Preparation Of Polypeptides From Rapeseed Meal By Magnetic Field Assisted Solid-State Fermentation With Mixed Strains And The Establishment Of Near Infrared Spectroscopy Detection Model

Rapeseed meal is a high-quality plant protein resource.However,denaturation of protein during processing affects digestion and utilization of rapeseed meal;in addition,rapeseed meal also contains tannins,phytic acid,and glucoside thioglucoside(thioglycoside)and other anti-nutritional substances and toxic components,seriously limiting its utilization.Some studies have shown that solid-state fermentation with microorganisms not only could convert denatured proteins in rapeseed meal into easily absorbed peptides,but also could degrade the anti-nutritional factors in it.However,traditional solid-state fermentation of rapeseed meal requires autoclaving of raw materials,resulting in high production costs.In addition,the fermentation efficiency of traditional method is low,and the key indicators of the fermentation process cannot be monitored in real-time,those disadvantages also hinder the application of solid-state fermentation technology of rapeseed meal in the production on a large scale.In this study,multi-strain mixed fermentation method was employed to inhibit the growth of miscellaneous bacteria and increase the polypeptide content for non-sterilized rapeseed meal fermentation.Alternating magnetic field stimulation technology was used to strengthen the fermentation process of rapeseed meal and to improve the fermentation efficiency.Near-infrared spectroscopy detection models for key indicators such as polypeptide content and pH during the magnetic field-assisted fermentation process were build,which provide a theoretical basis for the subsequent research on real-time monitoring technology for solid-state fermentation of rapeseed meal.The main contents and results of this study are as follows:(1)Research on the preparing technology of rapeseed meal polypeptides by solid-state fermentation with mixed bacteria.Taking the content of rapeseed polypeptide as the index,the effect of single-strain,double-strain and triple-strain fermentation of Bacillus subtilis,Candida tropicalis and lactic acid bacteria was studied.It was found that the highest yield of polypeptides,i.e.6.12%,was achieved in triple-strain fermentation.The effects of fermentation time,fermentation temperature,inoculum amount and feed-liquid ratio on the fermentation were studied by single-factor and response surface tests,and the optimal fermentation conditions were 3 d,40℃,1:1.2 g/m L ratio(rapeseed meal : water),15% inoculum,and the peptide content of rapeseed reached 7.325%.The pH value decreased after fermentation,and the titratable acid content reached 85.54±0.32 g/kg,which was49.30% higher than that of rapeseed meal raw materials;the content of glucosinolates was 22.775±0.275 μmol/g,which was 46.20% lower than that of rapeseed meal.(2)Study on the variations of microbial flora during the preparation of rapeseed meal polypeptides by solid-state fermentation of mixed strains.The changes of microbial flora during the fermentation process of mixed strains were studied by high-throughput sequencing technology.For the bacterial flora,the abundance of lactic acid bacteria first decreased and then increased,the abundance of Bacillus subtilis increased continuously,and the abundance of other bacteria was always reduced.For the fungal flora,Candida tropicalis had a flora abundance of more than75.77%,occupying the absolute advantage proportion in the whole process of fermentation.Thus,by studying the changes of microflora during the fermentation of mixed strains,it can be concluded that the mixed fermentation without sterilization can inhibit the growth of miscellaneous bacteria and increase the content of rapeseed polypeptides of rapeseed meal.(3)Study on the preparation of rapeseed meal polypeptides by solid-state fermentation with mixed strains enhanced by alternating magnetic field.The optimal conditions were obtained by single-factor and orthogonal tests as follows: magnetic field intensity of 60 GS,magnetic field intervention time at the 22 nd h of fermentation,magnetic field treatment duration of 5 h,and the content of peptide reached 8.15% at the end of fermentation of rapeseed meal.In addition,through the analysis of rapeseed protein structure and amino acid composition,it is found that rapeseed protein structure became smaller,particle sizes were more uniform,and total amino acid content increases after magnetic field treatment.It was shown that the application of magnetic field stimulation could further improve the yield of rapeseed polypeptides and improve the quality of fermented rapeseed meal.(4)Preliminary study on the mechanism of solid-state fermentation of rapeseed meal enhanced by magnetic field based on metagenomics.The metagenomics techniques were used to study the rapeseed meal after the magnetic field treatment.By exploring of the microbial community structures of rapeseed meal at the levels of boundary,phylum,class,order,family,genus and species,it was found that magnetic field treatment did not change the microflora composition of microorganisms in the fermentation process at different classification levels,but the abundance of microbial floras would be affected.In addition,KEGG annotation showed that the ABC transporter,oxidative phosphorylation,glycolysis/gluconeogenesis pathway,glycine/serine/threonine metabolism,and cysteine/methionine metabolic pathway of microorganisms changed after magnetic field treatment;among them,ABC transporter protein,glycolytic/gluconeogenesis pathway,and glycine/serine/threonine metabolism were upregulated in abundance,while oxidative phosphorylation and cysteine/methionine metabolism pathways were downregulated in abundance.The changes in glycolysis/gluconeogenesis pathway,glycine/serine/threonine metabolism,cysteine/methionine metabolism pathway may be related to the appearance of ADP-specific phosphofructokinase,2,3-bisphosphoglycerate 3-phosphatase,Oxidoreductases,sarcosine dehydrogenase,L-threonine dehydrogenase,betaine-homocysteine transmethylase and cysteine lyase,the disappearance of the Transferases,cysteine synthase.In addition,variations of these enzymes increased the total and essential amino acid content of rapeseed meal by 0.93% and 2.51%,respectively,and caused the increases of glycine,methionine and threonine contents by 27.54%,20% and 15.69%,respectively.(5)Modeling of near-infrared spectroscopy of polypeptide content and pH during magnetic field-assisted rapeseed meal fermentation.The polypeptide content and pH during solid-state fermentation of magnetic field-assisted rapeseed meal were fitted using near-infrared spectroscopy detection,and the raw spectra were preprocessed using Standard Normal Variance(SNV),Savitzky-Golay(S-G),and Multiple Scattering Correction(MSC).Based on the Synergy interval partial squares(Si-PLS),it was found that for the pH model,the best model was established by the S-G preprocessing method.The Rc,RMSECV and Rp,RMSEP of the corrected and predicted models were obtained as 0.9557,0.227 and 0.9761,0.218,respectively;for the peptide content model,the MSC preprocessing method built the best model,and the Rc,RMSECV and Rp,RMSEP of the corrected and predicted models were obtained as 0.9661,1.776,0.9775 and 1.517.This result indicated that the established models were accurate and stable.The models provide the basis for the development of real-time monitoring technology for key indicators of the fermentation process of rapeseed meal.