. (2002).

crustaceans (Table X) (Hwang, 2003a,b; Hwang and Tsai, 1999; Tsai et al., 1995, 1996, 1997a,b, 2002). In A. floridus, 85% of the total toxicity was accounted for TTX and 15% by GTX1–4, whereas in A. germaini, 3% by TTX, 50% by GTX3, 7% by neoSTX and STX, and 40% by an unknown toxin. In D. reynaudi, 88% by TTX and 12% by GTX2, GTX4, and neoSTX, L. pictor occasionally has been causing human intoxication in Taiwan as well as Singapore, whose toxin was identified as TTX in the former country and palytoxin in the latter. In Z. aeneus and X. lividus, 82–83% of the total toxicity was accounted for TTX and 17–18% by GTX1–4. The toxic composition and toxicity in toxic crabs collected from diVerent areas are shown in Table XI (Hwang and Tsai, 1999). 7. Blue-ringed octopus Blue-ringed octopus Hapalochlaena (Octopus) maculosus (Family: Octopodidae) has so far been identified as the only lethal and TTX-bearing octopus. It is found in shallow coral and rock pools in the waters around Japan, Taiwan, Philippines, and Australia. This octopus probably produces a poison in the salivary glands, which seems to be used for hunting and defense purposes. Many researches attempted to elucidate the causative agent of human fatalities attributed to the bite from this species. Although, it was initially claimed to be the new toxin,

166

TABLE XI Species Xiphosuridae Carcinoscorpius rotundicauda Coenobitidae Birgus latro Xanthidae Zosimus aeneus

Atergatis floridus

Toxin

Approximate toxicity (MU/g)

Place

PSP

40

Thailand

TTX Unknown

10 1

Thailand South Pacific Ocean Ryukyu

STX, neoSTX, dcSTX, GTXs STX, neoSTX, GTXs STX, neoSTX, GTXs, TTX PSP, TTX STX, neoSTX, GTXs PSP TTX, GTX1–4 (minor) STX, neoSTX TTX and related compounds TTX, STX (minor) GTXs STX, neoSTX, GTX2 (minor) TTX TTX, GTX1–4 (minor)

2000 20 220 20 9 50 7 1400 20 50 180 63 10 8

Ryukyu Australia Philippines Philippines Fuji Palau Island Taiwan Ryukyu Ishigashi Island Miura Peninsula, Japan Chiba, Japan Fuji Australia Taiwan

D.-F. HWANG AND T. NOGUCHI

TOXIC COMPOSITION AND TOXICITY IN TOXIC CRABS

Platypodia granulosa Lophozozymus pictor

Atergatopsis germaini Demania alcala Demania reynaudi

Source: Hwang and Tsai (1999).

400 1400 5 6000 5 40 3000 8 3 100 9 16 2 4 3

Ryukyu Philippines Australia Singapore Taiwan Taiwan Philippines Philippines Taiwan Philippines Australia Ryukyu Australia Ryukyu Ryukyu

PSP or GTX1

4

Australia, Ryukyu

PSP

1

Australia, Ryukyu

TETRODOTOXIN POISONING

Demania toxica Eriphia sebana Eriphia scabricula Leptodius sanguineus Neoxanthias impressus Pilumnus vespertilio Portunidae Thalamita sp. Grapsidae Grapsus albolineatus

70% STX, 30% unknown toxin Palytoxin GTX2 Isomer of palytoxin TTX, GTX1–3 (minor) STX, GTX, TTX (minor) Palytoxin Palytoxin-like TTX (minor), GTX2–4, neoSTX, Unknown STX, neoSTX, GTX1, 2 STX, neoSTX, GTXs PSP PSP STX, neoSTX, GTXs

167

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D.-F. HWANG AND T. NOGUCHI

maculotoxin (Sutherland and Lane, 1969), finally it was identified as TTX (Sheumack and Howden, 1978). Its bite can paralyze the muscle much quicker than eating the puVer. The rapid response might be due to the prompt action of the intruded toxin to the neuron, in comparison to ingested toxin that ultimately aVects the brain through a long gastrointestinal pathway. An adult human may be killed within 20 minutes after the bite, if not treated immediately and properly. The toxin was found to exist not only in the salivary gland but also in the ovary of this octopus (Hwang et al., 1989; Sheumack et al., 1984). All octopuses generally have ink sac where a lot of ink is pooled. When they meet enemy or are attacked by them, they secrete a lot of ink from their sac and can conceal themselves to escape. Since they have a strong defense substance of TTX and need not use ink, the sac has lost above function. Significance why this blue-ringed octopus has TTX seems to use TTX for capturing preys or defensing itself from enemy. 8. Gastropods In December 1979, a paralytic poisoning by ingesting the digestive gland of trumpet shell C. sauliae (Family: Cymatiidae) occurred in Shizuoka Prefecture, Japan. The responsible toxin of poisoning was identified as TTX, and this was the first report of the occurrence of TTX in a gastropod (Narita et al., 1981). Since then, TTX was also detected in other gastropods such as Tutufa lissostoma, Natica lineata, N. vitellus, Rapana rapiformis, and R. venosa venosa from Japan and Taiwan (Cheng et al., 1996; Hwang et al., 1990a, 1991a,b,c, 1992a,b,c,d, 1994b; Jeon et al., 1984; Narita et al., 1984; Noguchi et al., 1981; Yasumoto et al., 1981). In 1980, specimens of Japanese ivory shell, Babylonia japonica (Family: Buccinidae) were collected from Fukui Prefecture, Japan and assayed (Noguchi et al., 1981). The highest toxicity (as TTX) recorded was 53 MU/g digestive gland. In 1981 and 1982, 22 of T. lissostoma (Family: Bursidae) specimens were collected in Shizouka Prefecture, 17 of them were toxic (Noguchi et al., 1984). Toxicity was detected in the digestive gland exclusively, with the highest score of 700 MU/g. The occurrence of TTX and anhydroTTX in a gastropod mollusk N. lineata (lined moon shell) was reported by Hwang et al. (1990a). The highest toxicity (720 MU/g) was found in the muscle, while the other parts including digestive gland were much less toxic (12–28 MU/g). Specimens showed a wide individual variation in toxicity. The other small toxic gastropods in Taiwan, including N. vitellus, Polinices tumidus, and Polinices didyma, contain higher amounts of TTX in the muscle, following with digestive gland and other tissues (Table XII). The gastropods Z. scalaris, N. clathrata, and Zeuxis suZatus contain TTX in all tissues. N. clathrata also contain minor amounts of PSP in the spring. On the other hand, the small

TETRODOTOXIN POISONING

169

TABLE XII THE TOXICITY AND TOXIN COMPOSITION OF SEVERAL GASTROPODS COLLECTED

Species Natica vitellus Natica alapailionis Natica lineata

Niotha clathrata Zeuxis scalaris Zeuxis castus-like Oliva miniacea Oliva mustelina Oliva hirasei Babylonia formosae Rapana rapiformis Rapana venosa venosa

Toxin composition

Approximate toxicity (MU per specimen)

Main toxin distribution

Collection place

TTX TTX TTX TTX TTX, TTX TTX TTX, TTX, TTX, TTX TTX TTX TTX TTX TTX

20 14 6 93 47 21 4 139 4 42 146 35 58 1 30 170

Muscle Muscle Viscera Muscle Muscle Muscle Viscera Viscera Viscera Viscera Muscle Muscle Muscle Viscera Viscera Viscera

Kaohsiung Pingtung Pingtung Kaohsiung Pingtung Chaiyi Tainan Pingtung Pingtung Pingtung Pingtung Pingtung Pingtung Pingtung Kaohsiung Ilan

PSP PSP PSP PSP

toxic gastropods O. miniacea, O. mustelina, and O. hirasei contain only TTX in the muscle, not in the digestive gland (Hwang, 2003a,b; Hwang et al., 2003). The incidents of gastropod poisoning have occasionally occurred in Taiwan (Hwang et al., 1995, 2002a,b, 2003, 2005; Shiu et al., 2003). Outbreaks of paralytic snail poisoning recently occurred in China. The epidemiological characteristics of this disease from an outbreak in Xhoushan City were recorded. Forty-two outbreaks of paralytic snail poisoning, involving 309 cases, occurred from 1977 to 2001 (Figure 3). Sixteen people (5.2%) died, 48 people (15.5%) required intubations, and 140 people (45.3%) required emergency hospital treatment as a result of these outbreaks. Outbreaks included multiple marine gastropod species, major species Z. samiplicutus, and occurred primarily during the summer (June–August) on 11 islands with high population densities. Peak numbers of outbreaks and amounts of gastropod toxicity occurred from 1978 to 1979 and from 1992 to 1994. Toxicity varied depending on specimen, region, and season. The toxin involved was identified as TTX (Shui et al., 2003; Sui et al., 2002). 9. Starfish Noguchi et al. (1982) first identified TTX in a starfish Astropecten polyacanthus, suggesting that this species is involved in intoxication mechanism in the TTX-bearing trumpet shell C. sauliae. Subsequently, two starfish

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D.-F. HWANG AND T. NOGUCHI

Astropecten latespinosus and Astropecten scoparius were also added to the list of TTX-bearing echinoderms (Maruyama et al., 1984, 1985; Miyazawa et al., 1985) from the results of toxicity examination of starfish collected from all over Japan. In the mouse assay, A. latespinosus and A. scoparius showed the toxicity scores of 13 and 5.7 MU/g, respectively. These findings made sure of TTX intoxication mechanism in gastropods to come from their food chain. Afterward, TTX study gave a clue to be expanded in many aspects. In 1985, TTX was also reported from starfish A. polyacanthus and A. scoparius from Seto Inland Sea, Japan, with the highest toxicity scores of 520 and 46 MU/g, respectively (Miyazawa et al., 1985). A. scoparius was found to contain TTX in Taiwan as well, with the highest toxicity of 354 MU/g in the viscera (Lin et al., 1998b). Toxin was characterized by HPLC and GC–MS analysis. GTX2–3 and STX (12%) were detected as the minor toxins. Main TTX intoxication mechanism of these carnivorous starfish is estimated to come from their food webs (Lin and Hwang, 2001). The toxicity of starfish was suddenly increased in September and reached to the maximum level in November. The highest level of toxicity was 16,821 MU per specimen. It was found that a significant amount of the toxin was there in the gonad of starfish. The starfish matured between October and November. The toxicity of gonad was increased as the maturity increases and reached to the top in November. The preyed animals in the digestive gland of starfish A. scoparius were mainly mollusks Bivalvia and Gastropoda. The amount of Gastropoda was marked by increase in number in October and November. The starfish A. scoparius might mainly accumulate high amount of TTX from smaller gastropod Umborium suturale and Natica pseustes. Furthermore, the starfish Astropecten vappa is reported to contain only TTX in Taiwan (Tsai et al., 2004). 10. Flatworms Miyazawa et al. (1986, 1987) first reported the occurrence of TTX in marine flatworms. Ten specimens of a flatworm Planocera multitentaculata were collected from the identical zone of the Seto Inland Sea, Ehime Prefecture, Japan and studied. The toxin was characterized as TTX by TLC, electrophoresis, and GC–MS analyses. The average lethal potency was 300 MU/g. In the same year, another flatworm Planocera reticulata was demonstrated to have TTX (Jeon et al., 1986). Anatomical distribution of TTX in P. multitentaculata from Hiroshima Prefecture, Japan was also examined (Asakawa et al., 2003), and it was found that the oviduct showed the highest toxicity (2000–3420 MU/g), followed by digestive organs (1400 MU/g) and others. The eggs laid by the flatworm showed an extremely high lethal

TETRODOTOXIN POISONING

171

TABLE XIII TOXICITY OF EGGS LAID BY THE FLATWORM P. MULTITENTACULATA

Date of collection

Place of collection

Weight of egg masses (g)a

May 8–18, 1985 May 6 May 22 May 22–June 1 June 3 June 3–8 June 16 June 22 June 22–July 5 July 5 July 5–13

Laboratory aquariumb Kata, Wakayama Iwashijima, Hiroshima Laboratory aquarium Iwashijima, Hiroshima Laboratory aquarium Asanami, Ehime Iwashijima, Hiroshima Laboratory aquarium Iwashijima, Hiroshima Laboratory aquarium

0.6 0.5 0.9 3.4 1.4 2.0 1.3 0.6 1.0 0.06 1.6

Toxicity (MU/g) 10,700 980 3660 3820 5600 2530 7600 8100 3810 1000 6600

Mean toxicity of parental worm 1160 170c 1330c – 700c – 220c 1210c 1350c – –

(Mean  S.E.: 5760  870) Combined sample in most cases. The parental flatworms reared in the laboratory aquarium have been collected from Iwashijima, Hiroshima. c Mean toxicity of flatworms collected from around the egg masses. Source: Miyazawa et al. (1986). a b

potency (5760  870 MU/g), with the highest score of 10,700 MU/g, which were 2–50 times higher than those of parental flatworm (Table XIII). The eggs could use such a high level of TTX for a defensive purpose against predators. Main TTX intoxication mechanism is also estimated to come from their food webs. In this case, the part of TTX in the flatworms is very much distinct to be a defense substance to prevent the eggs from predators. In this connection, according to Chinese literature, Chinese toxic puVer feeds on big flatworms. This puVer may be intoxicated with TTX through this food chain. Lin et al. (1998a) reported that the specimens of ‘‘torafugu’’ T. rubripes cultured in inland planes were nontoxic, but those in the coastal areas were toxic in the period between January and March in Taiwan. The toxin was found only in the liver and ovary of puVer. The toxic flatworm Stylochus orientalis was found to be the toxin source of cultured puVer. Except P. multitentaculata and S. orientalis, P. reticulata, Stylochus ijimai, and Koinostylochus spp. have been reported containing a high amount of TTX, but other species as Stylochoplana clara, Notoplana humilis, and Notocomplana koreana exhibited a low or trace toxicity (Jeon, 1985).

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11. Ribbon worms The occurrence of TTX in flatworm led scientists to extensive studies on nemertines, resulting in detection of TTX for the first time in two species of ribbon worm Lineus fuscoviridis and Tubulanus punctatus, with the highest score of 503 and 540 MU/g, respectively (Miyazawa et al., 1988). In addition to TTX, toxic TDA-like substance was detected from these ribbon worms. Kem (1976) isolated four polypeptides from the toxic ribbon worm Cerebratulus lacteus that showed a selective toxicity variety of animals, suggesting that the toxin is also used for a defensive purpose. Subsequently, the toxic mucus secretion of C. lacteus was shown to consist of cytotoxins and neurotoxins (Kem and Blumental, 1978). Later, Cephalothrix linearis from Shizuoka Prefecture, Japan was calculated to be a TTX-bearing species (Ali et al., 1990). The highest lethal potency was calculated to be 23,000 MU/g whole body, which is about 40 times greater than those scores of L. fuscoviridis and T. punctatus (Miyazawa et al., 1988). In C. linearis, the toxin was densely distributed in the proboscis (22,000 MU/g), a special device to prey food animals, followed by the other parts (13,600 MU/g). Its mucus secretion also contained TTX at a high level. This species showed rather wide individual and seasonal variations in lethal potency. The ribbon worms have ability to secrete a considerable amount of toxin when stimulated, suggesting that they utilize TTX for both defensive and oVensive purposes. The toxin secreted from C. linearis was mainly composed of a TDA-like substance, which showed same behavior as TDA in HPLC analysis. In this connection, Noguchi et al. (1991) purified a TDA-like substance from C. linearis and the flatworm P. multitentaculata, which exhibited a specific toxicity of 700 MU/mg. The TDA-like substance clearly diVers from TDA which is completely nontoxic. However, mass number of TDA-like substance is 320, not same as that of authentic TTX (319 dalton), suggesting that the TDA-like substance is a precursor of TTX or tautomer with Hþ. Between April 1998 and December 2001, during the surveillance of the toxicity of various marine fouling organisms in Hiroshima Bay, Hiroshima Prefecture, specimens of the ribbon worm Cephalothrix sp. showed very high toxicity of 25,590 MU/g whose main toxin was identified as TTX from various instrumental analyses. The component patterns agree with that of the C. linearis (Asakawa et al., 2003). 12. Annelids The occurrence of TTX, 4-epiTTX, and anhydroTTX in the annelid Pseudopotamilla occelata was detected by Yasumoto et al. (1989). During its investigation, the highest toxicity was recorded to be 24 MU/g. TTX as the

TETRODOTOXIN POISONING

173

TABLE XIV TOXICITY OF FOUR SPECIES OF ANNELID COLLECTED FROM THE SETO ISLAND SEA, JAPAN

Species

Body weight (g)

Highest toxicity (MU/g)

Ratio of toxic specimens

Lepidonotus helotypus Halosydna brevisetosa Hermenia acanthopeltis Harmothoe imbricata

0.7–1.9 0.3–0.4 0.3 2.1

110 14 20 4

8/14 2/3 1/1 1/1

Source: Yasumoto et al. (1989).

major toxin was identified by HPLC and TLC methods. Later Lepidonotus helotypus, Halosydna brevisetosa, Hermenia acanthopeltis, and Harmothoe imbricata were also detected as TTX-bearing annelids (Table XIV). L. helotypus showed the highest score (110 MU/g), which was accounted for mainly by TTX and partly by the TDA-like substance. 13. Arrowworms TTX was also detected in several species of marine arrowworm (zooplankton), such as Eukrohnia hamata (Family: Eukrohniidae), Parasagitta elegans, Flaccisagitta scripassae, and Flaccisagitta enflata (Family: Sagittae), and Spadella angulata (Family: Spadallidae) (Thuesen et al., 1988). Arrowworms could utilize TTX to paralyze their food organisms. They are carnivorous in nature and represent a vital position in food web to intoxicate the higher animals. 14. Red calcareous alga Paralytic toxicity was detected in a red calcareous alga Jania sp. from Okinawa, Japan and Gambier Islands, French Polynesia (Kotaki et al., 1983; Yasumoto et al., 1989). The highest toxin concentration was 44 ppb. The Okinawa Jania sp. contained anhydroTTX as the major toxin, along with TTX and 4-epiTTX as the minor, while the French one showed TTX as the major toxin. In the above red alga, TTX content is very low and therefore its origin may come from TTX-contaminated bacterium. 15. Dinoflagellate Kodama et al. (1996) identified TTX in cultured cells of Alexandrium tamarense, a notorious PSP-producing dinoflagellate. TTX was identified by HPLC-fluoro metric analysis and fast atom bombardment mass spectrometry (FABMS).

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D.-F. HWANG AND T. NOGUCHI

TTX accounted for about 0.1% of the total toxicity of the dinoflagellate cells. However, the occurrence of TTX in A. tamarense should be rationalized more clearly. 16. Bacteria During the last two decades a number of reports have been published, showing that many bacteria contained TTX. In 1986, the occurrence of TTX and anhydroTTX in Vibrio sp. from the intestines of a xanthid crab A. floridus was first demonstrated by Noguchi et al. (1986b). In its experiment, four strains of Vibrio sp. isolated from the contents of the intestines of A. floridus were grown in flask containing 500-ml water with 5 g each of phytone peptone and NaCl (pH 7.2), and incubate at 25 C for 10 days. The harvested cells from one strain showed a considerable amount of toxin (30 MU/flask). The presence of TTX and anhydroTTX in the cell extract was confirmed by HPLC. Ultraviolet (UV) spectrometry and GC–MS analysis indicated the presence of C9-base in the alkaline decomposed compounds of the harvested bacterial cells. In the same year, Yasumoto et al. (1986) also isolated two bacteria Shewanella alga and Alteromonas tetraodonis from the red calcareous alga Jania sp. and cultured them. TTX was detected in their culture broth. In 1987, Vibrio alginolyticus isolated from the starfish A. polyacanthus and puVer T. snyderi contained TTX (Narita et al., 1987; Noguchi et al., 1987). In the study of Noguchi et al. (1987), 26 of 33 strains of aerobic and facultative anaerobic bacteria that were isolated from the puVer were found to belong to genera of Vibrio. By instrumental analysis, TTX was detected in all strains of V. alginolyticus. Simidu et al. (1987) cultured a number of strains of marine bacteria and screened for toxin by HPLC and GC–MS analyses. Results showed that 12 of the 24 strains, which included most strains of Vibrionaceae, produced TTX and/or related substances (Table XV). Attempts were also made to isolate TTX-bearing bacteria from the blueringed octopus O. maculosus from the Philippines (Hwang et al., 1989). The results showed that 16 of the 22 isolated strains produced TTX and/or related substances. Six of the 16 strains produced TTX and/or related substances. Six of the 16 strains were classified into the genera of Vibrio, Pseudomonas, and two each of Alteromonas and Bacillus. The capability of TTX production by the bacteria Shewanella putrefaciens isolated from the intestine of a puVer T. niphobles was reported by Matsui et al. (1990). After being injected with the culture broth, the mice showed typical symptoms of TTX intoxication. A 25 ml of broth contained 15 MU of the toxin which was identified as TTX by GC–MS analysis.

TABLE XV ANALYSIS OF TTX IN BACTERIAL CELLS

HPLC Tetrodotosin

AnhydroTTX

GC–MS

Vibrio alginolyticus ATCC 17749 Vibrio alginolyticus NCMB 1903 Vibrio anguillarum NCMB 829 Vibrio anguillarum NCMB1291 Vibrio costicola (V. costicolus) NCMB 701 Vibrio fischeri NCMB 1281 Vibrio fischeri (Photobacterium fischeri) NCMB 1381 Vibrio harveyi (Aeromonas harveyi) NCMB 2 Vibrio marinus Ps 207 Vibrio parahaemolyticus ATCC 17802 Vibrio parahaemolyticus NCMB 1902 Vibrio parahaemolyticus ATCC 17802 Photobacterium phosphoreum NCMB 844 Aeromonas hydrophila NCMB 89 Aeromonas hydrophila NCMB 89*d Aeromonas salmonicida ATCC 14174 Aeromonas salmonicida ATCC 14174*d Plesiomonas shigelloides ATCC 14029 Escherichia coli IAM 1268 Escherichia coli IAM 1268*d Alteromonas communis IAM 12914 Alteromonas haloplanktis IAM 1218 Alteromonas nigrifaciens IAM 13010 Alteromonas undina IAM 12922 Alteromonas vaga IAM 12923

– – – – – – – – – – – –  – –    – – – –  – 

þ þ þ þ þ     þ þ þ þ þ  þ  þ    –  – –

þ þ þ þ þ –  þ  þ þ þ þ – – þ þ þ –   –  – 

175

þ, clearly detected; , diYcult to detect; –, not detected; *d, cultivated in a freshwater medium. Source: Simidu et al. (1987).

TETRODOTOXIN POISONING

Bacterial strains used

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D.-F. HWANG AND T. NOGUCHI

Hwang et al. (1994c) isolated bacteria representing four predominant genera Vibrio, Aeromonas, Flavobacterium, and Pseudomonas from the Taiwanese lined moon shell N. lineata. The genera Vibrio composed of more than 46% of the total population. Presence of TTX and/or related substances in V. alginolyticus and Aeromonas spp. was confirmed by HPLC, UV, and GC–MS analyses. Later, some bacteria, such as V. alginolyticus, Vibrio parahaemolyticus, Aeromonas sp., Pseudomonas spp., and Plsiomonas sp. from a gastropod N. clathrata, were demonstrated to produce TTX and/or related substances (Cheng et al., 1995). On the other hand, Kogure et al. (1988) examined some 50 strains of bacteria isolated from the marine sediments at 4000-m depths from Tokyo Bay, Japan. TTX concentration in the cells ranged from 0.13 to 0.43 MU/g wet sediment (Kogure et al., 1988). Sediments collected at the 4000-m depth showed a higher amount of TTX. V. alginolyticus was reported to produce more amount of TTX when cultured at facultative anaerobic condition than aerobic condition (Lin, 1999). The above findings indicate that marine bacteria belonging to the genus Vibrio, more specifically V. alginolyticus, are more frequently and abundantly available in toxic marine animals and more capable of producing TTX and/or related derivatives than others. B. TTX ELABORATOR

In the past several decades we have made tremendous progress in the analysis, chemistry, pharmacology, and biology of TTX. The wide distribution of TTX in a variety of organisms, along with marked individual, regional, and seasonal variations in toxin concentration, led us to controversies of the endogenous or exogenous origin of TTX. Kim et al. (1975) pointed that the ability to synthesize TTX is a coincidental genetic development in certain animals. Yotsu et al. (1992b) demonstrated that the tree frog A. oxyrhynchus retained a high level of toxicity when raised in a controlled environment for more than 3 years. The skin extracts of the hatched-raised frog (A. varius) from the eggs demonstrated the presence of TTX (Daly et al., 1997). Nagashima et al. (1999) examined subcellular distribution of TTX and its derivatives in the puVer liver and indicated the possible endogenous origin of TTX. However, the most important observation regarding individual, local, and seasonal variations of toxicity in TTX-bearing animals are not yet comprehensively rationalized by the endogenous theory. Because an animal produces toxin endogenously, there are usually no significant individual, local, and seasonal variations in toxicity. However, TTX-bearing animals are often found to show significant variations depending on the individual, location, and season.

TETRODOTOXIN POISONING

177

In contrast, there have been a few attractive documents in favor of the exogenous origin of TTX. Meanwhile, several attempts were made to confirm the exogenous speculation and it was postulated that bacteria are responsible for the production of TTX (Noguchi et al., 1987; Yasumoto et al., 1986). In support of this postulation, two possible TTX intoxication routes have been described so far. First, some bacteria produce TTX in the sediment and then the toxin is transferred to higher animals through the food web. Second, the host animals accumulate TTX through the symbiotic way. These ideas have been generated and justified from the discovery of TTX-producing bacteria in various toxic host animals and in marine sediments as well, as described in the previous section. There have been also a few documents containing diVerent approaches in support of the exogenous (food chain) hypothesis. Toxicity of all livers and partly muscles, gonads, and other viscera of above 5000 puVer fish (T. rubripes) cultured for 1–3 years in surrounding nets in eight Japanese prefectures were found to be nontoxic. When nontoxic puVer fish produced were reared with each TTX-containing diet of 0.5 and 4 MU TTX per gram fish body per day, respectively, they became toxic in 100 days after dosage of 4 MU/g fish body per day. These results show that puVer fish are intoxicated from the food chain (Noguchi, 1988). Debris of starfish was detected in the intestine of the trumpet shell C. sauliae, another TTX-bearing animal. Subsequently, Noguchi et al. (1982) demonstrated that the starfish, which the trumpet shell prefers as food, also contain TTX. The erratic distribution of TTX in Atelopus frog (Kim et al., 1975) and goby (Noguchi et al., 1971) suggested the possible accumulation through the food chain. In respect of diVerent feeding habits of the diverse TTX-bearing animals living in various environments, however, TTX accumulation mechanism should be rationalized more clearly. As illustrated in this chapter, number of TTX-bearing organisms has been increasing, especially since 1970. Despite the occurrence of TTX in a wide diversity, the mechanism of the induction of toxicity in TTX-bearing animals and the dynamic state of TTX and its analogues have not been completely described. However, considering that all TTX-bearing organisms live in sea or freshwater all or partly through their life, the mechanism of their TTX intoxication seems to be more or less involved in their ecological factor such as water. Information on the influence of environmental parameters, temperature, and water on toxin accumulation and toxin principle are also sorely lacking. Studies along these lines can be progressed. In regard with transmission of TTX through the food web, TTX in puVers seems to come directly from their food, such as toxic gastropods, flatworm, and starfish. TTX commonly accumulates in the eggs of puVers,

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presumably transmitted from the mother, since the toxin generally disappears from the larvae 1 week after fertilization. In the wild puVer, the toxin usually reappears from the larvae after about 2 weeks probably through ingestion of toxic organisms. Indeed, when larvae puVers initially possessing TTX during the first week after fertilization are cultured and fed a TTX-free diet, they do not become toxic. Thus, TTX intoxication mechanism in puVers is likely to occur through the food web, though intestinal bacteria in some species of these fish reportedly produce negligible amounts of the toxin. PuVers, however, seem inclined to seek TTX in their diets. Ingested toxin is initially accumulated in the liver, followed by the skin and other tissues. Fish, except those possessing TTX, such as puVers and the tropical goby Y. criniger, do not accumulate TTX even when toxin-containing diets are fed to them at sublethal doses (Ebesu et al., 2000; Hwang, 2003a,b; Miyazawa and Noguchi, 2001; Noguchi et al., 2004). Other TTX-bearing animals such as gastropods and starfish probably accumulate their toxins through the food web as well. In the trumpet shell (C. sauliae), TTX is ingested through its prey. During elucidation on the TTX transmission mechanism for this animal, starfish such as A. polyacanthus, A. latespinosus, and A. scoparius on which the gastropod feeds were determined to be links in the food web leading to TTX intoxication (Ebesu et al., 2000; Hwang, 2003a,b; Miyazawa and Noguchi, 2001). These toxic starfish are widely distributed in Japan. TTX-containing starfish probably derive the toxin through their diets. The ivory shell (B. japonica) is likely intoxicated with TTX by ingesting toxic puVers that fishermen discard in the sea. TTX intoxication mechanisms through food web links have already been established by model experiments (Ebesu et al., 2000; Hwang, 2003a,b; Miyazawa and Noguchi, 2001). From these studies, the intoxication method is hypothesized as illustrated in Figure 4. The initial source of TTX found in most animals, however, is believed to be marine bacteria. Toxic animals accumulate TTX through the food chain as the main route, minor from bacteria. C. MECHANISMS OF TTX INFESTATION TO ANIMALS

Various toxic tetraodontid species are globally distributed (Ebesu et al., 2000; Hwang, 2003a,b; Miyazawa and Noguchi, 2001). However, these fish are consumed only in certain parts of the world. Areas that have reported TTX poisoning cases due to consumption of toxic puVer include Japan, Taiwan, China, Hong Kong, Thailand, Korea, Singapore, Malaysia, Bangladesh, Australia, the United States, Kiribati, Papua New Guinea, and Fiji. Other organisms found to possess TTX are listed in Table II. Like puVers, these animals are found worldwide: Japan (gobies, blue-ringed octopuses, various

TETRODOTOXIN POISONING

TTX-producing marine bacteria

m asitis m or sy is) bios

Flatworm Ribbonworm Arrowworm Xanthid crab Small gastropod

(Par

TTX in sediment

Vibrio alginolyticus Vibrio damsela Staphylococcus sp. Bacillus sp. Pseudomans sp., and so on (Parasitism or symbiosis)

TTX dissolved in sea water or absorbed on and precipitated with planktonic carcass, and so on

179

Star fish

Pufferfish Tropical goby Large gastropod

FIG. 4 Proposed mechanism of TTX accumulation in marine animals.

gastropods, starfish, xanthid crabs), Taiwan (gobies, various gastropods, xanthid crabs, starfish), Philippines (goby), Bangladesh (rhacophoridid frog, horseshoe crab), Costa Rica and Panama (Atelopus frogs), Australia (blue-ringed octopus), the United States (newts), and Thailand (horseshoe crab). PuVers were long believed to be the exclusive source of TTX (Ebesu et al., 2000; Hwang, 2003a,b; Hwang and Lu, 2003; Miyazawa and Noguchi, 2001). The toxicity of puVer is related to their spawning season, with the highest toxicity levels occurring between March and June for Japanese puVers, though the pattern varies to some degree among species. The toxin is concentrated in the ovary, liver, and often, the skin, making the female puVer more toxic than the males, especially during spawning season. The parts of the puVer considered less toxic are the musculature, fins, and testes. Although muscle tissue may be toxic in at least three puVers, T. pardalis, L. lunaris, and C. patoca, no fatal cases of tetrodotoxication have yet been reported due to eating the meat of puVer as sashimi, or sliced raw fish, in Japan. On the other hand, the dishes known as ‘‘chiri,’’ fillets partially cooked in a stew of skins, livers, intestines, or testes, and ‘‘kimo,’’ partially cooked livers, have been associated with much fatal intoxication. The origin of TTX in TTX-bearing animals was often debated to be either endogenous or exogenous. In 1964, however, this toxin was unexpectedly

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detected in the California newt (T. torosa) (Mosher et al., 1965), thus, setting the controversy. In this organism, TTX was concentrated in the skin, ovary, muscle, and blood. Since then, TTX has been found in a variety of animals (Table II). Other newt species, including Taricha rivularis, T. granulosa, T. vulgaris, Triturus cristatus, T. alpestris, Triturus marmoratus, N. viridescens, C. pyrrhogaster, and C. ensicauda, were also discovered to possess TTX in almost the same anatomical distribution as that of T. torosa (Wakely et al., 1966; Yotsu et al., 1990a). In 1971, TTX was found in the skin, viscera, and muscle of a goby (Y. criniger), inhabiting the Amami and the Ryukyu Islands, Japan. This goby is also distributed in the Philippines and Taiwan, where it has been implicated in several human poisoning cases. In addition, TTX was detected in the skin of Atelopus frogs inhabiting Costa Rica and Panama. In 1978, TTX was isolated from the posterior salivary gland of the blueringed octopus (O. maculosus), which mainly inhabits northern Australia. Human TTX cases are occasionally reported in this area due to envenomation by O. maculosus. This octopus also occasionally appears in middle to south Japan. The octopus secretes TTX from the posterior salivary gland to paralyze its prey. In 1979, a serious poisoning incident due to ingestion of the trumpet shell (C. sauliae), which is widely consumed, occurred in Shimizu, Shizuoka, Japan. The causative agent was isolated from the digestive gland and identified as TTX (Narita et al., 1981). Two similar food poisoning incidents followed in Wakayama and in Miyazaki, Japan, in 1982 and 1987, respectively. The toxic carnivorous gastropods are as follows: the frog shell or ‘‘oonarutobora’’ (T. lissostoma), ‘‘hanamushirogai’’ (Zeuxis siquijorensis), and ‘‘araregai’’ (N. clathrata) in Japan and N. lineata (Hwang et al., 1991a,b) in Taiwan also contain TTX in their digestive glands. In addition, in 1980, TTX was detected in the ivory shell (B. japonica), collected in Fukui, Japan. Mass food poisonings due to ingestion of the eggs of the horseshoe crab sporadically occur in Thailand (Kanchanapongkul and Krittaya, 1995), where during early 1996 they caused about 20 deaths. TTX in the eggs was later determined to be the cause of death. Xanthid crabs (A. floridus, Z. aeneus), flatworms (Planocera spp.), and ribbon worms (Nemertinea) have also been shown to possess TTX. Presently, distribution of TTX is known in only a limited number of organisms (Table II). Toxicity data on TTX-bearing animals collected show more or less toxicity in them irrespective of local and individual variations of toxicity. All of them are found to be carnivorous. TTX in them seems to come directly from their food, such as in puVers, toxic gastropods, flatworms, and starfish, and is accumulated in species-specific organ(s).

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Since TTX is distributed in a variety of invertebrates and vertebrates and there is a wide individual variation of toxin content, even among members of the same species, the origin of TTX was deduced to be a universal organism such as a microbe. The reason why TTX-bearing animals struggle to accumulate TTX may be diVerent dependent on species. However, in the case of puVers, newts, flatworms, and horseshoe crabs, TTX may play a defensive role against predators, especially to prevent their eggs from the enemies. In other animals, TTX may be used as the oVensive substance to immobilize and capture prey by secretion or envenomation of the toxin from the proboscis in ribbon worms or salivary glands in the blue-ringed octopus. Using instrumental HPLC and GC–MS analyses for TTX, an intestinal bacteria isolated from a toxic xanthid crab (A. floridus) collected from Shimoda, Japan, was discovered to produce TTX (Noguchi et al., 1986b). This bacterium was also noted as analogous to Vibrio fischeri. Soon after, V. alginolyticus and several other Vibrio spp. isolated from the intestines of the puVer T. snyderi, the starfish A. polyacanthus, the blue-ringed octopus O. maculosus, and the horseshoe crab C. rotundicauda were also demonstrated to produce TTX using similar analytical techniques. Simidu et al. (1987) showed that 12 of 24 type culture strains of marine bacteria tested clearly produced TTX or related substances (Table XV). Yasumoto et al. (1989) and Matsui et al. (1990) ascertained that A. tetraodonis isolated from a calcareous alga (Jania sp.) and S. putrefaciens isolated from a puVer (T. niphobles) produced TTX and anhydroTTX, respectively. Typical TTX production by Vibrio group VIII (Hashimoto et al., 1990) isolated from the intestines of A. floridus was demonstrated through instrumental analyses for TTX as well as cell toxicity assays. An appreciable amount of TTX (30 MU/flask) was produced by Vibrio group VIII strain (Table XVI). TABLE XVI ANALYSIS FOR TTX AND RELATED SUBSTANCES IN THE EXTRACTS OF SEVERAL VIBRIO GROUPS OF BACTERIA ISOLATED FROM INTESTINES OF ATERGATIS FLORIDUS SPECIMENS COLLECTED FROM KOJIMA IN ISHIGAKI ISLAND, OKINAWA

HPLC Vibrio group

Lethal potency (MU)

TTX

AnhydroTTX

GC–MS

I III V VIII

– – – 30

þ þ  þ

– – – þ

þ

Source: Hashimoto et al. (1990).

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D.-F. HWANG AND T. NOGUCHI

TTX

AnhydroTTX

4-epi TTX

0

10

20

Retention time (minutes)

FIG. 5 HPLC of TTX fraction forms Vibrio group VIII isolated from a Kojima specimen (upper) and of authentic TTX (lower) (Noguchi et al., 1986b).

The TTX fraction showed two peaks (TTX and anhydroTTX) in HPLC analysis, and in GC–MS analysis for the toxin (C9-base) exhibited mass fragment ions at m/z 407 (molecular peak), 392 (base peak), and 376, all of which are specific to trimethylsilylated C9-base derived from authentic TTX (Figures 5 and 6). It is recognized from the above evidence that many bacteria can produce TTX and/or its derivatives. TTX productivity in vitro, however, seems largely dependent on culture conditions. The optimal culture conditions for maximum toxin yield by TTX-producing bacteria remain unknown. Such bacteria may produce minimal amounts of TTX in the intestines of fish and invertebrates. In addition, on the basis of the following observations it is presumed that the carnivorous TTX-bearing organisms are able to accumulate the toxin from TTX-producing bacteria:

TETRODOTOXIN POISONING

183

 Trumpet shells can become highly intoxicated by ingesting toxic starfish (Noguchi et al., 1982).  The debris of small gastropods is often found in the digestive ducts of toxic puVers (Jeon et al., 1984).  Cultured puVers (nontoxic) can become intoxicated by feeding on the liver of toxic puVers and do not become intoxicated by feeding on a nontoxic diet in net cages (Noguchi et al., 2004).  The starfish accumulated a lot of TTX from small toxic gastropods (Lin and Hwang, 2001).  Cultured puVers were intoxicated when the toxic flatworm appeared in the aquaculture pond (Lin et al., 1998a). However, during elucidation of several food poisoning cases due to ingestion of TTX-bearing organisms, TTX intoxication mechanism of causative organisms was found to come from their food chains. They were confirmed from their designed model and were reasonably proved to come from their TTX-containing diet. There have been a few attractive documents in favor of the exogenous

392 407 376

Relative intensity (%)

100 392 80 60

407 281

40

318 20 250

300 350 400 Mass spectrum (m/z)

392 407 376 9

11 13 15 Retention time (minutes)

Relative intensity (%)

100

450 392

80 60 407

40 20

59

100

203

318

200 300 400 Mass spectrum (m/z)

FIG. 6 GC–MS of TTX fraction of Vibrio group VIII from a Kojima specimen. GC, left; MS, right (Noguchi et al., 1986b).

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D.-F. HWANG AND T. NOGUCHI

origin of TTX. Meanwhile, several attempts were made to confirm the exogenous speculation and it was postulated that bacteria are responsible for the production of TTX (Noguchi et al., 1986b, 1987; Yasumoto et al., 1986). In support of this postulation, there are two possible TTX intoxication routes described so far. First, some bacteria produce TTX in the sediment and then the toxin is transferred to higher animals through the food web. Second, the host animals accumulate TTX through the symbiotic way. These ideas have been generated and justified from the discovery of TTX-producing bacteria in various toxic host animals and in marine sediments as well, as described in the previous section. TTX is one of the best-known notorious marine toxins that occasionally cause human intoxication, including fatality. Poisoning incidents owing to TTX have been almost exclusively associated with ingestion of toxic puVer and gastropod, especially from the waters of the Indo-Pacific Ocean regions. The toxicosis is characterized by the onset of symptoms of the victim. The treatment of the illness is mainly based on the symptoms of the patient. More fruitful treatment can be provided if the causative toxin is identified. Detection and determination of TTX are therefore essential not only for diagnosis and treatment purposes, but also for making quarantine rule and public awareness. Quantitative and/or qualitative detection of TTX in a sample are/is performed by several chemical and biological methods, as described below. D. TTX DETECTION METHOD

1. Mouse bioassay The features of detection methods for TTX are shown in Table XVII. Some methods including bioassay, HPLC, and liquid chromatography–mass spectrometry (LC–MS) are usually used to qualitatively and quantitatively detect TTX, but other methods including GC–MS, IR, and NMR are usually used to qualitatively detect TTX. Among them, LC–MS is the most powerful and sensitive tool for qualitatively and quantitatively determining TTX. Mice have been commonly used for determination of toxicity for TTX. To know the concentration of TTX in a sample of interest, the mouse bioassay is used and described above. Mouse bioassay is also used to identify an unknown toxin extract in comparison with a TTX-specific dose–death time relationship curve. A series of test solutions are prepared by diluting the unknown toxin extract with 0.1% acetic acid. Aliquots of each test solution are intraperitoneally injected into a group of mice. Using the median value of their death time at each dilution level, the dose–death time curve is drawn, which provides the nature

TABLE XVII THE FEATURES OF DETECTION METHODS FOR TTX

Detection system

Detection limit

Features

References

Bioassay HPLC

Mice Fluoromonitor

0.2-mg TTX 0.03-mg TTX

Death time Intensity

TLC Electrophoresis Capillary isotachophoresis UV spectroscopy GC–MS

Fluorescent spot Fluorescent spot Potential stand

2-mg TTX 2-mg TTX 0.25-mg TTX

Spot mobility Spot mobility Ion mobility

Hwang and Jeng, 1991; MHW, 1991 Nagashima et al., 1987; Yasumoto et al., 1982

Spectrometer Mass spectrometer

Qualitative Qualitative

IR spectrometry FABMS LC–MS

IR spectrometer Mass spectrometer Mass spectrometer

Qualitative Qualitative 0.05-ng TTX

Alkali decompose Alkali decompose þ ion-monitoring Functional group Ion-monitoring Ion-monitoring

ESI-TOF/MS NMR spectrometry

Mass spectrometer NMR spectrometer

Qualitative Qualitative

Ion-monitoring Proton spectrum

Cytotoxicity test

Microscopic examination ELISA

3 nmol/liter

Cell death

2 ng/ml

Monoclonal antibody

Immunoassay

Shimada et al., 1983 Suenaga and Kotoku, 1980 Narita et al., 1981 Onoue et al., 1984 Noguchi et al., 1991 Hwang et al., 2005; Tanu and Noguchi, 1999 Tanu et al., 2001 Nakamura and Yasumoto, 1985; Tsuruda et al., 2001; Yotsu-Yamashita, 2001 Hamasaki et al., 1996; Kogure et al., 1988 Matsumura and Fukiya, 1992; Watabe et al., 1989

TETRODOTOXIN POISONING

Method

185

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D.-F. HWANG AND T. NOGUCHI

of the toxin since the dose–death time curve is specific to toxin. For example, Hashimoto and Noguchi (1971) preliminarily identified goby toxin as TTX with considerable accuracy, using the dose–death time curve (Figure 7). Hwang et al. (1990a) also identified gastropod toxin as TTX using the dose–death time curve. Although the mouse has been the animal of choice for determination of TTX, it is not without controversy due to some potential drawbacks related to the bioassay, such as low accuracy caused by the individual variation inherent to biological system, lack of specificity, inconvenience of purchasing a particular size of mice of specific strain, and recent international movement of prevention from cruelty to animals. These limitations have spawned the development of several alternative chemical methods, with the goal of obtaining comprehensive tests surpassing the mouse assay, as illustrated under the following sections (Noguchi and Mahmud, 2001). 2. High-performance liquid chromatography HPLC methods have been explored for both qualitative and quantitative analyses of TTX and its analogues by many researchers. The fluorometric HPLC method is mainly composed of a high-pressure piston pump with a

Amount of toxin (MU)

9 Goby toxin (24 µg/g) Goby toxin (0.22 µg/g)

7

TTX STX 5

3

1 5

10 15 Death time (minutes)

20

FIG. 7 Dose–death times curve for goby toxin, TTX, and saxitoxin (STX) (Hashimoto and Noguchi, 1971).

TETRODOTOXIN POISONING

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syringe loading sample injector, stainless column, reaction pump for delivering reaction reagent, fluoromonitor, and chromatorecorder for calculation of peak area. Toxin is first separated from the contaminants with a buVer system on a column packed with either silica gel C-18 or ion exchange resin, and the toxin eluate is then mixed with NaOH. The toxin is converted into fluorescent compounds and then passed through a tube placed in an aluminum block oven. Eventually, when the fluorescent compounds are passed through a fluoromonitor equipped with a lamp, their retention times of the toxin and fluorescence intensities are recorded, showing chromatogram in the chromatorecorder. Toxins are identified from the retention times of the authentic TTXs. In quantitative analysis of HPLC, the detection limit of the authentic TTX is about 0.03 mg. Until now, several attempts have been made to detect TTX and its analogues under diVerent conditions of HPLC, and a number of advances in our understanding of the biochemistry of TTXs are a direct result of these developments. Briefly, some promising methodologies can be described here. In early 1980s, a fluorometric continuous TTX analyzer was constructed (Yasumoto et al., 1982). In this system, the toxin was first separated from contaminations on a column of a weak cation exchange gel (Hitachi Gel 3011C, Tokyo) with a 0.06 mol/liter citrate buVer solution (pH 4.0), and toxin concentrations of above 8 MU/g were detected. Due to the poor performance in TTX analogues, improved analyzer was constructed later by Yotsu et al. (1989). The improved method could detect TTX derivatives including 6-epiTTX, 4,9-anhydro-6-epiTTX, 4-epi-11-deoxyTTX, 11-norTTX-6(R)-ol, 4-epiTTX, 4,9-anhydroTTX, 11-deoxyTTX, and 4,9-anhydro-11-deoxyTTX isolated from puVer and newt specimens. TTX derivatives were separated on a Develosil column (1.0  25 cm2) with 0.06 mol/liter heptafluorobutyric acid in 0.001 mol/liter ammonium acetate buVer (pH 5.0). An amount of 4 mol/liter NaOH was used to produce fluorescent compounds. Separation of TTX from 6-epiTTX is, however, considered as the major achievement by this improved analyzer. This analyzer is especially useful for monitoring tropical animals, which contain a considerable amount of 6-epiTTX. Reversed-phase ion-pairing HPLC method has also been the system of choice by many researchers for the fastest and eYcient analysis of TTX and its analogues, where heptanesulfonic acid (HAS) is used as counterion (Nagashima et al., 1987). In this method, the detection reagent for TTX and related substances does not react with any PSP component if present in the contaminant sample. Table XVIII represents a reversed-phase HPLC condition according to the method of Nagashima et al. with slight modification (Arakawa et al., 1994), being used to analyze partially purified Japanese newt (C. pyrrhogaster) poison, TTXs (Figure 8) (Tsuruda et al., 2002).

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TABLE XVIII OPERATING CONDITIONS OF HPLC FOR THE ANALYSIS OF NEWT TOXINS TTXs

HPLC system

D-7480 Hitachi

Column (4.6  250 mm2, GL Sci., Inc., Japan) Column temperature Mobile phase

Inertsil ODS-3 30 C 60-mM ammonium phosphate buVer (pH 5.0) containing 10-mM HAS and 2% acetonitrile 4-M NaOH 0.8 ml/minute 110 C Excitation 384 nm, emission 505 nm

Reagent Flow rate Reaction temperature Detection Source: Tsuruda et al. (2002).

b

a

a d

e c

0

10

20

c b d

e

30

0

10

20

30

Retention time (minutes)

FIG. 8 HPLC of authentic TTXs (left) and the Japanese newt (C. pyrrhogaster) toxin (right). a, TTX; b, 6-epiTTX; c, 4-epiTTX; d, 4,9-anhydro-6-epiTTX; e, 4,9-anhydroTTX (Tsuruda et al., 2002).

TETRODOTOXIN POISONING

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3. Thin-layer chromatography In TLC analysis, TTX is spotted onto a silica gel-60 F254 precoated plate (Merck, Darmstadt). The plate is developed in three diVerent solvent systems of pyridine–ethyl acetate–acetic acid–water (15:5:3:4), 3-butanol–acetic acid– water (2:1:1), and 1-butanol–acetic acid–water (12:3:5) solvent in a sealed container. The solvent rises by capillary action and an ascending chromatographic separation is obtained. The plate is then sprayed with 10% KOH followed by heating at 100 C for 10 minutes. The toxin is visualized as a yellow fluorescent spot under UV light (365 nm). In TLC analysis, the Rf values of TTX are around 0.70, 0.45, and 0.20 with pyridine–ethyl acetate– acetic acid–water, 3-butanol–acetic acid–water, and 1-butanol–acetic acid– water solvent, respectively. It is also possible to detect TTX on the TLC plate using the Weber reagent that gives pink spot of the toxin. In TLC, the detection limit is about 2 mg of TTX (10 MU). TLC is a useful technique in those laboratories where HPLC and other costly analytical systems are not available. 4. Electrophoresis Electrophoresis is a relatively simple and rapid method with high resolution detection of polar compounds like TTX. When 1 ml of TTX (10 MU, corresponding to 2 mg) is applied onto a 5  18 cm2 cellulose acetate membrane (Chemetron, Milano), the ion molecules of TTX move toward the cathode with a mobility (Rm) clearly smaller than that of authentic STX. The analysis is performed for 30 minutes in an electrolytic buVer solution of 0.08 mol/liter TrisHCl (pH 8.7), under the influence of an applied electric field with a constant current of 0.8 mA/cm width. The toxin is visualized in the same manner as described for TLC. 5. Capillary isotachophoresis Capillary isotachophoresis is a rapid, accurate, and potential detection technique for TTX. A small amount of TTX in contaminated extracts can be determined by this method (Shimada et al., 1983). It is performed using a cationic system, as TTX exists as cation under acidic and neutral conditions. Conditions for capillary isotachophoresis composed of 5 mmol/liter potassium acetate (pH 6.0) as an electrolyte, containing 0.2% Triton X-100 and 0.5 volume of dioxane, and 10 mmol b-alanine adjusted to pH 4.5 with acetic acid as a terminating electrolyte. When TTX is applied to isotachophoretic analyzer (Shimadzu IR-2A) equipped with a potential gradient 0.32, it is eventually monitored by the detector. PU is expressed as (PGS-PGL), where PGS, PGL, and PGT stand for potential gradient values for sample, leading ion, and

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D.-F. HWANG AND T. NOGUCHI

terminating ion, respectively (Miyazaki and Katoh, 1976). The quantitative detection limit by this method is about 0.25 mg of TTX (1.2 MU). 6. UV spectroscopy In UV spectroscopy, TTX is generally determined by irradiating a crude toxin with UV light. A small amount of TTX is dissolved in 2 ml of 2 mol/liter NaOH and heated in a boiling water bath for 45 minutes. After cooling at room temperature, the solution is examined for the UV absorption spectrum, characteristic to the C9-base, 2-amino-6-hydroxymethyl-8-hydroxyquinazoline which should have been derived from TTX and/or related substances, if present (Suenaga and Kotoku, 1980). In the analysis, UV spectrum of the alkalidecomposed compounds of TTX appears as a shoulder at around 276 nm (Tanu and Noguchi, 1999), indicating the formation of the C9-base (Figure 9), specific to TTX or related substances.

240 260 280 300 Wavelength (nm)

240

260 280 300 Wavelength (nm)

FIG. 9 UV absorption spectra of alkaline hydrolyzates of authentic TTX (left) and horseshoe crab (C. rotundicauda) toxin (right) (Tanu and Noguchi, 1999).

TETRODOTOXIN POISONING

191

7. Gas chromatography–mass spectrometry GC–MS analysis is an indirect method to detect TTX in a crude extract which is diYcult to purify for other advanced analysis. In this method, TTX and its derivatives (0.2 ml with 25 MU) are dissolved in 2 ml of 2 mol/liter NaOH and heated in a boiling water bath for 45 minutes. After being cooled at room temperature, the alkali-decomposed compounds are adjusted to pH 4.0 with 1 mol/liter HCl and extracted thrice with three volumes of 1-butanol. The extracts are combined and evaporated to dryness in vacuum, and to the residue is added a mixture of N,O-bis acetamide, trimethylchlorosilane, and pyridine (2:1:1), in order to derive trimethylsilyl (TMS) ‘‘C9-base’’ compounds. The derivatives are then submitted to GC–MS analysis. The column temperature is maintained from 180 to 250 C at a rate of 5 C/minute. The flow rate of inlet helium carrier gas is maintained at 20 ml/minute. The ionizing voltage is usually kept at 70 eV with the ion source temperature at 200 C. An example of MS of the selected ion-monitored chromatograms (SIM) from the TMS derivatives of alkali-decomposed puVer T. oblongus poison TTX (‘‘C9-base’’) is shown in Figure 10. Sharp fragment ions appear at m/z 407 (parent peak), 392 (base peak), and 376, indicating the presence of the quinazoline skeleton in the toxin (Narita et al., 1981). 8. IR spectrometry

100

50

Relative intensity (%)

Relative intensity (%)

IR spectrometry is the analytical technique for determination of functional groups of TTX. IR spectra of TTX are measured in a KBr pellet, using a JASCO model IR-S spectrophotometer (Tokyo) (Onoue et al., 1984). Figure 11 represents an IR spectrum of puVer toxin, showing characteristic absorption in 3360 (OH), 3200, 1660 (guanidium), 1610 (COO–), and 1075 cm1.

100

392

75

407 203

320

376

0 100 200 300 400 Mass spectrum (m/z)

500

50

392

407

75 203

320

376

0 100

200 300 400 Mass spectrum (m/z)

500

FIG. 10 Mass spectra of C9-base-(TMS)3 derivative from a marine puVer toxin (left) and authentic TTX (right) (Narita et al., 1981).

D.-F. HWANG AND T. NOGUCHI

Transmission (%)

192

3600

2800

2000

1800

1600

1400

1200

1000

800

600

Wave number (cm−1)

FIG. 11

IR spectrum of puVer toxin (Onoue et al., 1984).

Although the spectrum in IR analysis is presumed to be complex, it is a helpful tool for identification of TTX. 9. Fast atom bombardment mass spectrometry FABMS is a direct method for the qualitative confirmation of TTX. The analysis is carried out on a JEOL JMS DX-300 mass spectrometer (Tokyo) equipped with a JEOL DA-5000 data system (Noguchi et al., 1991). Xenon is used to provide the primary beam of atoms, the acceleration voltage of the primary ion being 3 kV. Scanning is repeated within a mass range of m/z 100–500. In this analysis, about 0.1 mg of TTX and glycerol as matrix are placed on the sample stage of the mass spectrometer, mixed well, and submitted to the ion chamber of the spectrometer (FAB). Both positive and negative mass spectra of TTX are then measured. As shown in Figure 12, TTX shows (M þ H)þ and (M þ H  H2O)þ ion peaks at m/z 320 and 302, respectively, in the positive mass spectrum, and an (M – H)– peak at m/z 318 in the negative spectrum. Secondary ion MS, performed by a Hitachi M-80B mass spectrometer equipped with a Hitachi M-0101 data system, presented essentially the same result as shown by FABMS. Extensively purified sample is required for the successful application of this method. 10. Liquid chromatography–mass spectrometry LC–MS is developed for the detection of TTX with considerable accuracy (Shida et al., 1998). In this method, combined HPLC–MS is performed using Hitachi M-1000 system coupled to a mass spectrometer. HPLC system is

100

100

183 (2G − H)−

Relative intensity (%)

Relative intensity (%)

TETRODOTOXIN POISONING

50 318 (M − H)− 0

100

FIG. 12 1991).

200 300 400 Mass spectrum (m/z)

500

193

185 (2G + H)+

50

(M + H)+ (M + H − H2O)+ 320 302

0 100

200 300 400 Mass spectrum (m/z)

500

Positive (right) and negative (left) FAB mass spectra of TTX (Noguchi et al.,

equipped with an ODS-3 (1.5  150 mm) column. Acetonitrile (50%, flow rate 70 ml/minute) is used as mobile solvent. The eZuent from the column is split to provide flow to the ion-spray interface. An example of mass spectra of a brackish water puVer toxin in LC–MS analysis is shown in Figure 13 (Tanu and Noguchi, 1999). In the MS, a protonated molecular ion peak (M þ H)þ appeared at m/z ¼ 320 showing the molecular weight of the toxin of 319, in good accordance with that of TTX. Recently, we found that the combination of C-18 Sep-Pack cartridge and Ultrafree micro centrifuge filters with LC–MS (California) is very useful in detecting TTX from the urine and blood samples for poisoned patients for diagnosis of TTX food poisoning. The detection limit of TTX in biological sample was 12.5 nM (equivalent to about 3.9 ng/ml or 0.02 MU/ml) by LC–MS (Hwang et al., 2005). 11. Electrospray ionization-time of flight/mass spectrometry ESI-TOF/MS is a valuable technique for determination of TTX, although it is not widely used so far in marine toxin determination. In this analysis, a portion of purified TTX (less than 0.05 mg) is dissolved in a small amount of 1% acetic acid, and added to 50% aqueous methanol. ESI-TOF/MS is taken on a Micromass Q-Tof Mass Spectrometer (Tokyo). Recently, TTX in a tree frog Polypedates sp. extract has been successfully analyzed by ESI-TOF/MS analysis (Tanu et al., 2001). As shown in Figure 14, in the spectrum of the toxin, protonated molecular ion peak (M þ H)þ appeared at m/z ¼ 320.1103, suggesting the molecular weight of the toxin to be 319.1025 which agrees well with that of authentic TTX (C11H17N3O8 ¼ 319.1016).

D.-F. HWANG AND T. NOGUCHI Relative intensity (%)

194

(M + H)+ 100

320 119 160

201

249

279

0 100

200 300 Mass spectrum (m/z)

400

FIG. 13 Mass spectrum of chromatogram at 8.6 minutes from a brackish water puVer (T. nigroviridis) toxin in LC–MS analysis (Tanu and Noguchi, 1999).

(M + H)+ 320.1103

Relative intensity (%)

100

302.1229 0 100

200

300

400

500

600

700

800

Mass spectrum (m/z)

FIG. 14 ESI-TOF/MS analysis of a frog (Polypedates sp.) toxin: y-axis, relative intensity; x-axis, mass spectrum at m/z (Tanu et al., 2001).

12. 1H NMR spectrometry 1

H NMR has been playing an important role as a complementary method to determine the absolute configuration of TTX and its derivatives. At present 13 derivatives of TTX have been isolated, whose 1H NMR data are presented by various investigators (Endo et al., 1988; Nakamura and Yasumoto, 1985; Tsuruda et al., 2001; Yasumoto et al., 1988; Yotsu et al., 1990a,b, 1992a,b,c; Yotsu-Yamashita, 2001). In 1H NMR analysis, the lyophilized powder of purified toxin is dissolved in 0.6 ml of 4% acetic-D3 acid-D (99.5% purity) in D2O and placed in an

TETRODOTOXIN POISONING

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OH 10

HO

+ H2N

2

O O H H 5 4 OH H 11CH OH H3 11 2 N S2 6 7 N H 1 HO 8 OH H H H 9

TTX

11 8

4a 7

4

5

6

5

9

4

3 2 Chemical shift (ppm)

1

0

FIG. 15 1H NMR spectrum of a brackish water puVer (T. nigroviridis) toxin (Mahmud et al., 1999a).

NMR tube. Tetramethylsilane (Me4Si) is used as an external standard. The spectrum is taken on a Varian Unity Plus 500 spectrometer at 500 MHz. Figure 15 exhibits signals at 2.03 (s, CH3COOD), 2.34 (d, J ¼ 9.6 Hz), 3.97 (s), 4.05 (s), 4.26 (s), 4.29 (s) and 5.44 (d, J ¼ 9.4 Hz) ppm. C4-H was coupled with C4a-H with a spin-spin-coupling constant of almost same of 9.4 and 9.6, respectively, which is a typical characteristic of TTX. 13. Cytotoxicity test Cell bioassays are eVective biological screening tools for a wide range of drugs, chemicals, and toxic compounds. The utility and design of these in vitro methods are described by the mechanism of action associated with the compounds of interest and the availability of appropriate target cell lines. For the quantitative measurement of a sodium channel blocker TTX, even at low level of detection (
3 nmol/liter) in marine bacteria, the tissue culture bioassay (TCBA) has been used (Kogure et al., 1988). This assay is based on the ability of sodium channel-blocking toxins to antagonize the

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D.-F. HWANG AND T. NOGUCHI

combined eVects of two chemicals, veratridine and ouabain, on neuroblastoma cell lines (ATCC, CCL-131). Veratridine (0.075 mmol/liter) and ouabain (1 mmol/liter) cause the cells to round up and die subsequently. Cells continue to grow in the presence of TTX, as TTX counteracts the veratridine. The amount of toxin is estimated from the linear relationship of the relative abundance of living cells and the concentration of toxin in the samples. The results are scored by visually noting the morphology of a significant number of cells under microscope. The sensitivity of this method is much higher than that of the mouse assay. This method is, however, not always convenient for routine bioassays, because it is time consuming, and needs some experience to distinguish dead cells from living cells during microscopic examination. To replace the time-consuming and subjective cell-counting procedure, TCBA in combination with a water-soluble tetrazolium salt 2-(4-iodophenyl)-3(4-nitriphenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) was improved for the quantitative measurement of TTX, using a microplate reader (Kogure et al., 1988). WST-1 is used as an indicator of living cells. TTX concentrations can be measured between the range of 2 and 70 nmol/liter by this method. WST-1 TCBA is also a useful assay for the detection of TTX in a supernatant of bacterial culture and its sensitivity is several times higher than that of TCBA as reported by Hamasaki et al. (1996). This method seems to be promising, owing to the omission of both washing and dissolving steps, which allows greater reproducibility of the assay. 14. Immunoassay Immunoassays are commonly used as inexpensive, sensitive, and highly selective methods for the detection and quantification of a wide variety of drugs and other substances of biomedical significance (Raybould et al., 1992). For TTX detection, several immunoassay techniques have been developed so far, without much success. Watabe et al. (1989) developed an enzyme-linked immunosorbent assay (ELISA) system by raising a monoclonal antibody (Mab) against a TTX derivative TDA. In this approach, TDA was conjugated with bovine serum albumin (BSA) and injected intraperitoneally into Balb/c mice. Spleen cells of mice were then isolated and fused with myeloma cells X63-Ag8-6.5.3, and cloned. Mab was produced in ascites fluid in the mouse by cloned cell and its detection capability for TTX was 0.03–100 mg/well (0.3–1000 mg/ml). This method had, however, low sensitivity. Huot et al. (1989) reported the production of two anti-TTX Mabs. Binding of these antibodies was only partially inhibited (48% and 25%) by free TTX at a concentration of 50 ng/ml, showing, however, a low performance. Raybould et al. (1992) developed a rapid, significantly sensitive and specific competitive inhibition enzyme immunoassays (CIEIAs) for TTX

TETRODOTOXIN POISONING

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detection and quantification in biological samples, by using a newly developed Mab from mice. In this CIEIA approach, Mab detected TTX with sensitivities at IC50 and IC20 of 6–7 ng/ml, respectively. Other group reported the competitive enzyme-linked immunoassay (EIA) for TTX determination, although these methods required a long time for the analysis (Matsumura, 1995a,b; Matsumura and Fukiya, 1992). Recently, a Mab against TTX has been developed from Balb/c mice immunized with TTX– BSA conjugate, by which a rapid (generally takes 30 minutes) and highly sensitive EIA system has been established for quantitative analysis of TTX. In this promising method, Mab can detect TTX at concentrations of 2–100 ng/ml. This method is considered as a useful tool for monitoring TTX in puVer fish or other seafoods. Using this highly specific Mab, a useful immunoaYnity column chromatography has also been developed for isolation and identification of TTX from the urine samples of a poisoned patient (Kawatsu et al., 1997, 1999). This chromatographic technique is performed in combination with fluorometric HPLC. The maximum recovery rate of TTX by this immunoaYnity is reported to be 88%. The detection limit of TTX is 2 ng/ml of the urine. For diagnosis of TTX poisoning patients, this chromatographic method might be used as a potential tool. E. CHEMISTRY OF PUFFER TOXIN

PuVer toxin generally distributes toxic puVers. Japanese people have a habit to eat puVers’ muscle and liver for a long time and, accordingly, have encountered food poisoning, sometimes resulting in death. On account of many and serious poisonings due to ingestion of puVer in Japan, many Japanese scientists have, so far, studied on its chemical and pharmacological characterizations for a long time. Dr. R. Tahara succeeded in partial purification of the puVer toxin in 1909 and named it as TTX after its scientific name. The absolute structure of TTX was elucidated by three groups of scientists: Goto et al. (1965), Tsuda et al. (1964), and Woodward (1964) in 1964, and the synthesis of racemic TTX was further reported by Kishi et al. (1972). In 1999, synthesis of (–)-5,11-dideoxyTTX (Yotsu-Yamashita et al., 1999), together with its 4-epiTTX (Yasumoto and Michishita, 1985) and 4,9-anhydroTTX (Yotsu et al., 1989) analogues, was achieved by Nishikawa et al. (1999) as the first stereocontrolled total synthesis of TTX analogues. The TTX origin deriving from bacteria was also reported by two groups. Since then, 4-epiTTX (Tsuda et al., 1964), 4,9-anhydroTTX (anhydro-epiTTX or anhydroTTX) (Goto et al., 1965), and TDA (Woodward, 1964) were isolated from the puVer as minor components. In the acidic aqua, 4-epiTTX and 4,9-anhydroTTX are in equilibrium with TTX. Crystalline TTX forms hemilactal type (Woodward, 1964) while in the acidic aqua, TTX forms protoned hemilactal type with bilateral ions where the hemilactal equilibrates

198

D.-F. HWANG AND T. NOGUCHI

with the hydroxy lactone (Woodward, 1964). Other TTX derivatives have already been isolated and characterized as shown in Figure 2. Pure TTX is insoluble in water and organic solvents. However, in strongly acidic aqua it is soluble and stable while in alkaline one it is unstable. Molecular weight is 319. It does not show clear melting point and beyond 200 C, it changes into black color. TTX has a guanidium function in its molecule. Specific toxicity of TTX is 5000–8000 MU/mg where 1 MU is defined as amount of TTX that can kill a male ddY strain mouse of body weight 20 g in 30 minutes, corresponding to about 0.2 mg of authentic TTX. MLD50 for human is about 2 mg. This toxicity is almost equivalent to that of STX, a typical component of PSP which is one of the most potent neurotoxins among toxins with low molecular weight. Monoclonal TTX antibody to detect TTX with high sensitivity has been developed by two groups (Kawatsu et al., 1997, 1999) and consequently, microdistribution of TTX in several tissues of the puVer (Mahmud et al., 2003a,b; Tanu et al., 2002) and the newt (Tsuruda et al., 2002) was elucidated. However, antibody to cure TTX-poisoning patients remains to be developed. TTX is supposed to bind with some high molecular weight substance such as protein or to occur as water-soluble precursor(s) instead of free TTX since free TTX is water insoluble and if alone, TTX cannot distribute the cell of TTX-bearing organisms. F. PHARMACOLOGY OF TTX

1. Introduction TTX is a potent neurotoxin originally found in the ovary and liver of puVer, followed by some tissue(s) in TTX-bearing animals such as newts, gobies, frogs, gastropods, and so on. From the recent investigations, it has now become abundantly clear that origin of TTX is exogenous from a food web in the puVer. The pharmacology of TTX had been studied for a long time especially in Japan to prevent food poisoning since puVers are regarded as the most delicious fish among Japanese. On the basis of previous literatures, TTX is well known to play the selective and potent blocking action on the sodium channel (Ebesu et al., 2000; Narahashi, 2001; Narahashi et al., 1960). Recently, its cellular and molecular mechanism of action has been extensively studied. The fact is equally important that TTX has since then been used extensively as a chemical tool in the laboratory for the purpose for studying the sodium channel, other ion channels, and various aspects of membrane excitability and synaptic transmission. On the basis of the achievements of recent pharmacological studies for TTX, TTX is suggested to play a role of defense and oVense against enemy

TETRODOTOXIN POISONING

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TABLE XIX TTX RESISTIBILITY IN TTX- AND NON-TTX-BEARING ORGANISMS

Species TTX-bearing organisms Xanthid crab Atergatis floridus Tropical goby Yongeichthys criniger Japanese newt Cynops pyrrhogaster PuVer fish Toxic Takifugu niphobles Takifugu pardalis Takifugu rubrips (culture) Generally nontoxic and rarely toxic Lagocephalus wheeleri Lagocephalus gloveri Liosaccus cutaneus Nontoxic Boxfish Non-TTX-bearing organisms General fish Oplegnathus punclatus Oplegnathus fasciutus Girella punctata Land animal Mouse

MLD (MU/20 g) 1000 >300 >10,000 700–500 500–550 300–500 15–19 19–20 13–15 0.9–1.3 0.8–0.9 0.8–1.8 0.3–0.5 1

Source: Saito et al. (1984).

and pathogenic microorganisms in TTX-bearing animals, resulting in utilization as potent pain-stopper medicine for rheumatism, neuralgia, and heavy cancer, and is expected as a promising anesthetic. TTX-bearing organisms such as puVers, gobies, xanthid crabs, newts, and gastropods have very high resistibility against TTX in comparison with that of non-TTX-bearing organisms such as fish, mice, and so on, as shown in Table XIX (Koyama et al., 1983; Saito et al., 1984). TTX-bearing organism should be qualified to have high TTX resistibility on TTX intoxication from a food chain. When puVers are cultured in a net during growing up to adult since fertilization, puVer grown are never intoxicated with TTX. However, such nontoxic puVers keep high TTX resistibility yet and can be intoxicated if TTX-containing foods are fed to them as shown in Table XX. On the other hand, in a case of non-TTX-bearing fish, which have not high TTX resistibility even though less than MLD50 amount of TTX-containing foods continues to

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D.-F. HWANG AND T. NOGUCHI

TABLE XX TTX INFESTATION TO NONTOXIC CULTURED PUFFER FISH BY FEEDING ON TTX-CONTAINING LIVER OF WILD PUFFER FISH

Dosage of TTX

Toxicity score of liver (MU/g)

Feeding periods (days)

0.5-MU/g body weight per day

4-MU/g body weight per day

20 40 60 80 100 120 140 200 240

<4 <4 <4 <4 11 29 37 70 70

<4 6 90 95 100 140 210 420 480

be fed to them for a long time, they never accumulated TTX in the liver and exclude or decompose it. These diVerent phenomena between animals may come from each inherited quality. The reason why they have high resistibility against TTX has been suggested to have a lot of special TTX-resistant (TTX-R) sodium channels, diVerent from those of non-TTX-R organisms. As being suggested, two diVerent types of sodium channels were surely found. One, TTX-R sodium channel is surely 3300 times higher TTX-R than that of another ‘‘TTX-sensitive sodium channels.’’ However, since TTX-R sodium channels distribute not only puVers but also non-TTX-bearing organisms, its number in puVers seems to be not richer to deserve high TTX resistibility. Therefore, species-specific mechanism of masking and/or modulating TTX in TTX-bearing organisms seems to be equipped in the TTX-bearing animals. Probably functional group involved in toxicity in TTX structure may bind to some high molecular weight substance which is reversible. Herein, the above high molecular weight substance is suggested to occur only in TTX-bearing organisms. On stimulation, TTX is secreted alone and/or with TTX-binding substance and plays a role of defense and/or oVense substance. Such a TTX-binding high molecular weight substance seems to function as follows: The shore crab, Hemigrapsus sanguineus, is highly resistant to TTX. To elucidate the mechanism of TTX resistance, its body fluid was examined for neutralizing eVects against TTX (Shiomi et al., 1992). When the body fluid was injected into mice together with TTX, the lethal activity of TTX was greatly reduced. However, the body fluid did not counteract the lethal eVect of PSPs. The body fluid contains TTX-binding,

TETRODOTOXIN POISONING

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high molecular weight substances (>2,000,000) that are responsible for the neutralizing activity against TTX. 2. Mechanism of action on sodium channels Although TTX blocking the nerve and muscle conduction had been known for many years (Kao, 1966; Narahashi, 1974), it was in 1960 that the sodium channel was focused as the possible action site of TTX. As known, TTX could block the action potential without any eVect on the resting membrane potential and the delayed rectification because of the activity of potassium channels. Hence, TTX is suggested to block the sodium channel (Narahashi et al., 1960). This hypothesis was extensively elucidated by the voltage clamp technique using lobster giant axons (Narahashi et al., 1964). Figure 16 is an example of such experiment. Sodium currents are completely and reversibly blocked by TTX while potassium currents are unchanged. Nowadays, TTX has become a popular chemical tool in the laboratory and is used as a useful biochemical agent. A lot of studies have been conducted about TTX action, including the possible eVects on sodium channel receptors and other ion channels and dynamical mechanism of sodium channel blocks. There have been published reviews describing the mechanism of action of TTX

mA/cm2 15 10 5 0 −5

Control

8-12-71-Bo

TTX 3 3 10−7 M 5 minutes

Washing

0

2

4

28 minutes

6 ms

FIG. 16 Families of membrane currents associated with step depolarization (10-mV steps) in a squid giant axon before and during external applications of 3  10–7 M TTX and after washing with toxin-free medium. Note that TTX blocks transient sodium currents without any eVect on steady-state potassium currents (Narahashi, 2001).

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D.-F. HWANG AND T. NOGUCHI

(Catterall, 1980, 1992, 1995, 2000; Narahashi, 1974, 1988a,b, 2001). Meanwhile, the structure and function of voltage-gated sodium channel have been clearly demonstrated in the past few years. The sodium channel consists of various subunits, depending on the tissue of origin. The a subunit is the major and most important component because expression of the a subunits of brain or skeletal muscle sodium channels in Xenopus oocytes without the b subunits yields functional sodium channels, although these are activated and inactivated more slowly than the sodium channels in native neuron or muscle. Only when coexpressed with b1 and b2 subunits, normal channel gating is observed (Isom et al., 1992, 1995; Schreibmayer et al., 1994). The channel from mannalian brain is a complex of an a subunit (260 kDa), a b1 subunit (36 kDa), and a b2 subunit (33 kDa). The channel from skeletal muscle contains only the a and b1 subunits; in the channel from electric eel electroplax, only the a subunit is required for ion conductance. 3. Molecular structure of sodium channels The a subunit contains four homologous domains (I–IV) and each domain consists of six a-helical transmembrane segments (S1–S6) (Catterall, 1992, 1995; Guy and Seetharamulu, 1986; Narahashi, 2001). A loop that dips into the transmembrane region of the protein between transmembrane segments S5 and S6 of each domain is considered to form a channel pore. Within the pore, a set of four amino acids (aspartic acid, glycine, lysine, and alanine), occupying equivalent positions in each of the four domains, come together to form a narrow bottleneck and are likely intimately involved in channel conductance and ion selectivity (Denac et al., 2000). The S4 segments carry multiple positive charges by a number of conserved arginine or lysine residues occupying every third position along the chain interspersed by two hydrophobic residues. These charges physically traverse the membrane in a corkscrew manner in response to the electric field, and are believed to form the voltage sensor. The negative internal transmembrane electric field pulls these charges into the membrane, locking them into a ‘‘cocked’’ position. Current models indicate that these positive charges be stabilized within the membrane segments. Depolarization releases the S4 segments to ‘‘unscrew’’ outward, initiating a conformational change that opens the channel (Catterall, 2000). A small cytoplasmic loop between domains III and IV, containing the hydrophobic amino acids isoleucine, phenylalanine, and methionine, comprises the fast inactivation gate. These amino acids act as a ‘‘tethered ball,’’ which physically blocks the pore by binding to receptor residues in the cytoplasmic opening in response to conformational changes linked to channel activation.

TETRODOTOXIN POISONING

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The b subunits could indirectly modulate channel gating and ion conductance by stabilizing relevant a subunit conformations. They also possess immunoglobulin-like folds in their secondary structure analogous to celladhesion molecules. So they may interact with extracellular proteins through these folds and then influence the location and density of sodium channels in neural tissues (Bonhaus et al., 1996; Catterall, 2000). The sodium channel is the target for several classes of natural neurotoxins. These toxins may aVect channel function in a variety of ways by binding to specific receptor sites on the a subunit and interacting with specific portions of the channel protein. These toxins have been invaluable probes for channel structure and function and their specific sites have, in some cases, been localized on the channel protein. The inactivation gate is located in the inner loop between S6 of domain III and S1 of domain IV; transmembrane segment S4 of each domain contains voltage sensors; and several phosphorylation sites by protein kinase and protein kinase C are identified in the inner loops between domains I and II and between domains III and IV (Figure 17).

l

ll

lll

ψ

IV

H3N+

ψψ 1 2 345

ψ

ψ

ψ

ψ

H3N+

β2 subunit

α subunit

ψ

β1 subunit

6

1 2345

6

1 2345

6

h

−OOC

Voltagesensing S4 transmembrane segment H3N+

1 234 5

6

Outside Membrane Inside

P

COO−

COO−

Inactivation

P

− +

P P

P

P

Substrate for phosphorylation by cAMP−dependent protein kinase

P

Substrate for protein kinase C

P

Modulation

FIG. 17 Subunit of voltage-gated Naþ channel a subunit consists of the four homologous domains (I–IV). The polypeptide chains are represented by continuous lines in each segment ion of the channel protein (Catterall, 2000; Narahashi, 2001).

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D.-F. HWANG AND T. NOGUCHI

4. TTX-R sodium channels Most sodium channels in neurons and skeletal muscle fibers possess high sensitivity to TTX block. The IC50 values are in the order of nmol/liter. While those possessing less sensitivity or resistibility to TTX have also been found in various tissues. Denervated skeletal muscle and cardiac muscle represent classical cases. The IC50 values for TTX block are 1 mmol/liter for rabbit Purkinje fibers (Cohen et al., 1981), 9 mmol/liter for rat myocardial cells (Matsuki and Hermsmeyer, 1983), and 14 mmol/liter for pig papillary muscle (Baer et al., 1976). These IC50 values are approximately two to three orders of magnitude higher than those for TTX-sensitive (TTX-S) sodium channels of neutral tissues and skeletal muscle. Interestingly, TTX-R sodium channels are also found to be present in various neurons, including bullfrog sensory neurons (Campbell, 1988; Guo and Strichartz, 1990), garter snake sensory neurons (Jones, 1986), group C sensory neurons (Bossu and Feltz, 1984), rat nodose neurons (Ikeda and Schofield, 1987; Ikeda et al., 1986), and human and mouse dorsal root ganglion (DRG) neurons (Kostyuk et al., 1981; Matsuda et al., 1978; McLean et al., 1988; Schwartz et al., 1990; Yoshida et al., 1978). In recent decade, TTX-R sodium channels were further focused because they are related to C fibers, which transmit pain sensation to the brain. If a chemical acts on TTX-R sodium channels but does not act on TTX-S sodium channels, this chemical is supposed to be a useful antinociceptive. The fact indicated that capsaicin blocked unmyelinated C fibers of human sural nerve in vitro and there was a good correlation between the sensitivity of C fibers to capsaicin and their resistance to TTX. The C fiber action potentials were found to be completely blocked by capsaicin in the presence of TTX (Grosskreutz et al., 1996). The characteristics of TTX-S and TTX-R sodium channels of rat DRG neurons are as follows (Narahashi, 2001; Ogata and Tatebayashi, 1992; Roy and Narahashi, 1992). The IC50 values for TTX block are 0.3 nmol/liter and 100 mmol/liter for TTX-S and TTX-R sodium channels, respectively. Thus, there is a 300,000-fold diVerence in TTX sensitivity between the two types of sodium channels. The kinetics of sodium current are markedly diVerent, TTX-S sodium currents are faster than TTX-R sodium currents in both activation and inactivation phases. Significant diVerences are also found in the activation and inactivation voltages. The potentials for 50% activation of sodium current are –26 mV for TTX-S and –15 mV for TTX-R channels and those for 50% inactivation are –70 mV for TTX-S and –40 mV for TTX-R channels. TTX-S and TTX-R sodium channels are known to have significant diVerences in sensitivity to certain chemicals. Both chemicals lead and cadmium

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decreased the sodium currents and changed more activation in TTX-R than TTX-S sodium channels. The lead-induced changes of the potential for 50% conductance are 15 mV and 25 mV, and the lead-induced decreases in maximum conductance are 8% and 55% for TTX-S and TTX-R sodium channels, respectively. The cadmium-induced changes of the potential for 50% conductance are 25 mV and 21 mV, and the cadmium-induced decreases in maximum conductance are 16% and 69% for TTX-S and TTX-R sodium channels, respectively. On the other hand, synthetic pyrethroid insecticides tetramethrin and allethin were found to significantly aVect the sensitivity of TTX-S and TTX-R sodium channels (Ginsburg and Narahashi, 1993; Tatebayashi and Narahashi, 1994). Tetramethrin reveals an enormous tailing current responsible for terminating a depolarizing pulse in a concentration-dependent manner, while causing almost no eVect on the peak sodium current. Pyrethroids modulate the individual sodium channel by prolonging the open time and by inhibiting the activation (Chinn and Narahashi, 1986; Yamamoto et al., 1983), and a method has been developed to calculate the percentage of sodium channels modified by pyrethroids (Tatebayashi and Narahashi, 1994). Comparison of TTX-S and TTX-R sodium channels for their percentages of tetramethrin modification as a function of concentration is given in Table XXI (Narahashi, 2001). TTX-R sodium channels are 30–100 times more sensitive to tetramethrin than TTX-S sodium channels. Sodium channels from diVerent organs have been cloned and classified. Some structural aspects of sodium channels are being disclosed

TABLE XXI PERCENTAGES OF THE FRACTION OF TTX-S AND TTX-R SODIUM CHANNELS MODIFIED BY VARIOUS CONCENTRATION OF TETRAMETHRIN

Modification of sodium channels (%) Tetramethrin (mM)

TTX-S

TTX-R

0.01 0.03 0.1 0.3 1.0 3.0 10.0

0 0.24  1.25  3.53  7.70  12.03 

1.31 5.15 15.35 35.48 57.82 74.85 81.20

0.10 0.13 0.66 1.20 1.89

Each value indicates the mean  S.E.M. (n ¼ 4). Source: Narahashi (2001).

 0.28  0.30  0.79  2.70  2.29  1.23  1.57

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(Catterall, 1992, 1995; Goldin, 1999; Marban et al., 1998; Plummer and Meisler, 1999). Sodium channels in the rat brain are classified into type I, II, IIA, and III, which comprise the a, b1, and b2 subunits, and all are sensitive to TTX block (IC50 values in the order of nmol/liter). In adult skeletal muscle, ml or SkM sodium channels are found to contain a and b subunits. They are sensitive to nmol concentrations of TTX and also sensitive to the blocking action of m-conotoxin GIIIA. In the heart and denervated skeletal muscle, HI or SkM2 sodium channels are found to be resistant to TTX with IC50 values in the order of 2–6 mmol/liter. In neuroendocrine and peripheral neurons, PN1 sodium channels are also sensitive to TTX with IC50 values in the order of nmol/liter. In dorsal root and trigeminal ganglion neurons, SNS or PN3 and SNS2 or PN5 sodium channels are found. Among them, SNS sodium channels are insensitive to TTX with IC50 values in the order of 30 mmol/liter or more, whereas SNS2 sodium channels are resistant to TTX with IC50 values in the order of 1 mmol/liter. 5. Site of action and binding of TTX Chemical compounds that block the sodium channels can be classified into several groups based on binding sites (Catterall, 1992). TTX, STX, and m-conotoxin bind to site 1 of sodium channels causing channel block. Batrachotoxin, grayanotoxins, veratridine, and aconitine, which prolong the sodium channel opening, bind to site 2. Site 3 is bound by sea anemone and a-scorpion toxins (class 1), which also cause a prolongation of sodium channel opening. b-Scorpion toxins of classes 2 and 3 bind to site 4, resulting in modulation of channel gating. Brevetoxins and ciguatoxin bind to site 5, prolonging sodium channel opening. Pyrethroids which also prolong sodium channel opening and modulate the gating bind to site 6. Binding site 1 of sodium channels for TTX locates in short segments SS1 and SS2 that connect transmembrane segments S5 and S6 of each domain (Catterall, 2000). This part plays as the selectivity filter for various ions, and TTX blocks the selectivity filter in the outer pore of sodium channels (Hille, 1975). Amino acids with negative charge are present in analogous positions in all four domains. These amino acids are supposed to form outer and inner rings that serve as the binding site for TTX (Catterall, 2000). Cardiac sodium channels are 200–1000 times less sensitive to TTX than TTX-S neuronal sodium channels. This diVerence is caused by various amino acids. The brain and skeletal muscle sodium channels have phenylalanine and tyrosine, respectively, in the P-loop of domain I, whereas the cardiac sodium channels contain cysteine in the corresponding position (Backx et al., 1992; Heinemann et al., 1992; Penzotti et al., 1998; Satin et al., 1992; Sunami et al., 2000). The sodium channels of DRG (PN3/SNS) are more resistant to TTX than

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the cardiac sodium channels (Roy and Narahashi, 1992). These channels contain serine in domain I to substitute phenylalanine in cardiac sodium channels (Sivilotti et al., 1997). G. THERAPEUTIC APPLICATION OF TTX

Since TTX was found to eVectively block sodium channel, it has been conducted for the possible therapeutic applications. One possible application is a neuroprotective drug in the treatment of ischemic damage of the brain that follows stroke. Whereas the onset of stroke is accompanied by a rapid local damage in the brain, infarction spreads from the point of damage slowly. Thus, there is a therapeutic window during which damages to the peri-infarct area can be prevented or minimized by administration of a neuroprotective drug (Koroshetz and Moskowitz, 1996). Ischemia causes membrane depolarization, which in turn evokes repetitive discharges. At the nerve terminals, these discharges open calcium channels, causing influx of Ca2þ which in turn releases neurotransmitter. At the glutamatergic synapses, the released glutamate activates the NMDA receptor channel through which Ca2þ ions enter. The increase in intracellular Ca2þ concentration kills the neuron. Therefore, the presynaptic sodium channels, the presynaptic calcium channels, and/or the NMDA receptors may be blocked to prevent or minimize the ischemic damages. TTX is eVective in mitigating ischemic damages caused by occlusion of vessels in rat hippocampus (Yamasaki et al., 1991) and those caused by exposure to veratridine in cerebellar neurons and hippocampal neurons (Lysko et al., 1994). Focal microinjection of TTX reduces neurological deficits and tissue loss after spinal cord injury. In rats subjected to a standardized weightdrop contusion, the injection of TTX into the injury site was eVective in attenuating the damages to the large diameter axons (Rosenberg et al., 1999). TTX is also eVective in blocking electrographic seizures in vitro (Burack et al., 1995). Stimulus train-evoked seizures were blocked for several hours after localized injection of 50 mmol/liter TTX in rat hippocampal slices, whereas responses to single stimulus were minimally altered by TTX. TTX injections that blocked electrographic seizures were nearly always located in CA2/3 stratum radiatum and/or stratum lacunosum-moleculare. When applied in the perfusion medium, TTX was eVective in blocking electrographic seizures at low concentrations (5–20 nmol/liter). TTX was also eVective in preventing posttraumatic epileptogenesis in rats (Graber and Prince, 1999). Evoked epileptiform field potentials were observed in the injured cortex, and thin sheets of Elvax polymer containing TTX implanted over lesions were eVective in decreasing evoked epileptiform potentials.

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To find out drugs that could recover the intoxication caused by TTX, some attempts were made. 4-Aminopyridine at 1–2 mg/kg (i.m.) was found to be eVective in guinea pigs to restore the toxin-induced diaphragmatic block, bradypnea, bradycardia, and depressed cortical activity (Chang et al., 1996, 1997). TTX was a good anesthetic to the rabbit eyes without causing any systemic toxicity when operating an excimer laser keratectomy (Chang et al., 1997). Topically applied TTX at 1 or 10 mmol/liter was shown to be a long-acting anesthetic in the rabbit cornea (Schwartz et al., 1998a,b). Subcutaneous injections of TTX with epinephrine were eVective for prolonged local anesthesia of poreutaneous sciatic nerve in rats. The median eVective concentration of TTX was 11.5 mmol/liter and reversible blocks lasted over 13 hours (Kohane et al., 1998). Recently, TTX is found to have a renoprotective action in a rodent model for ischemia reperfusion in jury that contributes significantly to posttransplant graft dysfunction (Garvin et al., 1999). TTX was also useful to protect the kidney from warm ischemia in uninephrectomized rats (Li et al., 1992). A Mab against TTX has been developed. Mice were injected i.p. with 1.5 MU of TTX, and 3 minutes later 100 mg of antibody immunoglobulin G (IgG) were injected through the tail vein. A 100% survival was observed (Matsumura, 1995a). The Mab was highly specific for TTX, and no crossreaction to TTX derivatives and paralytic shellfish toxins, and could neutralize TTX in vitro (Matsumura, 1995b). The ability of TTX-specific Mab to confer passive protection against lethal TTX challenge was investigated (Rivera et al., 1995). The Mab, T20G10, was specific for TTX, less reactive with anhydroTTX, and unreactive with TDA. T20G10 specifically inhibited TTX binding, but had no eVect on STX binding. T20G10 prevented death of mice orally administered with TTX.

IV. HIGHLIGHT OF VIEWPOINT A. PROPOSED PROGRAMS OF NEW FOOD INDUSTRY FOR PUFFER

1. Production of nontoxic puVer by new developed biotechnology The issue ‘‘how to make wild puVer safe to eat’’ has been carefully elucidated in the section on prevention. Now, harvest of wild puVer in Japan has recently outstandingly decreased on account of their overfishing since 10 years ago. Instead, puVer culture has increased resulting in 80% of total marketed puVer. For their culture, the puVer parents are paired with toxic wild female and nontoxic cultured male puVer. The eggs fertilized with nontoxic sperms are toxic

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in the beginning and become nontoxic larvae after 2 weeks of hatching. These larvae grow into adult by feeding nontoxin diet in surrounding nets. These adult puVers are nontoxic. PuVer fan can eat each part of them with safety. 2. Revival of a traditional food puVer liver ‘‘Kimo’’ Since Japanese Public Health Administration prohibited serving puVer liver, ‘‘kimo’’ as food in 1983, ‘‘kimo’’ (a traditional food) fans have been anxious to revive safe ‘‘kimo.’’ The kimo harvested in the above culture will be available for revival of a traditional food (‘‘kimo’’), which is expected to activate Japanese fishery industry because puVer livers have been discarded so far (Noguchi et al., 2004). B. METHOD OF SPECIES IDENTITY FOR PUFFER BY GENOME TECHNIQUES

1. Introduction Species identification of seafood product is important for the implementation of the labeling regulations as set by many countries. These regulations to prevent the substitution of some commercially important fish can be eVectively achieved when species-specific data of all fish species are available. Genome and protein techniques are the two valuable methodologies that can be used for fish species identification. These methods can prevent adulteration of toxic puVer species as nontoxic puVer one. Hence, the development of genome and protein techniques for eVective identification of puVer species is critically needed. Recently, molecular methods based on nucleic acids amplification by means of polymerase chain reaction (PCR) have been well established and applied to the field of species identification. Usually, PCR is coupled with other techniques that are capable of detecting diVerences in the sequences of the PCR amplification products. With the PCR products, restriction fragment length polymorphism (RFLP), or single-strand conformation polymorphism (SSCP) could achieve analyses. Species identification can also be accomplished by using the other means of PCR reaction, such as random amplified polymorphic DNA (RAPD). All these techniques use gel-based patterns to facilitate species identification. They could be divided into two categories based on the targets: the RAPD is directed at multiple gene locations, while the RFLP and SSCP are generally aimed at only one or very few gene locations (Hold et al., 2001). These methods for species identification are chiefly based on mitochondrial cytochrome b gene (Quinteiro et al., 1998; Russell et al., 2000), mitochondrial 12S rRNA (Comesana et al., 2003), mitochondrial 16S rDNA

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(Karaiskou et al., 2003a), mitochondrial ND-1 gene (Politov et al., 2000), mitochondrial a-actin gene (Fernandez et al., 2000), and nuclear 5S rDNA (Carrera et al., 2000; Karaiskou et al., 2003a,b). Mitochondrial DNA is the primarily employed genetic tool, and one of its advantages is the high copy numbers of the mitochondrial genome (Mackie et al., 1999), compared to nuclear genome within a cell. The great advantage of the DNA techniques is that there is satisfactory information to be recognized by the primers although the DNA degrades on processing. The principal theory and practical application of these methods for species identification are concisely discussed below. 2. Direct sequence analysis Direct sequencing of species-specific DNA is currently the most reliable approach for species identification. However, it was regarded as a labor-intensive and high-priced technique (Chapela et al., 2003). The most important advantage of this technique is that the results are not aVected by intraspecific variability (Bossier, 1999). Moreover, it is not necessary to investigate the data from all reference samples because the sequence of nucleotide residues can be compared to those in the international database GenBank. This technique has been successfully applied in identifying many fish species, including cephalopod (Chapela et al., 2003), caviar (Ludwig et al., 2002), tuna (Lockley and Bardsley, 2000; Takeyama et al., 2001), Trachurus species (Karaiskou et al., 2003a,b), sole and halibut (Cespedes et al., 2000), puVer (Cheng et al., 2001; Hsieh, 2003; Hwang et al., 2002a; Song et al., 2001), flatfish (Comesana et al., 2003), anchovy and sardine (Jerome et al., 2003; Sebastio et al., 2001), hake (Quinteiro et al., 2001), and salmon (Russell et al., 2000). The complete DNA sequence also provides an invaluable tool for accomplishing genetic studies among taxa and phylogenetic relationships for closely related fish species (Hsieh, 2003; Karaiskou et al., 2003a,b). When this technique was applied to identify the tuna and bonito species of canned products using the amplified product of a 59-bp short fragment, 9 of the 11 samples were correctly diVerentiated (Unseld et al., 1995). The increased use of nucleotide sequences in PCR and direct sequencing for elucidating species-specific genes or full genome information in vertebrates has made international databases available, complementary for subsequent comparative studies in species identification of fish. 3. PCR-RFLP At present, PCR-RFLP is the most common DNA-based technique used for fish species identification. Scientists draw the greatest attention to mitochondrial cytochrome b gene. The species-specific gene appears to have substantial inter- and intraspecies variation in its original nucleotide sequence, and the

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level of variation within species is much less than between species (Mackie et al., 1999). The technique is considered as an easy, rapid, and relatively cheap tool in identifying fish species. PCR-RFLP allows for the amplification of a conserved region of DNA sequence using PCR and the detection of the specific patterns after treatment with restriction enzymes, usually 4–6 bp in length (Hold et al., 2001; Sun and Lin, 2003). Generally, the shorter recognition sequence of enzyme will generate greater number of fragments (Sotelo et al., 2001). Direct sequencing technique is generally considered to be the preliminary step for RFLP analysis. The generated sequences are then utilized for examination of potential restriction sites of nucleases that could be applied for the diVerentiation of each species. Finally, the species-specific patterns of samples obtained after the cleavage by restriction enzymes could be detected and compared after electrophoresis (Ludwig et al., 2002). In recent years, many reports showed the great eVectiveness of PCR-RFLP analysis in diVerentiating raw fish species (Cespedes et al., 2000; Comesana et al., 2003; Fernandez et al., 2000; Karaiskou et al., 2003a,b; Politov et al., 2000; Russell et al., 2000; Sebastio et al., 2001; Takeyama et al., 2001) and cooked fish or seafood products (Chapela et al., 2003; Hold et al., 2001; Jerome et al., 2003; Ludwig et al., 2002; Mackie et al., 1999; Quinteiro et al., 2001). Except for the 12S rRNA (Cespedes et al., 2000; Comesana et al., 2003), 16S RNA (Colombo et al., 2002), a-actin (Fernandez et al., 2000), control region, and ND-1 gene (Politov et al., 2000; Quinteiro et al., 2001), most of these publications concentrate on the mitochondrial cytochrome b gene. As to the severely degraded products like sterilized food, the mitochondrial control region is the most suYcient for the identification of fish species (Mackie et al., 1999; Quinteiro et al., 2001). PCR-RFLP was shown to achieve the recognition of mixed species of canned tuna fish, while PCR-SSCP was not (Mackie et al., 1999). With this technique, a 96% success rate was achieved for authentic identification from a total of 120 examinations (Hold et al., 2001). Typically, the use of two or more restriction enzymes was adequate to accurately discriminate fish species. In some cases, PCR-RFLP did not allow precise identification of all the test fish species (Colombo et al., 2002; Sotelo et al., 2001). So far, intraspecific variation has not been a serious problem when using this technique for fish species identification, although this possibility cannot be excluded. 4. PCR-SSCP SSCP is a fast, easy to perform, and well-suited technique for detecting even single base changes in short DNA fragments (Hayashi, 1996). PCR is initially applied to amplify the region of interest. The resultant DNA is usually heated in a denaturing solvent and separated as single-strand DNA

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by electrophoresis in a nondenaturing polyacrylamide gel electrophoresis (PAGE) (Orita et al., 1989). In comparison with PCR-RFLP and RAPDPCR, PCR-SSCP is the best method to reflect the entire nucleotide sequence of the sample of interest (Orita et al., 1989). From a total of 72 cases in identifying canned tuna fish, only 7 cases were incorrectly identified (Rehbein et al., 1999). The PCR-SSCP of mitochondrial 12S rRNA allows for the identification of grouper species, wreck fish, and Nile perch fish (Asensio et al., 2001). The weakness of the technique is that both the references and test samples need to be run on the same gel. In addition, limited information on SSCP patterns is available (Rehbein et al., 1997). 5. RAPD-PCR PCR is frequently used to amplify a particular DNA of interest. However, the RAPD analysis, also known as arbitrary primed PCR (AP-PCR), uses short primers amplifying fragments of DNA which are essentially unknown to the scientists (Dinesh et al., 1996). It is a relatively faster, cheaper, and simpler technique for use than PCR-SSCP and PCR-RFLP (Mackie et al., 1999). It can be done without having adequate information about the genome of the studied species. Reproducibility is an encountering problem, but the use of strict PCR conditions and high quality of template DNA helps prevent the main drawback of this technique. Four of 20 10-mer random oligonucleotide primers were applied to identify part of mussel species using RAPD method. Further use of the designed primers based on the information could lead to achieving complete identification of the mussel species (Rego et al., 2002). Callejas and Ochando (2001) used 10 random primers to create RAPD markers for identification of barbell fish. However, this approach failed to identify canned tunas and bonitos (Mackie et al., 1999). The use of RAPD in 16S rRNA gene failed to identify all the test puVer fish species because a low-level variation in DNA sequence was observed between T. rubripes and T. pseudommus (Song et al., 2001). 6. PuVer genome The international Human Genome Project that aims to identify and characterize all human genes in order to facilitate the knowledge of human biology has provided a driving force to vertebrate genomics research (Lander et al., 2001; Venter et al., 2001). Identifying coding and regulatory sequence is a major challenge of the postsequencing era of the human genome project. Comparative genomics, the comparison of genomic sequences from diVerent species, is a good approach to interpret the human genome. Therefore, several other vertebrate genomes are being investigated to gain insight into their biology and to serve as models. These models include mouse, rat,

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chicken, frogs, and fish such as the zebrafish, medaka, and fugu (Amaya et al., 1998; Beier, 1998; Venkatesh et al., 2000; Waterston et al., 2002). Among them, the ‘‘torafugu,’’ T. rubripes, has the smallest vertebrate genome of about seven to eight times smaller than human’s but has a similar repertoire of genes. Its genes are densely packed with short intergenic and intronic sequences and contain less than 10% repetitive DNA (Brenner et al., 1993). Its usefulness in the discovery of conserved regulatory elements has already been elucidated. Hence, the puVer genome is a good complement to the genetic studies in other vertebrates (Elgar et al., 1996; Venkatesh et al., 2000). The whole genome of T. rubripes was identified as 365 Mb (Aparicio et al., 2002). The authors also highlighted that three-quarters of the predicted human proteins have strong match to fugu proteins, implying that there exists an interesting linkage between human and fugu proteomes (Aparicio et al., 2002). For the most updated puVer fish genome sequences, some Web sites such as http://fugu.hgmp.mrc.ac.uk, http://www.fugu-sg.org, and http://www.jgi.doe.gov/fugu are available. C. METHOD OF SPECIES IDENTITY FOR PUFFER BY PROTEIN TECHNIQUES

Due to morphological similarities, the manufacturers and consumers may have diYculty in distinguishing nontoxic L. gloveri from L. lunaris, a species that accumulates lethal level of TTX in its muscle. Therefore, serious food poisoning incidents due to ingestion of toxic puVer fish or the toxic dried dressed fish fillets have occasionally occurred in Taiwan. From the viewpoint of food protection and public safety, it is critical to accurately identify the nontoxic puVer fish from the toxic species. Sodium dodecyl sulfate-PAGE (SDS-PAGE), native isoelectric focusing (N-IEF), and immobilized pH gradients-two-dimensional electrophoresis (IPG-2DE) were employed to validate the feasibility of using these techniques to develop species-specific muscle protein profiles for puVer species identification. Species-specific bands of sarcoplasmic, myofibrillar, SDS-soluble, and urea-soluble proteins were found in the molecular weight region below 30 kD of the SDS-PAGE patterns. Coomassie blue/silver double staining provided better protein banding patterns for discrimination of diVerent puVer species than Coomassie blue staining alone. In some cases, especially for Coomassie blue-stained sarcoplasmic proteins, the density of the protein profiles facilitates precise identification. The use of double staining to stain SDS-soluble proteins seemed to be the best combined approach for identifying puVer species when using the SDS-PAGE method (Chen and Hwang, 2002; Chen et al., 2002a,b). The SDS-PAGE pattern of 1% SDS extract with double staining and the concentration of low-molecular-weight proteins are shown in Table XXII. In the region above 19.5 kD, the pattern was almost the same

214

FOLLOWING DOUBLE STAINING AND DETERMINED BY DENSITOMETRY

Molecular weight (kD) 30.5 30.2 29.3 28.4 28.0 26.8 24.9 24.2 23.9 22.8 21.9 19.5 18.9 17.0

Relative amount (%) LW

LG

4.9 – – – 3.6 – 4.0 – – 2.6 4.0 3.3 – 2.2

3.9 – – 3.5 – – 5.4 – – 3.8 1.8 2.9 – –

 0.5a

 1.2a  1.2ab  0.4b  1.4b  1.0ab  0.3a

LL  0.8a  0.8  0.8a  1.0a  0.6c  0.4b

3.9  – – – 3.8  – – 3.7  – 2.5  5.5  2.5  – 1.9 

LI 1.0a

0.6a 0.8 0.9ab 0.8b 0.4b 0.3a

4.0  – – – 3.7  – 3.8  – – 4.2  5.7  4.2  – 1.0 

TO 0.6a

1.0a 0.6b 1.0a 0.8b 0.3a 0.2b

– 3.6 2.3 – 1.5 3.8 – – 4.1 – 5.0 – 3.6 –

TX  1.0a  0.6a  0.2b  1.0a  0.6a  1.3b  0.6

– 4.0  3.7  – 0.4  2.8  – – 3.4  – 6.2  2.3  – –

SP 0.6a 1.0a 0.2c 0.6a 0.7a 0.8b 0.8b

– 1.3 – – 3.6 1.1 – – 4.1 2.0 8.2 – – –

 0.3b  1.0a  0.2b  0.9a  0.5b  0.7a

D.-F. HWANG AND T. NOGUCHI

TABLE XXII COMPOSITION PERCENTAGE OF 1% SDS EXTRACT WITH LOWER MOLECULAR WEIGHT (30.5 kD) FROM SEVEN PUFFER SPECIES

– – 4.2  0.3b – 3.1  0.5a – – 3.5  0.4a –

– – 6.5  0.5a – 1.5  0.3b – 2.8  0.5 – –

– – 2.4  0.5c – 2.8  0.7a – – – 3.7  0.7

– – 2.5  0.2c 1.5  0.4 – – – 2.3  0.6b –

– – 2.4  0.5c – 3.8  0.5a 2.8  0.5a – – –

– – 1.6  0.3d – 3.1  0.5a 1.3  0.3b – – –

1.6  0.8  – – 0.7  2.0  – – –

0.3 0.2 0.2c 0.4a

LW, Lagocephalus wheeleri; LG, Lagocephalus gloveri; LL, Lagocephalus lunaris; LI, Lagocephalus inermis; TO, Takifugu oblongus; TX, Takifugu xanthopterus; SP, Sphoeroides pachygaster. Data are mean  S.D. of triplicate assays, and ‘‘–’’ represents the relative amount being less than 0.1%. Valves in a rank not followed by a same letter are significantly diVerent. Source: Chen and Hwang (2002).

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16.6 15.0 13.9 11.5 10.3 8.7 7.7 7.4 7.1

215

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D.-F. HWANG AND T. NOGUCHI

as that of Coomassie blue staining. In the double-stained region of 17.0–7.4 kD, the characteristic protein bands for each puVer were as follows: 17.0, 13.9, 10.3, and 7.4 kD for L. wheeleri; 13.9, 10.3, and 7.7 kD for L. gloveri; 17.0, 13.9, and 10.3 kD for L. lunaris; 17.0, 13.9, 11.5, and 7.4 kD for L. inermis; 13.9, 10.3, and 8.7 kD for T. oblongus and T. xanthopterus; and 16.6, 15.0, 10.3, and 8.7 kD for Sphoeroides pachygaster. T. oblongus and T. xanthopterus could be diVerentiated by the presence of 18.9 kD and 19.5 kD protein bands, respectively. Evaluation of the native IEF patterns showed that the majority of watersoluble puVer muscle proteins fell in the region with isoelectric point (pI) values of 5.85–8.65. The characteristic species-specific protein bands were found in all the three regions of pI 3.50–5.20, 5.85–6.55, and 7.35–8.15. Among them, the pI 3.50–5.20 was the most suitable region for identifying species-specific proteins. Coomassie blue staining was shown to be more adequate than silver-staining method for revealing the protein profiles for the identification of puVer fish species. Table XXIII listed the characteristic bands in the three clustered regions for each of the six puVer species. The species-specific bands at the acidic region of pI 3.50–5.20 were pI 4.18 and 4.90 for L. wheeleri; 5.02 for L. gloveri; 5.09 for L. lunaris; 4.20 and 4.50 for L. inermis; 4.13 for T. oblongus; and 4.59 for L. sceleratus. At the region of pI 7.35–8.15, the species-specific protein bands were pI 7.47, 7.63, and 7.80 for L. wheeleri; 7.38, 7.47, 7.63, and 7.82 for L. gloveri; 7.69, 7.86, and 7.97 for L. lunaris; 7.50, 7.77, and 7.99 for L. inermis; 7.77 and 7.99 for T. oblongus; and 7.44, 7.51, and 7.68 for L. sceleratus. Many of the numerous protein bands in the third region of pI 5.85–6.55 also provided good diVerential characteristics for species identification. Therefore, the Coomassie bluestained native IEF gels, which contain many sharp and species-specific protein bands, can be used as a valuable tool for puVer species identificaiton. In comparison between Coomassie blue staining and silver staining, silverstaining IEF gels produced high levels of background staining. Hence, native IEF is a feasible tool for identifying puVer species with either Coomassie blue stain or silver stain (Chen et al., 2003). In viewing the IPG-2DE patterns, it was noted that puVer muscle proteins that fell in the region with pI values of 3.5–7.0 and molecular weights of 7.4–45.0 kD were good for species comparison. The more acidic proteins of lower molecular weights showed species-specific characteristics. Therefore, species identification of puVer can be achieved from the comparison of IPG-2DE protein pattern profiles (Chen et al., 2004). Aside from conventional electrophoretic methods, capillary electrophoresis (CE) is considered as a novel electrophoretic technique for protein separation with advantages of being easy, rapid, automatic, using trace amount of test samples, and qualitative as well as quantitative within one analysis

Fish species

Protein content

pI 3.50–5.20

pI 5.85–6.55

Lagocephalus wheeleri Lagocephalus gloveri Lagocephalus lunaris Lagocephalus inermis Takifugu oblongus Lagocephalus sceleratus

7.2 6.2 6.3 7.4 5.9 10.4

4.18, 4.90 5.02 5.09 4.20, 4.50 4.13 4.59

5.90, 6.06, 5.90, 5.89, 5.92, 6.14,

Source: Chen et al. (2003).

6.01, 6.12, 6.01, 6.01, 6.01, 6.29,

6.08, 6.19, 6.45, 6.45, 6.09, 6.41,

pI 7.35–8.15 6.17, 6.41, 6.49, 6.49 6.33, 6.48,

6.28, 6.40, 6.43, 6.47 6.49 6.54 6.46, 6.53 6.55

7.47, 7.63, 7.80 7.38, 7.47, 7.63, 7.82 7.69, 7.86, 7.97 7.50, 7.77, 7.99 7.77, 7.99 7.44, 7.51, 7.68

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TABLE XXIII PROTEIN CONTENTS (mg/ml) AND THE SPECIES-SPECIFIC BANDS OF WATER-SOLUBLE PUFFER FISH PROTEINS WITH COOMASSIE BLUE STAINING

217

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(Grossman et al., 1989). CE is a powerful tool for the separation of proteins and peptides since it could identify diverse protein products by only one amino acid residue. The main constitutes of a CE instrument are highvoltage power supply, eluent system, capillary column, detector, and data processor (Gordon et al., 1988). It not only has the reliable separation theory from conventional electrophoresis but also the excellent separation resolution from chromatography (Kuhr, 1998). When applied for species identification, the pH of the eluent solvents (LeBlanc et al., 1994), the protein amounts injected to the system (Gallardo et al., 1995), and the packing material of the capillary column (Crockford and Johnston, 1995) all play important role in achieving success. CE also allows for precise identification of fish species that have been frozen, stored at diVerent temperatures for several months (Larrain et al., 2002; LeBlanc et al., 1994). When compared to SDS-PAGE, the CE and 2DE had more discriminatory power for identifying the species of myosin light chains in tilapia (Crockford and Johnston, 1995). The CE profiles of the peptides and amino acids were eVectively used to detect adulteration of shark fin (Chou et al., 1998). SDS-capillary gel electrophoresis (SDS-CGE) has been established as a powerful qualitative and quantitative technique in separating fish myofibrillar proteins, and thus is considered as an alternative to classic SDS-PAGE for fish species identification (Sotelo et al., 2000). The application of CE for puVer identification is worthy of study. The focus of basic molecular research will gradually move from genes/ genomes to proteins/proteomes because genome sequencing cannot define all protein components and their functions (Vihinen, 2001). To actually respond to the genes, the proteome referred to the systematic identification of the total protein complement of the genome must be done. Approaches for proteome analysis primarily include three protocols: 2DE, protein microchemistry, and bioinformatics. First of all, the IPG-2DE, which is the most commonly used procedure currently, is used to find out target proteins. Then matrix-assisted laser desorption time-of-flight mass spectrometry (MALDTOF-MS) is used to characterize protein spots. Sequence tagging with tandem MS (MS–MS) can be employed to further identify the proteins. Finally, the established proteome results were compared with genomic and proteomic information from databases on the web (Babnigg and Giometti, 2004). So far, the application of proteomic protocols for the investigation of fish has been limited. Inspection of international databases for DNA and protein information shows that the relative amount of protein sequence information in aquatic vertebrate research is merely 4% of that of total DNA sequences (Pineiro et al., 2003). Although the whole sequences of puVer genome has been established (Aparicio et al., 2002), publications related to their proteomic information are still not available.

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D. TTX AS ATTRACTANT AND HIBERNATION AGENT

We recently investigated the attracting eVect of small dose of TTX on eight toxic small snail species (P. didyma, N. lineata, N. vitellus, Z. suZatus, N. clathrata, O. miniacea, O. mustelina, and O. hirasei) and two nontoxic snail species (Pomacea canaliculata and Satsuma bairdi). It was found that all toxic snails showed significantly positive relationship between comparative attracting variation and the toxicity reported, and the relationship was a linear line (Hwang et al., 2004). The relationship between TTX resistance ability and the toxicity also has a positive correlation. However, the nontoxic species showed the negative response. The more toxic snail appeared to be the more preferable to TTX, indicating that TTX is an attractant for toxic snails (Figure 18). Previous papers (Hwang et al., 1990b,c, 1992b) have pointed that toxic gastropod possessed high-resistant ability to TTX and secreted the toxin as defense or attack agent. Matsumura (1995c) found that TTX was mostly distributed in the surface of puVer eggs, and might act as a pheromone to attract the male puVer. Meanwhile, Arakawa et al. (2003) investigated microdistribution patterns of TTX in the tissues of three species of puVer T. vermicularis, C. patoca, and T. steindachneri, and a Japanese

Comparative attracting variation (percentage per hour)

6

b 4

2

cc

b Oliva miniacea Natica vitellus Oliva hirasei

b

b

a Zeuxis sufflatus

a Natica lineata

Niotha Polinices clathrata didyma

Oliva mustelina

Y = 5.895X + 3.443 r = 0.806

0 50

d Satsuma bairdi d Pomacea canaliculata

−6

150

200

250

Snail toxicity (µg TTX per specimen)

−2

−4

100

Naticidae Nassariidae Olividae Camaenidae Ampullariidae

FIG. 18 The comparative attracting variation of TTX to toxic and nontoxic snails (Hwang et al., 2004).

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newt C. pyrrhogaster by means of a Mab-based immunoenzymatic technique under a light microscope. The distribution patterns in puVers varied in respect of species. In the skin sections of T. vermicularis, TTX was visualized at diVerent shapes of glands in the epidermis layer, while in case of C. patoca and T. steindachneri, neither such gland nor gland-like apparatus was apparent, and TTX was mainly detected in sacciform cells. Similarly, in the ovary section of T. vermicularis, TTX was visualized at nucleus, yolk vesicles, and/or yolk granules in various stages (late pari nucleolus stage, yolk granule stage-I, and yolk granule stage-II) of oocytes. In C. patoca, TTX was localized at the connective tissues and in the nucleus of some perinucleolar oocytes. On the other hand, observation of the newt skin sections demonstrated that TTX was distributed at immature glands in juvenile and at the granular cells composing of granular and mixed glands in adult specimens. No specific stain of TTX was recognized in larval section. A duct-like structure extending from the gland toward super epithelial layer was visualized in the skin sections of both T. vermicularis and C. pyrrhogaster. When stimuli by wiping with gauze (‘‘handling stimulus’’) were given, these animals secreted an applicable amount of TTX from the skin, suggesting that they have a gland of TTX to secrete it toward the body surface possibly as a biological defensive agent. Hence, the biological significance of TTX in toxic animals should be further studied. It is mysterious that TTX is assumed to act as major active agent of zombe powder used to kill people and then revive them. Haitian wizards usually use toxic puVer as a material of zombe powder, while the phenomenon of zombe is still a riddle or witchery. It is necessary to elucidate how much of toxin amount can let a person into hibernation and how to control time can let a person revive. If these problems become clear, TTX would be an important drug like anesthetic for healing serious burn wound, conducting organ transplantation, and intending space flight travel in future. On the other hand, circadian rhythms are driven by a pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus in mammals. Some reports have indicated that TTX reversibly abolishes the expression of circadian rhythm (Welsh et al., 1995; Yamaguchi et al., 2003). Noguchi and Watanabe (2005) further showed that TTX resets the clock. Hence, the neurophysiological role of TTX in human needs further study. V. SUMMARY This chapter on TTX poisoning begins with a brief description to puVer poisoning and then elucidates the origin, toxicology, chemistry, and pharmacology of TTX. The identification methods of puVer species are also

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presented. TTX is especially hazardous among marine toxins in the world, especially in Asian countries. TTX causes traditional poisoning incidents due to ingestion of toxic puVer in Japan, Taiwan, and China. Its poisoning incident continues to occur in spite of tendency of decrease yet. EVective medical treatment has not so far been established for puVer poisoning. Along this way, some attempts were made to prepare an antibody against experimental animals, with some promising results. Intoxification mechanism of TTX-bearing orgainsms such as puVer, gastropod, and so on was found to come from a food web, resulting in further discovery of them. Accordingly, the distribution was enlarged, though the number was limited. The structure of TTX was clearly elucidated in 1964. At present, it is demonstrated that TTX is composed of more than 10 components, which diVer from each other in side chain as well as in specific toxicity. TTX has long been detected by the mouse bioassay method, which is simple but not specific for this toxin. Recently, instrumental analyses have been widely applied to various natural substances such as HPLC, GC–MS, and HPLC–MS. TTX blocks sodium channels distributed on the cell membrane, and inhibits sodium ion flow in and out of the cell, resulting in paralysis of the muscle concerned. Applying the mechanism of action, TTX is often used as a specific pharmacological tool in studying nerve/muscle physiology. Furthermore, to prevent from intoxification due to ingesting the toxic puVer products, the DNA and protein techniques have been ultilized to identify the puVer species. REFERENCES Ali, A.E., Arakawa, O., Noguchi, T., Miyazawa, K., Shida, Y., and Hashimoto, K. 1990. Tetrodotoxin and related substances in a ribbon worm Cephalothrix linearis Nemertean. Toxicon 28, 1083–1093. Amaya, E., OYeld, M.F., and Grainger, R.M. 1998. Frog genetics: Xenopus tropicalis jumps into the future. Trends Genet. 14, 253–255. Aparicio, S., Chapman, J., Stupka, E., Putnam, N., Chia, J.M., Dehal, P., ChristoVels, A., Rash, S., Hoon, S., Smit, A., Gelpke, M.D., Roach, J., et al. 2002. Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes. Science 297, 1301–1310. Arakawa, O. 1988. Studies on paralytic toxins in crabs of the family Xanthidae. Ph.D. Thesis University of Tokyo. p. 130. Arakawa, O., Noguchi, T., Shida, Y., and Onoue, Y. 1994. Occurrence of 11-oxotetrodotoxin and 11-nortetrodotoxin-6R-ol in a xanthid crab Atergatis floridus collected at Kojima, Ishigaki Island. Fish. Sci. 60, 769–771. Arakawa, O., Mahmud, Y., Tanu, M.B., Tsuruda, K., Okada, K., Kawatsu, K., Hamano, Y., Takatani, T., and Noguchi, T. 2003. Micro-distribution of tetrodotoxin in puVers and newts. In ‘‘Proceedings of International Scientific Symposium on Marine Toxins and Marine Food Safety’’ (D.F. Hwang and T. Noguchi, eds), pp. 57–65. National Taiwan Ocean University, Keelung. Asakawa, M., Toyoshima, T., Ito, K., Bessho, K., Yamaguchi, C., Tsunetsuge, S., Shida, Y., Kajihara, H., Mawatari, F., Noguchi, T., and Miyazawa, K. 2003. Paralytic toxicity in the ribbon

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MARINE BIOTECHNOLOGY FOR PRODUCTION OF FOOD INGREDIENTS ROSALEE S. RASMUSSEN AND MICHAEL T. MORRISSEY Seafood Laboratory, Department of Food Science and Technology Oregon State University, Astoria, Oregon 97103

I. Introduction II. Sources of Marine-Derived Food Ingredients A. Macro- and Microalgae B. Extremophiles C. Marine Sponges D. Fish and Seafood By-Products III. Marine-Derived Food Ingredients A. Photosynthetic Pigments B. Lipids C. Polysaccharides D. Proteins E. Enzymes IV. Conclusions References

The marine world represents a largely untapped reservoir of bioactive ingredients that can be applied to numerous aspects of food processing, storage, and fortification. Due to the wide range of environments they survive in, marine organisms have developed unique properties and bioactive compounds that, in some cases, are unparalleled by their terrestrial counterparts. Enzymes extracted from fish and marine microorganisms can provide numerous advantages over traditional enzymes used in food processing due to their ability to function at extremes of temperature and pH. Fish proteins such as collagens and their gelatin derivatives operate at relatively low temperatures and can be used in heat-sensitive processes such as gelling and clarifying. Polysaccharides derived from algae, including algins, carrageenans, and agar, are widely used for their ability to form gels and act as thickeners and stabilizers in a variety of foods. Besides applications in food processing, a number of marine-derived ADVANCES IN FOOD AND NUTRITION RESEARCH VOL 52 # 2007 Elsevier Inc. All rights reserved

ISSN: 1043-4526 DOI: 10.1016/S1043-4526(06)52005-4

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compounds, such as omega-3 polyunsaturated fatty acids and photosynthetic pigments, are important to the nutraceutical industry. These bioactive ingredients provide a myriad of health benefits, including reduction of coronary heart disease, anticarcinogenic and anti-inflammatory activity. Despite the vast possibilities for the use of marine organisms in the food industry, tools of biotechnology are required for successful cultivation and isolation of these unique bioactive compounds. In this chapter, recent developments and upcoming areas of research that utilize advances in biotechnology in the production of food ingredients from marine sources are introduced and discussed. I. INTRODUCTION The term biotechnology is associated with a number of meanings. In a broad sense, it can be defined as ‘‘any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use’’ (FAO, 2000). However, to some, biotechnology fits into a narrower definition restricted to ‘‘the commercial application of living organisms or their products, which involves the deliberate manipulation of their DNA molecules’’ (USDA, 1994). While this chapter will include some discussion of genetic research, it will be largely focused on the broader meaning of biotechnology, exploring new advances in the controlled manipulation and utilization of marine organisms for the production of food ingredients. Although the marine world represents nearly three-fourths of the Earth’s surface, it is one of the most underutilized biological resources, containing a vast array of organisms with unique biological systems and characteristics. Marine organisms such as macro- and microalgae, sponges, fish, and bacteria have all developed diverse and unique characteristics that allow them to survive under conditions with varying degrees of salinity, pressure, temperature, and illumination. Thanks to the tools of marine biotechnology, molecules that promote survival in marine environments have begun to be identified and methods of extraction are being developed and improved upon. In 2003 alone, over 650 new marine compounds were isolated from marine microorganisms and phytoplankton, green algae, brown algae, red algae, sponges, coelenterates, bryozoans, mollusks, tunicates, and echinoderms (Blunt et al., 2005), and the majority of marine organisms, mostly microorganisms, remain unidentified (Colwell, 2002; USDA, 1995). Biomolecules derived from marine organisms are useful to the food industry in a number of applications, including eYcient food production under unique conditions such as low temperature or high pressure; providing added nutritional benefits to foods; and/or using ‘‘natural’’ pigments, preservatives, or flavors. As shown in Table I, some major categories of marine-derived food

TABLE I MARINE-DERIVED FOOD INGREDIENTS

Health benefits and other advantages

Category

Food ingredient

Application

Major marine sources

Photosynthetic pigments

Carotenoids: b-carotene, astaxanthin, and lutein

Natural food colorings, nutraceutical agents, farmed salmon pigmentation

Microalgae: D. salina, S. maxima, C. protothecoides, C. vulgaris, and H. pluvialis

Vitamin A precursors, antioxidants, anticarcinogenic, anti-inflammatory, natural pigments

Carotenoids: fucoxanthin

Stimulation of UCP1, resulting in metabolic thermogenesis Natural food colorings

Seaweed: U. pinnatifida

Possible antiobesity eVect and reduced risk of type II diabetes Natural source of pigmentation Anticancer activity, natural source of pigmentation

Phycobilins: phycoerythrin phycocyanin Chlorophylls

Lipids

Natural food and beverage colorants

Omega-3 fatty acids: SDA, EPA, DHA

Nutraceuticals, fish oil capsules, fortification of livestock, feed and infant formula

Sterols

Aquaculture feed

Red and blue-green algae: e.g., P. cruentum Aquatic plants and bacteria: e.g., S. platensis and A. flos-aquae

Fish (e.g., salmon, sardine, tuna, herring), microalgae (Navicula spp., N. frustulum, B. sinensis, P. tricornutum, C. cohnii, A. carteri, G. simplex, G. cohnii, C. minutissima, P. cruentum, Thraustochytrium, Schizochytrium), fungi (phycomycetes), extremophiles, macroalgae (Rhodophyceae, Bryophytes), krill (E. superba), transgenic terrestrial plants Microalgae (thraustochytrids)

Numerous health benefits (e.g. visual and neurodevelopment, reduce risk of cardiovascular problems, ameliorate diseases such as arthritis and hypertension)

References Ben-Amotz, 1993; Gregory, 1996; Guerin et al., 2003; Maeda et al., 2005; Ramirez and Morrissey, 2003; Shi and Chen, 2001; von Elbe and Schwartz, 1996 Maeda et al., 2005 Borowitzka, 1993; Roman et al., 2002 Bhattacharya and Shivaprakash, 2005; Chernomorsky et al., 1999; de Oliveira RangelYagui et al., 2004; Donaldson, 2004; Egner et al., 2001; Kay, 1991; Sarkar et al., 1994 Borowitzka, 1993; Cohen et al., 1988; Horrocks and Yeo, 1999; Jiang et al., 2001; Kendrick and Ratledge, 1992; Nettleton, 1995; Radwan, 1991; Sijtsma and de Swaaf, 2004; Ursin, 2003; Venugopal and Shahidi, 1995; Yap and Chen, 2001; Yongmanitchai and Ward, 1989 Borowitzka, 1993; Lewis et al., 1999

(continued)

TABLE I (continued) Category

Food ingredient

Application

Major marine sources

Health benefits and other advantages

References

Polysaccharides

Phycocolloids: algins

Thickener, stabilizer, and emulsifier in foods such as salad dressings, ice cream, jam, and mayonnaise Gel formation and coatings in the meat and dairy industry

Brown seaweed (S. confusum, L. japonica, E. maxima, L. pallida, M. angustifolia)

Water soluble, stable at high temperatures, high viscosity

BeMiller and Whistler, 1996; FAO, 2004; Ohshima, 1998; Sutherland, 1996; Tseng, 2001

Red algae (K. alvarezii, E. denticulatum, B. gelatinum)

BeMiller and Whistler, 1996; FAO, 2004; Ohshima, 1998; Renn, 1993; Tseng, 2001; Vlieghe et al., 2002

Phycocolloids: agar

Gel formation and food gums

Red algae (Gelidium, Grateloupia, Gracilaria, Hypnea, Gigartina)

Anti-HIV activity and anticoagulant properties, water soluble, high viscosity, stable over wide pH ranges Water soluble, ability to gel aqueous solutions at low concentrations

Fucans/fucanoids

Potential use as nutraceuticals

Cell walls of brown algae, sea urchin eggs, sea cucumbers

Exopolysaccharides from cyanobacteria Exopolysaccharides from extremophiles

Emulsion stabilizers, bioflocculants Unique thickening, gelling, stabilizing, suspending, coagulating, filmforming, and water retention properties

Cyanobacteria (C. capsulata, Nostoc, Cyanothece) Extremophiles (Pseudoalteromonas, Alteromonas, Vibrio, H. mediterranei)

Phycocolloids: carrageenans

Anticoagulant, antithrombotic, anti-inflammatory, antiviral, cellular antiproliferative, and adhesive activities Unique and unusual properties Unique properties: unusual gelling, high metal-binding and thickening capacities, resistance to high salinities, temperatures, and pH

BeMiller and Whistler, 1996; FAO, 2004; Freile-Pelegrin and Murano, 2005; MarinhoSoriano and Bourret, 2005; Ohshima, 1998; Renn, 1993 Berteau and Mulloy, 2003; Kuznetsova et al., 2003; Mourao, 2004

de Philippis et al., 2001 Guezennec, 2002; Herbert, 1992

Chitin, chitosan, and their derivatives

Proteins

Collagen

Gelatin

Albumin

Gelling agents, antimicrobial activity, edible protective films, clarification and deacidification of fruit juices, emulsifying agents, nutraceuticals, water purifiers, and others Edible casings in the meat industry (e.g., sausages)

Stabilizer, texturizer, or thickener in ice cream, jam, yogurt, cream cheese, margarine, confectionaries, and lowfat foods; clarifiers (e.g., isinglass) in beverages such as wine, beer, cider, and vinegar; film- and foam-forming agents Replacement for egg albumin as a whipping, suspending, or stabilizing agent

Crustaceans (shrimp, crab, lobster, prawn, krill), lactic acid bacteria (L. plantarum)

Increase dietary fiber, reduce lipid absorption, antitumor, bactericidal and fungicidal activities

Haard et al., 1994; Rao and Stevens, 2005; Shahidi and Abuzaytoun, 2005; Shahidi et al., 1999; Synowiecki and Al-Khateeb, 2003

Fish (albacore tuna, silver-line grunt, bigeye snapper, brown-backed toadfish, hake, trout, lingcod, catfish, rainbow trout, yellow sea bream, common horse mackerel, tiger puVer, and others) Fish, especially cold-water (pollock, cod, haddock, hake, cusk)

Can be extracted from processing by-products

Jongjareonrak et al., 2005; Noitup et al., 2005; Senaratne et al., 2006

Forms gels at low temperatures, isinglass has been shown to prevent and treat chronic atrophic gastritis

Choi and Regenstein, 2000; Djagny et al., 2001; GomezGuillen et al., 2002; Haard et al., 1994; Norland, 1990; Xu et al., 2004

Mollusks, crustaceans, low-fat fish

High flexibility and strength, health benefits (e.g., anticoagulant, antioxidant) Alters the cell structure of some bacteria, does not coagulate under heat, natural preservative High productivity, favorable nutritional and functional properties

Haard et al., 1994; Nicholson et al., 2000; Ockerman and Hansen, 1988

Protamine

Antibacterial agent, preservative in fruits, rice, and confectionaries

Fish spermatozoa (e.g., herring and salmon milt)

Protein powders

Mariculture and animal feed, use as functional ingredients and nutritional supplements

Extremophile (Dunaliella), fish processing by-products (e.g., arrowtooth flounder and herring)

Islam et al., 1986; Ohshima, 1998; Potter et al., 2005 Herbert, 1992; Sathivel et al., 2004

(continued)

TABLE I (continued) Category

Food ingredient

Application

Major marine sources

Enzymes: Digestive proteases

Gastric proteases (e.g., pepsins, gastricsins, chymosins)

Cold renneting milk, fish feed digestion aid

Fish viscera (Atlantic cod, carp, harp seals, American smelt, sardine, capelin, salmon, mackerel, orange roughy, palometa, tuna)

Serine and cysteine proteases (e.g., trypsins, chymotrypsins, collagenases, elastases, cathepsin B)

PPO inactivation (preventing unwanted color changes in food products such as shrimp and fruit), food processing (lowtemperature protein digestion, meat tenderizing, curing of Herring, squid fermentation) Numerous uses in the fats and oils industry (e.g., production of omega-3enriched triglycerides) Processing and fermentation of tea, coVee, raisins, and prunes

Pyloric ceca, pancreatic tissues, intestines, hepatopancreas (stomachless bone fish, sardine, capelin, cod, cunner, salmon, anchovy, palometa, Atlantic white croaker, carp, hybrid tilapia, herring, spiny dogfish, rainbow trout, crustaceans, mollusks, short-finned squid)

Replace HCl for converting chitin into oligomeric units Creates protein cross-links to improve rheological properties of gels, i.e., surimi, gelatin

Enzymes

Lipases

Polyphenol oxidases (e.g., tyrosinase, polyphenolase, phenolase, catechol oxidase, cresolase, catecholase) Chitinolytic enzymes Transglutaminase

Health benefits and other advantages

References

Demonstrate catalytic activity at lower temperatures, thus minimizing unwanted chemical reactions and bacterial growth Demonstrate catalytic activity at lower temperatures, thus minimizing unwanted chemical reactions and bacterial growth

Shahidi and Janak Kamil, 2001; Simpson, 2000

Atlantic cod, seal, salmon, sardine, Indian mackerel, red sea bream, and others

Higher specificity for omega-3 fatty acids

Shahidi and Janak Kamil, 2001; Shahidi and Wanasundara, 1998

Crustaceans, microorganisms

Have higher activity at lower temperatures, as compared with terrestrial counterparts

Haard et al., 1994; Whitaker, 1996

Digestive tracts of fish, shellfish and shellfish waste, squid liver; octopus saliva Red sea bream, rainbow trout, atka mackerel, walleye, pollock liver, scallop muscles, botan shrimp, squid

Less harsh than HCl and results in more consistent products Strengthens gels with protein cross-linkages

Shahidi and Janak Kamil, 2001

Eichler, 2001; Gudmundsdottir and Palsdottir, 2005; Haard and Simpson, 2000; Haard et al., 1994; Raksakulthai and Haard, 2001; Shahidi and Janak Kamil, 2001; Simpson, 2000

Ashie and Lanier, 2000; Chen et al., 2003; Ohshima, 1998; Shahidi and Janak Kamil, 2001

Extremophilic enzymes

Red algae enzymes in the starch degradation pathway (e.g., a-1,4glucan lyase and others)

Production of the natural sugar 1,5-anhydro-Dfructose and the antifungal compound microthecin

Red algae (genera Gracilariales)

Production of compounds that exhibit antioxidant, antimicrobial, antiblood-clotting, and/or antitumor properties Active at moderate to high temperatures (45–100
C or more)

Yu, 2005

Thermophilic enzymes (e.g., heat-adapted a-amylase, glucoamylase, cellulase, chitinase, pectinase, b-galactosidase, xylose isomerase, pullulanase, neutral proteases, lipases, and serine and acid proteases) Psychrophilic enzymes (e.g., cold-adapted alcohol dehydrogenase, a-amylase, b-galactosidase, lipase, aspartate chitinase, pectinase, transcarbamylase, Ca2þ–Zn2þ protease, citrate synthase, b-lactamase, malate dehydrogenase, subtilisin, triose phosphate isomerase, and xylanase) Alkaliphilic enzymes (e.g., cyclomaltodextrin glucanotransferase, extracellular b-mannanases, pectinolytic enzymes, xylanases)

Baking and brewing, food processing, production of natural sweeteners, use in transesterification and oligosaccharide, peptide, and phospholipid syntheses

Thermophiles

Beer, wine, and dough fermentation, cheese production, milk and fruit juice processing

Psychrophiles (e.g., Antarctic and Arctic microorganisms)

High activity at low to moderate temperatures (5 to þ20
C), can replace mesophilic counterparts, reduce residual heat coagulation that commonly occurs during cheese production

Cavicchioli et al., 2002; Gerday et al., 2000; Gomes and Steiner, 2004; Herbert, 1992

Low-cost production of cyclodextrins from starch, guar gum hydrolysis, treatment of pectin-containing eZuent, production of wheat and rice straw

Alkaliphiles (e.g., Bacillus sp.)

Active under moderate to high alkalinities

Gomes and Steiner, 2004; Herbert, 1992

Eichler, 2001; Gomes and Steiner, 2004; Herbert, 1992

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ingredients used commercially are photosynthetic pigments, polyunsaturated fatty acids (PUFAs), sterols, polysaccharides, proteins, and enzymes. Many marine-based food ingredients fall under the category of nutraceuticals, which are bioactive substances with medicinal characteristics or added health benefits such as anticancer or anti-inflammatory activity. Fortification of foods with nutraceuticals has become an increasingly popular method for providing nutritional food products to health-conscious consumers. Marinebased nutraceuticals are already an active industry in Japan and Europe, and the US market has experienced significant growth over the past decade. According to Ohr (2005), consumer awareness of marine-based nutraceuticals has been growing due to reports on their extensive health benefits such as enhanced antioxidant activity and immunity. Some examples of marine nutraceuticals currently marketed in the United States include products such as fish and algal oils rich in omega-3 fatty acids, chitin and chitosan, fish and shark liver oil, marine enzymes and chondroitin from shark cartilage, sea cucumbers, and mussels. Omega-3 fatty acids are well known for their wide range of health benefits, including reduced risk of cardiovascular disease and enhanced brain development in infants, while chondroitin has been shown to have anti-inflammatory and anticancer properties. Marine-based food ingredients and nutraceuticals can be derived from a vast array of sources, including marine plants, microorganisms, and sponges, all of which contain their own sets of unique biomolecules that allow them to thrive in their respective habitats. Another growing source for marine-based food ingredients has been fish and seafood by-products resulting from postharvest processing. This chapter will cover current applications of marine biotechnology in the production of food ingredients from marine plants, animals, microorganisms, and processing by-products. In Section II, the major sources of marine-based food ingredients will be introduced and in Section III, specific biomolecules that have applications in the food industry will be discussed.

II. SOURCES OF MARINE-DERIVED FOOD INGREDIENTS A. MACRO- AND MICROALGAE

The term alga refers to a plant or plantlike organism from one of several phyla of mostly aquatic, chlorophyll-containing nonvascular organisms. Algae are divided into two general categories—macroalgae, such as red, yellow-green, green, and brown algae, and microalgae, such as blue-green algae. According to Chen and Jiang (2001), there may be over 50,000 species of algae worldwide. Humans utilize algae as a source of health food, food ingredients, and

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high-value chemicals and pharmaceuticals. Recently, there has been a renewed interest in developing high-quality products from algae, with algae showing great potential as a source of bioactive substances. Some cultivated marine microalgae, such as Spirulina, Dunaliella, Chlorella, and Crypthecodinium cohnii, have exhibited promising nutraceutical properties, including the presence of b-carotene, omega-3 fatty acids, and antioxidants (Molyneaux and Lee, 1998). The algal biotechnology industry is growing, with an aquaculture sector that produces large amounts of seaweeds, such as Laminaria, Porphyra, and Gracilaria, and microalgae, including Dunaliella and Spirulina. Additionally, the utilization of algae-derived colloids (phycocolloids) such as algin, agar, and carrageenan has developed into an important industry (Tseng, 2001). Global production of aquatic plants for 2002 was 11.6 million tons, generating US$6.2 billion, with the highest production coming from Japanese kelp (Laminaria japonica) (4.7 million tons) followed by Nori (Porphyra tenera) with 1.3 million tons (FAO, 2004). Despite the growing promise of algae as a source of food ingredients, the industry has developed with only varying amounts of success and its biotechnological potential remains to be fully exploited (Chen and Jiang, 2001; Ramirez and Morrissey, 2003). A major setback is in achieving eYcient production methods, as the traditional means of cultivating algae using open systems brings with it many limitations, and new closed-system cultivation technologies (photobioreactors) are not yet fully developed. Additionally, extraction of food ingredients from cultivated algae in a profitable manner can be quite challenging. Recent advances in transgenic work with algae oVer interesting and promising alternatives to traditional production methods. In this section, biotechnologically important species of macro- and microalgae as a source of food ingredients will be reviewed, along with major developments and challenges facing the industry. 1. Macroalgae Macroalgae, or seaweeds, provide a wide range of food and food ingredients, with an estimated total annual production value of US$5 billion. Wild and farmed seaweed combined amount to an annual usage of 7.5–8 million tons (wet seaweed), with increasing algae cultivation in response to growing demands. Cultivation of macroalgae now contributes to over 90% of the global seaweed demand, with the remainder being naturally harvested (FAO, 2004). There are four major classes of macroalgae: Rhodophyta (red algae), Phaeophyta (brown algae), Chlorophyta (green algae), and Cyanophyta (blue-green algae) (Renn, 1993). Some specific commercially important cultivated seaweeds and seaweed products include the brown seaweed

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L. japonica, wakame from the brown seaweed Undaria pinnatifida, Hizikia from Hizikia fusiforme, and the high-value product Nori, worth about US$16,000/dry ton, from the red seaweed Porphyra sp. (FAO, 2004). The Laminaria cultivation industry is one of the largest producers worldwide of the hydrocolloid algin, providing 13,000 tons per year (Tseng, 2001). Biotechnological advances regarding macroalgae cultivation include establishment of cell and tissue cultures that can biologically synthesize desired compounds, such as eicosanoids, on a large scale under a controlled environment (Rorrer et al., 1998). For example, by feeding Laminaria saccharina, a specific diet rich in linoleic and g-linolenic acids, production of three desired bioactive hydroxy fatty acids [15-hydroxy-5,8,11,13-eicosatetranenoic acid from arachidonic acid; 13-hydroxy-9,11-octadecatetraenoic acid from stearidonic acid (SDA); and 13-hydroxy-9,11-octadecadienoic acid from linoleic acid] was increased up to 400% (Rorrer et al., 1998). According to Rorrer et al. (1998), results of their studies with macroalgae demonstrate that biotechnology for controlled production of bioactive substances from cell and tissue cultures of marine seaweeds utilizing bioreactor systems holds great promise. 2. Microalgae Microalgae are the most primitive and simply organized members of the plant kingdom, with the majority existing as small cells of about 3–20 mm, and a few species organized into simple colonies (Ramirez and Morrissey, 2003). This group of microorganisms is extremely diverse and a rich source of potential bioactive ingredients such as vitamins, pigments, fatty acids, sterols, and polysaccharides (Borowitzka, 1993; Grobbelaar, 2004; Kay, 1991; Yap and Chen, 2001). There is great potential for use of microalgae in production of food ingredients, as they are photoautrophic microorganisms that can grow on a very simple culture medium containing seawater, nitrate, phosphate, trace amounts of certain metals, and carbon dioxide (Luiten et al., 2003). Thanks to development of new production and environmental technologies, biotechnology of microalgae has grown in importance (Volkman, 2003), with well-established microalgae production plants in Taiwan (Chlorella), Thailand (Spirulina), the United States (Spirulina, Dunaliella), Australia (Dunaliella, Chlorella), Israel (Dunaliella), Czechoslovakia (Scenedesmus), and Mexico (Spirulina), and smaller operations in Cuba, Iran, India, China, Vietnam, Chile, France, Spain, and South Africa (Ben-Amotz, 2004; Borowitzka, 1993). Cultivation of microalgae oVers several advantages over use of conventional higher plants, including high growth rates, high uptake and release rates promoted by a large surface to volume ratio, strains that can tolerate extreme conditions, no need to obtain high-quality agriculture soils, possibility for high-density growth using

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closed photobioreactors with semicontrolled parameters, and highly valued end-products (Grobbelaar, 2004). Some products obtained from microalgae include b-carotene, astaxanthin, vitamins C, A, E, H, B1, B2, B6, and B12, supplements in health foods, PUFAs, and viscosifier gums from polysaccharides (Luiten et al., 2003). Commercially produced microalgal oils have been incorporated into infant milk formulations and used as dietary supplements and food additives (Volkman, 2003). Despite the great potential for production of food ingredients from microalgae, only a few species are being cultivated on a large scale. Some noteworthy strains include Aphanizomenon, Nostoc, Spirulina (Arthrospira), Chlorella, Dunaliella salina, and Haematococcus pluvialis (Grobbelaar, 2004; Kay, 1991; Pulz, 2001; Richmond, 2004). The cyanobacteria Aphanizomenon is a relatively new source of microalgae for human consumption that is harvested in temperate lakes of the United States and sold as a health food and dietary supplement (Kay, 1991; Pulz, 2001). Large-scale commercial production of Aphanizomenon flos-aquae began in the early 1980s in Oregon. This Aphanizomenon strain is desirable as a health food source because it is high in protein and rich in vitamins, minerals, carotenoids, and phycobiliproteins (Hu, 2004). A. flos-aquae has also been reported to promote digestion and to have immune-enhancing eVects along with anti-inflammatory activity (Jensen et al., 2001). Despite the economic advantages to natural cultivation of A. flos-aquae, there exist numerous inconsistencies along with the possibility of contamination. Therefore, future use of photobioreactors may provide a safer, more controlled environment for harvesting this biomass (Hu, 2004). Nostoc is tolerant to extreme environments and is found throughout the world in places such as hot springs, the polar region, and deserts. Nostoc, which has been a part of the Chinese diet for 2000 years, is recognized as a healthy food owing to its low fat content and high levels of protein and natural pigments (Danxiang et al., 2004). The two Nostoc species of the most economic value are N. flagelliforme and N. commune, which are harvested in several Asian and South American countries. Spirulina is a photoautotrophic blue-green alga that commonly flourishes in brackish, saline lakes with extremely high pH (Mao et al., 2005). This cyanobacterium appears to be one of the most important microalgal species utilized by humans: it has been used as food for thousands of years by the Aztecs and Mayans (Wang et al., 2005) and for centuries by African populations near the alkaline lakes of Chad and Niger (Grobbelaar, 2004). In the later part of the 1970s, the first commercial production plant for Spirulina was established, and Spirulina is now cultivated around the world using open raceway ponds, with major facilities in the United States (Hawaii and California), China, Taiwan, and Japan (Hu, 2004; Pulz, 2001). Spirulina is

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widely produced and generally recognized as safe (GRAS) approved Spirulina is presently available for use in foods and supplements (Mao et al., 2005; Ohr, 2005). Spirulina has great potential as a nutrient source, as it is high in protein (60–70% depending on strain) (Mao et al., 2005) and contains significant levels of vitamins, minerals, essential fatty acids, and antioxidants such as carotenoids (especially chlorophyll) and phycobiliproteins (de Oliveira Rangel-Yagui et al., 2004; Hu, 2004; Liu and Cao, 2001). Spirulina has also been reported to have numerous health benefits. Chronic or subchronic treatment with Spirulina has been suggested to reduce lipid peroxidation, increase antioxidant levels, reduce reactive nitrogen species toxicity, and reduce cholesterol levels (Wang et al., 2005). Spirulina reduces potential brain damage from strokes and other neurological disorders (Wang et al., 2005), and daily 2-g supplements of Spirulina reduced allergy symptoms in allergic rhinitis patients (Mao et al., 2005). According to Mao et al. (2005), Spirulina exhibits anti-inflammatory activity thanks to the compound c-phycocyanin, which is a pigment commonly found in blue-green algae that also has antioxidant activity. Two of the most well-known species are Spirulina (Arthrospira) platensis and Spirulina (Arthrospira) maxima. Chlorella is a freshwater, unicellular green microalga that is widely used as a food supplement in Japan and around the world. Mass commercial cultivation of Chlorella for use as a health food supplement has taken place for over 35 years, with a more recent application in mariculture feed (Iwamoto, 2004). Many strains of Chlorella can be grown heterotrophically, allowing for production of a high-quality powder without contamination. Chlorella supplements are taken in the form of tablets, capsules, liquid, or as food additives. Claims for health benefits of Chlorella include improved immune function and improved control of hypertension, fibromyalgia, and ulcerative colitis (Halperin et al., 2003). Dunaliella is a motile green alga with a cell volume ranging from 50 to 1000 mm3. This ovoid biflagellate predominates in aquatic systems with salinity contents of 10% or higher and may be the most halotolerant eukaryotic organism known, as it can survive at salinities ranging from 0.2% to 35% (Ben-Amotz, 1993). There is great biotechnological potential for use of Dunaliella in the production of antioxidants such as b-carotene, ascorbic acid, and tocopherol. When stressful culture conditions (i.e., nitrogen deficiency, high light intensity, and high NaCl) are induced, the production of these antioxidants has been reported to reach 13.1%, 2.5%, and 1.2%, respectively (El Baz et al., 2002). The halotolerance of Dunaliella allows for outdoor cultivation under high salt conditions with minimal contamination from other bacteria, while its high b-carotene content helps to protect it from solar irradiation (Ben-Amotz, 1993). Carotenoids are generally the main products from cultivation of Dunaliella and they are sold in three

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major forms: Dunaliella powder, b-carotene extract, and dried Dunaliella for use in feeds (Ben-Amotz, 2004). Dunaliella cultivation takes place in Australia using large unstirred shallow ponds, in the Ukraine with natural ponds, and in the United States and Israel using raceway ponds (Borowitzka, 1993; Pulz, 2001). Numerous smaller facilities are located around the world in Mexico, Cuba, Chile, India, Iran, Taiwan, and Japan. H. pluvialis is a freshwater, green, unicellular alga that is used extensively for production of the orange-red pigment astaxanthin in both open and closed culture systems (Grobbelaar, 2004; Orosa et al., 2005; Pulz, 2001). In order to produce astaxanthin from Haematococcus using a large-scale outdoor system, the algae are first grown under conditions that promote rapid growth. Next, environmental stressors, such as nitrogen starvation, introduction of compounds that prevent cell division and high light intensity, are used to induce carotogenesis (Guerin et al., 2003; Orosa et al., 2005). Then the cells are harvested by settling and centrifuging, the cell biomass is cracked to improve the bioavailability of astaxanthin, and then the extract is dried and encapsulated or the astaxanthin is further extracted from the biomass (Guerin et al., 2003). It has been reported that use of malonate as a carbon source can increase carotenoid yields by up to 13-fold (Orosa et al., 2005). Haematococcus-derived astaxanthin has value as a nutraceutical and as a source of pigment in aquaculture feed, and Haematococcus has been approved by the FDA as a dietary supplement ingredient (Cysewski and Todd Lorenz, 2004). 3. Algae cultivation Although microalgae have numerous potential biotechnological applications for use in the food, cosmetics, and pharmacy industries, several setbacks remain for achieving eYcient production rates (Pulz, 2001). The main limitation for microalgae and microalgal products to reach their economic potential is the need for closed culture systems in the form of closed photobioreactors, which are costly to run because of high light requirements and slow growth rate of some organisms (Ramirez and Morrissey, 2003). Open systems have traditionally been the dominating method for mass production of cultured microalgae; however, these systems are in contact with the air and have significant drawbacks, such as evaporative losses, diVusion of CO2 to the atmosphere, an ongoing possibility of contamination and pollution, and light limitation in the high layer thickness (Pulz, 2001). Open systems are also dependent on weather and climate and therefore quality of products can be highly variable (Sijtsma and de Swaaf, 2004). IneYcient use of light energy and limited growth rates can have undesirable consequences such as slow cell growth rates and production of secondary metabolites

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(Hejazi and WijVels, 2004). Therefore, in order to create a profitable industry, it seems necessary to improve production methods for microalgae. A hopeful solution to open systems is the use of closed systems (closed photobioreactors) in which all the important culture conditions are controlled and regulated. Closed systems present the opportunity to reduce the risk of outside contamination, reduce CO2 loss, and provide reproducible cultivation conditions such as regulated water and temperature parameters (Pulz, 2001). However, as already mentioned, closed systems are costly to run due to light requirements and the often slow cell growth of microalgae. One biotechnological advancement is the concept of ‘‘milking’’ microalgae for their secondary metabolites. This method allows for reuse of the same culture and continuous removal of desired compounds, and would be a way to compensate for the low productivity often experienced with algal cultures (Hejazi and WijVels, 2004). One of the main cultivation systems utilized on a large scale is the shallow open raceway pond, which is used for production of D. salina at high salinity, S. platensis at high alkalinity, and Chlorella sp. (Luiten et al., 2003). Additionally, some strains, such as Chlorella, Nitzschia, Cyclotella, and Tetraselmis, can be grown heterotrophically in fermenters. Heterotrophic growth makes use of glucose as a source of both carbon and energy, and it is generally less costly and easier to regulate as compared to photoautotrophic cultivation. Crypthecodinium and Chlorella are both grown on a large-scale using fermentative technology (Apt and Behrens, 1999). 4. Algal transgenics Although still in its beginning stages, algal transgenic biotechnology shows great potential for more eYcient and specialized production of food ingredients. The possibility of using transgenic algae as ‘‘cell factories’’ is an appealing new technology that could be utilized for the production of compounds such as carotenoids, PUFAs, and enzymes (Leon-Ban˜ares et al., 2004). Genetic work with algae thus far has resulted in the establishment of molecular tools and sequence information that can be used to integrate transgenes into select strains of algae, especially in the case of the green algae Chlamydomonas reinhardtii and Volvox carteri and the diatom Phaeodactylum tricornutum (Walker et al., 2005). However, the term ‘‘algae’’ refers to a wide range of organisms with varying biological systems, and the methods of transformation developed for one type of algae may not function in another. Therefore, more work is necessary to standardize methods of transgenic introduction and expression in diVerent types of algae. For a thorough review of the genomic information available for select algae, see Grossman (2005).

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Transgenes can be integrated into algae via nuclear or chloroplastic transformation. Both methods have several advantages and disadvantages. For example, chloroplastic transformation allows for high levels of expression without foreign gene silencing, while nuclear transformation provides a greater range of possibilities in the areas of algal metabolism and protein expression (Leon-Ban˜ares et al., 2004). Nuclear transformation is a routine, standardized procedure in several species of microalgae, with reports of stable transformations in diatoms, dinoflagellates, and chlorophytes, while strains of red, green, and euglenoid algae have all been successfully transformed using chloroplastic techniques (Dunahay, 1996; Walker et al., 2005). In the following sections, specific genetic advances among groups of microand macroalgae will be highlighted along with their potential applications in the food industry. a. Diatoms. Diatoms are an abundant and diverse group of unicellular heterokont (flagellated) algae that dominate the phytoplankton of cold, nutrient-rich waters. Diatoms are known to contain the antioxidants fucoxanthin and chlorophyll along with a silica-based cell wall (Graham and Wilcox, 2000). The metabolic pathways involved in the generation of the silica cell wall are largely unknown and show potential for the discovery of novel enzymes and proteins that may prove to be commercially useful (Leon-Ban˜ares et al., 2004). Over the last decade there has been significant progress in the genetic study of diatoms, with developments in genetic techniques and increasing numbers of species with genomes that have been sequenced and transformed with foreign genes (Montsant et al., 2005; Walker et al., 2005). Although transgenic diatoms have not yet been used in commercial applications, there are several promising areas of research with the potential for development into large-scale industries. One example is in the manipulation of algal lipid metabolism pathways to produce specific oils such as the production of biodiesel from transgenic diatoms (Dunahay, 1996). This type of transgenic lipid manipulation could also be valuable in the production of PUFAs from microalgae. Another interesting development has been the conversion of the obligate photoautotroph P. tricornutum into an organism capable of heterotrophic growth (Zaslavskaia et al., 2001). This was achieved by transformation of the diatom with a human glucose transporter gene. The ability to convert photoautotrophs to heterotrophs could be valuable to algal biotechnology because the cultivation of algae dependent on light can be expensive and ineYcient. b. Dinoflagellates. After diatoms, the next most important eukaryotic primary producers in marine coastal waters are the dinoflagellates, a group of unicellular algae that contain chlorophyll, carotenoids including b-carotene,

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and PUFAs (Graham and Wilcox, 2000; Walker et al., 2005). Although there is potential for use of dinoflagellates in algal transgenics, these organisms have very large genomes that make gene sequencing impractical. Despite setbacks, gene transformation has been successfully carried out in two species of dinoflagellates, Amphidinium and Symbiodinium, and expressed sequence tag (EST) operations are underway (Lohuis and Miller, 1998; Walker et al., 2005). c. Green algae. Chlorophytes that have experienced significant progress in the field of transgenics include Chlorella sp., C. reinhardtii, Haematococcus sp., and Dunaliella sp. Some of these species have been considered for use as cell factories in the production of valuable food ingredients or pharmaceuticals such as proteins. Plant-derived proteins are GRAS approved, as they show low risk of contamination due to viruses, prions, or bacterial endotoxins (Franklin and Mayfield, 2004). The green alga Chlorella has experienced major advancements in nuclear transformation, with promising applications in the production of valuable proteins and peptides. Some examples include production of antimicrobial and bioinsecticide peptides and human and fish growth hormones for use in aquaculture (Walker et al., 2005). C. reinhardtii is a freshwater, eukaryotic alga whose genome is now publicly available (along with only two other eukaryotic algal genomes) (Montsant et al., 2005). C. reinhardtti has numerous favorable attributes that make it an interesting candidate for use as a cell factory in the production of valuable proteins: it can be grown either heterotrophically or phototrophically, it can be cultivated on a large scale, and stable transgenic lines can be created relatively quickly (Franklin and Mayfield, 2004). Major biotechnological advances have taken place in the nuclear transformation of C. reinhardtii, with potential applications in the food and pharmaceutical industries. For example, C. reinhardtii produces an antigenic protein of the pathogenic bacteria Renibacterium salmoninarum, which causes kidney disease in salmonids. When this antigenic protein was fed to trout and rabbits, it promoted generation of antibodies against the bacterium (discussed in Leon-Ban˜ares et al., 2004). Although C. reinhardtii shows promise in the field of transgenic algal protein production, several challenges remain, including reducing the silencing of foreign gene expression and enhancing translation in chloroplast-based expression (Walker et al., 2005). In addition to transgenic advances in Chlorella sp. and C. reinhardtii, there has been great eVort to develop the biotechnological tools required to research and exploit the commercially important green microalgae H. pluvialis and D. salina. These microalgae are the world’s major suppliers of natural astaxanthin and b-carotene, respectively, and successful DNA

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transformation has been carried out in both species. Research into the metabolic pathways and regulatory mechanisms involved in the production of these antioxidants could result in transgenic technologies designed to develop commercial strains with increased antioxidant productivity. Although gene transformation in these species has been met with limited success, in part due to the high salt environment required by Dunaliella sp., progress involving transformation with foreign genes coding for hygromycin resistance and the human hexose/Hþ symporter has shown promising results (Walker et al., 2005). In a recent example of biotechnological applications for microalgae, the Dunaliella species D. bardawil was mutated and strains rich in the carotenoids phytoene and phytofluene were selected for growth in small outdoor ponds in Israel. These strains were gradually brought to large-scale growth in open raceways for commercial carotenoid production (Ben-Amotz, 2004; Werman et al., 2002). d. Macroalgae. Although macroalgae are commercially important producers of food and phycocolloids such as alginates, agars, and carrageenans, transgenic research in this field remains far behind that for land plants and microalgae. However, the first macroalgae genome project was announced in the brown alga Ectocarpus siliculosus (Peters et al., 2004) and genetic work has been carried out with Laminaria and Porphyra. Recent advances in the field of plant transgenics include genetic engineering of the chloroplast genome, which allows for numerous advantages over nuclear transformation, including increased expression levels of transgenes, use of operons, and more precise and predictable loci (Walker et al., 2005). Further research into chloroplast transgenics could lead to more eYcient production of desired compounds, such as polysaccharides, from macroalgae cultivation. B. EXTREMOPHILES

Extreme aquatic environments are oftentimes inhabited by microorganisms that have developed unique biological properties in order to survive and thrive under conditions outside the tolerance levels of most living things. This diverse group of organisms, referred to as extremophiles, includes bacteria, cyanobacteria, algae, and yeasts that exhibit high tolerances for certain conditions such as extreme salinities, temperatures, pressures, radiation levels, and heavy metal concentrations (Herbert, 1992). Biomolecules such as enzymes isolated from extremophiles can be highly useful in the food industry due to their unique activities under abnormal conditions, and it has been widely accepted that extremophiles have strong potential to be valuable resources for use in biotechnology (Fujiwara, 2002; Guezennec, 2002; Herbert, 1992). Additionally, the discovery of deep-sea

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hydrothermal vents has revealed an increasing number of new bacterial species with incredible diversity and the ability to produce novel biomolecules such as enzymes, polysaccharides, and other bioactive molecules with potential commercial importance in the food industry (Guezennec, 2002). For more extensive reviews on extremophiles and their biotechnological potential, please see Gomes and Steiner (2004), Guezennec (2002), Fujiwara (2002), Cavicchioli et al. (2002), and Eichler (2001). C. MARINE SPONGES

Marine sponges are lower invertebrate animals belonging to the phylum Porifera. They have porous skeletons composed of double-walled cell colonies that become permanently attached to underwater surfaces. From a biotechnological perspective, marine sponges represent a rich source of new, undiscovered bioactive natural products. The Porifera phylum contains approximately 15,000 diVerent species with diverse growth forms (Belarbi et al., 2003). Lower invertebrates, such as sponges, possess a far greater diversity of certain lipid components, such as fatty acids, sterols, and other unsaponifiable compounds, as compared with higher-up animals. Biotechnological interest in sponges began in the early 1950s with the discovery of unknown nucleosides such as spongothymidine and spongouridine in the marine sponge Cryptotethya crypta. These nucleosides were found to be the basis for synthesis of cytosine arabinoside (Ara-C), the first marine-derived anticancer agent (Luiten et al., 2003). Since then, more than 5300 diVerent products (about one-third of all marine natural products) have been isolated from sponges, with hundreds more being discovered every year (Blunt et al., 2005; Luiten et al., 2003; Wang et al., 2003). Most of the bioactive compounds from sponges have antibiotic, antitumor, anti-inflammatory, antiviral, immune suppressive, antifouling, or antimalarial properties. Besides nucleosides, sponges have also been found to be a source of compounds such as bioactive terpenes, sterols, cyclic peptides, alkaloids, fatty acids, peroxides, and often-halogenated amino acid derivatives (Luiten et al., 2003). As explained by Osinga et al. (1999) and Belarbi et al. (2003), a number of these metabolites are actually produced by endosymbiotic microorganisms that live in the sponge tissue rather than by the sponges themselves. Although sponge mariculture was attempted in the late nineteenth and early twentieth centuries, it has not yet proven to be very lucrative, as little is known about how to replicate the sponge’s natural environment and life cycle (Luiten et al., 2003). Additionally, the bioactive compounds of interest are often only produced in trace amounts by sponges. However, recent developments using a primmorph system may facilitate production of bioactive compounds from sponges. Primmorphs are very densely packed,

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spherical sponge-cell aggregates (1-mm diameter) that are produced by gently agitating a dissociated cell suspension. In vitro cell culture of certain sponges, such as Geodia cydonium, Dysidea avara, and Suberites domuncula, for use in producing bioactive compounds is a developing area of research, and use of the primmorph system has allowed for the discovery of basic mechanisms involved in cell proliferation and programed cell death (Le Pennec et al., 2003). According to Le Pennec et al. (2003), sponge cell culture using the primmorph system is available for biotechnological applications. Additionally, the possibility of ‘‘milking’’ primmorphs (as discussed previously with microalgae) has been suggested as a potential technology to increase metabolite production (Muller et al., 2000). Two additional recent biotechnological methods for production of metabolites from sponges include ex situ culture and sponge-cell culture. In ex situ culture, functional sponges are grown outside of the sea and they are fed powdered substrates, guaranteeing a product of consistent quality; however, growth rates remain low compared to functional sponge growth in the sea. Optimization of growth rates may be achieved with increasing knowledge of the biology of Poriphera. Sponge-cell culture has shown promising growth rates and corresponding metabolite production, but a major setback with this method has been large amounts of contamination. Current work has been focused on developing methods to discriminate between sponge cells in culture and contaminants, with promising results. In a comparison of metabolite production using various methods, including chemical synthesis, wild harvest, mariculture, primmorphs, ex situ culture, sponge-cell culture, and genetic modification, it was determined that optimal techniques vary depending on the compound of interest. Mariculture proved to be the most economically feasible production method, while ex situ culture shows promise for the future production of valuable compounds that require close monitoring of growth parameters. Wild harvest was determined to be less feasible due to the logistics and environmental issues of removing massive amounts of sponges from their natural marine habitats. Due to the often small concentrations of metabolites present in sponges, cell culture was also not a highly recommended production technique. Use of primmorphs did not appear feasible either, as the amount of biomass required to form the primmorphs was larger than the biomass necessary for direct extraction of the desired metabolites (Sipkema et al., 2005). The use of genetic modification for production of specific metabolites is an interesting alternative to more traditional methods such as mariculture, cell culture, and chemical synthesis. Transfer of genetic material from a sponge host into bacteria that are easier to grow could allow for increased production of desired metabolites at a much lower cost. However, the majority of the metabolites of interest are not simply proteins, but molecules that are

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formed as a result of complex biochemical pathways involving numerous intermediate compounds and enzymes. It has been predicted that future production methods will likely involve transfer of genetic material into bacteria, followed by bacterial fermentation to produce metabolite precursors and then chemical synthesis of the final product (Sipkema et al., 2005). D. FISH AND SEAFOOD BY-PRODUCTS

Production of food ingredients from fish and seafood by-products is a growing area of interest, as it is a way to reduce processing waste and more eYciently utilize raw materials. Along with increased commercial production of fish and shellfish worldwide, there has been an increase in the amount of fish processing discards, at times amounting to up to 70–85% of the total weight of the catch (Shahidi, 1994). For example, in Quebec it has been estimated that 90% of all shrimp landings are processed and 70–75% of the raw material ends up as processing waste (Goldsmith et al., 2003). Fish processing discards have traditionally been dumped inland, incinerated, or hauled to the ocean (Shahidi, 1994; Suresh and Chandrasekaran, 1998). However, thanks to technological advances, a higher proportion of fish and seafood by-products are being utilized to meet growing demands for fish meal. Also, new product development and extraction of commercially important biomolecules from underutilized bycatch and processing discards is an important area of research (Okada and Morrissey, 2007; Shahidi and Janak Kamil, 2001). Numerous countries have expressed heightened interest in this area of work because it could help to reduce waste, thereby catering to ethical and environmental concerns over processing discards, and it could result in the development of valuable, natural marine-derived products (Gudmundsdottir and Palsdottir, 2005; Ohshima, 1998; Okada and Morrissey, 2007). For example, in Japan by-products have been used for the production of a number of commercially successful biomolecules, such as chitin and chitosan, PUFAs, growth hormones for aquaculture, protamines for use as antibacterial agents in food, and some pharmaceutical compounds (Ohshima, 1998). Some additional food ingredients obtained from fish and seafood include proteins in the form of albumin and gelatin, and a number of valuable enzymes with unique properties. One approach to extracting valuable compounds from shellfish processing waste is through the use of marine microorganisms in a procedure referred to as solid state (substrate) fermentation, or SSF. SSF may prove to be an economically advantageous tool for the production of certain compounds from marine waste. For example, the marine fungus Beauveria bassiana can be used to produce chitinase from chitinous prawn waste. Without the fungus, this conversion step normally accounts for 12% of the

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total production cost of chitinase; however, use of SSF may result in a more economically favorable method for obtaining chitinase, with a maximum yield of 248 units/g initial dry substrate reported after 5 days of incubation (Suresh and Chandrasekaran, 1998). III. MARINE-DERIVED FOOD INGREDIENTS A. PHOTOSYNTHETIC PIGMENTS

Photosynthetic pigments are bioactive compounds used by autotrophs, such as plants, algae, and cyanobacteria, to capture solar energy for photosynthesis. Due to the fact that each pigment captures light only over certain wavelength ranges, autotrophs use multiple pigments in order to absorb more of the sun’s energy. These photosynthetic pigments fall into three major categories: carotenoids, phycobilins, and chlorophylls (Table I). 1. Carotenoids Carotenoids, which are present in all plants and many photosynthetic bacteria, represent photosynthetic pigments in the red, orange, or yellow wavelengths. Nature’s most widespread pigments, carotenoids are linear polyenes that function both as light energy harvesters and as antioxidants that inactivate reactive oxygen species formed by exposure to light and air (von Elbe and Schwartz, 1996). Of the approximately 600 known carotenoids, about 50 have been shown to exhibit some provitamin A activity, which is their primary beneficial role in the diet of humans and animals (Gregory, 1996; von Elbe and Schwartz, 1996). As potent antioxidants and vitamin A precursors, carotenoids have been suggested to have protective activity against cancer, aging, ulcers, heart attack, and coronary artery disease (Li and Chen, 2001). Carotenoids are commonly used in food products as food-coloring or nutraceutical agents, and they can either be produced synthetically or derived from natural sources. Microalgal production of carotenoids such as b-carotene and astaxanthin is an attractive area of research, as they are valuable bioactive ingredients and can be present at relatively high concentrations in some algal cells. Strains of algae that are currently being investigated for use as natural producers of commercial carotenoids include D. salina, S. maxima, Chlorella protothecoides, Chlorella vulgaris, and H. pluvialis (Ramirez and Morrissey, 2003). a. b-Carotene. The carotenoid that is most commonly found in plant tissues and has been reported to exhibit the most provitamin A activity is b-carotene (Gregory, 1996; von Elbe and Schwartz, 1996). High accumulations

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of b-carotene among marine plants and algae are species specific and involve the combination of several stress factors such as high light intensity, limited nitrates, and high salt concentrations. Cultivated algae can therefore be induced to produce more b-carotene by controlling certain environmental growth conditions. A major marine producer of b-carotene is the extremely halophilic microalgae D. salina, which is the most b-carotene-enriched eukaryotic organism known (Ben-Amotz, 1993; El Baz et al., 2002). Although producers of microalgal b-carotene may find it hard to compete economically with synthetic production plants, natural b-carotene (a mixture of cis and trans isomers) has been reported to be more biologically active than the synthetically produced, fat-insoluble, crystallizable, all-trans-b-carotene (Ben-Amotz, 1993). Additionally, microalgal-derived b-carotene can be marketed as a ‘‘natural’’ food additive for products catered toward consumers interested in buying organic and natural foods. b-Carotene derived from Dunaliella has been marketed commercially in several forms, such as an extract in edible oils (containing 1.5–30% b-carotene) and dried Dunaliella powder in capsules or tablets (containing 5% b-carotene). These b-carotene-rich powders can be used in the health food and pharmaceutical industries to prepare naturally colored products (e.g., margarine) and to provide antioxidant activity for cancer prevention (Borowitzka, 1993). Additionally, b-carotene can be used in the aquaculture industry as a natural pigment in fish tissues. Despite promising advances in algal production of b-carotene, numerous disadvantages remain. For one, harvesting the algae is a complicated procedure, as the biomass growth is rarely higher than 1 g/liter and the density of the algal cells is nearly identical to that of the growth medium. Also, most of the world relies on synthetically produced b-carotene, which is easily produced in large amounts, making it quite challenging for cultivators of microalgae to compete (Borowitzka, 1993). b. Astaxanthin. Another important carotenoid that can be derived from marine sources is the orange-red pigment astaxanthin. Astaxanthin is the major carotenoid pigment present in aquatic animals and the primary pigment responsible for the pink color of salmon flesh and the reddish color of shrimp and lobster exoskeletons following heating (astaxanthin is blue when complexed with proteins prior to heating) (von Elbe and Schwartz, 1996). Astaxanthin cannot be synthesized by animals and is obtained through consumption of carotenoid-containing plants and algae (Guerin et al., 2003). With an antioxidant activity up to 10 times stronger than other carotenoids (including b-carotene, canthaxanthin, and lutein) (Miki, 1991), astaxanthin provides protective activity against cancer, inflammation, and UV light (Guerin et al., 2003). The health benefits of astaxanthin along

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with its strong coloring properties make it an important potential ingredient for use in the nutraceutical, cosmetics, food, and feed industries (Guerin et al., 2003; Miki, 1991). Microalgae-derived astaxanthin supplements have been available to the public since about 2000 (Guerin et al., 2003), and astaxanthin in commercial form (Zanthin) was just approved by the FDA in June of 2005 as a dietary supplement with a patent for use in slowing development and ameliorating the eVects of diseases of the eye and/or central nervous system (Ohr, 2005). Many astaxanthin supplements, including Zanthin, are derived from the green microalgae H. pluvialis, which is cultivated at an industrial scale and is the richest known source of natural astaxanthin, producing more than 30 g astaxanthin per kilogram of dry biomass in commercial operations (Guerin et al., 2003). In addition to its use in dietary supplements, H. pluvialis can be added to aquaculture feed for enhanced pigmentation of salmon flesh (Molyneaux and Lee, 1998). However, farmed salmon-fed carotenoid pigments are labeled as ‘‘color added,’’ which can cause confusion among consumers. Naturally derived astaxanthin is a highly valued bioactive compound and has experienced rapid market growth. However, there are several inherent disadvantages to microalgal production of astaxanthin. For example, H. pluvialis is a freshwater alga, making open-air cultivation diYcult due to high risk of contamination from undesirable organisms (Borowitzka, 1993). Additionally, optimal conditions for astaxanthin production change throughout the growth cycle of Haematococcus. Although current producers of natural astaxanthin have a hard time competing with synthetically produced astaxanthin, there are hopes that with increased cultivation and extraction technologies and rises in public demands for natural foods (e.g., naturally pigmented farmed salmon), microalgal production of astaxanthin will become more economically feasible and profitable. A promising new extraction technology that could increase the commercial success of natural astaxanthin is use of the milking process with a two-phase bioreactor for continuous removal of astaxanthin from Haematococcus (Hejazi and WijVels, 2004; Pulz, 2001). c. Other carotenoids. Naturally derived carotenoids other than b-carotene and astaxanthin do not appear to be a very promising field of algae cultivation thus far. However, work with the microalgae C. protothecoides resulted in the development of a three-step cultivation process for high yield of the carotenoid lutein (Shi and Chen, 2001). The cultivation procedure involves a fed-batch system, in which nutrients are supplemented to achieve high algal growth, followed by a nitrogen-limiting stage and then maintenance of the culture at elevated temperatures. The latter two steps in the process increase environmental stress, thus promoting carotogenesis (i.e., lutein synthesis).

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Another interesting carotenoid with potential commercial value is fucoxanthin, from the edible seaweed U. pinnatifida (Maeda et al., 2005). Fucoxanthin has been reported to be of potential use in treating obesity and reducing the risk of certain diseases, such as type II diabetes, due to its ability to promote expression of the uncoupling protein UCP1. UCP1 stimulates metabolic thermogenesis, a process by which metabolism leads to production of heat rather than adenosine triphosphate (ATP) production and excess fat accumulation. Although UCP1 is primarily found in brown adipose tissue and humans store most of their fat as white adipose tissue, there is hope that fucoxanthin may promote expression of UCP1 in the white adipose tissue as well. Rats and mice that were fed a diet rich in fucoxanthin were reported to have significantly increased expression of UCP1 and weight reductions in the abdominal white adipose tissue (Maeda et al., 2005). 2. Phycobiliproteins Phycobiliproteins are protein-pigment complexes, such as phycoerythrobilin (red) and phycocyanobilin (purple to deep blue), which can be easily isolated from eukaryotic algae (e.g., red algae, glaucophytes, and cryptomonads) or cyanobacteria (Apt and Behrens, 1999; Borowitzka, 1993). Aquatic autotrophic organisms contain additional photosynthetic pigments such as phycobiliproteins because more than 10 m below the water surface light wavelengths for some colors are almost completely absorbed (Voet et al., 1999). Phycobiliproteins, which generally make up 1–10% of the dry weight of algal biomass, have a high market value but a small market size (Skulberg, 2004). They possess a number of qualities with potentially valuable commercial applications. For example, they are able to form stable conjugates with numerous compounds, such as biotin and antibodies, they are fully water soluble, and they can emit fluorescence (Apt and Behrens, 1999). Phycobiliproteins are utilized as natural food colorings in products such as chewing gums, dairy products, jellies, and ice sherbets in Japan, Thailand, and China (Borowitzka, 1993; Roman et al., 2002). 3. Chlorophylls Chlorophylls are green pigments that can be found in any plant, alga, or cyanobacterium that carries out photosynthesis. Chlorophylls are primarily used in the food industry as natural colorants in foods and beverages (de Oliveira Rangel-Yagui et al., 2004). Additionally, chlorophylls and their derivatives exhibit anticancer activity in their ability to bind carcinogenic hydrophobic compounds such as polycyclic aromatic hydrocarbons, heterocyclic amines, and aflatoxin. The resulting complexes are less bioavailable and

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are therefore excreted from the body rather than absorbed (Chernomorsky et al., 1999; Donaldson, 2004; Egner et al., 2001; Sarkar et al., 1994). Although the majority of industrial chlorophylls are extracted from vegetable sources, there is a growing interest in developing the biotechnological tools necessary for production of chlorophylls from microalgae. Microalgal chlorophyll production can be carried out using fermentation processes, which allow for several advantages over traditional methods, including the potential for continuous cultivation as well as rapid multiplication and growth of microorganisms (de Oliveira Rangel-Yagui et al., 2004). Some possible microalgal sources of chlorophylls include Spirulina sp., such as S. platensis, and A. flos-aquae (Bhattacharya and Shivaprakash, 2005; de Oliveira Rangel-Yagui et al., 2004; Kay, 1991). B. LIPIDS

1. Marine-based long-chain PUFAs (LC-PUFAs) PUFAs are essential structural components of cell and organelle membranes, contributing to regulation of membrane properties such as fluidity, structure, phase transitions, and permeability (Yap and Chen, 2001). Marine-based LC-PUFAs have 20 or more carbons with 2 or more double carbon bonds, and they are classified by the position of the first double bond from the methyl (omega) terminus. Of particular interest are the omega-3 LC-PUFAs (n-3 LC-PUFAs) in which the first double bond is located at the third carbon from the methyl terminus. n-3 LC-PUFAs can contain up to six double bonds and cannot be synthesized by animals. The most well-studied marine n-3 LC-PUFAs are eicosapentaenoic acid (EPA), with 20 carbons and 5 double bonds, and docosahexaenoic acid (DHA), with 22 carbons and 6 double bonds (Sijtsma and de Swaaf, 2004). These two bioactive ingredients have been of increasing interest lately due to research revealing their beneficial eVects on many aspects of human health such as reducing risk factors associated with cardiovascular problems, assisting visual and neurodevelopment, and ameliorating diseases such as arthritis and hypertension (Bao et al., 1998; Grimm et al., 2002; Horrocks and Yeo, 1999; Hu et al., 2002; Leaf et al., 2003; Nettleton, 1995). Although a precursor to EPA and DHA, called a-linolenic acid (ALA, 18:3), can be obtained from terrestrial plant sources, it is converted to EPA and DHA very ineYciently by the human body (Pawlosky et al., 2001; Salem et al., 2003). The market for omega-3 fatty acid nutraceuticals in Europe and Japan has long been established, and the US market is experiencing rapid growth. Although most Western diets do not include enough n-3 LC-PUFAs, advances in marine biotechnology are helping to incorporate these by use

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of fish oil capsules, fortification of livestock and aquaculture feed to produce omega-3-enriched farmed fish, eggs, and milk, and addition of DHA to infant formula (Sijtsma and de Swaaf, 2004). Marine-based oils used in the food industry as omega-3 dietary supplements and in functional foods include algal oil, cod liver oil, sardine oil, tuna oil, and salmon oil (Ohr, 2005). Many of these marine oils, along with fish oil marketed as omega-3 concentrate, have been GRAS approved by FDA, provided that the combined intake of DHA and EPA does not exceed 3 g/person/day. Although fish oil supplements have long been popular in Europe and Japan, a more attractive option for many in the food industry is to enrich everyday foods with n-3 LC-PUFAs (Garcia, 1998). Some examples of foods that have been marketed as omega-3-enriched products are bread and margarine (Europe); cakes, pasta, and dog food (United Kingdom); and infant formula (Japan) (Garcia, 1998). Major applications, sources, and benefits of n-3 LC-PUFAs are summarized in Table I. a. Microalgae as a source of n-3 LC-PUFAs. Algae are believed to be the primary manufacturers of n-3 LC-PUFAs in the marine food chain and the only plant source available for EPA and DHA (Ackman et al., 1964; Ohr, 2005). Although the current contribution of n-3 LC-PUFAs derived from microorganisms to the market is very low, EPA and DHA have been found at high levels in various species of marine micro- and macroalgae with relatively high oxidative stability compared to fish oils (Bajpai and Bajpai, 1993; Sijtsma and de Swaaf, 2004). Some examples can be found in several species of diatoms, dinoflagellates, and thraustochytrids. Diatoms generally contain fairly high levels of EPA (15–30% of total fatty acids) and no DHA. Some examples of diatoms rich in EPA are Navicula pelliculosa (freshwater) and the marine diatoms Nitzschia frustulum, Navicula incerta, and Biddulphia sinensis (reviewed in Yap and Chen, 2001). The diatom P. tricornutum has been reported to contain more than 35% of its total fatty acids as EPA (Borowitzka, 1993; Yongmanitchai and Ward, 1989). Alternatively, dinoflagellates, such as C. cohnii (nonphotosynthetic), Amphidinium carteri, Gymnodinium simplex, and Gyrodinium cohnii, have high potential for use in commercial production of DHA, which ranges from 12% to 51% of the total fatty acids in these organisms (Yap and Chen, 2001). Microalgal n-3 LC-PUFA production technologies. Although the basic production of bioactive compounds from marine microorganisms was already discussed in Section II.A.3, advances in cultivation of microalgae specifically for production of n-3 LC-PUFAs will be explored here. To begin with, oil obtained from bioreactor cultivation of select microorganisms is referred to as single-cell oil (SCO). The main application of SCO biotechnology, in which there have been several successes and increasing industrial

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interest, is in producing high-value products such as n-3 LC-PUFAs (Sijtsma and de Swaaf, 2004). However, numerous challenges remain, including optimization of algae culturing methods, bioreactor operations, and omega-3 extraction and isolation procedures. Maximum omega-3 production can be induced by altering growth conditions: lipids found in actively growing and dividing algae contain high percentages of n-3 PUFAs (Yap and Chen, 2001). For example, under optimal culture conditions, the microalga Chlorella minutissima can produce an EPA content of up to 45% of its total fatty acids and is therefore a promising source for commercial production of EPA (Seto et al., 1984). Large-scale production of EPA was reported using the red alga Porphyridium cruentum with controlled cell concentrations and temperatures (Cohen et al., 1988). For a thorough review of EPA production from microorganisms, see Bajpai and Bajpai (1993). One interesting alternative to use of photobioreactors is the possibility of growing heterotrophic organisms on organic substrates, which eliminates the need for light, a limiting factor in mass production of algae, and allows for higher cell densities (Sijtsma and de Swaaf, 2004). However, there are several setbacks that need to be overcome, such as the limited number of heterotrophic species available, the risk of contamination due to the rich media required, the slow growth rates of microorganisms, and the need to reduce production costs to well below the market price of omega-3 fatty acids (Sijtsma and de Swaaf, 2004). Some promising heterotrophic marine organisms are the thraustochytrids, C. cohnii, and P. tricornutum (following genetic transformation) (Domergue et al., 2002; Sijtsma and de Swaaf, 2004; Volkman, 2003). Thraustochytrids, which are taxonomically aligned with heterokont algae, have promising applications in DHA production because they can be grown on an industrial scale using fermentation technology and they yield high levels of DHA under dense biomass concentrations (Barclay, 2006; Barclay et al., 1994; Lewis et al., 1999). Some examples include Thraustochytrium and Schizochytrium, which have been reported to contain between 25% and 60% of total fatty acids as DHA, predominantly as triglycerides or oils (Kendrick and Ratledge, 1992). Schizochytrium can contain over 70% of its body weight as lipids and is currently used for commercial production of concentrated DHA oil and dried microalgae, some of which are used as a poultry feed additive to produce omega-3enriched eggs and for enriching rotifers and brine shrimp prior to feeding them to cultured finfish larvae or shrimp (Barclay and Zeller, 1996; Lewis et al., 1999; Sijtsma and de Swaaf, 2004; Volkman, 2003). Additionally, it has been suggested that thraustochytrid oils, which contain mostly DHA, oVer an advantage over many fish-derived oils used in the feed industry

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because many aquaculture species require proportionally more DHA than EPA (Lewis et al., 1999). Accompanying the ongoing research developments involving thraustochytrids have been a number of approved US patents, with the most recent being a procedure outlining fermentative growth of Schizochytrium and Thraustochytrium for the production of high levels of n-3 PUFAs to be used in food products or aquaculture feed (Barclay, 2006). C. cohnii is known to accumulate especially high amounts of DHA (30–50% of total fatty acids) with other PUFAs only present at trace amounts (Jiang et al., 2001). With the addition of vitamin B12 and tryptone as a nitrogen source, C. cohnii produced up to 64% DHA and had a higher specific growth rate and biomass concentration (Jiang et al., 2001). C. cohnii is being cultivated in large-scale bioreactors with well-controlled parameters [pH, temperature, air flow, pressure, dissolved oxygen (DO), agitation] for use as a commercial source of DHA (Sijtsma and de Swaaf, 2004). However, a complicating factor is the production of excess polysaccharides by C. cohnii, leading to an increased viscosity and a strong decrease in oxygen transfer, thereby impeding the development of high-density cell biomass for DHA production (de Swaaf et al., 2001). One hopeful solution is use of a commercial polysaccharide hydrolase, which could be used to lower the viscosity of the culture and decrease the amount of stirring required (de Swaaf et al., 2003). Also, although the extracellular polysaccharides produced are byproducts in DHA production, they could potentially be used in the food industry for functional food products (Sijtsma and de Swaaf, 2004). Another possibility for more eYcient production of DHA from C. cohnii is the use of milking technology (discussed in Section II.A.3), in which the cells would first be grown according to normal growth conditions and then they would be stressed in order to produce higher concentrations of DHA, which would be continuously removed from the system (Hejazi and WijVels, 2004). Similar to ‘‘milking’’ is the idea of semicontinuous culturing in which microorganisms are held at steady state with renewal every 24 hours. Semicontinuous culturing under nitrogen-limiting conditions was reported to increase the percentage of EPA in total fatty acids among the marine microorganisms P. cruentum, P. tricornutum, and Isochrysis galbana, with diVerent culture strategies being recommended for the diVerent species (Otero et al., 1997). Further work with a strain of I. galbana has revealed important information regarding optimal lighting conditions (i.e., photon flux density and photoperiods) for maximum biomass production (Tzovenis et al., 2003). b. Fish as a source of n-3 LC-PUFAs. Although uni- and multicellular marine plants such as phytoplankton and algae are the primary sources of n-3 LC-PUFAs, high levels can be found in the tissue of many marine fish

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as a result of transfer through the aquatic food chain. The flexible, unsaturated properties of n-3 LC-PUFAs help fish to maintain membrane fluidity in cold environments (Shahidi and Wanasundara, 1998). A major source of omega-3 fatty acids for human consumption has traditionally been fish such as salmon, sardine, mackerel, menhaden, anchovy, tuna, and herring (Sijtsma and de Swaaf, 2004; Yap and Chen, 2001). Although incorporation of n-3 LC-PUFAs into foods and beverages is a growing area of interest, a major challenge remains in the high oxidative susceptibility of these unsaturated fatty acids, which can result in strong fishy odors and flavors (Garcia, 1998). Some additional challenges to the industry are related to oil quality and purification. Marine fish oil can vary in quality depending on factors such as fish species, season, and catch location, and it is a complex mixture of fatty acids that has to undergo purification steps in order to isolate DHA and/or EPA (Sijtsma and de Swaaf, 2004). Liver oil from cod and halibut carries with it the risk of vitamins A and D overdose, which can cause toxic eVects, and the risk of increased cholesterol and saturated fatty acid intake (Shahidi and Wanasundara, 1998). In attempts to avoid the possible negative side eVects of capsule consumption, concentrated forms of n-3 LC-PUFAs are being developed. These are preferred over crude marine oils for use in pharmaceuticals and food enrichment, as they allow for increased intake of DHA and EPA while keeping the intake of other lipids minimal (Shahidi and Wanasundara, 1998). An important area of research is development of technologies that allow for eYcient, cost-eVective, high-quality extraction of omega-3 oils. Numerous methods have been established for concentration of n-3 LC-PUFAs, including adsorption chromatography, fractional or molecular distillation, enzymatic splitting, low-temperature crystallization, supercritical fluid extraction, and urea complexation; however, each has its own drawbacks and thus far only a few are suitable for mass production (Shahidi and Wanasundara, 1998). According to Shahidi and Wanasundara (1998), there is growing industrial interest in use of enzymatic methods due to the potential benefits of obtaining the concentrated oils in acylglycerol form. A promising new n-3 LC-PUFA extraction technique called the pH-shift method may prove beneficial in the industry because no heat is required, thereby limiting decomposition reactions and oxidative damage that normally occur during extraction (Morrissey and Okada, 2005). Due to a growing interest in the bioactive properties of n-3 LC-PUFAs, there is an increasing demand for purified concentrates for use as dietary supplements and in functional foods; however, it is believed that traditional sources will be insuYcient in meeting this demand (Bajpai and Bajpai, 1993; Lewis et al., 1999). In addition to development of improved methods of omega-3 extraction from fish oils, a possible solution is the use of alternative,

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sustainable sources of omega-3 fatty acids such as marine microalgae, fungi, extremophiles, macroalgae, and krill (Sijtsma and de Swaaf, 2004; Yap and Chen, 2001). c. Fungi as a source of n-3 LC-PUFAs. Another promising source of n-3 LC-PUFAs is the class of algae-like fungi called phycomycetes. These lower marine fungi, especially those from the Mortierella genus (e.g., M. alpina, M. elongata, M. ramanniana, and M. isabellina), are known to produce high levels of either g-linolenic acid, arachidonic acid, or EPA (Yap and Chen, 2001). Another type of phycomycete (Phythium sp.) produces EPA at high yields, while other species, such as Entomophthora obscura and Phytophthora infestans, have been reported to produce significant levels of DHA. d. Transgenic organisms as a source of n-3 LC-PUFAs. An alternative biotechnological approach to maximizing production of n-3 LC-PUFAs is the use of genetic enhancement. For example, it was shown that P. tricornutum could be converted into a heterotrophic organism through the insertion of a glucose transporter gene (discussed in Section II.A.4) (Zaslavskaia et al., 2001). Use of this type of fermentation technology could prove to be economically beneficial over photoautotrophic growth because production of n-3 LCPUFAs would no longer be dependent on light, but rather on glucose supply. Work in plant genomics has led to the development of terrestrial plants capable of producing significant amounts of SDA, an n-3 PUFA that is a precursor to EPA (Ursin, 2003). Traditionally, terrestrial plants have been unable to synthesize marine omega-3 fatty acids such as EPA and DHA, but rather they are known for their ability to produce the n-3 PUFA ALA. As discussed previously, ALA is converted to EPA and DHA very ineYciently by the human body, with the rate-limiting step being a reaction involving n-6 fatty acid desaturase. In research led by the food biotechnology company Monsanto, the genes for n-6 and n-12 desaturases isolated from the marine fungi M. alpina were introduced into canola seed, along with the n-15 fatty acid desaturase from canola (Brassica napus). The resulting transgenic canola lines accumulated SDA at up to 23% of total lipids and more than 55% of total lipids was in the form of omega-3 fatty acids (ALA þ SDA). As a metabolic intermediate between ALA and EPA, SDA can be converted to EPA with much greater eYciency than ALA. Clinical trials demonstrated that SDA could increase plasma EPA levels at a rate three to four times greater than ALA and one-third as eVectively as dietary EPA (James et al., 2003). Use of transgenic land-based plants in the commercial production of n-3 LC-PUFAs is a novel technology that could greatly increase worldwide consumption of omega-3 fatty acids. However, challenges remain in the consumer acceptability of genetically modified products and in the reduced

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eVectiveness of dietary SDA compared to dietary EPA in raising plasma EPA levels. In another gene transfer study, three enzymes necessary for EPA production were cloned from the genomes of P. tricornutum and Physcomitrella patens and then expressed in the yeast Saccharomyces cerevisiae (Domergue et al., 2002). Although the biosynthetic pathways of arachidonic acid and EPA were reconstructed in the yeast, a high number of side products were reported. The ability of P. tricornutum to accumulate high levels of EPA with few side products is therefore thought to be indicative of a highly eVective regulatory system that is not present in S. cerevisiae. These results show the importance of understanding not only biosynthetic pathways but also regulatory mechanisms in order to successfully use gene transfer technology for the production of n-3 LC-PUFAs. The genes that code for the two enzymes necessary for the conversion of EPA into DHA have been identified and cloned from the genomes of the marine microalgae Pavlova and Isochrysis (Pereira et al., 2004). These genes were expressed in yeast and conversion of EPA to DHA was successfully carried out. This study marks the final step in the elucidation of the biosynthetic pathway and isolation of enzymes necessary for production of DHA. Future work in this area may lead to commercial production of DHA using genetically enhanced microorganisms. e. Extremophiles as a source of n-3 LC-PUFAs. Some deep-sea bacteria have been found to contain large amounts of EPA and DHA, presumably to allow their membranes to be fluid and adaptive to extreme temperatures and pressures (Yap and Chen, 2001). For example, psychrophiles, including some species of yeast, fungi, and microalgae, can accumulate up to 80% of their biomass as lipid, mostly in the form of triglycerides (Herbert, 1992). Additionally, psychrophilic PUFA synthesis is carried out under low temperatures using conventional fermentation technology, resulting in faster production rates and more consistent product quality and yields as compared with use of mesophilic phototrophs. For these reasons, psychrophilic PUFA production is considered by some to be a potentially lucrative field of work. Some promising organisms are Mortierella sp. such as M. alpina, which can produce EPA as 15% of total extractable fatty acid at 12
C (Bajpai and Bajpai, 1993), and the microalgae P. tricornutum and C. minutissima, which produce significant amounts of EPA at low temperatures (Herbert, 1992). f. Macroalgae and mosses as a source of n-3 LC-PUFAs. Some macroalgae and mosses are known to produce fairly high yields of PUFAs; however, eYcient culturing systems remain to be established. For example,

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macroalgae in the class Rhodophyceae contain relatively high amounts of C20 PUFAs such as arachidonic acid and EPA; however, owing to their size they are almost impossible to culture under controlled conditions (Yongmanitchai and Ward, 1989). Another potential source of PUFAs is Bryophytes, which are lower plants such as Mnium, Polytrichum, Marchantia, Matteuccia, Bryum, Sphagnum, Ctenidium, and Pogonatum spp. (Radwan, 1991). One species of moss, Pogonatum urnigerum, can yield more than 70% of its total fatty acids as EPA; however, culturing of this moss is not profitable as a result of low cell proliferation (Yap and Chen, 2001). g. Krill as a source of n-3 LC-PUFAs. Krill are small, shrimp-like crustaceans, the most abundant being the Antarctic krill, Euphausia superba (Venugopal and Shahidi, 1995). This zooplankton is rich in EPA and DHA, and contains potent antioxidants such as astaxanthin, vitamins A and E, and a novel flavonoid (Bunea et al., 2004). It has been suggested that the combination of n-3 LC-PUFAs with phospholipids in krill oil facilitates passage through the intestinal wall, thus improving the bioavailability of the omega-3 fatty acids (Sampalis et al., 2003). Krill oil has been shown to alleviate/reduce some of the symptoms of premenstrual syndrome and help manage hyperlipidemia by reducing levels of glucose, total cholesterol, triglycerides, low-density lipoprotein (LDL), and HDL as compared to both fish oil and a placebo (Bunea et al., 2004; Sampalis et al., 2003). Krill oil is sold as a dietary supplement to benefit the cardiovascular system (Ohr, 2005). 2. Sterols Sterols are membrane lipids that are essential to all eukaryotic organisms and are synthesized by microeukaryotes and some bacteria (Lewis et al., 2001; Volkman, 2003). Although microalgae are a rich source of commercially important bioactive molecules from the sterol biosynthetic pathway, significantly more research is necessary in order to develop the biotechnological tools for their exploitation within the food industry. Thus far, the only commercial use of sterol-containing microalgae has been as feed for aquaculture stocks, such as crustaceans and mollusks, which lack the mechanism to synthesize sterols (Volkman, 2003). A major limitation for the use of microalgae-derived sterols in the food industry is that algal cells typically have a low sterol content, amounting to less than 0.1% of the dry weight (Borowitzka, 1993; Volkman, 2003). Therefore, in order for microalgal production of sterols to be economically beneficial, the bioactive molecules would need to command a high market price. Numerous unusual and unidentified sterols have been reported in microalgae that could have potentially valuable uses in the food industry (Borowitzka, 1993; Lewis et al., 2001).

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A DHA-producing strain of thraustochytrids was found to produce 20 sterols, some of which were the same as those found in common food plants and 7 of which were unknown (Lewis et al., 2001). The study also showed that environmental conditions, such as temperature and DO, and culture age can influence the sterol content and profiles of the thraustochytrid strain analyzed. A maximum dry weight percentage of sterols of 0.32% was reported 2 days prior to reaching peak biomass concentration in cultures grown at 20
C with high DO (Lewis et al., 2001). C. POLYSACCHARIDES

Polysaccharides are polymers of simple sugars (monosaccharides) linked together by glycosidic bonds. They have numerous commercial applications in products such as adhesives, paper, paints, foods, and beverages. Algae are a well-established source of hydrocolloids, which are used extensively in the food industry as thickeners, stabilizers, and emulsifiers (FAO, 2004; Tseng, 2001). Although red and brown algae are the most prominent sources of marine-derived polysaccharides, a major limiting factor in the commercial production of algal polysaccharides has been the high costs involved in cultivating algal biomass (Borowitzka, 1993). Alternative sources that are being investigated include cyanobacteria and extremophiles living in deepsea vents. An additional marine-derived polysaccharide that will be discussed is chitin, which is found abundantly in the exoskeletons of crustaceans and has numerous potential uses in the food industry. For a summary of marine polysaccharides discussed in this section, along with their applications, sources, and benefits, see Table I. 1. Polysaccharides from algae a. Hydrocolloids. Hydrocolloids are carbohydrates that dissolve in water to form a viscous solution. Phycocolloids (hydrocolloids extracted from algae) are a growing industry, with about 1 million tons of seaweed harvested annually for hydrocolloid extraction (FAO, 2004). The three major phycocolloids used as commercial food ingredients are algins, carrageenans, and agar. These are used for applications such as thickening aqueous solutions, forming gels, forming water-soluble films, and stabilizing products such as ice cream (FAO, 2004; Tseng, 2001). Algins are extracted from brown seaweed and are available in both acid and salt form. The acid form is referred to as alginic acid and it is a linear polyuronic acid. The salt form, alginate, is an important cell wall component in all brown algae, constituting up to 40% of the dry weight of algal biomass (Graham and Wilcox, 2000). Algins are extracted commercially from several

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diVerent species such as Sargassum confusum and the cultivated plant L. japonica (Ohshima, 1998; Tseng, 2001). Despite a limited number of successes, brown seaweed has proven too expensive to cultivate and most algins are extracted from wild algal strains, such as those found abundantly oV the coast of South Africa and commercially gathered for algin extraction and exportation (Ecklonia maxima, Laminaria pallida, L. pallida var. schinzii, and Macrocystis angustifolia) (FAO, 2004; Stirk, 2004). The high numbers of carboxyl groups that make up alginic acid allow it to combine easily with cations and, once converted into its salt form, it can be used to provide high viscosity at low concentrations (BeMiller and Whistler, 1996; Ohshima, 1998). The high viscosity of sodium alginate or propylene glycol alginate solutions and the fact that they remain stable during pasteurization and cooking make them valuable natural food ingredients that can serve as thickeners, emulsifiers, and stabilizers in foods including salad dressings, ice cream, jam, and mayonnaise (BeMiller and Whistler, 1996; Ohshima, 1998). Carrageenans are a group of biomolecules composed of linear polysaccharide chains with sulfate half-esters attached to the sugar units, giving them an overall negative charge and preventing precipitation at low pH. These properties allow carrageenans to dissolve in water, form highly viscous solutions, and remain stable over a wide pH range. There are three general forms of commercially available carrageenans (kappa, lambda, and iota), each with their own gel-forming abilities (Renn, 1993). They are extracted from red algae using dilute alkaline solutions to produce the sodium salt of carrageenan. In the past, carrageenan was extracted from wild seaweeds such as Chondrus crispus (Irish moss); however, advances in biotechnology have resulted in the successful cultivation of several carrageenan-containing species, especially Kappaphycus alvarezii and Eucheuma denticulatum from the Philippines and Betaphycus gelatinum from Hainan (FAO, 2004; Tseng, 2001). The primary applications for carrageenans in the food industry are in dairy and processed meat products (Ohshima, 1998). Carrageenans can form gels with milk and water and can be used to coat meats to help retain moisture, seasonings, and flavors and to serve as a protective barrier (BeMiller and Whistler, 1996). From a human health perspective, it has been reported that carrageenans have antihuman immunodeficiency virus (HIV)-1 activity and some anticoagulant properties (Vlieghe et al., 2002). A third marine-derived hydrocolloid of importance to the food industry is agar, which is a mixture of polysaccharides with similar structural and functional properties as carrageenan (BeMiller and Whistler, 1996). Agar, which is composed of 70% agarose and 30% agaropectin, is extracted from red algae such as Gelidium, Grateloupia, Gracilaria, Hypnea, and Gigartina

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(FAO, 2004; Ohshima, 1998). Agars are commercially important in the food industry due to their capacity to gel aqueous solutions at low concentrations and for their use as food gums (BeMiller and Whistler, 1996; Renn, 1993). Although it is the oldest phycocolloid produced in China, agar is currently the lowest in production compared with algin and carrageenan (Tseng, 2001). Methods to produce agar from cultivated seaweeds using tanks and ponds have been developed, but so far they have proven to be economically unsuccessful (FAO, 2004). However, Gracilaria dura from the Mediterranean Sea was reported to be a commercially viable source of agar, yielding 32–35% agar with high gel strength that can be further improved with alkali treatment (Marinho-Soriano and Bourret, 2005). Another strain of Gracilaria growing oV the coast of the Yucatan Peninsula, G. crassissima, was also reported to produce a good quality agar and to be a possibly exploitable source of commercial grade agar (Freile-Pelegrin and Murano, 2005). b. Fucans/fucanoids and other polysaccharides. Fucans are a group of polysaccharides primarily composed of sulfated L-fucose, with less than 10% other monosaccharides. They are found widely in the cell walls of brown algae (Phaeophyceae), but not in green, red, golden, or freshwater algae or in terrestrial plants. Besides brown algae, the only known reported sources of fucans are the egg jelly coats of sea urchins and the body walls of sea cucumbers. For an excellent review of the sources, structures, functions, and biological properties of fucans, the reader is referred to Berteau and Mulloy (2003). Although the major physiological purposes of fucans are not thoroughly understood, they are known to possess numerous biological properties with potential human health applications. Fucoidans, or fucans from brown algae, have been reported to exhibit anticoagulant, antithrombotic, antiviral, and cellular antiproliferative and antiadhesive activities, as well as having an eVect on the inflammatory and immune systems. These properties make fucanoids an attractive alternative to the mammalian-derived anticoagulant, heparin, which is more likely to carry with it infectious agents such as prions or viruses (Berteau and Mulloy, 2003; Kuznetsova et al., 2003; Mourao, 2004). Additionally, fucanoids can make up more than 40% dry weight of isolated algal cell walls and can easily be extracted using either hot water or an acid solution (Berteau and Mulloy, 2003). The toxicity of fucanoids from L. japonica was tested in rats and no adverse eVects were reported at levels of 300 mg/g body weight per day; however, significantly prolonged blood-clotting times were observed at three times that level and higher (Li et al., 2005). Although fucanoids have yet to be exploited in the food industry, the fact that they are easy to isolate and have numerous health benefits gives them the potential to serve as valuable bioactive ingredients in natural health foods.

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Another group of sulfated polysaccharides with potential use as nutraceutical agents have recently been isolated from the Chlorophyta Ulva pertusa. These polysaccharides were reported to aVect levels of low- and high-density cholesterols and triglycerides in the plasma and serum of mice, with the resulting conclusion that they have great potential for use in preventing ischemic cardiovascular and cerebrovascular diseases (Pengzhan et al., 2003a,b). 2. Exopolysaccharides from cyanobacteria Cyanobacteria are considered by some to be a promising source of exocellular polysaccharides (de Philippis et al., 2001). Certain strains of cyanobacteria are known to contain large amounts of released exocellular polysaccharides, which consist of a relatively large number of monosaccharides and display unique, unusual properties (de Philippis et al., 2001). Cyanobacterial polysaccharides have demonstrated the potential to be used for the stabilization of emulsions or as bioflocculants; however, further research is necessary for commercial development. Polysaccharides produced by Cyanospira capsulata, one Nostoc strain, and two Cyanothece strains were reported to have viscosity values comparable to or above those for aqueous solutions of xanthan gum under similar concentrations (de Philippis et al., 2001). 3. Exopolysaccharides from extremophiles Extremophiles such as deep-sea bacteria contain polysaccharides with a wide variety of chemical and physical properties that are oftentimes not present in or are variations of the more traditional, terrestrial plant-derived polysaccharides (e.g., thickening, gelling, stabilizing, suspending, coagulating, filmforming, and water retention) (Guezennec, 2002). Exopolysaccharides (EPSs) secreted by deep-sea hydrothermal microorganisms have been identified in Pseudoalteromonas, Alteromonas, and Vibrio. One strain of Alteromonas was found to produce an anionic EPS with potential use as a thickening agent, while other Alteromonas strains produced polymers with qualities such as unusual gelling properties, significant thickening ability, and high metal-binding capacity (reviewed in Guezennec, 2002). Additionally, halophiles such as Halobacterium mediterranei have been reported to contain EPSs with highly favorable rheological properties and resistance to high salinities, temperatures, and pH (Herbert, 1992). Although EPSs from extremophiles exhibit unique and potentially valuable properties as food ingredients, their commercial application in biotechnology and industry will ultimately be dependent on factors such as yield, price, and markets (Guezennec, 2002).

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4. Chitin and chitosan Chitin is a homopolymer of N-acetyl-D-glucosamine residues and is a major structural component in the exoskeletons of crustaceans, mollusks, arthropods, and the cell walls of numerous fungi and algae. Owing to its widespread presence in both terrestrial and aquatic organisms, chitin is second only to cellulose as the most abundant biopolymer on the Earth (Shahidi and Abuzaytoun, 2005). On a dry weight basis, shrimp, crab, lobster, prawn, and crayfish have been reported to contain between 14% and 35% chitin, while deproteinized dry shell waste of Antarctic krill contains approximately 40% crude chitin (Haard et al., 1994). Crustaceans are the primary sources of chitin used in industry. Chitin can be extracted from shellfish and crustacean waste by mixing with a dilute acid to induce demineralization, followed by a deproteinization step in a hot alkaline solution (Synowiecki and Al-Khateeb, 2003). Once isolated, chitin can be deacetylated to create chitosan, a large cationic polymer with numerous commercial applications in the food, pharmaceutical, and waste treatment industries. Chitosan can be used in meat preservation, as it inhibits growth of spoilage bacteria in foods (Darmadji and Izumimoto, 1994). Chitosan has been sold as a weight loss supplement due to the belief that it absorbs and binds fat, inhibits LDL cholesterol, and boosts HDL cholesterol; however, studies on its eVectiveness in this area have been inconsistent (Ohr, 2005; Shahidi and Abuzaytoun, 2005). The majority of the chitosan produced in Japan is used to treat wastewater in the food industry, as it can remove water-soluble proteins with high biochemical oxygen demands (Ohshima, 1998). Additionally, chitin derivatives have numerous properties that could be further utilized commercially, such as ability to form gels, high capacity for adsorption, polyelectrolyte properties, reactive functional groups, biodegradability, and antitumor, bactericidal, and fungicidal activities (Synowiecki and Al-Khateeb, 2003). Although chitosan is used in wound healing (i.e., sutures and poultices), there exist a variety of food applications for chitin, chitosan, and their derivatives, including use as antimicrobial agents, edible films, additives (e.g., for clarification and deacidification of fruit juices or emulsification), nutraceuticals (e.g., increasing dietary fiber, reducing lipid absorption), and water purifiers (Shahidi and Abuzaytoun, 2005; Shahidi et al., 1999). Despite the various potential applications of chitosan, there are several drawbacks. For example, the costs of production often outweigh the economic benefits of its application. Extraction yields for chitosan from waste are generally very low (3–5% of raw material) and production is limited by seasonal variations in crustacean harvesting. Also, additional waste streams are created during the alkali deproteinization of chitin during isolation (Ludlow, 2001; Synowiecki and Al-Khateeb, 2003). One interesting

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new technology for extraction of chitin that oVers an alternative to the more harsh chemical methods traditionally utilized is the use of the lactic acid bacteria Lactobacillus plantarum. Fermentation of shrimp waste with Lactobacillus results in production of a solid portion of chitin and a liquor containing shrimp amino acids, minerals, and pigments (Rao and Stevens, 2005). D. PROTEINS

Proteins from marine sources show promise as functional ingredients in foods because they possess numerous important and unique properties such as film and foaming capacity, gel-forming ability, and antimicrobial activity (Table I). Some of the most prevalent marine proteins used in foods are collagen, gelatin, and albumin, all of which can be extracted from fish and seafood by-products. The protein protamine has also shown promise for use in the food industry as a natural antibacterial preservative. Collagen is a connective tissue protein found in skin, bones, cartilage, and ligaments. It has been reported to contribute to up to 30% of the total proteins in animals and it can be isolated from both terrestrial and marine sources (Senaratne et al., 2006). Collagen is used widely in the food, cosmetic, and pharmaceutical industries. A major food application is in the meatprocessing industry as edible casings for products such as sausages. The most widely used form of collagen (type I) was extracted from the skins of two diVerent fish species, albacore tuna (Thunnus alalunga) and silver-line grunt (Pomadasys kaakan), and compared to bovine type I collagen (Noitup et al., 2005). Interestingly, the fish skin type I collagens were less stable in that they had lower denaturation temperatures and lower levels of hydroxyproline, a cross-link promoter. In another study, collagen isolated from the skin of brown backed toadfish (Lagocephalus gloveri) was reported to have a significantly lower denaturation temperature compared to porcine collagen (Senaratne et al., 2006). These diVerences between land-based and marine collagens are presumably due to the diVerences in habitats of the organisms. The distinct qualities of marine collagens are useful in the food industry in products that require formation of gels or casings at low temperatures. Also, collagen can be extracted from fish processing by-products in an eVort to reduce and utilize marine discards. Some additional marine sources of collagens include bigeye snapper, hake, trout, lingcod, catfish, rainbow trout, yellow sea bream, common horse mackerel, and tiger puVer, among others (Jongjareonrak et al., 2005; Noitup et al., 2005). Gelatin is a protein product formed by the partial hydrolysis of collagen. It has a unique gel-forming ability and is used in the food industry as a stabilizer, texturizer, thickener, or foaming agent in ice cream, jam, yogurt,

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cream cheese, margarine, marshmallows, bakery products, and low-fat foods. Traditionally, gelatin has been derived from beef or pork; however, gelatin can also be extracted from marine sources for commercial use in foods (Choi and Regenstein, 2000). Marine gelatin can be derived from the skins of flatfish, such as sole and megrim; cold-water fish species including pollock, cod, haddock, hake, and cusk; or alternative sources such as squid and octopus, by either acid or alkaline treatment methods (Djagny et al., 2001; Gomez-Guillen et al., 2002; Norland, 1990). Although many properties of marine and land-based gelatins are similar, marine gelatins tend to have lower melting points and form weaker gels at relatively low temperatures (Choi and Regenstein, 2000; Haard et al., 1994; Leuenberger, 1991). These diVerences are likely due to the lower levels of proline and hydroxylproline found in marine gelatins compared to land-based sources (Gomez-Guillen et al., 2002). Marine gelatins are excellent emulsion stabilizers, crystal growth inhibitors, and foam and film-forming agents, and can serve as edible protective coating materials or clarifiers (Djagny et al., 2001; Haard et al., 1994; Norland, 1990). A particularly important application is isinglass, a high-grade gelatin made from the swim bladders of fish. Isinglass is widely used as a commercial clarifier in beverages such as wine, beer, cider, and vinegar due to its ability to induce aggregation of yeast and other insoluble particles (Hickman et al., 2000). In addition to its clarifying properties, isinglass was reported to prevent and treat symptoms of chronic atrophic gastritis in rats (Xu et al., 2004). Albumin is a blood plasma protein synthesized in the liver. It is a flexible protein that will readily change shape as a result of ligand binding or changes in environmental conditions; however, the presence of disulfide bridges provides strength and allows albumin to easily regain its original structure. Albumin has exhibited several properties that make it beneficial to human health, such as antioxidant and anticoagulatory activities and ability to maintain microvascular integrity (Nicholson et al., 2000). Although it is typically derived from egg whites, albumin can also be isolated from mollusks, crustaceans, and low-fat fish (Ockerman and Hansen, 1988). In order to extract albumin from marine sources, minced flesh is cooked in acid and the resulting digested protein is pressed, ground, extracted, digested with sodium hydroxide, and neutralized with lactic acid to produce a mixture of mostly polypeptides that is spray-dried for use in industry. Marine-derived albumin can be used as a replacement for egg albumin as a whipping, suspending, or stabilizing agent (Haard et al., 1994). Also of interest is protamine, a simple, cationic protein consisting largely of arginine residues. Protamine associates with DNA in the place of histones in spermatozoa and can be extracted from the spermatic cells of fish, birds, and mammals (Ohshima, 1998; Potter et al., 2005). Commercial marine

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sources include salmon and herring milt. The major application of protamine in the food industry thus far has been as a preservative in products such as fruits, rice, and confectionaries. Protamine is a promising antibacterial agent in foods because it does not coagulate under heat and can kill or significantly inhibit the growth of some bacteria, disrupting the cell envelopes of both Gram-negative and Gram-positive bacteria (Islam et al., 1986; Johansen et al., 1997). The antibacterial eVect of protamine is slightly reduced in food matrices due to interferences such as nonspecific binding to negatively charged food particles and the presence of divalent cations (Ca2þ and Mg2þ) (Pink et al., 2003; Potter et al., 2005). Fortunately, these inhibitory eVects have been shown to be reduced by altering the electrostatic properties of protamine (Potter et al., 2005). A protein mixture similar in composition to soy meal can be commercially extracted from the extremophile Dunaliella for use in mariculture and animal feed. The industrial-scale growth of Dunaliella can turn out protein extract at about 100-times greater productivity than that reported in agriculture and 50-fold greater than in fish farming (Herbert, 1992). Protein powders can also be extracted from the processing discards of fish such as arrowtooth flounder (Atheresthes stomias) and herring (Clupea harengus). These freezedried powders have been reported to possess favorable nutritional and functional properties, exhibiting desirable mineral levels and amino acid profiles along with high fat adsorption and emulsifying capacities (Sathivel et al., 2004). E. ENZYMES

1. Marine enzymes: Introduction and sources Enzymes are bioactive compounds with the ability to transform other molecules, making them valuable biotechnological tools for use in the food and feed industries. As ingredients in food, enzymes can influence factors such as processing, storage, spoilage, and safety. Thanks to advances in biotechnology, the use of marine-derived enzymes in food applications has grown into a promising field of research (Table I) (Diaz-Lopez and Garcia-Carreno, 2000; Venugopal and Shahidi, 1995). Excellent reviews with in-depth discussions of the use of marine-derived enzymes available include Okada and Morrissey (2007), Shahidi and Janak Kamil (2001), and Haard and Simpson (2000). Owing to its vast diversity of organisms and habitats, the marine world is a rich source of unique and valuable enzymes with potential applications in the food industry. Many marine-derived enzymes have physical, chemical, and/or catalytic properties unparalleled by their terrestrial counterparts. For example, most marine enzymes have cold-adapted properties that are useful in food and

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feed processing, such as their ability to have high catalytic activity at low temperatures and to be inactivated at moderate temperatures (Diaz-Lopez and Garcia-Carreno, 2000). Additionally, marine-based enzymes are valuable as food ingredients and in food processing due to their specificity, diverse properties, salt tolerance, and high activity at mild pH (Okada and Morrissey, 2007; Shahidi and Janak Kamil, 2001). Taken together, these properties allow for production of food without the undesired side eVects and by-products resulting from use of enzymes that operate under more extreme conditions such as higher temperatures. Use of enzymes in the food industry also has benefits over chemical or mechanical methods, which are oftentimes harsher and more damaging to a product (Shahidi and Janak Kamil, 2001). The main challenges for use of marine-derived enzymes are limited availability depending on harvest; instability of raw material; and potentially poor economic advantages depending on technologies for extraction, potential markets, and quality of by-products (Haard et al., 1994). A potential biotechnological approach to these challenges is in the transfection and overexpression of fish enzyme genes in select marine microorganisms (Simpson, 2000). Major sources of marine enzymes are by-products produced as a result of fish and shellfish processing such as the viscera, heads, skin, bones, exoskeletons, and shells. Specific sources for diVerent marine enzymes are listed in Table I. Some novel sources of enzymes with unique properties include extremophiles and red algae. 2. Marine enzymes: Applications Traditionally, marine-based enzymes have been used in a limited number of products, that is, fish sauce, cured herring, or fish protein hydrolysate; however, more recent uses include the accelerated production of other products such as PUFAs and improving processing techniques such as the removal of skin, scales, and membranes from fish; purification and cleaning of roe for caviar production; extraction of carotenoproteins from shellfish processing waste; substituting rennet during cheese manufacturing; removal of the oxidized flavor from milk; ripening and fermentation of fish product (for fish sauce); and preparation of fish protein hydrolysates and concentrates (Diaz-Lopez and Garcia-Carreno, 2000; Shahidi and Janak Kamil, 2001; Venugopal and Shahidi, 1995). 3. Marine enzymes available for use in the food industry There are numerous marine-derived enzymes available for use in the food industry (Table I). Although an attempt will be made to discuss the major categories of important marine enzymes, a large number of enzymes are

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available and the reader is referred to the reviews mentioned in the beginning of the Section III.E.1 for further information. Enzymes that have been isolated from the marine world for use in the food industry include digestive proteolytic enzymes such as gastric, serine, and cysteine or thiol proteases; lipases; polyphenol oxidases (PPOs); chitinolytic enzymes; muscle proteases; transglutaminase; extremophilic enzymes; and a novel red algae enzyme. a. Digestive proteases. Proteases are enzymes that cleave the peptide bonds in proteins. Although they are considered to be the most important and widely used group of enzymes in the food industry, proteases extracted from marine organisms are used in limited amounts, in part due to a lack of basic knowledge regarding these specific enzymes and also due to consumer attitudes toward their source—fish and seafood discards (Simpson, 2000). However, digestive proteases from marine sources have received growing interest from researchers and food processors owing to their high enzymatic activities at low temperatures and an increasing availability of raw materials such as viscera (Okada and Morrissey, 2007). Fish viscera are a rich source of digestive enzymes, such as pepsin, trypsin, chymotrypsin, and gastricsin, and numerous researchers have been developing methods for their recovery on a large scale (Haard et al., 1994; Reece, 1988). Work is being carried out around the world in countries such as Japan, Great Britain, and Denmark, with specific examples being the Icelandic Fisheries Laboratory, which has developed a way to recover trypsin-like enzymes from cod viscera (Stefansson and Steingrimsdottir, 1990) and Marine Biochemicals (Tromso, Norway), which has developed industrial-scale methods for the recovery of trypsin, pepsin, chymotrypsin, alkaline phosphatase, and hyaluronidase from fish viscera (Almas, 1990). An alternative new technology is the utilization of microorganisms to produce enzymes from marine sources, for example cold-adapted proteases from marine invertebrates have been successfully expressed in yeast (Kristjansdottir and Gudmundsdottir, 2000). Due to their ability to hydrolyze proteins, proteases can significantly alter the texture of food products. One of the most economically important applications of proteases is in the tenderizing of meat after rigor mortis (Whitaker, 1996). They can also be used to enhance the texture of cereals and baked products, remove membranes from organs and egg sacks, thereby improving drying and quality of egg products, ripen cheeses, remove skin from fish and squid, and recover bone proteins (Haard et al., 1994; Simpson, 2000). In shrimp, proteases can be used to loosen shells from the meat, recover flavor compounds for use in surimi-based and cereal-based extrusion products, and recover carotenoprotein (up to 80% of the protein and 90% of the astaxanthin in shell waste) (Haard et al., 1994). Stomachless marine organisms, such as crayfish, cunner, and puVer, also contain digestive

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proteases that can be used to inactivate PPO and/or pectin-esterases in fruit juices (Shahidi and Janak Kamil, 2001). Three of the most widespread types of digestive proteases found in the marine world are acid/aspartyl (gastric) proteases, serine proteases, and cysteine or thiol proteases. Acid/aspartyl proteases are found in the stomachs of animals and are therefore active under acidic conditions and inactive in alkaline environments (Simpson, 2000). Three main types of gastric proteases are pepsins, gastricsins, and chymosins. Pepsins are aspartic endopeptidases that have been found in the gastric fluid of numerous marine and freshwater species (Shahidi and Janak Kamil, 2001). Cold-adapted pepsins from Atlantic cod (Gadus morhua) are being recovered in Norway, with commercial applications in the cheese industry for cold renneting milk and in the fish feed industry to assist in digestion (Simpson, 2000). Gastricsin is a gastric protease that has been identified in marine organisms and has similar properties as pepsin. A third type of acid protease of interest is the rennin chymosin, which is typically present in the digestive compartment of young ruminant stomachs. Chymosin has been identified in the stomachs of marine organisms such as carp and harp seals (Shahidi and Janak Kamil, 2001). Cheddar cheese prepared with seal chymosins was reported to have higher sensory scores compared to cheese made with calf rennet (Simpson, 2000). Digestive serine proteases are present in the pyloric ceca, the pancreatic tissues, and the intestines of animals and have been reported in numerous species of Archaea (Eichler, 2001). Serine proteases are inactive at acidic pH and have high activity under neutral to slightly alkaline conditions (Simpson, 2000). Although fish serine proteases are quite similar to their mammalian counterparts, they have been reported to be more active under alkaline rather than neutral conditions (Shahidi and Janak Kamil, 2001). Some of the most well-known serine proteases from marine sources include trypsins, chymotrypsins, collagenases, and elastases. Trypsin-like enzymes can be found in both cold- and warm-water marine organisms such as stomachless bone fish (Carassius auratus gibelio), sardines, and others (Shahidi and Janak Kamil, 2001). They can inactivate enzymes such as PPO, giving them potential use in the food industry for preventing undesired color changes in PPO-containing products such as shrimp and fruit (Haard et al., 1994). Trypsins and other digestive proteases have also been isolated from crustaceans and mollusks in an organ called the hepatopancreas, which is a combination of the mammalian liver and pancreas (Shahidi and Janak Kamil, 2001). A popular marine source for trypsins is the Atlantic cod, which contains cold-adapted enzymes that have catalytic activity between 4 and 55
C and are sensitive to inactivation by autolysis, low pH, and/or moderate heat (65
C or above). Research conducted over the past two decades has helped to develop industrial methods for extraction

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of cold-adapted trypsins from fish processing by-products such as the pyloric cecum of cod. These trypsins have promising applications in areas of food processing that require protein digestion at low temperatures in order to avoid undesirable chemical reactions and bacterial contamination (Gudmundsdottir and Palsdottir, 2005). For example, cod trypsins have been used for lowtemperature curing of herring (Matjes) and in squid fermentation, thereby accelerating the ripening process which traditionally takes about 1 year (Haard et al., 1994; Simpson, 2000). Another type of serine protease, elastase, is produced by the pancreas and is meant for the digestion of proteins such as elastin, a fibrous protein found in connective tissues. Elastase is an intestinal protease that operates at alkaline pH and has been isolated from marine animals such as carp, catfish, and Atlantic cod (Shahidi and Janak Kamil, 2001; Simpson, 2000). A fourth category of marine serine proteases is the digestive collagenases, which have been isolated from the digestive organs of many fish and from the hepatopancreas of marine invertebrates such as crab, prawn, and lobster (Haard et al., 1994; Shahidi and Janak Kamil, 2001). Collagenases are thought to be one of the principal compounds responsible for flesh mushiness observed in the seafood industry following handling and storage; therefore, these enzymes have potential applications as meat tenderizers in the manufacturing of high-quality meat and meat products (Haard et al., 1994). Like the gastric proteases, digestive cysteine or thiol proteases are active at acidic pH and inactive at basic pH. They are important components of the hepatopancreas of many marine crustaceans, and are responsible for over 90% of the protease activity in the hepatopancreas in short-finned squid (Illex illecebrosus) (Raksakulthai and Haard, 2001). Cathepsin B is one example of a marine-derived digestive thiol protease. Only a few marine sources have been identified for cathepsin B, including surf clam (Spisula solidissima), horse clam (Tresus capax), and mussel (Perna perna L.) (Simpson, 2000). b. Lipases. Lipases are mainly produced in the pancreas and catalyze the hydrolysis of triglycerides into free fatty acids, mono and/or diglycerides, and glycerol at the lipid-water interface. Marine sources of lipases include Atlantic cod, seal, salmon, sardines, Indian mackerel, red sea bream, and others. Lipases have valuable applications in the food industry because they have distinct specificities and they are able to catalyze processes such as esterification, hydrolysis, and exchange of fatty acids in esters (Shahidi and Wanasundara, 1998). These characteristics provide numerous opportunities for use of marine lipases in the fats and oils industry such as the production of triglycerides enriched with n-3 LC-PUFAs. Production of these triglycerides using commercially available lipases from nonmarine sources is

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generally diYcult because nonmarine lipases are either less specific than marine lipases or they have diVerent specificities than desired (Shahidi and Janak Kamil, 2001). A marine lipase isolated from Atlantic cod was shown to preferentially hydrolyze LC-PUFAs over shorter-chain fatty acids (Lie and Lambertsen, 1985), and lipase-assisted hydrolysis of seal blubber oil and menhaden oil has been used for enrichment of acylglycerols with n-3 LC-PUFAs (Shahidi and Wanasundara, 1998). c. Polyphenol oxidases. PPOs, including tyrosinase, polyphenolase, phenolase, catechol oxidase, cresolase, and catecholase, are found in plants, animals, and a number of microorganisms. They are responsible for the postharvest brown discolorations that appear in certain crustaceans, fruits, and vegetables, and for the brown and black colors of products such as tea, coVee, raisins, and prunes (Whitaker, 1996). These dark pigments are a result of the PPO-catalyzed oxidation of diphenols to form quinones, which then undergo further oxidation and polymerization. Traditionally, plant-derived PPOs have been used in tea fermentation; however, marine PPOs are actually better suited for this process because they are cold-adapted and therefore have higher activity at low/moderate temperatures compared with their terrestrial counterparts. In addition to extraction of PPOs from crustacean by-products, use of genetic engineering to produce PPOs from marine microorganisms has also been suggested (Haard et al., 1994). d. Chitinolytic enzymes. In nature, chitinolytic enzymes are utilized for degradation of chitin during molting of insects and crustaceans and as digestive aids, as they are able to disrupt the exoskeleton of prey, allowing access to the soft inner tissues. The genes for these chitinolytic enzymes have been cloned from several diVerent organisms (Shahidi and Abuzaytoun, 2005). Chitinases have been identified in the digestive tracts of numerous fish, in shellfish and shellfish waste, and in squid liver and octopus saliva. Chitin degradation enzymes have also been identified in the hyperthermophilic archaea Thermococcus chitonophagus (Andronopoulou and Vorgias, 2004) and in the marine bacterium Bacillus sp. LJ-25 (Lee et al., 2000). Chitinases have a wide range of potential applications in the food industry; for example, they can replace hydrochloric acid in the conversion of chitin into commercially available oligomeric units, resulting in products with more consistent characteristics (Shahidi and Janak Kamil, 2001). e. Transglutaminase. While most enzymes utilized in the food industry are responsible for the breakdown of specific compounds, transglutaminase is unique in that it is able to modify protein functions by promoting crosslinks (Ashie and Lanier, 2000). There exist numerous marine sources for

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transglutaminase, including red sea bream, rainbow trout, atka mackerel, walleye, pollock liver, muscles of scallop, botan shrimp, and squid (Shahidi and Janak Kamil, 2001). A major commercial application of transglutaminase is in cross-linking proteins for the production of surimi, thereby improving the rheological properties of the protein gel (Ohshima, 1998; Shahidi and Janak Kamil, 2001). Recently, a microbial transglutaminase was reported to catalyze gel formation from gelatin. Unlike typical gelatinbased gels, transglutaminase-catalyzed gels were thermally irreversible, presumably due to the strong cross-linking ability of this enzyme. Addition of chitosan was reported to promote gel strength and results in more rapid formation of gels (Chen et al., 2003). f. Extremophilic enzymes. Enzymes produced by extremophiles can have valuable applications in the food industry owing to their ability to function under a diverse range of extreme conditions. For example, thermophilic enzymes can operate within a temperature range of 45–100
C or more. These enzymes have current use in the production of natural sweeteners and also have potential applications in reactions involving transesterification and synthesis of oligosaccharides, peptides, and phospholipids. Specific examples include: enzymes that interact with carbohydrates, such as a-amylase, glucoamylase, cellulase, pectinase, b-galactosidase, xylose isomerase, and pullulanase; neutral (fungal) proteases for baking and brewing; and lipases and acid proteases for use in food processing (Herbert, 1992). Psychrophilic enzymes, which operate within temperatures ranging from 5 to þ20
C, also have numerous advantages for use in the food industry: they have high activity at low to moderate temperatures, they are easily inactivated with heat, and bacterial contamination and competing reactions are reduced at low temperatures (Gerday et al., 2000; Herbert, 1992). Specific cold-adapted enzymes that have been isolated from Antarctic and Arctic microorganisms include alcohol dehydrogenase, a-amylase, aspartate transcarbamylase, Ca2þ–Zn2þ protease, citrate synthase, b-lactamase, malate dehydrogenase, subtilisin, triose phosphate isomerase, and xylanase (Gerday et al., 2000). Psychrophilic enzymes can be useful as replacements for their mesophilic counterparts in processes such as beer and wine fermentation and cheese production. Cold-adapted amylases, proteases, and xylanases are useful to the dough industry for reducing fermentation time and improving textural properties. Psychrophilic enzymes can replace calf rennet in manufacturing cheese, thereby reducing residual heat coagulation. Some examples of commercially available extremophilic rennet agents are: Marzyme IIÒ and ModilaseÒ . The cold-adapted form of b-galactosidase is useful to the milk industry because it allows for the breakdown of lactose

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into glucose and galactose at lower temperatures, thereby reducing bacterial activity and speeding productivity (Herbert, 1992). Another potential commercial application of extremophiles is in the use of the alkaliphilic form of the enzyme cyclomaltodextrin glucanotransferase for production of cyclodextrins (CDs) from starch. CDs are widely used in the food industry as emulsifying, foaming, and stabilizing agents, and the possibility for their low-cost commercial production using alkaliphilic Bacillus sp. has been demonstrated. Bacillus sp. alkaliphiles also have been reported to contain extracellular b-mannanases, which have potential in the food industry because they can hydrolyse products that contain mannan, such as guar gum. Another product of Bacillus sp. alkaliphiles is pectinolytic enzymes, which have been used to treat pectin-containing eZuent at an orange-canning operation. Alkaliphiles are also known to synthesize xylanases, which could be used to produce valuable products from plant residues such as wheat and rice straw (Herbert, 1992). g. Enzymes from red algae. Recent research has revealed a type of red marine algae (genera Gracilariales) that produces enzymes important in the starch degradation pathway, including a-1,4-glucan lyase, which catalyzes the formation of the natural sugar 1,5-anhydro-D-fructose (AF). AF, which is also present in seaweeds and edible mushrooms, is a versatile molecule that is a precursor to compounds with antioxidant, antimicrobial, and/or anti-blood-clotting and antitumor properties (Yu, 2005). Researchers also isolated an enzyme in the starch degradation pathway that is known to convert AF into the antifungal compound microthecin. IV. CONCLUSIONS Marine biotechnology for the production of food ingredients has experienced rapid growth and shows great potential for the future. The number of food ingredients that can be derived from marine sources is ever-increasing thanks to advances in the biotechnological tools utilized for their identification and extraction. These resulting components can be used in a variety of applications, such as fortification/nutraceuticals, natural pigments, stabilization, antimicrobial food coatings, and in the development of more eYcient and natural food processing techniques. Despite the number of opportunities that the tools of marine biotechnology provide for production of food ingredients, numerous challenges remain. In order to maximize profits in the production of marine-derived compounds, cultivation techniques for select marine organisms must be

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refined and made more eYcient. Also, in many cases, there lies the challenge of finding niche markets for marine-derived ingredients in which they can economically compete with or surpass their synthetic counterparts. Although recent developments in genetics show promising results, aquatic plant transgenic research remains far behind that for terrestrial plants. Overall, the field of marine biotechnology for production of food ingredients is still in its developing stages, with many challenges to overcome; however, there is great potential and what seem to be unlimited opportunities for growth and progress. REFERENCES Ackman, R.G., Jangaard, P.M., Hoyle, R.J., and BrockerhoV, H. 1964. Origin of marine fatty acids. Analysis of the fatty acids produced by the diatom Skeletonema costatum. J. Fish Res. Bd. Can. 21, 747–756. Almas, K.A. 1990. Utilization of marine biomass for production of microbial growth media and biochemicals. In ‘‘Advances in Fisheries Technology and Biotechnology for Increased Profitability’’ (M.N. Voigt and J.R. Botta, eds), pp. 361–373. Technomic Publishing Co., Lancaster, PA. Andronopoulou, E. and Vorgias, C.E. 2004. Isolation, cloning, and overexpression of a chitinase gene fragment from the hyperthermophilic archaeon Thermococcus chitonophagus: Semi-denaturing purification of the recombinant peptide and investigation of its relation with other chitinases. Protein Expr. Purif. 35, 264–271. Apt, K.E. and Behrens, P.W. 1999. Commercial developments in microalgal biotechnology. J. Phycol. 35, 215–226. Ashie, I.N.A. and Lanier, T.C. 2000. Transglutaminase in seafood processing. In ‘‘Seafood Enzymes’’ (N.F. Haard and B.K. Simpson, eds), pp. 147–166. Marcel Dekker, Inc., New York. Bajpai, P. and Bajpai, P.K. 1993. Eicosapentaenoic acid (EPA) production from microorganisms: A review. J. Biotechnol. 30, 161–183. Bao, D.Q., Mori, T.A., Burke, V., Puddey, I.B., and Beilin, L.J. 1998. EVects of dietary fish and weight reduction on ambulatory blood pressure in overweight hypertensives. Hypertension 32, 710–717. Barclay, W. 2006. Schizochytrium and thraustochytrium strains for producing high concentrations of omega-3 highly unsaturated fatty acids. US patent 7022512. Barclay, W. and Zeller, S. 1996. Nutritional enhancement of n-3 and n-6 fatty acids in rotifers and Artemia nauplii by feeding spray-dried Schizochytrium sp. J. World Aqua. Soc. 27, 314–322. Barclay, W., Meager, K.M., and Abril, J.R. 1994. Heterotrophic production of long chain omega-3 fatty acids utilizing algae and algae-like microorganisms. J. Appl. Phycol. 6, 123–129. Belarbi, E.H., Gomez, A.C., Chisti, Y., Camacho, F.G., and Grima, M.E. 2003. Producing drugs from marine sponges. Biotechnol. Adv. 21, 585–598. BeMiller, J.N. and Whistler, R.L. 1996. Carbohydrates. In ‘‘Food Chemistry’’ (O.R. Fennema, ed.), pp. 157–223. Marcel Dekker, Inc., New York. Ben-Amotz, A. 1993. Production of beta-carotene and vitamins by the halotolerant alga Dunaliella. In ‘‘Marine Biotechnology, Volume 1: Pharmaceutical and Bioactive Natural Products’’ (D.H. Attaway and O.R. Zaborsky, eds), pp. 411–417. Plenum Press, New York. Ben-Amotz, A. 2004. Industrial production of microalgal cell-mass and secondary products—major industrial species: Dunaliella. In ‘‘Handbook of Microalgal Culture: Biotechnology and Applied Phycology’’ (A. Richmond, ed.), pp. 273–280. Blackwell Publishing, Oxford.

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FRUITS OF THE ACTINIDIA GENUS ICHIRO NISHIYAMA Department of Food and Nutrition, Komazawa Women’s Junior College Inagi, Tokyo 206-8511, Japan

I. Introduction II. Actinidia Species and Cultivars A. A. deliciosa B. A. chinensis C. A. arguta D. A. rufa E. Other Actinidia Species III. Fruit Components A. Sugar and Sugar Alcohol B. Organic Acids C. Vitamin C D. Calcium Oxalate E. Pigments F. Actinidin G. Other Components IV. Allergenic Properties V. Health Benefits VI. Perspectives Acknowledgments References

Kiwifruit is the most well-known crop in the genus Actinidia. Although Actinidia fruit sales in the international market are dominated by a single kiwifruit cultivar Actinidia deliciosa ‘‘Hayward,’’ there are a considerable number of cultivars and selections in the genus that have widely diverse shape, size, and hairiness. They also oVer a wide variation in sensory attributes such as flesh color, flavor, and taste, and in nutritional attributes such as the vitamin C level and carotenoid content. The level of actinidin, which is a cysteine protease in kiwifruit, also varies greatly among cultivars. This chapter reviews available information related to several important components, allergenic properties, and health benefits of Actinidia fruits. ADVANCES IN FOOD AND NUTRITION RESEARCH VOL 52 # 2007 Elsevier Inc. All rights reserved

ISSN: 1043-4526 DOI: 10.1016/S1043-4526(06)52006-6

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I. INTRODUCTION Among the fruits of the Actinidia genus, kiwifruit is the most well-known, and is one of the few crops marketed worldwide. At the beginning of the nineteenth century, kiwifruit merely grew wild in China. In 1904, kiwifruit seeds were brought to New Zealand for the first time (Ferguson, 2004). Subsequently, it was domesticated in New Zealand and launched onto world markets in the 1960s. The kiwifruit industry has made rapid progress since then and kiwifruit are now common and easily obtainable throughout the year. Kiwifruit is one of only four new fruit crops introduced to international trade in the twentieth century. They are now grown in many countries, notably Italy, China, New Zealand, Chile, France, Greece, Japan, and the United States. The industry’s remarkable progress might be attributed to the success of a single cultivar, ‘‘Hayward’’ (Ferguson, 1999). ‘‘Hayward’’ fruit have an attractive emerald green flesh and fine flavor, which is sometimes described as a mix of strawberry, banana, and pineapple. Until recently, the cultivar name ‘‘Hayward’’ was often taken as synonymous with kiwifruit as most consumers are not aware of other varieties. However, not all of the attributes of ‘‘Hayward’’ are perfect. Fruits of some Actinidia cultivars are much sweeter than those of ‘‘Hayward,’’ have much higher vitamin C content, or have much higher contents of carotenoids such as b-carotene and lutein. In 2000, the yellow-fleshed fruit of a novel cultivar ‘‘Hort16A’’ were released as ‘‘ZESPRITM GOLD Kiwifruit’’ from New Zealand into the world market. They shattered the common perception that the flesh of kiwifruit is absolutely green. Since then, the production of ‘‘Hort16A’’ has grown dramatically and is likely to account for 17–18% of New Zealand kiwifruit exports (Belrose, Inc., 2006). Quite recently, fruits of Actinidia arguta, which is closely related to kiwifruit, have become commercially available (Williams et al., 2003). These fruits are sold under commercial names, such as ‘‘Baby kiwi,’’ ‘‘Kiwi berry,’’ or ‘‘Grape kiwi,’’ because they are grape-sized fruits with a completely hairless skin. In addition to the cultivars described above, several Actinidia cultivars have already been launched onto markets on a small scale. Moreover, development of new Actinidia cultivars of commercial potential is now in progress in several countries including New Zealand, Italy, China, Korea, and Japan. New and quite diVerent Actinidia fruit, with widely diverse size, shape, hairiness, flesh color, nutritional value, and flavor, are anticipated for introduction to future international trade. The kiwifruit industry is now in a

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transition period from domination by only one variety, ‘‘Hayward,’’ into increasing varietal diversity, which oVers greater choice to the consumers. These novel and/or minor cultivars, however, are probably unfamiliar to most people, even to those interested in food science; most readily available information on kiwifruit or on Actinidia fruits refers to ‘‘Hayward.’’ In this chapter, several cultivars representative of Actinidia species are presented. The available information concerning several important components, allergenic properties, and health benefits of Actinidia fruits are also reviewed.

II. ACTINIDIA SPECIES AND CULTIVARS The genus Actinidia is composed of 76 species and about 120 taxa in all (Ferguson and Huang, 2007). In international trade today, the term kiwifruit is taken as including two distinct Actinidia species: A. deliciosa and A. chinensis. Recently, fruit of A. arguta have made an entry into international trade. Other Actinidia species have commercial potential, or are important as useful genetic resources for cultivar development by interspecific hybridization techniques. A. A. DELICIOSA

The most common type of kiwifruit is A. deliciosa. Fruit of this species has a dull-brown skin with dense hair. Its flesh is translucent and bright green, which contrasts against its white core and black seeds. ‘‘Hayward’’ (Figure 1) is the most commercially available cultivar. Therefore, it is the standard cultivar against which the quality of a new cultivar is evaluated. The mature fruit have a moderate sugar–acid balance and their flavor is considered by many to be superior. The most important advantage of this cultivar is the remarkably long storage life of the fruit, which enables exports by ship to distant markets (Ferguson, 1999). ‘‘Koryoku’’ (Figure 1) is a seedling produced by open pollination of ‘‘Hayward.’’ The fruit are long and cylindrical; they have dense, easily shed hair. Total soluble sugar in its mature fruit is higher than that in ‘‘Hayward.’’ ‘‘Ryoku’’ in the cultivar name means ‘‘green’’ in Japanese. Consistent with that name, the green flesh color is deeper than that of ‘‘Hayward.’’ The carotenoid content of ‘‘Koryoku’’ fruit is much higher than that of ‘‘Hayward.’’ ‘‘Sanryoku’’ (Figure 1) is an interspecific hybrid of A. deliciosa ‘‘Koryoku’’
A. chinensis. The fruit has a distinctive bulletlike shape: a long cylindrical shape with a pointed end. The flesh is lime-green. Density of the hair on

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FIG. 1

Fruits of Actinidia species.

the surface is sparse, like A. chinensis fruit described below. Soluble solid contents of ripe fruit are 16–19%. B. A. CHINENSIS

A. deliciosa and A. chinensis are very similar species. They had been classified as the same species until 1984, when they were determined to be two distinct species by Liang and Ferguson (1984). The variant with smooth-skinned, almost hairless fruit retained the original name A. chinensis, and the hairyfruited variant took the name A. deliciosa (Ferguson, 2004). The A. chinensis

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fruit are usually, although not always, yellow-fleshed, whereas those of A. deliciosa have green flesh without exception. ‘‘Hort16A’’ (Figure 1) is a cultivar developed by HortResearch in New Zealand. The fruit have a characteristic shape with a protrusion of the stylar end, which is a so-called ‘‘beak.’’ The fruit have a bright-yellow flesh, and are marketed under the commercial name ‘‘ZESPRITM GOLD Kiwifruit.’’ ‘‘Hort16A’’ fruit are currently grown under license to ZESPRI International in countries of the Northern hemisphere such as Italy, Japan, Korea, France, and the United States. ‘‘Sanuki gold’’ (Figure 1), which is a hybrid of A. chinensis ‘‘Kuimi’’ and a male line of the same species, was produced recently by Kagawa Agricultural Experiment Station in Japan. It produces applelike globose fruit with a deep yellow flesh. The fruit are remarkably large. The most prominent property of ‘‘Sanuki gold’’ is its exceptionally high vitamin C content (Table II). ‘‘Jintao’’ is a yellow-fleshed kiwifruit developed by the Wuhan Institute of Botany in China (Huang et al., 2002). The name ‘‘Jintao’’ means ‘‘golden peach’’ in China. The fruit are long and cylindrical. ‘‘Jintao’’ is being commercialized under the name ‘‘Kiwigold’’ by the Kiwigold Consortium in Italy. ‘‘Hongyang’’ (Figure 1) is a selected clone from a red-fleshed A. chinensis resource collected from Henan Province in China. The fruit of ‘‘Hongyang’’ have a deep red color around the core. The red and yellowish-green appearance of the transverse section of the fruit is particularly striking and decorative. The fruit have a sweet taste with a mean soluble solids concentration of 19.6% when ripe (Wang et al., 2003). ‘‘Hongyang’’ fruit are now marketed under the name ‘‘Red sun.’’ ‘‘Chuhong’’ is a novel red-fleshed cultivar that was released oYcially in 2005 from China. The average soluble solid content of ripe fruit is 16.5%, with a maximum of 21%. The fruit are long or flat, and elliptical (Zhong et al., 2007). C. A. ARGUTA

A. arguta is a species with high cold hardiness: it is sometimes called ‘‘hardy kiwi.’’ It can grow well in places where A. deliciosa or A. chinensis vines cannot survive. A. arguta produces smooth and hairless grape-sized fruit, weighing 5–15 g. The skin is edible, so these fruit are consumed whole. A disadvantage of the fruit of this species is that the storage life and shelf life of the fruit are more limited than those of either A. deliciosa or A. chinensis (Fisk et al., 2006). ‘‘Ananasnaya’’ (Figure 1), which is sometimes called ‘‘Anna’’ in abbreviation, is the most widely grown A. arguta cultivar in United States and Chile. The fruit are sweeter than ‘‘Hayward’’ fruit. They are bite-sized and completely fuzzless, thus they are marketed under the name ‘‘Baby kiwi’’ or

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‘‘Kiwi berry.’’ Although the cultivar name ‘‘Ananasnaya’’ has come into wide use, it has engendered some confusion. The true ‘‘Ananasnaya’’ is possibly a hybrid of A. arguta and A. kolomikta selected many years ago by Michurin

TABLE I VARIOUS ACTINIDIA GENOTYPES

Species genotype

Color of flesh

A. deliciosa Hayward Green Bruno Green Abbott Green Elmwood Green Koryoku Deep green A. deliciosa
A. chinensis Sanryoku Lime green A. chinensis Jiangxi 79–1a Yellow Golden king Yellow Kuimib Yellow Sanuki gold Deep yellow Kobayashi39 Yellow Hongyangc Yellow, partly red Hort16Ad Yellow A. rufa Awaji Deep green Nagano Deep green A. arguta Hirano Green Gassan Green Issai Green Mitsuko Green Ananasnayae Green A. arguta
A. deliciosa Kosuif Deep green Shinzan Deep green

Density of hairs

Average fruit weight (g)

Dense Dense Dense Dense Dense

99.0 101.8 79.8 116.0 98.7

Sparse or absent

107.0

Sparse or Sparse or Sparse or Sparse or Sparse or Absent Sparse or

97.8 136.6 102.5 166.1 106.9 77.6 104.4

absent absent absent absent absent absent

Absent Absent

9.4 16.4

Absent Absent Absent Absent Absent

6.5 11.3 7.8 10.1 6.5

Absent Absent

37.8 20.6

Synonymous with ‘‘Koshin’’ or ‘‘Red princess.’’ Synonymous with ‘‘Applekiwi’’ or ‘‘Kaimitsu.’’ c Synonymous with ‘‘Rainbow red.’’ d Known commercially as ZESPRITM GOLD Kiwifruit. e Known commercially as ‘‘Baby kiwi.’’ f Recent study using RAPD analysis suggested A. rufa, not A. arguta, is involved in the parentage. Values are means  SD (n ¼ 24). a b

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(Ferguson, 1999). In this chapter, however, the name ‘‘Ananasnaya’’ is used for this A. arguta cultivar. ‘‘Kosui’’ (Figure 1) is a cultivar released from Kagawa Agricultural Experiment Station in Japan. The fruit have an intermediate size between that of kiwifruit and A. arguta fruit (Table I). The average soluble solid content of the ripe fruit is 17%, with a maximum of 21%. ‘‘Kosui’’ is reported to be an interspecific hybrid between A. arguta ‘‘Issai’’ and A. deliciosa ‘‘Matua.’’ However, a study using random amplified polymorphic DNA (RAPD) analysis suggested the possibility that A. rufa is involved in the parentage of this cultivar (Kokudo et al., 2003). Recently, several novel A. arguta cultivars have been developed. They include ‘‘Hortgem Tahi,’’ ‘‘Hortgem Toru,’’ ‘‘Hortgem Wha,’’ and ‘‘Hortgem Rua’’ which were developed in New Zealand (Williams et al., 2003), and ‘‘Chiak’’ in Korea (Jo et al., 2007b). These fruit might enter the international trade arena in the near future. D. A. RUFA

In Japan and Korea, A. rufa is distributed wild (Ferguson, 1991). Mature fruit have hairless brown skin and weight of 8–20 g. A. rufa fruit have several unique characteristics, such as high quinic acid and low protease contents (Kim et al., 2007; Nishiyama et al., 2007a). These characteristics make A. rufa valuable genetic resources for crossbreeding of Actinidia plants. E. OTHER ACTINIDIA SPECIES

The A. eriantha fruit are long and cylindrical. The fruit surface is densely covered with white villose. They have easily peeled skin, and the flesh is jade green. Although A. eriantha fruit are typically tart and not so palatable, a new cultivar, ‘‘Bidan,’’ which was developed quite recently in Korea, is reportedly sweet (Jo et al., 2007a). Several desirable attributes of another species, A. kolomikta, are its coldhardiness, precocity, and extraordinary high vitamin C content. For those reasons, it might be a useful genetic resource for cultivar development in Actinidia species. Other Actinidia species are presently underutilized, but they remain potentially useful genetic resources. Characteristics of other species are detailed elsewhere by Huang et al. (2003) and Ferguson and Huang (2007).

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III. FRUIT COMPONENTS A. SUGAR AND SUGAR ALCOHOL

Sugar content is a basic parameter in evaluating fruit market quality attributes. For rough estimation of total soluble sugar content, total soluble solids are measured using refractometry as a convenient method. The total soluble solid content of mature ‘‘Hayward’’ fruit juice is 13–15%. The levels of soluble solids in eating-ripe fruit are generally higher in ‘‘Koryoku,’’ ‘‘Sanryoku,’’ ‘‘Hongyang,’’ and ‘‘Kosui,’’ compared with ‘‘Hayward,’’ although it might be aVected by several environmental factors. Compositions of soluble sugars and sugar alcohols in kiwifruit have been determined using HPLC (Pe´rez et al., 1997), gas chromatography (Sanz et al., 2004), and electrochemical biosensors (Esti et al., 1998). The main soluble sugars in ‘‘Hayward’’ fruit are glucose and fructose, whereas sucrose is present in smaller amounts. Glucose and fructose are present in almost equal amounts. The concentrations of glucose and fructose are approximately 3–5 g/100 g fresh weight (FW); that of sucrose is 0.7–1.5 g/100 g FW (Pe´rez et al., 1997; Sanz et al., 2004). ‘‘Hayward’’ fruit also contain myo-inositol, a hexahydric sugar alcohol. The myo-inositol level in the ‘‘Hayward’’ fruit, which was reported to be 153 mg/100 g FW, is higher than commonly consumed fruits, including orange, grapefruit, and mandarin orange (Sanz et al., 2004). The sugar composition in A. chinensis fruit resembles that of ‘‘Hayward.’’ Glucose and fructose are the predominant soluble sugars in fruits of most A. chinensis genotypes (Esti et al., 1998). The composition of soluble sugars in A. arguta fruit diVers greatly from those in A. deliciosa and A. chinensis fruit. A. arguta fruit contain 5.0–9.5 g/100 g FW sucrose as a predominant soluble sugar, and glucose and fructose at concentrations of 0.8–2.0 g/100 g FW (Nishiyama et al., in preparation). The diVerence in soluble sugar composition between A. arguta fruit and kiwifruit might cause a diVerence in the sweetness of these fruit even if they have the same soluble sugar content. In addition, A. arguta fruit contain myo-inositol at high concentrations of 0.65–1.05 g/100 g FW (Nishiyama et al., in preparation). Klages et al. (1997) suggested that part of the myo-inositol in the fruit might be synthesized in the fruit, whereas some of myo-inositol might be translocated from the phloem as a minor component. These fruit are considered to be the richest dietary source of myo-inositol among commonly consumed foods including fruits, vegetables, beans, grains, nuts, fish, and meats (Clements and Darnell, 1980). Although the nutritional significance of myo-inositol has not been established, it is classified as a member of vitamin B complex and the high content of myo-inositol might be a strength of A. arguta fruit.

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B. ORGANIC ACIDS

Acidity of fruits is an important factor that aVects the sensory characteristics because the taste of fruits is determined primarily by the sugar–acid balance. Importance of some organic acids, such as citric and malic acids, to the production of energy in the body is well established. In addition, high fruit acidity might play a preventive role against proliferation of microorganisms and might contribute toward their long storage life. Ascorbic acid is an important component of the fruit both as vitamin C and as an antioxidant factor. For that reason, it will be addressed separately in the next section. The major organic acids in ‘‘Hayward’’ fruit are citric, quinic, malic, and ascorbic acids. Among them, citric and quinic acid concentrations are higher than those of malic and ascorbic acids. The respective concentrations of citric, quinic, and malic acids in eating-ripe ‘‘Hayward’’ fruit are 0.99–1.29, 0.74–1.18, and 0.08–0.19 g/100 g FW (Marsh et al., 2003, 2004). According to MacRae et al. (1989), these organic acids are not distributed uniformly among ‘‘Hayward’’ fruit. Citric acid is highest in the inner cortex and quinic acid is highest in the outer cortex. The core has the lowest total acid content in the fruit. These organic acids cause diVerent perceptions of acidity. At equivalent molar concentrations, quinic acid has a greater impact on the perception of acidity than malic or citric acids (Marsh et al., 2003). The proportion of these organic acids appreciably changes during fruit ripening depending on the storage temperature (Marsh et al., 2004). Therefore, the fruit taste might be influenced by storage conditions. In other A. deliciosa, A. chinensis, and A. rufa fruits, the compositions of organic acids are fundamentally equivalent to those in ‘‘Hayward.’’ In A. arguta fruit, the concentrations of quinic acid are 0.60–0.71 g/100 g FW: somewhat lower than that in ‘‘Hayward’’ (Nishiyama et al., in preparation). The predominant organic acids in commonly consumed fruit are citric and/or malic acids. Actinidia fruits are unusual in that quinic acid levels are similar to that of citric acid as the predominant organic acid. Although the nutritional importance of quinic acid has not been established, it is an intensely interesting compound because it is a key intermediate for aromatic compound biosynthesis via the shikimic acid pathway. Moreover, quinic acid is a component of several potent free radical scavengers, including chlorogenic acid. C. VITAMIN C

Humans, in common with other primates, are incapable of vitamin C biosynthesis. For that reason, this nutrient must be obtained from dietary sources. Vitamin C is well known to be essential for capillary and blood

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vessel integrity and cartilage and bone development because it is required for collagen biosynthesis (Ball, 2005). Aside from its role as a vitamin, vitamin C has various physiological and pharmacological eVects. Vitamin C improves the bioavailability of iron by facilitating its absorption in the intestine. It also prevents formation of carcinogenic nitrosamines in the stomach (Bender, 2003). Vitamin C acts as a radical trapping antioxidant and plays an important role in the defense against cellular damage by oxidants (Ball, 2005). Therefore, increased intake of vitamin C is associated with reduced risks of certain diseases, including cancer and cardiovascular diseases. The most active form of vitamin C, L-ascorbic acid (AA), is a labile substance that is readily oxidized to L-dehydroascorbic acid (DHAA), mainly through activity of L-ascorbate oxidase and reaction with oxygen in the presence of heavy metal ions and light (Gregory, 1996). Although DHAA itself does not exhibit vitamin C activity, its biological activity has been considered to be equivalent to AA because it can be converted readily in the human body into AA by either NADPH or glutathione-dependent reductases (Bender, 2003). Vitamin C activity is lost when DHAA is further oxidized to 2,3-diketo-L-gulonic acid because of the irreversibility of this reaction (Gregory, 1996). Therefore, vitamin C content in food is usually expressed as the sum of AA and its partially oxidized form, DHAA. Table II shows the contents of vitamin C, which is designated as the sum of AA and DHAA, in the fruits of several Actinidia cultivars (Nishiyama et al., 2004b). ‘‘Hayward’’ fruit contain 65.5 mg/100 g FW vitamin C. The 2000 Dietary Intake values for vitamin C are 75 mg/day for women and 90 mg/day for men. Therefore, intake of one average-sized ‘‘Hayward’’ fruit a day supplies about 70–90% of the required vitamin C. Among cultivars of A. deliciosa, ‘‘Abbott’’ fruit show the lowest vitamin C concentration; fruit of ‘‘Bruno’’ show the highest. Actinidia fruits are an excellent dietary source of vitamin C. Vitamin C contents in Actinidia fruits vary from less than 10 mg/100 g FW to more than 2000 mg/100 g FW (Huang et al., 2003). Among the species, the highest vitamin C content is observed in A. latifolia fruit (671–2140 mg/100 g FW), followed by A. eriantha fruit (500–1379 mg/100 g FW), although they are not commercial crops (Huang et al., 2003). The vitamin C contents of A. deliciosa, A. chinensis, and A. arguta fruits are distributed respectively in the ranges of 50–250, 50–420, and 81–430 mg/100 g FW (Ferguson, 1991; Ferguson and MacRae, 1991; Huang et al., 2003; Nishiyama et al., 2004b; Rassam and Laing, 2005). In most cultivars of A. chinensis, vitamin C contents tend to be higher than A. deliciosa. For example, the vitamin C content of ‘‘Hort16A’’ fruit is about 1.6 times that in ‘‘Hayward’’ fruit. Among the A. chinensis fruits,

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TABLE II CONCENTRATION OF VITAMIN C IN FRUIT OF ACTINIDIA GENOTYPES

Species genotype A. deliciosa Hayward Bruno Abbott Elmwood Koryoku A. deliciosa
A. chinensis Sanryoku A. chinensis Jiangxi 79-1 Golden king Kuimi Sanuki gold Kobayashi 39 Hongyang Hort16A A. rufa Awaji Nagano A. arguta Hirano Gassan Issai Mitsuko A. arguta
A. deliciosa Kosui Shinzan

Concentration (mg/100 g fresh weight)a 65.5  80.0  29.2  47.0  39.9 

14.2 16.8** 5.5** 4.4** 4.0**

75.0  9.0 73.7  144.2  157.1  205.8  129.1  64.4  103.7 

19.9 16.5** 36.4** 19.8** 16.2** 10.0 13.1**

25.5  3.3** 47.1  6.4** 37.3  141.0  184.6  150.6 

Ratio to Hayward 1.00 1.22 0.45 0.72 0.61 1.15 1.13 2.20 2.40 3.14 1.97 0.98 1.58 0.39 0.72

10.9** 24.2** 23.4** 33.0**

0.57 2.15 2.82 2.30

40.9  9.4** 99.8  32.1**

0.62 1.52

Values are means  SD of 12 experiments. **: Significantly diVerent vs ‘‘Hayward’’ at p < 0.01. Reprinted with modification from Nishiyama et al. (2004b, Table III) with permission. Copyright (2004) American Chemical Society. a

‘‘Sanuki gold’’ fruit contain an exceptionally large amount of vitamin C. The concentration is more than three times that in ‘‘Hayward.’’ Average-sized ‘‘Sanuki gold’’ fruit are 1.7 times larger than that of ‘‘Hayward.’’ Therefore, they contain about five times more vitamin C than ‘‘Hayward.’’ In A. rufa fruit, vitamin C contents are low among the Actinidia fruits. However, they are excellent sources of vitamin C among commercially available fruit. The vitamin C contents in fruit of A. arguta and its interspecific

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hybrids show wide variation within species. Vitamin C contents are extremely high in ‘‘Gassan,’’ ‘‘Issai,’’ ‘‘Mitsuko,’’ and ‘‘Shinzan’’ fruit. In contrast, ‘‘Hirano’’ and ‘‘Kosui’’ fruits contain low levels of vitamin C. Because Actinidia fruits contain high levels of vitamin C, they are attractive materials for nutritional juice production. However, high vitamin C can cause nonenzymic browning in juice products during storage (Wong and Stanton, 1989). D. CALCIUM OXALATE

Kiwifruits of all varieties contain appreciable amounts of oxalate, which causes unpleasant oral irritation when ingested. They contain insoluble calcium oxalate as raphide crystals in idioblast cells. The fine, double-pointed needlelike crystals are the main cause of mechanical irritation of mucous membranes in the mouth (Perera et al., 1990). The raphide crystals are usually embedded in mucilage within the idioblast cells in intact tissue, but they are released after the cells are broken oV from tissue damage by chewing. When the kiwifruit flesh is processed into puree or nectars, more idioblast cells are broken and needlelike crystals are dispersed throughout the product, engendering more intense irritation than that by nonprocessed flesh. Dried or lyophilized fruit also cause unpleasant irritation because the mucilage in the idioblast cells shrinks during drying and some of the sharp crystals protrude from the dried mucilage matrix (Perera et al., 1990). Microscopic observation revealed that most raphide-containing idioblast cells are located in the locular region adjacent to the seed in A. deliciosa (Perera et al., 1990), A. chinensis, A. rufa, and A. arguta fruits (Watanabe and Takahashi, 1998). Concurrent with those observations, quantitative analyses by Rassam and Laing (2005) showed that the inner pericarp contains the highest concentrations of oxalate, and the core and outer pericarp contain much lower levels among the edible parts of A. chinensis fruit. Oxalate is also observed at high concentrations in kiwifruit skin. The presence of high concentrations of oxalate in locules and skin support the idea that the component serves as a defensive factor to protect seeds and the fruit itself (Rassam and Laing, 2005; Rassam et al., 2007). The extent of the irritation caused by kiwifruit might be aVected by several factors, including oxalate concentration, the shape of the raphides, the presence of cysteine protease ‘‘actinidin’’ (Boyes et al., 1997), and the fruit acidity. Considerable variation exists among species in oxalate content and in the raphide shape. Total oxalate concentrations in fruits are estimated to be 37–65 mg/100 g FW in A. deliciosa (Perera et al., 1990), 18–45 mg/100 g FW in A. chinensis (Rassam and Laing, 2005), and 5.0–8.5 mg/100 g FW in A. arguta (Watanabe and Takahashi, 1998). The length of raphides in A. deliciosa fruit is

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0.4–0.5 mm, and 0.2–0.3 mm in A. chinensis, A. arguta, and A. rufa (Watanabe and Takahashi, 1998). However, the diVerence in the irritant sensation caused by the fruits of these species has not been quantified. Synergic eVects of actinidin and acidity on calcium oxalate-induced irritation remain to be evaluated. Aside from the unpleasant sensation, oxalic acid might cause adverse nutritional and pathological eVects. Ingested oxalic acid binds calcium, and reduces its absorption in the intestine, resulting in a reduced bioavailability of calcium (Noonan and Savage, 1999). It is also considered that ingestion of a high oxalic acid diet can be a risk factor for kidney stone formation. However, oxalate concentrations in the Actinidia fruits described above correspond to only 1–10% of that in spinach on a weight-for-weight basis. Therefore, antinutritional properties of oxalate in Actinidia fruits seem to be negligible. Nevertheless, development of new Actinidia cultivars with lower oxalate content is expected, both for sensory and in nutritional aspects. E. PIGMENTS

Actinidia fruits contain several types of pigment, including chlorophylls and carotenoids. The pigments in the fruits were initially studied using spectrophotometric methods or thin-layer chromatography (Fuke et al., 1985; Possingham et al., 1980). Recently, however, analyses of the pigments are performed exclusively by HPLC using a reversed-phase column. Thus far, chlorophylls and their degradative products, lutein, b-carotene, and some other carotenoids in the fruits, have been determined in several Actinidia species (Cano, 1991; Fuke et al., 1985; McGhie and Ainge, 2002; Montefiori et al., 2005; Nishiyama et al., 2005). The pigment profiles in fruit of A. deliciosa closely resemble those in green leaves, but at much lower pigment concentration. Anthocyanins have also been determined in fruits of some Actinidia genotypes (Montefiori et al., 2005; Seager, 1997). 1. Chlorophyll Many fruits including tomato, orange, citrus, banana, and paprika are known to change color from green to yellow, orange, or red during ripening. This phenomenon is usually attributed to chlorophyll degradation and carotenoid synthesis, which are induced by ethylene (Schoefs, 2005). Therefore, chlorophyll is generally scarce in ripe fruit. Instead, yellow or red colors predominate due to the presence of carotenoids and anthocyanins. In contrast, chlorophylls in A. deliciosa fruit must be retained during ripening, giving them a rare deep green flesh even when ripe. Moreover, the low concentrations of chlorophyll pigments in their pericarp persist for several months during storage of the fruit. Wide variation exists in chlorophyll contents between and within species (Nishiyama et al., 2005, 2007). The chlorophyll content in A. deliciosa fruit

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TABLE III CONCENTRATION OF CHLOROPHYLLS IN FRUIT OF ACTINIDIA GENOTYPES

Concentration (mg/100 g fresh weight)a Species genotype

Chlorophyll a

A. deliciosa Hayward 1.12  0.20 Bruno 1.02  0.18 Abbott 0.92  0.15 Elmwood 1.28  0.15 Koryoku 1.84  0.57* A. deliciosa
A. chinensis Sanryoku 1.59  0.19 A. chinensis Jiangxi 79-1 Tracesb Golden king 0.10  0.03** Kuimi 0.20  0.10** Sanuki gold 0.07  0.02** Kobayashi39 0.26  0.09** Hongyang 0.53  0.17* Hort16A 0.07  0.05** A. rufa Awaji 2.83  0.20** Nagano 2.41  0.18** A. arguta Hirano 2.55  0.89** Gassan 2.41  0.65** Issai 2.32  0.78** Mitsuko 3.00  0.90** Ananasnaya 2.68  0.67** A. arguta
A. deliciosa Kosui 1.99  0.22** Shinzan 3.15  0.55**

Chlorophylls aþb

Chlorophyll b

Chlorophylls a þ b

Ratio to Hayward

0.53 0.44 0.41 0.59 0.90

1.65 1.46 1.33 1.87 2.74

1.00 0.88 0.81 1.13 1.66

 0.11  0.07  0.10  0.08  0.23**

    

0.31 0.25 0.24 0.22 0.80*

0.74  0.06

2.33  0.25

1.41

Traces Traces 0.07  0.04** Traces 0.08  0.05** 0.20  0.07** Traces

– – 0.27  0.14** – 0.34  0.14** 0.73  0.24* –

– – 0.16 – 0.21 0.44 –

1.37  0.08** 1.18  0.09**

4.20  0.28** 3.59  0.28**

2.55 2.18

1.07 0.98 0.99 1.21 1.20

3.62 3.39 3.31 4.21 3.88

1.17** 0.83** 1.06** 1.19** 0.80**

2.19 2.05 2.01 2.55 2.35

2.91  0.40** 4.39  0.79**

1.76 2.66

 0.28**  0.19**  0.29**  0.29**  0.16**

0.92  0.19** 1.24  0.23**

    

Values are means  SD of eight experiments. Concentration below the limit of detection (<0.05). * , **: Significantly diVerent vs ‘‘Hayward’’ at p < 0.05 and p < 0.01, respectively. Reprinted with permission from Nishiyama et al. (2005, Table III). Copyright (2005) American Chemical Society. a b

ranges from 1.33 to 2.74 mg/100 g FW (Table III). The concentrations of chlorophyll in A. deliciosa fruit are about 2% or less than those of spinach, which contains 80–110 mg/100 g FW chlorophyll (Nishiyama, unpublished data). Of the A. deliciosa cultivars, ‘‘Koryoku’’ is the only one that showed a

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significantly higher chlorophyll concentration than ‘‘Hayward.’’ The high chlorophyll concentration in ‘‘Koryoku’’ fruit is considered to be the main reason for its deep green flesh color. In contrast to A. deliciosa, fruit of A. chinensis are usually, although not always, yellow-fleshed. As implied by their flesh color, the chlorophyll content in A. chinensis fruit is much lower than those in A. deliciosa fruit (Table III): chlorophyll concentrations in those fruits range from less than 0.05 to 0.73 mg/100 g FW. The low chlorophyll concentration in A. chinensis fruit is considered to be associated with the transition of chloroplast to chromoplast during fruit ripening (McGhie and Ainge, 2002). Fluorescence microscopic observation indicates that the remaining chloroplasts in A. chinensis fruit are localized mainly in the outermost region of the outer pericarp (Nishiyama et al., unpublished data). The chlorophyll concentrations in A. rufa, A. arguta, and their interspecific hybrid fruits are much higher than that in ‘‘Hayward.’’ The chlorophyll contents of the fruits are 1.76–2.66 times that of ‘‘Hayward’’ on a weight-for-weight basis (Table III). It is well established that chlorophyll molecules are converted easily into olive-brown pheophytins in acidic conditions. Degradation of the pigments in the acidic environment is markedly accelerated by heating (von Elbe and Schwartz, 1996). Kiwifruits contain high levels of organic acids. Thus, the chlorophyll in the fruits is readily degraded by intrinsic acidity when the fruits are macerated. Consequently, kiwifruit puree spontaneously loses its green color. Processing of kiwifruit through thermal treatment thoroughly degrades chlorophyll (Cano and Marı´n, 1992). Freezing and thawing processes also induce serious degradation of chlorophyll molecules in the kiwifruits (Cano and Marı´n, 1992; Venning et al., 1989). Therefore, kiwifruit lose their green color, which is the most prominent feature of the fruit, when they are canned or processed into juice, jam, or sauce (Robertson and Swinburne, 1981). Although freeze-drying is a good option to stabilize the green color of the fruit, the application of the method is limited. The degradation of chlorophyll during kiwifruit processing is a serious issue to be solved. 2. Carotenoids Actinidia fruits contain several types of carotenoids, including violaxanthin, neoxanthin, lutein, and b-carotene, which are identical components to those generally found in green leaves (Cano, 1991; McGhie and Ainge, 2002; Montefiori et al., 2005; Nishiyama et al., 2005, 2007). These carotenoids are important both as colorants and as health-promoting constituents. After ingestion, b-carotene is converted in vivo into vitamin A, which is necessary for normal retinal function. Carotenoids have potent antioxidant activity

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(Young et al., 2004), and a higher dietary intake of carotenoids is associated with a lower risk of cardiovascular disease, cataract, age-related macular degeneration, and certain types of cancer (Block et al., 1992; Giovannucci, 1999; Granado et al., 2003; Kritchevsky, 1999; Rock, 2004; Seddon et al., 1994; Sesso and Gaziano, 2004; van Poppel, 1996). A considerable variation exists in carotenoid contents among species. The concentrations of lutein and b-carotene in ‘‘Hayward’’ fruit are 0.418 and 0.088 mg/100 g FW, respectively. Among A. deliciosa fruit, the fruits of ‘‘Koryoku’’ and ‘‘Sanryoku’’ have markedly higher lutein concentrations than ‘‘Hayward’’ (Table IV). Lutein concentrations of commonly consumed fruits are estimated to be less than 0.15 mg/100 g FW (Hart and Scott, 1995; Tee and Lim, 1991). Therefore, A. deliciosa fruits are the richest dietary source of lutein among commonly consumed fruits. The high lutein contents of A. deliciosa fruit are probably attributable to the remaining chloroplasts in the fruit. This idea is strongly supported by the fact that a significant positive correlation exists between the concentration of lutein or b-carotene and that of total chlorophyll (Nishiyama et al., 2005). In most A. chinensis genotypes, the lutein contents are much lower than that in ‘‘Hayward’’ and other green-fleshed cultivars, whereas the b-carotene contents are almost of the same or of a slightly higher level than that in ‘‘Hayward’’ (Table IV). These data suggest that the yellow flesh color of A. chinensis is mainly attributable to the absence of chlorophyll from the fruit instead of an abundance of these carotenoids. As implied by the higher chlorophyll content, both lutein and b-carotene contents in A. rufa, A. arguta, and their interspecific hybrids are much higher than those in ‘‘Hayward.’’ The respective concentrations of lutein and b-carotene in these fruits are 0.736–1.082 mg/100 g FW and 0.143–0.285 mg/100 g FW (Table IV). The data indicate that these fruits are outstanding dietary sources of lutein among fruits. Particularly, A. arguta fruit are an extremely convenient lutein source because they have a soft and edible skin, which has a higher lutein concentration than the pulp. Therefore, whole A. arguta fruit contain about 1.4 times more lutein than those without skin on a weight-for-weight basis (Nishiyama et al., 2007). Additional information about the contents of other carotenoids including violaxanthin, neoxanthin, zeaxanthin, and b-cryptoxanthin in Actinidia fruits are available elsewhere (Cano, 1991; McGhie and Ainge, 2002; Montefiori et al., 2005). Carotenoids are chemically more stable than chlorophyll. Freezing and thawing of the food causes little change in the carotenoid content. Thermal processing has minimal eVect on the carotenoid content (Boileau and Erdman, 2004; Updike and Schwartz, 2003). However, thermal treatments of food induce isomerization of carotenoids from trans to cis isomers, thereby decreas-

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TABLE IV CONCENTRATION OF LUTEIN AND b-CAROTENE IN FRUIT OF ACTINIDIA GENOTYPES

Lutein Species genotype

(mg/100 g fresh weight)a

A. deliciosa Hayward 0.418 Bruno 0.434 Abbott 0.398 Elmwood 0.569 Koryoku 0.897 A. deliciosa
A. chinensis Sanryoku 0.691 A. chinensis Jiangxi 79-1 0.107 Golden king 0.087 Kuimi 0.152 Sanuki gold 0.117 Kobayashi39 0.144 Hongyang 0.404 Hort16A 0.155 A. rufa Awaji 0.926 Nagano 0.876 A. arguta Hirano 0.786 Gassan 0.746 Issai 0.799 Mitsuko 0.933 Ananasnaya 0.762 A. arguta
A. deliciosa Kosui 0.736 Shinzan 1.082

b-Carotene Ratio to Hayward

(mg/100 g fresh weight)a

Ratio to Hayward

 0.082  0.063  0.101  0.104  0.138**

1.00 1.04 0.95 1.36 2.15

0.088 0.094 0.085 0.093 0.150

1.00 1.07 0.97 1.06 1.70

 0.056**

1.65

0.110  0.015

1.25

 0.026**  0.015**  0.032**  0.012**  0.025**  0.072  0.030**

0.26 0.21 0.36 0.28 0.34 0.97 0.37

0.115  0.121  0.097  0.150  0.081  0.123  0.066 

0.015 0.017 0.019 0.037** 0.007 0.016 0.008

1.31 1.38 1.10 1.70 0.92 1.39 0.75

 0.050**  0.061**

2.22 2.10

0.177  0.012** 0.145  0.017**

2.01 1.65

 0.209**  0.140**  0.178**  0.214**  0.132**

1.88 1.78 1.91 2.23 1.82

0.224  0.227  0.247  0.245  0.285 

0.049** 0.048** 0.019** 0.033** 0.041**

2.54 2.58 2.80 2.78 3.23

 0.074**  0.172**

1.76 2.59

0.143  0.007** 0.269  0.022**

1.62 3.05

    

0.013 0.014 0.009 0.017 0.036**

Values are means  SD of eight experiments. **: Significantly diVerent vs ‘‘Hayward’’ at p < 0.01. Reprinted with permission from Nishiyama et al. (2005, Table IV). Copyright (2005) American Chemical Society. a

ing provitamin A activity (von Elbe and Schwartz, 1996). Regardless of isomerization, processed products such as jams and juices made from Actinidia fruits are promising dietary sources of these carotenoids.

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3. Anthocyanins Most people recognize that the kiwifruit flesh color is green or yellow, thus the presence of red pigments in kiwifruits is surprising. Although red pigments occur in the inner pericarp of fruits of several genotypes in A. deliciosa and A. chinensis (Montefiori et al., 2005), ‘‘Hongyang’’ (Wang et al., 2003) is the only commercially grown cultivar. Recently, a novel red-fleshed cultivar ‘‘Chuhong’’ was released from China (Zhong et al., 2007). Development of new cultivars of red-fleshed A. chinensis and A. deliciosa has now progressed mainly in China and New Zealand. The red pigment is water-soluble, and turns pale blue under alkaline conditions. Consequently, the red pigment in the Actinidia fruits has been believed to be anthocyanins. Liquid chromatography-mass spectrometry analyses revealed that the major anthocyanins are cyanidin 3-O-xylo-(1-2)galactoside in A. chinensis fruit and cyanidin 3-O-galactoside and cyanidin 3-O-glucoside in A. deliciosa fruit (Montefiori et al., 2005). Anthocyanin compounds have been reported to exhibit antioxidative (Pool-Zobel et al., 1999; Tsuda et al., 1996), antimutagenic (Yoshimoto et al., 1999, 2001), antidiabetic (Jayaprakasam et al., 2005; Matsui et al., 2002), and anticarcinogenic (Hou et al., 2004; Kamei et al., 1995) activities. Although anthocyanins in red-fleshed kiwifruit oVer potential health benefits, those benefits should not be overestimated because anthocyanin concentrations in kiwifruit are only 10% or less than those in whole blueberry fruits (Montefiori et al., 2005). Instead, the principal benefit of these pigments in red-fleshed kiwifruit is aesthetic, to foster commercial appeal. Attractiveness of ‘‘Hongyang’’ fruit was actually evidenced by an experimental market method (Jaeger and Harker, 2005), which showed that consumers were willing to pay a considerable price premium for the fruit. F. ACTINIDIN

It is a well-established household fact that raw kiwifruit prevent setting of gelatin-based food such as table jelly and bavarois. This phenomenon is attributable to large amounts of protease in the fruit. The protease was first characterized by Arcus (1959) and named ‘‘Actinidin’’ after the generic name ‘‘Actinidia.’’ Later, biochemical and enzymological properties of actinidin (EC 3.4.22.14) were studied extensively by several investigators. Actinidin is a cysteine protease that requires a free sulfhydryl group for activity (Arcus, 1959; McDowall, 1970). This protease is composed of 220 amino acid residues with molecular mass of 23,500 (Carne and Moore, 1978). The amino acid sequence of the enzyme shows considerable homology with papain, a well-known cysteine protease in immature papaya fruit (Carne and

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Moore, 1978). The conformation of the polypeptide chain is also remarkably similar to that of papain (Baker, 1977). Actinidin is thus classified in a member of the papain superfamily, which includes papain and bromelain, and the mammalian cathepsins B, K, and L. The optimal pH of actinidin is about 4 when using food proteins such as gelatin (Arcus, 1959) or myofibrillar proteins (Nishiyama, 2001) as the substrate. The optimal pH is around 6 when the enzyme activity is measured with synthetic peptides as the model substrates (Boland and Hardman, 1972; Boyes et al., 1997; McDowall, 1970; Sugiyama et al., 1997). Regardless of this accumulated knowledge, nothing is known as yet about the physiological function of actinidin in fruits. In the biochemical field, ‘‘actinidin’’ is oYcially designated as ‘‘actinidain’’ because it is recommended by the International Union of Biochemistry and Molecular Biology (IUBMB) Enzyme Nomenclature List. However, the name ‘‘actinidin’’ is used widely all over the world in other fields including food science, horticulture, and medicine. It might become a cause of some confusion. 1. Spatial distribution in the fruit Distribution of actinidin within kiwifruits has been reported in some Actinidia fruits (Boyes et al., 1997; Lewis and Luh, 1988a; Pre´stamo, 1995). The actinidin activity is observed mainly in outer and inner pericarp regions; very little activity is detected in the skin and the core. Longitudinal distribution of the enzyme has not been reported. 2. Changes during fruit growth and ripening Concentrations of actinidin in kiwifruits were reported to increase rapidly during their growth. Therefore, immature thinned fruit contain only a small amount of actinidin, and are not utilizable as raw materials for producing actinidin products. The protease activity in the fruits doubles or triples during postharvest ripening (Lewis and Luh, 1988a). This phenomenon contrasts against the fact that papain in immature papaya fruit decreases to trace amounts during their maturation. 3. EVects on food proteins Kiwifruit generally contain a large amount of actinidin. For that reason, they cannot be used satisfactorily as a food ingredient for protein-based foods. Particularly, gelatin is susceptible to actinidin (Arcus, 1959). Therefore, thermal treatment of kiwifruit flesh or juice is required to inactivate the protease prior to addition to gelatin jelly or bavarois. Heat treatment of

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kiwifruit, however, inevitably degrades its fresh flavor, bright green color, and nutrients such as vitamin C. To solve that problem, Funaki et al. (1996) adopted oryzacystatin, an edible inhibitor for cysteine protease occurring in rice seed, for preventing hydrolysis of gelatin by actinidin. This method is of potential usefulness, but it has not become widespread. Perhaps it is simpler and easier to replace gelatin with some polysaccharide gelling agent such as agar or carageenan. The proteolytic enzymes of plant origin, including papain, bromelain, and ginger proteases, have proteolytic eVects on myofibrillar proteins and/or collagen. Therefore, these enzymes are used for meat tenderization (Lawrie, 1998). Among these enzymes, papain is the most widely used as a meat tenderizer. However, the enzyme treatment of meat tends to overtenderize the surface, producing a mushy texture, while leaving the interior unaVected (Lawrie, 1998). Actinidin also possesses proteolytic activity on muscular proteins. It is useful as an eVective meat tenderizer (Lewis and Luh, 1988b). Although the commercial utilization of actinidin as a meat tenderizer remains quite limited, it oVers potential advantages. According to Nishiyama (2001), actinidin shows unique pH-dependence on hydrolysis of myofibrillar proteins (Figure 2). For pH of 3–4.5, actinidin thoroughly hydrolyzes all myofibrillar proteins, including myosin heavy chain and actin, in a nonspecific manner. In contrast, for pH of 5.5–8, actinidin hydrolyzes myosin heavy chain into fragments, whereas it showed little or no proteolytic eVect on actin (Nishiyama, 2001). In contrast,

FIG. 2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of pork and chicken myofibrillar proteins treated with purified actinidin at various pH values. MHC, myosin heavy chain; A, actin. Reprinted with permission from Nishiyama (2001, Figs. 2 and 5A).

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papain or bromelain nonselectively hydrolyzes all myofibrillary proteins in a wide pH range (Nishiyama, 2001). These results suggest that overtenderization of the meat surface can be controlled, to some extent, by regulating the pH condition in which the meat is treated with actinidin. Further experiments are necessary to elucidate the advantages of actinidin as a meat tenderizer. Although actinidin has no collagenase activity, it cleaves the aterocollagen molecule of yellowfin tuna at specific sites inside the interstrand crosslinking peptide (Morimoto et al., 2004). Sugiyama et al. (2005) also reported that kiwifruit juice degrades nonhelical domains of beef collagen. These eVects of actinidin on collagen probably aid meat tenderization. EVects of kiwifruit juice on the organization of collagen fiber in muscular tissue was also confirmed by immunohistochemical studies using anticollagen antiserum (Nishiyama, 2000). Actinidin also hydrolyzes a-casein and b-casein in milk (Nishiyama and Oota, 2002; Yamaguchi et al., 1982). Bachmann and Farah (1982) demonstrated the occurrence of a bitter taste in mixtures of milk proteins and raw kiwifruit, which was attributed to a caseinolytic protease in kiwifruit splitting casein into bitter peptides. 4. Utilization for enzyme supplement Actinidin vigorously hydrolyzes food proteins including myofibrillar proteins, gelatin, and milk proteins. Therefore, kiwifruit are expected to have a digestion-promoting eVect. Actually, in New Zealand, currently marketed kiwifruit-derived dietary supplements such as ‘‘Zylax’’ and ‘‘Kiwi Crush’’ were developed as digestive enhancers. At pH 2 or below, however, actinidin exhibits low proteolytic activity and is rapidly inactivated (Nishiyama, 2001). Therefore, the digestion-promoting eVect of kiwifruit must be highly dependent on the gastric pH. 5. DiVerences among cultivars Varietal diVerences in actinidin contents were first reported by Pre´stamo (1995), and slight diVerences were observed among four Actinidia cultivars. Although Boyes et al. (1997) observed considerable variation in actinidin levels among Actinidia genotypes, most plant materials were not commercially grown cultivars but conserved strains. The first striking discovery about a commercially grown cultivar was that ‘‘Hort16A’’ fruit contain only a trace amount of actinidin (Nishiyama, 2000). Subsequently, wide variation in actinidin levels was found to exist among Actinidia genotypes (Nishiyama and Oota, 2002).

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TABLE V ACTINIDIN CONCENTRATION AND PROTEASE ACTIVITY IN FRUIT JUICE FROM ACTINIDIA GENOTYPES

Species genotype A. deliciosa Hayward Bruno Abbott Elmwood Koryoku A. deliciosa
A. chinensis Sanryoku A. chinensis Jiangxi 79-1 Golden king Kuimi Sanuki gold Kobayashi39 Hongyang Hort16A A. rufa Awaji Nagano A. arguta Hirano Gassan Issai Mitsuko Ananasnaya A. arguta
A. deliciosa Kosui Shinzan

Actinidin concentration (mg/ml)

Protease activity (nmol pNA released/min)

2.91  0.18 3.13  0.12 3.89  0.14** 3.05  0.14 4.35  0.11**

6.34  5.59  10.2  5.75  11.9 

3.16  0.24

7.66  0.29

2.80 2.55 5.74 6.10 2.60 ND ND

5.47  0.17 5.92  0.21 13.3  1.56* 15.1  0.77** 6.73  0.92 0.44  0.02** 0.42  0.02**

 0.13  0.06  0.28**  0.53**  0.20

0.78 0.23 0.99* 0.18 0.53**

ND ND

0.84  0.15** 0.82  0.12**

10.7  1.36** 10.6  1.61** 1.64  0.55** 9.27  2.55** 5.35  1.02**

166  7.38** 115  25.4** 25.7  7.63** 114  29.6** 113  1.8**

ND 4.95  0.18**

0.38  0.04** 208  4.38**

*p < 0.01; **p < 0.001 vs the corresponding value of Hayward; ND, not detected. Each value represents mean  SE of at least six experiments. Reprinted with modification from Nishiyama and Oota (2002, Table II), Nishiyama et al. (2004a, Table II), and Yamanaka et al. (2004, Table II) with permission.

In A. deliciosa, A. chinensis, and their interspecific hybrid, actinidin concentrations in the fruit juice range from trace amounts to 6.1 mg/ml (Table V and Figure 3). ‘‘Abbott,’’ ‘‘Koryoku,’’ ‘‘Kuimi,’’ and ‘‘Sanuki gold’’ exhibit considerably higher actinidin concentration compared to ‘‘Hayward.’’ In contrast, ‘‘Hongyang’’ and ‘‘Hort16A’’ fruit contain very little actinidin. In A. rufa fruit, the actinidin content is below the detection limit. In A. arguta

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315

FIG. 3 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of actinidin in fruit juice of Actinidia genotypes. The arrow indicates actinidin.

and its interspecific hybrids, actinidin concentrations in fruit juice are markedly higher in ‘‘Hirano,’’ ‘‘Gassan,’’ ‘‘Mitsuko,’’ ‘‘Ananasnaya,’’ and ‘‘Shinzan’’ than that in ‘‘Hayward.’’ In contrast, ‘‘Kosui’’ fruit contain only trace amounts of actinidin. However, the low content of actinidin in ‘‘Kosui’’ fruit might be an inherited trait from some A. rufa genotype because a study using RAPD analysis suggested that A. rufa, not A. arguta, is involved in the parentage of this cultivar (Kokudo et al., 2003). Because the amount of actinidin in the fruits might aVect the taste (Boyes et al., 1997), digestion-promoting eVect, allergenic properties (Pastorello et al., 1998), and characteristics for processing the fruit, it is probably an important index of fruit quality. Fruits of cultivars with higher actinidin content are expected to have higher digestion-promoting eVects. In addition, they are promising as raw materials for industrial manufacture of actinidin products such as a meat tenderizer and a digestive enhancer. On the other hand, fruits of cultivars with trace amounts of actinidin are suitable as ingredients of protein-based foods. Although the eVects of actinidin on the taste of kiwifruit have not been well established, it is speculated that the enzyme causes a tingling sensation in the lips, mouth, tongue, and throat. Therefore, the higher actinidin content might, to some extent, adversely aVect the fruit taste. However, fruits with high actinidin levels, such as those of ‘‘Sanuki gold’’ and A. arguta, apparently do not cause a more irritating sensation than ‘‘Hayward’’ fruit. Further experiments are necessary to clarify the relationship between actinidin concentration and taste of the fruit.

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Whereas actinidin in ‘‘Hayward’’ fruit has been studied intensively, the analogous protease in A. arguta fruit has rarely been studied biochemically. The protein with an apparent molecular mass of 24,500 in A. arguta fruit (Figure 3) is detectable using immunoblot analysis using antiactinidin antiserum, which does not bind to the analogous proteases such as papain and bromelain (Nishiyama and Oota, 2002). Therefore, it is tentatively regarded as actinidin at the present time. However, a distinct diVerence exists in enzymological properties of these two proteases. The protease activity in the juice of A. arguta and its interspecific hybrid fruits is 18–33 times that of ‘‘Hayward,’’ even though actinidin concentration is merely 1.7–3.7 times greater (Nishiyama et al., 2004a; Table V). Moreover, a purified protease in ‘‘Shinzan’’ fruit exhibits decidedly diVerent specificities toward synthetic peptide substrates compared to actinidin purified from ‘‘Hayward’’ fruit (Nishiyama et al., 2004a; Table VI). Actinidin reportedly consists of two (McDowall, 1973) or six (Sugiyama et al., 1996) closely related components. Therefore, both the ‘‘purified’’ actinidin from ‘‘Hayward’’ and that from ‘‘Shinzan’’ might consist of several isozymes. If it was the case, the diVerence in the substrate specificity between actinidin and the protease from ‘‘Shinzan’’ might be explained using the diVerence in the proportion of each isozyme. Otherwise, the protease in A. arguta fruit must be regarded as a distinct protease that is related closely to actinidin; the enzyme should perhaps be given a diVerent name such as ‘‘argutain.’’ G. OTHER COMPONENTS

Among fresh fruits, ‘‘Hayward’’ fruit are considered to have large amounts of vitamin E (1–2 mg/100 g FW). Although vitamin E possesses health benefits such as antioxidant activity, kiwifruit do not seem to be a good TABLE VI COMPARISON OF SUBSTRATE SPECIFICITY BETWEEN ACTINIDIN AND A PROTEASE PURIFIED FROM ‘‘SHINZAN’’

Specific activity of protease (nmol pNA released/min/mg protein) Substrate

Actinidin

Protease from Shinzan

Bz-Arg pNA Pyr-Phe-Leu pNA Bz-Phe-Val-Arg pNA

1.22 35.5 1460

14.8 367 420

Reprinted with modification from Nishiyama et al. (2004a, Table III) with permission.

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dietary source of the vitamin because it is probably localized in the seeds (Ferguson and Ferguson, 2003). Kiwifruit are a rich source of dietary fiber among fresh fruits and vegetables. ‘‘Hayward’’ fruit contain about 2–3 g/100 g FW dietary fiber, which is probably responsible for the mild laxative properties of the fruit (Rush et al., 2002). Kiwifruit are also a rich dietary source of potassium. Dietary potassium has been associated with prevention of hypertension, apoplexy, and osteoporosis. An average-sized ‘‘Hayward’’ fruit contains 200–300 mg potassium, which supplies about 10–15% of the daily requirement. Varietal diVerences in the contents of these health-associated components have thus far not been fully investigated. IV. ALLERGENIC PROPERTIES Acute allergy to kiwifruit was first described by Fine (1981), and kiwifruit have now become a major elicitor of plant food allergies (Lucas et al., 2003; Mills et al., 2004). It is also reported that kiwifruit allergies are often crossreactive with other allergies such as those to pollens, rye, hazelnut, chestnut, banana, and avocado (Lucas et al., 2003). Pastorello et al. (1998) revealed that the major allergen of A. deliciosa ‘‘Hayward’’ is actinidin, which is designated Act c 1 according to the allergen nomenclature. This finding engendered the speculation that fruit of ‘‘Hort16A,’’ which contain extremely low levels of actinidin, are much less allergenic than those of ‘‘Hayward.’’ However, it is not as simple as that. A study (Bublin et al., 2004) revealed that ‘‘Hort16A’’ fruit contain chitinase-related protein as a novel allergen in addition to allergens that are commonly found in ‘‘Hayward’’ fruit: phytocystatin and thaumatin-like protein (Act c 2). It has also been demonstrated that patients who are allergic to ‘‘Hayward’’ fruit face a high risk of allergy to ‘‘Hort16A’’ (Lucas et al., 2005). A recent in vitro study has suggested that some kiwifruit-allergic individuals might suVer allergic cross-reactions if they consume raw A. arguta fruit (Chen et al., 2006). These results give rise to the speculation that ‘‘Hongyang’’ and ‘‘Kosui’’ fruits might not be free from allergenic risk even though they contain very low concentrations of actinidin. Allergenic properties of each Actinidia cultivar should be examined carefully in future studies. Although Actinidia fruits are consumed principally as a fresh fruit, they are also used as a component in some processed foods including beverages and jam. Fiocchi et al. (2004) demonstrated that heat-treatment and homogenization of ‘‘Hayward’’ fruit extremely, but not completely, reduces their allergenicity. Reduced allergenicity of A. arguta pulp by heat-treatment has

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also been suggested in results of an in vitro IgE binding study (Chen et al., 2006). In contrast, lyophilization does not seem to be able to reduce kiwifruit allergenicity (Mempel et al., 2003). V. HEALTH BENEFITS Kiwifruit contain several health-beneficial constituents. Among the components, antioxidants such as vitamin C, vitamin E, carotenoids, and polyphenolics are considered to be the main factors responsible for health-promoting properties of kiwifruit. Although antioxidant activities of these components have been well established through in vitro studies, direct eVects of kiwifruit consumption on oxidative stress in human cells has been scarcely studied. Collins et al. (2001, 2003) have shown that kiwifruit supplementation provides dual protection against oxidative DNA damage, enhancing antioxidant levels and stimulating cellular DNA repair. A similar eVect of kiwifruit consumption on cellular protection against DNA damage has been shown by Rush et al. (2006). These results suggest that consumption of kiwifruit protects against a kind of DNA damage that has been shown to cause mutations through miscoding (Shibutani and Grollman, 1994) and that therefore might be responsible for initiating carcinogenesis (Collins et al., 2003). Kiwifruit might also provide protective eVects against cardiovascular diseases. According to Duttaroy and Jørgensen (2004), consuming two or three kiwifruits per day for 28 days significantly reduces platelet aggregation response and plasma triacylglycerol levels in human volunteers. The antiplatelet potential of the kiwifruit is probably unrelated to its antioxidant activity, and the antiplatelet factor(s) in the kiwifruit remain to be elucidated. Kiwifruit apparently have laxative eVects and relieve constipation. However, reliable scientific data on the laxative eVect of kiwifruit remain limited. Rush et al. (2002) showed that, for elderly persons, ingestion of an adequate amount of kiwifruit enhances various parameters of laxation, including frequency and ease of defecation, stool bulk and softness. This laxative eVect of kiwifruit is probably caused mainly by dietary fiber in the fruit (Rush et al., 2002). VI. PERSPECTIVES As a consequence of active development of new Actinidia cultivars in several countries, some novel fruits are anticipated soon in international markets.

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These fruits, in combination with the preexisting varieties, show a wide diversity of sensory and nutritional attributes such as taste, shape, hairiness, flesh color, vitamin C level, and carotenoid content. This will provide greater choice for the consumers. They will also vary in storageability and transportability, which are of particular concern to producers and retailers. In addition, we have to pay attention to the diVerences in allergenic properties of these fruits. The extensive genetic diversity within the genus Actinidia ensures cultivar development by conventional selection and breeding methods without using genetic modification technology, which would be likely to engender consumer anxiety. Among the Actinidia fruit, A. arguta seems to be a highly promising crop. They are small and smooth-skinned. Therefore, they are edible whole. They are ultimately an easy-to-eat fruit that promises consumers’ convenience. Moreover, they are a rich dietary source of b-carotene, lutein, and myoinositol in addition to vitamin C. For the expansion of production and marketing of the A. arguta fruit, however, their short storage life will have to be overcome through future development. Their storage life might be improved by treatment with 1-methyl cyclopropene, which retards fruit ripening by blocking ethylene action. Although kiwifruit are marketed mostly as fresh fruit at present, an increased utilization of processed fruits is expected. Among the processed products, kiwifruit juice seems to be the most promising because consumers can conveniently enjoy health-promoting benefits of the fruit. Kiwifruit are diYcult to process, but progress in processing technologies including membrane separation and osmotic distillation will make it possible to manufacture safe and stable beverage products that retain the nutrients and flavor of kiwifruit. Of course other Actinidia fruits such as those of A. arguta can be good raw materials for highly nutritious juice products. ACKNOWLEDGMENTS I am grateful to Dr. Ferguson in HortResearch in New Zealand for providing valuable literatures. REFERENCES Arcus, A.C. 1959. Proteolytic enzyme of Actinidia chinensis. Biochim. Biophys. Acta 33, 242–244. Bachmann, M.R. and Farah, Z. 1982. Occurrence of bitter taste in mixtures of milk proteins and kiwi fruit (Actinidia chinensis). Lebensm. Wiss. Technol. 15, 157–158. Baker, E.N. 1977. Structure of actinidin: Details of the polypeptide chain conformation and active site ˚ resolution. J. Mol. Biol. 115, 263–277. from an electron density map at 2.8 A

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Sugiyama, S., Ohtsuki, K., Sato, K., and Kawabata, M. 1997. Enzymatic properties, substrate specificities and pH-activity profiles of two kiwifruit proteases. J. Nutr. Sci. Vitaminol. 43, 581–589. Sugiyama, S., Hirota, A., Okada, C., Yorita, T., Sato, K., and Ohtsuki, K. 2005. EVect of kiwifruit juice on beef collagen. J. Nutr. Sci. Vitaminol. 51, 27–33. Tee, E.-S. and Lim, C.-L. 1991. Carotenoid composition and content of Malaysian vegetables and fruits by the AOAC and HPLC methods. Food Chem. 41, 309–339. Tsuda, T., Ohshima, K., Kawakishi, S., and Osawa, T. 1996. Oxidation products of cyanidin 3-O-b-Dglucoside with a free radical initiator. Lipids 31, 1259–1263. Updike, A.A. and Schwartz, S.J. 2003. Thermal processing of vegetables increases cis isomers of lutein and zeaxanthin. J. Agric. Food Chem. 51, 6184–6190. Venning, J.A., Burns, D.J.W., Hoskin, K.M., Nguyen, T., and Stec, M.G.H. 1989. Factors influencing the stability of frozen kiwifruit pulp. J. Food Sci. 54, 396–400, 404. van Poppel, G. 1996. Epidemiological evidence for beta-carotene in prevention of cancer and cardiovascular disease. Eur. J. Clin. Nutr. 50(Suppl.), S57–S61. von Elbe, J.H. and Schwartz, S.J. 1996. Colorants. In ‘‘Food Chemistry Third Edition’’ (O.R. Fennema, ed.), pp. 651–696. Marcel Dekker, Inc., New York. Wang, M., Li, M., and Meng, A. 2003. Selection of a new red-fleshed kiwifruit cultivar ‘Hongyang’. Acta Hortic. 610, 115–117. Watanabe, K. and Takahashi, B. 1998. Determination of soluble and insoluble oxalate contents in kiwifruit (Actinidia deliciosa) and related species. J. Jpn. Soc. Hortic. Sci. 67, 299–305. Williams, M.H., Boyd, L.M., McNeilage, M.A., MacRae, E.A., Ferguson, A.R., Beatson, R.A., and Martin, P.J. 2003. Development and commercialization of ‘Baby Kiwi’ (Actinidia arguta Planch.). Acta Hortic. 610, 81–86. Wong, M. and Stanton, D.W. 1989. Nonenzymic browning in kiwifruit juice concentrate systems during storage. J. Food Sci. 54, 669–673. Yamaguchi, T., Yamashita, Y., Takeda, I., and Kiso, H. 1982. Proteolytic enzymes in green asparagus, kiwi fruit and miut: Occurrence and partial characterization. Agric. Biol. Chem. 46, 1983–1986. Yamanaka, M., Oota, T., Fukuda, T., and Nishiyama, I. 2004. Varietal diVerence in actinidin concentration and protease activity in fruit juice of Actinidia species. J. Jpn. Soc. Food Sci. Technol. 51, 491–494. (in Japanese). Yoshimoto, M., Okuno, S., Yoshinaga, M., Yamakawa, O., Yamaguchi, M., and Yamada, J. 1999. Antimutagenicity of sweetpotato (Ipomoea batatas) roots. Biosci. Biotechnol. Biochem. 63, 537–541. Yoshimoto, M., Okuno, S., Yamaguchi, M., and Yamakawa, O. 2001. Antimutagenicity of deacylated anthocyanins in purple-fleshed sweetpotato. Biosci. Biotechnol. Biochem. 65, 1652–1655. Young, A.J., Phillip, D.M., and Lowe, G.M. 2004. Carotenoid antioxidant activity. In ‘‘Carotenoids in Health and Disease’’ (N.I. Krinsky, S.T. Mayne, and H. Sies, eds), pp. 105–126. Marcel Dekker, Inc., New York. Zhong, C.H., Wang, Z.Y., Bu, F.W., and Peng, D.F. 2007. Selection of a new red-fleshed kiwifruit cultivar ‘Chuhong’. Acta Hortic. (in press).

INDEX

A Abees sweet potato, dietary fiber contents of, 13 Acetyl CoA carboxylase (ACC-1), 90 Acid/aspartyl proteases, 279 Actinidia arguta fruits, 294 Actinidia deliciosa, 293 Actinidia eriantha, 299, 302 Actinidia genus fruits allergenic properties, 317 components, 299–316 actinidin, 310–316 calcium oxalate, 304–305 organic acids, 300–301 pigments, 305–310 sugar and sugar alcohol, 299–300 vitamin C, 301–303 health benefits and, 317–318 lutein and b-carotene, 309 perspectives, 318–319 species and cultivars, 295–299 A. arguta, 297–299 A. chinensis, 296–298 A. deliciosa, 295–296, 298 A. eriantha, 299 A. kolomikta, 299 A. rufa, 298–299 Actinidia kolomikta, 297 Actinidin in Actinidia genus fruits, 310–316 actinidin concentration and protease activity, 314 cultivars, differences among, 313–316 enzyme supplement and, 313 food proteins, effects on, 311–313 fruit growth and ripening, changes during, 311

protease from Shinzan and, 316 spatial distribution in fruit, 311 Acute encephalitis, CDV infection and, 63 Ad-2, 91 Ad-5, 65, 68, 90–91 effect of infection on, 66 human obesity, association with, 66 induced obesity, 90–91 mechanism of, 91 mechanism of action, 67, 91 Ad-31, 91 Ad-36, 65, 68, 87–91 animal models, 88–89 effect of infection on, 65 human obesity, association with, 65, 89 induced obesity, 87–90 mechanism of, 89–90 mechanism of action, 67, 89–90 Ad-37, 65, 67, 91 effect of infection on, 65 human obesity, association with, 65 mechanism of action, 67 Adenoviruses, 65, 68, 84–90 SMAM-1, 65, 68, 85–87 type 5, 65, 68, 90–91 type 36, 65, 68, 87–90 type 37, 65, 90 Adenovirus type 5, see Ad-5 Adenovirus type 36, see Ad-36 Adenovirus type 37, see Ad-37 Adipogenic pathogens, see also individual pathogens effects of, 64–66 mechanism of action, 67–68 Aeromonas sp., 176 Albumin, 275 Alexandrium tamarense, 173–174

326

INDEX

Algae cultivation, 249–250 fucans/fucanoids and other polysaccharides from, 271–272 hydrocolloids, polysaccharides from, 269–271 polysaccharides from, 240–241, 269–274 transgenics, 250–252 diatoms, 251 dinoflagellates, 251–252 green algae, 252–253 macroalgae, 253 Alicyclobacillus acidoterrestris, 128 Allergenic properties, of Actinidia genus fruits, 317 Alteromonas, 272 Alteromonas tetraodonis, 174, 181 Ambystoma tigrinum, 159 Amphidinium, 252 Amphidinium carteri, 262 Amukeke chips, 37–38 a-Amylase influence, SPS syrup and, 26–27 Annelids tetrodotoxin in, 172–173 toxicity of, 173 Anthocyanins, 2–3 in Actinidia genus fruits, 308–310 health benefits associated with, 16 refrigerated fruit juices, contents of, 119–120 in sweet potato, 14–16 Antioxidant activity, in refrigerated fruit juices, 120–122 Aphanizomenon, 247 Aphanizomenon flos-aquae, 261 Apple juice, see also Refrigerated fruit juices consumption of, 104 Aromatic profile, of refrigerated fruit juices, 113–114 Arothron firmamentum, 158 Arothron hispidus, 158 Arothron manilensis, 158 Arothron mappa, 158 Arothron nigropunctatus, 158 Arothron reticularis, 158 Arothron sp., 152 Arothron stellatus, 158 Arrowworms, tetrodotoxin in, 173 Arthrospira, 247 Aspergillus fischeri, 131

Astaxanthin, in marine-derived food ingredients, 239, 258–259 Astropecten latespinosus, 170, 178 Astropecten polyacanthus, 169, 170, 174, 178, 181 Astropecten scoparius, 170, 178 Astropecten vappa, 170 Atelopus chiriquiensis, 161 Atelopus oxyrhynchus, 161, 176 Atelopus varius ambulatorius, 161 Atelopus varius varius, 160 Atelopus zeteki, 162 Atergatis floridus, 163–166, 174, 181 Atergatopsis germaini, 163–165, 167 Autoimmune thyroiditis, RAV-7 and, 76–77 Avian leukosis viruses, 73, 75 Avian retroviruses viruses, 73 Ayamurasaki sweet potato, antioxidative and radical scavenging activities, 15 B Baby kiwi fruit, see Actinidia genus fruits Babylonia formosae, 169 Babylonia japonica, 168 Bacillus sp., 174, 281, 283 Bacteria, tetrodotoxin in, 174–176 Bacteroides thetaiotaomicron, 83–84 Batata, see Sweet potato BDV, see Borna disease virus (BDV) Beauregard sweet potato protein contents of, 9 starch composition and, 23–24 Beauveria bassiana, 256 Beniazuma sweet potato, protein contents of, 9 Betaphycus gelatinum, 270 Beverage, from sweet potato, 40 Biddulphia sinensis, 262 Birgus latro, 166 Blue-ringed octopus, tetrodotoxin in, 165, 168 Body mass index (BMI) in humans Ad-36 and, 89 CP and, 77–78 SMAM-1 and, 86 Boniato, see Sweet potato Borna disease virus (BDV), 64, 67, 81–83, 92 effect of infection on, 64 human obesity, association with, 64 induced obesity, 64, 67, 81–83 mechanism of, 82–83 mechanism of action, 67, 82–83

INDEX Brachycephalus ephippium, 161 Bread supplemented with SPF development and storage stability of, 33–34 dough enhancers, 33–34 macroscopic and sensory evaluation of, 33 Bryum, 268 Byssochlamys fulva, 131 Byssochlamys nivea, 131 C Calcium oxalate, in Actinidia genus fruits, 304–305 Camote, see Sweet potato Candida lambica, 129 Candida pelliculosa, 127 Candida sake, 129 Canine distemper virus (CDV), 63–64, 67, 69–73, 92 acute encephalitis, 63 alters hypothalamic integrity, 71 cytokine production, 72–73 down regulates long leptin receptor and increases leptin, 71 melanin-concentrating hormone, 71–72 effect of infection on, 64 F gene mRNA levels, 70 of genus Morbillivirus, 63 hit-and-run effect, 62, 70, 72 human obesity, association with, 64 induced obesity, 63, 69 mechanism of, 70–73 mechanism of action, 67, 70–73 neuropeptides and, 72 nucleoprotein transcripts, 70 obesity impact, 69 on brains and reproductive organs, 69 on catecholamine levels, 69, 72 on insulin levels, 69 on leptin levels, 69, 71 on lipid and fat cells, 69 on lipogenesis, 69 on tyrosine hydroxylase, 69 replication in rain, 70 Canthigaster ribulata, 157 Capillary isotachophoresis, detection method for TTX, 185, 189–190 Carcinoscorpius rotundicauda, 152, 162, 166, 181 b-Carotene, 2–3 in marine-derived food ingredients, 239, 257–258

327

in sweet potato, 10–13, 16, 31 Carotenoids in actinidia genus fruits, 307–309 in marine-derived food ingredients, 239, 257–261 astaxanthin, 239, 258–259 b-carotene, 239, 257–258 refrigerated fruit juices, contents of, 117–119 Catecholamine levels, CDV obesity impact on, 69, 72 CDV, see Canine distemper virus (CDV) CEBP a gene, 90 CEBP b gene, 90 Cephalothrix linearis, 172 Cephalothrix sp., 172 Cerebratulus lacteus, 172 Charonia sauliae, 148, 168–139, 177 poisoning due to digestive gland of, 147–148 Chelonodon patoca, 158, 179, 219–220 Chick Embryo Lethal Orphan virus (CELO), 85, 91 China, puffer poisoning cases in, 148–152 Chinese potato, see Sweet potato Chitin and chitosan, polysaccharides from, 273–274 Chitinolytic enzymes, 281 Chlamydia pneumoniae (CP), 66, 68, 77–78 coronary artery stenosis and, 78 coronary heart disease (CHD) and, 77 effect of infection on, 66 higher BMI and, 77–78 human obesity, association with, 66 mechanism of action, 67, 78 Chlamydia pssittaci, 78 Chlamydia trachomatis, 78 Chlamydomonas reinhardtii, 250, 252 Chlorella minutissima, 263, 267 Chlorella protothecoides, 257, 259 Chlorella sp., 245–248, 250, 252 Chlorella vulgaris, 257 Chlorophyll in actinidia genus fruits, 305–307 in marine-derived food ingredients, 239, 260–261 Chymotrypsins, 279 Cilera abana, see Sweet potato Coarse inginyo, 37 CODEX, General Standard for Fruit Juices and Nectars, 104 Coenobitidae, 166

328

INDEX

Collagen, 274 Collagenases, 279–280 Color, of refrigerated fruit juices, 122–124 Colostethus inguinalis, 161 CP, see Chlamydia pneumoniae (CP) Crypthecodinium, 250 Crypthecodinium cohnii, 245, 262–263 Cryptotethya crypta, 254 Ctenidium, 268 Cyanobacteria, exopolysaccharides from, 272 Cyanospira capsulata, 272 Cyanothece, 272 Cyclotella, 250 Cynops ensicauda, 159, 180 Cynops pyrrhogaster, 159, 180, 220 Cytokine production, and CDV, 72–73 Cytomegalovirus, 77 Cytotoxicity test, detection method for TTX, 185, 195–196 D Demania alcala, 167 Demania reynaudi, 163–165, 167 Demania toxica, 167 Diatoms algae transgenics, 251 Dietary fiber, in sweet potato, 13–14, 31 Digestive proteases, 278–280 Dinoflagellates algae transgenics, 251–252 tetrodotoxin in, 173–174 Diodon hystrix, 152 Dunaliella bardawil, 253 Dunaliella salina, 247, 250, 252, 257–258 Dunaliella sp., 245–246, 248–249, 252–253, 258, 276 Dysidea avara, 255 E Ectocarpus siliculosus, 253 Elastase, 279–280 Electrophoresis, detection method for TTX, 185, 189 Electrospray ionization-time of flight/mass (ESI-TOF/MS), 161–162 TTX, detection method for, 185, 193–194 Endomycopsis fibuligera, 36 Entomophthora obscura, 266 Enzyme-linked immunoassay (EIA), 197 Enzyme-linked immunosorbent assay (ELISA) system, 196

Enzymes chitinolytic, 281 digestive proteases, 278–280 extremophilic, 282–283 food industry and, 277–283 lipases, 280–281 marine-derived food ingredients, 242–243, 276–283 polyphenol oxidases, 281 from red algae, 283 transglutaminase, 281–282 Eriphia scabricula, 167 Eriphia sebana, 163, 167 Escherichia coli, in refrigerated fruit juices, 130–131 ESI-TOF/MS, see Electrospray ionization-time of flight/mass (ESI-TOF/MS) Ethanol, SPF fermentation to, 36 Eucheuma denticulatum, 270 Eukrohnia hamata, 173 Eupenicillium, 131 Euphausia superba, 268 Exopolysaccharides cyanobacteria and, 272 extremophiles and, 272 Extremophiles marine-derived food ingredients sources, 253–254 polysaccharides from, 272 sources of LC-PUFAs, 267 Extremophilic enzymes, 243, 282–283 F FABMS, see Fast atom bombardment mass spectrometry (FABMS) Fast atom bombardment mass spectrometry (FABMS), 173–174 TTX, detection method for, 185, 192–193 Fat cells, CDV obesity impact on lipids and, 69 Fatty acids contents, of refrigerated fruit juices, 112–113 Fatty acid synthetase (FAS), 90 F gene mRNA levels, and CDV, 70 Fiber one cereal, 34–36 Fine patterned puffer, poisoning due to liver of, 147–148 Fish LC-PUFAs and, 264–266 marine-derived food ingredients and, 256–257

INDEX Flaccisagitta enflata, 173 Flaccisagitta scripassae, 173 Flatworms tetrodotoxin in, 170–171 toxicity of eggs laid by, 171 Flavonoids contents, of refrigerated fruit juices, 119–120 Flavor, of refrigerated fruit juices, 113–114 Food industry, enzymes and, 277–283 Food ingredients production, marine biotechnology for, 237–284 Food Standards Agency, UK, 108 Free amino acids contents, of refrigerated fruit juices, 112–113 French-fries, from sweet potato, 41–42 Frogs, tetrodotoxin in, 160–162 Fruit and vegetables, see also Refrigerated fruit juices consumption of, 105 epidemiological studies, 105–106 protective effect of, 105–106 Fungi, sources of LC-PUFAs, 266 Furan formation, in refrigerated fruit juices, 111–112 Fusarium, 129 G G. crassissima, 271 Gas chromatography-mass spectrometry (GC-MS), 111 detection method for TTX, 185, 191 Gastropods poisoning due to digestive gland of, 147–148 tetrodotoxin in, 168–169 toxicity and toxin composition of, 169 GC-MS, see Gas chromatography-mass spectrometry (GC-MS) Gelatin, 274–275 Gelidium, 270 Geodia cydonium, 255 Geotrichum sp., 129 Gigartina, 270 ‘‘Giza 69’’, 13 GLUT-4, 90 Gobies intoxication in uremic patient in Taiwan, 151–152 local variation of toxicity in, 161 tetrodotoxin (TTX) in, 159–161

329

GPDH gene, 90 Gracilaria, 245, 270–271 Grapefruit juice, see also Refrigerated fruit juices consumption of, 104 Grape kiwi fruit, see Actinidia genus fruits Grapsus albolineatus, 167 Grateloupia, 270 Green algae transgenics, 252–253 Guava juice, see also Refrigerated fruit juices consumption of, 104 Gut microbiota, 66–67, 83–84 effect of infection on, 66 human obesity, association with, 66 induced obesity, 83–84 mechanism, 84 mechanism of action, 67, 84 Gymnodinium simplex, 262 Gyrodinium cohnii, 262 H Haematococcus pluvialis, 247, 249, 252, 257, 259 Haematococcus sp., 252, 259 Halobacterium mediterranei, 272 Halosydna brevisetosa, 173 Hapalochlaena maculosa, 180 Hard red spring wheat (HRSW), 33 Harmothoe imbricata, 173 Hayward fruit, see Actinidia genus fruits Hazard Analysis and Critical Control Points (HACCP) system, 106 Health benefits, of Actinidia genus fruits, 317–318 Helicobacter pylori, 77–78 Hemigrapsus sanguineus, 200 Hermenia acanthopeltis, 173 High-performance liquid chromatography (HPLC), 151 operating conditions for analysis of newt toxins, 188 TTX, detection method for, 185–188 HIPEF-treated juice, 118–119, 124 Hit-and-run effect of CDV, 70, 72, 92 Hizikia fusiforme, 246 1 H NMR spectrometry, see NMR spectroscopy Hort16A fruit, see Actinidia genus fruits HPLC, see High-performance liquid chromatography (HPLC) Human adenoviruses, 85

330

INDEX

Human food systems SPF production and utilization for, 28–38, 30, 34 sweet potato starch utilization in, 18–29 (see also SPS syrup) sweet potato utilization as value-added product in, 17–18 Human Genome Project, 212 Human obesity association with ad-5, 66 ad-36, 65 ad-37, 65 BDV, 64 CDV, 64 CP, 66 gut microbiota, 66 infections in, 92–93 inflammation and, 92–93 macrophage colony-stimulating factor (MCSF), 93 RAV-7, 64 scrapie agent, 65 SMAM-1, 65 Hydrocolloids, polysaccharides from, 269–271 Hydroponic sweet potato starch (HSPS) syrup, 27–28 Hyperglycemia, in ME7-infected mice, 80 Hyperlipidemia, RAV-7 induced, 74, 77 Hypnea, 270 Hypothalami, of CDV infected rats, 70–71 Hypothalamic integrity, and CDV, 71 I IL-b transcripts, 73 IL-6 transcripts, 73 Immunoassay, detection method for TTX, 185, 196–197 Infectobesity concert, 62 Insulin levels, CDV obesity impact on, 69, 90 IR spectrometry, detection method for TTX, 185, 191–192 Isochrysis galbana, 264 J Jania sp., 173 Japan, puffer poisoning cases in, 147–148 Japanese potato, see Sweet potato J6/66 sweet potato composition of starch processed from, 23–24 protein contents of, 9

K Kappaphycus alvarezii, 270 Kara-imo, see Sweet potato Kenya, sweet potato processing and utilization in, 44–47 Kiwi berry fruit, see Actinidia genus fruits Kiwifruit, see Actinidia genus fruits Kloeckera apis, 127, 129 Koganesengan sweet potato, protein contents of, 9 Koinostylochus sp., 171 Krill, sources of LC-PUFAs, 268 Kumar, see Sweet potato Kumara, see Sweet potato Kunuzaki, from sweet potato, 40 L Lactobacillus brevis, in refrigerated fruit juices, 129 Lactobacillus plantarum, 274 in refrigerated fruit juices, 129–130 Lagocephalus gloveri, 157, 213, 216 Lagocephalus inermis, 157–158, 216 Lagocephalus lunaris, 157–158, 179, 213, 216 Lagocephalus sceleratus, 157, 216 Lagocephalus wheeleri, 157, 216 Laminaria, 245–246, 253 Laminaria japonica, 245, 246, 270–271 Laminaria saccharina, 246 LC-MS, see Liquid chromatography-mass spectrometry (LC-MS) Lepidonotus helotypus, 173 Leptin levels CDV obesity impact on, 69, 71 and leptin receptor, CDV down regulates, 71 Leptodius sanguineus, 167 Lineus fuscoviridis, 172 Lipases, 280–281 Lipids CDV obesity impact on fat cells and, 69 marine-based long-chain PUFAs (LC-PUFAs), 239, 261–268 extremophiles as source of, 267 fish as source of, 264–266 fungi as source of, 266 krill as source of, 268 macroalgae and mosses as source of, 267–268 microalgae as source of, 262–264

INDEX transgenic organisms as source of, 266–267 marine-derived food ingredients, 239, 261–269 sterols, 268–269 Lipogenesis, CDV obesity and, 69 Liquid chromatography-mass spectrometry (LC-MS), 310 detection method for TTX, 185, 192–194 Lophozozymus pictor, 163–165, 167 Lymphotropic RNA virus, 63 M Macroalgae algae transgenics, 253 and mosses, sources of LC-PUFAs, 267–268 Macrophage colony-stimulating factor (MCSF), in human obesity, 93 Mandazis, from sweet potato, 46–47 Mango juice, see also Refrigerated fruit juices consumption of, 104 Marchantia, 268 Marine-based long-chain PUFAs (LC-PUFAs) lipids, 239, 261–268 production technologies, 262–264 sources of extremophiles as source of, 267 fish, 264–266 fungi, 266 krill, 268 macroalgae and mosses, 267–268 microalgae, 262–264 transgenic organisms, 266–267 Marine biotechnology, for food ingredients production, 237–284 Marine-derived food ingredients categories of, 239–243, 257–283 enzymes, 242–243, 276–283 lipids, 239, 261–269 photosynthetic pigments, 239, 257–261 polysaccharides, 240–241, 269–274 proteins, 241, 274–276 sources of, 244–257 algae cultivation, 249–250 algae transgenics, 250–252 extremophiles, 253–254 fish and seafood byproducts, 256–257 macroalgae, 245–246 macro- and microalgae, 244–253 marine sponges, 254–256 microalgae, 246–253

331

Marine enzymes food industry and, 277–283 chitinolytic enzymes, 281 digestive proteases, 278–280 enzymes from red algae, 283 extremophilic enzymes, 282–283 lipases, 280–281 polyphenol oxidases, 281 transglutaminase, 281–282 sources of, 276–277 Marine organisms, 238 Marine sponges, sources of marine-derived food ingredients, 254–256 Matobolwa, from sweet potato, 38 Matteuccia, 268 Measles virus (MV), 63 Melanin-concentrating hormone (MCH), 69 CDV down regulation, 71–72 Michembe from sweet potato, 38 Microalgae, sources of LC-PUFAs, 262–264 Mineral contents, in sweet potato, 13 Mnium, 268 Morbillivirus, CDV of genus, 63 Mortierella alpina, 266–267 Mortierella elongata, 266 Mortierella isabellina, 266 Mortierella ramanniana, 266 Mouse bioassay, detection method for TTX, 184–186, 196 N Nassarius glans, 149 Nata de coco dessert, 17 Natica alapailionis, 169 Natica lineata, 168–169, 176, 180, 219 Natica pseustes, 170 Natica vitellus, 149, 168–169, 219 Navicula incerta, 262 Navicula pelliculosa, 262 Neosartorya fischeri, in refrigerated fruit juices, 131 Neoxanthias impressus, 167 Neuropeptides expression and CDV, 72 Neurotropic negative-stranded RNA virus, 63 Newts local and sexual variation of toxicity in, 160 tetrodotoxin (TTX) in, 159–160 Nigeria, traditional utilization of sweet potatoes in, 6 Niotha clathrata, 149, 168–169, 176, 219

332

INDEX

Nitzschia, 250 Nitzschia frustulum, 262 N-3 LC-PUFAs, see Marine-based long-chain PUFAs (LC-PUFAs) NMR spectroscopy, detection method for TTX, 185, 194–195 Nonenzymatic browning evaluation, in refrigerated fruit juices, 111–112 Nostoc, 247, 272 Nostoc commune, 247 Nostoc flagelliforme, 247 Notocomplana roreana, 171 Notophthalmus viridescens, 159, 180 Notoplana humilis, 171 Nucleoprotein transcripts, of CDV, 70 Nutrients, in refrigerated fruit juices, 105 O O. maculosus, 174 Oliva hirasei, 146, 149, 169, 219 Oliva miniacea, 149, 169, 219 Oliva mustelina, 149, 169, 219 Omega-3 LC-PUFAs, see Marine-based long-chain PUFAs (LC-PUFAs) On-the-go food, 17 Orange-fleshed sweet potatoes, source of provitamin A, 10 Orange juice, see also Refrigerated fruit juices consumption of, 104, 106 Organic acids, in Actinidia genus fruits, 300–301 Osteopetrosis, 73 P Prachaeturichtys palynena, 160 Paralytic shellfish poisons (PSPs), 152 Paramesotriton hongkongensis, 159 Paramyxoviruses, 63 Parasagitta elegans, 173 Passion fruit juice, see also Refrigerated fruit juices consumption of, 104 Patents, on sweet potato products, 40–41 Pectinesterases activity, of refrigerated fruit juices, 124–126 Penicillium, 129 Phaeodactylum tricornutum, 250–251, 251, 262–264, 266–267 Photosynthetic pigments carotenoids, 239, 257–261

astaxanthin, 239, 258–259 b-carotene, 239, 257–258 chlorophylls, 239, 260–261 marine-derived food ingredients, 239, 257–261 Physcomitrella patens, 267 Phytophthora infestans, 266 Pigments in Actinidia genus fruits, 305–310 anthocyanins, 308–310 carotenoids, 307–309 chlorophyll, 305–307 Pilumnus vespertilio, 167 Pineapple juice, see also Refrigerated fruit juices consumption of, 104 Planocera multitentaculata, 170–171, 170–172 Planocera reticulata, 170–171 Platypodia granulosa, 167 Plsiomonas sp., 176 Pogonatum sp., 268 Pogonatum urnigerum, 268 Polinices didyma, 168, 219 Polinices tumidus, 168 Polypedates sp., 161 Polyphenol oxidase, 281 refrigerated fruit juices, inactivation in, 126–127 Polysaccharides from algae, 240–241, 269–274 fucans/fucanoids and other polysaccharides from, 271–272 hydrocolloids, 269–271 chitin and chitosan, 273–274 cyanobacteria, 272 extremophiles, 272 marine-derived food ingredients, 240–241, 269–274 Polytrichum, 268 Pomacea canaliculata, 219 Porphyra sp., 245, 246, 253 Porphyridium cruentum, 263–264 PPAR-g gene, 90–91 Pro-opiomelanocortin (POMC) cell bodies, loss of, 71 Protamine, 275–276 Proteases, 278–280 Proteins marine-derived food ingredients from, 241, 274–276 in sweet potato leaves and roots, 8–10, 31

INDEX Provitamin A, orange-fleshed sweet potatoes and, 10 Pseudoalteromonas, 272 Pseudomonas sp., 176 Pseudopotamilla occelata, 172, 174 Psychrophilic enzymes, 282 Puffer edible part of, in Japan, 147 food industry programs for, 208–220 new developed biotechnology and, 208–209 traditional food puffer liver kimo, revival of, 209 nontoxic production, 208–209 poisoning cases in Japan, 147–148 Taiwan and China, 148–152 poisoning deaths, 142 species, 156–158 direct sequence analysis approach, 210 genome techniques, identification by, 209–213 PCR-RFLP technique, 210–211 PCR-SSCP technique, 211–212 by protein technique, 213–218 RAPD-PCR technique, 212 tetrodotoxin in, 156–158 toxicity of, 147–148, 156 Taiwanese puffer, 157 toxin chemistry, 197–198 traditional food puffer liver kimo, 209 TTX infestation to nontoxic cultured, 200 R Rain, CDV replication in, 70 Rapana rapiformis, 168–169 Rapana venosa venosa, 168–169 RAV-7, 64, 67, 73–77 autoimmune thyroiditis, 76–77 effect of infection on, 64 human obesity, association with, 64 induced hyperlipidemia, 74, 77 induced obesity, 74–75 specificity of, 75–76 mechanism of induced changes, 67, 76–77 stunting and, 74 Ready-to-eat breakfast cereals (RTEBC), 34–36 Ready-to-eat sweet potato breakfast cereal physical properties and sixth graders’ acceptance of extruded, 35–36

333

sensory characterization by descriptive analysis of, 34–35 Red algae, enzymes from, 283 Red calcareous alga, tetrodotoxin in, 173 Redigobius caninus, 160 Refrigerated fruit juices anthocyanins contents, 119–120 antioxidant activity, 120–122 aromatic profile of, 113–114 carotenoids contents, 117–119 color, 122–124 consumer preferences for, 104 consumption of, 104 E. coli, 130–131 fatty acids contents, 112–113 flavonoids contents, 119–120 flavor of, 113–114 free amino acids contents, 112–113 furan formation in, 111–112 inclusion in healthy diet, 104 Lactobacillus brevis, 129 Lactobacillus plantarum, 129–130 market for, 104 Neosartorya fischeri, 131 nonenzymatic browning evaluation, 111–112 nutrients in, 105 pectinesterases activity, 124–126 physicochemicals and quality characteristics of, 108–111 polyphenol oxidase inactivation, 126–127 processing, 107–108 quality and safety issues, 104–131 quality parameters, 106–107 Salmonella enteritidis, 131 Staphylococcus aureus, 131 vitamin A contents, 117–119 vitamin C concentration in, 114–117 yeast, 127–129 Renibacterium salmoninarum, 252 Rhodotorula rubra, 129 Ribbon worms, tetrodotoxin in, 172 Rous-associated virus-7 (RAV-7), see RAV-7 S Saccharomyces cerevisiae, 128, 267 Salmonella enteritidis, in refrigerated fruit juices, 131 Sargassum confusum, 270 Satamu sweet potato, 6 Satsuma bairdi, 219

334

INDEX

Satsuma-imo, see Sweet potato Scenedesmus, 246 Schizochytrium, 263–264 Scrapie agent effect of infection on, 65 human obesity, association with, 65 induced obesity, 65, 67, 78–81 mechanism of, 80–81 mechanism of action, 67 Seafood byproducts, 256–257 Shewanella alga, 174 Shewanella putrefaciens, 174, 181 Sillago japonica, 160 SMAM-1, 65, 68, 85–87, 92 effect of infection on, 65 human obesity, association with, 65 induced obesity, 85–87 mechanism of, 87 mechanism of action, 67, 87 Snail poisoning outbreaks and patients, in Zhoushan City, 150 Sodium channels mechanism of action on, 201–202 molecular structure of, 202–203 TTX-R, 204–206 Soursop juice, see also Refrigerated fruit juices consumption of, 104 Space missions, sweet potato vegetarian products intended for, 38–40 Spadella angulata, 173 Species and cultivars, of Actinidia genus fruits, 295–299 A. arguta, 297–299 A. chinensis, 296–298 A. deliciosa, 295–296, 298 A. eriantha, 299 A. kolomikta, 299 A. rufa, 298–299 Species identification for puffer by genome techniques, 209–213 direct sequence analysis approach, 210 PCR-RFLP technique, 210–211 PCR-SSCP technique, 211–212 puffer genome technique, 212–213 RAPD-PCR technique, 212 by protein technique, 213–218 SPF, see Sweet potato flour SPF/whole-wheat bran (SPFWWB) cereal, 34–36 Sphagnum, 268

Sphoeroides pachygaster, 216 Spirulina (Arthrospira) maxima, 248, 257 Spirulina (Arthrospira) platensis, 248, 250, 261 Spirulina sp., 245–248, 261 Sporamins A and B proteins, in sweet potato, 9 SPS, see Sweet potato starch (SPS) SPS syrup a-amylase influence, 26–27 enzymatic hydrolysis into glucose syrup, 25 pH effects and concentration times on functional properties of, 26 physicochemical and viscometric properties of, 25–26, 29 sensory and consumer evaluation of, 27–28 Staphylococcus aureus, in refrigerated fruit juices, 131 Starfish, tetrodotoxin in, 169–170 Sterols, marine-derived food ingredients in, 268–269 Stunting, and RAV-7, 74 Stylochoplana clara, 171 Stylochus ijimai, 171 Stylochus orientalis, 171 Suberites domuncula, 255 Sugar and sugar alcohol, in Actinidia genus fruits, 299–300 Suioh sweet potato greens, nutritional contents of, 8–9 Sweet potato, see also SPF (sweet potato flour) anthocyanins, 14–16 antioxidative and radical-scavenging activities, 14–15 beverage, 40 biochemical composition of, 7–16 b-carotene in, 10–13, 16 dietary fiber, 13–14 French-fry-type product from, 41 growing season, 5 health benefit from antihypertensive and antidiabetic properties of, 15–16 in human diets, 6–7 kunuzaki, 40 leaves and roots, 5 mineral contents, 13 nutritional composition of, 7–16 origin of, 4 patents regarding products, 40–41 per capita consumption of, 17 potential in Ugandan food system, 42–44 potential products, 38–42

INDEX processing and utilization in Kenya, 44–47 production of, 7–8 products intended for space missions, 38–40 protein in leaves, 8–9 in roots, 9–10 scientific classification, 4 sporamins A and B proteins in, 9 as staple food, 2, 13 starch bulk ingredients from cultivars of, 23–24 composition and properties of, 23–24 effects of processing technology on yield and quality, 23, 25 utilization in human food systems, 18–29 starch syrup influence of a-amylase on physical properties and consumer acceptability of, 26–27 pH effects and concentration times on functional properties of, 26 physicochemical and viscometric properties of, 25–26, 29 sensory and consumer evaluation of, 27–28 starch utilization [see Sweet potato starch (SPS)] textural measurements and product quality of restructured French-fries from, 41–42 utilization as value-added products, 17–18 varieties, 11–12 vegetarian products intended for space missions, 38–40 vitamin A deficiency (VAD) and, 10–11 yam and, 4–5 Sweet potato flour (SPF) breadmaking properties of, 32–33 bread supplemented with development and storage stability of, 33–34 dough enhancers, 33–34 macroscopic and sensory evaluation of, 33–34 fermentation to ethanol, 36 genetic variation in color of, 36–37 like products, 37–38 preparation of, 36 processed in agroecological sites, and small-scale processing technologies, 37

335

production and utilization for human food systems, 28–38 protein content of, 30–31 quality characteristics of, 30–31 quality evaluation of, 37 ready-to-eat sweet potato breakfast cereal, 34–36 use as coloring materials, 14 use in wheat-based composite flour products, 36–37 Sweet potato leaves and roots, 5 proteins in, 8–10 Sweet potato starch (SPS) bulk ingredients from cultivars of, 23–24 composition and properties of, 23–24 effects of processing technology on yield and quality, 23, 25 production procedures for, 21–22 scanning electron micrographs of granules of, 19 syrup a-amylase influence, 26–27 pH effects and concentration times on functional properties of, 26 physicochemical and viscometric properties of, 25–26, 29 sensory and consumer evaluation of, 27–28 utilization in human food systems, 18–29 Symbiodinium, 252 T Tachypleus gigas, 162 Taiwan goby intoxication in uremic patient in, 151–152 poisoning incident due to ingestion of unknown fish in, 150 puffer poisoning cases in, 148–152 Takifugu chinensis, 157 Takifugu chrysops, 156 Takifugu exascurus, 157 Takifugu flavidus, 156–157 Takifugu niphobles, 150, 157–158, 174 Takifugu oblongus, 152, 157, 191, 216 Takifugu obscurus, 156–157 Takifugu pardalis, 156, 158, 179 Takifugu poecilonotus, 156, 158 poisoning due to liver of, 147–148 Takifugu porphyreus, 156

336

INDEX

Takifugu pseudommus, 157, 212 Takifugu rubripes, 156, 171, 212–213 Takifugu snyderi, 156, 174, 181 Takifugu stictonotus, 157 Takifugu vermicularis, 157, 219–220 Takifugu xanthopterus, 157, 216 Talaromyces flavus, 131 Taricha granulosa, 159, 180 Taricha oregon, 159 Taricha rivularis, 180 Taricha torosa, 159, 180 Tetraodon alboreticulatus, 157–158 Tetraodon fangi, 158 Tetraodon nigroviridis, 157–158 Tetraodon ocellatus, 157 Tetraodon steindachneri, 158, 219–220 Tetraodontiformes, see Tetrodotoxin (TTX) Tetraselmis, 250 Tetrodotoxication symptoms, 152–154 Tetrodotoxin (TTX) 4,9-anhydro type, 145 bearing organisms, 156–176 annelids, 172–173 arrowworms, 173 bacteria, 174–176 blue-ringed octopus, 165, 168 dinoflagellate, 173–174 flatworms, 170–171 frogs, 160–162 gastropods, 168–169 gobies, 159–161 horseshoe crab, 162–164 newts, 159–160 puffer, 156–158 red calcareous algae, 173 ribbon worms, 172 starfish, 169–170 toxin accumulation from TTX-producing bacteria, 182–183 TTX resistibility in, 199 xanthid crabs, 163–167 causative agent, 156–208 chemical structure, 143–145 detection methods for, 184–197 bioassay, 185–186 capillary isotachophoresis, 185, 189–190 cytotoxicity test, 185, 195–196 electrophoresis, 185, 189 ESI-TOF/MS, 185, 193–194 FABMS, 185, 192–193

GC-MS, 185, 191 HPLC, 185–188 immunoassay, 185, 196–197 IR spectrometry, 185, 191–192 LC-MS, 185, 192–194 mouse bioassay, 184–186, 196 NMR spectroscopy, 185, 194–195 TLC, 185, 189 UV spectroscopy, 185, 190 distribution in animals, 146 elaborator, 176–178 hemilactal type, 144 hibernation agent, as attractant and, 219–220 infection to animals, mechanisms of, 178–184 infestation to nontoxic cultured puffer fish, 200 lacton type, 144 mechanisms of accumulation in marine animals, 179 pharmacology of, 198–207 mechanism of action on sodium channels, 201–202 molecular structure of sodium channels, 202–203 site of action and binding of TTX, 206–207 TTX-R sodium channels, 204–206 poisoning, 145–156 due to ingestion of puffer, 142 due to liver of finepatterned puffer, 147–148 incidents in Japan, 143–148 incidents in Taiwan and China, 148–152 incidents in world, 143–152 prevention, 155–156 symptoms and signs, 152–154 treatment, 154–155 site of action and binding of, 206–207 tautomers, 143 therapeutic applications, 207–208 Thalamita sp., 167 TH cell bodies, loss of, 69, 71 Thermococcus chitonophagus, 281 Thin-layer chromatography (TLC), detection method for TTX, 185, 189 Thraustochytrium, 263–264 Tissue culture bioassay (TCBA), 195 TLC, see Thin-layer chromatography (TLC) 3T3-L1 preadipocytes, 90 TNF-a transcripts, 73

INDEX Transgenic organisms, sources of LC-PUFAs, 266–267 Transglutaminase, 281–282 Triturus alpestris, 159, 180 Triturus cristatus, 180 Triturus marmoratus, 180 Triturus vulgaris, 159, 180 Tropical fruit juices, consumption of, 104 Trypsin, 279 TTX, see Tetrodotoxin (TTX) Tubulanus punctatus, 172 TU-82-155 sweet potato, composition of starch processed from, 23–24 Tutufa lissostoma, 168 Tylototriton andersoni, 159 Tyrosine hydroxylase (TH), see TH cell bodies U Ubhatata, see Sweet potato Ugandan food system, sweet potato potential in, 42–44 Ulva pertusa, 272 Umborium suturale, 170 Undaria pinnatifida, 246, 260 V Vibrio alginolyticus, 174, 176 Vibrio fischeri, 181 Vibrio parahaemolyticus, 176 Vibrio sp., 174, 181, 272 Vitamin A contents, of refrigerated fruit juices, 117–119 Vitamin A deficiency (VAD), sweet potato role in fight against, 10–11, 47–48 Vitamin C

in actinidia genus fruits, 301–303 concentration in refrigerated fruit juices, 114–117 Volvox carteri, 250 W White-fleshed sweet potato roots, 5 Whole-wheat bran (WWB) cereal, 34–36 X Xanthias lividus, 164–165 Xanthid crabs tetrodotoxin (TTX) in, 163–167 toxicity and toxin composition of, 165, 167–168 Xiphosuridae, 166 ‘‘Xushu 18’’ sweet potato, 24 Y Yam b-carotene content, 4–5 growing season, 5 sweet potato and, 4–5 Yeast, in refrigerated fruit juices, 127–129 Yongeichthys criniger, 159, 178 Yongeichthys nebulosus, 151, 160 Z Zanthin, 259 ZESPRITM GOLD kiwifruit, see Actinidia genus fruits Zeuxis castus-like, 169 Zeuxis samiplicutus, 149, 169 Zeuxis scalaris, 149, 168–169 Zeuxis sufflatus, 168, 219 Zosimus aeneus, 163–166

337