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1.1 Background of the Study
The microbiota plays a fundamental role on the induction, training, and function of the host immune system. It is recognized that the gastrointestinal microbes of homeothermic animals serve several functions, such as digestion and development of the mucosal system, angiogenesis, and as a protection barrier against disease (Schiffrin and Blum, 2002; Stevens and Hume, 1998). The majority of these microbes are present in the digestive tract communities. They contribute to the harvest of dietary nutrients that would otherwise be inaccessible (Backhedet al., 2004; Sonnenburget al., 2005). The potential benefit of the microbes is to influence the absorption of some nutrients, such as lipids. Thus, the establishment of a microbe is a key component in order to health by competitive mechanisms and immune system development and maturation. By facilitating the growth of protective bacteria the growth of protective bacteria that will compete against the opportunistic pathogens for food and adhesions sites to intestinal membrane, they can prevent the pathogens entering the body.
An important aspect is the specificity of the host response, which depends on the bacteria species colonizing the digestive tract. Therefore, it is important to know the composition of these microbes in fish and their effects, which different factors could have on it, especially in the case of fish culture with high economical interest. In return, the immune system has largely evolved as a means to maintain the symbiotic relationship of the host with these highly diverse and evolving microbes. When operating optimally, this immune system-microbiota alliance allows the induction of protective responses to pathogens and the maintenance of regulatory pathways involved in the maintenance of tolerance to innocuous antigens. However, in high-income countries, over use of antibiotics, changes in diet, and elimination of constitutive partners, such as nematodes, may have selected for a microbiota that lack the resilience and diversity required to establish balanced immune responses. This phenomenon is proposed to account for some of the dramatic rise in autoimmune and inflammatory disorders in parts of the world, where our symbiotic relationship with the microbiota has been most affected.
A promising alternative approach for controlling fish diseases is the use of probiotics or beneficial bacteria, which control pathogens through a variety of mechanisms. The use of probiotics, in human and animal nutrition, is well documented (Rinkinenet al., 2003) and recently, has been applied to aquaculture (Irianto and Austin, 2002a). The term probiotic means “for life,” originating from Greek words “pro” and “bios” (Gismondoet al., 1999). The concept of probiotic was originally used by Lilley and Stillwell (1965) to mean a substance(s) that stimulates growth of other microorganisms (Chukeatirote, 2002). Parker (1974) modified the definition to “organisms and substances which contribute to intestinal balance”. Fuller (1992) revised the definition as, “A live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance”. This definition has put forward the importance of live cells as the essential component of a potential probiotic and its clears the confusion created by the use of term “substance”. However, an effect in intestinal microbial balance has been defined and demonstrated only in few cases. This noted by Tannock (1997), and he proposed the following definition “living microbial cells administered as dietary supplement with the aim of improving health”.
Bacillus subtilishas been shown to possess antitumor and immunomodulatory activities. Some studies have demonstrated that B. subtilisand spores of B. subtilisact as probiotics by promoting the growth and viability of the beneficial lactic acid bacteria in the intestinal tracts of humans and some animals. Typically, the lactic acid bacteria (LAB) and Bacillus sp. have been widely used and researched for human and terrestrial animal purposes, and LAB and Bacillus sp. are also known to be present in the intestine of healthy fish (Ringo and Vadstein, 1998 and Hagiet al., 2004). Interest in LAB and Bacillus sp. was stemmed from the fact that they are natural residents of the human GIT with the ability to tolerate the acidic and bile environment of the intestinal tract. Bacillus sp. has been shown to possess adhesion abilities, produce bacteriocins (antimicrobial peptides) and provide immunostimulation.
Probiotics exert several beneficial effects on human health, including interaction with the immune system, production of antimicrobial substances, enhancement of the mucosal barrier function and competition with enteropathogens for adhesion sites (Boesten and De Vos, 2008). Nowadays, the unravelling of the molecular mechanisms underlying these beneficial effects is an attractive field for gut microbiologists. Among the different extracellular compounds responsible for these processes, proteins secreted and released into the environment by probiotic bacteria (extracellular proteins) might mediate certain interactions, since they would be able to interact directly with mucosal cells, such as epithelial and immune cells (Sanchez et al., 2009a). Evidence of a direct interaction between bacterial extracellular proteins and the human immune system is mainly available for commensal and pathogenic species. Theoretically, it is possible that certain extracellular proteins secreted by probiotic bacteria might also reach the gut mucosa, acting as molecular effectors responsible for downstream responses in mucosal cells. The grey mullets belonging to the family Mugilidae constitute an important group of fishes. They are distributed in all the temperate and tropical regions of the globe. They inhabit the coastal and brackish waters and are very important as food fish (FAO, 2005). The entire fish caught is marketed fresh, dried, salted and frozen. They are also smoked and marketed after a press and dry process. In addition, the role of mullets represents an important economic resource in some Mediterranean countries. Mullets also are used in Chinese medicine and are widely cultivated in freshwater ponds in south eastern Asia (FAO, 2005). They are euryhaline and often can penetrate lagoons and estuaries, migrating back to the sea to spawn.
Most probiotic microorganisms belong to the lactic acid bacteria (LAB) viz. Lactobacillus spp., Enterococcus spp. and Bifidobacterium. Most of the species of the genus Lactobacillus are part of human and animal commensal intestinal flora (Zoetendal et al., 2006) and consists of a physiologically and genetically diverse group of rod-shaped, Gram-positive, nonpigmented, non-spore forming, catalase negative, facultative aerobic to anaerobic LAB with intense applications in fermented food production industry (Azcarate-Peril and Raya, 2001). These microorganisms are also called friendly bacteria and are generally recognized as safe (GRAS) microorganisms. The use of probiotics and other immune stimulants as dietary supplements can enhance innate defense and resistance to pathogens during periods of stress. Probiotics may also improve nutrient availability due to exogenous enzymes secreted into the host intestine or to endogenous enzymes available into the bacterial cells and released when they are lysed by the effect of the acidic environment of host’s stomach. Both types of enzymes may increase the digestive activity and degradation of diet compounds of the tested animal, even if not digestible by its own enzymatic machinery. In that vein, gut microbiota helps to conver nutrients into energy, but can also produce some essential nutrients, such as vitamins; hindering the microbial colonization by pathogens either by competitive exclusion for space or through the production of antimicrobial metabolites as bacteriocins produced by some Lactobacillus of most importance is to mention the immune modulatory capacity of the microbiota in early response, by activating the immune system of the host (Vrieze, 2010). Immune enhancement against various pathogens in order to maintain the health status of fish remains as a significant task in the present scenario. Extracellular proteins from the probiotic strains isolated from the gut of fishes remains as the thrust area of research in the current scientific forum. To date, putative extracellular proteins of probiotic bacteria have mainly been predicted by bioinformatics means, usually through the identification of certain domains, such as signal peptide or cell wall anchoring motifs via, sequence homology (Barinovet al., 2009). Only few of them was experimentally identified and partially characterized (Sanchez et al., 2009b; Van Pijkerenet al., 2006). With this as key focus, the present study emphasizes on the isolation, identification of probiotic strains from the gut of mullet fish, M. cephalus, immune enhancement in M. cephalususing the identified probiotic strain, followed by purification, characterization of extra-cellular proteins secreted by the identified probiotic strain and in silicoanalysis of the same.




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