eChapters

Introduction

One of the greatest challenges facing the world is how to meet the nutritional needs of a growing human population that is projected to hit 10 billion by 2050. To meet the additional food demands imposed by the nearly 30% population increase, global food production has to increase by as much as 50% (FAO, 2017). This problem is further multiplied due to the imminent threat of an acute shortage of space to grow food, degradation of arable land, limited resources, climate change etc. in traditional ways (Yang and Kim, 2020). Between 1984 and 2016, India faced depletion of arable land at a rate of 0.12 % per year, which means we are left with land to live on, but shortage of food. Hence, to tackle these problems, there is a need to adopt intensive, environmentally friendly, multidimensional and sustainable food production systems like aquaponics (Dijkgraaf et al., 2019).

Aquaponics is a sustainable method of growing fish, and vegetables as well as f lowers (Mohapatra et al., 2020). It is becoming quite popular with individuals as well as commercial farming entrepreneurs, and governments interested in modern farming for year-round food production in different climatic conditions. Moreover, with this type of versatile farming, we can grow considerably more food with less water, land and labour than the traditional farming systems.

Fisheries: A glance

The fisheries sector is a complex enterprise that includes capture and culture based fisheries, including mariculture, coastal aquaculture, freshwater aquaculture, and cold-water fish culture that contribute to the country’s food, health, economy, exports, employment, and tourism. Fisheries and aquaculture continue to provide millions of people with food, nutrition, revenue, and a means of subsistence. Fish is an inexpensive and high-quality source of animal protein, making it an excellent choice for reducing hunger and malnutrition in the country. In 2020, global aquaculture production reached a new high of 122.6 million tonnes, including 87.5 million tonnes of aquatic animals valued at USD 264.8 billion. Inland aquaculture produced 54.4 million tonnes, while marine and coastal aquaculture made 68.1 million tonnes. Over time, the Indian fishing industry evolved and became a significant socioeconomic asset for the country (FAO, SOFIA, 2022) India is the world’s third-largest producer of fish and aquaculture, accounting around 16% of total inland and 5% of total marine fish production. In 2021 22. The overall marine and inland fish production in India was 162. 48 lakh tonnes, with 121.21 lakh tonnes and 41.27 lakh tonnes coming from the inland and marine sectors, respectively. The fishing industry is vital to the national economy and is a major contributor to the country’s foreign exchange gains. India’s fish production has risen from 56.56 lakh tonnes in 2000-01 to 162.48 lakh tonnes in 2021-22.The majority of fish produced is for human consumption, and this proportion is predicted to increase from 89% in the base year (2016-18) to 91% by 2028 (OECD/FAO, 2019). In 2021-22, frozen f ish, cuttlefish, and squid contributed 6%, 3%, and 5% of total marine product export value, respectively.

Introduction

Aquaculture has emerged as a viable business model having high growth potential for the entrepreneurs and start-ups in India. The rate of adoption of the aqua-business models for new age entrepreneurs has been very high in recent times. Some of the viable business models that are technology based and scalable have been attractive to the entrepreneurs are in the indoor aquaculture system like RAS, Biofloc systems. It offers many advantages like low space requirements, easy monitoring, higher level of intensification, easy management, short supply chain to the urban markets etc. These indoor aquaculture system are broadly defined as peri-urban aquaculture system offering high growth potential to the entreprenuers in India. The chapter provides a broad outline of the opportunities and recent trends of entrepreneurial developments through peri-urban aquaculture.

Entrepreneurship is the risk taking ability and readiness to venture into a new economic enterprise, along with all its challenges in order to make profit. One of the examples of entrepreneurship is the starting of new businesses in any field with business potential. In other words, entrepreneurship is the creation or extraction of economic value (Diochon et al. 2011; Gaddefors et al. 2017; Alvarez, et al. 2001). It is also viewed as a change, generally entailing risk beyond what is normally encountered in starting a business, which may include other values than simply economic ones. A person gets the benefits as he invests in one or more businesses, bearing most of the risks and availing most of the rewards. According to Shane (2000), entrepreneurship comprises both “enterprising individuals” and “entrepreneurial opportunities”[4]. An entrepreneur is generally seen as an innovator, a source of innovative ideas, services, and business.

Introduction

The Himalayan and peninsular regions of India comprise of diverse coldwater resources in terms of upland rivers, fast flowing streams, high, mid and low altitude natural lakes and reservoirs (Singh and Akhtar, 2015; Singh and Sarma, 2017; Sarma et al, 2018). The Indus, the Brahmaputra and the Ganga basins in the Indian Himalayan Region (IHR) is a domicile of over 4 % of the country’s population having rich repository of biodiversity, a natural habitat of over 8000 species of flowering plants, 816 tree species, 675 edibles, nearly 1740 species of medicinal plants and about 258 species of fish (Singh and Akhtar, 2015; Pramanik and Bhaduri, 2016; Singh and Sarma, 2017). IHR mainly encompasses the states of Jammu & Kashmir, Himachal Pradesh, Uttarakhand, Sikkim and Arunachal Pradesh where the diverse natural resource-base and wide climatic range, harbour huge inhabitants of endemic, indigenous and exotic coldwater fish species extending an enormous scope of developing upland farming practices (Singh and Sarma, 2017; Singh, 2020), game and ornamental f isheries of large extent (Zaidi et al., 2018). These can provide an excellent opportunities for augmenting livelihood security of upland people residing in the Himalayan terrain, where opportunities to earn revenue is difficult and hard to adopt the farming practices. Today, the Himalayan states offer a unique value proposition in coldwater fisheries to harness its potential. There is an increasing trend of coldwater fish production from the present level of 52,084 MT to 90 thousand MT by FY 2024-25 boosting current productivity from ~1 ton/ha to 3 ton/ha. Coldwater fisheries in the country is now occupying a key place among India’s freshwater fishes, contributing significantly to the nutritional security and job creation in rural hilly areas. More than 1.40 lakh fishermen in India directly depend on coldwater fisheries in hilly areas. In Himalayan region, aquaculture and fisheries represent an essential, often irreplaceable source of high quality animal protein crucial to the balance of diets in marginally food secure communities. The aquatic resources in the IHR are quite valuable for the development of aquaculture and fishery for food, sport, recreation and employment. In this paper, the author has attempted to present possible opportunities in coldwater aquaculture and fisheries which has great promises of future for job creation and nutritional security.

Introduction

Aquaculture and Fisheries remain important sources of food, nutrition, income, and livelihoods for hundreds of millions of people around the world including in India. Total fisheries and aquaculture production reached a record of 214 million tonnes in 2020, comprising 178 million tonnes of aquatic animals and 36 million tonnes of algae (FAO 2022). Global aquaculture production continued to grow amid the worldwide spread of the COVID-19 pandemic. Global aquaculture production consisted of; 87.5 million tonnes of aquatic animals predominantly used as food for human consumption; 35.1 million tonnes of seaweeds and other micro-algae for both food and non-food uses. Global consumption of aquatic foods has increased significantly. In 2019, global aquatic food consumption was estimated at 158 million tonnes and consumption increased at an average annual rate of 3.0 percent from 1961, compared with a population growth rate of 1.6 percent. In India, the total fish production during the year 20-21 is estimated at 14.73 Million Metric Tonnes (MMT) with a contribution of 11.25 MMT from the inland sector and 3.48 MMT from the marine sector. The fisheries sector contributes about 1.24% to the gross value added (GVA) and 7.28% to the agricultural GVA. In India, the changing consumer preferences, advancements in technology and income growth are the real drivers that augur well for further expansion and demand of food from aquatic origin. Against this backdrop, this sector is poised for unprecedented growth and opening various avenues of entrepreneurship.

Introduction

Fisheries and Aquaculture remains to be an important source of food, nutrition, income, and livelihood to millions of people. Fish is an affordable and good source of animal protein, a good option to reduce hunger and malnutrition in the country. India has bountiful resources comprising inland, brackish and marine water along with diverse fish and shellfish species. Indian Fisheries sector evolved gradually over the years and became an important socio economic attribute for the nation. India is the third-largest fish and aquaculture producing country and accounts for about 16% of total inland and 5% of total global marine fish production respectively. In 2021-22, India’s total marine and inland fish production stood at 16.24 million metric tonne (MMT), which includes 12.12 MMT and 4.13 MMT from inland and marine sectors, respectively. The annual growth rate in the marine sector was observed at around 18.7 %, whereas inland sector achieved more than 7.76 % during 2021 22 ( Table 17.1).The fishery potential of the country has been estimated to the tune of 22.31 MMT out of which Marine fisheries potential is 5.31 MMT and Inland fishery potential is 17 MMT. (Table 17.3).

Marine fisheries activities are spread along the country’s long coastline (8118 km) and in the vast Exclusive Economic Zone (EEZ 2.02 million km2). The inland resources are in the form of rivers and canals (0.19 million km), f loodplain lakes (1.0 million ha), ponds and tanks (2.43 million ha), reservoirs (3.15 million ha), brackish water (1.18 million ha), saline/alkaline affected areas (0.24 million ha), and such others etc., covering about 9 million ha, providing livelihood to 16 million (Table 17.2). In this direction sea cage culture may play an important role in rapid expansion of seafood production at global level. In present scenario, due to limited availability of fish fingerling we may not expand sea cage culture to the optimum level. Therefore, need of hour is to strengthen the available infrastructure and other needed backward and forward support system enhancing for fish seed production apart from developing market linkages with diversification of species.

I always feel I am very lucky and got a chance to live in this beautiful God gifted planet by blessings of God and whatever God fixed for me I am trying my best to do so that I can fulfil my journey and reach to my destine and finally will back to the same place where I came. My father was a Botany Lecturer, he retired from his job in the year 2009 and my Mother is a house wife. I have one brother and two sisters. All are married now and me my misses, my son (Calyx) staying at my native village with my parents after I came back from Indian Agricultural research Institute and my brother and two sisters are staying at Bhubaneswar. I have a very small family staying happily at my village. I have completed my schooling at my village school which is just 1 km away from my home. My village Garh Guatira is belongs to Nimapara block of Puri district of Odissha and it is just 50 km away from Bhubaneswar. My village is very small which is surrounded by agriculture field specially 90% area is under rice cultivation. So I spent my childhood with my natural village climate and maximum time I spent in the village surroundings with its natural activities. I got first class in Botany (Hons) with distinction from the Nimapara college and it was one of the best moments for me, at least one step I moved towards my destination.

An Overview of Fish and Shellfish Health Management in Aquaculture in India

Aquaculture in India in particular freshwater fish culture comprised mainly of Indian major carp cultivation has a long history. Since 1980s commercial carp culture expanded, particularly in Andhra Pradesh and in few years became an Asia model. Then on, it is slowly and steadily expanding to different parts of the country particularly Punjab, Haryana, West Bengal, Orissa and Tamil Nadu and Karnataka. Of late, culture of Pungasius (Basa) has come up on large scale in AP. In addition, there is also growing small scale commercial cultivation of catfish, murrels, tilapia and freshwater prawn(Macrobrachium rosenbergii). Aquarium fish breeding is also expanding to establish itself as an industry in India.

Brackish water culture has been practiced traditionally in India in Pokkali fields of Kerala, Bheries of West Bengal and Ghaznis of Karnataka since hundreds of years. From 1990 onwards, commercial shrimp culture came into existence along the coasts of India. High export value, support from government and short culture period encouraged private entrepreneurs to pursue shrimp farming in a big way. There is vast potential for fish culture in brackish water and at the present efforts are on to popularize culture of Asian seabass, mullets, Eutroplus and milk fish. Mariculture is still at infancy but increasing at a slow and steady rate.

Aquaculture health management has to be viewed from the incidences and outbreak of diseases due to growth and expansion of the industry. Aquaculture has expanded tremendously in the last three decades, growing at 6-7 % annually. Contribution of fish production from aquaculture in the country is around 60 % compared to 10 % it was 40 years ago. In few years to come, aquaculture contribution may go up to 80 % of total fish production. Today 2.43 m ha of freshwater and 1.15 m ha of brackish water are under culture. In addition, 8.62 m. ha of irrigated inland area mainly in Haryana, Maharastra and Karnataka which have become saline and unsuitable for agriculture are slowly being reclaimed for aquaculture. Farmers are growing acclamatized saltwater fish and shrimp spp in inland saline waters and a shift like this may favour pathogens with different virulence to cause disease. Overall, it can be seen that in addition to increased conventional areas in fresh and brackish water, aquaculture is being practiced in unconventional areas like saline soils. Performance of fish and shellfish in these non conventional water bodies in the long run needs to be studied from their health point of view.

Diseases are among the greatest deterrents to the sustained production in aquaculture. White spot disease (WSD) in penaed shrimp and epizootic ulcerative syndrome (EUS) in fresh and brackish water fishes are the well known devastating diseases with serious impact in aquaculture. Aquaculture medicine which broadly encompasses prevention and management of diseases in cultured aquatic organisms becomes a vital requirement for a sustained industry.

The four K’s essential for scientific aquaculture health management are knowledge about the disease process, knowledge about the pathogen, knowledge about the host and the knowledge about the environment. Disease development process is often complicated and involves host-pathogen- environment interactions. Knowledge about the pathogen, attaching and entering the host, deriving nourishment, reproduction, transmission, overcoming host defence barriers, etc are very essential. Susceptibility of a host to a pathogen is important which depends on host species, age, size, immuno competence and stress response. Besides knowledge about how the temporal and spatial aspects of environment stress the host and favour the pathogen to cause disease is vital

Disease Development Process

There exist a delicate balance between the host, pathogen and the environment. When this delicate balance gets upset, disease can result. Aquaculture environments can stress the host, favour the pathogen and result in disease development. To appreciate the process of disease development understanding the pathogenicity mechanisms of the pathogens, disease resistance mechanism of the host and the role of the environment is essential. Furthermore, role of various pathogens, their adaptive modifications, the functional importance of the target tissue, interaction between pathogen and host at the target tissue level, pathogenicity mechanisms of pathogens, etc are necessary to gain insight into process of disease development. Only when a pathogen can establish on or in the host, proliferate, overcome the non-specific and/or specific defense barriers of the host, produce the pathogenic factors, cause cellular and tissue damage, produce significant pathological changes, impair the function of the target tissue cause clinical disease often with mortality. This process is very complicated which often get accelerated by stress and environmental factors. However, the sequence of disease development will to a large extent depend on the nature and load of the pathogen (parasite, bacteria, fungi, virus), its intensity per unit area or unit weight of the host, size of the host and environmental factors.

An Overview of Parasitic Diseases in Asian Aquaculture

Parasitic induced diseases bring about significant economic losses in fish culture system. Estimated loss due to mortality and morbidity of fish due to parasitic infection is much more than that from microbial diseases. Protozoan ciliates (Ichthyopthirius, Trichodina) and flagellates (Ichthybodo, cryptobia) are some of the common ectoparasites of fish. Certain ectocommensal ciliates (Epistylis, Vorticella and Zoothamnium) attach to the external surface of fish leading to fouling. These protozoan ectoparasites are often associated with mortalities of younger stages of cultured fish. The situation becomes worst in water with low oxygen and high organic matter. Most extoparasitic protozoans have simple and short life cycle and reproduce by binary fission. Most protozoan ectoparasites are readily detected in direct microscopic examination of skin and gill scrapings. Histopathological changes in the integument following ectoparasitic protozoan infection are outcome of two counteracting cellular processes – hyperplasia of the epithelial cells, including mucus cells and chloride cells, versus a progressive cellular destruction. Cellular necrosis primarily occurs due to the feeding and attachment activity of the parasites. Generally, immersion therapy using suitable chemicals can control ectoparasitic protozoan infections

Disease caused by endoparasitic sporozoans are a serious threat to fish farms. These sporozoan spores present in the pond soil are normally ingested by the fish. The infective element of the spore (sporoplasm) is released in the gut. The sporoplasm reaches the target tissue using the vascular route. Once inside the target tissue, the vegetative trophozoites cause massive destruction of the target tissue and produce large spore containing cysts. There are two types of sporozoan infecting fish and shell fish namely myxosporidian and microsporidian. Myxosporidian occur in fish either as histozoic (intercellular tissue parasites) or as coelozoic (inside the lumen of various ducts and tubules) parasites. Microsporidians are intracellular obligatory parasites and lead to massive hypertrophy of infected cells (xenomas). There is no effective treatment for sporozoans. However, treating pond soil with chemicals and lime is the best option.

With increasing fish culture activities several bacterial diseases causing morbidity and mortality in fish have been reported in tropical and subtropical aquaculture systems. Common and major diseases are due to motile aeromonads, Vibrios and Pseudomonas. However, bacterial diseases of minor nature have also been reported from aquacultural systems. In the last two decades several new diseases due to Streptococcus sp, Edwardsiella and Ricketsia have also emerged.

Bacteria can cause disease either as primary pathogens or as secondary opportunistic invaders. Bacterial diseases in fish can be broadly classified as surface ulcerative, acute systemic and chronic granulomatous types (Table 1, Plates 4 and 5). Surface ulcerative types of diseases are characterized by hemorrhagic surface ulcers and are caused by species of Aeromonas, Pseudomonas, Vibrios, Flexibacteria and Myxobacteria. Surface ulcerative disease conditions at times develop to acute systemic disease. Acute systemic disease are characterized by the presence and proliferation of bacteria in internal organs like kidney, heart, spleen, blood and other visceral organs. These diseases produce significant necrotic changes in all affected organs and can cause mortality in a short time scale. Bacterial hemorrhagic septicemia caused by Aeromonas hydrophila is a major problem in carp farms. Chronic granulomatous type of disease conditions are characterized by the formation of granulomas or nodules with the initiating bacteria in the centre. These conditions will not produce mass mortality but can produce chronic low levels of mortality and can significantly reduce growth.

A. Bacterial Diseases in Shrimp

Bacterial diseases in hatcheries and farms are being increasingly recognised as major hurdles to successful shrimp farming (Plate 6). Among the several bacterial diseases, diseases due to Vibrio sp. (vibriosis) are common. As majority of the Vibrio are secondary opportunistic pathogens, problems related to vibriosis can be traced to stress, poor water quality and bad management. It is widely accepted that the ubiquitous opportunistic vibrios are always responsible for causing mortalities in shrimp which are already stressed by poor water quality, external fouling and primary pathogens like viruses.

Vibrios are gram negative, oxidase positive, motile rods. Most frequently reported species in hatchery situations include Vibrio harveyi, V. vulnificus, V. parahaemolyticus and V. alginolyticus. All species of cultured shrimp are susceptible to vibriosis under stressful condition.

The common clinical signs associated with vibriosis are: 1) high mortalities, particularly in PL and young juveniles shrimp, 2) Moribund shrimp appearing hypoxic and often coming to pond surface and edges, 3) Reddening of shrimp, 4) Shell and appendage necrosis with blackening, 5) Presence of luminiscence in affected shrimp in tanks and ponds.

Viruses are submicroscopic particles made up of nucleic acid covered with a protein coat. The nucleic acid is either RNA or DNA and not both. Protein coat is often covered with an envelope and depending on the presence or absence of an envelope, virus can be an enveloped or non enveloped virus. Size of the virus vary from 20-200 nm. Shape of the virus varies from a simple icosahedra to bullet shape to complex brick shape.

Virus are classified based on their nucleic acid (DNAor RNA) and protein profile. However, additional details on type of disease, host range, and geographic distribution of the virus are also used in classification. The classification of the virus is decided by the International committee on taxonomy of virus (ICTV).

Viruses are obligatory intracellular parasites requiring a living cell to replicate. As the virion is lacking its own metabolic machinery, it depends fully on the living host cell for machinery and raw materials for its metabolism and replication. This complex biology of the virus has made it difficult to clearly identify a viral process to design drugs for selective killing without damaging the host cell. Any attempt to kill the intracellular virus affects the host cell.

Replication of Virus in a Cell

There are two kinds of entry into and exit of virus from a cell: a) receptor mediated endocytosis (RME), b) direct entry by fusion. Non-enveloped virus enter the cell by RME (Fig. 1). RME of virus is similar to regular entry of other macromolecules into the cell. With this receptor mediated specified process, virus enter a cell in a vesicle which later fuses with lysosome. Inside the lysosome, viral particles undergo partial changes in protein coat due to digestion by lysosomal enzymes and move to cytoplasm. Finally, this uncoated nucleic acid replicates followed by synthesis of proteins and assembly of viral capsid proteins and genome to full blown virus particles. The site of assembly of the virus may be in the cytoplasm or nucleus of a cell which is referred to as inclusion body. Inclusion bodies which varies in size, shape, location and staining reaction can be used as a diagnostic feature for some viruses. Non- enveloped virus exit from a cell by damaging plasma membrane and hence clearly show cytopathic effect(CPE) in cell culture.

A. Viral Diseases of Shrimp

Shrimp farming which has grown to an industry in the last three decades is young compared to fish farming in the world. Yet the shrimp farming is facing serious problems with several types of viral diseases. Intensive culture in hatcheries and grow outs along with movement of stock across national and international borders of these animals with poor immune system has encouraged emergence of large number of viruses. During a short span of 30 years, 15 viruses of economic concern have been reported in shrimp culture of the Asia Pacific region. Shrimp viruses (Plate 8) can be broadly categorised into two groups:

1. Viruses infecting ectodermal (epidermis, hypodermal epithelium of fore and hindgut, nerve cord and nerve ganglia) and mesodermal (hematopoitic organs, antennal gland, gonads, lymphoid organ, connective tissue and striated muscle) origin tissues and replicating within the cytoplasm (YHV, TSV) and within the nucleus (IHHNV, WSSV), 2. viruses like MBV, HPV, BMNV, and BP infecting endodermal origin tissue and replicating within the nucleus of hepatopancreatic and mid gut epithelial cells. Lack of established shrimp cell line is the major drawback in study of shrimp viruses. Following are some of the common viruses causing diseases in shrimp hatcheries and grow out ponds in the Asia Pacific region.

Introduction

Oomycete and fungal diseases are considered as the second most economically impactful diseases in aquaculture, after bacterial diseases. However, among both the diseases, oomycetes are more common. The oomycetes are members of the Kingdom Chromista, which also includes diatoms, kelps, and brown algae. These group of pathogens form free-swimming zoospores and their cell walls are composed of cellulose and glycans rather than chitin. Fish-pathogenic oomycetes (Saprolegnia, Achlya, Aphanomyces and Branchiomyces), are commonly known as water moulds, are ubiquitously distributed in a variety of aquatic environments and ecological niches, and belong to the Order Saprolegniales. These pathogens have low host specificity and infect a diverse range of fishes. On the other hand, most of the fungi (Fusarium sp., Aspergillus sp., Candida sp., Paecilomyces sp., Penicillium sp. and Cladosporium sp.) responsible for infection in fish, form non-motile spores and their cell walls are made up of chitin. In addition, Ichthyophonus sp. an important pathogen which, superficially appears like fungus and produces spores and hyphae, belongs to a distinct clade of protistan parasites. Among the oomycete diseases, infection with A. invadans is considered as one of the most serious fish disease. Therefore, the disease is discussed elaborately.

Infection with Aphanomyces invadans: Infection with Aphanomyces invadans is popularly known as, Epizootic Ulcerative Syndrome (EUS). It has a complex infectious aetiology and is clinically characterised by the presence of invasive Aphanomyces infection and necrotising ulcerative lesions, typically leading to a granulomatous response (Plates 9 and 10). In an expert consultation held during the sidelines of the Fifth Symposium on Diseases in Asian Aquaculture (DAAV) at Gold Coast, Australia in November 2002, it was proposed to rename the disease as epizootic granulomatous aphanomycosis; however, the term EUS is still used popularly.

In the past several Non infectious diseases (NIDs) of fish and shellfish have been observed in natural waters. However, with expansion in fresh, brackish and marine aquaculture including aquarium keeping world wide there is a phenomenonal increase in incidences of NIDs and economic loss. Common Non infectious diseases (NIDs) in fish are 1.Gas bubble disease(GBD), 2. Body deformities(BD), 3.Nutritional diseases(ND) and 4. Disease due to harmful algal bloom(DHAB)

1. Gas Bubble Disease (GBD)

Gas bubble disease (GBD), is caused by supersaturation of gases in water. The gas involved is nitrogen in most cases and rarelycarbon dioxide and oxygen can cause GBD as these two gases are used by fish tissues and readily processed.Oxygen can cause gas-bubble disease at about 350 per cent air saturation, but nitrogen can cause the disease even below 118 per cent. GBD becomes prominent whenever there is a change in temperature, pressure and turbulance in aquatic environment, leading to disturbance in metabolism of f ishes. Bubbles of gas may form in the eyes, skin, gills, fins and within the internal organs where small bubbles may coalesce into larger bubbles.Fish absorb the excess gas, which forms bubbles in the small blood vessels, due to pressure changes inside their body. Gas bubble disease (GBD), is seen both in captive and wild fish. When bubbles affect internal organs, some fish may diesometimes without obvious clear clinical signswhile other fish may be chronically affected, and have a reduced appetite, activity and buoyancy. GBD can also cause secondary bacterial, viral or protozoan infections.All fish and shelfishspecies as well as amphibians and aquatic invertebrates are susceptible to GBD.Sensitivity of fish to GBD in terms of pathology and mortality varies with species and age.

Introduction

Fish is the cleanest animal, gets bath 24 hrs a day provided water is clean without any toxic material. Perhaps this was the scenario world over 5 decades ago. However, in the last few decades due to industrialisation, modernisation of agriculture and animal husbandry, human health care industry and city developments all have contributed to toxic material to water endangering the fish and the consumer, leave alone clean environment. In recent years, even intensive aquaculture also add several chemicals, pesticides, antibiotics contributing to pollution. Natural water bodies such as rivers, lakes, reservoirs, scoastal/marine waters are ultimate receivers of pollutants which also serve as the source of water for aquaculture. Although pollution control by treatment measures and awareness have improved over the years still there is a long way to achieve the clean water. Effect of toxins in aquaculture has multiple angles, differing from that of toxins on land animal. Fish depends heavily on water for respiration, physiology, reproduction, food and feeding and its very sustenance- as a supporting medium.

Aquaculture is expanding world over growing at 6-8% gradually involving more areas and species contributing a major share to total fish production with gradual decline in capture fisheries from the natural water bodies. Invariably, most of the natural water bodies are polluted with one pollutant or the other in the country. In addition, several pollutants are added to the system by various aquacultural practices. Although large quantity of information is available on aquatic toxicology, toxicology in aquaculture from health point view of fish and the consumer is meager and not focused. Against this background, it is very important to study, understand various toxicants, their mode of action, accumulation and effect on fish and the consumer. Toxicology in aquaculture is incomplete unless it includes or addresses safety of the consumers. Further, poisoning of fish in farms and wild deliberately or otherwise needs forensic studies/approach to address the issues.

Epidemiology is the study of diseases in a population in its natural setting. In epidemiology, the population is the patient and hence epidemiology is population medicine. All epidemiological studies are field based. In aquatic epidemiology, aquaculture ponds serve as the laboratory. This can be a great advantage over laboratory studies, which cannot be easily extrapolated to field conditions.

Diseases are among the greatest deterrents to the sustained production in aquaculture. Diseases such as Epizootic Ulcerative Syndrome (EUS) in fishes and White Spot Syndrome (WSS) in shrimps have devastated aquaculture with bad impact on productivity and profitability. Various diagnostic and health management tools are being used to devise disease control strategies. It is becoming increasingly difficult to manage pathogens that have become endemic. Epidemiological approaches which have been very successful in human and veterinary medicine are now being increasingly recognised as important weapons that can be used in aquaculture to formulate disease control programmes.

Concept of Cause for a Disease

Cause from an epidemiological perspective is interpreted in quite a wide sense. This is somewhat different to the more traditional view of the role of cause being restricted to etiological agents. An epidemiological definition of a cause of a disease is “an event, condition or characteristic that plays an essential role in producing an occurrence of the disease”. Epidemiologists avoid defining the word cause for any disease outbreak, but prefer to use words such as determinants, exposures and risk factors. Alternatively, they will categorise causes as direct or indirect; necessary or sufficient; and single or multiple rather than defining primary etiology.

Introduction

Aquaculture and Zoonotic Diseases

Diseases that can transfer from wild or domestic animals to humans are referred to zoonosis (Fig 1). Zoonosis also includes biotoxin producing agents from fish to human beings which is well documented. There are a number of pathogens which spread from fish and shellfish to human and other domestic animals causing diseases.There are also many other infectious organisms of fish origin that have not been reported but have potential to infect and harm man. Zoonotic diseases originated from fish and shellfish have been documented in the past mostly from wild caught fish and shellfish. However, in the last 30 years as the capture fisheries is declining and aquaculture is growing steadily worldwide, zoonotic incidences from the latter are increasing.

Aquaculture growing at 6-7% annually world over including India is contributing to 50-60% of total fish production today. Additionally, aquarium industry is booming worldwide. Therefore,incidences and scale of zoonosis due to aquaculture are quite different from that from capture or wild fisheries. Disease outbreaks in cultured fish are often by pathogens indigenous to aquatic environment. In aquaculture, disease outbreaks are associated with quality and quantity of nutrients and high stocking density which increase pathogen load leading to easy transmission to human. Apparently healthy fishes may also harbor bacterial pathogens, especially in their kidneys and intestines. World Health Organisation (WHO) considers many diseases in aquatic animals as emerging type. Emerging diseases appear in a population for the first time or it may have been existed before, but is now increasing rapidly. Usually information on zoonotic potential of emerging diseases is limited which is essential to disseminate to human health managers. Potential biological contamination of aquaculture products can occur from bacteria, fungi, viruses, parasites and biotoxins. The location of the farm, the species being farmed, water temperature, husbandry systems, postharvest processing, and habits in food preparation and consumption are among the main factors influencing the zoonotic risk associated with aquatic animals and their products.

Disease diagnosis refers to the various procedures and techniques used to identify the nature of disease and to precisely pinpoint the primary and secondary pathogens involved. Disease diagnosis is an integral part of health management. Proper diagnosis helps to adopt accurate therapy and avoids indiscriminate use of chemotherapeutics. Information on case history and clinical signs should be carefully used while examining samples for diagnosis. By following rational and scientific sampling protocols, one can hope to arrive at accurate diagnosis. Sampling methodology is key to the success of proper disease diagnosis. Dead fish/shrimp should never be used for any diagnostic purpose. Autolytic post-mortem changes, saprophytic invaders, spoilage organisms, etc. mask the actual etiological agents and can lead to false diagnosis.

The three commonly used conventional diagnostic approaches are fresh examination using microscope, microbiological methods and histopathology. With little facilities and infrastructure, fresh microscopical diagnostic approach can be done at the farm/hatchery site itself. Gross examinations along with simple skin and gill preparations examined under microscope can many a times give very good information about the disease and the etiology. All fresh preparations should be made from live or moribund fish and the preparations should be wet and moist. The fish meant for examination should be kept wet and not allowed to dry. Many of the commonly occurring external disease problems can be easily diagnosed by this method.

Systemic bacterial diseases need to be positively diagnosed by adopting the microbiological approach. Samples for bacterial isolations should be taken from kidney, blood, body cavity, etc. from live or moribund fish following strict aseptic conditions. In shrimp, it is ideal to use hemolymph for bacterial isolations. Dominant bacterial isolates are identified following standard procedures and their antibacterial sensitivity tested before recommending any antibacterial therapy. This method requires a minimum of 4-5 days.

Introduction

Development and standardisation of diagnostics for aquaculture health management have undergone great improvements over the last three decades. Diagnosis in aquaculture health management involves several stages starting from farm-level observations to conventional laboratory identification and histopathology to sophisticated modern molecular tools. Accordingly, diagnosis can be broadly categorised under three levels depending on the level of scientific development.

1. level-I detection system, farm site observation and record-keeping;

2. level-II system, conventional laboratory identification employing microbiology, parasitology, and histopathology and

3. level-III system, advanced and specialized immunological and biomolecular techniques.

The interaction of an antibody with an antigen forms the basis of all immunochemical techniques. Antibody binds specifically to an antigen at epitope or antigenic determinant level and this property has been used for development of immunodiagnosis. Furthermore, as antibodies react with antigen at ambient conditions and do not require sophisticated equipment ideal field test kits can be developed. Biomolecular tools using nucleic acid probes although very sensitive are costly and time-consuming requiring sophisticated equipment and trained personnel. Against this background, there is always a need for developing simple, low-cost, rapid yet sensitive farm- level diagnostics. Such field-level tests facilitate screening a large number of samples by a large number of poor and medium-sized farmers who constitute a majority in developing countries. Large-scale screening besides empowering farmers is considered to be ideal for effective health management. Antibody probes provide an ideal format for developing simple field-level diagnostics as antigens ideally react with antibodies at ambient conditions without requiring sophisticated equipment. Available technology for raising large quantities of monoclonal antibodies (MAbs) against pathogens has increased the scope for developing field detection kits in human, animal and agricultural health management. Several farmer-friendly detection systems also referred to as point-of-care (POC) detection method based on antibodies have been innovated in the recent past. POCT is defined as ‘medical diagnostic testing at the time and place of patient care.

Introduction

Nucleic acid based diagnosis is highly specific, sensitive and rapid method widely used in health management. The detection is mainly based on structure and sequence of bases of nucleic acid which are unique to an organism. In recent years, nucleic acid based diagnosis is becoming common and popular in fish and shell fish health management. Commonly employed nucleic acid based diagnosis are DNA hybridisation and Polymerase chain reaction (PCR).

Nucleic acids are made up of nitrogen bases. The primary sequence of bases in DNA or RNA have regions which are unique for a particular organism or gene. The sequence of DNA is composed of a series of four phosphorylated bases Adenine (A), thymidine (T), guanidine (G) and cytosine (C). Pairs of bases (A=T, or G=C) can form hydrogen bonds, which cause opposing strands of DNA to form a semi-stable bonding (hybridization or annealing) to one another. If the opposing strands of DNA match exactly, then the familiar double helix form of DNA results. In the following example, the opposing strands in (1) will form a double stranded, tightly bonded structure, and the DNA strands are described as complementary. In (2) the strands are non-complementary (opposing A=T and G=C base pairs do not line up) and the two strands will not bond.

DNA Hybridisation

The basic principle in DNA hybridization is that, if a strand of double helix of DNA is separated from its complement (by heat) it will bind (reanneal or hybridize) only to its complementary strand (or to a duplicate of its complementary strand). Two complementary RNA structure will hybridise in the same manner. Inaccurate and mismatched bindings will be unstable and fall apart.

The binding of one DNA strand to another is in some respect is analogous to binding of antigen and antibody. However, in the case of nucleic acid bindings, the precise molecular nature of binding (base pairing) is known and can be easily manipulated by choosing DNA strand of appropriate length and by incubating the strands in appropriate salt concentration, temperature. The stringency (faithfulness of the match) of hybridisation can be precisely defined and as a result coincidental cross reaction is virtually eliminated.

Introduction

Scope and importance

Aquaculture is breeding, rearing and husbandry of aquatic animals such as f ishes, crustaceans and molluscs for food, ornament and sports. It is a husbandry involving special animals, the cold blooded vertebrates and invertebrates, whose physiology, growth, metabolic activities and overall performance depend heavily on ambient aquatic temperature and environment. Therefore, husbandry and the health management of aquatic animals is quite different from that of the other land based warm blooded animals.

Aquaculture is one of the fastest growing food production sectors in the world including India. Three decades ago, few species were cultured in India mainly in inland waters and then aquaculture was contributing less than 10 % of total f ish production in the country. In the last 25 years, there is a rapid growth in aquaculture with annual rate of 7-8% with significant contribution (above 60%)to total fish production today. The number of species cultured today in India including ornamentals is close to 40. Besides, a good number of plant and animal fish food organisms are cultured to support the hatcheries and nurseries in aquaculture. The area under aquaculture is increasing steadily in inland and coastal brackish water while marine water is also being added up for aquaculture slowly and steadily. Very interestingly, non-traditional environment such as manmade saline soils are also explored successfully for aquaculture. Intensification of culture activity with high stocking density artificial feed and fertilizers is another dimension of aquaculture noteworthy. Large scale movement of aquatic animals and their raw or processed products across national and international borders is important from aquaculture production and health point of view. In general, aquaculture package of practices alter the very water in terms of its chemistry and microbial load and has significant negative impact on the health of fish in culture system.

Farming of fish and shellfish has gained significant grounds in several parts of the world. However, the major bottleneck to successful farming is disease in farmed aquatic animals. In recent years, lot of attention is being given to health management using various forms of immunoprophylactic measures such as vaccination and immunostimulation. To rationalise immunoprophylactic applications, it is vital to have insight into the specific and non-specific defense mechanisms of farmed aquatic animals. Through disease process studies it is very well known that a pathogen can cause disease only if it can overcome the non-specific and specific defence barriers of the host and successfully establish and proliferate. Non-specific and specific immune systems are the two main branches of the defense system. The immunity which an organism derives against a pathogen is the result of a delicate interaction and cooperation between the two systems.

Defense System of Fish

Surface Barriers as Local Immune System

The immune systems present in the surface structures of fish have their potential importance in local immune responses. Important surface barriers are skin, gills and gastrointestinal tract.

Introduction

Aquaculture is given major thrust in many countries including India not only as a source of good quality food of animal origin but also because of economic gains it brings to aqua-farmers, creation of employment and export trade. It has emerged as one of the fastest growing food producing sector sowing to intensification and diversification of culture systems. However, the success of the intensive aquaculture system depends on several factors including species cultured, type of feed, water quality, management practices and strategies adopted to prevent/ control disease problems. Further, the intensive aquaculture operation results in accumulation of wastes leading to water quality deterioration, oxygen depletion, build-up of toxic metabolites such as hydrogen sulphide, methane, ammonia and nitrites that cause stress to culture animals leading to susceptibility to diseases. Among the several challenges that seriously affect the growth and sustainability of aquaculture, the incidence of microbial diseases are considered as major threat. Thus, keeping cultured aquatic animals in good health becomes major requirement for the success of aquaculture.

Though application of chemicals and drugs are often considered necessary to maintain water quality and animal health, use of these, in the long run are known to cause harmful effects both to the animal and the environment. Further, accumulation of drugs and chemicals in tissues, development of resistance to drugs by pathogens, pathogen build up, destruction of useful microorganisms, deterioration of pond environment and toxicity issues have limited their use in aquaculture system. Under these circumstances several alternative strategies have been proposed as viable option for the success of aquaculture in the form of prophylactic measuressuch as use of probiotics, prebiotics, synbiotics, immunostimulants and bacterial vaccines. The term prophylaxis refers to all preventive steps taken including better aquaculture management practices that are adopted during a hatchery and farming operation to improve growth, minimize the load of pathogens and prevent occurrence of diseases.

Any technique that uses living organisms or substances from these organisms to make or modify a product or to improve plant or animal or to develop microorganism for specific uses, can be termed biotechnology. Biotechnology involves microbes or cells derived from plants and animals for efficient production of useful products for mankind. The use of microbes for production of alcohol and penicillin, to name a few, is well known. However, in the last 25 years, several novel organisms, yielding useful products have been created by genetic engineering. Today, there is a wide application of biotechnology in human medicine, agriculture and animal husbandry in the area of diagnosis prophylaxis and therapy.

Aquaculture industry has undergone a sea change world over in the last three decades. The important needs of the industry are development of fast growing disease resistant fish and shellfish varieties, development of cheap and effective vaccines, disease diagnostics, cell lines sand probiotics. Efforts on these lines have already begun since 3-4 decades and results are highly promising. Some of the biotechnologies employed in aquaculture health management (Plate 15) are fish cell lines, monoclonal antibodies and nucleic acids for diagnostics sub unit and nucleic acid vaccines for prophylaxis, transgenes for disease resistance, gene editing in fishes and their pathogens and probiotics for bioremedial measures.

A. Cell Lines for Health Management

Cell lines are transformed cells that are adapted well to grow and multiply outside the host in in vitro culture systems. Cells lines can be developed from fast growing tissues of young fish. There are two methods of developing cell lines (1) Explant method, (2) Enzymatic method. Cell lines are grown in sterile plastic or glass containers containing appropriate medium having nutrients salts, antibiotics, buffers, serum etc. Minimum essential medium (MEM) is the common medium employed for fish cell line. Cell lines are developed, characterised and deposited in national and international repositories, which can be procured for use in research and industry.

Fish are Sentient Animals

There is strong increasing evidence that finfish, like other vertebrates are sentient animals. This means that fish are self-aware, they can feel pain and distress, they have long term and short-term memory and to some extent, they can experience emotions.

Fish are intelligent, sensitive creatures and like many other animals, they explore, travel, socialise, hunt and play. Some species care for their young and use tools as humans do. Fish are sentient animals capable of suffering and feeling pain. Most fish have highly developed senses with excellent taste, smell, hearing and colour vision. Until fairly recently, many people didn’t realise that fish were sentient or feel pain, and the mental abilities of fish were given limited attention by the scientific community. Now, recent discoveries open up a new world of understanding. Far more complex than we ever realised, f ish live rich social lives: communicating; hunting cooperatively; and, in some cases, developing cultural traits. Consensus arrived in the mid-2000s around science demonstrating high-level pain perception in fish, and examinations of fish behaviour are beginning to reveal a picture of fish as complex, social and emotional beings. Appreciation of these facts are not widespread in civil society, but identified in time for the principle of fish welfare to be recognised by legislation by the European Union.

The term probiotic means “for life” and it is currently used to refer bacteria that are associated with beneficial effects for humans and animals. The credit for first observation made on the positive role of some selected bacteria was attributed to Eli Metchnikoff, the Russian born Nobel Prize recipient, worked at the Pasteur Institute in the beginning of the last century. Metchnikoff suggested that - “the dependence of the intestinal microbes on the food makes it possible to adopt measures to modify the microflora in our bodies and to replace the harmful microbes by useful microbes”. Interim Henry Tissier, a French paediatrician, observed that children with diarrhea in their stools had a low number of bacteria which are characterized by a peculiar Y shaped morphology. On the contrary, these “bifid” bacteria were abundant in healthy children. Based on these observations, Tissier suggested that “bifid” bacteria could be administered to patients with diarrhoea to help restore a healthy gut microflora. These two scientists were the first to give scientific intimation for the use of probiotics when even the term probiotics was not coined. Later in 1965, the term probiotics is coined by Lilly and Stillwell to describe substances produced by one microorganism, that stimulate the growth of another. Subsequently, Parker defined probiotics as organisms and substances that contribute to intestinal balance. Further, Fuller (1989) improved the Parker’s definition of probiotics as “live microbial feed supplements which beneficially affect the host animal by improving its intestinal microbial balance”. Havenaar and Huis in’t Veld (1992) extended the probiotic definition with the description: “A viable mono or mixed culture of microorganisms which, applied to animal or man, beneficially affects the host by improving the properties of the indigenous microflora”. At the end of the millennium, Salminen and his coworkers (1999) proposed the following definition: “probiotics are microbial cell preparations or components of microbial cells that have a beneficial effect on the health & well- being of host”. Whereas FAO/WHO (2001) defined probiotics as “living bacteria that, when administered in adequate amounts, confer a health benefit on the host”. This definition for probiotics is widely accepted.

Introduction

Probiotics, prebiotics and synbiotics are interrelated terms. Probiotic is discussed in detail in previous chapter. In this chapter, prebiotics and its effect on gut along with probiotics are emphasized. Prebiotics can be defined as non digestible food ingredients that stimulate the growth and/or activity of bacteria in the digestive system in ways claimed to be beneficial to health. They were f irst identified and named by Marcel Roberfroid in 1995. He defined prebiotic as a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon host well-being and health. Further, FAO defined prebiotic as “a non-viable food component that confers a health benefit on the host associated with modulation of the microbiota.”Whereas the term synbiotics refers to a product that contains both probiotics and prebiotics in the form of synergism. A synbiotic has been defined as “a mixture of prebiotics and probiotics that beneficially affect the host by improving the survival and implantation of live microbial dietary supplements in the gastrointestinal tract, by selectively stimulating the growth and activating the metabolism of one or a limited number of health promoting bacteria, and thus improving host welfare”. As described above, the combined use of prebiotics and probiotics is often described as synbiotic, but the United Nations Food & Agriculture Organization (FAO) recommends that the term “synbiotic” be used only if the net health benefit is synergistic.

Introduction

From earlier chapters, it is evident that Lactobacilli and Bifidobacteria are the two important genera that have potent applications in food microbiology and human nutrition due to their role in food and feed production, preservation and also due to probiotic potentialities. Most of the bacterial species are still unknown indicating that the identification and classification of bacteria is a hard task since the beginning of microbiology and consequently our knowledge about bacterial ecology is very low. Mean while the expectations about novel bacterial products are high. Therefore, bacterial identification has become a growing field of interest in microbiology. Since, Lactobacilli and Bifidobacteria genera include a large number of species and strains exhibiting important properties in an applied context, chiefly in the area of probiotics as food and therapeutics, an updated list of species belonging to these two genera, their phylogenetic relationships and other relevant taxonomic information are of great significance. The classification and identification of a probiotic isolate at strain level point out its typical habitat and origin. Moreover, the species, or genus name, may also indicates the strain’s safety and technical applicability for its use in probiotic products. Taxonomy helps to place a newly identified organism in a relevant group and enables the scientific communication. Further it facilitates the construction of databases that can be used for rapid identification of organisms at laboratory level easily. Thus, taxonomy of Lactobacilli and Bifidobacteria helps to study these organisms scientifically and allows characterizing and arranging them in an orderly manner which promote the scientific community to explore the benefits from those organisms for the betterment of mankind.

Introduction

Preceding chapters discuss the essentials of probiotics, their role in maintaining gut health in combination with prebiotics and significance of systematic classification of probiotic isolates. Further, to draw an idea how probiotics function in the gut, it is essential to know the interaction of probiotics with other microorganisms in the gastrointestinal milieu - the site of their probiotic action. Knowledge in the ecology of probiotic microorganisms in host environment such as gut gives an ideal picture of their functioning manner and the factors (both host and microbial) responsible for it.

Study of microbial ecology in the gastrointestinal tract is very difficult since gut harbors highly diversified microflora that varies not only at species level but also at individual level. The diversity of gut microflora in two healthy individuals of same species may exhibit variations in their composition and it may be influenced by geodemographic factors also. In this regard, the present chapter deals with the study of ecology of Lactobacillus- most commonly used probiotic bacteria in the gastrointestinal tract which in turn helps in framing selection criteria for screening of potential probiotics.

Recent studies on interaction between human and gut microflora discussed that co-evolution between microorganisms and their host have contributed for the development of a very complex microbial diversity in the gut. Highly diversified microflora in the human gut is a consequence of natural selection at two levels, top-down and bottom-up selection pressure. Hierarchy theory expounds that higher levels compel lower levels for possible organizational solutions i. e. organisms at the higher levels govern the structure or population dynamics of an ecosystem while on contrary lower levels control the structure of ecology when bottom-up level is the selection pressure. In case of interaction between human and gut microflora, being host human is at higher level in the hierarchy for selection and as mentioned above, top down selection pressure is driven by higher levels; host selects the beneficial microorganisms for functional redundancy leading to the development of a microbial community with divergent microbial divisions. Chances for lateral gene transfer also thought to influence the selection process and there by structure of the microbial community. On the other hand, competition among the members of host selected microbiota exert bottom up selection pressure resulting in the development of highly specialized genomes with functionally prominent set of genes. Natural selection at host level obviously favors functionally constant microbial communities which benefits the host with functional redundancy encoded in their genomes. Mean while bottom-up cell-level selection pressure aspire to favor niche specialization to avoid competition between the members of microbial community. In order to be a part of specialized niche, to become more potent microorganisms adapt a particular set of abiotic and biotic factors within the habitat by acquiring new sets of genes essential for their survival and thus escape the competition from decrepit microbial species.

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.

Introduction

It became clear from the discussions of previous chapters that probiotics perform metabolic functions such as fermenting indigestible dietary residues and endogenous mucus, production of vitamins, and absorption of minerals. They also play key role in intestinal epithelial cell proliferation and differentiation, and the homeostasis of the immune system. Further, probiotics are also known to involve in modifying gut pH, antagonizing pathogens through the production of antimicrobial compounds, competing with pathogens for recept or sites as well as for available nutrients and growth factors, stimulating immunomodulatory cells of the host, and produce a wide range of enzymes such as lactase, amylase, lipase, protease, phytase and so on. The molecular mechanisms of probiotics are largely unknown and opens avenue for research in probiotics. Moreover, current research in probiotics and health consciousness in the public lead to an increased interest towards probiotics from the public, researchers, governmental organizations (such as the WHO/FAO) and medicine and food companies. In this chapter, application of probiotics in treating various diseases and disorders are discussed.

Diversity of gut microbial community

The human microbiota (the collection of microbes that live on and inside us) consists of about 100 trillion microbial cells that outnumber our ‘‘human’’ cells 10 to 1, and that provide a wide range of metabolic functions that we lack. It has been estimated that they encode 100-fold more unique genes than our own genome. Furthermore, the gut microbes contribute to energy harvest from food, and changes of gut microbiome may be associated with bowel diseases or obesity. To understand and exploit the impact of the gut microbes on human health and well-being it is necessary to decipher the content, diversity and functioning of the microbial gut community. If we consider ourselves as superior organisms encompassing these microbial symbionts, by far the majority of genes in the system are microbial. In this sense, completing the human genome also requires the characterization of the microbiome (the collection of genes in the microbiota).

Methods of counting viable and non-viable bacteria in gut

Before the advent of genome sequencing and genome-wide analysis method, our knowledge of molecular characteristics of diverse gut microbial population was extremely limited. To obtain an estimate of cultivability, microbial ecologists generally compare microscopic counts with total viable counts. The total viable count made on a nonselective agar based medium estimates the number of colony forming units (cfu) per gram of sample. The finding that total viable counts are typically lower than total microscopic counts was thought to be due to the number of dead cells. Indeed, dead bacteria in feces may constitute up to one third of the total bacterial community. However, recent nucleic acid based studies indicated that a majority of bacteria in a variety of ecosystems are different from those described in culture.

Introduction

Let food be thy medicine and medicine be thy food- the age-old quote by Hippocrates, is certainly the tenet of today. Microbial cultures have been used for millenniums in food and alcoholic fermentations and in the past century have undergone scientific scrutiny for their ability to prevent and cure a variety of diseases. Later it became popular in improving human and farm animal nutritive values and recently in aquaculture also. The growing interest in the understanding the role of food in human health has moved from its primary role as a source of energy to the subtle action of biologically active food components on human health. Henceforth functional foods or nutraceuticals are of great demand in present time. The growing scientific evidence suggests that the food supplements containing beneficial bacteria can provide an array of benefits to the host. Now a day’s probiotic fermented foods are in great demand due to the awareness of beneficial aspects of probiotics among people. Several industries focused on the improvement of conventional fermented foods scientifically in order to fetch good market for their products. Particularly, animal feed companies and researchers have been looking for alternative products and strategies that can help to maintain animal gut health in order to prevent or reduce the prevalence of pathogens in the food chain. After understanding the mechanism and beneficial aspects of probiotics in detail at molecular level in earlier chapters, now it is relevant to discuss the applied part of probiotics. In this chapter, commercial importance of probiotics and probiotics derived products at industrial level are emphasized.

Introduction

Lactic acid bacteria (LAB) are characterized as Gram-positive cocci or rods, non-aerobic but aerotolerant, able to ferment carbohydrates for production of energy and lactic acid. Lactic acid bacteria includes different major genera: Lactococcus, Enterococcus, Lactosphaera, euconostoc, Melissococcus, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella. Other genera are Aerococcus, Microbacterium, Propionibacterium and Bifidobacterium. Through carbohydrate fermentation along with lactic acid, various organic acids are produced due to which there is a fall in pH level and creates unfavorable environment or inhibits growth of some undesired microorganisms. The low pH makes organic acids lipo -soluble, allowing them to invade into cytoplasm of pathogens by rupturing cell membrane. LAB are generally regarded as safe (GRAS), and have a significant role in the preservation of foods and fermented products. Under controlled conditions they can be used as natural competitive microbiota or as specific starter cultures. Antagonistic substance like bacteriocins are produced from lactic acid bacteria that inhibits the activity and growth of other bacteria and these bacteriocins are active against various pathogens in minimum concentration. These are antimicrobial peptides modified by post -translational mechanisms and exported to the extracellular medium. Till date about 239 bacteriocin producing LAB genome and 789 putative bacteriocin producing genes cluster are stored in the public databases. Many gram positive and gram negative bacteria in the environment produce bacteriocins.

Introduction

The gut microbiota is the complex and dynamic community of microorganisms that includes various bacteria, fungi and other microorganisms, proliferate and lead a symbiotic/mutual relationship with the host system. These microorganisms protect the host against pathogens by unique mechanisms, helps to maintain shape and strengthen the intestinal epithelium and boost immune response. Diverse bacterial metabolites can immigrate the host bloodstream and other parts of the body via various channels in humans and animals, such as gut-microbiota-brain, gut microbiota-skin, gut-vagina, gut liver, gut-bones etc. However, many aspects of modern lifestyle would alter the gut microbiota and lead to numerous diseases including inflammatory bowel disease, diabetes mellitus, obesity, and metabolic syndrome. Probiotics and other beneficial microbes exert many beneficial effects on their hosts by modulating the gut microbiota. While the term probiotic is reserved only for well-characterised strains with clinically proven health benefits, other beneficial microorganisms include, organisms responsible for the fermentation of foods viz. yogurt, kefir, kombucha, kimchi and many others that have been shown to have distinct health benefits. Interestingly, major group of these beneficial organisms belong to lactic acid bacteria.

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.