Food Structure

Volume 10 Number 4 Article 10

1-1-1991

Natural Systems for Preventing Contamination and Growth of Microorganisms in Foods

King-Thom Chung Memphis State University

Chris A. Murdock Memphis State University

Follow this and additional works at: https://digitalcommons.usu.edu/foodmicrostructure

Recommended Citation Chung, King-Thom and Murdock, Chris A. (1991) "Natural Systems for Preventing Contamination and Growth of Microorganisms in Foods," Food Structure: Vol. 10 : No. 4 , Article 10. Available at: https://digitalcommons.usu.edu/foodmicrostructure/vol10/iss4/10

This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Food Structure by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. FOOD STRUCTURE, Vol. 10 (1991), pp. 361-374 1046-705X/91 $3.00 +. 00 Scanning Microscopy International, Chicago (AMF O'Hare), IL 60666 USA

NATURAL SYSTEMS FOR PREVENTING CONTAMINATION AND GROWTH OF MICROORGANISMS IN FOODS

King-Thorn Chung and Chris A. Murdock

Department of Biology, Memphis State University Memphis, TN 38152

Abstract Introduction

Food-borne illness is a vast and costly worldwide Food-borne illness is a serious worldwide problem problem. Although complex, it may be divided into two in both developed and developing countries. The cost is major types of animal and human disease: intoxication enormous. For example, in Scotland, outbreaks of poul­ caused by toxic substances in food and secondly, try-borne salmonellosis are estimated to cost between infection, caused by microorganisms. Toxic substances 200,000 and 900,000 English pounds per year (186). In may be already present in foods, or they may be the U.S. A. food-borne diarrhea alone cost between $5 produced by microorganisms through contamination and billion and $17 billion each year (6). However, the cost proliferation in food. Prevention of the invasion of in terms of health and life cannot be fully measured. foods by microorganisms may be achieved by effective Today, there are more than 6.5 million cases of food­ use of intrinsic factors found in plants and animals. borne illness each year in the United States alone (119). These factors include pH, moisture content, oxidation­ Over 9,000 resulted in death in 1990 (111). reduction potential, nutrient content, biological This article reviews major types of food-borne structures, and antimicrobial constituents. A second type diseases and discusses the natural preservation systems of natural protection is that of microbial antagonism. which we believe have the most immediate potential for Research work discussed includes that done in the application to the food products industry. The methods author's laboratories involving tannic acid and microbial discussed will help control contamination and growth of antagonism against Listeria monocytogenes in milk. The food-borne microorganisms. safety of all natural mechanisms of defense is important economically and in public health. We predict that the Types of Food-Borne Diseases use of antimicrobials occurring naturally in foods and safe microbial antagonism will receive more attention in There are two basic types of food-borne diseases: future food safety research. a. intoxication; and b. infectious. Food-Borne Intoxication Key Words: Food safety, food-borne diseases, intrinsic Food-borne intoxication may be caused by toxic factors, extrinsic factors, antimicrobials occurring substances already present in certain foodstuffs, toxins naturally in foods , microbial antagonism, natural produced by microorganisms which contaminate the mechanisms, tannic acid. food, or substances added to food. Examples of food­ borne intoxication are numerous (139, 153). One exam­ ple is mycotoxins produced by molds. These are afla­ toxin, zearalenone, zearlenol, trichothecene, ochratoxin, citrinin, penicillic acid, patulin, sterigmatocystin, alter­ Initial paper received September 4, 1991 nariol methyl ether, mycophenolic acid, panitrem A, and Manuscript received December 30, 1991 "PR" toxin produced by three genera, i.e., Aspergillus Direct inquiries to K.-T. Chung sp., Penicillium sp., and Fusarium species (170). Three Telephone number: 901 678 4458 of these mycotoxins, i.e., aflatoxin B , sterigmatocystin FAX number: 901 678 3299 1 and ochratoxin A toxins were reported to be carcinogens for animals and possibly for human beings (92). Bacte­ rial toxins are also important source of food-borne intox­ ication. Enterotoxins produced by Staphylococcus aure­ us (132, 173), and neurotoxins produced by Clostridium botulinum (1, 89, 106, 133, 138) are two examples

361 King-Thorn Chung and Chris A. Murdock

(Table 1). Other sources of food-borne intoxication are distributed to large numbers people; 3. More meals from pesticides and food additives; their impact on eaten away from home; 4. More emphasis in these es­ health is certainly a public concern (23 , 30, 37). Food tablishments on salads, cold foods , and raw fruits and additives are often added to food to improve their ap­ vegetables that require extensive handling ( 119). pearance, flavor or as preservatives. For example, The current work force of nine million food han­ there are currently nine approved food colorants used in dlers is poorly paid and poorly educated (119). The food industries of the U.S.A. Five of these colorants relationship between low education levels, low socio­ are azo dyes (142). There are three thousand other azo economic status, and increased frequency of enteric dyes which are used in textile, paper, and leather indus­ infection is well recognized (119). Many establishments tries (122) , which can contaminate water supplies. are poorly supervised by health regulating authorities Many azo dyes such as Butter Yellow and Ponceau 3R and they often lack proper sanitary facilities . These can be anaerobically converted into aromatic amines by factors all contribute to the recent increases of incidence intestinal microflora ( 44-46, 87) . Many of these of food-borne diseases. aromatic amines are active mutagens and are very likely, Antibiotics are used either for the prevention of important carcinogens (43 , 51) . They also could be re­ animal diseases or as an additive to stimulate animal lated to our present high incidence of colon cancer (14 , growth (69, 91) . As a result, many food-borne patho­ 15, 85 , 185). It is worth mentioning that although more gens have become antibiotic-resistant. Evidence shows than 2 ,000 food additives exist, most of them probably that antibiotic-resistant Salmonella is the cause of many do not cause any food-borne intoxication. However, the salmonellosis outbreaks in the United States (91). The chronic and residual type of effects with mutagenic and prevalence of antibiotic-resistant food-borne pathogens carcinogenic potential cannot be completely ignored. may also be a factor in enhancing the seriousness of Food-Borne Infection food-borne diseases today. Food-borne infection is caused by ingestion of food containing viable bacteria which then grow and Prevention of Contamination and Growth of establish themselves in the host, resulting in illness. Microorganism in Foods Some of these pathogens occur in the gastrointestinal tracts of normal healthy animals and humans. Other mi­ The "best medicine" is always prevention. Nu­ croorganisms are ubiquitous in nature, occurring on soil merous methods were available to minimize microbial and vegetables, in animal wastes, and on animal carcass­ contamination of foods. Many of these simply stress es. Human skin surfaces and nasal passages also harbor available "physical" approaches (3 , 54). But they such bacteria as Staphylococcus species. Water supplies require recognition and implementation. may be contaminated with fecal materials which contain Several aspects of the storage environment affect some of these pathogens. Food and food utensils, air both the food and microorganisms. Examples are tem­ and dust also can be carriers for spread of these patho­ perature, relative humidity, and presence and concentra­ gens. It is extremely difficult to prevent such pathogens tion of different gases in the environment. These factors from entering raw foods. are referred to as "extrinsic" factors (98) . By control­ The major food-borne pathogens are: Salmonella ling "extrinsic" factors, one can extend the shelf-life of species, Listeria monocytogenes, Shigella species, foods. However, this usually requires expensive facil­ Campylobacter jejun.i , Clostridium perfrin.gen.s, entero­ ities and operations; and, it may not be feasible in some pathogenic Escherichia coli, Bacillus cereus, Vibrio areas where capital, personnel and equipment are species, Yersin.ia en.terocolitica, Plesiomonas shigel­ limited. loides and Aeromon.as hydrophila. Their major charac­ Plants and animals that serve as food sources have teristics, the types of food-borne diseases caused and evolved mechanisms of defense against the invasion and their association with foods are listed in Table 1. proliferation of microorganisms and some remain active Bacillus cereus and Clostridium perfrin.gens also produce in fresh food (98). These inherent food plant or animal enterotoxins; however, a large number of viable cells tissue elements are referred to as "intrinsic" factors must be consumed, which implies the release of toxins (properties) . Among them are pH, moisture content, ox­ in vivo , rather than in the food. In this review, we idation-reduction potential, nutrient content, biological consider them as causing food-borne infection rather structures and antimicrobial constituents (98). These than intoxication. factors serve as natural defense mechanisms against mi­ crobial contamination and proliferation during the devel­ Why Is Food-Borne Infection Serious Today? opment stages of food plants and animals. If we can make effective use of these factors , we can further en­ Food-borne infection has long been considered a hance the capability of preventing the contamination and "traditional" or even "common" problem. Why is it be­ growth of microorganisms in foods . Major "intrinsic" coming even more serious today? There are many rea­ factors are briefly reviewed below. sons such as the following: 1. Greater variety of foods; pH: 2. Mass production and international distribution of Most microorganisms normally grow at a pH foods, allowing for low-level contaminated foods to be value around 7, while few grow below 4.0. The pH of

362 Preventing Contamination and Growth of Microorganisms in Foods

TABLE 1. Characteristics of Important Food Borne Pathogens

Name of Pathogens Type of Food-Borne Gram A * Major type of References Disease Stain w Food Associated

Salmonella species Food-borne infection 0.95- Beef, pork, turkey 5, 32, 66, (Salmonellosis) 0.99 chicken, ice cream, milk 71' 88, 126

Listeria species Food-borne infection + milk, cole slaw, cheese 27, 29, 40, 62, 80, (Listeriosis) 116, 120, 143, 145, 15 7' 15 8' 15 9' 160

Shigella species Food-borne infection salads, seafood 31' 163, 167, (Shigellosis) 174, 181

Campylobacter jejuni Food-borne infection poultry, pork, beef, lamb 61, 63, 72, 168 (Campylobacteriosis) clams, cakes, raw milk

Enteropathogenic Food-borne infection 0. 96 cheese, sandwiches 39, 73, 149 Escherichia coli (Gastroenteritis) hamburgers

Staphylococcus aureus Food-borne intoxication + 0.86 milk, cream sauces, salads, pud-131, 176 (Gastroenteritis) dings, custards, bakery products

Clostridium perfringens Food-borne infection + meat, poultry 77' 110 (Gastroenteritis)

Clostridium botulinum Food-borne intoxication + 0.94 meat, fruit, vegetables, 1, 89, 106, honey, syrups 133, 138

Bacillus cereus Food-borne infection + cereal, corn, corn starch, 4, 47, 65, 99, (Gastroenteritis) mashed potatoes, vegetables, 121, 127, 137 meat products, puddings, soups, sauces, macaroni, cheese

Vibrio species Food-borne infection 0.94 seafood, oysters, crabs, eggs, 25, 41, 42, 67, ( Gastroenteritis) asparagus, potatoes, crawfish, 82, 83, 125, 135 shrimp, clams Yersinia enterocolitica Food-borne infection cakes, meats, seafood, vege- 64, 84, 112, 128 (Gastroenteritis) tables, beef, lamb, pork, milk

Plesiomonas Food-borne infection 0.97 fish, shellfish 123 shigelloides (Gas troen teri tis)

Aeromonas hydrophila Food-borne infection fish 9 (Gastroenteritis)

* Aw: water activity (minimum value for growth)

363 King-Thorn Chung and Chris A. Murdock many foods such as fruits, drinks, vinegar and wine (fer­ the types of micro-organisms in those foods (98). mented products), fall below the level at which bacteria Nutrient Content normally grow. Proper pH is excellent for maintaining Many foods have a variety of energy sources, ni­ the keeping quality of these products. The natural acidi­ trogen sources, minerals, vitamins and related growth ty of many foods can be considered as nature's way of factors required for microbial growth. For energy protection from microbial invasion. Artificial adjust­ sources, food-borne microorganisms may utilize sugars, ment of pH of certain foods may also be a way to protect alcohols and amino acids. Some are able to use compli­ them. cated carbohydrates such as starch, cellulose, pectin, Moisture Content hemicellulose as a source of energy. A few of them can Drying or desiccation is one of the oldest methods use fat as an energy source. The primary source of ni­ of preserving foods. Available water is usually defined trogen for food-borne microorganisms are amino acids. in terms of water activity (aw). In foods, the~ is de­ Some microbes can utilize nucleotides, peptides or pro­ fined by the ratio of vapor pressure of food substrate to teins. Growth factors such as Vitamin Bare required by the vapor pressure of pure water at the same tempera­ many food-borne Gram-positive bacteria. Foods lacking

, ture, i.e., ~ = p/p0 where p = vapor pressure of solu­ these growth factors may limit the growth of these type tion, and Po = vapor pressure of solvent (usually water). of bacteria (98) The addition of salt (NaCl) or sugar lowers aw. For ex­ Protective Biological Structures ample, a NaCl solution of 22%, has an ~ of 0. 86. The natural covering of some foods provides lim­ Most spoilage bacteria do not grow below a = ited protection against microbial contamination and 0.91, while some fungi can grow as low as aw = 0~70. spoilage. Examples are: testa of seeds, outer coverings With respect to food-poisoning bacteria, Staphylococcus of fruits, the shell of nuts, the hide of animals and shells aureus can grow at an aw = 0.86; Clostridium botulinum of eggs (98). Type E, 0.97; Escherichia coli, 0.96; Clostridium botu­ linum Type A and B, 0.94; and Vibrio parahaemolyticus, Antimicrobials Occurring Naturally in Foods 0.93 (98). Many naturally occurring substances in the food The aw of most fresh food is above 0. 99; some plants and animals have been shown to have antimi­ foods have lower values, for example: the aw for liver crobial activity. These antimicrobial substances contrib­ sausage is 0. 96; salami 0. 82-0.85; dry fruit, 0. 72-0. 80; ute greatly to the stability of some foods against attack honey 0.75 and marmalade, 0.82-0.92. (12). by microorganisms. A recently detailed review (20) of Oxidation-Reduction Potential (0/R; Eh) these antimicrobials occurring in plants and animals showed that there are several categories of these The oxidation-reduction potential (0/R or Eh) of antimicrobials in foods: a substrate is defined as the ease with which the sub­ Enzymes and Proteins: Conalbumin and avidin strate loses or gain electrons. When an element or com­ in the egg white are inhibitory to microorganisms. Con­ pound loses an electron, it is said to be oxidized; where­ albumin, which binds with ir~n, makes it unavailable for as a substrate that gains an electron becomes reduced. use by microorganisms. It is inhibitory to both Gram­ When electrons are transferred from one compound to negative and Gram-positive bacteria. Micrococcus and another, a potential difference is created between the Bacillus species are particularly sensitive to conalbumin ~wo. This difference is measured by an appropriate (26). Avidin can bind biotin and thus can inhibit the Instrument and expressed in the term of millivolts (m V). growth of microorganisms which have a strict require­ A highly oxidized substrate would have a positive Eh ment for biotin. while a reduced substrate a negative Eh. Another iron binding protein, lactoferrin in bo­ Microorganisms display varying degrees of sensi­ vine milk, is inhibitory to the growth of Bacillus tivity to the oxidation-reduction potential of their growth subtilis, B. stearothermophilus and Escherichia coli medium (98). Some bacteria, such as Clostridium re­ (136, 147). The casein fraction of milk also has been quire reduced conditions for growth. They are called demonstrated to inhibit B. stearothermophilus (7). Part anaerobes. Others, such as Bacillus species require of this inhibitory action is due to lactoferrin present in positive Eh for growth and are regarded as aerobes. the casein fraction. Some such as lactobacilli and streptococci, actually grow Lysozyme in both milk a11d eggs is inhibitory to better under slightly reduced conditions, and are regard­ several organisms including Listeria monocytogenes, ed as microaerophilic. Some bacteria have the capacity Campylobacter jejuni, Salmonella typhimurium, Bacillus to grow under either aerobic (positive Eh) or anaerobic cereus, and Clostridium botulinum (95). The mechanism (negative Eh) conditions, and are called facultative anaerobes. Most molds and yeasts associated with food ~f action of this enzyme involves hydrolysis of ~-1 ,4- hnkage between N-acetylmuramic acid and N-acetylglu­ are aerobic, but a few tend to be facultative anaerobes. cosamine layer of the bacterial cell wall. Usually Gram­ Plant foods, especially plant juices, have Eh values from +300 to +400 mv. Solid meats have an Eh n~~ative bact~ria are resistant to lysozyme, but suscepti­ blllty can be mduced following sodium chloride and eth­ value of -200 mv while cheeses have Eh values of -20 to ylenediamine tetraacetate treatments which weaken the -200 mv. Different Eh values of foods may influence outer membrane (178). Lysozyme has received more

364 Preventing Contamination and Growth of Microorganisms in Foods attention recently and is considered to have a good TABLE 2. Sources of Organic Acids in Foods potential as a food preservative because it is specific for (from reference 20) bacteria and harmless to humans. Lactoperoxidase in bovine milk is an effective Organic Acids Food Sources antimicrobial enzyme when combined with thiocyanate (SCN-) and hydrogen peroxide (147). This is called the Citric Acid Citrus Fruits lactoperoxidase system. Thiocyanate is widely distribut­ Succinic Acid Asparagus, Broccoli, Sugar Beets , Rhu­ ed in animal tissues and secretions (148). Hydrogen barb, Fermented Cheese and Vegetables peroxide is readily produced by catalase-negative bacte­ ria such as Lactobacillus or can be added exogenously. Malic Acid Fruits and Vegetables Apples The lactoperoxidase system is active against H20 2-pro­ Tartaric Acid Grapes and Pineapples ducing bacteria such as Lactobacillus and Streptococcus spp., and also Gram-negative catalase positive organisms Benzoic Acid Cranberries , Raspberries , Plums , including Pseudomonas spp. and Escherichia coli (24). Prunes, Cinnamon, Cloves It was recently demonstrated (162) that the lactoperoxi­ Lactic Acid Sauerkraut, Pickles , Olives, Meats , dase system delayed but did not prevent the onset of the Cheeses exponential growth of Listeria monocytogenes Scott A culture. Propionic Acid Swiss cheeses The lactoperoxidase system can form various oxi­ dizing products such as hypothiocyanate, cyanosulfurous double bond to c14• c16 and cl8 fatty acids, increase acid and cyanosulfuric acid, all of which show anti­ antimicrobial activity. Generally, Gram-negative bacte­ microbial activity (148). Several possible mechanisms ria are less susceptible than Gram-positive bacteria to of inhibition have been postulated including reduced the inhibitory effects of fatty acids. This was possibly oxygen uptake, reduced lactate production by fermenta­ due to the fact that the lipopolysaccharide layer of tion organisms, inhibition of key metabolic enzymes Gram-negative bacteria was resistant to fatty acids such as hexokinase, glyceraldehyde-3-phosphate-dehy­ penetration into cells and thereby conferred resistance drogenase and D-lactate dehydrogenase, inhibition of glu­ ( 161). As with organic acid, the undissociated form of cose uptake, cytoplasmic damage with leakage of ions fatty acid molecules is responsible for the antimicrobial and ultraviolet absorbing materials and inhibition of activity. Therefore, the antimicrobial activity of fatty nucleic acid and protein syntheses (162). acid is favored by a reduction in pH. Organic acids: Organic acid, whether present in The mechanisms for the antimicrobial activity of foods naturally, or occurring as a result of fermentation , fatty acids have been studied by a number of investiga­ also have antimicrobial properties (Table 2). Examples tors. Fatty acid caused a reduction of oxygen uptake are citric acid, succinic acid , malic acid tartaric acid and induced leakage of amino acids (76). Fatty acid also ' ' benzoic acid, lactic acid and propionic acid. Mecha- altered cell membrane permeability or uncoupled the nisms of action involve direct pH reduction of the sub­ electron transport chain of specific protein responsible strate, depression of the internal cellular pH by ioni­ for A TP (adenosine triphosphate) generation and nutrient zation of the undissociated acid molecules or disruption transport to the cell (161) . It was suggested (75) that of substrate transport by alteration of cell membrane fatty acid caused the inhibition of membrane transport, permeability, or inhibition of nicotinamide adenine resulting in nutrient deprivation. It was also demon­ dinucleotide (reduced form) [NADH] oxidation (20, 74). strated (81) that inhibition of Staphylococcus aureus was Generally the undissociated form of organic acid mole­ due to increased membrane permeability. cules is responsible for the antimicrobial activity. There are problems with using medium-chain fatty Medium-chain fatty acids: Medium-chain fatty acid as food preservatives. For example, solubility is acids, both saturated and unsaturated, occur in plant and one of the main difficulties. There are also antagonists animal fats. Seed fats contain low levels of saturated in food , such as albumin , starch, and cholesterol, which fatty acids, while marine oils are high in unsaturated interfere with the antimicrobial activity of fatty acids fatty acid (58). Medium-chain fatty acids, containing 12 (132). However, fatty acids may still have potential as to 18 carbon atoms are effective antimicrobial agents. microbial inhibitors for slightly acid foods and some It has been reported (103) that lauric acid, myristic and foods which do not have suitable preservatives. It was palmitic acid (C ,C ,and C , respectively) are effec­ proposed (103) that concurrent use of multiple approved 12 14 16 food additives is a good way for food preservation. tive inhi.bito~s of bact~ria and both caproic acid (C 10) and launc acid are active against yeast. Fatty acids, will therefore, be used more often as anti­ Chain length, degree of saturation and geometric microbial food agents. configuration are all important factors affecting anti­ The naturally occurring fatty acid esters of su­ microbial activity of fatty acids. For Gram-positive crose and other polyhydric alcohols have also been re­ bacteria, saturated fatty acids with 12 carbons as well as ported to possess antimicrobial properties (19, 52, 104, 105, 115). Glycerol monolaurate, for example, was de­ th~ monounsaturated palmitoleic acid (C 16 : 1) and linoleic acid are most effective (102). Addition of one cis monstrated to be inhibitory to Vibrio parahaemolyticus (19) , Aspergillus niger, Penicillium citrinum, Candida

365 King-Thorn Chung and Chris A. Murdock

Allinase 2 CH 2 =CH-CH 2 -SO-CH 2 -CHNH 2 -COOH + H20 CH 2 :CH-CH 2 -505-CH 2CH =CH 2 + 2 CH 3 COCOOH + 2NH 3 Alliin Allicin Pyruvic Acid Ammonia

Figure 1. Enzymatic Conversion of Alliin to Allici n. uti/is, and Saccharomyces cerevisiae ( 104). Sucrose di­ caprylate and sucrose monolaurate have also been shown to inhibit growth of Gram-negative and Gram-positive bacteria as well as fungi (104, 105). ~ Plant Essential Oil Components: Extracts from + DCOOH plants and plant parts used as flavoring agents in foods HO'¢ and beverages possess antimicrobial activity. Typical 0 0 examples are the Allium species: A. sativum (garlic), A. CH2CH20H Oleuropeln (B-3,4-dlhydroxy- Elenollc acid cepa (onion) and A. porrum (leek) (20). Effects of ex­ phenylethyl alcohol) tracts from garlic and onion on food-borne bacterial pathogens have been reported (59 , 100, 114 , 155 , 176). Several species of food spoilage yeasts and molds were aglycone HO~ ¥ also inhibited by garlic (53 , 124, 172) . The principal 9 antimicrobial component of garlic has been demonstrated y N to be allicin (2-propenyl-2-propenethiol sulfinate) (169). CH2CH200C COOCH3 The intact garlic bulb does not contain allicin , but rather + 2 Glucose alliin (S -allyl-L-cysteine-S-oxide), which is hydrolyzed by allinase to yield allicin, pyruvic acid and ammonia Figure 2. Hydrolysis of Oleuropein. (Figure 1). Other plants used as herbs and spices in food in ­ Humulones and Lupulones: Certain major con­ clude: achiote, allspice (pig menta) , almond (bitter) , stituents of hop resin such as humulones (humulone, angelica, basil (sweet) , bay (laurel), bergamot, calmus, cohumulone, adhumulone) and lupulones (lupulone, cananga, caraway, cardamom, celery, cinnamon, citron­ colupulone and adlupulone) were reported to exhibit ella, clove, coriander, dill, elecampane, fennel , garlic, antimicrobial activities (20). The mechanism of inhibi­ ginger, lemon, licorice, lime, mace, mandarin , marjo­ tion is not known at the present. ram, musky bugle, mustard , nutmeg, onion, orange, ore­ Oleuropein: The phenolic components of ethyl gano, paprika, parsley, pennyroyal, pepper, peppermint , acetate extracts of the green olive including oleuropein rosemary, sage, sassafras, spearmint, star anise, tar­ and aglycone of oleuropein were shown to be inhibitory ragon (estragon), thyme, turmeric, verbena and win­ to Lactobacillus plantarum, Leuconostoc mesenteroides, tergreen (20) . Certain antimicrobial compounds in these and fungi Geotrichum candidum and Rhizopus spp. plants which have been identified are thymol {5 -methyl- (101) . In studying the antimicrobial properties of ol­ 2-(1 - methylethyl)-phenol} , cinnamic aldehyde europein and products of its hydrolysis (70), it was dis­ (3 -phenyl-2-propenal) , and eugenol {2-methoxy-4-(2- covered that the crude oleuropein extract per se was in­ propenal) phenol}. These antimicrobial compounds are hibitory only to 3 of 17 species screened, i.e. , Bacillus often in the essential oil fraction (20) . subtilis, Staphylococcus aureus and Pseudomonas Pigments and Related Compounds: It has been solanacearum, but the acid hydrolysis product of the ex­ shown (117, 118) that proanthocyanidine in cranberries tract was inhibitory to 11 bacteria including Lacto­ is inhibitory to Saccharomyces boyancis, and that pelar­ bacillus plantarum WSO, L. brevis 50, Pediococcus gonidin-3-monoglucoside and its degradation products cerevzszae 39, Leuconostoc mesenteroides 42, are inhibitory to E. coli and Staphylococcus au reus (86). Staphylococcus aureus, Bacillus subtilis, Salmonella Anthocyanins were shown to be inhibitory to microbes typhimurium , Pseudomonas solanacearum, P. (140, 187). Also demonstrated was that monoglucosides lachrymans , Erwinia carotovora, Xanthomonas of cyanidin, pelargonidin and delphinidin were inhibito­ vesicatoria. Using lactic acid producers, i.e., L. ry to certain bacteria (141) . The mechanism of inhibito­ plantarum, L. brevis, Pediococcus cerevisiae and ry action of anthocyanins is not fully understood. One Leuconostoc mesenteroides for test organisms, it was possibility is that anthocyanin has chelating ability, found that oleuropein was not inhibitory, but two of its which makes metal ion unavailable to enzymes. The ad­ hydrolysis products, the aglycone and elenolic acid were dition of magnesium and calcium could reverse bacterial inhibitory (Fig. 2). The aglycone of oleuropein and inhibition by malvidin-3-monoglucosides (166). It has elenolic acid were much more inhibitory when the broth also been reported (38, 183) that anthocyanins had an medium contained 5% NaCl (70). inhibitory effect on certain bacterial enzymes. Caffeine: Caffeine is present in coffee, tea and Chlorophyll ide a, a degradation product of chloro­ cocoa beans, and has been demonstrated to inhibit the phyll a, has also been shown to inhibit the growth of growth and mycotoxin production by several Aspergillus Bacillus subtilis, E. coli and Pseudomonas fluorescens and Penicillium species (33, 34, 113) at 1 mg/ml. (17). Caffeine uncoupled the regulation of glycolysis and

366 Preventing Contamination and Growth of Microorganisms in Foods

Figure 3. Hydrolysis of Tannic Acid. 0

OH + HO OH +Glucose OR Tannic Acid Gallic Acid (R-Digallic Acid) 0 Ellegic Acid glucogenesis m Saccharomyces cerevzszae (175). parasiticus (11). Growth inhibition of A. parasiticus by caffeine was Hydroxycinnamic Acid Derivatives: Many high­ shown (35) to be due to an alteration of purine metabo­ er plants and plant parts including vegetables, fruits, lism, but the inhibition of aflatoxin synthesis was not grains and nuts contain hydroxycinnamic acid derivatives due to an inhibition of AMP (adenosine monophosphate) which are antimicrobial. These include caffeic, chloro­ phosphodiesterase or a chelation of key metal ions. It genic, p-coumaric, ferulic and quinic acids (56). was suggested (36) that caffeine may limit the uptake of Tannins are present in strawberries, grapes, ap­ glucose, and demonstrated (179) that growth of ples, tea and possibly other plant products. Tannins are Lactobacillus plantarum was inhibited by caffeine. The also widely distributed in barks and nuts, sometimes ap­ mechanism of action is not well understood, but caffeine proaching ten percent of the weight of these structures is known to depress bacterial DNA synthesis. The (130). It has been reported that tannins are inhibitory to compound also may form caffeine-phenolic acid Salmonella senftenberg (18). Inactivation of coxsackie­ complexes which are inhibitors of bacteria. virus, echovirus, reoviruses, herpes viruses and polio­ Theophylline and Theobromine: Theophylline viruses by tannins has also been reported (108). (1 ,3-dimethylxanthine) and theobromine (3 , 7-dimethyl­ Vegetable tannins are commonly called tannic xanthine) are methylated derivatives of xanthine, and are acids, and basically are groups of phenolic compounds. present in tea, cocoa beans and cola nuts. Although in­ They can be divided into two categories: hydrolyzable stant tea infusion has a bactericidal effect on several and condensed tannins. The hydrolyzable tannins can be food -borne bacteria including Salmonella typhimurium, divided into two classes: gallotannins and ellagitannins. E. coli, Staphylococcus aureus and B. cereus, no evi­ Upon hydrolysis, the gallotannins yield gallic acid and dence has indicated that theophylline and theobromine glucose while ellagitannin yield ellagic acid and glucose are antimicrobial (180). Both theophylline and theobro­ (Figure 3). Condensed tannins contain little if any mine have little anti-aflatoxigenic activity. We have carbohydrate. found that theophylline and theobromine are not inhibito­ We tested the antimicrobial activity of tannic acid ry to many species of food -borne bacteria (Chung, et and found that it is inhibitory to many food-borne bacte­ al. , unpublished results). ria including Aeromonas hydrophila , Escherichia coli, Phytoalexins: When the living plant tissue is Listeria monocytogenes , Salmonella enteritidis, Staphy­ subjected to miCrobial infection or stress, such as injury lococcus aureus, and Streptococcusfaecalis, while gallic or physiological stimuli, low molecular-weight antimi­ acid and ellagic acid are not (Chung, et al., unpublished crobial compounds are synthesized. This group of com­ results). It seems that esterification is important for the pounds are called phytoalexins. For example, phaseollin antimicrobial activity. If tannic acid is hydrolyzed, the produced by green beans (150), has been shown to be ester bond is broken to release gallic acid and/or ellagic toxic to fungi but not to bacteria (20). Other phyto­ acid (Fig. 3) and the antimicrobial activity is lost. This alexins produced by various types of beans include may be what happens in tannin-containing plants or coumestrol, kievitone, genestein, and other isoflavo­ fruits such as grapes or strawberries. Tannic acid serves noids. Garden peas produce pi so ten (20). Carrot roots as a natural antimicrobial agent to protect against micro­ produce isocoumarin, eugenin and related phenolic com­ bial infection. When the plants or fruits mature, tannic pounds which are antimicrobial (156). In demonstrating acid is hydrolyzed and releasing gallic acid, ellagic acid the antimicrobial effect of carrot phytoalexin 6-methy­ and glucose. Glucose gives the sweet taste of the fruit oxymellein, results indicated that the compound interacts and the antimicrobial activity of tannic acid is diminish­ with cell membranes and disturbs the membrane-asso­ ed. This is a good natural mechanism against microbial ciated functions in Candida albicans (2). 6-methoxymel­ contamination of foods. If we can manipulate this sys­ lein was also shown to inhibit the growth of both fungi tem properly, we may be able to extend the shelf life of and bacteria (109). Carrot seed oil contains geraniol, certain food products. For example, the hydrolysis of terpineol and other oxygenated acyclic and monocyclic tannic acid can be manipulated to match the commercial terpinoids, which inhibited the growth of Aspergillus readiness of fruit consumption, or it could be added to certain food products to extend shelf-life.

367 King-Thorn Chung and Chris A. Murdock

It is worth noting that tannins, ellagic acid, gallic fusion (BBHI). Also, lactic acid bacteria affected the acid and methyl gallate have been used as antioxidants growth of Staphylococcus aureus, Yersinia enterocoli­ (130, 171). Many beneficial effects of ellagic acid and tica, Salmonella typhimurium, Salmonella enteritidis and tannic acid have been reported (8 , 50, 93, 94, 146). Bacillus cereus in vacuum packed bologna type sausage Tannic acid is categorized as a "Generally Recognized as (134). Safe" (GRAS) food additive from the code of federal Since Listeria monocytogenes has recently become regulation (60). It is allowed to a maximum of 400 ppm a major concern to the food industry, attention has been in frozen dessert/mixed soft candies; 150 ppm in bever­ given to antagonism against this organism in foods. The ages, non-alcoholic beverages and basal gelatins, pud­ growth of L. monocytogenes was somewhat inhibited in ding and fillings; 130 ppm in hard candies; 100 ppm in medium in the presence of Streptococcus cremoris (Lac­ baked goods, baking mixes; and 10 ppm in meat prod­ tococcus lactis subsp. cremoris) or Streptococcus lactis ucts (60). The antimicrobial and antioxidant properties (Lactococcus lactis subsp. lactis) (182). Also, bacterio­ of tannic acid, the antimutagenic and anticarcinogenic cin-producing Pediococcus species were inhibitory to L. potential of tannic acid and their hydrolyzed products, monocytogenes during the manufacture of fermented will undoubtedly be well received by consumers when its semidry sausage (16). efficiency and safety are known. The potential for uti­ More work has been done on lactic acid bacteria lization of this natural product to defend against micro­ such as Lactobacillus, Lactococcus (group N Streptococ­ bial spoilage in food systems is therefore very cus), Leuconostoc and Pediococcus. This is partly be­ promising. cause lactic acid fermentation has been a good method of Additional Plant Antimicrobials: There are food preservation. We now learn that lactic acid bacte­ many other plant components which also exhibit antimi­ ria are capable of producing inhibitory substances, inclu­ crobial activity. For example, magnalol and honokiol, ding hydrogen peroxide, diacetyl, and bacteriocins (55, the components of Magnoliae Cortex, showed antibacte­ 107). One bacteriocin designated as pediocin AcH was rial activities against Gram-positive bacteria including isolated (21) from Pediococcus acidilactici strain H. It Staphylococcus au reus and Lactobacillus spp. (129). is a small peptide with a molecular weight of 2,700. Sorbic acid from the berries of the mountain ash tree Pediocin AcH inhibited the growth of several food-spoil­ (Rowanberry) are antimicrobial (57). Sorbic acid and its age bacteria including Aeromonas hydrophila, Bacillus potassium, calcium or sodium salts are collectively cereus, Brothothrix thermospacta, Lactobacillus known as sorbates. Sorbates have been used in foods as leichmanni, L. plantarum, L. viridescens, Leuconostoc effective inhibitors of fungi, including those that pro­ mesenteroides and Pseudomonas putida as well as the duce mycotoxins, and certain bacteria (13 , 28, 151 , 164, food-borne pathogens, Staphylococcus aureus , Clostri­ 165). Extracts from the Chinese Nutgall showed anti­ dium perfringens and Listeria monocytogenes. The pos­ microbial activity (184). Chinese medicinal plants such sible mode of action of pediocin AcH was associated as Tin Men Chu , Siu Mao Heung, and Sey Lau Pai in­ with non-specific binding to the non-specific receptor, hibit the growth of many food-borne bacteria (48). The lipoteichoic acid and attachment to specific receptors. chemical compositions of these extracts are not totally This causes loss of membrane integrity and resulting loss understood. Medicinal plants which have been used as of cytoplasmic materials and dividing capability (22). folklore medicine include licorice, ginger, paeony root, Another bacteriocin, nisin, produced by Lactococcus gassia and many others, all of which may contain anti­ lactis has been well characterized and is approved for microbial components (90). More thorough survey and some uses as a food preservative (68, 97). The primary additional studies will certainly be necessary to disclose target of nisin was reported to be the cytoplasmic mem­ basic components of such antimicrobial agents and their brane (152, 154). A number of other bacteriocins such modes of action against microorganisms involved in as Lactacin B is produced by Lactobacillus acidophilus food-borne diseases. ( 10); Lactocin 27 by Lactobacillus helviticen LP 27 Microbial Antagonism in Foods ( 177) and Pediocin P A -1 by Pediococcus acidilactici PAC 1.0 (79, 143) have also been reported. Studies Foods are generally good microbial environments. along this line seem to be promising since no ill effects Usually more than one type of microorganism present in on human health by foods properly fermented with lactic a particular food. Microbial antagonism has been docu­ acid bacteria has generally been reported. mented in various kinds of foods, especially fermented We have also been interested in studying the in­ products (96). For example, the growth of salmonellae teraction of L. monocytogenes with other bacteria, parti­ and staphylococci were retarded in foods when cultured cularly in milk. We isolated a Gram-positive coccus with lactic streptococci (78). The shelf-life of both from Bulgarian style cultured milk and found that growth refrigerated ground and mechanically deboned poultry of L. monocytogenes was significantly inhibited in the meat was extended by 2 days using the resting cells of presence of this unidentified culture (Figure 4). Data starter cultures, Pediococcus cerevisiae (Accel) and show that this inhibition is not due to pH exclusively, Lactobacillus plantarum (Lactacel DS) (144). Both of nor due to the nutrient depletion caused by this organ­ these organisms inhibited the growth of Psudomonasflu­ ism. It is probably due to some factor or factors pro­ orescens and P. putrefaciens in buffered brain heart in- duced by this organism ( 49).

368 Preventing Contamination and Growth of Microorganisms in Foods

- 1010 then will we be able to make full and proper use of these ::l I compounds. An example is tannic acid. If we can add u.. j I I _... (.) "" tannic acid in proper amounts to food products, we may - 8 be able to extend the shelf-life of many products. When t: 1 0 I z I a product is ready for consumption, a hydrolytic enzyme ::l /)' ~ I I (tannase) can be added to release the ellagic acid and 1 0 v I lactic acid bacteria and a number of bacteriocins are z ~ 0 I ~ I I known, further research is needed to develop molecular _J "' r's- I biological techniques to produce large amount of these 0 I ~ I (.) 100 bacteriocins on an industrial scale. A better understand­ 0 3 6 9 12 15 18 21 ing of this kind of microbial interaction in food systems DAYS may lead to better methods of food preservation. Devel­ opment of natural means of protection will have the ad­ vantages of better product quality, greater economy and -&-LISTERIA ALONE IN MILK will build public confidence. The natural mechanisms discussed in this article, especially the use of the anti­ -e-LISTERIA IN MIXED CULTURE IN MILK microbials occurring naturally in foods and safe micro­ bial antagonisms, we predict, will receive more attention -+-ISOLATE ALONE IN MILK in future food safety research. References Figure 4. Co-Culture of Listeria monocytogenes with an unknown isolate in milk. The regular milk was pasteur­ 1. Abrahamson K, Riemann H. (1971). ized by incubation in a 65 oc water bath for 45 minutes. Prevalence of Clostridium botulinum in semi-preserved Seventy-five ml of each of these milk were transferred meat products. Appl. Microbiol. 2.1., 543-544. into 250 ml sterile Erlenmeyer flask and inoculated with 2. Amin M, Kurosaki F, Nishi A. (1988). Carrot L. monocytogenes alone, the unknown isolate alone, and phytoalexin alters the membrane permeability of also L. monocytogenes and unknown isolate together at Candida albicans and multilamellar liposomes. J. Gen. the final concentration of about 103/ml each. The inocu­ Microbiol. 134, 241-246. lated milk were then incubated at 15 °C. Colony 3. Anderson ME, Huff HE, Marshall RT, Stringer forming units (CPU) were done every 3 to 4 days. CPU WC, Naumann HD. (1983). Automated beef carcass of L. monocytogenes were performed on selective cleaning systems. U.S . Department of Agriculture Listeria media (to one liter of water add phenylethyl­ Utilization Research Report No. 7 , 28p. alcohol agar, 35.5 g; glycine anhydride 10 g; lithium 4. Anonymous. (1968) . Food-borne outbreaks, chloride 5 g; and moxalactam 200 mg). CFU of the un­ annual summary. 1968. U.S. Department of Health, known isolate were performed on trypticase soy agar. Education and Welfare. National Communicable Disease Center, Atlanta, GA. Microbial antagonism is present in many food sys­ 5. Anonymous. (1986). Salmonellosis outbreaks, tems. If we can effectively use this phenomenon to Hillfarm Dairy, Melrose Park, Illinois, Final Task Force counteract spoilage organisms, in fact this would become Report, Sept. 13, 1985. a welcome natural mechanism against microbial contam­ 6. Archer DL, Kvenberg JE. (1985). Incidences ination of foods. More research work is obviously and cost of food-borne diarrheal disease in the United needed in this area. States. J. Food Prot. 48, 887-894. Conclusion 7. Ashton DH, Busta FF. (1968). Relief of casein inhibition of Bacillus stearothermophilus by iron, As reviewed in this article, there are many possi­ calcium, and magnesium. Appl. Microbiol. ~. 628-635. ble mechanisms in food plants and animals which can be 8. Athar M, Khan WA, Mukhtar H. (1989). Ef­ utilized to prevent contamination by and proliferation of fect of dietary tannic acid on epidermal, lung, and fore­ microorganisms. We refer to these mechanisms as in­ stomach polycyclic aromatic hydrocarbon metabolism trinsic factors. These factors may be much less expen­ and tumorigenesis in Sen car mice. Cancer Res. 49, sive to use than extrinsic factors to control the contam­ 5784-5788. ination and growth of microorganisms in foods. Parti­ 9. Baman SI. (1980). Aeromonas hydrophila as cularly attractive is the more effective use of antimicro­ the etiological agent in severe gastroenteritis: report of bials occurring naturally in foods. We need to better a case. Am. J. Med. Techno!. 46, 179-181 understand the basic antimicrobial mechanisms of the 10. Barefoot SF, Klaenhammer TR. (1984) components and how they work in food systems. Only Purification and characterization of the Lactobacillus

369 King-Thorn Chung and Chris A. Murdock acidophilus bacteriocin Lactacin B. Antimicrobial Agents Effect of potassium sorbate on some organisms associated Chemotherapy 26, 328-334. with cottage cheese spoilage. J . Milk Food Techno!. 25 , 11 . Batt C , Solberg M , Ceponis M. (1983) . Effect 318-323. of volatile components of carrot seed oil on growth and 29. Bradshaw JG , Peeler JT, Corwin JJ , Hunt JM, aflatoxin production by Aspergillus parasiticus. J. Food Tierney JT, Larkin EP, Wedt RM. (1985). Thermal Sci. 48 , 762-764, 768. resistance of Listeria monocytogenes in milk. J . Food Prot. 12. Belitz HD , Growsch W . ( 1987). Food 48 , 743-745 . Chemistry. Spring-Verlag, New York, pp. 3-5. 30. Branen A , Davidson PM , Salminen S. (1990). 13 . Bell TA , Etchells JL, Borg AF. (1959) . Influ­ Food Additives. Marcel Dekker, New York. ence of sorbic acid on the growth of certain species of 31. Bryan FL. (1979). Infections and intoxications bacteria, yeasts and filamentous fungi. J. Bacterial. 77 , caused by other bacteria. In: Foodborne Infections and 573-580. Intoxications , 2nd Ed ., Riemann H , Bryan FL (eds.) , 14. Berg MJ , Howell MA , Silverman SJ. (1973). Academic Press , New York, pp . 211-297. Dietary hypothesis and diet-related research in the etiology 32. Bryan FL. (1981) . Current trends in foodborne of colon cancer. Health Serv. Rep. 88 , 915-924. salmonellosis in the United States and Canada. J . Food 15 . Berg JW , Howell MA. (1974). The geographic Prot. 44, 394-401. pathology of bowel cancer. Cancer 34, 807-814. 33 . Buchanan RL, Tice G , Marino D. (1981 ). Caf­ 16. Berry ED, Liewen MB , Mandigo RW , Hutkins feine inhibition of ochratoxin A production. J . Food Sci. RW .(1990). Inhibition of Listeria monocytogenes by bac­ 47 , 319-321. teriocin-producing Pedio coccus during the manufacture of 34. Buchanan RL, Harry MA, Gealt MA . (1983). fermented semidry sausage. J. Food Protect. 53 , 194-197. Caffeine inhibition of sterigmatocystin, citrinin and patulin 17 . Beuchat LR , Lechowich RV , Schanderl SH, Co production. J. Food Sci . 48 , 1226-1228. DYC , McFeeters RF. (1966). Inhibition of bacterial 35. Buchanan RL, Hoover DG , Jones SB . (1983). growth by chlorophyllide a. Quart. Bull., Mich . State Caffeine inhibition of aflatoxin production: mode of action. Univ. Agri . Exp . Sta. 48, 411 -416. Appl. Environ. Microbial. 46, 1193- 1200. 18. Beuchat LR , Heaton EK. (1975). Salmonella 36. Buchanan RL, Lewis DF. (1984). Caffeine survival on pecans as influenced by processing and storage inhibition of aflatoxin in synthesis: probable site of action. conditions. Appl. Microbial. 29 , 795-801. Appl. Environ. Microbial. 47 , 1216- 1220. 19. Beuchat LR. (1980). Comparison of anti-Vibrio 37 . Burke JA . (1985). Chemical contaminants in activities of potassium sorbate, potassium benzoate, and food s: some analytical considerations. J . Assoc. Off. Anal. glycerol and sucrose esters of fatty acids. Appl. Environ. Chern. 68, 1069-1073 . Microbial. 39 , 1178-1182. 38. Carpenter JA, Wang Y-P , Power JJ . (1967). 20. Beuchat LR , Golden DA. (1989). Antimicro­ Effect of anthocyanin pigments on certain enzymes. Proc. bials occurring naturally in foods . Food Techno!. 1.1.Ul, Soc. Exp . Bioi. Med. 124, 702-706. 134-142. 39. Carter AO , Borczyk AA , Carlson JA , Harvey 2 1. Bhunia, AK , Johnson MC , Ray B. (1988). Puri­ B, Hockin JC , Karmali MA, Krishnan C , Korn DA , Lior fication and antimicrobial spectrum of a bacteriocin H. (1987). A severe outbreaks of Escherichia coli produced by Pedio coccus acidilactici. J . Appl. Bacterial. 0157:H7-associated hemorrhagic colitis in a nursing home. 65 , 261 -268 . New Eng . J . Med. 317, 1496-1500. 22 . Bhunia, AK , Johnson MC , Ray B, 40. Centers for Disease Control , Atlanta, Georgia. Kalchayanand N.(1991 ) . Mode of action of pediocin AcH (1985) . Listeria outbreak associated with Mexican-style from Pedio coccus acidilactici H on selective bacterial cheese-California. Morbidity Mortality Weekly Rept. 34, strains. J. Appl. Bacterial. 70, 25-33 . 357-359. 23. Biehl ML, Buck WB . (1987) . Chemical 41 . Centers for Disease Control. (1986a). Toxigenic contaminants: their metabolism and their residues . J. Food Vibrio cholerae 01 infection - Louisiana and Florida. Prot. 50, 1058-1073. Morbidity Mortality Weekly Rept. 35 , 606-607. 24. Bjorek L , Rosen CG , Marshall V , Reiter B. 42 . Centers for Disease Control. (1986b). Cholera (1975). Antimicrobial activity of the lactoperoxidase in Louisiana - update. Morbidity Mortality Weekly Rept. system in milk against Pseudomonads and other Gram­ 35 , 687-688. negative bacteria. Appl. Microbial. 30, 199-204. 43. Cerniglia CE, Freeman JP, Franklin W , Pack 25 . Blake P, Weaver RE, Hollis DG. (1980) . LD . (1982). Metabolism of azo dyes derived from ben­ Diseases of humans (other than cholera) caused by Vibrios . zidine, 3 ,3 '-dimethylbenzidine and 3 ,3 ' -dimethoxyben­ Ann. Rev. Microbial. 34, 341-367. zidine to potentially carcinogenic aromatic amines by 26. Board RG. (1969). The microbiology of the intestinal bacteria. Carcinogenesis 3_ , 1255-1260. hen' s egg. In: Advances in Applied Microbiology, Vol. 11 , 44. Chung K-T, Fulk GE, Egan M . (1978) . Reduc­ Periman D (ed.), Academic Press, New York, pp . 245- tion of azo dyes by intestinal anaerobes. Appl. Environ. 281. Microbial. 35, 558-562. 27. Brackett RE. (1988). Presence and persistence 45 . Chung K-T, Fulk GE, Andrews AW . (1981) . of Listeria monocytogenes in food and water. Food Mutagenicity testing of some commonly used dyes. Appl. Techno!. 42(4) , 162-164. Environ. Microbial. 42 , 641-648. 28. Bradley RL, Harmon LG, Stine CM. (1962). 46. Chung K-T. (1983). The significance of azo-

370 Preventing Contamination and Growth of Microorganisms in Foods reduction in the mutagenesis and carcinogenesis of azo Techno!. 42(4), 199-200. dyes . Mutat. Res. 114, 269-281. 66. Doyle MP, Cliver DO. (1990) . Salmonella. In: 47. Chung K-T, Sun HL. (1986). Distribution and Foodborne Diseases, Cliver DO (ed .), . Academic Press, characteristics of Bacillus cereus from rice in Taiwan. J. New York, pp. 185-204. Food Sci. ll, 1208-1212. 67. Eyles MJ, Davey GR, Arnold G, Wane HM . 48. Chung K-T, Thomasson WR, Wu-Yuan CD. (1985) . Evaluation of methods for enumeration and (1990). Growth inhibition of selected food-borne bacteria, identification of Vibrio parahaemolyticus in oysters. Food particularly Listeria monocytogenes, by plant extracts. J. Techno!. 37(7), 302-304. Appl. Bacterial. 69, 498-503. 68. Federal Register. (1988). Nisin preparation: 49. Chung K-T, Murdock C , Layne J. (1991) . Inter­ affirmation of GRAS status as a direct human food action of Listeria monocytogenes with other bacteria in ingredient. Code of Federal Regulations, Title 21 . part milk. Abstracts of 91st Annual Meeting of the Amer. Soc. 184. 53: 11247-11251. for Microbia. , Washington DC, p. 39 (abstract, copy 69. 1983 Feed Additive Compendium. vol. 21 . available from K-T Chung). Published by The Miller Publishing Company , 50. Cliffton EE. (1967) . Effects of ellagic acid on Minneapolis, Minnesota, pp. 162-312. coagulation mechanisms and of traumatic bleeding. Am. J . 70. Fleming HP, Walter WM Jr. , Etchells JL. Med . Sci. 254, 483-490. (1973) . Antimicrobial properties of oleuropein and 51. Combes RD, Haveland-Smith RB . (1982). Are­ products of its hydrolysis from green olives. Appl. view of the genotoxicity of food , drug and cosemetic Microbial. 26, 777-782. colours and other azo , triphenylmethane and xanthine dyes. 71. Flowers RS . (1988). Salmonella. Food Techno!. Mutat. Res . 98, 101-248. 42(4), 182-185. 52. Conley AJ , Kabara JJ . (1973) . Antimicrobial 72.Foster EM . (1986). New bacteria in the news. A action of esters ofpolyhydric alcohols . Antimicrob. Agents special symposium. Campylobacter jejuni. Food Techno!. Chemother. 1_, 501-506. 40(8), 20. 53. Conner DE, Beuchat LR. (1984) . Effects of 73 . Frank JF. (1988) . Enteropathogenic Escherichia essential oils from plants on growth of food spoilage coli. Food Techno!. 42(4) , 192-193. yeasts . J. Food Sc. 49 , 429-434. 74. Freese E , Sheu CW, Galliers E. (1973). 54. Crouse JD , Anderson ME, Naumann HD. Function of lipophilic acids as antimicrobial food (1988). Microbial decontamination and weight of carcass additives. Nature 241, 321-325. beef as affected by automated washing pressure and length 75 . Freese E . (1978) . Mechanisms of growth oftime of spray . J. Food Prot. 21,471 -474. inhibition by lipophilic acids. In: The Pharmacological 55 . Daeschel MA . (1989) . Antimicrobial substances Effect of Lipids, Kabara JJ (ed .), Am. Oil Chern. Soc. , from lactic acid bacteria for use as food preservatives. Champaign, Ill , p . 123. Food Techno!. 1lal, 164-166. 76. Galbraith H, Miller TB. (1973) . Effect of long­ 56. Davidson PM , Branen AL. (1981) . Antimicro­ chain fatty acids on bacterial respiration and amino acid bial activity of non-halogenated phenolic compounds. J. uptake. J. Appl. Bacterial. 36, 659-675 . Food Protect. 44, 623-632, 647. 77 . Genigeorgis C. (1986) . Problems associated 57. Davidson PM , Juneja VK. (1990). Antimicrobi­ with perishable processed meats. Food Techno!. 40(4), al agents. In : Food Additives , Branen AL, Davidson PM , 140-154. Salminen S (eds .), Marcel Dekker, New York, pp. 83-137. 78. Gilliland S E , Speck ML. (1972) . Interactions 58. DeMan JM . (1980) . Lipids. In : Principles of of starter cultures and food borne pathogens: lactic Food Chemistry , A VI Pub . Co. Inc., Westport, Conn., p. streptococci versus staphylococci and salmonellae. J. Milk 35-85 . Food Techno!. 35, 307-310. 59. DeWit JC , Notermans S , Gorin N, 79 . Gonzalez CF, Kunka BS . (1987) Plasmid­ Kampelmacher EH. (1979) . Effect of garlic oil or onion oil associated bacteriocin production and sucrose fermentation on toxin production by Clostridium botulinum in meat in Pedioco ccus acidilactici. Appl. Environ. Microbial. 53, slurry . J. Food Protect. 42 , 222-224. 2534-2538. 60. Direct Food Substances Affirmed as Generally 80. Gray ML. (1960) . A possible link in the Recognized as Safe, Code of Federal Regulations, Title 21 relationship between silage feeding and listeriosis. J. Am. (1989 ed .), pt. 84, p. 1097. Vet. Assn. 136, 205-208. 61. Doyle MP, Shoeni JL. (1986) . Isolation of 81. Greenway DLA , Dyke KGH. (1979). Mecha­ Campylobacter jejuni from retail mushrooms. Appl. nism of the inhibitory action of linoleic acid on the growth Environ . Microbial. ll, 449-450. of Staphylococcus aureus. J. Gen. Microbial. 115 , 233- 62 . Doyle MP. (1988) . Effect of environmental and 245 . processing conditions on Listeria monocytogenes. Food 82 . Hackney CR, Kleeman E , Ray B, Speck M . Techno!. 42(4) , 169-171. (1981) . Adherence as a method for differentiating virulent 63. Doyle MP. (1988) . Campylobacter jejuni. Food and avirulent strains of Vibrio parahaemolyticus. Appl. Techno!. 42(4), 187-188. Environ. Microbial. 40, 652-658. 64. Doyle MP. (1988) . Yersinia enterocoliticca. 83 . Hackney CR, Dicharry AA. (1988) . Seafood­ Food Techno!. 42(4), 188-189. borne bacterial pathogens of marine origin. Food Techno I. 65 . Doyle MP. (1988) . Bacillus cereus. Food 42( 4), 104-109.

371 King-Thorn Chung and Chris A. Murdock

84. Hanna MO, Stewart JC , Zink DL, Carpenter mechanism of the antimicrobial action of oleuropein. J . ZL, Vanderzant C . (1977). Development of Yersinia Appl. Bacterial. 35 , 559-567. enterocoliticca on raw and cooked beef and pork at 102. Kabara 11 , Swieczkowski OM, Conley AJ, different temperatures. J. Food Sci. 42, 1180-1184. Truant JP. (1972). Fatty acids and derivatives as anti­ 85. Hanszel W , Berg JW, Seg i M . ( 1973). Large­ microbial agents . Antimicrob. Agents Chemother. 2., 23- bowel cancer in Hawaiian Japanese. J. Natl. Cancer Inst. 28. iL 1765-1779. 103 . KabaraJJ . (1983) . Medium-chain fatty acids 86. Hartman GD. (1959). The effect of anthocyanin and esters. In: Antimicrobials in Foods, Branen AL, pigment, pelargonidin-3-monoglucoside, and its heat Davidson PM (eds.), Marcel Dekker, New York, p. 109. degradation products on growth of selected bacteria. M .S. 104. Kato N, Shibasaki I. (1975) . Comparison of Thesis, University of Georgia, Athens. antimicrobial activities of fatty acids and their esters. J. 87. Hartman C P , Andrews AW, Chung K-T . Ferment. Techno I. 53 , 793-801 (Kakko Kogaku Zasshi, ( 1979). Production of mutagen from ponceau 3R by a Japan) . human intestinal anaerobe. Infect. Immun. , 23 , 686-689. 105. KatoN. (1981). Antimicrobial activity of fatty 88 . Hauschild AHW , Bryan FL.(1980). Estimate of acids and their esters against film-forming yeast in soy cases of food and waterborne illness in Canada and the sauce. J. Food Safety .3. , 121-126. United States. J. Food Prot. 43 , 435-440, 446 . . 106. Kautter DA , Lilly T Jr. , Solomon HM , Lynt 89. Hauschild AHW, Hilsheimer R . (1980) . Inci­ RK . (1982) . Clostridium botulinum spores in infant foods : dence of Clostridium botulinum in commercial bacon. J . a survey. J . Food Prot. 45, 1028-1029. Food Prot. 43 , 564-565 . 107. Klaenhammer TR. (1988). Bacteriocins of 90. Hikino H . (1985). Recent research on oriental lactic acid bacteria. Biochimie 70, 337-349. medicinal plants. In: Economic and Medicinal Plant 108. Konowalchuk J , Spiers 11. (1976). Antiviral Research, Vol. I, Wagner H , Hikino H , Farnsworth NR , activity of fruit extracts. 1. Food Sci. !1_,1013-1017. (eds.), Academic Press , pp. 53-86. 109. Kurosaki F , Nishi A. (1983) . Isolation and an­ 91. Holmberg SD, Wells JG, Cohen ML.(1984) . timicrobial activity of the phytoalexins 6-methoxymellein Animal-ta-man transmission of antimicrobial-resistant from cultured carrot cells. Phytochemistry 22, 669-672. Salmonella : investigations of U.S. outbreaks , 1971-1983. 110. Labbe RG . (1988). Clostridium p erfringens. Science 225 , 833-835. Food Technol. 42(4), 195-196. 92 . Hsieh DPH. (1989). Carcinogenic potential of 111. Langseth L. (1990) . Food safety issues in the mycotoxins in foods . In: Food Toxicology: A Perspective 1990s. Food Safety Notebook l, 31 -34. on the Relative Risks, Taylor SL, Scanlan RA (eds.), 112. Leistner L , Hechelmann H , Albeit R. (1975). Marcel Dekker, New York, pp . 11 -30. Nachweis von Yersinia enterocolitica in Faeces und Fleisch 93 . Huang MT, Wood AW , Newmark JM , Sayer von Schweinen, Rindern und Geflugel (Identification of JM , Yagi H , Jerina OM , Canney AH.(1983) . Inhibition of Yersinia entero co litica in feces and meat of swine, bovine the mutagenicity of bay-region diol epoxide of polycyclic and fowl). Fleischwirtschaft. 55 , 1599-1602. aromatic hydrocarbons by phenolic plant flavonoids . 113 . Lenovich LM . (1981 ). Effect of caffeine on Carcinogenesis (London) 1, 1631 -1637. aflatoxin production on caeca beans. J . Food Sci . 46, 655- 94. Huang MT, Chang RL, Wood A W , Newmark 656. HL, Sayer JM , Yagi H, Jerina OM , Canney AH. (1985). 114. Mantis AI , Karaioanoglou PRG , Spanos PG , Inhibition of the mutagenicity of beta-region diol epoxide Panetos AG. (1978). The effect of garlic extract on food of polycyclic aromatic hydrocarbons by tannic acid poisoning bacteria in culture media. 1. Staphylo coccus hydroxylated anthraquinones and hydroxylated cinnamic aureus. Lebensm, Wiss . Technol. 11, 26-28. acid derivatives. Carcinogenesis (London), Q. , 237-242. 115. Marshall DL, Bullernan LB . (1986) . Anti­ 95 . Hughey VL, Johnson EA. (1987). Antimicrobial microbial activity of sucrose fatty acid ester emulsifiers. J . activity of lysozyme against bacteria involved in food Food Sci. ll, 468-470. spoilage and food-borne disease. Appl. Environ. 116. Marth EH . (1988) . Disease characteristics of Microbial. 53 , 2165-2170. Listeria monocyrogenes. Food Technol. 42(4), 165-168. 96. Hurst A. (1973). Microbial antagonism in 117. Marwan AG , Nagel CW. (1986) Characteriza­ foods. Can. Inst. Food Sci. Technol. J. Q. , 80-90. tion of cranberry benzoate and their antimicrobial 97 . Hurst A. (1981) . Nisin. Advances in properties. 1. Food Sci . ll, 1069-1070. Microbiology 27 , 85-123. 118. Marwan AG , Nagel CW. (1986). Microbial 98 . Jay JM. (1986) . Modern Food Microbiology . inhibitors in cranberries. J. Food Sci. ll, 1009-1013. 3rd Ed ., Van Nostrand Company , New York, Chapters 2- 119. Marvick C. (1990). Disease pattern changes 3 , pp . 13-60. with food system. J. Amer. Med. Assoc. 264, 2858-2859. 99. Johnson KM. (1984). Bacillus cereus foodborne 120. McLaughlin J . (1987). Listeria monocytogenes: illness-update. 1. Food Protect. 47 , 145-153. recent advances in the taxonomy and epidemiology of 100. Johnson MG, Vaughh RH. (1969) . Death of listeriosis in humans. J. Appl. Bacterial. 63 , 1-11. Salmonella typhimurium and Escherichia coli in the 121. Melling J , Capel BJ , Turnbull PCB, Gilbert presence of freshly reconstituted dehydrated garlic acid RJ . (1976) . Identification of a novel enterotoxigenic activ­ onion. Appl. Microbial. li, 903-905 . ity associated with Bacillus cereus. 1. Clin. Pathol. 29 , 101. Juven B, Henis Y , Jacoby B. (1972) . The 938-940.

372 Preventing Contamination and Growth of Microorganisms in Foods

122. Meyer U. (1981). Biodegradation of synthetic MK (1960) . Anthocyanins. II. Action of anthocyanin organic colorants. In: Microbial Degradation of Xeno­ pigments and related compounds on the growth of certain biotics and Recalcitrant Compounds, Leisinger T, Cook microorganisms. Food Technol. 14, 626-632. AM, Hutter R, Nuesch J (eds.), FEMS Symp. 12, Academ­ 141. Pratt DE, Powers JJ, Somaatmadja D. (1960). ic Press, 371-383. Anthocyanins. I. The influence of strawberry and grape 123. Miller ML, Koburger JA. (1985). Plesiomonas anthocyanins on the growth of certain bacteria. Food Res. shigelloides: an opportunistic food and waterborne £lli.l, 26-32. pathogen. J. Food Prot. 48, 449-457. 142. Prival JJ, Davis VM, Peiperl MD, Bell SJ. 124. Moore GS , Atkins RD. (1977). The fungicidal ( 1988). Evaluation of azo dyes for mutagenicity and and fungistatic effects of an aqueous garlic extraction on inhibition of mutagenicity by methods using Salmonella medically important yeast-like fungi. Mycologia 69, 341- typhimurium. Mutat. Res. 266, 247-259. 348. 143. Puce MJ, Vedamuthu ER, Kunda BS, 125. Morris J, Black R. (1985) . Cholera and other Vandenbergh PA. (1988) . Inhibition of Listeria vibrioses in the United States. New Eng. J . Med. 312, monocytogenes by using bacteriocin PA-l produced by 343-350. Pediococcus acidilactici PAC 1.0. Appl. Environ. 126. Morris GK. (1986). Salmonella. In: Progress Microbial. 54, 2349-2353. in Food Safety, Cliver 0 , Cochrane BA (eds.), Food Res. 144. Raccach M, Baker RC, Regenstein JM, Mulnix Inst. , Univ. of Wisconsin, Madison, pp. 46-50. EJ. (1979). Potential application of microbial antagonism 127. Mortimer PR, McCann G. (1974). Food poi­ to extended storage of a flesh type food . J. Food Sci . 44, soning episodes associated with Bacillus cereus in fried 43-46. rice. Lancet l, 1043-1045. 145. Ralovich B. (1984). What is the role of food­ 128. Moustafa MK , Ahmed AA-H, Marth EH. stuffs in the transportation of Listeria. In: Microbial (1983) . Occurrence of Yersinia enterocoliticca in raw and Association and Interaction in Food , Kiss I, Deak T , Incze pasteurized milk. J . Food Prot. 46, 276-278. K (eds.) , Akademiai Kiado, Budapest (Proceedings of the 129. Namba T , Hattori M , Tsunezuka M , 12th International IUMS-ICFMH Symposium, Budapest, Yamagishi T, Konishi K. (1982) . Studies on dental caries Hungary, July 12-15, 1983), D . Reidel Publishing prevention by traditional Chinese medicine. part III. In Company, Boston, pp . 99-105. vitro susceptibility of a variety of bacteria to magnolol and 146. Ratnoff OD, Crum JD. (1964). Activation of honokiol, the component of Magnoliae cortex. Hageman factor by solutions of ellagic acid. J. Lab. Clin. Shoyakugaku Zasshi 36, 222-227. Med . 63, 359-377. 130. Namiki M. (1990) . Antioxidants/ antimutgens 147. Reiter B . (1978) . Review of the progress of in food . Critic Reviews in Food Science and Nutrition 29 , dairy science: antimicrobial systems in milk. J . Dairy Res. 273-300. 45, 131-147. 131. Newsome RL. (1988) . Staphylococcus aureus . 148. Reiter B, Harnulv G. (1984) . Lactoperoxidase Food Technol. 42(4) , 194-195. antimicrobial system: natural occurrence, biological 132. Nieman C. (1954). Influence of trace amounts functions , and practical applications. J. Food Prot. 47 , of fatty acids on the growth of microorganisms . Bact. Rev . 724-732. l.R, 147-163. 149. Remis RS , MacDonald KL, Riley LW , Puhr 133. NFPA/CMI. (1984). Botulism risk from post­ ND , Wells JG , Davis BR, Blake PA, Cohen ML. (1984) . processing contamination of commercially canned foods in Sporadic cases of hemorrhagic colitis associated with metal containers. Natl. Food Processors Assoc./Container Escherichia coli 0157:H7 . Ann. Intern. Med. 101 , 623- Mfrs . Inst. J. Food Prot. 47 , 801-816. 626. 134. Nielsen H-JS , Zeuthen P. (1985) .Influence of 150. Rizk A-F, Wood GE. (1980) . Phytoalexins of lactic acid bacteria and the overall flora on development of leguminous plants. CRC Crit. Rev. Food Sci. Nutr. U, pathogenic bacteria in vacuum-packed cooked emulsion­ 245-295. style sausage. J. Food Prot. 48, 28-34. 151. Robach MC. (1980). Use of preventives to 135. Oliver J. (1985) . Vibrio: An increasingly control microorganisms in foods. Food Technol. 3400), troublesome genus . Diagn. Med . ,8. , 43-48. 81-84. 136. Oram J D , Reiter B. (1968). Inhibition of 152. Rulr E , Sahl HG. (1985) Mode of action of the bacteria by lactoferrin and other iron-chelating agents. peptide antibiotic nisin and influence on the membrane Biochem. Biophys. Acta. 170, 351-365. potential of whole cells and influence on the cytoplasmic 137. Ormay L, Novotry T. (1968). The significance and artificial membrane vesicles. Antimicrobial Agents of Bacillus cereus food poisoning in Hungary. Proceedings Chemotherapy 27 , 841-845. of the Sixth International Symposium on Food 153. Saha JG. (1969). Significance of organochlo­ Microbiology , Haalem, Netherland, p . 279. rine insecticide residues in fresh plants as possible 138. Pierson MD, Reddy NR.(l988). Clostridium contaminant of milk and milk products. Res. Rev. 26, 89- botulinum. Food Technol. 42(4), 196-198. 126. 139. Pohland AE, Wood GE. (1987). Occurrence of 154. Sahl HG, BrandisH. (1983). Efflux of low-Mr mycotoxins in food. In: Mycotoxins in Food, Krogh P substances from the cytoplasm of sensitive cells caused by (ed.), Academic Press, pp . 35-64. the Staphylococcin-like agent Pep-5. FEMS 140. Powers JJ, Somaatmadja D , Pratt DE, Hamdy Microbiological Letters .lQ, 75-79.

373 King-Thorn Chung and Chris A . Murdock

155. Saleem ZM , Al -Delaimy KS. (1982). Inhibi­ Methods for the isolation and enumeration of tion of Bacillus cereus by garlic extracts. J . Food Prot. 1_ , Staphylococcus aureus . In: Compendium of methods for 1007-1009. the microbiological examination of foods , 2nd ed. , Speck 156. Sarkar SK, Phan CT. (1979) . Naturally-occur­ ML (ed .) , Am. Soc. Microbial. Washington, DC , pp . 411- ring and ethylene-induced phenolic compounds in the 427 . carrot root. J. Food Prot. 42, 526-534. 174. Todd ECD. (1983) . Food-borne and water­ 157. Schlech WF, Lavigne PM, Bortolussi RA , borne disease in Canada 1977 Annual summary. 1. Food. Allen AC, Haldane EV , Wort AJ , Martin RS , Hightower Prot. 45 , 865-873. AW, Johnson SE, King SE, Nicholls ES , Broome CV. 175. Tortora P , Burlini N, Hanozet M, Guerritore (1983) . Epidemic listeriosis:evidence for transmission by A. (1982) . Effect of caffeine on glucose-induced food. New Eng. J . Med. 308, 203-206. inactivation of gluconeogenetic enzymes in Saccharomyces 158. Schlech W F. (1988). Virulence characteristics cerevisiae . A possible role of cyclic AMP. Eu. J . of Listeria monocyrogenes. Food Techno!. 42(4), 176-178. Biochem. 126, 617-622. 159. Seeliger HPR, Finger H. (1976). Listeriosis. 176. Tynecka Z , Gos Z. (1973). The inhibitory In: Infectious Diseases of the Fetus and Newborn Infant, action of garlic (Allium sativum L.) on growth and Remington JS , Klein JO (ed .) , W .B. Saunders Co., respiration of some microorganisms . Acta Microbial. Pol. Phildelphia, pp. 333-365 . Ser. , B. iW., 51 -62 . 160. Sharp JCM . (1987). Infections associated with 177. Upreti GC , Hinsdill RD . (1975). Production milk and dairy products in Europe and North America and mode of action of lactacin 27: Bacteriocin from a 1980-1985. Bull WHO , 65 , 397-406. homofermentative Lactobacillus. Antimicrobial Agents 161. Sheu CW , Freese E. (1973) . Effects of fatty Chemotherapy I , 139-145. acids on growth and envelope proteins of Ba cillus subtilis . 178. Vakil JR , Chandan RC, Parry RM , Shahani 1. Bacterial. ill, 516-524. KM . (1969). Susceptibility of several microorganisms to 162. Siragusa GR , Johnson MG.(l989). Inhibition milk lysozymes. J . Dairy Sci . 52, 1192-1197. of Listeria mono cytog enes growth by the lactoperoxidasc­ 179. Vanos V , Bindschedler 0 . (1985) . The micro­ thiocyanate-H20 2 antimicrobial system. Appl. Environ. biology of instant coffee. Food Microbial. I , 187-197. Microbial. 55 , 2802-2805 . 180. Vanos V , Hofstaetter S, Cox L. (1987) . The 163. Smith JL. (1987) . Shigella as foodborne microbiology of instant tea. Food Microbial. 1_ , 19-33. pathogen. J. Food Prot. 50, 788-801 . 181. Wachsmuth IK , Morris GK. (1989) . Shigella . 164. Sofos JN , Busta FF. (1981) . Antimicrobial In: Foodborne Bacterial Pathogens, Doyle MP (ed.) , activity of sorbates. J . Food Prot. 44, 614-622. Marcel Dekker, New York, pp . 448-461. 165 . Sofos JN , Busta FF. (1983). Sorbates. In: 182. Wenzel JM , Marth EH. (1991) . Behavior of Antimicrobials in Foods. Branen AL, Davidson PM, (eds .) Listeria monocytog enes in the presence of lactic acid Marcel Dekker, New York, p. 141. bacteria in an agitated medium with internal pH control. J . 166. Somaatmadja D , Powers 11 , Handy MK. Food Prot. 54, 183-188. (1964). Anthocyanins . VI. Chelation studies on anthocya­ 183 . Wheeler OR. (1966). The effects of malvidin- nins and other related compounds. 1. Food Sci. 29 , 644- 3-monoglucoside on purified enzymes from cell fractions 654. of Salmonella enteritidis. M .S. thesis . Univ . of Georgia, 167. Sours HE , Smith DG . (1980). Outbreaks of Athens . foodborne disease in the United States 1972- 1978. 1. Inf. 184. Wu-Yuan CD , Chen CY, Wu RT. (1988). Dis. 142, 122-125 . Gallotannins inhibit growth , water-insoluble glucan 168. Stern NJ , Henderson MP, Blankenship L, synthesis and aggregation of mutans streptococci. J . Dental Deibel KE , Doores S, Doyle MP, Ng H , Pierson MD , Res . 67 , 51 -55. Sofos JN , Sveum WH, Westhoff DC. (1985). Prevalence 185 . Wynder EL, Reddy BS . (1974) . Metabolic and distribution of Campyloba cter Jejuni and epidemiology of colorectal cancer. Cancer 34, 801-806. Campylobacter coli in retail meat. J . Food Prot. 48 , 595- 186. Yule BF, MacLeod AF, Sharp JC , Forbes GI. 599. (1988) . Costing of a hospital-based outbreaks of poultry­ 169. Stoll A, Seeback E . (1948) . Uber Allin, die borne Salmonellosis: Epidemiology and Infection. 100, 35- genuine Muttersubstanz des Knoblauchols Allium com­ 42. pound . I. (Alliin, the true mother compound of garlic oil) . 187. Zimmerman VC. (1959) . The influence of Helv. Chim. Acta 11. 189-210. anthocyanin pigments on certain bacteria in vitro . M .S . 170. Stoloff L. (1982). Mycotoxins as potential Thesis , University of Georgia, Athens. environmental carcinogens. In: Carcinogens and Mutagens in the Environment, Stitch H-F (ed.) , CRC Press, Boca Editor's Note: All of the reviewer's concerns were Raton, Florida, pp . 97-119. appropriately addressed by text changes, hence there 171 . Su 1-D , Osawa T , Kawakishi S, Namiki M. is no Discussion with Reviewers. (1987). A novel antioxidative synergist isolated from Osb erkia chinensis L. Agri . Bioi. Chern. 21. 3449-3450. 172. Tansey MR , Appleton JA.(1975) . Inhibition of fungal growth by garlic extract. Mycologia 67, 409-413. 173 . Tatini SR , Hoover DG , Lachica RVF. (1984) .

374