Study On The Preparation And Allergenicity Of Soybean Meal By Microbial Solid State Fermentation

Soybean meal is the byproduct of soybean oil extraction, and it has been an excellent plant protein source for its characteristic of high protein content and well-balanced amino acid profile. Currently, soybean meal was mainly used as animal feed in livestock, poultry or aquaculture, and rarely used in the food industry. However, soybean, as the raw materials of soybean meal, is one of eight major allergenic foods recognized by WHO/FAO, and the absorption and utilization of soybean meal nutrition in animals were reduced due to the presence of anti-nutritional factors, such as soybean protein allergens, which was the main factor that limited its application in animal feed. Recently, research has been mainly focused on microbial fermentation about the reduction in the anti-nutritional factors. However, little information is available on the effects of fermentation on the allergenicity of soybean meal. The aim of the study is to explore the effects of microbial solid fermentation on the potential allergenicity of soybean meal, which would provide the theoretical basis for development of high-quality, hypoallergenic fermented soybean meal product.Several works involved in this study included the following three aspects: Firstly, optimization of the solid state fermentation conditions for soybean meal. Secondly, allergenicity assessment of fermented soybean meal. Thirdly, analysis of physicochemical properties of fermented soybean meal. The main methods, results and conclusions are addressed as follows.1. The solid state fermentation of soybean meal was fermented by mixture of Bacillus subtilis, Lactobacillus casei and Yeast, and the conditions were optimized by single factor experiment based on analysis of molecular weight of the protein in fermented soybean meal using SDS-PAGE. It was concluded that the optimum fermentation conditions: water content was 60%, fermentation temperature was 30℃, inoculation quantity was 12%, inoculation ratio of Bacillus subtilis: Lactobacillus casei: Yeast was 2: 1: 1, the order of inoculation was inoculation of Bacillus subtilis 24 h after inoculation Lactobacillus casei and Yeast, and the total fermentation time was 72 h.2. The extraction methods of fermented soybean meal protein was improved, and the results showed that heating extraction methods can effectively prevent the degradation of soybean protein in fermented meal by microbial protease during extraction. It can more accurately reflect the degradation of soybean proteins in fermented meal based on SDS-PAGE electrophoresis.3. The allergenicity of fermented soybean meal was assessmented in vitro using competitive inhibition ELISA method. It indicated that the fermentation process can reduce the potential allergenicity of soybean meal through the analysis of binding ability of specific antibodies using rabbit polyclonal antibodies and human allergic serum. On the basis of the above, the sensitized mice model was established to evaluate the allergenicity of fermented soybean meal in vivo. It was found that the levels of specific IgE antibodies and mMCP-1 in mice serum of fermented soybean meal group were lower than those of the soybean meal group. It also indicated that the fermentation process could reduce the potential allergenicity of soybean meal.4. Partial physicochemical properties of the fermented soybean meal under optimal conditions were analyzed, and the main physicochemical indicators meet the requirements of the fermented soybean meal product used for animal feed. The molecular weight distribution of fermented soybean meal was analysed by size exclusion chromatography-high performance liquid chromatography(SEC-HPLC), and the results showed that the proportion of peptides molecular weight below 2 KDa amount to 47.57%. Peptides were further identified by liquid chromatography-mass spectrum(LC-MS), and it showed that 6 allergenic epitopes still existed in these peptides, including three human IgE binding epitope, namely β-conglycinin α-subunits N232-D383, glycinin G1 subunit G253-I265 and glycinin G2 subunit E169-S215, one rabbit Ig G binding epitopes located at glycinin G5 subunit G68-G98, and two pig IgG binding epitopes is glycinin G5 subunit A365-I375 and V153-A167, respectively. The results indicated that the fermented soybean meal in the study still have potential allergenicity.