Efficient Protease Gene Engineering Strains And Soybean Peptide Purification Process Of Antioxidant Optimization

Soybean peptide antioxidation of antioxidation, delay ageing, adjust the immune function, can be in such physiology to a certain extent within the body eliminate many physiological function obstacle, reduce the risk of alzheimer's disease. As the functional health food, has great development prospects. But because of enzymatic hydrolysis enzyme directly with commodity of soybean peptide antioxidant poor taste, has certain bitter. Soybean peptide product factory production oxidation of stagnation. Fermentation of soybean peptide bitter disappear, antioxidant and natural aromatic flavour of fermentation. Bacillus subtilis in this subject first, use for template genome genetic engineering method for preparation of high-yield soybean peptide antioxidant strains, and the protease optimized conditions of fermentation, and using the membrane of soybean peptide antioxidant purification. The main research contents and the results are as follows:(1)Bacillus subtilis genomic DNA in for template, according to apr E gene sequences of design, synthesis of primers, apr from Bacillus subtilis PCR method using the DNA genetic fragments from apr, and pGM-T carrier to intermediate cloning identification of sequencing, appraisal right for enzyme digestion, connection, transformation, constructing expression plasmid pET-22b-apr and to express apr plasmid expressing and identification of induction, the best, the best time to 4h induced by inducing temperature is 37℃.(2)Using fermentation with soy protein for the extraction of raw materials of soybean peptide and antioxidant in strong against oxidation, determined by single factor experiment, fermentation temperature, pH value, age inoculation quantity, liquid ratio, fermentation time of soybean peptide in antioxidant effects of oxidation, determine the optimal factors, and work on test results selected notable influence factors of four yuan quadratic regression orthogonal test, the combinatorial optimization by rotating SAS8.2 software for statistical analysis of test data processing and analysis of the preparation of soybean peptide antioxidant best fermentation conditions for fermentation time, for 18h,37℃, fermentation temperature for bed for 160r/min, speed of age for 38.65 h, pH, for 7.06.inoculated quantity 4.18%, than to 4.18/100.(3)Using hollow-fiber membrane ultrafiltration method of soybean peptide antioxidant purification, the author examines the pressure, temperature, pH, material concentration on the hollow fiber membrane interception rate, and according to the experimental results L9(33)orthogonal experiment, experiment design membrane purification soybean peptide antioxidant the best conditions for:pressure, temperature for 0.1 MPa 40℃, pH 7.0, material concentration 4.0%. According to the best condition with different molecular weight of purification hollow fiber membrane molecular weight gain different section of the micromolecule peptide, to fight oxidation value index, phase with iron-thiocyanate method for determination of potassium and optimizing the strongest antioxidant activity for the part 500Da-1000Da. In order to make the film for reuse, cleaning the membrane using L9(33)orthogonal experiment design, select the cleaning reagent, temperature, cleaning time three factors, with membrane recovery degree, determines the index for the best cleaning condition for film containing enzymes cleaning reagent:0.5%, cleaning, cleaning 40℃for temperature 40min, membrane recovery time for 70.31% degrees.