Advances in Food and Nutrition Research Volume 57

ADVISORY BOARDS KEN BUCKLE University of New South Wales, Australia MARY ELLEN CAMIRE University of Maine, USA ROGER ...

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ADVISORY BOARDS KEN BUCKLE University of New South Wales, Australia

MARY ELLEN CAMIRE University of Maine, USA

ROGER CLEMENS University of Southern California, USA

HILDEGARDE HEYMANN University of California, Davis, USA

ROBERT HUTKINS University of Nebraska, USA

RONALD JACKSON Quebec, Canada

HUUB LELIEVELD Global Harmonization Initiative, The Netherlands

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

(1989–1993)

STEVE L. TAYLOR

(1995–

)

Academic Press is an imprint of Elsevier 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA 32 Jamestown Road, London NW1 7BY, UK Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands Linacre House, Jordan Hill, Oxford OX2 8DP, UK First edition 2009 Copyright # 2009 Elsevier Inc. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material. Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. ISBN: 978-0-12-374440-1 ISSN: 1043-4526 For information on all Academic Press publications visit our website at elsevierdirect.com Printed and bound in USA 09 10 11 12 10 9 8 7 6 5 4 3 2 1

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

Pedro Bouchon

Department of Chemical and Bioprocess Engineering, Pontificia Universidad Cato´lica de Chile, Santiago, Chile (209) Paul Pui-Hay But

Food and Drug Authentication Laboratory, Department of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, P.R. China (1) Sheila Dubois

Bureau of Chemical Safety, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canada (235) Kari Dunfield

Department of Land Resources, University of Guelph, Guelph, Ontario, Canada (155) Wei Fan

Department of Food Science, University of Guelph, Guelph, Ontario, Canada (155) Zoe Gillespie

Bureau of Chemical Safety, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canada (235) Samuel Benrejeb Godefroy

Bureau of Chemical Safety, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canada (235) Alan R. Hipkiss

School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, The University of Birmingham, Edgbaston, Birmingham, United Kingdom (87) Ann Huber

Soil Resource Group, Guelph, Ontario, Canada (155) Ka Ho Ling

Food and Drug Authentication Laboratory, Department of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, P.R. China (1)

ix

x

Contributors

Azadeh Namvar

Department of Food Science, University of Guelph, Guelph, Ontario, Canada (155) Peter D. Nichols

CSIRO Marine and Atmospheric Research and Food Futures Flagship, Hobart, Tasmania, Australia; Food and Drug Authentication Laboratory, Department of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, P.R. China (1) Olga M. Pulido

Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada; Bureau of Chemical Safety, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canada (235) Mohsin Rashid

Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada; Professional Advisory Board, Canadian Celiac Association, Ottawa, Ontario, Canada (235) Karl J. Siebert

Food Science & Technology Department, Cornell University, Geneva, New York (53) Connie Switzer

Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada; Professional Advisory Board, Canadian Celiac Association, Ottawa, Ontario, Canada (235) Elizabeth Vavasour

Bureau of Chemical Safety, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canada (235) Keith Warriner

Department of Food Science, University of Guelph, Guelph, Ontario, Canada (155) Marion Zarkadas

Professional Advisory Board, Canadian Celiac Association, Ottawa, Ontario, Canada (235)

CHAPTER

1 Fish-Induced Keriorrhea Ka Ho Ling,* Peter D. Nichols,*,† and Paul Pui-Hay But*,1

Contents

2 2 3 3 6 6 9 9 11 13 14 15 15 15 18 18 19 21 22 23 23 25 27 27

I. Introduction A. Fish as food B. Keriorrhea C. Case reports and symptoms II. Fish Incriminated A. Escolar and oilfish B. Harmful effects C. Uses D. Supply E. Mislabeling and mishandling F. Global concerns III. Regulation and Litigation A. Regulation B. Litigation IV. Biochemistry and Toxicity A. Wax esters and their biological roles B. Toxicity C. Animal tests D. Human studies V. Identification and Detection A. Morphological and anatomical analyses B. Protein analysis C. DNA analysis D. Lipid analysis

* Food and Drug Authentication Laboratory, Department of Biology, The Chinese University of Hong Kong, { 1

Shatin, N.T., Hong Kong, P.R. China CSIRO Marine and Atmospheric Research and Food Futures Flagship, Hobart, Tasmania, Australia Corresponding author

Advances in Food and Nutrition Research, Volume 57 ISSN 1043-4526, DOI: 10.1016/S1043-4526(09)57001-5

#

2009 Elsevier Inc. All rights reserved.

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Ka Ho Ling et al.

VI. Wax Ester-Rich Fish and Other Potential Hazards A. Gempylidae family B. Other deep-sea fish C. Diacylglyceryl ether (DAGE)-rich fish VII. Discussion and Recommendations A. The rationale: To ban or not to ban? B. Recommendations VIII. Conclusions References

Abstract

30 30 30 39 40 40 41 44 45

Many deep-sea fishes store large amounts of wax esters in their body for buoyancy control. Some of them are frequently caught as by-catch of tuna and other fishes. The most noteworthy ones include escolar and oilfish. The accumulation of the indigestible wax esters in the rectum through consumption of these fish engenders discharges or leakage per rectum as orange or brownish green oil, but without noticeable loss of water. This physiological response is called keriorrhea, which is variously described as ‘‘oily diarrhea,’’ ‘‘oily orange diarrhea,’’ or ‘‘orange oily leakage’’ by the mass media and bloggers on the internet. Outbreaks of keriorrhea have been repeatedly reported across continents. Additional symptoms including nausea, vomiting, abdominal cramps, and diarrhea were complained by the victims. They are probably due to anxiety or panic when suffering from keriorrhea. Escolar and oilfish are banned from import and sale in Italy, Japan, and South Korea. Rapid detection of the two fishes is imperative to ensure proper labeling and safeguarding of the public before and after any keriorrhea outbreak.

I. INTRODUCTION A. Fish as food Fishes are an excellent source of proteins, polyunsaturated fats, vitamins, and other nutrients. The wide range of biodiversity in fishes allows a good selection of different forms, sizes, colors, tastes, and textures to fit one’s diet preferences. When served alone or in combination with various spices, other meats, and vegetables, and prepared by a range of culinary methods, there are unlimited ways to turn fish into the most enjoyable gourmet item. As compared to other sources of meat, consumption of fish has additional health benefits, which is most often associated with the presence of omega-3 long-chain (>C20) polyunsaturated fatty acids (o3 LC-PUFA). This provides protection against cancer of the alimentary tract, coronary heart diseases, stroke, and other disorders (Erkkila et al.,

Fish-Induced Keriorrhea

3

2004; Fernandez et al., 1999; He et al., 2004; Hu et al., 2002; Mozaffarian and Rimm, 2006; Norat et al., 2005). Fishes may also occasionally cause harm to health. When incompletely cooked or improperly handled, fishes can become a medium for transmission of parasites and diseases (Butt et al., 2004a,b). Allergens such as parvalbumins in fish muscles or even parasites such as Anisakis simplex in fish can cause allergic reactions (Du Plessis et al., 2004; Lehrer et al., 2003; Poulsen et al., 2003; Taylor et al., 2004; Wild and Lehrer, 2005). The safety of fish consumption is now a major consumer worry (Brewer and Prestat, 2002; Lyon, 2008; Senkowsky, 2004; Verbeke et al., 2008) and the news of poisoning after fish consumption is not infrequent. Certain components in fish including tetrodotoxin and ciguatera toxins are notorious for their toxic properties (Hashimoto and Fusetani, 1978; Kazuo, 1999; Lawrence et al., 2007; Lehane and Lewis, 2000; Miyazawa and Noguchi, 2001; Noguchi and Ebesu, 2001; Stommel and Watters, 2004; Ting and Brown, 2001). Mercury and other heavy metals and various contaminants, such as pesticides, other organochlorines, and antibiotics, accumulated in fish are a serious concern (Du Plessis et al., 2004; Guallar et al., 2002; Hightower and Morre, 2003; Kostyniak et al., 1998; Senkowsky, 2004). A common response to fish poisoning is diarrhea, often in the form of loose and watery stools accompanied with excessive water loss. However, in some special cases, the uncontrollable urge of bowel movements and discharges do not involve a noticeable loss of water. In those cases, oil is discharged or leaked through the rectum, and this type of poisoning responses is called keriorrhea or keriorrhoea.

B. Keriorrhea Keriorrhea specifically refers to the pathological symptom of involuntary passage or leakage of oil, or actually wax esters, through the rectum. This term was coined by Berman et al. (1981) based on the Greek words keri and diarroia, which mean ‘‘wax’’ and ‘‘to flow through’’, respectively. More specifically, they refer to the symptoms observed in cases developed after consumption of certain oily fish, wherein the oil discharged appears orange or brownish green in color, while little water is lost (Fig. 1.1). This ailment is variously described as ‘‘oily diarrhea’’, ‘‘oily orange diarrhea’’, or ‘‘orange oily leakage’’ by the mass media and bloggers on the internet.

C. Case reports and symptoms Outbreaks of keriorrhea have been reported in many continents, including Africa, America, Asia, Australia, and Europe (Table 1.1). However, few are recorded in the scientific or medical literature. Therefore, the

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FIGURE 1.1 Oil discharged after consumption of escolar. Reprinted with permission from Ruello (2004, Nick Ruello of Ruello and Associates Pty Ltd.).

actual number of affected people over the years are largely underestimated as the internet is floating with many more reports or communication about personal experiences of embarrassing oily diarrhea after consumption of fish. In most cases, these fishes came into the spotlight because of a large outbreak that involved a substantial number of people; otherwise, scattered occurrences are generally neglected. Australia has documented several keriorrhea outbreaks, allowing further tracing into the etiology and symptoms in patients. In South Australia, between 1997 and 1999, there were nine cases of gastrointestinal complaints after rudderfish consumption. In two episodes that took place in 1999, patients complained of diarrhea, often oily and orange colored, within hours of consumption. Through protein fingerprinting, the implicated fish was identified as escolar (Givney, 2002). Two outbreaks of diarrhea in 1999 and 2001 were reported to be associated with butterfish consumption in Victoria, Australia. The victims complained of diarrhea or yellow oily diarrhea. The fish causing the outbreaks in 1999 was identified as either escolar or rudderfish, and the one in 2001 as escolar (Gregory, 2002) Another outbreak of gastrointestinal illness occurred among attendees of a conference lunch in New South Wales, Australia, in October 2001. Analysis of the oil in the fish samples served revealed a high proportion of wax ester (96–97%) and showed close resemblance to the oil composition in escolar. A distinctive symptom reported by many ill persons was the presence of oily diarrhea. Investigators of the outbreak conducted a

TABLE 1.1

Selected cases of keriorrhea outbreak related to escolar and oilfish consumption

Fish involved

Location

Date

No. of people affected

Oilfish

South Korea

May 2007

N/A

Toronto, Canada Hong Kong SAR, China

February 2007

N/A

January 2007

600þ

Victoria, Australia Sydney, Australia Victoria, Australia Victoria, Australia California, USA New South Wales, Australia South Australia Cape Town, South Africa

August 2001

5

January 2001

9

November 1999

11

November 1999

10

11 August 2003

42

October 2001

20

October 1999

N/A

1989

N/A

Escolar

* Escolar could be possibly involved in a few cases. ** The fish involved were reported as escolar but under the scientific name Ruvettus pretiosus.

Remarks

References

White tuna sushi at restaurants Mislabeled as cod or sea bass Mislabeled as cod

Fuga (2007)

Mislabeled as butterfish Fish curry in a canteen Mislabeled as butterfish Mislabeled as butterfish Served in a buffet Mislabeled as rudderfish or butterfish Mislabeled as rudderfish Mislabeled as rudderfish

CBC (2007); Jacquet and Pauly (2008) *Ling et al. (2008a,b); Jacquet and Pauly (2008) **Gregory (2002) Leask et al. (2004) **Gregory (2002) **Gregory (2002) Feldman et al. (2005) Yohannes et al. (2002)

Givney (2002) Berman et al. (1981)

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telephone interview of the cohort of conference attendees using a standard questionnaire. Out of 44 attendees, 20 (46%) became ill following the conference. The median incubation period was 2.5 h (range 1–90 h). The most common symptoms reported were diarrhea (80% including 38% reporting oily diarrhea), abdominal cramps (50%), nausea (45%), headache (35%), and vomiting (25%). None of the food or beverages consumed was significantly associated with the illness. However, all individuals who consumed fish became sick, but not those who did not (four persons). Among those who consumed fish, the following potential risk factors did not have a significant association with the illness: body mass index (BMI), age, health status, and the amount of fish consumed (Yohannes et al., 2002).

II. FISH INCRIMINATED A. Escolar and oilfish Outbreaks of keriorrhea are reported in consumers who admitted having consumed various fishes (e.g., Atlanta cod, butterfish, cod, ruddercod, or rudderfish). However, so far, almost all episodes can be traced to two varieties: escolar and oilfish.

1. Biology Escolar and oilfish belong to the Gempylidae (snake mackerel) family in the order Perciformes (Alexander et al., 2004; Nakamura and Parin, 1993). There are currently 24 species under 16 genera in Gempylidae, and they are all found in the marine environment (FishBase, 2008). All species in this family usually occur in very deep waters in tropical and subtropical seas (Nakamura and Parin, 1993). They have elongated and compressed body with isolated finlets after the anal and dorsal fins (Nakamura and Parin, 1993). Escolar (Lepidocybium flavobrunneum Smith) is also called black oilfish (Fig. 1.2A and B). It is a large fish (up to 2-m long, but usually 150-cm wide) covered with small cycloid scales and smooth skin (Fishbase, 2008). It has a faint but highly undulating lateral line on the semifusiform body, which changes color from dark brown to almost black with age. In addition, a prominent keel flanked with two small oblique ridges, one on each side of the keel, is present on the caudal peduncle (Nakamura and Parin, 1993; Pauline, 1980). A single species of escolar is generally recognized. However, Brendtro et al. (2008) recently analyzed the mitochondrial control region and flanking tRNA sequences for 225 escolar specimens collected from six sites at different locations of the Atlantic and Pacific Oceans. Their results revealed two distinct clades, one for the


7

10 cm A

B

10 cm C

D

FIGURE 1.2 (A) Escolar (Regulatory Fish Encyclopedia, U.S. FDA, 1993–2008, reprinted with permission); (B) Line drawing of escolar (Nakamura, 1995, Food and Agriculture Organization of the United Nations, reprinted with permission). (C) Oilfish (Regulatory Fish Encyclopedia, U.S. FDA, 1993–2008, reprinted with permission); (D) Line drawing of oilfish (Schneider, 1990, Food and Agriculture Organization of the United Nations, reprinted with permission).

Atlantic and the other for the Pacific (plus four from South Africa) samples. A previous study by Collette et al. (1984), interestingly, also noted differences between escolar in the Atlantic and the Indo-Pacific regions, their vertebral counts being 31 and 32, respectively. There is a possibility that two species or subspecies of escolar may be warranted (Brendtro et al., 2008). Oilfish (Ruvettus pretiosus Cocco) is also called castor-oil fish (Fig. 1.2C and D). It is a large fish (up to 3-m long, but usually 100–150 cm wide) possessing a dark brown semifusiform body and a single, usually obscure, lateral line. Its lower jaw slightly protrudes beneath the upper jaw (Bettoso and Dulcic, 1999; Fishbase, 2008). Rows of spiny tubercles on the cycloid scales make the skin of oilfish very rough. A layer of porous blubber-like tissue is observed after the scales are removed (Gudger and Mowbray, 1927). In addition, a rigid and scaly abdominal keel is located on the ventral contour of the fish (Bettoso and Dulcic, 1999; Bone, 1972; Nakamura and Parin, 1993). Escolar and oilfish are commonly found at depths between 100–800 m and 200–1100 m, respectively (Nakamura and Parin, 1993). As in many

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Ka Ho Ling et al.

deep sea fish, both escolar and oilfish lack a swim bladder. Their buoyancy in water is achieved by the storage of large amounts of low-density lipid, particularly in the dermis, flesh, and the bones of the skull (Bone, 1972). The stored lipid provides a sufficient lift to make them neutrally buoyant. Oilfish, at rest, hangs in the water heads up at 45 to the horizontal (Bone, 1972).

2. Chemistry Both escolar and oilfish possess considerable lipid in the body, accounting for approximately 20% of their wet weight (Cox and Reid, 1932; Gudger, 1925; Mori et al., 1966c; Nichols et al., 2001; Ukishima et al., 1987). The major components contributing to more than 90% of the total oil content are indigestible wax esters (Alexander et al., 2004; Ruiz-Gutierrez et al., 1997; Yohannes et al., 2002). Other lipid classes (hydrocarbon, triacylglycerol, sterol, and polar lipid) are present in small or negligible quantities. Moreover, both escolar and oilfish have high levels of histidine (8–11 mg/g) in their muscles (Feldman et al., 2005; Kan et al., 2000; Leask et al., 2004). A large and essentially unpredictable variability in the total oil and wax ester content of escolar was found with no clear correlation between the fish size and oil content. Also, no significant difference was found between the oil content of fish on the west coast and east coast of Australia, at least for specimens collected in summer (Ruello, 2004).

3. Effect of cooking on oil content and composition Ruello & Associates Pty. Ltd. (Ruello, 2004) conducted a study for the Australian Government Department of Health and Ageing. A comparative study was made on baking and grilling escolar. Far more water than oil is lost during cooking as the total oil content on a wet-weight basis actually increases. Moreover, the cooking method has little effect on the oil composition; wax esters remain as the predominant oil class in the samples tested. The perception for the reduction of the volume or potency of the oil/wax ester in escolar by heavy grilling or other normal cooking method is unfounded. The notion of correct and incorrect cooking methods (with regards to keriorrhea) and that one cooking method is better than another (e.g., grilled vs. baked) is equally unfounded. Battering and frying are unlikely to raise the risk of keriorrhea. There was no evidence to support the common perception that wax esters can be ‘‘grilled out’’ of the fish or otherwise substantially reduced by normal cooking methods. Cooking actually ‘‘concentrates’’ the oil as water is expelled from the flesh. Freezing the (raw) fillet for as long as 9 months also failed to noticeably reduce the capacity of escolar to induce keriorrhea (Ruello, 2004).


9

B. Harmful effects Escolar and oilfish have long been known to possess purgative effects because of their high oil content, accounting for approximately 20% of their wet weight (Cox and Reid, 1932; Gudger, 1925; Mori et al., 1966c; Ukishima et al., 1987). Substantial amounts of indigestible wax esters have been incriminated as the cause of keriorrhea and other acute gastrointestinal symptoms, such as abdominal cramps, nausea, headache, and vomiting in susceptible individuals (Alexander et al., 2004; RuizGutierrez et al., 1997; Yohannes et al., 2002). Therefore, the wax esters in these two fish are regarded as a natural toxin called gempylotoxin (FDA, 2001a). Moreover, both escolar and oilfish have high levels of histidine in their muscles (Feldman et al., 2005; Kan et al., 2000; Leask et al., 2004). If they are refrigerated improperly, bacteria can multiple and convert the histidine into histamine also termed scombrotoxin (FDA, 2001b), which can lead to cardiovascular, gastrointestinal, and neurological disorders (FDA, 2001b; Feldman et al., 2005; Leask et al., 2004).

C. Uses Escolar and oilfish have been variously used as food. Oilfish has been traditionally used by Polynesians and Melanesians as a purgative medicine (Cox and Reid, 1932; Gudger, 1925). In the Union of the Comoros, an island nation in the Indian Ocean, oilfish is targeted as a food source and caught by local people regularly (Helfman et al., 1999; Stobbs and Bruton, 1991). In Bermuda, oilfish is used as food and is acclaimed as an excellent eating (Gudger and Mowbray, 1927). In the Canary Islands and other seafaring regions in Spain, the fish is also used as a folk medicine in drinking broth made from the bones to relieve constipation (Raisfeld and Patronite, 2006; Ruiz-Gutierrez et al., 1997). About 10 tons of escolar and oilfish were sun-dried and consumed annually in Japan before their sale was prohibited (Mori et al., 1966c). Although described by health authorities as a purgative in Taiwan, they are openly sold and served as sashimi and fish steaks under the name You-yu (literally oilfish) (Fig. 1.3). Their roes (Fig. 1.4) are pressed and dried in Tungkang (Dunggang), Taiwan, and hailed as one of the three treasures of that little port as a new delicacy (Li, 2007). Compliments to the fish have come from many gastronomes. Berman et al., (1981) commended that escolar had a very agreeable flavor with a soft, butter-like texture. Some people regard the fish as a delicacy; for instance, Bykov (1983) remarked in his book that ‘‘The taste qualities of this fish (oilfish) are high. It is an excellent table fish.’’ Raisfeld and Patronite (2006) praised it (escolar) as a dream fish, like toro (tuna), but cheaper.

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FIGURE 1.3 You-yu sold as skinless and boneless fillet at fish market in Tungkang, Taiwan. The sample was identified as escolar using DNA sequencing by the authors (photo provided by authors).

The two fish are banned in Italy, Japan, and the Republic of Korea. In the rest of the world, they are generally not regarded as suitable for catering. As a result, they are frequently marketed and labeled as other more expensive commercial fish but at lower prices. There are various suggestions for their exploitation, with the hope that their unusually high levels of wax esters could be utilized for the good of mankind. Wax esters derived from orange roughy and oreo dories have been included in various industrial processes like cleaning and degreasing (Nichols et al., 2001). Wax esters enriched in o3 LC-PUFA can be absorbed by rats, and wax esters are less prone to oxidation and can be better formulated than liquid o3 derivatives (Gorreta et al., 2002). Thus, wax esters enriched in o3 can be a food supplement as there is increasing evidence that a diet high in o3 LC-PUFA may help prevent coronary heart diseases (Iacono and Dougherty, 1993), and high level of wax esters in escolar and oilfish can act as a potential source for this purpose. Wax esters from escolar were processed by deacidification, decolorization,


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FIGURE 1.4 You-yu roe retailed in Tungkang, Taiwan. One of the samples was identified as escolar using DNA sequencing by the authors (photo provided by authors).

hydrogenation, and distillation to obtain a semisolid wax at room temperature; the refined wax was tested safe and useful as a base for medicine and cosmetics (Ukishima et al., 1987). A further value-added process involved removal of most of the lipid from escolar meat by repeated alkaline washing to make a gel, which was tested and found to be a better raw material than the commercial bigeye snapper used currently for surimi (ground fish meat) production (Pattaravivat et al., 2008). Additionally, on the internet, there are discussions on the possibility of using escolar and oilfish for slimming or weight reduction. The value of this application is doubtful as only the oil (wax esters) would be discharged.

D. Supply As escolar and oilfish are widely distributed in the tropical and temperate seas, they are frequently caught and marketed as a result of by-catch with other commercially important species (Mori et al., 1966c; Tserpes et al., 2006). It was reported that about ten tons of by-catch oilfish and escolar were sun-dried and consumed annually in Japan before the sale prohibition was implemented (Mori et al., 1966c). Up to 400 tons of escolar are caught annually in Australia (Shadbolt et al., 2002). In 2003, escolar catch accounted for up to 16,501 tons of total by-catch species in longline fishing conducted by the Southern and Western Tuna and Billfish Fishery

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(Lynch, 2004). In a report on the by-catch of tuna longliners from a South Korean observer program, escolar and oilfish ranked the second (20.8%) and third (15.6%) most common by-catch fish species, respectively (Yang et al., 2005). According to the US National Marine Fisheries Services, annual landings of escolar varied between 40 and 80 tons from 1999 to 2007, while that of oilfish increased from 32.4 tons in 1999 to 216.3 tons in 2007 (Figs. 1.5A and B) (NMFS, 2008). Taiwan has a decade-long record of oilfish harvest, and the annual catch increased 15-fold from 2,700 tons in 1999 to near 42,000 tons in 2007 (Fig. 1.6). A 90 80

Quantity (in ton) Price per ton (100US$/ton)

70 60 50 40 30 20 10 0

1999 2000 2001 2002 2003 2004 2005 2006 2007 Year

B 250 Quantity (in ton) Price per ton (100US$/ton)

200

150

100

50

0

1999 2000 2001 2002 2003 2004 2005 2006 2007 Year

FIGURE 1.5 (A) The landing quantity and price of escolar in the US from 1999 to 2007. (B) The landing quantity and price of oilfish in the US from 1999 to 2007. Data from NMFS (2008), National Marine Marine Fisheries Services Annual Landings Database.


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45,000 Quantity (in ton)

40,000

Price per ton (NT$/ton)

35,000 30,000 25,000 20,000 15,000 10,000 5000 0

1999

2000

2001

2002

2003 Year

2004

2005

2006

2007

FIGURE 1.6 The landing quantity and price of oilfish in Taiwan from 1999 to 2007. Data from Taiwan Fisheries Agency (1999–2007, Taiwan Fisheries Yearbook).

In the market, escolar is often deep skinned, and the skin and red muscle are discarded. The longitudinal portion of muscle tissues is cut parallel to the backbone and then into chunks or blocks of white skinless and boneless fillets. In sushi bars or fish markets, escolar chunks are cut into slices and served as sashimi in Hong Kong and Taiwan under the names of Bai-yu-tuan and You-yu, respectively. Escolar of smaller sizes may be cut into transverse sections through the backbone, and the cutlets are retailed with the skin on (Ruello, 2004). Oilfish, on the other hand, is more often retailed as cutlets. The scales on the outside of the fish are removed, leaving big grayish quadrangular patterns of the skin remaining on the cutlets.

E. Mislabeling and mishandling Escolar and oilfish are of low commercial values because of their kerriorrheic properties. They are considered as ‘‘not suitable for catering’’ or even banned from sale in various countries. However, they are commonly marketed as a result of their substantial by-catch with tuna and swordfish (Shadbolt et. al., 2002; Tserpes et al., 2006). According to the European Communities (Labelling of Fishery and Aquaculture Products) Regulations 2003 (S.I. No. 320 of 2003), L. flavobrunneum and R. pretiosus must be marketed as escolar and oilfish, respectively, and no other commercial names can be used alternatively. Yet, both species are usually mislabeled as sea bass, butterfish, rudderfish, white tuna, or codfish either

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Ka Ho Ling et al.

intentionally or accidentally. Under these circumstances, outbreaks of keriorrhea associated with consumption of escolar or oilfish have been repeatedly reported in several continents (Berman et al., 1981; Feldman et al., 2005; Givney, 2002; Gregory, 2002; Jacquet and Pauly, 2008; Leask et al., 2004; Ling et al., 2008a,b; Shadbolt et al., 2002; Waldman et al., 2006). Besides wax ester poisoning, escolar and oilfish are common candidates for histamine poisoning, which has a greater affect than keriorrhea and can cause more serious manifestations. Escolar and oilfish contain high levels of histidine in their muscles (Feldman et al., 2005; Kan et al., 2000; Leask et al., 2004). If these fish are inadequately refrigerated, bacteria can multiply and convert histidine into histamine, also termed scombrotoxin (FDA, 2001b). This conversion often happens when large numbers of unsold fish steaks are stocked over time to avoid food inspection in case of a related keriorrhea outbreak. The fish may reappear later in markets, but at that time, the steaks may be contaminated and not suitable for consumption. Scombrotoxin, like wax esters, is heat stable and is not destroyed by cooking; it can lead to cardiovascular, gastrointestinal, and neurological disorders (FDA, 2001b; Feldman et al., 2005; Leask et al., 2004). Therefore, detection of wax esters could help prevent more severe food poisoning from happening once any keriorrhea outbreak is reported (Ling et al., 2008a,b).

F. Global concerns Australia, Canada, the United States, and a majority of member states of the European Union have issued special guidelines toward trading and consumption of the two fish. Italy and Japan have banned their import and sale (Alexander et al., 2004) before 2007. However, outbreaks of keriorrhea are still reported occasionally across continents. Recently, an outbreak of over 600 cases of keriorrhea occurred in Hong Kong toward the end of 2006 (Chong, 2007; Chung, 2007a; Connolly et al. 2007; Jacquet and Pauly, 2008; Ling et al., 2008a,b). The packages of oilfish cutlets were mislabeled as codfish (Chong, 2007; Ling et al., 2008a,b). Escolar, on the other hand, was found offered as sushi or sashimi under the name of snowfish or white tuna (Chung, 2007b; Ling et al., 2008a,b; Mok, 2007). In February 2007, similar fish cutlets were found in Chinatown in Toronto, Canada, and resulted in a keriorrhea mini-epidemic there. Three months later (May 2007), the oily fish was found sold as white tuna sushi in South Korea and several cases of illness were reported, leading to the South Korean government prohibiting the use of oilfish and escolar for human food and banning any import of the fish in August 2007 (Stenhouse, 2007). Escolar- or oilfish-related illness has long been recognized, yet the problems have never been eradicated and still occur repeatedly (Shadbolt et al., 2002). The public always express great concern over


15

this problem; for instance, the oilfish scandal in 2006 awoke the Hong Kong public to this food safety issue and there were strong demands for regulating and identifying these two fish (Goh, 2007). Indeed, voices to completely ban the fish are on the rise. The industry, however, holds an opposite opinion and insists that the two fish are not toxic and are suitable for catering provided that certain guidelines are followed. In addition, there are great individual differences in terms of susceptibility. Some people consider the fish as a delicacy and enjoy the fish without problems, while others experience frequent keriorrhea (Ruello, 2004). Therefore, it is a dilemma for the responsible agencies as to whether to ban the two fish. As a result, different countries have different rules and the rules also change with time making it a nightmare to food safety and control agencies in case of further outbreaks.

III. REGULATION AND LITIGATION A. Regulation Because of the differential impact of escolar- or oilfish-related problems around the world, and also variation in individual susceptibility, different governments continue to promulgate only modest regulation on both fish. Only three countries, Japan, South Korea, and Italy, completely ban the trading and import of the two fish, while other countries only issue special guidelines or warnings toward them (Table 1.2). It therefore remains that the majority of countries do not have any regulations for the two fish.

B. Litigation Although outbreaks of keriorrhea have repeatedly occurred worldwide, it is rare to find cases of prosecution or litigation. However, recently in Hong Kong, a supermarket chain was fined over selling oilfish mislabeled as codfish (Lau, 2007; Wong and Lam, 2007). In that case, a total of 14 complaints were received by the Hong Kong Centre for Food Safety regarding diarrhea or serious stomach upsets after consumption of fish cutlets labeled as cod purchased from the supermarket chain. It was later confirmed that the so-called cod fish was actually oilfish. The Food and Environmental Hygiene Department, HKSAR, later initiated prosecution toward the supermarket chain. Finally, the supermarket chain pleaded guilty of selling food not of the substance expected by consumers and was fined HK$45,000 for mislabeling oilfish as cod and ordered to pay the cost of laboratory tests. In Hong Kong, sale of oilfish is not regulated because it is not considered poisonous. The magistrate, however, still condemned

TABLE 1.2 Polices on escolar and oilfish in various countries or cities Country/city

Authority

Action/recommendation

References

Australia

Queensland Health, Queensland Government Canadian Food Inspection Agency

No ban. Recommended that the fish are not suitable for catering

Queensland Health (2008)

No ban. Recommended to choose smaller portion sizes and prepare the fish in a way to reduce oil content No ban. Cautioned the Danish fish importing companies and issued cooking and storage recommendations No ban. Issued opinion from advisory group. Recommended a notification to public of the potential health risks and proper preparation practices No ban. Published information concerning potential problems in connection with the consumption of the fish No ban. Issued guidelines on labeling and handling the fish

CFIA (2007)

Canada

Denmark

Danish National Food Administration

European Union member states

European Food Safety Authority, European Union

German

German federal Institute for Risk Assessment

Hong Kong

Centre for Food Safety

Alexander et al. (2004)



WGNCO (2007)

N/A Japanese Ministry of Health and Welfare Department of Health

Banned import and sale Banned import and sale

Alexander et al. (2004) Kawai et al. (1985)

No ban. Recommended the fish are not suitable for catering

Singapore

Agri-Food and Veterinary Authority

Sweden

Swedish National Food Administration

United Kingdom

Food Standards Agency

USA

Food and Drug Administration

No ban. Issued notification of the potential health risks and required proper labels No ban. Cautioned the Swedish National Fish Trade Association and issued cooking and storage recommendations No ban. Issued notification of the potential health risks and mislabeling problems of the fish Reversed to no ban. Advised against the sale of the fish in intrastate/interstate commerce, and requested warning labels

Macau Disease Control Centre (2007) Chua (2007)

Italy Japan Macau


Statham (2003)

Yohannes et al. (2002)

18

Ka Ho Ling et al.

the supermarket chain for contravening food safety regulation (Public Health and Municipal Services Ordinance, Hong Kong Laws Chapter 132) by failing to ensure its products were safe and labeling accurate, and pointed out that the supermarket had committed very serious offence in selling, without warning, a product that was generally unsuitable for human consumption.

IV. BIOCHEMISTRY AND TOXICITY A. Wax esters and their biological roles Wax esters are carboxylic esters consisting of a fatty acid esterified to a fatty alcohol (Fig. 1.7), wherein both the acids and alcohols can be either saturated or unsaturated (Kolattukudy, 1976). Wax esters are present in different organisms, from the seeds of jojoba to the head oil of sperm whale (Busson-Breysse et al., 1994; Spencer et al., 1977; Takagi et al., 1976). Wax esters serve a variety of biological functions; for instance, they are used as energy reserve in seeds and roes, provide buoyancy in dinoflagellates and pelagic invertebrates, and prevent water loss as in the waxy layer on the cuticle of insects ( Joh et al., 1995; Nelson et al., 2000; Phleger, 1998). Although wax esters can be found across different taxa, the major muscle lipid components of most fish species, including many commercially important fish, are triacylglycerols and phospholipids. Wax esters, in contrast, are considered less common lipid components, and where they occur in deep-sea fish species provide a way to enhance buoyancy (Bone, 1972; Lee and Patton, 1989). The source of such a high level of wax esters (up to 20% of wet weight) in escolar and oilfish is still unknown, but may possibly be formed by similar mechanisms as in another wax esterrich fish, orange roughy (Hoplostethus atlanticus Collett). The ability to synthesize large amounts of long-chain alcohols is the key to determine whether a marine animal produces wax ester or triacylglycerols as its major neutral body lipid (Lee and Patton, 1989). In a study of wax esters synthesis in Euchaeta norvegica, which is a wax ester-rich zooplankton, radio-labeled glucose or alanine was given to the organism and most of the radioactivity in the wax esters was detected in the alcohol moiety, implying that fatty alcohols are synthesized de novo from nonlipid precursors, while the fatty acids in wax esters are sourced from dietary fatty O R

C

O

R’

FIGURE 1.7 The basic structure of a wax ester (R ¼ fatty acid chain; R0 ¼ fatty alcohol chain).


19

acids (Henderson and Sargent, 1980). Sargent et al. (1983) in their study of orange roughy suggested that the wax esters could be produced by (a) de novo biosynthesis of 20:1 and 22:1 fatty acids that are then reduced to fatty alcohols, (b) chain elongation and desaturation of shorter chain dietary fatty alcohols and fatty acids to yield long chain fatty acids that are finally reduced to alcohols, or (c) or modification of dietary fatty alcohols and acids. In orange roughy, wax esters are stored extracellularly (Phleger and Grigor, 1990). Extracellular wax esters serve only for buoyancy. The storage of wax esters could be superior to that of triacylglycerols under certain physiological situations. A unit volume of wax esters provide approximately 70% more upthrust than the same volume of triacylglycerols in seawater with a density of 1.025 g/cm3 (Sargent, 1978). In addition, wax esters are essentially noncompressible, and are superior to a gas-filled swim bladder, during vertical migration (Phleger et al., 1999). Escolar and oilfish, which both lack a swim bladder, could travel vertically at depths between 100–800 m and 200–1100 m, respectively (Nakamura and Parin, 1993), and wax esters provide them with better buoyancy control. High concentrations of wax esters in the fish skin, like the function of wax ester-rich blubber in whale, help insulate them from the freezing deep-sea environment. Indeed, oilfish, similar to orange roughy, has the highest oil content (32.3%) in the integument (Bone, 1972).

B. Toxicity It was reported that most people experience keriorrhea without bowel cramps or abdominal discomfort, implying that the frequent passage of oil in most people is caused by the lubricant effect of the oil, but not by an irritant effect as in the case of toxic substances in ordinary diarrhea (Du Plessis et al., 2004). Oilfish was previously called ‘‘castor-oil fish’’ based on an erroneous report that its oil was composed of 13% hydroxyoleic acid (Gudger, 1925) and a mistaken test that falsely indicated a similar purgative ability between the fish oil and castor oil (Cox and Reid, 1932). Unlike wax esters, hydroxyoleic acid, which is the purgative chemical in castor oil, causes diarrhea by an irritant effect on the bowel instead of the lubricating and pooling effects of wax esters to the rectum (Berman et al., 1981). Indeed, studies revealed that hydroxyoleic acid is unlikely to be present in oilfish (Mori et al., 1966c; Nevenzel et al., 1965). Wax esters are not destroyed or decomposed during cooking. Resistance to digestive enzymes, such as lipase, and a low melting point (in oily state at human body temperature) results in pooling of large amount of these lubricant wax esters in the rectum leading to keriorrhea. In a strict sense, wax esters are not completely indigestible in mammals. Wax esters are hydrolyzed by lipases at a very slow rate and the products, especially fatty alcohols are only slowly absorbed. Therefore,

20

Ka Ho Ling et al.

dietary wax esters are little absorbed and the majority is excreted in a mixture of wax esters (altered or unaltered) and fatty alcohols. Rats that were fed with single small doses of cetyl palmitate (major wax ester in spermaceti) had triacylglycerols present in the lymph (Munk and Rosenstein, 1891). Large doses of mutton-bird oil (mainly as cetyl and oleyl oleates) fed to rats were partially absorbed (Carter and Malcolm, 1927). It was also true for cats that cetyl esters and cetyl alcohols were found in the feces (Carter and Malcolm, 1927). Moreover, rats on a diet with 15% jojoba oil (rich in wax esters) absorbed 70% and excreted the remainder as wax esters and free alcohols, while a purgative effect was observed for this type of diet (Savage, 1951). Again, partly absorbed and partly excreted wax esters and free alcohols were observed in rats fed with feeds containing 15% of oleyl palmitate (Hansen and Mead, 1965). There is no specific wax ester digestive lipase in mammals. Hydrolysis of wax esters is carried out by the same lipase that also acts on triacylglycerols. Savary (1971) showed that purified mammalian pancreatic lipase is 10–50 times slower in hydrolyzing wax esters than triacylglycerols. The reasons for a much slower hydrolysis of wax esters by lipase are product inhibition and hydrophobicity. The hydrolyzed products, fatty alcohols and fatty acids, form an oil or solid phase in water. The products diffused only slowly out of the oil-water interface and the bulk of insoluble reaction mixture cause ‘‘product smothering’’ to the water-requiring lipase (Lee and Patton, 1989). Moreover, the large amount of slowly absorbed, thus accumulated, fatty alcohols could reverse the reaction and result in synthesis of wax esters. Hansen and Mead (1965) showed that synthesis of waxes can occur in the intestine of rats fed with oleyl alcohol (fatty alcohol). The hydrophobic nature of wax ester molecules can hamper their interaction with the active site of lipase (Lee and Patton, 1989). All of these properties engender a very ineffective hydrolysis of wax esters, and if a large dose of wax esters is consumed, much of the wax esters are passed through the intestines without digestion and absorption. Therefore, a pooling of large amounts of these lubricant wax esters in the rectum leads to keriorrhea. Data from one escolar-associated outbreak found no correlation between BMI, age, health status, and amount of fish consumed to the severity and occurrence of symptoms, while other factors, such as variability in wax ester content in different fillet cut depths, could be relevant (Yohannes et al., 2002). Unlike some fish species, such as herring, which have uniform muscle oil content along the body (Brandes and Dietrich, 1953), muscle oil content in escolar and oilfish is not evenly distributed. Bone (1972) found that muscle oil content in oilfish increases from 14.5% (near vertebral column) to 24.7% (near the skin). A similar trend was also observed by Ruiz-Gutierrez et al. (1997) with higher oil content found in subcutaneous muscles than the periosteum. However, the lipid profile for


21

dorsal and ventral muscle portions is much the same (Ruiz-Gutierrez et al., 1997). Moreover, there is a tendency that muscle oil content is higher at the anterior part and decreases toward the posterior end (Bone, 1972). The highest oil content (32.3%) is found in the integument (Bone, 1972). Wax ester content in a fish species also could vary with environmental factors, such as catch location, diet of the fish and physiological factors, such as gender and reproductive stage (Nichols et al., 2001). For instance, wax ester content in mullet increased sharply in the roes during the reproductive season (Iyengar and Schlenk, 1967). Seasonal variation of wax ester content may account for the fact that a majority cases of oilfishrelated outbreaks in Australia occurred between August and November (Doyle, 2002). Although painless, keriorrhea was frequently reported as the only symptom associated with escolar consumption (Berman et al., 1981; Ruello, 2004). Other reports, however, recorded much severer symptoms, such as abdominal cramps, nausea, headache, and vomiting, after escolar consumption (Yohannes et al., 2002). Yohannes et al. (2002) indicated that scombroid (histamine) poisoning was unlikely the reason for the severer symptoms in the 2002 outbreak, given that the onset was very rapid (45 min) while symptoms common for scombroid poisoning, including fever, flushing, and rapid pulse rate, were not detected.

C. Animal tests A limited number of studies have been performed with animals. In one case, two cats were fed 20 g of escolar flesh each. The smaller cat weighing 530 g exhibited diarrhea with frequent watery stools 4 h after consumption, while the larger animal weighing 810 g did not show any diarrhea (Mori et al., 1966c). Due to the limited sampling size and the experimental design, the divergent responses could be due to a difference in sensitivity or dosage level; the smaller cat consumed approximately 50% more flesh (0.038 g flesh/g body weight) than the larger one (0.025 g flesh/g body weight) in terms of the flesh consumed per unit body weight. Toxicity of the wax esters from escolar and oilfish was also assessed in rats (Mori et al., 1966c). A total of 20 rats were divided into five groups. The test groups were fed daily with rice, casein, yeast, and salt mixture together with escolar flesh (7.5 g), oilfish flesh (7.5 g), escolar oil (1.5 g), or oilfish oil (1.5 g), while the control group was fed the same without the flesh or fish oil. Signs of seborrhea, with oil smudging on hairs, mouth, and belly, was observed on the second day of feeding in all, except the control, groups. All 16 rats fed with either flesh or fish oil showed diarrhea and 13 of them died within 10 days. Seborrhea is another long-term side effect of eating the oily fish. Wax esters are released through the sebaceous gland of the skin, blocking the

22

Ka Ho Ling et al.

pores and potentially interfering with metabolism. Seborrhea and acute fatality was reported in animals fed with various wax esters (Matsuo, 1962), but not by Hansen and Mead (1965), where rats were fed with oleyl palmitate. Two studies conducted on orange roughy, another deep-sea fish rich in wax esters, can be illuminating. Rats were fed for 28 days a diet containing various amounts of flesh from orange roughy. Rats on a low dose diet of less than 360 g orange roughy/kg body weight (30 g wax esters/kg body weight) displayed no observable difference from the control groups on a wax ester-free diet. In the group fed with 540 g orange roughy/kg (44 g wax esters/kg), persistent oiliness around the anus and fecal smudges on food pots were observed. The hairs over the whole body became heavily coated with oil and the excretion of oily feces was observed in the groups on a diet of 720 and 1430 g orange roughy/kg. All rats on 2870 g orange roughy/kg (233 g wax esters/kg) diet died within 11 days ( James and Treloar, 1984). The second study was on pigs fed with orange roughy flesh at 6.7 g/kg/day (equivalent to a daily fish meal of 500 g for an average person of 75 kg body-weight) over an extended period (starting from live weight at 20 kg until 80 kg). All pigs stayed healthy and gained weight throughout the whole period. No purgative effect was observed ( James and Body, 1986). Based on these results, the two reports concluded that normal consumption of small portions of orange roughy by humans generally would not cause any serious health problem. An estimate of the safety window can also be made by taking into consideration the fact that escolar and oilfish have three to four times more wax esters than orange roughy (Nichols et al., 2001).

D. Human studies No clinical studies are available, except three reports that described volunteer tests on the responses after consumption of escolar or oilfish (Berman, et al., 1981; Cox and Reid, 1932; Ruello, 2004). In the study by Cox and Reid (1932), one of the authors ingested an ounce (about 28 g) of extracted oilfish oil (equivalent to about 140 g of oilfish flesh) and reported no symptoms. In the second study (Berman et al., 1981), two of the authors consumed about 500 g of baked escolar flesh. They found the fish to have a very agreeable flavor with a soft, butter-like texture. After 12 symptom-free hours, oil began to be passed per rectum. It was difficult to contain the oil that was pooling in substantial quantities in the lower rectum, and therefore frequent evacuation was necessary. Approximately 10 ml of oil were passed on one occasion. The oil was clear orange or green in color, inoffensive in odor, and not on most occasions contaminated significantly by fecal material. The authors did not experience any bowel cramps or visceral discomfort, suggesting that the discharges were


23

caused by the lubricant effect of the oil, rather than by any irritant action. The oil passed per rectum was further analyzed and confirmed as wax esters, suggesting that wax esters were not digested in humans and passed through the gastrointestinal tract unchanged. In the third report (Ruello, 2004), the author and his wife consumed 140–260 g of baked or grilled escolar flesh on seven occasions with remarkably different experiences and varying degrees of painless keriorrhea. The author experienced a very mild to heavy form of keriorrhea after each meal of fresh or frozen fish, while his wife experienced only one episode of mild keriorrhea. According to the author, it was typical keriorrhea and painless passage of oil per rectum, with no diarrhea (watery liquid feces) at any stage. He also highlighted the potential for embarrassment from stained clothing arising from the unanticipated passage of oil as an aerosol with flatulence (or when at the toilet). Although the limited and informal records may not be conclusive, the author did emphasize that the one larger portion of 260 g did lead to ‘‘an unsettled lower intestine’’ and discomfort at 6 h after consumption and stronger oil flow, whereas the smaller portions triggered milder keriorrhea with an onset time at 13 h post ingestion. The author also added that, in a casual trial by nine informed volunteers on 150–180 g of escolar fillet, only three reported mild doses of orange oil discharge for about two days after the meal. Based on the three volunteer trial reports and the summaries of outbreak episodes, it may be concluded that typical keriorrhea resulting from consumption of reasonable portions of escolar or oilfish fillet will lead to oily discharges generally without warning 1–36 h after ingestion. Frequent urges for bowel movements occur due to the lubricant qualities of the indigestible wax esters accumulated in the rectum. These symptoms are generally not accompanied by other discomfort nor diarrhea. Such responses after intake of escolar or oilfish flesh are not obligatory and vary among people due to individual sensitivity. There is a possibility that other symptoms such as stomach cramps, loose bowel movements, diarrhea, headache, nausea, and vomiting, as reported in various outbreaks, are incidental or due to embarrassment, anxiety, and panic induced by keriorrhea. It is also likely that diarrhea is a wrong descriptive used by confused victims suffering from keriorrhea.

V. IDENTIFICATION AND DETECTION A. Morphological and anatomical analyses Oilfish is usually sold as cutlets with integument and bone still attached (Fig. 1.8). Bone (1972) studied the musculature of oilfish and found that it possesses a high proportion of white muscle (80%) and little red muscle in

24

Ka Ho Ling et al.

FIGURE 1.8

Oilfish cutlet. (photo provided by authors).

FIGURE 1.9 Oilfish fillet. (Regulatory Fish Encyclopedia, U.S. FDA, 1993–2008, reprinted with permission).

the myotomes (Figs. 1.8 and 1.9). Oil stored in bone is not infrequent in fish, but the high oil content in oilfish skeletal elements (21.1% in vertebral bone and 30.5% in frontal bone) is remarkable (Bone, 1972). The bone structure is indeed a girder system enclosing oil sacs, making the bone significantly less dense than water (Bone, 1972). Consumers are advised not to suck the bones because of the notably high oil content (Nordhoff, 1928). The integument of oilfish is covered with characteristic ctenoid scales with scattered pores of various sizes (Fig. 1.10) (Bone, 1972). The pores are connected to a large system of subdermal space, which can be observed in the transverse section of the fish (Bone, 1972). The characteristic integument of oilfish, if not removed, can assist a trained eye to


25

FIGURE 1.10 (A) Oilfish integument; (B) close up of oilfish integument (photos provided by authors).

identify the fish. In contrast, escolar is often sold as fillets without skin (Fig. 1.11), making it difficult to reach a definitive identification. When the whole fish is available, the highly undulating lateral line in escolar is a distinguishing characteristic (Figs. 1.2B and 1.12), which was used for identification purposes in an outbreak of histamine poisoning (Kan et al., 2000). An additional characteristic for the two fish is that they are exceptionally oily to the touch.

B. Protein analysis Polyacrylamide gel electrophoresis (PAGE) and cellulose acetate membrane electrophoresis (CAME) were applied to distinguish escolar and oilfish from 27 commercial fish based on muscle protein differences (Ochiai et al., 1984). Myogen fractions from the muscles were subjected

26

Ka Ho Ling et al.

FIGURE 1.11 Escolar sashimi purchased in Japanese restaurant in Hong Kong (photo provided by authors).

FIGURE 1.12 Fillet from escolar (Regulatory Fish Encyclopedia, U.S. FDA, 1993–2008, reprinted with permission).

to either PAGE with Coomassie brilliant blue staining or CAME with Ponceau 3R staining to visualize the protein profile. The gel was also stained for lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities to look for characteristic patterns to identify escolar and oilfish. Ochiai et al. (1984) concluded that the myogen protein could be used to distinguish escolar and oilfish from other fish, while both dehydrogenase (LDH and MDH) did not give species-specific pattern. Moreover, PAGE is said to be better than CAME for the purpose. However, if fish is processed by cooking or sun-drying, the species-specific proteins could be denatured (Carrera et al., 1999).


27

C. DNA analysis In contrast to the heat-labile proteins, DNA is relatively stable and can be tested in samples heated up to 120 C (Lenstra, 2003). It is, unlike proteins, less affected by physiological conditions, environmental factors, storage, and processing (Shaw et al., 2002). DNA sequences are now widely used for species identification in DNA barcoding (Ratnasingham and Hebert, 2007). Species identification can be made by sequence searches on public sequence databases, such as GenBank (www.ncbi.nlm.nih.gov) and BOLD (www.barcodinglife.org). DNA sequencing, which determines the actual nucleotide types and arrangements in amplified DNA fragments, is used to differentiate escolar and oilfish from other commonly marketed fish (Ling et al., 2008a,b). Four mitochondrial DNA regions, namely 12S rRNA gene, 16S rRNA gene, cytochrome b gene, and cytochrome oxidase subunit I (COI) gene, were sequenced and the results confirmed that some codfish samples on the market were actually oilfish. The four regions were used to construct four neighbor-joining (NJ) trees and they were all useful in distinguishing escolar and oilfish and differentiating the two fish from other fish (Fig. 1.13). DNA sequencing was also successful when applied to cooked oilfish samples.

D. Lipid analysis Escolar and oilfish contain a mixture of wax esters with different carbonchain length, mainly C32, C34, C36, and C38, formed by combining different fatty acids and fatty alcohols. The dominant fatty acids in escolar and oilfish wax esters are the monounsaturated fatty acids (Table 1.3), namely oleic acid (18:1o9) and eicosenoic acid (20:1o9), while the dominant fatty alcohols are saturated and monoenoic fatty alcohols (Table 1.4), known as cetyl alcohol (16:0) and oleyl alcohol (18:1o9). PUFA, which are trace components in muscle wax esters, are commonly found in wax esters from roe, they include 20:4o6, 20:5o3, 22:5o3 and 22:6o3. These differences could be due to the functional role in muscle for providing buoyancy, while that of roe is to store energy and key essential PUFA for fry development (Lee and Patton, 1989). Wax esters are more hydrophobic than analogous triacylglycerols. For each hydrocarbon chain in triacylglycerols there is one hydrophilic ester group, whereas in wax esters there is one-half of an ester group (Lee and Patton, 1989). Thus, long-chain wax esters are classified as nonpolar lipids with triacylglycerols being somewhat more polar. This small difference in polarity is applied to separate wax esters from triacylglycerols and other lipids using chromatography (Lee and Patton, 1989). The unusually high levels of wax esters in escolar and oilfish allow thin-layer chromatography (TLC) or gas chromatography to be applied to differentiate the

28

Ka Ho Ling et al.

R1 C4 E2 C3 100 A3 A2

Oilfish group

E1 A1 R2

99

E3 E5 E6 E7 100 A6

Escolar group

A4 A5 G3 W1 86

H2 88 100

H1 E4

M1 C1 100

C2

Other commonly marketed fish samples

C5 B1 100

B2 G1

J1 100 S1 S2 T1

70 100

T2 G2

0.02

FIGURE 1.13 The neighbor-joining (NJ) trees for 12S rRNA gene sequences from 34 fish samples. Escolar and oilfish could be distinguished from other commonly marketed fish (including cod, sea bass, salmon, catfish, swordfish, halibut etc.) (Ling et al., 2008a,b; data from authors).

two fish. Ling et al. (2008a,b) used TLC to rapidly differentiate escolar and oilfish from other wax ester-absent fish. In that study, lipid was extracted by hexane and applied to silica gel plates. The plate was developed in a


TABLE 1.3

16:1 18:1 20:1 a

The major fatty acids in the wax esters of escolar and oilfish muscle Fatty acids (R chain in Fig. 1.7)

Escolar (%) a (a and b)

Palmitoleic acid Oleic acid Eicosenoic acid

1.1/2 64.2/80 24.7/15

Oilfish (%) (a and c)

a

1.8/3.7 72.1/47.7 10.9/6.9

Data as in a: Mori et al. (1966c); b: Berman et al. (1981) and c: Ruiz-Gutierrez et al. (1997).

TABLE 1.4

14:0 16:0 16:1 18:0 18:1 20:1 a

29

The major fatty alcohols in the wax esters of escolar and oilfish muscle Fatty alcohols (R0 chain in Fig. 1.7)

Escolar (%) a (a, b, and c)

Oilfish (%) a(a)

Myristyl alcohol Cetyl alcohol Palmitoleyl alcohol Stearyl alcohol Oleyl alcohol Eicosenol

3.1/3/2.6 33.7/43/52.6 4.1/5/3.9 10.4/10/8.9 24.6/16/24.9 11.2/15/2.7

2.5 48.1 7.4 5.3 29.5 1.5

Data as in a: Mori et al. (1966c); b: Berman et al., (1981); and c: Nichols et al., (2001).

glass tank lined with filter paper and saturated with xylene, which was the mobile phase for resolving nonpolar lipids. After development, the plate was oven-dried and sprayed with 40% sulfuric acid in ethanol: anisaldehyde (9:0.1), and heated at 100 C until color was observed. A characteristic spot at Rf ¼ 0.6, which belongs to the nonpolar wax esters, was found only in escolar and oilfish (Fig. 1.14). Because wax esters are heat-stable, cooked oilfish samples showed an identical TLC spot to the untreated oilfish samples. Nichols et al. (2001) analyzed the nonsaponifiable lipids of escolar and oilfish by gas chromatography and TLC-flame ionization detection and revealed that the lipid class profiles and the wax ester-derived fatty alcohol profiles readily distinguish the two fish from other wax ester-rich fish, such as orange roughy and six species of deepsea oreos. The lipid class and fatty alcohol profiles of escolar and oilfish are very similar, although oilfish has higher levels of 18:1o9 and 16:1o7, and lower levels of 18:0 than escolar (Nichols et al., 2001). Based on the subtle differences in lipid class and fatty alcohols profiles, two unknown fillet samples associated with an outbreak of keriorrhea was traced to escolar (Nichols et al., 2001).

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Ka Ho Ling et al.

1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18 19 20

21 22 23 24 25 26

FIGURE 1.14 Thin layer chromatogram of oil extracted from 26 fish samples observed under visible light. Only oilfish (lanes 1–7, 11) and escolar samples (lanes 8–10) showed a characteristic spot at Rf ¼ 0.6 (Ling et al., 2008a,b; data from authors).

VI. WAX ESTER-RICH FISH AND OTHER POTENTIAL HAZARDS A. Gempylidae family Escolar and oilfish are the only species of their respective genera (Alexander et al., 2004; Nakamura and Parin, 1993), and there are another 22 species in the same family, Gempylidae (Table 1.5). Species in this family share similar characteristics and thus these species may contain indigestible wax esters in their muscle. There is evidence that the presence of wax esters is an environment-based characteristic rather than a phylogeny-based character; for example, the deeper-living members in Mycophidae have higher wax ester contents than the epipelagic species of the same family (Nevenzel et al., 1969). However, examination of the lipid content and composition in other species of the Gempylidae family, except escolar and oilfish, is limited, and further investigation appears warranted to both inform industry, health authorities, and government agencies and also to safeguard the public.

B. Other deep-sea fish Fish with more than 10% wax esters in the total lipids of body tissues are uncommon. When higher levels of wax esters are found in epipelagic fish species, they are mainly stored in roe and the body lipids of these fish


TABLE 1.5

A list of the 24 species under the family Gempylidae Species name

Common name

Diplospinus multistriatus Epinnula magistralis Gempylus serpens

Striped escolar

4

Lepidocybium flavobrunneum

Escolar

5

Nealotus tripes

6

Neoepinnula americana Neoepinnula orientalis Nesiarchus nasutus Paradiplospinus antarcticus Paradiplospinus gracilis

Black snake mackerel American sackfish

1 2 3

7 8 9 10

12 13

Promethichthys prometheus Rexea alisae Rexea antefurcata

14 15 16 17 18

Rexea bengalensis Rexea brevilineata Rexea nakamurai Rexea prometheoides Rexea solandri

11

Domine Snake mackerel

Remarksa

Consumed as food Sold frozen, as sausages or fish cake Flesh oily and may have purgative properties

Sackfish Black gemfish Antarctic escolar Slender escolar

Roudi escolar

Consumed as food Of no fishery interest Limited distribution near Namibia and western South Africa Reports of ciguatera poisoning

N/A Long-finned escolar A bycatch of deepwater prawn trawl fishery in Australia Bengal escolar Short-lined escolar Nakamura’s escolar Royal escolar Utilized as a food Silver gemfish Good edible quality and especially tasty when smoked (continued)

31

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Ka Ho Ling et al.

TABLE 1.5

Species name

Common name

Paxton’s escolar

20

Rexichthys johnpaxtoni Ruvettus pretiosus

21

Thyrsites atun

Snoek

22 23

Thyrsitoides marleyi Thyrsitops lepidopoides

Black snoek White snake mackerel

24

Tongaichthys robustus

Tonga escolar

19

a

(continued)

Oilfish

Remarksa

The flesh is very oily, with purgative properties Highly commercial, marketed fresh Rarely caught since 1980. Good for smoked fish and fish and chips

According to information listed in FishBase (2008).

have no or negligible levels of wax esters. Wax esters, however, are stored in the muscle and other body tissues in deep-sea fish. Wax esters have lower specific gravities than triacylglycerols, and their viscosities are much less influenced by temperature and pressure variations; these properties make wax esters superior to triacylglycerols or the presence of a swim bladder for buoyancy control in deep-sea fish. Therefore, it is not unusual to find high levels of wax esters in deep-sea fish. For example, orange roughy, which is a deep-sea species with high levels of wax esters (Table 1.6) (90–97% of total lipids), is commonly available in the market (Fig. 1.15). Wax esters are mainly found in the skin of orange roughy, and the removal of skin and superficial flesh (deep skinning) significantly reduces the amount of oil present. However, deep-skinned orange roughy still contain 5.5% total lipids of which as much as 93% is indigestible wax esters (de Koning, 2005). Ruello (2004) mentioned that an informant had oily discharge after eating orange roughy, and he himself experienced mild keriorrhea 38 and 60 h after consuming 300 g of this fish. A note was published in the Hong Kong Medical Journal to alert the medical professionals to this fish when dealing with sensitive patients (But et al., 2008). In Myctophidae (lantern fish family), many members contain large amounts of wax esters in the body (Table 1.6). Species in this family are well-known for their diel vertical migrations, traveling between 10 m (at night) to 3000 m (at day time), and they are abundant and small in

TABLE 1.6

Wax ester-containing fish species

Familya

Species name [valid name]b

Arripidae

Armpits trutta [Arripis trutta] Bathylagus antarcticus

Bathylagidae Carangidae Chlamydoselachidae Coryphaenidae Gadidae

Gempylidae

Seriola aureovittata [Seriola lalandi] Chlamydoselachus anguineus Coryphaena hippurus Melanogramus aeglefinus [Melanogrammus aeglefinus] Lepidocybium flavobrunneum Ruvettus pretiosus

Body part analyzedc

Wax esters (% of total lipid)

References

Roe

26

Bledsoe et al. (2003)

Muscle

5

Roe

42

Reinhardt and van Vleet (1986) Joh et al. (1995)

Liver

58

Roe Roe

36 15

Muscle

89

Muscle

92

Shimma and Shimma (1970) Lee and Patton (1989) Bledsoe et al. (2003)

Matsumoto et al. (1955), Nevenzel et al. (1965), Mori et al. (1966c) Cox and Reid (1932), Nevenzel et al. (1965), Mori et al. (1966c) (continued)

TABLE 1.6

(continued)

Familya


Body part analyzedc


Gonostomatidae

Cyclothone acclinidens Cyclothrone ataria

Whole body Whole body

29 70

Whole body

22

Whole body

17

Lee and Patton (1989)


33 20

Howellidae

Cyclothrone pallidae [Cyclothone pallida] Cyclothrone pseudopallidae [Cyclothone pseudopallida] Cyclothrone signata Gonostoma gracile [Sigmops gracilis] Howella sp.

Lee and Patton (1989) Kayama and Nevenzel (1974) Lee and Patton (1989)

Latimeridae Lotidae

Latimeria chalumnae Lota lota

Whole body Muscle Adipose tissue Roe

16 93 97 80–85

Lee and Patton (1989) Kayama and Nevenzel (1974) Patton et al. (1977)

Lutjanidae Merlucciidae

Lutjanus campechanus Macruronis novaezelandiae [Macruronus novaezelandiae] Merluccius capensis Merluccius hubbsi

Roe Roe

18 32

Nevenzel et al. (1966) Kaitaranta and Ackman (1981) Lee and Patton (1989) Bledsoe et al. (2003)

Roe Roe

25 28

Mori and Saito (1966a) Mendez et al. (1992)

References

Moridae

Mugilidae

Myctophidae

Laemonema morosum

Muscle

50

[Laemonema longipes] Lotella phycis

liver Liver

60 30

Podonema longipes [Laemonema longipes] Pseudophycis bacchus [Pseudophycis bachus] Mugil cephalus

Liver

25

Roe

26

Roe

67

Komori and Agawa (1954, 1955), Ueno et al. (1955) Komori and Agawa, (1953) Hayashi and Yamada (1976) Bledsoe et al. (2003)

Mugil japonicus [Mugil cephalus] Benthosema glaciale Centrobranchus chaerocephalus Electrona antarctica

Roe

70

Iyengar and Schlenk (1967), Spener and Sand (1970) Mori and Saito (1966a)


55 15

Lee and Patton (1989) Patton et al. (1977)

Muscle

62

Electrona carlsbergi

Whole body

7

Gonichys barnesi [Gonichthys barnesi] Gymnoscopelus braueri Gymnoscopelus nicholsi

Whole body

12

Reinhardt and van Vleet (1986) Reinhardt and van Vleet (1986) Patton et al. (1977)

Whole body Muscle

61–90 20

Phleger et al. (1999) Reinhardt and van Vleet (1986) (continued)

TABLE 1.6

(continued)

Familya

Nomeidae Notosudidae Nototheniidae


Body part analyzedc


References

Whole body

56–94

Phleger et al. (1999)

Krefftichthyes anderssoni [Krefftichthys anderssoni] Lampanyctus ritteri [Nannobrachium ritteri] Myctophum nitidulum Neocyttus helgae Neocyttus rhomboidalis Oreosoma atlanticum Protomyctophum bolini

Muscle

87

Nevenzel et al. (1969)

Whole body Muscle Muscle Muscle Muscle

12 30 22 9 8

Stenobrachius leucopsarus Symbolophorus evermanni Triphoturus mexicanus Cubiceps gracilis Scopelosaurus sp. Pleuragramma antarcticum

Muscle Whole body Muscle Whole body Whole juvenile Muscle

90 10 74 47 22 48

Patton et al. (1977) Bakes et al. (1995) Bakes et al. (1995) Bakes et al. (1995) Reinhardt and van Vleet (1986) Nevenzel et al. (1969) Nevenzel et al. (1969) Nevenzel et al. (1969) Lee and Patton (1989) Lee and Hirota (1973) Reinhardt and van Vleet (1986)

Muscle Muscle Muscle Muscle

52 76 62 85

Bakes et al. (1995) Mori et al. (1966b) Bakes et al. (1995) Ackman et al. (1972)

Percidae

Allocyttus niger Allocyttus verrucosus Pseudocyttus maculates Paralepsis rissoi [Arctozenus risso] Perca fluviatilis

Roe

80–85

Rachycentridae Sciaenidae

Rachycentron canadum Cynoscion nebulosus

Roe Roe

36 40

Scombridae

Euthynnus alletteratus Scomber australasicus Argyropelecus hawaiiensis [Argyropelecus sladeni] Astronesthes sp. Eustomias sp. Tetrogonurus cuvieri [Tetragonurus cuvieri] Hoplostethus gilchristi [Hoplostethus atlanticus] Hoplostethus islandicus [Hoplostethus atlanticus]

Roe Roe Whole body

31 26 41

Kaitaranta and Ackman (1981) Lee and Patton (1989) Iyengar and Schlenk (1967) Lee and Patton (1989) Bledsoe et al. (2003) Lee and Hirota (1973)

Whole body Whole body Whole body

20 10 40

Patton et al. (1977) Lee and Hirota (1973) Lee and Patton (1989)

Muscle

97

Mori et al. (1978)

Muscle

90

Kaufmann and Gottschalk (1954)

Oreosomatidae Paralepididae

Sternoptychidae Stomiidae Tetragonuridae Trachichthyidae

Part of the table modified from Lee and Patton (1989, Table 1) with updated information. a According to information listed in FishBase (2008). b Species name cited as the one listed in the original literature, valid name is based on FishBase (2008). c Whole body included the whole specimen, but some studies excluded guts to eliminate lipids in food.

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FIGURE 1.15

Orange roughy fillet retailed in Hong Kong (photo provided by authors).

size (FishBase, 2008). They are occasionally found in fish markets. Other deep-sea fish families that have high levels of wax esters in their muscle, such as Oreosomatidae (oreo family) and Gonostomatidae (bristlemouth family), are mostly of limited fishery interest (FishBase, 2008). The predominant lipid components in fish roes, like muscles, are triacylglycerols or phospholipids. Yet certain fish species have high levels of wax esters in their roes but not in muscles (Table 1.6) (Bledsoe et al., 2003). Wax esters are present in the roe oil, but not in other muscle and intestine tissues in amber fish (Seriola aureovittata) ( Joh et al., 1995). Wax esters are specifically located in mullet roes (Mugil caphalus) and nowhere else in the fish (Lyengar and Schlenk, 1967). The wax esters in roes, like those in muscles, may play a role in buoyancy, permeability control,


39

insulation, or as an energy reserve (Kaitaranta and Ackman, 1981). However, these wax esters may also be, specifically in roe, acting as a fatty acid reservoir for modifying structural lipids after fertilization (Bledson et al., 2003). The functional differences between the wax esters in muscles and roes could be attributed to the structural differences of wax esters in roes, in which a much higher content of PUFA are present in roes in comparison to those in muscles (Iyengar and Schlenk, 1967; Kalogeropoulos et al., 2008). Indeed, fish roes, such as mullet roes, has long been consumed (Bledson et al., 2003; Kalogeropoulos et al., 2008) and the roes from escolar and oilfish are advertised as high-priced souvenirs from Tungkang, Taiwan (Fig. 1.4). However, the high level of wax esters in these fish roes may cause keriorrhea if too much is consumed.

C. Diacylglyceryl ether (DAGE)-rich fish It was reported in Japan that students in a primary school suffered diarrhea and oil leakage after consumption of Stromateus maculatus (Iida, 1971; Sato et al., 2002). Sato et al. (2002) analyzed the lipid composition of S. maculatus and found DAGE as the major lipid class in muscle (55% of total lipids), but no wax ester was detected. They further conducted an acute toxicity test and found dose-dependent toxicity responses. On day 2 of administration, the results showed significant reduction in body weight (4.4 1.7 g), high diarrhea rate (43%), and high mortality rate (4/7) in mice given DAGE equivalent to 1/40 of their body weight. The toxicity was even higher when DAGE and triacylglycerol was given together. However, this dose is equivalent to 0.43 g of DAGE in a mouse of 17 g and 1.5 kg of DAGE in a man of 60 kg. The dose tested is far beyond normal human consumption. DAGE is widely distributed in various fish (Mori et al., 1972). Some fish, however, possess extraordinary high levels of DAGE in their muscles (Table 1.7) (Endo et al., 2001; Iida, 1971; Mori et al., 1972; Nichols et al., 2001; Sato et al., 2002). DAGE is likely used in deep-sea fish to achieve buoyancy (Endo et al., 2001). Little is reported on the toxicity of DAGE. Capsules of DAGE derived from the livers of deep sea dogfish, however, are widely marketed as neutraceuticals for human consumption (Nichols et al., 2001). DAGE is not wax. In a strict sense, DAGE excretion should not be classified as keriorrhea, which is coined specifically for wax excretion, and thus is beyond the coverage in this review. Further studies, however, are recommended concerning Stromateus maculatus and its DAGE-rich lipid and possible incidences of illness. If a broader descriptive is needed for medical description of oil leakage including wax esters and other oils, ladiorrhea, where ‘‘ladi’’ stands for oil in Greek, can be used.

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Ka Ho Ling et al.

TABLE 1.7 Diacylgylceryl ether (DAGE)- or gylceryl ether (GE)-rich fish species DAGE/GE (% of total lipid)

Family

Species name

Centrolophidae

Centrolophus niger Centrolophus sp. Tubbia sp.

14.7–92.5

Cubiceps gracilis Stromateus maculatus

25.4 20.3

Nomeidae Stromateidae

27.7 2.2–82.1

References

Nichols et al. (2001) Mori et al. (1972) Nichols et al. (2001) Mori et al. (1972) Mori et al. (1972)

VII. DISCUSSION AND RECOMMENDATIONS A. The rationale: To ban or not to ban? The incidence of keriorrhea is unlike other food-poisoning cases. Only some people have a reaction after eating the fish. Escolar and oilfish have been traditionally used for food and as a purgative medicine (Cox and Reid, 1932; Gudger, 1925; Gudger and Mowbray, 1927; Helfman et al., 1999; Raisfeld and Patronite, 2006; Ruiz-Gutierrez et al., 1997; Stobbs and Bruton, 1991). Some connoisseurs of good foods even gave them high compliments as dream foods (Bykov, 1983; Raisfeld and Patronite, 2006). Their flesh and roes are regarded as a delicacy in Taiwan (Li, 2007). Despite the shocking animal study that saw 13 out of 16 rats fed daily with high doses of escolar or oilfish die within 10 days (Mori et al., 1966c), it is very unlikely that normal human consumption at any single time could be life-threatening. Indeed, the responses to eating the fish are highly variable and unpredictable due to differences in individual susceptibility and variation in wax ester content between servings. However, the rationale is that people do not all tolerate the same food in the same manner, and some people may have medical conditions, such as food allergies, that preclude them from consuming certain items. Therefore, countries that consider the two fish dangerous and ban them should check fish on the market constantly to avoid illegal sale, while countries that consider the two fish safe should check for mislabeling to ensure that people know what they consume and, at the same time, require a proper warning sign be shown. In either case, food safety is of utmost concern whether the fish is banned or not. The highest food safety standards can


41

be achieved through education and inspection, together with appropriate penalties that deter either illegal sale or mislabeling.

B. Recommendations 1. Labeling The problem of misidentification and mislabeling of escolar and oilfish occurs throughout the entire supply chain. Escolar and oilfish are named differently in different places. Moreover, the fish are mislabeled differently across countries, for example, as butterfish and rudderfish in Australia and as sea bass in Britain. In response to keriorrhea outbreaks, a technical advisory group was established in Australia to assist the fishing industry and consumers in identification and labeling. The common names of ‘‘escolar’’ and ‘‘oilfish’’ are endorsed for L. flavobrunneum and R. pretiosus, respectively. The advisory group also distributed pictures of fish species responsible for keriorrhea to the industry and included both fish in the Australian Seafood Handbook. Similarly, the Hong Kong Centre for Food Safety issued Guidelines on Identification and Labeling of Oilfish/Cod with a view to regulating the naming, labeling and handling of the two fish (hereafter known as HK Guidelines; WGNCO, 2007). In the HK Guidelines, retailers are advised to indicate designated common names on their packages, in which case R. pretiosus and L. flavobrunneum should both be labeled as ‘‘oilfish’’ and no other names are allowed. In Britain, both L. flavobrunneum and R. pretiosus are labeled the same as ‘‘escolar’’ according to UK Food Standards Agency. To facilitate a proper communication between authorities in different countries, standard common names should be designated for L. flavobrunneum and R. pretiosus, as has occurred in Australia.

2. Risk assessment and education Many food science and public health professionals are not well-informed of fish-induced keriorrhea. The public and health professionals should be educated on the potential health threat related to exotic food, such as escolar and oilfish. User-friendly brochures or leaflets are useful to the general public, especially vulnerable groups including pregnant women and people suffering from cardiovascular and gastrointestinal illness, on the possible consequences arising from escolar and oilfish consumption. After all, well-informed consumers are less likely to panic once affected and in turn, they could better describe their symptoms and take proper action, such as seeking help and reporting to pertinent authorities, once symptoms are noted. A prompt response to any keriorrhea outbreak allows physicians and sensitive consumers to be alerted to possible hazards and facilitates rapid diagnoses. Time is an essence to the problem before it becomes widespread. The seafood and catering industry is

42

Ka Ho Ling et al.

another key player in preventing such outbreaks from becoming more prevalent. Industry should be educated on identifying problematic fish and use correct seafood names with proper labels and warning signs. Moreover, the catering industry should be educated on the appropriate manner to handle the fish so as to reduce the level of offensive wax esters and to avoid bacterial growth, which will lead to scrombrotoxin. In short, any education program needs to include the industry, consumers, health professionals, and food safety agencies in order to establish a social norm for appropriate preventive and remedial measures.

3. Warnings and handling In countries where escolar and oilfish are not banned from sale, the display of warnings and proper handling procedures should be made compulsory whenever the fish are available. In the HK Guidelines (WGNCO, 2007), retailers are advised to display supplementary warning statements, such as: (i) this fish can cause digestive discomfort to some individuals; (ii) if you are pregnant, have bowel problems, or malabsorption, you are advised not to consume this fish; (iii) if you are eating this fish for the first time, consume only a small portion; (iv) if you experience gastrointestinal symptoms after eating this fish, do not consume the fish in future; and (v) seek medical advice if symptoms persist. Both British (Food Standards Agency) and Singaporean (Agri-Food and Veterinary Authority) authorities recommend cooking methods, such as grilling, in order to reduce the oil content. In 1999, the Swedish and the Danish National Food Administrations required cooking recommendations, including cooking these fish in such a way that most of the fat could be separated from the dish and the cooking liquid must not be used for preparation of sauce, to be available where the fish are offered for sale (Alexander et al., 2004). Ruello (2004), however, did not find much difference by grilling or baking. Moreover, people are advised to remove or avoid sucking the bones because of their notably high oil content (Nordhoff, 1928). It was also recommended to keep the storage time short because of the high content of histidine (Alexander et al., 2004). For health professionals in consultations with patients complaining of diarrhea, it is pertinent to ask ‘‘Is it oily?’’ and ‘‘Have you consumed fish?’’ If answers are confirmative, the case should be reported to food inspection agencies. To follow up on the alert, food inspection agencies should collect the expelled oil sample, a residual sample of the cooked fish (if available), and any uncooked fish from the same source to check for wax esters. If wax esters are detected, DNA analysis should be applied, if possible, to identify the source species. Concurrently during the inspection process, food safety agencies should trace back to the supply source of the fish to see if proper labels were in place. If not,


43

warnings should be issued to the public, healthcare professionals and fishery industry.

4. Detection and inspection Any sound policy needs proper inspection to facilitate implementation. A good authentication method is clearly necessary to check against substitution, misidentification, and mislabeling. This is particularly important at an initial stage of a keriorrhea outbreak, to stop further circulation of incriminated samples at outlets and sources and to prevent the situation from escalating- to political uproar. The recent situation in Hong Kong was illuminating. In fall 2006, oilfish cutlets were labeled as codfish but at lower prices. Initially, scattered cases of keriorrhea and other complaints surfaced through the mass media. In response to public inquiry, the agency responsible for food monitoring merely advised citizens to buy from reliable sources and recommended the public to be cautious in purchasing fish. That recommendation could hardly curb the spread of keriorrhea and soon over 600 cases were recorded by mid-January 2007. The general public in Hong Kong was panicking and various political parties organized demonstrations demanding for proper government actions (Fig. 1.16). To help round up mislabeled items and offer a tool to the fish industry, the team led by the senior author announced in early February 2007 a rapid TLC detection method validated by molecular sequencing and GC-MS (Goh, 2007; Ling et al., 2008a,b). The uproar then subsided. Authentication methods, including morphological, anatomical, protein, DNA, and lipid analyses, can be utilized in differentiating escolar and oilfish from other commonly marketed fish. Accuracy is of utmost importance. Cost and time are also important factors in screening large numbers of fish samples, either for routine inspection or after an outbreak. The simple, rapid, and inexpensive TLC method developed by Ling et al. (2008a,b) serves as a good example for differentiating escolar and oilfish from other commonly marketed fish, which contain no wax ester. It is applicable to cooked or excreted samples as wax esters are heatstable and indigestible by humans. The whole process, from lipid extraction to staining, took less than 30 min while the equipment involved is readily available in most analytical laboratories. Because each TLC plate can accommodate more than 20 samples, and multiple plates can be run simultaneously, the method is space-saving and suitable for screening large sample sets. The method, however, cannot distinguish escolar from oilfish nor differentiate the two fish from other wax ester-containing fish. Other more thorough protocols, such as DNA sequencing, can be the next step in confirming the precise sample identity.

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Ka Ho Ling et al.

FIGURE 1.16 Demonstration demanding immediate government actions against oilfishinduced keriorrhea epidemic in Hong Kong (photo of the Democratic Alliance for the Betterment and Progress of Hong Kong, reprinted with permission).

VIII. CONCLUSIONS The escolar- and oilfish-related problems are global in scope and respect no national boundaries. Differences in opinions, lack of clinical data, confusing labeling systems, and expensive detection methods are all factors that have contributed to the prevalence of the problem over decades. Albeit the uncertainty, like playing Russian roulette, it is clear that some people consume the fish without any notable response while some others experience serious keriorrhea. It is important to regulate the fish, either by complete banning or designated labeling, and educate the general public for the potential risks regarding escolar and oilfish consumption. Keriorrhea is likely not restricted to escolar and oilfish, and other wax ester-rich fish, known or unknown, may also pose a threat to consumers. It is therefore crucial to inform health professions of the distinctive symptoms of expelled oil droplets. Lastly, the academia and research agencies should continue to investigate the lipids of various fish for the possible identification of those species with unusually high content of wax esters, given that fish are the largest group of vertebrates and a significant part of human diet.


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REFERENCES Ackman, R. G., Hooper, S. N., Epstein, S., and Kelleher, M. (1972). Wax esters of barracudina lipid: A potential replacement of sperm whale oil. J. Am. Oil Chem. Soc. 49, 378–382. Alexander, J., Autrup, H., Bard, D., Carere, A., Costa, L. G., Cravedi, J.-P., Di Domenico, A., Fanelli, R., Fink-Gremmels, J., Gilbert, J., Grandjean, P., Johansson, N., et al. (2004). Opinion of the scientific panel on contaminants in the food chain on a request from the commission related to the toxicity of fishery products belonging to the family of Gempylidae. Eur. Food Saf. Authority J. 92, 1–5. Bakes, M. J., Elliott, N. G., Green, G. J., and Nichols, P. D. (1995). Variation in lipid composition of some deep-sea fish (Teleostei: Oreosomatidae and Trachichthyidae). Comp. Biochem. Physiol. 111B, 633–642. Berman, P., Harley, E. H., and Spark, A. A. (1981). Keriorrhea—The passage of oil per rectum—After ingestion of marine wax esters. S. Afr. Med. J. 59, 791–792. Bettoso, N. and Dulcic, J. (1999). First record of the oilfish Ruvettus pretiosus (Pisces: Gempylidae) in the northern Adriatic Sea. J. Mar. Biol. Assoc. UK 79, 1145–1146. Bledsoe, G. E., Bledsoe, C. D., and Rasco, B. (2003). Caviars and fish roe products. Crit. Rev. Food Sci. Nutr. 43, 317–356. Bone, Q. (1972). Buoyancy and hydrodynamic functions of integument in the castor oil fish, Ruvettus pretiosus. Copeia 1, 78–87. Brandes, C. H. and Dietrich, R. (1953). The distribution of fat in the bodies of herrings. Veroffentl Inst Meeresforsch Bremerhaven 2, 109–121. Brendtro, K. S., McDowell, J. R., and Graves, J. E. (2008). Population genetic structure of escolar (Lepidocybium flavobrunneum). Mar. Biol. 155, 11–22. Brewer, M. S. and Prestat, C. J. (2002). Consumer attitudes toward food safety issues. J. Food Saf. 22, 67–83. Busson-Breysse, J., Farines, M., and Soulier, J. (1994). Jojoba wax: Its esters and some of its minor components. J. Am. Oil Chem. Soc. 71, 999–1002. But, P. P. H., Ling, K. H., and Cheng, S. W. (2008). Orange roughy is rich with indigestible wax esters. Hong Kong Med. J. 14, 246. Butt, A., Aldridge, K., and Sander, C. (2004a). Infections related to the ingestion of seafood. Part I: Viral and bacterial infections. Lancet Infect. Dis. 4, 201–212. Butt, A., Aldridge, K., and Sander, C. (2004b). Infections related to the ingestion of seafood. Part II: Parasitic infections and food safety. Lancet Infect. Dis. 4, 294–300. Bykov, V. P. (1983). ‘‘Marine Fishes: Chemical Composition and Processing Properties.’’ Amerind Publishing, New Delhi, India. Carrera, E., Garcia, T., Cespedes, A., Gonzalez, I., Fernandez, A., Hernandez, P. E., and Martin, R. (1999). Salmon and trout analysis by PCR-RFLP for identity authentication. J. Food Sci. 64, 410–413. Carter, C. L. and Malcolm, J. (1927). Observations on the biochemistry of ‘‘mutton bird’’ oil. Biochem. J. 21, 484–493. CBC (Canadian Broadcasting Corporation). (2007). Canadians Fall Ill After Eating Mislabeled Oily Fish. Canadian Broadcasting Corporation, Canada. http://www.cbc. ca/health/story/2007/02/23/oilfish.html. Accessed Nov. 5, 2008. CFIA (Canadian Food Inspection Agency). (2007). ‘‘Facts on Escolar.’’ Canadian Food Inspection Agency, Canada. http://www.inspection.gc.ca/english/fssa/concen/ specif/escoe.shtml. Accessed Nov. 5, 2008. Chong, W. (2007). Sale of oilfish to be curbed. The Standard 26 Jan. Chua, L. H. (2007). ‘‘Potential Health Issues Associated with Consumption of Escolar and Oilfish.’’ Agri-Food and Veterinary Authority, Singapore. http://www.ava.gov.sg/NR/rdonlyres/ 191531A0-5689-4DD9-9648-AE283FD78656/14747/Cir_FoodTradersEscolarFish1.pdf. Accessed Nov. 5, 2008.

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CHAPTER

2 Haze in Beverages Karl J. Siebert

Contents

I. II. III. IV.

The Physics of Haze Visual Perception of Haze Causes of Hazes in Beverages Diagnosing Haze Problems A. Microscopy B. Chemical analysis C. Enzyme treatment V. Protein–Polyphenol Haze A. Nature of haze-active (HA) protein B. Nature of HA polyphenols C. Nature of protein–polyphenol interaction D. Effects of conditions on particle size and haze intensity E. Particle size effects on sedimentation and filtration operations F. The effects of pH and alcohol on haze G. Time course of haze formation H. Beverage differences VI. Analyses Related to Protein–Polyphenol Haze Formation A. Predictive haze tests B. HA protein C. HA polyphenol VII. Preventing or Delaying Haze Development A. Cold maturation B. Ultrafiltration

54 57 58 59 59 60 60 60 60 63 65 70 72 72 73 74 75 75 75 75 76 76 77

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C. Adsorbents D. Enzymes VIII. Summary References

Abstract

77 80 81 82

Beverages such as beer, wine, clear fruit juices, teas, and formulated products with similar ingredients are generally expected by consumers to be clear (free of turbidity) and to remain so during the normal shelf life of the product. Hazy products are often regarded as defective and perhaps even potentially harmful. Since consumers are usually more certain of what they perceive visually than of what they taste or smell, the development of haze in a clear product can reduce the likelihood of repeat purchasing of a product and can have serious economic consequences to a producer. Hazes are caused by suspended insoluble particles of colloidal or larger size that can be perceived visually or by instruments. Hazes in clear beverages can arise from a number of causes, but are most often due to protein–polyphenol interaction. The nature of protein–polyphenol interaction and its effect on haze particles, analysis of haze constituents, and stabilization of beverages against haze formation are reviewed.

I. THE PHYSICS OF HAZE The phenomenon of haze or turbidity in beverages occurs when light passing through a sample is deflected or scattered by suspended particulate matter. An observer perceives the scattered light and, as a result, the sample appears turbid. While particles larger than colloidal size can scatter light, these usually settle out and do not form stable systems. Stable systems must either have particles of a density similar to the suspending liquid or have particles that are sufficiently small for the ambient thermal energy to keep them suspended indefinitely. The latter are called colloids, which by most definitions range between 1 and 1000 nm (largest dimension). Ambient temperatures provide energy that results in Brownian motion. This produces random collisions of solvent molecules with particles that are sufficient to keep small particles with somewhat greater density than a solvent in suspension indefinitely. While light scattering can be observed with a photometer (an instrument in which a light source, a sample, and a detector are in a straight line; see Fig. 2.1), light that fails to reach the detector could either have been scattered or absorbed. For this reason, scattering is typically observed with an instrument in which the detector is placed at some angle to the incident light beam (see Fig. 2.2). The two most frequently

Haze in Beverages

Lamp

Monochromator or filter

Sample cell

55

Transmitted light detector

FIGURE 2.1 The light path in a photometer. 90 Detector Forward scatter detector

Lamp

Lens

FIGURE 2.2

Sample cell

The light path in a turbidimeter.

used types of static light scattering instruments have detectors at a narrow angle (generally in the 11–25 range) or at 90 . The latter are also called nephelometers. In the case of instruments designed to measure very high turbidities, there may also be a backscatter detector; however, the levels of turbidity typically found problematic in clear beverages are considerably lower than this level. With most light scattering instruments, the amount of light scattered in colored solutions is underestimated because some of the scattered light is absorbed. In order to make color-corrected haze measurements or haze-corrected color measurements, an instrument that simultaneously measures scattering and transmission is required. A ratio between the two observations is then used to produce the desired result. Turbidimeters can employ white light, light passed through an optical filter, or monochromatic (laser) light. Because the relationship between the wavelength of light and the size of the particles affects scattering, instruments that use different light sources (e.g., white light vs. white light passed through a green filter) inevitably give different results. Most photometric instruments employ filters or monochrometers to select light of a narrow wavelength range; this certainly impacts the results. The intensity of scattered light depends on a number of parameters including the size, shape, and concentration of the suspended particles,

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the angle at which the scattering is observed, the wavelength of light in relation to the particle diameter, and the refractive indices of the particle and the solvent. The physics of this for spheres much smaller than the wavelength of light was described by Strutt (who later inherited the title of Lord Rayleigh) in 1871 (Strutt, 1871). Rayleigh later developed the form shown in Eqs. (1) and (2), which express the scattered light intensity at any angle relative to the incident beam, where Iy is the intensity of the scattered light at angle y, r is the particle radius, and l0 is the wavelength of light used (Thorne and Svendsen, 1962). 2 3 (1) Iy ¼ 3 ðsin z z cos zÞ z z¼

4pr y sin 0 2 l

(2)

The Mie theory (actually Mie’s solution to Maxwell’s equations for spheres) can be applied to spherical particles that are smaller than, similar in size to, and larger than the wavelength of light used (Mie, 1908). With particles much larger than the wavelength, the Mie theory can be simplified to the Fraunhofer theory. The mathematics of scattering is complicated for other than spherical shapes, and that is why the assumption that particles are spherical is often made. Many anecdotal accounts state that turbidity measured with narrowangle scattering is oversensitive to large particles, while that measured at 90 is oversensitive to small particles (Siebert, 2008). This can lead to ‘‘invisible hazes’’ that are perceptible visually but not with a turbidimeter, and vice versa. According to both the Rayleigh and Mie theories, light scattering intensity is very strongly influenced by the relationship of particle size to the wavelength of light used, with larger particles scattering light much more intensely than small particles at narrow angles (Gales, 2000; Siebert, 2008). With 90 scattering, small particles scatter substantially more intensely than larger ones. Light scattering results are highly influenced by the method of instrument calibration (Gales, 2000; Siebert, 2008). This is frequently done using formazin, which is very different in size and shape from yeast (often the largest particles encountered in a sample) or fine-particle haze (the smallest). Commercial culture yeasts are usually polyploid and larger than diploid strains, with oval shapes and mean diameters on the order of 10 mm. Fine particle hazes in filtered beverages are typically below 1 mm. Process samples or those that develop haze in package can have intermediate-sized particles. Calibration of a turbidimeter with particles of the same size as those measured should give correct results at any angle. However, the sizes of particles found in beverage samples are frequently not known in advance and may be bi- or

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even trimodal. For example, both yeast and fine-particle haze are present in samples of fermented beverages during processing. For a fixed number of colloidal-size spherical particles, 90 scattering intensity appears to be essentially proportional to the particle radius squared (Siebert, 2000); this was attributed to the particle cross-sectional area. Temperature can affect haze in several ways. Lowering temperature can result in reduced solubility of marginally soluble substances and may lead to a higher concentration of particles. This is responsible for the phenomenon known as ‘‘chill haze.’’ Typically, warming a sample will dispel most of the turbidity provoked by chilling. On the other hand, elevated temperatures can speed interactions between substances that form insoluble particles, leading to more rapid haze development.

II. VISUAL PERCEPTION OF HAZE As with instrumental turbidity measurements, the conditions under which humans observe light scattering also impact results. As expected, the geometry of the viewing system (the angle between the light beam and the observation) has a large influence (Gales, 2000). Visual observations are nearly always made with white light, but differences in the light source (e.g., incandescent, photoflood, or fluorescent lamps) presumably have some effect on the results. It has been observed that the particle size and concentration as well as the illumination intensity, solution color, and viewing background influence visual perception of turbidity. Studies were carried out with a sensory panel using polymer spheres with a number of diameters in the range 0.15–10.3 mm, each suspended in different colored solutions. Thresholds were determined using the Ascending Method of Limits. When expressed as weight or number concentration, the thresholds varied greatly, but when expressed as turbidity measured at 90 they were quite similar, regardless of the particle size or solution color (Carrasco and Siebert, 1999; Fleet and Siebert, 2005). With bright illumination, thresholds ranged from 0.21 to 2.19 nephelometric turbidity units (NTU). Surprisingly, reducing illumination intensity led to generally lower thresholds (greater sensitivity) up to a point, but further reductions produced higher thresholds (Fleet and Siebert, 2005). It appears that lower illumination results in less reflection from the sample container, making it easier to perceive the scattered light. Using light-colored rather than black viewing backgrounds led to much higher thresholds (Fleet and Siebert, 2006). This appears to be due to the difficulty in seeing scattered white light against a light-colored background. Suprathreshold particle suspensions were evaluated using Magnitude Estimation (ME) and Sensory Descriptive Analysis (Carrasco and Siebert,

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1999). Equations predicting ME or instrumental turbidity as a function of sample characteristics were developed. Principal Components Analysis was applied to the Descriptive Analysis results and this indicated that panelists responded to only two fundamental properties, degree of cloudiness and homogeneity/nonhomogeneity. Only the larger particles studied caused much change on the second axis, leading to the conclusion that when modest numbers of large particles are present, samples take on a nonuniform appearance (Carrasco and Siebert, 1999; Siebert, 2008).

III. CAUSES OF HAZES IN BEVERAGES Hazes in clear beverages (beer, wine, clear fruit juices, tea, etc.) can be caused by a variety of phenomena. Processing problems can lead to particles from filter media (such as diatomaceous earth) or adsorbents. These are not normal occurrences and can usually be readily discovered and their cause addressed. Grape juice and wine can contain tartrate particles that arise from tartaric acid in grapes forming salts with various cations. Often, this leads to regular crystals. Cool storage and additions of seed crystals or salts facilitates settling out of tartrate precipitates during processing (Jackson, 1994). Grains typically contain oxalates. As a result, grain-based beverages (such as beer) can develop oxalate crystals (mainly calcium salts). The standard practice of adding gypsum (CaSO4) to brewing water (for a number of reasons) leads to crystallization and precipitation of calcium oxalate during processing (Rehberger and Luther, 1999). Fragments of plant raw materials (e.g., grape skins or fruit pulp) can in some cases pass through a process and enter the final product. If this occurs due to a processing problem, it is generally transitory and can be addressed by refiltering the product. Microorganisms (yeast or bacteria), which may be either culture organisms added intentionally or contaminants, if not removed by filtration or sedimentation can lead to turbidity. The organisms themselves, in sufficient numbers, scatter light. The growth of some organisms alters product chemistry and may cause formation of unsightly hazes, ropes, or strings. In some cases, microbial cell fragments may arise and can be particularly problematic. For example, the disc centrifuges often used to remove yeast after brewery fermentations are known to produce shearing forces that break off yeast cell wall fragments (Siebert et al., 1987). Agitation of yeast by other means is also problematic (Lewis and Poerwantaro, 1991; Stoupis et al., 2003). In beer, the resulting particles resist sedimentation and impair filtration. Sucrose syrups from either beet or cane origin are used in some formulated products or added to coffee beverages at the point of sale

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(in the form of flavored sugar syrups). Occasionally, these products develop flocs (large gauzy-appearing structures that float in the product). A number of causes have been associated with these, but most authors have attributed this to associations between positively charged proteins and negatively charged polysaccharides that form either under acidic conditions or in products containing ethanol (Clarke et al., 1978; Foong et al., 2002; Morel du Boil, 1997). Polysaccharide–protein interaction has also been reported in apple juice, between arabinogalactan and protein (Brillouet et al., 1996). A number of polysaccharides have been associated with beverage hazes or flocs. These include arabinans in red wine (Belleville et al., 1993), starch and mannan in wheat beers (Delvaux et al., 2000), betaglucans in beer (Jackson and Bamforth, 1983), and retrograded starch in apple juice (Beveridge, 1997). Polyphenols have been implicated in hazes of many beverages including white wine (Somers and Ziemelis, 1985), apple juice (Beveridge, 1997; Van Buren and Way, 1978), and beer (Gramshaw, 1969; Steiner and Stocker, 1969). Proteins have been associated with hazes in beer (Asano et al., 1982; de Clerck, 1969), red and white wine (Dizy and Bisson, 1999; Hsu et al., 1989; Pocock and Rankine, 1973; Sitters and Rankine, 1980; Waters et al., 1995), apple juice (Beveridge et al., 1998; Hsu et al., 1989), grape juice (Hsu and Heatherbell, 1987; Hsu et al., 1987), pear juice (Hsu et al., 1990), and kiwifruit juice (Wilson and Burns, 1983). While most of the previously mentioned causes of haze can create product defects, they do not normally occur if a process is carried out properly. The most frequent cause of haze in clear beverages is protein– polyphenol interaction (Bamforth, 1999; Siebert, 1999). This occurs normally and even when a beverage is properly stabilized, protein– polyphenol haze usually develops eventually. The objective is to delay its onset so that any haze produced is imperceptible until after a product’s intended shelf life.

IV. DIAGNOSING HAZE PROBLEMS A. Microscopy Light microscopy can be used to detect particles with regular shapes (e.g., crystals) and microbes like yeast and bacterial cells (Glenister, 1971). Microscopy can also detect some irregular particles such as diatomaceous earth or adsorbents (Glenister, 1974). It is much less informative with amorphous particle hazes. The use of specific stains can, however, provide useful information. An excellent book by Glenister (unfortunately no

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longer in print) describes stains and microscopic techniques that are useful for characterizing beer hazes of various origins (Glenister, 1975). A fluorescent tag (fluorescein isothiocyanate) attached to the lectin Concanavalin A is useful in staining yeast cell wall fragments (Siebert et al., 1981). Concanavalin A specifically binds to mannan, which is prominent in yeast cell walls.

B. Chemical analysis Chemical analysis of haze materials isolated from a beverage must be interpreted with caution because composition is often not well-related to cause. For example, beer hazes typically contain a high proportion of carbohydrate, with a modest amount of protein, and little polyphenol (Belleau and Dadic, 1981; Siebert et al., 1981). In order to prevent or delay haze formation, however, it is not necessary or helpful to remove carbohydrate. Reducing the amount of either protein or polyphenol typically has that effect. As a result, it appears that the large amount of carbohydrate found in the haze was coagulated with or adhered in some way to the protein–polyphenol haze backbone.

C. Enzyme treatment Treatment with specific enzymes has sometimes been used to diagnose haze problems (Siebert et al., 1981). Conclusions of the effects must be tempered by considering that enzyme preparations may have small amounts of unspecified enzyme contaminants.

V. PROTEIN–POLYPHENOL HAZE A. Nature of haze-active (HA) protein Only proteins that contain proline bind polyphenols. Asano et al. (1982) demonstrated that the haze-forming activity of a protein is roughly proportional to the mole percentage of proline it contains (see Fig. 2.3). DNA has codes for exactly 20 amino acids. If each of these were equally present in a protein, there would be 5 mol% of each one. In fact, most proteins have much less proline than this. There are a few exceptions. Casein has about 8 mol% proline and the grain prolamins (proline-rich, alcoholsoluble proteins) are even higher. Hordein, the barley prolamin, contains about 20 mol% proline. As a result, it readily forms haze with polyphenols and is the main beer haze-active (HA) protein. Hordein contains even more glutamine (Q) than proline (P), and often these amino acids are adjacent in the protein (see Fig. 2.4). In fact, the sequence P-Q-Q-P occurs

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Haze forming capacity

120 100 80 60 40 20 0

0

20

40 60 80 Mole % proline

100

FIGURE 2.3 The relationship between the proline content of a polypeptide and its haze-forming activity with catechin based on data from Asano et al. (1982).

Q Q Q P F P Q Q P I P Q Q P Q P Y P QQ P Q P Y P Q Q P F P P Q Q P F P Q QP V P Q Q P Q P Y P Q Q P F P P Q Q PF P Q Q P P F W Q Q K P F P Q Q P P FG L Q Q P I L S Q Q Q P C T P Q Q T P LPQ-

FIGURE 2.4 Partial amino acid sequence of barley hordein (source of haze-active protein in beer); P ¼ proline and Q ¼ glutamine.

repeatedly. The adjacent location of proline and glutamine appears to provide unusually strong polyphenol binding. Some relatively prolinerich proteins (PRPs) have been found in apple juice (5 mol%) (Wu and Siebert, 2002) and grape seeds (9.5 mol% proline) (Wu and Lu, 2004). Even higher proline contents have been reported in salivary PRPs; these can contain 40–45 mol% proline and also have a substantial amount of glutamine. This protein binds ingested polyphenols, which precipitates the PRPs and removes the lubrication these normally provide. The result is the sensation of astringency (Green, 1993; Haslam and Lilley, 1988). The number of different amino acids actually found in proteins is greater than the 20 in the DNA code. This is because some of them are produced by posttranslational modification. Hydroxyproline, for example, is not coded in DNA. Proline is inserted in the peptide chain and the hydroxy group is later added to the peptidically liked proline to form hydroxyproline. Although hydroxyproline is found in some proteins that are known to be haze active, such as gelatin, polyhydroxyproline (the synthetic homopolymer of hydroxyproline) forms no haze with

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polyphenols (Siebert et al., 1996c). The polyphenol binding of gelatin (and similar proteins) appears to be entirely due to the proline they contain. Free proline does not form haze with polyphenols (Siebert et al., 1996a); it appears that only peptidically linked proline can do that. Proline differs from all the other coded amino acids in having a secondary amine group that participates in a peptide bond (see Figs. 2.5 and 2.6). Because of its ring system, proline is more rigid than most amino acids and cannot form an alpha-helix structure. As a result, it leads to a more open, less compact protein, which provides better access to polyphenol-binding sites than a more compact protein structure (Hagerman and Butler, 1981). Only one other amino acid (but not one of the coded 20), when peptidically linked, has been shown to bind polyphenols. This is sarcosine (N-methyl glycine), which has a secondary amine like proline (see Fig. 2.7), but lacks a ring system. Polysarcosine has been shown to bind to polyphenols and produce haze (Hagerman and Butler, 1981; Siebert and Lynn, 2008). So it appears that the essential R

O H N

N H

N H O

FIGURE 2.5

R

Segment of a peptide composed of alpha-amino acids.

N N N

FIGURE 2.6

O

O

Segment of a peptide composed of proline.

O N N

N O

FIGURE 2.7 Segment of a peptide composed of sarcosine.

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feature for polyphenol binding is a peptidically linked secondary amine rather than a nitrogen-containing ring system.

B. Nature of HA polyphenols The polyphenol molecular features that lead to attachment to proteins are generally understood. Simple phenols, that is, phenols containing a single hydroxy group on an aromatic ring, essentially do not bind to proteins (Eastmond and Gardner, 1974). In experiments in which various polyphenols were combined with bovine serum albumin, the energy released upon binding was observed (McManus et al., 1985). The binding energy was weak with m-diphenol, moderate with o-diphenol, and strong with the vicinal triphenol. So, two or more hydroxy groups on an aromatic ring are required and the binding is stronger when they are adjacent and when there are more hydroxy groups. One aromatic ring with two or more hydroxy groups constitutes one binding moiety. In order to cross-link two protein molecules, a polyphenol needs to have two such binding groups. ‘‘Single-ended’’ polyphenols (with only one binding moiety) can bind to proteins and have been shown to compete with HA polyphenols for binding sites in proteins under some conditions, inhibiting haze formation (Siebert and Lynn, 1998). The polyphenols in beer, fruit juices, and tea are typically members of the flavan-3-ols (see Fig. 2.8) and the proanthocyanidins constructed from them. Each of the flavan-3-ols has two asymmetric centers, at positions 2 and 3 (on the C ring). The more naturally prominent members of the flavan-3-ols are (þ)-catechin and ()-epicatechin (see Fig. 2.9). These two molecules are remarkably similar, differing only in the orientation of the hydroxy group at position 3; both are expected to have one moderately strong binding end (the B ring) and one weak binding end (the A ring). OH OH

HO

8 7 A

B O C 3

6 5

2

4

OH

OH

FIGURE 2.8

The basic flavan-3-ol structure.

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OH OH

O

HO

OH OH OH OH

O

HO

OH OH

FIGURE 2.9

The structures of (þ)-catechin (top) and ()-epicatechin (bottom).

However, one study showed substantial differences between them, with (þ)-catechin producing more haze than ()-epicatechin when combined with polyproline (the synthetic homopolymer of proline) at 25 C (Siebert and Lynn, 1998). Also prominent in some beverages are gallocatechin and epigallocatechin; in these compounds, an additional hydroxy group is located on the B-ring vicinal to the two already there (see Fig. 2.10). These molecules are expected to have one strongly binding end (the B ring with three vicinal hydroxy groups) and one weakly binding end. Proanthocyanidins are formed from flavan-3-ol ‘‘building blocks’’; although not truly polymers, it is convenient to refer to these as dimers, trimers, etc., indicating the number of flavan-3-ol ‘‘monomers’’ they contain. As the size and complexity of proanthocyanidins increase, their haze-forming activity increases (Asano et al., 1984; Hagerman and Butler, 1981; Mulkay and Jerumanis, 1983) and their solubility and likelihood of surviving processing decrease (Asano et al., 1984). As a result, beer and clear fruit juices have significant amounts of monomers and dimers but little of trimers (and higher). The prominent proanthocyanidin

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OH OH

O

HO

OH

OH OH OH OH

O

HO

OH

OH OH

FIGURE 2.10

The structures of gallocatechin (top) and epigallocatechin (bottom).

‘‘dimers’’ found in most fruit juices and beer are formed of two monomers connected from position 8 (on the A ring) of one monomer to position 4 (on the C ring) of the other. The prominent dimer in grape juice and wine is procyanidin B1 (catechin–epicatechin; see Fig. 2.11). In apple juice, procyanidin B2 (two epicatechins) predominates. The prominent dimers in beer are procyanidin B3 (two catechins joined together) and prodelphinidin B3 (a catechin and a gallocatechin joined together; see Fig. 2.11). Because of the additional hydroxy group in prodelphinidin B3, this compound is expected to be somewhat more haze active than procyanidin B3, and that has been shown to be the case (Mulkay and Jerumanis, 1983).

C. Nature of protein–polyphenol interaction The basic mechanism is that a polyphenol molecule with at least two binding sites attaches to two proteins and bridges them together. Additional polyphenol molecules attach this structure to additional protein molecules and eventually the complex grows so large that it is no longer soluble. At this point, it becomes a colloidal particle and scatters light. The

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particle may continue to grow until it is so large that Brownian motion can no longer suspend it; at that point it starts to sediment. Because warming can often disperse protein–polyphenol hazes, it is clear that covalent bonding is not involved in their formation. Asano et al. demonstrated that protein–polyphenol haze formation is inhibited by the nonpolar solvent dioxane and the hydrogen bond acceptor dimethylformamide (DMF), but not by a solution of sodium chloride (Asano et al., 1982).

OH OH

O

HO

OH OH OH HO

OH

O

OH OH OH OH

O

HO

OH OH OH HO

OH

O

OH OH

FIGURE 2.11 Continued

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67

OH OH

O

HO

OH OH HO

OH

O OH

OH OH OH OH

O

HO

OH

OH OH HO

OH

O OH

OH OH

FIGURE 2.11 The structures of the procyanidin ‘‘dimers’’ prominent in (from top to bottom) grape juice (procyanidin B1), apple juice (procyanidin B2), and beer (procyanidin B3 and prodelphinidin B3).

They concluded that the interaction involves hydrophobic and hydrogen bonding, but not ionic bonding. Siebert et al. demonstrated that preformed haze could be dispelled by adding dioxane or DMF, but adding sodium chloride solution actually increased the haze (Siebert and Lynn, 2008). Increased binding strength with increased ionic strength is a known effect

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of hydrophobic bonding (Oh et al., 1980). Hagerman and coworkers carried out a study in which two quite different types of polyphenols were each combined with bovine serum albumin; in one of these cases, the interaction was attributed to hydrogen bonding (Hagerman et al., 1998). Bianco et al. (1997) used NMR to measure the energy involved in polyphenol interactions with caffeine, a surrogate compound for peptidically linked proline. Based on the binding energy observed, the authors suggested that p-bonding occurs (in the case of peptidically linked proline, this would be manifested as stacking of the flat aromatic ring of the polyphenol with the relatively flat proline ring). A response surface model of the effects of HA protein concentration (gliadin, the wheat prolamin), HA polyphenol concentration (tannic acid, TA), alcohol, and pH on the amount of haze formed was constructed using a buffer model system (Siebert et al., 1996a). Figure 2.12 shows the effects of protein and polyphenol on haze predicted by the model at fixed levels of pH and alcohol. The model indicates that as protein increases at fixed polyphenol levels, the haze rises to a point and then starts to decline. Similarly, when polyphenol increases at a fixed protein level, the haze increases to a maximum and then declines. A conceptual model that accounted for this behavior was proposed (see Fig. 2.13) (Siebert et al., 1996c). It is assumed that there are a fixed number of polyphenol-binding sites in an HA protein, presumably related to the proline content, and an HA polyphenol molecule can attach to at the most two proteins (this is likely for steric reasons, even if a

160 140

80 60

Haze (NTU)

120 100

40 20 100

80 Ta nn 60 i 40 (m c aci g/L d )

20

500 400 300 in 200 iad ) Gl g/L 100 (m

FIGURE 2.12 Response surface model predictions of the effects of HA protein (gliadin) and HA polyphenol (TA) on the haze intensity in a model system at fixed levels of pH and alcohol. Reprinted with permission from Siebert et al. (1996a). Copyright 1996 American Chemical Society.

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[polyphenol] = [protein]

[polyphenol] < [protein]

[polyphenol] > [protein]

Polyphenol molecule

Protein molecule with fixed number of polyphenol binding sites (i.e., haze-active)

FIGURE 2.13 Concept of influence of protein–polyphenol proportion on particle size and haze. Reprinted with permission from Siebert et al. (1996c). Copyright 1996 American Chemical Society.

polyphenol has more than two parts of the molecule that can bind to protein). In some cases, a polyphenol may form an intramolecular bridge between two parts of a protein molecule, but this would not lead to haze. When there are similar numbers of polyphenol-binding sites in proteins and polyphenol-binding ends present in a system, a large network will form, corresponding to large particles and a lot of light scattering. When there is a high proportion of protein to polyphenol, polyphenols will have

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no difficulty finding sites in proteins to attach to and can readily join two protein molecules together. However, there will be few additional polyphenols available to join these protein ‘‘dimers’’ or ‘‘sandwiches’’ together. This will produce small particles with relatively little light scattering. With a polyphenol-rich system, most of the attachment sites in the proteins will be occupied by one end of an HA polyphenol molecule; however, few sites in other proteins will be available for the other end of the polyphenol to attach to. Small particles will again result, with little light scattering. This conceptual model was later verified with particle size analysis (Siebert and Lynn, 2000) (see Fig. 2.14). When each of the several concentrations of gliadin was combined with a fixed amount of TA in a model system, the particle sizes changed, and the largest particles were seen with a gliadin-to-TA concentration ratio (by weight) of 5:1, with smaller particles at higher and lower ratios. A similar pattern was seen when a fixed amount of gliadin was combined with various levels of TA. Once again, the largest particles were seen with intermediate ratios. The changes were striking in that they were not gradual shifts of a monomodal distribution. Rather, particles of one or two discrete sizes were present, depending on the protein-to-polyphenol ratio.

D. Effects of conditions on particle size and haze intensity A more detailed study was carried out with many more levels of protein and polyphenol than were used to construct the initial response surface model (Siebert and Lynn, 2000). The results (see Fig. 2.15) indicated that 16 14 Number %

12 10 8 6 4 2 0 0.1

0.6 3.8 23.6 145.6 Particle diameter (µm)

FIGURE 2.14 Particle sizes measured with 100 (&), 200 (d), 300 (▲), 400 (l), and 500 (▼) mg/L protein (gliadin) concentrations added to 40 mg/L TA. Reprinted with permission from Siebert and Lynn (2000). Copyright 2000 American Society of Brewing Chemists.

Haze in Beverages

50 Gliadin (mg/L)

5:1

Haze (NTU)

500

71

100 150

400

2:1 100

300

150 100

200 50

100 40

50 60 80 100 120 Tannic acid (mg/L)

140

FIGURE 2.15 Response surface predictions from haze intensity observations made with 30 combinations of gliadin and TA at pH 4.5. Reprinted with permission from Siebert and Lynn (2000). Copyright 2000 American Society of Brewing Chemists.

the basic concept of the relationship of protein-to-polyphenol ratio to haze intensity was correct, but that there was fine structure. Ridges of greater haze intensity were seen at 2:1 and 5:1 concentration weight ratios of gliadin to TA; these correspond to TA:gliadin molar ratios of 15:1 and 6:1, respectively. These ratios coincided with the larger size particles observed with particle size analysis. This indicates remarkably quantized behavior. With 30 different protein:polyphenol ratios, particles of only a few sizes were seen and changes were in the proportions of particles of different size.

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E. Particle size effects on sedimentation and filtration operations The dramatic changes in haze particle size seen with alterations in protein-to-polyphenol ratio in a model system, would, if this also occurs in real beverages, have profound effects on both sedimentation (e.g., cold maturation in a tank or centrifugation) and filtration operations.

F. The effects of pH and alcohol on haze The previously described response surface model was used to predict the effects of ethanol and pH on haze intensity at fixed levels of protein and polyphenol (Siebert et al., 1996a) (see Fig. 2.16). Changing the ethanol concentration at the pH of grape juice and wine (near 3) would appear to have little effect on haze intensity. Slightly above pH 4, increasing ethanol first decreased haze intensity and then increased it slightly. This is within the pH range of apple juice and beer. This effect was attributed to the known suppression of protein–polyphenol interaction caused by nonpolar solvents. Ethanol, which is semipolar, has been shown to decrease protein–polyphenol precipitation (Hagerman et al., 1998). At higher ethanol levels, haze tends to increase. This could be due to the well-known effect of ethanol in reducing protein solubility by competing for water. The effect of pH on haze intensity was striking (Siebert et al., 1996a) (see Fig. 2.16). When the pH rose from near 3 to slightly above 4, the haze intensity increased by a factor of 7 with the same amounts of protein and polyphenol. At higher pH, the haze intensity declined. While proteins

Alcohol (%v/v)

FIGURE 2.16 Response surface model predictions of the effects of pH and ethanol on haze intensity in a model system at fixed levels of protein and polyphenol. Reprinted with permission from Siebert et al. (1996a). Copyright 1996 American Chemical Society.

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often have least solubility near their isoelectric points, the calculated pI of gliadin is near 8 (Siebert, 2006) and the pKa of polyphenols is also high, typically of the order 9 or 10. The interaction of saliva protein and polyphenol also demonstrates a sharp maximum slightly above pH 4 (Siebert and Chassy, 2003). So presumably the behavior has something fundamental to do with the nature of the protein–polyphenol interaction.

G. Time course of haze formation The time course of protein–polyphenol haze development in many packaged clear beverages has a two-phase pattern (see, for example, Fig. 2.17). At first no observable change occurs for some time. After this, haze formation begins and follows an essentially linear development rate. This phenomenon has been reported in beer (McMurrough et al., 1992) as well as apple juice, grape juice, and cranberry juice cocktail (Siebert, 1999, 2006). While it is possible that soluble protein–polyphenol complexes could be formed during the initial stage and that these only grow large enough to become insoluble particles (which scatter light) during the second phase, the pattern has typically been attributed to changes in the polyphenol component that affect the development of the protein–polyphenol haze (see Fig. 2.18). Various authors have proposed that oxidation or polymerization of polyphenols enhances their combination with proteins and thus their haze-forming activity. The evidence here, however, is somewhat contradictory. Oxygen 18 added to beer reportedly ended up in the polyphenol fraction (Owades and Jakovac, 1966); this indicates that

140 120 Haze (NTU)

100 80 60 40 20 0 0

20

40 60 80 100 120 Time (days)

FIGURE 2.17 The haze development pattern in cranberry juice cocktail stored at 37 C. Reprinted with permission from Siebert (1999). Copyright 1999 American Chemical Society.

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Simple phenolics Polymerization (various mechanisms) Complex phenolics (”tannins”) + complex nitrogenous material Tannin-nitrogen complexes Combination and growth Haze material

Preformed complex phenolics Activation (e.g., by oxidation) Active phenolics + complex nitrogenous material Phenol-nitrogen complexes” Further activation and growth Haze material

FIGURE 2.18 Possible mechanisms of polyphenol polymerization or activation leading to haze development based on concepts from Gardner and McGuinness (1977).

polyphenol oxidation is occurring. It has been reported, however, that dimeric proanthocyanidins depolymerized rather than polymerized in wort and beer (Derdelinckx and Jerumanis, 1987). Radiolabeled epicatechin did not polymerize in beer to form dimers or trimers; however, labeled dimeric catechin was readily incorporated into beer haze (McGuinness et al., 1975). Some authors who looked for, but failed to find evidence of polymerization, attributed it instead to ‘‘activation’’ of some sort (Gardner and McGuinness, 1977). McMurrough et al. (1992) showed that reducing polyphenol concentration by treatment with polyvinylpolypyrrolidone (PVPP), a well-known polyphenol adsorbent (see later), led to a longer time before the start of haze formation and to a lower haze development rate once the process began. It is quite clear that some change in the polyphenols occurs that leads to the increase in the rate of haze formation.

H. Beverage differences In general, beer tends to be rich in HA protein and poor in HA polyphenol, while apple juice tends to have the opposite pattern (Siebert et al., 1996a). Grape juice is fairly low in HA protein and variable in HA polyphenol. White wines were uniformly low in HA protein, while red wines were quite variable (Siebert et al., 1996b). Vitis vinifera white wines had very low levels of HA polyphenols, while Vitis labrusca white wines had higher and vinifera–labrusca hybrids had intermediate levels (Siebert et al., 1996b). All red wines had high levels of HA polyphenols, and most had low levels of HA protein; the two exceptions were both hybrids.

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VI. ANALYSES RELATED TO PROTEIN–POLYPHENOL HAZE FORMATION A. Predictive haze tests Most producers of clear beverages employ forcing tests in which packages of the beverage are stored at elevated temperature for some time, often with agitation or periods of chilling (Bamforth, 1999; Berg, 1991). After the storage period, samples are withdrawn and their hazes are measured, either as is or after chilling. The test conditions are usually designed to produce results similar to those expected after much longer periods in the trade. A more rapid predictive test for beer is based on the addition of alcohol followed by chilling and then measuring haze (Chapon, 1973); this has generated useful results (McCarthy et al., 2005; Moll et al., 1976).

B. HA protein A number of approaches have been used to determine the amount of HA protein in a sample. The most successful of these is based on adding a fixed amount of TA to a sample (Thompson and Forward, 1969); after incubation, the turbidity is measured and the increase in turbidity observed is presumed to be proportional to the amount of HA protein in the sample. This method has the advantage that only substances able to form haze with polyphenols respond. The saturated ammonium sulfate precipitation limit (SAPL) method has also been widely used, but is far inferior in providing useful information (Berg et al., 2007; Siebert et al., 2005).

C. HA polyphenol A variation on the Thompson and Forward method was developed in which a HA peptide or peptide-like material (e.g., gelatin, gliadin, polyproline, or soluble polyvinylpyrrolidone) is added to a sample to induce haze in proportion to the amount of HA polyphenol it contains (Siebert et al., 1996a). This gives little response in beer, which contains very little HA polyphenol, and causes much larger haze increases in fruit juices and wine. Both of the tests based on provoking haze with a single addition have the disadvantage that the endogenous amounts of the complementary material influence results. For example, when adding TA to induce haze with HA protein, the amount of endogenous HA polyphenol will influence results. And similarly, when adding a protein-like material to provoke a response with HA polyphenols, the endogenous HA protein will affect results. The effects are small when measuring high concentrations of one species in the presence of small amounts of the other. In most beers, for example, which are high in HA protein and low in HA polyphenol, the

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results of a single-addition method are fairly accurate for HA protein and inaccurate for HA polyphenol. Titration provides an approach to measurement that is less affected by endogenous amounts of HA species. Methods that carry out titration manually or automatically have been widely used. The Tannometer instrument developed by Chapon (1993), which carries out automated turbidimetric titrations, has been widely used. The P-T Standard instrument of Schneider has been applied to beer (Schneider and Raske, 1997).

VII. PREVENTING OR DELAYING HAZE DEVELOPMENT It is normal to employ beverage-processing steps that lead to a reduction in the likelihood of haze formation, or at least a delay the onset of haze development beyond the intended shelf life of a product.

A. Cold maturation A traditional approach in many cases is prolonged cold storage followed by a sharp filtration, also carried out cold. Cold storage encourages formation and settling out of insoluble complexes. Reduced temperature decreases the solubility of some potential haze material and also reduces the energy from ambient heat that keeps particles suspended. As a result, some of the haze material is precipitated and left on the floor of the storage tank or taken out by the filter. The effect of cold maturation can be enhanced by the use of fining agents. These facilitate the formation of haze and precipitation of substances that, if not removed, could later give rise to haze. A number of substances have been used as fining agents, including the HA proteins gelatin and isinglass (Harding, 1979; Hickman et al., 2000), HA polyphenols such as TA, and some fine particles such as bentonite (Duncan, 1992) and colloidal silica (Hahn and Possmann, 1977) or silica sol (Goertges and Haubrich, 1992). Gelatin is often used to fine fruit juices (Bannach, 1984) and wine (Baldwin, 1992). Also often used for fruit juices and wine are bentonite or silica. Mixtures of two or more fining agents are frequently used. Gelatin, isinglass, TA, and colloidal silica are used in beer fining. Both isinglass and gelatin are derived from collagen proteins. Gelatin is largely from bovine or porcine skins, while isinglass is from the swim bladders of certain tropical fishes. Collagen is rich in both proline and hydroxyproline. Gelatin is generally thought to contain about 12% proline and a similar amount of hydroxyproline. While hydroxyproline does not participate in binding polyphenols (see earlier), it does facilitate a very open molecular structure, and this presumably aids access to the polyphenol-binding sites in the protein.

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TA is not a single well-defined compound, but rather a family of related compounds with some common structural features (Haslam, 1974). All TAs have some number of gallic acid (3,4,5-trihydroxybenzoic acid) moieties attached to a glucose molecule by ester linkages. Some of the galloyl groups can be attached to other galloyl groups (also by ester linkage) rather than the glucose. The structure of a particular TA (number and location of galloyl groups) depends on its natural source. Because galloyl groups have three vicinal hydroxy groups on an aromatic ring, they bind very strongly to HA proteins. It was shown that the strength of TA binding to proteins is a function of the number of terminal galloyl groups; that is, those with all three hydroxy groups available (Siebert, 1999). Apparently interior galloyls (those with one of the hydroxy groups occupied in an ester linkage) are not available to bind to proteins, probably for steric reasons. TA is a very strong HA polyphenol. This has benefits for use both as a reagent to measure HA protein and as a fining agent in beverage stabilization.

B. Ultrafiltration The removal of macromolecules by ultrafiltration has often been used in the production of clear fruit juices and wine (Girard and Fukumoto, 2000). This treatment removes both proteins and polysaccharides. Ultrafiltration through a 10,000 Da cut-off membrane has been shown to stabilize wines against haze formation (Flores, 1990). Because proteins are involved in beer (Evans and Sheehan, 2002) and champagne foams (Seneé et al., 1999), and these are desirable properties, ultrafiltration is not a suitable treatment for these products. Adsorbents that indiscriminately remove protein are unsuitable for the same reason.

C. Adsorbents Adsorbents that remove proteins or polyphenols are used to treat a number of beverages to delay the onset of haze formation. Protein adsorbents include bentonite and silica. Bentonite removes protein nonspecifically (see Fig. 2.19) and so is unsuitable for stabilizing beverages where foam is desirable (beer and champagne). Silica, on the other hand, has remarkable specificity for HA proteins while virtually sparing foam-active proteins in beer (Siebert and Lynn, 1997b) (see Fig. 2.20). Silica removes approximately 80% of the HA protein from unstabilized beer, while leaving foam-active protein nearly untouched at commercial treatment levels. This was shown to occur because silica binds to the same features in polypeptides that polyphenols do (peptidically linked proline; Siebert and Lynn, 1997b) (see the concept in Fig. 2.21). In contrast, in unstabilized apple juice, silica removes only on the order of 20% of the HA protein

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FIGURE 2.19 The effects on foam active (&) and HA (d) protein of treating unstabilized beer with bentonite. Reprinted with permission from Siebert and Lynn (1997b). Copyright 1997 American Society of Brewing Chemists.

5

FIGURE 2.20 The effects on foam active (&) and HA (d) protein of treating unstabilized beer with silica hydrogel. Reprinted with permission from Siebert and Lynn (1997b). Copyright 1997 American Society of Brewing Chemists.

even with very high treatment levels (Siebert and Lynn, 1997a). This difference is accounted for in Fig. 2.22; silica has limited effectiveness in polyphenol-rich beverages (especially fruit juices), where most of the polyphenol-binding sites in proteins are occupied by polyphenols, leaving few for silica to attach to (Siebert and Lynn, 1997a). Polyphenol adsorbents are mainly polyamides (Dadic, 1973). At one time various nylons were used, but PVPP is most frequently used today (McMurrough et al., 1997). The structure of PVPP (see Fig. 2.23) resembles that of polyproline (Fig. 2.6); both have five-membered, saturated, nitrogen-containing rings and amide bonds. As a result, it appears likely that PVPP binds to polyphenols in a similar manner to that of HA proteins. As with gliadin–TA binding,

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Protein molecule with fixed number of polyphenol binding sites (haze-active)

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FIGURE 2.21 Concept of silica binding to HA protein in beer. Reprinted with permission from Siebert and Lynn (1997b). Copyright 1997 American Society of Brewing Chemists.

DMF and dioxane both impede polyphenol binding to PVPP, while NaCl enhances it (Siebert and Lynn, 2008). So, both hydrogen and hydrophobic bonding appear to be involved, but not ionic bonding. While PVPP works even in protein-rich beverages, it is far less effective than in polyphenolrich beverages. PVPP removed at most half of the HA polyphenol from unstabilized beer (Siebert and Lynn, 1997b), but at high doses it took out 100% of the HA polyphenol from unstabilized apple juice (Siebert and Lynn, 1997a). This appears to be because much of the HA polyphenol in protein-rich beverages is attached to proteins at both ends and inaccessible to PVPP (see Fig. 2.24). In order for PVPP to bind to the polyphenol, the complex with protein must come apart, at least at one end. If PVPP binds to one end of a polyphenol molecule that is attached to protein at the other end, then the PVPP treatment could remove some HA protein and there is some evidence that this occurs (Siebert and Lynn, 1997b). In high-polyphenol, low-protein beverages, the vast majority of the HA polyphenol is readily accessible (see Fig. 2.25).

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Protein molecule with fixed number of polyphenol binding sites (i.e. haze-active)

Polyphenol molecule

Silica gel type adsorbent

FIGURE 2.22 Concept of silica action in a polyphenol-rich beverage. Reprinted with permission from Siebert and Lynn (1997a). Copyright 1997 American Chemical Society.

O N

FIGURE 2.23

N

O

Structure of a segment of polyvinylpyrrolidone or PVPP.

PVPP is commonly used to remove undesirable brown or pink pigments from wine (Jackson, 1994). However, because much of the color of red wine is due to polyphenolic compounds, treatment with PVPP or other polyamides can diminish the red color and so must be carefully controlled. Additions of gelatin or egg white (egg albumin has about 3.6 mol% proline) have traditionally been used to more gently remove some polyphenol from red wines to ‘‘soften’’ astringency.

D. Enzymes At one time, broad spectrum proteolytic enzymes (mainly papain and bromelain) were widely used to delay or minimize haze formation in beer (de Clerck, 1969). The enzymes cleaved protein chains, that when

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Polyphenol adsorbent

FIGURE 2.24 Concept of PVPP action in beer. Reprinted with permission from Siebert and Lynn (1997b). Copyright 1997 American Society of Brewing Chemists.

cross-linked by polyphenols, led to smaller and more soluble complexes that resulted in less haze. These enzymes were inexpensive and effective in stabilizing beer against haze formation. Unfortunately, the enzymes also attacked foam proteins, seriously impairing beer foam. This often led to the use of a foam stabilizer (typically propylene glycol alginate) to at least partially offset the damage. Most major brewers replaced enzyme (and foam stabilizer) use with adsorbent treatments. Recently, proteolytic enzymes that cleave peptide bonds only adjacent to proline were introduced (Lopez and Edens, 2005). Since proline is involved in the polyphenol-binding sites and there is little proline in the foam-active proteins, these enzymes are specific for haze proteins and do little damage to foam proteins.

VIII. SUMMARY This review has summarized knowledge of the phenomena of haze development in clear beverages. The most frequent cause of haze is from the interaction of PRPs with polyphenols that have at least two binding

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Polyphenol molecule


Polyphenol adsorbent

FIGURE 2.25 Concept of PVPP action in a polyphenol-rich beverage. Reprinted with permission from Siebert and Lynn (1997a). Copyright 1997 American Chemical Society.

locations. Beverages are generally stabilized against haze formation with fining agents or adsorbents that remove one or another of the HA species, or with enzymes that attack the HA proteins. The nature of a beverage (protein-rich or polyphenol-rich) impacts the effectiveness of particular protein and polyphenol adsorbents.

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McGuinness, J. D., Eastmond, R., Laws, D. R. J., and Gardner, R. J. (1975). The use of 14 C-labelled polyphenols to study haze formation in beer. J. Inst. Brew. 81, 287–292. McManus, J. P., Davis, K. G., Beart, J. E., Gaffney, S. H., Lilley, T. E., and Haslam, E. (1985). Polyphenol Interactions. Part 1. Introduction; Some observations on the reversible complexation of polyphenols with proteins and polysaccharides. J. Chem. Soc., Perkin Trans. II 1429–1438. McMurrough, I., Kelly, R., and Byrne, J. (1992). Effect of the removal of sensitive proteins and proanthocyanidins on the colloidal stability of lager beer. J. Am. Soc. Brew. Chem. 50, 67–76. McMurrough, I., Madigan, D., and Kelly, R. J. (1997). Evaluation of rapid colloidal stabilization with polyvinylpolypyrrolidone (PVPP). J. Am. Soc. Brew. Chem. 52, 38–43. Mie, G. (1908). [Contributions to optics of hazy media, especially colloidal metal solutions.] Beitra¨ge zur Optik tru¨ber Medien, speziell kolloidaler Metallo¨sungen. Ann. Phys. (Leipzig) 330, 377–445. Moll, M., That, V., Schmitt, A., and Parisot, M. (1976). Methods for predicting the colloidal stability of beer. J. Am. Soc. Brew. Chem. 34, 187–191. Morel du Boil, P. G. (1997). Refined sugar and floc formation. A survey of the literature. Int. Sugar J. 99, 310–312, 314. Mulkay, P. and Jerumanis, J. (1983). [Effects of molecular weight and degree of hydroxylation of proanthocyanidins on the colloidal stability of beer.] Effets du poids moleculaire et du degre d’hydroxylation des proanthocyanidines sur la stabilite colloidale de la biere. Cerevisia 8, 29–35. Oh, H. I., Hoff, J. E., Armstrong, G. S., and Haff, L. A. (1980). Hydrophobic interaction in tannin–protein complexes. J. Agric. Food Chem. 28, 394–398. Owades, J. L. and Jakovac, J. (1966). Study of beer oxidation with O18. Proc. Annu. Meeting Am. Soc. Brew. Chem. 180–183. Pocock, K. F. and Rankine, B. C. (1973). Heat test for detecting protein instability in wine. Aust. Wine Brew. Spirit Rev. 91, 42–43. Rehberger, A. J. and Luther, G. E. (1999). Wort boiling. In ‘‘The Practical Brewer,’’ ( J. T. McCabe, ed.), pp. 165–199. Master Brewers Association of the Americas, Wauwatosa. Schneider, J. and Raske, W. (1997). The protein–polyphenol balance in the brewing process. Measured by nephelometric opto-electronical rapid methods. Brauwelt Int. 15, 228–234. Seneé, J., Robillard, B., and Vignes Adler, M. (1999). Films and foams of Champagne wines. Food Hydrocoll. 13, 15–26. Siebert, K. J. (1999). Effects of protein–polyphenol interactions on beverage haze, stabilization, and analysis. J. Agric. Food Chem. 47, 353–362. Siebert, K. J. (2000). Relationship of particle size to light scattering. J. Am. Soc. Brew. Chem. 58, 97–100. Siebert, K. J. (2006). Haze formation in beverages. LWT Food Sci. Technol. 39, 987–994. Siebert, K. J. (2008). Visual versus instrumental perception of haze – A review. Tech. Q. Master Brew. Assoc. Am. 45, 90–98. Siebert, K. J. and Chassy, A. W. (2003). An alternate mechanism for the astringent sensation of acids. Food Qual. Pref. 15, 13–18. Siebert, K. J. and Lynn, P. Y. (1997a). Mechanisms of adsorbent action in beverage stabilization. J. Agric. Food Chem. 45, 4275–4280. Siebert, K. J. and Lynn, P. Y. (1997b). Mechanisms of beer colloidal stabilization. J. Am. Soc. Brew. Chem. 55, 73–78. Siebert, K. J. and Lynn, P. Y. (1998). Comparison of polyphenol interactions with PVPP and haze-active protein. J. Am. Soc. Brew. Chem. 56, 24–31. Siebert, K. J. and Lynn, P. Y. (2000). Effect of protein/polyphenol ratio on the size of haze particles. J. Am. Soc. Brew. Chem. 58, 117–123.

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Siebert, K. J. and Lynn, P. Y. (2008). On the mechanisms of adsorbent interactions with hazeactive protein and polyphenol. J. Am. Soc. Brew. Chem. 66, 46–54. Siebert, K. J., Stenroos, L. E., and Reid, D. S. (1981). Characterization of amorphous-particle haze. J. Am. Soc. Brew. Chem. 39, 1–11. Siebert, K. J., Stenroos, L. E., Reid, D. S., and Grabowski, D. (1987). Filtration difficulties resulting from damage to yeast during centrifugation. Tech. Q. Master Brew. Assoc. Am. 24, 1–8. Siebert, K. J., Lynn, P. Y., Clark, D. F., and Hatfield, G. R. (2005). Comparison of methods for assessing colloidal stability of beer. Tech. Q. Master Brew. Assoc. Am. 42, 7–12. Siebert, K. J., Carrasco, A., and Lynn, P. Y. (1996a). Formation of protein–polyphenol haze in beverages. J. Agric. Food Chem. 44, 1997–2005. Siebert, K. J., Lynn, P. Y., and Carrasco, A. (1996b). Analysis of haze-active polyphenols and proteins in grape juices and wines. In ‘‘4th International Symposium on Cool Climate Viticulture and Enology,’’ pp. VII-18–VII-21. Rochester, New York. Siebert, K. J., Troukhanova, N. V., and Lynn, P. Y. (1996c). Nature of polyphenol–protein interactions. J. Agric. Food Chem. 44, 80–85. Sitters, J. H. and Rankine, B. C. (1980). Identification of hazes and deposits in wine. Aust. Grapegrower Winemaker 17. Somers, T. C. and Ziemelis, G. (1985). Flavonol haze in white wines. Vitis 24, 43–50. Steiner, K. and Stocker, H. R. (1969). [Polyphenols and cold stability of beer.] Polyphenole und Ka¨ltestabilita¨t des Bieres. Tageszeitung fuer Brauerei 66, 550. Stoupis, T., Stewart, G. G., and Stafford, R. A. (2003). Hydrodynamic shear damage of brewer’s yeast. J. Am. Soc. Brew. Chem. 61, 219–225. Strutt, J. W. R. (1871). On the scattering of light by small particles. Philos. Mag. 41, 447–454. Thompson, C. C. and Forward, E. (1969). Towards the chemical prediction of shelf life. J. Inst. Brew. 75, 37–42. Thorne, R. S. W. and Svendsen, K. (1962). Particle size of beer turbidigens and its influence on nephelometry. J. Inst. Brew. 68, 257–270. Van Buren, J. P. and Way, R. D. (1978). Tannin hazes in deproteinized apple juice. J. Food Sci. 43, 1235–1237. Waters, E. J., Peng, Z., Pocock, K. F., and Williams, P. J. (1995). Proteins in white wine, I: Procyanidin occurrence in soluble proteins and insoluble protein hazes and its relationship to protein instability. Aust. J. Grape Wine Res. 1, 86–93. Wilson, E. L. and Burns, D. J. W. (1983). Kiwifruit juice process using heat treatment techniques and ultrafiltration. J. Food Sci. 48, 1101–1105. Wu, L. C. and Lu, Y. W. (2004). Electrophoretic method for the identification of a haze-active protein in grape seeds. J. Agric. Food Chem. 52, 3130–3135. Wu, L. C. and Siebert, K. J. (2002). Characterization of haze-active proteins in apple juice. J. Agric. Food Chem. 50, 3828–3834.

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3 Carnosine and Its Possible Roles in Nutrition and Health Alan R. Hipkiss

Contents

I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV. XVI. XVII. XVIII. XIX. XX.

Introduction Carnosine Metabolism Carnosine and Neurological Activity Carnosine and Other Tissues Possible Functions of Carnosine Control of pH Carnosine and Chelation of Zinc and Copper Ions Carnosine and Aging Carnosine and the Causes of Aging Proteotoxicity and Aging Carnosine, Oxygen Free Radicals, and Oxidative Stress Carnosine and Nonenzymic Protein Glycosylation (Glycation) Carnosine and Proteolysis of Altered Proteins Carnosine and Gene Expression Carnosine, Anticonvulsants, and Aging Carnosine and Dietary Restriction-Mediated Delay of Aging Carnosine, Regulation of Protein Synthesis, and Aging Carnosine and Corticosteroids Carnosine and Age-Related Pathology Carnosine, Diabetes, and Secondary Complications

89 91 91 92 92 92 93 93 94 95 96 97 99 100 102 103 108 109 110 110

School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, The University of Birmingham, Edgbaston, Birmingham, United Kingdom Advances in Food and Nutrition Research, Volume 57 ISSN 1043-4526, DOI: 10.1016/S1043-4526(09)57003-9

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XXI. XXII. XXIII. XXIV. XXV. XXVI. XXVII. XXVIII. XXIX. XXX. XXXI. XXXII. XXXIII. XXXIV.

Carnosine and Neurodegeneration Alzheimer’s Disease Parkinson’s Disease Carnosine and Ischemia Carnosine and Osteoporosis Carnosine and Cataractogenesis Carnosine and Deafness Carnosine and Cancer Carnosine and Wound Healing Carnosine and Immune Function Carnosine, Calcium, and Heart Failure Carnosine and Autistic Spectrum Disorders Carnosine and Blood Pressure Carnosine and Consumption of Alcoholic Beverages XXXV. Carnosine and High Fructose Foods and Drinks XXXVI. Carnosine and Dialysis Fluids XXXVII. Possible Ways to Increase Tissue Carnosine Levels: Physiological Regulation XXXVIII. Possible Ways to Increase Tissue Carnosine Levels: Dietary Supplementation XXXIX. Is there any Evidence that Changes in Dietary Carnosine Have any Effects in Humans? XXXX. Would Vegetarians Benefit from Carnosine Supplementation? XXXXI. Deleterious Effects of Carnosine XXXXII. Conclusions References

Abstract

112 114 116 119 120 120 121 121 122 122 123 123 124 124 125 125 126 128 129 130 131 132 133

The dipeptide carnosine has been observed to exert antiaging activity at cellular and whole animal levels. This review discusses the possible mechanisms by which carnosine may exert antiaging action and considers whether the dipeptide could be beneficial to humans. Carnosine’s possible biological activities include scavenger of reactive oxygen species (ROS) and reactive nitrogen species (RNS), chelator of zinc and copper ions, and antiglycating and anticross-linking activities. Carnosine’s ability to react with deleterious aldehydes such as malondialdehyde, methylglyoxal, hydroxynonenal, and acetaldehyde may also contribute to its protective functions. Physiologically carnosine may help to suppress some secondary complications of diabetes, and the deleterious consequences of ischemic–reperfusion injury, most likely due to antioxidation and carbonyl-scavenging functions. Other, and much more speculative, possible functions of carnosine considered include transglutaminase inhibition, stimulation of proteolysis mediated via effects on proteasome activity or induction of protease and

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stress-protein gene expression, upregulation of corticosteroid synthesis, stimulation of protein repair, and effects on ADP-ribose metabolism associated with sirtuin and poly-ADP-ribose polymerase (PARP) activities. Evidence for carnosine’s possible protective action against secondary diabetic complications, neurodegeneration, cancer, and other age-related pathologies is briefly discussed.

I. INTRODUCTION Carnosine (b-alanyl-L-histidine) and related compounds, homocarnosine and anserine, together with N-acetylated forms (see Fig. 3.1 for structures), are common dipeptides found in mammals, birds, and fish (Abe, 2000; Bonfanti et al., 1999; de Marchis et al., 2000; Lamas et al., 2007; Tsubone et al., 2007). One remarkable feature of these compounds is that they are often found at relatively high concentrations (Table 3.1). The highest value reported for terrestrial mammals is that of the middle gluteal muscle of the thoroughbred racehorse which contained over 100 mmol of carnosine per kg dry weight of muscle (Dunnet and Harris, 1997). It has recently been reported, however, that the carnosine plus anserine levels in turkey breast muscle can exceed 200 mM (Jones et al., 2007). It is a valid generalization that there is more carnosine in anaerobic, glycolytic, white muscle than in red, aerobic, muscle (Table 3.2).

O HO NH

O

O

H N

H N

HO N

NH

O

N

NHR NH2 R = H CAR R = COCH3 N-CAR

HCAR O

O N

N HO

HO O

NH

N

O

NH NH2

NH2 ANS

N

BAL

FIGURE 3.1 Structures of carnosine (CAR), N-acetylcarnosine (N-CAR), homocarnosine (HCAR), anserine (ANS), and balanine (BAL).

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TABLE 3.1 Carnosine and anserine concentrations in common animal species (from Aristoy and Toldra, 2004)

Pig

Beef Lamb

Loin Ham Neck Loin Neck Shoulder Neck

Chicken Pectoral Turkey Salmon Trout Sardine

Leg Wing

Carnosine (mg/100 g)

Anserine (mg/100 g)

313 449 186 375 201 39.3 94.2 180 63 66 0.53 1.6 0.1

14.5 22.9 10.7 59.7 25.4 31.5 119.5 772 233 775 589 344 1.33

TABLE 3.2 Carnosine content varies according to tissue (from Purchas et al., 2004 and Cornet and Bousset, 1999) Animal tissues/muscle

Beef

Lamb

Pig

Cheek Heart Liver Semitendinosus muscle Longissimus muscle Semitendinosus muscle Triceps brachii muscle Massetuer muscle Trapezius muscle Longissimus dorsi muscle

Carnosine (mg/100 g)

Anserine (mg/100 g)

42.9 32.6 77.5 452 491 356 251 38 147 268

6 6 6

Carnosine is also associated with nervous tissues, including the brain, where it is concentrated especially in the olfactory lobe (Bonfanti et al., 1999; de Marchis et al., 2000). However, human cerebral spinal fluid contains homocarnosine but no carnosine (Huang et al., 2005).


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II. CARNOSINE METABOLISM Carnosine is synthesized from b-alanine and L-histidine by the enzyme carnosine synthase, a reaction which also requires ATP. Studies using primary cell culture have indicated that the dipeptide is synthesized by muscle cells, glial cells and oligodendrocytes (Bauer, 2005). Although carnosine is found enriched in neurons, especially those of the olfactory lobe, it appears that these cells are capable of taking up the dipeptide following its release from glial cells in which it is synthesized. Not unexpectedly, carnosine synthesis is subject to some form of metabolic regulation; synthesis of the dipeptide is reduced when astroglia-rich cultures are treated with dibutyryl cyclic AMP and other agents which activate cyclic AMP-dependent protein kinases (Schulz et al., 1989). It has been suggested that changes in carnosine synthesis accompany morphological differentiation in muscle and astroglia (Bauer, 2005). Carnosine can be acetylated at its amino terminus to form N-acetylcarnosine, although the enzyme responsible has not been characterized. It has also been reported that N-acetyl-carnosine is readily de-acetylated in the tissues (Barbizhayev, 2008). A phosphorylated form of carnosine has also been described (Quinn et al., 1992), but again little more is known about the enzymes responsible or its function.

III. CARNOSINE AND NEUROLOGICAL ACTIVITY Animal studies have shown that carnosine can affect neurological function, not surprising given that fact the dipeptide is synthesized by the brain and that specific transporters for it are present in the choroid plexus (Teuscher et al., 2004), part of the blood–brain barrier. One possible role for carnosine within the neuronal system is modulation in glutamatergic sensory neurons (Bonfanti et al., 1999). For a detailed discussion of carnosine’s function within the mammalian brain, the reader is referred to the fine review by Bonfanti et al. (1999). The kidney brush border also possesses a carnosine transport system and there is evidence that kidney also contains an active carnosinase (Sauerhoefer et al., 2005). There is also evidence that carnosine can influence sympathetic nervous activity in kidney (Tanida et al., 2005) as well as brown (Tanida et al., 2007) and white adipose tissue (Shen et al., 2008). Other studies have shown that carnosine has antidepressant activity in rats (Tomonaga et al., 2008). In chicks, carnosine induces hyperactivity (Tsuneyoshi et al., 2007) whereas its reverse structure (L-histidinyl-balanine) has sedative and hypnotic effects (Tsuneyoshi et al., 2008). The mechanisms involved in remain obscure however.

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IV. CARNOSINE AND OTHER TISSUES Although carnosine seems to be primarily associated with the brain and innervated tissues such as muscles (skeletal and heart) at least in the rat (Aldini et al., 2004), carnosine has been reported to be present in the eye lens (Quinn et al., 1992), which suggests that its function might not be restricted nervous tissue.

V. POSSIBLE FUNCTIONS OF CARNOSINE Although carnosine was discovered over 100 years ago, much remains to be revealed about its functions; indeed carnosine and homocarnosine have been described as forgotten and enigmatic dipeptides (Bauer, 2005). There are numerous examples of protective actions of carnosine against a variety of insults mediated by discrete entities (oxygen free radicals, reactive nitrogen species, glycating agents, deleterious aldehydes, toxic metal ions) as well as ameliorating conditions associated with aging. Carnosine has been shown to protect various cells against ischemia–reperfusion injury, for example in rat liver (Fouad et al., 2007; Fujii et al., 2003), kidney (Kurata et al., 2006), heart (Alabovsky et al., 1997; Lee et al., 1999; Zaloga and Siddiqui, 2004), and brain (Dobrota et al., 2005). Protective activity exerted by carnosine has also been observed with respect to diabetes, osteoporosis, neurodegeneration, wound healing and loss of vision, and hearing and immune function. Possible biochemical functions (Quinn et al., 1992) of carnosine include control of pH, immunostimulant, wound healing agent, antioxidant, metal-ion chelator, carbonyl scavenger, and antiglycator (Table 3.3). The evidence for some of these proposals is highly varied, however.

VI. CONTROL OF pH The most convincing proposal is that carnosine plays one or more roles in control of intracellular hydrogen ion concentration (Abe, 2000; VaughanJones et al., 2006). Carnosine is an effective physiological buffer; it is presumed that this property explains its predominant association with white, glycolytic, muscles which possess relatively few mitochondria and thereby generate lactic acid. Not only may carnosine, also possible in its acetylated form, help to directly suppress the rise in hydrogen ion concentration but its ability to activate the enzyme carbonic anhydrase (Temperini et al., 2005) would increase bicarbonate buffer capacity. These properties may help explain carnosine’s protective action in ischaemia, a condition associated with severe intracellular acidosis.


TABLE 3.3

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Possible Homeostatic properties of carnosine

Buffer Hydroxyl radical scavenger Antioxidant Chelator of copper and zinc ions Aldehyde/carbonyl scavenger Antiglycator Stimulates nitric oxide synthesis Stimulates proteolysis Activates carbonic anhydrase Upregulates synthesis of oxidized protein hydrolase (OPH) Suppresses protein cross-linking Reacts with protein carbonyls Suppresses AGE reactivity May participate in protein deglycation May participate in histone deacetylation May participate in repair of isoaspartate residues May stimulate synthesis of stress proteins

VII. CARNOSINE AND CHELATION OF ZINC AND COPPER IONS Carnosine is an avid chelator of metal ions (Baran, 2000). Complexes with calcium, copper, and zinc ions have been described (Trombley et al., 2000). It is possible, therefore, that carnosine could exert some sort of control of calcium metabolism in muscle tissue (heart or skeletal). It is also likely that the dipeptide controls the availability of zinc ions in neuronal tissue, especially the olfactory lobe where both carnosine and zinc are enriched (Bakardjiev, 1997; Bonfanti et al., 1999; Sassoe-Pognetto et al., 1993). Zinc–carnosine complexes, called polaprezinc, are also effective in the repair of ulcers and other lesions in the alimentary tract (Matsukura and Tanaka, 2000).

VIII. CARNOSINE AND AGING It has been previously suggested that carnosine might possibly be an antiaging agent (Boldyrev et al., 1999a; Hipkiss, 1998; Hipkiss et al., 2001). This suggestion was based on (i) a report of observations made in Australia around 1990, but finally published in 1994, that carnosine could

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not only delay senescence in cultured human fibroblasts but also reverse the senescent phenotype by promoting what appeared to be rejuvenating effects (McFarland and Holliday, 1994, 1999), and (ii) a substantial body of work from Russian laboratories in the 1980s and 1990s on the dipeptide’s antioxidant activity and protective functions towards heart and kidney ischaemia (see Boldyrev, 1993; Boldyrev et al., 1993, 1995, 1997; Quinn et al., 1992; and references therein). Later work showed that growing fibroblasts with carnosine also protected telomeres against shortening (Shao et al., 2004). Antiaging effects of carnosine were subsequently observed in senescence-accelerated mice and fruit flies (Yuneva et al., 1999, 2002). That carnosine seems to be specifically associated with longlived, postmitotic tissue, such as muscle and nerves, is at least consistent with the idea that the dipeptide does not compromise cell survival and may help ensure longevity.

IX. CARNOSINE AND THE CAUSES OF AGING The explanation of the causes of aging remains somewhat controversial. Most, but not all, biogerontologists reject the idea that aging is a genetically programed process along the lines of growth and development, because the majority of animals in the wild die from predation, starvation or disease, before they age significantly. Hence the selection of genes specifically programing aging would not be of any evolutionary advantage. The consensus of opinion is that aging is the result of a breakdown of molecular homeostasis, due to the chronic effects of the forces of instability (endogenous and exogenous) to which all cells and organisms are continuously subjected. In other words, organisms have evolved to survive long enough to reproduce their genes, during which time entropic events must be either controlled or their effects eliminated. Consequently, the changes which we refer to as aging are thought to have no evolutionary significance, but result from the eventual failure of longevity genes whose functions ensure survival for sufficient time for the organism to reproduce successfully. That there is a frequent correlation between organism longevity and the ability to resist certain external stresses, such as heat or irradiation, is consistent with this idea. Various possible mechanisms have been proposed to explain aging. These include environmental and endogenous factors which affect an organism’s ability to survive by causing genetic changes (e.g., DNA damage and telomere shortening), altering gene expression, increasing oxidative stress, compromising energy provision and promoting the


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accumulation of altered proteins. There is evidence which suggests that carnosine has at least the potential to ameliorate to some degree most of these possible causes of aging (Hipkiss, 1998).

X. PROTEOTOXICITY AND AGING At a biochemical level the most common symptom of aging and its related pathologies is the accumulation of altered or abnormal proteins (Dalle-Donne et al., 2003; Hipkiss, 2006a; Levine, 2002). It should be pointed out that abnormal proteins are normally formed continuously, intracellulary, and extracellularly, and they originate from biosynthetic errors (gene expression is not 100% perfect) and postsynthetic damage due to the actions of deleterious endogenous and exogenous agents (e.g., oxygen and glucose). It is generally thought that the age-related accumulation of aberrant polypeptides is a consequence of the decline in functional activity of the gamut of homeostatic process (e.g., DNA repair, proteolysis, antioxidant enzymes), because the molecules which carry out these fun ctions are themselves also subjected to the same range of insults which they should normally prevent or eliminate. Indeed it is becoming increasingly apparent that molecular overengineering of certain of these homeostatic gene products can indeed increase both stress-resistance and organism longevity. Furthermore, not only does the accumulation of aberrant polypeptides result in loss of function of the normal gene products, but these altered molecules also appear to possess gain of function toxicity, mostly due to their aggregation, oligomerization, and cross-linking potential. Certain aberrant proteins also induce oxygen free radical generation. Among the better understood resultant effects of altered protein accumulation are compromised proteolytic activities, inflammation (or sometimes called ‘‘inflammaging’’), and induction of the stress response. An obvious contentious question is: does aging causes proteotoxocity, or does proteotoxicity causes aging? Most likely the answer is ‘‘yes’’ to both alternatives, simply because proper control of protein metabolism (synthesis and degradation) is essential for viability. Table 3.4 lists the possible areas in which carnosine could theoretically exert some protective, homeostatic effects which suppress cellular and/or organism aging, by the dipeptide mostly acting at the postsynthetic level to suppress formation of altered proteins. However, it is also possible that, by scavenging oxidative and glycoxidative agents, carnosine could inhibit gene modification and thereby prevent synthesis of altered gene products and general DNA damage.

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TABLE 3.4 Hypothetical mechanisms of carnosine’s antiaging activity

Suppresses altered protein accumulation by: Scavenging hydroxyl radicals and hypochlorite ions Scavenging endogenous and exogenous toxic aldehydes and glycating agents Reacting with protein carbonyls/AGEs Inhibiting transglutaminase activity Slowing protein synthesis Activates altered protein elimination by: Stimulating expression of oxidized protein hydrolase Stimulating NO synthesis which upregulates proteasome activity Stimulating synthesis of stress/chaperone proteins Participating in protein deglycation Participation in SIRT metabolism (gene silencing) by: Accepting acetyl groups released by SIRT-mediated histone deacetylation Scavenging (acetyl)-ADP-ribose units generated following NAD cleavage which accompanies SIRT-mediated histone deacetylation Protects telomeres against shortening Upregulates release of corticosterone

XI. CARNOSINE, OXYGEN FREE RADICALS, AND OXIDATIVE STRESS Over 50 years ago Harman (1956) proposed the so-called ‘‘oxygen free radical theory of aging.’’ This theory proposed that much age-related damage to proteins, lipids, and DNA was caused by incompletely reduced oxygen atoms, that is, oxygen free radicals. It has often been assumed that mitochondria are the principle source of these reactive oxygen species (ROS) because of the organelles’ intimate association with oxygen. However, it should be noted that ROS can be produced elsewhere within the cytosol and extracellularly too. ROS are not exclusively deleterious as they are also involved in cell signaling, although there are enzymes such as superoxide dismutase, catalase, and various peroxidases, in the cytosol and mitochondria, which provide defense against excessive ROS generation. There is evidence that carnosine can also exert antioxidant activity inhibiting oxidation of lipids (Bogardus and Boissonneault, 2000; Decker et al., 2001; Nagasawa et al., 2001) and proteins (Boldyrev et al., 1999a,b; Guiotto et al., 2005a; Hipkiss et al., 1998a;


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Kang et al., 2002; Kim and Kang, 2007; Quinn et al., 1992). The dipeptide can scavenge hydroxyl radicals (Tamba and Torreggiani, 1999), which are the most damaging ROS. Hydroxyl radicals are generated from hydrogen peroxide in the presence of bivalent metal ions such as copper; hydrogen peroxide is formed by the action of superoxide dismutase on superoxide anions. Carnosine can also scavenge at least two other deleterious ROS, the hypochlorite anion (OCl) (Hipkiss et al., 1998a; Quinn et al., 1992), which is formed from superoxide and chlorine ions by the action of myeloperoxidase, and peroxynitrite (ONOO) (Fontana et al., 2002) which is formed by the reaction of superoxide with nitric oxide. Consequently, it is theoretically possible that carnosine could prevent damage mediated by these ROS in vivo.

XII. CARNOSINE AND NONENZYMIC PROTEIN GLYCOSYLATION (GLYCATION) Other sources of age-related macromolecular damage are metabolic aldehydes and ketones. And the best investigated example is the chemical process, originally described in cooking, called the Maillard or browning reaction. This process, originally termed nonenzymic protein glycosylation, but now called glycation, involves the reaction of a reducing sugar such as glucose with an amino group of a protein, eventually producing a highly complex brown product, now known as advanced glycation endproducts (AGEs) (Suji and Sivakami, 2004). In fact, it turns out that glucose is the least reactive of all the common metabolic sugars due to the fact that its aldehyde group is 99.99% unavailable for reactivity because of the predominant cyclic form of the glucose molecule. Other common sugars such as galactose and fructose are much more reactive than glucose; indeed diets high in galactose and fructose are frequently employed experimental tools to induce diabetes-like symptoms in laboratory animals (Wang et al., 2008). It has also been found that certain metabolic intermediates and their by-products, if present in excess, can glycate proteins (Brownlee, 1995; Thornalley, 1999), DNA (Barea and Bonatto, 2008), and amino lipids (Lankin et al., 2007) very rapidly indeed to generate products very similar to those found in senescent cells and organisms. As described above, carnosine was shown in the 1990s to exert antiaging effects in cultured cells, and the question arose about the mechanism (s) involved. When this work was initiated, carnosine was usually regarded as an antioxidant (Kohen et al., 1988) but as other and better antioxidants did not exert the antiaging/rejuvenating effects on cultured fibroblasts, this suggested that additional activities were necessary to explain its actions. It was suggested that carnosine’s structure resembled preferred protein glycation sites and it was demonstrated that the

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dipeptide did indeed possess antiglycating activity (Hipkiss et al., 1995a). It inhibited protein glycation, subsequent cross-linking (Hipkiss et al., 1998a), and formation of AGEs induced by a variety of reactive aldehyde and carbonyl compounds (glucose, deoxyribose, ribose, fructose, dihydroxyacetone, malondialdehyde, acetaldehyde, formaldehyde, and methylglyoxal) (Brownson and Hipkiss, 2000; Hipkiss and Chana, 1998; Hipkiss et al., 1998b, 2002). These observations have been confirmed and greatly extended by other workers (Burcham and Pyke, 2006; Gugliucci et al., 2002; Seidler, 2000; Seidler et al., 2004; Yan and Harding, 2005; Ukeda et al., 2002; see also references in Hipkiss, 2005), who have detailed the chemistry of the various carnosine–carbonyl adducts generated (Aldini et al., 2002, 2005; Carini et al., 2003; Liu et al., 2003). A carnosine–carbonyl adduct, formed between the dipeptide and hydroxynonenal, has been isolated from oxidatively stressed biological tissue (Orioli et al., 2005) and, furthermore, detected in rat urine (Orioli et al., 2007). Given that tissue levels of carnosine are generally higher in humans than in rodents (Hipkiss and Brownson, 2000), it is anticipated that similar adducts will be detected in human tissues. Having shown that carnosine can suppress the reactivity of low molecular weight carbonyl compounds by simply reacting with the deleterious glycating agents, we suggested that carnosine could react with carbonyl groups generated on macromolecules such as aminolipids and proteins following oxidation or glycation. Using radiolabeled carnosine, we showed (Brownson and Hipkiss, 2000), at least at the test-tube level, that the dipeptide could indeed react with protein-bound carbonyl groups, and the term ‘‘protein carnosinylation’’ was coined. As yet, however, no evidence for the presence of ‘‘carnosinylated’’ protein has been obtained from biological tissue. However, protein g-glutamyl-carnosine adducts have been detected (Kuroda and Harada, 2002) in animal muscle. There are a number of differing explanations that could account for the formation of these adducts. The g-glutamyl-carnosine adduct may derive from the reaction of carnosine with the transient carbonyl group generated during the spontaneous deamidation of a glutamine residue (Kuroda and Harada, 2002). Another possibility is that adduct formation results from the action of transglutaminase on a protein glutamine residue and carnosine producing a g-glutamyl-carnosine residue in the protein; subsequent proteolysis would release the free g-glutamyl-carnosine adduct. A third explanation is the reaction of carnosine with glutamate semialdehyde, formed following ROS-mediated oxidation of a protein arginine residue; subsequent hydrolysis again releasing the free adduct. As noted above, although no ‘‘carnosinylated’’ protein has been detected, a ‘‘carnosinylated’’ lipid has been detected in human muscle (Schroder et al., 2004), possibly arising from the reaction of an oxidized (amino)-lipid


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with carnosine. Indeed, carnosine has been shown to inhibit MG-induced glycation of LDL and formation of foam cells in vitro (Rashid et al., 2007). There is one study which investigated the effects of carnosine on formation of protein carbonyls in cultured cells following exposure to glucose degradation products present in sterilized peritoneal dialysis fluids. It was shown that preexposure of cultured mesothelial cells to 20 mM carnosine suppressed ROS generation and formation of protein carbonyls induced by the glucose degradation products (Alhamdani et al., 2007a,b). The mechanism by which this protection was exerted remains uncertain, however; possibilities are (i) the extracellular carnosine may simply react with the deleterious carbonyl compounds extracellularly; (ii) intracellular carnosine reacts with the carbonyl compounds thereby preventing their interaction with intracellular macromolecules; and (iii) carnosine reacts with protein carbonyls forming ‘‘carnosinylated’’ proteins. This problem should not be difficult to resolve experimentally. Deleterious protein cross-linking can also be induced by reactive nitrogen species (RNS) such as peroxynitrite ONOO formed by the reaction of superoxide with nitric oxide (NO). The cross-links are formed between tyrosine residues following nitration by peroxynitrite (Sitte, 2003). Carnosine appears to play roles not only in NO generation but also in protection against excess NO production by inducible nitric oxide synthetase (NOS), thereby preventing ONOO-mediated protein modification (Fontana et al., 2002). Evidence for a carnosine–NO adduct has also been published (Nicoletti et al., 2007).

XIII. CARNOSINE AND PROTEOLYSIS OF ALTERED PROTEINS As noted above, accumulation of altered protein forms is a common feature of aging, which can be explained by either, or both, increased generation of the aberrant polypeptides or a decrease in cellular ability to eliminate them by selective proteolysis (Hipkiss, 2006a). In the past few years, much evidence has emerged showing that cell senescence is accompanied by decreases in either, or both, proteasome- and autophagymediated proteolysis (Bergamini et al., 2007; Bulteau et al., 2006; Carrard et al., 2002; Donati, 2006; Martinez-Vicente and Cuevo, 2007; Ngo and Davies, 2007; Vernace et al., 2007a). Indeed, upregulation of either, or both, proteasome and autophagic activity has been shown to delay onset of the senescent state in cultured cells (Bergamini et al., 2007; Chondrogianni and Gonos, 2007; Donati, 2006; Hansen et al., 2008; Vernace et al., 2007b). Quite why these proteolytic activities decline during aging is uncertain; possible explanations include inhibition of proteasome activity by cross-linked proteins (Carrard et al., 2002; Ding and Keller, 2001), and accumulation of lipoprotein cross-linked material

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(lipofuscin) in the autophagosomes (Dahlmann, 2007; Tatsuta and Langer, 2008). Also it should be noted that participation of chaperone proteins is necessary for the recognition, delivery, and degradation of altered proteins (Leidhold and Voos, 2007; Otto et al., 2005; Rakwalska and Rospert, 2004). This may help explain why increased expression of certain chaperone proteins can extend life span in some organisms as well as protect against heat and other stressing agents (Liao et al., 2008 Morrow and Tanguay, 2003; Rattan, 2006). Although carnosine’s ability to suppress both formation and reactivity of some of the age-associated macromolecular modifications (e.g., protein–protein cross-links and protein AGEs) could contribute to its apparent antisenescent effects, these prophylactic actions cannot by themselves explain the dipeptide’s apparent rejuvenating activity towards cultured human fibroblasts observed by McFarland and Holliday (1994, 1999). It is possible that carnosine may stimulate proteolysis. We obtained preliminary evidence that protein breakdown is increased in ‘‘old’’ fibroblasts when cultured with carnosine (Hipkiss et al., 1998b), while Bharadwaj et al. (2002) showed that the dipeptide stimulated proteolysis of HIF-1a protein in cultured cardiomyocytes. Carnosine was also shown to stimulate neutral (nonlysosomal) protease activity in cell-free extracts from rat brain (Bonner et al., 1995). Intracellular elimination of aberrant polypeptides mostly, though not exclusively, involves the proteasomes, and recent evidence suggests that nitric oxide can stimulate proteasomal activity (Kotamraju et al., 2006; Thomas et al., 2007). It is possible that carnosine can upregulate NOS as it has been suggested that carnosine itself is the source of NO rather than arginine (Alaghband-Zadeh et al., 2001). Therefore, in addition to decreasing the formation of glycated and cross-linked protein which can inhibit proteasomal activity, carnosine may actually stimulate proteasomal function to improve the elimination altered protein forms. This proposal should be easy to test using cell culture and in tissues of aging animals fed a carnosine-enriched diet.

XIV. CARNOSINE AND GENE EXPRESSION Carnosine can affect gene expression. Ikeda et al. (1999) showed that carnosine markedly upregulates vimentin synthesis in cultured rat fibroblasts, while an association between carnosine and vimentin, a cytoskeletal, intermediate filament protein has been noted in glial cells and neurons (Bonfanti et al., 1999). Interestingly, it has also been shown that the protease, oxidized protein hydrolase (OPH), is coexpressed with vimentin in COS cells (Shimizu et al., 2004). Thus, it is at least possible that carnosine could induce synthesis of OPH in the cultured human fibroblasts and thereby increase the cellular ability to eliminate oxidized


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polypeptides. While much more needs to be done to confirm or refute many of these proposals, they could help to explain carnosine’s rejuvenating actions of cultured human fibroblasts, particularly as increased protein turnover is a well-recognized antiaging strategy (Hipkiss, 2003). However, it should be noted that excessive proteolysis may contribute to the aging phenotype as in the case of age-related muscle wastage or sarcopenia. It has recently been reported that vimentin is very readily and specifically glycated in cultured human fibroblasts (Kueper et al., 2007). The biological significance of this observation is at present uncertain but the fact that carnosine seems to mimic preferred glycation sites and apparently promotes expression of a protein (vimentin) which is itself highly susceptible to glycation may not be entirely coincidental and should be explored. Other studies have shown that activated macrophages secrete vimentin, an intermediate filament protein, which may play a role in bacterial killing and generation of oxidative metabolites (Mor-Vaknin et al., 2003). It is possible that the released vimentin helps to quench any excess glycoxidation species that are generated by activated leukocytes. It has recently been shown that senescent human fibroblasts accumulate a particular stress protein modified by glycation (Unterluggauer et al., 2008). It was found that heat cognate protein Hsc70 appears to be a target for selective glycation in senescent fibroblasts. One conjectures therefore whether the generation of the highly glycated Hsc70 protein (Hsc70AGE) is part of a triggering or sensing mechanism for the induction of stressinduced senescence, and, furthermore, whether carnosine’s antisenescence effects might be related to its ready glycation thus sparing the Hsc70 protein from modification. A form of rejuvenation of the senescent phenotype occurs in phorbol ester-treated U937 leukemia cells and that changes in proteolytic activity and vimentin expression are involved (Hass, 2005). It has been known for some time that the enzyme poly-ADP-ribose-polymerase-1 (PARP-1) plays an important role in suppressing cellular senescence and the response to cellular stress. Recent studies have shown that PARP-1 can associate with and strongly stimulate the 20S proteasomes, an activity which is involved in the selective degradation of oxidized proteins (Selle et al., 2007). It appears that PARP-1 catalyzes the synthesis of poly-ADPribose (from NADþ units) and becomes attached to various acceptor proteins located in the nucleus. One conjectures whether there is any parallel between the metabolic changes which accompany the rejuvenation phenomenon described above, and carnosine’s apparent ability to control vimentin expression, its proposed effects on proteolytic activity, its potential to react with glycating agents such as ADP-ribose, and its ability to induce cellular rejuvenation in cultured human fibroblasts described by McFarland and Holliday (1994, 1999).

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XV. CARNOSINE, ANTICONVULSANTS, AND AGING Another form of protein dysfunction which accompanies aging is the spontaneous deamidation of asparagine residues which can result in the generation of isoaspartate residues in proteins. The enzyme proteinisoaspartate-methyltransferase (PIMT) plays an important role in the repair of isoaspartate residues converting them into the normo-form via formation of a cyclic succinimide intermediate (Zhu et al., 2006). A recent study has shown that hydroxylamine can selectively cleave this intermediate generating a normal C-terminal fragment plus an N-terminal fragment with either an aspartyl-N-hydroximide or an aspartyl dihydroxamate residue at its C-terminal end (Zhu and Aswad, 2007). It is interesting that hydroxylamines can, like carnosine, delay senescence in cultured human fibroblasts (Atamna et al., 2000) and react with carbonyl groups (Hipkiss, 2001). Given its basic nature, one therefore speculates whether carnosine could similarly cleave peptide bonds at isoaspartate residues, possible even in vivo, and this action may further contribute to the dipeptide’s antiaging activity. Such action could generate carnosine adducted to N- and C-terminal protein fragments which would be easy to detect, should they exist. Studies in aging models using nematode worms have shown that anticonvulsants (valproic acid, volpramide, trimethadione, and ethosuximide) extend life span (Evason et al., 2005; Hughes et al., 2007). Some anticonvulsants also upregulate carnosine levels in mouse brain and homocarnosine levels in human brain (Petroff et al., 1998, 2006). Both carnosine and homocarnosine also have anticonvulsant activity in mice, rats, and humans (Jin et al., 2005; Kozan et al., 2008; Petroff et al., 1998; Wu et al., 2006; Zhu et al., 2007). It is also thought, however, that carnosine’s anticonvulsant action is exerted via a carnosine–histidine– histamine pathway (Zhu et al., 2007) activating histaminergic, GABAergic, and glutamicergic systems (Kozan et al., 2008). Whether there is any other connection between anticonvulsant activity and carnosine’s antiaging actions is obviously highly speculative. It may be relevant to note that epileptic seizures and a shortened life span, together with altered protein accumulation, are consequences of PIMTdeficiency in mice, while treatment with valproic acid, an anticonvulsant, partially suppresses these symptoms including effects on life span (Yamamoto et al., 1998). Conversely, PIMT overexpression can increase life span of Drosophila (Bennet et al., 2003). Furthermore, the chemistry of some anticonvulsants (ethosuximide) resembles quite closely the structure of the succinimide intermediate formed during both asparagine deamidation and PIMT-mediated repair of isoaspartate residues. One conjectures whether there are any relationships between these


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observations particularly because PIMT levels are 50% downregulated, posttranslationally, in epileptic human hippocampus (Lanthier et al., 2002), leading to the accumulation of aberrant tubulin. Another speculation which can be offered in this context is whether carnosine stimulates PIMT expression. It is known that small molecules regulate expression of PIMT mRNA. For example, R-()-deprenyl (Huebscher et al., 1999), lithium, and valproic acid (Lamarre and Desrosiers, 2008) can upregulate PIMT, while the cyclic tripeptide argininyl–glycyl–aspartyl inhibits PIMT expression (Lanthier and Desrosiers, 2006). One wonders therefore whether carnosine is an activator of PIMT expression functioning at either transcriptional or translational levels; both the isoaspartyl residue in the protein to be repaired and carnosine contain b-peptide bonds. It may only be coincidental that PIMT expression decreases with tumor malignancy (Lapointe et al., 2005) and carnosine can inhibit tumor cell growth (Holliday and McFarland, 1996). The biological role of PIMT involves the selective methylation of isoaspartate residues followed by a demethylation step to reform the succinimide intermediate. The demethylation causes the release of methanol which can be converted to formaldehyde and finally to formic acid, as demonstrated in rat brain preparations. It was found that S-adenosylmethionine (SAM), the methyl donor, caused formaldehyde levels to rise in the rat brain homogenates, thus suggesting that excessive formaldehyde may be a precipitating factor in Parkinsons’s disease (PD) (Lee et al., 2008). It is possible that carnosine could suppress formaldehyde toxicity by reacting with it to generate a carnosine–formaldehyde adduct. This should be a relatively easy experiment to perform to test this prediction.

XVI. CARNOSINE AND DIETARY RESTRICTION-MEDIATED DELAY OF AGING There is much evidence that caloric restriction (CR) can delay aging and onset of much age-related pathology in many species, and increase maximum life span (see Partridge and Brand, 2005; and references cited therein). Recent observations suggest that fasting periods, rather than a decrease in overall caloric intake per se, may be the cause of these effects (Goodrick et al., 1990; Mager et al., 2006; Masternak et al., 2005; Mattson and Wan, 2005). The mechanisms involved remain uncertain but they are currently thought to involve an interaction between gene expression and carbohydrate metabolism. Genetic studies have indicated that histone/ protein deacetylase (sirtuin) activities have important roles in the CR phenomenon (Guarente, 2000; Westphal et al., 2007). Coupled with the

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sirtuin-mediated deacetylation reaction is the conversion of NADþ to ADP-ribose and nicotinamide (Lin et al., 2000; Medvedik et al., 2007). There are a number of theoretical locations where carnosine might have some influence on sirtuin-mediated protein deacetylation and ADPribose metabolism. First, carnosine can behave as an acetyl acceptor by forming N-acetyl-carnosine. Secondly, ADP-ribose, a product of sirtuinmediated NAD-coupled protein deacetylation, is a potent glycating agent. Hence carnosine could be the ultimate acceptor for ADP-ribose by generating as adduct, ADP-ribosyl-carnosine. A third possibility concerns the polyADP-ribosylation of certain proteins, as a consequence of oxidative stress, carried out by a PARP, using NADþ as the ADP-ribose source. A number of studies have shown that PARP is involved in aging regulation and may protect cells against senescence (Burkle et al., 2005; Hass, 2005). Again, given the glycating potential of ADP-ribose and carnosine’s antiglycating and antiaging properties, one speculates on whether the dipeptide plays a role in either the formation or, following proteasomal activity on the poly-ADP-ribosylated proteins (Selle et al., 2007), the subsequent depolymerizition of the modified protein. However, no carnosine–ADP-ribose adduct has been reported. Another possible explanation of the effects of dietary restriction (DR) on the aging process might involve a decrease in glycolysis which inevitably accompanies DR-induced fasting periods (Hipkiss, 2006b). This could result in a decrease in the production of the highly deleterious glycolytic by-product, methylglyoxal (MG), whose reactivity towards proteins carnosine can inhibit (Brownson and Hipkiss, 2000; Hipkiss and Chana, 1998). It is possible that control of sirtuin activity by NADþ/NADH levels can also influence generation of altered proteins by increasing or decreasing MG generation. NADþ is essential for the conversion of the glycolytic intermediate glyceraldehyde-3-phosphate (G3P) to 1,3-diphosphoglycerate (1,3-DPG) by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which also yields NADH. In the ad libitum-fed (AL) condition, it is likely that NADþ levels would be low and NADH levels high due to continuous glycolysis. This would limit GAPDH activity and promote G3P accumulation, together with its immediate precursor dihydroxyacetone phosphate (DHAP). It is important to note that both G3P and DHAP are very effective glycating agents which can readily modify protein amino groups, etc. More importantly, however, both G3P and DHAP can spontaneously decompose into MG, the exceedingly toxic glycating agent that may also be responsible for much of the protein/lipid glycation observed during hyperglycaemic conditions. Indeed there are a number of observations suggesting that MG can induce ROS production and many of the deleterious physiological and biochemical changes characteristic of the aged phenotype (Cantero et al., 2007; Desai and Wu, 2008; Dhar et al., 2008; Han et al., 2007;


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Jia and Wu, 2007; Schalkwijk et al., 2008; Vander Jagt, 2008; Yamawaki et al., 2008; also see Hipkiss, 2006b, 2008a; and references therein). Carnosine’s ability to react with MG (Brownson and Hipkiss, 2000; Hipkiss and Chana, 1998) could conceivably contribute to inhibiting the deleterious effects of this highly reactive endogenous glycating agent, especially in tissues where glycolysis is extensive or persistent. In DR conditions during the fasting periods, NADH would be metabolized by the mitochondria and NADþ regenerated, thus allowing G3P oxidation, preventing DHAP build-up and MG production, and thereby decreasing the incidence of protein and lipid glycoxidation. This model (see Fig. 3.2) would also explain the so-called oxygen paradox where increased mitochondrial function (aerobic metabolism) is found to be beneficial with respect to aging and many related conditions (Hipkiss, 2008a). It is also possible that protein AGEs have a major role in affecting aging in animal models. Cai et al. (2008) have recently demonstrated that an oral glycotoxin (protein AGE made by treatment of albumin with MG) can substantially abolish many of the beneficial effects that CR exerts on aging, at least in mice. They found that the presence of the AGE (MGmodified protein) in the diet of CR mice promoted an age-related increase in oxidative stress similar to that observed in animals fed ad libitum; the life span of the AGE-treated mice was also decreased to that observed in the ad libitum-fed mice. It was concluded that as normal laboratory mouse food contains large amounts of protein AGEs, any reduction of food intake will automatically decrease the AGE load. It therefore follows, somewhat controversially, that the explanation of the beneficial effects of CR on organism life span may have little to do with decreased calorie intake, but instead reflects the effects of AGEs, exogenous, and endogenous, via MG generation. It has been proposed (Hipkiss, 2007b, 2008a,b,c) that dietary restriction, induced by CR or intermittent fasting, will decrease MG formation and thereby lower endogenous AGE generation, compared to animals fed ad libitum in which MG levels are likely to be raised due to increased frequency of food intake. Many other studies have suggested that MG is a major source of metabolically generated AGEs which in turn can affect organism life span, especially as MG can provoke many of the deleterious changes associated with aging (see Hipkiss, 2008a,b,c; and references therein). For example, mutation in the gene coding for triose phosphate isomerase which provokes the accumulation of the MG precursor dihydroxyacetone phosphate, induce a shortened life span in Drosophila (Celotto et al., 2006; Gnerer et al., 2006). Defects in the enzyme responsible for the detoxification of MG, glyoxalase-1, shortens life span of the Caenorhabditis elegans (Morcos et al., 2008), while overexpression of glyoxalase-1 can extend life span in the nematode (Morcos et al., 2008). There is a substantial body of

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Glucose DHAP + G3P NAD+

MG

Glycation ROS

MG

NADH 1, 3 DPG ATP Pyruvate Mitochondrial dysfunction

Acetyl –CoA O2

NADH

ATP Carnosine CO2 H2 O

NAD+ Mitochondrion Sirtuin activity

MG-carnosine adduct

Nicotinamide Controls gene expression to maintain juvenile character A speculation on how carnosine and caloric restriction may modulate ageing

FIGURE 3.2 A speculation on how dietary restriction and carnosine might regulate methylglyoxal (MG) production and consequent generation of altered proteins which characterize the aged state. Increased frequency of glycolysis decreases NADþ availability and increases likelihood of MG generation and protein modification. Caloric restriction and/or aerobic exercise decreases NADþ consumption and increase NADH oxidation to facilitate NADþ regeneration to maintain juvenile character via NADþdependent sirtuin activity and decreased MG generation.

evidence suggesting that increased MG production has a causal role in much diabetes-associated pathology (see Ahmed and Thornalley, 2007; and references therein) The variation in tissue susceptibility to these and other aging-induced changes may partly result from differing levels of those molecules (glutathione, polyamines, carnosine, creatine, pyridoxamine, glyoxalases 1 and 2) which normally exert protective activity against glycoxidating agents such as MG. It is also possible that intermittent glycolysis could be hormetic by upregulating synthesis of some of these defense molecules (Hipkiss, 2007b). Interestingly, carnosine has also been described as a


Methylglyoxal (MG) induced deleterious protein-AGE

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Possible protection by carnosine

Protein-NH2 + CH3COCHO + Carnosine (MG)

Carnosine-MG adduct (excreted?)

Protein-MG adduct

Protein-CO-AGE (protein carbonyl)

+ Carnosine

Protein-CO-carnosine adduct (carnosinylated-protein) (inert lipofuscin) (degraded?)

Protein-NH2

Protein-protein–AGE (cross-linked; resists proteolysis; inhibits proteasomes; induces ROS)

FIGURE 3.3 Schematic showing possible sites of intervention by carnosine during formation of cross-linked methylglyoxal-modified proteins. AGE, advanced glycation end-product.

glyoxalase mimetic (Battah et al., 2002), in addition to its other protective activities. Nevertheless, carnosine’s ability to protect against MG toxicity may be important physiologically especially with respect to the changes responsible for aging and related pathologies (see Fig. 3.3). Intracellular carnosine concentration may be subject to metabolic regulation. Destruction of the dipeptide by carnosinase is stimulated by citrate (Vistoli et al., 2006), thus raising the possibility that inhibitory molecules could be created to prevent destruction of the dipeptide in sera. Carnosine’s synthesis by carnosine synthetase is downregulated by raised cAMP levels (Schulz et al., 1989), at least in astrocytes. Thus, high glucose concentrations could lower cAMP levels and hence stimulate carnosine synthesis. It is interesting that many of the studies using model organisms to study how aging is delayed by genetic, physiological, and dietary means have a common feature, that is the upregulation of mitogenesis

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(Anderson et al., 2008; Bonawitz et al., 2007; Cunningham et al., 2007; Guarente, 2008; Hipkiss, 2008b; Lopez-Lluch et al., 2008; Rodgers et al., 2008; Soh et al., 2007). This effect is also induced by increased aerobic exercise in mammals where many of the symptoms of aging are suppressed. One possible explanation is that the stimulation of synthesis of mitochondria also increases synthesis of the necessary chaperone proteins which are required for maintenance of protein quality in the growing organelle and the cytosol. Hence, as these proteins also participate in the elimination-altered proteins arising from postsynthetic damage, it is likely that the increased ability to recognize and degrade the aberrant proteins due to erroneous protein synthesis will also improve overall cellular stress-resistance and enhance longevity. Carnosine, when complexed with zinc ions, has been shown to stimulate expression of certain stress proteins, for example, Hsp72 (Odashima et al., 2002, 2006; Mikami et al., 2006; Wada et al., 2006). Furthermore, carnosine may also stimulate synthesis of the stress hormone corticosterone (discussed below) which may in turn upregulate expression of a number of stress proteins. Although very hypothetical these suggestions are relatively easy to test. That the stress protein Hsc70 becomes preferentially glycated in senescent cells and that synthesis of vimentin, another readily glycated protein, is induced by carnosine is perhaps indicative of a relationship between these factors; for example, could upregulation of vimentin synthesis compete with Hsc70 glycation and help delay onset of stress-induced premature senescence?

XVII. CARNOSINE, REGULATION OF PROTEIN SYNTHESIS, AND AGING It has recently been shown that carnosine can also exert suppressive effects on mRNA translation initiation (Son et al., 2008); the dipeptide inhibited interleukin-8 mRNA translation by suppressing phosphorylation of initiation factor eIF4E in peroxide-activated intestinal epithelial cells and Caco-2 cells. eIF4E Phosphorylation is required for effective mRNA translation, which explains the observed carnosine-mediated decreased synthesis of the proinflammatory cytokine. Carnosine also inhibited phosphorylation of other regulatory proteins ERK1/2 and p38 MAP kinase (Son et al., 2008). This may be important as Davis et al. (2005) have shown that accelerated aging can be suppressed by inhibiting p38 MAP kinase phosporylation. The mechanism by which carnosine suppresses kinase phosphorylation is unknown, but it could be a consequence of decreased glycoxidative damage within the cell, due to the presence of carnosine, rather than direct participation of the dipeptide in the signaling pathway.


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Defective eIF4E limits mRNA translation initiation and results in life span extension in C. elegans since studies show that aging can be delayed by partial inhibition of protein synthesis. Studies in C. elegans revealed that senescence was delayed, stress resistance enhanced, and life span extended in eIF4E mutants (Pan et al., 2007; Syntichaki et al., 2007). Similarly, life span of C. elegans was extended and stress resistance enhanced when translation was inhibited where synthesis of eleven ribosomal proteins was suppressed using inhibiting-RNAs (RNAi) (Hansen et al., 2007). Certain ribosomal protein defects also have beneficial effects on yeast longevity (Chiocchetti et al., 2007). Explanation of these effects is uncertain (Kaeberlein and Kennedy, 2007), but it is possible that a lower rate of bulk protein synthesis, resulting from decreased translation initiation frequency, also lowers synthesis of error-proteins (Hipkiss, 2007c). This lowered production of biosynthetic aberrant proteins could directly lower the load that the chaperone and proteolytic apparatus must deal with: the chaperone/proteolytic apparatus is responsible for the elimination of altered protein generated postsynthetically as well as those formed by biosynthetic errors. Compared to normal gene products, error-proteins are more readily glycated and oxidatively damaged by ROS (Dukan et al., 2000; Fredriksson et al., 2006) than the normal gene products, thus the mutant organisms would generate fewer protein carbonyls, that normally characterize the senescent state (Stadtman, 1992) than the wild type. Consequently, the decreased level of biosynthetic error-proteins would not only decrease formation of protein carbonyls but also increase the relative availability of chaperone and proteolytic activities for the recognition and elimination of altered proteins arising from deleterious postsynthetic modification (Hipkiss, 2007a). Interestingly, methionine restriction (40% and 80%) also delays aging in rodents (Miller et al., 2005; Naudi et al., 2007). Methionine is the initiating amino acid in protein biosynthesis; therefore, this could again indicate that decreased translation initiation is an effective antiaging strategy by decreasing biosynthetic formation of error-proteins, similar to the effects of the defective eIF4E initiation factor in nematodes outlined above (Hipkiss, 2008c). It is possible that because carnosine’s also has inhibitory effects on eIF4E activity and slows protein synthesis, the beneficial effects on fibroblast senescence and life span could be mediated via a similar mechanism in human cells.

XVIII. CARNOSINE AND CORTICOSTEROIDS A recent study has shown that intracerebroventricular carnosine administration stimulates corticosterone release in chick brain (Tsuneyoshi et al., 2007). Studies performed some 30–40 years ago showed that

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hydrocortisone or cortisone (Cristofalo and Kabakjian, 1975; MacieiraCoelho, 1966) have positive effects on the growth and life span of cultured human fibroblasts. These findings have recently been reactivated where the beneficial effects of glucocorticoids towards cultured human fibroblasts have again been demonstrated (Kletsas et al., 2007). Given that carnosine can also affect fibroblasts life span in a positive manner (McFarland and Holliday, 1994), it is at least conceivable that carnosine’s action is mediated via glucocorticoid upregulation. A recent study at the whole animal level has revealed mixed results however, Caro et al. (2007) showed that 4 weeks chronic treatment with corticosterone decreased markers of lipid peroxidation but protein glycoxidation and oxidative damage to mitochondrial DNA were both increased in rat liver. Clearly, this is another research area which should be explored.

XIX. CARNOSINE AND AGE-RELATED PATHOLOGY Accumulation of altered protein forms, particularly protein carbonyl groups, is not only the most common biochemical signature of aging (Levine, 2002) but such aberrant polypeptides are associated with many age-related diseases (Dalle-Donne et al., 2003) as well. As carnosine has the potential to intervene in a number of processes that possibly contribute to the phenomenon we call aging, particularly where generation of altered proteins is involved, it follows that the dipeptide may have some beneficial effects with respect to either the causation or progression of those age-related conditions which also involve accumulation of aberrant protein forms. Table 3.5 lists the possible conditions against which carnosine might exert some therapeutic effects. It should be emphasized that this list is, for the most part, purely speculative and considerable amounts of work needs to carried out to verify or eliminate these suggestions.

XX. CARNOSINE, DIABETES, AND SECONDARY COMPLICATIONS The secondary complications of diabetes include cardiac and circulatory disorders, peripheral neuropathy, cataractogenesis, and stroke. Over the past decade it has become increasingly evident that much diabetesassociated pathology derives from hyperglycaemia where glucose, or more likely its metabolites and by-products, chemically modify intracellular and extracellular proteins and aminolipids via the process called glycation (Ahmed and Thornalley, 2007; Goh and Cooper, 2008; Magalhaes et al., 2008; Singh et al., 2001; Vlassara and Palace, 2002). The process is termed nonenzymic glycosylation or glycation to distinguish it


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TABLE 3.5 Potential age-related conditions against which carnosine could be explored therapeutically

Diabetes and diabetic complications Ischemia Neurodegeneration Osteoporosis Deafness Slow wound healing High blood pressure Heart disease Cataractogenesis

from the enzyme-mediated attachment of sugars to proteins or lipids required for proper cell function/distribution. It should be noted that glycation of proteins mediated by glucose is relatively slow, but other common sugars such as galactose or fructose are much more rapid. Furthermore, there is much evidence that certain metabolic intermediates of glucose catabolism (via glycolytic pathway) can almost immediately glycate intracellular and extracellular proteins. As noted above, MG is particularly damaging (Cantero et al., 2007; Desai and Wu, 2008; Dhar et al., 2008; Gomes et al., 2008; Kalapos, 1999; Mirza et al., 2007; Nakayama et al., 2008; Wang et al., 2007; Yander, 2008; Yao et al., 2007) and many studies have suggested that MG is the primary source of much of the deleterious protein glycation which is responsible for diabetic complications (see Rabbani and Thornalley, 2008; Wang et al., 2008 for recent reviews). Hence there has been an extensive search for agents which possess antiglycating activity which may be employed to suppress AGE formation and attendant diabetic complications. It was suggested some time ago that carnosine might be a candidate antiglycating agent for the control of secondary diabetic complications (Hipkiss, 1998; Hipkiss et al., 1995a,b). Carnosine may help suppress some features of aging at cellular and whole organism levels, possibly by inhibiting the reactivity of ROS and deleterious aldehydes including formation of AGEs (Hipkiss et al., 2002). It is theoretically possible that the dipeptide is beneficial towards those conditions where formation of protein AGEs plays important and most likely causal roles. Even in complication-free diabetics, the levels AGE precursors such as MG and glyoxal are elevated in their sera (Han et al., 2007). So it is likely that carnosine and other carbonyl scavengers might exert beneficial effects towards diabetes and its secondary complications (Hipkiss, 2005). Lee et al. (2005) have indeed demonstrated that dietary carnosine suppresses a number of diabetic complications in mice.

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There is also evidence indicating that low cellular carnosine levels are associated with diabetes (Gayova et al., 1999; Nagai et al., 2003). A recent study has shown that carnosine can inhibit formation of glycated lowdensity lipoprotein which normally provokes formation of foam cells associated with circulatory disorders which characterize diabetic complications (Rashid et al., 2007). Furthermore, carnosine can decrease blood pressure in rats (Nagai et al., 2003; Tanida et al., 2005) and possesses vasodilatory activity (Ririe et al., 2000); hypertension is another consequence of diabetes. It has been shown that the toxic effects of fructose and high glucose levels on blood pressure might be mediated via the generation of MG (Wang et al., 2006). MG not only induces peroxynitrite production in vascular smooth muscle cells (Chang et al., 2005) but also plays a causative role hypertension (Wu, 2006). Clearly, if carnosine does indeed scavenge MG in vivo, then it could exert protective effects towards hyperglycemia-induced hypertension. Another complication of diabetes is the loss of peripheral neuronal function. It has recently been shown that carnosine and its zinc complex can ameliorated progressive diabetic neuropathy in mice (Kamel et al., 2008), although the zinc–carnosine complex was more effective than the uncomplexed dipeptide. An interesting observation is that serum AGE levels are higher in diabetic vegetarians compared to diabetic omnivors (KrajcovicovaKudlackova et al., 2002). This may be because of the absence of carnosine in vegetarian diets, although a raised intake of fructose by vegetarians is an alternative explanation. More evidence that carnosine may possess therapeutic potential comes from studies on carnosinase in mice and humans. These studies have shown that higher levels of this enzyme are associated with diabetic end-stage kidney disease in humans, whereas a lower activity seemed to be protective (Freedman et al., 2007; Janssen et al., 2005). A further study using diabetes-prone mice, in which serum carnosine or carnosinase levels were manipulated, showed that the onset of diabetic complications was enhanced when carnosinase activity was increased by transgenic modification with the human carnosinase gene CN1; diabetes was milder and delayed in animals supplemented with carnosine (Sauerhofer et al., 2007).

XXI. CARNOSINE AND NEURODEGENERATION There is evidence from animal studies that carnosine can affect brain function/activity (Tanida et al., 2007; Thio and Zhang, 2006; Tomonaga et al., 2004, 2005, 2008; Tsuneyoshi et al., 2007, 2008); furthermore, the dipeptide is protective against a number of neurotoxic agents, for


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example, N-methyl-D-aspartate (NMDA) (Shen et al., 2007a,b), copper (Hornung et al., 2000), zinc (Kawahara et al., 2007), ROS (Kim and Kang, 2007; Kim et al., 2004), and RNS (Calabrese et al., 2005). There is also substantial evidence suggesting that carnosine is protective against ischemia in the brain and induced seizures; both of these are discussed separately below. Many neurodegenerative diseases are age-related, consequently it is possible that factors which delay general aging could also delay onset of neurodegeneration. Furthermore, many neurodegenerative diseases have at least one feature in common, that is the accumulation of altered proteins, a feature characteristic of aged cells generally. These aberrant protein forms are found as tangles and amyloid plaque in Alzheimer’s disease (AD), as Lewy bodies in PD and inclusion bodies in Huntington’s disease (HD) (Bossy-Wetzel et al., 2004). From first principles, general explanations for their occurrence are either increased production of altered proteins or their decreased clearance from the tissue. The likelihood that neurodegeneration is accompanied by dysfunction of either the ubiquitin/proteasome system (Paul, 2008) and/or the autophagic apparatus (Bandhyopadhyay and Cuervo, 2007) has been recognized for some time. The possibility that carnosine might stimulate proteolytic activity by, for example, activating proteasome function by increasing nitric oxide synthesis (Thomas et al., 2007), or whether it affects autophagy or chaperone formation/activity, should be explored. It has long been suggested that ROS may play causal roles in these diseases and in the production of the aberrant protein molecules (de Arriba et al., 2006). In addition, protein damage inflicted by RNS has also been suggested. As discussed above, carnosine has been shown to possess antioxidant activity and also to react with RNS as well, hence the molecule has the potential to be considered as a theraupeutic agent (Calabrese et al., 2008). There are a number of findings suggesting that agents that facilitate elimination of protein carbonyls (by either proteolytic elimination or by enzymically mediated chemical reduction) may suppress neurodegenerative conditions in model systems (Botella et al., 2004). Consequently, as carnosine may also react with protein carbonyls, it is theoretically possible that it could suppress formation and/the reactivity of protein carbonyls in the brain. Whether carnosine participates in carbonyl reductase activity has not been investigated but it is also a reasonable speculation. There are numerous examples where carnosine has been demonstrated to be protective activity against neurotoxic agents. For example, the dipeptide was shown to protect the mitochondria of cultured astroglial cells against nitric oxide-induced damage (Calabrese et al., 2005). Carnosine was also shown to protect neurofilament-L against oxidative damage, aggregation, and formation of dityrosine induced by hydrogen

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peroxide and cytochrome c (Kim and Kang, 2007; Kim et al., 2004). Carnosine was shown to modulate the neurotoxic effects of copper and zinc (Hornung et al., 2000). In the following sections, the possible roles for carnosine as protective agents in specific neurodegenerative conditions are discussed.

XXII. ALZHEIMER’S DISEASE The causal events in AD are much discussed but increased oxidative/ glycoxidative damage is acknowledged to play a role. One common altered protein form which accompanies AD is a small peptide fragment called amyloid-b-peptide (Ab-peptide). Ab-peptide is generated from a larger protein called amyloid precursor protein (APP) via the action of two proteases. It appears that an enzyme (insulin degrading enzyme or IDE) which normally cleaves the Ab-peptide in the middle declines with age (Caccamo et al., 2005; Farris et al., 2005; Qiu and Folstein, 2006). The reason for IDE’s decline in activity is unknown; possibilities include inactivation by ROS, RNS, or glycating agents, decreased gene expression and preferential usage in insulin metabolism. Glycoxidation events have a role in neurodegenerative disorders, and some recent papers have proposed that MG may be directly involved (Bar et al., 2002; Luth et al., 2005; Munch et al., 1997, 2003; Pamplona et al., 2005, 2008; Reddy et al., 2004; Yan et al., 1994). Other reactive aldehydes such as lipid oxidation products, hydroxynonenal, malondialdehyde, and acrolein are additional sources of protein damage. It is theoretically possible that carnosine or related structures could react with these deleterious aldehydes and thereby suppress their damaging effects towards proteins (Hipkiss, 2007a). It may be significant that a low serum carnosine level has been reported to be associated with AD (Fonteh et al., 2007). Furthermore, a raised level of protein AGEs in cerebral spinal fluid (CSF) (Ahmed et al., 2005; Luth et al., 2005; Shuvaev et al., 2001; Yamagishi et al., 2005) is associated with AD, while homocarnosine levels in CSF generally decline markedly with age (Huang et al., 2005; Janssen et al., 2005). It may also be significant that carnosine is enriched in the olfactory lobe (Barkardjiev, 1997; Bonfanti et al., 1999; Sassoe-Pognetto et al., 1993) and a loss of a sense of smell may be an early symptom of neurodegeneration (Ghanbari et al., 2004; Kovaks, 2004). Given carnosine’s homeostatic properties outlined above, it is at least worth considering whether carnosine or homocarnosine possess therapeutic potential toward AD (Hipkiss, 2007a), especially as the choroid plexus possesses a carnosine (homocarnosine) transport protein which may control CSF homocarnosine levels (Teuscher et al., 2004). While carnosine is absent from human CSF, one speculates that homocarnosine might act as an antiglycating agent in CSF (Hipkiss, 2007a). The


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age-related decline in CSF homocarnosine levels (Jansen et al., 2006) could at least partially explain the observed increase in glycated proteins CSF of AD patients, as well as the strong relationship between aging and AD. Many of the proteins which accumulate during neurodegenerative conditions may also become cross-linked by transglutaminase (Andringa et al., 2004; Junn et al., 2003; Karpuj and Steinman, 2004; Selkoe et al., 1982), an enzyme which cross-links the glutamine side chain to a lysine e-amino group. Transglutaminase protein is also associated with many of the inclusion bodies characteristics of AD, PD, etc. (Junn et al., 2003). It is theoretically possible that carnosine could substitute for the lysine residue e-amino group in the transglutaminase reaction (Hipkiss, 2007a) to generate g-glutamyl-carnosine as a hypothetical reaction product. While no such linkage has been reported (or sought) in neuronal tissue from neurodegenerative brain, the predicted g-glutamyl-b-alanyl-histidine products have been isolated from animal muscle tissues (Kuroda et al., 2000). However, as discussed above, it is possible that such structures might be derived from the spontaneous deamidation of glutamine residues in close proximity to carnosine or from the reaction of the dipeptide with oxidatively induced glutamic semialdehyde. Carnosine’s copper and zinc ion-chelating activity may also contribute to suppression of neurodegenerative conditions (Hipkiss, 2005). Zinc has been reported to be associated with the amyloid which accumulates in AD brain (Bush and Tanzi, 2002; Danscher et al., 1997; Religa et al., 2006), while copper ion-mediated oxidation of neuronal proteins may accompany both AD and PD (Smith et al., 2006). Carnosine has been found to protect cultured neurons against zinc-induced death (Kawahara et al., 2007). Cell culture studies have shown that carnosine is protective against the toxicity of the Ab-peptide which accumulates in the AD brain. The dipeptide prevented Ab-peptide (1–42)-induced glutamate release, but increased expression of the NMDA receptor. It was proposed that carnosine’s protective activity was exerted via regulation of glutamate release and independent of the carnosine–histidine–histamine axis (Fu et al., 2008). Similarly, Boldyrev et al. (2004c) found that carnosine suppressed the cytotoxicity of Ab42 in cerebellar granule cells independently of the dipeptides’ effects on calcium metabolism and ROS generation. Preston et al. (1998) also showed that the toxicity of the Ab42-related peptide fragment (25–35) towards rat brain endothelial cells was inhibited by carnosine and related structures, although the mechanism involved was not investigated. There is one explorative study investigating whether there is any correlation between serum carnosinase levels and dementia. The findings, using a small sample size, indicate that while there was no significant

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difference between control patients and those suffering from mixed dementia or AD, carnosinase activity was higher in patients who regularly exercised (Balion et al., 2007).

XXIII. PARKINSON’S DISEASE Parkinson’s disease is a neurodegenerative condition associated with the loss of dopaminergic neurons in a region of the brain called the substantia nigra pars compacta. The cause of PD is unknown but it seems that the substantia nigra is particularly susceptible to oxidative damage which in turn induces mitochondrial dysfunction and increased production of ROS, accompanied by the accumulation altered protein species which form aggregates called Lewy bodies. A major component of Lewy bodies is a protein called a-synuclein, an abundant presynaptic protein, but other proteins are present in these structures including ubiquitin, transglutaminase, and a number of heat-shock proteins. It appears likely that oxidative events including mitochondrial dysfunction play a major role in PD. Among the deleterious agents thought to be involved are peroxynitrite and hydroxyl radicals (Yokoyama et al., 2008). As noted above, carnosine has been shown to inhibit protein damage mediated by peroxynitrite and hydroxyl radicals in astroglial cells (Nicoletti et al., 2007). There is some evidence that carnosine can suppress some of the oxidative damage associated with PD using a model system, and possibly inhibit fibrillization of a-synuclein (Herrera et al., 2008). In order to investigate PD in animal models, one approach is to use a chemical called 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a known neurotoxin which induces symptoms similar to PD in animals and humans. When this compound was injected into senescence-accelerated mice (SAMP1), the animals demonstrated short-term tremor, weight loss and pronounced rigidity. Changes in the brain were observed including increased levels of protein carbonyls, lipid hydroperoxides, and monoamine oxidase-B activity. However, if these MPTP-treated animals were also treated with carnosine (100 mg/kg) for 14 days, the weight loss and rigidity were decreased, as were the levels of protein carbonyls, lipid hydroperoxides, and monoamine oxidase-B activity in their brains (Boldyrev et al., 2004a,b,c). These observations seem to suggest that carnosine suppresses some of PD-like changes induced by MPTP. At present it is unknown whether carnosine is similarly beneficial in humans. It has recently been shown that oxidative damage to glyceraldehyde dehydrogenase (GDH), an important glycolytic enzyme, occurs in the frontal cortex in PD patients (Gomez and Ferrer, 2009). Not only would this limit ATP synthesis and generation of many necessary metabolic


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intermediates, but would also increase formation of MG. As noted above, MG is a highly toxic agent which rapidly reacts with available protein and lipid amino groups, and can provoke mitochondrial dysfunction and can induce many of the biochemical symptoms of aging. As also noted above, carnosine has been shown to protect proteins against MG-induced modification. a-Synuclein is a major component of the Lewy bodies which accompany PD. There is an extensive literature about the roles of a-synuclein mutations and its metabolism, modification, and modes of aggregation (Bisaglia et al., 2009). There is some evidence that carnosine can inhibit a-synuclein oligomerization in a model system (Kang and Kim, 2003; Kim et al., 2002). Another paper has shown that an early event in Lewy body diseases is formation of adducts between a-synuclein lysine amino groups and malondialdehyde (MDA), a lipid peroxidation product, in the substantia nigra and frontal cortex (Dalfo and Ferrer, 2008). Many years ago it was shown that carnosine inhibited both MDA-mediated toxicity in cultured neuronal cells and formation of protein carbonyls and protein cross-linking (Hipkiss et al., 1997). It appears that the a-synuclein contains intramolecular cross-links possibly mediated by tissue transglutaminase (Andringa et al., 2004; Muma, 2007; Ruan and Johnson, 2007). As noted above there is a theoretical possibility that carnosine may be a competitive inhibitor for tissue transglutaminase which could prevent formation of the a-synuclein crosslinks which may prevent Lewy body formation and proteasome inhibition. There is evidence that proteasomal function is compromised in PD (McNaught et al., 2003), which could promote accumulation of a-synuclein, etc. and consequent inclusion body formation. Mutations in the genes for at least two proteins, Parkin and ubiquitin carboxyterminal hydrolase L1, which are components of the ubiquitin–proteasome system (Pallancke and Greenamyre, 2006) are associated with PD. Additionally, mutations in the gene for a protein, termed Pink1, which may have a role in mitochondrial function and appears to associate functionally with Parkin (Clark et al., 2006), are also associated with PD. One possibility is that Pink1 regulates a mitochondrial protease HtrA2 also called Omi (Plun-Favreau et al., 2007) which may be involved in protein quality control. Whether stimulation of the proteasomal system and/or chaperone-mediated autophagy by carnosine-induced increased stress protein synthesis is beneficial to the compromised proteolytic apparatus is unknown. A recently proposed explanation of PD involves the formation of adducts between dopamine and the products of the peroxidation of arachidonic acid and docosahexanoic acid (Liu et al., 2008b). At least two compounds, hexanoyl-dopamine and propanoyl-dopamine, derived

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from arachidonic acid and docosahexanoic acids respectively, are cytotoxic to cultured neuronal cells via ROS production and mitochondrial dysfunction. It is conceivable that carnosine could substitute for dopamine as the dipeptide does exhibit antioxidant activity. This suggestion predicts that hexanoyl-carnosine and propanoyl-carnosine might be generated. Interestingly it has recently been reported that dietary supplementation of young pigs with docosahexanoic acid provokes a decrease in muscle carnosine levels (Li et al., 2008), which may indicate some sort of relationship between the dipeptide and unsaturated fatty acids and is consistent with the idea that carnosine and docosahexanoic acid peroxidation products form adducts together. Although there is no cure for PD, a common treatment to maintain dopamine supply, and hopefully slow down the degeneration, involves using L-dopa as a source of dopamine. It has been reported, however, that some of the monoamine oxidase-generated oxidative metabolites of L-dopa are neurotoxic, most likely due to the generation of aldehyde groups on them (Burke et al., 2004). Given carnosine’s avidity for a variety of metabolic aldehydes (discussed above), it is theoretically possible that the dipeptide could react with 3,4-dihydroxyphenylglycolaldehyde (dopal), the product of monomine oxidase activity on L-dopa, especially as L-dopa treatment in rats elevates dopal levels in their brains 18-fold (Fornai et al., 2000). Combining carnosine treatment with L-dopa therapy in PD subjects has recently been examined in a study using 36 patients (Boldyrev et al., 2008). It was found that carnosine treatment (1.5 g/day) significantly improved a number of neurological symptoms including decreased rigidity of the hands and legs, and increased hand movement and leg agility. At a biochemical level, it was found that the level of protein carbonyls in blood plasma was decreased after 30 days carnosine treatment; furthermore, the levels of red cell Cu/Zn-superoxide dismutase were increased in the carnosine-treated PD patients. While these results are very encouraging with respect to the efficacy of carnosine with respect to PD and possible other neurological conditions involving aldehydes and ROS species, they require much larger and extensive trials to confirm these findings. Nevertheless, they do seem to indicate that carnosine can exert some therapeutic benefit despite the presence of serum carnosinases. There has been another study where carnosine has been employed in combination with a source of dopamine. Sozio et al. (2008) chemically linked the b-amino group of carnosine to an L-dopa precursor and then measured the release of dopamine over a 12-h period in rats. It was found that when presented as a co-drug the level of tissue dopamine was retained at higher levels compared to when the animals were given free L-dopa. There were no major effects of Parkinsonian behavior noted,


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although given the very brief time over which these experiment was carried out, this is not unexpected.

XXIV. CARNOSINE AND ISCHEMIA Evidence that carnosine possesses anti-ischemic activity emerged from Russian studies some years ago (see Stvolinsky and Dobrota, 2000 and references therein). Since then even more encouraging evidence has been obtained; the dipeptide has therapeutic potential against a number of ischemic conditions in brain, liver, heart, and kidney. Studies of brain ischemia or strokes using animal models (Gallant et al., 2000; Rajanikant et al., 2007; Tang et al., 2007; Yasuhara et al., 2008) have shown that carnosine is protective, even when added after the ischemic injury (Dobrota et al., 2005; Rajanikant et al., 2007; Tang et al., 2007). As part of the possible explanations for its protective action, it has been suggested that carnosine’s ability to scavenge the lipid peroxidation products hydroxynonenal (Tang et al., 2007) and malondialdehyde (Dobrota et al., 2005) help to compensate for any ischemia-induced deficit in antioxidant activity. It has also been shown in the postischemic mouse brain that carnosine treatment, 2 h following the experimental stroke, caused a decrease in ROS levels and matrix metalloproteinase protein levels and activity, whereas glutathione levels were preserved (Rajanikant et al., 2007). It appeared that carnosine treatment 2 h following the experimental stroke was also effective in decreasing infarct area, but when added after 4 h carnosine was ineffective, indicating a therapeutic window if carnosine is to be considered for treatment of strokes in humans. Interestingly, a recent study has shown that the presence of bestatin, an inhibitor of the enzyme carnosinase which cleaves the dipeptide into its constituent amino acids, histidine, and b-alanine, suppressed the efficacy of carnosine in the mouse brain stroke model (Min et al., 2008). Indeed the presence of bestatin increased stroke severity but did not raise cerebral carnosine levels. This may indicate that conversion of the dipeptide into histidine and b-alanine is required for efficacy; alternatively bestatin may have been exerting other unidentified effects. The carnosine analogues anserine and N-acetyl-carnosine were much less effective than carnosine in decreasing stroke infarct size. Homocarnosine, however, does protect cultured neuronal cells against ischemia (Tabakman et al., 2004). Protective effects have also been observed against ischemia in liver (Fouad et al., 2007), heart (Alabovsky et al., 1997), and kidney (Fujii et al., 2005, 2003; Kurata et al., 2006). Despite these clear observations of efficacy, the underlying mechanisms responsible for carnosine’s effects remain uncertain but presumably include its antioxidant and carbonyl-scavenging

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activities and possible actions on matrix metalloproteinases (Rajanikant et al., 2007) and histamine receptors (Kurata et al., 2006).

XXV. CARNOSINE AND OSTEOPOROSIS It is possible that regulation of protein glycation, and formation of protein AGEs, can affect osteoporosis (Hein, 2006). Some recent studies suggest that glycation can affect bone’s mechanical property (Shiraki et al., 2008; Tang et al., 2009) possibly by provoking deleterious changes in osteoblast function (Franke et al., 2007). Whether dietary carnosine would affect glycation of bone proteins is an obvious question which has been addressed by a Japanese group who have produced evidence suggesting the carnosine–zinc complexes are therapeutic in terms of bone loss in animal models and humans (Kishi et al., 1994; Sugiyama et al., 2000; Yamaguchi and Kishi, 1993; Yamaguchi and Matsui, 1996). It is suggested that the carnosine–zinc complex both stimulates bone formation by osteoblasts and decreases bone resorption by the osteoclasts (Yamaguchi, 1995; Yamaguchi and Kishi, 1995a). The mechanisms involved remain obscure, but it appears that in cultured mouse marrow cells the carnosine–zinc complex inhibits osteoclast cell formation, when present at between 106 and 104 M, by inhibiting the action of transforming growth factor-b (Yamaguchi and Kishi, 1995a) and parathyroid hormone, possibly by interfering with calcium signaling (Yamaguchi and Kishi, 1995b). It is also possible that the zinc–carnosine complex enhances the anabolic effects of estrogen on osteoblasts (Yamaguchi and Matsui, 1997). It is clear from these observations that carnosine, when complexed with zinc, may have beneficial effects towards control of osteoporosis but many more studies, including double-blind trials in humans, are required before any unequivocal statement of its efficacy can be made.

XXVI. CARNOSINE AND CATARACTOGENESIS A Russian group headed by Barbizhayev have produced a substantial body of work emphasizing carnosine’s potential for the treatment of lenticluar cataracts in humans (Barbizhayev, 2008; Barbizhayev et al., 2004). In particular, Barbizhayev has suggested (Barbizhayev et al., 2001) that N-acetylcarnosine, which is unsusceptible to the action of serum carnosinase, might be useful as a prodrug as the acetyl group is apparently readily cleaved intracellularly to release carnosine which then exerts its anticataractogenic effects, most probably via a combination of antioxidant and antiglycating activities. It is thought that there is little carnosinase activity in the eye lens. The use eyedrops containing


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N-acetyl-carnosine over trial periods of 2 and 6 months were reported to alleviate vision deficiency (lens opacity, visual acuity) associated with cataractogenesis, compared to placebo group. The improvements were sustained for 24 months (Barbizhayev, 2004, 2005).

XXVII. CARNOSINE AND DEAFNESS Production of ROS is associated with deafness in animals and humans. It appears that carnosine can suppress loss of hearing induced by antibiotics and other agents, although it is uncertain as to the precise mechanisms involved (Zhuravskii et al., 2004a,b). Early studies had shown, however, that carnosine exhibited excitatory activity to the afferent fibers in the lateral line organ of frogs (Mroz and Sewell, 1989; Panzanelli et al., 1994) which may indicate an evolutionary role of the dipeptide in sound detection.

XXVIII. CARNOSINE AND CANCER Antineoplastic activity of carnosine was first reported more than two decades ago (Nagai and Suda, 1986). L-Carnosine’s ability to kill cultured transformed cells (3T3 cells and HeLa cells), selectively, was found to be dependent on the absence of pyruvate in the growth medium (Holliday and McFarland, 1996, 2000); D-carnosine was nontoxic to HeLa cells. When pyruvate or certain other metabolic intermediates (oxaloacetate and a-ketoglutarate) were present in the growth medium, the toxic effects of carnosine towards the transformed cells were inhibited, but citrate, isocitrate, succinate, fumarate, and malate had no effects upon carnosine’s ability to kill the cells. The explanation of carnosine’s toxicity towards transformed cells is very uncertain. It is possible that carnosine may be inhibiting glycolysis by reacting with glyceraldhyde-3-phosphate, and thereby limiting the supply of metabolic precursors and possibly ATP, whereas the addition of pyruvate, oxaloacetate, and a-ketoglutarate enables these limitations to be overcome. It should be pointed out that many transformed cells are highly dependent on glycolysis for their ATP supply and are more sensitive to agents that interfere with this pathway. The presence of dietary carnosine in vitamin E-deficient rats was found to increase mammary tumor latency, while not affecting tumor incidence (Boissoneault et al., 1998). Another beneficial effect of carnosine in relation to cancer has recently been reported: carnosine was shown to inhibit metastasis of hepatocarcinoma SK-Hep-1 cells (Chung and Hu, 2008). Unlike the effects reported above, carnosine did not affect the viability of these cells but instead the dipeptide inhibited cell migration and invasion. The mechanism responsible apparently involves a decrease

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in extracellular matrix metalloproteinase-9 (MMP-9), an activity necessary for tumor invasion and angiogenesis. However, carnosine did not directly affect MMP-9 activity. Instead the dipeptide appears upregulate expression of the antimetastatic gene nm23-H1, whose gene product inhibits MMP-9 gene expression, and thereby suppresses synthesis of this activity necessary for tumor invasion and metastasis.

XXIX. CARNOSINE AND WOUND HEALING One problematic aspect of the aged organism is slower wound healing. There is evidence that carnosine can have beneficial effects here (Roberts et al., 1998). When carnosine is complexed with zinc to form ‘‘polaprezinc,’’ it behaves as an antiulcer drug which also possesses wound-healing activity (Nagai et al., 1986). Investigation of the possible mechanisms involved has revealed that the zinc–carnosine complex may stimulate synthesis of insulin-like growth factor-1 (Watanabe et al., 1998) and decreased secretion of interleukin-8 (IL-8), due to suppression of IL-8 mRNA expression in gastric epithelial cells. Polaprezinc also downregulated NF-kB activation by a number of activators suggesting overall anti-inflammatory action (Shimada et al., 1999), but did not involve modification of prostaglandin E2 production in gastric epithelial cells (Arakawa et al., 1990). Further investigation revealed that zinc–carnosine induced expression of the stress protein Hsp72 while inhibiting NF-kB activation in colonic mucosa (Odashima et al., 2002, 2006). These observations may help explain the beneficial effects of polaprezinc in rodent and human gut (Mahmood et al., 2007). Polaprezinc also ameliorated aspirininduced mucosal injury in rats (Naito et al., 2001) most probably by inhibiting the increase in neutrophil myeloperoxidase via inhibition of TNF-a expression. Vimentin is thought to play a role in wound healing (Mor-Vaknin et al., 2003), and carnosine has been shown to stimulate vimentin expression in rat fibroblasts (Ikeda et al., 1999). Therefore, it is possible that this provides an additional mechanism for carnosine’s beneficial effects on wound healing.

XXX. CARNOSINE AND IMMUNE FUNCTION There is some evidence suggesting that carnosine can upregulate immune function. Carnosine’s ability to react with hypochlorite anions (Formazyuk et al., 1992; Quinn et al., 1992) generated in activated leukocytes via the myeloperoxidase reaction, suggests that the dipeptide may limit hypochlorite-mediated oxidation in vivo (Pattison and Davies, 2006)


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and moderate neutrophil function (Tan and Candish, 1998). There is also some evidence that carnosine can suppress contact hypersensitivity in mice, but the mechanisms involved have not been studied in detail (Reeve et al., 1993a,b).

XXXI. CARNOSINE, CALCIUM, AND HEART FAILURE Carnosine occurs in cardiac muscle at concentrations between 2 and 10 mM (Roberts and Zaloga, 2000). Heart failure is thought to be associated with dysregulation of myocardial calcium metabolism resulting in contractile failure. There is evidence that carnosine can improve cardiac contractility, possibly via its effects on regulation of intracellular calcium levels, in a concentration-dependent manner (Zaloga et al., 1996). Studies in rats have shown that carnosine increases the levels of free calcium ions while also increasing the sensitivity of the contractile proteins to calcium (Batrukova and Rubstov, 1997; Roberts and Zaloga, 2000; Zaloga et al., 1997). It is unknown if there is any relationship between heart failure and myocardial carnosine levels in humans patients, although it is known that tissue carnosine levels are decreased in animals suffering from trauma and chronic infection which are associated with impaired cardiac contractility (Roberts and Zaloga, 2000).

XXXII. CARNOSINE AND AUTISTIC SPECTRUM DISORDERS Autism and Asperger’s syndrome are regarded as pervasive developmental disorders. Autism is a neurological disorder associated with impairment of language, cognition, and socialization, whereas Asperger’s syndrome is an autistic condition not associated with language delay or intellectual impairment. The causes of these conditions are unknown. In a double-blind, placebo-controlled, trial it was found that that carnosine supplementation improved the behavior, communication, and socialization in children with autistic spectrum disorders (Chez et al., 2002). The mechanism responsible for these effects is very uncertain, but it has been hypothesized that increased oxidative stress may be associated with autism (Chauhan and Chauhan, 2006; Chauhan et al., 2004; Yorbik et al., 2002), and that polymorphisms in the gene coding for the aldehydescavenging enzyme glyoxalase 1 could be a susceptibility factor (Junaid et al., 2004). Other workers have questioned this conclusion (Rehnstrom et al., 2008; Sacco et al., 2007; Wu et al., 2008). However, a recent finding by Fujimoto et al (2008) has shown that expression of glyoxalase mRNA in white blood cells correlated inversely with the onset of depression in bipolar disorder patients, compared to controls. It is possible that changes

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in glyoxalase expression in neocortex tissue may play a role in autism (Sacco et al., 2007) as increased protein AGEs have been detected in postmortem autistic brain ( Junaid et al., 2004). These observations may underline carnosine’s effects on the autistic children as the dipeptide possesses antioxidant activity and is protective against methylglyoxalmediated protein modification. However, it should be emphasized that there have been no other published reports of the beneficial effects of carnosine towards autistic spectrum disorders.

XXXIII. CARNOSINE AND BLOOD PRESSURE There is evidence that carnosine is a vasodilator (Ririe et al., 2000) and thus can lower blood pressure (Niijima et al., 2002; Tanida et al., 2005). It has been shown that carnosine promotes synthesis of nitric oxide (Nicoletti et al., 2007; Tomonaga et al., 2005), a well-known dilator of blood vessel walls. Also carnosine can inhibit angiotensin-converting enzyme (ACE) activity (Hou et al., 2003; Nakagawa et al., 2006) possibly via effects on cGMP and nitric oxide, which again points to the possibility that the dipeptide or carnosine-enriched foods could be explored to combat raised blood pressure in humans.

XXXIV. CARNOSINE AND CONSUMPTION OF ALCOHOLIC BEVERAGES Consumption of alcoholic drinks leads to the generation of acetaldehyde in the tissues, predominantly the liver but also in the brain. Acetaldehyde can react with protein amino groups to generate carbonyls with the potential for cross-linking to other macromolecules. It is thought that acetaldehyde generation is a major source of ‘‘hangovers’’ experienced following excessive alcohol consumption. Given carnosine’s ability to react with acetaldehyde and protect cultured human fibroblasts and lymphocytes against its toxicity (Hipkiss et al., 1998a) as well as prevent cross-linking between protein and DNA (Hipkiss et al., 1995b), it has been suggested that ingestion of carnosine, either as a supplement or as a high-carnosine food (meat), could be an effective way to prevent ‘‘hangovers’’ (Hipkiss, 1998), as well as protecting the brain and other tissues against alcohol-induced glycoxidative damage. This should be relative easy to test. Beneficial effects of carnosine have been described with respect to ethanol-induced liver injury in mice (Liu et al., 2008a,b). It was found that following 3 weeks of ethanol treatment (present in drinking water), subsequent exposure to carnosine decreased liver malondialdehyde


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levels by around 40% compared to ethanol-treated animals. The dipeptide also promoted a decline in indices of cell damage (release of liver enzymes), increased glutathione content and catalase and glutathione peroxidase activities, and downregulated expression of inflammatoryassociated cytokines (IL-6 and TNF-a).

XXXV. CARNOSINE AND HIGH FRUCTOSE FOODS AND DRINKS There has been much interest in the metabolic effects of fructose and whether its consumption should be restricted due to the sugar’s potential deleterious effects with respect to diabetes-associated phenomena (Abdel-Sayred et al., 2008; Brown et al., 2008; Le and Tappy, 2006; Miller and Adeli, 2008). In particular, fructose glycates proteins far more readily glucose to generate protein AGEs, and as a consequence, could possibly be responsible for increasing the incidence of type-2 diabetic complications ( Jia and Wu, 2007; Lo et al., 2008). Additionally, fructose is also a ready metabolic source of methylglyoxal which, as already described, is a highly deleterious agent which is thought to be a major causal agent of AGE formation and therefore much of the secondary complications of type-2 diabetes. As carnosine can react with methylglyoxal and fructose to prevent their damaging effects on proteins at the test-tube level, one might consider whether increasing tissue carnosine levels might be beneficial in high fructose diets. However, no such study has yet been carried out to test whether any of these ideas are justified. A further observation has recently been revealed in that high fructose consumption by men can increase the risk of gout due to an increased production of uric acid (Choi and Curhan, 2008; Gao et al., 2008). Consequently it would be interesting to determine whether carnosine ameliorates uric acid production in humans.

XXXVI. CARNOSINE AND DIALYSIS FLUIDS Treatment of kidney failure involves dialysis using heat-sterilized dialysis fluids. Because the dialysis fluid contains glucose, the heating inevitably generates glucose degradation products such as methylglyoxal, glyoxal, and acetaldehyde, which are well recognized for their ability to induce AGEs on protein targets. Hence dialysis with aldehyde-containing dialysis fluid will not be expected to improve kidney health, but exacerbate the kidney dysfunction. Because of carnosine’s ability to protect proteins against aldehydic glycating agents, the possibility that the dipeptide may decrease reactivity of the AGE precursors was explored

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(Alhamdani et al., 2007a,b). It was shown that heat-treated peritoneal dialysis fluid compromised the viability of cultured human peritoneal mesothelial cells, whereas the additional presence of carnosine in the incubation medium considerably enhanced cell viability, and markedly decreased cell-associated protein carbonyl groups and ROS generation. These observations obviously suggest that carnosine could be employed to either remove deleterious glucose degradation products from dialysis fluid prior to use, or that the dipeptide could be added to dialysis fluid to suppress the reactivity of the protein damaging agents. However, in the latter case it is uncertain whether patients’ kidneys would be able to deal with (i.e., selectively excrete) the putative aldehyde–carnosine adducts.

XXXVII. POSSIBLE WAYS TO INCREASE TISSUE CARNOSINE LEVELS: PHYSIOLOGICAL REGULATION There have been relatively few studies of age-related changes in tissue carnosine levels despite the fact that the initial observation of the dipeptide’s ability to suppress some features of senescence were made more than 15 years ago. Carnosine levels have been reported to decline with age in the rats ( Johnson and Hammer, 1992; Stuerenburg and Kunze, 1999) and human muscle (Stuerenburg and Kunze, 1999). More recently, Tallon et al. (2007) found evidence of carnosine’s age-related decline in human muscle fibers. On the assumption that increasing tissue levels of carnosine might be beneficial in terms of aging and some of its related conditions, this can be achieved either by physiological regulation or by dietary supplementation. Muscle carnosine levels are generally higher when accompanied by intense exercise in fast-twitch type II fibers compared to slow-twitch type I fibers. It has been found that muscle levels of the dipeptide can be increased following resistance exercise in humans (Hill et al., 2006) and there are also reports that very high levels of carnosine are present in highly trained race-horses (Harris et al., 1990). In humans, the carnosine content of vastus lateralis muscle is generally high in sprinters and body builders (Tallon et al., 2005); 8 weeks intensive training resulted in a doubling of the carnosine content of the vastus lateralis muscle (Kim et al., 2005). In athletes involved in explosive/intense muscle exercise, it is likely that the raised carnosine levels are required as physiological buffers. Hence it is possible that raising carnosine levels may improve muscle performance by increasing buffer capacity. In an attempt to increase carnosine levels by dietary means, but circumventing the effects of serum carnosinase, increasing b-alanine intake has been investigated


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(Harris et al., 2006). Studies have shown that the availability of b-alanine may limit carnosine synthesis, histidine being generally available metabolically. Therefore, it was suggested that dietary supplementation with b-alanine could raise carnosine synthesis in the tissues and studies on human subjects have shown that b-alanine is effective in raising muscle carnosine levels (Harris et al, 2006). Furthermore, b-alanine supplementation for 4 and 10 weeks increased vastus lateralis carnosine content by 58% and 80%, respectively, in subjects subjected to high-intensity cycling. A similar study using Vietnamese sports-science students showed that b-alanine supplementation promoted an increase in muscle carnosine concentration (Kendrick et al., 2008), although there were no improvements in any of the exercise parameters measured. In a double-blind randomized study of 26 elderly subjects, aged between 55 and 92 years, it was found that b-alanine supplementation for 90 days improved muscle endurance (physical working capacity) by 28%, presumably due to the increased synthesis of carnosine (Stout et al., 2008). It is interesting that while no beneficial effects in terms of muscle performance were observed in young subjects (Kendrick et al., 2008), whereas in the elderly, improvement was detected (Stout et al., 2008), presumably due to the lower tissue carnosine levels which limit performance in old muscles. It should be pointed out that subjects consuming b-alanine as a supplement (40 mg/kg body weight) experienced symptoms of flushing, skin irritation and prickly sensations for up to 1 h, first of the ears, forehead and scalp and then the trunk, arms, hands, spine, and buttocks. Lowering the b-alanine dose to 10 mg/kg body weight effectively eliminated these symptoms (Harris et al., 2006). Interestingly, consumption of chicken broth (enriched in carnosine) containing the equivalent of 40 mg/kg body weight b-alanine did not induce any of the unpleasant symptoms, but carnosine was not detected in the plasma of these subjects. Whether it is possible to raise carnosine levels in human brain is unknown. One study in rats has shown that oral administration of chicken extract (a major source of carnosine in humans too) did provoke an increase in brain carnosine levels: a single dose of the chicken extract led to an increase in carnosine levels within 30 min in plasma, but 1 or 2 h duration were required for increased levels of carnosine to be observed in the cerebral cortex, hypthalamus, and hippocampus (Tomonaga et al., 2007). It is uncertain whether these effects result from direct uptake of the carnosine from plasma or a consequence of de novo synthesis. In a study using senescenceaccelerated mice (SAMP8), it was found that oral supplementation with creatine provoked, at 25 weeks of age, a transient 88% increase in muscle carnosine content, accompanied by a 40% increase in anserine content, which coincided with an improvement in resistance to contractile fatigue (Derave et al., 2008). At 60 weeks, no differences were detectable between the creatine-supplemented and control animals in terms of their muscle

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carnosine and anserine levels. The mechanism responsible for this effect is uncertain but could involve an upregulation of carnosine synthesis or its decreased catabolism, but why either of these is influenced by creatine supply remains unclear. This study also showed that muscle carnosine declines by 45% with age (from 10 to 60 weeks) in the control SAMP8 mice. Relatively little is known of the factors that control carnosine synthesis, although as mentioned above, the enzyme involved, carnosine synthetase, does appear to be regulated by cyclic AMP (Schulz et al., 1989) which suggests the possibility that conditions which lower cyclic AMP levels may increase carnosine synthesis. Thus, increased glucose metabolism via glycolysis might be accompanied by an upregulation of carnosine synthesis. It is possible that this suggested relationship is beneficial due to carnosine’s ability to suppress the reactivity of a major deleterious byproduct of the glycolytic pathway, methylglyoxal (MG) (discussed above). The primary enzyme responsible for carnosine’s hydrolysis into b-alanine and histidine is carnosinase, an activity which is stimulated by citrate (Vistoli et al., 2006). These observations raise the possibility that intracellular carnosine levels may be subject to metabolic regulation due to the effects of major metabolic intermediates and effectors on the enzymes responsible for its synthesis and degradation. Carnosine levels in the tissues have been seen to decline following trauma and during chronic infection (Fitzpatrick et al., 1980). It is interesting that pathological states are associated with decreased cardiac function possible due to problems with cardiac muscle contraction. It has been suggested that decreased carnosine levels may play a role in decreased muscle contractivity in a number of disease states including congestive heart failure (Roberts and Zaloga, 2000).

XXXVIII. POSSIBLE WAYS TO INCREASE TISSUE CARNOSINE LEVELS: DIETARY SUPPLEMENTATION While carnosine is absorbed intact from the gut, the presence of serum carnosinase is frequently cited as an impediment to the dipeptide’s potential efficacy. However, studies have shown that serum carnosine levels are raised at least temporarily, up to 4–5 h, following a carnosinecontaining meal (Antonini et al., 2002; Park et al., 2005). Such studies indicate a window of opportunity for carnosine administration. One approach to overcoming the carnosinase effect would be to employ a carnosinase inhibitor such as bestatin, although undoubtedly there would be some side effects. The fact that carnosinase has been shown to be upregulated by citrate (Vistoli et al., 2006) may permit the design of specific inhibitory molecules. Another approach would be to employ a form of carnosine which is resistant to carnosinase attack such as


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N-acetyl-carnosine or the decarboxylated form carcinine. In fact N-acetylcarnosine has been proposed as a prodrug to treat cataracts in the eye lens, as the acetyl group is readily removed intracellularly (Barbizhayev et al., 2004) (see section on cataracts for more details). An alternative way to evade serum carnosinase activity would be to introduce carnosine via nasal administration (Hipkiss, 2005). This route may be particularly appropriate for raising carnosine levels in the brain as the olfactory lobe is normally enriched in the dipeptide. Synthesis of carnosine analogues resistant to carnosinase attack is another method which has been employed in an attempt to circumvent the problem of serum carnosinase (Bellia et al., 2008; Cacciatore et al., 2005; Calcagni et al., 1999; Guiotto et al., 2005b). Some of the resultant structures with the b-alanine replaced by 2,3-diaminoproprionic acid residue and with acetyl groups on either of its amino groups were shown to not only resist attack by carnosinase but also inhibit the enzyme’s ability to cleave carnosine, while still retaining hydroxyl radical-scavenging activity and preventing peroxynitrite-mediated tyrosine nitration (Cacciatore et al., 2005). Other structures synthesized have included sulfonamidopseudopeptides, tauryl-histidine, tauryl-1-methylhistidine, and tauryl-3methylhistidine (Calcagni et al., 1999), while another Italian laboratory generated cyclodetrin conjugates of carnosine, attached via the dipeptide’s b-amino group to either carbon-3 or carbon-6 of the glucose moiety. These glycosidic derivatives were resistant to carnosinase attack, and seemed to be better inhibitors of copper-induced lipid peroxidation than the parent dipeptides, carnosine, and anserine (Bellia et al., 2008). There were no reports on the efficacy of the cyclodextrin conjugates with respect to carnosine’s antiglycating activity, however.

XXXIX. IS THERE ANY EVIDENCE THAT CHANGES IN DIETARY CARNOSINE HAVE ANY EFFECTS IN HUMANS? There have been few studies on carnosine consumption in humans. Due to the documented presence of carnosinase in blood, many scientists have assumed that the dipeptide’ survival would be relatively short due to its rapid hydrolysis by the enzyme. Nevertheless, a study by Gardner et al. (1991) showed that plasma carnosine levels peaked at over 180 mg/ml, 0.5 h after intake of a beverage containing 3 g of carnosine. Maximal carnosine levels in urine occurred within 2 h. In another study, Park et al. (2005) showed that ingestion of cooked ground beef containing 248 mg of carnosine led to plasma carnosine concentration rising from essentially 0 to around 30 mg/ml within 3.5 h and thereafter rapidly declining such that none was detectable 2 h later. These studies indicate

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that, despite the presence of serum carnosinase, ingestion of carnosine can lead to raised levels of the dipeptide in blood which could then lead to effects on tissue carnosine. Studies of the effects of carnosine consumption on humans have been rare. One study showed that, following intake of a bolus of carnosine (450 mg), serum total antioxidant activity was increased by 11% 1 h after ingestion (Antonini et al., 2002). Such an increase in antioxidant function is consistent with the dipeptide’s recognized antioxidant activity. It is possible that human brain function can be affected by dietary carnosine as it has been shown that the dipeptide (two 400 mg doses per day) can modulate the behavior, socialization, and communication skills of autistic children (Chez et al., 2002 and discussed above). Homocarnosine, which also possesses anticonvulsant activity in humans, is found in human CSF. It is unknown whether the sevenfold age-related decline in homocarnsine levels in human CSF (Huang et al., 2005; Janssen et al., 2005) contributes to the onset or progression of any age-related pathology. However, as protein AGEs accumulate in CSF of AD patients (Ahmed et al., 2005; Shuvaev et al., 2001), one has to at least consider whether there is a causal relationship between these observations (Hipkiss, 2007a). Carnosine (400 mg/day) together with omega-3-fatty acids (eicosapentaenonic acid) was employed in a study of dietary effects on dyslexic children. In this study there were no significant effects of the dietary supplements on a range of language skills and behavior problems (Kairaluoma et al., 2008). There is some evidence that carnosine supplementation can restore sense of taste in humans. A report from Japan states that polaprezinc (a zinc–carnosine complex) is frequently effective in treating patients experiencing taste disorders (Ikeda et al., 2005). Experiments with zincdeficient rats showed that polaprezinc was effective in restoration of taste bud proliferation (Hamano et al., 2006). Polaprezinc has also been shown to be beneficial in treatment of ulcers and other gut lesions (discussed above) and in inhibiting some of the changes surrounding osteoporosis (also discussed above).

XXXX. WOULD VEGETARIANS BENEFIT FROM CARNOSINE SUPPLEMENTATION? The possibility that vegetarian diets, deficient in carnosine, could be somewhat deleterious in the ability to suppress aldehyde-induced protein modification and AGE has been discussed (Hipkiss, 2005, 2006c). Indeed, Harris et al. (2007) showed that muscle carnosine levels were reduced by up 50% in vegetarian subjects. There is one report of


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increased AGEs in vegetarian type-2 diabetics’ sera (KrajcovicovaKudlackova et al., 2002). This may be due to the absence of carnosine in the vegetarian diet, but an alternative explanation could be that the increased fructose in the vegetarian diet increases AGE formation. It should be pointed out that many plants contain high levels of aldehyde scavengers which are probably present to protect plant proteins against glycating sugars such as fructose, and which could be exploited as dietary antiglycating agents.

XXXXI. DELETERIOUS EFFECTS OF CARNOSINE Carnosine is usually regarded as being almost nontoxic (Sato et al., 2008). However, there are some indications that the dipeptide can have deleterious effects. It has been known for a long time that humans with mutations in the gene coding for serum carnosinase show high levels of the dipeptide in their blood which is accompanied by neurological dysfunction (Gjessing et al., 1990; Wassif et al., 1994; Willi et al., 1997). This may suggest that elevated serum carnosine or a failure to cleave the dipeptide elsewhere has deleterious effects, although there have been claims made that elevated levels of serum carnosine is not in itself a problem. There is one rather obvious possible way in which carnosine could be deleterious should the dipeptide prove to be an inhibitor of serum transglutaminase activity (Hipkiss, 2007a). Inhibition of this enzyme would suppress the development of cross-linking between fibrin molecules and could therefore compromise blood clotting. However, this remains a speculation at this stage. There is one report suggesting that carnosine affects spermatogenesis in senescence-accelerated mice (SAMP1), reducing cell yield and increasing destructive changes in spermatogenic epithelium in the testicular tubules (Gopko et al., 2005). However, the same research group had earlier stated (Zakhidov et al., 2002) that carnosine did not modify the incidence of chromosome mutations in spermatogenic cells in these animals. There is also a paradoxical situation in the aging-resistant SAMR1 mice. In 2002 it was stated that carnosine increased the count of aberrant spermatogonia in the SAMR1 animals (Zakhidov et al., 2002), whereas it was later reported that in these animals, carnosine treatment resulted in no increase in the incidence of aberrant spermatogia (Gopko et al., 2005). Hence the significance of these observations is uncertain, but there have been no other reports of deleterious effects of carnosine. Nevertheless, it cannot be arbitrarily assumed that carnosine may not be without toxicity in some systems or organs.

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XXXXII. CONCLUSIONS Studies using model systems, cell culture, and animals have indicated that carnosine possesses a range of potential homeostatic functions which together may help to suppress many of the biochemical changes to macromolecules which accompany aging and a number of related pathological conditions. Especially relevant is carnosine’s carbonyl-scavenging ability which may prove to be particularly important in suppressing formation of protein carbonyls and those cross-linked protein species which inhibit proteasomal elimination of altered proteins. Carnosine may also stimulate nitric oxide synthesis and thereby increase proteasome activity, as well as upregulate synthesis of another protease, OPH. There is also some evidence that carnosine’s other properties could also contribute to its antiaging activities include antioxidant, wound healing agent, aldehyde scavenger (including methylglyoxal), copper and zinc chelator, heatshock protein inducer, anti-inflammatory agent, and antiepileptic agent. Much more work is required to explore all these proposals and speculations. As aging does appear to be multifactorially controlled, it is perhaps not unsurprising that a pluripotent agent such as carnosine might exert antiaging effects via more than one mode of action. Evidence of carnosine’s efficacy towards human health is relatively sparse in comparison with the range of effects observed in model systems and in animals. The most intensely investigated is cataractogenesis, although predominantly undertaken by a single research group. There is also relatively strong evidence that, in its zinc complex form (polaprezinc), carnosine has positive effects on in repair of gut lesions such as ulcers. While a good case can be made for the use of carnosine in alleviating the deleterious effects of ischemic conditions, especially stroke-related events, there is little or no direct evidence as yet that the dipeptide is efficacious in human patients. This is also the position for the neurodegenerative diseases, AD and PD, although there is evidence from one research group showing that carnosine does have beneficial effects on Parkinson’s patients undergoing treatment with L-dopa. The beneficial effects of carnosine on children with autistic spectrum disorders have been described by only one research group, so this needs verification. While a case can be made that carnosine may be useful in controlling the secondary complications associated with type-2 diabetes, there is no direct evidence that increased carnosine consumption suppresses their development in humans. Clearly, much more work is required to verify or refute the many proposals made of carnosine’s efficacy towards human health. Whether such studies will be undertaken is in doubt simply because of the nonpatentability of the molecule and therefore it is unlikely to generate large


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monetary benefit to any company or institution. However, if one were to try to determine whether carnosine does prevent many of the unpleasant effects of ‘‘hangovers’’ following excess ethanol consumption, one would anticipate that there would be no difficulty in finding volunteers. Science can sometimes be fun as well as intellectually challenging.

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Yasuhara, T., Hara, K., Maki, M., Masuda, T., Sanberg, C. D., Sanberg, P. R., Bickford, P. C., and Borlongan, C. V. (2008). Dietary supplementation exerts neuroprotective effects in ischemic stroke model. Rejuvenation Res. 11, 201–214. Yokoyama, H., Kurolwa, H., and Yano R Araki, T. (2008). Targeting reactive oxygen species, reactive nitrogen species and inflammation in MPTP neurotoxicity and Parkinson’s disease. Neurol. Sci. 29, 292–301. Yorbik, O., Sayal, A., Akay, C., Akbiyik, D. I., and Sohmen, T. (2002). Investigation of antioxidant enzymes in children with autistic disorder. Prostglandins Leukot. Essent. Fatty Acids 67, 341–343. Yuneva, M. O., Bulygina, E. R., Gallant, S. C., Kramarenko, G. G., Stvolinsky, S. L., Semyonova, M. L., and Boldyrev, A. A. (1999). Effect of carnosine on age-induced changes in senescence-accelerated mice. J. Anti Aging Med. 2, 337–342. Yuneva, A. O., Kramarenko, G. G., Vetreshchak, T. V., Gallant, S., and Boldyrev, A. A. (2002). Effect of carnosine on Drosophila melanogaster lifespan. Bull. Exp. Biol. Med. 133, 559–561. Zakhidov, S. T., Gopko, A. V., Semenova, M. L., Mikhaleva, Y. Y., Makarov, A. A., and Kulibin, A. Y. (2002). Carnosine modifies the incidence of genetically abnormal sex cells in the testes of senescence-accelerated mice. Bull. Exp. Biol. Med. 134, 78–80. Zaloga, G. P. and Siddiqui, R. A. (2004). Biologically active dietary peptides. Mini Rev. Med. Chem. 4, 815–821. Zaloga, G. P., Roberts, P. R., and Nelson, T. E. (1996). Carnosine: A novel peptide regulator of intreacellular calcium and contractility in cardiac muscle. New Horiz. 4, 26–35. Zaloga, G. P., Roberts, P. R., Black, K. W., Lin, M., Zapata-Sudo, G., and Sudo, R. T. (1997). Carnosine is a novel peptide modulator of intracellular calcium contractility in cardiac cells. Am. J. Physiol. 272, H462–H468. Zhu, J. X. and Aswad, D. W. (2007). Selective cleavage of isoaspartyl peptide bonds by hydroxylamine after methyltransferase priming. Anal. Biochem. 364, 1–17. Zhu, J. X., Doyle, H. A., Mamula, M. J., and Aswad, D. W. (2006). Protein repair in the brain, proteomic analysis of endogenous substrates for protein L-isoaspartyl methyltransferase in mouse brain. J. Biol. Chem. 28, 33802–33813. Zhu, Y. Y., Zhu-Ge, Z. B., Wu, D. C., Wang, S., Liu, L. Y., Ohtsu, H., and Chen, Z. (2007). Carnosine inhibits pentylenetetrazol-induced seizures by histaminergic mechanisms in histidine decarboxylase knock-out mice. Neurosci. Lett. 416, 211–216. Zhuravskii, S. G., Aleksandrova, L. A., Sirot, V. S., and Ivanov, S. A. (2004a). Natural antioxidant L-carnosine inhibits LPO intensification in structures of the auditory analyzer under conditions of chronic exposure to aminoglycoside antibiotics. Bull. Exp. Biol. Med. 138, 361–364. Zhuravskii, S. G., Aleksandrova, L. A., Ivanov, S. A., Sirot, V. S., Lopotko, A. I., and Zhloba, A. A. (2004b). Protective effect of carnosine on excitable structures of the auditory apparatus in albino rats with acute acoustic trauma. Bull. Exp. Biol. Med. 137, 98–102.

CHAPTER

4 Recent Advances in the Microbial Safety of Fresh Fruits and Vegetables Keith Warriner,* Ann Huber,† Azadeh Namvar,* Wei Fan,* and Kari Dunfield‡

Contents

I. Introduction II. Outbreaks Linked to Fresh Produce III. Characteristics of Pathogens Recovered from Salad Vegetables A. Pathogenic E. coli B. Shigella C. Salmonella D. Campylobacter E. Listeria monocytogenes F. Aeromonas hydrophila G. Endospore-forming bacteria H. Enteric viruses I. Human pathogenic protozoa IV. Transmission of Human Pathogens in Manure, Soil, and Water to the Vegetable Production Chain A. Manure and biosolids B. Irrigation water C. Soil D. Transport of human pathogens within the environment V. Interaction of Pathogens with Fresh Produce A. Survival in the phyllosphere

156 157 160 160 164 164 165 166 166 166 167 167 168 169 171 176 177 179 179

* Department of Food Science, University of Guelph, Guelph, Ontario, Canada { {

Soil Resource Group, Guelph, Ontario, Canada Department of Land Resources, University of Guelph, Guelph, Ontario, Canada


#


155

156

Keith Warriner et al.

B. Colonization of the rhizosphere C. Internalization of human pathogens in growing plants D. Genetic and physiological factors VI. Interventions to Enhance the Safety of Fresh Produce A. Biocontrol of human pathogens VII. Conclusions and Future Research References

Abstract

180 181 186 188 191 192 192

Foodborne illness outbreaks linked to fresh produce are becoming more frequent and widespread. High impact outbreaks, such as that associated with spinach contaminated with Escherichia coli O157:H7, resulted in almost 200 cases of foodborne illness across North America and >$300 m market losses. Over the last decade there has been intensive research into gaining an understanding on the interactions of human pathogens with plants and how microbiological safety of fresh produce can be improved. The following review will provide an update on the food safety issues linked to fresh produce. An overview of recent foodborne illness outbreaks linked to fresh produce. The types of human pathogens encountered will be described and how they can be transferred from their normal animal or human host to fresh produce. The interaction of human pathogens with growing plants will be discussed, in addition to novel intervention methods to enhance the microbiological safety of fresh produce.

I. INTRODUCTION The fresh-cut market has experienced rapid growth within the last decade and an estimated 6 million packs of bagged produce are sold within North America each day (Doyle and Erickson, 2008; Jongen, 2005). The driving force behind the rapid growth of the fresh produce is the desire of consumers to lead a healthy lifestyle along with the convenience of preprepared products. Consumers have become accustom to all-year-around availability of fruit and vegetables with the convenience of prepacked products that require minimal preparation. To meet consumer demand, the primary production has shifted away from local farmers to highly centralized centers, which in the case of North America, are located in Mexico, California, and Florida (Doyle and Erickson, 2008). Indeed, California alone supplies over 70% of all the leafy greens (e.g., lettuce, spinach) consumed within North America (FDA, 2002). Centralization of production has brought many benefits to the consumer such as relatively cheap produce, consistent quality, and all-year-round availability. However, the downside of centralized production is that when foodborne illness outbreaks occur, they are typically widespread and involve a high number of

Microbiological Safety of Fresh Produce

157

cases (Gorny, 2006). Although consumers in the course of an outbreak frequently turn to organic or locally grown produce, there is no evidence that this poses any less risk compared to ‘conventionally’ produced crops (Arthur et al., 2007; Loncarevic et al., 2005). In this respect, it cannot be concluded that the rapid rise in foodborne illness outbreaks are linked to centralized production alone. It is noteworthy that the increase in the fresh-cut (bagged salad) market has coincided with the increase in foodborne illness cases. Here, the produce is cut or shredded which provides entry points for pathogens, in addition to releasing nutrients to support microbial growth. Modified Atmospheric Packaging (MAP) reduces spoilage by aerobes but can enhance the virulence of pathogens such as Escherichia coli O157:H7 (Chua et al., 2008). In addition, the higher proportion of vulnerable, susceptible, people within a population along with increased surveillance and high prevalence of virulent pathogens within the environment are further reasons to consider for the rise in foodborne illness outbreaks (Arthur et al., 2008; FDA, 2001; Sewell and Farber, 2001).

II. OUTBREAKS LINKED TO FRESH PRODUCE There has been a rapid rise in foodborne illness outbreaks linked to fresh produce (Fig. 4.1). The pathogens of main concern are Salmonella and E. coli O157:H7 although, in principle, a diverse range of pathogenic microbes can contaminate fresh produce at any point in the chain. There has been several high profile foodborne illness outbreaks associated with fresh produce with sprouted seeds, tomatoes, and leafy greens remaining the most prominent (Table 4.1) (Doyle and Erickson, 2008). The underlying reasons for why specific produce types have been implicated in the majority of outbreaks can, in part, be explained by the market volume Outbreaks

3500

90 80 70 60 50 40 30 20 10 0

3000 Cases

2500 2000 1500 1000 500 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006

0

Outerbreaks

Reported cases

Year

FIGURE 4.1 Foodborne illness outbreaks linked to fresh produce from 1990 to 2006. Source: Centre for Science in the Public Interest (2008).

TABLE 4.1

Outbreaks linked to fresh produce

Date

Pathogen

Produce

Comments

December 2005 February 2006

Salmonella Salmonella

Mung bean sprouts Alfalfa sprouts

February 2006 June 2006 July 2006 August 2006

Salmonella E. coli O121:H19 Salmonella Salmonella

Alfalfa sprouts Lettuce Fruit salad Alfalfa sprouts

September 2006 September 2006 October 2006 October 2006 October 2006 November 2006 November 2006 November 2006 April 2007 August 2007 April 2008

E. coli O157:H7 Clostridium botulinum E. coli O157:H7 E. coli O157:H7 Salmonella E. coli O157:H7 E. coli O157:H7 Salmonella Salmonella Shigella sonnei Salmonella

Spinach Pasteurized carrot juice Lettuce Lettuce Tomatoes Lettuce Lettuce Peanut butter Lettuce Baby carrots Cantaloupe

Canada, 618 confirmed cases Canada, sprout recall due to suspected contamination Australia, 100 confirmed cases of salmonellosis United States, 4 confirmed cases United States and Canada, 41 confirmed cases United States, sprout recall due to suspected contamination United States, 205 confirmed cases; 3 deaths United States and Canada; 6 cases

June 2008 September 2008 September 2008 November 2008 December 2008

Salmonella E. coli O157:H7 Salmonella Salmonella Salmonella

Tomatoes/peppers Lettuce Alfalfa sprouts Basil Alfalfa sprouts

Canada; 30 confirmed cases Canada; recall due to suspected contamination United States; 183 cases United States; 81 confirmed cases United States; 71 confirmed cases United States; 481 confirmed cases UK, recall for suspected contamination Canada, 4 cases Canada, United States and Mexico, 64 confirmed cases United States and Canada, 1442 confirmed cases United States and Canada; 134 confirmed cases United States, 14 confirmed cases UK, 32 confirmed cases United States, recall for suspected contamination


159

(Anonymous, 2006; Thunberg et al., 2002; Valentin-Bon et al., 2008). However, there is a growing body of evidence supporting the hypothesis that certain pathogens are adapted to persist on different produce types (Bassett and McClure, 2008). For example, foodborne illness outbreaks linked to tomatoes have commonly implicated Salmonella (Barak and Liang, 2008; Greene et al., 2008) (Table 4.1). The same pathogen is also associated with cantaloupes, sprouted seeds, and lettuce (Arthur et al., 2007; Mohle-Boetani et al., 2008; Sivapalasingam et al., 2004). E. coli O157: H7 has been associated with sprouted seeds, lettuce, apples ( juice), and spinach ( Jablasone et al., 2005; Sivapalasingam et al., 2004; Valentin-Bon et al., 2008). Parsley is prone to contamination from Shigella and soft fruit with enteric viruses such as Hepatitis A (Anonymous, 1999; Jacobson et al., 2004; Mataragas et al., 2008; Naimi et al., 2003; Peterson et al., 1983; Reller et al., 2006; Seymour and Appleton, 2001; Wu et al., 2000). A more curious associated is the link between basil and Cyclospora (ChacinBonilla, 2007; Sivapalasingam et al., 2004). The underlying reasons for such associations remain obscure as is many aspects on the interactions of human pathogens with fresh produce. Although foodborne illness outbreaks linked to fresh produce have been recorded over 30 years, there has been a rapid increase in the number of cases recorded (Fig. 4.1). In 2005, the largest outbreak of salmonellosis linked to mung bean sprouts occurred within Ontario (Table 4.1). The implicated Salmonella serovar was Enteritidis, which is more commonly associated with poultry and raw eggs. How the S. Enteritidis strain became associated with the mung beans sprouts remains open to speculation although one major sprout producer within the province was targeted as the cause of the outbreak. The inability to trace human pathogens implicated in fresh produce foodborne illness outbreaks back to the original source is not uncommon and the ‘‘smoking gun’’ is rarely found. This can be attributed to the relatively short shelf life of fresh produce, which is often discarded by the time an outbreak is identified. The lack of traceability of produce is a further contributing factor that makes identification of specific sources problematic. However, the 2006 E. coli O157:H7 outbreak linked to baby spinach America was unique in that the strain of the pathogen was recovered from the infected people, spinach within unopened bags, and the farm where the crop was cultivated (Cooley et al., 2007). The route of spinach contamination was considered to be through the transfer of E. coli O157:H7 from a cattle ranch near the field via infected wild pigs that found access to the crop through a broken fence. Yet, it is noteworthy that a survey of the Salinas valley in the summer of 2006 found a high prevalence of E. coli O157:H7 within the area, suggesting the actual route could have been via contaminated irrigation water (Cooley et al., 2007). Other notably outbreaks linked to fresh produce occurring in 2006 included lettuce, sprouts, cantaloupes, and Clostridium botulinum linked

160


to pasteurized carrot juice (Doyle and Erickson, 2008) (Table 4.1). The latter was of specific interest given that the neurotoxin levels detected in contaminated product were the highest ever recorded (FDA, 2006). As with the majority of fresh produce outbreaks the exact sequence of events that led to the six cases of botulism within the United States and Canada remains unexplained. One of the largest foodborne illness outbreaks linked to fresh produce occurred primarily within the southern states of the US in 2008. The initial cause of the Salmonella Saintpaul outbreak was identified as tomatoes (Centers for Disease Control and Prevention (CDC), 2008; Lang, 2008). However, the failure to recover the Salmonella serovar from tomatoes shifted focus to peppers and cilantro (common ingredients of salsa) from Mexico. The outbreak lasted over 80 days and resulted in over 1400 cases being recorded (CDC, 2008). The actual number of cases is probably 10–30 times this figure given that the majority of illness outbreaks go unrecorded. Despite the number of cases involved in the Salmonella Saintpaul outbreak, no specific source or ‘‘smoking gun’’ was identified. However, the outbreak, like those within recent years, highlighted key deficiencies within the fresh-cut chain. Specifically: Inability to control the dissemination of human pathogens within the

environment;

Failure of post-harvest interventions to remove field acquired

contamination;

Lack of traceability to track contaminated produce back to the source.

The knowledge gaps associated with the microbiological safety within the fresh produce chain are significant. Despite the large body of research devoted to fresh produce, there still remains much to be known about the survival of pathogens within the environment and nature of interactions with growing plants. To understand the nature of human pathogen interactions with plants and survival in the environment, it is informative to provide a brief overview of the characteristics of those implicated in the majority of recorded outbreaks (Table 4.2).

III. CHARACTERISTICS OF PATHOGENS RECOVERED FROM SALAD VEGETABLES A. Pathogenic E. coli Nonpathogenic (generic) E. coli is a normal inhabitant of the gastrointestinal tract of humans and animals. However, some E. coli strains have now acquired virulence factors enabling them to cause disease of the gastrointestinal, urinary, or central nervous system. Pathogenic E. coli can be

TABLE 4.2

List of pathogenic bacteria and symptoms

Pathogen

Bacterial pathogens Aeromonas hydrophilia

Bacillus cereus

Incubation period

Infectious dose and symptoms

Significant sources

Unknown, symptoms can last for several weeks 6–15 h diarrheal type 0.5–6 h emetic

Gastroenteritis, septicemia, cellulitis, colitis, and meningitis Dose: Unknown but thought to be high (>109 cfu) Diarrheal type: watery diarrhea, abdominal cramps Emitic: vomiting, occasional abdominal cramps and/or diarrhea. Dose >106 cfu Diarrhea which may be watery or sticky and can contain blood. Guillain–Barre syndrome. Dose: >500 cfu Neurotoxin affects nervous system leading to lethargy, weakness and breathing difficulty. LD50 3 ng/kg Severe abdominal pain and diarrhea which is initially watery but becomes grossly bloody. Hemorrhagic colitis. > 100 cells Flu-like symptoms that may develop into septicemia, meningitis and encephalitis. Still birth or abortion in pregnant women. >104 Nausea, vomiting, abdominal cramps, diarrhea, fever, and headache. >103

Water, sewage

Campylobacter jejuni

2–5 days

Clostridium botulinum

18–36 h

Escherichia coli O157:H7

24–48 h

Listeria monocytogenes

1–90 weeks

Salmonella

24–48 h

Soil, starchy grains

Manure especially derived from poultry production. Raw poultry Soil, sediments, water

Manure from ruminants, sewage, raw beef Manure, sewage, soil, silage

Manure, soil, wild and domestic animals, sewage. Raw meat especially poultry (continued)

TABLE 4.2

(continued)

Pathogen

Incubation period

Infectious dose and symptoms

Significant sources

Shigella sonnie

12–50 h

Abdominal pain; cramps; diarrhea; fever; vomiting; blood, pus, or mucus in stools; tenesmus >10 cells

Manure, sewage

24–48 h

Enteric viruses Norwalk Like Virus (Norovirus) Hepatits A

Cryptosporidium parvum

2–4 days

Nausea, vomiting, diarrhea, and abdominal pain Sudden onset of fever, malaise, nausea, anorexia, and abdominal discomfort, followed by jaundice Dose: >20 virons Enteric protozoan Severe watery diarrhea

Cyclospora cayetanensis Giardia lamblia

7 days

Dose: >10 cells Diarrhea which can last up to 6 weeks

7 days

Diarrhea which can last 1–2 weeks

10–50 days

Dose: >1 cysts Adapted from FDA Bad Bug Book (2008).

Sewage, infected food handlers and water

Domestic animals, manure, sewage, infected food handlers and water Sewage and water Sewage, manure, wild and domestic animals, infected food handlers and water


163

subdivided into five different categories based on the type of clinical condition they cause although all share common linkages. All pathogenic E. coli stains follow a similar strategy of infection by colonizing the intestinal mucosal cells. The mode in which illness occurs varies between the different pathogenic E. coli types. ETEC and EaggEC produce enterotoxin, EIEC invades the epithelial cells with EPEC and EHEC adhering to the cell and modifying cellular activity. Although all pathogenic E. coli represent a significant health risk, those belonging to the EHEC group are of most concern, especially E. coli O157: H7 (Weiner and Osek, 2007). The reason for the high virulence of EHEC is through the production of Shiga-like toxins (verotoxin or verocytotoxin). The genes for Shiga toxin are believed to have been horizontally transferred to E. coli from Shigella via bacteiophage. There are two toxins (encoded by Stx 1 and Stx 2) that act by cleaving a single adenine residue from 28S rRNA belonging to the ribosomal subunit resulting in the shutdown of protein synthesis. The kidney is rich in receptors for attachment of E. coli O157:H7 and consequently toxicoinfection by the bacterium can be accompanied by renal failure (HUS syndrome) (Table 4.2). Although E. coli O157:H7 is considered the most significant EHEC strain, it must be noted that other non-O157 Shiga-toxin producing types such as O111, O145, O113, O103, O91, O26, and O104 also exist (Bower, 1999). Collectively, all E. coli possessing toxin genes are categorized as Shiga-toxin E. coli or STEC. However, the presence of stx genes is only one of several virulence factors required to cause illness (McNally et al., 2001). It is interesting to note that E. coli O157 and non-O157 serotypes associated with animals contain only half the virulent factors compared to those of clinical isolates ( Johnson et al., 2004). Therefore, the most virulent STEC have a tendency to be harbored by humans or introduced to animals in contact with sewage ( Johnson et al., 2004). The main source of E. coli O157:H7 is from the manure of ruminants (cattle, sheep) and sewage (Chase-Topping et al., 2008). Other livestock and wildlife have lower frequency of carriage. Although the estimates of STEC vary seasonally, and between herds, approximately 2–100% of cattle harbor E. coli O157:H7 (Hancock et al., 1997). In a 12-month abattoir study in Great Britain, Milnes et al. (2008) determined the fecal carriage of STEC O157 to be 4.7% in cattle, 0.7% in sheep, and 0.3% in swine. Conversely, in another British study, Hutchison et al. (2005) isolated E. coli O157:H7 in 13% of fresh cattle manure, 21% of fresh sheep manure, and 12% in fresh swine manure. ETEC, EIEC, and EAggEC have been previously recovered from contaminated vegetables (Robins-Browne, 2007; Scavia et al., 2008) and are a major cause of diarrhea, especially in infants. The three groups of pathogenic E. coli can be water or foodborne although typically transmitted through person-to-person contact. EPEC, a further type of pathogenic

164


E. coli, is almost exclusively transferred via person-to-person contact although it has also been implicated in sporadic cases of foodborne illness (Ochoa et al., 2008).

B. Shigella Shigella sonnei has been implicated in several vegetable related foodborne illness (Table 4.1) outbreaks although it is normally associated with person-to-person contact (Solodovnikov et al., 2008). Although E. coli O157:H7 and Shigella share pathological traits, the latter is less tolerant to environmental stress (Islam et al., 1996). Therefore, in the majority of cases, infected food workers are considered the primary source of Shigella. However, outbreaks of foodborne illness associated with lettuce contaminated at preharvest with Shigella have occurred. The most notable was an outbreak involving contaminated iceberg lettuce imported from Spain into the United Kingdom, Norway, and Sweden. Subsequent investigations identified that irrigation water contaminated with human sewage was the source of the pathogen (Kapperud et al., 1995).

C. Salmonella The genus Salmonella includes over 2700 serovars, 200 of which are commonly associated with human illness with S. Typhimurium and S. Enteritidis being the most prevalent (Franz and van Bruggen, 2008). Salmonella is carried within the gastrointestinal tract of wild animals, poultry, pigs, and humans. However, Salmonella recovered from vegetables typically belong to less common serotype groups, for example Newport or Montevideo (Franz and van Bruggen, 2008). There is concern with regard to the distribution of multidrug-resistant Salmonella within the food chain. It is commonly believed that the use of antibiotics as animal growth promoters has led to the prevalence of resistant serovars. Yet, through studying the epidemiology of Salmonella, it has become evident that the overprescription of antibiotics along with misuse (e.g., failure to complete a course of the drug) has played a significant role in the emergence of resistant strains (Kelly et al., 2004). The possible use of antibiotics to suppress plant pathogens has been considered as a possible route by which Salmonella and other human pathogens can acquire resistance. Although this may seem unlikely, it is interesting to note that streptogramin-resistant Enterobacter faecium has been previously isolated from bean sprouts (Snary et al., 2004). Similar to E. coli, the main transmission route of Salmonella to vegetables is through fecal contamination, cross-contamination and food handling. In a 12-month abattoir study, Milnes et al. (2007) determined the fecal carriage of Salmonella to be 23.4% in swine 1.4% in cattle, and


165

1.1% in sheep. Hutchison et al. (2004) isolated Salmonella from 8% of fresh cattle manure, 8% of swine manure, 18% of poultry manure, and 8% of sheep manure; levels in stored manure were lower. Salmonella have been isolated from a broad range vegetables especially sprouted seeds (Brandl, 2006; Johnston et al., 2005). An interesting feature of Salmonella associated with vegetables (and other environmental sources) is the tendency to have low virulence compared to those isolated from clinical sources (Herikstad et al., 2002; Olsen et al., 2001; Sivapalasingam et al., 2004). Evidence is accumulating to suggest that genes present within Salmonella enhance the survival of the pathogen outside the host environment. Significantly, mutants of Salmonella lacking such genes have higher virulence than their parent strain (Winfield and Groisman, 2004). Therefore, a number of Salmonella appear to have enhanced their survival in the environment at the expense of virulence. However, this does not imply of course that Salmonella associated with fresh produce represents a low risk.

D. Campylobacter Campylobacter jejuni is a normal commensal of the gastrointestinal tract of poultry, pigs, and cattle. In a 12-month abattoir study, Milnes et al. (2007) determined the fecal carriage of thermophilic Campylobacter to be 54.6% in cattle, 43.8% in sheep, and 69.3% in swine. Hutchison et al. (2004) isolated Campylobacter from 13% of fresh cattle manure, 14% of swine manure, 19% of fresh poultry manure, and 21% of fresh sheep manure; levels in stored manure were significantly lower. Human carriers also represent significant vehicle by which the pathogen can be transferred to foods. Campylobacter is notoriously fastidious and has very specific growth conditions. The bacterium can survive for short periods outside the host environment but not to the same extent as Salmonella and E. coli (Alter and Scherer, 2006; Garenaux et al., 2008; Mihaljevic et al., 2007). However, despite such fragility, C. jejuni, and to a lesser extent Campylobacter coli, has been the main cause of gastroenteritis for several years (Janssen et al., 2008). This is likely due to the low infectious dose (104 tita levels E. coli O157:H7 in radish sprouts Internalization of Salmonella into tomatoes by inoculating stems or blossom of plants Colonization of gfp labeled E. coli O157: H7 at cut edges of leafy greens Internalization of E. coli O157:H7 within lettuce seedlings cultivated in soil or hydroponically Internalization of MS2 coliphage into growing cress plants Internalization of E. coli O157:H7 into lettuce seedlings Internalization of nonpathogenic E. coli into cabbage seedlings Internalization of E. coli O157:H7 and Salmonella into growing Arabidopsis plants when pathogens were introduced into soil Internalization of E. coli in spinach plants cultivated in soil or hydroponically

Sand layer between inoculated soil and phyllosphere

Oron et al. (1995)

Surface sterilization using 0.2% HgCl Immersion in 70% ethanol for 2 min

Itoh et al. (1998) Guo et al. (2001)

No surface sterilization Confocal microscopy to detect gfp label No surface sterilization Confocal microscopy to detect gfp label

Takeuchi and Frank (2000)

Inoculated soil was overlaid with agar

Kirkham et al. (2002)

No surface sterilization Confocal microscopy to detect gfp label 80% ethanol

Solomon et al. (2002)

No surface sterilization Confocal microscopy to detect internalized populations

Cooley et al. (2003)

20,000 ppm sodium hypochlorite

Warriner et al. (2003a)

Wachtel et al. (2002)

Rafferty et al. (2003)

Internalization of E. coli and Salmonella in sprouting mung beans No internalization of E. coli O157:H7 in spinach plants when subjected to physical or biological damage Internalization of E. coli O157:H7, Salmonella and L. monocytogenes in growing plants No internalization of Salmonella in tomato fruit when inoculum was applied to the roots of growing plants Aggregation of Salmonella in stomata and cut cuticle cracks Enhancement of internalization by E. coli O157:H7 in Arabidopsis if coinoculated with Wausteria paucula Internalization of Salmonella in parsley leaves Internalization of Salmonella but not L. monocytogenes when introduced onto the roots of 4-week-old barley plants Internalization of E. coli O157:H7 into mature lettuce plants when introduced onto the roots of plants

183


Warriner et al. (2003b)

2000 ppm sodium hypochlorite

Hora et al. (2005)


Jablasone et al. (2005)

No surface sterilization Screening for Salmonella in ripened fruit

Jablasone et al. (2004)

No surface sterilization

Duffy et al. (2005)


Cooley et al. (2006)


Lapidot and Yaron (2007)

1% chloramine T

Kutter et al. (2006)

No surface sterilization. Perforated polypropylene sheet used to physically separate the inoculation site (roots) from the aerial leaves

Bernstein et al. (2007)

(continued)

TABLE 4.6

(continued)

Comments

Method used to assess internalization

Researchers

Internalization of E. coli O157:H7 and Salmonella in hydroponically cultivated lettuce Internalization of different Salmonella serovars into tomatoes when introduced onto the plant blossom No internalization of E. coli O157:H7 via roots when applied to soil No internalization of E. coli O157:H7 in leaves when inoculated into the phyllosphere of growing lettuce plants No internalization of E. coli O157:H7 or Salmonella when introduced into the water for irrigating 3–33-day posttransplanted lettuce plants Internalization of E. coli O157:H7 overexpressing curli into lettuce leaves when applied as a surface inoculum

1% silver nitrate

Franz et al. (2007)

2000 ppm calcium hypochlorite

Shi et al. (2007)

Surface sterilization using 80% ethanol followed by 0.1% HgCl2 Surface sterilization using 80% ethanol followed by 0.1% HgCl2

Zheng et al. (2008)

Surface sterilization using 80% ethanol followed by 0.1% HgCl2

Erickson et al. (2008)

70% ethanol and visualization using bioluminescent marker

Tanner et al. (2008)

Zheng et al. (2008)


185

Attempting to demonstrate internalization of human pathogens within growing plant tissues is problematic. The traditional approach is to surface sterilize plant material with sanitizers (e.g., sodium hypochlorite, peracetic acid, ethanol) and recover the subsequent bacteria. However, spores (fungal and bacterial) and biofilms are resistant to sanitizers leading to false positive results (Reissinger et al., 2001). Penetration of sanitizer into the internal tissue of plants is a further problem and can potentially lead to an underestimation of endophyte numbers. Therefore, to overcome such limitations, several researchers have taken the approach of carefully inoculating the roots of plants without contacting the leaves which are subsequently screened for the presence of the target pathogen (Franz et al., 2007). The addition of a physical layer (e.g., sand) between the roots and phyllosphere is a further approach to prevent contamination being introduced on, as opposed to within, leaves (Franz et al., 2007). However, the potential for contamination being introduced onto the external surface of leaves exists thereby reducing the confidence that pathogens detected are truly internalized. An alternative to culturing techniques is the application of cell labeling exploiting green fluorescent protein (gfp) in combination with laser confocal microscopy (Table 4.6). Gfp is a protein originally isolated from the jellyfish Aequorea victoria. The key benefit of gfp is the ability to fluoresce under UV light in the absence of an energy source or other cellular cofactors, thereby enabling in situ visualization with minimum disruption to cell physiology. The gene encoding for gfp can be readily inserted and expressed in bacterial cells using plasmid vectors. However, for the plasmid to be retained and replicated within the host cell, selective pressure (typically using an antibiotic) needs to be applied. Therefore, when studying plant:microbial interactions over extended periods, selective agents cannot be used and hence the gfp phenotype can be readily lost. A further limitation to gfp labeling is the need for a high cell density in order to visualize bacteria using confocal microscopy. Clearly, if the tagged bacteria are present in low numbers then locating cells within plant tissue is unlikely. A further method to visualize the presence of internalized bacteria is through the use of glucuronidase (GUS) activity stain. The GUS stain is based on the cleavage of a chromagenic substrate (e.g., 5-bromo-4-chloro3-indoyl-b-D-glucuronide; X-GLUC) that can be directly visualized as a blue/green precipitate within plant tissues. Therefore, if the target cell is present the chromagen accumulates and hence has greater sensitivity compared to gfp labels. The GUS technique has been used extensively to study plant:bacteria interactions based on gus gene insertion into the target bacterium (Wilson et al., 1995). GUS activity is not present in plants or a wide range of bacteria (Wilson et al., 1995), although it is expressed in the majority (>96%) of known generic E. coli strains (Liang et al., 2005).

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Warriner et al. (2003b,c) have used GUS in situ staining to demonstrate the internalization of generic E. coli in spinach and bean sprouts.

D. Genetic and physiological factors The finding of a diverse population of endophytes within plants may be unexpected given that the endogenous plant defenses function to guard against microbial invasion. The inducible defenses are commonly activated in response to phytopathogens to contain the site of infection and prime the plant against further microbial attack. At the site of infection the hypersensitive response (HR) is induced, which releases an oxidative burst to cause localized necrosis. At the same time, the plant hormones (e.g., salicylic acid) are circulated to other parts of the plant to activate the systemic acquired resistance (SAR) which primes the defenses. It is thought that endophytic bacteria enhance the resistant of plants to phytopathogens by inducing SAR. The ingress of opportunistic saprophytes into the plant activates the localized induced resistance (LIR) (Newman et al., 2001) which releases antimicrobials within the localized area to suppress saprophytic activity without leading to necrosis (Esposito et al., 2008). Hence, microbes that become established as endophytes do not induce HA and LIR within plants. One strategy developed by phytopathogens and endophytes is to lose flagella or shield Lipopolysaccharide (LPS) which prevent detection, hence activation by plant defenses (Gozzo, 2003; Liu et al., 2007). Enteric bacteria have been found to differ with respect to the ability to become integrated into the endophytic microflora of alfalfa roots (Dong et al., 2001). A strain isolated from maize, Klebsiella pneumoniae 342, colonizes the interior of several host plants in higher numbers compared to S. Typhimurium and E. coli K12 (Dong et al., 2003). However, a Salmonella mutant lacking flagella and Type III secretion system could readily colonize the roots of alfalfa and become integrated into the endophytic microflora. However, restoration of either phenotype reduced the ability of Salmonella to colonize roots (Dong et al., 2003). It was thought that the lack of flagella and Type III secretion system prevented the activation of the salicylic acid response or independent response which in turn failed to induce PR1 promoter, and hence release of antimicrobials (Iniguez et al., 2005). The question of if enteric pathogens found naturally in the environment shed their outer surface structures to enhance internalization into plants remains unclear. Nevertheless, it is possible that the ability to prevent activation of plant defenses may explain the interstrain variability that exists with respect to pathogen interactions with plants. Although plant and animal pathogens infect different hosts, there are several similarities in the strategies employed (Buckhout and Thimm, 2003). For example, Type III secretion systems can be found in both plant and animal pathogens. The Type III secretion system is


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essentially a microtube by which the invading bacterium attaches to the surface of the host cell. Chemicals and proteins are delivered through the Tir III protein to sequester defense mechanisms and reprogram host cell activity. Of course, this does not imply that a plant pathogen would cause disease in animals. Indeed, to date only Ps. auruginosa PA14 is known to cause disease in both animals and plants (Plotnikova et al., 2000). Nevertheless, it is conceivable that traits in phytobacteria associated with plant interactions could be present in enteric pathogens such as E. coli O157:H7. It is known that broad host range bacteriophage that infects Pseudomonas and E. coli O157:H7 can transfer genetic traits between these two genera (Hendrix, 1999; Muniesa et al., 2003). An example of phytobacteria sharing genes with enteric pathogens has been found in Ps. syringae pv. maculicola. The phytopathogen posses a b-lactamase which protects the bacterium against preformed defenses in Arabidopsis. The gene encoding for the enzyme, donated as sax (Survival on Arabidopsis eXtract), has been identified in a range of Ps. syringae pathovars but absent from nonphytopathogenic strains. From comparative homology studies, the gene shows a high level of similarity to an uncharacterized gene in E. coli O157:H7 (Crooks and Lamb, 2001). Whether the expression of sax within E. coli O157:H7 enhances persistence within plants has yet to be established but does point to an adaptive response of human pathogens to survive outside of the host environment. However, this view remains contentious with many researchers in the field, suggesting that the interaction of human pathogens with plants is a passive process being comparable to any oppertunistic saprophyte (Doyle and Erickson, 2008). Yet, as previously indicated, there is accumulating evidence to support the hypothesis that human pathogens have evolved specialized mechanisms for becoming established and persisting on plants. It has been observed that pathogens such as Salmonella and E. coli O157:H7 preferentially attach to cut surfaces and natural openings such as stomata, whereas common epiphytes such as Ps. fluoresecens colonize the intact plant tissue (Li et al., 2008; Melotto et al., 2006; Seo and Frank, 1999; Takeuchi and Frank, 2001). It is known that attachment of human pathogens is an active process which requires the bacterium to be in a viable state although the actual internalization process can be passive (Solomon and Matthews, 2006). It is thought that cell surface components such as cellulose, flagella, pilli, and Type III secretion systems all play a role in cell attachment (Barak et al., 2005; Zogaj et al., 2001). Several genes and mechanisms have been identified as being involved in attachment of human pathogens to plants. These mechanisms include curli, fimbriae, adhesins, and capsule production (Barak et al., 2005, 2007).

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From microarray studies it has been demonstrated that virulence genes are upregulated in S. Typhimurium during colonization in response to lettuce root exudates (Klerks et al., 2007) which aid attachment to the plant tissue. It is also observed that genes involved in sugar– phosphate metabolism are also upregulated which is thought to attract the enteric pathogen to cut edges of damaged leaves (Klerks et al., 2007). The presence of structures on the plant cell walls has also been proposed. This hypothesis is supported by the fact that attachment of pathogens such as E. coli O157:H7 and Salmonella is plant specific. For example, attachment of pathogens is more frequently observed with Brassicaceae compared to lettuce, carrots, or tomatoes (Barak and Liang, 2008). Collectively, studies to date support the view that human pathogens have adapted to colonize plants as a means of persisting between animal or human hosts.

VI. INTERVENTIONS TO ENHANCE THE SAFETY OF FRESH PRODUCE Postharvest washing of vegetables remains the key intervention to remove field acquired contamination. There have been numerous papers and reviews on the relative performance of different sanitizers including hypochlorite, chlorine dioxide, and peroxyacetic acid (Allwood et al., 2004; Gonzalez et al., 2004; Ibarra-Sanchez et al., 2004; Koseki and Isobe, 2006; Rodgers et al., 2004; Romanova et al., 2002). From reviewing the literature, typical log reductions achieved for Salmonella on lettuce cover a wide range: peroxyacetic acid 1.7 (Hellstrom et al., 2006), acidified sodium chlorite 3.1 (Inatsu et al., 2005), chlorine dioxide 1.53 (Inatsu et al., 2005), ozone 5.6 (Rodgers et al., 2004), and electrolyzed water 1.0 (Koseki and Isobe, 2006; Koseki et al., 2003). However, on natural contaminated produce the log count reductions achieved are typically 1–2 log cfu/g regardless of the sanitizer applied (Doyle and Erickson, 2008). The limitation of postharvest washes can be attributed internalized populations or those within biofilms on the surface of produce (Gonzalez et al., 2004; Koseki et al., 2003). The heavy organic loading of wash water can also readily neutralize sanitizers such as hypochlorite. This can be addressed to a degree by operating at a set oxidation–reduction potential (ORP). Here, the amount of chlorine introduced into the water is increased in the presence of organic matter to maintain the chlorous acid levels within the wash. However, even with ORP-controlled systems the LCR is not significantly improved compared to when chlorine washes alone are applied (Guentzel et al., 2008). Postharvest washing does not only provide a low level of confidence of decontaminating produce but is also thought to result in


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cross-contamination between fresh produce batches. It is known that contaminated flume water is a potential source of contamination of Salmonella for fruits such as tomatoes (Zhuang et al., 1995). Luo (2007) reported that lettuce washed in recycled chlorinated water with high total solids content spoiled more rapidly compared to samples washed in fresh chlorinated water. More direct evidence for cross-contamination via wash water was reported by Ilic et al. (2008). The researchers evaluated the effect of commercial wash process on the coliform and E. coli counts associated with spinach. The finding of the study was that the proportion of samples positive for coliforms increased from 53% to 79% following washing (Ilic et al., 2008). It is possible that the increase in coliform prevalence was caused by the uptake of wash water caused by a temperature differential effect. It is known that warm produce placed into cold water results an influx of wash water (hence contamination) into the inner vascular system of leafy greens and fruit (Bolton et al., 2002; Fukumoto et al., 2002; Ibarra-Sanchez et al., 2004). To overcome the problem of infiltration, it is recommended to use warm water for washing produce (FDA, 1998). However, this has the adverse effect of warming the produce, thereby accelerating plant autolysis and growth of spoilage microbes or even human pathogens. Surface pasteurization of produce using steam, hot water, or chlorine dioxide gas was shown to enhance the reduction of microbial loads on hard surface produce (Stringer et al., 2007). However, delicate produce such as leafy vegetables can be damaged by the steam process (Allwood et al., 2004; McWatters et al., 2002; Sy et al., 2005). A study conducted by Sapers and Sites (2003) showed that cantaloupe treated with hot (80 C) 5% hydrogen peroxide for 3 min reduced E. coli and Salmonella populations with no signs of tissue damage after 26 days of storage at 4 C. However, some disadvantages with heat treatments for produce occur. Heat treatment can reduce microbial loads on produce but has little effect if contaminated after heating. A study by Conway et al. (2005) showed that injured apples had larger microbial loads of pathogens when heat treated. This was most likely due to the damage of enzymes that are the plant’s defense system against invasion of microorganisms. Overall, the quality of commodities such as fresh cut lettuce diminishes during extended storage after heat treatment of more than 3 min. Irradiation of produce has been shown to be effective in reducing microbial contamination where the maximum dosage level is 1.0 kGy for fruits and vegetables (Bari et al., 2005; Gomes et al., 2008; Niemira, 2007). An irradiation dose of 1.0 kGy treatment decreased mesophilic bacteria in Mexican salads (Erickson, 2008). However, irradiation can cause changes in pectin structure leading to texture loss and hence shelf life. Also, viruses seem to be relatively resistant to irradiation treatment and relative to vegetative cells, suggesting that doses delivered to inactive

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pathogens (e.g., Salmonella) would be insufficient to kill NLV (Gomila et al., 2008). High hydrostatic pressure (HHP) processes have been used mainly for sauces or seafood and proven effective at reducing microbial populations without adverse effects on product quality (Considine et al., 2008; Brinez et al., 2006). HHP treatment causes bacterial inactivation by damaging the cell membrane, which affects membrane permeability and intracellular enzyme inactivation and possibly ruptures the plant cell wall (Kniel et al., 2007). Although HHP have proven to be one of the most effect decontamination intervention strategies, there is the potential of causing disruption to plant tissue leading to accelerated spoilage (Basak and Ramaswamy, 1998; Butz et al., 2002; Pre´stamo and Arroyo, 1998). Pressure-induced damage to plant tissue results from the stress and strains imposed on the cell walls which subsequently results in loss of texture (Fuchigami et al., 1995; Hartmann and Delgado, 2004; Kidmose and Martens, 1999). However, it has also been reported that HHP treatment can enhance the texture of vegetables through de-esterifying pectin via the activity of pectin methyltransferase (PMT) which facilitates calcium binding to stabilize plant cell walls (Fennema, 1996; Sila et al., 2004). UV light has been considered as an alternative to sanitizer-based systems for decontaminating fresh produce (Bialka and Demirci, 2008). Unlike chemical sanitizers, UV does not leave residues and the product does not need to be dried thereby providing energy savings (Bialka and Demirci, 2008). UV light can be divided into three classes: UV-A (400–320 nm), UV-B (320–280 nm), and UV-C (5 log reductions of human pathogens and viruses (MS2 phage surrogate) on leafy greens (Hadjok et al., 2008; Xie et al., 2008).

A. Biocontrol of human pathogens Control of human pathogens at the primary production level is problematic due to its open nature. It has been proposed that decontamination of irrigation water through ozonation or chlorination is an option although the cost and effect on the plant microecology are obvious limitations (Ajwa et al., 2002; Rojas-Valencia et al., 2004). A more practical approach is to use biocontrol strategies whereby antagonistic microbes are introduced into the rhizosphere to reduce or inhibit pathogens. To date the majority of biocontrol strategies have been focused on controlling phytopathogens (Vassilev et al., 2006). The use of biocontrol strategies to control human pathogens is an emerging area but has yet to be studied in detail. It has been previously reported that Enterobacter is antagonistic against Salmonella introduced onto Arabidopsis and lettuce (Cooley et al., 2003, 2006). Although the mechanisms are unknown, it is thought that Enterobacter can compete more effectively for nutrients utilized by Salmonella to support growth and persistence. Fett et al. (2006) introduced a Pseudomonas fluorescens strain (isolated from the rhizosphere of wheat) into the soak water of Salmonella inoculated alfalfa seeds. When the seeds were sprouted that sprouts with Ps. fluorescens had 5 log lower Salmonella levels compared to controls (Fett et al., 2006). Matos and Garland (2005) introduced a cocktail of bacteria (unknown composition) obtained from germinated sprouts to alfalfa seeds inoculated with Salmonella. The Salmonella counts on sprouts derived from seed treated with biocontrol agent were 5.7 logs lower compared to controls. However, because the bacterial cocktail was directly obtained from sprouts, attempting to reproduce the composition on a commercial scale could be problematic. The application of bacteriophage has been evaluated for controlling human pathogens although with various degrees of success (Guenther et al., 2008; Hudson et al., 2005). Bacteriophages are viruses that infect and replicate within bacterial hosts. Advantages of phages are that they are specific, self-perpetuating, and self-limiting. Although widely used in Eastern Europe, bacteriophages have only recently being approved for food applications. In 2006, the Food and Drug Administration (FDA) approved a L. monocytogenes-specific phage preparation (LMP-102) for use as an antimicrobial agent against L. monocytogenes contamination of ready-to-eat foods (Guenther et al., 2008; Lang, 2006; Leverentz et al., 2001). In relation to fresh produce, Abuladze et al. (2008) evaluated a cocktail (designated ECP-100) of phages to control E. coli O157:H7 on a variety of vegetable types. The researchers reported a 94–99% reduction of E. coli O157:H7

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introduced onto tomatoes and complete inactivation with spinach inoculated with 14,000 cfu of the pathogen. However, it is possible that infection and phage replication occurred during cultivation of survivors given that phage numbers on the fresh produce samples remained the same (Abuladze et al., 2008). Leverentz et al. (2004) reported a 2–3 log reduction of Salmonella on melon although complete elimination of the enteric pathogen was not observed.

VII. CONCLUSIONS AND FUTURE RESEARCH Despite the increased awareness of food safety issues surrounding fresh produce, the number and frequency of foodborne illness outbreaks continue to rise. It is evident that the centralization of production coupled with the growth in the bagged salad market are significant factors to explain the incidence of foodborne illness linked to fresh cut produce. However, environmental factors also play a significant role especially in terms of manure management in disseminating human pathogens to water courses and/or soil. The adaption of human pathogen strains to grow and persist on plants remains a relatively unexplored area. In many aspects, the adaption of human pathogens to plants would make sense given that the microbes need to survive in the environment between infecting hosts. The finding that human pathogens can contaminate growing crops in the field and persist through to consumption is of concern and interventions above simple postharvest washing have to be considered. One strategy of interest is through implementing biocontrol methods that can control human pathogens at the primary production level. With regards to postharvest control, it can be envisaged that more effective decontamination treatments will be adopted such as AOP or more likely irradiation. However, regardless of technological advances there will always be a role of GAP (Good Agricultural Practice) and GMP (Good Manufacturing Practice) for enhancing the microbiological safety of fresh produce.

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CHAPTER

5 Understanding Oil Absorption During Deep-Fat Frying Pedro Bouchon

Contents

I. Food Deep-Fat Frying: A General Overview A. The deep-fat frying process B. Heat and mass transfer during deep-fat frying C. Structure development during frying II. Nutritional Aspects of Food Deep-Fat Frying A. Frying oils and oil degradation B. Consumption of fried food and human health III. Oil Absorption A. Kinetics of oil absorption B. Factors affecting oil absorption C. Oil absorption reduction References

Abstract

One of the most important quality parameters of fried food is the amount of fat absorbed during the process, which undermines recent consumer trends toward healthier food and low-fat products. In order to obtain a product with a low fat content, it is essential to understand the mechanisms involved during the frying process, so that oil migration into the structure can be minimized. To get such an understanding, this chapter briefly describes the frying process from technological and scientific perspectives. First, it gives a general overview of the frying process and describes the most important quality attributes of fried food. Thereafter, it centers on key nutritional aspects, particularly on the effect of excessive oil consumption on human health, oil degradation,

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Department of Chemical and Bioprocess Engineering, Pontificia Universidad Cato´lica de Chile, Santiago, Chile Advances in Food and Nutrition Research, Volume 57 ISSN 1043-4526, DOI: 10.1016/S1043-4526(09)57005-2

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and toxic compounds generation in fried food. Finally, this chapter discusses the most important factors affecting oil absorption, oil absorption kinetics, and different strategies that may be adopted to decrease oil content.

I. FOOD DEEP-FAT FRYING: A GENERAL OVERVIEW Deep-fat frying, also known as immersion frying, is one of the oldest and most common unit operations used in the preparation of food. The process was first developed around the Mediterranean area due to the influence of olive oil there, but today numerous processed foods are deep-fat fried because of the unique flavor–texture combination imparted to the food (Varela, 1988). Certainly, fried products are of great importance to the food industry because of their popularity among consumers and the huge quantities of fried food and oils that are used at industrial and commercial levels. A critical aspect of deep-fat fried food is the high amount of oil that is absorbed during the process, reaching in some cases 40% of the total food product weight. Numerous studies have revealed that excess consumption of fat is a key dietary contributor to coronary heart disease and perhaps cancer of the breast, colon, and prostate (Browner et al., 1991), imposing an alert to human consumption. Despite this, consumption of oils and fats is still high. For instance, in the United States, consumers eat four or more snacks a day and consume more than 6.5 billion pounds of snack food annually. As such, salty snacks account for slightly over half of the total snack sales and are consequently a large part of the American diet (Mintel International Group Ltd, 2006). A wide variety of food materials can be used to produce fried products, including vegetables, meat, dairy, and grains. Key growth categories are those that offer the most product variations adhering to convenience, flavor, and health trends. In terms of health, interests in salty snack products that are organic or all natural, low-calorie, low-fat, lowcarbohydrate, low-sodium, or offer health-promoting benefits such as elimination of trans fat are in greater demand by consumers. Although consumers are interested in healthier snack products, they are not willing to sacrifice flavor. Intense and full-flavor snacks remain an important trend in the salty snack market (Mariscal and Bouchon, 2008).

A. The deep-fat frying process Deep-fat frying can be defined as a process of cooking food by immersing them in edible oil at a temperature above the boiling point of water, and therefore, may be classified as a dehydration process (Farkas, 1994). Frying temperatures usually range between 130 and 190 C, but most

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common frying temperatures are in the 170–190 C range. Deep-fat frying is a complex unit operation involving high temperatures, significant microstructural changes to both the surface and the body of the food, and simultaneous heat and mass transfer resulting in flows in opposite directions of water vapor (bubbles) and oil at the surface of the piece as depicted in Fig. 5.1 (Bouchon et al., 2003). The high temperatures of the frying oil lead to the evaporation of water at the surface of the food. Due to evaporation, water in the external layers of the product moves to the surrounding oil and surface drying occurs, inducing crust formation. Additionally, oil is absorbed by the food, replacing part of the water (Mellema, 2003). One of major aim of deep-fat frying is to seal the food surface while immersing the food into the oil bath so that its flavor and juices can be successfully retained within the food. As a matter of fact, most of the desirable characteristics of fried food are derived from the formation of a composite structure: a dry, porous, crisp, and oily outer layer or crust and a moist cooked interior (Bouchon and Aguilera, 2001). Frying technology is important to many sectors of the food industry, including the suppliers of oils and ingredients, fast-food shop and restaurant operators, industrial producers of fully fried, par-fried, and snack foods, and manufacturers of frying equipment (Blumenthal, 1991). Deepfat fryers basically consist of a chamber where heated oil and food are placed and the size depends on their use. Accordingly, the frying equipment is divided into two broad categories: (1) batch frying equipment, normally used in catering restaurants and small plants and (2) continuous fryers, which are used on an industrial scale to process large amounts of

Heat transfer

Mass transfer

Crust

Core

Conduction:

Water:

Convection:

Water vapor: Oil:

FIGURE 5.1 Schematic diagram of simultaneous heat transfer (left-hand side of the figure) and mass transfer (right-hand side of the figure) during deep-fat frying (with the courtesy of M. C. Moreno).

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food. Fryers normally operate under atmospheric conditions; however low- or high-pressure conditions may be used.

1. Fried food A wide spectrum of fried food is available in the market. They are usually classified into three categories: (1) thin products, such as potato, tortilla, and banana chips, (2) thick products, such as French fries, and (3) battered/breaded food, such as fish fingers. Thin products are nearly fully dehydrated (moisture content lower than 5%), a requirement for shelf-life stability, which is around 2 months. Their fat content is high, achieving up to 40% w.b. (Dobraszczyk et al., 2006). Oil stability is the key factor during storage, rather than fungal spoilage, because of the development of offflavors. Thick as well as buttered/breaded products have a higher water content (ranging between 30% and 50% w.b.) and lower oil content. Frozen par-fried French fries may have oil content as low as 5% w.b.; however, these products must be either oven-cooked or go through a second frying step before consumption, which necessarily increases the fat intake per portion (there is further dehydration and, if fried, additional oil intake). In fact, when using a second frying stage, the final oil content is generally higher than in fresh fried products. Doughnuts are also an extremely popular fried food category. They have a high oil content that ranges from 15% to 20% w.b., but about 10% of the fat is used in the preparation of the dough. Battered and breaded foods (fish/chicken) contain similar oil contents of around 15–20% w.b. A critical aspect of these products is the contrast between the crispy and oily outer layer and the soft cooked interior (Dobraszczyk et al., 2006).

2. Frying equipment Modern batch fryers are constructed with high-grade stainless steel to avoid oxidation catalysis. Usually, the operators immerse and remove the baskets manually from the oil, but new equipment may include an automatic basket-lift system. The device may consist of one or more chambers with an oil capacity ranging from 5 to 25 l, and oil may be directly heated through electricity, gas, or fuel (Dobraszczyk et al., 2006). Important factors to consider when selecting a batch fryer are power source, speed of temperature recovery, and safety. The simplest heating system consists of gas flames directly placed underneath the bottom of the vessel. Oil can also be directly heated through an electrical resistance heater that may be installed few inches above the bottom of the fryer, allowing the arrangement for a cool zone at the bottom of the vessel where debris can fall, minimizing oil damage. This is a clear advantage compared to the previous heating system, where the provision of a cold zone under the heaters is not possible (Rossell, 1998). New developed high-efficiency fryers include turbo-jet infrared burners that use up to 40% less energy than


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the standard gas-fired fryers with the same capacity (Moreira et al., 1999). In order to increase shelf life, avoid smoking, charring, and off-flavor development, oil filtration and removal of food scraps are essential practices that need to be carried out every day. New equipment can also have a built-in pump filtration unit for the removal of sediments (Kochhar, 1998). Continuous fryers are used to process large amounts of food, having a throughput that varies from 250 to 25,000 kg product per hour (Moreira, 2006). These are automated machines that consist of a frying vessel where oil is maintained at the desired temperature, a conveyor belt that transports the food through the oil (the product is often pushed through the bath by means of a screen and/or paddles), and an extraction system that eliminates the fumes, primarily made up of moisture and a fine mist of fatty acids (Dobraszczyk et al., 2006; Moreira et al., 1999). The oil may be heated directly using a battery of gas burners or an electric heater in the frying vessel, or by means of an external heat exchanger where oil is continuously pumped through. Some continuous fryers are designed with multiple heating zones along the fryer that can be adjusted separately, providing optimal temperature control to improve product quality. Continuous fryers may be also provided with an indirect oil heating system unit. In those systems, oil is heated by pumping a heated thermal fluid into a tube arrangement immersed in the oil bath (Dobraszczyk et al., 2006; Moreira et al., 1999). It is important to mention that in continuous fryers, the oil that is constantly absorbed by the fried product needs to be replaced with fresh oil continuously. The amount of fresh oil added to the vessel is the oil turnover, defined as (weight of oil in the fryer)/(weight of oil added per hour) (Banks, 1996), and therefore represents the time needed to replace all the oil contained in the equipment. Fast oil turnovers are desired since they preserve the oil quality better. Normally, the oil turnover is kept between 3 and 8 h (Kochhar, 1998). Deep-fat fryers may also operate at a higher pressure (932 psi). These devices have been developed to meet particular needs primarily in certain catering outlets, especially those devoted to chicken frying because of the uniform color and improved texture (higher moisture content) conferred to products. Pressure fryers may reduce the frying time considerably, but they can also increase frying oil deterioration rate since steam retention within the fryer increases free fatty acids content (Moreira et al., 1999). Another technology that is being increasingly adopted is vacuum frying, which consists of a deep-fat frying process carried out in a closed system under pressure well below the atmospheric levels (preferably lower than 1 psi), making it possible to reduce substantially the frying temperature due to water boiling-point depression. The low temperatures employed and minimal exposure to oxygen account for most of its benefits, which include natural color, flavor, and nutrient preservation

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(Mariscal and Bouchon, 2008; Shyu and Hwang, 2001), as well as oil quality protection (Shyu et al., 1998) and reduction of toxic compound generation (Granda et al., 2004). The equipment was first developed by Florigo B.V. during the sixties to produce high-quality chips; however, due to the improvement in blanching technology and in raw material quality, the use of this technology almost disappeared (Moreira et al., 1999). Nowadays, vacuum frying technology is being used to maintain natural colors, flavors, and nutrients in high added-value products, such as vegetables and fruits (Dueik and Bouchon, 2009).

B. Heat and mass transfer during deep-fat frying From an engineering perspective, deep-fat frying can be defined as a unit operation where heat and mass transport phenomena occur simultaneously. Convective heat is transferred from the frying media to the surface of the product, which is thereafter conducted within the food. Mass transfer is characterized by the loss of water from the food as water vapor and the movement of oil into the food (Singh, 1995). Water evaporation initiates at the surface of the product after initial heating occurs, and the boiling point of the interstitial liquid is reached, which is slightly higher than the boiling point of water. After the initial surface water is lost, water starts escaping vigorously and heat transferred through natural convection gives path to a forced convection regime due to the high turbulence associated with nucleate boiling. As frying progresses, the evaporation front moves toward the interior of the product and a dehydrated crust layer is formed, whose temperature rises above the boiling point of water. It is important to note that the maximum surface temperature only approaches that of the frying oil, remaining 10–15 C below it, because of the heat transfer resistance at the boundary oil layer in contact with the surface of the food (Fig. 5.2). On the other hand, the temperature inside the food material (known as core region), where liquid water is still there, is restricted to values around the boiling point of the liquid. As frying proceeds, the water loss rate decreases (falling-rate period) leading to bubble-end point, that is, when water escape stops (Singh, 1995). This is actually the processing condition that must be fulfilled in chip like products (crisps in the U.K.), where a maximum of 5% moisture content is permitted. Mass transfer during frying is not only characterized by the movement of water in the form of vapor from the food into the oil, but also by the movement of oil into the food. Frying is a dehydration process where water escape leaves empty spaces within the crust structure, which in turn determines the volume available for oil absorption. In fact, the amount of oil uptake has been shown to be directly proportional to

Temperature (⬚C)


Surface T⬚:

Model

Experimental

Centre T⬚:

Model

Experimental

180

215

R2 = 0.886

140

R2 = 0.992 100 60 20 0

100

200

300

Temperature (⬚C)

Time (s) 180

R2 = 0.934

140

R2 = 0.989

100 60 20 0

100

200

300

Temperature (⬚C)

Time (s) R2 = 0.918

180 140

R2 = 0.983 100 60 20 0

100

200

300

Time (s)

FIGURE 5.2 Predicted and experimentally determined temperatures (mean values) when frying a raw potato cylinder at 155 C (top), 170 C (middle), and 185 C (bottom); from Bouchon and Pyle (2005a).

the amount of moisture lost, as will be discussed in the following sections (Gamble et al., 1987). One of the key parameters that distinguishes frying from other unit operations is the high heat transfer rates that are achieved, which are far higher than those found during baking and drying. Heat transfer rates to the surface of the food will depend on the thermal properties and chemical composition of the frying medium, and on the turbulence generated by the vigorous vapor escape. Several authors have attempted to measure natural convective heat transfer coefficients mainly using a metal transducer. Evidently, the absence of water vapor surrounding the metal piece

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yields a natural convective heat transfer coefficient that is different from that when a food is undergoing frying and that is only meaningful during the early stages of frying when few bubbles are present. Using the lumped capacity method with a spherical aluminum transducer, Miller et al. (1994) estimated natural convective heat transfer coefficients for canola oil, palm oil, corn oil, and soybean oil at 170, 180, and 190 C. They obtained values ranging from 250 to 280 W/(m2 K). Using a similar approach, Moreira et al. (1995a) estimated the natural convective heat transfer coefficient for soybean oil, which was 280 W/(m2 K) when heated at 190 C. Comparable values were obtained by Bouchon and Pyle (2005a), who determined natural convective heat transfer coefficients of 262, 267, and 282 W/(m2 K) when heating palm olein at 155, 170, and 185 C, respectively. Several studies have attempted to estimate boiling convective heat transfer coefficients for immersion frying. Hubbard and Farkas (1999) obtained maximum average values of 610, 650, and 890 W/(m2 K), when frying potato cylinders at 120, 150, and 180 C, respectively, which are far higher than natural convective ones. In addition, they found that the time to reach these maxima decreased as the frying temperature increased and that the convective heat transfer coefficient gradually decreased to 300 W/(m2 K) over the duration of the process. Costa et al. (1999) reported maximum average values of 443 and 650 W/(m2 K) and average values of 353 and 389 W/(m2 K), after approximately 5 min when frying French fries at 140 and 180 C, respectively. Interestingly, they explained that the heat transfer coefficient might be expected to be position dependent due to the difference in turbulence occurring at different locations in the product. In fact, Sahin et al. (1999) found differences when determining the boiling convective heat transfer coefficient at the top and bottom surfaces of potato slices during frying (150–190 C). Contrary to what might be expected, they determined higher coefficients at the bottom surface (450–480 W/(m2 K)) as compared to the top surface (300–335 W/(m K)) until crust was formed. They attributed this to the fact that a strong insulating effect was produced by the vigorous escape of bubbles at the top surface, while at the bottom surface bubbles remained in a single layer, providing a lower resistance. Bouchon and Pyle (2005a) estimated boiling convective heat transfer coefficients for increasing frying times at different temperatures, which ranged approximately from 260 to 600 W/(m2 K), similar to those found by Costa et al. (1999) and Sahin et al. (1999). They adjusted a first-order kinetic model to experimental data to describe the change with frying time, which they used when testing a mathematical model of the process. Overall, all studies have found that convective heat transfer coefficients are up to two or three times greater that those measured in the absence of bubbling. This research has made it possible to get a better


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understanding of the different heat transfer rates and vapor escaping regimes encountered during the different periods of frying.

C. Structure development during frying The structure of the crust and core regions of fried food products is the result of several alterations, most of which occur at the cellular and subcellular levels. Aguilera and Gloria (1997), using fast freezing after frying and cryosectioning, demonstrated that three distinct microstructures exist in finished fried commercial French fries: (1) a thin outer layer (approx. 250 mm) formed by the remnants of cell walls of cells damaged by cutting; (2) an intermediate layer of shrunken intact cells, which extends to the evaporation front; (3) the core with fully hydrated intact cells containing gelatinized starch. Microstructural changes in the core region are similar to those occurring during simmering since this inner structure is restricted to temperatures below the boiling point of water. Main changes include starch gelatinization (in starchy products), softening of the middle lamellae (which is greatly responsible for the so-called mealy texture), and protein denaturation. Microstructural changes at the crust are certainly more aggressive than those occurring in the inner structure due to the exposure to temperatures well above 100 C (in atmospheric deep-fat frying). Besides the physical damage produced when the product is cut, chemical and physical changes include starch gelatinization and subsequent dehydration, protein denaturation, breakdown of cellular adhesion, water evaporation and rapid dehydration of cells located in the forming crust, and oil uptake itself (Bouchon and Aguilera, 2001). Desired organoleptic properties, particularly textural ones, are a direct consequence of these microstructural changes. A chip must be firm and snap easily when deformed, emitting a crunchy sound (Krokida et al., 2001a), whereas in thick products the better the contrast between a rich and soft inner structure and a crispy outside, the better the product (Moreira, 2006). Firmness is often related to starch swelling and gelatinization, as well as to the stability of pectic substances of the cell wall and middle lamellae. The importance of microstructural changes occurring during deep-fat frying has been greatly recognized when studying oil absorption mechanisms. In fact, as commented by Baumann and Escher (1995), the explanation of factors affecting oil uptake needs to be validated by a structure analysis in relation to the location of oil deposition and to the mechanism of oil adhesion to the surface. Numerous studies have shown that oil uptake during deep-fat frying is confined to the surface region of the fried product and in cavities and open pores within the two outer layers that constitute the crust (Bouchon et al., 2001; Farkas et al., 1992; Keller

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A

C

B

150 mm

A 90 mm

C

B

FIGURE 5.3 Orthogonal sections (A, B, and C) of a reconstituted image obtained by CLSM, showing the oil location in the crust of a fried potato; from Bouchon and Aguilera (2001).

et al., 1986; Saguy et al., 1997). Bouchon and Aguilera (2001) and Pedreschi et al. (1999) used noninvasive confocal laser scanning microscopy to study oil location directly in fried potatoes, where they observed that oil seemed to flow through the passages that imposed the lowest resistance and was concentrated in concave shells around the cells, with no presence of oil in their interior (Fig. 5.3). Microstructural evidence plus the fact that oil uptake is related to the amount of moisture lost, are key aspects to consider the microstructure of the crust region (mean pore size, connectedness, and permeability) as the single most important product-related determinant for the final oil uptake into the food (Bouchon et al., 2001). Specific aspects related to oil absorption kinetics will be discussed in Section III.

II. NUTRITIONAL ASPECTS OF FOOD DEEP-FAT FRYING One of the most important quality parameters of fried food is the amount of fat absorbed during the process, which is incompatible with recent consumer trends toward healthier food and low-fat products (Bouchon


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and Pyle, 2004). In fact, current nutrition recommendations point to a reduction of total dietary fats, including trans and saturated fatty acids. In addition, important nutritional compounds degrade during the process, and toxic molecules may generate either in the foodstuff or in the frying oil itself, whose intake should be at least limited.

A. Frying oils and oil degradation Food can be fried in a wide range of fats and oils, which include vegetable oils, shortenings, animal fats, or a mixture thereof. Most important criteria used to select frying oils are long frying stability, fluidity, bland flavor, low tendency to foam or form smoke, low tendency to gum (polymerize), oxidative stability of the oil in the fried food during storage, and certainly price (Kochhar, 1999). Saturated fatty acids provide a greater stability in frying applications, but they are undesirable from a nutritional standpoint (Sanibal and Mancini-Filho, 2004). Conversely, oils high in polyunsaturated fatty acids show lower thermo-oxidative stability than rich monoenoic unsaturated fatty acids or saturated fatty acids oils (Kita et al., 2005). Antioxidants such as tertiary butyl hydroquinone (TBHQ), butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) may be added to improve oil stability; however, some of them are limited or prohibited under certain regulations. TBHQ is regarded as the best antioxidant for protecting frying oils against oxidation and, like others, it provides carry-through protection to the finished fried product (Dobraszczyk et al., 2006). Most popular oils used for frying are palm oil and its fractions, sunflower oil (especially high-oleic sunflower oil), rapeseed (canola), and soybean oils. The last two have a high level of linolenic acid (8–10%), making them vulnerable to oxidation and off-flavor development, and therefore, can be slightly hydrogenated for industrial frying. This procedure can also be applied to sunflower oil and may be attractive when requiring a high polyunsaturated-to-saturated ratio for dietary purposes (Rossell, 1998). Olive oil has excellent attributes, which make it suitable for frying, that is, a low level of polyunsaturated fatty acids and a mixture of phenolic antioxidants that make it resistant to oxidation. However, extravirgin and virgin olive oils are far too expensive for industrial use, converting refined solvent-extracted olive oil as a plausible candidate for certain industrial frying operations (Dobraszczyk et al., 2006). Animal fats, despite their high level of saturated fatty acids, may also be used in deepfat frying due to the characteristic flavor imparted to the food and/or low cost. In turn, fish oils are rarely used as frying medium, since their high level of long-chain polyunsaturated fatty acids makes it prone to oxidation (Rossell, 1998).

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Care must be taken when selecting frying oils, since they undergo thermal, oxidative and hydrolytic degradation due to their exposure to elevated temperatures in the presence of air and moisture (Kita et al., 2005). Water, which is released from the foodstuff, attacks ester linkages of triacylglycerols giving rise to di- and monoglycerides, glycerols, and free fatty acids, free molecules that are more susceptible to oxidative and thermal degradation than when esterified to the glycerol molecule (Choe and Min, 2007). Oil thermal oxidation to form peroxides takes place by loss of hydrogen in the presence of trace metals, heat, and light, giving rise to hydroperoxides. Hydroperoxides are compounds that are not stable under deep-fat frying conditions and may undergo fission to produce a wide variety of secondary lipid peroxidation products, including aldehydes, ketones, and other carbonyl-containing compounds (Mahungu et al., 1999). These compounds contribute to the volatile fraction of degraded frying oils, determining the development of off-flavors in the fried product (Melton et al., 1994; Subramanian et al., 2000). In addition, dimers, oligomers, and polymers may be formed, giving rise to excess darkening, increasing oil viscosity, and decreasing smoke point of the frying oil (Choe and Min, 2007; Mahungu et al., 1999). No-calories fat substitutes, such as sucrose polyesters (Olestra), which are synthesized from sucrose and fatty acid methyl esters, have been widely studied and several snacks fried in this medium are available in the market place. This product has no calories since digestive enzymes are not able to break it down due to structural impairment. A major disadvantage that prevents a wide acceptance of this product is related to the gastrointestinal discomfort that may be caused to some individuals (Dobraszczyk et al., 2006, p. 104).

B. Consumption of fried food and human health Fat has a strong influence on the palatability of fried foods. The inclusion of cooking fat into the crusty surface, which is developed during the frying process, helps in building up the crunchiness that is highly appreciated by consumers. On the other hand, the linkage between overconsumption of fat and several diseases has been well-documented. Oil consumption, especially saturated fat, is considered to be one of the key dietary contributors to diseases like obesity, coronary heart disease, cancer, diabetes, and hypertension (Saguy and Dana, 2003). In addition, several studies have provided strong evidence that trans fatty acids increase plasma concentration of low-density lipoproteins and reduce the concentration of high-density ones (Ascherio and Willett, 1997). Trans fatty acids are produced during hydrogenation, a process that is commonly used to increase thermal stability of frying oils, but they can


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be also generated during high thermal processing, such as deep-fat frying (Choe and Min, 2007). It is estimated that diet-related diseases cost the society over US$250 billion annually in medical expenses and loss of productivity (Anand and Basiotis, 1998). As a consequence, consumer trends are moving toward healthier food and low-fat products, creating the need to reduce the amount of oil in end products. Despite such market forces, the consumption of snack food is increasing in developed and developing countries, and fried products still contain large amounts of fat varying from 5% in frozen French fries to up to 40% in potato chips (Dobraszczyk et al., 2006). Due to the large contribution of fried foods to the total saturated and trans fatty acids intake, the use of healthier oil sources offers immense potential to favorably alter population fat intake (Minihane and Harland, 2007). In relation to cancer, there is some evidence that highly oxidized and heated fats may have carcinogenic characteristics. HNE (4-hydroxy-2trans-nonenal), a secondary lipid peroxidation product derived from linoleic acid oxidation, has assumed particular interest because it has shown cytotoxic and mutagenic properties. Its toxicity, as well other secondary lipid peroxidation products (HHE: 4-hydroxy-2-trans-hexenal and HOE: 4-hydroxy-2-trans-octenal), is explained through the high reactivity with proteins, nucleic acids, DNA, and RNA. Research links them to different diseases such as atherosclerosis, Alzheimer’s, and liver diseases (Seppanen and Csallany, 2006). Research is rapidly progressing, but results are still not conclusive. In addition to generation of toxic compounds in the frying oil, toxic molecules may be generated in foodstuff. In April 2002, Swedish scientists sounded an alarm when they discovered that certain cooked food, particularly potato chips and French fries, contained high levels of acrylamide, a chemical compound that is listed by the World Health Organization (WHO) as a probable human carcinogen (Mitka, 2002). This substance has been shown to be produced when food is heated above 120 C due to a reaction between amino acids and reducing sugars (Mottram et al., 2002). WHO has not yet called for any reduction in food containing high levels of acrylamide; however, several studies that aim at reducing its content in fried food are now available (Dueik and Bouchon, 2009). Acrylamide may be converted into glycilamide by living organisms, a compound that is thought to be considerably more toxic than acrylamide; however, little research is yet available in the scientific literature (Besaratinia and Pfeifer, 2004; Koyama et al., 2006). Other heat-induced harmful compounds may be found in certain food. Among them, we can find droxymethylfurfural in carbohydrate-rich foods and heterocyclic amines in protein-rich foods. An in-depth discussion about toxic compound generation may be found in Dueik and Bouchon (2009).

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III. OIL ABSORPTION As explained in previous sections, frying is a complex unit operation involving simultaneous heat and mass transfer, leading to the removal of water from the food and oil absorption at the surface of the piece. It is characterized by the existence of two different regions, the crust and the core, separated by a moving evaporation front, which propagates inward as frying progresses. A key quality factor of fried food is the amount of oil uptake, which should be minimized. In order to obtain food products with a low fat content, it is essential to understand the mechanisms involved during the frying process, especially the kinetics aspects, so that oil migration into the structure can be minimized.

A. Kinetics of oil absorption Even though it is not fully understood when and how the oil penetrates into the food structure, it has been shown that most of the oil is confined to the surface region of the fried product (Bouchon et al., 2001; Farkas et al., 1992; Keller et al., 1986; Saguy et al., 1997) and there is strong evidence that it is mostly absorbed during the cooling period (Aguilera and Gloria, 1997; Bouchon et al., 2003; Moreira et al., 1997; Ufheil and Escher, 1996). For that reason, it is believed that during frying, after initial heating occurs, the vigorous escape of water vapor would generate a barrier to prevent oil migration into the porous crust and as a consequence oil absorption would be limited during most of the immersion period. As a result, oil uptake would mainly result from the competition between drainage and suction into the porous crust once the food is removed from the oil and cools down, being essentially a surface-related phenomenon. The mechanism of oil absorption was first explained by Gamble et al. (1987). They suggested that the largest amount of oil was pulled into the product when it was removed from the fryer because of the vacuum effect due to steam condensation. Accordingly, they suggested that oil absorption depended on the amount of water removed and on the way moisture was lost. In 1996, Ufheil and Escher (Ufheil and Escher, 1996) studied the dynamics of oil uptake during deep-fat frying of potato slices using a fat-soluble and heat-stable dye (Sudan Red B). They determined that most of the oil was absorbed when the product was removed from the oil bath and proposed that oil uptake was primarily a surface phenomenon, involving equilibrium between adhesion and drainage of oil upon removal of the product from the oil. Matz (1993), when focusing on postfrying cooling kinetics, determined that potato chips only absorbed 15% of the total oil when they were rapidly removed from the fryer, while their temperature was still rising. Moreira et al. (1997) determined that


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only 20% of the total oil content of tortilla chips was absorbed while they were immersed in the oil bath and that almost 64% of the total oil content was absorbed during postfrying cooling, the rest remaining on the surface (outer layers of the product). Later, Bouchon et al. (2003) combined and adapted the methods developed by Ufheil and Escher (1996) and Moreira et al. (1997) and were able to distinguish three different oil fractions when frying potato cylinders (155, 170, and 185 C), that is, (1) structural oil (STO), which represents the amount of oil absorbed during frying, (2) penetrated surface oil (PSO), which represents the amount of oil suctioned into the food during cooling following its removal from the fryer, and (3) surface oil (SO), that is, the oil that remains on the surface. A schematic diagram showing these oil fractions is presented in Fig. 5.4. Results showed that only a small amount of oil was able to penetrate during frying since most of the oil was picked up at the end of the process, suggesting that oil uptake and water removal were not synchronous phenomena. After cooling, oil was located either on the surface of the product or was suctioned into the porous crust microstructure, with an inverse relationship between them for increasing frying times. According to experimental facts, several approaches have been used to describe and model oil absorption. Moreira and Barrufet (1998) explained the mechanism of oil absorption during cooling in tortilla chips in terms of capillary forces. This hypothesis was supported by experimental results, where they determined that oil uptake occurred during the first 20 s of cooling, that is, when the temperature was still above the condensation temperature (100 C). Ni and Datta (1999) developed a multiphase porous media model to predict energy transfer, water loss, and oil absorption during frying. They assumed that vapor and air transport took place through convection and diffusion, whereas liquid phase transport (water and oil) was mediated by convective and capillary flows. In their model, they

Surface oil

Structural oil Penetrated surface oil

FIGURE 5.4 Diagram showing the three locations of oil in the product microstructure after deep-fat frying; from Bouchon et al. (2003).

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considered that oil absorption could take place during the immersion period as water moved out from the food, and therefore, they did not take into account oil absorption during postfrying cooling, as determined experimentally by several authors. In recent years, Bouchon et al. (2005b) developed a modified form of the Washburn equation, where they expressed the pressure difference needed to initiate oil infiltration during postfrying cooling (Patm – Ppore > 0), as a function of capillary pressure and vapor pressure, enriching the total driving pressure. A schematic diagram showing the capillary penetration phenomena when having different arrangements is shown in Fig. 5.5. If a reference datum plane (h ¼ 0) is set at the bottom of each of the capillaries shown in the previous Fig. 5.5, the expression for the total driving force, that is the piezometric pressure difference along the penetration length h for a capillary with an upward (þrgh cos a) or a downward (rgh cos a) orientation, can be represented by following equation (Bouchon and Pyle, 2005b), DP ¼ P2 P10¼ Patm Ppore

1 2s cos y rgh cos aA ¼ Patm @PV r

(1)

where P2 is piezometric pressure at the bottom of the capillary (Pa);P1 , piezometric pressure at the liquid side of the meniscus (Pa); Pv, vapor pressure (Pa); Patm, atmospheric pressure (Pa); r, radius of the capillary (m); s, oil surface tension (N/m); y, contact angle (rad); r, oil density (kg/m3); g, acceleration due to gravity (m/s2); h, oil penetration distance (m); and a, angle between normal and vertical axes (rad). Patm Pv h θ

1

h

2

Patm

θ 2

1 α Pv α

FIGURE 5.5 Schematic diagram showing the capillary penetration phenomena when having different arrangements. Left: upward configuration, the action of gravity restricts capillary penetration. Right: downward configuration, the action of gravity enhances capillary penetration; from Bouchon and Pyle (2005b).


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Since Ppore depends heavily on vapor pressure, this is expected to occur after some cooling takes place. As Ppore decreases and vapor condenses, the pressure difference is expected to force the oil within the structure. The condensation mechanism should predominate in thick samples and short frying times, since in thinner samples or longer frying times, moisture loss rate may diminish considerably (and therefore Ppore), allowing oil absorption to begin early. Oil drainage upon removal of the food from the oil bath certainly plays an important role, since this defines the surface oil layer to be sucked upon cooling. It has been suggested that surface roughness may importantly increase the surface area, enhancing oil absorption (Saguy et al., 1998). In an effort to quantify the irregular conformation of the surface, Pedreschi et al. (2000) and Rubnov and Saguy (1997) have used fractal geometry, confirming the significant role of crust roughness in oil absorption. Wetting properties are certainly important since they affect the oil capability to drain. Pinthus and Saguy (1994) used a fundamental approach based on surface chemistry to describe the relationship between the initial interfacial tension between a restructured potato product and various frying media, and the medium uptake during deep-fat frying. They found that total oil uptake was higher for lower initial interfacial tensions, showing a power relationship. This result suggests that a lower interfacial tension between the fluid and the solid would increase wetting adhesion and, therefore, increase the total oil content, reflecting the importance of the wetting phenomena. In addition, the authors found a linear relationship between medium uptake and s.cosy, suggesting the importance of capillary displacement in the mechanism of medium uptake. As can be seen, the surface properties of the product and the physical and chemical properties of the frying media are extremely relevant to the oil uptake mechanisms. Blumenthal (1991) noticed the importance of oil surface tension during deep-fat frying, and developed what he called the ‘‘surfactant theory of frying.’’ He explained that several classes of surfactants are formed during frying of food as a result of the degradation of the frying oil itself or as a result of the reactions occurring between the food components and the oil. These compounds act as wetting agents, reducing the interfacial tension between the food and the frying oil, causing increased contact between the food and the oil and finally producing excessive oil absorption by the fried product. In fact, Tseng et al. (1996) evaluated the effect of oil degradation on the thermal and physical properties of soybean oil, and they determined how the quality attributes of tortilla chips were affected by oil degradation. They found that surface tension decreased and viscosity increased significantly with oil degradation, a fact that may well affect oil tendency to drain. Fracturability,

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moisture content, and oil uptake of tortilla chips were not significantly affected by the oil degradation time. However, after allowing the chip to cool down, only 19% of the total oil was on the surface of the chips fried in fresh oil, while 49% remained on the surface of those fried in degraded oil. Also as frying oil degrades, polymer formation increases oil viscosity, affecting its tendency to drain. Extended discussions about oil absorption mechanisms are reviewed by Mellema (2003) and Zaiifar et al. (2008).

B. Factors affecting oil absorption There has been much research to examine the different factors affecting oil absorption during frying and many empirical studies have correlated oil absorption measurements with process and/or product characteristics. According to the oil absorption mechanisms explained in the previous section, some factors that may be relevant to the amount of oil absorbed will be now presented.

1. Moisture content The amount of oil uptake has been shown to be directly proportional to the amount of moisture lost. Several studies claim that higher initial moisture content results in an increased oil uptake; however, oil absorption seems to be better related to the amount of water loss than to the initial moisture content (Gamble et al., 1987). As explained in the previous section, it is well-established that oil absorption will occupy the empty space left by water, which in turn determines the maximum available volume for oil absorption. The effective water vapor transport through the forming crust is, therefore, an important parameter that affects water escape and probably oil uptake, and as explained by Saguy et al. (1998), diffusion rate is markedly affected by the mechanical properties of the product and the crust. Because of the aforementioned relationship between moisture loss and oil absorption, many studies aim at reducing initial water content in order to decrease the uptake. The effectiveness of these pretreatments though, which is usually achieved through drying, is not due to a reduction of the moisture content on its own (as commonly believed), but due to the structural changes occurring at the surface of the food, which reduce surface permeability (Moreno and Bouchon, 2008).

2. Crust microstructure It has been found that a decrease in the initial porosity in the food may reduce oil absorption (Pinthus et al., 1995). However, as explained by the authors, crust formation plays an additional and fundamental role as soon as frying commences. As the moisture turns to steam and exits the product, it leaves behind a sponge-like tunnel network, which constitutes the oil reservoir. In accordance, the microstructure of the crust region,


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which is formed while the food is cooking in the frying oil, has been pointed out as the single most important product-related determinant for the final oil uptake into the product (Bouchon et al., 2001). In fact, pore development (Thanatuksorn et al., 2007) and pore size distribution (Saguy et al., 1998) have been found to directly influence oil absorption during frying. Some natural ingredients are added to reduce oil uptake because of their film-forming capability and/or because they reduce the porosity of the external layers. In formulated products, the permeability of the outer layer of the product depends on the thickness of the sheeted dough since it determines the structural resistance to vapor escape. A stronger and more elastic network can result in a less permeable outer layer that may act as an effective barrier against oil absorption (Bouchon and Pyle, 2004).

3. Product geometry The surface area of the food plays an important part in oil uptake. As explained previously, oil absorption is a surface phenomenon involving equilibrium between adhesion and oil drainage as the product is removed from the fryer. Therefore, products with a greater surface-to-volume ratio will absorb more oil as revealed by the linear relationship found between exposed surface area and amount of oil uptake (Gillat, 2001). Also, several studies have shown that oil absorption decreases with increasing product thickness (Baumann and Escher, 1995; Gamble and Rice, 1988; Selman and Hopkins, 1989). Surface roughness is another factor that can result in an increased oil uptake, since it not only impairs oil drainage but also increases overall surface area (Saguy et al., 1998).

4. Frying oil temperature and frying time These two process parameters are closely related since products must be fried until they reach certain final moisture content, so a lower oil temperature implies a longer frying time. A clear influence of oil temperature on oil absorption has not been found. Gamble et al. (1987) found no correlation between oil temperature and oil content when frying potato slices, but concluded that a lower oil temperature resulted in lower oil content in the early stages of frying with a greater difference between 145 and 165 C than between 165 and 185 C. Similarly, Moreira et al. (1997) determined higher differences in oil absorption between 130 and 160 C than between 160 and 190 C. In addition, Moreira et al. (1995b) determined that the oil absorption rate was unaffected by the oil temperature when frying tortilla chips and that a frying temperature of 190 C gave a higher oil content (35%) than a frying temperature of 155 C. Nonaka et al. (1977) also found that oil content in French fries increased with increasing frying temperature. Bouchon et al. (2003), in deep-fat frying potato cylinders, showed that total oil absorption is a temperature-independent

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process for short frying times (1 min at 155, 170, and 185 C). For longer frying times, they found that oil content of potato cylinders fried at 155 C was significantly lower than those fried at 170 and 185 C, but no difference was found between the two higher temperatures. Krokida et al. (2000), when frying potato strips for increasing time (from 0.3 to 20 min), also concluded that a lower oil temperature resulted in a lower oil content for long frying times (over 3 min), the difference being higher as frying proceeded. They also found that equilibrium moisture content varied as the oil temperature increased from 150 to 190 C. In accordance, they concluded that the lower oil content could be explained by the lower moisture loss and not necessarily as an effect of the oil temperature itself. They also determined that oil content increased for increasing frying times, especially for thinner products. The effect of frying time in the amount of oil absorbed may be related to the microstructure developed during frying, as previously explained. Pinthus et al. (1995) concluded that crust porosity increased linearly with frying time and, as the crust structure has been demonstrated to play a significant role in the oil uptake, a thicker porous crust would lead to higher oil content. It is important to point out that some conclusions about the effect of oil temperature on oil uptake may be biased by the way results are expressed. Some researchers have reported that oil absorption results as a percentage of the total weight of the product, that is, a wet basis. Conclusions must be analyzed with care in these situations since when frying at a higher temperature for the same frying time, a higher dehydration results. When results are expressed on wet basis (w.b.), there is a systematic reduction in the basis as the water content diminishes. When oil uptake results are measured as a percentage on a dry-weight basis (d.b.) and the solids remain constant throughout the whole process, it may provide a consistent basis for comparison (Moreno and Bouchon, 2008).

5. Oil type and deterioration The influence of the oil type and quality on oil absorption and residues absorbed by fried foods is widely documented (e.g., Blumenthal, 1991; Blumenthal and Stier, 1991; Krokida et al., 2000; Nonaka et al., 1977; Pokorny, 1980). No relationship has been found between oil type and oil absorption; however, it has been shown that an increase in the initial interfacial tension between oil and restructured potato products decreases oil absorption (Pinthus and Saguy, 1994). Further, as mentioned earlier, oil degradation produces surfactants that act as wetting agents, which also increase the absorption (Blumenthal, 1991). Food materials leaching into the oil, breakdown of the oil itself, and oxygen absorption at the oilair interface contribute to change the pure triglyceride oil into a mixture of hundreds of compounds. These materials increase heat transfer and also reduce the surface tension between the


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food and the oil (Blumenthal, 1991). These surface-active agents may have a pronounced effect on fat absorption. By improving the wetting capabilities of oil and reducing the surface tension, these surfactants may lead to a higher oil uptake (Saguy et al., 1998). Also, oil viscosity increases as a result of dimer and polymer formation in aging oils (Blumenthal, 1991). A higher viscosity would make oil drainage from the product surface difficult, increasing the oil taken up.

C. Oil absorption reduction Oil reduction in deep-fat-fried products may be obtained through prefrying and/or postfrying treatments. Prefrying treatments are mainly based on the marked effect that the crust microstructure has in oil absorption, and mainly intend to reduce surface permeability. Postfrying treatments aim to remove surface oil before postcooling suction begins. Drying (microwave, hot-air treatment, baking) prior to frying is shown to be effective in oil uptake reduction in several food products (Gamble and Rice, 1987; Krokida et al., 2001a; Moreno and Bouchon, 2008). It is important to note that the effectiveness of these pretreatments is not due to a reduction of the moisture content on its own (as commonly believed), but due to the structural changes occurring at the surface of the food, which reduce surface permeability. Interestingly, osmotic dehydration had also been extensively reported as an effective pretreatment in oil absorption reduction, whose effectiveness greatly depends on the solution employed (Bunger et al., 2003; Krokida et al., 2001b; Moyano and Berna, 2002). However, as revealed in a recent study, the decrease in oil absorption has been shown to be really due to the increase in solid content occurring during the osmotic dehydration process, rather than a reduction in the amount of oil taken up (Moreno and Bouchon, 2008). Actually, the study demonstrated that osmotic predehydrated samples may absorb as much oil as freeze-dried samples. In addition, during the last decade, much attention has been given to the use of hydrocolloids with thermal gelling or thickening properties, such as methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), long-fiber cellulose, corn zein, and alginates, among others, to inhibit oil uptake (Albert and Mittal, 2002; Garcıá et al., 2002; Mellema, 2003). The hydrocolloid mixture can be added to the product in several ways: (1) added directly in the formula, such as in doughnuts and restructured potato products, (2) included in the batter or breading, (3) sprayed on the product as a solution (Pinthus et al., 1993). A pioneering work in relation to direct incorporation was carried out by Pinthus et al. (1993), where they determined that the addition of HPMC and powdered cellulose to doughnuts and falafel balls reduced oil absorption, being more effective with HPMC as an oil barrier. In a more

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recent study, Bouchon and Pyle (2004) examined the oil-absorption capacity of three different restructured potato chips during deep-fat frying using low-leach potato flake and native or pregelatinized potato starch. Interestingly, they found that the product containing native potato starch as an ingredient picked up the lowest amount of oil when sheeted into a thick chip, whereas it absorbed the largest amount of oil when sheeted into a thin chip. They explained that thin chips are vulnerable to high levels of stretching, as they result in highly permeable and brittle outer surfaces susceptible to oil infiltration. In contrast, thick restructured potato chips could withstand higher steam pressures due to their stronger solid structure, which was not ruptured. In addition, a flat and smooth outer surface was obtained, which allowed oil to drain easily from the surface and was less vulnerable to oil absorption, as revealed qualitatively by SEM and quantitatively by reflective confocal microscopy. Recently, Gazmuri and Bouchon (2009) studied the oil absorption capacity of a restructured matrix made with different proportions of native wheat starch and vital wheat gluten, which were either directly fried or fried after a predrying step. Results showed that gluten had a predominant role in the structure, making the dough more elastic and less permeable to oil absorption. Interestingly, they found that even though predried products with high gluten content had higher moisture content before frying, they absorbed the lowest amount of oil, suggesting that oil uptake is not clearly related to the amount of moisture lost but rather to the product microstructure. Even though some results can be found in relation to restructured sheeted products, most research has been focused on coatings and batters. The effectiveness of a coating depends on its mechanical and barrier properties, which in turn depend on its microstructure and composition, along with food substrate characteristics (Garcıá et al., 2002). Mallikarjunan et al. (1997) explained that when edible films made of cellulose derivatives are used, a protective surface layer is formed due to thermally induced gelation above 60 C, which inhibits fat absorption. They determined that an edible coating made of MC was more effective than corn zein and HPMC when applied on mashed potato balls. Williams and Mittal (1999) also determined a higher reduction in oil uptake when applying an edible film of MC, compared to HPMC and gellan gum films, on a pastry mix. Similarly, Garcıá et al. (2002) determined that MC was more effective than HPMC when applied on the surface of potato strips and dough discs. Albert and Mittal (2002) evaluated the effect of several edible coatings applied to the surface of a pastry mix. They determined that soy protein isolate (SPI), whey protein isolate (WPI), and MC were the most effective coatings. Postfrying treatments, such as hot air (Nonaka et al., 1977) and superheated steam drying (Kochhar, 1999) have been shown to be effective in


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oil uptake reduction. These processes are aimed to remove surface oil before postcooling suction takes place. The equipment, know as ‘‘low-fat box,’’ is mounted at the discharge end of the fryer and it removes the fat excess from the recently fried food normally using superheated steam at 150–160 C. The oilvapor mixture that is obtained is then filtered, and the oil is pumped back to the fryer. Units range from batch strippers for pilot plant to continuous production units and they can reduce oil content by up to 25% (Kochhar, 1999). Following the same principle, in common continuous production lines the excess fat is removed by passing the product immediately after emerging from the fryer over a vibrating screen, which allows the fat to drain off (Dobraszczyk et al., 2006). The effect of vacuum frying in terms of oil uptake reduction is still not clear, but it appears to be instrumental in reducing oil absorption (Mariscal and Bouchon, 2008).

REFERENCES Aguilera, J. M. and Gloria, H. (1997). Determination of oil in fried potato products by differential scanning calorimetry. J. Agric. Food Chem. 45, 781–785. Albert, S. and Mittal, G. S. (2002). Comparative evaluation of edible coatings to reduce fat uptake in a deep-fat fried cereal product. Food Res. Int. 35, 445–458. Anand, R. S. and Basiotis, P. P. (1998). Is total fat consumption really decreasing? Fam. Econ. Nutr. Rev. Retrieved July 2008 from. http://findarticles.com/p/articles/mi_m0EUB/ is_3_11/ai_53885191. Ascherio, A. and Willett, W. (1997). Health effects of trans fatty acids. Am. J. Clin. Nutr. 66, 1006S–1010S. Banks, D. (1996). Food service frying. In ‘‘Deep Frying’’ (E. G. Perkins and M. D. Erickson, eds.), pp. 245–257. AOAC Press, Champaign. Baumann, B. and Escher, E. (1995). Mass and heat transfer during deep fat frying of potato slices: I. Rate of drying and oil uptake. Lebensmittel-Wissenschaft Technologie. 28, 395–403. Besaratinia, A. and Pfeifer, G. (2004). Genotoxicity of acrylamide and glycidamide. J. Natl. Cancer Inst. 96, 1023–1029. Blumenthal, M. M. (1991). A new look at the chemistry and physics of deep-fat frying. Food Technol. 45, 68–71. Blumenthal, M. M. and Stier, R. F. (1991). Optimization of deep-fat frying operations. Trend. Food Sci. Technol. 2, 144–148. Bouchon, P. and Aguilera, J. M. (2001). Microstructural analysis of frying of potatoes. Int. J. Food Sci. Technol. 36, 669–676. Bouchon, P. and Pyle, D. L. (2004). Studying oil absorption in restructured potato chips. J. Food Sci. 69, E115–E122. Bouchon, P. and Pyle, D. L. (2005a). Modelling oil absorption during post-frying cooling. II Solution of the mathematical model, model testing and simulations. Trans. Inst. Chem. Eng. C Food Bioproducts Process. 83, 253–260. Bouchon, P. and Pyle, D. L. (2005b). Modelling oil absorption during post-frying cooling. I Model development. Trans. Inst. Chem. Eng. C Food Bioproducts Process. 83, 261–272. Bouchon, P., Hollins, P., Pearson, M., Pyle, D. L., and Tobin, M. J. (2001). Oil distribution in fried potatoes monitored by infrared microspectroscopy. J. Food Sci. 66, 918–923.

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CHAPTER

6 Introduction of Oats in the Diet of Individuals with Celiac Disease: A Systematic Review Olga M. Pulido,*,† Zoe Gillespie,* Marion Zarkadas,‡ Sheila Dubois,* Elizabeth Vavasour,* Mohsin Rashid,‡,} Connie Switzer,‡,} and Samuel Benrejeb Godefroy*

Contents

I. Introduction II. Methods A. Pivotal in vivo clinical studies on the effect of oats in patients with celiac disease and dermatitis herpetiformis (Table 6.1) B. Nonpivotal studies testing the effect of oats in patients with celiac disease by in vitro methods or serology (Table 6.2) III. Results A. Pivotal in vivo clinical studies on the effect of oats in patients with celiac disease and dermatitis herpetiformis B. Nonpivotal studies testing the effect of oats in patients with celiac disease using in vitro methods or serology C. Other studies relevant to the effect of oats in patients with celiac disease

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* Bureau of Chemical Safety, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, { { } }

Ontario, Canada Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada Professional Advisory Board, Canadian Celiac Association, Ottawa, Ontario, Canada Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada


#


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IV. Discussion A. Evidence based on pivotal and nonpivotal studies B. Evidence based on other reviews on the safety of oats C. Biochemistry and taxonomy of oats relevant to its potential toxicity D. Benefits of the consumption of oats V. Conclusions VI. Appendix I A. Summary of pivotal in vivo clinical studies testing the safety of oats in patients with celiac disease or dermatitis herpetiformis (Table 6.1) B. Summary of nonpivotal studies testing the effect of oats in patients with celiac disease by in vitro methods or serology (Table 6.2) C. Other studies relevant to the effect of oats in patients with celiac disease Acknowledgments References

Abstract

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Celiac disease is an immune-mediated disease, triggered in genetically susceptible individuals by ingested gluten from wheat, rye, barley, and other closely related cereal grains. The only treatment for celiac disease is a strict gluten-free diet for life. This paper presents a systematic review of the scientific literature on the safety of pure oats for individuals with celiac disease, which historically has been subject to debate. Limitations identified within the scientific database include: limited data on long-term consumption, limited numbers of participants in challenge studies, and limited reporting about the reasons for withdrawals from study protocols. Furthermore, some evidence suggests that a small number of individuals with celiac disease may be intolerant to pure oats and some evidence from in vitro studies suggests that an immunological response to oat avenins can occur in the absence of clinical manifestations of celiac disease as well as suggesting that oat cultivars vary in toxicity. Based on the majority of the evidence provided in the scientific database, and despite the limitations, Health Canada and the Canadian Celiac Association (CCA) concluded that the majority of people with celiac disease can tolerate moderate amounts of pure oats. The incorporation of oats into a gluten-free diet provides high fiber and vitamin B content, increased palatability, and beneficial effects on cardiovascular health. However, it is recommended that individuals with celiac disease should have both initial and long-term assessments by a health professional when introducing pure oats into a gluten-free diet.

Introduction of Oats in the Diet of Individuals with Celiac Disease

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I. INTRODUCTION Celiac disease is an autoimmune disorder in which ingestion of gluten causes damage to the small-intestinal mucosa in genetically susceptible individuals (Fasano et al., 2008; Green and Cellier, 2007; Losowsky, 2008; Presutti et al., 2007). Celiac disease is also known as celiac sprue or glutensensitive enteropathy. The clinical presentation of celiac disease is highly variable. In addition to the intestinal symptoms, celiac disease is associated with various extraintestinal complications. It is, therefore, considered as a multisystem disorder (Briani et al., 2008). Patients with celiac disease also have an increased risk of developing other autoimmune disorders, such as type I diabetes mellitus (Barton and Murray, 2008; Gianfrani et al., 2008). In children, celiac disease can be associated with growth failure and delayed puberty (Tully, 2008). Furthermore, the symptoms and associated conditions of celiac disease can vary greatly in number and severity resulting in frequent delays in diagnosis, and/or misdiagnosis. Common examples of misdiagnoses include: irritable bowel syndrome, chronic fatigue syndrome, and fibromyalgia (Catassi and Fasano, 2008; Cranney et al., 2007; Rashid et al., 2005). Dermatitis herpetiformis1 is a condition of the skin that is also triggered by the ingestion of gluten in genetically susceptible individuals and is considered a dermatological form of celiac disease (Abenavoli et al., 2006; Alaedini and Green, 2005; Briani et al., 2008; Losowsky, 2008). Gluten is a generic name given to storage proteins in wheat, barley, rye, and other closely related cereal grains. It is the gluten in wheat flour that binds and gives structure to bread, baked goods, and other foods, making it widely used in the production of many processed and packaged foods. For individuals with celiac disease, these proteins trigger an inflammatory injury in the absorptive surface of the small intestine resulting in malabsorption of protein, fat, carbohydrate, fat-soluble vitamins, folate, and minerals, especially iron and calcium (Koning, 2008; Tye-Din and Anderson, 2008; Wieser and Koehler, 2008). Celiac disease is a lifelong condition. If celiac disease is not diagnosed early and treated with a strict gluten-free diet, it can be associated with serious complications, including: osteoporosis, lymphoma, and infertility in both men and women (Bianchi and Bardella, 2008; Fasano et al., 2008; Freeman, 2008; Green and Cellier, 2007; Malandrino et al., 2008; Mangione, 2008; Pastore et al., 2008; Pellicano et al., 2007; Pope and Sheiner, 2009). A small-intestinal biopsy is necessary to confirm the diagnosis of celiac disease (Dickson et al., 2006; Haines et al., 2008). However, the advent of 1

Throughout this publication, when not otherwise specified, dermatitis herpetiformis is included under the general term celiac disease.

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new diagnostic serological tests, particularly for anti-endomysial and antitissue transglutaminase antibodies (Alaedini and Green, 2008; Fasano et al., 2008; Gianfrani et al., 2008; Hill and Holmes, 2008; Hopper et al., 2008), has now estimated the worldwide prevalence of celiac disease to be between 1 in 100 and 200 individuals (Catassi, 2005; Catassi et al., 2007b; Fasano et al., 2003; Harrison et al., 2007; National Institute of Health, 2004). Certain groups of people have markedly elevated risks of developing celiac disease. First-degree relatives of individuals diagnosed with celiac disease have a 10–20% increased risk of developing celiac disease ( Jolobe, 2008). A high prevalence of celiac disease is also found in individuals with Down syndrome and IgA deficiency (Presutti et al., 2007). Presently, the only treatment of celiac disease is a strict lifelong exclusion of wheat, rye, barley, and other related cereal grains2 from the diet (Akobeng and Thomas, 2008; Buchanan et al., 2008; Catassi et al., 2007a; Ciclitira et al., 2005; Collin et al., 2007; Guandalini, 2007; Hopman et al., 2008; Kupper, 2005; Zarkadas and Case, 2005; Zarkadas et al., 2006). The amount of gluten that can be tolerated varies amongst people with celiac disease. Some patients tolerate an average of 34–36 mg gluten/day without any clinical manifestations of celiac disease, while others who consume approximately 10 mg gluten/day developed mucosal abnormalities (Akobeng and Thomas, 2008; Catassi et al., 2007a). Although there is no evidence to suggest a single definitive threshold for a tolerable gluten intake, there is evidence that a daily gluten intake of

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