FOOD HYGIENE I

BASICS IN FOOD HYGIENE

lecture notes

Faculty of Veterinary Science, Szent István University

Department of Food Hygiene

2007

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Writen by: dr PÉTER. LACZAY full profesor, Head of Departmentdr. OLIVÉR REICHART associated professordr. Peter Szekely Kormoczy senior scientific associate

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Contents

1. BASIC TERMS, FOODBORNE HAZARDS AND ADVERSE HEALTH-EFFECTS 1.1. Basic terms (definitions, concepts)1.2. History of food hygiene1.3. Purpose and tools of food hygiene1.4. Hazards and adverse health effects of food origin

2. MULTIPLICATION (GROWTH) AND DEATH OF MICROORGANISMS2.1. Rules of microbial multiplication2.2. Effect of environmental factors on the growth of microbes2.3. Rules of microbial cell death

3. PATHOGEN MICROORGANISMS IN FOOD3.1. Occurrance of microorganisms in food, infection and contamination of food by

microorgansisms3.2. General aspects of pathogenicity3.3. Bacteria3.4. Viruses3.5. Prions3.6. Parasites3.7. Regulation of the microbiological aspects of food safety

4. FUNDAMENTALS OF CHEMICAL-TOXICOLOGICAL FOOD SAFETY4.1. Chemical contaminants in food, (public)health-damaging effects4.2. Principles of the regulation4.3. Veterinary drug preparations, banned active substances4.4. Pesticide residues4.5. Contaminants of environmental origin4.6. Contaminants of technological origin4.7. Contaminants of biological origin4.8. Toxic substances of natural origin4.9. Control of chemical contamination in food

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1. BASIC TERMS, FOODBORNE HAZARDS AND HEALTH-DAMAGES1.1. Basic terms„The supply of people with safe and healthy foodstuffs is one of the most important public health aspect by which a nation is able to improve its public health status and consequent economical development of a country” (WHO, 1999).

Food is one of the most important object in preserving or improving public health but in contrast, it is one of the most important potential carrier of health hazards. Food contains nutrients that is vital for the existence and development of human beings but about 70 percent of health damaging agents are also taken up by food. As a basic requirement, food should contain, in appropriate ratio, the necessary proteins, carbohydrates, lipids, macro- and microelments, vitamins and other active substances for supporting vital functions but it should not contain pathogen microorganisms and other harmful biological agents or chemical and physical contaminants. Further expectations are the deliciousness of food altogether with the satisfaction of consumers’ expectations and requirements of the related legal regulations concerning also the outer appearance, packaging and self-life of foodstuffs.

These requirements and expectations are fully covered by two terms such as food safety and and food quality.

Food safety, ensured all along the process of primary production, food manufacture, storage and dsitribution, guaranties that the foodstuff will not be hazardous for health of the consumer if it is prepared and consumed appropriately. In other words, this term indicates the consumers’ safety as a result of food is being free of pathogen microorgansims and other harmful biological agents, chemicals or these are present only in legally acceptable quantities in the consumed foodstuffs.

Therefore, food safety results in offering wholesome food which can be consumed without public health risk.

Food quality means the sum of those properties of foodstuff that satisfy the expected demands of consumers and related offical prescriptions. In other words, food quality means that the composition, internal content, sensory properties, keepability, packaging and labelling of foodstuff comply with the prescriptions and consumers’ expectations. Thus, quality includes quality prescriptions and consumer demands, expectations related to food nutritional-physiological (composition, internal content), sensory (freshness, colour, taste, flavour, consistency) and suitability (processing level, keepability, packaging, labelling) properties, values.In summary, food safety basically is frameworked by legal requirements/regulations while food quality is supported by obligatory or recommended prescriptions and consumers’ expectations

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indicating characteristic properties and demands. Supposing that these are present and satisfied, respectively, the foodstuff becomes acceptable.

Biological Chemical Physical Nutritional-physiological

Sensory Suitability

Bacteria Drugs -Glass particles Proteins, amino acids

Freshness Degree of procssing

Víruses Pesticides Metal particles Carbohydrates, lpids

Flavour, colour, odour,

Keepability

Parasites Environmental contaminans

Bone splinters Macro- and microelements

Juiceness Packaging

Fungi Technological contaminants

Others Vitamins and other materials

Consistency Labelling

Prions Biological contaminantsNatural substances

Food hygiene is the sum of conditions and regulations which are necessary to ensure food safety and suitability all along the food chain. This definition is derived from the general food hygiene principles described by the FAO/WHO Codex Alimentarius Committee and it is also included in the food hygiene Regulation of European Union (852/2004/EU). The definition means that food hygiene is a system of conditions and tools targeting the establishment of food safety and suitability. This goal can be reached by observing the food hygiene prescriptions and regulations and by the coressponding (official) control. Thus, food hygiene as tool is the system of requirements and control in order to establish food safety.Food suitability means that the food is acceptable for human consumption because it is not spoiled but wholesome and nutritive. Altogether, it emphasises the nutritional-physiological and sensory value of the food. The spoilage itself (e.g. stuffiness, mouldness, rancidity, saurness, superficial ropiness/slimeness or changes in consistency), especially in early phase, usually does not carry health hazard and it basically is a quality problem. Occasionally, from the normal-natural constituents degradation products can be formed (e.g. biogen amines) during spoilage and these substances possess health damaging potential, consequently food safety concern.In summary, the food hygiene practice serves primarly food safety but influences also the food quality.

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Acceptibility of foodstuffs

Safety Quality

1.2. History of food hygieneThe cleaness and innocuity of consumed food was an important demand already in the historical societies. According to the religious Egyptian prescriptions, it was the task of priests to inspect the meat of sacrified animals intended for human consumption and those had to be physically sound, healthy and clean. The inspected animals were stamped on their horn. Pigs were considered as unclean animal and altogether with cattle that was a saint species, they were not intended for human consumption. Jewish priests also inspected the sacrified and slaughtered animals and they were released for human consumption if proved to be physically sound and healthy. They considered pigs and horses as unclean species. They distinguished kosher (fit) and trefa (unfit) meat. Phonicians did also not consume pig and cattle meat, mohamedans did not eat pigmeat.The term hygiene was borne in the Greek culture (in latin fonetically: higiénia). Hygiea is the Godness of health in Greek mythology. She was the daughter of Asklepiosnak who is the God of (medical)treatment. The knowledge of identifying of diseased animals and unfit meat developed much during the era of ancient greeks and romans and correspondingly, the consumption of pigmeat became a general habit. In Athen and Rome already an extended slaughter and meat industry functioned with well equiped slaughterhouses and meat halls.Later, following the fall of Roman Empire, a significant decline was experienced. By the 13 th

Century, however, along with growing meat consumption, butcher guilds were formed. The slaughter industry was controlled by guild masters and selling of spoiled meat was severily fined. Slaughters were regulated, the LawBook of Buda (1241) ordered that slaughterman of Buda may slaughter only during light period of the day in the common abbatoir at bank of the Danube. Later, in the 17th-18th Centuries several documents mentioned that towns and counties already prescribed the obligatory practice of ante mortem inspection of slaughter animals. The real advance in history of food inspection was in the second half of 1800’ years, when a new interdsciplinary profession, the meat inspection was developed rooting from the medical and veterinary sciences. Initially, medical doctors were involved but by the end of the 19th Century, the rapidly developing veterinary science engulfed the new discipline and gradually developed it into a complex of food hygiene. At turn of the Century and in the first decade of the 20th Century, the animal markets and public slaughterhouses at Székesfehérvár served as professional and organisational centres. The cattle (1872), pig (1902) and horse (1905) slaughterhouses were important establishments also on European scale and were driven by veterinarians. From 1908, ministerial regulation made obligatory the meat inspection in Hungary and it declared that the master of meat inspection was the veterinarian. Later, the well known regulation of „100000” (100.000/1932 FM Regulation) introduced the rules of bacteriological meat inspection and further specified of meat hygiene. Milk hygiene was also started developing. Following a transient decline after the Second World War, from the mid sixties, the discipline of food hygiene started rapidly advancing. The scope of food-control was getting wider and gradually embarrassed the hygiene control of production. On course of this

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developmment, by the mid seventies, the earlier end-product control was transformed into process-control that was extended to the whole production process.

In the domestic veterinary education, food hygiene initially was performed in the framework of epidemiolgy (1875) (Medical-Officer’s Meat-Inspection, Vilmos Zlamál), and from 1888 Ferenc Hutyra already lectured meat inspection as an autonom discipline and the slaughterhouse parcticals were also introduced. Later, from the end of the first decade of the 20 th Century, the education of milk hygiene was also initiated. Thereby, the regular education and practice of the two main disciplines of food hygiene, the meat and milk hygiene were started (Albert Breuer, Géza Semsey). In the university education of food hygiene an important cornerstone was the establishment of the independent Department of Food Hygiene the in 1949 (Vilmos Csiszár). Since then, the Department has been responsible for the graduate education of the more and more complex discipline of food hygiene and also for the post-gradual education of specialists in food hygiene and food microbiology.

1.3. The purpose and tools of food hygieneThe food hygiene related conditions and rules indicate the requirements which are necessary of producing, manufacturing, distributing, controlling and examining of safe food. Consequently, these are the main tools of creating and guaranteeing food safety. In other words, food safety is a primary aim in achieving public or consumers’ health protection and food hygiene is the principal (but not exclusive or single) tool in realisation of the purpose. As already has been discussed, the requirements of food hygiene ensure also the suitability of foodstuffs maintaining the spoilage-free, nutritive and healthy condition of food. The primary responsible for food safety is the producer. This responsibility is extended further to undertakings (disregarding if retail or wholesale) participating in storage, distribution, catering and in certain degree also to the consumer. Thereby, the responsibility is divided, but the interest is common in preserving safety of food up to the consumer. Food industrial establishments are able to ensure the safety of their products by observing the food hygiene conditions and rules and by elaborating and running self-control system based on overall risk analysis extended to the whole process.The State controls through official food-control activity if food hygiene rules are observed or not. This check is directed to the foodstuff itself examing its safety for the consumer but the activity of the primary producer, manufacturer and distributor is also controlled (as well as the establishments themselves and equipment) if those satisfy the required conditions appropriate for producing and keeping (maintaining) safe food. The sufficiently frequent official control based on professional, consequent and homogenous principles reflects the responsibility of the State in protectig the public health of a Country.

During the recent years the application of principles and rules of food hygiene have been extended to the whole food chain including the primary production. The potential (micro)biological and chemical hazards arising in primary production (e.g. in plant and feed

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production, animal farming, milking, egg production, aquaculture, etc.) are greatly influencing the products of animal origin that are without further processing and also those that are manufactured from primary production derived raw materials. These latter should be free of pathogen microbes and toxic chemicals or should contain them in legal, tolerable quantities.

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FOOD CHAIN

To guarantee the health of production animals and the safety of food of animal origin (basic products of fresh meat, fresh egg, fresh milk, etc.) demands the fine harmonisation of epidemiological, feeding, animal hygiene, official administrative and food hygiene activities. The food production animals must be housed from time of birth according to the corresponding principles of food hygiene in order to achieve the desired safety and suitability. Observing the related principles and rules is the obligation and responsibility of the farmer (undertaker). His activity is supported by private veterinarian on contractual basis and the fulfilment of requirements for the production of safe raw material is controlled by official veterinarian. Thereby, the satisfaction and control of the principles and requirements of food hygiene also in the primary production are two-sided. The primary responsible for food safety is the producer (farmer) and the State controls and facilitates the process by means of regulations and guidelines altogether with official audits and inspections.

The historical natural, direct confidence between producer (seller) and consumer is supplemented-substituted-mediated by the Sate. Recently, the power and influence of consumers has significantly been growing and this process is coupled by certain loss of confidence. As a reaction, to re-gain consumers’ confidence, the production and distribution side is getting more and more integrated up to the level of integrated production systems with high level and efficiency of self-control. As we can see, the former two-sided interaction is going to be further diversificating.

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FEED INDUSTRY

ANIMAL FARM

ABBATOIR, FOOD PRODUCING, PROCESSING UNITS

WHOLESALE UNITS

REATIL UNITS, CATERING, PUBLIC CATERRING

CONSUMER

1.4. Hazards and adverse health effects of food originSeventy percent of hazardous agents adversely influencing the health of consumers are getting into the consumers’ organism by food, and the remainder quantity by drinking water and air. The foodborne infective and toxic agents can be clasified into 5 main groups:

- Microbiological agents- Chemical agents- Physical contaminants- Radioactive contaminants- Risk factors of new technological, biotechnological origin

Microbiological risk agents are bacteria, viruses, moulds and fungi, parasites and prions. The most important foodborne causatives of clinically manifested diseases are the bacteria but the number of cases caused by viruses are also increasing in number. Among bacteria the most importants are the zoonotic ones, such as Salmonella spp., Camplyobacter sp. and Yersinia sp., furthermore the Listeria monocytogenes, the verotoxin producing E. coli, and the toxin producing strains of Staphylococcus aureus (though, these latters usually are of human origin). The zoonotic agents are originating primarly from raw materials, basic products derived from infected animals (primary infection) and the infected food mediates them into the body of consumers. The basis of protection against them is the reduction of the infection rate to a minimum in production animals at the primary production level. Microbiological hazards may arise also from humans handling foodstuffs (mainly viruses, streptococci and staphylococci), but may origin also from the environment (Listeria). In these cases, the secondary infection inducing causatives contaminate foodstuffs in later phases of the food chain (manufacture, distribution, catering, public catering, households). The consequence of conatamination may be:

- the development of a corresponding foodborne disease- the spoilage of the product, a shorter keepabilty- change in characteristic properties of the product (colour, odour, taste, consistency).

The majority of chemical contaminants are taken up by plants (feed) or by the production animals or contaminate their surfaces during the primary production in the farm. A smaller portion of chemical contaminants are forming during food processing or are carried by additives. Chemical hazardous agents contaminating during the primary production are the residues of veterinary drugs and pesticides, miscellenous environmental contaminants (e.g. toxic metals, dioxines, polychlorinated biphenyls), contaminants of biological origin (mycotoxins, marine and fresh water biotoxins, histamine), furthermore toxic substances of natural contents (cyanoglycosides, nitrites, alkaloids, etc.). Chemicals able to adversely influence human health may be formed also during food industrial processing or during

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preparing food but they also can be released from production-technical-tools or from packaging materials (e.g. polycyclic aromatic carbohydrates, nitrosamines). The mal-application of additives during industrial processing may also result in contamination (colorants, preservatives, artificial sweeteners, antioxidants). Disregarding from a few exceptions, the adverse health conditions develop slowly (chronically) and without detectable, characteristic symptoms, therefore, their recognition (diagnosis) and the verification of the causal relationship mostly is very difficult or impossible.Other, physical contaminants may be found in foodstuffs (glass spliters, metal fittings, etc.) These may be derived from break of lamps, from machines, equipment (crew, metal fittings) and from packaging materials (jar, metal box, can).Majority of radioactive isotopes transferred by food into the organism are natural of origin and this may be supplemented by an additional human activity related small quantity. In case of food, the principal isotopes to be considered are 137Cs and 90S.Poor experience is available concerning potential hazards associated with new technologies, biotechnologies, irradiated food and genetically modified food (aimed to satisfy changing consumer demands, to establish longer shelf-life or creating new properties).Consumption of infected or contaminated foodstuffs may cause clinically manifested diseases (food-infection, food-toxicosis) and may induce several other kind of health-damages (teratogen, mutagen, carcinogen effects, immunosuppression, allergy, bacterial resistance and intestinal microflora deviation) According to the estimation of the World Health Organisation, the number of diseases which may be coupled to food consumption is continuously increasing. Yearly, it affects 10-30% of human population also in developed countries. In Hungary this may represent 1-3 million cases (usually not notified).Most of the clinical cases are food-infections when the causative is a microorganism present in the foodstuff (bacterium, virus or parasite). In food infection the foodstuff is not only a passive mediator, carrier of bacteria in transporting them into the organism but a medium, thereby it actively influences the microbial multiplication and virulence, consequently the development and severity of infection is also affected. Considering that these patients generate further potential infections by releasing the microbes, it is not a sufficient measure to prohibit the consumption of the infected foodstuff but epidemiological measures are also necessary to be taken for preventing the spread of infection.A smaller portion of clinical cases is real food-poisoning caused by toxin produced by microorganism, or toxin naturally present in the foodstuff or exogen toxic substance. Now, the foodstuff is only a passive mediator of toxic substances and by prohibiting the furher consumption of the incriminated foodstuff, the spread of the disease can be prevented.In certain causatives (e.g. Clostridium perfringens) induced diseases, both the multiplication and the actually produced toxin are playing role in the pathomechanism (toxico-infection).The foodborne clinical diseases usually characterised by gastrointestinal symptoms (vomiting, diarrhoea, abdominal pain). Occasionally, especially in children and elderly people and in immunocompressed individuals, other organic affections and compliactions may occur. The

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usually short incubation period allows the recognition of the causative relationship with the consumption of food and the causative agent can be detected in the food-remainder.Many health damages caused by chemical substances are developing slowly, through years or decades, and the causal relationship can hardly be detected.

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2. MULTIPLICATION AND DEATH OF MICROORGANISMS2.1. Multiplication rules of microorganismsIn a closed system, under the complex influence of appropriate environmental factors (medium, temperature, pH, etc.) the multiplication of microorganisms is analogous to the autocatalytic processes. (Closed system means that following the inoculation of the medium with microbe-culture no nutritives/metabolites are added or removed).At multiplication of microorganisms the change of cell-mass or cell-concentration in function of time (the multiplication rate) is propotional to the prevailing (actual) cell-mass or cell-concentration.

dx/dt = ·x

where dx/dt is the multiplication rate, is the specific multiplication rate, x is the biomass or cell-concentration, t is the time.The cell-concentration can be measured directly (filtration, dry weight of the biomass), or indirectly by a parameter that is proportional to the cell-mass (optical density, ATP-, protein-content, etc.). These methods are suitable for the determination of total biomass and are unable to distinguish between living and dead cells. The living cell concentration determined by the plate counting method is always lower than the total cell concentration, this latter includes both the dead (non-multiplicating) and living cells.The multiplication rules described below are applicable only for determination of cell-mass, the cell-concentration can be determined only if the cell-concentration and cell-mass are closely proportional. This proportionality is true for bacteria and yeasts and only rarely for moulds.The microbial multiplication is characterised by the growth curve which demonstrates the change of cell-concentration in function of time (Figure 2.1.).

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0

1

2

3

4

5

6

0 5 10 15 20 25

time (h)

Abso

rban

ce1. 2.

3. 4. 5.

Figure 2.1.: Growth curve of Saccharomyces cerevisiae

According to the specific multiplication rate, (μ), the growth curve can be divided into five characteristic phases.

1. Lag Phase There is no apparent growth, the inoculate is adapting to the new environment, microbes synthetise the new enzymes required for utilising the substrates, they repair lesions of earlier injuries (e.g. freezing, drying, osmotic shock, etc.).The specific multiplication rate: μ = 0.

2. Accelerating PhaseA portion of microbes already are multiplicating. The specific multiplication rate is:0 < < max.

3. Exponential PhaseThe genetically determined growth rate is maximum in this phase but is controlled by environmental factors.The specific multiplication rate: = max, constant value.

The value of μ is constant in this phase, therefore the change of cell-concentration can be calculated by separation and integration of the dx/dt = ·x differential equation.

x = x0·exp (·t)

In food microbiology, the cell-concentration usually is determined by cultivating methods resulting in living-cell count (N). Using logarithmic form in the exponential phase:

log10N = log10N0 + ·t

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The above equation shows that in the exponential phase, the growth curve has a linear section. From the slope of this linear section (/2,303) the specific multiplication rate can be calculated:.

µ = 2.303∙slope

From the specific growth rate (μ), the doubling time of cell-concentration (the generation time, tg) can be determined:

tg = ln 2 / = 0.693 /

Calculation of cell-concentration using the generation time:

N = N0·2t/tg

Similarly to the specific multiplication rate, the generation time is also genetically determined and controlled by environmental factors. Under optimum conditions, the doubling time of the main microbe-groups of food-hygienic concern are as follows:

Bacteria: 20 – 30 minutesYeasts: 2 hoursMoulds: 4 – 6 hours

4. Decelerating PhaseDue to exponential growth, one or more components of the nutrient start to be depleted, or toxic-inhibitory metabolites are accumulating and consequently, the specific multiplication rate decreases.The specific multiplication rate: max. > > 0

5. Stationary PhaseDue to the depletion of key-nutrient-component(s) in the culture medium, and/or the accumulation of inhibitory substances, the multiplication is arrested. Sometimes in this phase the slow growth and death of microbe population are in equilibrium and the value of cell-concentration is unchanged.The specific multiplication rate: = 0.

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(6. Destruction Phase)

The stationary phase is followed by the destruction phase where the measure of cell-number decrease is higher than the actual multiplication rate. This phase will be discussed in framework of death rules, it is considered not belonging to the growth curve related events.

From the equation of growth law (dN/dt = ·N) the living cell concentration (e.g. the change of the number of pathogen microorganisms in stored foodstuffs) can be calculated, assuming that the value of the specific multiplication rate (μ) influenced by environmental factors is known. If it is, then we have a possibility to predict the living cell concentration in function of time. This prediction is the subject of the predictive microbiology which is one of the most dynamically developing part of food science.

The concept of predictive microbiology is a detailed knowledge of the responses (multiplication, death, production of metabolites, toxins, etc.) of microbes to environmental conditions determined by mathematical modelling. It enables objective evaluation of the effect of processing, distribution and storage operations on the microbiological condition and consequently on food safety.

2.2. Effect of environmental factors on the growth of microbesThe metabolism and multiplication is the result of biochemical reactions taking place in the cells. The environmental factors affect these reactions influencing the equilibrium developing betwen the two sides of plasma and cytoplasmic membranes. Reasonably changing the environmental parameters, we can control the microbial multiplication both favorably or unfavorably. Facors affectig the microbial growth are summarised in Table 2.1.

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Table 2.1.Factors affecting the multiplication of microbes in food

__________________________________________________________________Intrinsic factors Implicit factors

Nutrients Specific multiplication rateWater activity SynergismpH and buffer-capacity AntagonismRedox-potential CommensalismAntimicrobial constituentsAntimicobial structure

Extrinsic factors Processing factorsRelative humidity SlicingTemperature WashingGaseous atmosphere Packaging

IrradiationHeat treatment

__________________________________________________________________

It should be emphasised that the effect of intrinsic and extrinsic factors summarised in Table 2.1. can only be manifested (totally or partially) if they are able to affect the microorganisms directly. The multiplcation-influencing effect of extrinsic factors are becoming apparent following the development of equilibrium in food (temperature, water activity, partial oxygen tension). Similarly, the intrinsic factors also need direct interaction with microbes for developping optimum effect (e.g. microorganisms embedded into lipid droplets cannot utilise water soluble nutrients).

2.2.1. Effect of substrate (nutrient) concentration on multiplicationThe growth rate of microorganisms is determined by the concentration of an essential nutrient that is present in the relative minimum concentration. The relationship between the substrate concentration (S) and the specific multiplication rate is similar to the substrate dependence of enzyme-catalised reaction rates. At low concentrations of substrate, the specific multiplication rate increases with the increase of concentration, however, this relationship shows saturation property (Figure 2.2.). The Monod equation of the saturation is similar to the Michaeis-Menten equation used for enzyme reactions:

= max·

where S is the substrate concentration, Ks is the half-saturation coefficient, (the substrate concentration belonging to the max/2 value). The Ks values usually are very low, in case of

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components being the source of carbon and energy are fallen into the concentration range of 10-5 M.Over the critical concentration the growth rate is independent of the substrate concentration (Skr) and has the maximum value (μmax). This is the case in most foods, disregarding from some very fastidious microorganisms, the quantity of nutrients (both macro- and microcomponents) is sufficient to achieve maximum multiplication rate (too high substrate concentrations may already be inhibitory)

max

max/2

KS Scr S

Figure 2.2.: Effect of substrate concentration on the multiplication rate

2.2.2. Effect of the water activity on the growthMetabolic processes in cells require free water in liquid form. The reactions in the cytoplasm are taking place in aqueous medium and the water is not merely a solvent but it is also a special reagent. The cytoplasmic membrane is permeable to water molecules and the direction of movement is determined by the difference of transmembrane chemical potential of water. The flow of water, similarly to the movement of other molecules, is directed from the site of higher chemical potential toward the lower one. Equation expressing the chemical potential of water (w):1

w = wo + RT ln aw + VmP + mgh

where wo is the normal potential, R is the gas constant, T is the absolute temperature, , aw is

the water activity, Vm is the partial molar volume of water, P is pressure, g is the gravitational constant, h is the height (relative to the reference level).Practically, in microbial dimension, under normal condition, the effect of pressure and gravity is negligible and the chemical potential is controlled by the water activity:

1 µ not to be confused with theconventionally identically marked specific multiplication rate.

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w = wo + RT ln aw

The relationship between water activity and osmotic pressure (Posm):

Posm = ·ln aw

From the aspect of food microbiology, the movement of water is determined by the difference in water activity or osmotic pressure. The water as solute tends to move from the higher water activity site (lower osmotic pressure) toward the lower water activity site (more concentrated, higher osmotic pressure).

The water activity of a solution is determined at identical temperature by the ratio of the partial vapour pressure (P) (developing above the solution) and vapour pressure (P0) of clear water. The value of water activity is between 0 and 1.

aw = P / P0

The water activity of solutions, in case of non-dissociable solved substances can be calculated also from the mol numbers:

aw = Nw / (Nw+NS)

where Nw is the mol number of water, Ns is the mol number of the dissolved substance.In case of dissociable substances, in measure of dissociation, the water activity reducing effect of dissolved substances is increasing.

Due to the flow of water in a closed system, sooner or later an equilibrium is developing between the solution and the surrounding atmosphere. In equilibrium the water activities of the solution and the atmosphere are equal, therefore the water activity value of the solution can be calculated from the equilibrium relative humidity (ERP%):

aw = ERP% / 100

Microorganisms are able to take up water necessary for their growth only above a certain minimum-water activity of the environment. The minimum water activity demands of the different microbe groups are summarised in Table 2.2..

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Table 2.2. Minimum water activity demand of microbial growth

_______________________________________________________Microbe group Minimum water activity demand_______________________________________________________Most Gram-negative bacteria 0,97Most Gram-positive bacteria 0,90

Halophilic bacteria 0,75Most yeasts 0,88

Osmophylic yeasts 0,62Most filamentous fungi 0,80

Xerotolerant fungi 0,71Xerophilic fungi 0,61Xeromyces bisporus 0,60

_______________________________________________________

The characteristic water activity values of some foods is demonstrated in Table 2.3.

Table 2.3.Water activity of some foods

____________________________________________________Food aw

____________________________________________________Fresh vegetables, meat, milk, fish 0,98<

Cooked meat, bread 0,95 – 0,98Cured meat, ham, cheese 0,91 – 0,95Dry cheese, salami 0,87 – 0,91Flour, rice, beans, cereals 0,80 – 0,87Jams 0,75 – 0,80Dried fruits, caramels 0,60 – 0,75Spices, milk powder 0,20 – 0,60

____________________________________________________

Practically, apart from a few osmophilic yeasts and xerophilic fungi, below 0.7 water activity there is no microbial multiplication, therefore from part of food microbiology, this value is considered as critical water activity level. However, it is important to emphasize that even if active growth is restricted, microorganisms can survive for a long time at very low water activities and are frequently stored in culture collections in this form.Dried foods are microbiologically stable until their water activity is less than 0.7. During storage, in an atmosphere with high relative humidity (RH is over 70%), the water moves from

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the environment into the food, increasing its water activity above the critical value of 0.7, resulting in microbiological spoilage. The relationship between water activity and water content is very sensitive to temperature. At the same water content, the water activity increases with temperature, as it is demonstrated by the sorption isotherms in Figure 2.3.

0

0,2

0,4

0,6

0,8

1

0 5 10 15 20

Water content (%)

Wat

er a

ctiv

ity

T1

T2

T3

T1>T2>T3

Figure 2.3.: Sorption isotherms

2.2.3. The effect of pH on multiplicationThe acidity and alkalinity of the environment, characterised by its pH value has a profound effect on the activity and stability of macromolecules also in enzymes and on the rate and direction of transport processes. Thus, it is not surprising that the metabolism and growth of microorganisms are influenced by pH.In general, bacteria grow fastest in the pH range of 6.0-8.0, yeasts 4.5-6.0, and filamentous fungi 3.5-4.0. The most important exceptions in food microbiology are the acid producing bacteria, such as lactobacilli and acetic acid bacteria, with a pH optimum of 5.0 and 6.0. Most foods are slightly acidic, the alkaline pH usually indicates protein decomposition or rotting.The acidity of a product can determine its microbial ecology, and the rate and character of its spoilage. For example, plant products classed as vegetables generally have a moderately acidic pH and soft-rot producing bacteria such as Erwinia carotovora, and pseudomonas play a significant role in their spoilage. In fruits, however, a lower pH prevents bacterial growth and spoilage is dominated by yeasts and moulds. The pH of post-rigor mammalian muscle is around 5.5 (lower than in fish-meat of 6.2-6.5) and this contributes to the longer shelf life of mammalian meat. The ability of relatively low pH to reduce microbial multiplication plays a role in the preservation of foods with acetic acid and lactic acid.

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2.2.4. The effect of redox-potential on microbial multiplicationOxidation-reduction reactions occur as a result of transfer of electrons between atoms or molecules. In biological systems the most general form of redox-reaction is the hydrogen transfer that includes redox pairs, protons (H+) and electrons (e-):

[Oxidant] + H+ + ne- = [Reductant]

where [Oxidant] indicates the oxidated form, [Reductant] indicates the reduced form, H + is the hydrogen ion, n is the number of electrons in the transfer process.In living cells the electron and hydrogen transfer reactions are structured into electron transport systems contributing to energy generation by oxydative phosphorylation. The tendency of a medium to accept or donate electrone, to oxidise or to reduce, is termed its redox potential (Eh), referred to the normal hydrogen electrode and is measured in the form of potential difference against an external reference by an inert metal electrode, usually platinum, immersed in a medium. The measured Eh value is also infuenced by the relative proportions of oxidized and reduced species and the concentration (activity) of protons present in the reaction. The relationship of a single redox-couple is expressed by the Nernst equation.

where E0 is the normal potential of the reaction, R is the gas constant, T is the absolute temperature, F is the Faraday constant, n is the electron transfer number (number of electrons transferred in the reaction).The tendency of an atom or molecule to accept or donate electrons is expressed as its standard redox potential (E0). A large positive E0 value indicates that the oxidized species of the couple is a strong oxidizing agent and the reduced form is only weakly reducing. A large negative E0 indicates the reverse case. The normal potential of some important redox-couples is sumarised in Table 2.4.

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Table 2.4.Some important redox-couples and their standard potential

---------------------------------------------------------------------------Couple E0 (mV)

---------------------------------------------------------------------------½ O2/H2O +820Fe3+/Fe2+ +760

Cytochrome C ox/red +250Dehydroascorbic acid/ascorbic acid +...80Methylene blue ox/red +...11

Pyruvate/lactate -190Glutathion oxid./Glutathion red. -230NAD+/NADH -320

---------------------------------------------------------------------------

From the Nerst equation, it is clear that the hydrogen ion activity affects the redox potential. At 25 °C for every unit decrease in the pH, the redox potential increases by 58 mV. The high redox potential of fruit juices are largely a reflection of their low pH, as it is demonstrated by the redox potentials shown in Table 2.5.

Table 2.5.Redox potential and pH of some foods

-------------------------------------------------------------------------------------------Eh (mV) pH

-------------------------------------------------------------------------------------------Fresh meat (post rigor) -200 5,7Fresh minced meat +225 5,9Cooked sausages and canned meat -20 –150 ca. 6,5Wheat (whole grean) -320 –360 6,0Potato tuber ca. –150 ca. 6,0Spinach +74 6,2Pear +436 4,2Grapes +409 3,9Lemon +382 2,2

-----------------------------------------------------------------------------------------

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Table 2.6.Factors influencing the redox potential of foodstuffs

--------------------------------------------------------------------Presence of redox couples

Ratio of oxidized to reduced formspH of the medium

Redox buffer-capacityOxygen availability

Microbiological acivity---------------------------------------------------------------------

From factors influencing the redox potential of foodstuffs (Table 2.6.), the presence of redox couples, the ratio of oxidized and reduced forms, the effect of the pH of medium, can be explained using the Nerst equation. The logic of buffer-capacity is analogous to the pH-buffer capacity. It indicates the potency of a medium to compensate the redox potential changes inducing effects.

The oxygen, which is present in the air at a level of 21%, is usually the most influential redox couple in food systems. It has a high E0 value in water medium (+820 mV), therefore it possesses powerful oxidizing capacity. If sufficient air is present in food, the high positive potential results in an equilibrium of the participant redox couples shifted largely into the oxidized state.Thus, the intrinsic factor of redox potential is strictly linked with the extrinsic factor of storage atmosphere. Increasing the access of air to a food material by chopping, grinding, or mincing will increase its redox potential value. Similarly, exclusion of air as in modified vacuum packaging or canning, will reduce the redox potential of the product.Microbial growth in a food reduces the redox potential of the environment (medium). This is usually ascribed to a combination of oxygen depletion and the production of reducing compounds such as hydrogen by the micro-organisms. Oxygen depletion appears to be the principal mechanism; as the oxygen content of the medium decreases, parallel the redox potential declines from value of 400 mV at air saturation by about 60 mV for each tenfold reduction in the partial pressure of oxygen. Redox potential of the medium exerts an important elective effect on the microflora of a foodstuff. Although, microbial growth can occur over a wide range of redox potential, individual micro-organisms are conveniently classified into one of the four basic groups on the basis of the redox range over which they can grow and their response to oxygen.

Obligate or strict aerobs are those micro-organisms that are respiratory, generating most of their energy from oxidative phosphorylation and using oxygen as the terminal electron acceptor in the process. Consequently they have a requirement for oxygen and a medium with

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high redox potential (moulds, pseudomonads, Pseudomonas fluorescens, grow at Eh over 100 mV, Bacillus subtilis, growth at Eh over –100 mV).

Obligate anaerobes tend to grow only at low or negative redox potentials and often require oxygen to be absent. For many anaerobes, oxygen exerts a specific toxic effect, which is linked to the inability of obligate or aero-intolerant anaerobes to scavenge and destroy toxic products of molecular oxygen such as hydrogen peroxide (H2O2) and, more importantly, the superoxide anion radical (O2

-). They lack the enzymes catalase and superoxide dismutase (Clostridia). The redox potential range of their multiplication is below -300 mV.Facultative anaerobs are able to grow in both aerobic and anaerobic environments. According to the actual redox potential of the environment, they generate energy by aerobic respiration or anaerobic fermentation (Enterobacteria, Yeats).Aerotolerant anaerobes are incapable of aerobic respiration, but can nevertheless grow in the presence of air (Lactic acid bacteria). They can generate energy only by fermentation and lack both catalase and superoxide dismutase, but are able to grow in the presence of oxygen because they have a mechanism for destroying superoxide, based on the accumulation of millimolar concentration of manganase.

2.2.5. Interpretation of the effect of pH and redox potential on microbial growth

Several essential cell functions such as ATP synthesis in bacteria, active transport of nutrient components and cytoplasmic regulation occur at the cell membrane and are dependent on potential energy stored in the membrane in form of a proton motive force. This force is an electro-chemical potential difference developed by the active separation and translocation of protons from the membrane interior to the external environment during the oxidative metabolism at the initial phase of in the electron-transport chain. When the outpumped protons return into the cell along the proton-gradient, the stored energy (in form of electrochemical potential difference) is released. This energy is used for the active transport, ATP synthesis and cytoplasmic regulation.Protons have positive charge, so their movement through the membrane results in electric and chemical (pH) potential difference.This electrochemica potential difference (Δμ) according to Mitchell’s chemiosmotic theory:

where E and pH are the electric potential- and pH differences betwen the two sides of membrane. Some typical values of the proton motive force are summarised in Table 2.7.

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Table 2.7.Some typical chemiosmotic potential differences

Mitochondrium E. coli ChloroplastpH 1,4

Acidic outside2

Acidic outside3,5

Acidic insideE (mV) 140

Positive outside70

Positive outside0

Gpm (kJ/mol H+) 22,3 18,5 20,2

Introducing the term of free-enthalpy difference between the two sides of membranes, the so called proton-motive force (built up at active translocation of protons through memmbranes and released at moving back along the proton gradient):

Gpm = F· = F·(Eo – Ei) – 2.303·R·T·(pHk – pHi)

where indices o and i mean the outer and inner sides, respectively and F is the Faraday’s constant.Separating the inner and external parameters of the proton motive force:

Gi = F·Ei – 2,303·R·T·pHi

Go = F·Eo – 2,303·R·T·pHo

The proton motive force between two sides of membranes:

Gpm = Go – Gi

Acording to the homeostasis of the living cell, the inner redox potential and pH move in a narrow range, therefore the value of G i is realtively constant, while Go is affected by the redox potential and pH of the environment and can be manipulated. Every effects reducing the proton motive force decrease (or destruct) the metabolic activity of cells.

Effect of high external pHThe external [H+] and Eh are low. The chemical potential () of protons in the environment is less than in the internal side of the membrane. The environment removes the protons from the cells’ surfaces.

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Effect of low external pHThe external [H+] and Eh are high. The chemical potential in the environment is high enough to prevent the action of proton pump.

The preservative effect of weak lypophilic acids (benzoic-, sorbic-, proprionic acid) could be interpreted on this way as well. If the external pH is low, the dissociation equilibrium favours to the undissociated molecules, and they are able to pass through the membrane. In the cytoplasm at the relatively high pH (around 7.5 in neutrophils) they dissociate and produce protons. If the external concentration of the weak acid is high enough, the cell cannot compensate the interior effect of protons, cytoplasmic pH drops and the chemiosmotic system does not work.

2.2.6. Effect of the temperature on the growthMicrobial growth can occur over a wide temperature range from about, -8 up to 100 °C at atmospheric pressure. The most important requirement is that water should be present in liquid state. Within the temperature range of their growth, each organism can be characterised by a minimum, optimum and maximum growth temperature. (Table 2.8.).

Table 2.8.Cardinal temperatures of microbial growth (°C)

----------------------------------------------------------------------------------------------------------Minimum Optimum Maximum

---------------------------------------------------------------------------------------------------------Thermophiles 40 – 45 55 – 75 60 – 90Mesophils 5 – 15 30 – 40 40 – 47Psychrotrophs -5 - +5 25 – 30 30 – 35(facultative psychrophils)Psychrophils -5 - +5 12 – 15 15 – 20(obligate psychrophils)----------------------------------------------------------------------------------------------------------

Minimum temperature Below this temperature the micro-organism does not grow. During the storage below the minimum temperature of growth, although small destruction could occur, but most of the microbes survive the cold storage. The cold storage (chilling and freezing) are widely used processes in food preservation. Optimum temperature At this temperature the specific growth rate (µ) is maximum (under defined environmental conditions) and the generation time is minimum value.

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Maximum temperature Over this temperature there is no growth. Storage over the maximum temperature results in microbial destruction. The higher the temperature, the greater the destruction rate. The heat destruction of microorganisms is the most important procedure in food preservation (pasteurization, sterilization). The so called cardinal temperatures are greatly characteristic for a microorganism, but their value are markedyl influenced by environmental factors (medium, pH, aw, redox potential). As a rule, the viability and resistance of microorganisms to the environmental factors is the greatest at its optimum temperature. Apart from this temperature, the micro-organism become fastidious and the minimum water activity requirement increases and the resistance to the environmental effects decreases. (Figure 2.4.).

0,8

0,85

0,9

0,95

1

0 10 20 30 40 50

Temperature (°C)

Wat

er a

ctiv

ity re

quire

men

t

P. expansum A. flavus

Figure 2.4.: The effect of temperature on the minimum water activity requirement of the growth of Penicillium expansum and Asprgillus flavus

In food microbiology the mesophilic and psychrotropic organisms are generally of greatest importance. Mesophiles, with temperature optimum around 37 °C , are frequently of human or animal origin and include many of the more common foodborne pathogens such as Salmonella, Staphylococcus aureus, Clostridium perfringens.As a rule, mesophiles grow more rapidly at their optimum than psychrotrops, so spoilage of perishable products stored in the mesophilic growth range is faster than spoilage under chill conditions.

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Among the micro-organisms capable of grow at low temperatures, two groups can be distinguished such as: the true or strict psychrophiles („cold-loving”) have optima of 12-15 °C and will not grow above about 20 °C. Psychrotrophs or facultative psychrophiles can grow at the same temperatures as strict psychrotrophs but have higher optimum and maximum growth temperatures. This tolerance of a wider range of temperature means that psychrophs are found in a more diverse range of habitants and consequently are of greater importance in the spoilage of chilled food.

Thermophiles are generally of far less importance in food microbiology, although thermophile spore formers such as certain Bacillus and Clostridium species (Bacillus stearothermophilus, Clostridium thermosaccharolyticum) could cause spoilage in tropical cans.

2.2.6.1. Effect of chilling on microorganismsChilled foods are stored at temperature close to, but above their freezing point, typically at 0-5 °C. Chill storage can change both the nature of spoilage and the rate at which it occurs. There may be qualitative changes in spoilage characteristics as low temperatures exert a selective effect preventing the growth of mesophiles and leading to a microflora dominated by psychrotrophs. At low temperature the lag period increases and growth rate decreases. Though psychrotrophs can grow in chilled foods but their multiplication rate is reduced and the onset of spoilage is delayed. The ability of micro-orgaisms to grow at low temperature appears to be particularly associated with the composition and architecture of the plasma membrane. As temperature is lowered, the plasma membrane undergoes a phase transition from liquid crystalline state to a rigid gel in which solute transport is severely limited. The temperature of this transition is lower in psychrotrophs and psychrophiles largely as a result of higher levels of unsaturated and short chain fatty acids in their memebrane lipids. If some micro-organisms are allowed to adapt to growth at lower temperatures they increase the proportion of these components in their membranes.

There seems to be no taxonomic restriction on psychrotrophic organisms which include members of yeasts, moulds, Gram-negative and Gram-positive bacteria.Though mesophiles cannot grow at chill temperatures, they are not necessarily killed. Fast chilling will produce an early developing phenomenon known as cold-shock which causes death and injury in a proportion of the population but its effects are not predictable in the same way as heat processing. The principal mechanism of cold shock appears to be damage of membranes caused by irreversible phase changes in the membrane lipids which create hydrophilic pores through which cytoplasmic contents can leak out.

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2.2.6.2. Effect of freezing on microorganismsFoods begin to be frozen somewhere in the range of -0.5 to-3 °C. The freezing point is lower than that of pure water due to solutes present. As water is converted to ice during freezing, the concentration of solutes in the unfrozen water increases, decreasing its freezing point and water activity. The decreased temperature results in lower water activity even in pure water/ice system as it is shown in Table 2.9.

Table 2.9.Effect of freezing on water activity of pure water/ice

-------------------------------------t (°C) aw

-------------------------------------0 1

- 5 0.953-10 0.907-15 0.864-20 0.823-40 0.680

-------------------------------------

The effect of reduced water activity will be manifested at time of storing of the already frozen food at higher temperature favouring to microbial multiplication (above -10 °C). Under this condition not the normal flora or spoilage inducing microbes are multiplicating in the product but moulds and yeasts which are psychrophils and tolerate low water activity.

Accordingly, on the surface of meat and poultry stored in the temperature range of -5 - -10 °C black spots or white spots may appear due to the activity of Cladosporium herbarum (white spots) or Sporotrichum carnis, or Thamnidium elegans.Micro-organisms are affected by each phase of the freezing process. During cooling down, a portion of the population is subject of to cold-shock and death. At freezing temperature further death and injury occur. Initially, ice is formed mainly extracellularly, intracellular ice formation is promoted by more rapid cooling. The ice crystalls may mechanically damage cells and the generated high extracellular osmotic pressure (low water activity) will dehydrate cells. Changes in the ionic strength and pH of the water phase as a result of freezing will also disrupt the structure and function of numerous cell components and macromolecules which depend on these factors for their stability.Cooling down to the storage temperature will prevent any further microbial growth if the temperature has dropped below -10 °C. Finally, during storage there will be an initial decrease in viable numbers followed by slow decline over time. The lower the storage temperature, the slower the death rate. As with chilling, freezing will not render an unsafe product safe, because

30

its microbial lethality is limited and preformed toxins will persist. Frozen chickens can be considred, after all, an important source of Salmonella.Survival rates after freezing will depend on the precise conditions of freezing, the nature of food material and the composition of the microflora, but have been variously recorded as ranging between 5 and 70 percent. Bacterial spores are virtually unaffected by freezing, most Gram-positive bacteria are relatively resistant and Gram-negatives show the greatest sensitivity. While frozen-storage does reliably inactivate higher oganisms such as pathogenic protozoa and parasitic worms, food materials often act as cryoprotectants for bacteria, thereby bacterial pathogens may survive for long periods in frozen state. In one extreme example, Salmonella has been successfully isolated from ice-cream stored at -23 °C for 7 years.The extent of microbial death is also determined by the rate of cooling. Maximum lethality is observed with slow cooling where, although there is little or no cold shock experienced by the organisms, exposure to high solute concentrations is prolonged.Survival is greater with rapid freezing where exposure to these conditions is minimized. Rapid freezing with liquid nitrogen as cooling medium, is an efficient tool in preservation processes of micro-organisms.

2.3. Rules of destruction of microorganismsWhen the environment has a lethal effect on the micro-oganisms (high temperature, irradiation, disinfectants) cells are injured, their growth ability is lost and are killed because of key cellular components are destroyed and cannot be replaced. Dead cells are not able to multiplicate and directly cannot be detected by culturing. The examination of death rate is based on the detection of survival rate. Considering that multiplication is greatly depended on environmental factors, in evaluation of death rates the careful selection of appropriate culture medium and culturing conditions play a crucial role.The generaly accepted view is that the destruction rate of microbes can be described on the analogy of first-order chemical processes. At a given lethal effect the rate of death is directly proportional to the concentration of living cells. Upon a given and constant intensity lethal effect, the change in the number of survived cells:

dN/dt = -k·N

where N is the actual number of living cells, dN/dt is the rate of destructin, k is the specific destruction rate, or death rate coefficient. Similarily to the exponential phase of growth, the death of microbes caused by a lethal effect of constant intensity is an exponential process. The concentration of living cells exponentially decreases by time:

N = N0·exp (-k·t)

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Turn on logarithmic form:

log10N = log10N0 - ·t

which is the equation of the survival curve.

The survival curve represents the relationship between the logarithm of the survival cell concentration and the lethal dose (Figure 2.5.). If the intensity of the lethal effect (temperature, disinfectant concentration, activity of irradiation) is constant, the survival curve is linear. The death rate coefficient (k) can be calculated from the slope of this curve.In microbiologcal practice instead of the death rate coefficient, the decimal reduction time (D) is used.Decimal reduction time (D) is the time for the surviving population to be reduced by 1 log cycle (one magnitude). The survival fraction is 1/10 of the initial living cell concentration (Figure 2.5.) and the died fraction is 9/10. In case of irradiation, instead of decimal reduction time, the decimal reduction dose is used.

lg NConstant intensity lethal effectThe values of environmental factors are constatnt

1

D

time (dózis)

Figure 2.5.: Survival curve and decimal reduction time (D)

The relationship between the heat destruction rate (k) and decimal reduction time (D): D = 2.303/k.

The measure of resistance of micro-organisms to the lethal effect is the decimal reduction time, which is highly affected by the environmental factors (temperature, water activity, pH, redox potential). The higher the intensity of the lethal effect, the shorter the decimal reduction time. In food industry, there are three methods for the destruction of microbes: heat treatment,

32

irradiation and disinfection. The application of these procedures will be introduced in the next chapter, in this section we shall deal with the theoretical base of these methods.

2.3.1. Kinetic principles of heat treatmentHeat treatment is the most important technique in food preservation. The purpose of heat treatment technology is to reach microbiological safety without degradation of vitamins, sugars, proteins, etc. In foods. The planning of a proper heat treatment in the industry is based on the Bigelow model introduced in 1921, which describes the thermal death time (TDT) as a function of temperature.Thermal death time (TDT) is the time necessary for n decimal reductions of the population. The survival fraction is 10-n . As decimal reduction time means 1 decimal reduction in survivors, TDT = n D. According to the Bigelow model, the relationship between the thermal death time and temperature:

lg TDT = a - ·T

The relationship is the equation of the heat destruction curve, which has a slope of -1/z. Figure 2.6. represents this curve, which has two important parameters: z and F values.

lg TDT

1

z

lg F

121.1 T (°C)Figure 2.6.: Heat destruction curve, z and F values

z value: is the temperature change (increase), which results in a tenfold (1 log) change (decrease) in TDT. Matematically it is the negative reciprocal of the slope of destruction curve.

F value: is the thermal death time (TDT) at 121.1 °C (250 F).

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The z value is the measure of the temperature dependence of heat destruction rate, similarly to the Q10 value (applied in chemical reaction kinetics), which gives the effect of a 10 °C temperature increase on the chemical reaction or thermal death rate.

Q10 = kT+10 / kT

The connection between the Q10 and z values: Q10 =

The z values of vegetative cells are between 3 and 7 °C, with an average of 5 °C, in case of bacterial endospores is about 10 °C, while the physical and chemical changes in foods have a z value of 30-32 °C.

Substituting the average z values of thermal death into the equation for calculating Q10:

Vegetative cells: Q10 = 100Endospores: Q10 = 10Chemical reactions: Q10 = 2

Comparing the Q10 values of heat destruction to that of the chemical reactions, it is apparent that the temperature dependence of thermal death is far more higher than that of chemical reactions or physical processes. Increasing the temperature with 10 °C, the rate of chemical reaction will be twofold, while the heat destruction rate grows up to 10-100-fold. This recognition has led to the introduction of the UHT (ultra high temperature, 135-140 °C for only 1 second) technology in heat treatment.At high temperature, the death rate of microbes is greatly accelerated, therefore very short exposure time is sufficient to reach microbiological safety. During this short time, the important components of the product will not changen significantly.

12 D conceptFood microbiological safety requirement is for every cases when the growth and toxin production of Clostridium botulinum is possible (pH>4,5, no preservative additives present) that the heat treatment has to result a minimum of 12 log unit reduction in the Clostridium botulinum spores. According to this requirement, in the sterilisation process the heat treatment time means minimum 12 D of Clostrdium botulinum.

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Time equivalent of the heat treatment (F0 value)The heat treatments in food industry, apart from a few exeptions of liquid heat treatment technologies, are not isotherm processes. They consist of heating, temperature keeping and cooloing phases. Destruction of microbes takes place in each section and the result is the sum of the partial destructions. In order to compare the several heat treatment processes, their microbiological efficency is given by an isotherm heat treatment for F0 minutes at 121.1 °C, referring to the thermal destruction of Clostridium botulinum.For example, if the F0 value of a heat treatment process (independently of its temperature-time profile) is 2.8 minutes, it means that the effect of the heat treatment on Clostridium botulinum is equal to the effect of a 2.8 minutes long heat treatment at 121.1 °C temperature.The calculation of the F0 value is based on the establishment of temperature-time profile of the heat treatment and determination of each partial destruction value using the equation of the thermal death curve, finally integration of the partial destructions. Do not confuse F and F0.

The F value is a microbiological feature and it means the thermal death time at 121.1. °C.

The F0 value is a technological feature and it indicates that the effect of the actual heat treatment on Clostridium botulinum spores is equivalent to F0 minutes heat treatment at 121.1 °C.

Concerning that the 12D121.1 of Clostridium botulinum spores is 2.52 minutes, the food microbiological safety requirement for a sterilization process is that F0 ≥ 2.52 minutes.

Example for sterilizationCans of 1 kg (1000 g), with an initial concentration of Clostridium botulinum spores of (N0) 103/g is heat treated. Afer heat treatment (F0 2,52 minutes), the survivor fraction must be reduced by minimum 12 order of magnitude.

N = 10(3-12) = 10-9/g

This concentration of spores means that 1 living spore of Clostridium may occur in 109 g end-product. As 109 g = 106 kg, the probability of finding of 1 infected can is equal to 10 -6. Only 1 can from 1 million.

Heat resistance of microorganismsAccording to their heat resistance, microorganisms can be classified in two groups: vegetative organisms and endospore-forming bacteria (Bacilli, Clostridia).The vegetative organisms including the vegetative form of the endospore forming bacteria have a relatively low heat resistance. Most of them can be destructed by pasteurization, which means a heat treatment below 80 °C. Some heat resistant ascospore forming moulds

35

(Byssochlamys fulva, Neosartoria fisheri) have high heat resistance and sometimes cause problems in tomato juice and soft drinks. Their elimination claims a heat treatment close to 100 °C for 20 minutes.The endospores of bacilli and clostridia mainly due to their very low water activity, are far more heat resistant than the vegetative cells. Thermophiles produce the most heat resistant spores while those of psychrotrophs and psychrophiles are the most heat sensitive. The bacterium endospores can be killed applying heat treatment over 100 °C.There are significant diferences in the heat resistance and also in heat dependence of thermal death rate between the vegetative forms and spores. The vegetative forms not only can be destructed at lower temperatures, but their thermal death rate increase in function of the applied heat increase is much steeper than in bacterial endospores. By comparing of Q10

values, it can be concluded that a heat increase of 10 °C is coupled with 100 times increase in thermal death rate of vegetative forms in contrast to the 10 times increase of death rate of endospores.The heat resistance of some microrganisms of food safety importance is shown in Table 2.10.

Table 2.10.Microbial heat resistance

-----------------------------------------------------------------------------------------------Vegetative microbes (z 5 °C) D (minute)-----------------------------------------------------------------------------------------------Salmonella sp. D65 0.02-0.25Salmonella senftenberg D65 0.8-1.0Staphylococcus aureus D65 0.2-2.0Escherichia coli D65 0.2-2.0Yeast and moulds D65 0.5-3.0Lysteria monocytogenes D60 5.0-8.3Campylobacter jejuni D55 1.1-----------------------------------------------------------------------------------------------

Bacterial endospores (z 10 °C) D121 (minute)-----------------------------------------------------------------------------------------------B. stearothermophilus 4 – 5 ***Cl. thermosaccharolyticum 3 – 4 ***Desulfotomaculum nigrificans 2 – 3 ***B. coagulans 0,1Cl. botulinum Types A & B 0.1-0.2Cl. sporogenes D80 0.1-0.3Cl. botulinum Type E D110< 1 sec-----------------------------------------------------------------------------------------------

*** Heat resistance is greater than in case of Cl. botulinum

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As it is shown in Table 2.10., some bacterial endospores (indicated with ***) have sigificantly higher heat resistance than that of Cl. botulinum A and B.The spores of these microbes can survive the heat treatment with F0 = 2.52 minutes equivalent and may cause spoilage. Considering that these are not pathogen microorganisms, the risk affect only quality with economical consequences but is still unacceptable for the producer. To prevent this type of risk, in case of natural products (without preservative additives), the time equivalent of the sterilisation process is more than 2.52 minutes. Some typical F0 values are summarized in Table 2.1.1.

Table 2.11.Some typical F0 values for canned foods

-------------------------------------------------------------------Foodstuffs F0 (minute)-------------------------------------------------------------------Asparagus 2-4Beans in tomato sauce 4-6Carrots 3-4Peas 4-6Milk pudding 4-10Meats in gravy 8-10Potetoes 4-10Mackerel in brine 3-4Meat loaf 6Chocolate pudding 6------------------------------------------------------------------

The environmental factors affect the heat resistance of microorganisms. The effect of pH and water activity on heat destruction rate is similar for all microbes, while the effect of redox potential depends on the kind of microorganism.

Effect of pH on the heat resistanceThe heat resistance is the highest at the pH optimum of growth. Apart from this optimum, in acidic or alkaline range, the special heat detruction rate (k) increaes. The effect of pH on thermal death time is similar to the pH dependence of the acid-base catalyzed chemical rections.

Effect of water activity on the heat resistanceInvestigations of the effect of water activity on heat resistance resulted in similar conclusions: Heat resistance increases with decreasing water activity for every micro-organisms. The high heat resistance of baterial endspores is due mailny to their low water activity (about 0.37). An

37

important practical consequence of this relationship is that killing of microorganisms applying dry heat requires much higher temperature than heat-killing in moisture (steam) atmosphere.

2.3.2. Kinetic basis of chemical disinfectionThe kinetic description of the antimicrobial effect of a disinfectant is similar to the first order chemical reactions as it was introduced in section 2.3. and demonstrated in Figure 2.5. The death rate coefficient (k) can be determined from the survival curve according to Figure 2.5. The concentration dependence of the death rate coefficient is expressed by the following equation::

where k2 and k1 are the death rate coefficients belonging to c2 and c1 concentrations, and n is the concentration-exponent. The equation gives the change of the death rate as the nth

power of the concentration-quotient.According to the exponential relationship, the logarithm of the death rate coefficient is a linear function of the logarithm of the concentration. The slope of the curve is the concentration exponent. Figure 2.7. demonstrates the realtionship.

log k

n =

log c

Figure 2.7.: Effect of disinfectant concentration on the death rate coefficient

Concentration exponents of antimicrobial agents may range from 1 (formaldehyde, quaternary ammonium bases and mercury compounds) up to 6 (phenols) and 10 (alcohols). To a certain extent, the concentration exponent may be indicative of the mechanism of action. In case of low values (n=1-2), the concentration exponent could be interpreted as the stoichiometric coefficient of the lethal reaction between the disinfectant and a critical stucture-molecule of the cell. At high n values, the lethal effect is probably not stoichiometric. The concentration-

38

exponent gives important information on the sensitivity of the disinfectant to dilution. The higher the concentration-exponent, the higher the sensitivity to dilution For example, in case of n=2 (which is a common value for industrial disinfectants), a three-fold dilution results in 32=9-fold decrease in the activity. That means that the contact time necessary to the same destruction is 9-times higher than at the original concentration. The effect of the same dilution on the activity of alcohol is 310=59049.The equation of the disinfectant concentration dependence of killing time of microbes (belonging to the same death rate) was established by Watson in 1908. It is an useful tool in planning of disinfecting processes.

(where t is the time to kill microbes at concentration c, and n is the concentration exponent)If the concentration exponent is known, it is possible to calculate from one concentration-time data pair any other concentration-time combinations:

In practice, according to the standard European tests for antimicrobial activity, the concentration of a disinfectant is acceptable if it results 5 log degradation in 5 minutes. That means that the decimal reduction time (D) of the population treated with the antimicrobial agent is not longer than 1 minute.

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3. PATHOGEN MICROORGANISMS IN FOODSTUFFS3.1. Occurrance of microorganisms in foodstuffs, infection and contamination of foodstuffs by microorganisms 3.1.1. Occurrance, importance (incidence, significance)Miscellenous microorganism can be detected almost anywhere in Nature. Their basic medium mostly is the soil but are widely distributed also in natural waters, in the air, in plants, on surfaces of animal and human body, furthermore in organs with natural direct connection to environment (e.g. in digestive system and respiratory tracts). Being mainly heterotroph organisms, microorganisms require appropriate carbon hydrogen- and nitrogen-containing compounds for their synthetic and energy-generatig proccesses. These organic materials are available in dead (perished) plants and animals in great quantities. By decomposing and transforming the organic materials, microbial carbon, nitrogen and other natural compounds play a fundamental role in the natural circulation and in creation of the life-conditions of higher organisms. Foodstuffs are components of this natural circulation and most of them are excellent media of microorganisms.Body surfaces of higher organisms and their organs with natural orificies to the environment may contain microorganisms in high number. Majority of these microorganisms are harmless saprophytes and compose the natural microflora of the skin, airways, digestive and urinary&genital tructs. Some species of these common epithelial habitants, however, under certain conditions are able to get into the circulation of the host animal and can induce disease. Microorganisms of exogenous origin may also invade the circulation and consequently the tissues. Both processes result in the infection of the food producing animal and the intravital infection of food of animal origin. A part of pathogen microorganisms can induce not only the disease of the host food producing animal but also the human consumer who is eating the infected food of animal origin (zoonotic pathogens). Pathogen microorganisms may be present in foodstuffs not only due to intravital infection but also on occasion of post mortem contaminations from several other ways and sources.Only a minor part of microorganisms are able to cause diseases. They mostly are saprophytes and can multiplicate under favourable conditions both in fresh and processed foodstuffs and will be present in high number. On course of their metabolic activity, they degrade and transform the components of the foodstuffs, thereby the nutrient-composition and sensory properties of foodstuffs will be changed. This occasionally may cause poorer quality or even, spoilage (e.g. proteinase containing microbes induced rotting or fermentation&acidification caused by carbonhydrate degrading ones).The metabolic activity of micobes is not always harmful. Certain foodstuffs will gain their desirable characteristic properties by metabolites released from the natural microbial habitants or added species. Foodstuffs, produced by useful microbes are for example the fermented milk products, several cheeses, raw sausages and other fermented meat products furthermore, foodstuffs of plant origing produced by alcoholic fermentation (sour krout, pickled cumumber).

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Species belonging mainly to the genus of Lactobacilli and applied as probiotics are also belonging to the useful microbes. These can grow in foodstuffs (especially in fermenetd milk products) but the purpose of their application is not to change the quality of the food but favourably influence the composition of intestinal flora. In spite of some positive applications, the contamination of foodstuffs with microorganisms is not desirable but harmful. The study of certain contamination-indicating, i.e. indicator microorganisms serves the recognision of possible safety risk coupled to the actual contamination. The (non-pathogenic) E. coli for example is the habitant of the normal intestinal microflora. Its occurrance in foodstuffs, however, indicates fecal contamination. Fecal contamination of foodstuffs, nevertheless, may include pathogen bacteria, viruses and parasites. Indicator microorganisms can indicate also the efficiency of preservative procedures (e.g. heat treatment). The determination of total count in foodstuffs may also have indicator role (e.g. indicating general hygiene conditions or in heat-treated or chilled foodstuffs).

3.1.2. Infection, contaminationA basic task of food-hygiene is the prevention of infection or contamination of food by microorganisms. Intravital infection of raw materials of animal origin represents a direct risks for consumers. The primary infection of meat, milk or eggs is usually due to systemic disease of the animal organism (bacteraemia or septicaemia) (alternatively, milk infection may be the consequence of mastitis caused by galactogenically invading bacteria).In animals, classic, clinically characteristicly manifested disease inducing zoonotic causatives (e.g. tuberculosis, brucellosis) were successfully eradicated in the past decades. In general, this greatly reduced the incidence of intravital infections by zoonotic pathogens but symptomless carriers may harbour other zoonotic agents in the intestines from where they occasionally may cause systemic diseases by getting into the circulation through the intestinal wall (translocation) upon the effect of stress (transport, slaughter conditions). Apparently, the appropriate pre-slaughter treatment and humane slaughter of animals is important both of animal welfare and meat safety/quality aspects. At primary infection, in contrast to secondary contaminations on the surfaces, the pathogens are postitioned also in deep of the tissues (e.g. in bulky musculature). At these sites the temperature on chilling can be reduced below 20 °C only during 5-6 hours. Therefore, the low number of translocated and distributed microorganims (<102 CFU/g), are able to multiplicate above disease inducing threshold number.The secondary contamination of foodstuffs may occur during slaughter, primary and secondary processing, storage, distribution or preparation in kitchens. A worth to mention practical source of secondary (cross)contamination of foodstuffs of animal origin with zoonotic pathogens is the injury of the intestines at slaughter and further (cross)contamination during primary and secondary processing.Simultaneously, from the fecal microflora spoilage inducing microbes are also contaminating the body surfaces of the slaughtered animal (carcasses and internal organs), such as

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(Enterobacteriaceae), Lactobacillus, Enterococcusok, ceratin Bacillus and Clostridium species). Fresh milk and egg-shell contamination may also due to fecal origin.Soil microorganisms are responsible mainly for the contamination of plants (vegetablées, spices, fruits). In contrast to the compostion of fecal microflora, the soil flora consits of aerob and anaerob spore forming microbes and fungi but on fertilized fields microorganisms of fecal origin may also enrich the contaminating spectrum.Further important secondary contamination sources of foodstuffs of animal and plant origin are the contacting contaminated surfaces of rooms, equipment, tools, the air, insects, rodents, birds, and the personnel. Food industrial workers may serve as mediators of cross-contamination and are important sources of food-contaminating human pathogens (e.g. Salmonella Typhi, S. Paratyphi, shigellák, Streptococcus pyogenes, Staphylococcus aureus, calici-, polio- and hepatitis-víruses).

3.2. General characterisitics of pathogenicityThe pathogenicity of microbes basically is the consequence of their infectivity, growth potential and toxin producing ability. The measure of pathogenicity, the disease inducing ability is best termed as virulance. The virulant causative is able to evoke diseaase also in hosts with normal resistance or immunity. Low number of virulent cells are sufficient for inducing disease (e.g. the infective dose of E. coli O157 serotype is 10 cells, while other E. coli strains would need an infecdtive dose of million cells).The selective binding of pathogen microbes to the hosts, the access to nutritives necessary for their multiplication and the breakdown of hosts’ resistance are the function of so called virulance factors. These are the adhesines that facilitate of binding, the invasines promoting the penetration into cells and the toxins that paralyse cell functions. Most causatives possess multiple virulance factors tha determine the host and tissue specificity and the character of cell pathology.Toxins may be regarded as the most efficient virulance factors. There are two main groups of exo- and endotoxins. Most exotoxins are proteins that can be inactivated by heat treatement except the heat-stable exotoxin of Staphylococus aureus. Further typical exotoxin examples are the botulotoxin produced by Clostridium botulinum and the shiga toxin (verotoxin) produced by Shigella dysenteriae. This toxin is able to destroy the epithelial cells of the large intestine while one gramm of botulotoxin can kill 1 million people. Endotoxins are the lipopolysaccharides of the outer cell-membrane of Gram-negative bacteria. They are thermophils, remain bound to the cells and are less active than the exotoxins. Mycotoxins are the main pathogenicity factors of moulds and for example the proteases are the virulance factors of yeasts.The manifestation of pathogenicity is greatly depends on the host’s resistance and on environmental factors. Physiological (e.g. age, pregnancy) and pathological (chronic diseases, immunosuppressive effects) conditions reduce the resistance of hosts against causatives. Environmental factors while significantly influence the growth potential and metabolic activity of

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microorganisms, correspondingly affect the risk of development (incidence and severity) of foodstuffs mediated diseases (see also in Chapter 5).Consumption of foodstuffs that contain microorganisms may cause diseases of two types: infectious or toxicoses. On course of infections, the causative is ingested with the foodstuff by the consumer, multiplicate in the intestines and affect the mucosa, occasionally it may cause systemic infection. Characteristic examples are infections caused by Salmonella or Campylobacter species, viral gastroenteritis or toxoplasmosis. During toxicosis, the causative is multiplicating in the foodstuff and produce toxin. The toxin is taken up by the consumer in the foodstuff and induce disease (e.g. botulism, enterotoxicosis induced by S. aureus). An intermediate form is the toxico-infection, when the causative is taken up by the foodstuff and multiplicate in the intestines while produce disease-inducing toxin (pl. Clostridium perfringens).

3.3. BacteriaMajority of foodborne diseases are caused by bacteria or their toxins. Concerning their source, they may originate from food-production animals (zoonotic pathogens), personnel handling foodstuffs (human pathogens) or from the environment (e.g. listeria, C. botulinum). Primary infection or secondary contamination are the ways of bacteria to foodstuffs. Az élelmiszerbe, illetve annak felületére juthatnak elsődleges fertőződés vagy másodlagos Heat treatment appiied during food processing or preparation of meal, bacteria usually can be killed or inactivated (except the enetotoxin produced by S. aureust). Human disease may, therefore, be developed at consuming fresh (raw or inappropriately heat-treated foodstuffs, meals.

3.3.1. Salmonella entericaSalmonellas are intestinal, Gram-negative bacteria (Enterobacteriaceae family), 2-5 m long, mostly ciliated rods. In selective, differentiating media following 27 hours incubation period, they form colonies of 1-2 mm diameter, slightly convex, smooth egded and transparent while after 48 hours incubation, they appear in colonies of slightly bigger, radiating forms, sorrounded by a slimy ditch. Salmonellas do not decompose lactose, saccharose, salycin, adonite and carbamide but decompose glucose, mannite, maltose and sorbite, occasionally decompostion is accompanied with gas production. Most serotypes generate hydrogen-sulfite, reduce nitrite from nitrate and do not favour for indole formation from tryptophane. According to their three main antigens (O-, H-, Vi-antigens), already more than 2500 serotypes are know. More than 99 percent of serotypes are belonging to a single species (S. enterica), and there are six subspecies within the species. Serotypes, important in food-microbiology, are belonging into the Ist subspecies (S. enterica subsp. enterica). In order not to confuse subtypes’ names with binominal species names, subtypes’ names are written with capital letter initiatives but not with italic (S. enterica subsp. enterica serovar Enteritidis vagy röviden Salmonella Enteritidis).

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3.3.1.1. Occurrance, infection of foodstuffs, contamination of foodstuffsSalmonellas are the potential inhabitants of intestines of humans and animals. Certain serotypes are present and induce disease only in definite hosts (e.g. S. Typhi, S. Paratyphi és S. Sendai in humans, S. Typhisuis in pigs, S. Gallinarum és a S. Pullorum in certain poultry species only). Majority of serotypes are facultative pathogen and are able to evoke disease both in humans and animals. These serotypes present in the digestive system cause mostly symptomless carrier conditionin animals, occasionally paratyphus while in humans gastroenteritis or septicaemia will be developed. Salmonellas are released from the animal or human organism to the environment by faeces (into waters, soil, plants), where can survivre for a long period but do not multipicate. The infection of raw materials of animal origin (meat, milk, eggs) may be intravital but mostly due to faecal secondary contamination. The faecal contamination of foodstuffs and waters may be the consequence of slaughter, milking, egg-production hygiene and effluent-treatment anomalies and personnal hygiene.

3.3.1.2. Growth characteristics, resistance to exogen factorsSalmonellas are able to multiplicate in the range of 6 oC - 47 oC temperature (optimum: 35-37 oC) and of 3.8-9.5 pH (optimum is close to neutral). At extreme values, growth is possible only if the other multiplication factors are optimal. The actually desirable minimum pH-value depends also on the quality of the acid (e.g. at identical pH, the growth inhibitory effect of acetic acid is more marked than that of citric acid). For this reason, at mayonaise preparation, the necessary 4.5 pH-value must be adjusted using acetic acid. Water activity less than 0.94 reliably inhibit the multiplication of salmonellas, but bacteria can survive for a long time. Accordingly, salmonellas can survive in milk and egg powder, paste, spices and choclate for months or years.The heat-resistance of salmonellas can be characterised by D60oC=0.1-2 min and D65,5oC=0.02-0.3 min values. In order to make a foodstuff salmonella-free under plant/production conditions, usually heat treatment of seven-times D-value duration is necessary. In practice, this means a heat treatment that ensures at least 70 °C core tempearture for 1 minute. These data, however, are valid only for products with high water activiry, at lower aw-value, the heat-resistance may be much higher. For example in milk-chocklet (aw0.5), the D70oC-value of S. Typhimurium may be 12-18 hours. To kill salmonellas in dried egg products, the usually applied temperature-time combination (64 oC; 2,5 min) for pasteurization of egy-juice is far not sufficient. A significantly longer duration of heat treatment is necessary (e.g. storage at 55 oC for 2-3 weeks). Preliminary, slight heat treatment may significantly increase the heat-resistance of salmonellas (and that of other bacteria). A sublethal heat treatment at 48 oC for 15-30 minutes, increases the heat-resistance of S. Enteritidis up to 2-3 times of the original value. The mechanistic background probably is the synthesis of heat-shock proteins and change in the cell-membrane structure by insertion of saturated fatty acids.

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3.3.1.3. Foodborne diseasesMost of the human salmonelloses are caused by zoonotic serotypes of animal origin taken up by consuming foodstuffs. Nevertheless, human pathogen serotypes without animal pathogenicity may also been mediated by foodstuffs. All serotypes that potentially can cause paratyphus but ususally being present in symptomless carrier form in animals are able to induce diseases in humans. The human disease cases more frequently are caused by S. Enteritidis (a serotype thought to be adapted to humans), more rarely by S. Typhimurium. The incidence of human cases caused by the other serotypes (such as S. Infantis isolated usually from poultry, especially from broilers) is much more rare. The human diseases primarly are induced by infected or contaminated foodstuffs of animal origin. According to Hungarian statistics, more than half of the disease cases are caused by foods prepared using eggs, mainly floating islands, cold buffet kitchen preparations, cakes, pastes but frequently by contaminated, non-appropriately roasted meat (mostly poultry), fresh/raw meat-products, more rarely fresh milk, fish, shells, and contaminated, non-washed vegetables. (3.1. table).

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3.1 TableFoodborne bacterial diseases

Pathogen Infective dose Symptoms Mediator foodstuffSalmonella spp. 103-105 cell General nause, fever,

vomiting, diarrhoea, rarely septicaemic symptoms, arthritis

Meals prepared with eggs, raw, non-appropriately heat treated meat, meat preparations, fish, shells, vegetables

EnterohaemorrhagicE. coli (EHEC)

101-102 cell Watery, later bloody diarrhoea, definite abdominal pain, haemolysis, acute renal insufficiency (HUS)

Raw, not well done beef, fresh milk, salads, drinking water

Yersinia enterocolitica 104-105 cell Abdominal pain, diarhhoea (children), appendicitis like symptoms (adults)

Raw, non-appropriately roasted pork, fresh milk, vegetables

Shigella spp. 101-102 cell Profuse, watery, later bloody diarrhoea, abdominal pain, fever

Not-zoonosis, any kind of foodstuffs

Vibrio parahaemolyticus 105-107 cell/g Abdominal pain, diarrhoea (occasionally bloody)

fish, shells, crabs

Campylobacter spp. 102-103 cell Definite abdominal pain, watery or bloody diarrhoea, fever, occasionally compications

Raw, not appropriately roasted poultry meat, fresh milk, drinking water

Listeria monocytogenes >102 cell/g Flue-like symptoms, meningitis, meningoencephalitis, abortion, still-born

Fresh seep and goat milk, milk products, raw meat and meat products, fish, shells, vegetables, fruits

Staphylococcus aureus 106 cell/g (0,1-0,2 g/g toxin)

Vomiting, diarrhoea, abdominal pain

Food toxicoses, foodstuffs containing egg with enterotoxin, boiled paste, fresh milk, milk products, raw sausages, pork-cheese

Clostridium botulinum 0,1-1 g toxin General nausea, abdominal pain, visual disturbances, difficulties in speaking and engulfing, respiratory paralysis

Food toxicoses, mainly home-made cans containing botulinum toxin, processed preparations, expired ready to eat meals, fish, shells, crab-salads

Clostridium perfringens 103-105 cell Profuse, watery diarrhoea, abdominal pain

Mostly meat meals stored warm for a longer period

Bacillus cereus 105-107 cell

105-108 cell/g

Fever, watery diarrhoea, abdominal painvomiting, rarely diarrhoea

Meat meals, soups, dressings

Rice meals, pastes

Eggs can be infected germinatively (ovogen infection) but salmonellas (and other causatives) usually are getting into the egg through the pores of egg-shell following fecal contamination. This potential penetration is greatly inhibited by the glycoprotein layer covering the shell. Washing and mechanical cleaning remove the protective layer and facilitates the penetration of causatives. The more porous duck egg shell structure promotes the penetration of salmonellas, therefore duck egg must not be used for preparation of foodstuffs.

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The contamination of meat is primarily owing to fecal contamination as a consequence of imperfectness of slaughter technology. It can be detected most frequently in poultry slaughter tecnology because it is highly automated and running with very high line-speed (See also in chapter 9).The uptake of 103-105 bacterial cells with food usually is able to induce disease in humans, i.e. the bacterium first must multiplicate in the foodstuff. Following this, salmonellas further grow in the human small intestines and are bound to the eptithelial cells and heat-labile enterotoxin and cytotoxic protein are released into the intestinal lumen evoking diarrhoeic and other symptoms of enteritis. The multiplicating bacteria can move into the lymphatic and blood vessels and may cause systemic infection up to septiceamia.Symptoms, usually appear 6-24 hours after the consumption of the contaminated foodstuff in forms of heasache, general nausea, transient higher body temperature or fever and diarrhoea (non bloody). Persisting diarrhoea may cause significant fluid loss and life-threatening condition that should be prevented by treatment in hospital.The measure of foodstuff contamination (i.e. the initial count of bacteria taken up by the host) can significantly influence the duration of incubation period and severity of disease. The lethality mostly is 0,1%, but especially in elder, above 65 years old people, it may be several times higher. Diseased indivuduals, first of all in the initial-transient phase of the infection, are releasing the bacteria in great number in the faeces and this usually is over by 1-2 weeks (this period may be much longer in children).

3.3.2. Escherichia coli E. coli is a representative member of the Enterobacteriaceae family. According to DNA-homology, the E. coli and the four Shigella species are belonging to a single species, but based on biochemical and epidemiological differences, they are sorted into different genera. Members of the E. coli species usually are ciliated (rarely are without cilia) 2-3 m long rods and the surface of most genera are equiped with fimbriae.They form colonies of 1-2 mm diameter in normal agar under both aerobe and anaerobe conditions during 24 hours. The colonies are decomposing lactose and are red in lactose-litmus agar, while in bromide-thymolblue-lactose agar they are yellow. E. coli produce indole from tryptophane, acid and gas from from lactose (certain genera not or slowly decompose lactose) and are positive in methyl-red test. They can be differentiated biochemically from the closely related coliforms by the IMVIC tests (indole, methyl-red, Voges-Proskauer and citrate reactions)l (3.2. table).

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3.2. tableThe pattern of IMVIC tests in the presence of different enterobacteria

Species Indole Methyl-red Voges-Proskauer

Citrate

Escherichia coli + + - -Shigella -/+ + - -Salmonella - + - +/-Citrobacter - + - +Klebsiella -/+ - + +Enterobacter - - + +Proteus +/- + - +/-Yersinia -/+ + - -

The E. coli strains can be sorted into miscellenous serotypes on the basis of O-, K-, H- és F-antigens. Considering that the antigens may occur in any combination, a great number of serotypes are existing.

3.3.2.1. Occurrance, pathogenicityE. coli is a common habitant of the intestines of humans, warm-blooded animals and amphibia and is the natural component of the aerobe and facultative anaerobe bacterial flora. As high as 109 cell/g can be found in human faeces therefore, it is the main indicator of fecal contamination. Majority of E. coli strains are harmless but some are facultative pathogens. These latters, based on their virulance characteristics, K or O:H serotypes, mechanism of their pathomechanism and the pattern of clinical symptoms are classified into four main groups (3.3. table).

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3.3. tableGrouping of Escherichia coli strains based on their pathogenicity

Group Pathology, virulance factors

Morbidity Symptoms Zoonosishumans animal

EnteropathogenE. coli (EPEC)

Severe damaging of epithelial microvilli

+ + Watery diarrhoea -

EnteroinvasiveE. coli (EIEC)

Necrosis of epithelia in large intestinal mucosa

+ + Bloody diarrhoea (dysenteric-like)

-

EnterotoxicE. coli (ETEC)

Production of enterotoxins (ST, LT)

+ + Watery diarrhoea -

Enterohaemorrhagic E. coli (EHEC)

Producion of verotoxins (cytotoxins)

+ + Haemorrhagic colitis, hemolysis, uraemia (HUS)

+

ST = heat-stabile, LT = heat-labile, HUS = haemolytic uremic syndrome

Certain serotypes of E. coli may cause disease both in humans and animals, manifested mainly in form of enteritis, less frequently septicaemia or other symptoms. Neveretheless, E. coli infections in humans usually induced by other serotypes than in animals, thus, the EPEC, EIEC and ETEC strains are not considered to be zoonotic. The verotoxin producing EHEC strains occurring for example in the intestines of cattle can be transferred into the human organism by foodstuffs and may evoke severe, zoonotic diseases. The disease in humans is characterized by the development of haemorrhagic enterocolitis, fllowed occasionally by haemolysis and consequent uremia (haemolytic uremic syndrome: HUS). These same strains may cause haemorrhagic enterocolitis also in calves and they may be present in the intestines of other food-production animal species in form of symptomless carriers (cattle, small ruminants, swine, poultry). A characteristic representative of the verotoxin-producing strains is the O157:H7 serotype, that was isolated from hamburger in the USA at 1982 on occasion of a hamburger induced epidemic ("hamburger disease"). Human infections are due to the consumption of fresh or not well-done meat contaminated during slaughter (mainly beef and ground beef) and (fecal-contaminated) fresh milk, vegetables, salads.

3.3.2.2. Growth characteristics, heat-resistanceE. coli is a characteristically mesophil bacterium, that can multiplicate in the temprature range of 7-50oC (optimum around 37oC). The highest rate of growth is at close to neutral pH, but under otherwise optimum conditions it will multiplicate down to pH 4.4. Usually the acid-resistance of the verotoxin-producing strains is higher than that of other E.coli strains. The minimum necessary water avtivity value for growth is 0.95. Its heat-resistance is not marked,

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the related D60oC-value is 0.1 min. The heat-resistance of the verotoxin-producing strains is higher, the corresponding D60oC-value is 2-2.5 min.

3.3.2.3. Human diseases The infective dose of EHEC strains is very low, already 10-100 bacteria are able to induce disease in humans. Generally, the incubation period is 1-2 days (sometimes 3-8 days), followed by the appearance of abdominal pain, and initially watery, later bloody diarrhoea, meantime, the abdominal pain gradually becomes more intensive. The lack of fever differentiates the disease from other types of inflammatory colitis. The occurrance of enterocolitis is more frequent in adults and it may become fatal in elderly people.The haemoytic uremic syndrome can be developed mostly in children. In Europe and North America EHEC strains are thought to be the principal causatives of the acute renal insufficiencies of children. In 10% of children below 10 years old infected with verotoxin producing E. coli strains, HUS is developing with high, 3-5% lethality. The disease is characterized by haemolytic anaemia, trombocytopenia and acute renal insufficiency, occasionally initially appearing bloody diarrhoea.

3.3.3. Yersinia enterocoliticaThe Yersinia genus of Enterobacteriaceae family includes three human pathogen species, namely the Y. pestis, a Y. pseudotuberculosis és a Y. enterocolitica. The latter two can evoke disease also in animals but foodborne disease (food infection) is caused only by the Y. enterocolitica. The Y. enterocolitica is short, 1-2 m long rod Gram-negative bakterium. It is facultative anaerobe, catalase-positive and oxydase-negative.Based on their somatic (O)- and flagellar (H)-antigens, the Y. enterocolitica strains are sorted into serotypes and, according to their biochemical properties, into biotypes (3.4. table).

Table 3.4. Characteristics of the different bio-and serotypes of Y. enterocolitica

Biotypes Serotypes Characteristic host Symptoms Occurrance1 08 humans gastroenteritis North America2 09 humans

swinegastroenteritissymptomless

Europe, JapanEurope, Japan

3 01 chincilla septicaemia Europe4 03 humans

swinegastroenteritissymptomless

Europe, JapanEurope

5 02 hares, goat septicaemia Europe

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The human diseases in Europe are caused caused by the 4/03 and less frequently the 2/09 bio/serotypes. Swine is a symptomless carrier of these strains and bacteria are present primarily in the intestines but may occur in other animal species such as ruminants, hare, games). Human diseases are caused by consuming of fresh or not well-done pork contaminated during slaughter, more rarely fresh milk contaminated during processing or fertilized, non-washed vegetables.

3.3.3.1. Growth characteristics, heat-resistanceY. enterocolitica is a psychrofil bacterium, that can readily multiplicate in a wide temperature range (-1oC - 40oC). The optimum growth temperature and pH is 29oC and pH 7-8, respectively (minimum pH is 4.2 and minimum water activity is 0.96.). It is heat-sensitive but there is a significant difference between heat-resistance of strains (the D-value is between 1-60 sec at 63oC).

3.3.3.2. Human diseasesDiseases due to Y. enterocolitica infection can be developed following 1-10 days incubation mainly in children younger than 5-7 years old. Symptoms are abdominal pain for 1-2 weeks accompanied by slight increase in body temperature and diarrhoea. Vomiting is rare. Occasionally, the acute ileitis and the inflammation of mesenterial lymph nodes results in symptoms similar to observable in case of appendicitis. These latter symptoms characterize the disease in adult. Rarely, the infectrion is coupled with extraintestinal complications (e.g. arthritis, erythema nodosum).

3.3.4. Enterobacter spp.The members of the Enterobacter genus belonging to the Enterobacteriaceae family are living in the soil and (surface)waters but may occur also in intestines and airways of humans and animals. E. cloaceae and E. aerogenes may play a role in human pathology of the intestinal tract (enteritis, enterocolitis), urinary tract infections (cystitis, pyelonephritis), upper airways inflammations (bronchitis) and of septic processes (meningitis purulenta). Specifically in neonates, E. sakazakii may induce septicaemia, meningitis, accompanied with high (40-80%) letality. These latter infections occasionally are owing to the consumption of breast-milk replacers contaminated with the bacteria.The members of this genus are rods of vividly advancing by means of peritrich cilia, biochemically are very active including the decomposition of lactose and are positive in the Voges-Proskauer test. E. sakazakii often produces yellow pigment. Its growth optimum is at 37oC, but it still intensively multiplices at 23-25oC (its generation time at this temperature is 40 min) and cannot grow at 4oC. It is relatively resistant to heat, at 60oC its D-value is 2.5 min. The virulance factors of E. sakazakii are less known but a part of its strains may produce enterotoxin.

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The infective dose of E. sakazakii is low; in breast-milk replacer already in a quantity of <3 cell/100 g may cause disease. After dissolving the powder form of breast-milk replacer product, the resulting fluid form is an excellent culture medium for the multiplication of E. sakazakii if it is kept at 25-45oC temperature. For this reason, if the dissolved replacer is not consumed within a short time, it must be chilled back to 4-8oC and be stored at this temperature. The prepared fluid replacer may be stored for 4 hours at ambient temperature. E. sakarakii, among the members of the Enterobacter genus, is the least heat-sensitive. To kill at 60oC, several m inutes heat-exposure is necessary, but at 70oC it will be killed fast.

3.3.5. Shigella spp.The members of the Shigella genus, the S. dysenteriae, S. flexneri, S. boydii and the S. sonnei are 2-3 μm long, non-ciliated rods. They are the causatives of the human dysentery and do not occur in production animals. The source of contamination of foodstuffs are the nfected and discharging humans, sometimes as vectors, the flies. Shigellas are mezophil bacteria, growing at 10-45oC temperature and at an optimum of pH 6-8 values, respectively. Below pH 4.5, they are perished.The significance of the miscellenous species is changing according to different regions. The epidemic level dysentery in developing countries is caused by the S. dysenteriae, in Hungary most of the sporadic cases are induced by the S. sonnei. The symptoms are appearing some days after the infection in form of severe, watery diarrhoea, followed by bloody defecation, abdominal pain and fever. In organisms with appropriate immune condition, the disease spontaneously disappear within 3-5 days. The dysentery can be fatal in infants and in elderly people. S. sonnei usually evoke a less severe form accompanied with watery diarrhoea.

Vibrio spp.Vibrios are relatives of enterobacteria, and are 1.5-3 m long rods, Gram-negative, facultative anaerobe, fermentative, and in contrast to enterobacteria, they are oxydase-positive bacteria.More than 30 species of Vibrio genus are known, most of them are iiving int he sea-water and they can be found in the digestive tract and surfaces of marine living creatures. Certain species, however, are the habitants of fresh waters. Majorities are harmless saprophites, but pathogens and facultative pathogens are also represented.From Vibrio species, the V. cholerae is the causative of human cholera. It is spread by the infected drinking water. In several developing countries, in people living under poor hygiene conditions, the incidence of the diseasea and mortality is high. The disease in Europe occurs only sporadically/exceptionally.The natural reservoir of V. parahaemolyticus are the sea water and marine fish, molluscs. In humans, it may cause food infection after consumption of infected, fresh or undercooked fish, shells, crabs. Symptoms are gastroenteritis, rarely septicaemia. In Japan people happily consume fresh fish, consequently, about 70% of food-infections are caused by V. parahaemolyticus.

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The causative is able to multiplicate in the temperature range of 5-43 oC (optimum 37oC), but usually it cannot grow below 10oC. Under favourable conditions its multiplication rate is extremely high, the generation time is only 11 minutes. The optimum pH is a slight alkalic (7.5-8.5) but it can grow at up to pH 11. Vibrios usually are acid-sensitive but the V. parahaemolyticus may multiplicate at down to pH 4.8-5.0. It is halophyilic, resisting to 8-9% NaCl concentration (optimum 2-3%) and is slightly heat-resistant with a D60oC-value of 0.7 min, (i.e. at 60oC temperature, it will be killed by a 1 minute heat-treatment).The infective dose is realtively high of 105-107 cell/g. The pre-condition of the pathogenicity is the ability of haemolysin-production (Kanagawa-positive strains). The development of the disease is manifested in diarrhoeic symptoms following an usual 12-24 hour incubation period, and the symptom is accompanied by abdominal pain, rarely slight fever and vomiting or dysentery-like bloody diarrhoea. The disease mostly is recovered spontaneously in 2-5 days.

3.3.7. Campylobacter spp.Campylobacter originally were classified into the family of vibrios, todays, however they form a separate family of gamma-proteobacteria. Until the begining of the1970s, the members of the family such as C. fetus and C. jejuni were considered as causatives of only animal diseases (abortions, enteritises, hepatitis). By the development of culture methods, campylobacters could be isolated also from the faeces and food commodities, and became apparent that C. jejuni and less importantly, C. coli after taken up by food, are able to induce mianly diarhhoeic and occasionally more severe extraintestinal symptoms. Todays, in most countries, campylobacter is the most frequent cause of food-infections.They are curved (campylo=curved) rod or S shaped, ciliated at one or both ends, 2-5 m long, Gram-negative, microaerophil bacteria. For growth, campylobacter require 3-6% oxygen and 2-10% carbon dioxide. They are oxydase-positives, do not decompose carbon-hydrate, and they gain their energy-supply from amino acids and organic acids (e.g. pyruvate).

3.3.7.1. Occurrence, contamination of foodstuffsMost of Campylobacter species are saprophytes, but some of them are facultative pathogens.Campylobacte fetus subspecies fetus may cause abortion in sheep, less frequently in cattle. Rarely, it may induce illness in humans in form of septicaemia and abortion. C. jejuni and C. coli are natural components of intestinal flora of several mammalian species. They may cause sporadically abortion or pneumonia in ruminants and enteritis in new-bornes. In hens at early laying period, Campylobacters can multiplicate into the circulation and may evoke diarrhoea and hepatitis and infrequently, consequent extraintestinal complications. Humans can be infected by consuming not well-done poultry meat, less freqently meat of mammals contaminated during meat-processing at slaughterhouses or by feacal contaminated fresh milk, shells, mushroom, fresh vegetables and drinking water. The surface of fresh, pre-chilled poultry meat usually is contaminated in 20-75%-C by C. jejuni. In contrast to salmonellas, Campylobacter species cannot invade eggs germinatively and by drying on the shell, they quickly perished.

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3.3.7.2. Growth characteristics, heat-resistanceCampylobacters are thermophyl bacteria, the optimum of growth of C. jejuni and C. coli is at 42oC, and the minimum is at 30-32oC. The thermophyl character and the widespread avian intestinal distribution of these bacteria can be considered as an adapation to the body temperature of birds (41-42oC). Nevertheless, campylobacters are sensitive also to higher temperartures and are killed at 55oC applied for 1 minute (D55oC1 min).Though, they do not multiplicate at lower temperarture but can survive for a longer period of weeks-months while chilling or freezing. They are sensitive to dryness with a minimum water activity requirement of 0.99 and usually are perished soon in the environment but survive for weeks in surface waters. In spite of being sensitive to pH reduction, campylobacters are able to multiplicate at as low pH as 4.9.

3.3.7.3. Human diseasesThe foodborne diseases are caused primary by C. jejuni, and rarely by C. coli. Most of the causative strains are directly of animal origin and are getting into the human consumers’ organism with foodstuffs contaminated by faeces. Occasionally the food industrial personnel also can serve as contamination source.Corresponding to the thermophil character of the bacterium, the majority of human diseases are developing in summer times mainly in children of 1-4 but, according to epidemiological data, also between 15-24 years old ages.The infective dose of the causative is low, already 100-500 (sometimes 10-100) cells can induce diease. The virulance factors and the consequent pathomechanism are not completely known but the colonisation promoting adhesins and the epithel-damaging cytotoxic toxins may play a role in the processSymptoms are appearing after a usual 3-5 days (sometimes 1-11 days) incubation period in form of definite abdominal pain, fever and diarrhoea. Vomiting is rare. The faeces contains 10 6-109/g bacteria, it is often evely smelling and it may be bloody or watery, dysenteric-like. The gastrointestinal symptoms occasionally are preceded by nausea (headache, dizziness, fever) outlasting for 1 day. The disease is over after 5-8 days but the causative may be released for 2-3 weeks. The complications are unusual but as in salmonellosis, reactive arthritis may be developed. Campylobacter infection may contribute to the development of severe neurological symptoms or to polyneuropathia and polyradiculoneuritis manifested in respiratory paralysis or to Guillan-Barré syndrome (GBS).Beyond theCampylobacter genus, the Campylobacteriaceae family includes the members of the Arcobacter and Helicobacter genus. The Arcobacter species are aerotolerants and can grow even at 15oC temperature. In spite of that Arcobacter strains are relatively often isolated from the surface of poultry meat, less frequently from pork and beef, the number of food infections of Arcobacter origin is very low. A. butzleri, following the intake of contaminated food, may cause severe diarrhoe, occasionally septicaemia in children and in

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immunocompressed adults (in adults with diminished resistance). The rate of incidence is higher than was diagnosed earlier.Helicobacter pylori, after colonising the human gastric mucosa, can induce chronic gastritis and consequent gastric and duodenal ulcer that can be terminated in adenocarcinoma. It survives only in the human gastric and duodenal mucosa, in the environment it will be died soon.

3.3.8. Listeria monocytogenesListeria are short (1-2 m), ciliated Gram-positive rods. Pathologically, only the L. monocytogenes is significant. it is an important zoonotic bacterium that, in most cases following its intake by infected or contaminated foodstuffs, may cause severe diseases in animals and humans. The bacterium is aerobe, facultative anaerob, catalyse-positive, esculine hydrolysing and arginine non-hydrolysing agent that generates acid from glycose or maltose and does not decompose mannite or xylose. Its pathogenic ability corresponds to the haemolytic and lypolytic activities and toxic effects of the monocytosis inducing factor and lipoid compounds, respectively found in the cell-wall.

3.3.8.1. Occurance, pathogenicity, infection and contamination of foodstuffsThe bacterium is widely distributed in the soil, in natural waters, effluents, on plants, in the intestianl tract of healthy humans and animals (mainly in ruminants and birds). Its natural medium is the soil, from where it is taken up in soil-contaminated feed by animals or in soil-contaminated foodstuffs by humans. Humans can also be infected by intaking of infected or contaminated foods of animal origin.A L. monocytogenes is a facultative pathogen. Humans, domesticated and wild mammals and birds are susceptible. It can induce disease mainly in young or pregnant animals, especially in ruminants and rabbits, occasionally in poultry. In ruminants it may cause mastitis and by secreting into the milk, it may result in primary, intravital infection of milk. Usually, however, it is in the intestinal tract symptomless and may induce secondary contamination of fresh milk and meat (contaminated during slaughter or secondary processing).The majority of foodborne human diseases are caused by infected or contaminated fresh milk (especially sheep and goat milk) and fresh milk-based milk products and by contaminated fresh meat and fresh-meat based meat products (raw sausages, salami), fish, shells, furthermore vegetables and fruits contaminated by soil. The moderate acidity plant foodstuffs (e.g. sauerkrout and different vegetables, including those produced from sprouted/germinated seeds) may carry bacteria and may mediate human diseases.Bacteria may occur also in the intestines of healthy humans and can be released by the faeces. The probability of this occurrence was surveyed in healthy slaughterhouse workers and in patients hospitalised on other reasons and it was between 1-5% indicating that workers potentially can contaminate the foodstuffs.3.3.8.2. Multiplication characteristics and resistance

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L. monocytogenes is a psychrophyl and halophylic bacterium with a temperature optimum of 30-35oC, but it is able to multiplicate in the wide range of 0-42oC, i.e. also at frigo-temperature (<5oC). Under chill temperature the growth rate is slow, its generation time is above 12 hours. The multiplication is inhibited below pH 5.5 but it is able to grow, depending on the type of the acidifying compound, down to pH 4.4. Being halophylic, it can grow in medium containing 10% NaCl and it preserves its vitality in the presence of 16% NaCl at pH 6.0 up to 1 year. The bacterium usually survives freezing and forms biofilm on the surfaces.Its heat-resistance is similar to the other non-sporulating Gram positives having some minutes and some seconds values of D60oC-, and D70oC, respectively. The generally applied fast pasteurization of milk in practice (72oC, 15-40 sec) reduces the surviving microbe number by at least 5 magnitudes, resulting in an acceptable safety if the initial bacterial load of fresh milk is sufficiently low.

3.3.8.3. Human diseasesIn spite of the wide-spread environmental distribution, the bacterium relatively rarely causes human disease. The manifested cases, however, can be very severe with significant lethality of 20-30%-os. The estimation of the minimum infective dose alway carry a certain uncertainity and this is specifically true for the L. monocytogenes. Considering that in the mass listeria-coupled food-infection cases, the bacterial count of the foodstuffs was always above 10 2 cell/g, the minimum infective-dose is thought to be relatively high.Clinical manifestations occur mainly in children, in the elderly, in individuals with lower resistance and first of all in pregnant women. In these latter, the risk of infection is 10-12 times higher than the usual.The symptoms are appearing late after the ingestion of the infected foodstuff (by 10-18 or 3-70 days). Thus, the incriminated foodstuff often canot found already at time of symptoms’ manifestation. In contrast to food-infections caused by intestinal bacteria, the L. monocytogenes induced diseases are characterised by extraintestinal symptoms, in mild cases a flue-like condition, in the more severe cases meningitis, meningoencephalitis, occasionally endocarditis. In preganat women most often flue-like symptoms can be seen with fever, headache, occasionally with abdominal pain to which the transplacental infection of the foetus can be associated and abortion, premature birth or still-borne take place. In infected infants, days or weeks after birth septicaemia, granulomatosis infatiseptica, pneumonia, or meningitis can be developed with significant (25-40%) mortality. The infection is characterized by highly elevated monocyta number in the blood after which the causative is named as monocytogenes.

3.3.9. Staphylococcus aureusStaphylococci are Gramm-positive, spherical, aerobe, facultative anaerobe bacteria. They are habitants of the skin of humans and animals, the upper respiratory and intestinal tracts, the mucosa of urinary tract and genitalia, but also of the soil, natural fresh waters and plants.

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More than 30 species are belonging to the Staphylococcus genus from which the most important pathogens are the coagulase positives, first of all the S. aureus and S. intermedius. The coagulase-negatives mostly are saprophytes but some may cause dermatitis (e.g. S. hyicus, S. epidermidis) and S. intermedius induces purulent processes mostly in carnivores. In food production animals principal pathogen is the S. aureus.

3.3.9.1. Occurrence, pathogenicityS. aureus is widely distributed in the environment, and is harboured also by most domestic animals. More than 50% of healthy human individuals are carrying the causative mainly on the surfaces skin and the epithel of the respiratory tracts, usually as harmless saprophyte. The bacterium, however, is facultative-pathogen and in case of reduced resistance of the organism, it may initiate local inflammatory processes (e.g. dermatitis, tonsillitis), or septicaemia.S. aureus strains may induce purulent, mostly local inflammation in animals, mastitis in ruminants, septicaemia, arthritis, and dermatitis in birds. Strains pathogenic in animals typically are not pathogenic in humans and vica versa, human pathogens are not pathogens in animals though occasionally may colonize the organism and in cattle may cause mastitis.The food safety importance of Staphylococci is coupled to the multiplication of certain strains in foodstuffs and production of heat-stable enterotoxin. Following the ingestion of sufficient amount of the enterotoxin, it causes food toxicosis in the consumer manifested in acute symptoms within a few hours.There are seven enterotoxin types, most frequently the A and D types can cause toxicosis. The enterotoxins are small molecular weight polypeptides and they resist to the activity of intestinal proteases and are very heat-resistant being inactivated only by long lasting boiling (D100=1-3 hours, D120=10-40 minutes).

3.3.9.2. Multiplication characteristics, toxin productionS. aureus is a mesophyl bacterium, the optimum of growth and toxin production is around 37oC. It can multiplicate until 6oC, but the toxin production is ceased below 10oC. Similarly, it is able to produce toxin only within a narrower pH range than is necessary for growth (Table 3.5.), and below pH 6, the bacterium produces a very few toxin. The toxin production at optimum micro-ecological growth conditions usually is intensive, but there might be very marked differences.

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Table 3.5.Ecological parameters of the multiplication and toxin production of S. aureus

Parameter Multiplication Toxin productionRange Optimum Range Optimum

Temperature (oC) 6-48 35-37 10-45 35-40pH 4.0-9.8 6.0-7.0 4,5-9,0 6.0-7.0NaCl (%) 0-20 0.5-4.0 0-20 0,5Water activity 0.83 - >0.99 0.98 - <0.99 0.86 - >0.99 >0.99Redox-potential -200 mV -

>+200 mV>+200 mV ? >+200 mv

It is a halophylic bacterium, certain strains are able to grow even in the presence of 20 % NaCl and may produce toxin. Correspondingly, they are tolerant to the water activity of the medium. The minimum water avctivity value for growth and toxin production is 0.86 and 0.83, respectively (evidently, under this condition their multiplication rate is very low with a generation time of >5 hours, and only a minute quantity of toxin is produced). Its heat-resistance is normal, the D-values measured at the fast and slow pasteurization temperature of milk (62oC and 72oC) are characteristically 1-2 minutes and 5-10 seconds, respectively.

3.3.9.3. Development of food-poisoning, symptomsThere are two basic conditions of the development of food poisoning: the contamination of foodstuff followed by the multiplication of the pathogen bacterium and its toxin production in the foodstuff.The contamination can be the result of intravital infection, e.g. mastitis caused by S. aureus, when mastitis-inducing strains multiplicating in the milk are producing toxin in the animal. Though, historically this was the first bibliographic example for S. aureus caused food poisoning, in practice this is rather sporadic because the mastitis-inducing strains very exceptionally produce enterotoxin.The poisonous quantity of toxin in foodstuffs usually is 100-200 ng/g, but in the case of A-type, lower quantities can be toxic. The source of contamination typically are the carrier humans, who directly (by the airways’ excreta, or by infected wounds, dermatitis) or more frequently indirectly (via contaminated human hand) contaminate the foodstuffs. Therefore, in the prevention a specifically important factor is the personal hygiene.In order to reach the infective dose of the toxin, the bacteria must be multiplicated in the foodstuff in great number (>105-106/g). The fresh meat and fresh-meat-based meat products (e.g. sausages) can be secondary-infected by toxin-producing strains of animal origin during processing. This option is also infrequent.S. aureus is a relatively undemanding microorganism, it is able to grow in the most miscellenous foodstuffs and this is promoted by the high protein and water content of food and

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its temperature of around 37oC. A mixed microflora, however, can significantly slow down, even can inhibit the multiplication (see also in Chapter 5).Consequently, human disease, food poisoning are caused by egg-containing foods such as, creams, foams, pastries, noodles/pasta contaminated by strains of human origin, especially if following preparation they are kept at room temperature (i.e. without refrigeration) for a long time. Less frequently, fresh/raw meat-products, headcheese (brawn), fresh milk and fresh milk based milk products may be the source of food poisoning (formerly, most food poisoning were due to the consumption of ice-cream, but it is over since it is made from powder).Ingesting the foodstuff containing sufficent amount of toxin, the symptoms are developing within a short time (2-4 hours) in forms of nausea, vomiting, gastric spasm and diarrhoea. The course of the disease is fast, the symptoms usually are passed in 1-2 days, In severe cases, because of the significant fluid loss, the patient should be hospitalised.

3.3.10. Clostridium botulinumClostridia are Gramm-positive, anaerobe, thick rod shaped, spore forming bacteria. Their natural habitat is the soil but they usually can be found also in the human and animal intestinal tract. Clostridia can induce disease only if on any reason, they are getting into the tissues from the intestines or soil and there multiplicate or if their toxin is absorbed from the intestines.From food safety aspect, the important clostridial species are the severe food poisoning inducing C. botulinum and the toxico-infectious C. perfringens.

3.3.10.1. Occurrence, classificationThe vegetative forms and spores of C. botulinum are widely distributed in the nature and are multiplicating under anaerobe conditions (e.g. in soil, mud, in the intestinal tract of mammals, birds and fish). Spores are extremely resistant to heat and ionising rays but the germinatig spores are already sensitive to heat. The germinated, vegetative cells are multiplicating under anaerobe (microaerophyl) conditions and in optimum micro-climate (ecology) (temperature, humidity) they become numerous and produce toxin.Strains can be sorted into 7 types based on their produced toxin (A, B, C, D, E, F, G). A C and the D types can be found in the soil and in the intestinal tract of animals and in animals almost always these types are the causatives of diseases. Humans are diseased mainly by the A, B and the E types, less frequently by the F type. The G type is also living in the soil, but it generates toxin only in minute amount. In foodstuff type G has not been detected up to now. The C. botulinum strains can be grouped into four biotypes based on their molecular-biological, biochemical and ecological properties. (Table 3.6.).

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Table 3.6.Classification of C. botulinum strains

Group Toxin-

productions

Biochemical activity Growth conditions Heat resistance Pathogenicity

Proteolysis Lipolysis Sacharose-

decomposition

Min. temp Opt.

hőm.

Min.

pH

Min.

aw

Sótűrés D100

min

D121

min

I. A, B, F + + - 10-12oC 35-40oC 4.6 0.94 10% 30 0,1-0,2 Humans

II. B, E, F - + + 3-5oC 25-30oC 5.0 0.97 5% <0.1 <0,001 Humansr

III. C, D - + - 15oC 40oC 3% 0.1-1 Animals

IV. G +/- - - 12oC 37oC 3% 0.8-1,2 -

Strains belonging to group I are mesophyls, they do not grow below 10oC, therefore are not important in chilled foodstuffs. In the same time, their spores are the most heat-resistant ones (D121oC= 0.1-0.2 min). The members of the group possess definite proteolytic activity resulting in organoleptic change in foodstuffs (scraping, cheesy taste) but spices can hide this abnormality or it is hardly realised if consumed cold.Strains of group II are psychrophyls, they may be grown, enriched and produce toxin also in chilled foodstuffs (minimum temperature 3oC). They are more sensitive to the pH, water activity and salt concentration of the medium than microbes of group I and the heat resistance of the spores are also lower (D100oC<0.1 min). The growth rate and toxin production are slow at chill temperature. At 3.3oC, they require 1-2 months for producing poisoning quantity of toxin but at 5oC, already 15 days are sufficient. In Hungary, the human pathogen strains of C. botulinum are belonging to group II.

3.3.10.2. Toxin production, mechanism of action, contamination of foodstuffsToxins produced by C. botulinum strains, the botulinum toxins (botulotoxins) are the known most potent organic toxins, their lethal dose in adults is altogether 0.1-1.0 g. The toxins are high molecule mass proteins (150 kDa), that are released into the environment after the bacterial cell destruction (protopla smatoxins). At this time, the toxins are relatively inactive (prototoxins) and are activated by proteolytic enzymes, thereby their potency is multiplicated for several-hundred times.The activating enzyme can be produced by the bacterium itself (proteolytic strains), or the activation may take place in the host’s organism by help of the costumers’ digestive enzymes (trypsine). This latter process characterises the toxins produced by strains belonging to group II. In both cases following proteolysis, the active neurotoxin consists of a light (L) and a heavy (H) chain, which are coupled by a disulfite bridge.This toxin is a neurotoxin. Following its binding to the receptors of the presynaptic membrane of motor neurons at the cholinerg nerve endings, it is taken up by the neuron through endocytosis (at this phase, it already cannot be inactivated by antitoxin). Through the L-chain, the toxin specifically inhibits the release of neurotransmitter, while the H-chain, as endopeptidase, damages the synaptic vesicular peptides and prevents the release of

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acetylcholine. The consequence is the flacid paralysis of skeletal muscles. The effect of the toxin is irreversible.The toxin production, under appropriate ecological conditions, takes place in the foodstuffs. The toxin is not produced in living organisms, therefore the disease cannot spread from animal to man or from man to man.The foodstuffs are contaminated by the causative originating mostly from the soil, in case of fish and shells from the waters. Thr germinated vegetative cells under anaerobe conditions (microaerophil) coupled with appropriate temperature, pH and water activity, are multiplicating and producing toxin. The most favourable conditions for this purpose are present in cans. The botulism was the classic living example of food poisoning until the 1920s years when the sterilisation parameters were determined relative to the measure of heat resistance of C. botulinum spores and the 12D-principle was introduced (see the details in Chapter 6). Since than, the number of botulism cases have been dramatically reduced but did not disappear. Todays, botulism primarily may due to the consumption of home-made, not well-cooked meals or not sufficiently heat-treated processed foods, such as plant cans, salads, home-slaughter-based meat-products (headcheese, white pudding, sausages, raw, smoked ham, etc.) and mushroom preparations, marinated fish, crab salads, shells and over-stored ready to eat meals. The toxin is heat-sensitive, it is inactivated within 10 minutes at 80oC. Thus, appropriate cooking, roasting will destroy the toxin with good probability. Disease always is the consequence of consuming not appropriately heat treated meals and foodstaffs.

3.3.10.3. Human diseaseThe classic symtoms of botulism usually are appearing 12-36 hours (extremes are: 6 hours - 10 days) after the consumption of the foodstuff containing the toxin. In the initial, feverless phase of the disease, depression, sweating, muscle pain, abdominal discomfort, occasionally vomiting occur and in a short time, the specific neurotoxic effects are manifested.Owing to the damage of nerve endings in the muscles of eyes, the ability of distance-estimation is disturbed, and strabism, diplopia, problems in reading, mydriasis (altogether vision disturbances) can be detected. Due to the affection of the pharyngeal muscles, mouth dryness, disturbances in swallowing and sound formation, hoarseness, larynx paralysis may be observed. In the final stage, the developing flaccid paralysis (functional paralysis) causes respiratory paralysis and cardiac arrest, ultimately death. Without appropriate treatment, 10-20% of the cases are fatal within 24 hours. The clinical tretment is based on the administration of antitoxin serum and if possible on the removal of toxin from the stomach.In Hungary, yearly 2-8 C. botulinum toxin-induced food poisonings occur often with fatal outcome.In the prevention, the principal acts are observing the general hygiene rules, the kill of spores and the inactivation of already produced toxin. The consumption of home-made cans, meat products originating from home-slaughtered animals always requires higher caution than those that are coming from regulated industrial production applying controlled preservative procedures. Considering that the toxin is heat-sensitive, by boiling the meals for about 10

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minutes furthermore, foodstuffs kept in frigo for several days should be boiled again in order to inactivate the toxin with good probability. We should have suspect for C. botulinum infection if the package of the foodstuff show the sign of gas generation, the top is bulged, damaged. Under oxygen-deprived conditions, strains belonging to group II (prevalent in Hungary) cause fermentation and acidification of the foodstuff. Should not consume such a foodstuff ! Once the normal can is opened, it should be consumed immediately or should be chill-stored for maximum 2 days.Beyond the classic, foodstuff mediated manifestations, C. botulinum sporadically may cause wound-infection in humans, and toxico-infection in infants. In case of infant-botulism, the causative is able to multiplicate in the intestinal tract and to produce toxin. The development of this disease form is possible because in infants the normal inhibitory intestinal flora that could prevent the growth of C. botulinum (as it occurs in later ages) is missing yet. The most important members of this flora are the Enterococcus strains.

3.3.11. Clostridium perfringensIn humans, by infecting the sites of injuries and wounds, the bacterium induces gas-gangrene accompanied with oedema and gas-production, occasionally necrotic enteritis may develop. In animals, it induces enterotoxaemia. The bacterium can multiplicate in food and getting into the intestines, while sporulating, eneterotoxin is produced causing food toxicosis manifested in accumulation of intestinal fluid and consequent diarrhoea.

3.3.11.1. Occurrence, classificationBased on their toxin production the C. perfringens strains can be classified into five types (A, B, C, D, E). The A type of C. perfringens is responsible for the food toxicosis and it may induce gas-gangrene in humans by producing alpha toxin. The C type can produce alpha and beta toxins, this latter toxin may evoke intestinal mucosal necrosis. The natural source of C. perfringens strains is the soil, the B, C, D and E types are primarily living in the intestines of animals, and they only transiently may appear in the soil. The spores of C. perfringens A often can be detected in the faeces of healthy humans in a quantity of <103/g.It is the C. perfringens A which is multiplicating in the intestines and while sporulating produces toxin and causes food toxicosis in humans.

3.3.11.2. Growth chracteristics, toxin production, foodstuffsThe bacterium is multiplicating at 12-50oC. The growth rate is very fast at the optimum temperature range of 43-47oC, the generation time is only 7 minutes. The acid and salt resistance of the vegetative form is relatively weak and their water activity demand is 0.97 (the minimum and optimum pH-values of growth are 5.0 and 6.0-7.5; respectively, the presence of 6% NaCl is inhibitory). The heat-resistance of the vegetative cells is similar to the other non-sporulating bacteria (D60oC: a few minutes). The inter-strain heat-resistance of spores show marked variability; the D100oC value is characteristically some minutes, but it may be much shorter or longer (0.3-40 perc).

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In order of developing food toxicosis, the causative must multiplicate in the foodstuff up to 106 cell/g. After consuming the foodstuff that contains high number of the microbe, the vegetative forms are sporulating in the small intestines, meantime they may produce enterotoxin (only <1% of the strains can produce toxin in measurable quantity).The toxin is a heat-labile protein and it is a constituent of the spore capsule and is released into the intestinal tract at the lysis of the sporangium. Sporadically, low quantity of toxin production may occur also in the foodsuff, it is without any significance in the pathogenesis of in the human disease. The toxin is bound to specific membrane receptors of the epithelial cells and in short time it increases the membrane permeability by forming pores resulting in Na+, Cl- and fluid loss of the epithelial cells and of the whole organism (enterosorptio).The microbes usually are taken up by man consuming meat but any foodstuff can serve as source of the disease in which the spores can survive and, due to mal-storage, the vegetative forms appear and grow in high number. The spores contaminating the food may be of soil origin or at slaughter processing of faecal origin. In the imperfectly heat-treated foodstuffs the vegetative forms are germinating from the surviving spores and fastly multiplicating. This growth may be continued also in the intestinal tract, the patient may release the causative in high number (106/g) for 3 days. In the intestinal tract bacteria are sporulating under anaerobic conditions while toxin production takes place a few hours after ingesting the contaminated food.

3.3.11.3. Human diseaseThe symptoms are appearing 8-12 (occasionally 24) hours after ingesting the contaminated food in form of nausea, abdomnal painand profuse, watery diarrhoea (vomiting is rare). The sympoms are alleviating in a few hours and mostly spontaneously are disappearing in 1-2 days without medical treatment. The infective dose is high, disease will develop following the uptake of 106-108 vegetatíve cells.In prevention of C. perfringens toxico-infection, beyond the minimalisation of contamination, the principal measure is the urgent consumption of the prepared food and/or their chilling within 2-3 hours below 10oC. Especially favourable bacterial multiplication condition is the long duration storage at 30-50oC and this must be prevented because owing to the short generation time, from 100 germs under optimum temperature condition, >105 cells will develop during 2 hours.The beta-toxin of C. perfringens C (necrotising toxin) may cause severe haemorrhagic, necrotic enteritis. In the pathogenesis the disturbance of trypsin production plays a role. Trypsin is able to inactivate the toxin in the intestines. In contrast to the discussed toxico-infection, this is not a food safety issue.

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3.3.12. Bacillus cereusBacteria belonging to the Bacillus genus are rod shaped, Gram-positive, aerobe or facultatve anaerobe, spore forming. With exeption of B. anthracis, their sources are the soil, but they can be found also in the air, natural waters, on plants and on the skin and in intestinal tract of animals and humans. From the several species of the genus, only the B. anthracisnak has primary pathological importance. Most specie are saprophytes and widely disributed in the environment. B. cereus is also a member of he group, it is a common inhabitant of soil, its spores often can be found in low quantity in foodstuffs and in addiives. Certain strains are able to multiplicate in soil-contaminated foodstuffs, they produce toxin and may cause food-toxicosis manifested in vomiting, diarrhoea. B. cereus is a facultative anaerobe, 3-5 long, 1 m wide rod shaped bacterium. It can multiplicate in the 8-55oC temperature range (optimum 28-35oC), the minimum pH- and aw-values are 5.0, and 0.95, respectively.It can induce two kind of diseases, accompnied by voiting and diarrhoea. Main characteristics are summarised in table 3.7.

Table 3.7.Characteristics of food-toxicoisi caused by B. cereus

Parameter Type of diarrhoea Type of vomitingInfective dose 105-107 105-108/gToxin production In small intestines In foodstuffsType of toxin protein peptideInactvation 56oC, 5 min 126oC, 90 minIncubation time 8-16 h 0.5-5 hDuraton of disease 12-24 h (a few days) 6-24 hSymptoms fever, watery diarrhoea, abdominal pain vomiting, rarely diarrhoea Mediator foodstuffs meats, soups, souces, milk products Rice foods, pastes

The two disease forms are caused by different enterotoxins. In the diarrhoeic form, the toxin primarly is produced in the intestinal tract. It is heat and acid sensitive and its effect is similar to which is casued by the toxin produced by C. perfringens. The emetic toxn is cyclic peptide, it is acid and heat resistant. The toxin is produced in the foodstuff, its effect is mediated by 5-HT 3

receptors and consequent n. vagus stimulation. The diarrhoeic disease is due to the ingestion of meat meals, soups, souces, someimes milk products. The votiming form primarly is caused by rice meals and pastes. A frequent source can be the spore-contaminated seasons.Both disease forms are characterised by relatively mild symptoms which usually spontaneously disappear in a short time. Concerning the prevention, consider the description of food-toxicosis of C. perfringens.

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3.4. VirusesViruses are a special class of microorganisms, their structure and way of multiplication are basically different from those observable in other living organsims. They usually are smaller (20-300 nm) than the other microorganisms, and contain only one kind of nucleic acid (RNA or DNA). They are not able to grow or divide only strictly to multiplicate (megsokszorozódással szaporodnak, azaz növekedni, osztódni nem képesek). Viruses are obligate parasites, they can multiplicate only in living cells. For this reason, the number of viruses in the environment, including foodstuffs, natural waters, surfaces is never increasing but usually decreasing in fuction of environmental conditions. Recently the ratio of viral origin gastrointestinal infectious diseases is increasing. The viruses mostly spread by direct or indirect human contact but foodstuffs are also important mediators. Statisics indicate that in the USA more than one-third of the foodstuffs mediated diseases are caused by viruses and in Europe, including Hungary, the number of recognised number of viral diseases is also continuously increasing. This tendency primarily is backgrounded by the development of the diagnostic possibilities but the technological changes in food production, the mass production and altered consumer habits are also play a role.

3.4.1. Classification and characterisation of viruses spread by foodstuffs and/or by waterThe food-related viruses can be classified according to several aspects such as the source of infection (human or animal origin), physico-chemical properties, and the consequent clinical symptoms and pathological findings. In practice, these latter two aspects seem to be most useful resulting in the following differentiation:a) Viruses causing gastrointestinal diseasaes- rotaviruses (A, occasionally C, and B groups)- astroviruses (1-8. serotypes)- adenoviruses (enteral adenoviruses = 40-41. serotypes)- human caliciviruses (e.g. norovirus, sapovirus)- other viruses (e.g. coronaviruses)b) Hepatitis viruses- hepatitis A virus (HAV)- hepatitis E virus (HEV)c) Viruses inducing other diseases- tick encephalitis virus- enteroviruses (polio-, coxsackie, echoviruses, certain types of enterovius).Majority of the listed viruses evoke diseases only sporadically, but the caliciviruses and the hepatitis A virus are frequent pathogens. Diseases caused by noroviruses (belonging to the caliciviruses) are the second most frequent viral infections.Viruses are able to survive for a long time in foodstuffs and surface waters (even for months) and they may survive the preservative treatments, for example of freezing or mild heat-

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treatment (e.g. at 60oC for 30 min). Potent, virucide disinfecting agents (e.g. oxydizing agents, inorganic and organic halogen derivatives) are able to inactivate them.

3.4.2. Contamination of foodstuffs, products most frequently mediating viral infectionsPrincipally any foodstuffs can be contaminated and may mediate viral causatives, but the most frequent sources of viral infection are the foodstuffs consumed raw or only minimum-heat treated. Viruses are contaminating foodstuffs mainly as consequence of primary or secondary faecal contamination. In the primary case, the foodstuff is already contaminated at site of the production (e.g. raw/fresh vegetables, salads, fruits, shells). Fruits consumed without washing (e.g. raspberry, strawberry) are often contaminated by irrigation-water while the effluent (sewage) is the source of contamination of shells (it is the principal cause of viral enteritis in countries where the consumption of shells is preferred).Contamination, however, may arise secondary during processing or preparing the food. In this case, the source of infection is the faeces of infected man and it actually is mediated by contacting the hand of the person or the working surfaces of the establishment. The basic background of foodborne viral diseases is the severe violation of pesonal hygiene. Accordingly, the analysed cases of foodborne viral diseases indicate that those mostly were mediated by hand-made foodstuffs (e.g. sandwiches, salads, ready to eat meals).It must be mentioned, however, that in animals the pathogenic viruses can get into the milk during viraemia and by consuming this milk, also into the human organism. For example the virus of tick-encephalitis is able to infect humans consuming the fresh milk of goat, sheep, and cow or soft-cheeses made from the infected milk.The symtoms of the viral enteral diseases are appearing only after the incubation period of several days or 1-2 weeks while the virus is present in the faeces of infected person already during the incubation period and it can be released for days after disappearing of acute symptoms.

3.4.3. Calicivirus-infectionsTodays the most important foodborne viral diseases are the calicivirus-caused onces. The small, non-capsulated, one–thread RNA type viruses belonging to the Caliciviridae family have two human genera:

The Norwalk-like virus (LV), earlier was termed as "small, round, structured viruses" (SRSV), recently has been named as noroviruses, and

The Sapporo-like viruses or "classic" caliciviruses, recently called as sapoviruses.Along with the development of molecular methods, the number of recognised virus strains are continuously increasing, and they usually are named after their geographic location.All over the world, independently of age, human caliciviruses are causing 30-40% of viral gastroenteritis epidemics and more than 75% of the cases within epidemics. A humán calicivírusok világszerte, életkortól függetlenül a virális eredetű gastroenteritisek 30-40%-át, az ilyen járványokban pedig az esetek több mint 75%-át okozzák. The infection is spread primarly by faeces-contaminated

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foodstuffs and/or water. The intrapopulation circulation of caliciviruses is facilitated by their potential release both in the pre- and- post-symptomtic periods. Owing to partially the short term developed immunity and partially to the high degree of genetic diversity of the virus, the patients can repeatedly be diseased during their life.The human caliciviruses are relatively resistnt to environmental impacts and they may induce disease being present already in as low as 10-100 particle numbers. The average incubation period is short (24-48 hours). In most cases, the principal clinical symptom is vomiting and the disease is fastly over, patients are recovered within 1-3 days. Complications are rare, occasionally the fast and high fluid loss may lead to hospitalisation especially in case of young children and elderly people. Vaccine is not available, the basis of prevention is the observation of the hygiene rules.

3.5. PrionsPrions are low molecular weight peptides (28-30 kDa). They can be found in the normal neural cells and also in other kind of tissue cells of the animal and human organisms. They may play role in signal transducton mechanisms but first of all in maintaining the normal, specific structure, thereby function of neurons and other cell types. The production of prions are coded by PrP-genes that are found in the genetic material of cells also under nomal conditions. The proteolytic enzymes are able to decompose the normal prion proteins (PrP c) in framework of the cell metabolism.Infection or spontaneous mutation may result in the production of abnormal prion proteins (PrPsc). The abnormal prion with changed spatial structure and/or amino acid sequence, will transform/distorse the normal prions of the host. This process is extremely slow takng several years to be manifested. The abnormal prions are proteolytic enzyme-resistant and accumulating in the neuronal (and perhaps in other) cells, they cause funcional disturbances, degeneration (vacualisation: resulting in spongiform histological appearance in brain), then necrosis. For this reason, the developed pattern that will lead to death of the host is termed as transmissible spongiform encephalopathy (TSE).

3.5.1. Human and animal diesasesTransmissible spongiform encephalopathies are occurring in both animals and humans. In animals the bovine spongiform encephalopathy (BSE), the scrapie in sheep, the transmissible mink encephalopathy (TME), the chronic wasting disease (CWD) of elks and deers and the feline spongiform encephalopathies (FSE) of ruminants in zoo and living in the wild, and of felines are belonging here. Similar diseases in humans are the Creutzfeldt-Jakob Syndrome (CJD), and the new variant of this disease identified first in 1996 (nvCJD), the very sporadically occurring (about 1 case/10 millions man/year) Gertsmann-Sträussler-Scheinker syndrome, the fatal familiar insomnia, furthermore the kuru that is related to ritual cannibalism (since its prohibition the case number is greatly reduced).

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The most prevalent form of human prion diseases is the CJD. It is sporadic in 85% of cases, 10-15% has familiar background and less than 5% is iatrogen of origin. The affection is fatal, death occurs usually 4 months after the appearance of clinical symptoms (characterized by progressive dementia, myotonic seizures, cerebellar ataxia, disorinetation, disturbances in speach and vision). This form of the disease is not corresponding to the spongiform encephalopathies occurring in animals. In contrast, in 1996 first in Great Britain, a new variant of CJD (nvCJD) was identified in humans and this form is considered to be of BSE-origin. It was proved that the human infection was mediated by meat contaminated with brain- and spinal cord, with certain internal organs and nerve tissues, therefore BSE has become an animal disease of special importance also form aspects of public health and food-safety. BSE was identified in epidemic form in Great Britain first (1986). Cattle were fed by meat and bone meals obtained from small ruminants infected with scrapie and dead cattle. Additionally, in the by-product industry the technology was changed, this type of raw material was rendered at lower temperature and a solvent step was abonded, thereby the heat-resistant prions were left intact resulting in the development of epidemic BSE.The BSE prions practically identical to the human prions causing nvCJD, but they differ from the scrapiae prions. It is probable that abnormal prions causing BSE could be formed earlier also spontaneously and sporadically but that time BSE was not identified as a separate disease entity. The natural infection with prions takes place orally. Prions can reach the lymphatics, spleen, spinal cord and brain through the M-cells (these cells are without microvilli and surface hydroxylase enzymes) of the Peyer-plaques at the distal portion of the ileum. Prions may already appear in the central nervous system in mid term of the incubation period. These infected organs represent the special risk materials exposed during the slaughter of ruminants (SRM).The incubation time of BSE is long, typically 3-5 years. Only a few animal become diseased in a herd. The BSE prion does not spread horisontally, but in the perinatal period it may reach the calves. The disease changes the behaviour and posture of cattle (apprehension, hyperesthesia and locomotor ataxia. In spite of good appetite progressive loss of condition and milk yield, high stepping of forelegs with hindquarter incoordiantion and difficulty in turning, aggression, kicking violently,, increased noise may produce startled looks, ear twitching and muscle tremors, excessive licking of the coat with rubbing and scratching without pruritus, without fever, finally increased weakness, falling and inability to rise). The duration of clinical course is from 2 weeks to some months, sometimes more than a year.

3.5.2. Occurrence of BSE prions in the body-tissuesBSE prions are present primarly in the central nervous system, but in artificially infected calves prions could be detected also in the tonsils, the distal ileum and in the mesenterial lymph nodes. No prion has been found in the milk or muscle even in clinical cases. The occurrence of prions in different tissues and body-fluids in scrapie infected sheep and BSE infected cattle is shown in Table 3.8.

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Table 3.8.Miscellenous tissues and body-fluids infected with prions in case of scrapie or BSE

Tissue Scrapie (sheep) BSE (cattle)Brain, spinal cord +++ +++Spleen, lymph nodes ++ -Tonsils ++ +Proximal ileum ++ -Distal ileum ++ ++Peripheral nerves, adrenals, nasal mucosa

+ -

Pancreas, liver, placenta +(?) -Marrow +(?) ?Muscle, kidneys, heart, blood, milk, faeces

- -

+++ = High, ++ = medium, + = slight, +(?) = occasionally, ? = single data, uncertain

The BSE prion can be transmitted to sheep experimentally and the caused disease is very similar to scrapie regarding the clinical appearance and tissue distribution pattern. Naturally this has never found in sheep (naturally infected goats with BSE prion, however, has already been identified), but its potential occurrence and the presence of infective prions in the lympho-reticular tissues and in the peripheral nerves must carefully be observed. Pig or poultry could not be artificially infected orally.

3.5.3. Resistance, protection/preventionThe resistance of prions inducing transmissible spongiform encephalopathies is very high. To kill the abnormal prions the application of at least 133oC with 20 minutes exposure time is necessary.The transmissible spongiform encephalopathies are not curable in man or animals. The protective strategy in preventing infection of humans with foodstuffs, especially against BSE brought about basic changes in the feeding of cattle (the prohibition of feeding proteins of mammalian origin except milk and milk-powder) in the slaughter technology (prevention of meat contamination by prevention of mechanical injury of brain and spinal cord including the requirement of removal of spinal cord in one piece), in handling at slaughterhouse and destroying the specific risk materials (the introduction of the term of specific risk materials, SRM, the separated collection and handling of these materials, and their destroyment) and in general, the higher guarantee of traceability in the food-chain (homogenous recording system).

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3.6. ParasitesSeveral parasites of one-cell and multicell type ones can be ingested by humans with water and foodstuffs and they may cause occasionally severe diseases. Their occurrence is worldwide but are more prelevant at subtropic and tropic territories.

3.6.1. ProtozoaCertain one-cell type parasites can induce foodborne diseases by intravitally infected meat of food-production animals. Theses parasites are the toxoplasmas (Toxoplasma gondii), the sarcosporidia (Sarcocystis suihominis, S. bovihominis), furthermore, by secondary infection of drinking water, occasionally different foodstuffs, the giardia (Giardia lamblia), the aemobas (Entamoeba histolytica), and the cryptosporidia (Cryptosporidium parvum).The life cycle of protozoa is sometimes complex, including two or more host organisms, and a part of the life cycle may be a cyst that serves the survival following the release from the host. Man may be an intermediate host (e.g. toxoplasmosis), in other cases the final host (e.g. sarcocystosis). The most important protozoa and the main characteristics of the induced human infestations are described in Table 3.9.

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Table 3.9.Most important protozoa and the induced diseases in humans

Human diseases Causative Source of infection SymptomsToxoplasmosis Toxoplasma gondii Raw, not weel-done meat containing cysts,

vegetables, fruits and drinking water contaminated with cysts

Pregnant women: abortion, stillbirth, damages of fetus, otherwise usully is symptomless, sometimes generalised infection with organ manifestations (e.g. brain)

Sarcocystosis Sarcocystis suihominis, S. bovihominis

Raw meat, not well done, containing sporocysts

Inappatence for 1-2 days, vomiting, diarrhoea, abdominal pain, sweating, and after 2-3 weeks, diarrhoea for 1-3 weeks

Giardiosis Giardia lamblia Drinking water contaminated with human faeces or drinking water, foodstuffs contaminated with faeces of dogs and cats

Chronic diarrhoea, abdominal pain, disturbances in absorption

Amoebiasis Entamoeba histolytica Drinking water, foodtuffs contaminated with human faeces

Ameobic dysentery: haemorrhagic diarrhoea, abdominal pain, anaemia, abscesses in brain

Crystosporidiosis Crystosporidium parvum Drinking water, foodstuffs contaminated with human faeces

Diarrhoea (occasionally haemorrhagic), abdominal pain, exsiccosis

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3.6.2. WormsCertain helminths as zoonotic agents intravitally infestating animals may cause the primary infection of meat and consequently severe foodborne diseases of humans. First, Trichinellas should be mentioned (Trichinella spiralis), furthermore those metacestodes of Taenia species, of which the final host is the man (Taenia saginata: cysticercus bovis; T. solium: c. cellulosae). Equally important zoonotic causatives are the Echinococcus granulosus and Echinococcus multiloculari but they cause secondary contamination of foodstuffs with faeces of infected animals followed by the infection of humans.The non-zoonotic causatives, such as the ascaris species (Ascaris lumbricoides), and trichuris species (Trichuris trichuria) can induce diseases in humans when raw, non-washed vegetables, fruits contaminated by human faeces are ingested.The main properties of the most important helminths and the induced diseases are summaried in table 3.10.

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Table 3.10.The most important helminths and the induced diseases

Human diseases Causative Source of infection SymptomsTrichinellosis Trichinella spiralis Raw or not well-done pork- and boar meat,

raw sausagesSymptomless or excitatory sympoms, vomiting, diarrhoea (enteral phase), headache, fever, muscular pain (migration of larvae)

Taeniosis (Cysticercosis)

Taenia saginata, T. solium (cysticercus bovis, c. cellulosae)

Raw or not-well-done cysticercous meat , non-washed vegetables, fruits contaminated with human faeces, watered with sewage

Nausea, abdominal pain, restlessness

Echinococcosis Echinococcus granulosus Drinking water, foodstuffs contaminated with dog faeces

Depending on the migration of larvae, organ-related symptoms (liver, CNS, eyes)

Echinococcus multilocularis Foodstuffs, drinking water contaminated with faeces of red fox (polar fox), sometimes dog, cat

Its larvae (Echinococcus alveolaris, E. multilocularis) cause tumor-like lesions in the liver of intermediate hosts (man, ruminants, horse)

Ascaridosis Ascaris lumbricoides Foodstuffs, drinking water contaminated with human faeces

Migration of larvae: fever, cough, pneumonia. In enteral phase: abdominal pain, obliteration of bile ducts, ileus

Trichuriosis Trichuris trichuria Vegetables, fruits contminated with human faeces., direct contact with faeces

Symptomless or in massive infection: fever, mucous, haemorrhagic diarrhoea, anaemia

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3.7. Microbiological food-safety regulation3.7.1. European Community RegulationIt is a fundamental food-saferty principle that non-safe foodstuff with public health hazard is unacceptable and must not be placed on the market (Regulation 178/2002/EC). This principle includes also the microbiological safety aspects requiring that foodstuffs should not contain micro-organisms or their toxins or metabolites in quantities that present an unaccepatble risk for human health. In order to prevent differing interpretations, it was appropriate in the European Union to establish harmonised safety criteria and corresponding standards on the acceptability of food, in particular as regards the presence of certain pathogenic micro-organisms. (Regulation 2073/2005/EC).The Regulation distinguish food-safety and technological hygiene criteria. The food-safety (selected) criteria determine the microbiological acceptability of the marketed foodstuff by applying microbiological limit values. The technological hygiene criteria indicate the appropriate operation of production technology, without direct reference to the end-product.According to the Regulation, in each phase of the food production, processing, marketing it is the task and responsibility of the food bussiness operators to satisfy the requirements of technological hygiene and to guarantee the appropriateness of the product to the corresponding food-safety criteria during its shelf-lifte. The compliance with microbiological criteria is checked by the competent authority by controlling the documentation and activity of the producer and also by testing the product in official laboratory.If a food product does not satisfy the safety criteria, its marketing must be prohibited or it is withdrawn from the market. (If the product has not yet in retail market, it is possible to re-process it by applying a technology that removes the given hazard.) If the technological hygiene criteria are not met, the producer must apply improving actions in the technology to ensure compliance but this does not affect the marketing of the product.Table 3.11. demonstrates some characteristic food-safety and technological hygiene requirements.

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Table 3.11.Some food-safety and technological hygiene criteria

Foodstuff Micro-organismsk/their toxin, metabolites

Requirementsn c m M

Food-safety criteriaReady to eat foods (which facilitate the growth of L. monocytogenes)

L. monocytogenes 55

00

100 cfu/gb

Not present in 25 gc

Meat products consumed raw Salmonella 5 0 Not present in 25 gc

Cheese, butter, cream madem from fresh or thermised milk Salmonella 5 0 Not present in 25 gc

Ready to eat foodstuffs containing fresh egg Salmonella 5 0 Not present in 25 gc

Cheese, milk powder, whey-powder Staphylococcus enterotoxin 5 0 Not present in 25 gc

Technological hygiene criteriaSplitted broiler poultry, turkey Salmonella 50 7 Not present in sample taken

from 25 g neck skinGround meat Number of aerobe microbes

E. coli55

22

5x105 cfu/g50 cfu/g

5x106 cfu/g500 cfu/g

Pasteurised milk Enterobacteriaceae 5 2 <1 cfu/ml 5 cfu/mla) n=sample number; m=threshold value of acceptability; M=threshold value of unaccepatability; c=border range; just acceptable number of contaminated samples fallen between values of m and Mb) if the producer can certify to the Authority that the product during its shelf life the contamination was along below 100 cfu/g (cfu, colony forming unit)c) It is the criterium to be fulfilled, if the above requirement is not met

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3.7.2. National regulationsIn Hungary, before the implementation of the related EU Regulation (01 January 2006), the acceptable measure of microbiological contamination of foddstuffs was regulated by the Decree of the Ministry of Public Health (4/1998. (XI.11.). Following the on-going modifiation of the Decree, it may contain only supplementary regulation for foodstuffs produced in the territory of the Hungarian Republic. The supplementary regulation includes requirements on certain technological hygiene aspects for controlling the production technology, furthermore requirements of zero tolerance to certain specified pathogens (pl. Brucella, Campylobacter, Mycobacterium, verotoxin-producing E. coli, Shigella, Yersinia enterocolitica). These latter criteria are shown in Table 3.12.

Table 3.12.Pathogens influencing the judgement of foodstuffs

According to the Decree of 4/1998 (11.11.) Ministry of Public Health

Specified pathogens that are not acceptable in the tested sample (threshold value: 0)Bacteria and their toxins:

Brucella species,Campylobacter speciesjok (in ready to eat foodstuffs),Clostridium botulinum,Coxiella burnetii,Verotoxin-producing E. coli, S. aureus enterotoxin,Francisella tularensis,Mycobacterium species,Salmonella typhi and Salmonella paratyphi A, B, C,Shigella species,Vibrio cholerae,Yersinia enterocolitica,

And other, here not listed bacteria, approved as pathogen.Viruses:

Hepatitis,Rotavirus,Norwalk and Norwalk-like viruses.

Protozoa:Entamoeba histolytica,Giardia lamblia,Toxoplasma gondii,Sarcosporidium species,

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Cryptosporidium and other protozoonsWorms:

Cysticercus/Taenia,Echinococcus,Trichinella spiralis.

It is important to emphasize that the above described points may be applied only on the National level as a supplementary regulation. The National technological hygiene criteria may be more severe than the Community ones, because this will not result in commercial restrictions against the free movement of goods. New food-safety criterium, however, can be introduced on the National level only if the member State can scientifically prove its validity, it is accepted by the European Food Safety Office, and it is put into the related Community Regulation.

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4. BASICS OF CHEMICAL-TOXICOLOGICAL FOOD-SAFETY4.1. Chemical contaminants in foodstuffs, health damaging effectsThe number of recorded chemical substances is well above 10 millions, from which 70-80 thousand the number of chemicals that may be in direct contact with humans. Majority of these substances potentially may occur also in foodstuffs and may adversely influence the health of consumers. The classification of foodborne health damaging substances and the induced adverse effects can be seen in Tables 4.1. and 4.2., respectively.

Table 4.1.Chemical compounds in food with public health hazards

Added, residual and environmental materials Harmful compounds of natural originResidues and environmental

contaminantsAdditives

Veterinary drug residuesPesticide residuesContaminants of environmental originContaminants of technological originContaminants of biological origin

PreservativesTechnological additivesTaste improving and aromatics

Colouring agents

Alkaloids (e.g. solanine, morphine)CyanoglycosidesMethyl-alcoholNitratesCertain nutritives (e.g. lactose, phenylalanine)Toxins of plant origin (e.g. toxins of moulds)

Table 4.2.Health damaging effects of chemical compounds occurring in foodstuffs

Clinical toxicosis Microtoxicosis Latent toxic effects: mutagen, teratogen, carcinogen effects Immunosuppressive effect Allergenic effects Damaging effect on intestinal flora (dysbacteriosis) Bacterial resistance potentiating effect Other, special effects (e.g. aplastic anaemia)

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4.2. The background of regulationsConsidering the high number, widespread use, occurrence and generation of chemical substances, the complete freedom from chemical contamination of foodstuffs is practically impossible. In order to protect consumers’ health, the (exposing) quantity of chemicals must be reduced in a measure that can prevent the development of health damage during their lifelong intake. This purpose can be achieved by establishing so called limit values based on data originated from risk analysis and evaluation carried out by international expert bodies/commitees. This means the obligatory prescription, observation and control of tolerable upper limit concentrations of residues in foodstuufs which are without public health hazard.The principle regulatory prescriptions of chemical contaminants in food can be found in Regulation 1881/2006/EC. In harmony with this regulation, but undiscussed issues are regulated on National level in Decree 17/1999. (VI.16.) Ministry of HealthSeparate rules contain the tolerable MRLs (maximum residue limits) of veterinary drugs in foods of animal origin, the tolerable concentrations of herbicide residues in foods and raw materials of plant origin, furthermore the tolerable level of radioactive contamination of foodsatuffs. A separate law describes the rules of monitoring-examination of health-hazardous residues in fooddstuffs of animal origin.In Hungary, the rules corresponding to food additives, materials used in direct contact with foodstuffs, furthermore the sampling and analytical test methods of chemical substances are described in the Hungarian Codex Alimentarius (Table 4.3.).

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Table 4.3.Principle prescriptions and rules regulating the chemical-*toxicological safety of

foodstuffs

Regulation/prescription Regulated issuesRegulation 1881/2006/ECDecree 17/1999. (VI.16.) Ministry of Health

Contaminants of biological origin (mycotoxins) Contaminants of environmental origin (toxic metals,

dioxins and PCB-s, prohibited pesticides) Contaminants of technological origin Pesticide residues in foodstuffs of animal origin

(authorised substances) Harmful materials of natural origin (pl. solanine, cian-

hydrogen, methanol, nitrites and nitrates)Regulation 2377/90/EEC Residues of veterinary preparations

Active subsatnces prohibited to use for treatment in production animals

Regulation 396/2005/EC

Decree 34/2004. (IV.26.) Ministry of Health

Herbicide residues in foodstuffs of plant and animal origin Pesticide residues in foodstuffs of animal origin

Decree 5/2002. (II.22.) Ministry of Health – Ministry of Agriculture

Herbicide residues in foodstuffs and raw materials of plant origin

Regulation 3954/87 EURATOM Radioactive contaminationDecree 10/2002. (I.23.) Minisitry of Agriculture

System of residue monitoring for - banned/anabolic active substances, and preparations - veterinary drug preparations, and contaminating materials

Prescriptions of the Hungarian Codex Alimentariusi

Food additives Materials used in direct contact with foodstuffs Test methods, sampling

4.3. Veterinary drug preparations, banned active substancesOne of the basic condition of chemical safety of foodstuffs of animal origin is that they must not contain drug residue(s) in consumers’ health threatening quantities. For this reason, in treatment of food production animals only those acive substances and vehicles (in form of products) may be used for the residues of which (following their application) in the edible tissues of the animals and in products (milk, eggs, honey) official tolerable (from aspects of public health and sometimes also industrial processing) limit values (Maximum Residue Limit = MRL) have been established.In the European Union these limit values are determined by the Committee of Veterinary Medicinal Products (CVMP) based on the several times modified Regulation 2377/90/ECC and observing the proposals of The Joint Expert Committee of FAO and WHO (Working Committee on Veterinary Medicinal Residues In Foodstuffs).

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The evaluated active substances, groups of active substances are classified into 4 groups (Annex I-IV). Those substances are belonging to the first group for which final MRLs are established. In the second group those substance are found for which the elaboration of MRL is not necessaary. For the members of the third group only provisional MRLs are established because important data are absent. No MRL available for substances of the fourth group (not possible to establish ADI/MRL and/or not required/initiated). The members of this latter group are prohibited to use in production animals (banned substances) for any purpose (medical treatment, prevention, growth promotion). There are, however, anabolic substances which may be used for treatment but not for growth promotion (steroid and polipeptide hormons, furthermore, beta-agonists.

4.3.1. Banned substancesMain groups of the banned substances and anabolic compounds are shown in Table 4.4.

Table 4 Most important active substances not permitted for treatment of production animals

Active compounds RemarkChloramphenicolNitrofuranes (e..g. furazolidone)Nitroimidazole (e.g. dimetridazolel, ronidazole, metronidazole)Stilbene and its derivativesSteroid drugs)Grow a youngormon (e.g. BST, PST)ThyreostaticsBeta-agonists e.g.(clenbuterol, cimaterol)Antibacterial growth promoters (e.g. avoparcin, zinc-bacitracin, virginiamcin)

Except for therapeutical and zootechnic purposes

Except for therapy

Among the banned substances, the intake of chloramphenicol causes ten times increase in the rate of manifestation of fatal aplastic anaemia (a severe disturbance in haematopoesis) in sensitive consumers. Seventy per cent of patients suffering in this disturbance will die within some months (in spite of careful medical care), in survivors the incidence of leucaemia will be increased. Considering that the effect practically is independent from the dose (not dose-dependent), the measure of daily acceptable intake (ADI) by the consumer cannot be determined, consequently the related MRL cannot be calculated.

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The residues of the 5-nitrofurane-derivatives and the nitroimidazols may induce tumors in sensitive consumers. Similarily, the synthetic stibene-derivatives are also carcinogens (e.g. diethyl-stilbeostrol: DES). In children of women who are consuming DES-containing foodstuffs, the incidence of mammary carcinoma was significantly increased. Stilbenes are also belonging to the banned substances.

4.3.2. Compounds used only for therapeutic purposes (hormons, beta-agonists)The natural steroid hormons (androgens, oestrogens, gestagens) produced in the animal and human organisms and their synthetic derivatives (e.g. trenbolone, melengestrole) furthermore the myco-oestrogen zearanol are anabolics but they must not be used as growth promoters. In spite of the severe legal ban, they may illegally be applied in animal farming of several countries, e.g. by implanting the active-substance pellet into the connective tissue of skin behing of ears. Eating foodstuffs containing the residues of anabolic hormones, oestrogen or androgen efffect can develop (depending on the type of the hormon) in consumers. The repeated intake of these hormons may result in disturbances in the development of secondary sexual characteristics, infertility, abortion, teratogenic defects. In the Member states of the European Union and in Third Countries exporting foodstuffs of animal origin into the EU, prohibited the use of biotechnologically produced polypeptide-hormons (growth hormons, BST, PST) for growth promotion.The beta-agonists, especially clenbuterol were the most „popular” growth promoters in the 1990s. These compounds, by stimulating the beta-adrenoreceptors (primarily 2), therapeutically beneficially relax the smooth muscle of the bronchi and the uterus (registered preparation is available also in Hungary for bronchodilation in respiratory diseases of horses). Beta agonists mixed in feed and fed continuously can increase the protein synthesis and the decomposition of lipids, reduce the protein catabolism resulting in higher protein/lipid ratio in muscle. To achieve the described effects, higher dose than the therapeutic one is necessary, consequently in the edible tissues (especially in the kidney and liver) the accumulation of residues occur in quantities representing risk for the consumer’s health. Due to this mal-practice, in the past 10-12 years in Spain, France and Italy several severe clinically manifested human disease cases occurred (CNS excitation, tachycardia, general muscle-pain) mostly following the consumption of calf-liver (less frequently veal). These toxicoses, outlasting for 1-3 days and mostly requiring hospitalisation, were caused the consumption of liver or veal containing 0.2-1 g/g clenbuterol-residue.

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4.3.3. AntibioticsThe application of those antibiotics as growth promoters that used also in human therapy (e.g. penicillins, tetracyclines) was banned in most European countries at the begining of the 1970s. Following this, specifically for feeding purpose antibacterial growth promoters were developed and placed into the market (e.g. avoparcin, zinc-bacitracin, virginiamycin, flavophospholipol, avilamycin). These antibiotics are not used in human therapy, teherfore for a long time they were considered that their application is without significant influence on the antibiotic-sensitivity of human pathogens.In the past decade, the significant increase of antimicrobial resistance of important human pathogen bacteria (e.g. Staphylococcus aureus, pneumococcusok, members of the Enterobacteriaceae family), furthermore zoonotic agents (e.g. Salmonella spp., Campylobacter spp.) called again the attention to the potential risks of use of antibacterial growth promoters. It was found that Enterococcus strains (Gram-positive intestinal indicator microorganisms) that can be isolated from the intestinal tract of clinically healthy slaughter animals (including poultries) are prone to transfer transpozons, plasmids carrying the genetic codes of resistance-mechanisms. This horisontal transfer is directed to bacteria regardless to bacterial species living in the surrounding environment. Thereby, the resistance mechanisms developed in animals’ intestinal bacteria are transferred to bacteria living in the human intestinal tract. Accordingly, a relationship was established between the widespreadl use of the glycopeptide antibiotic avoparcin as gowth promoter and the incidence of occurrence of the structurally related vankomycin-resistant Enterococcus faecium strains. Bacteria becoming resistant and contaminating the foddstiuff of animal origin can get into the intestinal tract of human consumers. While multiplicating in the intestines, they may transfer the resistance to human pathogen strains which represent potential infection source of severe, hardly treatable diseases.As a consequebce, the application of avoparcin as growth promoter was banned in Denmark at 1995, next year in Germany and subsequent year in the other countries of the European Union and Hungary. Later, considering the potential risk of development of cross-resistance to antibiotics used in human therapy, the application of all antibiotics in growth promotion was banned in the member states of the European Union including Hungary.From safety point of view, among the registered veterinary drug preparations used for prevention or treatment, the antibacterial ones are especially important. The presence of their residues in the edible tissues and animal products (e.g. milk) may represent potential allergic, intestinal flora damaging and bacterial resistance inducing effects. A special issue is the residue contamination at site of injection following the application of long-acting parenteral injection preparations.

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Among antibiotics, principally penicillins are the allergens, already 10 UI penicillin-residue is able to evoke allergic reaction in atopic individuals. In spite of low toxic potential of penicillins, due to the allergic potential, their MRLs in edible tissues and milk are low (50 and 4 g/kg, respectively) compared to other active substances.Thr observation of the withdrawal time for milk is specifically important also in preventing the inhibitory effect of penicillins in the starter cultures applied in milk-product manufacture (0.01 IU/ml concentration is already inhibitory: 1 IU benzyl-penicillin = 0.6 g).In order to prevent the intestinal-flora damaging effect, on course of risk analysis (carried out by the mentioned Joint Expert Committee) while the toxicological characteristics are evaluated, the potential adverse microbiological consequences are also considered. Accordingly, beside the calculation of the Acceptable Daily Intake (ADI) for humans based on the corresponding toxicological results, the microbiological ADI is also calculated. The effects of antibacterial residues are evaluated on the composition of intestinal flora, metabolic activity, barrier function in inhibiting colonisation, furthermore the effect on the selection (pressure) of resistant strains. The highest dose level yet without effect is determined for each effect (No Observed Effect Level = NOEL) and from these, the microbiological ADI value is calculated.At determining the MRLs from the toxicological and microbiological ADIs, the lowest value (respresentig the highest safety for consumers) is used for further calculations. The less toxic antibacterial substanaces which have potent effects also against anaerobe bacteria, the lower value usually belongs to the microbiological ADI. Among the microbiological effects, the inhibition of colonisation barrier is outstandingly important because it may promote/facilitate the multiplication of important foodborne zoonotic-pathogens (e.g. Salmonella- and Campylobacter-strains). The prevention of development of antimicrobial resistance by sufficiently low intake of antimicrobials is also important.In preventing the discussed undesirable effects, special attention should be paid to the application of the so called long-acting products and the to the residues enriched at the site of injection. At the site of injection, occasionally visually observable oedame and hard nodules are developping, in which the antibiotics are captured in active form and high concentration. During meat inspection, these sites must be excised and condem and the edible tissues must be sampled for laboratory residue determinations. If the sample is positive for banned substance(es) or the authorised substance is present in concentrations over the related MRL, the whole carcase is unfit for human consumption and a survaillance program is initiated at the site of origin. (see also in chapter 4.9.1.).The illegal use of banned substances or neglecting the observation of the coresponding MRL value of authorised drug preparations or the presence of certain contaminating chemicals in foodstuffs of animal origin of (e.g. pesticides, toxic components, mycotoxins) are examined in the framework of nation-wide monitoring program carried aoutby the competent authority

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and based on random sample selection. Beyond the monitoring activity, the competent authority is carrying out ad hoc control examinations.

4.4. Pesticide residuesPesticides represent residues of chemical substances applied in plant protection, furthermore in extermination of rodents and insects and they may be present on the surface or inside of foodstuffs. The majority of pesticides are the herbicides, the quantity of which has been significantly reduced in Hungary during the past 10-15 years. The number of varieties of the active subsatnces is also restricted but the less selective compounds that are toxic also to the higher organisms including man are still used. Understandable why the observation of the related withdrawal times, similarily to the veterinary drug preparations, is so important.Among the high number of pesticide active substances, the chlorinated hydrocarbons and less importantly the other insecticides are worth to mention because of their potential presence in foodstuffs of animal origin and and their toxicological profile. The residues of other herbicides such as weed-killers and fungicides may be important contaminants in foodstuffs of plant origin but usually cannot be detected in foodstuffs of animal origin.The chlorinated hydrocarbons are highly lipid soluble, and the mostly are persistant in the environment and are prone for accummulation in the in living organisms. Certain representatives of the group (e.g. DDT, the beta-isomer of hexachlor-cyclohexane) are magnificating (enriched) in the food-chain, reaching espcially high concentrations in the maternal milk. The DDT and the β-HCH can be present in the maternal milk about 30 times higher concentration than in the cow-milk.The use of the definitely persistant chlorinated hydrocarbons, such as DDT was already banned in 1968, but the residues of the substances and their degradation products still can be detected in the fat-tissues of the wild animals. The detectable quantities, however, have been greatly reduced by the 90s years. The concentrations of total DDT (parent compound and metabolites) was only between 0.01-0.4 mg/kg, and this is significantly lower than the tolerable 1 mg/kg threshold value. In case of slaughter animals, the measure of DDT contamination was <0.1 mg/kg in the 90s years and since then this value, similarily to the contamination in games, further reduced. There is an interesting relationship between the time of contamination with DDT and in the ratio of DDT-parent compound and its DDE- metabolite in fat tissues. The DDE/DDT ratio is about one in freshly contaminated samples, by time it is gradually increasing reaching one magnitude change during 20 years (>10). This way, based on the DDE/DDT ratio, the time of the contamination can be determined.Among the less persistant chlorinated hydrocarbons, the gamma isomer of the hexachlor-cyclohexane (Lindan) and especially the endosulphane compound are more toxic to warm-blooded animals than DDT is but neither are prone for accumulation or biomagnification. Their detectable quantities in slaughter animals in Hungary is <0.01 mg/kg for years and this

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is less by at least one magnitude than the related tolerable threshold value (Lindan: 1 mg/kg, endosulphane 0.1 mg/kg). In animals living in the wild, the measure of contamination is also well below the tolerable value.The organophosphates and the insecticid carbamates are less persistant in the environment than the chlorinated hydrocarbons and they mostly decompose fast in the environment and in the animal organisms. They are, however, usually are more toxic for warm-blooded animals and man (they may cause acute toxicosis). The organophosphates and the insecticid carbamates are cholinestares inhibitory compounds, the inhibition can be developed in organophosphates following repeated intake (biological accumulation). Their tolerable threshold values for foodstuffs of animal origin are ranging between 0.01-0.05 mg/kg, the analytically detectable (LOD) quantities (if detectable anyway) are lower by one magnitude. In contrast, in foodstuffs of plant origin (mostly in early products), they can be detected more frequently above the tolerable value.The pyrethrines and their synthetic derivatives, the pyrethroids are potent insecticides. They are less toxic for mammals and birds (fish are very sensitive) and regularly are fastly decomposed in the environment and in animal organisms. Owing to their definite insecticide property and low toxicity, they are frequently used also in veterinary practice. In foodstuffs of animal origin, pyrethrines or pyrethroids have not been detected over the detectable level (0.01 mg/kg) following plant-protection or veterinary applications.Up to now in Hungary, residues of other herbicides (weed killers, fungicides), or insecticides and rodencides have not been detected in foodstuffs of animal origin.In contrast to the zero detectable pesticide contamination in foodstuffs of animal origin, in 1-2 percent of the marketed products of plant origin, residues of mainly fungicides being over the tolerable limit were found during the recent years in Hungary. In import foodstuffs of plant origin, (mostly in seasons) however, several times the residues of already banned but in certain tropic developing countries still used herbicides have been detected (e.g. DDT, aldrin, dieldrin, HCB).

4.5. Contaminants of environmental orginFrom the great number of potential environmental contaminants, the most important food related ones are the toxic heavy metals and the mettalloids such as cadmium, lead, mercury and arsenic, furthermore the polychlorinated compounds (derivatives). This latter group involves the dibenzo-dioxins and -furans, the polychlorinated biphenyls and certain banned peresistant chlorinated hydrocarbons (discussed earlier). A part of these compounds (e.g. heavy metals) are also natural components of the environment, but the consumer primarily uptake them as food contaminants generated by human activities (industrial, agricultural and domestic ones, transportation and waste combustion).

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In order to protect consumers’ health, the acceptable/tolerable maximum daily, weekly, and monthly (this latter in case of cummulative-type substances) intake (TDI/TWI/TMI) for the principal contaminants are determined, respectively. These quantities are without health-damaging effect even if continuously taken for life. The international organisation responsible to the determination of the tolerable intake quantities based on risk analyses (Joint Expert Committee of FAO/WHO, JECFA) often is able to erect only provisional values (PTDI/PTWI, PTMI) because of imperfect set of data. The Committee periodically re-evaluate/revise these provisional values in the mirror of new toxicological and epidemiological data and if necessary change them.Taking into consideration of the tolerable intake values determined by the JECFA, countries or group of countries (e.g. the European Union) are determining threshold values in foodstuffs which are the basis of official control. It should be kept in mind that in case of contaminants of environmental origin (considering their unordered formation and uncontrollable application), the risk probability factor is higher than in case of offically well controlled preparations discussed earlier. This is specifically true for the miscellenous polychlorinated contaminants.Many of the environmental contaminants are very persistant, they are decomposing only very slowly, through several decades. These are termed also as POP-substances (persistent organic pollutants, POP). The group includes the already discussed pesticides of persistent chlorinated hydrocarbons (e.g. DDT, aldrin, dieldrin), furthermore several polychlorinated industrial ingredients (e.g. polychlorinated biphenyls, hexachlor-benzol), and organic contaminants (e.g. polychlorinated dibenzo-dioxins and -furanes). The polyaromatic hydrocarbons (PAHs) are also can be classified into the group of POP and we are going to discuss these contaminants as of technological origin in Chapter 4.6.1.

4.5.1. Toxic heavy metals, metalloids4.5.1.1. Cadmium, leadCadmium is the most dangerous heavy metal. It damages the function of several organs and organ systems including the testicles, the liver, and the metabolism of the skin and bone but the nephrotoxic effect is the most important component. A part of the toxic effects can be attributed to its zinc antagonistic effect. Cadmium is taken up by humans via the food or less importantly by inhalation at smoking. It is a natural component of the environment (mainly in ores containing zinc) but it can be enriched as a result of human activity, in phosphate- fertilizers contaminated by the metal, in the smoke and the effluent of metal-processing plants, and released during the combustion of fossil fuels.The dissolution of cadmium into the soil and into the sediment of natural waters is facilitated by the drop of pH (acidification). The relased cadmium is taken up through their roots by plants and are stored in plant tissues. In contrast to the lead, the cadmium primarily can be

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found in the plant tissues, therefore, the contamination cannot be diminished by even careful washing. In water biotops (e.g. in sea) following the pH-dependent dissolution of cadmium from the mud and being also present as floating suspended particlesa are taken up mainly by shells and crabs.In food production animals (mammals, birds), the majority of cadmium is in the kidneys and liver. The main sources of human uptake are the kidney and liver of slaughter animals, the shells and crabs, furthermore, the oily seeds and vegetables. The average concentrations in kidneys are 100-1000 g/kg, in liver 10-100 g/kg, in meat and fruits <10g/kg, in fish 20 g/kg, in shells 200-1000 g/kg. The daily average cadmium uptke by non-smoking individuals is 10-35 g. Smoking is increasing the daily uptake by (daily 20 cigarets) 2-4 g. Cadmium taken up by drinking water is usually <2 g.After taken up into the intestinal tract, about five percent (3-7%) of cadmium is absorbed but in iron-defficient individuals, the absorption rate may reach the 15-20%. The absorbed cadmium is bound to metallothionein in the liver, and it is gradually released into the blood and is filtrated in the kidneys and is reabsorbed in the proximal tubules. In the tubular cells, the toxic Cd2+ is released from the protein binding and by gradually accumulating it causes irreversible nephropathy. The critical cadmium level in the cortex of kidneys that induces tubular dysfunction in 10% of persons is estimated to be 200 mg/kg. This quantity can be taken up by ingestion of daily 175 g cadmium through 50 years. The uptake of daily 100 g cadmium could result in a critical overload in 2% of a regular population.In order not to overcome the 50 mg/kg renal cadmium concentration and taking into account the average 5% absorption of the metal, and the very slow excretion and its cummulative character, its PTWI value was defined as 7 g/kg by The Joint Expert Committee of WHO/FAO. In the Member States of the European Union, the average measure of the weekly cadmium uptake ranges between 2.8-4.2 g/kg, and this is 40-60% of the PTWI value. The tolerable maximum cadmium and other heavy-metal content of foodstuffs of animal origin in Hungary is described in Table 4.5.

Table 4.5.Maximum tolerable concentrations of heavy metals in food of animal origin

Food/product Maximum concentration (mg/kg)Cd Pb Hg As

Fresh meat (cattle, sheep, swine, poultry) 0.05 0.1 N.a.a N.a.Edible offals (catle, sheep, swine, poultry) 0.5-1.0 0.5 N.a. N.a.Fish meat 0.05-0.1 0.3 0.5-1.0 N.a.Crabs, shells 0.5-1.0 0.5-1.5 0.5 N.a.Fresh game meat and products 0.1 0.5 0.5 1.0Meat products 0.1 0.15 0.03 0.2Cow milk N.a. 0.02 N.a. N.a.Butter 0.02 0.02 0.02 0.1Cheese 0,05 0.1 0.02 0.3

a not available

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In foodstuffs of animal origin, the number of official measures in cases of heavy metal threshold/tolearble level violations are greatly reduced in the past 10 years in Hungary (it is already only <0.2%). Earlier, the over-the-tolerance-levels were detected in the kidneys of pigs and cattle, but by the reduction of age of slaughter animals, the number of objections was also reduced from the second part of the 1990s., Several times were detected intolerable levels, however, in foodstuffs of plant origin, (in oily seeds, oil, season paprika, and sometimes in vegetables).Lead is the oldest known environmental contaminant heavy metal. Its toxic effect, similarily to cadmium, is very complex, the most important in humans are the haem-synthesis inhibitory and the central nervous system (CNS) damaging effects and kids are most sensitive than adults. In children already as low as <10 g/100 ml lead concentration inhibits the activity of delta-aminolevulinacid-dehydratase that is important in the haem-synthesis. Anaemia, manifesting also in CNS clinical signs appears only at >40 g/100 ml blood concentrations. In children, the most important CNS symptom is the reduction of the mental power. Results of high number of studies show that the increase of blood lead concentration from 5 g/100 ml value to 20 g/100 ml level is accompanied by about a 5 point IQ reduction. Another CNS finding is the occasionally developing polyneuritis.Lead can get into the human organism by contaminated food, water and air. The ordinary level of lead in soil is 5-100 mg/kg, but in heavily contaminated soil it may surpass even the 10.000 mg/kg-concentration. The most important sources of environmental lead contaminations are the transportation and industrial activity. In the past 10-15 years, by reducing, later terminating of using added lead in fuels and the regulated reduction of industrial emmission, the measure of lead contamination and the corresponding lead concentrations in human blood significantly dropped in developed countries. According to a survey, in the USA, the average blood-lead concentration of the population was 12.5 g/100 ml at the end of 70s and this diminished to 2.8 g/100 ml by the begining of 90s. A similar but a lower rate of decline was observed also in the European countries. The lead much less intensively dissolved from the soil than the cadmium, therefore it is not deposited in the tissues of plants but appear as plant surface contamination. In the animals, it initially accumulating in tissues having good blood supply (liver, kidneys), followed by accumulation in bones from which as a result of calcium metabolism it continuously can be released into the blood.In human adults the absorption of lead from the gastrointestinal tract is only 10%, but in children, it may be up to 50%. Following absorption, it initially is deposited in the red blood cells, liver, kidneys, finally it is accumulating in the bones. Its half-life in soft tissues is 1-2 months, in bones already is 25-30 years. Its toxic effects are well known and the neurotoxic

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effects are the most crucial and important also in determining the measure of the tolerable weekly uptake. JECFA determined the PTWI value of lead originating from any possible sources as 25 g/kg. An European common consumer may ingest about 20 g/kg by foodstuffs. In Hungary, the estimated weekly lead-intake is 13.1 g/kg, this is about the half of the PTWI value.The lead content of the Hungarian foodstuffs has been gradually reduced in the past 10 years. This drop is due to, beside the moderate control of potential general industrial and environmental contaminations, modernisations in the food-industry, the gradual replacing of selded to welded cans, the diminsihed use of machinary, equipment and packaging materials coated with lead releasing tin. The lead contamination in foodstuffs of animal origin, in accordance with the general tendency, is also significantly reduced. The 5-6% positivity at the begining of the 1990 years in muscle and liver samples of pig and cattle has practically beeen reduced to zero during 10-15 years.

4.5.1.2. Mercury, arsenicMercury in its elementary form, and in forms of inorganic and organic compounds is widely present in the environment. It generally acn be detected in the air (in a usual concentration of <10 ng/m3) and in the natural waters at 10-50 ng/l concentraions. The mercury content of foodstuffs is mostly low (<0,01 g/g) and this low concentration is in elementary form or in inorganic compounds. Fish and shells are special because in these species the concentration of mercury might be much higher and most cases (>90%) is present in form of methyl-mercury. The mostly industrial origin inorganic mercury compounds released into the surface fresh waters and seas are bio-methylated through microbial activity and this methyl-mercury is accumulated in low-ranked water creatures and later ingested by shells and fish.Due to the decribed magnification in the nutrition chain, the methyl-mercury is enriched in the organisms and it can reach specifically high concentrations in predatory fish such as in shark, sword-fish, catfish and hake/pike. The usual concentration of methyl-mercury in fish is <0.4 mg/kg, but in predatory-fish it may be several times higher. The majority of people consume a daily maximum of 20-30 g, but at certain regions the measure of fish consumption may be as high as daily 400-500 g. Accordingly, the methyl-mercury intake of food origin can be ranged between 0.2 and 3-4 g/kg.The lipid-soluble methyl-mercury is fastly and almost fully absorbed from the consumers’ alimentary tract, easily penetrates the blood-brain barrier and is accumulating in the CNS. It is neurotoxic and specifically damages the cerebellar granular cells, furthermore the auditory and visual cortices. It easily can penetrate also the placenta and causes severe fetal developmental disturbances. In a sea-side settlement of Japan, the consumption of heavily contaminated fish with methyl-mercury caused a mass-toxicosis resulting in 46 deaths at the 1950s (Minamata-disease). In foetuses of mothers with less severe symptoms, grave

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developmental abnormalities were detected indicating the high fetal sensitivity to the neurotoxic effect of methyl-mercury. In order to protect the developping foetuses, the earlier tolerable weekly intake of 3.3 g/kg methyl-mercury was reduced to 1.6 g/kg in 2003.In Hungary the detected mercury content of foodstuffs is very low. Evidently, the highest concentrations can be found in fish but mercury contamination over the tolerable level (0.5-1 mg/kg) has never been detected in domestic or import fish and fish-products during the past years. In contrast, several foreign publications reported over the tolerable intake level of mercury contamination in benetic fish at the nearby seas (e.g. Adriatic sea), indicating the importance of the official control of import fish.Arsenic, coupled to other metals, almost anywhere can be found in the nature in low quantities. Its elementary form practically is not toxic, but its organic and inorganic compounds of 3-and 5 valents are toxic. The inorganic substances (e.g. arsenic-trioxide) are potent poisons, the organic derivatives (e.g. arsenic-acid) are less toxic. It occurs in form of arsenate in the soil and surface waters. The arsenic content of the natural waters usually is 1-2 g/l, but in certain regions (mainly due to earlier vulcanic activity) the arsenic concentration of the drinking water can be higher even by thousand times. At certain parts of Hungary (e.g. Békés-County), the natural arsenic content of spring-water is rather high, more than 100 g/l (the new threshold value is 10 g/l), for this reason arsenic is removed.The concentraton of arsenic in foodstuffs regularly is low, <0.25 g/g. Certain marine fish and shells are exempt, these organisms are building the arsenic into organic molecules, e.g. into arseno-betain, arseno-choline, arsenic sugar-derivaties and thir arsenic content may become higher. These organic arsenic compounds, however, are less toxic and are quickly excreted from the human organsims. According to measurements, the ratio of organic derivatives usually is 95 % and altogether 2-5% is the ratio of toxic inorganic arsenic compounds in the often high total arsenic content of fish and shells. The weeky tolerable intake of inorganic arsenics is 15 g/kg (WHO). There is no PTWI-value for the organic derivaties from which fish consuming individuals weekly may ingest more than 300-350 g but direct health damaging effect has not been recorded. In Hungary no tolerable intake level is prescribed for arsenic (neither organic nor inorganic compounds) in fish, fish-products. Before the implementation of the new regulation, between 2000-2002, in 11 % of import fish and crabs (within: 32% of predatory fish) the total arsenic content was over the formerly prescribed threshold value (determined by the Laboratory of the Food and Feed Safety Directorate of the Hungarian Central Agricultural Office).

4.5.2. Polychlorinated organic contaminantsAmong the several, different organic contaminats that are potentially present in foodstuffs, from part of the health/safety of the consumer, the most important are the polychlorinated diaromatic carbohydrates. In the practice, simply these altogether are termed as dioxins.

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More precisely, three distinct family of compounds are included, namely the polychlorinated dibenzo-p-dioxines (PCDD), the polychlorinated dibenzo-furans (PCDF) and certain polychlorinated biphenyls (PCB). More than 400 compounds are belonging to the three groups that may be different concerning their physico-chemical properties and especially their biological effects.

4.5.2.1. DioxinsThe group of the most important „dioxins”, from point of view of food-toxicology, includes 29 compounds (7 dibenzo-dioxins, 10 dibenzo-furans and 12 bifenyls). Their toxicological properties and biochemical mechanism of action are similar to the most toxic basic-molecule of 2,3,7,8-tetrachlor-dibenzo-p-dioxins (TCDD). Their biological-toxicological potencies, however, are different. To fracilitate their comparability in toxicity and consequent influence on consumers’ health and calculatability of tolerable intake levels for consumers, the use of toxicity equivalency factors have been introduced (TEF). The toxic potency of 2,3,7,8-TCDD is considered to be 1, and the potential toxic potency of other compounds are related to this one. The toxicity equivalency of some characteristic dioxin-like compounds determined by The Joint Committee of WHO are shown in Table 4.6.

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Table 4.6.Toxicity equivalency factors of some dioxins, dibenzofuranes and PCBs with dioxin-

like effect(WHO TEF 2005)

Congeneer Toxicity equivalency factorDibenzo-p-dioxins2,3,7,8-TCDD1,2,3,7,8-PentaCDD1,2,3,4,7,8-HexaCDD1,2,3,4,6,7,8-HeptaCDDOCDD (okta-)

11

0.10.1

0.0003Dibenzo-furans2,3,7,8-TCDF2,3,4,7,8-PentaCDF1,2,3,7,8-PentaCDF1,2,3,4,7,8-HexaCDDOCDF

0.10.30.050.1

0.0003Polychlorinated biphenyls3,3’,4,4’,5-PentaCB (PCB 126)3,3’,4,4’,5,5’-HexaCB (PCB 169)3,3’,4,4’-TetraCB (PCB 77)2,3,3’,4,4’,5,5’-HeptaCB (PCB 189)

0.10.03

0.00010.0003

The demonstrated data indicate that the toxic potencies of the different compounds are lower than that of TCDD. By applying the equivalency factors, the measured quantities of the given compounds in the environment or in foodstuffs can be converted into "TCDD-equivalency" (TEQ), and this is the basis of risk-assessments and derived tolerable value determinations.Dioxins are typical contaminants formed during the (imperfect) combustion of organic materials (e.g. waste-incineration, forest fires) or during the production of certain polychlorinated aromatic compounds (e.g. pentachlor-phenol used for wood-preservation or chlorinated phenoxy-aceticacid herbicides). These are almost insoluble in water, extremely persistant in the environment and are ready to accumulation in the animal and human organisms and to biomagnification in the food-chain.The consumer can uptake these compounds first of all by ingesting contaminated plants (vegetables, fruits), or by consuming meat, offals, milk, eggs of animals fed by contaminated feed, furthermore by fish meat and fishery products. Nighty percent of the uptake of dioxin-like compounds is performed with contaminated foodstuffs and 80 percent within this is with

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foodstuffs of animal origin. As environmental contaminants, they may contaminate the most different kind of natural materials (e.g. additives used in food industry), representing another type of risk to the consumer. They are readily absorbed from the alimentary tract and are accumulating in the fat tissues. Dioxins are able to disturbe of several biochemical mechanisms, they are immunotoxic, teratogens and carcinogens. The carcinogen effect is manifested mainly in the liver.The tolerable weekly intake level (TWI) of dioxins and PCBs with dioxin-like effect is 14 pg TEQ/kg.The European Committee accepted a strategy plan in 2001 for the reduction of dioxin-like materials in the environment, feed and in foodstuffs. As a part of the plan, tolerance threshold values were prescribed for fresh meat and meat products (ruminants, pigs, poultry), for liver, fish-meat és fishery products, for milk and milk-products, for eggs, egg-products furthermore for fats of animal origin, plant oils and fish oils intended for human consumption. These values were valid only for dioxins and not for dioxin-like PBC. Since then, sufficient number of data accumulated also for dioxin-like PCBs opening the possibility for establishing a common tolerable value for both.The tolerable maximun levels (in fat) of dioxins in foodstuffs of animal origin usually is 1-3 pg TEQ/g, in fishmeat (in the wet mass), and in liver. liver products the tolerable maximum level is 4-6 pg TEQ/g, (zsírra, halaknál nedves tömegre vonatkoztatva). The sum of the tolerable values for dioxins and dioxin-like PCBs are usually 1.5-2 times of the tolerable value of dioxin itself.The dioxin contamination of different kind of foodstuffs gradually declined in the 1990s years. According to West-European data, the regular dioxin content of meat and milk-products was <0.1 pg/g, eggs contained <0.2 pg/g, while in fishmeat it was <0.5 pg/g. At special areas, however, e.g. in certain fish species at the Baltic sea (hering, salmon) the contamination of environmental origin is much higher and the resulted dioxin levels may exceed the tolerable value.According to the data of the European Committee, the present dioxin contamination of foodstuffs in the Member States is resulting in a daily average uptake of 30-100 pg/day. An additional daily uptake is the estimated 60-110 pg quantity of the dioxin-like PCBs. These data reflect the average, the effective uptakeby by certain members of the population may be 2.5-times higher. Beyond the tolerable threshold values, the European Committee determined those concentrations („intervention levels”) above which the existence of an actual contamination source should be suspected. The identification and elimination of these sources is the basis of further potential reduction of the dioxin-exposition.

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4.5.2.2. Polychlorinated biphenylsThe group of the polychlorinated biphenyls (PCBs), beyond 12 dioxin-like PCBs that possess dioxin-like effect, includes high number of further less toxic compounds without dioxin-like toxicity. Earlier, the PCBs were widely used in different industrial processes (in heat-transfering and hydraulic systems, transformators and condensators, etc.), but their application was banned in developed countries in the 1980s because of the potential of high environmental contamination.

4.6. Contaminants of technological originChemical compounds potentially damaging the consumers’ health and applied in food-industrial technologies or formed in the foodstuffs during the technological processes are belonging into this group including contaminants getting into the foodstuffs during ordinary contact from production and technical objects, packaging materials. Considering that the use of these materials is strictly regulated and the conditions of their formation are well-known, their health damaging potential is essentially lower than that of the formerly discussed contaminants of environmental origin.On course of technological processess, however, potentially carcinogen compounds are also formed which may play a role in the pathogenesis of human tumour development. These substances are for example, the polycyclic aromatic hydrocarbons (PAHs), the heterocyclic amines (HCA-k) and the nitrosamines. According to certain opinions, about one third of malignant tumorous diseases may be linked somehow to foodstuffs, or malnutrition. The detectable quantities of genotoxic compounds in foodstuffs regulary are very low per component, but their simultaneous presence and activity in concenrt may promote the development of tumours in humans.

4.6.1. Polycylice aromatic hydrocarbone, heterocyclic aminesThe PAH substances are generated during the imperfect combustion of organic materials (e.g. wood, oil, coal/carbon). Todays, approximately 100 such compounds are known and one quarter of these are proved carcinogens including the widely known 1,2-benzpyrane.The main mediators of human PAH exposition are the air, foodstuffs, drinking water and tobbaco smoke. Foodstuffs are contaminated by smoke-gases present in the air and for example by smoking, heat treatment, drying procedures during which foodstuffs are direct contact with combustion products. The PAH-contamination is exceeding the 10 g in foodstuffs of plant origin, especially in vegetables possessing large leaves, consequently grand surfaces (e.g. lettice). In plant oils the PAH-concentration may reach even the 10-20 g/kg value. The PAH-contamination of foodstuffs of animal origin generally is very low (<1 g/kg). Due to smoking, the concentration of PAH in meat and specifically in fish may reach

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the 50-100 g/kg level. Similarily high PAH concentration may be developed at charcoal grilling of meat (>100 g/kg) if the meat is in direct contact with flame or if the melting fat is contacting glowing wood.The measure of daily PAH intake with foodstuffs is estimated to be about 3 g/nap (0.06 g/kg). The majority of this quantity is taken up by contaminated foodstuffs of plant origin and only a minor portion by foodstuffs of animal origin. This quantity usually is smaller than the quantity taken up by tobacco smoke (1 box/day cigaretta results in 2-5 g). The health damaging potential of food-mediated PAH is subject of debate.The measurement of benzpyrane concentration serves as marker for the carcinogen PAH contamination of foodsuffs in the European Union. According to the related regulations in force, the benzpyrane content of fats, oils and fishmeat must not exceed the threshold value of 2 g/kg-ot, while in smoked meat and meat products and fish the threshold value of 5 g/kg. The threshold quantity in nutrients and baby foods is 1 g/kg.During roasting of meat and fish, specifically if they are roasted abruptly at high temperature (>200oC) and for a longer time, genotoxic heterocyclic amines may also be formed. Their concentration is usually highest in grilled meat. Their estimated daily intake is mostly <0.1 g, which in itself probably is without health risk but may increase the health risk of other genotoxic materials of nutritional origin.

4.6.2. NitrosaminesThe third big group of carcinogen compounds of technological origin potetially present in foodstuffs are the nitrosamines (in general the N-nitrose substances). These are originating from the reaction of secondary amines containing =N-NO-group and of nitrites (specifically nitric acid). Approximately 80 seems to be carcinogen from the about 100 known nitrosamines from which the dimethyl-nitrosamine is the most widespread and most toxic.They can get into the human organism through two ways, partlally exogenously with foodstuffs, and partially by forming endogenously in the alimentary tract. This latter may be realised by the reaction of secondary or tertiary amines and nitrites in the stomach (pH 1-3) (see also in Chapter 4.8.2.).The most important exogenous nitrosamine sources are the cured and roasted meats, fish (especially the grilled on charcoal ones), smoked meat products, fish and cheeese, ceratin beers and cereal alcohols (e.g. whisky). In the recent two decades, the nitrosamine content of pickled meat and fish has been reduced due to the diminished authorised quantity of nitrite usable for curing. The nitrosamine concentration of cured products mostly is <10 g/kg, which taking daily 100 g consumption means daily <1 g intake. Much more nitrosamine can be drinken by beers, the consumption of 1 litre may result in up to 5 g nitrosamine intake. Considering our present consumption pattern, Hungarian consumers ingest an average of <1g nitrosamine. This quantity probably is without direct health-damaging potential but

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regarding the possible interactiion with other genotoxic materials present in the organisms, we should endevourfor the further reduction of their intake.

4.7. Contaminants of biological originAmong chemicals present in foodstuffs with potential public health hazard, we have discussed the food-toxicological importance of veterinary drug and pesticide residues and contaminants of environmental and technological origin. The main characteristics of substances belonging to the former group is that they are present in tissues of animals and plants (serving as potential foodfoodstuffs or raw materials) following a targeted and authorized application. In other words, their application is regulated and their residuological innocuity is garanteed by observing the determined and prescribed withdrawal time. In contrast, the contaminants of environmental origin (e.g. toxic heavy metals and miscellenous polychlorinated organic substances) may be the natural components of the environment or can be accumulated there under less controlled conditions. Consequently, the risk of contamination of food is essentially higher than that of the officially controlled veterinary drug and pesticide preparations or of potentially harmful materials formed during the technological processes or during the preparation of food.In the followings, we are going to discuss the food-toxicological importance of contaminants of biological origin. This group involves all health damaging chemical compounds which are formed by the activity of microorganisms in foodstuffs or in their raw material of animal or plant tissue origin or by eaten by animals.The contaminants of biological origin in foodstuffs can be sorted into four main groups:

Mycotoxins produced by moulds, Marine and fresh water biotoxins produced by microscopic algae, Histamine and other biogen amines formed by microbial decarboxylation, Bacterial toxins.

In spite of that bacterial toxins as chemical compounds are belonging to the chemical contaminants of biological origin, on traditional reason, we are discussing them with the microbial contaminants. Therefore, in the followings, we are going to evaluate the food-toxicological importance of mycotoxins, marine and fresh-water biotoxins, histamine and other biogen amines and neglect bacterial toxins.

4.7.1. MycotoxinsMycotoxins are the secondary metabolic products of moulds. The toxin producing moulds are widely distributed in the nature and they can be detected in the soil, in different plants and agricultural products. To multiplicate, they need oxygen, appropriate temperature and humidity. About two hundres species out of the several thousand moulds are considered of being important toxin producers. The characteristics of approximately 20 toxins or groups of

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toxins are known in detail from which the members of four groups exhibit/show public health importance such as the aflatoxins, ochratoxins, patulin and fusariotoxins (Table 4.7.).

Table 4.7.Main characteristics of mycotoxins of public health importance

Group Compounds Toxin producing moulds Occurrance Toxic effectsAflatoxins B1, B2, G1, G2,

M1, M2

Aspergillus flavus, A. parasiticus, A. nomius, hydroxylated metabolites

Oily seeds, corn, cereals, soya, spices, milk, milk products

carcinogen, hepatotoxic, immunosupressive

Ochratoxins Ochratoxin A Penicillium verrucosumAspergillus species

Cereals, coffe-, cacao- and soya been, grapes, wine

carcinogen, teratogen, nephrotoxic, immunosupressive, neurotoxic

Patulin Aspergillus and Penicillium species

Apple, other fruits, vegetables, apple juice

Enzyme inhibition, genotoxic, oedema inducing

Fuzariotoxins trichotecens T-2 and HT-2

toxin, DON, DAS

Fusarium species Cereals (wheat, barley, ryes, oats, rice), cereal products

Protein synthesis inhibition, hemato- and immunotoxic necrotic

Zearalenon F-2 toxin Fusarium species Cereals (corn, wheat, barley, rice)

Oestrogenic effect, fertility disturbances, infertility, damaging of spermatogenesis

Fumonizines B1, B2, B3 Fusarium moniliforme, other Fusarium species

Corn and corn-based foodstuffs

Nephro- and hepatotoxic, pulmonary oedema, encephalomalacy, oesophagus and liver carcinoma (?)

Moulds may cause spoilage, nutritional devaluation of foodstuffs and their toxins are able to induce several kind of health damaging effects. The most importan are the carcinogen, teratogen, immunosuppressive and neurotoxic effects.Mycotoxins are getting into the consumers’ organism by contaminated foodstuffs of plant origin and by foodstuffs of animal origin deriving from animals fed with feed contaminated with the toxin (primarily with milk, eggs and offals). The principal source of mycotoxin contaminations are the foodstuffs of plant origin; the appearance and concentrations of mycotoxins in foodstuffs of animal origin is depending on the absorption, distribution, biotransformation and excretion of toxins in the animal organisms, specifically in the edible tissues and milk, eggs. Bibliographic data show that aflatoxins, ochratoxin A and deoxynivalenol are readily absorbed from the digestive tract while the absorption of fumonizin B1 is poor.Among the well absorbing toxins, the aflatoxins are rapidly metabolised in the liver (they are covalently bound to the macromolecules and they severily damage them); among metabolites, the hydroxylated derivatives partially are excreted also by the milk (the letters of M1 and M2 are indicating milk) and they are representing food-safety risk. The half-life of ochratoxin A is much longer, therefore in internal organs of animals consuming feed

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contaminated by the toxin, mainly in kidneys and (in descending concentrations) in muscle, liver and fat residue accumulation can be developed. Deoxynivalenol (and probably diacetoxyscirpenol and T-toxin also trichotecenes) are fastly metabolized and exctreted in short time from the organism, thereby in practice they are without residue concern.Among food of animal origin with food safety importance, the potentially mycotoxin-contaminated milk is especially important. Aflatoxin B1, ochratoxin A and possibly zearalenon may represent safety risk because they may appear in the milk in unchanced form or as active metabolites (aflatoxin M1, zearalenol). The inactivation of mycotoxins in food by ordinary heat-tretamnet procedures is extremely difficult because these usually are heat-stable compounds. The principal method of protection is therefore, the prevention of contamination of foodstuffs and raw materials.

4.7.1.1. AflatoxinsAflatoxins are mycotoxins produced mainly by the Aspergillus flavus and Aspergillus parasiticus species. The four most important toxins are sorted into B and G groups (blue and green) based on their fluorescency under UV-light. From the A B1, B2, G1 and G2 metabolites növényi terményekben the B1 and G1 toxins are the prelevant in grains. The 4-hydroxy-metabolites, biotransformed from B1 and B2 toxins in the organism of cow that consumed feed contaminated by the toxins, are termed also as milk toxins of M1 and M2. Usually 1-3 % of the toxin present in the feed is secreted in the milk but this ratio may individually be different as well as can be changed in the same individual periodically.Aflatoxins are potent toxins. They are genotoxic carcinogens, and the B1 toxin is the most potent. Comparing to B1 toxin, the carcinogen potential of M1 is lower by approximately one magnitude. Their main target is the liver but on sustained exposure, they are immunsuppressive already at lower doses.Aflatoxin producing mould species my occur also in Hungarian grains and most of them have toxin producing potential. Fortunately, our climate is not favoring to the toxin production (it requires long-lasting temperature of >30oC and >80-85% humidity), therefore Hungarian food of plant origin are practically free of aflatoxin. In contrast, the continous control of import feedstuffs, especially of the oily seeds is very important (peanut, pistacia, nuts). Most frequently the peanut and pistacia consignments are contaminated. The import of this latter was transiently prohibited into the European Union from Iran in the mid 1990 because of the high contamination over the tolerable level.Considering the food of animal origin, the potential aflatoxin (M1) contamination of milk and milk products is significant. A linear interrelationship was detected between the B1

contamination of feedstuffs and M1 content of milk.To prevent to surpass of the tolerable level (0.05 g/kg) of aflatoxin M1 contamination in milk, the daily B1 uptake by milking cow should not be more than 40 g/nap. Supposing a

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daily 12 kg/cow concentrate consumption, this means a maximum aflatoxin feed contamination of 3.4 g/feed kg. In respect of aflatoxin B1 contamination of cow-feed and consequent M1 contamination of milk, the highest risk is the use for feeding of press-cake (a by-product oil manufacture from oily seeds) containing about 85% of total aflatoxin. Since in Hungary the use of peanut for

feeding of animals is prohibited, the probability of aflatoxin M1 contamination of milk is low. Aflatoxin M1 in fresh milk and in milk poducts is relatively stabile, it cannot be inactivated by pasteurasation. It is not soluble in milk-fat, therefore at manufacture of milk products, especially of skimmed milk, it is accumulated in the way, etc.On course of butter making, about 10% of M1 appear in the cream and only 10% in the butter, the majority of toxin is concentrated in the buttermilk. It is relatively stabile also in acidic milk products, and is concentrated in milk powder.Concerning food of animal origin, tolerable aflatoxin (M1) maximum level is existing only for milk and milk products (0.05 g/kg). Foodstuffs of plant origin for direct human consumption have a more strict tolerable level (4 g/kg of total aflatoxin, and 2 g/kg B1) versus products that will be selected and cleaned before use (10 or 15 g/kg total aflatoxin, and 5 or 10 g/kg B1).In Hungary, almost 4-7 thousand food samples of plant origin are examined yearly for mycotoxin including aflatoxins, ochratoxin, fuzariotoxins and patulin. In the recent years, over the tolerable levels of aflatoxin primarily was found in samples of, peanut, import ground paprika and rice. Mycotoxin contamination has not been detected in the majority of food of animal origin.

4.7.1.2. OchratoxinsOchratoxins are biologically very active mycotoxins primarily produced by Aspergillus and Penicillium strains. Under continental climate the toxin production of Penicillum verrucosum strains is important. They are multiplicating only at 30C temperature and 0.80 water activity and are the main source of ochratoxin-contamination of cereals in Mid and North Europe. Since at these teritories, the grain is widely used also as animal feed, the toxins of P. verrucosum may appear in certain animal tissues, especially in pig kidney and liver. Under subtropic and tropic climates, miscellenous Aspergillus strains are responsible for the toxin production (A. ochraceus, A. alutaceus, A.carbonarius).Concerning biological activity and toxicity, the most important ochratoxins are the chlorine atom containing ochratoxin A, which most frequently and in the highest quantity occurs in cereals, beens (coffee-, caco- and soya been) furthermore in grapes and in wines. The toxin may be formed also under the Hungarian climatic conditions and it occasionally may reach sigificant levels in feedstuffs. Certain ochratoxin-producing strains are able to

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synthetise also other mycotoxins (e.g. citrinin, penicillin-acid), the presence of which may influence the toxicity.Ochratoxin A is slowly but relatively well absorbed from the gastrointestinal tract and it is accumulating in the highest concentration in the kidneys, in less quantity in the liver, muscle and fat of animal and human organisms. It appears also in the milk in form of ochratoxin (a metabolite formed in the rumen of ruminants) and it is less toxic than the parent compound.Ochratoxin A is definitely nephrotoxic in mammals, causing tubulo-nephrosis and kidney fibriosis. The proximal tubule is the primary site of its cytotoxic and carcinogen effect. E Most sensitiuve species is the pig and in less measure the laboratory rodents. In rodent the the carcinogen effect requires higher dose than the nephrotoxic one. The toxin can penetrate the placenta and it is embryotoxic and teratogen and is immunosuppressive and neurotoxic in higher doses in animal experiments.It is nephrotoxic also in humans and earlier it was the supposed causative of the so called balcanic endemic nephropathy (a tumorous urinary disease) manifested mainly in the countryside population in Romania and former Yugoslavia. Recent epidemilological and clinical data have not been supported this assumption, therefore other nephrotoxic agents may also be involved in the pathogenesis.The established value of tolerable weekly intake of ochratoxin A is (PTWI) 100 ng/bw kg. According to collected data, the average weekly ochratoxin intake in Europe is 45 ng/bwkg, this is less than the half of the tolerable dose. This total value is distributed in cereals and wines (approximately 25, and 10 ng/bwkg, respectively) in grape juice and coffee (each 2-3 ng/bwkg), in pigmeat and offals (1.5 ng/bwkg) and in the other potential sources such as dried fruits, cacao, tea, milk, etc. (1 ng/ttkg).In the European Union the tolerable ochratoxin A level in cereals, in products of cereal origin, raisin, rosted coffee, wines and grapes is in the range of 2-10 g/kg. There is no prescribed value for fooodstuffs of animal origin.In Hungary, during the recent years ochratoxin in over the tolerable level has been found only in coffee samples. The half of the examined foodstuffs of plant origin, however, contained ochratoxin in below the tolerable level (usually 1-4g/kg), indicating a regular potential dietary exposition also in Hungary.

4.7.1.3. PatulinPatulin is produced by several Aspergillus and Penicillium moulds, and they most often induce the spoilage of apples. They may occur also in other fruits, vegetables, cereals, and occasionally ont he surface of meat products kept in frigo. Practical importance, however, only have the contamination of apple and apple juice.Following its discovery, patulin was intended to be applied as an antibiotic, but its toxicity also for higher organisms was realised soon. According to animal experiments, patulin

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increases the permeability of vascular capillaries and may induce oedema. It exhibits high affinity to sulphhydryl groups, thereby inhibiting the function of several enzymes. The genotoxic effect may be addressed to this property (it inhibits the DNA synthesis). Patulin does not accumulate int he organism, therefore its established tolerable intake level is daily 0.4 g/bwkg.In the European Union tolerable threshold value is existing only for fruit juices, alcohols, solid apple products (25-50 g/kg). In Hungary, during the recent years relatively few samples were examined for patulin but the number of contaminated samples fallen over the 25 g/kg value was high. This finding indicates that on course of apple processing, the so called brown-spoilage fruits should be removed because this abnormality is caused by the patulin-producing P. expansum.

4.7.1.4. FusariotoxinsAflatoxins, ochratoxins and patulin are belonging to the storage-moulds, the fusariotoxin producing Fusarium moulds are belonging to the so called fieldland-moulds, i.e. their optimum multiplication conditions primarily are found on the tillage (plough-land). Fusarium moulds are widely distributed in all over the world and most of them are toxin producer. They are able to synthetise several kind of toxins under a relatively wide range of temperature, including the Hungarian climate conditions. The members of three groups are important such as trichotecens, zearalenon and fumonizines.The group of trichotecen-skeletal toxins include more than 50 chemical congeeners with complex chemical structures. Their most important representatives are the T-2 toxin, the HT-2 toxin, deoxynivalenol (DON), tnivalenol, the diacetoxyscirpenol (DAS) and the fusarenon-x. The T-2, the HT-2 toxin and DON are the most significant food-contaminants. They are frequently occurring on cereals (wheat, corn, barley, rye, oat, rice). The DON in itself or altogether with other trichotecens, or with zearalenon can be detected in almost any grain-products.They are protein synthesis inhibitory, haemato and immuno toxic compounds which may contribute to the higher incidence of infective diseases. Ingesting in higher quantity, they are able to induce vomiting, diarrhoea, necrosis in the mouth and oesophagus, contact-inflammation of the skin, furthermore the drop in leucocyte count. Fusariotoxins are heat-resistant. During the milling process, however, their quantity is greatly reduced due to the removal of the surface contamination on course of hulling before milling. They are not present in food of animal origin in significant quantities because animals refuse to consume feed significantly contaminated with trichotecens and the ingested toxin avidly metabolised in and fastly excreted from the animals’ organism.The provisory maximum tolerable daily intake level of DON is 1 g/bwkg, while of T-2 and HT-2 toxins (individually and altogether) it is 6 g/bwkg.

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According to the related regulation of the European Union, bread, paste and grains-intended-directly-for-human-consumption may contain maximum 500-750 g/kg DON, while flour and milling products may contain 300 g/kg T-2 and HT-2 toxins. In the Hungarian grain products the T-2 and HT-2 toxins only sporadically are present but the DON-contamination occasionally is significant.Zearalenon (F-2 toxin) is different from the trichotecens in chemical composition and biological effects. It is a characteristically oestrogenic mycotoxin. The effect is specifically manifested in sows exhibiting oedematous endometrium accompanied with intensive cell-proliferation. Functional asynchrony is appearing in the function of the uterus and ovaries, the inplantation of ovum is inhibited, by preventing pregnancy, infertility and in chronic cases, multiplex ovarial cysts may be developed. The spermiogenesis is also damaged by the F-2 toxin.Humans are probably also sensitive to the effects of zearalenon, however, the toxin-related clinical manifestations have never been identified. Hungarian scientific results inndicate that zearalenon is specifically bound to human receptors and the mycotoxin could be detected in children showing early pubertic phenomenons.The tolerable intake level of zearalenon is 0.5 g/bwkg.Zearalenon is present in cereals as surface contamination and it is accumulating in the bran during the milling process.Tolerable threshold levels are existing int he European Union for flour, bran, bread, pate and biscuit (50-75 g/kg). According to the results of Hungarian examinations, F-2 toxin is rarely occurring in domestic cereal products.Fumonizins are the members of a relatively new group of mycotoxins, and based on their biological effects, they are considered as „aflatoxins of the 90s years”. They primarily are produced by the Fusarium moniliforme, but other Fusarium species are also able to syntheise the toxin. The mould can multiplicate under conditions present both on the fieldlands and storage-sites and they produce toxin during humid conditions following hot and dry periods. The toxin-generating moulds are distributed all over the world, first of all on maize. The contamination of damaged corn of maize can be specifically intensive.Out of the existing more than ten types, three (B1, B2, B3) occur most frequently and the B1 is the most important one.Fumonizins may cause miscellenous diseases in animals resulting in leukoencephalomalacia in horses (ELEM, equine leukoencephalomalacia), pulmonary oedema and emphysema in pigs (the so called fattening pulmonary oedema, PPE, porcine pulmonary oedema), liver and renal damage in rats. They may play a role in the pathogenesis of oesophageal and liver carcinoma.

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Fumonizins are concentrated in the embryo and seed-coat of plants. The treatment of surface of seed, boiling in alkaline, washing and chilling are suitable methods for reducing the toxin concentration.The tolerable daily intake level of fumonizin B1, B2 and B3 toxins individually and altogether is 2 g/bwkg. The estimated dietary daily intake of European consumers is about 0.1 g/kg. Consumers who are regularky eating maize-based products, this value is about 1.0 g/kg. In the European Union, the tolerable threshold value for maize-based products, fluor, groats, oil is 400-1000 g/kg.

4.7.2. Marine and fresh-water biotoxinsThose are about 40 out of the several thousands microscopic marine algae species which are producing miscellenous biotoxins. The toxin containing algae are primarily taken up by bivalve molluscs, occasionally by fish, thereby the toxin may become a part of the human food chain generatig potential hazard for the human consumer. Algae in fresh water also may produce toxin but their public health importance is essentially lower. Toxins produced by microalgae, are termed also as phycotoxins.

4.7.2.1. Marine biotoxinsToxin producing algae are world-wide distributed both at continental and warm climate areas but a given species and its actual high multiplication altogether with the enriched presence of its toxin is characteristic for a given area. Public heath hazard due to the consumption of marine molluscs and fish contaminated by the toxin was restricted to costal areas close to the territories of the algae, todays, however it has become imternational.Among the marine phycotoxins, practically the different shell-toxins and the ciguatera toxin accumulated in fish are important. The most important toxins belonging to the former group are the paralytic shell poison (PSP), athe diarrhoic shell poison (DSP), the amnesic shell poison (ASP) and the neurotoxic shell poison (NSP). The group of ciguatoxins includes at least 20 compounds with polyether structure produced by bentic and epiphyte dinoflagellae. The most important properties of the major toxins are shown in Table 4.8.

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Table 4.8.Major marine biotoxins

Name Source of human disease

Toxic effects Maximum level*(related to molluscs

or fishmeat kg)

Occurrance

Paralytic shellfish poison= PSP, principal component: saxitoxin)

Marine molluscs(mainly bivalve

molluscs)

inhibition of membrane conductivity, respiratory paralysis (human lethal dose: 20-80 g/bw saxitoxin-equivalent)

800 g/kg World-wide, continental and tropic areas

Diarrheic shellfish poison= DSP, complex toxin-group; okadaic-acid, dinofisistoxins, yessotoxins, etc.)

Molluscs(mainly bivalve

molluscs)

vomiting, diarrhoea, abdominal pain and usually recovery in 3 days (certain components are carcinogens?)

Okadaic-acid, dinofisis toxins 150 g/kg

yessotoxins: 1 mg/kg

Canada, South-Amerika, Japan, West-Europe, Skandinavia

Amnesic shellfish poison=ASP; domoic-acid

Marine molluscs vomiting, diarrhoea, disturbances in orientation, amnesia

20 mg domoic-acid/kg Mainly the West-costal parts of USA, Canada,

Japan, North-West-costal part of Europe

Neurotoxic shellfish poison=NSP; brevetoxin)

Marine molluscs vomiting, diarrhoea, paraesthesia, motor incoordination, asthma-like symptoms

800 g/kg**(brevetoxin-equivalent)

Floride, New-Zeeland

Ciguatera toxins (ciguatoxin, maitotoxin)

Tropic and subtropic fish

vomiting, diarrhoea, muscle pain, paraesthesia, circulatory disturbances

- Floride, Bahamas, Caribbean-sea, East costal part of Africa,

Asia

* Regulation 853/2004/EC** FDA recommendation

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Some natural marine biotoxins are very potent poisons (e.g. already 1-4 mg PSP can be lethal); but their presence in marine molluscs or fish is without any organoleptic abormality. They cannot reliably be inactivated by heat-treatment. Therefore, the basis of protection is the prevention, first of all the regulation of farming and harvesting of bivalve molluscs. The corresponding prescriptions are described in the EU Regulation of 853/2004/EU. This Regulation includes the puiblic health requirements along with the threshold values of toxins for marine molluscs (Details in Chapetr 19.2.). They are examined by biological (mouse) and instrumnetal analytical means (LC-MS/Ms).

4.7.2.2. Fresh-water biotoxinsOccasionally, the eutrophycation based high degree alga multiplication may cause public health problem. The causatives are the potentially toxin-producing blue-algae that often termed as cyanobacteria. These toxins, in contrast to the above discussed marine biotoxins, are not accumulated in vectors but they are acting by direct contact with the human organism, e.g during swimming (contacting with the skin) or by the drinking water.The toxins of the blue algae have different bilogical activity (e.g. hepato- and neurotoxins, lipopolysacharides) and the most important is the liver damaging biotoxin produced by the Microcystis aeruginosa. The direct contact with blue-algae may evoke allergic dermatitis, occasionally more severe hypersensitivity reactions/symptoms. There are no reliable data on the possible symptoms following oral ingestion/intake of the toxins.

4.7.3. Histamine (scombrotoxin)The biogen amine histamine present in animal tissues has potent biological actions and normally is synthetised from histidine by the histidine-decarboxylase enzyme. The enzyme is produced also by certain bacteria while multiplicating. The enzyme interating with free histidine found in tissues with high protein content and certain foodstuffs, produces histamine (microbial decarboxylation). The meat of certain fish species (especially macrelas, sardinia and tunafish) contains significant quantity of free histidine, from which big amount of histamine can be formed by histidine-decarboxylase of bacterial origin.Since bacterial multiplication primarily is temperature dependent, the basic method of prevention of histamine production is the chilling of fish within 6-9 hours after catching and its storage at <4oC temperature or freezing up to time of consumption.The activity of histidine-decarboxylase is preserved even after the death of bacteria and also under chill condition or it is reactivated fast following freezing and

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defrosting. This is the reason why the inhibition of growth of the enzyme producing bacteria is so important.Beside, histamine, in rotting fishmeat other biogen amines (e.g. putrescine, cadaverine) can be formed and may adversly affect the health of consumers.Histamine and also tyramine (potentially is equally harmful for human health) may be formed from tyrosin by microbial decarboxylation during ageing of certain cheeses or in wine.The maximum tolerable concentrations of histamine is regulated altogether with the microbiological criteria in foodstuffs in Regulation of 2073/2005/EC. Accordingly, fishery products, made from fish species containing high amount of histidine (especially macrela, hering, sardella), may not contain histamine over 100 mg/kg during their shelf-life, and the histamine concentration in 2 out of 9 examined sample may be fallen between 100 és 200 mg/kg. Fishery products manufactured by enzymatic ageing in salt solution, the maximum tolerable value may the double of the previously described ones. (See also in Chapetr 19.1.2.).In Hungary the histamine content of the examined fish and cheese samples is ususally appropriate, pickled fish-cans, however, occasionally may contain over-the threshold-concentrations of histamine.

4.8. Toxic materials of natural originThose natural components of foodstuffs are belonging here which in original quantities or after enrichment may be hazardous for public health. Basically, they can be sorted into two groups. One group of compounds are nutritives (also) with harmful effect, the members of the other group are harmful compounds without nutritive property.

4.8.1.Nutritive materialsNutritive compounds may cause health damage mostly in the presence of inherited or acquired enzymopathia (partial or full lack of a metabolic enzyme), consequently the nutritive compound cannot participate in the metabolism. Characteristic examples are the lactose intolerance, galactosaemia, phenylketonuria or coeliacia (gluten-sensitive-enteropathia). In these conditions, the life-long diet of patients must include lactose, phenylalanin or -gliadin -free foodstuffs. In contrast to the previous compounds, the eruka-acid and trans-fatty acids are hazardous to any consumers. Eruka acid may occur in rape-oils at higher quantity. Trans-fatty acids are formed during processing of oil (hardening and hydrogenisation in manufacturing of margarin) and similarily to the saturated fatty acids, they have cholesterin level increasing effect.

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4.8.2. Toxic natural ingredients without nutritive propertyThe non-nutritive toxic compounds(ingredients) are natural poisonous components of certain raw materials. High number of compounds with different biological activities are belonging here. From part of food safety, the solanin, cyanohydrogen, methyl alcohol, morphin and its derivatives, furthermore nitrates and nitrites are the most important components.Solanine. Chemically, it is a glycoalkaloid which can be hydrolysed to sugar (solanose) and an alkaloid of solanidine. Solanidine is the toxic component with a saponine like effect: locally it is tissue irritant, following absorption it causes haemolysis and atropin-like neuronal symptoms. Solanine can be found mainly in potatoes (primarily in the green, germ-like tubers), but it is formed also below the skin of apple, turkey egg and tomato. Maximum tolerable value is for potato: 180 mg/kg.Ciyanhydrogen (hidrogen-cyanide). It is present in certain plants in glycoside bound as cyanoglycoside. Cyanoglycosides practically are not poisonous but enzymatically cyanhydrogen can be released from cyanoglycosides which is strongly toxic. The cianidion is bound to tissue oxydative enzymes containing tri-valent iron, thereby it is going to bind to cytochrome-oxydase and inhibits the tissue oxydative processes resulting in cytotoxic anoxia.From food safety point of view, the seed cyanoglycoside content of stone-fruits (almonds, apricots, cherries, plums) is important. These seeds contain the prunazin or amigdalin cyanoglycosides, from which at injury of the seed the released emulzin enzyme is splitting the cyanhydrogen. The consumption of ten pieces of raw, bitter almond may be lethal for children and the consumption of 15-20 seeds may induce severe poisoning in adults. On course of industrial processing, the cyanoglycosides can be decomposed by appropriate treatment. There are maximum tolerable values for sweet industrial products containing stone-fruits (e.g. marcipan), for fruit compots, wines, and alcohol (pálinka) preparations: 10 mg/kg, 1 mg/l, and 20-40 mg/l. Methyl-alcohol (methanol). It is present in alcoholic drinks in low quantity. Organoleptically (smell, taste), it cannot be distinguished from ethanol. For this reason it is used for adulteration of alcolic drinks, sometimes resulting in mass toxicosis. Methanol is metabilised in the human organism partially similarly to ethanol:

alcohol-dehydrogenase aldehyde-dehydrogenase folate dependentMethanol Formaldehyde Formic ac id CO2+H2O

slow fast very slow

In man, both alcohols are oxydated by alcohol-dehydrogenase into the appropriate aldehyde (ethanol → acetaldehyde, methanol → formaldehyde), the oxydation of methanol, however, is much more slower. Aldehydes, formed in the first oxydation,

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are oxydated further again by a common enzyme, the aldehyde dehydrogenase, into the appropriate acid (acetaldehyde → acetic acid, formaldehyde → formic acid), fastly in both cases. Acetic acid is without toxicological significance because in form of acetyl-CoA it enters the citrate cycle and is transformed into CO2, and water. The folate-dependent oxydation of formic acid, however, is very slow.The narcotic alcohol itself and the acidosis furthermore, the retinal damage inducing formic acid are the reponsible for the toxic effect of methanol. In man, the lethal dose of methanol is 30-100 ml, but already 8-10 ml may cause severe toxicosis. The methanol content must be maximum 0.2% of the ethanol content in commercial palinka, brandy (cognac), and liquor.Morphin and derivatives. Morphin and related alkaloids, such as codein, tebain, narcotin, narcein and papaverine are biologically very active compounds and widely used in medical treatment. They are the main components of opium obtained from the dried milk of green poppy capsule. The morphin, codein and tebain (fenantrene-derivatives) are inhibitors of the central nervous nsystem (CNS), the narcotin, narcein and papaverine (isochinoline derivatives) are smooth muscle relaxants. The scsalding water of poppy capsules contain about 2-4 mg morfphin. Its lethal dose in adults is 200-400 mg. Tolerable threshold values are established for poppy seed, it is 30 mg/kg for morphin, and 20 mg/kg for the other alkaloids.Nitrates. Nitrates are the common constituents of the soil, surface fresh and marine waters, plants and animal tissues. They are easily converted into nitrites through microbial activities. Nitrites are vasodilator and methaemoglobin forming compounds. Reacting with secondary and tertiary amines, they may converted into nitrosamines. Methaemoglobin may be formed physiologically in small quantities (<10%). In contrast, if 20% of haemoglobin is converted into methaemoglobin, hypoxaemic symptoms are appearing, at 60-80% conversion lethal hypoxia is developing.Nitrosamines may be formed in the stomach of human organisms (pH 1-3), the process need preformed nitrites to be present. Nitrites are syntheised from nitrates in the intestines as a result of baterial activities. Nitrates are occurring in green plants, certain species are intensively accumulate nitrates and several factors may increase the nitrate content of plants (intensive N-artificial fertilizing, the lack of certain trace elements, long-lasting drought, etc.).From part of consumers’ health, the nitrite/nitrate content of drinking water is also important. In drinking water mainly nitrites are present but, specifically in dug wells, a major part of nitrates microbically is converted into nitrites. Tolerable threshold value of nitrates in foodtsuffs is existing for spinach and common lettuce (200 mg/kg), while in case of nitrites the existing thershold value is 10 mg/kg for baby foods only.

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4.9. The control of chemical contamination of foodstuffsThe site of the potentially consumer-health damaging chemical contamination of foodstuffs basically is the primary production, the farm (residues, contaminants of environmental origin, majority of contaminants of biological origin, toxic components of natural origin). Accordingly, in preventing and control of chemical contamination, the primary production has outstanding importance, such as the animal farming and plant cultivation, or the food production animal and products of animal origin (fresh milk, eggs, honey, live bivalve molluscs, snail, fishery products), furthermore the plant products (cereals, fruits, vegetables, season-plants, etc.).

4.9.1. Basic principlesThe chemical-toxicological safety of foodstuffs (as part of the overall food saftey) primarily is the reponsibility of the food industrial undertaker (producer, manufacturer). The producer may market live animal, products of animal or plant origin which have not been treated with banned drug/chemical and the corresponding withdrawal time was observed. The responsibles of the food processing establishments must do their best in order to receive/accept animals or products (as raw material) for which the producer in the previous step of the food chain took the responsibility that those were not treated with banned substance and the withdrawal times were observed and staisfied their duty on recordings and information supply. The state contributes to the guarrantee of safety by elaborating and implementing the related legal framework and controlling the adherence to those rules. In Hungary the elaboration of the corresponding regulation primarily is the function of the Ministry of Public Health while the control is the duty of the Ministry of Agriculture and Country Development.Basically, two systems of control are operating, the national residue monitoring program, and the random official control. In Hungary, the Ministry of Agriculture and Country Development is responsible for the former and the terriorial food control authorities are responsible for the latter activity.

4.9.2. Monitoring systemsThe basic component of the control is the National monitoring system based on random sampling of foodstuffs of both animal and plant origin. In Hungary, the operation of the system is the function of the Food and Feed Safety Directorate of Central Agricultural Office (foodstuffs of animal origin) and the Plant, Soil and Agro-Environmental Protection Directorate of Central Agricultural Office (foodstuffs of plant origin).

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The examinations are carried out following the corresponding yearly study plan that is elaborated in the previous year and approved by the EU. The study plan includes the list of chemicals to be determined, the name of study samples, the frequency of sampling, the list of analytical laboratories involved, the tolerable concentrations prescribed in the corresponding regulations, and the official measures in case of positives when banned substance or higher than the tolerable concentration is detected in foodstuffs of animal or plant origin.

4.9.2.1. Foodstuffs of animal originOn course of monitoring of foodstuffs of animal origin, the samples are taken from live animal, feedstuffs, drinking water, body fluids, excreta, animal tissues and products of animal origin. The subject of monitoring are cattle, pig, sheep, goat, solipeds, poultry, farmed water animals, eggs, honey, furthermore, snail, rabbit, gamemeat and farmed game. The study items (compounds) are belonging to two groups. In group „A” are the anabolics and the compounds banned for using in food production animals. In group „B” are the veterinary drug preparations (products) and miscellenous contaminant compounds. Concerning group „A” compounds, the purpose of official inspection is to discover of banned application while in case of group „B” compounds the aim is to detect potential poor professional use or abuse and check if the withdrawal time was observed or not. Higher portion of samples must be examined for compounds of group „A”.The number of examined samples in the actual year is determined taking into consideration respectively the number of animals slaughtered in the previous year and production rate. The minimum animal number is 0.4% of the slaughtered cattle, 0.05% of slaughtered pigs, and after each 200 tonnes in a year, 1 broiler chick or 1 turkey sample but minimum 200 samples per year.A portion of samples are taken at the farm (group „A”), the other portion of samples are taken at the slaughterhouse (groups „A” and „B”). Sampling is the duty of the official vet. Samples must be transported frozen and urgently into the laboratory.The laboratory examination is going on two levels: screening and confirmative. In case of group A-compounds always, in case of group B-compounds only when detected positivity at screening, confirmatory examination must be done.If it is confirmed that banned substance was used, based on decision of the authentic authority, the animals will be killed without compensation and the flock/herd will be under veterinary control and a further official sampling program is initiated.If over the maximum residue level (MRL) was detected, marketing restriction is ordered for the farm and more severe examination is carried out. In case of repeated

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occurrence, the live animals and products originating from this same farm must be inspected more intensively for 6 months..At the slaughterhouse, when suspected that the withdrawal time was not observed for the animal intended for slaughter, the slaughter must be postponed until receiving the negative result of the laboratory examination. In case of positive result, however, the animal is unfit for human consumption. The meat, offals and wastes of animals that were treated with banned substances, must be destroyed as high (infection) risk material.

4.9.2.2.Processed and unprocessed products and foodstuffs of plant originConsidering the chemical contamination of agricultrural products and foodstuffs of plant origin, the pesticide residues are the most important contaminants. Accordingly, in this product group, the National monitoring program is primarily directed to the detection of possible pesticide residues and a minor part of the program intends to examine the heavy metal, nitrate, nitrite content of unprocessed agricultural products of plant origin. In Hungary, the planning and execution of monitoring examinations are the function of the Plant, Soil and Agro-Environmental Protection Directorate of Central Agricultural Office and of the County Plant- and Soil Protection Directorates.In Hungary, the number of authorised active substances is about 270, and in the framework of monitoring examinations about 200-220 compounds can be checked. This is a good ratio also in international practice/comparison.The examinations include the domestic and import products and plant based processed foodstuffs (baby foods and drinks, flours, muesli, and fruit drinks). In case of domestic products, the inspection of the prescribed ordinary use of pesticides and the sampling are carried out at site of cultivation and in framework of inspections at the market sites. The examination of import products includes foodstuffs originating primarily from third countries and done at the borders furthermore in framework of import market inspections at wholesalers and at the big market-chains. Reasonably, the sampling of domestic products is carried out during harvesting at site of cultivation and at the market sites while in case of import products it is done at the borders and at the big wholesale market chains.In Hungary, in the framework of the monitoring system about 3600-3700 samples are examined yearly, and approximately 50% of these samples are domestic fresh vegetables and fruits (a minor portion also cereals), 40% are unprocessed import products and the remainder about 10% is plant based processed foodstuffs. In year of 2006, 1.6 percent of the examined domestic unprocessed products contained residues above the tolerable threshold level. The quantity of objectionable samples of import products (presence of residue in higher concentration than the tolerable value or the detection of pesticide not authorised in Hungary) was 3.4%. Higher than the

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usual objection/rejection ratio occurred in case of tropical fruits, fine (dessert) grapes, tomato and paprika.The monitoring system is supplemented with random, targeted examinations, usually in framework of more intensive official inspections (e.g. seasonal ones at summertime or Christmas time) and on suspect.

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