Study On Mixed Fermentation Of Rapeseed Meal Feed With Probiotics

China's rapeseed meal is rich in resources.Rapeseed meal has the advantages of rich crude protein content and balanced amino acid composition.It has high application potential as a plant protein feed.However,due to the existence of various anti-nutritional factors in rapeseed meal,the feeding value of rapeseed meal is largely restricted.In this paper,High-efficiency strains of bacteria for rapeseed meal fermentation were selected and combined probiotics were applied to ferment rapeseed meal to improve the quality of rapeseed meal.In this paper,47 strains were isolated from sources such as rapeseed meal fermentation silage using selection medium containing glucosinolates.Using the degradation ability of the main anti-nutritional factor glucosinolates of rapeseed meal and the activity of protease as indicators,two probiotic strains,Y8 and GYB6,were screened for the efficient fermentation of rapeseed meal.The two strains were identified as Bacillus amyloliquefaciens and Bacillus subtilis by morphological characteristics,physiological and biochemical characteristics and 16S r DNA sequence analysis.B.subtilis,B.amyloliquefaciens and 6 strains of probiotics preserved in the laboratory were used forsingle bacteria fermentation.The degradation rate of glucosinolate X(%),peptide increase rate Y(%),tannin degradation rate Z(%),phytic acid degradation rate M(%),crude fiber degradation rate W(%)were used asthe evaluation indices,the weighting method to take N was used as the comprehensive evaluation index(N=0.5×X+0.2×Y+0.1×Z+0.1×M+0.1×W).The results showed that B.amyloliquefaciens had the highest degradation rate of the glucosinolate in rapeseed meal.The degradation rate was 53.89%.The peptide increase rate of rapeseed meal after fermentation was the highest.It was 88.89%.Saccharomyces cerevisiae had the highest degradation rate of phytic acid and tannin in rapeseed meal.The degradation rate was 36.31%and 23.01%,respectively.B.subtilishad the highest degradation rate of crude fiber in rapeseed meal.The degradation rate was 37.48%.According to above indicators and comprehensive evaluation indicators,B.amyloliquefaciens,B.subtilis and S.cerevisiae were selected forfurther mixed fermentation.In order to establish a synergistic way to ferment rapeseed meal with the three strains,the two strains were first used to ferment.The results showed that B.subtilis and S.cerevisiae were mixed in one-step fermentation,and the degradation rate of glucosinolate and the growth rate of peptide in rapeseed meal were the highest,which achieved the synergistic effect.The stepwise fermentation effect of B.amyloliquefaciens with S.cerevisiae and B.subtilis respectively,was better than one-step fermentation.Therefore,the method of the three bacterium combination to ferment rapeseed meal was to inoculate B.subtilis and S.cerevisiae first and then inoculate B.amyloliquefaciens.First,the optimal ratio of three bacteria mixed fermentation was determined.Results showed that the optimal ratio of B.subtilis:S.cerevisiae:B.amyloliquefaciens was2:3:1.Through single factor and orthogonal experiments,the optimal fermentation temperature,feed-water ratio,fermentation time and inoculation amount of three kinds of probiotic were explored to determine the best process of mixed solid-state fermentation of rapeseed meal.The results showed that the optimal conditions for solid fermentation of rapeseed meal by B.subtilis,S.cerevisiae,and B.amyloliquefaciens were inoculation amount 30%,fermentation temperature37?,feed water ratio 1:1.2.First B.subtilis and S.cerevisiae was inoculated for 48 h fermentation and then B.amyloliquefaciens was inoculated for 48 h.The nutrient components,anti-nutritional factor components,probiotic bacterial counts and sensory indexes of rapeseed meal before and after fermentation were analyzed and determined.After the mixed fermentation with B.amyloliquefaciens,B.subtilis and S.cerevisiae,the crude protein content of rapeseed meal increased from 33.16%to 38.56%,and the amino acid content increased by 18.35%.According to SDS-PAGE analysis,the macromolecular protein in rapeseed meal was degraded to a small molecule proteins,and the rapeseed peptide content increased by nearly 3 times.After fermentation,the degradation rate of the main anti-nutritional factor glucosinolate was 99.04%,Glucosinolate was almost completely degraded.The amounts of isothiocyanate and oxazolidine thione was also greatly reduced,with degradation rates of 71.32%and 83.87%,respectively.The degradation rates of other anti-nutritional factors,such as tannin,phytic acid and crude fiber silk was 30.24%,43.09%and39.73%,respectively.The biomass of probiotic bacteria in fermented rapeseed meal is 3.84×10~8CFU/g.The quality of rapeseed meal has been greatly improved.