Selection Of Highly Nattokinase Productive Strains And Applied Research

In this study，screening of highly nattokinase productive strains strains，purebredand mixed fermentation fermentation was carried out for further application．Firstly，a nattokinase high productive strain of Bacillus subtilis BN-05(the NKactivity reached2647.19IU/g) was isolated by combined efforts of fibrinolyticactivity based isolation method and gene amplification screening method，then it wasvalidated to Apply physiological-biochemical characteristic and the molecularidentification of16S rDNA．Secondly，to optimize the production of fermentation substrates nattokinase (NK)fermentation conditions was chosen germinating soybean which was rich in γ-GABA(233.56mg/100g dry weight)．Screening the principal factors were fermentationtemperature，inoculates the quantity and adding amount of Mg2+by the method ofplackett-Burman on the basis of single factor preliminary experiment．To determinethe optimal level of each parameter value which were15min of the steaming time，55%water content，30g/250mL of the sample loading capacity，35℃fermentationtemperature，7%inoculation scale，36h fermentation time，0.20%Mg2+addingthrough the central composite design to establish a regression model for enzymeproduction．In these factors，interactions between addition amount of Mg2+andinoculation quantity are the most prominent for the enzyme producing ability．TheNK activity reached6918.76IU/g through this process of fermentation．Thirdly，to improve tasting of the natto by mucormycosis MS-07and bacteriaBN-05for mix fermenting．Ascertaining the optimal process parameters which werefermentation time at58.62h，temperature of27.38℃，inoculates the quantity at5.92%and PH values at5.02to enhance the activity of elastase by means of centralcomposite design on the basis single factor test．By optimization of the technologicalconditions，the production of Protease achieved to207.605U/g．Mixed fermentationof bacteria and mucor through orthogonal experiment to optimize process parameterson basis of pure breed fermentation．The optimum fermentation conditions were asfollows：30℃fermentation temperature30℃，6%inoculum size，24h incubationtime，and1:1bacteria ratio with the target of NK activity．Under the adoptedcondition，the NK activity achieved3426.45IU/g，amino nitrogen content of productscome to1.08%，and this products also had a nice pleasing smell．Finally，the products of mixed fermentation were rich in NK and Monaclin K toreduce blood fat，cholesterin and prevent and relieve thrombotic diseases with creating for the current consumers’ taste．With Monacolin K index to raise functionalsubstance content，the optimal fermentation parameters of Monascus products werefurther determined through the single-factor and orthogonal tests．After two dayslactic acid macerating，water in fermentation medium was40%．Then disinfected，made cool，inoculated M1and GIM3.439co-fermentation．After two days offermentating，it was inoculated yeast seed broth which was cultivating48hours infermentation substrate． The culture conditions were yeast inoculums1.5%，fermentation temperature30℃and cultivation28days．The Monacolin K offermentation products detected were1.22mg/g，and the detection of fermentationproducts Citrinin content and aflatoxin B1were respective0.10mg/kg and3.4μg/kgin a limited range．The technology for improving the product content of Monacolin Kand NK through solid fermentation with mixed medium was studied．The processingparameters of preparing Monascus products were1/4peeled and fragmentation ofsoybean，2.00%carbon source，35%initial water content，1.50%nitrogen source．TheMonacolin K and γ-GABA content of the products were1.615mg/g and20.11mg/g．In the meantime, nattokinase activity was3228IU/g．The products of citrininand aflatoxin B1content were0.08mg/kg and3.7μg/kg through safety evaluation．...