| Vitamin K2(MK-7)is a high value fat-soluble vitamin,which is a polyene compound composed of 2-methyl-1,4-naphthoquinone ring and 7 isoprene unit side chains.It plays an important role in preventing osteoporosis,arterial calcification,cardiovascular disease,Parkinson’s disease,and is widely used in medicine,health care products and other fields.Bacillus natto is a commonly used strain for vitamin K2 production by fermentation,but its ability to synthesize vitamin K2 still needs to be further improved,and the information about what happens during the fermentation of Bacillus natto is not clear.All these factors hinder the fine and rational regulation of fermentation and the further improvement of industrial production level.Therefore,a strain of Bacillus natto ND-1-A27,which was bred in our previous research for MK-7 production,was selected as the research object in this work.Through the optimization of fermentation process and the addition of different surfactants,the ability of the strain to produce MK-7 was improved.And through analyzing the differences of metabolites under different fermentation conditions,ten metabolites with significant differences which might be related to the production of MK-7 by Bacillus natto ND-1-A27 were identified.The main research work is as follows:(1)The medium composition and fermentation conditions for the synthesis of MK-7 by Bacillus natto ND-1-A27 were investigated.In this paper,the carbon and nitrogen sources and salt ions in fermentation medium were screened and their concentration gradient was tested.On the basis of the optimal medium,the culture conditions were optimized.The effects of fermentation temperature,rotation speed,initial p H of culture medium,filling amount and inoculation amount on the production of MK-7 by Bacillus natto ND-1-A27 were investigated.The optimal medium components after optimization are as follows: glycerol 70 g/L,soy peptone 90 g/L,yeast powder 30 g/L,dipotassium hydrogen phosphate 0.06 g/L,magnesium sulfate 0.08 g/L.The conditions of optimal culture are as follows: 3% inoculation volume,initial p H of 8.0,filling volume of 20 m L/250 m L,40°C,120 rpm for 6 d.Under the above optimum process,the concentration of MK-7 reached 25.94±0.8 mg/L,which was 110.55%higher than that of the pre-optimization process(12.32±1.3 mg/L).(2)The effects of surfactants on the fermentation of Bacillus natto ND-1-A27 for MK-7production were explored.Surfactants can interact with the cell membrane of the bacteria,affect the growth of the strain and cell morphology,and change the permeability of the cell membrane.In this paper,the effects of surfactant on the growth,cell morphology and fatty acid content of MK-7 fermentation by Bacillus natto ND-1-A27 were studied.The results showed that the addition of 0.1% Triton X-100 in the fermentation system could promote the growth of the strain,and the concentration of MK-7 reached 37.96±1.5 mg/L.The surface of the cell becomes rough and has small pores,and the content of saturated fatty acids in the cell membrane decreases while the content of unsaturated fatty acids increases,which increase the fluidity of the cell membrane and promote the transport of substrates and product secretion.(3)The metabolites of Bacillus natto ND-1-A27 fermentation under different fermentation systems were analyzed by metabonomic method.The results showed that the relative contents of 104 metabolites were different under the two conditions of adding surfactant or not,among which 43 metabolites had significant differences(VIP≤5,P-Value≤0.05 and PPM≤3).Further induction and analysis of the metabolic pathways of the above significantly different metabolites showed that 25 of the different metabolites were related to the metabolism of amino acids,and 2 of the different metabolites were related to the metabolism of microorganisms in different environments.Among them,the relative contents of mesoconate,dihydroxyzeatin,N(α)-acetyl-L-2,4-diaminobutyrate,N-acetyl-D-tryptophan,N-a-acetylcitrate,4-guanidyl butyrate,p-formylaminobenzoic acid,pseudouracil nucleoside,4-oxobenzamide decreased(2R,3R)-3-Methylglutamyl-5-semialdehyde-N6-lysine and citric acid gradually with the prolonging of fermentation time.It is speculated that these 10 different metabolites may promote the synthesis of MK-7. |
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