Study On Preparation Of Soybean Meal With Low Antigenicity By Fermentation Assisted Enzymatic Hydrolysis

Soybean meal is a by-product of soybean oil extraction.Its protein content is up to 43%⁴6%,and it is rich in bioactive substances such as isoflavones and phospholipids,as well as fat,sugar,minerals and other nutrients.It is a very high quality.Livestock and poultry plant protein feed ingredients.However,due to the presence of antigenic proteins in soybean meal,it can cause allergic reactions in young animals.The symptoms are diarrhea,intestinal inflammation and growth stagnation,and even cause animal death,which limits its application in young animal feed to a certain extent.Therefore,reducing the antigenic protein activity in soybean meal is a hot topic in animal nutrition research at home and abroad in recent years.At present,fermented soybean meal and enzymatically hydrolyzed soybean meal are increasingly favored for their high nutritional value,rich in active ingredients,low production cost and safe and non-polluting properties.This thesis intends to explore a processing method of low antigen soybean meal by combining microbial fermentation and protease hydrolysis.This thesis is divided into three parts:?1?Screening for probiotics with the best effect of degrading antigen protein by SDS-PAGE,degree of hydrolysis,ELISA,etc.;?2?Screening by SDS-PAGE,degree of hydrolysis,ELISA,etc.The protease with the best effect on degrading the antigen protein is obtained;?3?The optimal conditions for the synergistic fermentation of the soybean meal by the enzyme enzyme are determined by the response surface method.The main research methods and conclusions are as follows:?1?Different extraction methods were used to extract the protein in soybean meal,and the protein electrophoresis band integrity of fermented soybean meal was used as a screening index.A better protein extraction method was selected:weigh potassium dihydrogen phosphate?KH₂PO₄·2H₂O?1.2g,disodium hydrogen phosphate?Na₂HPO₄·12H₂O?14.5g,dissolved in distilled water and dilute to 1000mL,take100mL of the solution after constant volume,add 48g urea,0.31g DTT and dissolve to make denaturing extraction buffer,pre-80?After heating for 5 min,6 mL of soybean meal was added thereto,ultrasonically extracted for 35 min,centrifuged,and the supernatant was taken,and dialyzed overnight in a dialysis bag with a molecular weight cut off of 3500D to collect proteins.?2?Fermented soybean meal by Lactobacillus plantarum,Lactobacillus casei,Bacillus subtilis,Bacillus licheniformis and Aspergillus oryzae,which are classified into solid state fermentation and liquid fermentation according to the ratio of material to water.The protein bands were determined by SDS-PAGE and the OPA method was used to determine the degree of protein hydrolysis of soybean meal,the quantitative detection of soy antigen protein?Glycinin and?-Conglycinin?in the hydrolysate and the antigenicity of the two antigenic proteins by ELISA.Variety.The results showed that the best fermentation strain was Bacillus subtilis,the fermentation condition was1:1,the inoculum was 1mL 1×10⁸cfu/mL,and the fermentation was carried out at 37?for 12h.At this time,the degree of hydrolysis of soybean meal was 3.88%,the degradation rate of Glycinin in soybean meal was 82.4%,and the degradation rate of?-Conglycinin was 88.5%.According to the total amino acid content,the essential amino acid content in the soybean meal after solid-state fermentation of Bacillus subtilis for 12 hours was about 194 mg/g.?3?Neutral protease,alkaline protease,pepsin,flavor protease and papain were used to hydrolyze soybean meal.Protein bands were analyzed by SDS-PAGE.The degree of protein hydrolysis of soybean meal was determined by OPA method.Soybean antigen protein?Glycinin and?-Conglycinin?Quantitative detection and ELISA assay for antigenic changes in these two antigenic proteins.The results showed that pepsin was added at 0.5%of the substrate mass,and hydrolysis was carried out at pH 2,37?for 60 min as the optimum reaction condition.At this time,the degree of hydrolysis of soybean meal was 10.7%,the degradation rate of Glycinin in soybean meal was 79.3%,and the degradation rate of?-Conglycinin was 87.6%.According to the total amino acid content,the essential amino acid content in soybean meal was about 170 mg/g after pepsin hydrolysis for 60 min.?4?Using response surface methodology to optimize the conditions for the preparation of low antigenic soybean meal by co-fermentation of Bacillus subtilis and pepsin,and obtain the optimal fermentation conditions for the addition of 0.825%soybean meal quality pepsin to soybean meal.After adjusting the pH to 2,at 37?Enzymatic hydrolysis for 88.2min.After that,1mL of Bacillus subtilis with a concentration of 1×10⁸cfu/mL was inserted into the medium,and the fermentation was allowed to stand in a 37?incubator for 17.52h.At this time,the degradation rates of Glycinin and?-Conglycinin were 78.6%and 40.5%,respectively.