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Food Biotechnology Notes:, , Unit 1:, Intrinsic and Extrinsic parameters of foods that affect microbial growth, In most cases, micro-organism utilizes our food supply as a source of nutrient for their growth. This in the, course can result in deterioration (decay) of food. The organism not only deteriorates the food but may also, pose risks of disease to the human being on consumption of such contaminated food. However, the growth of, microorganisms in food may be affected by several factors like pHysical, chemical and biological. These factors, can broadly divide into two categories i.e., Intrinsic Parameters:Intrinsic parameters are natural or inherent properties of food. These parameters greatly affect the number and, types of microorganism that will colonize the food and food product. Intrinsic parameters affect only, microorganisms, not to the food itself. Intrinsic parameters of food include:pH:Every organism has a minimal, maximal and optimal pH for growth. Some organism can growth better at low, pH or acidic pH, some can grow in alkaline pH and while other grow at somewhat neutral pH. PH influence, both the growth rate and types of organism that will predominant the food. In general yeast and mold are more, acidic tolerant than bacteria., Moisture content or water activity (aw):Micro-organism (Mos) have an absolute demand for water, however, the exact amount of water needed for, growth of microorganisms varies. This parameter helps us to understand the movement of water from the, environment to the cytoplasm or from cytoplasm to the environment. The water requirement of microorganisms, is expressed in pHysical form; called water activity (aw).Water activity is the ratio of the vapor pressure of water, present in food substrate (solution) to the vapor pressure of pure water at the same temperature., , aw=P/Po, Minimal water activity requirement for different microorganisms:-, , 1., 2., 3., 4., , Most bacteria 0.91, Most yeast 0.88, Most mold 0.80, HalopHilic bacteria 0.75

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5. XeropHilic fungi 0.65, 6. OsmopHilic yeast 0.60, Oxidation/Reduction potential (O/R potential):Oxidation/reduction (OR) potential of a substrate (food) may be defined as ease of with which substrate loss or, gain the electron. OR potential is usually written as Eh and expressed in term of millivolt (mv)., Aerobic organism including Micrococcus, Pseudomonas, Bacillus, Acetobacter etc. Required +ve Eh value and, anaerobes including Clostridium, Bactericides etc. Required –ve Eh value. The redox potential of food is, influenced by its chemical composition as well as specific processing and treatment. Also, OR potential is, influenced by storage condition., OR potential of some foods are:, , Raw meat:- +ve 200mv, , , , Raw minced meat:-+ve 225mv, , , , Cheese:- -ve 200mv, , , , Plant/fruit juice:- -ve 300mv to +ve 400mv, , Nutrients contain:The kinds and proportional of nutrient in food are all important in determining which micro- organism, (microorganisms) is most likely to grow. In general, the simple compound is utilized first by the measuring, microorganisms., Anti-microbial constituents:Some foods possess naturally occurring substances which influence the activity of invading microorganisms,, for example:1. Plant, -Clove:-Essential oil, Eugenol, -Garlic:-Allicin, -Common:-Cinnamic, aldehyde, -Sage:-Eugenol, Thymol; -Mustard oil:-Allyl isothiocyanate, -organic:-Thymol and isothermal

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-Fruits, vegetables, tea:-Hydrocinnamic acid and derivatives, 2-Animal, - Cow’s milk:-Lactoferrin, conglutinin, lacto-peroxidase system., -Egg:-Lysozyme, 0vatransferrin (inhibit Salmonella enteritidis), Biological structure:The natural covering of some foods provides excellent protection against the energy of micro- organism and, spoilage of food by such microorganisms. Natural covering of food like,, -Testa of seed, -Shell of egg/nuts, -peel of fruits/vegetable, -Hide of animal may limit the entry of microorganisms, Extrinsic Parameters:Extrinsic parameters are environmental factors, in which food and food products are kept. Extrinsic parameters, substrate independent and affects both micro-organism (mos) as well as food .Unlike intrinsic parameters,, extrinsic parameters can be maintain and regulated well. The extrinsic parameters includes:1. Temperature of storage:Temperature of storage is highly important parameters that affect the spoilage of highly perishable food. Mos, are reported to grow between -340c to 1000c and each organism exhibit a minimum, optimum and maximum, temperature for growth and these are known as cardinal temperature., 2. Relative humidity:Humidity is the concentration of water vapor in the atmosphere. Relative humidity is the ratio expressed as the, percentage of moisture in air to the moisture present in food under the saturation condition at temperature and, pressure., 3. Presence and concentration of gases:Presence of different gases and its varying concentration may significantly affects the colonizing mos on the, food i.e. Surface spoilage are prevented by altering gaseous composition. Oxygen is one of the most important, gases which affect both food products as well as Mos.

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Oxygen gas when come in contact with food, influence redox potential of food and finally the microbial growth., Presence and activity of micro-organism:Inhibition or destruction of one population of mos by presence of other population of mos present in same, habitat is the microbial interference. Some mos produced substances/metabolites (like secondary metabolites),, that are either lethal or inhibitory to others., , Unit operation:, In chemical engineering and related fields, a unit operation is a basic step in a process. Unit operations involve, a physical change or chemical transformation such as separation, crystallization, evaporation, filtration,, polymerization, isomerization, and other reactions. For example, in milk processing, homogenization,, pasteurization, chilling, and packaging are each unit operations which are connected to create the overall, process. A process may require many unit operations to obtain the desired product from the starting materials,, or feedstock’s., Role & significance of microorganism:, Because human food sources are of plant and animal origin, it is important to understand the biological, principles of the microbial biota associated with plants and animals in their natural habitats and respective roles., Although it sometimes appears that microorganisms are trying to ruin our food sources by infecting and, destroying plants and animals, including humans, this is by no means their primary role in nature. In our present, view of life on this planet, the primary function of microorganisms in nature is self-perpetuation. During this, process, the heterotrophs carry out the following general reaction:, All organic matter, (carbohydrates, proteins, lipids, etc.), Energy + Inorganic compounds, (Nitrates, sulfates, etc.), This, of course, is essentially nothing more than the operation of the nitrogen cycle and the cycle of other, elements. The microbial spoilage of foods may be viewed simply as an attempt by the food biota to carry out, what appears to be their primary role in nature. This should not be taken in the teleological sense. In spite of, their simplicity when compared to higher forms, microorganisms are capable of carrying out many complex, chemical reactions essential to their perpetuation.

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Unit 2nd, Spoilage of food, They can be defined as any visible or invisible change which can makes food or product derived from food, unacceptable for human consumption. Spoilage of food not only causes health hazard to the consumer but also, cause large economic losses. Spoilage not only leads to loss of nutrients from food but also cause change in, original flavor and texture. It is estimated that about 25% of total food produced is spoilt due to microbial, activities only despite range of preservation methods available. Thus the spoilage of food is not only a health, hazard but also carry lot of economic significance too., In total, the food spoilage is considered a complex pHenomenon whereby a combination of microbial and, biochemical activities take place. Due to such activities, various types of metabolites are formed which aid in, spoilage. The detection of these metabolites helps in detection of spoilage.

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APPARENT HEALTH BENEFITS OF FERMENTED MILKS:, The topic of health-promoting effects of certain fermented foods and/or the organisms of fermentation are, beset by findings both for and against such effects. Some studies that appear to be well designed support, health benefits; however, other equally well-designed studies do not. The three areas of concern are the, possible benefits to lactose-intolerant individuals, the lowering of serum cholesterol, and anticancer activity., Lactose-intolerant individuals:, The condition is due to the absence or reduced amounts of intestinal lactase, and this allows the bacteria in the, colon to utilize lactose with the production of gases. The breath hydrogen, Test for lactose intolerance is based on the increased levels ofh2 produced by anaerobic and Facultative, anaerobic bacteria utilizing the non-absorbed lactose. A large number of investigators have found that lactose, malabsorbers can consume certain fermented dairy products without harmful effects., Lowering of serum cholesterol:, Large dietary intakes of yogurt were found to lower cholesterolemia, and the findings suggested that yogurt, contains a factor that inhibits the synthesis of cholesterol from acetate. This factor may be 3-hydroxy-3methylglutaric acid and/or orotic acid plus thermopHilus milk and methanol solubles of thermopHilus milk on, liver cholesterol, and the investigators found that both products significantly reduced liver cholesterol levels, compared to controls., Anticancer activity:, Use of yogurt and yogurt extracts shows effect against a sarcoma and a carcinoma. The enzymes assayed were, B-glucuronidase, nitroreductase, and azoreductase because they can convert indirectly acting carcinogens to, proximal carcinogens. It may prove to be significant in colon cancer where the body evidence supports a role, for diet., , Dehydrated foods, Food drying is a method of food preservation that works by removing water from the food, which inhibits the, growth of bacteria and has been practiced worldwide since ancient times to preserve food. The food so produce, is known as dehydrated food. Drying (dehydrating) food is one of the oldest and easiest methods of food, preservation. Dehydration is the process of removing water or moisture from a food product. Removing, moisture from foods makes them smaller and lighter.

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What types of food can be dehydrated?, , , Beef. Biltong. Chicken. Chicken Jerky. Fish. Fish Jerky. Ground Beef Jerky. ..., , , , Lamb. Meat. Pork Jerky. Turkey. Venison Jerky., , , , Okra. Onion. Peas. Plums (prunes) Potato. Potato (Hash Brown) Potato (instant potato flakes) ., , , , Chili (Powder) Garlic (Powder) Ginger. Herbs. Herbs 2. Lavender., , , , Bean Bark. Bread Crumbs. Broth. Cheese. Cottage Cheese. Eggs., , Dehydrated vegetables store well if hermetically sealed in the absence of oxygen. The planning on a storage life, of 8-10 years at a stable temperature of 70 degrees F. They should keep proportionately longer if stored at cooler, temperatures same as with many other foods, dried fruit have both good and bad aspects. Dried fruit can boost, your fiber and nutrient intake and supply your body with large amounts of antioxidants. However, they are also, high in sugar and calories, and can cause problems when eaten in excess., Industrial food dehydration is often accomplished by freeze-drying. In this case food is flash frozen and put into, a reduced-pressure system which causes the water to sublimate directly from the solid to the gaseous phase., Although freeze-drying is more expensive than traditional dehydration techniques, it also mitigates the change, in flavor, texture, and nutritional value. In addition, another widely used industrial method of drying of food is, convective hot air drying. Industrial hot air dryers are simple and easy to design, construct and maintain. More, so, it is very affordable and has been reported to retain most of the nutritional properties of food if dry using, appropriate drying conditions., Study of the microbiology of dehydrated, 17 different kinds of dried soups from 9 different processors had total counts of less than log 5.00/g. These, soups included chicken noodle, chicken rice, beef noodle, vegetable, mushroom, pea, onion, tomato, and others., Some of these products had total counts as high as log 7.30/g, and some had counts as low as around log 2.00., , ENTERAL NUTRIENT SOLUTIONS (MEDICAL FOODS):, Enteral nutrient solutions (ENS), also known as medical foods, are liquid foods administered by tube. They are, available as powdered products requiring reconstitution or as liquids. They are generally administered to certain, patients in hospitals or other patient care facilities but may be administered in the home. Administration is by, continuous drip from enteral feeding bags, and the process may go on for 8 hours or longer, with the ENS at, room temperature. Enteral foods are made by several commercial companies as complete diets that only require, reconstituting with water before use or as incomplete meals that require supplementation with milk, eggs, or the, like prior to use. ENS-use preparations are nutritionally complete, with varying concentrations of proteins,

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peptides, carbohydrates, and so forth, depending on patient need., , The microbiology of ENS has found the products to contain varying numbers and types of bacteria and to be, the source of patientinfections. The most frequently isolated organism was StapHylococcusepidermidis, with, Corynebacterium, Citrobacter,and Acinetobacter spp. From a British study, enteral feeds yielded 104-106, organisms/ml, with coliforms and Pseudomonas aeruginosa as the predominant types., A medical food is defined as a food which is formulated to be consumed or administered entirely under the, supervision of a physician and which is intended for the specific dietary management of a disease or condition for, which distinctive nutritional requirements, based on recognized scientific principles, are established by medical, evaluation. This particular category of food has gained traction recently and many companies are looking to, develop an innovative medical food. However, FDA narrowly defines the conditions under which a medical food, may be marketed:, , 1. It must be a specially formulated and processed product (as opposed to a naturally occurring foodstuff, used in its natural state) for the partial or exclusive feeding of a patient by means of oral intake or enteral, feeding by tube;, 2. It must be intended for the dietary management of a patient who, because of therapeutic or chronic, medical needs, has limited or impaired capacity to ingest, digest, absorb, or metabolize ordinary, foodstuffs or certain nutrients, or who has other special medically determined nutrient requirements, the, dietary management of which cannot be achieved by the modification of the normal diet alone;, 3. It must provide nutritional support specifically modified for the management of the unique nutrient needs, that result from the specific disease or condition, as determined by medical evaluation;, 4. It must be intended to be used under medical supervision; and, 5. It must be intended only for a patient receiving active and ongoing medical supervision wherein the, patient requires medical care on a recurring basis for, among other things, instructions on the use of the, medical food.,  In terms of ingredients that may be used in medical foods, the ingredients should be either:, (1) A food additive used in accordance with FDA’s food additive regulations;, (2) A color additive used in accordance with the color additive regulations;, (3) A substance that is generally recognized, by qualified experts, to be safe under the conditions of its, intended use (GRAS); or;, (4) A substance that is authorized by a prior sanction issued by FDA.

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Cheese:, Cheese is a highly proteinous food made from the milk of some herbivores. Cheese is, believed to have originated in the warm climates of the Middle East some thousands of, years ago, and is said to have evolved when milk placed in goat stomach was found to, have curdled. The scientific study and manipulation of milk for cheese manufacture is, however just over a hundred years old. About a thousand types of cheese have been, described depending on the properties and treatment of the milk, the method of, production, conditions such as temperature, and the properties of the coagulum, and the, local preferences., , (a) Standardization of milk, The quality of the milk has a decided effect on the nature of cheese. Cheese made from, skim milk is hard and leathery; the more fat a cheese contains the smoother its feel to the, palate. The fat/protein ratio is often adjusted through fat addition in order to yield a, cheese of consistent quality. In the US, pasteurization (High Temperature Short Time), or (Long Temperature Short Time) must be given to milk to be in certain types of, cheeses, such as cottage or cream cheese. For others the milk need not be pasteurized., But must be stored at for at least 60 days at 2°C. Pasteurization gives a better control, over the processes of cheese production. However, the organisms present in raw milk, are important during the ripening processes. The milk may also be homogenized by, forcing it at high speed through small orifices to, Reduce the milk fat globules for use in producing soft cheeses., , (b) Inoculation of pure cultures of lactic acid bacteria as starter cultures, In the past, lactic acid was produced by naturally occurring bacteria. Nowadays they are, inoculated artificially, by specially selected bacteria termed starters. Indeed lactic acid, formation is necessary in all kinds of cheese. For cheese prepared at temperatures less, than 40°C strains of Lactococcus lactis are used. For those prepared at higher, temperatures the more thermopHilic Streptococcus thermopHilus, Lactobacillus, bulgaricus, and Lact. Helveticus are used., Lactic acid has the following effects:

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(i) It causes the coagulation of casein at pH 4.6, the isoelectric point of that protein,, which is used in the manufacture of some cheeses, e.g. Cottage cheese., , (ii) It provides a favorably low pH for the action of rennin the enzyme which forms the, curd from casein in other types of cheeses., , (iii) The low pH eliminates proteolytic and other undesirable bacteria., (iv) It causes the curd to shrink and thus promotes the drainage of whey., (v) Metabolic products from the lactic acid bacteria such as ketones, esters and, aldehydes contribute to the flavor of the cheese., C) Adding of rennet for coagulum formation, The classical material used in the formation of the coagulum is ‘rennet’ which is derived, from the fourth stomach, abomasum or veils of freshly slaughtered milk-fed calves, Rennet is produced by soaking and/or shredding air-dried veils under acid conditions, with 12-20% salt. Extracts from young calves contain 94% rennin and 6% pepsin and, from older cows, 40% rennin and 60% pepsin. Rennin (chymosin) is the enzyme, responsible for the coagulation of the milk. Pepsin is proteolytic and too high an amount, of pepsin can result in the hydrolysis of the coagulum and a resulting low yield of, cheese, and a bitter taste may result from the amino acids., , (d) Shrinkage of the curd, The removal of whey and further shrinkage of the curd is greatly facilitated by heating, it, cutting it into smaller pieces, applying some pressure on it and lowering the pH. In, many types of cheeses, such as Parmesan, Emmenthal and Gruyere, there is a stage, known as ‘scalding’ in which the temperature can be as high as 56°C in the preparation., Acid produced by the lactic starters introduce elasticity in the curd, a property desirable, in the final qualities of cheese., , (e) Sa1ting of the curd and pressing into shape, Salt is added to most cheese varieties at some stage in their manufacture. Salt is, important not only for the taste, but it also contributes to moisture and acidity control., Most importantly however it he1ps limit the growth of proteiolytic bacteria which are, undesirable. The curd is pressed into shape before being allowed to mature.

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(f) Cheese ripening, The ripening or maturing of cheese is a slow joint microbiological and biochemical, process which converts the brittle white curd or raw cheese to the final full-flavored, cheese. The agents responsible for the final change are enzymes in the milk, in the, rennet and those from the added starter microorganisms as well as other microorganisms which confer the special character of the cheese to it., , High gravity brewing (HGB):, It is a brewing technique that begins with the production of wort that has a higher sugar, concentration. Because there are more fermentable sugars, a higher-alcohol beer can be, produced. This allows the brewer to blend the beer down to its saleable alcohol content, later in the process, using special de-aerated water (water with no oxygen dissolved into, it). Producing a more concentrated wort and beer will increase a brewery’s capacity., Who practices this technique?, , Although some microbreweries now practice HGB, it is a technique that is mostly used, by larger breweries. This is not to say that microbreweries are not brewing high gravity, worts to make higher alcohol beers, because they are – the only difference being that, they are not blending it to a lower alcohol level at the end of the process., What are the benefits of HGB?,  In a competitive market, HGB can significantly increase capacity through tank, and equipment utilization. The result is less capital and operating costs with, savings in fuel, electricity, water and refrigeration., Using HGB will give the brewer substantial capacity gains, as we’ve mentioned, but it, will also:, , , , , Improve consistency of final product – particularly in terms of the alcohol, level., Improve colloidal stability – a higher concentration of proteins and tannins, makes for higher precipitation rates.

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What are the disadvantages of HGB?, Creating a higher-alcohol brew requires a slightly longer fermentation, as you’d expect., However it also:, , , decreases the brew house yield and hop utilization, , , , requires a slightly longer lautering time, , , , slightly reduces foam stability, , What does an HGB brewer have to keep in mind?, There are a few things that a high gravity brewer must keep in mind. Many of these can, be easily managed by tweaking the process and understanding its limits., The first consideration is FLAVOUR:, HGB can result in a higher concentration of volatile aromatics produced by the yeast., Flavor changes can pose a problem when a brewer is trying to match their HGB product, to an existing lower gravity beer., The, , second consideration is, , YEAST :, , HGB, and higher alcohol content in general, can have a negative impact on yeast health, – depending on the tolerance your yeast strain has for higher alcohol levels., HGB equipment:, There is a price-tag associated with HGB – which is perhaps one of the reasons HGB is, used mostly by large brewers., Getting rid of diacetyl:, With the exception of a very few notable lagers, diacetyl is considered completely, unacceptable in the style.The interesting thing about the obsession with diacetyl in, modern brewing, especially among lager brewers, has nothing to do with diacetyl’s, association with bacterial contamination., Beers containing diacetyl, whether the consumer knows it or not, are satiating and make, beer drinkers feel full. This translates to decreased beer consumption during a beer, drinking session and a corresponding drop in beer sales. My tips to minimize diacetyl, are:, 1. Use a 3-day diacetyl rest for ales and a 7-day diacetyl rest for lagers following, primary fermentation.

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2., , Select a, , yeast strain not, , noted for, , diacetyl, , production., , 3. Minimize air pick-up during racking, filtration and packaging because oxygen, causes alpHa- acetolactate, , in, , beer, , to, , convertto, , diacetyl., , 4. Keep a clean brewery and use clean yeast since lactic acid bacteria and, Pediococcus can both cause diacetyl problems in beer., , Unit 3rd, MICROBIOLOGICAL EXAMINATION OF SURFACES:, The need to maintain food contact surfaces in a hygienic state is of obvious importance., The Primary problem that, or, , utensils, , has to be overcome, , when examining, , surfaces, , for microorganisms is the removal of a significant percentage of, , the resident biota. The most commonly used methods for surface assessment in food, operations are presented below:, , 1. Swab/Swab-Rinse Methods:, Swabbing is the oldest and most widely used method for the microbiological, examination of surfaces not only in the food and dairy industries but also in hospitals, and restaurants either cotton or calcium alginate swabs are used. If one wishes to, examine given areas of a surface, templates may be prepared with openings, corresponding to the size of the area to be swabbed, for example, 1 in2 or 1 cm2., 2. Contact Plate, The replicate organism direct agar contact (RODAC) method employs special Petri, plates, which are poured with 15.5-16.5 ml of an appropriate plating medium, resulting, in a raised agar surface. When the plate is inverted, the hardened agar makes direct, contact with the surface Agar Syringe/"Agar Sausage" Methods, By this method, a 100-ml syringe is modified by removing the needle end to create a, hollow cylinder that is filled with agar. A layer of agar is pushed beyond the end of the, barrel by means of the plunger and pressed against the surface to be examined. The, exposed layer is cut off and placed in a Petri dish, followed by incubation and colony, enumeration., 3. Direct Surface

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A number of workers have employed direct surface agar plating methods, in which, melted agar is poured onto the surface or utensil to be assessed. Upon hardening, the, agar mold is placed in a Petri dish and incubated. It was used successfully to determine, the survival of Clostridium sporogenes endospores on stainless-steel surfaces., 4. Ultrasonic Devices, Ultrasonic devices have been used to assess the microbiological contamination of, surfaces, but the surfaces to be examined must be small in size and removable so that, they can be placed inside a container immersed in diluent. Once the container is placed, in an ultrasonic apparatus, the energy generated effects the release of microorganisms, into the diluent., 5. Spray Gun, A spray gun method was based on the impingement of a spray of washing solution, against a circumscribed area of surface and the subsequent plating of the washing, solution. Although the device is portable, a source of air pressure is necessary. It was, shown to be much more effective than the swab method in removing bacteria from meat, surfaces, 6. Swab/Agar Slant, The method involves sampling with cotton swabs that are transferred directly to slants., Following incubation, slants are grouped into one-half logi0 units based on estimated, numbers of developed colonies., , METABOLICALLY INJURED ORGANISMS:, When microorganisms are subjected to environmental stresses such as sublethal heat, and freezing, many of the individual cells undergo metabolic injury, resulting in their, inability to form colonies on selective media that uninjured cells can tolerate. Whether a, culture has suffered metabolic injury can be determined by plating aliquots separately, on a nonselective and a selective medium and enumerating the colonies that develop, after suitable incubation. The colonies that develop on the nonselective medium, represent both injured and uninjured cells, whereas only the uninjured cells develop on, the selective medium. The difference between the numbers of colonies on the two media, is a measure of the number of injured cells in the original culture or population.

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The existence of metabolically injured cells in foods and their recovery during culturing, procedures is of great importance not only from the standpoint of pathogenic organisms, but for spoilage organisms as well Injury of foodborne microorganisms has been shown, by a large number of investigators to be induced not only by sublethal heat and freezing, but also by freeze drying, drying, irradiation, aerosolization, dyes, sodium azide, salts,, heavy, , metals,, , antibiotics,, , essential, , oils,, , and, , other, , chemicals,, , such, , as, , ethylenediaminetetraacetic acid (EDTA) and sanitizing compounds., , Enumeration and Detection of Food-borne Organisms:, The examination of foods for the presence, types, and numbers of microorganisms, and/or their products is basic to food microbiology. In spite of the importance of this,, none of the methods in common use permits the determination of exact numbers of, microorganisms in a food product. Although some methods of analysis are better than, others, every method has certain inherent limitations associated with its use. The four, basic methods employed for "total" numbers are as follows:, , 1. Standard plate counts (SPC) for viable cells, 2. The most probable numbers (MPN) method as a statistical determination of viable, cells, , 3. Dye reduction techniques to estimate numbers of viable cells that possess reducing, capacities, , 4. Direct microscopic counts (DMC) for both viable and nonviable cells, , CONVENTIONAL STANDARD PLATE COUNT:, By the conventional SPC method, portions of food samples are blended or, homogenized, serially diluted in an appropriate diluent, plated in or onto a suitable agar, medium, and incubated at an appropriate temperature for a given time, after which all, visible colonies are counted by use of a Quebec or electronic counter. The SPC is by far, the most widely used method for determining the numbers of viable cells or colony-

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forming units (cfu) in a food product., , MEMBRANE FILTERS:, Membranes with a pore size that will retain bacteria (generally 0.45 um) but allow water, or diluent to pass are used. Following the collection of bacteria upon filtering a given, volume, the membrane is placed on an agar plate or an absorbent pad saturated with the, culture medium of choice and incubated appropriately. Following growth, the colonies, are enumerated. Alternatively, a DMC can be made. In this case, the organisms, collected on the membrane are viewed and counted microscopically following, appropriate staining, washing, and treatment of the membrane to render it transparent., These methods are especially suited for samples that contain low numbers of bacteria., The overall efficiency of membrane filter methods for determining microbial numbers, by the DMC has been improved by the introduction of fluorescent dyes., , MICROSCOPE COLONY COUNTS:, Microscope colony count methods involve the counting of micro colonies that develop in, agar Layered over microscope slides. The first was that of Frost, which consisted of, spreading 0.1 ml Of milk-agar mixture over a 4-cm2 area on a glass slide. Following, incubation, drying, and staining, micro-colonies are counted with the aid of a microscope., , MOST PROBABLE NUMBERS:, In this method, dilutions of food samples are prepared as for the SPC. Three serial, aliquots or Dilutions are then planted into 9 or 15 tubes of appropriate medium for the, three- or five-tube method, respectively. Numbers of organisms in the original sample, are determined by use of standard MPN tables. The method is statistical in nature, and, MPN results are generally higher than SPC results. Among the advantages it offers are, the following:, o It is relatively simple., o Results from one laboratory are more likely than SPC results to agree with those, from another laboratory.

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o Specific groups of organisms can be determined by use of appropriate selective, and differential media., o It is the method of choice for determining fecal coliform densities., o Among the drawbacks to its use are the large volume of glassware required, (especially for the five-tube method), the lack of opportunity to observe the, colonial morphology of the organisms, and its lack of precision., , DYE REDUCTION:,  Two dyes are commonly employed in this procedure to estimate the number of, viable organisms in suitable products: methylene blue and resazurin. To conduct, a dye-reduction test, properly prepared supernatants of foods are added to, standard solutions of either dye for reduction from blue to white for methylene, blue and from slate blue to pink or white for resazurin. The time for dye, reduction to occur is inversely proportional to the number of organisms in the, sample., , DIRECT MICROSCOPIC COUNT:,  In its simplest form, the DMC consists of making smears of food specimens or, cultures onto a microscope slide, staining with an appropriate dye, and viewing, and counting cells with the aid of a microscope (oil immersion objective). Dmcs, are most widely used in the dairy industry for assessing the microbial quality of, raw milk and other dairy products., , Bioassay and related methods:,  After establishing the presence of pathogens or toxins in foods or food products,, the next important concern is whether the organisms/toxins are biologically active., For this purpose, experimental animals are employed where feasible. When it is, not feasible to use whole animals or animal systems, a variety of tissue culture, systems have been developed that, by a variety of Responses, provide information, on the biological activity of pathogens or their toxic products.

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WHOLE-ANIMAL ASSAYS:, Mouse Lethality:,  This method was first employed for foodborne pathogens around 1920 and, continues to be an important bioassay method. To test for botulinal toxins in, foods, appropriate extracts are made and portions are treated with trypsin (for, toxins of nonproteolytic Clostridium botulinum strains). Pairs of mice are, injected intraperitoneally (IP) with 0.5 ml of trypsin-treated and untreated, preparations. Untreated preparations that have been heated for 10 minutes at, 1000C are injected into a pair of mice. All injected mice are observed for 72, hours for symptoms of botulism or death. Mice injected with the heat, preparations should not die because the botulinal toxins are heat labile., Specificity in this test can be achieved by protecting mice with known botulinal, antitoxin, and in a similar manner, the specific serologictype of botulinal toxin, can be determined., , Suncus Murinus,  This small animal has been used in Japan as an experimental model for, emesis research using a variety of drugs, and it has been shown to, respond to cereulide, the emetic toxin of Bacillus cereus. Suncus murinus, is referred to as the Japanese house shrew, and adults do not exceed 100, g in weight. For experimental use, those weighing 50-80 g are used., Suckling (Infant) Mouse,  This animal model was introduced by Dean et al.12 primarily for, Escherichia coli enterotoxins And is now used for this and some other, foodborne pathogens. Typically, mice are separated from their mothers, and given oral doses of the test material consisting of 0.05-0.1 ml with, the aid of a blunt 23-gauge hypodermic needle. A drop of 5% Evans, blue dye per milliliter of test material may be used to determine the, presence of the test material in the small intestine. The animals are, usually held at 25°C for 2 hours and then killed. The entire small, intestine is removed, and the relative activity of test material is

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determined by the ratio of gut weight to body weight (GW/BW)., Rabbit and Mouse Diarrhea:,  Rabbits and mice have been employed to test for diarrheagenic activity of, some foodborne pathogens. Employing young rabbits weighing 500-800, g, inoculated orogastrically with approximately 1010 cells of Y, enterocolitica suspended in 10% sodium bicarbonate. Diarrhea developed, in 87% of 47 rabbits after a mean time of 5.4 days. Bacterial colonization, occurred in all animals regardless of dose of cells. Enterocolitica. The, animals were given inocula of 109 cells/ml in peptone water, and fresh, drinking water was allowed 24 hours later. After 2 days, feces of mice, were examined for signs of diarrhea., Monkey Feeding:,  The use of rhesus monkeys (Macaca mulatto) to assay staphylococcal, enterotoxins was developed in 1931 by Jordan and mcbroom.27 Next to, humans; this is perhaps the animal most sensitive to staphylococcal, enterotoxins. When enterotoxins are to be assayed by this method, young, rhesus monkeys weighing 2-3 kg are selected. The food homogenate,, usually in solution in 50-ml quantities, is administered via stomach tube., The animals are then observed continuously for 5 hours. Vomiting in at, least two of six animals denotes a positive response., CELL CULTURE SYSTEMS:,  A variety of cell culture systems are employed to assess certain pathogenic, properties of viable Cells. The properties often assessed are invasiveness,, permeability, cytotoxicity, adherence adhesion binding, and other more, general biological activities. Some cell cultures are used to assess various, properties of toxins and enterotoxins., Human Mucosal Cells,  Human buccal mucosa cells (about 2 x 105 in phosphate buffered saline), are mixed with 0.5 ml of washed E. Coli cells—2 x 1ml. The mixture is, rotated for 30 minutes at room temperature. Epithelial cells are separated, from the bacteria by differential centrifugation, followed by drying and

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staining with gentian violet. Adherence is determined by microscopic, counting of bacteria per epithelial cell., Human Fetal Intestine,  By this adherence model, human fetal intestine (HFI) cells are employed, in monolayers. The monolayers are thoroughly washed, inoculatedwith a, suspension of V parahaemolyticus, and incubated at 37°C for up to 30, minutes. Adherence is determined by the microscopic examination of, stained cells after washing away unattached bacteria. All strains of, Vparahaemolyticus tested adhered, but those from food-poisoning cases, have higher adherence ability than those from foods., , Common food borne diseases:,  Although a number of different infectious diseases may be contracted from foods, under certain circumstances, there are those that are contracted exclusively or, predominantly from the consumption of food products.,  Two examples of the former are hemorrhagic colitis and listeriosis; and of the, latter, botulism and staphylococcal food poisoning.,  Anthrax and brucellosis are two diseases that have in decades past been, contracted from eating diseased animals, but, with the prevalence of these, diseases being so low, they are rarely if ever contracted via the foodborne route.,  The recognized foodborne pathogens include multicellular animal parasites,, protozoa, fungi, bacteria, viruses, and possibly prions Pathogens may be, transmitted from contaminated feces via the fingers of unsanitary food handlers,, by flying or crawling insects, or from water.,  While this route is not as common for syndromes such as staphylococcal food, poisoning, it is the primary route of infection for the foodborne viruses and, entero-pathogenic protozoa and bacteria,  Much new information has been obtained during the past decade on the specific, mechanisms used by foodborne pathogens to cause human disease, and this is, especially true of the gram negative bacteria.

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 Beyond their role in intestinal fluid accumulation (diarrhea), not much more has, been learned about the enterotoxins that are produced by gram-negative bacteria., Their role in host cell invasion and subsequent pathogenesis seems minimal, , Air sampling,  Airborne bacterial and fungal cells and spores may be present in droplets as bioaerosols, as very small individual particles that stay suspended for long periods, or as larger clumps and aggregates that settle rapidly onto surfaces.,  They can be an important source of infection in medical facilities and can, contaminate sensitive manufacturing operations, but regular monitoring of, airborne microorganisms is sometimes neglected.,  Monitoring airborne microorganisms is therefore a key component of, environmental monitoring in many sectors and a range of technological solutions, has been developed to help operators achieve an effective monitoring, programme, not just in the pharmaceutical sector, but in hospitals, food factories, and a variety of other environments., Technology,  There are two principle means of monitoring the microbiological population of, the air, passive monitoring and active sampling. Both have a part to play, but, active sampling methods have become an essential environmental monitoring, tool, especially in the pharmaceutical and medical device sectors., Passive monitoring,  Passive monitoring is usually done using ‘settle plates’ – standard Petri dishes, containing appropriate (usually non-selective) culture media that are opened and, exposed for a given time and then incubated to allow visible colonies to develop, and be counted. Settle plates are very limited in their application since they are, only really capable of monitoring viable biological particles that sediment out of, the air and settle onto a surface over the time of exposure. They will not detect, smaller particles or droplets suspended in the air and they cannot sample specific, volumes of air, so the results are not quantitative.

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Active monitoring,  Active monitoring requires the use of a microbiological air sampler to, pHysically draw a known volume of air over, or through, a particle collection, device and there are two main types:, , 1) Impingers, •, , Impingers use a liquid medium for particle collection. Typically, sampled air is, drawn by a suction pump through a narrow inlet tube into a small flask, containing the collection medium., , •, , This accelerates the air towards the surface of the collection medium and the, flow rate is determined by the diameter of the inlet tube. When the air hits the, surface of the liquid, it changes direction abruptly and any suspended particles, are impinged into the collection liquid., , •, , Once the sampling is complete the collection liquid can be cultured to enumerate, viable microorganisms. Since the sample volume can be calculated using the, flow rate and sampling time, the result is quantitative, , 2) Impactors, •, , Impactor samplers use a solid or adhesive medium, such as agar, for particle, collection and are much more commonly used in commercial applications than, impingers, largely because of their convenience., , •, , In a typical impactor sampler air is drawn into a sampling head by a pump or, fan and accelerated, usually through a perforated plate (sieve samplers), or, through a narrow slit (slit samplers)., , •, , This produces laminar air flow onto the collection surface, often a standard agar, plate or contact plate filled with a suitable agar medium., , •, , The velocity of the air is determined by the diameter of the holes in sieve, samplers and the width of the slit in slit samplers., , •, , When the air hits the collection surface it makes a tangential change of direction, and any suspended particles are thrown out by inertia, impacting onto the, collection surface.

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•, , When the correct volume of air has been passed through the sampling head, the, agar plate can be removed and incubated directly without further treatment., , •, , After incubation, counting the number of visible colonies gives a direct, quantitative estimate of the number of colony forming units in the sampled air., ,  It is essential that air samplers are properly validated and regularly calibrated, to ensure accuracy. There are a number of points to consider:, , , Physical efficiency of the sampler – the relative efficiency of the sampler in, collecting particles over a range of sizes., , , , Biological efficiency – the relative efficiency of the sampler in collection of, microorganisms on a surface or in a liquid so that they are viable and can be, counted., , , , Validation of the instrument for its intended application and environment., , , , The flow rate of the sampler – with large sample sizes, the flow rate of air, through the sampling head is critical to the accuracy of the result., , Unit 5th, Indicators of Food microbial quality:,  The use of microorganisms and/or their products as quality indicators is, presented in Chapter 20, along with the use of coliforms and enterococci as, safety indicators. Indicator organisms may be employed to reflect the, microbiological quality of foods relative to product shelf life or their safety, from foodborne pathogens. In general, indicators are most often used to assess, food sanitation., , INDICATORS OF PRODUCT QUALITY:,  Microbial product quality or shelf-life indicators are organisms and/or their, metabolic products whose presence in given foods at certain levels may be used, to assess existing quality or, better, to predict product shelf life. When used in, this way, the indicator organisms should meet the following criteria:, , , They should be present and detectable in all foods whose quality (or lack

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thereof) is to be assessed., , , Their growth and numbers should have a direct negative correlation with, product quality., , , , They should be easily detected and enumerated and be clearly, distinguishable from other organisms., , , , They should be enumerable in a short period of time, ideally within a working, day., , , , Their growth should not be affected adversely by other components of the, food flora., ,  In effect, microbial quality indicators are spoilage organisms whose increasing, numbers result in loss of product quality. Total viable count methods have been, used to assess product quality. They are of greater value as indicators of the, existing state of given products than as predictors of shelf life because the, portion of the count represented by the ultimate spoilers is difficult to ascertain., Overall, microbial quality indicator organisms can be used for food products that, have a biota limited by processing parameters and conditions where an, undesirable state is associated consistently with a given level of specified, organisms., Organisms, Acetobacter spp. Fresh ciaer Bacillus spp., Byssochlamys spp., Clostridium spp., Flat-sour spores, Geotrichum spp., Lactic acid bacteria, Lactococcus lactis, Leuconostoc mesenteroides, Pectinatus cerevisiiphilus, "Pseudomonas Putrefaciens", Zygosaccharomyces bailii, , Products, Bread dough, Canned fruits, Hard cheeses, Canned vegetables, Fruit cannery, sanitation, Beers, wines, Raw milk (refrigerated), Sugar (during refinery), Beer; Butter, Yeasts Fruit juice, concentrates, Mayonnaise, salad dressing

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INDICATORS OF FOOD SAFETY:,  Microbial indicators are employed more often to assess food safety and sanitation, than quality. Ideally, a food safety indicator should meet certain important criteria., It should:, , • be easily and rapidly detectable, • be easily distinguishable from other members of the food biotech have a history, of constant association with the pathogen whose presence it is to indicate, , • always be present when the pathogen of concern is present, • be an organism whose numbers ideally should correlate with those of the, pathogen of concern, , • possess growth requirements and a growth rate equaling those of the pathogen, • have a die-off rate that at least parallels that of the pathogen and ideally persists, slightly longer than the pathogen of concern, , • be absent from foods that are free of the pathogen except perhaps at certain, minimum numbers In the historical use of safety indicators, however, the, pathogens of concern were assumed to be of intestinal origin, resulting from, either direct or indirect fecal contamination., ,  Thus, such sanitary indicators were used historically to detect fecal contamination of, waters and thereby the possible presence of intestinal pathogens., ,  The first fecal indicator was Escherichia coli., Metabolites, Cadaverine and putrescine, Diacetyl, Ethanol products, Histamine, Lactic acid, Trimethylamine (TMA), Total volatile bases (TVB), Total volatile nitrogen (TVN);, Volatile fatty acids, , Applicable Food Product, Vacuum-packaged beef, Frozen juice concentrate, Apple juice, fishery, Canned tuna, Canned vegetables, Fish, Seafood’s, Butter; cream

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Fig. Idealized relationship between an indicator organism and the relevant pathogen(s). The indicator, should exist in higher numbers than the pathogen during the existence of the latter., , PREDICTIVE MICROBIOLOGY/ MICROBIAL MODELING:,  The presence/absence of indicator organisms as noted above is used to predict, food safety. If a safety indicator is absent, the product is regarded as being safe, relative to the hazard for which the indicator is used. On the other hand, a, product can have extremely low numbers of a safety indicator and yet not pose a, hazard. The latter is true for many foodborne pathogens such as entero-toxigenic, staphylococci. When low numbers of an indicator or pathogen are present, it is, important to know how either will behave in a food product over time. This, future behavior calls into question the multiple parameters that affect the growth, and activity of microorganisms in foods, and if predictions are to be made about, the fate of low numbers of pathogens in a given product, how the pathogens and, these parameters interact needs to be handled.,  Microbial modeling or predictive microbiology is a rapidly emerging sub, discipline that entails the use of mathematical models/equations to predict the, growth and/or activity of a microorganism in a food product over time. The, predictive or modeling aspect is not new, for it is embodied in heat-process, calculations in the canning of low-acid foods under temperature effects,

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predicting the growth of an organism for a single parameter is not too difficult.,  Difficulty arises when multiple parameters are involved, as relatively few studies, have been conducted to determine their interplay on organisms The effective, application of predictive microbiology requires the selection of appropriate, models to reflect the effect of growth parameters. Among the many models that, have been proposed and tested are two kinetic models—the nonlinear Arrhenius, and Belehradek types. The former is applied with the dependent variable, expressed as in rate, whereas with the latter square-root model, the dependent, variable is expressed as V rate., , Foodborne Intoxication, 1. Clostridium Botulinum – Found,  Widely distributed in nature; soil and water on plants and intestinal tracts of, animals and fish. Grows only in little or no oxygen., Mode of Transmission:, •, , Bacteria produce a toxin that causes illness. Improperly canned foods, garlic, in oil, vacuum- packed and tightly wrapped food., , Symptoms:, , , Toxins affect the nervous system. Symptoms usually appear in 18 to 36 hours,, but can sometimes appear as few as four hours or as many as eight days after, eating. Double vision, droopy eyelids, trouble speaking and swallowing, and, difficulty breathing may occur. Can be fatal in three to 10 days if not treated., , 2. Clostridium Perfringens- Found,  Soil, dust, sewage, and intestinal tracts of animals and humans. Grows only in, little or no oxygen.

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Mode of Transmission:, •, , Also called "the cafeteria germ" because many outbreaks result from food left for, long periods in steam tables or at room temperature. Bacteria destroyed by cooking,, but some toxin-producing spores may survive., , Symptoms:, , , Diarrhea and gas pains may appear eight to 24 hours after eating; usually last, about 1 day, but less severe symptoms may persist for one to two weeks., , 3. Staphylococcus Aureus – Found:,  On the skin, infected cuts, pimples, noses, and throats., Mode of Transmission:, , • From people to food through improper food handling. Multiply rapidly at room, temperature to produce a toxin that causes illness., Symptoms:, , , Severe nausea, abdominal cramps, vomiting, and diarrhea can occur one to six hours, after eating; recovery within two to three days—longer if severe dehydration occurs., , Fecal Indicator bacteria:,  They are types of bacteria used to detect and estimate the level of fecal, contamination of water. They are not dangerous to human health but are used to, indicate the presence of a health risk.,  Each gram of human feces contains approximately ~100 billion (1×1011), bacteria.[1] These bacteria may include species of pathogenic bacteria, such as, Salmonella or Campylobacter, associated,  In, , addition,, , feces, , may, , with gastroenteritis., , contain pathogenic viruses, protozoa and

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parasites. Fecal material can enter the environment from many sources including, waste water treatment plants, livestock or poultry manure, sanitary landfills,, septic systems, sewage sludge, pets and wildlife.,  If sufficient quantities are ingested, fecal pathogens can cause disease. The, variety and often low concentrations of pathogens in environmental waters, makes them difficult to test for individually.,  Public agencies therefore use the presence of other more abundant and more, easily detected fecal bacteria as indicators of the presence of fecal, contamination., Types,  Commonly used indicator bacteria include total coliforms, or a subset of this, group, fecal coliforms, which are found in the intestinal tracts of warm blooded, animals.,  Total coliforms were used as fecal indicators by public agencies in the US as, early as the 1920s. These organisms can be identified based on the fact that they, all metabolize the sugar lactose, producing both acid and gas as byproducts.,  Fecal coliforms are more useful as indicators in recreational waters than total, coliforms which include species that are naturally found in plants and soil;, however, there are even some species of fecal coliforms that do not have a fecal, origin, such as Klebsiella pneumoniae.,  Perhaps the biggest drawback to using coliforms as indicators is that they can, grow in water under certain conditions.,  Escherichia coli (E. Coli) and enterococci are also used as indicators The, presence of fecal contamination., , , , Criteria for indicator organisms, Current methods of detection, o 1 Membrane filtration and culture on selective media, o 2 Fast detections using chromogenic substances, o 3 Application of antibodies, o 4 IMS/culture and other rapid culture-based methods, o 5 Gene sequence-based methods

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Water Quality Standard:, Drinking Water Quality Standard:,  World Health Organization Guidelines for Drinking Water Quality state that as, an indicator organism Escherichia coli provides conclusive evidence of recent, fecal pollution and should not be present in water meant for human consumption.,  In the U.S., the EPA Total Coliform Rule states that a water system is out of, compliance if more than 5 percent of its monthly water samples contain, coliforms., Recreational standards,  Early studies showed that individuals who swam in waters with geometric mean, coliform densities above 2300/100 ml for three days had higher illness rates.[4] In, the 1960s, these numbers were converted to fecal coliform concentrations, assuming 18 percent of total coliforms were fecal. Consequently, the National, Technical Advisory Committee in the US recommended the following standard, for recreational waters in 1968: 10 percent of total samples during any 30-day, period should not exceed 400 fecal coliforms/100 ml or a log mean of 200/100, ml (based on a minimum of 5 samples taken over not more than a 30-day, period).,  Despite criticism, EPA recommended this criterion again in 1976, however, the, Agency initiated numerous studies in the 1970s and 1980s to overcome the, weaknesses of the earlier studies. In 1986, EPA revised its bacteriological, ambient water quality criteria recommendations to include E. Coli and, enterococci., , PRINCIPLES UNDERLYING THE DESTRUCTION OF MICROORGANISMS, BY IRRADIATION:,  Several factors should be considered when the effects of radiation on microorganisms are, considered. These are discussed in the following subsections.

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Types of Organisms:,  Gram-positive bacteria are more resistant to irradiation than gram negatives. In general,, spore formers are more resistant than non-spore formers. Among spore formers, Paenibacillus larvae seem to possess a higher degree of resistance than most other aerobic, spore formers. Spores of Clostridium botulinum type A appear to be the most resistant, of all clostridial spores., Numbers of Organisms:,  The numbers of organisms have the same effect on the efficacy of radiations as in the, case of heat, chemical disinfection, and certain other phenomena: The larger the number, of cells, the less effective is a given dose., Composition of Suspending Menstrum (Food):,  Microorganisms in general are more sensitive to radiation when suspended in, buffer solutions than in protein-containing media., , Presence or Absence of Oxygen:,  The radiation resistance of microorganisms is greater in the absence of oxygen, than in its presence. Complete removal of oxygen from the cell suspension of, Escherichia co//has been reported to increase its radiation resistance up to, threefold. 56 The addition of reducing substances such as sulfhydryl compounds, generally has the same effect in increasing radiation resistance as an anaerobic, environment., Physical State of Food:,  The radiation resistance of dried cells is, in general, considerably higher than that for, moist cells. This is most likely a direct consequence of the radiolysis of water by, ionizing radiations, which is discussed later in this chapter. Radiation resistance of, frozen cells has been reported to be greater than that of non-frozen cells. Some of the

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researchers found that the lethal effects of gamma radiation decreased by 47% when, ground beef was irradiated at -196°C as compared to 00C., Age of Organisms:,  Bacteria tend to be most resistant to radiation in the lag phase just prior to active cell, division. The cells become more radiation sensitive as they enter and progress through, the log phase and reach their minimum at the end of this phase., , PROCESSING OF FOODS FOR IRRADIATION:,  Prior to being exposed to ionizing radiations, several processing steps must be carried, out in much the same manner as for the freezing or canning of foods., Selection of Foods:,  Foods to be irradiated should be carefully selected for freshness and overall desirable, quality.,  Especially to be avoided are foods that are already in incipient spoilage., Cleaning of Foods,  All visible debris and dirt should be removed. This will reduce the numbers of, microorganisms to be destroyed by the radiation treatment., Packing,  Foods to be irradiated should be packed in containers that will afford protection against, post irradiation contamination. Clear glass containers undergo color changes when, exposed to doses of radiation of around 10 kGy, and the subsequent color may be, undesirable., Blanching or Heat Treatment,  Sterilizing doses of radiation are insufficient to destroy the natural enzymes of foods. In, order to avoid undesirable post irradiation changes, it is necessary to destroy these, enzymes. The best method is a heat treatment that is, the blanching of vegetables and, mild heat treatment of meats prior to irradiation.

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RADAPPERTIZATION, RADICIDATION, AND RADURIZATION, OF FOODS:,  Initially, the destruction of microorganisms in foods by ionizing radiation was referred, to by terminology brought over from heat and chemical destruction of microorganisms., Although microorganisms can indeed be destroyed by chemicals, heat, and radiation,, there is, nevertheless, a lack of precision in the use of this terminology for radiationtreated foods. Consequently, in 1964 an international group of microbiologists, suggested the following terminology for radiation treatment of foods., 1. Radappertization is equivalent to radiation sterilization or "commercial sterility," as it, is understood in the canning industry. Typical levels of irradiation are 3(MK) kGy., 2. Radicidation is equivalent to pasteurization— of milk, for example. Specifically, it, refers to the reduction of the number of viable specific nonspore- forming pathogens,, other than viruses, so that none is detectable by any standard method. Typical levels to, achieve this process are 2.5-10 kGy., 3. Radurization may be considered equivalent to pasteurization. It refers to the, enhancement of the keeping quality of a food by causing substantial reduction in the, numbers of viable specific spoilage microbes by radiation. Common dose levels are, 0.75-2.5 kGy for fresh meats, poultry, seafood, fruits, vegetables, and cereal grains., , EFFECT OF IRRADIATION ON FOOD QUALITY:,  The undesirable changes that occur in certain irradiated foods may be caused directly by, irradiation or indirectly as a result of post irradiation reactions. Water undergoes, radiolysis when irradiated in the following manner: radiolysis, , 3H2O • H + OH + H2O2+ H2,  In addition, free radicals are formed along the path of the primary electron and react with, each other as diffusion occurs. Some of the products formed along the track escape and, can then react with solute molecules. By irradiating under anaerobic conditions, offflavors and off-odors are somewhat minimized due to the lack of oxygen to form, peroxides. One of the best ways to minimize off-flavors is to irradiate at subfreezing

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temperatures. The effect of subfreezing temperatures is to reduce or halt radiolysis and its, consequent reactants., ,  Other than water, proteins and other nitrogenous compounds appear to be the most, sensitive to irradiation effects in foods. The products of irradiation of amino acids,, peptides, and proteins depend on the radiation dose, temperature, amount of oxygen,, amount of moisture present, and other factors. The following are among the products, reported: NH3, hydrogen, CO2, H2S, amides, and carbonyls. With respect to amino, acids, the aromatics tend to be more sensitive than the others and undergo changes in, ring structure. Among the most sensitive to irradiation are methionine, cysteine,, histidine, arginine, and tyrosine. The amino acid most susceptible to electron- beam, irradiation is cystine; Johnson and Moser32 reported that about 50% of this amino acid, was lost when ground beef was irradiated. Tryptophan suffered a 10% loss, whereas, little or no destruction of the other amino acids occurred. Amino acids have been, reported to be more stable to gamma irradiation than to electron- beam irradiation. The, irradiation of lipids and fats results in the production of carbonyls and other oxidation, products such as peroxides, especially if irradiation and/or subsequent storage take place, in the presence of oxygen. The most noticeable organoleptic effect of lipid irradiation in, air is the development of rancidity. It has been observed that high levels of irradiation, lead to the production of "irradiation odors" in certain foods, especially meats. In, addition to flavor and odor changes produced in certain foods by irradiation, certain, detrimental effects have been reported for irradiated fruits and vegetables. One of the, most serious is the softening of these products caused by the irradiation-degradation of, pectin and cellulose, the structural polysaccharides of plants In green lemons, however,, ethylene synthesis is stimulated upon irradiation, resulting in a faster ripening than in, controls. Among radiolytic products that develop upon irradiation are some that are, antibacterial when exposed in culture media., , STORAGE STABILITY OF IRRADIATED FOODS:,  Foods subjected to radappertization doses of ionizing radiation may be expected to be as, shelf stable as commercially heat-sterilized foods.

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 There are, however, two differences between foods processed by these two methods that, affect storage stability:, , • Radappertization does not destroy inherent enzymes, which may continue to act,, and some post irradiation changes may be expected to occur. Employing 45 kGy, and enzyme- inactivated chicken, bacon, and fresh and barbecued pork,, Heiligman30 found the products to be acceptable after storage for up to 24, months. Those stored at 700F were more acceptable than those stored at 1000F., The effect of irradiation on beefsteak, ground beef, and pork sausage held at, refrigerator temperatures for 12 years these foods were packed with flavor, preservatives the appearance of the meats as excellent after 12 years of storage., A slight irradiation odor was perceptible but was not considered objectionable., The meats were reported to have a sharp, bitter taste, which was presumed to be, caused by the crystallization of the amino acid tyrosine. The free amino nitrogen, content of the beefsteak was 75 and 175 mg %, respectively, before and after, irradiation storage, and 67 and 160 mg % before and after storage, respectively,, for hamburger., , •, , Foods subjected to radurization ultimately undergo spoilage from the surviving, biota if stored at temperatures suitable for growth of the organisms in question., The normal spoilage biota of seafoods is so sensitive to ionizing radiations that, 99% of the total biota of these products is generally destroyed by doses on the, order of 2.5 kGy. Ultimate spoilage of radurized products is the property of the, few microorganisms that survive the radiation treatment., , HIGH-PRESSURE PROCESSING:,  The use of high-pressure processing (HPP) or Pascalization to reduce or destroy, microorganisms in foods dates back to 1884. In 1899, Hite successfully used hydrostatic, pressures to improve the keeping quality of milk, and in 1914 he demonstrated the, susceptibility of fruit borne organisms to hydrostatic pressures., ,  Thus, the utility of this process to control microorganisms and preserve foods has a long, history but only recently has it received much study.

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 The current interest is due apparently to consumer demands for minimally processed, foods, and to the lower cost and greater availability of processing equipment. HHP, treatments may be applied at room temperature, and with the exception of some, vegetables, shape, color, and nutrients of most foods are not affected., ,  About 10 HPP-treated foods, including fruit purees, jams, fruit juices, and rich cakes,, have been commercially available in Japan since the early 1990s. To carry out HPP,, high-hydrostatic pressures (HHP) are used, and one needs a suitable mechanical, chamber (steel cylinder) and pressure pumps to generate pressures of several hundred, megaPascal (MPa) (1 MPa = 10 atm; 100 MPa = 1 kbar)., ,  Come-up and come-down times for pressure are important, and rates of 2-3 MPa/s are, not uncommon. After the food is placed in suitable containers and sealed, the food, packs are placed in the cylinder containing a low-compressibility liquid such as water., ,  Pressure is generated with a pump, and it may be applied continuously (static) or in an, oscillatory manner. For the latter, two to four pressure cycles may be applied with, varying holding periods for each cycle. In a study on the inactivation of, Zygosaccharomyces bailii, continuous and oscillatory treatments were compared and, the latter was found to be the more effective., , PULSED ELECTRIC FIELDS:,  This physical method consists of the application of short pulses (microseconds) of high, electric fields to foods placed between two electrodes. It is a non-thermal process, similar in this regard to HHP described above.,  The lethal effect is essentially a function of pulse intensity, pulse width, and pulse, repetition rate. Pulsed electric field (PEF) generation requires a pulsed power supply, and a treatment chamber in one study, a 99% decrease in E. coli numbers was produced, by square-wave after 100 microseconds at 7°C compared to 93% by the exponentially, decaying method.,  Among the general properties and features of PEF as applied to foods are the following:, , • Gram-negative bacterial cells are more sensitive than gram positives or yeasts., • Vegetative cells are more sensitive than spores.

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• Microbial cells are more sensitive in the log phase of growth than in the stationary, phase., , • Cell death by PEF appears to be due to disruption of cell membrane function and, by electroporation (production of pores in membranes by the electric current). It, has been suggested that bacterial inactivation by PEF may be an "all or nothing", event since sublethal injury could not be detected., , • Overall, the antimicrobial effects of PEF are functions of electric field strength,, treatment time, and treatment temperature, with cells being more sensitive when, treated at higher temperatures., , , , In a study that compared PEF with HHP and heat for controlling ascospores of Z bailii, in fruit juices, two pulses of 32 to 36.5 kV/cm reduced vegetative cells 4.5 to 5 and, ascospores 3.5 to 4 log cycles, and this compared with a nearly 5- log reduction of, vegetative cells by HHP but only a 0.5 to 1 log reduction of ascospores with a 5- minute, treatment at 300 MPa.32 Overall, two pulses of 32 to 36.5 kV/cm reduced vegetative, cells or ascospores 3.5 to 5 log cycles for each of the five juices tested. The ascospores, were five to eight times more heat resistant than the vegetative cells., , ASEPTIC PACKAGING,  In traditional canning methods, non-sterile food is placed in non-sterile metal or glass, containers, followed by container closure and sterilization.,  In aseptic packaging, sterile food under aseptic conditions is placed in sterile containers,, and the packages are sealed under aseptic conditions as well.,  Food & Drug Administration approved the use of hydrogen peroxide for the sterilization, of flexible multilayered packaging materials used in aseptic processing systems.,  In general, any food that can be pumped through a heat exchanger can be aseptically, packaged. The widest application has been to liquids Treatment Time is Log10 CFU/ml, such as fruit juices, and a wide variety of single serve products of this type has resulted.,  The technology for foods that contain particulates has been more difficult to develop,, with microbiological considerations only one of the many problems to overcome.,  In determining the sterilization process for foods pumped through heat exchangers, the, fastest-moving components (those with the minimum holding time) are used, and where

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liquids and particulates are mixed, the latter will be the slower moving.,  Heat penetration rates are not similar for liquids and solids, making it more difficult to, establish minimum process requirements that will effectively destroy both organisms and, food enzymes. Some of the advantages of aseptic packaging are as follows:, o Products such as fruit juices are more flavorful and lack the metallic taste of, those processed in metal containers., o Flexible multilayered cartons can be used instead of glass or metal containers., o The time a product is subjected to high temperatures is minimized when, ultrahigh temperatures are used., o The technology allows the use of membrane filtration of certain liquids., o Various container headspace gases such as nitrogen may be used.,  Among the disadvantages are that packages may not be equivalent to glass or metal, containers in preventing the permeation of oxygen, and the output is lower than that for, solid containers. The spoilage of aseptically packaged foods differs from foods in, metal containers. Whereas hydrogen swells occur in high-acid foods in the latter, containers, aseptic packaging materials are nonmetallic. Seam leakage may be, expected to be absent in aseptically packaged foods, but the permeation of oxygen by, the nonmetal and non-glass containers may allow for other types of spoilage in lowacid foods., , MANOTHERMOSONICATION(THERMOULTRASONICATION),  When bacterial spores are simultaneously exposed to ultrasonic waves and heat, there is, a reduction in spore resistance.,  The effect is greatest when the two treatments are simultaneous, although some, reduction in resistance occurs when ultrasonication is carried out just before heating.,  This phenomenon has been studied by workers in Spain and designated, manothermosonication (MTS) or thermoultrasonication.,  In addition to spores, MTS has been shown to be effective in reducing the thermal, resistance of the enzymes peroxidase, lipooxygenase, and polypHenol oxidase.,  In an early study of the effect of MTS on heat resistance using quarter-strength Ringer's

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solution, the D value at 1100C of a B. cereus strain was reduced from 11.5 to ~1.5, minutes, and that of a B. licheniformis strain from D value at 99°C to 3 minutes.,  In a later study using whole milk and two strains of B. subtilis, D values at 10O0C were, reduced from 2.59 to 1.60 for one strain, and from 11.30 to 1.82 minutes in another., Comparable z values were 9.12-9.37 and 6.72-6.31, respectively.,  Ultrasonication was carried out at 20 kHz and 150 W. The z value results seem to, confirm the minimal effect that MTS has on z values.