Breeding A High Yield Strain Of MK-7 And Study On Fermentation And Intracellular Extraction Technology

Menaquinone-7（MK-7）,one of the important forms of vitamin K₂,has the characteristics of high biological activity,long half-life period in the blood.It has significant effects in preventing osteoporosis,cardiovascular and cerebrovascular sclerosis and in the treatment of Parkinson’s disease,etc.In recent years,it has attracted a lot of attentions in the fields of food health care and biological medicines.In comparison with the extraction method with low production efficiency and the chemical method with complicated route and great harm to the environment,the microbial fermentation method with green environmental protection,simple process and easy availability of raw materials has become a more promising industrial production method of MK-7.However,at present the microbial fermentation method faces the problems of low yield and low extraction efficiency of MK-7.How to improve the production capacity of the strain and the extraction rate of MK-7 is the key to promote its industrial production.Therefore,in this paper,a strain with the ability to produce MK-7 was screened from natto and was biologically identified.After being confirmed that it was Bacillus subtilis,it was used as a mutagenized strain,after going through the complex mutagenesis of ultraviolet（UV）and atmospheric and room temperature plasma（ARTP）,a mutant strain with high production of MK-7 was obtained and then its cultivation and fermentation process were optimized.On this basis,the extraction technology of intracellular MK-7 of Bacillus subtilis was explored,and a relatively complete and effective MK-7 fermentation production process and intracellular extraction process were finally obtained.The main findings are as follows:（1）Mutagenesis breeding of high-yielding MK-7 strain.The original stain Bacillus subtilis Y-9 with the production capability of MK-7 was screened from commercially available natto,and the mutant strains A-52 with high production and stable heredity were selected by UV+ARTP complex mutagenesis technology.The production of MK-7 was 22.5 mg/L,which was 129.5%higher than the original strain.This strain is deposited in the China Industrial Microorganism Strain Collection and Administration Center（CICC）with the deposit number of CICC 25137.（2）Optimization of cultivation and fermentation conditions for MK-7 produced by Bacillus subtilis A-52.The cultivation and fermentation process of MK-7 produced by strain A-52 was optimized by Box-Behnken experimental design and response surface analysis method.The medium components after optimization included glycerol 58 g/L,soybean peptone43 g/L,chlorine sodium phosphate 0.4 g/L,dipotassium hydrogen phosphate 0.6 g/L,magnesium sulfate 0.5 g/L and calcium chloride 0.2 g/L;The optimal fermentation conditions were liquid volume 20 m L（100 m L conical flask）,inoculum amount 3%（v/v）,initial p H 8.0,and fermentation time 7 days.The yield of MK-7 under these conditions was 51.1 mg/L,which was 127.5%higher than that before optimization.（3）The exploration of the extraction process of intracellular MK-7 in Bacillus subtilis A-52.On the basis of comparing the intracellular MK-7 extraction effect of ultrasonic,enzymolysis,and ultrasonic-assisted-enzymatic hydrolysis,the process parameters of ultrasonic-assisted-enzymatic hydrolysis were further optimized with the orthogonal experimental design.The optimized extraction process was:ultrasonic power of 100 W,ultrasonic duty cycle of 40%,enzyme addition of 8.0%（v/w）,and treatment time of 4 h.The yield of intracellular MK-7 in strain A-52 under these conditions was 0.72 mg/g and the purity of MK-7 was 45.7%...