eChapters

Introduction

Cell surface components such as adhesins, polysaccharides, and proteins play major roles in the adherence of probiotic bacteria to the intestinal epithelium, interactions that might lead to pathogen exclusion and immunomodulation of host cells. Probiotic bacteria have the ability to produce exocellular polymers called Exopolysachcharides (EPSs). These exocellular polymers present in the surface of many lactic acid bacteria, including Lactobacilli.The adhesive properties of lactobacilli are directly linked to their surface properties which are influenced by the structure and composition of their cell wall. The cell envelope of lactobacilli, bacteria, is composed of the bilipidic plasma membrane with embedded proteins encompassed by the cell wall. The bacterial cell wall consists of a thick multilayered sacculus made of peptidoglycan (PG) with teichoic acids (wall teichoic acids (WTA) and/or lipoteichoic acids (LTA)), exopolysaccharides (EPS), proteinaceous filaments called pili, and proteins that are anchored to the cell wall through different mechanisms (Figure 1).

Some species of lactobacilli possess an additional paracrystalline layer of proteins surrounding the PG layer, referred to as the S layer. These macromolecules together may play crucial roles in determining species and strain-specific characteristics of lactobacilli by influencing host-microbe interactions and microbial adaptations to the changing host environment. Several studies implicate that cell surface components, either individually or collectively, involve in host-microbe interactions. Lactobacilli beneficially affect the host in many ways by producing abundant variety of EPSs through the activity of extracellular glycosyl transferases. Approximately 30 species of lactobacilli are described as EPS producers. Among them, the best known are L. casei, L. acidophilus, L.brevis, L. curvatus, L. delbrueckii bulgaricus, L. helveticus, L. rhamnosus, L. plantarum, L. johnsonii, etc.

Ex.No.1. MICROBIAL SPOILAGE OF FRUITS

Fruits are living structures that continue to perform metabolic reactions and sustain physiological processes for a considerable length of time after harvest. The post-harvest infection of fruits by microorganisms may cause serious losses of fruits both in terms of quantity and quality. Initial infection which may be the direct result of mechanical or insect damage is followed by invasion of fruit tissues by secondary infection by common saprophytic organisms. . Many bacteria and fungi, for instance, are involved in decay after harvest. Typical fungi include Alternaria, Botrytis, Colletotrichum, Fusarium, Monilia, Penicillium, and Rhizopus. These fungi are generally weak pathogens, in that they usually infest only weak or damaged fruit. Molds are the most predominant spoilage organisms in fruits. Fungal rots in fruits like apple show brown or cream coloured areas.

Bacterial soft rot caused by Erwinia carotovora is wide spread in fruits that are not very acid. Fruits like straw berries, melons are in direct contact with moist soil and become infected from that source.

Introduction
This method consists of mixing the microorganisms in a small amount of India ink or nigrosine and spreading the mixture over a clean slide. These two pigments are not really bacterial stain and do not penetrate the microorganisms. These pigments make the background dark thus making organisms transparent against dark background. This method is useful for determining cell morphology and accurate size as no shrinkage of cells due to heat.
Materials
1. Bacterial culture (B. subtilis)
2. Microscopic slides (Clean, Grease Free)
3. Inoculating needle
4. Cedar wood oil
5. Microscope
6. Xylene

A. Gram’s Staining
Introduction
The most widely used staining procedure in microbiology is the Gram stain, discovered by the Danish scientist and physician Hans Christian Joachim Gram in 1884. Gram staining is a differential staining technique that differentiates bacteria into two groups: gram-positives and gram-negatives. The procedure is based on the ability of microorganisms to retain color of the stains used during the gram stain reaction. Gram-negative bacteria are decolorized by the alcohol, losing the color of the primary stain, purple. Gram positive bacteria are not decolorized by alcohol and will remain as purple. After decolorization step, a counterstain is used to impart a pink color to the decolorized gramnegative organisms.
The Gram stain procedure enables bacteria to retain color of the stains, based on the differences in the chemical and physical properties of the cell wall.
1. Gram positive bacteria: Stain dark purple due to retaining the primary dye called Crystal Violet in the cell wall. Example: Staphylococcus aureus
2. Gram negative bacteria: Stain red or pink due to retaining the counter staining dye called Safranin

1. Introduction

India is a land of large varieties of fruits and vegetables due to its vast soil and climatic diversity. India is the second largest producer of fruits and vegetables in the world, with an annual production of fruits 45.5 Million tonnes and vegetables 90.8 Million tonnes. It contributes 10.23% and 14.45% of the total world production of fruits and vegetables (F&V) respectively (NHB, 2010). Though fruits and vegetables are grown only on 7–8% of gross cultivated area, these contribute more than 18.8% of the gross value of agricultural output and 52% export earnings from total agricultural produce. These have a high export potential of 20-30 times more foreign exchange per unit area than cereals due to higher yields and higher price available in the international market (Sen et al., 2012). According to estimates, the per capita consumption of fruits and vegetables in India is only around 46g and 130g which are far below the stipulation of a minimum of 92g and 300g respectively as recommended by the Indian Council of Medical Research and National Institute of Nutrition. The low availability is mainly due to considerably high post-harvest losses. About 15-20% of the fruits and vegetables are wasted every year in the country, due to poor post-harvest management and lack of infrastructural facilities for processing and storage, amounting to a revenue loss of over Rs. 625 billion.

The sensorial, nutritional, and organoleptic quality of fresh produce starts deteriorating after harvest as a result of soon after harvest the altered plant metabolism and microbial growth. The quality deterioration is the result of produce respiration and transpiration, senescence, ripening-associated processes, wound-initiated reactions, development of postharvest disorders, microbial proliferation leading to postharvest quality loss. Therefore, it is necessary to understand the respiration and ripening processes and the physiological and biochemical changes during ripening. The best way to reduce respiratory metabolism is to reduce the storage temperature. All biological processes proceed more slowly at lower temperature (Yahia, 2007). In general, O₂ levels must be reduced to less than 10 % to see a reduction in the rate of respiration. Respiration rate is temperature dependent; so, as the temperature reduces the amount of oxygen required also drops. Therefore, oxygen levels must be substantially reduced at low temperature for it to affect the rate of respiration. It must however, be ensured that O₂ levels do not fall below extinction point as anaerobic respiration and decay may occur. Tolerance to low O₂ varies between species and cultivars and is also time dependent. Commodities may be able to tolerate low O₂ for short periods of time. Low O₂ can also be tolerated when CO₂ is low.

1. Algal Biomass

Algae is defined as heterogeneous assemblage of organisms that range in size from tiny cells to giant seaweeds, thus algae mostly are photosynthetic species which include eukaryotes and prokaryotic Cyanobacteria (blue-green algae) and live in aquatic habitats. They are ubiquitous in nature and are found in variety of environments ranging from soil, sand, marshes, brackish water, sea and fresh water. Most of them are capable of manufacturing their own food.

Microalgal research is offering wide potential in food biotechnology and is gaining more attention because of presence of wide range of bioactive and nutraceutical properties and added health benefits.The increasing population is one of the major growing concerns of today’s world. To meet the nutritional requirement of the rising population, researchers are looking for optional sources for food which are easy to cultivate, cost effective and produce large amount of bioactive compounds useful to prevent major diseases. Chlorella is unicellular green alga that is a potential source of nutrients like carotenoids, minerals, vitamins, and polyunsaturated fatty acids. Especially, omega-3 fatty acid and carotenoids have received considerable interests due to their roles in the prevention of chronic diseases. Lutein and zeaxanthin from Chlorella have been shown to be associated with eye health and function. Beta-carotene is known to reduce the risk of CVDs and certain cancers. Animal studies also demonstrated that Chlorella supplementation reduced the serum cholesterol levels.

Similarly, the blue green microalgae Spirulina, possesses diverse biological activities and nutritional significance. Regular consumption of Spirulina fortified food improves immune system by increasing phagocytic activity of macrophages, stimulating the production of antibodies and cytokines, increase accumulation of NK cells into tissue and activation and mobilization of T and B cells. Spirulina have also shown to perform regulatory role on lipid and carbohydrate metabolism by exhibiting glucose and lipid profile correcting activity in experimental animals and in diabetic patients. They were also found to be active against several enveloped viruses including herpes virus, cytomegalovirus, influenza virus and HIV. They are capable to inhibit carcinogenesis due to antioxidant properties.

    1.    Wohler (1828) synthesized the first organic compound, urea from inorganic components.

    2.    Emil Fischer (1894) demonstrated the specificity of enzymes and the lock and key relationship between enzyme and substrate.

    3.    Michaelis and Menten (1913) developed kinetic theory of enzyme action

    4.    Watson and Crick (1953) Proposed the double-helical model for DNA structure

    5.    Ingo potrykus (1999) Golden rice- rich in b-carotene.

    6.    Glyoxysomes : Glyoxysomes are temporary in that they occur during transient periods in the life cycle of a plant such as in certain beans and nuts which store fats in their seeds as energy reserves. Glyoxysomes appear in the first few days after seed germination in endosperm cells and associate closely with lipid bodies. They disappear after the storage fats are broken down and converted into carbohydrate.

23.1 Introduction
Various products can also be produced by emulsions that contain fat globules. For instance, cream is produced when milk contains concentrated fat. This cream can be further processed to produce butter, ghee, and butteroil. “Milk fat, ghee, butter oil, anhydrous milk fat, and anhydrous butter oil are fatty products that are exclusively derived from milk or products obtained from milk, or both, through processes that result in the almost complete removal of water and milk solids, not fat.” The method of manufacturing (FSSAI standards) has notably influenced the flavour and physical structure of ghee.

23.2 Types of Fat Rich Dairy Products
23.2.1 Cream or Malayi
“Cream is a yellowish component of milk that naturally rises to the surface when milk is allowed to stand. It is rich in fat globules.” Cream is mechanically separated in the dairy industry. The size of the fat globules is diminished as a result of homogenisation of cream, which renders the product less suitable for whipping.
Cream can be defined as
1. That portion of milk, which is rich in milk fat
2. That portion of milk into which has been gathered and which contains large portion of milk fat
3. When milk fat is concentrated into a fraction of the original milk that portion is called cream.

Fat-rich dairy products such as cream, makhan, butter, butter oil and ghee have played a pivotal role in growth and development of dairy industry in India as well as abroad from the time immemorial. The reference of makhan and ghee has been found in old Indian scriptures. Makhan was popularized by Lord Krishna about 5000 years before. In the traditional Indian dietary regimen, milk fat in the form of malai (cream), makhan (freshly churned butter) contributes significantly towards nourishment of people of almost all ages.

Indian dairy industry, over the years, has been converting surplus milk for the manufacture of fat-rich dairy products, especially ghee and butter with skimmilk as a byproduct because of technological and economic reasons. More than one-third of the total milk production is being utilized for the production of ghee and butter wherein milk lipids, the most expensive constituents of milk are conserved and preserved. Milk lipids play many diverse roles, some of which are essential for human health. Many of the desirable flavour and textural attributes of dairy products are due to their lipid components, consequently, butterfat has, traditionally, been highly valued. Significance of fat can also be realized from the fact that the consumer’s perception of food quality is largely based on the perceivable rich taste. Unfortunately, milk lipids are subject to chemical and enzymatic alterations which can cause flavour defects referred to as oxidative and hydrolytic rancidity, respectively. The storage stability of high-fat products is strongly influenced by these changes.

Procedure: Measurement of smoke point of given oils: Heat the given oils in a deep frying pan till fumes come out from oil. Record the temperature when the fumes come out and noted it as a ‘smoke point’. Cool the oil by keeping oil pan at room temperature. After cooling repeat the heating of same oils 2nd and 3rd time to measure the smoke point and record the observations

Introduction

Fermentation is one of the oldest food preservation techniques that can be traced back thousands of years. Fermentation is referred as one of method of biopreservation as it leads to extending the shelf life of foods that involve the use of microorganisms and their metabolites. (Ross et al., 2002) For millennia, the fermentation of food by using micro organisms has been used as a technique for ensuring shelf life extension and improving the quality characteristics, texture and flavour of food products as well as to develop new innovative products. The major bacteria used in food fermentation worldwide are lactic acid bacteria (LAB). LAB is a multitude of genera including Lactobacillus, Lactococcus, Streptococcus, Leuconostoc, Pediococcus, and Enterococcus. Lactic acid bacteria constitutes a diverse group of bacteria that enjoys the status of “generally regarded as safe” (GRAS) and “qualified presumption of safety” (QPS) by the Food and Drug Administration (FDA) and European Food Safety Authority (EFSA), respectively. In this respect, starter microorganisms are known to produce a wide array of antimicrobial and protein-acoustic compounds to inhibit the growth of undesirable flora in foodstuffs resulting in shelf life extension. (Hill et al., 2017).

INTRODUCTION

For thousands of years fermentation processes were largely natural phenomena exclusively identified with food preservation throughout the world. Fermentations can play a significant role in meeting the ever growing food requirements of world population providing benefits in the form of innovative food products with improved nutritional value and food quality along side better safety. Fermentations can be used to address a large variety of food related problems and in the development of processes to meet the demand for food. In the wake of estimations made by international organizations that global food demand and population would increase by two-fold by the year 2050, much emphasis has been placed over the ways and means to increase food production and preservation.

Introduction

Wine is a very unique fermented beverage. It was an essential part of various religious and cultural ceremonies in ancient civilizations; and continues to be considered as a ‘drink for elite’. The reason is that wine was considered as ‘Gift of God and ‘God’s nectar’ in religious text books, and medicinal value of the beverage is well supported by modern science. Wine is a beverage well tagged with its geographical origin. This concept originally comes from French concept of terroir; which explains that a varietal wine from a defined region possess distinctive qualities. Wine makers of the past enjoyed this definition of quality for better marketing opportunities. Today, globalization and advancement in wine science created empowered consumers who could clearly discriminate quality of wines. In addition to a product with high sensory qualities, consumers expect a healthful beverage produced in an environmentally sustainable manner. Enology research projects focus on such aspects; and this chapter is a glance on enology advancements and proven health benefits of wine, such as cardiovascular health gut health, dental health etc.

Introduction

Fermented foods are those foods which have been subjected to the action of microorganisms or enzymes that cause desirable biochemical changes and significant modification of the food (Campbell-Piatt, 1994). Fermented foods are usually produced using ingredients of plants or animals in combination with bacteria or fungi. The bacteria or fungi are either sourced from cultures maintained by humans or from the environment. The origins of fermented foods go back many thousands of years, with early evidence of the alcoholic fermentations of barley to beer and grapes to wine. Evidence suggests that fermented foods were consumed 7,000 years ago in Babylon (Battcock and Azam-Ali, 1998).Throughout the world, the fermented foods are used by the people. It plays a major role in the diet in many developing countries. Several traditional fermented foods are prepared in rural areas of many countries.

2.1. The Story of Fermentation

Human progress depends on man’s tact to tame the forces of nature. It calls for knowledge. Prior to Age of Science, our fund of such knowledge was gained by a process of trial, error and accidental discovery. Progress was due not so much to man’s understanding as it was to his unquenchable spirit, miraculous ingenuity and indomitable persistence.

At last, in the utmost urgency of struggle for a longer, healthier and more satisfying existence, humanity taught itself the scientific method, which has changed the world, and will change it more in the coming years. This chapter deals with one of the most momentous and far reaching of all scientific achievements: the ability to harness microscopic living creatures in the service of mankind. Thanks to the wonders of fermentation technology and biochemical engineering developed by scientists and skilled technicians in industrial, governmental and university laboratories around the world, billions of microbes are now at work in industry, producing substances that give us better food $#x0026; drink, better clothing $#x0026; shelter and better medicines. Thus, the human race has entered a new era of health, comfort and longevity.

In the Beginning Art without Science: The cave man discovered that meat allowed to stand a few days was more pleasing to the taste than meat eaten soon after the kill. The cave man’s earliest descendants learned that intoxicating drinks could be made from grains and fruits.

Fermentation plays a major role in our diet and give the basic foods like bread, yoghurt, wine and cereal and legume based traditional fermented foods. The development of fermented food products can be considered as one of the greatest achievements of human civilization and goes back thousands of years. It arose from the necessity to conserve foods, to make them more digestible and also more enjoyable. During the centuries, man learned by trial and error to conduct the fermentation processes by changing physical and chemical parameters in the basic food ingredients. It took much longer, however, to discover the cause of fermentation processes: only towards the end of the 19th century it was demonstrated, that baker’s yeast is the cause of alcoholic fermentation. Through the development of the technique of "pure microbial culture", the basis for a controlled use of microbes in food industry was established.

The unique feature of fermented foods is the vital role of microorganisms that bring about essential bio-trasformations of the substrates during fermentation contributing number of desirable properties such as improved product shelf life, enriched diet with improved flavor and texture, increased safety, enriched nutritional supplement and probiotic functions. Fermentation is a chemical reaction carried out by many types of microorganisms to obtain energy. Fermentation is a process for production of a product by means of mass culture of microorganisms. In fermentation, microorganisms breakdown complex organic compounds into simpler substances. Although chemical changes and microbial growth usually mean food spoilage, in some cases fermentation is desirable and microorganisms are actually added to foods. For example, in the production of beer, wine, and other alcoholic beverages, yeasts convert sugar into ethyl alcohol and carbon dioxide. In the making of yogurt and cheese, lactic acid bacteria convert lactose, a sugar found in milk, to lactic acid. Alcohol, acids, and other compounds produced in fermentation act as preservatives, inhibiting further microbial growth. In addition to its use with alcoholic beverages, cheese, and yoghurt, fermentation is used to produce yeast bread, soy sauce, cucumber pickles, sauerkraut, and other traditional fermented food products. The variation in agroclimatic conditions prevailing in India and diverse form of dietary culture of various ethnical groups produced traditional fermented foods such as idli, dosa (rice-legume based), vada, papad, wari, balle (legume based), bhatura, jelabi, nan (wheat based), paneer, shrikhand (milk based).

Introduction

Eradication of malnutrition and hunger (Sustainable Development Goal, SDG-2) are the major challenges to humanity. Availability of sufficient nutritionally balanced food is the prerequisite for ensuring global food security. Food security is also critical for achievement of SDG 1 (poverty eradication) and SDG 3 (health and well-being) (UNDP, 2019). Changing climatic conditions accompanied by global warming not only acts a major threat for achievement of SDGs but could actually lead to the reversal of the progress already made. The escalating global temperature has jeopardized the food security throughout the world. A great decrease in the global crop yield has been suspected even with a 2°C rise in temperature. On field, it is often associated with crop nutrient loss, less accumulation of micronutrients and low availability of micro-minerals like iron and zinc. Additionally, the effect of the rainfall and temperature has also been observed. The global food prices are projected to increase by 3–84% by 2050 due to the fluctuations in rainfall and temperature (Hoegh-Guldberg et al., 2018).

Millets are the small seeded grains which belong to family Grassacae/Poaceae. Traditionally, millets are used for the food and feed purpose and are among the oldest cultivated crops. The millet species of commercial importance are proso (Panicum miliaceum), foxtail (Setaria italica), barnyard (Echinochloa esculenta), finger (Eleusine coracana), little (Panicum sumatrense) and pearl (Pennisetum glaucum) in different regions of the world. In India, foxtail millet (priyangava), barnyard millet (aanava) and black finger millet (shyaamaka) have been mentioned even in the Yajurveda texts of 4500 BC (Morya et al., 2017). The records of consumption of foxtail and proso millet are as old as 2000 to 1000 BC in China. Cultivation of foxtail millet in Korean peninsula is documented around 3,500–2,000 BC. Foxtail millet is the oldest of all the cultivated millets and is believed to be originated in Southern Asia. From there, it was spread to Europian nations. In the west, cultivation of millets majorly started after 1850 (ICRISAT, 2019)

Introduction

Fermentation of food dates back to ancient practice of wine making. Fermented foods reflect culture and tradition of folklore and emphasis their close association with nature. Various methods employed for fermenting foods aimed in food preservation to reduce wastage; however lactic acid fermentation enhanced nutrition content and improved organoleptic properties. Experimental evidences on benefits on health and wellness on consumption of fermented foods are being reported. Fermentation process used for preparation of indigenous or traditional foods often depend entirely on natural microflora on the surface of raw material and containers used; Practices of fermenting foods are passed on as traditional know how to generations as a legacy. The fermented food manufacturers rely on commercial starter cultures, and highly reliant on indigenous materials as microbial collections are unique and highly variable according to region and products. Therefore, isolation of novel organisms, development of newer products and interactions are being explored using variety of fermented foods obtained through traditional processes

Introduction

The art of fermentation process is developed as far back as thousand years ago without understanding the role of microorganism in it. It is believed that the process of fermentation originates in Indus valley during 7000 – 8000 BC. Archives of harappan civilization showed that different clay utensils were used for the fermented product and the agriculture and animal husbandry were fully developed during that period. It is evident from the artifacts of Egypt and middle east that the practice of fermentation was common in that part (Farnworth, 2003). The earliest practice of cheese making is originated from Falcate between Tigris and the Euphrates rivers in Iraq, 8000 years back. Later, Egyptians developed the use of yeast in wine and bread making during 4000 BC (Ross et al., 2002). Further, Sumarians prepared beer from fermented barley during 1750 BC. Earlier the fermenting process was an artisan way without any knowledge of microorganism. However, the main turning point was in 1608, when Antony Van Leeuwenhoek first established the use of a microscope and revealed the existence of microorganisms. Another, major finding was Pasteurization by Louis Pasteur in 1861 AD and this was the first report that documented the role of microorganism in fermentation. Later, there was a shift observed in the production of fermented food from local communities to large scale industry. The health effect of fermented food was first reported by Roman historian Pliny, who reported that fermented milk is beneficial for gastrointestinal complains. Further, a French paediatrician proved that Bifidobacteria in food prevents infants against infection (Grill et al., 1995).