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Proteins are composed of amino acids and are large molecules that play a crucial role in various biological processes. They are considered the fundamental building blocks of life due to their abundance in the body, constituting approximately 60% of the dry weight of cells. These complex biomolecules are intricately folded and twisted strands of amino acids, actively participating in vital life processes, including metabolism, locomotion, immune response intercellular signaling, and molecular identification. As macronutrients proteins are present in all living organisms and directly contribute to numerous metabolic pathways. It is important to note that proteins are unique to each organism, exhibit species specificity, and vary within different organs of the same organism. With 22 different amino acids, the properties of a protein molecule are determined by the specific arrangement of these amino acids. While plants can synthesize all the necessary amino acids for protein production, animals rely on external sources for certain amino acids. Proteins consist of amino acids connected through peptide bonds, formed by the linkage between the amino (NH2 ) group of one amino acid and the carboxyl (COOH) group of another amino acid.

Amino acids are organic molecules made up of carbon (C), hydrogen (H), oxygen (O), and nitrogen (N). However, sulfur (S) and selenium (Se) are also found in some cases. Amino acids are essential organic compounds that come together to create proteins, thus making them the fundamental building blocks of proteins. These biomolecules play vital roles in various biological and chemical processes within the human body and are essential for the growth and development of an individual.

Amino acids consist of an acidic group (-COOH), a basic group (-NH2 ), and a side chain (R) or a hydrogen (H) atom, all bonded to the carbon atom. The features of amino acids are due to the side chain because they differ only in their side chain. All amino acids have the same basic structure except proline, which has a cyclic structure.

Various forms of amino acids like alpha (α), beta (β), gamma (γ) or delta (σ) form are found. The position of the amino group (-NH2 ) determines the form of the amino acid, i.e., if the NH2 group is attached to the α-carbon, it is called α-amino acid; if β-carbon, it is called β-amino acid, and so on. Mostly, α-Amino acids are found as the numbering of amino acids is done from the carboxylic group (-COOH).

Lipids are a class of organic compounds composed of hydrogen (H), carbon (C), and oxygen (O) atoms. These classes of biomolecules come together to create the fundamental building blocks that shape and govern the structure and functionality of living cells. The term lipid was used by the German biochemist Wilhem Bloor in 1943. Lipids are heterogeneous groups of substances that are insoluble in polar (water) and soluble in non-polar (ether, benzene, and chloroform) solvents. Lipids are the esters of fatty acids and their derivatives. Lipids play a crucial role in both the plant and animal domains. They are widely distributed in plants, but lipids are primarily stored in animal adipose tissues.

Classification of lipids

Lipids are classified into four different groups, as shown below:

1. Simple lipids

2. Complex lipids

3. Derived lipids and

4. Miscellaneous lipids

Carbohydrates are the macronutrients consisting of carbon (C), hydrogen (H), and oxygen (O) atoms. In simpler terms, carbohydrates can be defined as polyhydroxy aldehydes or ketones or substances, which yield them on hydrolysis. The general formula of carbohydrates is Cn (H2 O)n , where n is the number of carbon atoms. In nature, carbohydrates are the most abundant biological molecules. In the human diet, carbohydrates are one of the main nutrients that provide energy to the body. The green plants, through the process of photosynthesis, synthesize the carbohydrates.

Classification of carbohydrates

Carbohydrates are also known as saccharides, originating from the Greek word sakcharon, which means sugar. However, not all saccharides have a sweet taste. Carbohydrates can be classified into three distinct groups: monosaccharides, oligosaccharides, and polysaccharides. The number of sugar units determines this classification.

Monosaccharides

Monosaccharides, or simple sugars, are the most basic carbohydrates. They are the monomers that serve as the building blocks for forming polymers or complex carbohydrates. Monosaccharides are the most simple carbohydrates possessing carbonyl groups, i.e., aldehyde (CHO) and ketone (C=O) groups with two or more hydroxyl (OH) groups. Monosaccharide has the generic formula Cn (H2 O)n, where n is the number of carbon atoms. Monosaccharides are sugars that cannot be hydrolyzed further to form simple carbohydrates. These crystalline solids without color dissolve in water but remain insoluble in non-polar solvents.

Introduction and Scope of Biochemistry

Biochemistry is the field of study that focuses on studying chemical reactions occurring within living organisms. It is a specialized branch that combines the principles of both chemistry and biology. In order to comprehend the molecular mechanisms behind every biological event, this multidisciplinary f ield incorporates elements of biology, chemistry, and physiology. The scope of biochemistry extends from the study of small molecules and their interactions within cells to the analysis of complex metabolic pathways, enzyme kinetics, and the structure and function of biomolecules.

One of the main goals of biochemistry is to elucidate the chemical reactions that take place in living systems and understand how they regulate key biological processes. Biochemists investigate the structure, function, and properties of various biomolecules, such as proteins, carbohydrates, lipids, and nucleic acids. It helps us to study how these biomolecules interact and contribute to the overall functioning of cells, tissues, and organisms.

Biochemistry also plays a vital role in various areas of research and practical applications. For instance, in medical biochemistry, scientists study the molecular basis of diseases to develop diagnostic tests and therapeutic interventions. They investigate the biochemical mechanisms underlying genetic disorders, cancer, metabolic, and infectious diseases. Thus, understanding the biochemical basis of diseases can lead to the development of targeted and personalized treatments.

Vitamins are naturally occurring, vital nutrients that are needed in small amounts. They are important for bone and tissue health, wound healing, growth and development, immune system function, and other biological processes. These vital organic substances serve a variety of biochemical purposes. Vitamins, like minerals, are not biosynthesized by the body. Consequently, in order to maintain our health, we must obtain them from the food we eat or, in rare circumstances, supplements.

Vitamins in Milk

Together with water- soluble vitamins like vitamin C and B complex and provitamins, milk also contains a variety of fat- soluble vitamins like A, D, E, and K. Some of these vitamins do, however, have very low concentrations.

Vitamin A (Retinol)

Plant foods contain carotenoids, which are the precursors of vitamin A, whereas animal foods provide preformed vitamin A in the form of retinyl esters.

The primary constituents of milk are water, fat, protein, sugar (lactose), minerals, vitamins, and enzymes. Milk is a thin emulsion made up of two phases: A continuous aqueous colloidal phase and a dispersed phase of oil or fat. It is an opaque white liquid containing fat in an emulsion, protein, and a few minerals in a colloidal suspension, and lactose along with soluble proteins and a few minerals in a true solution. The physical characteristics of milk are comparable to those of water, but they are altered by the degree of dispersion of the colloidal and emulsified components as well as the presence of different solutes, such as salts, lactose, and protein, in the continuous phase.

Physical Appearance, Color and Optical Properties

Fat globules and casein micelles scatter light, giving the appearance of turbid and opaque milk. The way that molecules scatter light has an impact on optical characteristics. When the wavelength of light and the particle’s magnitude coincide, light scattering happens. Consequently, light with shorter wavelengths is scattered by smaller particles and vice versa. Because casein micelles scatter blue light more than red light at shorter wavelengths, skim milk appears slightly blue. Cow’s milk has a creamy color because of beta- carotene, a carotenoid precursor to vitamin A. The greenish tinge in whey is due to the presence of riboflavin. Turbid and opaque due to scattering of light mainly by protein micelles and fat globules.

Electrophoresis

The mobility of ions in an electric field is the basis for the separation method known as electrophoresis. Ions that are negatively charged move toward a positive electrode, while those that are positively charged move toward a negative electrode. Usually, one electrode is biased either positively or negatively, and the other is at the ground for safety reasons. Ions can be distinguished from one another based on their size, shape, and overall charge, which determine their migration rates.

Poly Acrylamide Gel Electrophoresis (PAGE)

Introduction

Electrophoresis is an electrochemical process in which substances with a net electric charge migrate under the influence of an electric current. This migration occurs in an agar gel or a liquid- filled buffered matrix such as cellulose acetate. Positively charged substances travel toward the cathode (negative electrode), while negatively charged substances go toward the anode (positive electrode). Different substances move at different rates depending on their charge. This movement is called electrophoretic mobility.

Proteins are charged molecules. They have a positive as well as a negative charge. Among proteins, particularly caseins, they have different contents. The charge density also varies depending on the molecular weight. Proteins have a net negative charge at a pH above 4.6, and hence they move towards the anode (positive electrode) when subjected to an electric field under alkaline conditions. Due to differences in the charge of individual protein fractions, their migration rates also differ and are hence separated into distinct bands. These bands are stained with the dye amido- black, destained with 7% acetic acid, and observed for the electrophoretic pattern.

Food adulteration is a global issue, and because there are insufficient regulations and oversight, developing nations are more vulnerable to it. But in many nations, food adulteration has gone unnoticed. Contrary to popular assumption, milk adulterants can, regrettably, present major health risks that result in fatal diseases. Modern milk adulteration involves increasingly complex techniques, necessitating state- of- the- art research to identify the adulterants. Because milk contains so many nutrients that are needed by both adults and infants, it is referred to as the ideal food. In terms of protein, fat, carbohydrates, vitamins, and minerals, it is among the best sources. Regretfully, milk adulteration is a very common problem worldwide. Potential causes could be the gap between supply and demand, the perishable nature of milk, the low purchasing power of the consumer, and the absence of appropriate detection tests. Because there is insufficient oversight and inadequate law enforcement, the situation is substantially worse in developing and underdeveloped nations. While quantitative detections of adulterants in milk are varied and complex, qualitative detections can be carried out simply using chemical reactions. The type of quantitative detection methods used depends on the milk’s adulterants.

Typical Adulterants in Milk

The addition of vegetable protein, milk from different species, whey addition, and watering are the main adulterants in milk that are known to be economically motivated. There is no serious health risk associated with these adulterations. Some adulterants, though, are too dangerous to ignore. Urea, formalin, detergents, ammonium sulfate, boric acid, caustic soda, benzoic acid, salicylic acid, hydrogen peroxide, sugars, and melamine are a few of the main adulterants in milk that have been shown to have serious negative health effects.

The terms “cultured dairy foods,” “cultured dairy products,” and “cultured milk products” are other terms for fermented dairy products. Lactobacillus, Lactococcus, Leuconostoc, and other lactic acid bacteria, along with yeasts in certain products, are used to ferment milk to make these dairy products. The product’s shelf life is extended through fermentation, which also improves the product’s taste and milk’s digestibility. There is a wide variety of fermented milk products available, each with unique flavors and attributes. The following categories apply to fermented milk products, based on the type of fermentation.

Changes in Nutrients Resulting from Fermentation

Fermentation lowers the lactose content in fermented milk products; however, if the starting milk is concentrated or fortified, the finished product’s lactose content may be higher than that of liquid milk.

The composition and content of protein and total amino acids are comparable to those of raw milk. Casein degradation is small compared to cheese, accounting for not more than 1% of the total protein. While some products of casein degradation are used by the starter microorganisms, others build up as free amino acids in the fermented kinds of milk. The amounts of free amino acids that build up differ depending on the species. Generally, proline is increased in large quantities, followed by serine, alanine, valine, leucine, and histidine. As a result of continuous starter culture activity, the level of free amino acids increases during storage. A limited degree of lipolysis occurs during the manufacture of fermented milk. The amount varies depending on the type of species, but the pattern and concentration of free fatty acids are both impacted. During fermentation and the breakdown of amino acids, trace amounts of volatile fatty acids are also produced.

Being an essential component of the rich Indian past, traditional dairy products and a sophisticated classification of confections have played a significant role in the Indian economy. Depending on how it is processed, it has significant social, cultural, and economic significance. These products have been developed over a long period using a combination of experience, traditional wisdom, and culinary skills. India is emerging as the highest milk- producing country in the world with an annual production of 221.1 million tons in the year 2021–22 (Source: Basic Animal Husbandry Statistics, MoFAHD, DAHD, GoI). An estimated 50% of the milk produced in India is used to make a range of traditional dairy products, including paneer, dahi, ghee, khoa, and shrikhand. The product is not only well- established in the Indian market, but it also has significant export potential due to the large Indian diaspora living all over the world.

All native milk products that have developed over time using locally accessible technologies are referred to as traditional Indian dairy products or Indian indigenous milk products. The Indian masses’ economic, social, religious, and nutritional well- being are greatly influenced by traditional dairy products.

Classification of Traditional Indian Dairy Products

Traditional dairy products in India are typically made, packaged, and/or sold using time- honored methods without the use of machinery or meticulous documentation of the procedure. These products do, however, have several technological advantages, including their widespread appeal, reduced cost of production, straightforward manufacturing process, adoption at the rural level or collection center, large profit margin, and abundance of employment opportunities. These products are categorized according to the manufacturing principle.

One of the most consumed and well- known milk products in the Indian subcontinent is ghee. It is a by- product of tradition, prepared both at organized dairy plants and the household level for a well- established market. Ghee is traditionally made from the milk of cows or buffalo. The tetrapyrrole pigments biliverdin and bilirubin are responsible for the distinctive color of buffalo ghee. On the other hand, ghee is golden yellow due to the presence of carotenoids in cow’s milk. Ghee’s composition changes according to the kind and feeding habits of the animal and can differ from place to place and season to season.

In addition to, being a substantial source of fat- soluble vitamins, essential fatty acids, and other growth- promoting components, ghee is a rich source of energy. Its value as food for humans is enormous. Additional attributes related to medicine are present in ghee. Ghee can improve one’s physical and mental attributes and treat eye conditions like ulcers. Ghee is used in religious and ceremonial rites in addition to being consumed.

Composition

Ghee is a complex lipid composed of mixed glyceride which makes up 99–99.5% of its total content. In addition, it has trace amounts of calcium, magnesium, phosphorus, iron, copper, and fat- soluble vitamins, tocopherols, carbonyls, hydrocarbons, carotenoids, free fatty acids, phospholipid, sterols, and sterol esters

The process of making dried milk involves eliminating water from milk, primarily through spray or roller drying. It is regarded as a significant sector of the dairy industry and is made from whole cow, buffalo, or standardized milk. Skim milk powder (SMP) is made from milk after the fat has been removed whereas whole milk powder (WMP) is made from whole cow or buffalo milk

FSSA (2006) Standard Of Milk Powder According to FSSA (2006), Milk powder is the product obtained by partial removal of water from the milk of cows and/or buffalo. The fat and/or protein content of the milk may be adjusted by the addition and/or withdrawal of milk constituents in such a way as not to alter the whey protein to casein ratio of the milk. It shall be of uniform color and shall have pleasant taste and flavor, free from off-flavor and rancidity. It shall also be free from vegetable oil/fat, mineral oil, thickening agents, added flavor, and sweetening agents. It may contain food additives. It shall conform to the microbiological requirements prescribed by FSSA (2006).

Water activity, high storage temperatures, longer than usual storage times, high product moisture content, high processing temperatures, and container headspace are some of the variables that affect the Maillard reaction in dried milk.

Casein and whey proteins are the major proteins present in milk. Unlike casein, whey proteins are heat-labile. Whey protein fractions present in milk are α-lactalbumin, β-lactoglobulin, bovine serum albumin, immunoglobulin, euglobulin, and lactoferrin. β-lactoglobulin and κ-casein interact during heat treatment. When milk is refrigerated for longer than 24 h, it forms γ-casein and protease-peptone fractions.

Commercially, one of the most significant milk products is ice cream. It is a frozen product which is usually made from milk or cream that has been f lavored with fruits like peaches or strawberries, and spices like vanilla or cocoa, or with sugar or a substitute. Stabilizers are sometimes combined with food coloring. Ice cream mix typically contains 10.0% milk fat, 11.0% solids not-fat, 14.0% added sugar, and 0.3% additives like an emulsifier, stabilizer, f lavor, and color substances.

Standard of Ice Cream

According to FSSA (2006), ice cream, kulfi, chocolate ice cream or softy ice cream means the product obtained by freezing pasteurized milk prepared from milk and/or other products derived from milk with the addition of sweetening agents and fruit products, egg products, coffee, cocoa, ginger, and nuts. It may also contain chocolate, and bakery products such as cake or cookies as a separate layer and/or coating. It may be frozen hard or frozen to a soft consistency. It shall be free from artificial sweeteners. It shall have a pleasant taste and smell free from flavor and rancidity. It may contain food additives. It shall conform to the microbiological requirements as given in FSSA (2006).

Concentrated milk products are obtained through partial removal of water which increases the dry matter content of milk. The increase in dry matter content in concentrated milk is proportional to the degree of concentration. The milk concentrate may be prepared from either full-cream whole milk or fat-free skim milk.

Depending on the nature of raw material and other non-milk additives concentrated milks are classified into four broad categories.

1. Sweetened condensed milk

2. Evaporated milk or unsweetened condensed milk

3. Sterile milk concentrate

4. Reconstituted concentrated milk

Sweetened Condensed Milk

One of the earliest dairy products to be produced industrially is sweetened condensed milk. According to FSSA (2006), sweetened condensed milk is the product obtained by partial removal of water from the milk of cow/buffalo with the addition of sugar or a combination of sucrose with other sugars or by any other process that leads to a product of the same composition and characteristics. The fat and/or protein content of the milk may be adjusted by the addition and/or withdrawal of milk constituents in such a way as not to alter the whey protein to casein ratio of the milk being adjusted. It shall have a pleasant taste and flavor and be free from off-flavor and rancidity. It shall be free from any substance foreign to milk. It may contain food additives. It shall conform to the microbiological requirement as laid down by FSSA. The said product shall also conform to the following requirements

Cheese is made from milk, with the addition of cream, buttermilk, and/or whey. This basic raw material is also called cheese milk. Cheese consists mainly of casein (the major protein of milk), other milk proteins, fat, other milk components, and a certain percentage of water, which is bound. Proteins are precipitated by enzymes and/or acidification as well as heat and are separated by suitable processes from the aqueous phase. (whey, serum). Cheese is most often molded, pressed, and salted and has fungal and bacterial culture additions. colorants, herbs, spices, and other non-dairy ingredients are also added to the cheese. It is consumed either fresh or at different stages of ripening.

Worldwide, there are more than 2000 types of cheese, sometimes made by very different manufacturing processes. A classification can be based on several aspects and is done in different countries according to different criteria. A general classification can be made for three major groups:

a. Rennet or natural cheese: manufactured straight from milk by using proteolytic enzymes (rennet) and acid, with a more or less pronounced ripening process.

b. Fresh cheese or non-ripened Cheese: Quarg, fresh cheese, and white cheese are examples. Its manufacturing process is similar to that of rennet cheese manufacturing, but it has a high degree of acidity and is not subjected to a proteolytic ripening process.

c. Long-life cheese (processed cheese): it is more often made from rennet cheese and is textured by thermal treatment and made shelf-stable.

Chemistry of Creaming

After being drawn from animals, normal milk will form a cream layer on standing. Because of the difference in density between fat globules (920 kg m−3 at room temperature) and milk plasma (density 1030 kg m−3), the granules tend to rise. This causes creaming, which we often want to prevent and it enables milk to be separated into cream and skim milk. Creaming is much enhanced when the globules have been aggregated into flocculates, clusters, or granules.

Cream Ripening

This unit operation serves as an intensive preparation of the cream for the churning process. It is divided into two categories.

1. Physical ripening;

2. biochemical ripening The following criteria are influenced by the cream ripening:

• Consistency, firmness, and spreadability of butter.

• Basic water content of butter during the manufacturing process (bound water in butter, which cannot be removed by any mechanical treatment).

• Buttermilk fat content, which has to be minimized.

The melting and solidification behavior of fat depends on the attached fatty acid, which indicate the hardness of the butterfat.

Natural hardness is caused mainly by seasonal feeding of the milk animals. Winter feed with a great deal of dry feed or roughage yields a hard butterfat with a high percentage of saturated and short-chain fatty acids and few double bonds in the fat molecules.

Summer feeding (green grass) results in fat with longer chains and a higher percentage of unsaturated fatty acids with many double bonds. We therefore distinguish between summer fat and winter fat or summer cream and winter cream.

Types of Enazymes in milk

1. Endogenous and 2. Exogenous.

Three sources of enzyme in milk:

1. The blood via defective mammary cells.

2. Secretory cell cytoplasm. Some of which are occasionally entrapped with fat globules by the encircling MFGM.

3. The outer layer of MFGM is probably the principal source of endogenous enzymes in milk. The outer layer of MFGM is derived from the apical membrane of the secretory cell which in turn originates from the Golgi membrane and acts as the principal source of enzyme in milk.

General Characteristics of Milk Enzymes

1. Deteriorative character e.g. lipase (commercially most important enzyme in milk), proteinase, acid phosphatase, xanthine oxidase.

2. Preservative enzymes. e.g. superoxide dismutase, sulfhydryl oxidase.

3. As an indicator of the thermal treatment history of milk. Related enzymes are alkaline phosphatase, ϒ glutamyl transpeptidase, and lactoperoxidase.

4. Mastitis infection indicator. Related enzymes are catalase, n-Acetyl-β D-glucosaminidase, and acid phosphatase.

5. Antimicrobial activity.

6. Commercial source of ribonuclease and lactoperoxidase

Common Milk Enzymes and their Role in Dairy Industry

The salts of the milk include those constituents that are present as ions or in equilibrium with ions. The main salts of milk are phosphates, citrates, chlorides, sulfates, carbonates, and bicarbonates of sodium, potassium, calcium, and magnesium. Approximately 20 other elements are found in milk in trace quantities including copper, iron, lead, boron, manganese, zinc, iodine, etc. These ions are more or less associated with themselves and with proteins. Some metals act as catalysts in the oxidation of milk fat leading to oxidative rancidity and certain metals like Ca, P, Mg, Mo, Co, Fe, etc. are components of metalloproteins such as lactoferrin, lactoperoxidase, xanthine oxidase, and parts of phospholipids.

Distribution of Salts Between Casein Micelles and Serum

The phosphorus is present as orthophosphate, but part of it is bound to organic components like serine and threonine residues of casein, molecules of hexoses and glycerol, phospholipids, etc. Phosphorus is also found in the hydroxyapatite structure of the colloidal calcium phosphate in the micellar structure of casein. The sulfur content of milk is about 0.36 g/kg, but most of it is in the amino acid residues methionine and cysteine of the proteins. About 10% is present as inorganic sulfate.

Physical Equilibria Among Salts

Milk contains several elements but all of them are not entirely in a soluble state. Some minerals exist in colloidal and ionic states at the normal pH of milk. The compounds that are in soluble condition play an important role in keeping various milk constituents in stable condition. A balance exists between the components that are in a soluble state and those which are in colloidal state.

Milk lipids or milk fats exist as an emulsion of small spherical droplets called milk fat globules. These globules are very small in size but very large in number. The diameter of the milk fat globules ranges between 0.1 and 22 µm and the number varies between 1.5 and 3.0 billion globules/mL. The average size of milk fat globules ranges from 2 to 5 µm. The average fat globule size in cow and buffalo milk are 3.4 and 4.5 µm, respectively. The surface of the milk fat globules is coated with an adsorbed layer of material known as the milk fat globule membrane (MFGM). MFGM contains phospholipids and proteins in a complex form. MFGM has two main functions (a) fat globule stabilization and (b) preservation of the fat globule’s identity.

General Composition

Triacylglycerols (triglycerides) represent 97–98% of the total lipids in the milk of most species. The diglycerides probably represent incompletely synthesized lipids in most cases. Although phospholipids represent less than 1% of total lipids, they play an important role in being present mainly in the MFGM and other membranous material in milk. The principal phospholipids are phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin. Trace amounts of other polar lipids, including ceramides, cerebrosides, and gangliosides, are also present. Phospholipids represent a considerable percentage of the total lipid of buttermilk and skim milk reflecting the presence of proportionately larger amounts of membrane material in these products.

Carbohydrates are polyhydroxy derivatives of aldehydes or ketones and undergo all the organic reactions common to the carbonyl groups and hydroxyl groups. The principal milk carbohydrate is lactose. It is a disaccharide. Hydrolysis of lactose with enzyme β-galactosidase yields glucose and galactose.

Lactose is the major carbohydrate present in all types of milk. The first record of isolation of lactose was in 1663, by Bartolettus by evaporation of whey. Milk contains only trace amounts of other sugars, including glucose, fructose, glucosamine, galactosamine, neuraminic acid, and neutral and acidic oligosaccharides.

Lactose plays a significant role in milk and milk products that are

• It plays a major role in the production of fermented dairy products.

• It contributes to the nutritive value of milk and milk products

• It affects the texture of certain concentrated and frozen products;

• It is involved in heat-induced changes in the color and flavor of highly heated milk products.

Status of Lactose

Lactose content varies extensively in the milk of different species. Breed of animal, individuality factors, udder infection, and especially the stage of lactation regulates the lactose content in milk. The lactose concentration in milk decreases gradually and significantly during lactation; this behavior contrasts with the lactational trends for lipids and proteins, which, after a decrease during early lactation, increase significantly during the second half of lactation. Lactose synthesis is hampered during mastitis. Lactose, along with sodium, potassium, and chloride ions, plays the principal role in controlling the osmotic pressure of the mammary system. Thus, any change in lactose content is compensated by a change in the soluble salt constituents. The osmotic relationship between lactose and soluble salts during the secretion of milk partly explains the presence of high lactose in milk with a reduced ash content and vice versa (Table 1).

Bovine milk contains an average of 3.5% protein. The concentration of protein changes significantly during lactation. The first few days of lactation exhibit the main change in protein concentration. The main changes occur in whey protein fraction.

Distribution of Protein in Milk

80% casein and 20% whey protein (β lactoglobulin- 10%, α- lactalbumin- 5%, serum albumin, and immunoglobulin- 5%).

Fractionation of Milk Proteins Fractionation of milk protein is mainly done by acidification at pH 4.6 around 30 °C.

Almost 80% of the bovine milk protein precipitates at pH4.6, and this fraction yielded by precipitation is called casein.

The remaining soluble part of the protein is referred to as whey protein/serum protein/ non- casein protein. Wide variation is observed in the casein- whey protein ratio in different types of milk(protein synthesis is independent of animal diet)

Significant Difference Between Casein and Whey Protein

1. Casein precipitates at pH 4.6 but does not precipitate whey protein at that pH. This property of milk protein is commercially exploited for the preparation of industrial casein and certain varieties of cheese.

2. Chymosin or other proteolytic enzymes produce specific changes in casein resulting in coagulation in the presence of calcium. Whey protein does not undergo this type of coagulation.

3. Casein is very stable to high temperatures. They can withstand heating up to 140 °C for 20 minutes but whey protein is relatively heat labile. They are completely denatured by heating at 90 °C for 10 min.

4. Casein is a phosphoprotein containing an average of 0.85% phosphorous while whey protein does not contain any phosphorous. The phosphate of casein is an important contributor to remarkably high heat stability and calcium- induced coagulation of rennet- altered casein.

5. Casein contains very low sulfur (0.8%) whereas whey protein is relatively rich in sulfur (1%). The principle casein contains only methionine but whey protein contains a significant amount of cystine, cysteine, and methionine. These sulfur- containing amino acids are responsible for many changes in milk on heating.

6. Casein is synthesized only in the mammary gland and no other sources in nature. Some of the whey proteins β lactoglobulin, and α- lactalbumin are synthesized in the mammary gland but bovine serum albumin and immunoglobulin are not synthesized in the mammary gland but derived from blood.

7. The whey protein is dispersed in solution and has a simple quaternary structure. Casein has a complex quaternary structure and exists in milk as large colloidal aggregates.

Introduction

The mammary glands of female mammals secrete milk, a necessary food that is liquid and meets the newborn’s nutritional needs. All mammals consider milk to be a complete food for their newborns. It is considered a necessary food for humans and can be consumed as a product or as liquid milk. It contains almost all essential nutrients except iron, which is very low in milk. In essence, milk is an emulsion of protein and fat in water, supplemented with carbohydrates, vitamins, and minerals.

Definition of Milk

• Milk is a complex biological fluid that differs from species to species in both composition and physical properties. It is the full lacteal secretion of a mammary gland that is obtained by milking mammals for a minimum of 72 h after calving or until the milk is free from colostrum.

• FSSAI defines milk as the normal mammary secretion derived from the complete milking of healthy milch animals. It should be free from colostrum.

From a chemical perspective, milk is a dynamically balanced mixture that comes in different forms such as emulsion, colloidal suspension, and true solution. It also contains varying amounts of proteins, fats, carbohydrates, salts, and water.

Gross Composition of Milk

Milk composition varies among different mammals. The average chemical composition of milk in various species is given in Table 1. Buffalo milk and sheep milk contain higher amounts of fat, while human milk comparatively has the highest lactose content. Human milk has lower protein content as compared to milk of other species.

Microbial Resistance Mechanism

Huge use of antibiotics in nosocomical environment has lead to the occurrence of multidrug resistance mechanism by microorganism against particular antibiotics (Munita et al., 2016; Zhao et al., 2020). Main operation includes chemical modification, efflux pump through passivation, modification of drug targeting gene and systemic elimination of antibiotics as many of them can form biofilm (Rajput et al., 2018) which can show extreme resistance. Chemical and enzymatic mechanism can include inactivation, activity loss, degradation and derivatizaton of antibiotics chemical group (Liu et al., 2019). Best idea may include production of enzymes or they can also expel antibiotic to efflux pump. Antibiotics drug efflux resistance can be accomplished by drug efflux pump playing an important role in microbial resistance. A different type of efflux pump system has been identified that include lipophilic and hydrophilic efflux system targeting the drug at different chemical properties (Wasaznik et al., 2009; Kumar et al., 2012). Another strategy adopting in resistance mechanism is modification of drug targeting gene. This mechanism makes the changes in drug to lose its target through drug targeting gene modification. Another important resistance mechanism is effectiveness of antibiotics by interfering with the target site. Therefore, bacteria has evolved different mechanism that include target site protection. A bactericidal role played by beta lactum antibiotics in inhibition of mucopeptide synthase and bacterial penicillin binding protein (PBP) in cell wall transpeptidation reaction that prevents the formation of complete call wall and hence dies (Sauvage et al., 2016). Bacteria also developed its own strategies to cope up with the environmental stress that can also include human body. In course of gaining the advantage bacteria needs to compete for nutrients and to keep away from the molecules produced by other competing organism (Zhao et al., 2019). Recently researchers have found the importance of biofilm resistance in bacteria. Bacterial biofilm resistance produce by complex and systematic drug resistance mechanism.

Introduction

The epidemiological risk and hazards to global health security posed by AMR has been reiterating in several World Health Assembly (WHA) declarations and has been prioritized under the Global Health Security Agenda (GHSA). The Indian Ministry of Health & Family Welfare (MoHFW) has also identified AMR as one of the top priorities for collaborative work with WHO and the first National Action Plan on AMR has been implemented (2017-2022). India ranked fifth in antibiotic consumption in food producing animals (ruminants, pigs, and poultry) in 2010. Even though there has been a gradual reduction of 3.6% in antibiotic consumption during 2011-2019 (Koya et al., 2022), with mounting incomes and shifting in dietary patterns in favour of the consumption of animal protein, especially for poultry, the veterinary antibiotic utilization is projected to further rise by 312%, elevating India to the fourth rank by 2030 (Van Boeckel et al., 2015). FAO has already reported some of the peak resistance rates among common commensal and environmental bacteria against antibiotics like penicillin, cephalosporins, erythromycin and co-trimazoles in India (INFAAR). Resistance to the broad spectrum fluoroquinolones and third generation cephalosporin antibiotics is already more than 70% in Escherichia coli, Klebsiella pneumoniae, and, Acinetobacter baumannii and more than 50% in Pseudomonas aeruginosa. Even the microbial resistance to carbapenems, the last-resort antibiotics, is extremely high among gram-negative microorganisms (Sumanth et al., 2016). To overcome these incidences, it is impulsive to understand the microbe and drug interaction in host system.

Introduction

The study of infectious diseases, alongside other clinical and natural strengths, is going through fast change introduced on by the approach of reasonable next generation sequencing (NGS) advancements (Koser et al., 2012).These technologies become adopted as routine methods easily, and laboratory techniques are transformed throughout the process. The amount of genomic bacterial data generated is huge. As of this writing, for example, over 200,000 Salmonella genomes alone are in the public domain with hundreds being added weekly. A whole genomic DNA sequence characterizes the maximum practicable level of structural detail on the individuating traits of an microbial organism or population. It can therefore be used to provide more accurate microbial genome recognition, evolutionary relations and a detailed catalogue of epidemiologically important characteristics. This has a significant role to play for outbreak research, diagnosis and treatment of infectious diseases, microbiology and epidemiology (Allard et al., 2019). Besides, DNA sequences are also a universal dataset which can theoretically be used to determine any biological function. This means that antimicrobial resistant (AMR) can be detected and to track the development and spread of AMR bacteria in a hospital or the community.

Introduction

Until the 20thcentury, bacterial infections were the number one cause of human death. In the 1940s, antibiotics became the lifelines for treating serious infections that saved millions of lives. Despite the advantages derived from the use of antibiotics, resistance to these drugs emerged. Unfortunately, resistance to most antibiotics emerged in time-course even for the most powerful antibiotics developed.Resistance to antibiotics is a challenge to the scientific community in keeping up the pace of antibiotic discovery.World Economic Forum’s Global Risks Reporthas listed antibiotic resistance as one of the greatest threats (WEF, 2020).United Nations (UN) has warned that drug-resistant diseases could account for 10 million deaths each year by 2050. By 2030, antimicrobial resistance (AMR) could likelylead up to 24 million people into extreme poverty(IACG,2019). Currently, at least 7,00,000 people die each year due to drug-resistant diseases (IACG,2019). The emergence and widespread of antibiotic-resistant organisms is a global challenge and needs a multi-dimensional approach to address the fundamental issues to minimize drug-resistance.The Antibiotic Resistance Genes Database (ARDB) contains a repository of around 23,000 antibiotic resistance gene or protein sequences conferring resistance against over 240 antibiotics (ARDB). Computational Biology (Bioinformatics) has become one of the important tools in studying antimicrobial resistance. It employs a range of computation techniques to make predictions about the biological phenomenon.

Introduction

Resistance to antibiotics in the bacterial world is a genetically determined phenomenon that was first observed within a few years after the introduction of antibiotics for therapeutic purposes. In Nature, ARGs are believed to have originated about three billion years ago as an intrinsic defense mechanism in antibiotic-producing bacteria (Perry et al., 2016; Iskandar et al., 2022), and subsequently, evolved by complex and quantum jump trajectories to mobilize, diversify and interconnect all ecosystems in the micro-biosphere. Bacterial genomes exhibit plasticity and continue to evolve under the selection pressure of antibiotics and other stressors (Patel, 2016). The ARGs in circulation at present are thus a mixture of ancient and modern counterparts. Recently, the interplay between bacterial metabolism and antibiotic resistance has also been revealed (Zampieri, 2021).

The use of antimicrobials in health management, livestock, and food industry is driving the emergence of resistant microbes at a rapid pace (Wall et al., 2016; Adegoke et al., 2016; Hu et al., 2017). As a result, AR pathogens have spread globally to cause loss of lives, production, productivity, and negatively impact economic growth. Recently, AR pathogens in a large study have been found directly responsible for millions of deaths, and the figures are likely to increase in the coming decades (Murray et al., 2022). The pace of the emergence of AR can however be slowed down by improving human interventions. The global and local AR combat plans implemented during the past few years have enhanced the mass awareness of the pandemic problem (WHO, 2015; FAO, 2016; NAP, GOI, 2017; WHO, 2022; WOAH, 2022). These Action Plans on AR containment encourage avoiding of indiscriminate use of antibiotics for the treatment of sick humans and animals, recommend the ban on the use of antibiotics as growth promoters in food animals, promote AR surveillance and monitoring, and support the ‘One-Health’ strategy and application of the biosecurity principles for containment of the resistant microbes.

Introduction

Antibiotic-resistant bacteria are a major cause of health care-associated infections around the world, and resistance has also emerged in infections in the wider community. Infections caused by multi resistant organisms significantly increase morbidity, mortality, and health care costs. Molecular analyses have revealed that widespread multi resistance has commonly been achieved by the acquisition of preexisting determinants followed by amplification in response to selection. The capture, accumulation, and dissemination of resistance genes are largely due to the actions of mobile genetic elements (MGE), a term used to refer to elements that promote intracellular DNA mobility (e.g., from the chromosome to a plasmid or between plasmids) as well as those that enable intercellular DNA mobility.

Insertion sequences (IS) and transposons (Tn) are discrete DNA segments that areable to move themselves (and associated resistance genes) almost randomly to newlocations in the same or different DNA molecules within a single cell. Other elements, such as integrons (In), use site-specific recombination to move resistance genes between defined sites. As these types of MGE are often present in multiple copies indifferent locations in a genome, they can also facilitate homologous recombination(exchange of sequences between identical or related segments). Intercellular mechanisms of genetic exchange include conjugation/mobilization (mediated by plasmidsand integrative conjugative elements [ICE]), transduction (mediated by bacteriophages), and transformation (uptake of extracellular DNA). Interactions between thevarious types of MGE underpin the rapid evolution of diverse multi resistant pathogens in the face of antimicrobial chemotherapy. Primarily the most important and/ or topical elements in Gram-negative and Gram-positive bacterial species of particular concern clinically, namely, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (the so-called ESKAPE group) (Rice LB., 2008) as well as Escherichia coli (giving the ESKAPEE group) (Llaca-Díaz J.M. et al.,2012); nonetheless, many of the MGE types described are of course broadly relevant to many other bacterial taxa.

Antibiotics, often referred to as “magic bullets” in the battle against bacteria, stand out as one of the most remarkable medical breakthroughs of the 20th century. Their advent revolutionized the therapeutic landscape, consistently saving millions of lives from bacterial infections (WHO, 2020). Undoubtedly, antibiotics are a blessing to humanity, not merely for their medicinal applications but also for their diverse uses. They have long been employed in various contexts, including animal husbandry and production, serving as preventive measures in numerous underdeveloped and developing nations for decades (Majumder et al., 2020). A range of antimicrobial agents, including antibiotics, antifungals, antivirals, disinfectants, and food preservatives, function to either inhibit microbial growth or eradicate them altogether. Among these, antibiotics, specifically tailored to combat bacterial infections, are the most widely used class of antimicrobials. With their increasing and often improper use, microorganisms have developed antimicrobial resistance (AMR) (Murray et al., 2022).

Antimicrobial resistance is an inevitable evolutionary process observed in all microorganisms, and is driven by genetic mutations aimed at resisting the lethal pressures of selection. Bacteria, adapt to environmental selection pressures by developing resistance to antibacterial drugs, thereby diminishing the efficacy of these treatments (Murray et al., 2022). The widespread use of antibiotics, particularly in developing nations, further flares up the process, leading to significant repercussions such as increased rates of morbidity and mortality (WHO, 2020). The incidence and prevalence of antimicrobial resistant bacterial infections have reached alarming levels in the 21st century, posing a substantial threat to global public health akin to a silent pandemic. Antibiotic resistance knows no borders and can impact individuals of any age or gender, regardless of their location. In its present state, antimicrobial resistance (AMR) stands as one of the foremost threats, not only to global health but also to food security (Murray et al., 2022).

The response of any living cell to a xenobiotic (including antibacterial agent) is significantly affected by the prevailing local environmental conditions. In case of bacterial infection, it may include the presence of other microbes, no matter commensal or pathogenic, or biochemical alterations at that particular site of bacterial infection in host. All these host and bacterial factors play a critical role in the evolutionary responses of bacteria but usually go unconsidered while determining the microbial sensitivity to a particular antibiotic in a clinical setting. These also include the direct and indirect interspecies interactions that can affect the responses of individual bacterial species and surviving microbial communities to a particular antibiotic therapy. These interactions may significantly alter the qualitative and quantitative responses, potential phenotypic changes and the final trajectory of evolution of antimicrobial resistance. Due acknowledgement and plausible interpretation to these interactions may help to reduce the upsurge in AMR and its associated morbidity and fatality and pave path to more proficient clinical success.

The factors participating in drug- microbe interaction can be classified into two broad groups: host factors and microbial factors. The environmental milieu of the interaction is chiefly regulated by the gut microbiota with stressors causing deflections. The gastrointestinal bacteria amend host mental and physiological cadence via microbial metabolites such as butyrate, polyphenolic antioxidants, vitamins, and amines, affecting the overall ready perception and active rebuke to a stimulus. Lifestyle stressors such as fluctuating sleep and erratic eating patterns (western diet) disturb the host circadian system along with the gut microbiome. The overall impact is impaired detoxification (decreased conjugation of bile acids or increased production of hydrogen sulphide), substrate oxidation and energy regulation (reduced butyrate production) in the host affecting the overall health and productivity.

Globally, antimicrobial resistance (AMR) is an important public health and development threat (WHO, 2020). Being one of the top 10 global public health threats, the ever-increasing rates in the occurrence of drug-resistant bacterial infections have become a grave public health concern for the governments (Laxminarayan et al., 2013). Moreover, such infections would be difficult to treat and therefore can lead to worse clinical outcomes (Laxminarayan et al., 2013). Worldwide, each year about 700,000 people die from antimicrobial resistant infections and the mortality figures are projected to reach 10 million/ year by 2050 (O’Neill, 2016). The crude infectious disease mortality rate in India is more than 400 per 100,000 persons and at least 23,000 deaths in adults and 58,000 neonatal deaths per year are caused by AMR bacteria (Laxminarayan and Chaudhury, 2016). A 2017 estimate puts the cost of developing an antibiotic at around US$1.5 billion (Towse, et al. 2017). Meanwhile, industry analysts estimate that the average revenue generated from an antibiotic’s sale is roughly $46 million per year. “That’s tiny and nowhere near the.

Introduction

Antimicrobials, viz. antibiotics, antivirals, antifungals and anthelmintics are substances widely used to prevent and treat infections in humans, fish & prawn, livestock animals, and crops. In a situation where microorganisms such as bacteria, viruses, parasites or fungi become resistant to antimicrobial treatments to which they were previously susceptible, it is apprehended that antimicrobial resistance in the microbes has evolved. In fact, antibiotic resistant bacteria evade the effects of antibiotics and multiply in their own way leading to severe health consequences in human and animals. The menace is such that antimicrobial resistance (AMR) hinders treatment modalities targeted for infectious diseases. The hazards of antimicrobial resistance is frequently observed in various pathogenic and non-pathogenic organisms, viz. Salmonella spp., Campylobacter spp., Yersinia sp., E. coli serotype O157:H7, enterococci, Pasteurella sp., Actinobacillus sp. with varying severity (McEwen and Fedorka-Cray, 2002). Microorganisms that develop resistance to commonly used antimicrobials are referred to as superbugs. Globally it is a matter of great concern that it spreads rapidly and threatening the ability to treat common infectious diseases, resulting in prolonged illness, disability and death. Without any effective antimicrobial agent, treatment of infectious diseases, many medical procedures like cancer therapy, diabetes management, organ transplantation, and major surgery will not be possible. The microbes showing resistance to multiple antimicrobials are commonly known as multidrug resistance (MDR) organisms (Magiorakos et al., 2012).

Introduction

Today, antimicrobial resistance (AMR) has become a global issue, contributing to rise in mortality and cost of treatment. Antimicrobial resistance (AMR) is the resistance of a microorganism against an antimicrobial agent to which it was originally sensitive (WHO, 2013). Dispersion of antibiotics is done at large scale, in India even sometimes without any proper prescription. Animal Husbandry is not untouched and antibiotics are used to a great extent, which needs enforcement of strict government rules and regulations for their use in f ield conditions (PHFI, 2011).

Extended-spectrum β-lactamases (ESBLs) are enzymes that hydrolyse most of the β-lactam antibiotics and thus mediate resistance to penicillins, 3rd and 4th generation cephalosporins (Saravanan et al., 2018). Beta-lactamase inhibitors, inhibit these ESBLs such as clavulanic acid, sulbactam and tazobactam (Trott, 2013). The genes encoding for these enzymes are commonly found both on the chromosomes and plasmids among Enterobacteriaceae family (Kotsoana et al., 2019).

Among Enterobacteriacae, E. coli and Klebsiella species are main environmental pathogens associated with various illnesses such as acute bovine mastitis (Koovapra, 2015), pathogenic E. coli with diarrhoea and extra intestinal infections in humans and animals (Levine et al., 1987), K. pneumoniae with respiratory and urinary tract as well as bloodstream infections (Daehre et al., 2018). Resistance to extended-spectrum β-lactams occurs mainly in members of Enterobacteriaceae family through plasmids encoded ESBL genes. (Sharma et al., 2010).

Introduction

The antibiotics are low molecular weight natural, semi-synthetic, or synthetic substance that kills or slows the growth of germs while causing little or no harm to the host is considered an antimicrobial (Walsh, 2000). Antibiotics are the type of medicine produced by some microorganisms that kill or slow the growth of other microorganisms. It includes any of a large group of chemical substances, as penicillin or streptomycin, produced by various microorganisms and fungi, having the capacity in dilute solutions to inhibit the growth of or to destroy bacteria and other microorganisms, used chiefly in the treatment of infectious diseases (De Vetten et al., 2003). However today, a large number of bacteria are resistant to various antibiotics. Antibiotic resistance occurs when a bacterium becomes able to survive and multiply despite the presence of an antibiotic that would normally control or kill it. In other words, the bacteria become “resistant” and continue to grow even at therapeutic levels of the antibiotic. While antibiotic resistance is a natural phenomenon, it is accelerated by the selective pressure exerted when an antibiotic is used. Bacteria that are naturally resistant to the antibiotic have a better chance of survival compared to those that are susceptible. As the susceptible bacteria are killed or inhibited, resistant strains are favored and continue to thrive (Holme et al., 2006). Some resistance arises without human influence, as bacteria can naturally produce antibiotics against other microorganisms, resulting in a low-level, natural selection for antibiotic resistance. However, the current surge in antibiotic resistant bacteria is largely attributed to the overuse and misuse of antibiotics. One of the main issues facing public health is antimicrobial resistance, particularly in developing nations where access to and use of medications are easier than in developed nations, leading to a disproportionately higher rate of inappropriate antibiotic use and higher resistance levels (Ganguly et al., 2011). Antimicrobial resistance will make it more difficult to control infections in the community and make health care services less effective (Bhatia et al., 2010). It is well known that India has one of the highest rates of infectious disease burdens in the world, and that the country’s high rates of malnutrition and inadequate sanitation make these circumstances worse (http://www. resistancestrategies.org/wpcontent/uploads/2011/03/Indiareport).

Antimicrobial Resistance abbreviated as AMR is defined as the process by which microorganisms like bacteria, fungi, viruses, and parasites change over a period and become resistant to a drug to which they were earlier susceptible to. This makes the medicines ineffective for treatment overtime, thus making infections difficult to treat thereby increasing the risk of spread of diseases caused by them and severe illness which may lead to death. This in turn necessitates the need for development of newer antibiotics. However, there has been a sharp decline in the development and manufacture of new antibiotics in the recent years resulting in development of only few antibiotics in the past two decades. The increase in emergence of antibiotic resistance concurrent with decline in development of newer antibiotics poses a serious threat to health of both humans and animals. Every year about 700,000 deaths have been reported to occur due to resistant infections and this rate is likely to increase further with estimated infections due to resistant pathogens expected to reach over 50 million globally by 2050 (O’Neill, 2016). The worrisome trends of increasing antimicrobial resistance across the globe poses a serious threat to both animal and human health as AMR not only increases the morbidity and mortality rates but also increases the economic burden on health care (Zhu et al., 2022). This presents an extremely grave situation as the supply of new antibiotics is inadequate to cope with the increase of AMR pathogens. The unnecessary use of antibiotics globally especially in the livestock sector as growth promoter further helps in selective enrichment of AMR pathogens which further increases health risks. It is well known that resistant pathogenic and commensal bacteria can persist and spread within and between premises despite zero antimicrobial drug use. This may be explained due to maintenance, replication, and further dissemination of resistance genes by horizontal transfer of genes via natural ecosystem and can occur either by conjugation, transformation and transduction; and these mechanisms can rapidly spread AMR genes among microbial community in natural environment and human microbiome, where these resistant bacterial species may be selected due to presence of antimicrobial drugs (Zhu et al., 2022). The physical transfer of bacteria via movement of animals, workers, and equipment and ineffective cleaning and disinfection may also be responsible for increasing the AMR pathogens in the natural environments.

AMR has been prevalent in almost all European countries from past decades. A 2021-2022 report published by the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC) on AMR in zoonotic and indicator bacteria from humans, animals and food origin reported vast differences in antimicrobials resistance levels among the European countries, with comparatively higher AMR percentages in the southern and eastern parts of Europe (ECDC, 2022, 2023). The resistance levels ranged from very high for tetracyclines, sulfonamides and ampicillin, moderate for ciprofloxacin to low levels for third-generation cephalosporins (cefotaxime and ceftazidime) in human cases infected with Salmonella spp. in 2022 (ECDC, 2023). The trends are dynamic and over a decade of 2013 2022, the human isolates from 12 and 15 countries reported a statistically significant reduction in tetracycline and ampicillin resistance, particularly for S. typhimurium (ECDC, 2023). The ciprofloxacin and cefotaxime combination, a highest priority critically important antimicrobials (CIA), had very low and rare resistance levels for Salmonella isolates of humans and animal and their derived meat.

Livestock species wise variation was also observed in the resistance pattern in imported meat, being higher in poultry meat isolates compared to pig or cattle meat for Escherichia coli (ECDC, 2023). High to extremely high levels of f luoroquinolone resistance from Campylobacter jejuni and C. coli of both human and animal origin isolates exist, due to which the fluoroquinolones can no more be used as a treatment option for human Campylobacter infections (ECDC 2023). The combined occurrence of erythromycin resistance and ciprofloxacin in Campylobacter spp is considered of high public health importance, and the report mentioned that the combined resistance for these antimicrobials was higher in C. coli compared to C. jejuni from food-producing animals and humans. However, while the ciprofloxacin resistance showed an increase in over 13 study countries, seven countries reported reduced erythromycin resistance for C. jejuni. Overall, resistance to conventional antimicrobials was frequent in Campylobacter and Salmonella from humans and animals. Increasing trend in resistance to one of two CIA for treatment in humans has been observed in poultry-originated Salmonella serovars and Campylobacter while combined resistance was is low in few Salmonella serovars and C. coli isolates from humans and animals in some countries (ECDC, 2023).

Introduction to Antibiotic Discovery

Antibiotics are the drugs that are used to treat illnesses caused by bacteria in humans and animals. These act by killing or inhibiting the growth and multiplication of bacteria. Antibiotics were considered as “magic bullets” that selectively kills microbes responsible for disease causation, but at the same time would not affect the host (Zaman et al., 2017).

Louis Pasteur in 1877, first discovered the properties of antibacterial compounds with the inhibition of anthrax by saprophytic bacteria. In 1928, Alexander Fleming observed that the growth of Staphylococcus aureus in petri dishes was inhibited by substances produced by the fungus Penicillium chrysogenum, which led to the discovery of the first antibiotic, penicillin (Aminov, 2017). Selman Waksman, discovered multiple antibiotics from filamentous actinomycetes living in the soil and defined an antibiotic as “a compound made by a microbe to destroy other microbes”. Following the discovery of Penicillin, various antimicrobial compounds such as sulphonamides, aminoglycosides, tetracyclines, lipopeptides, oxazolidinones, glycopeptides, streptogramins, and quinolones were discovered in the “Golden era” of antibiotics during the period from the 1940s to the 1960s and most of the antibiotics still in current use were discovered during that period (Salam et al., 2023). Millions of metric tons of newer classes of antibiotics have been produced in last 60 years since its inception.

A laboratory is essential for research work in science and development. Most of the research institutes have dedicated laboratories for researchers and students to conduct experiments, perform research work, as well as to provide hands-on learning/training to students. There are certain rules and regulations laid down by the laboratory in-charge which need to be followed strictly for safety purposes. Some of the laboratories keep ‘safety manual’ for researchers to read before entering into the laboratory to conduct experiments. Whenever safety is compromised in a laboratory, there is increase in risk of unwanted incidents in the laboratory. Storing food/beverages in the same refrigerator where laboratory chemicals are kept may lead to disastrous effects on health. This chapter aims to provide instructions in brief on the things to be performed (Do’s) before and after entering onto a laboratory as well as the things never to be done (Don’ts) in a laboratory for the safety reasons.

Good Laboratory Practices (Do’s)

• P Wear apron/labcoat while working in the laboratory.
• P Use gloves/respiratory mask/fumehood while handling toxic/volatile/inflammable chemicals/reagents.
• P Use pippet or pippetor for safety in transfer of solutions.
• P Protect eyes, face and naked parts of the body from UV radiations. Use safety goggles or face mask/apron.
• P Keep your eyes away from Liquid Nitrogen and use cryogloves while handling it.
• P Arrange all the necessary materials/solutions before starting the experiment.
• P Periodically fumigate/ozonize culture and inoculation rooms.
• P Autoclave all contaminated materials before discarding.
• P Pre-treat gel or solution containing Ethidium Bromide (EtBr) before discarding into laboratory waste-disposal bin, and use gloves while handling them.
• P Burn all the transgenic plant materials in an incinerator after taking necessary observations.
• P Make entry in the designated log-book and clean the instrument after using it properly.
• P Switch-off the instrument after use or when not in use.
• P Wash hands with soap and remove the labcoat before leaving/going out of the laboratory7.
• P Ensure that switches for fans/lights, and water taps are off while leaving the laboratory.
• P Follow the biosafety guidelines suggested for handling and disposal of the hazardous chemical.

Introduction

Several hazardous chemicals, microbes, and techniques like recombinant DNA technology are used for research and development work in research Institutes and universities. Therefore, the researchers, technical, helping and supporting staffs as well as the laboratory outputs must be kept free from contaminations. Thus, biosafety is necessary to prevent unintentional exposure/ release of hazardous chemicals, microbes/pathogens, and toxins to the laboratory staff and environment. Following the biosafety guidelines and ethical considerations are essential to maintain safety standards for the researchers as well as the environment. This chapter deals with various biosafety measures and ethical issues to be strictly followed while working in a research laboratory. The required safety level needs to be determined as per the chemical/biological materials are used in the laboratory to keep the researcher/environment safe for sustainable research and developmental activities.

Biosafety

The term biosafety is used to describe the procedure and policies needed to ensure environmental and personal safety. Biosafety refers to the containment principles, technologies and practices that are implemented to prevent unintentional exposure to pathogens, toxins, or their accidental release into the environment. A fundamental objective of any biosafety program is the containment of potentially harmful chemical/biological agents as precautionary safety measures.

Why Biosafety?

With the increasing number of countries adopting molecular biology tools and techniques in life science research and development activities, especially in the areas of agriculture and medicine, the biosafety issues are gaining importance to ensure biological safety for the public and the environment. Conducting research in safe and sustainable manner is not only a personal requirement but essential collective efforts to ensure biological safety for a clean and safe environment. This requires rules, regulations, monitoring bodies, and awareness among the workers as well as the public. Therefore, biosafety per se is an integral part of laboratory research and requires awareness among the researchers so that biological safety can be well taken care from the grassroot level.

For a researcher, laboratory is another home where he/she spends much time every day. As we take care of cleanliness, safety, and design of our home, we must take care on these aspects of laboratory too. It is the responsibility of the laboratory personnel to ensure cleanliness/safety of the laboratory. Since every laboratory chemical and equipment have the potential to cause harms if not used with appropriate precautions. Hence, as a laboratory user, it is our responsibility to follow the basic safety/biosafety rules and regulations set up by the Principal Investigator of the laboratory, Institutional Biosafety Committee, as well as by other regulatory authorities, if any. We must be aware of the general/personal protective measures, cleanliness and laboratory waste disposal prior to joining a research laboratory. In this chapter we will briefly discuss about personal safety, chemical safety, cleanliness, waste disposal, etc.

A. Personal Safety

1. Personal Protective Equipment (PPE)

Personal protective equipment (PPE) is special arrangement to protect the worker from specific hazards because of using a hazardous substance. PPE does not reduce or eliminate the hazard, but protects the wearer from exposure to hazardous material. PPE should not be reused or brought outside the laboratory as these might increase the risk of spread hazardous material or contamination. Used PPE must be kept inside the laboratory in a designated waste storage area adjacent to the work area.

Introduction

Proper electrical wiring and earthing are crucial for laboratory safety and functionality. A laboratory houses several installations/equipment that demand stable/continuous power supply. Inappropriate or faulty wiring and earthing may lead to significant risks, including electrical shocks, damage to equipment, and even incidence of fire. Therefore, understanding the requirements of electrical wiring and earthing is essential for designing, construction, and maintenance of laboratory. This chapter aims to provide a basic knowledge of electrical wiring and earthing necessary for a laboratory. Importance of earthing in mitigating electrical hazards and ensuring integrity of electrical installations are also described.

Basics of Electrical Wiring

Basic understanding of electrical wiring is necessary for laboratory safety and efficient electrical functioning. The potential difference between two electrical points (negative and positive) is known as Voltage, which drives the flow of electric current. This is measured as volt (V). The rate of flow of electricity in a circuit is known as Current (I), which is measured as ampere (A). Current can be either direct (DC) or alternating (AC). Opposition/resistance experienced in the flow of current in circuit is known as Resistance (R) and it is measured in ohms (Ω). Higher the resistance means lesser the current flows at a given voltage. The rate at which electrical energy is transferred/utilized in an electric circuit is known as Power (P), which is measured in watt (W). The Power can be calculated as the product of voltage and current (P = V × I).

Introduction

We come across a variety of chemicals every day in a laboratory. While some of them are relatively safer, others are extremely hazardous. Thus, it is very important to know the potential hazards that can be caused by the chemicals present in the laboratory and the safety measures needed to be followed to avoid the hazards. Additionally, we should also be aware of the steps necessary to be taken on such hazardous situations. According to IUPAC, a hazardous chemical mean the chemical which has the potential to cause damage in various ways. In a laboratory, there are chemicals that might be irritant, carcinogen, respiratory fibrogen, systemic poison, asphyxiant, flammable, etc. which have the potential to cause hazard to the workers or environment. Therefore, it is important to use appropriate procedure to reduce the risk and protect the health/safety of laboratory personnel, the public and the environment.

Types of Hazards Associated with Chemicals

Chemical hazards: The hazards caused by chemicals can be broadly classified into three categories.

1. Health hazard: This is also known as toxic effects of chemical. The risk associated with a chemical in laboratory depends on the extent of exposure and the inherent toxicity of the chemical. Many chemical reactions form the product that might be much more toxic than the reacting chemicals. For example, inadvertent mixing of formaldehyde (a common tissue fixative) and hydrogen chloride results in the generation of bis(chloromethyl) ether, a potent human carcinogen. For most of the chemicals, the route of exposure (through skin, eyes, gastrointestinal tract, or respiratory tract) is more important (Figure 17.1) that must be considered during risk assessment.

Introduction

Flowering plants (Angiosperms) are identified by their morphological characteristics like flower, fruit and seeds that help their taxonomic characterization into different species, genera, families and orders. The floral characters which are used for taxonomic characterization of plants include the type of inflorescences, perianth structure, floral symmetry, arrangement of floral leaves, type of androecium, stamens, gynoecium, carpel, ovule, etc. The type of fruit and seed also play important roles in taxonomic classification. In this chapter we will briefly discuss about various morphological features of flowering plants (in botanical terms) that are useful in taxonomy.

Nomenclature of Plant

Nomenclature of plant is the formal and scientific naming of plants. It is related to but distinct from taxonomy for grouping and classifying plants and provide botanical name.

The scientific/botanical nomenclature started with Species Plantarum by Carolus Linnaeus in 1753. Botanical nomenclature is governed by the International Code of Nomenclature for algae, fungi, and plants (ICN), which replaces the International Code of Botanical Nomenclature (ICBN).

ICBN and ICN

International Code of Botanical Nomenclature, (ICBN) was adopted by the VIIth International Botanical Congress held in Stockholm, July1950. While the International Code of Nomenclature for algae, fungi, and plants (ICN) was adopted by the XVIIIth International Botanical Congress held in Melbourne, Australia in July 2011.

Introduction

Test of pollen viability (extent of pollen viability) is important in pollination experiments to ensure fertility/seed setting. It is also important in monitoring state of pollens during storage in ecological/ taxonomic studies as well as during research on pollen biochemistry, genetics, stigma interactions, and incompatibility studies. Pollen viability refers to the ability of pollen to accomplish post-pollination events to achieve fertilization. Pollen viability test has gained importance particularly in plant breeding and hybrid seed production experiments/ practices. In this chapter, you will be acquainted with the basics of pollen viability test. However, for more professional knowledge on the topic readers are suggested to refer specialized books.

Pollen Viability Test

It is important to know the extent of viability of pollens used in pollination experiment, in finding fertility of a plant of interest, to monitor state of pollen on storage, in ecological/taxonomic studies, as well as in research on biochemistry, genetics, stigma interaction, incompatibility assay, etc. of pollen. Several pollen viability tests have been developed time-to-time. It is recommended to initially use different tests for a given pollen type to find out the most appropriate method that provides reliable result on pollen viability. It is important to note that every procedure requires standardization of protocol according to the sample used. Following different tests are used for assessing pollen viability.

1. In-vitro Pollen Germination Test

It is a simple, speedy, and quantitative test for assessing pollen viability. In this method, a collection/sample of pollen is germinated in vitro and observed under microscope. The percentage of germinating pollens (producing pollen tube) after appropriate treatment and a given period of time is determined. The percentage provides an index of viability of the pollen sample.

Introduction

Seed is a living entity (mature ovule) consisting of a tiny plant in embryonic stage along with the stored food protected by an outer layer known as seed coat. Seed is an important biological input in agriculture, which is economically important for successful crop husbandry. Cotyledons serve as the source of nutrients (proteins, carbohydrates, and oils) that not only nurture the plant at the early developmental (seedling) stages of plant but also the vegetarian (heterotroph) organisms. Being one of the important inputs in agriculture, viability of seed needs to be assessed to know the germination percentage of seeds used to raise a crop. Since viability of seeds may deteriorate on storage depending on the storage conditions. Therefore, often it becomes important to test viability/germinability and vigour of seeds before being used for raising a crop or conducting an experiment. In this chapter, we briefly discuss about the different methods which can be used for testing the viability of seeds.

Seed Viability

Seed viability can be defined as the capability of the seed to germinate and produce a normal seedling within a specified period. In other words, seed viability indicates how many seeds in a lot are alive and could develop into plants under appropriate field conditions. It is very important that most of the stored seeds are capable of germinating when sown in a field. They must possess higher viability at the beginning of storage and maintain it over the storage. A seed with high initial viability survives longer in storage; however, the viability deteriorates slowly at first and then rapidly as the storage prolongs. It is important to know the duration of storage after which a considerable decline in viability occurs under given storage conditions so that to take necessary action to revive the seeds. Excessive deterioration in vianility of seeds may lead to loss of the seed/material/ germplasm.

Introduction

Tissue culture refers to the technique of growing cells, tissues, or organs under controlled, artificial conditions. This allows propagation of tissues from a small sample of cells/tissues under sterile conditions. Plant tissue culture is widely used for various purposes, including the production of disease-free plants, rapid multiplication of plants with desirable traits, genetic modification, conservation of rare or endangered species, etc. Tissue culture is a valuable technique because it enables consistent and large-scale production of plants, ensures genetic uniformity, and allows manipulation of plant biology at cellular level. In this chapter, we will discuss about the major types of tissue culture techniques and their advantages, disadvantages, as well as applications in brief.

Different Types of Tissue Culture

Callus culture involves induction and growing of unorganized, undifferentiated cell masses from an explant on a nutrient/culture medium. The part of plant used to induce callus is called explant which can be tissue, organ or part of a plant.

Organ culture is used to maintain or grow an entire organ or a part of organ in vitro. It preserves the structure and function of the organ for studies or transplantation.

Embryo culture involves isolating and growing embryos from seeds or ovules in a nutrient medium. It is used to bypass seed dormancy or rescue embryos from hybrid crosses that may not develop naturally.

Cell suspension culture involves growing dispersed cells or small cell clusters in a liquid medium under continuous shaking. This is used for large-scale production of cells, secondary metabolites, and studying cell biology..

Introduction

Different types of culture media are used for culturing microbes and plant tissues. Preparing as well as sterilizing them properly following appropriate method with safety for the desired purpose are important parts of research work. In this chapter, we will discuss briefly about different types of media, their composition, preparation and sterilization in laboratory for microbial or plant tissue culture.

Culture Medium

Ingredients mixed to prepare solid, liquid or semi-solid substance to support the growth of microorganism or plant tissue in vitro (outside the body) is called culture medium. Frequently, it becomes important to grow microorganism or plant/animal tissue in vitro for any or all of the following purposes:

• To study characteristic features or properties of microorganisms.

• To identify the causal organism(s) of infection in a clinical sample, so that appropriate remedy can be determined.

• To culture/multiply microorganism or plant/animal tissues in vitro for research or commercial purposes.

• To prepare/produce biological molecules, vaccines, antigens, proteins, metabolites, etc. in vitro for research or industrial purposes.

Introduction

Use of acid/base is common in laboratories for experiments like titration and proper safety in their handling laboratory is necessary. In this chapter we will briefly discuss about the safety measures necessary to be adopted while using acids/bases in laboratory for any experiment.

Handling of Strong Acid/Base

There are four major points that need to be taken care of while working with strong acids or bases that might be quite harmful if any accident happens.

1. Protect Yourself

Wear a lab-coat and acid-resistant gloves: Always use lab-coat/apron and make it sure that the sleeves properly cover your arms. More importantly, use gloves made of acid-resistant materials and also wear shoes to protect your feet while wearing long-pants/jeans. Female with long hair, must tie her hair properly (with rubber band) to avoid any disturbance caused by appearance in between.

Wear safety goggles: It is important to wear safety goggles to protect your eyes using larger and fitting goggles that cover front as well as sides of your eyes. Always use properly fitting and adjustable goggles because smaller/ loose goggles may not protect the areas around your eyes against accidental acid spills and it may fall down from your face, if it is too loose.

Locate emergency wash area: There must be an emergency washing area nearby the work place or laboratory containing lab spill management kits. All the workers in the laboratory must be aware of using the kits and emergency plans.

Neutralize acid: Don’t panic even if an acid or chemical spill happens. Stay calm and act as quickly as possible. In case an acid spills, neutralize it by using sodium bicarbonate so that they can be cleaned and disposed safely. If acid comes in contact of skin, wash it off under running tap water but do not use/ apply sodium bicarbonate on skin.

Introduction

Measurement of pH of a solution and preparation of different types/strength of buffers are common practices in a laboratory, particularly in the Biochemistry and Molecular Biology laboratories. Therefore, in this chapter you will be acquainted with the procedures of measuring pH of a solution as well as preparation of some of the commonly used buffers in a laboratory.

Potential or Power of Hydrogen Ion (pH)

Measurement of pH of a solution is measuring the molar concentration of hydrogen ion [H+] in the solution, which is an indication of the acidity or basicity of the solution. The symbol pH stands for “power of hydrogen” and numerical value for pH is just the negative logarithm of the molar concentration of hydrogen ions [H+]. The ionic product of water (Kw ) is the basis for the pH scale. The total hydrogen ion concentration from all the sources and it can be experimentally measured.

The pH scale designates the concentration of H+ (and thus of OH−) in an aqueous solution, which ranges between 1.0 M H+ and 1.0 M OH−. The term pH is defined by the expression

Thus, the ionic product of water makes it possible to calculate the concentration of H+, with given concentration of OH−, and vice versa. The value of 7 for pH of a neutral solution is not an arbitrarily chosen figure; it is derived from the absolute value of the ionic product of water. A solution having pH greater than 7 is basic, and the concentration of OH− is greater than that of H+. A solution having a pH less than 7 is acidic.