The Mutation Breeding Of High-yielding Rennin Strains And The Optimization Of Liquid Fermentating Rennin Condition By The Response Surface Method

This paper considered Quambalaria cyanescens which could produce rennin as original strain, purified it, and determined the time of making preparation of the young spores by microscope oservating.We studied the relationship between the death rate and different doses of UV radiation and DES acting time, mutated the young spores by ultraviolet or DES, screened the aberrant strain for which we did the genetic stability experiment, an- alyzed the milk-clothing activity （MCA） and proteolytic activity （PA）of fermen- tive liquid,and finally obtained the high-yielding strains that produced high MCA/PA ratio fermentive liquid.Taking the ideal strain for the seed, we determined the factors and level in the single factor experiment, considered the significant of different factors in the Placket-Burman experiment, analyzed the interaction of the significant factors in the Box-Behnken experiment, and eventualy gained fermentating conditions in which we could produce the most rennin.The main achievement of this research as follows:（1） Separated the strain which can produce rennin from others on the agar slab, disco -vered that many spores came into being on the 6^th from the day spores were lined on the PDA slant,and determined the time for making preparation of the young spores.（2） Studied the relationship between different doses of UV and DES and the deaths of the young spores,and found that the ideal dose was UV radiating 40min or DES acting 40 min.In that case the death rate both ranged from 70% to 80%.（3）Mutated the spore suspension in different way: UV mutating; DES mutating; firstly UV and secondly DES mutating; firstly DES and secondly UV mutating. Randomly picked up 30 strains and marked in different mutating way. Screened the aberrant strai- ns by shaking the flask, and did the genetic stability experiment,analyzed the MCA/PA ratio of formentive liquid.We came to the conclusion that DU12 was the ideal strain.（4） The single factor experiment indicated that:the best carbon resource was glucose;the proper nitrogen resource was peptone;yeast cream not only be could considered as nitrogen resource,but also growing substance;MnSO₄,Tween-80 and CaCl₂ could improve the level of formentive liquid quality. Furthermore,there were some factors that effected the formentive liquid quality, such as original pH, incubating temperature and time, circumrotating speed and so on.（5） The Placket-Burman experiment indicated that circumrotating speed, glucose and original pH were the significant factors that effect the production of rennin.The Box- Behnken experiment showed that the interaction between glucose and circumrotating speed was significant, so was the effect of circumrotating speed and original pH. How- ever, original pH impacted glucose unconspicuously. The condition optimized by res- ponse surface methodwas that: glucose 36g, yeast cream 6g,CaCl₂ 0.5g, MnSO₄ 2.5 mg, tween-80 0.8 g, original pH 5.0,adding distilled water until 1000mL and putting 50mL medium into 250mL triangle flask, seeding 1% spore suspension, temperature 30℃, ci- rcumrotating speed 188r/min, incubating 96h.In that case, the milk-clothing activity of fermentive liquid could reach 610.52 SU/mL.