Screening Of Strains That Inhibit The Growth Of Silage Spoilage Organisms And Its Effect On The Aerobic Stability Of Maize Silage

Silage is a kind of high quality feed made of green plants after fermented by lactic acid bacteria,which has certain sour aroma and rich nutritional value.However,after opening the cellar or bag,the silage is prone to secondary fermentation,which leads to the deterioration of feed flavor,the growth and propagation of aerobic bacteria such as yeast and mold,so that the silage deteriorates and deteriorates,and even some mycotoxin will be produced to affect the safety of the feed.Therefore,this study intends to isolate and screen a Bacillus strain that can inhibit secondary fermentation of silage,and verify its efficacy through whole corn silage.The main results are as follows:1.Firstly,"+" line method and plate confrontation method were used to screen out three strains of Bacillus that could effectively antagonize yeast and various molds from nearly 1,000 strains of Bacillus stored in "Hebei Feeding Microorganism Technology Innovation Center",and one strain with the best effect was obtained by re-screening through the aerobic stability test of fermented bran,named CY-1.The strain CY-1 was identified as Bacillus siamensis by morphological observation,16S rDNA sequence analysis and physiological and biochemical tests.2.Ammonium sulfate precipitation method,acid precipitation methanol dissolution method and ethyl acetate extraction method were used to extract bacteriostatic substances from the supernatant of the fermentation of strain CY-1.By comparing the size of bacteriostatic zone produced by Picha,the optimal extraction method was determined to use 60%ammonium sulfate precipitation supernatant.The crude extract desalting by dialysis can produce strong inhibitory effect on yeast,and has certain thermal stability,protease tolerance,pH and UV tolerance,which is preliminarily speculated to be lipopeptide bacteriostatic substances.3.The whole corn was chopped for silage,and the experiment was divided into two groups:test group(T)which added strain CY-1 fermentation liquid;The same volume of sterile water was added as control group(CK),and the feed quality of 45 days of silage samples in two groups was evaluated.The results showed that the contents of dry matter,crude protein of T at 45 days of silage were significantly higher than those of CK(p<0.05),and the pH content of T was significantly lower than that of CK(p<0.05),but lactic acid and acetic acid were not significant.The results of microbial diversity showed that the dominant phylum were Firmicutes and Proteobacteria,and the dominant bacteria were Companilactobacillus and Levilactobacillus.The addition of strain CY-1 could reduce the abundance of Klebsiella species,but the difference was not significant.It was found that the dominant phylum in the two groups were mainly composed of Ascomycota and Basidiomycota.The abundance of Candida(p<0.01),Issatchenkia(p<0.05),Picha(p<0.05)and Fusarium(p<0.01)of T decreased significantly.The results showed that strain CY-1 was able to inhibit the proliferation of various yeasts and molds.In summary,the results indicated that strain CY-1 could help improve the fermentation quality of silage.A total of 183 differential metabolites were discovered in the untargeted metabolomics study.Among the differential metabolites in T,indoleacrylic acid and lichenin were up-regulated,and the expression of 2-aminobenzoic acid was downregulated,indicating that strain CY-1 may inhibit yeast and mold by producing some lipopeptide compounds.4.Through the index of whole corn silage,it was found that Bacillus siamensis CY-1 could reduce the number of yeast and mold,reduce the rising rate of pH and temperature and reduce the loss of dry matter and crude protein after the opening,which was beneficial to improve the aerobic stability of whole corn silage.The analysis of microbial diversity showed that Candida was the main fungus leading to secondary fermentation of whole corn silage,and the addition of strain CY-1 could reduce the proportion of Candida after aerobic exposure.With the delay of exposure time,the Acetobactor increased gradually and the Lactobacillus decreased gradually in two groups,but the proportion in T was higher than that in CK.