Microbiology and Quality Assurance PDF

MICROBIOLOGY AND QUALITY ASSURANCE Unit 1 - Role and significance of microorganisms in food Expected Learning Outcome Microorganisms associated with aquatic environment and fish and their role in spoilage of fish. Historical development in food microbiology. Role of microorganisms in...

MICROBIOLOGY AND QUALITY ASSURANCE Unit 1 - Role and significance of microorganisms in food Expected Learning Outcome Microorganisms associated with aquatic environment and fish and their role in spoilage of fish. Historical development in food microbiology. Role of microorganisms in aquatic environment and sources of entry of microorganisms to foods. Introduction to fish microbiology Fish /aquatic foods contain a variety of microorganisms from different sources. These contaminants in food cause problems of spoilage and health risk to consumers. Fish are not sterile and the microflora includes natural flora of the waters from which fish are harvested and acquired transient flora from environment especially during handling, processing, storage etc. Inner tissue of healthy fish is sterile and microorganisms are mainly associated with outer slime, gills and intestine. Microbial load is higher in intestine followed by gills and skin. Natural microflora of fish varies depending on the habitat of fish (freshwater / marine / river/ water/ lake/ etc.), its feeding habit and life history stages. Generally, fresh warm water fish have more mesophilic bacteria than cold water fish. Transient flora include microorganisms entering the food from fish contact surface (crafts/gears/ baskets/ fish holds etc), the air, soil, water/ice used for washing, food handlers, packaging material and storage environment. Fish harvested from polluted water contain variety of microorganisms depending on nature of pollutant, and also human pathogens such as bacteria, fungi, viruses, protozoans, parasites etc. Once the fish dies the associated microorganisms affect quality due to spoilage. Thus, it is necessary to maintain the quality by destroying associated microorganisms and preventing growth of surviving microorganisms. The different preservation methods mainly aim at maintaining fish quality by reducing, killing or inactivating associated spoilage microorganisms. History of microorganisms in food The role of microorganisms in spoilage and food poisoning was realised only after the establishment of bacteriology or microbiology as a science during the early 18th century. However, adoption of several approaches to avoid/ reduce spoilage was known since early 1 civilization. The use of salted meat, fish, fat, dried animal skin and cereals was practiced by the Sumarians at about 3000 B.C. Romans were known for adopting preservation techniques for meat and also use of snow for perishables including prawns at around 1000 B.C. Smoking of meats and making of cheese and wines emerged during the same time. But the people at that time were not fully aware of nature of the preservation techniques and also the role of foods in the transmission of diseases and the danger of eating infected animal meat. A Kircher, a monk, was first person in 1658 to observe the decaying bodies, meat, milk and other substances and believed it to be due to invisible worms, thus suggesting the role of microorganisms in food spoilage. During that time the theory of spontaneous generation of life was gaining importance and L. Spallanzani in 1765 was first to oppose it with his experiment using beef broth which remained sterile after boiling and sealing. But he was not able to convince the proponents of spontaneous generation who felt that air was necessary for life to begin. Later in 1837, Schwann’s experiment of passing heated air in to the boiling broth caused it to remain sterile thus disproving spontaneous generation of life. But it was not until Pasteur’s well planned swan neck flask experiment that the theory of spontaneous generation of life was put to rest. The development of canning of meat in glass bottles by Nicholas Appert in 1809 had practical application in food preservation. Pasteur was the first person to appreciate and understand the presence and role of microorganisms in food. He demonstrated the role of microorganisms in souring of milk in 1837, and applied heat to destroy undesirable microorganisms in wine and beer, in about 1860. Involvement of of microorganisms in food spoilage and food poisoning has led to the development of preservation methods to arrest and kill microorganisms. The science of food microbiology has application in all kinds of food meant for human consumption and aims at ensuring the wellbeing of consumers. Role of microorganisms in nature and in foods The food of humans which is of plant and animal origin are naturally associated with microorganisms of several kinds. Microorganisms in their natural habitat play an important role in cycling of nutrients in the ecosystem. In the process of performing their primary role in nature, the microflora associated with food cause spoilage of foods meant for human consumption. Thus, the knowledge on types of microorganisms naturally associated with plant and animal foods helps to predict the microbial types that could be present at later stages of handling, storage and preservation of food. Depending on the nature of food and its habitat a variety of microorganisms are expected in the food and these may often affect the safety of food. Therefore, information on factors such as total number and types of microorganisms naturally present, types of microorganisms present in specific food, and ones which are not natural to the food 2 becomes necessary. This information becomes valuable in ascertaining the safety of food during different stages of processing, handling and storage. 1.3 Microorganisms in aquatic environment All surface waters such as ponds, lakes, rivers and oceans differ in their physical, chemical and biological characters. Depending on the nutrient status of water body, the microbial load varies with higher numbers encountering in eutrophic waters. Ground waters or subterranean waters generally have very low microbial load because of filtration effect of soil layers. Categories of microorganisms in natural waters The natural waters contain a variety of microorganisms. These include, Natural flora : Microorganisms natural to the water body and Transient flora : Microorganisms entering the water body from outside environment like from soil, air and through pollutants. Microorganisms in natural aquatic environment play an important role in nutrient recycling, and as primary producers and decomposers of organic matter. All the microorganisms present in a water body can be seen as surface flora of inhabiting organisms. These not only include spoilage organisms but also human pathogenic microorganisms especially in sewage contaminated waters. Primary source of microorganisms found in food The foods of plants and animal origin carry several microorganisms associated with their natural habitat. Plants carry typical micro-flora on their surface and also get contaminated from outside sources. Animals carry microorganisms on their surface and intestine, and also contain contaminants from surrounding environment. Through their excretions and secretions animals release microorganisms in to surrounding environment. Besides, both plants and animals carry pathogenic microorganisms capable of causing human illness. The food associated microorganisms are influenced by the availability of specific nutritional requirements and the environmental parameters. The primary sources of entry of microorganisms in to foods are from the soil, water, air, during handling, processing transportation and storage of foods. Soil Soil being the rich source of several kinds of microorganisms immediately contaminates the plants and edible plant parts, and the surface of animals with the soil associated microorganisms. As the soil particles are carried in to aquatic environment through wind, rain and other means contamination of water takes place with several soil micro-flora. Therefore, it is not uncommon to find several microorganisms both in soil and water environment. These soil derived microorganisms form part of the the microbial flora 3 involved in spoilage of foods of plant and animal source. Thus, there is a need to reduce the load of soil microorganisms in foods which can be achieved by removing the soil by washing the surface of foods with good quality water, and by avoiding contact with soil/ dust. Water Natural waters not only contain several microorganisms native to the aquatic environment but also from soil, raw/treated sewage and pollutants entering the water body. The microbial numbers and types vary in different water bodies depending on the nutrient status. Thus, all kinds of microorganisms found in water are likely to be associated with the aquatic organisms as surface flora. Use of such water for food processing will add microorganisms from water to food. Sewage waters containing human pathogenic microorganisms contaminate foods when such waters are used without proper treatment. The water used in food processing should meet agreeable chemical and bacteriological characteristics. Air Air contains several microorganisms which may get deposited on the food being processed and handled. Though the air does not contain natural flora of microorganisms, whatever microorganisms encountered are those associated with the suspended solid material and water droplets. The sources of microorganisms to air are from dust, dry soil, and water spray from natural surface waters, droplets of moisture from coughing, sneezing and talking by food handlers, from sporulating moulds growing on walls, ceilings, floor, foods and food ingredients. Thus, it is likely that the microorganisms persisting in air get deposited on the food being processed and contribute for microbial load and subsequent spoilage of food. The number of microorganisms present in air depends on factors such as extent of movement of air, sunshine, humidity, location and amount of suspended dust in air. Quiet air allows settling of microorganisms but the moving air brings in microorganisms and keeps them suspended. Thus, the number of microorganisms in air is increased by air currents caused by movement of people, by ventilation and by breeze. The rain or snow removes microorganisms from the air. 1.5 Micro-flora of food processing facility The nature of micro-flora in a food processing facility varies depending on the nature of food being processed. Hence characteristic microbial populations are encountered in different processing units. Also variations may be observed in microbial numbers from one area of processing plant to another. The microbial types present inside the processing plant are related to quality of air out side the plant and the microbial population levels are related to the level of activity of workers. Reducing microbial load in processing area There is a need to reduce microbial load in the processing area. This can be achieved by by installing filtration, chemical treatment and heat or electrostatic precipitation units, and 4 taking measures in preventing the build up after reducing the microorganisms. The build up of microorganisms in the processing area can be prevented by maintaining the positive pressure in food process area, installing filters in ventilating systems that prevent spread of microorganisms from one part of a plant to another and installing UV- irradiated air locks at doors to reduce the number of organisms carried by workers. Handling and processing Foods grown/cultured in natural environment containing specific groups of microorganisms are further contaminated by several microorganisms during harvesting, handling and processing. Further, addition of microorganisms to food may take place from; All food contact surfaces including equipments coming in contact with foods, packaging material, and from food handlers. Foods are also prone for microbial contamination during transportation and storage. Use of sewage contaminated water for washing foods being processed contaminates it with human pathogenic microorganisms All the microorganisms associated with food handlers enter the food during handling of food from hands, garments, body surface, hair etc under poor personnel hygiene practices. Therefore, it becomes necessary to prevent/reduce contamination and microbial build-up in foods during handling and processing so as to produce safe food with good keeping quality. 1.6 Significance of microorganisms in foods Microorganisms associated with food derive energy from food for those cell growth, maintenance and reproduction. Based on their function microorganisms associated with foods may be divided in to three general groups; Those causing spoilage or undesirable changes in the food Those producing desirable changes Those producing disease Based on the extent of stability to microbial invasion foods may be classified as; Perishable foods Ex. Fish and meat Semi-perishable foods Ex. Potatoes. Tomato Stable foods Ex. Cereals, Flour and Sugar The stable or semi-stable foods become unstable or perishable when the moisture content increases. 5 Factors affecting microorganisms in foods The survival and activity of microorganisms in foods depends on several factors namely, numbers and types of microorganisms present, type of food, treatments to which the food has been exposed, processing or storage treatments that the food receives, whether the food is to be consumed as it is or heated. The food associated microorganisms may have useful function, cause spoilage, cause health hazard and play no role or remain inert. The cases of spoilage, food-borne illnesses or useful activity results due to the growth and multiplication of the microorganisms. The inert microorganisms are those which do not find food environment favorable for their growth, and remain dormant without causing any changes in food. Causes for spoilage of food Spoilage of food usually occurs due to, Undesirable changes brought about by the microorganisms in the odour, colour, taste, texture and appearance of the food. Some microorganisms may not directly involve in spoilage but bring about changes in food that will facilitate growth of spoilage organisms. Ex. Bacteriophage attacking useful organisms and facilitating growth of undesirable organisms leading to spoilage. Useful organisms are those which by their activity or fermentation reactions facilitated by the microbial enzymes produce desirable changes in food (Ex. bakery, dairy, wine industries). 1.7 Microorganisms associated with food The presence of small numbers of microorganisms associated with foods may not cause any problem, but their unrestricted growth can result in spoilage or deterioration of the food making it unfit for consumption. The wide variety of microorganisms associated with foods is mainly saprophytic. They can not be avoided in food as these are derived from the environment in which the food is prepared or processed, and also difficult to eliminate completely. However, it is possible to reduce the number or decrease their activities by altering the environmental conditions. A variety of bacteria, molds and yeasts are important as food spoilage organisms. Important microorganisms involved in spoilage of fish are: Bacteria Gram Negative Bacteria Acinetobacter, Aeromonas, Alkaligens, Enterobacter, Flovobacterium, Moraxella, Photobacterium, Pseudomonas Vibrio etc 6 Gram Positive Bacteria Bacillus, Corynebacterium, Enterococcus, Listeria, Microbacterium, Clostridium, Staphalococcus, etc Fungi Aspergillus, Penicillium, Fusarium, Scouplariopis, Saprolegnia, Cladosporium, Alternaria etc. Yeasts Candida, Cryptococcus, Debaryomyces, Hansenula, Pichia, Rhodotorula, Trichosporon etc Unit 2 - Growth and survival of microorganisms in food Expected Learning Outcome Factors that affect the growth of microorganisms in food. Categorising these factors as intrinsic and extrinsic based on whether these are internal to the food or outside the food. Nature of these factors and their role in inhibiting or promoting microbial activity in food. 2.1. Factors affecting the growth of microorganisms in fish Foods meant for human consumption are rarely sterile, and contain several microorganisms. Microorganisms associated with food include natural micro-flora of raw material and organisms that are introduced during harvesting / handling, processing, storage and distribution. The microbial load in food depends on factors such as 7 Nature of the food Storage environment Properties of organisms in foods Effects of processing Generally, micro-flora associated with food has no effect and food is consumed without objection and no adverse consequences. But in some instances associated microorganisms manifest their presence leading to spoilage, food borne illness or Transformation of properties of food in a beneficial way (Ex. Fermentation). Factors affecting microbial growth in foods Microorganisms associated with food and their growth is affected by several factors. These are broadly grouped into 2 types. 1. Intrinsic factors :Includes physicochemical properties of food such as, Nutrients pH and buffering capacity Redox potential Water activity / moisture content Antimicrobial constituents 2. Extrinsic factors:Include conditions of storage environment such as, Relative avidity Temperature of storage Gaseous atmosphere Presence / activities of other organisms 2.1.1. Nutrient content Microorganisms associated with foods derive nutrients as source of energy, nitrogen, water, vitamins, minerals etc. for their growth and energy needs from foods. Nature of nutrient availability favor growth of different microorganisms. Ability of microorganisms to utilize nutrients favors their growth on foods, while inability to utilize nutrients reduces microbial 8 growth. Ex. Saccharolytic microorganisms grow well on cereals using carbohydrates as carbon source. Proteolytic bacteria grow well on fish and meat. Diverse microbial group are associated with fruits containing variety of sugars (sucrose, fructose etc) favoring a variety of microorganisms and yeasts. Generally, foods where nutrients are easily available (fish/meat) support high microbial load and activity. Foods with low water content are protected against microbial invasion. 2.1.2 pH and buffering capacity The acidity and alkalinity of an environment affects growth and metabolism of microorganisms as the activity and stability of macromolecules, enzymes and nutrient transport is influenced by pH. Generally, bacteria grow fast at pH 6-8. But bacteria that produce acids have optimum pH between pH 5 and 6 (Ex: Lactobacillus and Acetic acid bacteria). Yeast grows best at pH 4.5-6.0 and Fungi at 3.5 – 4.0. In low pH foods (Ex. Fruits), spoilage is mainly by yeasts and fungi than bacteria. Fishes with pH around neutrality (6.5-7.5) favour bacterial growth and spoil rapidly than meat (pH: 5.5 – 6.5). Ability of low pH to restrict microbial growth has been employed as a method of food preservation (Ex: use of acetic and lactic acid). Buffering capacity refers to the ability of foods to withstand pH changes. Microorganisms have ability to change pH of the surrounding environment to their optimal level by their metabolic activity. Decorboxylation of aminoacids releases amines which increases surrounding pH. Deamination of aminoacids by enzyme deaminases release organic acids causing decease in pH. Thus, protein rich foods like fish and meat have better buffering capacity than carbohydrate rich foods. 2.1.3. Moisture content (aw) Moisture content of the food affects microorganisms in foods, and the microbial types present in foods depends on the amount of water available. Water requirement for microorganisms is described in terms of water activity (aw) in the environment and is defined as the ratio of the water vapor pressure of food substrate to the vapor pressure of pure water at same temperature. aw = P/Po P = vapour pressure of water in substrate Po = vapour pressure of solvent (pure water) aw is related to relative humidity (RH) RH = 100 x aw Water activity of solutes and requirements of certain microorganisms 9 aw of pure water: 1.0 NaCl solution (22 %): 0.86 Saturated NaCl solution: 0.75 Bacteria generally require higher value of aw than fungi G – ve bacteria require higher aw than G +ve bacteria Most spoilage bacteria do not grow at aw below 0.91 Spoilage molds grow at aw of 0.80 Halophilic bacteria grow at aw of 0.75 Xerophilic and osmophilic yeasts grow at of 0.61 Microorganisms like halophiles, osmophiles and xerophiles grow better at reduced aw. Microorganisms can not grow below aw 0.60, and in such situations spoilage of food is not microbiological but due to chemical reactions (Ex: oxidation). Relationship between aw, temperature and nutrition Growth of microorganisms decreases with lowering of aw The range of aw at which the growth is greatest occurs at optimum temperature for growth The presence of nutrients increases the range of aw over which the organisms can survive 2.1.4. Redox potential (Oxidation – Reduction potential / O-R potential/ Eh) Microorganisms show varying degree of sensitivity to O-R potential (Eh) of growth medium. Redox reaction occurs as a result of transfer of electrons between atoms or molecules. O-R potential of a substrate is defined as the ease with which the substrate loses or gains electrons. Substrate is oxidized when it loses electrons and is called as good reducing agent. Substrates that gain electrons become reduced and are a good oxidizing agents. Cu ------> Cu + e Oxidation may also achieved by addition of oxygen. 2 Cu + O2 -------> 2 CuO Transfer of electros from one compound to another creates a potential difference between two compounds and is measured by an instrument and expressed as millivolts (mv). Highly oxidized substances have more electric potential (positive potential) and reduced substances negative electrical potential. Zero electric potential when oxidation and reduction are equal. 10 O/R protential of a system is expressed by Eh. Aerobic microorganisms require positive Eh for growth and anaerobes, negative Eh. Reducing conditions in food is maintained by -SH groups in meat and ascorbic acid and reducing sugars in fruits and vegetables. Factors influencing O/R potential of a food The characteristic of O/R potential of the original food. Poising capacity – (Resistance to change in potential of food) Oxygen tension of the storage atmosphere of food Access that the atmosphere has to the food Microbial activity Eh requirement of microorganisms Aerobic microorganisms require oxidized condition (+ Eh) for growth. Ex. Bacillus sp Anaerobes require reduced condition (-Eh). Ex. Clostridium sp Microaerophils are aerobes growing at slightly reduced condition. Ex. Lactobacillus, Campylobacter. Facultative anaerobes have capacity to grow both under reduced and oxidized condition. Eg. Yeasts. Plant foods have positive Eh (fruits, vegetables) and spoilage is mainly caused by aerobes (bacteria and molds). Solid meat and fish have negative Eh (-200 mv), and minced meat positive Eh (+200 mv). Microorganisms and Eh of food Microorganisms affect Eh of food during growth. Aerobes reduce the Eh of environment due to oxygen utilization. Growth medium becomes poorer in oxidizing and richer in reducing substances. Microorganisms reduce Eh by releasing metabolites. Hydrogen sulphide released by anaerobic microorganisms reacts with oxygen and creates reduced condition. Presence or absence of appropriate quantity of oxidizing/ reducing agents in the medium influences growth and activity of all microorganisms. 2.1.5. Antimicrobial constituents and barriers All foods have one or the other mechanism to prevent or limit potentially damaging effects by microorganisms through protective physical barriers to infection (Ex. skin, shell, and husk) 11 and antimicrobial components. Natural covering of some foods provide excellent protection against entry and subsequent damage by spoilage microorganisms. These include outer covering of fruits, outer shell of egg, skin covering of fish and meats. The outer covering is usually composed of macromolecules and these are resistant to degradation and create inhospitable environment for microorganisms due to low aw and shortage of readily available nutrients.The antimicrobial substances such as short chain fatty acids in animal skin and essential oils in plant surfaces help to prevent entry of microorganisms. Physical damage to outer barrier allows microbial invasion and cause spoilage. Some foods are resistant to attack by microorganisms and remain stable due to the presence of naturally occurring substances which have antimicrobial property. Many plant species possess essential oils which are antimicrobial. 2.2 Extrinsic parameters Extrinsic parameters refer to those properties of the environment to which the food is exposed. This includes the characteristics of the storage environment of the food which have profound effect on the food as well as the microorganisms associated with the food. The intrinsic factors of the food are influenced by the conditions of storage environment, and there by affect the quality of food. The environmental factors which have influence on the food associated microorganisms are; Storage temperature Relative humidity of storage environment Presence or absence of gases Presence and activities of other microorganisms. 2.2.1. Temperature of storage Storage temperature refers to temperature at which the food is handled and stored. Microorganisms growing over a wide range of temperature have been reported from food. The minimum temperature supporting the growth of microorganism was found to be well below the freezing temperature of water, and the highest temperature close to boiling temperature of water. However, no single microorganism is capable of growing over this wide temperature range. Thus, selecting a proper temperature for the storage of different food types helps in maintaing quality. Each microorganism exhibits minimum, optimum and maximum temperature at which growth occurs, called cardinal temperature. This temperature is characteristic for an organism and is influenced by factors such as nutrient availability, pH of growth medium, water activity etc. 12 Grouping of microorganisms based on temperature requirement for growth Microorganisms are grouped in to 4 broad types based on cardinal temperature. Minimum Optimum Maximum temperature temperature temperature Groups for growth for growth for growth (0C) (0C) (0C) Psychrotroph -5 to +5 25-30 30-35 s Psychrophiles -5 to +5 12-15 15-20 Mesophiles 5-15 30-40 40-47 Themophiles 40-45 55-75 60-90 Psychrophiles Microrganisms capable of growing at low temperature are called psychrophiles. These are further divided in to 2 types based on their optimum temperature for growth. Obligate psychrophiles (cold loving) – These have temperature optima of 12-15oc, but unable to grow above 20oc. These are confined to polar regions and deep marine environment. Psychrotrophs (facultative psychrophiles) These have same minimum temperature for growth as psychrophiles but have higher optimum and maximum growth temperature. Thus are found in most diverse habitats, grow well in refrigerated temperature and cause spoilage of chilled food. Ex: Alcaligenes, Cornybacterium, Flavobacterium, lactobacillus, Pseudomonas, Enterococcus etc. These bacteria grow well at 5-7oc (refrigerator temperature ) and cause spoilage of meat, fish, poultry, eggs etc. Mesophiles These grow well under normal temperature conditions of 30-40oC. As a rule mesophiles grow quickly at their optimum temperature than psychrotrophs. Thus the spoilage of food at mesophilic temperature range is rapid than at chill temperature. Mesophiles are found in most genera of bacteria. 13 Thermophiles These are high temperature loving microorganisms with the optimum temperature in the range of 55- 75oC. Thermophiles are generally less important in food microbiology. But thermophilic spore formers of the genus Bacillus and Clostridium cause spoilage of canned foods. Molds and yeasts Like bacteria, many molds and yeasts are associated with food. Many spoilage molds are able to grow over a range of temperature. Some molds grow at refrigerated temperature (Aspergillus, Cladosporium, Thamnidium Sp). Yeasts also grow and involve in spoilage of food under appropriate conditions. These grow over psychrophilic and mesophilic temperature range. Storage temperature and spoilage Temperature of storage is most important parameter affecting the spoilage of highly perishable foods (Ex. fish). Nature of food need to be considered while selecting storage temperature. Maintaining all foods at refrigerated temperature is not advisable as it affects the quality of food. Ideal temperature for storage of most vegetables is 10oc , banana: 13-17oc and refrigerator temperature for most foods. 2.2.2 Relative humidity (RH) Relative humidity of the food storage environment refers to percentage of moisture present in the atmosphere. RH and water activity are closely related and RH is measure of aw of the gas phase of atmosphere. RH = 100 x aw Effect of RH on food being stored RH of storage environment influences aw of foods and growth of microorganisms on food surface. Foods of low aw stored in high RH environment leads to transfer of water from environment to the food, increasing aw of food until equilibrium is reached. Condensation of moisture on food surface results in localized regions of high aw on surface and subsurface. This favors growth of microorganisms which were viable but unable to grow due to low aw, increases aw of immediate environment due to metabolic activity of microorganisms which produce water as end product of respiration. Thus microorganisms grow and causes spoilage of food which was considered safe microbiologically due to the growth of microorganisms requiring high aw. 14 Foods of high aw when stored in low RH environment lose moisture, become flaccid and unfit for consumption due to loss of quality. RH and spoilage of food RH and temperature of environment are related, and as the temperature increases RH decreases. Foods that are susceptible for spoilage by yeasts, molds and certain bacteria should be stored under low RH conditions. Improperly wrapped animal meat kept in refrigerator (high RH) undergone surface spoilage. Selection of suitable RH condition for storage is necessary to avoid surface microbial growth and maintain desirable qualities of food as food may lose/take up moisture under improper RH condition and lose its quality. 2.2.3. Presence and concentration of gases in the environment Exposure of foods to gases in the storage environment (gaseous environment) affects growth and survival of microorganisms in foods. Since exposure of food to oxygen favors growth of aerobic microorganisms, gaseous environment need to be modified to ensure reduced microbial activity and resultant spoilage. This approach is commonly employed in the preservation of fruits and vegetables. Gases used to control microorganisms in foods Carbon dioxide, ozone and nitrogen are most important gases used to control microorganisms in food. Several ready to eat foods are packed in the presence of these gases to reduce microbial activity and extend shelf life of packed foods. Such foods are referred to as Modified Atmosphere Package (MAP) foods. Carbon dioxide is single most important atmospheric gas used to control microorganisms in foods and is used in varying concentration depending on the type of food. Carbon dioxide in elevated pressure is also used in carbonated water and soft drinks. Molds and Gram negative microorganisms are more sensitive to CO2 than Gram positive bacteria. Lactobacilli are resistant to CO2.Yeasts show considerable resistance and tolerate high CO2 level but can cause spoilage of carbonated beverage (Ex. Brettanomyces sp.) Mechanism of inhibition Carbon dioxide mainly acts as bacteriostatic agent. But some microorganisms are killed by prolonged exposure.Mechanism of inhibition of CO2 is due to the formation of carbonic acid which lowers pH. Lowered pH affects physical properties of plasma membrane of microorganisms and affects solute transport, inhibits key enzymes, and reacts with amino 15 group of proteins causing changes in their property and activity. Ozone (O3) is also has antimicrobial properties and extends shelf life of certain fruits and vegetables foods. O3 concentration of 0.15-5 ppm is known to double the shelf life by inhibiting spoilage bacteria and yeast. 2.2.4. Presence and activities of other microorganisms The nature of microorganisms (microbial associations) encountered in foods can affect micro-flora of food which are called implicit factors. These include Properties of organisms present in food Response of these organisms to their environment Interaction with other organisms in food Effect of activities of microorganisms on food micro-flora Among several organisms present in food, microorganisms which find condition suitable (in food) for growth dominate over other organisms. Ex. Molds can grow on dry fish, but slowly, than bacteria. In fresh fish bacteria overgrow molds as conditions are most favourable. Faster growing bacterial growth is inhibited by low aw or low pH where moulds grow and cause spoilage. Some food borne microorganisms produce metabolite /substances such as antibiotics, bacteriocins, hydrogen peroxide, organic acids etc which are inhibitory /lethal to other microorganisms. Spoilage microorganisms can interact wherein growth of one favours the growth of others. Ex: In low aw food (grain) growth of few molds increases aw leading to growth of other xerophilic molds. One organism may increase nutrient availability to others by degrading complex food substrates to simple forms. Some microorganisms may remove inhibitory substances thereby permit the growth of others. Unit 3 - Enumeration of microorganisms in foods Expected Learning Outcome 16 Laboratory methods for the detection of spoilage and food poisoning microorganisms associated with food. Method to be adopted depending on the kind of microorganisms to be enumerated in food. 3.1 Introduction The raw, semi-processed and processed foods as well as the ingredients used in the preparation of processed foods may contain several microorganisms. The microbial examination of foods is generally done to know the presence or absence of specific microorganisms, types of microorganisms, number of microorganisms and their products in foods. The information on the microorganisms associated with food will help in; Estimating shelf life (suitability for human consumption) by determining microbiological quality of a food or constituent of food. Identifying the cause of spoilage or presence of pathogens, when food is implicated in food borne illness. Methods of enumeration Several methods are available for the enumeration of microorganisms from food. These can be broadly divided in to direct methods or indirect enumeration methods based on whether the microorganisms are counted directly or the products released by them is estimated. I. Direct enumeration methods a. Direct counting methods Direct microscopic count (DMC) Direct counting on membrane filters b. Culture based methods Plate count method Pour plate method Spread plate method Most probable number (MPN) technique II. Indirect enumeration methods a. Alternative Methods (chemical and physical methods) Dye reduction test 17 Electrical methods ATP determination b. Rapid Methods Immunological methods DNA/RNA methodology 3.2 Study of microorganisms in foods by conventional methods Enumeration of microorganisms in foods is traditionally performed by directly counting all microorganisms present in foods or by allowing them to develop in to colonies and then counting. 3.2.1 Direct counting methods Microorganisms present in the food can be counted by observing the food sample directly or retaining the microorganisms on a filter paper by filtering the sample and then observing under microscope. A. Direct microscopic count (DMC) DMC involves detecting the presence of microorganisms in food by microscopic observation. It is a simple method and easy to perform. DMC is performed by making a smear of food specimen/cultures on to microscopic slides, staining with appropriate dye and viewing and counting all cells using microscope under oil immersion objective. This method can be used only when microorganisms are present in large numbers (106/ml). It is commonly used in dairy industry for assessing microbial quality of raw milk and other dairy products. Advantages Rapid and easy enumeration Can be employed to any foods (Ex: dried/frozen foods) Simple to perform Cell morphology can be assessed Efficiency can be increased by using florescent probes Disadvantages Requires tiresome counting under microscope causing fatigue to analyst Counts both viable and non-viable cells Food particles may interfere with counting and mistaken for microorganisms 18 Cells may not be distributed uniformly (single cells/ clumps) Some cells may not take up stain and missed while counting DMC counts are always higher than standard plate count Requires dilution of sample B. Direct counting on membrane filters Membrane fillers with pore size smaller (0.45 um) than bacteria retain bacteria and the retained bacteria can be counted using microscope. Procedure involved Concentrating/collecting bacteria on polycarbonate filters by filtering known volume of homogenized sample. Staining and counting of retained bacteria. Placing the membrane on a nutrient agar media or absorbent pad saturated with culture media of choice, and incubating Following growth , colonies are counted Advantages Well suited for samples containing low numbers of bacteria Facilities concertinaing bacteria by filtering large volume of sample Only small volume of food samples can be used for a single membrane Efficiency of membrane filter method can be increased by staining with florescent dyes (Ex. acridine orange) and observing under epiflorescence microscope (DEFT: Direct Epiflorescence Filter technique). Viable cells fluoresce green and are counted. Non viable cells appear orange. Acridine orange is an metachromatic fluorochrome which binds to double stranded DNA of viable bacterial cells. Can be used to enumerate microorganisms from a variety of foods (fresh fish, meat, fish/ meat products, water samples etc). 3.2.2 Culture based methods Culture methods involve examination of microorganisms in food by encouraging them to multiply in a liquid or solid media. On solid agar media bacteria develop as colonies and counting such viable colonies gives microbial load in foods. Enumerating microorganisms by culture based methods can be done by using plate count methods or MPN technique. 19 Culture media A wide variety of media with varied composition capable of supporting the growth are available for the cultivation of different microorganisms. The composition of the media varies depending on; Group/type of microorganism to be studied Overall purpose of the study Whether to grow wide range of microorganisms or specific types Resusitation of damaged but viable cells Type of diagnostic information required Ex: General purpose media: Plate count agar Lactose broth: For Escherichia coli Seective media: Baird parker agarfor Staphylococcus Bismuth sulphite agar for Salmonella TCBS for Vibrios A. Plate count method This method is variously referred to as total plate count (TPC), standard plate count (SPC) or aerobic plate count (APC). It is most widely used conventional method for determining viable cells or colony forming units (CFU) in foods. SPC involves blending/ homogenizing the sample, serially diluting in appropriate diluent, plating in or on suitable agar media, incubating at appropriate temperature for a given time, and counting visible colonies as CFU. Principle involved SPC is based on the principle that each viable bacterial cells multiples and grows in to a visible colony. Thus, counting number of colonies gives an idea about bacterial cells present in a sample. Counts determined by taking average of replicate plates showing 30-300 colonies. Factors affecting SPC Sampling method employed. Distribution of microorganisms in food. Nature of food biota 20 Nutritional adequacy of plating media Incubation temperature and time Type of diluent used Presence of other competing organisms etc. Plating on selective media for specific organisms is limited by degree of inhibition and effectiveness of selective/ differential agents employed. SPC can be performed by pour plate method or spread plate method (surface plating method) a). Pour plate method Appropriate dilution of the sample (1 ml) is mixed with agar medium, allowed to set, incubated at appropriate temperature and colonies developed are counted. Here colonies develop both on surface and subsurface of agar plate. Proper mixing of sample with agar medium is necessary so as to get isolated colonies which can be done by 2 ways. One ml of appropriate sample dilution is added to petri-plate and about 15 ml of agar medium is added and mixed by rotating the plate in clockwise and anticlockwise direction. One ml of appropriate sample dilution is added to test tube containing about 15 ml of molten agar medium, mixed by rolling the tube between the palm and poured to petri-plates, allowed to set and incubated. b). Spread plate method Diluted sample (0.1 ml) is spread on the surface of pre-poured, hardened agar plates using glass rod, incubated at appropriate temperature and colony developing on surface counted. Advantages Suitable for heat sensitive psychrotrophs in food as they do not come in contact with molten agar. Enables providing colony features useful in presumptive identification especially on selective media. Favours strict aerobes on surface, but microaerophils grow slowly Disadvantage Problem of spreaders and colony crowding makes the enumeration difficult. 21 B. Most probable number (MPN) technique MPN is suited for enumerating the presence of low numbers of microorganisms in foods. This involves inoculating replicate tubes of appropriate liquid media (3 or 5 tube) with three different sample sizes/ dilutions of the material to be studied and incubating at appropriate temperature. Then the absence or presence of growth is observed and MPN table consulted to get probable number of organisms in the sample. MPN numbers are generally higher than SPC. Advantages Relatively a simple method and assay to perform Results are comparable from one laboratory to another Specific group of organism determined by use of specific media. Suitable for detecting organisms present in low numbers Method of choice for coliform detection Disadvantages Requires use of large number of glassware and large volume of sample Can not observe colony morphology Lack of precision 3.3 Study of microorganisms by alternate and rapid methods The types and number of microorganisms present in food sample can be determined either by measuring the metabolites released by the microorganisms and constituents of microbial cells employing physical /chemical methods, or by using rapid methods such as enzyme linked immunosorbant assay (ELISA) or polymerase chain reaction (PCR).ck of precision. 3.3.1 Alternative methods (physical and chemical methods) 1. Dye reduction test (DRT) DRT is mainly used in dairy industry for assessing overall microbial quality of raw milk. The number of viable organisms in a sample is determined by their ability to reduce the redox dyes. The redox dyes take up electrons from active biological systems and are coloured when oxidized and colorless when reduced. Commonly used redox dyes 22 Methylene blue: appears blue when oxidized, and colorless when reduced. Resazurin: appears blue when oxidized, and pink or white when reduced. Triphenyltetrazolium salt: appers different from above two dyes in reduced/ oxidized stste. It is colorless when oxidized, and coloured (red or maroon) when reduced due to formation of formzan. Procedure Food supernatant is added to standard dye solution, incubated ( Ex. 10 min in resazurin dye) and color observed. Extent of reduction is related to bacterial load. Time for reduction to occur is inversely proportional to number of organisms present in the sample. Advantages Simple, rapid and inexpensive method. Suitable for assessing quality of raw material at farm or dairy. Disadvantages Not all organisms are able to reduce dye equally. Not suitable for food that contain reductive enzymes. 2. Electrical methods Electrical method is a physical method and is one of the most widely used alternative methods for microbiological analysis of foods. Principle Growth of microorganisms in a liquid medium changes the chemical composition of the medium, which leads to changes in its electrical properties. Measuring changes in electrical properties forms the basis of determining the microbial load in a sample. This is done by measuring the electrical impendence. When microorganisms grow in a culture media, metabolite substances of low conductivity in to the products of higher conductivity, thereby decrease the impendence of the media. In broth culture, measurement of independence over time gives reproducible results for species and strains of microorganisms. It is capable of detecting organisms in the range of 10 to 100 cells. Generally, cell populations of 105-106 /ml are detectable in 3-5 hours, and 104-105/ml in 5-7 hours. The times noted are required for the organisms to attain the threshold of 106-107 cells/ml. Application Useful in assessing the quality of vegetables: 90-95% agreement has been found between impedence measurement and TPC, requires 5 hours to analyse and suitable for ground meat and other foods. 23 Microbiological quality of pasteurized milk was assessed by using impedence detection time of 7 hour or less, which is equivqlent to TPC of 104 /ml or more bacteria. 3. ATP measurement Adenosine triphosphate (ATP) is the primary source of energy in all living cells and universal agent for the transfer of free energy from catabolic process to anabolic process. ATP generally disappears within 2h after cell death. The amount of ATP per cell is generally constant (10-18 – 10-17 mole per bacterial cell which is equivalent to 4 x 104 M ATP/105 cfu of bacteria). ATP in exponentially growing bacterial cells is 2-6 nanomole ATP/mg dry weight (about 0.40 % of dry weight of bacteria). Thus the measurement of cellular ATP can be equated to individual groups of microorganisms. A linear relation is observed between microbial ATP and bacterial numbers Procedure ATP measurement is done by using firefly luciferin- luciferase system. In the presence of ATP, luciferase emits light which is measured by luminometer. Amount of light produced is directly proportional to the amount ATP added. Luciferase produces one photon of light on hydrolysis of 1 ATP molecule. ATP facilitates the formation of enzyme – substrate complex which is oxidized by molecular oxygen. Application ATP assay is proved to be suitable in foods for assessing microbial quality, and involves complex sample preparation procedures. One of the problem in ATP assay in foods is contribution from non-microbial ATP mainly from foods which can be removed by sample processing procedures. In meat non-microbial ATP removal involves centrifugation, use of cation exchange resin and filtration. Though this is a rapid and sensitive method not used for routine monitoring of microbial contamination in foods. However, suitable for monitoring hygiene in food processing plants. On the spot monitoring of food handling surfaces/ equipments can be done by swabbing a designated area and reading the relative light units (RLU) using lumonometer. The amount of ATP measured is of both microbial and non-microbial origin and the presence of both indicates poor hygiene. Thus it is valuable for monitoring purpose but not for indicating numbers of microorganisms. 4. Thermostable nuclease test It is a chemical method used to detect presence of Staphylococcus aureus, a food poisoning organism in foods by detecting thermostable nuclease. S. aureus involve in food poisoning by producing enterotoxin which is a neurotoxin. The enterotoxin producing S. aureus produce coagulate and nuclease enzyme, and a high correlation is observed between these two enzymes. Nuclease of S. aureus is more heat stable than nuclease of other Staphylococcus sp and other bacteria. Coagulase is not heat 24 stable and hence it is not used. Increase in cell numbers increases the extractable thermonuclease of staphylococcal origin. Presence of 0.34 units of nuclease corresponds to 9.5 x 10-3µg of enterotoxin by S. aureus. Thermostable nuclease assay was found to be as good as coagulase assay for toxigenic strains. All the foods that are contaminated with enterotoxin were found to contain thermostable nuclease at S. aureus level of 106/g of food. Nuclease is detectable in sample at S. aureus numbers of 105-106/ml. And enterotoxins is detectable at cell number >106/ml. Advantages Thermostable nuclease is heat stable hence persists in food even after the bacteria are destroyed by heat/chemicals etc. Thermostable nuclease is detectable faster (within 3 hrs) than enterotoxin. Nucleases are produced by enterotoxignic stains before the appearance of enterotoxin. Nuclease estimation does not require concentration of cultures in food, but enterotoxin detection requires concentration of samples. Nucleases are heat stable, like enterotoxins. 5. Limulus lysate test Limulus lysate test is a chemical method used to detect the presence of endotoxin in foods. Endotoxin is produced by pathogenic Gram negative bacteria which consist of lipopolysaccharide (LPS) layer and lipid A in their cell wall. LPS is pyrogenic and responsible for ssymptoms associated with infection by Gram negative bacteria. Limulus amaebocyte lysate (LAL) test uses lysate protein obtained from the blood (haemolymph) cells (amaebocytes) of horse shoe crab, Limulus polyphemous. The lysate protein is most sensitive substance known for endotoxin. LAL test is performed by adding aliquots of food suspensions to small quantities of lysate preparation and incubated at 370c for 1 hour. Presence of endotoxin causes gel formation of lysate material. LAL reagents can detect endotoxin as low as 1 pg of LPS. Incorporation of chromogenic substrate (p-nitroaniline), to endotoxin activated enzyme cleaves the substrate and releases free p- nitroaniline that can be read at 405 nm. The amount of chromogenic compound released is proportional to the quantity of endotoxin in the sample. By knowing the amount of endotoxin per cell of Gram negaticve bacteria (which is fairly constant), the total bacterial load can be determined from the quantity of endotoxin measured. Application LAL test is a good and rapid indicator of total number of Gram negative bacteria in 25 refrigerated foods (fish/meat) which are spoiled mainly by Gram negative bacteria. Advantages LAL test detects both viable and non-viable Gram negative bacteria in food sample. Found suitable for assessing microbial quality of milk/ milk products and raw fish. Gives quick result. Food of high LAL value need further tests by other methods. Foods with low LAL titre can be categorized as low risk relative to Gram negative bacteria. 3.3.2 Enumeration of microorganisms by rapid methods The conventional cultured based methods often fail to detect the presence of microorganisms in foods and also take long time for laboratory analysis. This may be due to their low cell numbers and loss of viability due to damage to cells. This can be overcome by applying culture independ methods involving the detection of bacterial cell components and metabolites by immunological and molecular based approaches. The commonly employed rapid tests in food microbiology are; Enzyme linked immunosorbant assay Polymerase chain reaction 1. Enzyme Linked Immunosorbent Assay (ELISA) ELISA or enzyme immunoassay (EIA) is used in food microbiology to identify the specific pathogens or toxins released by them. Detection of specific microorganism among the mixture of organisms is made easier by employing ELISA. This method based on the antigen (cell or toxin) – antibody reaction is highly specific and helps to detect when antigens are present in very low levels. Thus, ELISA involves specific reaction between the antigen, antibody and an enzyme. This reaction complex produces a colour in the presence of chromogenic substrate which is proportional to the amount of antigen present. The enzymes such as horse radish peroxidase and alkaline phosphotase are commonly used which release a dye (chromogen) when exposed to their substrate. Procedure The antigen to be detected is taken in a test tube or microtitre plate and incubated with the antiserum. The excess antiserum is washed and the enzyme labelled with anti-immunoglobulin is added. After washing, the enzyme remaining in the tube or microtitre well is assayed to determine the amount of specific antibodies in the initial serum. 26 The amount of peroxidase enzyme is measured by adding enzyme specific substrate and the colour developed is measured colorimetrically. Polymerase chain reaction: (PCR) Polymerase chain reaction (PCR) is a molecular biology technique employed in food microbiology to identify the presence of specific microorganism of interest especially when present in very low numbers by targeting the specific gene sequence. As the foods may contain pathogenic microorganisms which may be present in low numbers or injured by conditions of food processing, the recovery of such organisms by routine plating techniques is not possible. In such situations PCR based methods become very useful in identifying the target organism. The PCR for the detection of pathogens generally targets virulence associated genes such as ctx gene encoding cholera toxin for Vibrio cholerae 01 and 0139; tdh gene coding thermostable direct hemolysin for V. parahaemolyticus; hns and invE gene for Salmonella; stx1, stx2 and eae gene for enterohaemorrhagic E. coli. Principle of PCR The method is based on the amplification of target DNA sequences in the genomic DNA in the presence of specific primers, oligonucleotides, buffers and polymerase enzyme. Under the conditions of repeated heating and cooling cycles millions of copies of target DNA will be synthesized with in a short period of time. The amplified product is detected after performing electrophoresis on agarose gel and visualized under UV light after staining with ethydium bromide. Advantages PCR is highly specific as it can amplify a target DNA fragment of pathogen of interest against DNA of other organisms. PCR is highly sensitive as it is possible to amplify target sequence from samples having very few microbial cells of interest. PCR is a rapid test since the results can be obtained with in a few hours. It facilitataes use of food samples directly or after enrichment. Sample lysate or enrichment lysate can be use for extracting DNA for further PCR amplification. Unit 4 - Food preservation techniques and microorganisms Expected Learning Outcome Methods of killing microorganisms or preventing their growth in food so as to ensure preservation of food for future use. Principles of killing /inhibiting microorganisms associated with foods by preservation methods involving use of low temperature, high temperature, radiations and chemicals. 27 Importance of microbial endospores and cell aggregates on quality of processed foods. 4.1 Food preservation techniques and microorganisms The foods of plant and animal origin contain several microorganisms and these along with natural food enzymes become active soon after the death of animal and harvest of plant foods. The intrinsic characters of the food and the environmental conditions influence the type and extent of microbial activity leading to spoilage which is characterized by deteriorative changes in food quality making it unfit for human consumption. The different preservation techniques have mainly aimed at reducing or eliminating microorganisms in food thereby prolonging or preventing the spoilage of foods. The preservation of foods is commonly done by use of low temperature, high temperature, drying, radiation and chemicals. 4.1.1 Preservation of foods with low temperature Low temperature preservation of food is based on the principle of reducing the microbial activity by subjecting to low temperature condition. As all metabolic activities of microorganisms are catalyzed by enzymes, and enzyme reactions are dependent on temperature, the low temperature slows down enzyme activity, thereby brings about reduction in microbial activity. The reduced microbial activity prolongs shelf life of foods. Shelflife of temperate and tropical water fish Activity of microorganisms is known to decrease by two fold with every 10oC decrease in temperature. Fresh fish from temperate waters generally have prolonged shelflife than tropical fishes. This is because of the low ambient temperature in temperate waters coupled with low body temperature of fish, being cold blooded, lowers both enzymatic and bacterial growth, thereby reduces deteriorative change and helps in extending the shelflife. In tropical waters, the high ambient temperature and high body temperature of harvested fish promotes deteriorative changes by enzymes and microorganisms and thus hastens the spoilage. Methods of low temperature presentation Low temperature preservation is generally attained by employing three different temperature conditions. They are; Chilling temperature: keeping foods at 10-15oC (slightly above refrigerated temperature). Refrigerator temperature : Keeping foods at 0~7oC Freezer temperature: Storage of foods below -18oC. In Fisheries, fish are preserved by keeping at chilling temperature and by freezing. 4.1.1.a. Chill storage of fish Chill storage is a process by which temperature of fish is reduced close to freezing point of 28 water (0oC). This delays both biochemical and bacteriological processes, thus prolongs shelf life. Deteriorative changes are retarded as long as low temperature is maintained. This ensures preserving natural nutritional and functional properties of food. Factors influencing quality of chilled food Quality of chilled food depends upon factors such as; Raw material quality Method and duration of chilling Efficiency of storage method Chilling can be achieved by use of ice (crushed/flake ice) and use of homogenous coolant (cold air or cold liquid), and refrigerated temperature. Use of cold liquid may be in the form of chilled freshwater for light chilling or refrigerated seawater/brine to attain temperature of 0-1oC Bacteria associated with low temperature storage Several microorganisms are capable of surviving and growing at low temperature and cause spoilage. Bacteria capable of a growing at or below 7oC are widely distributed. Gram negative bacteria are more common than Gram positive. Psychrotrophs grow well at this temperature. Growth at temperature below 00C is caused mainly by yeasts and molds than bacteria because of low water activity. The lowest recorded temperature for growth of microorganisms in food is -34oC, by yeast. The composition of microorganisms associated with fish changes during chill storage. Proportion of mesophiles decrease and psychrophiles dominate. Common bacterial genera associated with chilling temperature condition of foods are; Acinetobacter, Aeromonas, Enterococcus, Pseudomonas, Vibrio, Erwinia, Moraxella, Enterobacter, Achromobacter, Flavobacterium, Micrococcus etc. Extension of shelf life varying from 6 days to 30 days for different fish species has been reported by ice storage. 4.1.1.b. Preservation by Freezing Freezing involves lowering of temperature of food to -20oC and storage at same temperature. At this temperature the water in food as well as in microorganism is converted to ice crystals which affect fluidity of cell. This ensures prolonged shelflife as microbial activity is completely stopped at this temperature condition. Freezing is achieved by 29 Quick freezing: where temperature is lowered to -20oC within 30 min. Slow freezing: where temperature is lowered to -20oC within 3~72 hours. Quick freezing is more advantageous than slow freezing in achieving product quality. During freezing water in food is converted to ice crystals of variable size. Freezing also brings about changes in properties of food such as pH, titratable acidity, ionic strength, viscosity, osmotic pressure, freezing point, O/R potential etc. These changes along with non-availability of water make the environment unsuitable for microbial growth and activity. Comparison of effect of freezing methods on microorganisms Quick freezing Slow freezing Small ice crystals formed Large ice crystals formed Break down of metabolic rapport and causes cell Suppresses microbial metabolism damage Brief exposure to adverse conditions Longer exposure to injurious factors No adaptation to low temperature Gradual adaptation Causes thermal shock to microbes No thermal shock effect Accumulation of concentrated solutes with No protective effect beneficial effects. Drip loss is less Drip loss is more Shelf life of frozen foods Frozen foods can be stored indefinitely without microbial spoilage, but not done so as they lose original flavour and texture after thawing. Thus frozen foods are assigned freezer life. Freezer life for frozen foods is determined based on texture, flavor, tenderness, colour and nutritional quality upon thawing and cooking. Freezer life of frozen stored food does not depend on microbiology of frozen foods. Effect of freezing on microorganisms Freezing causes sudden mortality immediately on freezing, varying with microbial species. The number of surviving microorganisms after freezing die gradually when stored in frozen condition. Decline in microbial number is rapid at temperature below freezing point (-2oC) than at lower temperature, and is slow below -20oC. Bacteria differ greatly in their capacity to survive during freezing. Generally cocci are more resistant than Gram negative bacteria. 30 Food poisoning bacteria are less resistant to freezing while, microbial endospores and toxins are not affected by freezing. 4.2. Food preservation by high temperature The high temperature preservation of food is based on destructive effect of heat on microorganisms, thereby extending shelf life of foods. High temperature refers to any temperature higher than ambient temperature applied to food. Preservation of foods by heat treatment can be done by two methods viz. pasteurization and sterilization. 4.2.1 Pasteurization Pasteurization refers to use of heat at the range of 60~80oC for a few minutes for the elimination/ destruction of all disease causing microorganisms, and reduction of potential spoilage organisms. Pasteurization is commonly used in the preservation of milk, fruit juices, pickles, sauces, beer etc. Pasteurization process which is commonly employed in milk preservation can be achieved by heating the milk at 63oC for 30 min, called low temperature long time (LTLT) process; or 72oC for 15 sec, called high temperature short time (HTST) process. This process destroys most heat resistant non-spore forming pathogens (Ex. Mycobacterium tuberculosis), all yeasts, molds, Gram negative bacteria and most Gram positive bacteria. 31 Organisms surviving pasteurization The pasteurization treatment does not bring about complete removal or destruction of microorganisms and some organisms survive pasteurization process. The surviving organisms are of two types; Thermoduric microorganisms Thermophilic microorganisms. Thermoduric microorganisms are those which can survive exposure to relativity high temperature but do not grow at these temperatures. Example: The non-spore forming Streptococcus and Lactobacillus sp can grow and cause spoilage at normal temperature. So, milk need to be refrigerated after pasteurization to prevent spoilage. Thermophilic organisms are those that not only survive high temperature treatment but require high temperature for their growth and metabolic activities. Example: Bacillus, Clostridium, Alicyclobacillus, Geobacillus etc. 4.2.2. Sterilization Sterilization or appertization refers to destruction of all viable organisms in food as 32 measured by an appropriate enumeration method. This process kills all viable pathogenic and spoilage organisms. However, organisms that survive are non-pathogenic and unable to develop in product under normal conditions of storage. Thus, sterilized products have long shelf life. Commercially sterile or commercial sterility is often used for canned foods to indicate the absence of viable microorganisms detectable by culture methods or the number of survivors is so low that they are of no significance under condition of canning and storage. Processing of food for preservation using high temperature depends on the physical nature of the food. Foods (solid or semisolid) are generally processed by packing in cans, sealing and then sterilized. Most liquid foods are sterilized, packed in suitable containers and sealed aseptically. Temperature and time of sterilization given to a food depends on the nature (pH, physical state, nutritional type etc) of the food being processed. 4.2.3. Heat resistance of microorganisms Heat resistance of microorganisms is related to their optimal growth temperature. Psychrophiles are most sensitive and thermophiles are most resistant to heat treatment. 33 Spore forming microorganisms are most resistant than non spare formers. Gram positive bacteria are more resistant than Gram negative bacteria. Yeasts and molds are fairly heat sensitive Spores of molds are slightly more heat resistant than vegetative cells. Heat resistance of spores Bacterial spores are more heat resistant than vegetative cells. Thermophiles produce more heat resistant spores. Since spore inactivation is the main concern in canned foods, high process temperature is used to achieve this. The heat resistance of bacterial endospore is due to their ability to maintain very low water content in the DNA containing protoplast. Presence of calcium and dipicolonic acid in high concentration in spores helps to reduce cytoplasmic water. Higher the degree of spore dehydration greater will be its heat resistance. Factors affecting heat destruction of microorganisms Several factors associated with microorganisms as well as their environment affect heat destruction of microorganisms. Water: Heat resistance of microorganisms increases with decrease in moisture/ water activity and humidity. This is due to faster denaturation of protein in presence of water than air. Fat: Heat resistance increases in presence of fat due to direct effect of fat on cell moisture. Heat protective effect of long chain fatty acids is better than short chain fatty acids. Salts: Effect of salt in heat resistance of microorganisms is variable, and depends on type of salt, concentration used, and other factors. Some salts (sodium salts) have protective effect on microorganisms and others (Ca2+ and Mg2+) make cells more sensitive. Some salts (Ca and Mg) increase water activity, while others (Na+) decrease water activity there by affecting heat sensitivity. Carbohydrates: Presence of sugars in suspending medium increases heat resistance of microorganisms due to decreased aw. Different sugars show varying effect. Heat resistance decreases in the order of; sucrose>glucose>sorbitol>fructose>glycerol. pH: Microorganisms are most heat resistant to heat at their optimum pH for growth (about pH 7-0). Increase or decrease in pH reduces heat sensitivity. Thus, high acid foods require less heat processing than low acid foods. Proteins: Proteins have protective effect on microorganisms. As a result high protein foods need a higher heat treatment than low protein foods to obtain similar results. Number of microorganisms: Larger the number of microorganisms, higher the degree of heat resistance. This is due to the production of protective substance excreted by bacterial cells, and natural variations in a microbial population to heat resistance. Age of microorganisms: Microorganisms are most resistant to heat in stationery growth and 34 least in logarithmic growth phase. Also old bacterial spores are more resistant that young spores. Growth temperature: Heat resistant of microorganisms increases with increase in incubation temperature, especially in spore formers. This is mainly related to genetic selection favoring growth of heat resistant forms. Cultures grown at 44oC are known to have three times more heat resistance than those grown at 35oC. Inhibiting compounds: Heat resistance of most microorganisms decreases in the presence of heat resistant microbial inhibitor such as antibiotic (nisin), sulfur dioxide etc. Heat and inhibiting substances together are more effective in controlling spoilage of foods than either alone. Time and temperature: Generally believed that the longer the heating time, greater the killing effect. But higher the temperature, greater will be the killing effect. Thus, as temperature increases, time necessary to achieve the same effect decreases. Also, the size and composition of containers affect heat penetration. 4.2.4. Thermal destruction of microorganisms The preservative effect of high temperature treatment depends on the extent of destruction of microorganisms. Certain basic concepts are associated with the thermal destruction of microorganisms.These include; Thermal death time (TDT) D- value Z- value F- value 12D concept Thermal death time (TDT) TDT is the time required to kill a given number of organisms at a specified temperature. Here, temperature is kept constant and the time necessary to kill all cells is determined. Whereas, thermal death point is the temperature necessary to kill given number of organisms in a fixed time, usually 10 min. But it is of less importance. TDT is determined by placing a known number of bacterial cells/spores in sealed containers, heating in a oil bath for required time and cooling quickly. The number of survivors from each test period is determined by plating on a suitable growth media. Death is defined as the inability of organism to form viable colonies after incubation. D-value (Decimal reduction time D-value is the time in minutes required at specified temperature to kill 90% of microorganisms thereby reducing the count by 1 log units. Hence D – value is the measure of death rate of microorganisms. It reflects the resistance of an organism to a specific temperature and can be used to compare the relative heat resistance among different 35 organisms/spores. D-value for the same organism varies depending on the food type. D -value is lower in acid foods and higher in presence of high proteins. Example: D 250oF (121.1oC) for B. stearothermophilus: 4-5 min C. botulinum: 0.1 – 0.2 min. D 95oC for B. coagulans: 13.7 min B. licheniformis: 5.1 min. Z - Value Z-value refers to degrees of Fahrheit required for the thermal destruction curve to drop by one log cycle. Z value gives information on the relative resistance of an organism for different destruction temperature. It helps to determine equivalent thermal process at different temperature. Example: If adequate heat process is achieved at 150oF for 3 min and Z -value was determined as 100F, which means the10oF rise in temperature reduces microorganisms by 1 log unit. Therefore, at 140oF , heat process need to be for 30 min and at 160oF for 0.3 min to ensure adequate process. F - Value F- value is the better way of expressing TDT. F- is the time in minutes required to kill all spores/vegetative cells at 250oF (1210oC). It is the capacity of heat process to reduce the number of spores or vegetarian cells of an organism. F – Value is calculated by Fo = Dr (log a-log b) Dr = Decimal reduction time (D value) a = initial cell numbers b = final cell numbers 12D concept 12D concept is used mainly in low acid canned foods (pH >4.6) where C. botulinum is a serious concern. 12D concept refers to thermal processing requirements designed to reduce the probability of survival of the most heat resistant C. botulinum spores to 10-12. This helps to determine the time required at process temperature of 121oC to reduce spores of C. botulinum to 1 spore in only 1of 1 billion containers (with an assumption that each container of food containing only 1 spore of C. botulinum). 4.3. Food preservation by drying Preservation of foods by drying involves reducing the activity of microorganisms and enzymes by reducing moisture content. Lowering of moisture content inhibits the activities of food spoilage and food poisoning microorganisms. It is a traditional method of preservation of food, simple and easy to perform. Drying is generally done by exposing the fresh/ semi processed foods to sunlight until desired moisture level in attained. 36 4.3.1. Fish drying Small fishes are dried whole without any processing while, large fish are dried by cutting open or cutting to small pieces to ensure faster drying. Fish may be salted or not salted before drying. After drying, fish are packed and stored for later consumption as human food. The storage stability or shelflife of dried fish depends on factors such as quality of the raw material used, extent of drying and storage conditions to which fish is exposed after drying. Categories of dried foods Dried foods are generally divided in to 2 categories based on moisture content. They are, Low moisture foods: These foods do not contain moisture level more than 25%, and have aw of 0.6 or less. These foods have better keeping quality and shelflife. Intermediate moisture foods (IMF): These contain 15-50% moisture and aw between 0.60 and 0.85.Ex: Dried fruits, cakes etc. 4.3.2. Effects of drying on microorganisms Drying reduces the microbial activity in foods to a large extent, and depends on the moisture level attained after drying. Thus the storage stability (spoilage) of dried foods depends mainly on the aw of food. Generally, bacteria require high aw for growth (> 0.90) compared to yeasts and molds. At aw of 8.0~0.85: Spoilage of dried foods is caused by a variety of fungi and spoilage occurs in 1-2 weeks period. At aw of 0.75: Delayed spoilage caused by few types of organisms (fungi). At aw of 0.70: Spoilage greatly delayed, may not occur during prolonged storage. At aw of 0.65: Very few organism can grow, and spoilage is unlikely for 2 years. Growth of most organisms is prevented at aw 6.5. The undissociated form is responsible for antimicrobial activity, and about 86% is in undissociated form at pH 4.0 while, only 6% at pH 6.0. Effective against molds, yeasts and also wide range of bacteria. Inhibition of molds is due to the inhibition of dehydrogenase activity. Among bacteria catalase positive cocci are more sensitive than catalase negative forms. Also aerobes are more sensitive than anaerobes. These are found to be effective against Staph aureus, Salmonella, Coliforms, Psychrophilic spoilage bacteria, Vibrio parahaemolyticus and others. 43 Sorbates are used to extend shelf life of products such as fresh fish, fresh poultry meat, perishable fruits, cheese, bakery products, fruit beverages etc. Also used along with nitrites in meat products to extend shelf life. Sorbates prevent growth of vegetable cells that are germinating from endospores. Mode of action of sorbates Preservatives such as sorbates, benzoates and propionates are lipophilic compounds and antimicrobial activity is due to undissociated form. There chemicals affect proton motive force of bacterial cells. Being lipophylic, act on cytoplasmic membrane of microorganisms and separate proton (H+) and hydroxyl ions. The H+ ions move outside the cell and cause acidic pH while the OH ions increase pH inside the cell near neutrality. At this pH sorbates inside the cell dissociate and cause lowering of intracellular pH. These results in weakening of transmembrane gradient required for transport for aminoacids to inside cell, thus adversely affecting membrane transport and causing subsequent cell death. 3. Propionates Propionic acid and its calcium and sodium salts are permitted in foods as mold inhibitor in bread, cakes, cheese and other foods. Effective in low acid foods and commonly used in bread to prevent ropiness in bread dough. The inhibitory action is mainly fungistatic than fungicidal. Mode of action is similar to benzoates and sorbates. The undissociated form more effective (88% is in undissociated form at pH 4.0, and 6.7% at pH 6.0). Permissible limit in foods is 0.32%. 4. Sulphur dioxide and sulphites Sulphur dioxide (SO2), sulphite (-SO3), bisulphite (-HCO3) and metabisulphite (-S2O5) are used to control microorganisms and insects in foods such as molasses, dried fruits, wine, fruit juices etc SO2 is used both as antimicrobial and agent and antioxidant. It is bacteriostatic at low pH condition at 100-200 ppm level and bacteriocidal at high concentration. Aerobic microorganisms are more sensitive than fermentative forms. Yeasts are less sensitive than molds, acetic acid bacteria and lactic acid bacteria. Molds (Botrytis sp) on grapes are controlled by periodic gassing with SO2. Bisulphites are used to destroy aflatoxins in foods and used at 200-300 ppm level. Mechanism of action Actual mechanism is not known. But one of the possible sexplanation for the antimicrobial activity is the formation of undissociated sulphurous acid or gaseous sulphur dioxide, the effect of which is seen in low acid foods. Due to its strong reducing power lowers the oxygen 44 tension below the level required by aerobic organisms, and by direct action on certain enzymes inhibits microorganisms. Metabisulphites affect vegetative cells during endospore germination. 5. Nitrites and Nitrates Sodium nitrite and sodium nitrate are commonly used in meat curing because they help to stabilize red meat colour, inhibit spoilage and food poisoningorganisms and contribute to flavour development. Many bacteria utilize nitrate and reduce it to nitrite. Nitrite is highly reactive and can serve as both oxidizing and reducing agent. Nitrite in acid environment ionizes to nitrous acid which further decomposes to nitric oxide. This nitric oxide reacts with myoglobin under reduced condition to produce desired red pigment, nitrosomyoglobin. Nitrites are effective against several food poisoning (Clostridium sp) and spoilage microorganisms. Period factor Formation of a substance or an agent which is ten times more inhibitory to Clostridium spp than nitrate alone when medium with added nitrite is heated is called perigo factor (named after the microbiologist Perigo, J A). Absence of botulism in cured, canned and vaccum packed meat and fish products is attributed to perigo factor. The inhibitory or antimicrobial effect results from heat processing or smoking of meat and fish products containing nitrite. Use of nitrite in these products is more for preventing food poising due to Clostridium rather than color and flavour development. Using at levels of 120 ppm causes antimicrobial effect, and 15-20 ppm helps in fixing colour and flavour development. Made of action Antimicrobial effect of nitrite is because of Inhibition of vegetative cell growth Preventing generation and growth of spores that survive heat processing/smoking during post processing storage. The antimicrobial effect of nitrite is due to its inhibition of non-heme, iron-sulphur enzymes. Inhibition of botulism by nitrite is due its effect on iron-sulphur enzymes thus preventing synthesis of ATP from pyruvate 6. Antibiotics Antibiotics are secondary metabolites produced by microorganism such as fungi (Penicillium) and bacteria (Streptomyces, Actionmycetes). These inhibit/kill wide spectrum of 45 microorganisms. Antibiotics are used extensively to treat, control and prevent human and animal disease. Use of antibiotics in foods to control spoilage organism started in 1950 with the use of tetracyclines in poultry. However, antibiotic use in food is not very popular because of risks involved. Factors to be considered while using antibiotic in foods Antibiotic agent should kill, and not inhibit the flora. Should ideally decompose in to harmless products Should be destroyed on cooking Should not be inactivated by food components or products of microbial metabolites Should not readily stimulate development of resistant strains Should not be used in food if used therapeutically or as animal feed additives. Antibiotics used in food Some of the antibiotics used in food are; Tetracyclines Subtlin Tylosin Nisin Natamycin Tetracyclines Among several antibiotics, chlorotetracycline (CTC) and oxytetracycline (OTC) are well suited to use in fresh foods. Tetracyclines are effective against microorganisms because they inhibit protein synthesis. CTC and OTC were approved as food preservatives in 1955 and 1956, respectively. These are heat sensitive and storage labile. Also used in human and animals to treat disease. These are used for extending shelf life of refrigerated fish and other seafoods, red meat, vegetables, raw milk and other foods. CTC is generally more effective than OTC in controlling spoilage flora. A dose of 7-10 ppm level is known to extend shelf life of refrigerated meat by 3-5 days. Use of CTC with sorbates extends shelf life of fish for up to 14 days. Antibiotics are used as feed supplement. Their use is restricted because the risks outweigh the benefits. Subtilin Subtilin is produced by Bacillus subtilis and is effective against Gram positive bacteria. This is stable to acid treatment and heat resistant (stable at 121oC for 30-60 min). Subtilin is 46 effective in canned foods at 5-20 ppm level in preventing germinating endospores. It is not used in human medicine and animal feed. The inhibitory effect of subtilin is because of its effect on membrane transport systems. Tylosin Tylosin is effective against Gram positive bacteria. It inhibits protein synthesis by combining with 50S ribosomal subunit. Used mainly in animal feeds, and also to treat some diseases in poultry. Nisin Nisin is a bacteriocin (not antibiotic) produced by some strains of Lactobacillus lactis, and is widely used in food preservation. Bacteriocins are small proteins which inhibit only closely released strains /species of Gram positive bacteria. Nisin is effective against Gram positive bacteria and ineffective against fungi and Gram negative bacteria. The inhibitory effect is due to disruption of cytoplasmic membrane leading to pore formation, thus affecting membrane transport system. Nisin is used in processed dairy foods. Use of nisin in low acid foods (vegetables) allows reduction in processing time and temperature. Used at 1% level in foods. Nisin is desired in food as preservative because it is non toxic, produced naturally by lactic acid bacteria, heat stable, excellent storage stability, destroyed by digestive enzymes, does not contribute for off flavour/odour, and has narrow spectrum of antimicrobial activity. Natamycin This antibiotic is isolated from Strptomyces natalensis and is effective against yeast and molds (antifungal). It is effective in controlling yeasts and molds at much lower concentration (1-25 ppm) than sorbates. These bind to sterols in cell membrane and disrupt selective membrane permeability. 4.6. Endospores and formation of cell aggregates Endospores are metabolically dormant stages observed in certain Gram positive bacteria as a survival strategy to overcome unfavourable environmental conditions. Spore forming bacteria are of significance in food industry because of their ablity to cause food spoilage and produce toxins which cause illness in humans. Among the bacteria, Clostridium botulinum, C. perfringens and Bacillus cereus are toxigenic while many species of spore formers cause spoilage of food. Endospores are produced inside bacterial cell by members of Gram positive Bacillus, Clostridium, Desulfotomaculum, Sporolactobacillus and Sporosarcina. Spore formation also called as sporulation or sporogenesis is part of the natural life cycle of bacteria. Spores differ from metabolically active and growing vegetative cells by their inert resting condition. Endospores vary in size, shape and position in the vegetative cells in different bacteria and are often useful in the identification of some species. 47 4.6.1. Endosopre formation Endospore formation is initiated by the vegetative cell under the conditions of nutrient depletion, especially the carbon and nitrogen source. The vegetative cell prepares for sporulation by transforming in to a committed sporulating cell called sporangium. The sporangium actively involves in synthesizing compounds required for spore formation. Most spore formers develop mature spore of complex structure within 6-8 hours. Sporulation usually appears in the late logarthemic phase of growth possibly because of nutrient depletion and accumulation of toxic metabolites. Structureof endospore The spore released at the end of sporulation from the mother cell is structurally, biochemically and physiologically different from vegetative cells. The inner core of the spore containing proteins and nucleic acids is surrounded by several layers of varying composition. These include core, spore cortex, spore coat, and outer exosporium. Exosporium: The outermost spore layer is the exosporium and it varies in size in different species. It is a thin, delicate covering made of protein, polysaccaharide and some lipids. Spore coat: Following exosporium is the thick and structurally complex spore coat of several layers consisting of proteins with unusual aminoacids. The spore coat protects the inner spore cortex from attack by lytic enzymes, and serves as barrier to oxidizing agents. This layer is not involved in offering resistance to spores from heat or radiation. Spore cortex: It is made of several layers of loosely cross linked peptodoglycan with calcium and dipicolinic acid. Core: Core is the central region of the spore and contains DNA, ribosomes, most enzymes, diaminopimelic acid and divalent cations and other macromolecules. Core is also characterized by low water content of 10-30% of vegetative cell which reduces the core cytoplasm to a gel like consistency. The dehydration is responsible for spore dormancy and offering resistance to variety of agents. The core cytoplasm also contains high concentration of small acid-soluble spore proteins (SASP) which bound tightly to DNA and protect it from damaging effect of ultraviolet light, dessication, and dry heat. The SASPs also finction as a source of carbon and energy during germnation of endospore. 4.6.2. Germination of spores The spores remain in dormant state for a varying period of time, even for several years. When conditions become favourable the spores break dormancy and enter in to a process called spore germination. Presence of water and certain specific chemicals such as aminoacids or inorganic salts and environmental stimulus initiate germination process by activating dormant hydrolytic enzymes from spore membranes. These enzymes digest the spore cortex and expose the core to water. The rehydrated core utilizes nutrients and grows 48 out of spore coat fully reverting back to vegetative cell. Generally spores germinate in to vegetative cells within a short period of about 90 minutes. 4.6.3. Resistance of spores Bacterial endospores are highly hardy structures capable of withstanding extreme heat, drying, freezing, radiation and chemicals that would readily kill vegetative cells. Ability to survive such harsh environmental conditions is attributed to several factors. The high heat resistance of spores is due to high content of calcium and dipicolinic acid which removes water and makes it dehydrated. The absence of free water offers heat resistance and thus protective effect on proteins and nucleic acids. The spore is metabolically inactive due to non availability of water which makes it resistant to further drying. Presence of thick and impervious cortex and spore wall offer resistance against radiation and chemical substances. 4.6.4. Cell aggregates Microorganisms occur either as single cells or as aggregates consisting of clumps or chains of cells. Microorganisms always grow attaching on to suspended organic and inorganic substances rich in nutrients. In food industry spoilage and pathogenic organisms grow on 49 food residues and serve as continuous source of contamination to food. Also, the bacterial cell aggregates are more resistant to cleaning and disinfection in food industry. As the microbial cells occur in several layers on food residues and surfaces containing nutrients, the cells in the inner layers are not exposed to disinfection treatment and hence survive better. Formation of bacterial cell aggregates need to be prevented in any food handling and processing environment by following good sanitary measures. Unit 5 - Foodborne pathogens Expected Learning Outcome Infective and intoxication type of food poisoning and organisms involved Gram positive and Gram negative bacteria involved in food poisoning, their distribution, activity, nature of food poisoning and preventive measures to control them. 5.1 Foodborne pathogens Several human pathogenic microorganisms are associated with food and water in their natural environment, and foods also get contaminated with pathogens during handling and processing. Thus, ingestion of food or water contaminated with pathogens leads to food poisoning affecting the health of consumers. Food poisoning Food poisoning is an illness caused either by the ingestion of toxin elaborated by the pathogens or from the infection of the host through the intestinal tract due to ingestion of live organisms.Food poisoning by bacteria can be of two types; Bacterial food intoxication 50 Bacterial food infections Bacterial food intoxication It is a food borne illness caused by the presence of a bacterial toxin formed in the food. It is caused due to consumption of food containing pre- formed toxin but not live bacteria. Two types of food intoxications are caused by bacteria. Botulism: Food poisoning caused by the ingestion of food containing toxin produced by Clostridium botulinum. Sataphlococcal intoxication: Food poisoning caused by the ingestion of food containing pre-formed toxins produced by Staphyllococcus aureus Bacterial food infections This is a food borne illness caused by the entrance of live bacteria into the body through ingestion of contaminated food. The illness mainly results from the reaction of the body to pathogen’s presence or their metabolites. Ex: Salmonellosis, C. perfringes illness, Cholera, V. parahaemolyticus infection etc Sources of Pathogens Seafood/ foods of aquatic origin have potential to cause human illness. The entry of bacterial pathogens to aquatic environment /consumers results from the following: Pollution of aquatic environment from sewage or animal wastes. Consumption of raw or semi processed contaminated food/ water. Infected domestic animals used for food. (Ex: Salmonella in poultry) Unsanitary practices of product handling through food handlers. Presence of naturally associated human pathogens in aquatic foods/ environment 5.2. Clostridium botulinum food intoxication Botulism is a food borne illness caused by the ingestion of food containing neurotoxin produced by Clostridium botulinum. It is a Gram positive rod shaped anaerobic spore forming soil bacterium which can grow in the pH range of 4.6 to 8.5. It produces highly potent exotoxin, a neurotoxin, and ingestion of a small quantity (few nanograms: 30-100 ng) can cause paralyses and death. Toxin types Seven neurological toxin types (Type A to G) have been recognized based on toxin specificity. Of these types A, B, E and F are pathogenic to man. These are further divided into 2 types. 51 Proteolytic types: Includes A, B, and F type toxin producers. These are heat resistant, NaCl tolerant, mesophilic and soil is the general habitat. Non-proteolytic types: Includes some strains of B, E, and F. these are heat sensitive, psychrotolerant, NaCl sensitive and aquatic environment is the natural habitat. Toxins produced by type A, B, E and F cause human botulism. Botulinal toxin is one of the most potent of all poisons and very low amounts (30 -100 ng) can cause death. Clostridium botulinum Type E C.Botulinum Type E is associated with fish and fishery products and is primarily of marine origin. It is ubiquitous in natural environment including marine sediment as well as animals, birds and fish intestine. Toxin production by Cl. botulinum depends on the ability of cells to grow in food and autolyse to release toxins. Factors influencing toxin production Toxin production depends on the factors influencing spore germination and growth. These include composition of food, moisture current, pH, O-R potential, salt content and temperature and storage conditions. In heat processed foods C.botulinum are of great significance, as under-serilization can lead to survival of spore, which under suitable conditions germinate and make food toxic. Even seafood held at low temperature (refrigerated temperature) the type E can grow (at about 3oC). But fail to grow under frozen temperature. For the intoxication to develop the preformed toxins should be consumed (food containing toxin). Food that has been eaten which was served without heating prior to serving can cause illness. The toxin is inactivated when heated at 60oC for 5 min. Botulism occurs rarely (incidence of botulism is not common) and most of the outbreaks are associated with fish (Type E). Symptoms of food poisoning Symptoms vary from mild illness to fatal condition within 24 hr, symptoms develop within 12-36 hr and include nausea and vomiting followed by neurological disorders like visual impairment, loss of normal function of mouth and throat, lack of muscle coordination, respiratory impairment leading the death. Onset of symptoms is rapid with type E botulism, while the severity is high with type A Treatment Only known method of treatment is by administration of antitoxin and is effective only when administered before onset of symptoms. 52 Conditions necessary for outbreaks Presence of spores of type A, B, E or F in food being canned or processed. Food in which the spores can germinate and clostridia can grow and produce toxin. Survival of spores because of inadequate heating in canning or inadequate processing. Condition (environmental/storage) after processing that will permit germination of spores. Insufficient cooking of food to inactive the toxin. Ingestion of toxin bearing food. Prevention of outbreaks Use of approved heat process for canned foods. Rejection of all swollen or spoiled canned foods. Refusal even to taste a doubtful food. Avoidance of foods that have been cooked held but not well heated. Boiling of suspected food for atleast 15 min to inactive toxin. 5.3. Staphylococcus food intoxication Staphalococcal food poisoning is caused by the ingestion of enterotoxin formed in food during growth of certain strains of Staphylococcus aureus. This bacterium produces an exotoxin called enterotoxin which causes gastroenteritis or inflammation of intestinal tract. S. aureus is a Gram positive cocci appearing as bunch of grapes. Most of the entertoxin producing strains are coagulase positive (coagulate blood plasma), produce thermostable nuclease, facultatively anaerobic and grow better aerobically than anaerobic condition. However, some coagulase negative strain also produces entertoxin. These produce six neurologically distinct entertoxin (A, B, C, C2 D and E) which differ in toxicity. Most food poising involves types A or D. Grow in the temperature range of 4–40oC with rapid growth taking placing in the temperature rage of 20-45oC. Source of staphylococcus to food Source of staphylococci to foods are mainly from human and domestic animals (primary reservoirs). Commonly encountered in skin, hair, nasal passage, wound and throat. of humans. Food handlers are main source of food contamination with S. aureus. Besides, equipment and food contact surfaces also contribute for contaminatin with this organism. Generally, conditions in foods that favour the growth of S. aureus (at about 40oC) are more susceptible for food poisoning. En