All the isolates grown on MRS agar medium were facultative anaerobic, Gram positive and having rods or cocci. The shapes of colonies were circular, irregular, convex, rough, smooth, and shiny. Isolates were then classified as endospore formers, catalase, oxidase, citrate utilizers, and producers of negative test of indole, and MR-VP tests (Silva et al., 2013). Probiotic bacteria are commonly found in the gastrointestinal tract of animals and humans. The isolated bacteria were adapted to grow optimally at 37oC. The temperature is an important factor which affects the bacterial growth. The isolated bacteria were capable of surviving within the temperature range of normal human gut (Pundir et al., 2013). Probiotic bacteria were able to tolerate 1–7% NaCl concentrations. NaCl is an inhibitory substance which inhibits the growth of bacteria. Probiotic bacteria would not be able to demonstrate the activity in NaCl concentrations, if probiotic bacteria were sensitive to NaCl (Adebayo-tayo and Onilude, 2008). The absence of essential enzyme β-galactocidase, lactose intolerant persons cannot metabolize lactose. When they consume milk, it creates the symptoms including cramping, abdominal pain and diarrhoea. If lactose passes through the small intestine, it is converted to gas and acid in the large intestine by the colonic microorganism (Lin et al., 1991).
Bile salt and pH are the good factors which dramatically affect bacterial growth. Acid tolerance is important not only for tolerating gastric stresses but also for enabling the strain to survive for longer periods in highly acidic foods without reduction in their number (Wang et al., 2010). Bacterial cell wall contains lipids and fatty acids which disrupt in duodenal part of the gut by bile salts, as its nature is like the detergent. Hence, survival in bile salts rather than acidic environment is an important property of probiotic bacteria which facilitates to efficiently perform its action in gut (Haung and Adams, 2004). In this study, E. faecium GMB24 and E. hirae SMB16 showed good survivability at varying concentrations of phenol, acidic and bile salts. Phenol resistance is an essential characteristic for survival of probiotic bacteria in gastrointestinal tract. Phenol may be produced in the intestine through the deamination of some aromatic amino acids derived from dietary and endogenous proteins by bacteria (Pinto et al., 2006). Antimicrobial activity is one of the most desirable criteria for probiotic bacteria as they produce antibacterial substances such as organic acids, hydrogen peroxide, bacteriocins for inhibiting the growth of pathogenic and spoilage microorganisms (Yuksekdag and Aslim, 2010). All the culture supernatants obtained from the different bacterial isolates showed activity against most of the indicator bacteria. One of the characteristics of an ideal probiotics is resistance against antibiotics mostly after antibiotic administration. Such resistance to a wide spectrum of antibiotics indicated the rapid establishment of desirable probiotics microbial flora in patients. Resistance to some antibiotics of the probiotic strains could be used for both preventive and therapeutic purposes in controlling the intestinal infections. Resistant probiotic bacteria lack the genes being transferred to the other bacterial population by conjugation (Zhou et al., 2012).
Safety is one of the most recommended criteria for evaluation for probiotics as given in the FAO/WHO (2002) guidelines. The gastrointestinal tract is lined with epithelium layer of cells and mucoid lining. The epithelial layer would break down by hemolysis activity, while mucoid lining would disrupt by gelatinase activity. These injuries interfere with the normal functioning of these very important linings across which many physiological substances are exchanged and would cause pathways for infections. Absence of hemolytic and gelatinase activity is the selection criteria for probiotic strains indicate the none-virulent nature of these bacteria. The PCA was useful in showing the relationship among variables themselves and with factors. The heat-map generated in this study clustered the probiotics based on their phenotype properties such as acid and bile tolerance and phenol resistance. This study demonstrates the utility of PCA and heat-map analysis in the segregation and selection of probiotic isolates from different phenotypes for their potentiality to identify a candidate probiotic strain.
The immunological models selected for the screening of modulatory activity of probiotics bacteria with neutrophil adhesion, heamagglutination antibody titer and delayed type hypersensitivity. Probiotic bacterial cells administered orally showed significant in vivo, immunomodulatory activity. In neutrophil adhesion test, the neutrophil, an end cell unable to divide and limited capacity for protein synthesis but capable of wide range of responses in particular chemotaxisis, phagocytosis and exocytosis (Elgert, 1996). Probiotic bacterial cells significantly increased the adhesion of neutrophils to nylon fibers which correlates to the process of margination of cells in blood vessels, which is mediated through the interactions of the β2 integrins present on neutrophils and neutrophils reaching the site of inflammation (Smith et al., 1989; Shinde et al., 1999). Probiotic bacterial cell at both the doses in rats has highly significant when compared to control indicating possible immunostimulant effect. This may be due to the up regulation of the β2 integrins; they adhere firmly to the nylon fibers (Miller et al., 1987). Hence, report suggests that oral administration of probiotics causes the stimulation of neutrophils at the site of inflammation. This may help in increasing the immunity of body against microbial infections.
Antibody molecules, a product of B lymphocytes and plasma cells, are essential to humoral immune responses. IgG and IgM are the major immunoglobulins, which are involved in the complement activation, opsonization, neutralization of toxins, etc. The development of humoral immune responses to SRBCs by probiotics, as evidenced by increase in the antibody titer in mice, indicates the enhanced responsiveness of T and B lymphocyte subsets involved in the antibody synthesis (Benacerraf, 1978).
The effect of probiotic bacterial cells by haemagglutination test on the humoral immunity system involves the interaction of B cells with the antigens and their consequent propagation and differentiation into antibody secreting cells. These antibodies bind to antigens and neutralize or facilitate its elimination by cross-linking to form latex that is more readily ingested by phagocytic cells (Ramanatha et al., 1995). This test involves dilution of serum sample and addition of SRBCs. When SRBCs added to the serum antibody then agglutination occurs because of formation of antibody which bridges with erythrocytes and settles at the bottom as latex but unagglutinated red blood cells appear in the well bottom as a button. If haemagglutination was detected in the serum wells but not in control wells, the result is recorded as titer. E. faecium GMB24 and E. hirae SMB16 at both the doses showed very significant effect on the circulating antibody titer.
The DTH responses directly correlate with cell-mediated immunity which involves the effector mechanisms carried out by T lymphocytes. These responses are critical to defense against infectious organism, tumor immunity, foreign grafts infection and delayed type hypersensitivity reaction. Therefore, increase in DTH reaction in mice in response to T-cell dependent antigen exposed the stimulatory effect of probiotics on T-cells.
The mechanism behind the raised DTH during the cell-mediated immunity responses could be due to sensitized T lymphocytes. When challenged by the antigen, they are converted to lymphoblast and secrete a variety of molecules including proinflammatory lymphokines, attracting more scavenger cells to the site of reaction (Xie et al., 2007). Different doses of E. faecium GMB24 and E. hirae SMB16 showed significantly increased DTH reaction as compared to control group. The present investigation suggests that the probiotics have an overall stimulatory effect on both humoral and cellular immunities.