Breeding Of Thermoprotease-producing Strain And Its Application In The Preparation Of Polypeptides From Rapeseed Meal By Solid-state Fermentation

Rapeseed meal is a by-product of rapeseed seeds oil extraction,which is considered as an important vegetable protein.Polypeptides,hydrolyzed from rapeseed meal,are reported to have the bioactivities of antioxidation,inhibitation the growth of tumor cells and antihypertension,etc.At present,rapeseed polypeptides are commonly prepared by hydrolysis with protease or fermentation with mesophilic microorganism.However,the cost of protease is high in the hydrolysis process,and it will cause a large amount of steam consuming for sterilization of fermentation substrates and equipments in mesophilic fermentation.Moreover,rapeseed protein is easily denatured during high temperature and high pressure sterilization treatment,which decreases the conversion efficiency of rapeseed protein.Thermophilic solid-state fermentation on rapeseed meal with thermoprotease-producing strain may effectively inhibit the growth of mesophilic microorganisms in fermentation substrate.This way makes the direct solid-state fermentation without sterilization be possible,which can greatly reduce costs,increase the utilization of substrates and raise the yield of rapeseed polypeptides in fermentation.In this study,thermophilic bacteria were firstly screened from rapeseed meal for efficient fermentation and microbial community structure analysis.The feasibility of preparation polypeptides through solid-state fermentation by thermophile was studied.Subsequently,the isolated bacteria were mutagenized by atmospheric and room temperature plasma?ARTP?to improve the bacterial protease activities,and the technological parameters in solid-state fermentation on unsterilized rapeseed meal were optimized by response surface methodology experiment for the maximum polypeptide yield.And the influence mechanism of ARTP on isolated bacteria for higher protease production was investigated in this study.The main contents and results of this paper are as follows:?1?The feasibility of thermophilic fermentation on unsterilized rapeseed meal was preliminarily explored by comparing the biomass in the medium of thermophilic and mesophilic solid-state fermentation.The results showed that thermophilic fermentation could significantly reduce the biomass of mesophilic microorganisms in the substrate.A thermophilic protease-producing strain named RM-2 was successfully screened from rapeseed meal by means of primary screening according to the strains'hydrolysis circle and re-screening of protease activity.The protease activity of RM-2 was 25.50 U/mL.RM-2 was identified to belong to Geobacillus stearothermophilus through 16S rRNA gene sequencing.?2?High-throughput sequencing technology was used to analyze the bacterial community composition and the abundance in the substrate of unsterilized rapeseed meal under different conditions,to further verify the feasibility of thermophilic fermentation and to determine the dominant strains in fermentation processing.The results of Alpha diversity analysis showed that the Operational Taxonomic Units?OTU?in the thermophilic fermentation substrate decreased by 37.5%compared with the mesophilic fermentation;and Geobacillus was found to be the dominant strain in the thermophilic fermentation with a relative abundance of 72.83%.Beta analysis showed that there were significant differences among samples,and Geobacillus was the bacterial species who significantly affected thermophilic fermentation.?3?Using Geoacillus stearothermophilus as the starting bacteria,mutants with high yield of thermoprotease production were screened by ARTP.The results showed that the lethality rate of ARTP mutagenesis could reach 90.5%under the conditions of ventilation rate of 10 SLM?standard litre per minute?,power of 100 W and mutagenesis time of 5 s.Under these conditions,a number of A75 mutant strain with higher protease activity and stable inheritance was screened by using the ratio of hydrolysis circle diameter to colony diameter?K value?as the primary screening index and protease activity as the secondary screening index.The protease activity of A75?32.09 U/mL?was increased by 25.62%compared with original strain RM-2.?4?Response surface methodology was used to optimize the technological parameters for polypeptides,which was prepared from unsterilized rapeseed meal through thermophilic solid-state fermentation by mutant strain A75.It was showed that the maximum polypeptide yield was 6.78%under the conditions of inoculum level 10⁷ CFU/20 g substrate,fermentation time 65 h,liquid-to-material ratio 1.6?g/mL?,fermentation temperature 55?C,wheat bran addition 20%?W/W?and MgSO₄ addition 0.1%?W/W?.The content of small molecule protein and total amino acid was higher than that of unfermented rapeseed meal by 15.17%and16.47%??5?The mechanism of ARTP mutagenesis of Geoacillus stearothermophilus was investigated preliminarily.Compared with the original strain RM-2 by scanning electron microscopy,the morphology of mutant strain A75 changed into short rod.343 SNP?Single Nucleotide Polymorphism?mutation sites were found in mutant strain A75,which involved in 121 genes by genome sequencing.Comparing the amino acid sequences encoded by those genes with the databases of GO?Gene Ontology?,KEGG?Kyoto Encyclopedia of Genes and Genomes?and eggNOG?evolutionary genealogy of genes:Non-supervised Orthologous Groups?,it was found that many genes were involved in metabolism of carbohydrates,the translocation and metabolism of amino acids,the composition of cell wall and membrane and the metabolism of nitrogen compounds.This research result demonstrated that the active particles generated in ARTP treatment could penetrate the cell membrane of Geobacterium stearothermophilus,which might change the genetic material and then affect the expression of protease in the bacteria.