Molecular Characterization and Dosage Application of Autochthonous Potential Probiotic Bacteria in Cirrhinus mrigala

Abstract

The present communication deal with the identification of probiotic strain isolated from the intestine of Cirrhinus mrigala (Mrigal) on the basis of phylogenetic analysis and nucleotide homology and was found similar to Bacillus cereus strain SL1. The strain was further investigated for its probiotic abilities; acid and bile tolerance, autoaggregation, coaggregation and hydrophobicity. A dietary experiment of 90 days was carried out to examine its effect on the growth, digestive enzyme activity and survival of C. mrigala. Four isonitrogenous and isocaloric diets were prepared and B. cereus was incorporated in the experimental diets; T1, T2 and T3 at three different levels in the proportion of 2 × 10 4 , 2 × 10 5 and 2 × 10 6 cells 100 g-1 of feed respectively, along with a control diet TC without probiotic. After 90 days, the group of mrigal fed on probiotic supplemented diets showed significantly (P<0.05) better growth, specific growth rate (SGR), feed conversion ratio (FCR) than those fed with basal diet (control) with best result for diet T2 (P<0.05). Values of digestive enzyme activities (protease, amylase and cellulose) and carcass protein were also significantly (P<0.05) higher and excretion of metabolites (ammonia and phosphate) were lower in groups fed with diet T2. When probiotic supplemented diet fed fish were challenged with pathogenic Aeromonas hydrophila by immersion for 10 days, all probiotic fed groups showed high survival in comparison to group fed on control diet without probiotic supplementation indicating immunostimulating effect of probiotic in diets of C. mrigala. 47 Journal of FisheriesSciences.com Journal abbreviation: J FisheriesSciences.com Bhatnagar et al., 11(2):046-056 (2017) and toluene, and whirled for 1 minute followed by determination of optical density of the water phase at 450 nm. Hydrophobicity was thus calculated following the formula: % Hydrophobicity=[(OD450 before-OD450 after)/OD450 before] × 100 Aggregation property: Auto aggregation and coaggregation assays The rate of aggregation of bacterial isolates was determined following the method of Re et al. (2000) as modified by Kos et al. (2003). To evaluate the auto aggregation, the selected bacteria were grown in nutrient broth at 37°C for 24 hour. Following the centrifugation at 5000 g for 15 minutes, cells were washed twice and suspended in phosphate buffer saline to attain a possible concentration of about 107-108 CFU ml-1. After mixing for 10 seconds, 4 ml of the cell suspension were incubated at room temperature for 5 hour to determine auto aggregation. Each hour 0.1 ml of upper suspension was transferred by 3.9 ml of phosphate buffer solution, and the optical density was measured at 600 nm. Percentage (%) auto aggregation was calculated by the equation: 1-(At/A0) × 100 where, At represents the absorbance at time t=1, 2, 3, 4 or 5, and A0 the absorbance at t=0. For determining coaggregation, the cell suspension was prepared in a similar manner. 2 ml aliquots of two cell suspensions were mixed together by whirling for 10 second. Along with, about 4 ml of each cell suspension was set aside as control group. After mixing and incubating at room temperature for 5 hour the absorbance was measured at 600 nm. Coaggregation was calculated following Handley et al. (1987): (Ax+Ay)/2-A(x+y)/Ax+Ay/2 × 100, where Ax and Ay represented absorbance of each of the two strains in the control tubes, and (x+y) their mixture. Acid and bile tolerance assays The tolerance to acid was evaluated using a modified method of Conway et al. (1987) and Pennacchia et al. (2004). In a nutrient broth cultures were grown at 37°C for 18 hours. 1 ml of this bacterial suspension was added into 9 ml of sterile phosphate buffer saline solution adjusted to pH 2.5 with 5 N HCl. The bacteria were incubated at 37°C for 0 and 3 hour, representing the time spent by food in stomach; while pH 6.0 served as a control group. Bile salt tolerance was performed following the method of Gilliland et al. (1984). 1 ml of bacterial suspension was transferred into 9 ml of sterile nutrient broth prepared with bile salt. About 0.3% of bile salt concentrate was applied while second culture with 0% bile was used as a control. This suspension was incubated on nutrient agar at 37°C and viable bacteria were counted after exposure of 0 and 4 hour, reflecting the time spent by food in the small intestine.