Thermoprotease-producing Strain And Its Application In Solid-state Fermentation Of Soybean Meal

Soybean meal（SBM）,with about 50%protein content,is an important source of plant protein.However,the existence of anti-nutritional factors（ANFs）and allergenic proteins severely restrict its use in food and animal feeds industries.Microbial fermentation is one of the efficient methods to improve the quality of SBM,but traditional mesophilic fermentation has the distinctive disadvantages of high energy consumption and risk of bacterial contamination.Thus,it is particularly important to reduce production cost,improve fermentation efficiency and ensure the safety of fermented product,besides quality assurance of fermented soybean meal（FSBM）.The purpose of this study is to screen a thermophilic protease-producing strain which could be directly fermented under the condition of SBM without sterilization.Optimizing the fermentation process and evaluating the safety and quality of fermentation products will be performed.Through the analysis of microbial community structure,the influence of high-temperature protease-producing strains on microbial diversity in soybean meal substrate during fermentation was investigated.The real-time monitoring dynamic model of soybean meal high temperature solid state fermentation（SSF）by near infrared spectroscopy was established.The main contents and results of this study are as follows:（1）Thirty-four strains with thermophilic protease-producing properties with different morphology were obtained.A strain named YYC4 was isolated by the primary screening of skimmed milk plate and the rescreening of fermentation SBM,which could be used to ferment SBM.The content of water-soluble protein in SBM was increased to 13.20%by YYC4 fermentation.Strain YYC4 was identified as Bacillus licheniformis by physiology,biochemistry and 16S r DNA examination.The growth curves of strain YYC4 at 30℃ and 60℃ indicated that YYC4 was a member of thermophiles.（2）Through response surface optimization,the optimal conditions of thermophilic SSF of unsterilized SBM by YYC4 strain were as follows:solid-liquid ratio 1:1.80g/m L,fermentation temperature 55℃,inoculation 10⁷ CFU/g water-containing substrate,relative humidity 80%,incubation 50 h,Mg SO₄ 0.12%（w/w）.The contents of crude and water-soluble protein in SBM reached 52.48%and 20.33%,respectively,and a increment of 279.73%with the water-soluble protein after YYC4 fermentation.Meanwhile,trypsin inhibitor（TI）activity was decreased by 61.05%.The safety of FSBM was verified by acute toxicity animal experiment by giving a maximum gavage dose of 10 g/kg body weight.The result showed that FSBM was safe and non-toxic by observation of mice activity,measurement of viscera index and analysis of serum biochemical and immunological indexes.（3）The fermentation effect of YYC4 strain on SBM was compared with that of Bacillus subtilis 10160 and Geobacillus stearothermophilus A75,indicationg that both YYC4 and 10160 strains had better performance.The physicochemical,structural and functional characteristics of fermented SBM by YYC4(FSBM_YYC4)were as follows:Physical and chemical properties:FSBM_YYC4 protein in vitro digestibility reached89.96%.The content of essential amino acids was 19.20 g/100 g.FSBM_YYC4 was rich in volatile components,such as phenols,pyrazines,alcohols,ketones and indoles,etc.SDS-PAGE analysis showed that YYC4 strain had strong ability to degrade antinutritional factors,and the fermentation product had good solubility,and producing proteins with a molecular weight of less than 10 k Da.The analysis of molecular weight distribution showed that YYC4 strain could degrade a large number of insoluble macromolecular proteins into water-soluble small molecular proteins,and the relative content of components with molecular weight less than 5 k Da was 16.46%.Structural characteristics:After fermentation by YYC4,theλmax of fluorescence spectrum of SBM protein shifted from 353 to 362 nm,the surface hydrophobicity（H₀）increased from 98.27 to 166.13,the content of sulfhydryl（-SH）and disulfide bond（S-S）increased from 3.26 and 20.77μmol/g to 4.88 and 27.95μmol/g,respectively.The infrared spectrum analysis showed that the content ofβ-sheet in the secondary structure of protein increased from 38.44%to 60.88%.The particle size decreased from 629.25to 216.90 nm,and the absolute value of Zeta potential was larger.Protein particles were significantly reduced and appeared more evenly dispersed by atomic force microscopy（AFM）.Scanning electron microscopy（SEM）analysis showed that the apparent morphology of FSBM was more heterogeneous and the surface was loose,in addition,the FSBM protein presented as irregular fragments and smaller protein particles.Functional characteristics:The contents of flavonoids and polyphenols in SBM were 1.34 and 5.32 mg/g after YYC4 fermentation,corresponding to a increment of152.83%and 133.33%.Scavenging DPPH,hydroxyl radicals（·OH）,ABTS and superoxide anion（·O₂^-）radicals abilities of SBM increased significantly from 8.52%,24.60%,36.21%and 12.26%to 16.32%,79.92%,66.85%and 22.26%after YYC4fermentation,respectively.After simulated digestion in vitro,abilities of SBM to scavenge above radicals reached 16.30%,37.44%,50.55%and 30.32%,while it was39.93%,92.60%,72.55%and 38.83%for FSBM_YYC4.Inhibition on angiotensin I-converting enzyme（ACE）increased from 44.89%to 63.79%after SBM fermentation by YYC4.And after simulated digestion,inhibition on ACE of SBM and FSBM_YYC4was increased to 73.10%and 80.57%respectively.Water and oil holding capacity of FSBM_YYC4 were 296.03%and 233.58%.In addition,FSBM_YYC4 had good emulsifying property and emulsifying stability,which were 42.38 m²/g and 157.90 min,respectively.Inconclusion,after YYC4 strain fermentation,the physicochemical and functional properties of soybean meal were significantly improved,and the protein structure was significantly changed.（4）During thermophilic SSF of SBM,pH increased gradually and the activities of neutral,alkaline,and acid protease reached 181.31,73.5,and 33.72 U/g,respectively,and cellulose and amylase activities reached 1.21 and 94.67 U/g.Analysis of the microbial community diversity showed that the number of OTU decreased by 52.12%after 50 h of fermentation with YYC4,and the number of OTU decreased by 26.36%after 50 h of fermentation without inoculation of strain compared with SBM.A decrease of 34.98%in the number of OTU after 50 h fermentation with inoculation YYC4 than that without inoculation was obtained.After YYC4 fermentation for 12 h,Bacillus licheniformis became the dominant strain in the substrate,and its abundance reached81.64%.Then,the microbial community structure tended to be stable with the further extension of fermentation time,and its final relative abundance of 56.42%was acquired.（5）The changes of pH,water content,water-soluble protein and trypsin inhibitor（TI）content during thermophilic SSF of SBM were monitored by near infrared（NIR）spectrometer.A prediction model was established based on the si-PLS measurement.R_C,R_P,RMSECV and RMSEP of the established correction and prediction model were（pH:0.9157,0.9781,0.312 and 0.169）,（water content:0.9886,0.9909,0.339 and0.313）,（water-soluble protein content:0.9127,0.9883,1.83 and 0.681）and（TI content:0.9896,0.9916,0.261 and 0.236）.It can be seen that the models have a good predictive ability and the real-time monitoring of each index during thermophilic SSF of SBM can also be achieved.