| Along with the rapid development of fish farming technology, high-density intensive farmed has become the mainstream of the aquaculture industry in recent years. Therefore, there is a growing demand for quality feed additives. However, high-density intensive farmed caused frequent fish disease. One thing led to another, it is quite serious that abusing of antibiotics and growth additives in aquaculture. The harmful residues of farmed fish not only threatened our food security, but also deteriorated the water environment seriously. Under such circumstances, it’s high time that we find a feed additive which is non-toxic, no-residue and quality. Long-term production practices and a large number of studies have shown that Bacillus spp. could not only inhibit the growth of common pathogenic bacteria, but also promote animal growth, improve animal digestion enzyme activity, and was one of the most potential sources of probiotics.According to the general screening methods for probiotics and criterion for Bacillus, Bacillus licheniformis strain KL6was isolated. The in-vitro probiotic evaluation of this stain was studied, and then the fermentation medium and conditions was optimized. Effects of Different concentrations of Bacillus licheniformis KL6on growth and digestive enzyme activities of Cyprinus carpio were studied finally.In the first instance,20Bacillus strains were isolated from the hot springs by culture plate dilute method, and then selected the one which had the highest activity of protease and amylase. The strain was classified as Bacillus licheniformis according to the results of the morphology of colony and thallus, some physiology biochemical tests and the analysis of16SrDNA sequences, named KL6.In the second place, resisting adversity property and in-vitro probiotic evaluation of Bacillus licheniformis KL6was studied. The strain KL6had antistatic ability to the Staphyloccocus aureus Rosenbach, and didn’t to the Escherichia coli and Salmonella. The strain KL6had strong high-temperature resistance. Survival rate of Bacillus licheniformis KL6was without a significant difference (P>0.05), after being treated at85℃in15min. The survival rate decreased by26.19%(P<0.05) for20min. In the acid tolerance trial, the survival rate in the treatment group was50.00%、13.20%、7.33%respectively for pH2,3and4gastric fluid treatment groups(P0.05). In the bile salt treatment, the survival rate was84.40%. On the basis of the results of this studies Bacillus licheniformis, KL6, had potential as microbial feed additives.In the third place, optimize fermentation medium and conditions by and4-factors and3-levels orthogonal and single-factor expts. The number of viable cell was influenced by four factors in order of significance as follows:MnSO4> NaCl> Yeast extract> Peptone, the number of spores was MnSO4> Peptone> NaCl>Yeast extract, and the optimum culture condition was:Peptone1%、Yeast extract0.5%、 NaCl1%、25%solution of MnSO40.2%. The suitable temperature of Bacillus licheniformis was37℃. The fermentation process of the fermenter should be stop at14～16h, at that time the number of viable cell was101.63×109per milliliter, and the rate of spores was97.11%。On the basis of the results, Bacillus licheniformis KL6was suitable for high-density fermentation of liquid. This experiment provides a theoretical basis to the development of microbial feed additives and large-scale fermentation which is based on KL6.Last but not the least, the feeding trial in Cyprinus carpio with KL6was studied. Cyprinus carpio was fed with the basal diet supplemented of different concentrations of Bacillus licheniformis KL6, and then Effects of Different concentrations of stain KL6on growth and digestive enzyme activities of Cyprinus carpio were studied finally. Compared with the control group, the rate of weight gain rate and the coefficients of bait of the tested groups of0.5%,1.0% Bacillus licheniformis additive were significant difference (P<0.05). The rate of weight gain rate were14.35%、15.24%higher and the coefficients of bait is73.20%,74.54%lower than the control group, respectively(P<0.05). The protease activities of chyme of the tested groups of0.5%,1.0%Bacillus licheniformis additive were43.26%、44.05%higher than the control group, respectively(P<0.05). The amylase activities of chyme of the tested groups of0.5%,1.0%,2.0%stain KL6additive were5.45%、5.06%、5.22%higher than the control group, respectively(P<0.05). The protease activities of hepatic-pancreas of the tested groups of0.5%,1.0%,2.0%Bacillus licheniformis additive were15.32%,14.23%,12.75%higher than the control group, respectively (P<0.05). Stain KL6didn’t have siginificant effect on the amylase activities of hepatic-pancreas. The protease activities of Mid-gut tissue of the tested groups of1.0%,2.0%Bacillus licheniformis additive were25.16%,25.59%higher than the control group, respectively (P<0.05). Stain KL6didn’t have significant effect on the amylase activities of Mid-gut tissue. Bacillus licheniformis KL6could significantly improve the growth and digestive enzyme activities.In summary, isolated Bacillus licheniformis KL6can be used as an high-temperature resistance feed additives of large-scale fish farming, which is cost-effective. It was suitable for high-density fermentation of liquid and large-scale cultivation. When the addition of stain KL6was1.0%for feeding Cyprinus carpio, it could significantly improve the growth and digestive enzyme activities. This experiment provides a theoretical basis to the strains as feed additives used in fish farming. |
PDF Full Text Request