| Bacillus subtilis M2 was cultured and proliferated with TSB（trypticase soy broth）medium,and its culture method,extraction,separation and purification of antibacterial products and fermentation of common animal feed raw materials were studied.The results are as follows:1.Under the same culture conditions,when Bacillus subtilis M2 is fermented by solid culture and liquid culture,there is little difference in the yield of antibacterial active products,but the liquid culture operation is simpler and faster.Moreover,when Bacillus subtilis M2 is cultured for 12 h at 37℃and 120 rpm under the condition of 5%inoculation,its fermentation supernatant can obtain stable and batch active products.2.Bacillus subtilis M2 was inoculated by liquid fermentation.After fermentation for 12 hours,the antibacterial active substances were extracted by adding 20%acetonitrile and 25%ammonium sulfate to the supernatant after centrifugation.After verifying that it has sufficient activity,the active extract was initially separated by C18solid phase extraction column,and the active acetonitrile eluent components with 25%concentration were collected and separated and purified by high performance liquid chromatography.After recovering the components at different times,it was found that obvious antibacterial activity was detected in the components at 17 min,18 min and20 min,which showed that the antibacterial activity product produced by Bacillus subtilis M2 was not a single compound,but a complex composed of multiple compounds.Finally,after the analysis of 17 min component,18 min component and25%acetonitrile eluent with strong activity by LC-MS,it is found that the mass charge ratio of group fragment molecules obtained by mass spectrometry of these three components is mostly between 100 and 600,and the groups with group mass charge ratio of 102,217 and 261.1 appear in the three components.It is speculated that they may be important groups constituting antibacterial product molecules.3.Through the fermentation of soybean meal feed with Bacillus subtilis M2 and the optimization of fermentation conditions,the results showed that Bacillus subtilis M2 was inoculated into sterilized soybean meal at the proportion of 5%,30%of double distilled water was added,and the laboratory fermented soybean meal was obtained by culturing in a constant temperature incubator at 37℃for 12 h.Then the laboratory fermented soybean meal was inoculated into untreated ordinary soybean meal at the proportion of 10%,and then 30%of double distilled water was added,The fermentation of soybean meal can be completed by fermentation at room temperature25℃for 16 h～24 h.Compared with unfermented soybean meal of the same variety,the content of crude protein and crude fat increased significantly（P<0.05）,and the content of crude fiber decreased significantly（P<0.05）. |
