Screening An Aspergillus Oryzae Mutant With Improved Protease Production And Its Influence On The Flavor Formation Of Soy Sauce

Aspergillus oryzae has been used in oriental traditional brewing foods such as soy sauce because of its ability to secrete abundant protease lines.Studies have shown that protease activity of A.oryzae has an important effect on protein utilization and soy sauce flavor in soy sauce fermentation.Mutagenic selection of high protease-producing Aspergillus oryzae has become an important way to improve the efficiency and quality of soy sauce production.The traditional mutagenesis selection methods of Aspergillus oryzae have problems such as low throughput,time consuming,long cycle time and inconvenient operation.In this study,Aspergillus oryzae 3.042 was selected as the original mutagenic strain,a mutagenic selection method for high proteinase producing Aspergillus oryzae was constructed,the type of high proteinase expression of the mutant strain was analyzed,and the effect of the mutant strain on soy sauce brewing quality was evaluated.The main findings are as follows:(1)Mutagenic selection for high production of protease Aspergillus oryzaeAfter mutagenesis by atmospheric pressure room temperature plasma(ARTP),a good mutagenesis library was constructed.The mutagenesis conditions were determined as 10 SLM of aeration,100 W of power and 100 s of time.The mutant libraries were optimally stained with Fluorescein diacetate(FDA)at a concentration of 100?g·m L^-1 for 20 min at room temperature and Propidium iodide(PI)at a concentration of 6?g·m L^-1 for 10 min at 4°C.The mutant libraries were also labeled with a fluorescent signal.The flow cytometer detects fluorescence signal emitted by live cells bound to the FDA dye and sets the single cell mode as the sorting mode to rapidly sort the live cells from the mutant library into high throughput well plates.After high throughput culture and high throughput assay the mutant strain with high protease activity is initially screened.Finally,after four rounds of mutagenesis screening,a high yielding strain with 145.62%increase in neutral protease activity compared to the starting strain was obtained with good genetic stability.(2)Identification of highly expressed protease types in mutant strains of Aspergillus oryzaeThe protein was extracted from the solid state fermentation at 72 h and dissolved in protein loading buffer.After determining the protein concentration,the loading volume was adjusted to10?L and protein electrophoresis was performed.Three bands with significantly deeper molecular weights around 17 k Da and 38 k Da than the starting strain were selected and identified by matrix-assisted laser resolved ionization time-of-flight mass spectrometry.The results showed that the increased proteases in H34 mainly included acidic protease(Aspergillopepsin-1),alkaline protease and neutral protease(Neutral protease 2 homolog AO090001000135).The m RNA expression levels of these proteases were subsequently measured by RT-q PCR,where the transcript levels of ALP,pep A and AO090001000135 were up-regulated by 45%,233%and 256%,respectively,relative to the starting strain,again supporting the increased expression of the three proteases at the molecular level.(3)Effect of Aspergillus oryzae mutant strains on the quality of soy sauce brewingH34 was applied to soy sauce mock brew in the laboratory.Soybean meal and bran were mixed at a ratio of 4:1,and a suspension of Aspergillus oryzae spores at a concentration of 10⁷cfu was added to the mixed mass at a mass fraction of 0.2%.30°C constant temperature incubation was maintained at a moderate level,and the spores were turned over at the right time until maturity.Comparing the differences in total nitrogen,amino acid nitrogen,organic acids,amino acids and flavor components in brewing soy sauce,the total nitrogen and amino acid nitrogen of the mutant strain brewing soy sauce increased by 6%and 6.8%respectively compared to the starting strain.The fermentation group of the mutant strain was better than the control group in all indicators,and there was no significant change in flavor presentation.Therefore,the results of the study provide excellent strains for soy sauce fermentation.The foundation is laid for improved protein utilisation in soy sauce as well as improved flavour in soy sauce brewing.