Study Of Fungal And Actions Of Related Fungal Enzymes On Low Denatured Defatted Soybean Meal

Low denatured defatted soybean meal(LDSM)is the raw material of a series of soybean protein products,such as soybean protein isolate,soybean protein concentrate,soybean tissue protein,defatted soybean protein powder and so on.At present,the fungal contamination of wheat,soybean and other raw grains has been paid more and more attention.However,the fungal contamination of LDSM,especially the effect of fungal enzymes on the quality of soybean protein has not been reported.This thesis begins with our exploration of an abnormal electrophoresis phenomenon of soybean protein.On this basis,the diversity and number of fungi infected by LDSM,the types and activity changes of fungal secretase,and the hydrolysis of main components in soybean meal by fungal secretase were revealed step by step.This study will provide a new thinking and operation dimension for the production process control and product stability improvement of LDSM and soybean protein.The main contents and results of this paper are as follows:Firstly,the fungal diversity and number of fungi in soybean,dehulled soybean and LDSM samples from major soybean protein manufacturers in China were investigated.The results showed that the diversity and number of fungi in whole soybean from different factories were similar,and soybean dehulling treatment could significantly reduce the number of fungi in soybean,but had little effect on fungal diversity.The fungal diversity of LDSM produced by different manufacturers was quite different,but Aspergillus accounted for a large proportion.At the same time,the effects of different simulated storage conditions(15℃-65% RH,15℃-85%RH,25℃-75% RH,35℃-65% RH,35℃-85 RH)on fungal diversity in LDSM were investigated.It was found that the similarity of fungal diversity in LDSM at 25℃-75% RH and35℃-85% RH was beneficial to the growth of Aspergillus,under other conditions,the effect of LDSM storage on fungal diversity was not obvious.Moreover,it was found that most of the fungi producing protease were Aspergillus.Then the fungal secretase of LDSM was studied.Firstly,the extraction method of fungal enzyme was optimized based on protease activity.It was found that deionized water,p H 8.0universal buffer(UB)and 0.1mol/L dithiothreitol(DTT)solution had better extraction effect.At the same time,the enzyme activity was the highest when the extraction time was 5 minutes～10 minutes.Through the protease inhibitor addition experiment,it was found that the extracted protease was mainly alkaline serine protease.It was found that the protease activity was significantly positively correlated with the number of Aspergillus,β-glucosidase activity and lipase activity were significantly positively correlated with the number of Ascomycetes.The phospholipase activity was positively correlated with the number of Aspergillaceae.The activities of these four enzymes were negatively correlated with the number of Fusarium fungi.The acid soluble fraction S of LDSM and the alcohol extract P of acid insoluble fraction P were analyzed by proteomics,and 519,139 fungal proteins were identified respectively.In component S,20 proteases(11 extracellular enzymes and 9 intracellular enzymes),7glycosidases and 5 lipases/phospholipases were identified.Based on the analysis of molecular function,9 of the 11 extracellular proteases had serine endopeptidase activity,one proteolytic enzyme had both metallodipeptidase and polypeptidase activity,and the other protease had aspartate endopeptidase activity.Seven fungal glucosidase were predicted to have β-glucosidase,α-L-arabinofuranosidase,xylan 1,4-β-xylosidase,β-galactosidase,mannoseoligosaccharide glucosidase,maltose α-glucosidase and raffinose α-galactosidase,respectively.Lipase and phospholipase are predicted to have the activities of sterol esterase,triglyceride lipase,lysophospholipase and phospholipase B.In component P,6 proteases,2glycosidases and 2 lipases/phospholipases were identified.The proteins,lipids and soybean isoflavones in LDSM were obviously hydrolyzed by fungal enzymes.A strain of Aspergillus ochraceus was screened from stored LDSM,and an alkaline serine protease with molecular weight of about 75 k Da was isolated and purified from its fermentation broth.First of all,the protease was active only in the presence of activators,including alkyl sulfates,alkyl sulfonates and fatty acids.The activation ability of saturated fatty acids decreased sharply with the increase of carbon chain length(p<0.05).The activation ability of monounsaturated C14 and C16 fatty acids and polyunsaturated linoleic acid was not sensitive to carbon chain length and was significantly higher than that of SDS(sodium dodecyl sulfate).Secondly,the activity of the protease was not affected in the environment of protein denaturant(8 mol/L urea and 6 mol/L hydrochloric acid)or organic reagent(methanol and ethanol).Third,the protease showed self-hydrolysis independent of activator and could be observed after heating at 60℃ for 2 minutes.The hydrolysis of Aspergillus ochraceus protease in the process of soybean protein extraction was investigated.It was found that the degree of protein hydrolysis increased with the increase of residual lipid content in LDSM and extraction of p H.A strain of Irpex lacteus was screened from stored soybean meal,and a kind of aspartic protease with molecular weight of about 35 k Da was isolated and purified from its fermentation broth.Through the SDS-PAGE analysis of the hydrolysates of natural soybean protein,it was found that the protease could selectively hydrolyze the α’,α,γ subunits of soybean 7s protein and the A3 and A4 peptide chains of 11 s,while the β subunits of 7s and A1,A2,A5 and B peptide chains of 11 s were not hydrolyzed.Furthermore,the position of the enzymatic hydrolysis sites of α’,α and β subunits on the peptide chain was confirmed by the method of peptides.Combined with the protein molecular structure database(https://www.uniprot.org/),it was found that the N-terminal acid extension region of α’ and α subunit and the C-terminal extension region of α’,α and β subunits were the action regions of white rake protease.After heating denaturation,all the subunits of soybean protein could be hydrolyzed non-selectively by protease,which further indicated that the selective hydrolysis was due to the sensitivity of the enzyme to the spatial structure of protein.The effects of protease hydrolysis on the acid stability and thermal stability of soybean protein were investigated.The results showed that enzymatic hydrolysis could significantly improve the acid stability and thermal stability of soybean protein,but there was no significant difference between selective hydrolysis and nonselective hydrolysis of soybean protein.Finally,protease extracts of soybean meal with different storage periods(0 days,40 days and 90 days)were added to fresh low-denatured soybean meal to investigate the effects of enzymatic hydrolysis on the extraction of soybean protein and the gel properties of extracted soybean protein.The results showed that the soybean protein isolated from soybean meal stored for 90 days had smaller particle size and higher surface potential than those stored for 0 days and 40 days,but the extraction rates of 7s,11 s and soybean protein isolates decreased significantly(p<0.05).In addition,the enzymatic hydrolysis of protease resulted in a significant decrease in the storage modulus(G’)and protein solution viscosity(p<0.05)of soybean protein thermal gel.