The Breeding Of Highly Protease And Amylase Producing Strain Isolated From Fermentated Soybean Paste

Soybean paste is one of the traditional fermented soybean products, it is made of soybean which is the main raw materials, and it is semi-flow stated fermented product by natural fermentated. During the fermentation of soybean paste, the microorganisms in soybean paste secrete enzymes, soybean protein fibre acting a series of biochemical reactions under the effection of microorganisms and enzymes. Therefore, soybean paste has intense aroma and its mouthfeel, color and sheen is appropriate. In the paper, accessing the high-yield Aspergillus oryzae strain secreted protease and amylase by mutation breeding, in order to providing strains resources in industrial production.The investigated materials are the 26 strains (Aspergillus oryzae) which is taking HDF-3 as the representative strain. They are isolated from the local bean past collected from Baiquan town Qiqihar Heilongjang province. The strain HDF-C14 was selected by transparent circle method and the method of determinating the enzyme activity, which is highly producing protease and amylase.The protease activity of the strain HDF-C14 is 839.36 U/mL, protease specific activity is 252060 U/g, amylase activity is 381.88 U/mL, amylase specific activity is 143560 U/g.This paper investigated the relationship of dosaged and lethality of HDF-C14 which is mutated by physico-mutagenesis of microwave and He-Ne laser. The research findings that HDF-C14 is insensitivity with microwave mutagenesis. But when the He-Ne laser exposure distance is 2 cm and irradiating HDF-C14 for 30min, the death rate of HDF-C14 is 73.24%, screening the mutants after He-Ne laser mutagenesis using the transparent circle method. Using NTG chemical mutagenesis mutated the strain HDF-C14-H8 which is mutated by mutagenesis laser of He-Ne and is high yield protease and amylase. When the mutagenic dose of NTG concentration is 0.4mg/mL, the mutation time is 30min, the death rate is 73.81%. Finally obtain destination mutate HDF-C14-HN5 by screening. The protease activity of HDF-C14-HN5 is 1122.06 U/mL, increased 133.68% comparing to the original strain HDF-C14, and amylase activity is 564.14 U/mL, increased 147.74% comparing to the original strain HDF-C14. Using the method of continuous passage of the HDF-C14-HN5 strain of genetic stability, the results showed that after 10 consecutive passages, enzyme activity was stable and sporulation of strain is robust, there's no significant change in strain. Vaccinated the HDF-C14-HN5 into the sauce koji to fermenting, after the fermentation of 72h to 84h, the protease activity became highest, it reach 1572.64U/g; the highest amylase activity reach 2815.23U/g.In the present paper, using the breeding methods in the strain highly producing protease and amylase isolated from traditional soybean paste through physical and chemical compound mutation, the strain is applied to the product soybean paste, improving soybean paste production levels.