eChapters

Introduction

The “plant disease triangle” is a fundamental concept in plant pathology that illustrates the interaction between three essential components necessary for the development of a plant disease. Firstly, the host plant which refers to the plant species or cultivar that is susceptible to the pathogen. Host plants provide the necessary environment and resources for the pathogen to establish, grow, and reproduce. Secondly, the pathogen which is any organism (such as fungi, bacteria, viruses, nematodes, or other microorganisms) that can cause disease in plants. Pathogens can infect host plants through various means, including direct contact, airborne spores, soil-borne organisms, or vectors such as insects. And thirdly, the environment which encompasses all external factors that influence the interaction between the host plant and the pathogen. This includes abiotic factors such as temperature, humidity, soil moisture, light, and nutrient availability, as well as biotic factors such as the presence of other organisms (competitors, predators, symbionts) that may affect disease development.

The plant disease triangle illustrates that the occurrence of a disease requires the presence and interaction of all three components: a susceptible host, a virulent pathogen, and a conducive environment. If any of these components is missing or unfavourable, disease development may be inhibited or prevented. Any change in ecosystem can affect plant diseases, because plant disease is the result of interaction between a susceptible host, virulent pathogen and favourable environment. If any of the three factors are altered, the progress of any one disease can change. Although interactions among the three factors must be matched, weather is an important variable due to its dynamic behaviour.

Introduction

Rising temperature, decreasing precipitation, increased frequency in drought and flooding, etc. is making us realize climate change is not some cooked up story. Still there are people who believes climate change is not real. By definition, Climate change is a long-term shift in global or regional climate patterns. This will affect the life of people in many forms: health, food, water, etc. (Table 1). Agriculture is a highly vulnerable field to such weather and climate changes, they can have significant direct and indirect impacts on farm productivity and profitability. Climate change will have adverse effect on crop physiology, growth and chemistry and thus it will cause negative impact on agricultural production. Because of the variation in climate, moisture availability may change, which limits vegetation activity/plant growth, increase in drought/ heat/flooding will the crop growth and yield. Elevated CO2 does not have a profound effect on C4 plants. On the contrary, tissue nitrogen concentrations are higher for C3 species under increased CO2 . Intergovernmental Panel on Climate Change (IPCC) estimates that climate change and elevated CO2 is expected to increase competitiveness of invasive weed. IPCC also estimates that with 1°C rise in global temperature, 5 to 10 per cent yield reduction of major food and cash crops (Pachauri et al., 2014).

Introduction

Climate change is a deadliest hazard to life on Earth. Global temperature is rising due to greenhouse gas emissions from the usage of fossil fuels, this result in widespread agricultural and fisheries failure, the extinction of hundreds of thousands of species, and whole towns becoming uninhabitable (Lindwall, 2022). Rising global temperatures are severely killing humans and trees, forcing half of all species to move, worsening water-borne and respiratory ailments in people, and jeopardising millions’ food and water security (Isaacs-Thomas, 2022). Addressing these concerns necessitates a worldwide collaboration to minimise greenhouse gas emissions and promote sustainable behaviours. Climate change affects agriculture production, lowers crop nutritional value, and increases the frequency of extreme weather events. These interruptions jeopardise food production, availability, and access, especially in vulnerable countries. Microbes play an important role in soil health, nitrogen cycling, and ecosystem stability. They impact a variety of soil activities, such as organic matter breakdown, nutrient mobilisation, and plant growth stimulation. However, climate change poses a substantial danger to microbial communities, possibly changing their activities and altering ecosystem dynamics (Wang et al., 2022). Transitioning to a low-carbon economy requires a global effort. Mitigation strategies include enhancing energy efficiency, adopting renewable energy sources, electrifying industrial processes, implementing efficient transportation systems, and introducing carbon taxes or emissions markets.

Introduction

Climate change is widely acknowledged as the most significant contemporary challenge facing humanity. According to a recent report from the Intergovernmental Panel on Climate Change (IPCC, 2022), the situation has deteriorated further, with 3.3 billion people worldwide highly susceptible to climate change. Current unsustainable development practices are escalating the exposure of ecosystems and populations to climate-related risks. Human activities and their impact on the climate and environment are leading to unprecedented extinctions of animals and plants, resulting in a loss of biodiversity and posing a threat to life on Earth. The diversity of microorganisms is fundamental to maintaining a healthy global ecosystem, as the microbial world serves as the life support system of the biosphere. Although the influence of human activities on microorganisms is less conspicuous and less well understood, a significant concern is that changes in microbial biodiversity and functions will impact the resilience of other organisms, affecting their ability to adapt to climate change. Microorganisms play crucial roles in carbon and nutrient cycling, as well as in the health of animals (including humans), plants, agriculture, and the global food web. Microorganisms inhabit all environments on Earth occupied by larger organisms and are the exclusive life forms in certain environments such as the deep subsurface and extreme habitats. Dating back to the origin of life on Earth at least 3.8 billion years ago, microorganisms are likely to persist well beyond any future extinction events. Despite their vital role in regulating climate change, microorganisms are seldom the focal point of climate change studies and are typically overlooked in policy development. The challenge lies in understanding their intricate role in the ecosystem due to their vast diversity and varied responses to environmental changes.

Introduction

Climate change may advance more rapidly than anticipated, introducing novel environmental conditions for cultivating horticultural crops. The rise in atmospheric CO2 levels from non-renewable energy sources combustion drives global average temperatures, altering weather patterns worldwide with regional variations (Stocker et al., 2013). Currently, certain regions are already experiencing the effects of extreme weather events such as heat waves or frost, while prolonged droughts pose a growing threat to global food security elsewhere. Climate changes may yield both positive and negative outcomes, but they will undoubtedly alter production conditions and product quality (Kaufmann and Blanke, 2017).

Elevated temperatures can enhance the ability of air to hold water vapor, leading to increased water demand. This process can elevate evapotranspiration rates, potentially depleting soil water reserves and causing water stress in plants during dry periods. Fruit production is particularly vulnerable to water stress, especially in regions where trees lack irrigation. Numerous studies indicate that water stress diminishes crop yields and hastens fruit ripening (Henson, 2008).

Introduction

Agriculture is the backbone of every country. It largely depends on climate and its changes. Over the last few years climate change has affected crops, cropping systems, patterns and the ecosystem hugely. Climate change is caused by greenhouse gases in the atmosphere, which results in global warming (Aydinalp and Cresser, 2008). The rising temperature, melting of glaciers, loss in vegetation cover, frequent occurrences of drought and floods are some of the issues that have raised concern among the environmentalists. The change in temperature and precipitation patterns alters the traditional growing seasons. Climate change is expected to influence crop and livestock production, hydrologic balances, input supplies and other components of agricultural systems (Adams et. al., 1998). Vulnerability of climate change depends on physical, biological and socio-economic characteristics. Low income populations dependent on isolated agricultural systems are particularly vulnerable to hunger and severe hardship. These populations are already barely food-sufficient, even the slightest decline in yields could be very harmful in these areas. The most negative effects are foreseen in dry land areas at lower latitudes and in arid and semi-arid areas, especially for those reliant on rain fed, non-irrigated agriculture (Aydinalp and Cresser, 2008). The rise in temperature cause stress in crops and reduce yields and productivity. Crops that are sensitive to temperature during the growth stages are affected a lot. A shift in growing season is being seen due change in temperature. Irregular and altered patterns of rainfall are severely affecting the crops and water supplies for livestock, which in turn has increased the occurrence of floods and drought, causing soil erosion, disrupting harvesting schedules. Occurrence of new pests and diseases are seen in the places having warmer temperature. Altered temperature has altered the life cycle of pests and intensity of infestations. Rising of sea water level and increased evaporation leads to intrusion of salt water into fresh water making irrigation water more saline harmful to crops. Livestock are sensitive to heat stresses which in turn reduce livestock productivity and increase mortality rates.

1.1 Introduction

Water scarcity is seen as a major constraint to intensify agriculture in a sustainable manner as an attempt to meet the food requirements of a rapidly growing human population. The ever-increasing human population, climate change due to increased emissions of greenhouse gases (GHGs), and intensification of agriculture, are putting severe pressure on the world’s two major non-renewable resources of soil and water, and thus pose a big challenge to produce sufficient food to meet the current food demand. The present world population of 7.3 billion people is predicted to grow to over 9 billion by 2050, with the majority of this population increase occurring in developing countries, most of which already face food shortages. A 70% increase in current agricultural productivity will be required to produce sufficient food if these human population growth predictions prove to be correct. In this context, concerted efforts are being made globally to improve the effectiveness of water which will be used for enhancing the production of irrigated crops. Additionally, efforts are also being made to improve water harvesting and water conservation in rain-fed agriculture.

The injudicious use of saline/brackish water is all too often associated with the development of soil salinity, sodicity, ion toxicity, and groundwater pollution. Because of these negative effects, it is important to have a better understanding of exactly how the quality of water influences the management of irrigated agriculture, especially in arid and semi-arid regions.

2.1 Introduction

Water is the most precious gift of the nature and it is also the most crucial element for sustainability of the life in the earth. India is the largest user of freshwater in the world. India’s total water consumption alone is the higher than any other continents. The Agricultural sector is the largest consumer of water followed by the domestic sector and the industrial sector. The role of irrigation water as one of the most essential inputs for crop cultivation can’t be denied. As a traditional productive input, it ensures production by acting as an agent of insurance against inadequate and inconsistent monsoon. Ultimately the outcome provides agricultural production stability. Thus, irrigation is of prime importance in cultivation of vegetable crops as it ensures favourable water balance within the root zone in addition to natural precipitation. It fulfils the crop- water demand and improves the crop production and effectiveness of other agricultural inputs. It is also an important limiting factor of crop yield, because of its association with several factors of plant environment, which directly influence the crop growth and development (Yaghi et al., 2013). The various irrigation methods under different system of irrigation differ with regard to extent of control, timeliness and adequacy of supply of irrigation water for crop cultivation. Consequently, the economic benefits and the costs due to these irrigation methods vary among different irrigation systems. The dominant method of irrigation practiced in large parts of the country is surface irrigation (basin, border and furrow) where the entire soil surface is almost f looded without considering the actual consumptive requirements of the crops. Frequent over or under irrigation create the problems of water stress or water logging leading to reduced irrigation efficiency (less than 30%).

3.1 Introduction

Groundwater constitutes about 89% of the total fresh water resources in the planet. But in recent years, due to over exploitation of ground water and erratic nature of monsoon, there has been depletion of ground water across the world. Depletion of ground water has reached to the extent that it is virtually impossible to get the water table back. Even though there is a possibility of recharge of water from the other areas, the process is very slow and may take one year to replenish one meter. In view of this management of ground water has become one of the most significant issues in recent times. Added to it, there are also environmental problems such as aqua for mining, salt water intrusion, stream base flow reduction etc. For several reasons the efficient management of ground water resources through market mechanism has become difficult. Against this context the present article attempts to analyze the need for sustainable ground water management in India. The article also briefly discusses the concept of sustainable ground water management, factors affecting ground water availability, different approaches towards developing and using available ground water without adversely affecting the hydro geological balance. Further, the paper highlights strategies for sustainable groundwater management, including development of aquifers, rainwater harvesting and artificial recharge methods. The article offers some relevant policy recommendations for sustainable groundwater management in India.

4.1 Introduction

Water pollution is the contamination of water by an excess amount of a substance that can cause harm to human beings and/or the ecosystem. The level of water pollution depends on the abundance of the pollutant, the ecological impact of the pollutant, and the use of the water. Pollutants are derived from biological, chemical, or physical processes. Although natural processes such as volcanic eruptions or evaporation sometimes can cause water pollution, most pollution is derived from human, land-based activities (Figure 4.2). Water pollutants can move through different water reservoirs, as the water carrying them progresses through stages of the water cycle (Figure 4.1). Water residence time (the average time that a water molecule spends in a water reservoir) is very important to pollution problems because it affects pollution potential. Water in rivers has a relatively short residence time, so pollution usually is there only briefly. Of course, pollution in rivers may simply move to another reservoir, such as the ocean, where it can cause further problems. Groundwater is typically characterized by slow flow and longer residence time, which can make groundwater pollution particularly problematic. Finally, pollution residence time can be much greater than the water residence time because a pollutant may be taken up for a long time within the ecosystem or absorbed onto sediment.

5.1 Introduction

Out of the many climatic events that influence the earth’s environmental fabric, drought is perhaps the one that is most linked with desertification. Drought is a natural hazard originating from a deficiency of precipitation that results in a water shortage for some activities or some groups and is often associated with other climatic factors (such as high temperatures, high winds and low relative humidity) that can aggravate the severity of the event. Drought differs from aridity in that the latter is restricted to low rainfall regions and is a permanent feature of the climate. Drought occurrences are common in virtually all climatic regimes.

Widespread and severe drought conditions in Asia, Latin America and the Caribbean in 2000 have raised serious concerns about the continuing vulnerability of the world community to extended periods of droughts and water shortages. In 2000, major droughts affected much of south-eastern Europe, the Middle East, and the area through central Asia to northern China. Especially hard hit was Afghanistan, Bulgaria, Iraq and the Islamic Republic of Iran and parts of China. In North America, months of above-average temperature coincided with below-normal precipitation through northern Mexico and much of the southern and western regions of the USA, leading to one of the worst wildfires in the past 50 years.

By August 2001, much of Western Asia, Central Asia, and the Middle East was suffering the third year of a continuing drought that severely reduced many countries’ crop yields. The countries most affected were Afghanistan, India, Islamic Republic of Iran, Pakistan, and Tajikistan.

6.1 Introduction

Water deficit stress is a serious and frequently encountered abiotic stress in the terrestrial surface. Its deleterious effects on plant growth and productivity are well documented. Plant responses to water stress are believed to be complex as these operate at various levels of plant organization. Several in built physiological and biochemical mechanisms provide resistance to plants against stress. An understanding of the processes linked to these mechanisms is vital for optimizing crop growth and productivity under stress. Plants respond and adapt to water stress by altering cellular metabolism, thus invoking stress tolerance. Alteration in endogenous concentrations of growth regulators along with accumulation of osmolytes, modifications in antioxidant cascade, changes in protein profiles and induction of gene expression in plants under stress are important characteristic metabolic changes that invoke stress tolerance at the cellular level. Alteration in endogenous concentrations of plant bio-regulators under stress helps plants through better turgor maintenance and efficient water usage by influencing stomatal functioning, hydraulic conductivity and morphological adaptation. Progress made in plant adaptation to water stress is an outcome of advances made in analytical techniques on endogenous growth regulator analysis and powerful and reliable molecular and genetic techniques.

7.1 Introduction

Water is one of the most essential natural resources of the planet. Water is also the most critical resource for sustainable development in most of the developing countries. The quantities that are needed for drinking and sanitation purpose of humans are relatively small, and much larger quantities of water are required for many other purposes. It is essential not only for agriculture, industry and economic growth of a country, but also it is the most important component of the environment and ecosystem, with significant impact on overall wealth and nature conservation. Agricultural and industrial activities critically depend on a sufficient amount of fresh water that is withdrawn from rivers, lakes and groundwater aquifers. Currently, the rapid growth of world population along with the extension of irrigation dependent agriculture, development of industrial sector and climate change are stressing the quantity and quality of the natural water systems. Due to the increasing problems, human have begun to realize that they can no longer follow “use and discard” methodology either with water resources or any other natural resources. As a result, the need for water management has become evident. Climate change has already started to affect the hydrological cycle and the availability of freshwater for agriculture. So, proper water management practices play crucial roles in the food production and the management of ecosystems. Over the last century global irrigated area has increased more than sixfold from approximately 40 million hectares in 1900 to more than 260 million hectares in 2000. Today more than 40% of the world’s food comes from the irrigated cropland which is 18% of the total cultivated crop land. Irrigated area is increased by almost 1% every year and the demand for irrigation water will increase by 13.6% by 2025 (Jensen, 1993). On the other hand about 8-15% of fresh water supply will be diverted from agricultural sector to meet the increased demand of domestic and industrial use. On the other hand the efficiency of irrigation is very low, only 65% of the applied water is used by the crop (Fig. 1). So, to overcome shortage of irrigation water for agriculture, it is essential to increase the water use efficiency in the crop field and to use marginal water for irrigation.

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.

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.

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).

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.

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.

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

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.

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.

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

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).

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.

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

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.

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.

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.

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.

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.

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.

A key challenge for the years to come is feeding a rapidly growing human population while lowering the impact of food production on the environment. This is particularly true for low and middle-income countries where the demand for animal protein is likely to rise and further increase several folds in existing environmental changes (e.g., droughts, floods, extensive wildfires) have in recent years led to major food crises. To achieve these goals, food production not only needs to be increased, but most of all, good husbandry practices must be followed to reduce its negative impacts on the environment. Several studies have suggested that shifting the human diet towards increased consumption of f ish and seafood could be a solution to the need for protein that would sustain human and environmental health. In fact, fish and seafood consumption is forecast to increase by 27% on the horizon of 2030, mostly sustained by the aquaculture sector, which is expected to grow by 62% during the same period. The aquaculture industry contributes significantly to livelihood, food security, and poverty alleviation, with over 100 million people estimated to rely on aquaculture for their living. Fish is a cheap source of animal protein, and the current per capita consumption of fish in India is around 9 kg per annum, which is compared to the 11 kg recommended by the World Health Organisation (WHO). Presently, the contribution of the fisheries sector to the gross domestic product (GDP) and agriculture GDP has been estimated to be 1.2 and 4.2%, respectively.

Clinical Symptoms

• Gasping for air

• Discoloured gills with trapped materials

• Swollen gills with enhanced mucous secretion

• White to grey spots on the gills

• Diseased fish gather near the outlet of the pond

• Fish become lethargic and remain on the surface waters

Causative Agent: Flavobacterium branchiophilum, Cytophaga spp., Flexibacter spp.

Host (Common sp.)

• Goldfish • Koi carp

• Cichlids • Fighter fish

• Gourami fish

Clinical Symptoms • External haemorrhages

• Pale gills • Ascites

• Destruction of tissues in the kidney, spleen, and liver

• Excessive mucus secretion

• Skin hemorrhages with edema of the tissues

• Skin ulceration around the mouth and base of the fins

Causative Agent: Rhabdovirus

Host (Common sp.)

• Common carp

• Koi carp

• Crucian carp

• Silver carp

• Bighead carp

• Grass carp

• Goldfish

Clinical Symptoms

• Anchor worms can be seen with the naked eye

• Frequent rubbing or “flashing”

• Localised redness

• Inflammation on the body of the fish

• Tiny white-green or red worms in wounds

• Breathing difficulties

Clinical Symptoms

• White/brown cotton-like foci on the surface of the skin and/or gills

• Peritonitis

• Extensive haemorrhage • Necrosis

• Adhesions

The tissue/cell culture lab has the requirement of maintaining an aseptic environment. There are several aspects to the design of good tissue culture facilities. Ideally, cell culture work should be conducted in a single-use facility. Therefore, cell culture lab unit should be separated from the rest of the lab area. It can be either separate lab or a closed partition in a big lab. All new material should be handled as ‘quarantine’ material in a separate area, and materials known to be free of contaminants should only be allowed inside the culture room. In addition, the work surfaces should be thoroughly cleaned and disinfected between activities.

For most of the work involving non-infectious primary culture and cell lines, the laboratory should be designated to at least BSL2 facility. The location of the Biosafety cabinet is the most important point to consider. When operated correctly, the cabinet will provide a clean working environment, whilst protecting the operator from aerosols. Environmental monitoring with Tryptone Soy Broth agar settle plates inside the biosafety cabinet is a good indicator of the cleanliness of a cabinet. There should be no growth of bacteria and fungi after 3-5 days of incubation.

Things required for cell culture

1. Lab – having negative pressure (HEPA filter)

2. Safety cabinet (Hood/laminar flow)

a) Biosafety level 1 (BSL-1) – Basic level of protection. Agents handled under this cabinet are NOT infectious.

b) BSL-2 – Appropriate for moderate risk agents. Most of the non infectious cell cultures are done under this facility.

Working Principle: An autoclave is a sealed device that creates a different environment from the outside world under extraordinarily high pressure (Auto + clave = automatic locking). It uses moist heat or saturated steam under pressure to kill microorganisms and heat-resistant endospores. Its function is compared to that of a pressure cooker, i.e., steam is used to create a high pressure environment. However temperature of autoclave or steam sterilizer is generally maintained at 121°C for 15-20 minutes that is achieved by raising the pressure inside the device. This high pressure increases the boiling point of water and therefore raises the temperature that is too high for the microbes to survive, so used for sterilization.

Types of Autoclave cycle

1. Liquid cycle: Liquids take time to sterilize as they have high heat capacity. It is based on the principle of pressurized steam at 15 psi, 121°C temperature for 20 minutes. It has latent heat, which is 7× heat 100°C. Liquids and biological waste require slow exhaust to prevent the boiling over of super-heated liquids. This excess temperature is enough to hydrolyze the proteins of bacteria, viruses, or any other contaminant and therefore kill it. However, wet heat is seven times more intense than dry heat, so it requires less temperature than the dry heat cycle of the autoclave.

2. Dry Cycle: Here, steam is the sterilization medium, which penetrates inside the load and makes contact with all the surfaces, so a fast exhaust cycle is used. It has a high temperature of 160°C for 60 minutes.

In the realm of cell culture, the quality and composition of the growth media and reagents play a pivotal role in growth and sustaining cells in vitro. The preparation of these essential components requires precision, attention to detail, and adherence to the strict protocols to ensure optimal cellular growth, viability, and experimental reproducibility. This introduction serves as a foundational overview of the critical aspects involved in the preparation of media and reagents for cell culture experiments. Furthermore, the preparation of media and reagents for cell culture necessitates adherence to stringent quality control measures to ensure consistency and reproducibility across experiments. Validation of media batches, sterility testing, and periodic quality assessments are essential practic

What is primary cell culture?

In vitro culture of cells freshly obtained from the tissue is called primary cell culture. Unlike that of cell line, primary cell culture grows for a limited period of time. Examples, are primary mammary epithelial cells, primary hepatocyte culture.

Aim: To prepare the primary cell culture from chicken embryos.

Materials required for the primary cell culture

• 70% alcohol

• Cotton swabs

• Small beaker 20-50 ml

• Forceps

• Straight and required curved,

• 9 cm Petri dishes

• 11 day embryonated eggs

• Balanced Salt solution (BSS)

• 250 ml conical flask

• Magnetic centrifuge tubes

• Haemocytometer

• Growth medium with serum

• Culture flasks

What is keratinocyte?

The epidermal layer consists of keratinocytes, melanocytes, Langerhans cells, and Merkel cells. Their major function is to produce Keratinocytes which are the most abundant cells in the epidermis, keratin protein. Keratinocyte synthesizes major structural components of the epidermal barrier through a programmed process of differentiation. They are present in the innermost layer of the epidermis (Eckert et al?, 1989). The epidermis is composed of several layers, stratum corneum (horny layer), stratum lucidum (clear layer), stratum granulosum (granular layer), stratum spinosum (prickle cell layer), and stratum basale (basal layer), from the outermost to the deepest layer respectively. The process of maturation and movement of the keratinocytes from the stratum basale to the stratum corneum is what we call epidermal differentiation.

Procedures for isolation of canine karatinocytes

When studying skin cells, isolating keratinocytes from tissue samples, such as skin biopsies, is a critical initial step. Here is a general guide on how to isolate keratinocytes:

What is dermal fibroblast?

Skin fibroblasts are a type of cell found in the dermis, the middle layer of the skin. They are the most common cells in connective tissue and play a crucial role in maintaining the structural integrity of the skin. Fibroblasts are responsible for synthesizing and secreting extracellular matrix proteins such as collagen, elastin, and fibronectin, which provide support and elasticity to the skin. Additionally, fibroblasts play a role in wound healing by proliferating and migrating to the site of injury, where they aid in tissue repair and scar formation.

Aim: To prepare the primary cell culture of fibroblasts from canine skin.

Tools and instrument required for the procedure

1. Tissue collection box with ice cubes - To prevent contamination and maintain the stable temperature

2. Surgical Gloves - To prevent contamination

3. Tissue collection tube with 1X PBS - To collect sample

4. Laminar flow hood - To work in a sterile environment and prevent contamination

5. Cell culture dish - To digest the skin

6. Sterile 1000 μL and 200 μL pipette and tips-For accurate measurement and transfer of cell suspension and culture media.

7. Forceps - Fine-tipped forceps for handling and manipulating tissue.

8. Scalpel and blade holder - Scalpel for the dissection of skin tissue.

9. Surgical scissors - Chopping of the skin tissue

10. Falcon 70 μm cell strainer - To filter out undigested tissue debris and obtain a homogeneous cell suspension.

11. 15 ml and 50 ml Centrifuge tubes - To collect and centrifuge the digested cell suspension.

What is subculture?

In cell culture, a process called subculturing, or passaging, keeps cells alive and growing. It involves taking cells from an existing container, removing the used medium, and placing them in a new container with fresh nutrients. This allows the cells to continue multiplying and creates more cells for research.

Adherent subculture protocol Cells nearing 80% coverage need splitting!

1. Warm things up: Get your media, detachment solution, and incubator ready at 98.6°F (37°C).

2. Safety first! Disinfect your work area and supplies inside a biosafety cabinet.

3. Gently rinse: Remove the old media and wash the cells with a salt solution at room temperature. Be careful not to dislodge the cells.

4. Detach the cells: Add a pre-warmed enzyme solution to detach the cells from the flask surface. Keep an eye on them to make sure it works (usually 2 minutes). Note: Not all cells need this enzyme, and it can be harmful to some.

5. Stop the enzyme: Once all the cells are detached, add a culture medium with serum to neutralize the enzyme.

6. Collect the cells: Transfer the cell suspension to a tube, rinse the flask with fresh medium to get all the cells, and add it to the same tube.

7. Spin it down: Centrifuge the cell suspension for 5 minutes at room temperature (1000 rpm).

8. Get ready for counting: Remove the liquid, loosen the cell clump, and resuspend the cells in a fresh medium at a good volume for counting.

9. Count your cells: Use a hemocytometer to count the cells and determine their health.

Cell counting is the fundamental techniques in various fields of biology, medicine and biotechnology.

Importance of cell counting

1. Research: Cell counting is a critical component of biological and medical research. It helps scientists understand the behavior of cells in various contexts, such as studying cell proliferation, differentiation, and response to stimuli.

2. Disease diagnosis: Monitoring various medical conditions, including infections, autoimmune diseases, cancer and blood disorders. Complete blood counts (CBCs) are routinely used to assess the levels of different blood cell types and assess disease conditions.

3. Stem Cell Research: Cell counting plays a significant role in stem cell research, where precise quantification of stem cells is necessary for various applications, including regenerative medicine and tissue engineering.

4. Cell Culture: Cell counting is integral part to determine seeding density. Seeding of cells to available surface area for growth is important consideration for successful culture.

5. Cell-Based Assay: The cell-based assays are used for drug screening, toxicity testing, and evaluating the efficacy of new compounds. Accurate cell counts are essential to ensure the reliability of assay results.

Introduction

Peripheral blood mononuclear cells (PBMCs) consist of chiefly of lymphocytes and monocytes. Purified PBMCs are used in vitro to evaluate a variety of functions of lymphocytes viz; a) proliferation to mitogenic (PHA, Con-A) stimulation, b) monitoring of prior sensitization in antigen recall assays by scoring lymphocyte proliferation, c) immunophenotyping for surface markers as well as intracellular molecules in monocytes and lymphocytes etc. Activation of monocytes/macrophages by small molecules, cytokines and pathogen components can also be monitored. PBMCs can also be used for a variety of structural and functional studies for addressing issues in human immunology such as scoring for apoptosis and production of cytokines as well as other mediators in vitro.

Background information

Blood is a liquid mixture comprised of plasma, cells and proteins. The liquid portion, plasma, makes up half of the blood volume, and cells the other half. Isolation of lymphocytes is a common procedure in immunology and cell biology that involves separating and collecting lymphocytes.

Introduction

The protocol below describes the use of cell freezing methods involving an electric -80°C freezer for the cryopreservation of cell lines. ECACC routinely use a programmable rate-controlled freezer. This is the most reliable and reproducible way to freeze cells but as the cost of such equipment is beyond the majority of research laboratories the methods below are described in detail. If large numbers of cell cultures are regularly being frozen then a programmable rate-controlled freezer is recommended.

Materials

• Freeze medium (commonly 90% FBS, 10% DMSO or glycerol

• 70% (v/v) alcohol in sterile water

• PBS without Ca2+/Mg2+ • 0.05% trypsin/EDTA in HBSS, without Ca2+/Mg2+

• DMSO

Introduction

Structural changes in host cells by the viral infection is referred as cytopathic effect (CPE) and the responsible virus is called cytopathogenic virus. As a virus hijacks a cell to make copies of itself, the cell goes through distinct changes in its chemistry and structure. These effects are easiest to see in lab grown cell cultures. The severity of the damage caused by the virus depends on the specific virus itself, the type of cell it infects, how many viruses infect each cell (MOI), and other factors.

The early stages of this damage, called cytopathic effects (CPE), can be spotted directly under a microscope without any dyes or special treatments. While various types of CPE exist in living cells, some signs of viral infection, like inclusion bodies, require staining the cells to be visible. We can classify viruses based on how quickly they cause visible damage (cytopathic effect) in cell cultures. Viruses are typically considered slow if it takes 4-5 days to see this effect in cultures with a low starting dose of virus (MOI). On the other hand, viruses are considered fast if the damage shows up in just 1-2 days under the same conditions. It is important to note that at a high MOI all CPE can occur rapidly. So all decisions about rate of CPE appearance should be based on the lowest MOI that produces CPE

Principle

Isolating the virus in a laboratory is the gold standard for diagnosing many viral infections. Virus isolation is a highly specialised technique used to diagnose viral infections and produce a stock of viruses for research purposes. The isolation of the virus relies on the suitability of the sample, the ability of the cells to allow entry, the successful replication of the virus, and the release of viral particles from the cells. The choice of cell lines is dependent upon the suspected viral disease and the availability of such cell lines in the laboratory.

Materials required

Equipment

a) Biosafety cabinet level 2 (B2)

b) Carbon Dioxide (CO2 ) Incubator (set to 37°C with a 5% CO2 in air atmosphere)

c) Refrigerated centrifuge

d) Refrigerator

e) Inverted Microscope

f) Water bath

g) -86°C/-80°C deep freezer

h) Autoclave

i) Hot air oven

j) Filtration Assembly

Principle

The one-step growth experiment refers to a scientific methodology. A study aimed at observing the molecular events that take place during viral reproduction. This elucidates the fundamental features of the virus replication process and determines the optimal multiplicity of infection and timing for virus harvest. The initiation of modern bacteriophage research can be traced back to the development of the one-step growth curve by Max Delbrück and Emory Ellis in 1939, which utilised the Escherichia coli-T4 bacteriophage system. One step growth curve consists of three clearly defined phases 1. Latent period refers to the initial phase of a process where no significant changes or activities are observed. 2. Burst or Rise period is the subsequent phase characterised by a sudden increase virus particle. 3. Plateau period is the following phase where a stable and constant level virus replication.

Materials required