Study On Soybean Meal Antibacterial Liquid Fermented By Aspergillus Oryzae And Its Antibacterial Mechanism

Antibiotics,as a drug used to treat bacterial infections,have made significant contributions in medical and other fields since their discovery.However,the overuse or abuse of antibiotics has led to the emergence of drug-resistant microorganisms as the enemy of the traditional antibiotic industry.At present,many countries have restricted or even banned the use of some antibiotics,and actively developed new antibiotics or antibiotics Substitutes.Antibacterial peptides are small-molecule peptides substances with antibacterial activity released by natural organisms to protect themselves from foreign pathogens.Antibacterial peptides which have become ideal substitutes for traditional antibiotics are derived from natural sources,will not pollute the environment and are not easy to produce drug resistance.More and more antimicrobial peptides have been discovered or prepared and applied to the field of medicine,food and cosmetics.This article takes soybean meal as the research object,combines bio-fermentation and ethanol separation method to prepare and enrich soybean antibacterial peptides.On the basis of clarifying its antibacterial properties and structural information,the antibacterial action mechanism of soybean antibacteroial peptide was initially explored to provide theoretical data and method guidance for the industrial preparation of soybean antibacterial peptides.In this paper,soybean meal was used as raw material,and Aspergillus oryzae was used as fermentation strain.The soybean meal antibacterial solution was prepared by microbial fermentation.E coli and S aureus were used as indicator bacteria,and the antibacterial ability was the main index to optimize the fermentation conditions of soybean meal.The results showed that the liquid fermentation method was more suitable for the preparation of soybean meal antibacterial solution.The optimal fermentation conditions were as followed:material-to-water ratio of 1:9,initial fermentation p H at 4.5,Aspergillus oryzae accession rate of 0.7‰,and glucose addition amount of 2.5%,fermented at 37°C for 42 h,and the influence of each factor on the antibacterial activity of the soybean meal antibacterial solution was in this order: fermentation temperature > material-to-water ratio > Aspergillus oryzae addition amount > fermentation time > initial fermentation p H > glucose addition amount.On the basis,the antibacterial rate curve of soybean meal antibacterial solution was obtained.The results showed that the antibacterial rate of soybean meal antibacterial solution at a concentration of60 mg/m L against E.coli and S.aureus were 52.06% and 40.89%,respectively,indicating that soybean meal antibacterial solution was a good potential antibiotic substitute.Ethanol solutions with different concentrstions were used to fractionate the soybean meal antibacterial solution,and the internal relationship between the physical and chemical properties and antibacterial activity of the separated components was systematically studied.The results showed that the final supernatant H-4-Q had the highest antibacterial rate,when the concentration was 60 mg/m L,the antibacterial rates against E.coli and S.aureus were69.98% and 54.11%,respectively,compared with the same concentration of the non-ethanol-treated soybean meal antibacterial solution,the antibacterial activity increased by34.42% and 32.33%,indicating that ethanol classification can significantly enrich antibacterial active ingredients.The composition analysis found that H-4-Q had the highest total nitrogen content,accounting for 33.87% of the original fermentation solution.The content of total sugar of H-4-Q was the lowest among the five components.The results of peptide molecular weight distribution showed that the peptide molecular weight of H-4-Q was lower than that of other components,1-3 k Da and <1 k Da peptides accounted for 40.02% and29.47%,respectively.The results of amino acid distribution showed the H-4-Q had the highest proportion of basic amino acids.UPLC-ESI-Q-TOF-MS/MS indentified 65 antibacterial peptides from H-4-Q.According to the different targets of bacteriocin,the effects of H-4-Q on bacterial cell wall,cell membrane and intracellular phosphorus metabolism were examined.The results showed that after H-4-Q treatment,the cell walls of the two bacteria were destroyed and the permeability was increased.The alkaline phosphatase activity of the experimental group of E.coli and S.aureus was 4.12 times and 3.84 times higher than that of the control group,respectively.The conductivity of the two bacterial culture fluids also increased significantly after treatment,and the higher the concentration of H-4-Q,the greater the increase in electrical conductivity,and the ?-galactose polymerase activity in the cell also increasedsignificantly.The ?-galactosidase activity of E.coli and S.aureus was 39.60 U and 34.88 U,respectively,and the cell membrane was also damaged to a certain extent.The results of phosphorus metabolism showed that H-4-Q could make both the bacterial phosphorus metabolism rate gradually decreased and eventually stopped,but E.coli was more sensitive to the effect of H-4-Q,and the phosphorus metabolism rate had been significantly reduced within 2 hours,indicating that H-4-Q can not only act on the cell wall and cell membrane of bacteria,but also affect the intracellular phosphorus metabolism to inhibit bacterial growth and reproduction.