Effect Of Bacillus Sbutilis Natto On Rumen Fermentation And Quantity Of Rumen Functional Bacteria In Vitro

This study was carried out to investage the effect of Bacillus subtilis Natto (BSN)with different dose levels and different components on rumen microbial fermentation andthe quantity of rumen functional bacteria by In vitro gas production technology and Invitro batch fermentation methods, witch objective was to investigate the effect andmechanism of BSN on rumen fermentation.Experiment Ⅰ: According to the biological qualification of hydrolyzed casein andcarboxymethylcellulose（CMC）of Bacillus subtilis, four strains which isolated from fourdifferent sources, autoclaved soybeans with inoculation of straw (DC), autoclavedsoybeans without any of starter microbes (DD), autoclaved soybeans with ofcommercially Natto (ND), and one strain of Bacillus sbutilis Natto-NY-3stored in our lab(NY), respectively, were screened by pour casein and CMC plate method, andindentificated by16srRNA. And then investigate the effects of DD, DC, ND and NYstrains on rumen fermentation in vitro. Medium pH was unaffected (P=0.883) byBacillus subtilis in all treatments, while ammonia-N and VFA were significantlyincreased compared with control (P <0.05). The molar proportion of acetate:propionatewas unaffected in DC, DD and ND treatment compared with control (P>0.05), whilesignificantly decreased（P <0.05）in and NY treatment. The results indicated that theBacillus subtilis can stimulated in vitro fermentation by increase the molar proportion ofVFA and change the rumen fermentation type by change the molar proportion ofacetate:propionate, and may be have negative effects on rumen nitrogen metabolism withthe increasing of ammonia-N.Experiment Ⅱ: Two in vitro batch fermentation trial was used to study the effectof the major constituents of BSN culture on diet digestibility and rumen fermentation.Experiment1was designed to investigate the effects of different treatments, live (LBS),autoclaved (ABS) Bacillus subtilis Natto and Bacillus subtilis Natto culture (BSC)without bacteria, incubate48h using the DAISYIIIncubator on diet digestibility andrumen fermentation.The experiment repeated once and sampled at6h,12h,18h,24h,36hincubated. Experiment2was designed to investigate the effects of different treatments,blank control (CTR) that no additive, LBS contains of a minimum of1011cfu live Bacillus subtilis Natto, ABS contains of a minimum of1011cfu autoclaved Bacillus subtilis Natto,BSC contains of1ml Bacillus subtilis Natto culture without bacteria, and BSM containsof1ml liquid fermentation medium before noculated Bacillus subtilis Natto, on rumenfermentation and ruminal microbiome using serum bottle, Four sets of bottles were sealedanaerobically under CO2atmosphere with butyl rubber stoppers and capped withaluminum. One set (0h) were sampling immediately after mixed, other sets wereincubated12h,24h,36h respectively. Each set contained five treatments, and for eachtreatment, four bottles were incubated. Compared with control, the IVTD was enhancedsignificantly (P <0.05) in all additive treatments while the NDF and ADF were nodifferent with control, the molar proportion of propionate of BSC treatment wasenhanced significantly (P <0.05), the ammonia-N and Total VFA were enhancedsignificantly (P <0.05) in all additive treatments, and the LBS, ABS treatment higherthan the BSC, BSM (P <0.05). The MCP of LBS and ABS were significantly increasedand BSC trend to increase (P=0.0801) compared with control, The molar proportion ofeach kind VFA of LBS were significantly increased (P <0.05), while acetate:propionatewas decreased significantly (P <0.05) compared with control. The molar proportion ofiso-butyrate, butyrate, iso-valerate and valerate of ABS treatment were increased (P <0.05) compared with control. The results indicated that live Bacillus subtilis Natto is themajor constituents to influence rumen fermentation, and autoclaved Bacillus subtilisNatto take second place, there was no difference between liquid fermentation mediuminoculated Bacillus subtilis Natto and before on rumen fermentation, ammonia-N andMCP.Experiment Ⅲ: This experiment was designed to investigate the effects ofdifferent treatments, blank control (CTR) that no additive, LBS contains of a minimum of1011cfu live Bacillus subtilis Natto, ABS contains of a minimum of1011cfu autoclavedBacillus subtilis Natto, BSC contains of1ml Bacillus subtilis Natto culture withoutbacteria, and BSM contains of1ml liquid fermentation medium before noculated Bacillussubtilis Natto, on ruminal microbiome through q-PCR technology incubate12h usingserum bottles. The results indicated that compared with control, the fold change relativeto control of S. Dextrinosolvens was significantly increased in LBS, ABS, BSM, BSCtreatment, the fold change relative to control of R. amylophilus in BSM and BSC treatments were significantly increased, while the fold change relative to control of P.Bryanti were decreased in ABS treatment. Compared with BSM treatment the foldchange relative to control of R. flavefaciens was decreased, while the fold change relativeto control of other rumen functional bacteria were no difference between BSM treatmentand BSC treatment.