Isolationand Identification Of Probiotics And Application For Its Fermented Soybean Meal On Production Of Gushi Chickens

Four strains, bile and acid-resistantBacillus cereus and Lactobacillus that were isolated fromthe nature and Saccharomyces cerevisiaeand Aspergillus oryzae that were identified and stored byour laboratory, were mixed in accordance with a ratio of5:2:3:1(Bacillus cereus5×106/g,Lactobacillus2×106/g, Saccharomyces cerevisiae3×106/g and Aspergillus oryzae1×106/g) inthe current study. The compound probiotics were then used to ferment soybean meal at30℃for72hour. Dynamic changes of both conventional and unconventional indicators were monitoredalong with the72h-fermentation. Subsequently, both the compound probiotics fermented soybeanmeal and ordinary soybean meal made corn-soybean meal diet were employed to feed1-day-oldGushi egg chickens for a30-day experiment. Later, the growth performance, immune function,intestinal development, quantity and stability of intestinal microbial flora communities andmanure odor improvement were set to compare between the two treatment.Experiment1: Screening and Identification of bile and acid-resistant Bacillus andLactobacillusHigh temperature-resistant Bacillusand acid-productionLactobacillus were isolated andseparatedfrom nature environment, like straw organic fertilizers and and soil etc.. Later, bile andacid-resistance Bacillus strains that was able to produce protease and amylase of high avtivityand bile and acid-resistantLactobacilluswere screened out through screening andre-screening.Besides,16S rRNA of the isolated Bacillus and Lactobacillus were amplified andaligned for identification, while the phylogenetic trees of the isolates were constructed usingMEGA4.0for analysis. Finally,1bile and acid-resistant Bacillus cereus strain with competence toproduce protease and amylase and and1bile and acid-resistant Lactobacilluswith strong capacityto produce acid were successfully screened out.Experiment2: Dynamic monitoring of nutritional values for compound probiotics fermentedsoybean mealThe stock preparation of Bacillus cereus and Lactobacillus were manipulated using platingtechnique for viable count, while that of Saccharomyces cerevisiae and Aspergillus oryzae sporeswere counted using a hemocytometer. The four strains were supplemented to raw soybean mealwith the ratio of5:2:3:1(Bacillus cereus5×106/g, Lactobacillus2×106/g, Saccharomycescerevisiae3×106/g and Aspergillus oryzae1×106/g) so as to start the72h-fermentation with amoisture ranged75%-90%at30℃. Ventilation and samples collection were operated in a sterileenvironment every8hours. Subsequently, dynamic changes of fermented soybean meal’sconventional and unconventional indicators were determined. The results showed that:1)Moisture content of the fermented soybean meal had no significant changes (P>0.05) during the 72h-fermentation.2) Crude ash content of that increased significantly (P <0.05) only at72ndhourof the72h-fermentation.3) Crude protein and acid-soluble protein content were significantlyincreased (P>0.05) to high level and last for the rest of the fermentation.4) Viable cells declinedabsolutely at first, yet evidently raised (P <0.05) after40thhour.5) pH values sharply reduced (P<0.05) to5.48since8thhour and maintained a ideal low pH range till the end of the fermentation.6) Only serine content of48h-fermented soybean meal significantly increased (P<0.05) while allamino acids content of72h-fermented soybean meal absolutely raised (P<0.05) except for that ofmethionine.7) Trypsin inhibitor content steadily declined (P<0.05) during the fermentation.8)Phytic acid content of that increased slightly at first and then lowered (P>0.05).9) Proteaseactivity had big variations in the fermentation process.10) Amylase activity significantly raised(P<0.05) since8thhour and, and then maintained a high level since then.11) Though antigenprotein degradation strengthened gradually yet not entirely.Experiment3: Production and application of compound probiotics fermented soybean mealon Gushiegg chickens1201-day-old Gushi eggchickens were randomly divided into two treatments, four replicateseach treatment, which were fed with corn-soybean meal diets compared usingraw and fermentedsoybean meal respectively.Growth performance indicators were determined every10days andfresh fecal samples were collected at the same time.4Gushi chickens were randomly select andput to death with4mL5%sodium pentobarbital injection for each treatment at10th,20th,25thand30thdays of the experiment. Each part of the intestine and contents of those segments wereremoved and collected in a sterile environment. Subsequently, length and weight of the separatedintestinal segments were determined as well. In addition, samples of wing blood was alsocollected from the4randomly selected Gushi chickens at30thday. The results showed that:1)Compound probiotic fermented soybean meal (CFSM) significantly increased(P<0.05) Gushichickens’final body weight and average daily gain of entire experiment, and also absolutelyimproved (P<0.05)Gushi chickens’digestibility of crude protein in the whole trial period.2)CFSM had measly changed (P>0.05) entire immune organ index and final serum IgGconcentration, yet significantly increased (P<0.05) jejunal sIgA content, relative expression levelof blood leukocytes IL6, IL8and that of cymphocyte subsets CD3+.3) CFSM sharplyincreased(P<0.05) Gushi chickens’ duodenum length.4）CFSM significantly increased (P <0.05) thepopulation of Gushi chickens’ duodenum Lactobacillus and ileum and cecumEnterococcus, yetraised that of cecumEscherichia coli at the same time. CFSM had stronger influence on prophaseduodenum and jejunum microflora than those of late stage with the same age, moreover, theintestinal microflora species tend to be a more stable state. Though CFSM had slightly impact on the balance of ileum microflora, the colony species of that were always stabilized. However,CFSM had greater impact on cecal microflora, the cecal microbial species similarity graduallyreduced to27.2%, yet the diversity of that growing day by day.5）CFSM significantly reduced (P<0.05) entire period nitrogen and final skatole content in chicken manure, while sharply decreased(P<0.05) indole content in chicken manure. The results above inferred that CFSM had agrowth-promoting effect on Gushi chickens, and also strengthened their immune function in a way.Besides, CFSM promoted the enrichment of beneficial intestinal flora within Gushi chickens, themicrobial community stability of duodenum, jejunum and ileum and the microbialcommunitydiversity of cecal. In a way, CFSM reduced the odor of Gushi chicken manure andimproved the breeding environment.