Effect Of Bacillus Subtilis Natto On Milk Performance, Ruminal Fermentation, And Functional Microbial Of Dairy Cows

Bacillus sublitis natto is one of subspecies of Bacillus subtilis and used to produce Natto, and has benefit functions for human and animals. This study aims to estimate the effect of B. sublitis natto strain RNLBSN002(BSN2)as safe and direct-fed bacteria for dairy cows on performance, and makes a preliminary exploration on its probiotic mechanism.Experiment 1 studied the growth traits, physiology and biochemistry character of BSN2 by describing growth curve, and detecting physiology and biochemistry indexes. Liquid culture condition and solid culture condition were optimized by orthogonal design and single factor design. The best liquid fermentation condition was at glucose 1%, sucrose 1%, soy peptone 6%, tryptone 4%, inoculant concentration 2%, 5 mL culture in 100 mL conical flask, shaking speed 180 rpm and 37℃. The small scale test was carried out at the same conditions but the different capacity of culture in 1000 mL conical flask. The best capacity was 400 mL, and the bacteria product reached 4.6×1010 cfu/mL. The solid fermentation condition was at NaC1 0.30%,sugar 5%,soy peptone 5%,distilled water 50%,pH 6.5, inoculant concentration 5%(v/w). After 36 h solid fermentation, the available bacteria cells reached the highest point 3.58×109 cfu/g and the bacterial protein was 4.77 mg/g. The fermented culture contains higher amino acid than fermented before, especially increased aspartic acid, threonine, serine, glutamic acid, alanine, valine and tyrosine 6.29, 7.32, 6.99, 5.08, 6.63, 6.28 and 6.81%, respectively (P < 0.05).In Experiment 2, Gas production technique was used to examine the effect of BSN2 on rumen fermentation in vitro, then made a prediction on the benefits used in dairy cows. There were 3 group: 1) control group (CK); 2) 0.3×1011 cfu/d BSN2 (groupⅠ); 3) 1.5×1011 cfu/d BSN2 (groupⅡ). Gas production, pH value, NH3-N concentration and VFA concentrations in vitro were detected in this experiment. The result showed that, compared with control group (CK), the gas production in groupⅡincreased by 11.89% (P < 0.05), the pH value decreased (P < 0.05), the NH3-N concentration increased by 16.07% (P < 0.05), and the concentrations of acetic acid, propionic acid and total VFA increased by 10.43%, 50.35% and 20.70% (P<0.05), respectively. The conclusion indicated that the dosage of BSN2 at 1.5×1011 cfu/d benefit ruminal fermentation and increased the metabolites in vitro.In experiment 3, the animal feeding trial was conducted to evaluate effects of BSN2 on the production performance of dairy cows. Thirty six dairy cows in early lactation were randomly assigned into 3 groups: Control, basal diet; Group 1, basal diet plus 0.5×1011 cfu/d of BSN2 per cow and Group 2, basal diet plus 1.0×1011 cfu/d of BSN2 per cow. During the 70-d treatment period the following were determined on individual cow: daily milk production and dry matter intake (DMI), and weekly milk composition. Compared with Control, uncorrected milk yield, 4% FCM, energy-corrected milk, milk protein and lactose production of Group 2 were 14.7, 17.0, 17.4, 18.8 and 15.1% higher (P < 0.05) than cows fed Control, but Log10 SCC was 5.5% lower (P < 0.05). It concluded that BSN2 could increase the milk production, improve the feed conversion efficiency, benefit for milk protein and lactose production, and decrease the number of somatic cells, and thus improve the quality of milk.In Experiment 4, four rumen-cannulated dairy cows were researched on to study the effect of BSN2 on production performance, rumianl fermentation and ruminal microflora, estimate the benefits of BSN2 on dairy cows as direct-fed microbials and make a preliminary exploration of its probiotic mechanism. Four rumen-cannulated dairy cows were fed with the treatment diets in different period. There were three period: 1) from 1 to 7 d, Control diet + 60 g soybean meal culture (Pre-trial); 2) from d 8 to 21, Control diet + 60 g soybean meal culture containing 1.0×1011 cfu/d BSN2 (Trial); 3) from d 8 to 21, Control diet + 60 g soybean meal culture (Post-trial). The result showed that the DMI was not significantly different among these three periods. Compared with the Pre-trial period, during the Trial period milk production, milk protein and lactose production significantly increased, ruminal pH decreased (6.64 vs. 6.46, P < 0.05), and NH3-N, total VFA, and molar proportion of propionate increased 37.7, 47.4, and 6.4% (P < 0.05), respectively. Molar proportion of acetate decreased 1.9% (P < 0.05), and the A/P ratio dropped (3.23 vs. 3.02, P < 0.05). Total ruminal bacteria, proteolytic and amylolytic bacteria, and protozoa in rumen enumerated by culture method all increased (P < 0.05) in the Trial period. Real-time PCR analysis was used to compare Pre-trial and Trial populations of cellulolytic species, including Butyrivibrio fibrisolvens, Ruminococcus flavefaciens and Ruminococcus albus, which were increased by 7.0, 4.5 and 9.6%(P < 0.05), respectively. Two starch-fermenting bacteria species, Streptococcus bovis and Ruminobacter amylophilus, were increased by 25.4% and 14.6% (P < 0.05), respectively. Megasphaera elsdenii was also increased 8.8% (P < 0.05). When BSN2 was fed, Prevotella bryantii and P. brevis were increased significantly. The total bacteria and ciliate protozoa also increased (P < 0.05). These results demonstrate that BSN2 can enhance some ruminal bacteria and protozoa growth as well as ruminal fermentation, thus improving milk production, and milk component production. Therefore, BSN2 is proved to have a potential for being used as a probiotic for dairy cows.