【作者】 邓露芳；

【导师】 王加启；

【作者基本信息】 中国农业科学院 ， 动物营养与饲料科学， 2009， 博士

【摘要】 纳豆枯草芽孢杆菌为枯草芽孢杆菌的亚种,具有广泛的益生作用。本研究旨在评价纳豆枯草芽孢杆菌(Bacillus subtilis natto strain RNLBSN002,BSN2)作为奶牛安全饲用微生物的应用效果,并初步探讨了其发挥益生作用的机理。试验一通过对BSN2生长曲线、生理生化指标和16S rDNA分子测定,研究了BSN2的生长特性、生理生化特性;通过正交试验和单因素试验,优化了BSN2液、固态发酵的培养条件。试验结果表明,通过菌落形态、生理生化指标和16S rDNA序列测定,可判断此菌为枯草芽孢杆菌属(Bacillus subtilis)。此菌液体发酵的最佳培养条件组合为葡萄糖1%、蔗糖1%、大豆蛋白胨6%、胰蛋白胨4%、接种量2%、装液量5 mL/dL、转速180 r/min和温度37℃。1000 mL发酵容器的最适装液量为400 mL,细菌产量达到4.6×1010 cfu/mL。此菌固体发酵以豆粕为基质,添加NaC1 0.30%、蔗糖5%、大豆蛋白胨5%,蒸馏水50%,pH 6.5,接种5%(v/w)种子菌液,培养36 h时活菌数达到最大3.58×109 cfu/g,菌体蛋白含量为4.77 mg/g。发酵产物氨基酸含量较未发酵豆粕培养基有总体提高趋势,其中天门冬氨酸、苏氨酸、丝氨酸、谷氨酸、丙氨酸和酪氨酸分别提高6.29、7.32、6.99、5.08、6.63和6.81%(P < 0.05)。试验二通过瘤胃体外发酵产气试验,研究了BSN2对瘤胃体外发酵的影响。本试验以不添加BSN2为对照,动态观测基础日粮中BSN2添加量分别为0.3×1011 cfu/d(处理Ⅰ)和1.5×1011 cfu/d(处理Ⅱ)对瘤胃液体外发酵产气量、pH、氨氮和各挥发性脂肪酸含量的影响。结果表明,处理Ⅱ与对照相比,产气量提高了11.89% (P < 0.05),瘤胃pH显著降低(P < 0.05),氨氮浓度提高了16.07% (P < 0.05),乙酸、丙酸和总挥发性脂肪酸浓度分别提高10.43%,50.35%和20.70%(P < 0.05)。说明BSN2培养物在基础日粮中的添加量为1.5×1011 cfu/d时能促进瘤胃微生物发酵,提高瘤胃代谢产物浓度。试验三通过动物饲喂试验,研究了BSN2对奶牛产奶性能的影响,评价此菌作为奶牛饲用微生物的效果。本试验选用36头泌乳早期荷斯坦奶牛,采用完全随机试验设计,分成3个处理组:1)对照组,基础日粮+ 60 g豆粕载体;2)处理Ⅰ,基础日粮+ 0.5×1011 cfu/d BSN2;3)处理Ⅱ,基础日粮+ 1.0×1011 cfu/d BSN2。试验期70 d,每头奶牛每天测定产奶量和采食量,每周采集牛奶样本进行乳成分测定。试验结果表明,与对照组相比较,处理Ⅱ在干物质采食量差异不显著的情况下,显著提高奶牛产奶量,4%乳脂校正乳产奶量和能量校正乳产奶量达14.7、17.0和17.4%(P < 0.05),显著提高乳中乳蛋白和乳糖含量达18.8%和15.1%(P < 0.05),乳中体细胞数log10值降低5.5%(P < 0.05)。说明BSN2具有促进奶牛奶产量,提高饲料转化效率,促进牛奶中乳蛋白和乳糖产量,降低体细胞数,改善牛奶品质的作用。试验四通过瘘管奶牛饲喂试验,研究了BSN2对奶牛产奶性能、瘤胃发酵和瘤胃微生物的影响,验证此菌作为奶牛饲用微生物的效果,初步探讨此菌发挥益生作用的机理。采用4头瘘管奶牛进行为期28 d的自体前后配对饲喂试验。分成3个处理阶段:1)试验期第1～7 d,饲喂基础日粮+ 60 g豆粕培养基,即对照组;2)试验期第8～21 d,饲喂基础日粮+ 1.0×1011 cfu/d BSN2,即处理组;3)试验期第22～28 d,饲喂基础日粮+ 60 g豆粕培养基,即停喂组。试验结果表明,不同处理期奶牛干物质采食量差异不显著。与对照期比较,BSN2处理期奶牛产奶量显著增加,奶中乳蛋白和乳糖产量增加(P < 0.05),瘤胃pH降低(6.64 vs. 6.46, P < 0.05),氨氮、总挥发性脂肪酸和丙酸摩尔比显著提高37.7、47.4和6.4%,乙酸摩尔比显著降低1.9%,乙酸/丙酸降低(3.23 vs. 3.02,P < 0.05)。传统培养法结果表明,与对照期相比,BSN2处理期奶牛瘤胃中瘤胃细菌、蛋白分解菌、淀粉分解菌和原虫数量显著增加;实时定量PCR结果表明,试验期奶牛瘤胃纤维分解菌,包括溶纤维丁酸弧菌、黄色瘤胃球菌和白色瘤胃球菌数量分别提高6.97%、4.51%和9.6%(P < 0.05),两种淀粉分解菌,牛链球菌和嗜淀粉瘤胃杆菌分别提高25.4%和14.6%(P < 0.05),乳酸利用菌埃氏巨球型菌也提高8.85%(P < 0.05)。普雷沃氏菌,包括布氏普雷沃氏菌、普雷沃氏菌短杆菌及普雷沃氏菌属均有显著提高,总细菌数和原虫纤毛虫数量显著提高。说明BSN2具有促进瘤胃某些瘤胃微生物种类生长的作用,促进瘤胃发酵,从而改善奶牛奶产量和乳成分产量。因此,BSN2具有作为奶牛饲用益生菌的潜力。更多还原

【Abstract】 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.更多还原