Screening And Optimization Of Fodder Antibacterial Lactic Acid Bacteria Co-culture And Resistance Analysis

Lactic acid bacteria are beneficial microorganisms in the digestive tract of animals.Lactic acid bacteria produced antibacterial substances with a broad spectrum,which seldom not cause drug resistance and any other characters easily.Furthermore,lactic acid bacteria draw much attention as new feed additives with trophic and health function.In this study,lactic acid bacteria posting antibacterial activity were chosen as the research objects.A combination of co-cultured lactic acid bacteria was screened according to the growth characteristics of lactic acid bacteria thalluses,which obtained co-cultivation efficient inhibition lactic acid bacteria co-culture combination.And the co-culture conditions of lactic acid bacteria co-culture combination were optimized to produce metabolic antibacterial substances.We also analyzed the sensitivity of lactic acid bacteria to conventional antibiotics and antagonist with conventional probiotics.The results provided a reference for the development and production applications of lactic acid bacteria as new feed additives.The main specific contents and the results were as follows:1.Screening of the antibacterial lactic acid bacteria co-culture combinationSelecting Streptococcus thermophilus,Lactobacillus acidophilus,Lactobacillus casei and Lactobacillus fermentum,with the antibacterial activity of the culture supernatant was measured respectively.It was found that the four lactic acid bacteria to Escherichia coli,Enterobacter cloacae,Staphylococcus aureus,avian pathogenic E.coli,Pseudomonas aeruginosa,Enterococcus faecalis,Salmonella paratyphi A,Salmonella pullorum,Salmonella paratyphi B,avian Pasteurella multocida the ten kinds of pathogenic indicator bacteria showed different inhibition.The four lactic acid bacteria strains posted broad-spectrum antimicrobial activity.So we selected the four lactic acid bacteria for producing antimicrobial substances co-culture study.On the basis of the growth characteristics of the four lactic acid bacteria while culturing separately and co-culturing of combinations.With the antibacterial activity and biomass as index,Streptococcus thermophilus,Lactobacillus acidophilus,Lactobacillus casei,Lactobacillus fermentum combinations of two co-culture and each individual culture comparative analysised,we screended co-culture combination of lactic acid bacteria high-yielding inhibitory substance.The results showed that under the same conditions,after the Lactobacillus casei and Lactobacillus acidophilus co-cultured,the biomass and antibacterial activity increased.The OD600 nm value was(5.667±0.045),and the bacteriostatic diameter was(19.53±1.15)mm.So the co-culture combination of Lactobacillus casei and Lactobacillus acidophilus were selected to be the co-culture complex bacteria.2.Lactobacillus casei and Lactobacillus acidophilus co-culture condition optimizationWith the antibacterial activity of Lactobacillus casei and Lactobacillus acidophilus co-culture supernatant as index,we screened cheap carbon source and nitrogen source as medium raw materials,and gained optimized fermentation medium: 1.32% glucose,0.68%sucrose,1.5% yeast extract,0.5% wheat bran,0.005% manganese sulfate,0.02%magnesium sulfate,0.05% anhydrous sodium acetate,0.2% dipotassium phosphate.On the basis of the optimized medium,the different factors were investigated in order to optimize the conditions for producing antimicrobial substances,such as incubation time,temperature,inoculums,medium volume.The results showed that when Lactobacillus casei and Lactobacillus acidophilus combination co-cultured for 30 h at 33 ? in the conditions of the inoculums 4%,medium volume 40%,the antibacterial activity came to maximum,while the bacteriostatic diameter being up to 24.87 mm.3.Analysis of resistance to antibacterial lactic acid bacteriaThe antagonistic experiments between lactic acid bacteria and several strains of probiotics showed that the Streptococcus thermophilus,Lactobacillus acidophilus,Lactobacillus casei,Lactobacillus fermentum with Saccharomyces cerevisiae and Pichia yeast Av BD9 had no obvious antagonistic behavior,while Bacillus subtilis,Bacillus licheniformis,Bacillus cereus had antagonistic effect to varying degrees.So the probiotics complex mixed using should be paid attention to select the appropriate composition method to achieve better application results.The four lactic acid bacteria strains showed varying degrees of sensitivity to eight kinds of conventional antibiotics.So we should pay attention to the deployment using of lactic acid bacteria in daily application and replenished lactic acid bacteria live agents timely.In summary,by the comparative analysis of biomass and antibacterial activity,Lactobacillus casei and Lactobacillus acidophilus co-culture combination were screened out,which posting broad-spectrum antimicrobial activity.We received Lactobacillus casei and Lactobacillus acidophilus co-culture conditions,and reduced the cost of raw materials medium to some extent,and simplified the production process,which laid the foundation for the application of lactic acid bacteria.The different sensitivity of lactic acid bacteria to the conventional antibiotics and the antagonistic with other conventional probiotics would to provide a reference of lactic acid bacteria as a feed additive in the application.