The Isolation, Identification, Screening And Application Of Lactic Acid Bacteria For Silage In Guizhou Area

Silage is an indispensable feed in animal husbandry,especially for ruminants.In this study,high grade lactic acid bacteria were isolated from natural fermented silage by MRS plate scribing separation method.The identified lactic acid bacteria were further screened by molecular biology,and the selected high grade lactic acid bacteria were added in To silage additives to be added to alfalfa silage.In the process of silage fermentation,the silage nutrient composition,silage quality,aerobic stability.The effects of different combinations and mixed lactic acid bacteria on the fermentation quality of local alfalfa were analyzed.The result is to provide a good lactic acid bacteria strain for improving the silage quality in Guizhou.The results are as follows:(1)130s of lactic acid bacteria were isolated from the collected natural silage in this experiment.The results were all bacillus.10 strains were identified after microscopic observation,Gram stain and catalase reaction identification and 16 S rDNA sequence analysis.The results appear as shown below: Lactobacillus rhamnosus,Lactobacillus plantarum,Bacillus subtilis,Lactobacillus casei,Lactobacillus farciminis,Lactobacillus acidophilus,Lactobacillus paracasei are homo-fermentative lactic acid bacteria;Lactobacillus buchneri,Lactobacillus hilgardii,Lactobacillus brevis are heterofermentative lactic acid bacteria.(2)The acid production rate of all purified lactic acid bacteria was determined.It was found that Lactobacillus rhamnosus strain BDy3-10 can decreasing the pH to below 4.0 in MRS liquid medium for 24 hours,so the acid-producing rate is faster;The growth rate of the strain was determined,and it was found that Lactobacillus buchneri strain TSy1-3 increased the optical density of MRS liquid medium at a wavelength of 600 nm to 3.015 within 24 h,indicating that it grows faster.(3)L.rhamnosus strain BDy3-10 and L.buchneri strain TSy1-3 were activated.They were divided in To three groups as lactic acid bacteria inoculation(LR treatment added L.rhamnosus strain TSy1-3 bacterial fluid;LB treatment added L.buchneri strain BDy3-10 bacterial fluid;The LR+LB treatment was added with the equal amounts of L.rhamnosus strain TSy1-3 and L.buchneri strain BDy3-10 mixed bacterial fluid)in the local alfalfa(Medicago sativa)silage.The results showed that there were no significant differences in crude protein(CP),crude fat(EE),crude fiber(CF)and crude ash(Ash)content in the lactic acid bacteria treatment.The LB treatment significantly reduced the neutral detergent fiber(NDF)and acid detergent fiber(ADF)content of silage alfalfa(P<0.05),and the soluble carbohydrate(WSC)content was also higher than other treatments(P<0.05).(4)LR treatment(pH 5.00),LB treatment(pH 5.07),mixed bacteria LR+LB treatment(pH 5.05)can significantly reduce the pH of alfalfa(P<0.05)than CK treatment(pH 5.61).The lactic acid production in the LR treatment was higher than that in the LB,LR+LB and CK treatment,and the acetic acid content was lower than that in the LB,LR+LB and CK treatment,and no butyric acid was detected in the silage process.It shows that adding L.rhamnosus strain BDy3-10 can improve the silage fermentation quality of local alfalfa.(5)The aerobic stability of the LB treatment was the longest,reaching 173 h,which was 52 h longer than the control treatment,while the aerobic stability of the LR and LR+LB treatments was also better than that of the CK treatment.The aerobic stabilization time was 129 hour and 144 hour,8 hour and 23 hour longer than the control treatment.It is indicated that the addition of L.buchneri strain BDy3-10 can improve the aerobic stability of alfalfa silage after exposure to air.In summary,considering the effects of different lactic acid bacteria addition methods on alfalfa silage,it is concluded that L.rhamnosus strain BDy3-10 and L.buchneri strain TSy1-3 are the best addition methods.