Screening Of Protease-producing Lactic Acid Bacteria And Its Application In Soybean Meal Fermentation

Soybean meal is one of the most widely used plant-derived protein feeds with high crude protein content and balanced amino acid composition,however,the high content of macromolecular proteins affects the absorption of nutrients in animals,which reduces the feed utilization rate,and even causes diarrhea in animals in severe cases.The current treatment methods for macromolecular proteins in soybean meal are mainly physical,chemical and biological fermentation,of which the fermentation is the most promising treatment method.The key to fermenting soybean meal is,first,the strain used,and second,the fermentation process.In this study,we used lactic acid bacteria(LAB)as the starting strain,and screened LAB for high yielding proteases,excellent physiological and biochemical properties,good tolerance to bile salts,gastrointestinal adverse environment and broad-spectrum bacterial inhibition,and applied it to soybean meal for fermentation.The aim is to provide strain resource that can be applied to soybean meal fermentation and reference for exploring soybean meal fermentation systems.Firstly,more than 500 LAB strains isolated from forage grass,silage and piglet manure,kept in the lab were used inoculate into solid MRS medium for 48 h.After 2generations of activation,initial and re-screening of protease-producing strains by the perforation and forin methods,respectively.Six strains with the largest proteolytic ring diameter and highest enzyme activity were found to be ZZUPF95,P15,P24,ZZUPF94,LAO53 and LAO49.By treatment with 0.2%bile salt concentration,simulated artificial gastric fluid(p H 3.0)and intestinal fluid(p H 7.5)for 3 h,the viable bacterial count of the above six strains remained at 10~7 cfu/m L.After temperature and p H growth range tests,it was found that all above strains could grow at 4?50?and p H 3.0?10.0,and inhibited Escherichia coli,Salmonella enterica,Staphylococcus aureus,Pseudomonas aeruginosa,Listeria monocytogenes,Micrococcus luteus and Bacillus subtilis.After16S r RNA and rec A gene sequence analysis,ZZUPF95 was identified as Weissella cibaria,P15,P24 and ZZUPF94 was Lactobacillus plantarum subsp.plantarum,LAO49 was Lactobacillus fermentum and LAO53 was Lactobacillus reuteri.Through the above screening,it was finally determined that strains ZZUPF95 and P15 with high protease activity,excellent physiological and biochemical properties,strong bacterial inhibition ability,bile salt resistance,high activity in the gastrointestinal tract and optimal integrative performance,were used as fermentation agents for next soybean meal.Secondly,strains ZZUPF95 and P15 were cultured to a logarithmic period and then added to commercially available soybean meal at 2%inoculation for soybean meal fermentation test.A total of 5 groups were set up:control(soybean meal without added strains),ZZUPF95 group(soybean meal+ZZUPF95),P15 group(soybean meal+P15),mixed group(soybean meal+P15+ZZUPF95)and acidic protease group(soybean meal+protease),respectively.After anaerobic fermentation at room temperature for 0,12,24,36,48,60,72 h and 7,12,18,30 d,respectively,the microbial content,fermentation quality,chemical composition and protein molecular degradation were analyzed for the corresponding fermented soybean meal openings.Among them,microbial content were analyzed by plate culture method,organic acids by HPLC,crude protein content of soybean meal by Kjeldahl nitrogen determination,crude fat by Soxhlet extraction,crude fiber by fiber determination and protein degradation by SDS-PAGE.From the results,it can be seen that all groups of fermented soybean meal reached the best at 60 h of fermentation,LAB became the dominant strain of the fermentation system,lactic acid content reached the highest value,macromolecular protein degradation from 80 to 20?30 Kda,crude protein and crude fat content increased while crude fiber content decreased.In ZZUPF95 group,the number of LAB reached 5.31×10~9 after 48 h fermentation,with the best degradation of macromolecular proteins,the molecular weight of protein drops from about 80 to 20?30 Kda.The crude protein content of P15 group increased by 25.48%compared to the control at a significant level,with a 35.92%decrease in crude fiber and a 16.2%increase in crude fat.The p H value of the mixed bacteria group was reduced to 4.5 at 72 h,and maintained at this level throughout the whole fermentation cycle.At the same time,the lactic acid content of the soybean meal fermentation system with ZZUPF95,P15 and both added at 30 d of fermentation was 1.988,1.724 and 2.125 mg/m L,while the control and protease group were 0.727 and 0.838 mg/m L,respectively.It can be seen that the soybean meal added with LAB had higher lactic acid level than that of the control group during 30 d fermentation.From the above,it can be seen that although the fermentation quality of the soybean meal was optimal at 60 h,LAB treatment groups still maintained high lactic acid levels at 30 d.Therefore,this study also provides a reference for extending the storage time of fermented soybean meal.