Microbial toxins 2331 Introduction

Section 2.3.3 deals with the way in which toxic substances produced in food and feed by microorganisms enter the pathway from raw material to consumer.

Microorganisms are ubiquitous. Any environment supporting higher organisms contains microorganisms too, while the converse is not true. Absence of microorganisms in an environment indicates that special or unusual conditions have occurred, such as heating and filtration for sterilization or preservation.

During food production, raw food materials of plant or animal origin are exposed to soil, water, air, machinery parts, packaging materials, human hands, etc. As these invariably carry microorganisms, all raw food materials have in principle been inoculated with a variety of microbes. The opportunity for these microorganisms to grow is determined by the food environment. Major environmental factors include availability of water (referred to as water activity or aw) and nutrients, temperature, pH, and presence or absence of atmospheric oxygen. Growth also depends very heavily on how long suitable environmental conditions prevail. The majority of naturally occurring microbial contaminators are unable to multiply, or succumb to other microbes in a food environment. However, even if an infective microorganism remains alive without multiplying, the food may serve as a vehicle to transfer it to the human body and cause illness. Microorganisms which multiply usually degrade the food components enzymatically and excrete their metabolites. In many cases, the resulting loss of structure, or formation of off-smells is regarded as spoilage. However, a wide variety of fermented foods are manufactured of which the desirable taste, flavor, and other properties are especially due to microorganisms and their metabolic activity.

Table 2.4 Food hazards: perception of the consumer versus epidemiological data

Cause Perception1 Relative importance2

Table 2.4 Food hazards: perception of the consumer versus epidemiological data

Cause Perception1 Relative importance2

Microbial contamination



Nutritional imbalance


Environmental contaminants



Natural toxins



Food additives



Others, e.g., packaging materials




1 Survey held in the Netherlands, 1990.

2 Ranking based on objective scientific criteria including the severity, incidence, and onset of biological symptoms.

1 Survey held in the Netherlands, 1990.

2 Ranking based on objective scientific criteria including the severity, incidence, and onset of biological symptoms.

Table 2.5 Food-borne bacterial pathogens and associated diseases



Incubation time (hours)

Duration of disease (days)









Escherichia coli




Yersinia enterocolitica




Campylobacter jejuni


3-5 (days)


Listeria monocytogenes




Vibrio parahemolyticus




Aeromonas hydrophila




Staphylococcus aureus

toxin in food



Clostridium botulinum

toxin in food



Clostridium perfringens

toxin in intestine



Bacillus cereusc

toxin in food



Bacillus cereusd

toxin in intestine



a Affects people with a predisposing factor; high mortality rate. b High mortality rate; complete convalescence takes 6-8 months. c Emetic type. d Diarrheal type.

a Affects people with a predisposing factor; high mortality rate. b High mortality rate; complete convalescence takes 6-8 months. c Emetic type. d Diarrheal type.

Section 2.3.3 deals with some harmful aspects of microbial food contamination, namely the production of toxic substances causing food-borne disease. Food-borne diseases Epidemiological evidence has shown that microbial contamination is a major risk factor associated with food consumption. However, the average consumer does not always realize this, and is, for example, more concerned about environmental contaminants in food. This discrepancy between the incidence of food-borne diseases and the perception of the consumer is illustrated in Table 2.4.

Food-borne diseases can be either food-borne infections or food-borne intoxications, depending on whether the pathogen itself or its toxic product (a microbial toxin or toxic metabolite, produced in the food) is the causal agent. Table 2.5 lists the most important bacterial food-borne pathogens.

Of all reported food-borne diseases with microbiological etiology which occurred in Canada in 1984, infections with Salmonella and Campylobacter spp. constituted 67% and 8%, and intoxications originating from Clostridium perfringens, Staphylococcus aureus and Bacillus cereus 16%, 7% and 1%, respectively.

Other microbial agents causing food-borne intoxications include toxins produced by fungi (mycotoxins) and by algae, and toxic metabolites such as biogenic amines and ethyl carbamate produced by bacteria and yeasts. The various causative factors of food-borne diseases are summarized in Figure 2.3. In Section the major food-borne toxins will be discussed. Although intoxications by biogenic amines and ethyl carbamate are of microbial origin, they can also be regarded as chemical poisonings.

food-borne diseases poisonings infections chemical intoxications enterotoxigenic invasive poisonings poisonous plant tissues poisonous animal tissues microbial intoxications algal toxins mycotoxins enterotoxins bacterial toxins toxic metabolites neurotoxins interactions with carbohydrate metabolism

Figure 2.3 Classification of food-borne diseases. Bacterial toxins According to the mechanisms underlying the effects of bacterial toxins, they can be classified as follows:


- membrane-affecting toxins (e.g., Staphylococcus aureus toxins, see )

- lesion-causing toxins (e.g., Clostridium perfringens and Bacillus cereus toxins, see Subsection

These toxins will be discussed in relation to the properties of the causative bacteria, the conditions favoring toxin production, as well as the structure and stability of the toxin. Their toxicity will be discussed in detail in Part 2 of this book. Sub-unit bacterial toxins. To this group belong the toxins produced by Clostridium botulinum. C. botulinum are motile, Gram-positive rod-shaped spore-forming anaerobic bacteria. C. botulinum is not one species, but a group of bacteria which are all capable of producing neurotoxins. Biochemically, C. botulinum is very similar to Clostridium sporogenes and Cl. novii. However, the latter do not produce toxins and are therefore not relevant here. According to the toxin they produce, there are eight types of C. botulinum: A, B, C1, C2, D, E, F, and G.

Toxicity and symptoms. Botulism, caused by the ingestion of food containing the neurotoxin, is the most severe bacterial food-borne intoxication known. The type A toxin is the most lethal. Types A, B, E, and F are toxic to humans; types B, C, and D to cattle; and types C and E to birds.

After an incubation period of 12 to 72 hours, symptoms may start with nausea and vomiting, followed by tiredness, headache, muscular paralysis, double vision, and respiratory problems, often with fatal results. The duration of botulism is 1 to 10 days, mortality is relatively high (30 to 65%). In most foods, botulinum spores are of no consequence unless they are able to germinate and produce the toxin. The exception is infant foods in which botulinum spores are potentially infective and may give rise to toxicogenesis in the infant intestine. A good example of this is infant botulism caused by contaminated honey.

Recent outbreaks involved yogurt with hazelnut (UK 1989: 27 cases, 1 death; type B), fermented seal oil (Canada 1989: 4 cases, 2 deaths; type E), white fish (1989: 8 cases, 1 death; type E), traditional Eskimo fish product (1984, 1989: type E), and infant botulism (19871989: 68 cases).

Chemical properties (structure and stability) of botulinum toxin. C. botulinum produces an intracellular protoxin consisting of a non-toxic progenitor toxin (a hemagglutinin with molecular mass approximately 500,000) and a highly toxic neurotoxin (molecular mass approximately 150,000). The protoxin is released upon lysis of the vegetative bacterial cell. The neurotoxin is formed by proteolytic degradation of the protoxin. This proteolysis is caused by C. botulinum (type A, and some B and F) proteolytic enzymes, or by exogenous proteases e.g., trypsin when non-proteolytic C. botulinum (type C, D, E, and some B and F) are involved.

Botulinum toxin is heat-sensitive (inactivated at 80°C for 10 minutes or 100°C for a few minutes). It is acid-resistant and survives the gastric passage. Botulinum toxin is an exotoxin: it is excreted by the cell, but most of it is released upon lysis of the cell after sporulation.

Environmental conditions. C. botulinum grows best at pH >4.6 at temperatures of approximately 37°C (type E at 30°C). The minimum temperature for growth is 12.5°C (type E at 3.5°C).

Type of food involved; prevention. At particularly risk is food of low to neutral pH (>4.5) which has undergone inadequate heating. Examples include home-preserved vegetables which carry soil-borne C. botulinum, but also meat and fish which are contaminated during slaughtering with C. botulinum originating from the intestines. Of increasing importance are chilled vacuum-packed foods which usually have had minimal heat treatment, and contain no preservatives other than any naturally occurring antimicrobial substances, and are not reheated or only mildly heated prior to consumption.

Preventive measures include adequate heat processing to reduce the number of C. botulinum spores with a factor 1012 (the "botulinum cook" or "12-D concept", with D being the time required for a tenfold reduction in the population density at a given temperature). The heat-resistance of the spores varies: D-values of 1 minute at 80°C (type E), 100°C (type C), or 113°C (type A and D). Germination of spores surviving the heat treatment can be prevented by the addition of nitrite, lowering the pH or the aw, addition of salt, thorough heating of food prior to consumption (the toxin is heat-labile) and refrigerated storage (less adequate for type E). Membrane-affecting bacterial toxins. A well-known example of a bacterium-producing membrane-affecting toxins is Staphylococcus aureus. S. aureus are non-motile, non-sporeforming, Gram-positive bacteria which can excrete enterotoxins in food. These enterotoxins show clear antigenic activity, and based on their antigenic properties, they are differentiated in A, B, C1, C2, C3, D and E. Most enterotoxicoses are caused by toxins A, or A and D. A characteristic of S. aureus is the formation of the enzyme coagulase which can cause the clumping (coagulation) of blood serum. However, coagulase-negative sta-phylococci have also been incriminated in food-borne gastroenteritis.

Toxicity and symptoms. A quantity of 1 to 25 |g of the enterotoxin is required to cause sickness in adult humans. After a very short incubation period (V2 to 6 hours), symptoms of staphylo-enterotoxicose include violent vomiting and diarrhea, sometimes followed by shock but no fever. Serious dehydration may result from the diarrhea. The duration of the illness is 24 to 72 hours, and the mortality is very low. Everyone who consumes the poisoned food, becomes ill ("maladie du banquet": buffet disease).

Chemical properties (structure and stability) of the enterotoxin. The extracellular enterotox-ins are a heterogenous group of globular proteins consisting of linear peptide chains. The molecular mass ranges from approximately 30,000 to 235,000. The enterotoxin is heat-resistant (it withstands boiling at 100°C for more than 1 hour).

Environmental conditions. Growth is possible at temperatures between 7 and 46°C (optimum is 37°C), pH 4 to 9 (optimum is pH 7), aw >0.86, and salt (NaCl) concentrations up to 15%. S. aureus is a facultative anaerobe, but grows better under aerobic conditions. It is a poor competitor: it hardly grows in the presence of competitive microflora. About 70% of S. aureus of human origin are able to produce enterotoxins. The environmental conditions required for toxin production include: temperature >12°C, aw >0.90, pH >4.6, aerobic conditions, and little microbial competition.

Type of food involved; prevention. S. aureus is a very common microorganism. About 30 to 50% of humans carry the organism in the mucous membrane of the nose and throat, or on the skin. Animals also carry S. aureus. Particularly with this microorganism, the human factor plays a very important role in the transfer to food. For instance, sneezing behind the hand increased the S. aureus load of a test surface from about 100 to >5000 per 25 cm2. The types of food favoring enterotoxin production include dairy cream, ice cream, cured meats (ham, sausages, meat pies), and opened canned foods (in which fast growth is possible without competition). See also Section 2.4.3 for this aspect. S. aureus growth and toxin production can be prevented by proper storage (refrigerated, or too hot for growth), heating (this is not of help if the toxin has already been produced), adequate personal hygiene, cleanliness, and good disinfection practice. Lesion-causing bacterial toxins. Two examples of this type of toxin-producing bacteria will be discussed: Clostridium perfringens and Bacillus cereus.

Clostridium perfringens. Clostridium perfringens are Gram-positive, anaerobic (aerotolerant) spore-forming rod-shaped bacteria. Several serotypes are distinguished (A, B, C, D, E, F) which produce different enterotoxins. Particularly, serotype A is associated with food-borne intoxications.

Toxicity and symptoms. Although enterotoxin formation in food (i.e., meat and poultry) may occur, still the incidence of C. perfringens food poisoning due to preformed enterotoxin in the food is rare. (Therefore, in Table 2.5, C. perfringens itself is listed as the causal agent.) A large number (>108) of vegetative C. perfringens cells need to be consumed to release sufficient enterotoxin. After an incubation period of 8 to 24 hours, abdominal cramps (much gas produced) and diarrhea with nausea but rarely vomiting can last for 24 hours. A number of enterotoxins have been found to damage the intestinal wall; the glucose resorption is inhibited and the bowel movement is stimulated. The mortality of serotype A poisoning (mainly in the US) is 3 to 4%; serotype C (Europe) is rarely fatal.

Chemical properties (structure and stability) of the enterotoxin. The toxins are protein-type enterotoxins (molecular mass approximately 34,000 dalton). Release of the enterotoxins in the intestine occurs during sporulation and lysis of the C. perfringens cells. The proteinous nature of the enterotoxin makes it rather heat-sensitive.

Environmental conditions. Growth can take place at 15 to 50°C (optimum 40°C), pH 5 to 8 and aw >0.93.

Type of food involved; prevention. C. perfringens causes mostly problems in meats. In the live animal, the microorganism can penetrate into the body through the intestinal wall. The thermal resistance of the spores varies from heat-labile (D-value 0.3 minutes at 100°C) to relatively heat-resistant (D-value 17.6 minutes at 100°C). The heat resistance also depends on the composition of the food. When contaminated meat receives inadequate heating (e.g., in the center of large pieces of roasted meat) or when the cooked meat is not sufficiently cooled prior to storage, germination of surviving C. perfringens spores may occur. Prevention measures include good hygiene, adequate meat heating (>65°C at the center) followed by refrigerated storage (<7°C).

Bacillus cereus. Bacillus cereus are Gram-positive spore-forming aerobic rod-shaped bacteria. They produce enterotoxins as well as several enzymes of pathogenic relevance, including lecithinase and hemolysin. Two different enterotoxins are known: type I and type II.

Toxicity and symptoms. Type I diarrheagenic enterotoxin occurs most frequently and is mildly toxic. After an incubation period of 8 to 16 hours, 50 to 80% of the consumers develop abdominal cramps and diarrhea which may last for 24 hours. Type II emetic enterotoxin is less common. After a short incubation period of 1 to 6 hours, violent vomiting occurs. Symptoms may last for 8 to 10 hours.

Chemical properties (structure and stability) of the enterotoxin. Type I is a proteinous enterotoxin (with molecular mass approximately 50,000). It is formed in the intestine (relatively long incubation period; large number of cells «106 required). This enterotoxin is heat-sensitive and, being a protein, undergoes degradation by trypsin. Type II is a toxin with molecular mass <5000. It is formed in the food during the logarithmic phase of bacterial growth. Type II is stable at pH 10 and is heat-resistant.

Environmental conditions. Growth can take place at 10 to 50°C (optimum 37°C), pH 5 to 9.

Type of food involved; prevention. Particularly cereal products contain B. cereus spores. There is no evidence that human factors are involved in the contamination. Cooking with cereal containing dishes followed by inadequate cooling enables germination of the spores that survived the heating. Type I toxin is associated with sauces, pastries, etc.; type II toxin is associated with cooked or fried rice. The main prevention measure is adequate and immediate cooling after cooking. This should be carried out in shallow layers enabling fast heat transfer; storage should be at <10°C. Immuno-active bacterial endotoxins. Endotoxins are found in the cell wall of Gram-negative bacteria. Examples of bacteria with active endotoxins are Salmonella abortus equi and Escherichia coli. The endotoxins can be released upon lysis of the vegetative cells.

Toxicity and symptoms. Endotoxins are capable of stimulating the immune system in a non-specific way, and causing inflammations. Symptoms of intoxication include fever, shivering, painful joints, and influenza-like complaints, lasting for approximately 24 hours.

Chemical properties (structure and stability) of the endotoxin. Immuno-active endotoxins consist of lipopolysaccharides (LPS) bound covalently to protein and lipid fractions (Figure 2.4). The polysaccharide part consists of a lipid A fraction and a long polysaccharide chain. The lipid A fraction is identical in almost all bacteria. In the polysaccharide chain, a central part and an O-chain are distinguished. The central part has a similar structure in many bacteria, but the O-chain is rather characteristic.

O - antigen

O - antigen

Figure 2.4 General structure of endotoxins.

Little is known about the covalently-bound protein. It is assumed that it is bound to the lipid A and is thus referred to as lipid A associated protein (LAP) or endotoxin protein (EP). The biological activity of the endotoxin is associated with its LPS part. LAP or EP appear to play a minor role. This might explain the different activities of endotoxins of various bacteria.

Environmental conditions. Endotoxins are released at the end of the growth curve, i.e., after death of the bacteria. Favorable conditions for growth of Gram-negative bacteria include pH 4.5, aw >0.99, and temperatures ranging from 15 to 40°C.

Type of food involved; prevention. In principle, any type of food can serve as a vehicle. The release of endotoxins may take place in the intestine as a result of a food infection. Preventive measures against food infections include avoidance of cross-contamination of cooked food with raw foods, adequate heating, refrigerated storage, and adequate personal hygiene. Mycotoxins General. Mycotoxins are secondary metabolites of fungi which can induce acute as well as chronic toxic effects (i.e., carcinogenicity, mutagenicity, teratogenicity, and estrogenic effects) in animals and man. Currently, a few hundred mycotoxins are known, often produced by the genera Aspergillus, Penicillium, and Fusarium.

Toxicity and symptoms. Toxic syndromes resulting from the intake of mycotoxins by man and animals are known as mycotoxicoses. Although mycotoxicoses have been known for a long time ("Holy Fire" in the Middle Ages in Europe caused by the mold Claviceps purpurea; "Alimentary Toxic Aleukia" in the Soviet Union in 1940 caused by Fusarium spp.; "Yellowed Rice Disease" in Japan caused by Penicillium spp.), the mycotoxin-induced disorders remained the neglected diseases until the early 1960s, when the aflatoxins were discovered. This discovery was followed by much scientific research on mycotoxins.

Chemical properties (structure and stability). The chemical structures of some important mycotoxins are shown in Figure 2.5. The mycotoxins that will be discussed below are chemically stable and resistant to cooking. Several other mycotoxins have been shown to be unstable in foods. As this strongly reduces their toxicity, these will not be discussed here.

Environmental conditions. Mycotoxin contamination of food and feed highly depends on the environmental conditions that lead to mold growth and toxin production. The detectable presence of live molds in food, therefore, does not automatically indicate that mycotoxins have been formed. On the other hand, the absence of viable molds in foods does not necessarily mean there are no mycotoxins. The latter could have been formed at an earlier stage prior to food processing. Because of their chemical stability, several mycotoxins persist during food processing, while the molds are killed.

O O Aflatoxin B1


O O Aflatoxin B1


O OH Patulin

O OH Patulin


OO Aflatoxin M 1










CH3 Lysergic acid

CH3 Lysergic acid

Type of food involved; prevention. Many foodstuffs and ingredients may become contaminated with mycotoxins. The occurrence of various mycotoxins in foods and feeds has often been reported. Since the discovery of the aflatoxins, probably no commodity can be regarded as absolutely free from mycotoxins. Also, mycotoxin production can occur in the field, during harvest, processing, storage, and shipment of a given commodity. Aflatoxins. The aflatoxins are the most important mycotoxins. They are produced by the molds Aspergillus flavus and Aspergillus parasiticus.

Toxicity and symptoms. Aflatoxins are potent toxins. They are well-known for their carcinogenicity. In view of occurrence and toxicity, aflatoxin B1 is the most important of them, followed by G1 > B2 > G2. Aflatoxin B1 is a very potent hepatocarcinogen in various experimental animal species including rodents, birds, fish, and monkeys. It appears that the aflatoxins themselves are not carcinogenic but rather some of their metabolites. Primary liver cancer is one of the most prevalent human cancers in the developing countries. Epidemiological studies carried out in the 1970s provide statistical support for the association of food consumption, contamination with aflatoxins, and incidence of hepatocellular carcinoma. It is now believed that there are combined actions of aflatoxins and hepatitis B virus infection leading to primary liver cancer. Due to worldwide commercial activities, the threat of aflatoxins to human health is not limited to those countries where the mycotoxins are produced. Moreover, the international trade in animal feed ingredients has contributed to the potential hazard for public health, because milk and dairy products may become contaminated with aflatoxin M1 (see Figure 2.5), the 4-hydroxy derivative of aflatoxin B1 formed in cows after ingestion of aflatoxin B1 with their feed. Aflatoxin M1 is also a suspect carcinogen, although its carcinogenic potency is probably less than that of aflatoxin B1.

Chemical properties (structure and stability). Aflatoxins are derivatives of coumarin. (The structure of coumarin can be found in Section

The most important types of aflatoxins are B1, B2, G1, and G2 (Figure 2.6). Aflatoxins are heat-stable and are hard to transform to non-toxic products. However, two methods of detoxication should be mentioned. First, the fate of aflatoxin B1 during food fermentation has been investigated in a variety of products. It appeared that fungi involved in food fermentation, for instance Rhizopus oryzae and R. oligosporus, are capable of reducing the cyclopentanone moiety, resulting in the formation of aflatoxicol A (Figure 2.7). This reaction is reversible. Under suitable environmental conditions (e.g., presence of organic acids), aflatoxicol A is irreversibly converted to its stereoisomer aflatoxicol B. Aflatoxicol A is approximately 18 times less toxic than aflatoxin B1.

In lactic fermentations at pH <4.0, aflatoxin B1 is readily converted to aflatoxin B2a (Figure 2.7) which is also less toxic. Both transformations thus reduce the toxicity, but the detoxication is not complete unless the lactone ring of the aflatoxin molecule is opened (Figure 2.8).

This would correspond to the loss of fluorescence at 366 nm. It has been found that loss of fluorescence correlates with reduced mutagenicity. Screening fungi for their ability to reduce the fluorescence of aflatoxin B1 solutions revealed that certain Rhizopus spp. were able to transform 87% of aflatoxin B1 into non-fluorescent substances of as yet unknown nature. A similar detoxication by opening of the lactone ring is achieved by treatment with ammonia (NH4OH) at elevated temperature and pressure, which is applied at industrial scale to detoxicate animal feed ingredients, e.g., groundnut press-cake. At high pH the lactone ring of the aflatoxin molecule is hydrolyzed.

Figure 2.6 Structures of the major aflatoxins.

Aflatoxin B1


Aflatoxicol A

Aflatoxin B1

HO O O Aflatoxin B2a

HO O O Aflatoxin B2a


Aflatoxicol A

Aflatoxicol B

Figure 2.7 Detoxication of aflatoxin B1.

Aflatoxicol B

Figure 2.7 Detoxication of aflatoxin B1.

Environmental conditions. The fungi grow best at approximately 25°C at high relative air humidity (>80%). Aflatoxins are produced both pre- and post-harvest, at relatively high moisture contents and relatively high temperatures.

Type of food involved; prevention. Aflatoxins can occur on various products, such as oilseeds (groundnuts), grains (maize) and figs. Problems with aflatoxin contamination

Figure 2.8 Detoxication of aflatoxin B1 by opening the lactone ring.

occur in industrialized countries (US) as well as in the developing countries in Latin America, Asia, and Africa. Aflatoxin M1 can be detected in low concentrations in milk samples from around the world, because of the high sensitivity of the current analytical methods. Prevention of aflatoxin contamination is achieved by discouraging fungal growth. Particularly, adequate post-harvest crop-drying is essential to reduce the chance of fungal growth. Deoxynivalenol. Deoxynivalenol (DON) is a mycotoxin belonging to the group of trichothecenes (see Figure 2.5). It is produced by Fusarium graminearum.

Toxicity and symptoms. The trichothecenes, including T-2 toxin, HT-2 toxin, diacetoxyscirpenol, neosolaniol, fusarenon-X, nivalenol, and DON, induce a wide variety of toxic effects in experimental animals: diarrhea, severe hemorrhages, and immunotoxic effects. DON occurs worldwide. The toxin is of particular interest in the zootechnic sector, because feeding pigs with DON may lead to economic loss due to refusal of the feed and vomiting.

Chemical properties (structure and stability). DON is quite resistant to conventional food processing conditions.

Environmental conditions. The Fusarium producer strains prefer high relative air humidity at moderate temperatures (10 to 30°C).

Type of food involved; prevention. Fusarium species particularly occur on grains, e.g., maize, wheat and rye, in the moderate climate zones. Ergot alkaloids. Ergot alkaloids are produced by Claviceps purpurea, which grows in the ears of grasses and cereals. The fungus forms sclerotia (2 to 4 cm large ergot kernels), which are the hibernation stage. During the harvest the sclerotia may end up between the cereal grains.

Toxicity and symptoms. Ergot alkaloids act particularly on the smooth muscles. Severe poisoning leads to constriction of the peripheral arteries, followed by dry gangrene of tissue and loss of extremities. Neurological disorders (itching, severe muscle cramps, spasms and convulsions, and psychological disorders) may also occur.

Chemical properties (structure and stability). The sclerotia contain derivatives of lysergic acid (Figure 2.5), the ergot alkaloids. Figure 2.9 illustrates the basic structure of these substances, taking ergotamine as an example. Ergometrine, ergotamine, and ergocristine are among the most important.

Environmental conditions. Ergot formation is favored especially in pre-harvest rye by high relative air humidity and temperatures of 10 to 30°C.

Type of food involved; prevention. Claviceps pupurea is common in pre-harvest grains. Consequently, a strict quality control of grain before milling is required. Taking into account the present-day grain quality assurance systems and its relatively high no-effect level, ergot is not considered a serious threat to human or animal health.


Figure 2.9 Structure of ergotamine.


Figure 2.9 Structure of ergotamine. Patulin. Patulin is mainly produced by Penicillium expansum, Penicillium patulinum and Byssochlamys nivea.

Toxicity and symptoms. Patulin causes hemorrhages, formation of edema, and dilatation of the intestinal tract in experimental animals. In subchronic studies, hyperemia of the epithelium of the duodenum and kidney function impairment were observed as main effects.

Chemical properties (structure and stability). The structure of patulin is shown in Figure 2.5. It is stable under conditions required for fruit juice production and preservation (see below).

Environmental conditions. Moderate temperatures, high moisture content, and relatively low pH (3 to 5) favor the growth of the fungi involved and patulin formation.

Type of food involved; prevention. The toxin occurs in vegetables and fruits (apples). Patulin is an indicator of bad manufacturing practice (use of moldy raw material) rather than a serious threat to human and animal health, as recent subacute and chronic toxicity studies have revealed. Thus, regulatory action based on safety evaluation would not be necessary. Sterigmatocystin. Sterigmatocystin is produced by Aspergillus versicolor and Aspergillus nidulans.

Toxicity and symptoms. Sterigmatocystin is considered to be a carcinogen. Experiments with animals have shown that it causes liver and lung tumors in rats and mice. In comparison to the doses that induce tumors in rats, sterigmatocystin appeared to be a less potent carcinogen than the very potent aflatoxin B1.

Chemical properties (structure and stability). Sterigmatocystin is structurally related to the aflatoxins (Figure 2.5) and is equally stable.

Environmental conditions. Among the factors stimulating fungal growth and toxin production on cheese are lactose, fat, and some fat hydrolysis products.

Type of food involved; prevention. The natural occurrence of sterigmatocystin in food is probably limited. However, investigations on the occurrence of sterigmatocystin in food are, as yet, also limited. Sterigmatocystin occurs occasionally in grains and the outer layer of hard cheeses, when these have been colonized by Aspergillus versicolor. The concentration of sterigmatocystin in the outer layer of contaminated cheeses decreases rapidly from outside to inside. Insufficient data are available on the occurrence of sterigmatocystin, for example, in grated cheese to allow an evaluation of the health hazard caused by this product. Zearalenone. Zearalenone is produced by some Fusarium species, i.e., Fusarium roseum and Fusarium graminearum.

Toxicity and symptoms. Zearalenone has estrogenic and anabolic properties. Pigs are among the most sensitive animals. The International Agency for Research on Cancer has placed zearalenone in the category "limited evidence of carcinogenicity."

Chemical properties (structure and stability). Zearalenone (Figure 2.5) is structurally related to the anabolic zeranol. Few data are available on its stability.

Environmental conditions. The conditions favoring zearalenone production are similar to those favoring DON formation, i.e., high relative air humidity at moderate temperatures.

Type of food involved; prevention. Zearalenone often co-occurs with DON in various grains, in particular maize and wheat. A risk assessment study on zearalenone carried out in Canada revealed that currently no adverse health effects are anticipated from zearalenone due to the intake of maize products. Other food sources such as wheat, flour, or milk may also contribute to the exposure to zearalenone. For the time being, no regulatory action has been recommended. Ochratoxin A. Ochratoxin A can be produced by both Aspergillus ochraceus and Penicillium viridicatum.

Toxicity and symptoms. Ochratoxin A is a potent nephrotoxin in birds, fish, and mammals. Ochratoxin A is also teratogenic in mice, rats, hamsters, and chickens. The primary target organ is the developing central nervous system. There is a hypothesis that a renal disease observed in some areas of the Balkan countries is associated with exposure to ochratoxin A.

Chemical properties (structure and stability). The structure of ochratoxin A is shown in Figure 2.5. It is a fairly stable substance which is not easily metabolized.

Environmental conditions. Ochratoxin A production in cereals is favored under humid conditions at moderate temperatures.

Type of food involved; prevention. Ochratoxin A occurs in grains and, following transfer, in the organs and blood of a number of animals, especially pigs. Recently, a risk assessment study on ochratoxin A has been published. (Limited) Canadian data on estimated human intakes indicate that the tolerable daily intakes, estimated from carcinogenicity data of ochratoxin A, have been exceeded occasionally. More data are required to estimate the dietary exposure to ochratoxin A and to assess the need for regulatory controls or other control mechanisms. The current concern about ochratoxin A has led the International Union of Pure and Applied Chemistry (IUPAC) to the recent launching of a project in which the worldwide occurrence of ochratoxin A in food and animal feed will be mapped. Toxic microbial metabolites Biogenic amines. The main producers of biogenic amines in foods are Enterobacteriaceae and Enterococci. Most lactic acid bacteria which are used to produce fermented foods do not produce significant levels of biogenic amines.

Toxicity and symptoms. Biogenic amines have a stimulatory or toxic effect on the consumer. The symptoms of intoxication, persisting for several hours, include burning throat, flushing, headache, nausea, hypertension, numbness and tingling of the lips, rapid pulse, and vomiting. Especially, histamine has been indicated as the causative agent in several outbreaks of food intoxication. A level of approximately 1000 ppm of total biogenic amines in food is supposed to elicit toxicity, but from a Good Manufacturing Practice (GMP) point of view, levels in food of 50 to 100 ppm, 100 to 200 ppm and 30 ppm for histamine, tyramine, and phenylethylamine, respectively, or a total of 100 to 200 ppm are acceptable. The toxicity of histamine appears to be enhanced by the presence of other biogenic amines found in foods that can inhibit histamine-metabolizing enzymes in the small intestine. Estimating the frequency of histamine poisoning is difficult because most countries have no regulations for histamine levels in foods, nor do they request notification of histamine poisoning. Also, because histamine poisoning closely resembles food allergy, it may often be misdiagnosed.

Chemical properties (structure and stability). Biogenic amines are a group of moderately toxic substances which can be formed in fermented foods, mainly by decarboxylation of amino acids (Figure 2.10).

Environmental conditions. The levels of biogenic amines increase with the presence of free amino acids (precursors), low pH of the product, high NaCl concentrations, and microbial decarboxylase activity.

Type of food involved; prevention. Biogenic amines are especially associated with lactic fermented products, particularly wine, cheese, fish, and meat. Also, very low levels occur in fermented vegetables (Figure 2.11).

Biogenic amines also occur naturally in fruits, vegetables, and fish; they may be produced by microbial decarboxylase activity. For instance, fresh fish (mackerel, tuna, skipjack) contain high levels of histidine which is readily decarboxylated to histamine by Gram-negative bacteria, e.g., Proteus morganii.


Ethylamine C2H7N

Formula CH3CH2NH2

Precursor Alanine

Putrescine C4H12N2

H2N (CH2)4 nh2


Histamine C5H9N3


H2N (CH2)5 NH2




H2N (CH2)5 NH2



CH2CH2NH2 Tyrosine



Phenylethylamine C8H11N



Tryptamine C10H12N2




Figure 2.10 Major biogenic amines.




100 ppm 1000 ppm >2000 ppm

100 ppm 1000 ppm >2000 ppm beer wines sausages gouda cheese brie / camembert blue cheese / gorgonzola terasi (fish paste)

yoghurt sauerkraut

Figure 2.11 Presence of biogenic amines in fermented foods in relation to health hazards.

In meat products, species of Enterobacteriaceae have been found to be associated with cadaverine formation, and lactobacilli with tyramine formation. Also, sauerkraut may contain varying levels of biogenic amines, due to the large variations in the naturally selected microflora. In cheese, Enterobacteriaceae, heterofermentative lactobacilli, and Enterococcus faecalis were shown to be associated with the production of up to 600 ppm of biogenic amines including phenylethylamines. Lactobacillus buchneri has been shown to be involved in cheese-related outbreaks of histamine poisoning. Pasteurization of cheese milk, good hygienic practice, and selection of starters with low decarboxylase activity are measures to prevent the accumulation of these undesirable products. Ethyl carbamate. Ethyl carbamate (urethane) is associated with yeast fermented foods and beverages.

Toxicity and symptoms. Ethyl carbamate is a mutagen as well as a carcinogen.

Chemical properties. The structure of the carbamic acid moiety of ethyl carbamate may originate from several substances including naturally occurring citrulline, and urea and carbamyl phosphate resulting from the metabolism of L-arginine and L-asparagine by yeast. In addition, vicinal diketones and HCN released from cyanogenic glycosides can act as precursors. Ethanol (the other precursor) is formed as a result of alcoholic fermentation by yeasts, or as one of the products of heterofermentative lactic acid fermentation.

Ethyl carbamate (Urethane)

Table 2.6 Occurrence of ethyl carbamate in fermented foods and beverages1

Number of

Average level


















Malt beverages




Bread, toasted




Soya sauce












Note: ND = not detectable.

1 Data found in literature.

Note: ND = not detectable.

1 Data found in literature.

Environmental conditions. Heat and light enhance the formation of ethyl carbamate.

Type of food involved; prevention. Ethyl carbamate occurs in a variety of fermented foods and beverages (Table 2.6).

In most countries there is no legislative limit value, but the Food and Agriculture Organization World Health Organization (FAO/WHO) suggest a level of 10 ppb for softdrinks, and the Canadian Government recommends 30 to 400 ppb for various alcoholic beverages. Research on wine and stone fruit (cherry, plum) fermentations indicate that reduction of the levels of the precursors by enzymatic treatment, selection of yeast strains, control of fermentation conditions, and treatment of the pH-adjusted fermented pulp with CuSO4 may be useful in keeping the ethyl carbamate levels at a minimum.

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