Breeding Of High Protease-producing Strain And Fermentation Metabolic Control In Aspergillus Type Douchi Koji

Douchi is a traditional fermented condiment which is very liked by people in China.However the production of douchi is mainly at natural fermentation. Long production cycle,uneven quality and low production efficiency are still widespread in douchi enterprise. In thisstudy, Aspergillus type douchi koji is the objective of this study, screening and breeding afermented strain in douchi koji and study the application of metabolic control technology inkoji making process, then optimize the technology for preparing koji through inductionmethod, at last fermentation kinetics models was established.(1) A higher protease-producing strain was selected within five different forms ofAspergillus isolated from Aspergillus type douchi koji by testing the alkaline protease activity,neutral protease activity and amino nitrogen with the purebred fermentation of douchi koji.Protease-producing strain was identified as Aspergillus oryzae through ITS rRNA sequenceanalysis. The Aspergillus oryzae as start strain and an excellent mutant strain was mutatedmultiply by UV and DES through separation, purification and cultivation. Its neutral proteaseactivity of258U/g, which was increased by22.9%compared to the original strain (210U/g),with stable hereditary character. This strain was used in the following study.(2) Study the effect of different addition of protein isolates and amino acids to increasethe protease activity in douchi koji. Different kinds of protein isolates and amino acids havedifferent effects on the protease activity. Neutral protease activity got367.7U/g comparedwith CK group (279.2U/g) increased31.7%and protease activity reached the top earlier oneday than CK group with the additions of3%supernatant prepared by acid precipitation fromblack bean. It can be deduced that some small molecules come from black bean protein whichis hydrolyzed by acid could be utilized by Aspergillus oryzae directly and the small moleculesis a synthetic inducer of the neutral protease. Protease activity was repressed in varyingdegrees with the additions of different kinds of amino acids.(3) The optimization of technology for preparing Aspergillus type douchi koji throughinduction method was discussed. The significant three effects, such as temperature,inoculation volume and relative humidity, were determined through single factor testcombined with Plackett-Burman design. The optimum technolgy was obtained by CentralComposite Design; the results were fermentation temperature28.5℃, inoculation volume4.97%, relative humidity77%, addition of acidic supernatant fluid3%, which was preparedby acid precipitation from black bean, and fermentation period5d. Under the optimum conditions, protease activity was383U/g compared with traditional technology (292U/g), itwas increased31.16%. It indicated that it is practicable to prepare Aspergillus type douchikoji through induction method. It will put forward the new ideas for douchi koji production.(4) Kinetics for preparing Aspergillus type douchi koji through protease inductionmethod was discussed. The results showed that cell growth of Aspergillus oryzae and neutralprotease formation were characterized as typical S-type curves and the kinetics models forthem were established according to the Boltzmann model. The optimal parameters of modelswere obtained through mathematical derivation and nonlinear regression. The two kineticsequations were built, which characterized the douchi koji batch fermentation process verywell. A good correlation between models calculated values and experimental data wasobserved, therefore, it will provide the theoretical basis for fermentation course control ofAspergillus type douchi koji production.