Primary Study Of Probiotic Characteristics And Identification Of 3 Bacillus Strains

In recent years,antibiotics have been commonly used in the aquaculture industry to treat intestinal bacterial pathogens in animals.However,long-term overuse of antibiotics often leads to resistance to pathogenic bacteria that can spread from animals to animasl and enter the environment.It causes environmental pollution and can be transmitted to humans through the food chain,posing a threat to human food safety and health.Nowadays,more and more breeders and researchers are aware of the hidden dangers caused by the abuse of antibiotics.At the sametime,our country has entered the post-antibiotic era,and people’s demand for safe and effective antimicrobial agents is also growing.Probiotics have the potential to become the first choice for replacing antibiotics because of their effective,environmentally friendly,green and healthy characteristics.In this study,11 strains of Bacillus were selected by separating and purifying the plant-sourced mixed strains in the laboratory firstly.Secondly,the acid-resistant test,bile-resistant salt test,antibiotic susceptibility test and enzyme-producing qualitative test were conducted to screen strains with excellent biological properties.Thirdly,the screened out strains were identified from two aspects: physiological and biochemical characteristics,and 16 S rDNA molecular identification.Finally,in vivo antioxidation test in Drosophila was conducted.The main results are as follows:(1)The strain identification results showed that 513-A is Bacillus pumilus,513-O is Bacillus gobiensis,and 513-N is Bacillus subtilis.None of them have drug resistance,and 513-N strains has good antibacterial activity against common pathogenic bacteria.The survival rates of 513-A,513-O and 513-N strains after treated with HCl of pH 2.0for 1h are 70.61%,59.43% and 89.27%,respectively;the survival rates of513-A,513-O and 513-N strains after 0.3% pig bile salts treated 1h were67.30%.87.50% and 78.82%,respectively.(2)The antibacterial test results showed that the 513-N strain has a good antibacterial activity in against Escherichia coli,Staphylococcus aureus,Shigella flexneri,Salmonella choleraesuis and Salmonella paratyphi B.(3)Quantitative extracellular enzyme production tests showed that513-A,513-O and 513-N strains all have the ability to produce cellulase and protease,which are both extracellular enzymes.Their cellulase production activities are 6.21 U/ml,8.37 U/ml and 50.21 U/ml,respectively.The activities of neutral proteases are 44.40 U/ml,56.52U/ml and 90.31 U/ml,respectively;the activities of alkaline protease were 43.67 U/ml,65.72 U/ml and 120.44 U/ml,respectively.The 513-N strain has the ability to produce amylase,and the amylase activity is156.49 U/ml.(4)The result of antioxidant test of Bacillus in Drosophila indicates that the different doses of 513-A,513-O and 513-N mixed Bacillus in the medium have different effects on the antioxidant activity of Drosophila.The female Drosophila’ SOD and CAT activities in the middle-dose group are 11.71% and 48.65% higher than those in the control group after28 days of feeding.And the MDA content is decreased by 29.34%.The male Drosophila’ SOD and CAT activities in the middle-dose group are9.29% and 46.71% higher than those in the control group.And the MDA content is decreased by 23.91%.Both of Bacillus subtilis and Bacillus pumilus have the function of improving feed utilization.Therefore,513-A,513-O and 513-N strains have the potential of candidate strains for animal feed additives.513-O and 513-N strains also have the potential to be industrial alkaline proteases producing strains.And for the first time,the Bacillus gobiensis is to found that have the ability to produce cellulase and protease.