Screening Of Aminopeptidase Producing Strains Based On ARTP Technology And Optimization Of Fermentation And Enzymolysis Conditions

Aspergillus oryzae is a kind of aerobic fungus.It produces various kinds of proteases,which is widely used in food,feed fermentation and other industries.Aminopeptidase is a kind of important protease produced by Aspergillus oryzae.It can hydrolyze protein or peptide into free amino acids,which determines the taste and flavor of processed food.Improving the ability of Aspergillus oryzae to produce aminopeptidase is of great significance in food processing.In this study,Aspergillus oryzae CICC2066 was used as the starting strain,which was mutated by atmospheric and room temperature plasma(ARTP).The optimal mutagenic condition was determined as spore concentration of 1×10³/m L,the time is 120 s,the power of the instrument is 120 W,and the air flow is 10 SLM.After screening,a high-yield aminopeptidase strain M₅was obtained,and its aminopeptidase activity was 245.3 U/m L.Compared with CICC2066,the activity of was 1.21 times,and the activity of enzyme was stable after 5 times of subculture.The growth index of M₅spore was detected by scanning electron microscope,plate culture and koji culture.It was found that M₅spore was larger than CICC2066,and its shape changed,and the growth rate was faster in the same period of culture.The fermentation conditions of aminopeptidase produced by strain M₅were optimized,Plackett-Burman experimental design was used to determine the significant factors affecting aminopeptidase production of Aspergillus oryzae,including p H value,culture temperature and the amount of beef paste.Through Box-Behnken experiment,it is determined that the optimal liquid fermentation conditions for M₅are p H 6.6,temperature 36?,and beef extract addition amount 1.8 g/L.Under this liquid fermentation condition,the aminopeptidase activity is 479.22 U/m L,which is relatively unremarkable.Increased by 298%before optimization.It was found that aminopeptidase was applied to the hydrolysis of pig bone,the hydrolysis efficiency of M₅aminopeptidase fermentation broth was 8%higher than that of CICC2066.After precipitation with ammonium sulfate and separation by G-75,aminopeptidase was obtained with a purification ratio of 7.5 times.The results of Box Behnken experiment showed that the optimum conditions for the hydrolysis of pig bone powder by aminopeptidase were p H 6.6,temperature 41?,enzyme dosage3.5%.Under these conditions,the hydrolysis efficiency was 0.56%,which was 16%the hydrolysis ability of aminopeptidase was also significantly improved.In this study,a strain of Aspergillus oryzae with high aminopeptidase production was successfully screened through ARTP technology,and the combination of the Plackett-Burman experiment and the Box-Behnken experiment can improve the metabolism of Aspergillus oryzae to produce ammonia by changing the external environmental conditions.Peptidase ability and its hydrolysis abilit,it provides a theoretical basis for the application of Aspergillus oryzae aminopeptidase in food.