Study On Chemical Orientation Screening The High Menaquinonem Yield Strains And Production Performance

Menaquinone(MK)is a fat-soluble vitamin K with many important physiological activity such as promoting blood clotting,preventing osteoporosis,treating the mental diseases and so on.It is an important micronutrient for the human body.Chemical synthesis was used as a traditional production method of menaquinone with the disadvantage of low yield,more by-product and high response requirements.Whereas the biological fermentation method has the advantages of high yield and low energy consumption and so on.This paper taken the advantage of Bacillus subtilis.as the strain producing menaquinone,established a method of screening producing menaquinone strain through chemical orientation.The following several aspect works had been carried out in this paper:1.The menaquinone standards MK-0,MK-1 and MK-4 were analysied by high performance liquid chromatography,the menaquinone absorption bands were obtained at245 nm,265 nm,325 nm,and the maximum absorption wavelength λmax was 245 nm.With Douchi as the screening raw material,29 strains of bacillus were isolated by preliminary screening of water bath heating treating.,and 16 MK-producer strains were screened by HPLC analyzing.Accroding to the analysis of high performance liquid phase mass spectrometry(LC-MS),the strain fermentation product was identified to be MK-7,and the reliability of the chemical screening method for isolateing strain was verified.2.The strain DC-1 was determined to be the target strains with the highest MK-7yield.Comprehensive the results of biological assay,the strain was identified as Bacillus subtilis.3.The effects of strain medium components on the production performance of menaquinone were discussed.The researchs showed that the types tegother with addition amounts of carbon source and nitrogen source had a great influence on the yield and variety of MK in fermentation products.When the carbon source was glycerin and the addition amount was 0.5 g/100 m L,the yield of MK-7 was 26.830mg/L.When nitrogen source were peptone,corn steep liquor,beef extract,compound nitrogen source(soybean flour: peptone 1:1),the strain of MK product diversity became stronger.And except MK-7produced a different structure of menaquinone: MK-2.And when corn steep liquor was used as nitrogen source with the additive amount of 4 g/100 m L,MK-7 and MK-2 yield were the highest,up to 32.280mg/L and 1.292mg/L respectively.4.Optimized the DC-1 strain best culture conditions for the production of MK-7.The single factor test showed that the shaker speed,the culture temperature and the culture time had great influence on the production performance of MK-7.When shaker speed was 0r/min,the maximum yield of MK-7 was 34.296mg/L;When culture temperature was 42 ℃,the maximum yield of MK-7 was 50.283mg/L;When culture time was 4 d,the maximum yield of MK-7 was 54.383mg/L.Further results of orthogonal experiment indicated that the best cultivation condition of DC-1 strain was shaker speed 30 r/min,culture temperature 39 ℃,culture time 3.5 d.The yield of MK-7 as high as 63.942mg/L,increased by 98.09% compared with that before optimum.