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Due to the globalization of the food supply there has been a need to strengthen food safety systems in and between the countries. In this regard the World Health Organization (WHO) has promoted efforts in improving the food safety from farm to plate which started on World Health Day, 7 April 2015. The World Health Day 2015 slogan was “From farm to plate, make food safe”. The informal food production and marketing system is still strong in most countries of the South-East Asia Region, for the enforcement of food safety regulations. Traditionally, the preferences have been for hot and well-cooked food. These habits are partially responsible for preventing foodborne infections. In many countries street food has become very popular as it is cheap. Hygienic conditions are improving, provided that potable water supply and clean facilities are ensured by municipal authorities. Many waterborne and diarrhoeal diseases could be prevented with the introduction of bottled drinking water. The awareness and education on food safety by the consumers has strengthened in the enforcement of food standards. This has improved the hygienic practices and has helped in the prevention of foodborne illnesses. The “WHO Five keys to safer food” serves as the basis for educational programmes to train food handlers and educate the consumers. These help in preventing the foodborne illnesses. The important points are to keep food surfaces clean, washing of all utensils, plates and cutlery, keeping the raw food separate from the cooked food. Cooking food thoroughly to the appropriate temperature, keeping food at safe temperatures, both for serving and storage and using safe water and raw materials.

Even after public concern the scientists feel that the GMO foods are quite safe. According to them the genetically-engineered crops are very safe to eat as much as the non-GE of the same crop. According to them the environment has improved by reducing the use of pesticides. GMO stands for genetically modified organism. Soy, corn, or other crops are very common as GM crops which are made by genetically engineered DNA. Many have come to the conclusion that GM foods are safe, healthy and sustainable. However, some people are against them. There are many benefits of GM crops like less water or pesticides to be used. Genetically engineered crops fight diseases to save the foods. At the same time the industries/ farmers that grow them are also benefitted. These foods have higher nutritional profiles, both in the US and abroad like corn which is one of the most prominent GMO foods. Soy is found in tofu, vegetarian products, soybean oil, soy flour and many other products. The other major crops taken for genetic modification are Papayas, Canola and Cotton. For dairy products a lot of work is being done on the genetic engineering of animals in a big way. This is going to be the most intractable environmental challenge of the 21^st Century. Presently, around 92% of the U. S. corn (GE), 94% of soybeans and 94% of cotton are genetically engineered.

When certain disease-causing bacteria or pathogens contaminate food, they can cause foodborne illness, often called “food poisoning”. Foods that are contaminated may not look, taste or smell any different from foods that are safe to eat. Salmonella, Campylobacter, Listeria and Escherichia coli are common bacteria causing foodborne illness. Some foodborne illnesses are mild where most people get better within a few days, but illness can sometimes be more severe and fatal. Bacillus cereus can form heat resistant spores. On cooking the food with these spores the heat resistant toxin is left at room temperature. Reheating or lightly cooking the food does not destroy these toxins. Most types of foodborne illnesses cause symptoms like nausea, vomiting, watery diarrhea, abdominal pain and cramps. Fever and/or the presence of blood or mucus in the stool are signs of bacterial infection that may require a medical consultation. Proper food handling is vital to prevent most foodborne illnesses and diseases. WHO has given keys towards safer food which should be followed. Unsafe food containing harmful bacteria, viruses, parasites or chemical substances, causes more than 200 diseases which range from diarrhoea to cancers. Foodborne and waterborne diarrhoeal diseases kill around 2 million people worldwide annually, including many children. A single foodborne bacterium can grow into more than two million bacteria in just seven hours under the right conditions. Foodborne pathogen called Listeria sp. can survive and sometimes grow on foods being stored in the refrigerator. Hepatitis A virus can cause long-lasting liver disease and spreads typically through raw or undercooked seafood or contaminated raw produce. It is estimated that more than 250 million people worldwide suffer food allergies. The boiling temperature (smoke point) of cooking oil is above 200 °C. The boiling point of water is 100 °C.

According to the World Health Day (WHO,2015) food contamination happens when food are corrupted with another substance. It can happen during the process of production, transportation, packaging, storage, sales and cooking process. The contamination can be physical, chemical and biological. Faulde et al., (2013) studied the infection caused to patients by mothfly

Food adulteration is an unethical and often criminal malpractice. It is very common in countries of the South-East Asia region. It frequently occurs where informal food production and marketing services are predominant and enforcement of food regulation is weak. The prohibited substances are either added or used to partly or wholly substitute healthy ingredients. This creates the impression of freshness in old food artificially. Adulteration in food is usually done for financial gain. The high target is spices due to the demand and also their high price. Sometimes it can happen due to lack of proper hygienic conditions of processing, storage, transportation and marketing. The consumer is either cheated financially or becomes a victim of illness or disease. In case, the consumers take a lot of precautions at the time of purchase of such products it would help them in avoiding the purchase of such adulterated food. The whole spices: Dirt, dust, other seeds; chili powder: brick powder, salt powder or talc, powder. In ghee/butter: vegetable ghee, animal fat, mashed potato, sweet potato, etc. ; ice cream and beverages:saccharin; honey: jaggery, sugar syrup. Food contaminants are not added to the foods. They are present in foods as a result of the production (crop farming, animal husbandry and aquaculture), manufacture, processing, preparation, treatment, packing, packaging, transport or holding of such food or as a result of environmental contamination. Usually it includes the presence of harmful chemicals or microorganisms in food, which if consumed can cause several debilitating illnesses. Many times, unknowingly, the contamination may occur during the production, processing, storage and marketing.

In this century the usage of plastics had become very common. However, it is important to know as to what kinds of plastics are used for food handling and storage and the health hazards caused by using it. It has been observed that plastic packaging plays a significant role for the enhanced shelf life and in the ease of storage and cooking for many foods. Some kinds of plastic materials which are widely used for handling and storage of food and water include polyethylene terephthalate (PET). It is used in soft drinks, water, sport drinks, ketchups, salad dressing bottles, peanut butter, pickle, jelly and jam jars. This plastic is strong, heat resistant, resistant to gases and acidic foods. It can be transparent or opaque. It does not leach any chemicals that are suspected of causing cancer or disrupting hormones and can also be recycled.

Usage of High density polyethylene (HDPE) has been found to be common in milk, water, juice bottles, yogurt, margarine tubs, grocery, trash and retail bags. High-density polyethylene is stiff and strong but is not heat stable (it melts at a low temperature). It does not leach any chemicals that have a role in causing cancer or disrupting the hormones. HDPE can be recycled. Low-density polyethylene (LDPE) is used into making films of various types, some bread and frozen food bags and also in the making of squeezable bottles. Low-density polyethylene is relatively transparent. Many of the films are not heat stable either and may melt the food in case it is touching it. However, polypropylene (PP) is more heat resistant, harder, denser and more transparent than polyethylene and is therefore used for heat-resistant microwavable packaging and sauce or salad dressing bottles. Polycarbonate is clear and heat resistant. It is also durable and often used to make refillable water bottles and sterilisable baby bottles, microwave ovenware, eating utensils, plastic coating for metal cans. Tiny amounts of bisphenol A are formed when polycarbonate bottles are washed with harsh detergents or bleach (sodium hypochlorite). At high levels of exposure, bisphenol A is potentially hazardous as it mimics the female hormone estrogen. In addition, polystyrene (PS) and polyvinyl chloride (PVC) are also used during food material transportation and handling in supermarkets. Modern food safe plastic bags are plasticizer-free which do not leach harmful chemicals into the food during cooking.

Today, organic and local foods are preferred as they use less pesticides or fertilizers. However, these foods, like others, can be exposed to harmful bacteria during the growing and harvesting process. The farmers and distributors must use good sanitary practices to minimize food contamination. Consumers should cook food properly. The street food vendors are a traditional and indigenous fast food approaching in most countries of the South-East Asia Region. As these are economical they cater to millions of consumers. Among safe street foods there are many contributing factors that are associated with the safety of street food. WHO had conducted a survey in 1993 covering 100 street foods in 100 countries around the world. It revealed that the major health threat comes from raw and undercooked food, infected food handlers and inadequate hygiene practices in processing and storing such food. If the street food is cooked well and is served hot, then the chances of food poisoning is less. The water quality, hygienic conditions and the level of cleanliness especially in the summer season are the major factors for food poisoning. However improvements in physical infrastructure and hygienic conditions are growing as consumers are also demanding quality food. Michael et al., (2017) have looked into organic versus conventional foods.

According to Futter (2001) it is very important the way we cook our food. If the cooking is inadequate it causes food poisoning. While cooking there can be cross contamination starting from raw foods. It includes hands or utensils which can cause food poisoning. Most foods, especially meat, poultry, fish and eggs should be cooked thoroughly to kill most types of food poisoning bacteria. Generally the food should be cooked to a temperature of 75 °C or hotter. When food is cooked, it should be eaten promptly, kept hotter than 60 °C or cooled, covered and stored in the fridge or freezer. Some people are more at risk from food poisoning than others. In order to buy safe food in the market like buying pre-cut produce as a half a watermelon or cut veggies from a salad bar, it is important to choose items that are refrigerated or surrounded by ice. They must be kept in separate bags. FDA (2019) has studied the contamination in lettuce. Shopping at a farmer’s market is a great way to get locally-grown, fresh fruit, vegetables, and other foods. However, safety of these produce needs to be checked. Many markets have their own food safety rules and vendors must comply with them along with the government regulations. Fruits and vegetables must be washed thoroughly under running water just before eating, cutting or cooking. Fruits and vegetables must never be washed with soap or detergent or using commercial produce washes. Even if it has to be peeled before eating, it is still important to wash it first. Any bacteria present on the outside of items like melons can be transferred to the inside when it is cut or peeled. They should be refrigerated within two hours after preparation. The juice or cider must be treated (pasteurized) to kill harmful bacteria. Pregnant women, children, older adults and people with weak immune systems should drink only pasteurized or treated juice. Raw milk can harbor dangerous microorganisms like Salmonella, E. coli, and Listeria. World India (2017) has looked into the food regulations. These can pose serious health risks. Pregnant women, older adults and people with weakened immune systems are at higher risk for illness caused by Listeria. One source for these bacteria is soft cheese made from unpasteurized milk. It is vital to check the label to make sure that it is made from pasteurized or treated milk. Make sure that eggs are properly chilled at the market. FDA requires that untreated shell eggs must be stored and displayed at 45°F. While buying eggs it needs to be ensured that the eggs are clean and the shells are not cracked. Meat should be properly chilled even in the market and kept in closed coolers with adequate amounts of ice to maintain cool temperatures. If it is brought in an insulated bag or cooler during transit it is very beneficial to keep the quality. It should be always kept separately from your other purchases, so that the juices from raw meat (which may contain harmful bacteria) do not come in contact with produce and other foods. Essays (2018) have written a very comprehensive overview of food safety.

It is a discipline of science that describes handling, preparation and storage of food that can prevent food-borne diseases. When there are two or more cases of a similar illness from the ingestion of the same common food it is known as a food-borne disease outbreak (FSN, 2018). In order to avoid potential health hazards, food safety is very important. This needs safety between industry, market and the consumer. For food safety the labelling on food material is vital. This includes the origin of food, steps taken for food hygiene, food additives, pesticide residues, policies on biotechnology, food guidelines for the management of governmental import, export inspection and certification systems for foods. The food should be safe in the market with safe preparation and delivery of the food for the consumer. Foegeding et al., (1996) have done an assessment of risk factors with food borne pathogens.Warnock et al., (2007) have reported intervention to food safety.

When pathogens are in the food they may be transmitted to the consumer. It may result in an illness or death of the person or other animals. The pathogens include bacteria, viruses, molds and fungus. These multiply in the food medium. Joon et al., (2012) have found pathogenic bacteria in meat products of Korea. The developed countries have high intricate standards for the preparation of food; however, in the developing countries there are less standards and enforcement of the standards which are followed. Apart from food another major source of infection is water, which is very critical in spreading many diseases. It is possible to prevent food poisoning if good water is used for all purposes. However, it becomes difficult as in the food supply chain a large number of persons are involved apart from the fact that pathogens can be introduced into foods no matter how much precautions are taken. Cowden (2000) has studied the notification on food poisoning to improve health. Frenkel et al., (2015) studied the amount of wastes from food processing industries.

Human beings have been concerned about the concept of food safety since time immorial. Around the globe there has been major improvement in food safety management in the last twenty years. Looking into the progress made on the ways to make food safe and how to make them free from pathogens, this book, will explain food hygiene, the journey of research been taken in food safety till date and the challenges that we are going to face in future to ensure food safety and its wholesomeness. It also includes the role and responsibilities of the various sectors of society, namely governments, food industry, consumers and academia.

In the beginning of the twentieth century many modern food safety policies have been established. In the packing of meat and in food processing industries a lot of work has been done (Sinclair 1906). Another second generation policy includes the outbreak in the United States in early 1990s’ of pathogen E. coli O157:H7 and the BSE scandal of the late 1990s in the UK Lopman et al., (2003) have discussed on viral outbreak in europe. Currently, the crisis has come due to the economic and technological transformations which have taken place in food and the food supply system. As these changes have many health risks the public is very concerned. Therefore, the control forms of safety regulation and food safety laws have come into the picture. These policies include continuous line inspection, visual product inspection and detailed specification of approved hygiene practices. There is a need of a public health-focused policy which works towards the preventive integrated management of foodborne hazards from farm to table. This includes the use of modern science based risk management tools which will increase the efficiency accurately towards public health. A lot needs to be worked out for better implications. Current efforts to modernize food safety policy around the world are being worked out by the use of risk analysis and cost-benefit analysis in public administration and also the quality management in industry. The role of public economics plays an important role in food safety policies. For the social benefits the policy makers need to take decision on the costs and strengthening of the risk assessment. Today there are globalized markets and this can influence the health globally. Emerging economies like China are industrializing and urbanizing rapidly like that of Europe and North America.

Fermentation

The term fermentation is loosely used to denote any process in which microorganisms are involved, regards of whether or not the organism is growing in the presence of molecular oxygen. Thus, it can be defined as 'an ATP generating metabolic process in which organic compounds serve both as electron donor (becoming oxidized) and electron acceptor(becoming reduced). Energetically, fermentations are poor energy yielding process and products have lower energy content then the substrates. Since there is no net oxidation in fermentation, the number of molecules of C, H and O remain the same in products as in the substrate.

14.1 Culture from Urine

Infection of urinary tract due to microorganisms affect patient of all age group and both sex., and they vary in severity from an unsuspected infection to a condition of severe systemic disease. Urine is a ordinarily excellent culture medium for the multiplication of common pathogen of urinary tract. Most bacteria found in the urine are due to contamination of skin flora during passage and they tend to increase greatly, exceeding 10⁶/ml. When the bacterial count exceeds 10⁵ per ml of urine it indicates significant bacteriuria and is indicative of a urinary tract infection. Infection of the urinary tract may be limited to a single tissue/ organ or they may spread upward and involve the entire urinary system. Escherichia coli is the most common urinary tract pathogen. Pseudomonas aeroginosa causes another important urinary tract infection, which is characterized by blue green pus. Cystitis (inflammation of urinary bladder), Pyelonephritis (inflammation of the kidney) are permanent diseases of urinary tract.

Urinary tract infections are generally characterized by bacteriuria, bacteria in the urine. Quantitative microbiological assay of urine can identify the probable pathogen. For this purpose, calibrated loop- direct streak method and pour plate method are routinely used. Now-a-days commercially prepared special plastic device called the urine dip slide and diagnostic urine–culture called Bacturcult is being used for isolation and identification of microorganisms from genitourinary tract infections. The Bacturcult is a sterile disposable plastic tube coated with a special medium on its inner side for detection and identification of bacteriuria.

With increasing industrialization, water sources available for consumption as potable water are being adulterated with industrial, animal and human wastes. As a result water has become a formidable factor in disease transmission. Polluted water usually contains large amount of organic matter which forms nutritional base for growth and multiplication of microorganisms. Usually the water with non-pathogenic organisms, like autotrophs and saprophytic heterotrophs, are not of major concern, but presence of intestinal contaminants of fecal origin are of great concern. These pathogens are responsible for intestinal infection such as bacillary dysentery, typhoid fever, cholera and paratyphoid fever.

The detection of all the intestinal pathogens on a routine basis would be a tedious and difficult task, however, it would be easy to demonstrate the presence of some of the nonpathogenic intestinal organisms like Escherichia coli and Streptococcus faecalis. These are always found in intestine but their detection confirms the contamination of water with fecal material. Such organisms are also called pollution indicator.

Viruses are smaller than bacteria and require living cell for their growth. They can be detected through the bacteria to which they parasitise. Viruses are associated with both eukaryotes as well as prokaryotes and contain either DNA or RNA; only few have both DNA and RNA.

Viruses that infect bacteria are called bacteriophage. They consist of head or capsid and a tail. The head may be round, oval, polyhedral and is made up of proteins while tail is hollow and provides an exit for nucleic acid to enter in bacterial cell. The tail also helps in attachment to the cell wall surface of bacteria. After injection of the nucleic acid into bacteria they multiply and form large number of bacteriophage particles which cause lysis of the bacterial cell wall releasing many bacteriophages. Phages that cause lysis are known as lytic phages and that do not cause lysis are called temperate phage. The most common lytic phage is E.coli – a coliphage.

Identification of enteric bacilli is of prime importance for controlling intestinal infections. This can be achieved by preventing contamination of food and water by a group of bacteria belonging to family – Enterobacteriaceae which are found in the intestinal tract of human beings and lower mammals. The microorganisms of this family are short, gram negative, non spore forming bacilli. Such common enteric bacteria are a) pathogens such as species of Salmonella and Shigella b) occasional pathogens like species of Proteus and Klebsiella and c) normal intestinal flora like species of Escherichia and Enterobactor.

10.1 Transformation of E. coli by electroporation Principle

Preparation of electro-competent bacteria is considerably easier than preparing cells for transformation by chemical methods. Bacteria are simply grown to mid –log phase, chilled, centrifuged, washed extensively with ice cold buffer containing 10% glycerol. DNA may be introduced immediately into the bacteria by exposing them to a short high voltage electrical discharge. Alternatively, the cell suspension may be snap frozen and stored at-70^oC for 6 months before electroporation, without loss of transforming efficiency.

Since E.coli cells are small, they require very high strengths (12.5- 15KV cm^-1) for electroporation as compared to those used to introduce DNA into eukaryotic cells. Electroporation is temperature dependent and is best carried out at 0-4°C.

The highest efficiency of transformation is obtained when the concentration of input DNA is high (1-10 μg/ml) and when the length and intensity of the electrical pulse are such that only 30-50% of the cells survive in the procedure. Under these conditions, as many as 80% of the surviving cells may be transformed. Higher frequencies of transformation (colonies/ molecules input DNA) are obtained when the DNA concentration is low (?10pg/ml).

The method outlined in this protocol is followed with most strains of E.coli and with plasmids <15kb in size. However, substantial variation in the efficiency of transformation between the strains of E. coli. has been reported. As in case with chemical transformation, linear plasmid DNAs introduced into E.coli by a pulsed electrical discharge transform very efficiently from 10 to 1000 fold but less efficiently than the corresponding closed circular DNA – perhaps because the exposed terminal of linear DNA are susceptible to attack by intracellular nucleases.

Antibiotics are produced by some true bacteria, actinomycetes and fungi that destroy or inhibit the growth of other microorganisms. Some antibiotics are synthesized or modified in the laboratory; however, their origins are always in living cells.

9.1 Assay of Antibiotics

Soil serves as the major repository of microorganisms some of which produces antibiotics. Four groups of soil microorganisms viz; Streptomyces, Bacillus, Penicillium and Cephalosporium represent three microbial types, namely, actinomycetes, true bacteria and molds respectively.

Following procedure may be applied to isolate antibiotic-producing microorganisms from soil:

Our exposure to wide variety of chemicals has increased markedly in the recent past. Most of these compounds are mutagenic as well as potential carcinogens. Emphasis is being laid to develop method for detection of environmental carcinogens so that their use can be avoided.

Despite the fact that mammalian cell structure and human enzymatic pathways differ from those in bacteria, the chemical nature of DNA is common to all organisms; this permits the use of bacterial test systems for rapid detection of possible mutagens.

The Ames test which was developed by Bruce Ames in 1970, at the University of California, U.S.A. is a mutational reversion assay employing several special strains of Salmonella typhimurium, each of which has a different mutation in the amino acid histidine biosynthesis operon.

S. typhimurium, the indicator organism, used in this test is the mutant strain that cannot synthesize histidine (His^-), lack the enzyme to repair DNA so that mutation shows up readily and has leaky cell wall that permit the rapid entry of chemicals. In addition, this S. typhimurium strain carries mutation enhanced plasmid, the combined effect of these characters increase the sensitivity of the organism to mutagenic treatments.

The term growth for bacteria and other microorganism usually refers to changes in the total population rather than an increase in size or mass of an individual organism. As the mechanism of conservation and dissipation of heat generated during metabolism lacks in bacteria, the enzyme system in bacteria gets directly influenced by temperature. The purpose of studying the bacterial growth curve is a) to become familiar with the population growth dynamics of bacterial culture and b) to plot a growth curve and determine the generation time of bacterial culture.

A number of methods are now available for measuring bacterial growth. Some important ones are

1)By colony counting or cell counting

2)By weighing the cells

3)By cell activity (turbidity method)

The science dealing with the measurement of size of any microscopically visible microorganism is known as micrometry which is based on the principle of physics. Before measuring a cell, the diameter of the microscopic field must be established by means of optic devices, namely an ocular micrometer and a stage micrometer.

a)Ocular micrometer : It is a circular disc that is placed in the eyepiece. The disc is graduated in to several small divisions marked from 0 to 100.It is placed inside the eyepiece of the microscope. The distance varies according to the objective of the microscope which can be easily determined by using a stage micrometer.

b)Stage micrometer : It is a glass slide graduated in 1 mm. Again 1mm is divided into 10 large divisions and 100 small divisions (10 x 10 = 100 divisions). These divisions are equally placed therefore 1 small division is equal to 0.01 mm or 10 μm.

Generally microorganisms are so small and colourless in their natural state that it becomes difficult to observe them clearly under microscope. Stains and dyes, which are colouring agents, are used by biologist for better understanding of the morphological and anatomical structures of the organisms under study. Stains are prepared with great care and specification to get satisfactory result, while dyes are not as exacting in their characteristics.

Dyes are of two types, natural and synthetic. The natural dyes are used for histological purposes, whereas the synthetic dyes for bacterial stain. Usually dyes which are derivatives of cyclic compound benzene of benzole, are prepared from one or more substances found in coal-tar, and so are called coal-tar dyes. Chemically dyes are organic compounds containing both chromophore and auxochrome groups linked to benzene rings. Chromophore group provides the compounds the property of colour and thus benzene containing chromophores are called chromogens. It has no affinity to combine with fibres or tissue and as such not called dye. For being a dye, compound should contain an auxillary group which provides the compounds the properties of electrolytic dissociation.

The dyes may be acidic, basic or neutral. The acidic dyes are eosin, acid fuchsine and picric acid whereas the basic dyes are safranin, methylene blue, crystal violet etc. The acidic dyes ionize to give the dye portion of molecule a negative electrical charge. They are salts of sodium, potassium, calcium, magnesium etc. The basic dyes ionize to give the dye portion of molecule a positive electrical charge and are salts of chlorides, sulphates or acetates.