乳酸芽孢杆菌益生素的制备及对断奶仔猪生产性能和血清学指标的影响
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摘要
本文对3株乳酸芽孢杆菌的产酶性能和芽孢形成率、3株菌共生发酵的液体培养基和半固体培养基的成分、3株菌共生发酵液的抑菌活性及胞外代谢产物的成分进行了试验研究,用断奶仔猪对乳酸芽孢杆菌益生素作了应用试验,为乳酸芽孢杆菌益生素的开发与应用奠定了基础。
用不同的产酶发酵培养基培养乳酸芽孢杆菌SC、SL1和NT株,通过测定待测粗酶液的酶活性表明:3株菌均产生蛋白酶和纤维素酶,不产淀粉酶、脂肪酶和β-甘露聚糖酶;在发酵72 h后乳酸芽孢杆菌SC、SL1和NT株的蛋白酶活力分别为1426.292、1449.29、1439.326 U/mL,纤维素酶活力分别为76.2、66.4、45.2 U/mL。通过测定发酵液pH值,确定3株菌在发酵12 h后产酸量达到平衡状态,并维持36 h。通过测定芽孢形成率,确定3株菌随着发酵时间的延长,芽孢形成率逐渐提高。
用牛津杯双层平板定量扩散法检测3株菌代谢产物的抑菌活性,结果表明:代谢产物有抑菌活性,对金黄色葡萄球菌有极强的抑制作用,对大肠埃希菌抑制作用较弱。通过GC/MS分析,表明3株菌胞外代谢产物中含有抑菌物质和芳香类物质,初步确定抑菌物质为:烯丙基苯酚、沙丁胺醇、2-异丁基-4-甲基噻唑;芳香类物质为:1-[4-(1,1-二甲基乙基)-2,6-二甲基-3,5-二硝基苯基]乙酮和开米司酮。
用豆粕、豆汁、玉米粉、麸皮、白糖、K2HPO4、MgSO4、MnSO4、NaCl等成分配制不同的液体发酵培养基,通过测定3株菌共生发酵液中的蛋白酶、纤维素酶、pH值和芽孢形成率筛选出适宜的液体发酵培养基配方为:10 %豆汁100 mL/L,白糖60 g/L,K2HPO4 1.5 g/L,MgSO4 0.6 g/L,MnSO4 0.1 g/L,NaCl 5 g/L,pH值自然。
用不同添加量的豆粕、麸皮、鱼粉、白糖和水分配制半固体发酵培养基,通过测定3株菌共生发酵物中的蛋白酶、纤维素酶、pH值和细菌总数筛选出适宜的半固体发酵培养基配方为:按豆粕600 g/kg、麸皮200 g/kg、鱼粉100 g/kg、白糖100 g/kg配制成固形物,按固形物与水的比例为1:1.2(g/mL)添加自来水,pH值自然。在3株乳酸芽孢杆菌共生发酵后的半固体发酵培养基中添加300 g/kg麸皮,搅拌均匀,45℃烘干,粉碎,测定其中的芽孢数,即为乳酸芽孢杆菌益生素。
选用45头28±2日龄杜长大三元杂交断奶仔猪,分为5组,每组设3个重复,每重复3头仔猪;对照组饲喂基础日粮;抗生素组在基础日粮中添加2 g/kg阿莫西林;3个试验组在基础日粮中添加乳酸芽孢杆菌益生素,添加量分别为5、10、20 g/kg;试验期为28 d。试验结果表明:10 g/kg益生素添加量的仔猪日增质量提高最多,比对照组增加11.54 %(P<0.05);20 g/kg益生素添加量的料质量比降低最多,比对照组减少3.95 %(P<0.01);乳酸芽孢杆菌益生素可使断奶仔猪血清葡萄糖和总胆固醇含量升高或降低,血清白蛋白和总蛋白含量在56日龄时极显著升高,血清IgG含量降低,血清IgA含量基本不变,粪便中大肠埃希菌数量降低。
本研究制备出了断奶仔猪使用的乳酸芽孢杆菌益生素,在断奶仔猪日粮中的适宜添加量为10 ~ 20 g/kg。
The experiment was conducted to study some performance of three lactate Bacillus strains, included its ability of producing enzyme and spore formation rate, the composition of liquid medium and semi-solid medium by three bacterial strains symbiotic fermentation, activity of bacteriostasis, components of extracellular metabolites and breeding experiment in weaned piglets. According to those studies that aim to establish base of the development and application about lactate Bacillus probiotics.
Lactate Bacillus strain SC, SL1 and NT were cultured by various ferment medium producing enzyme. By testing the activity of crude enzyme liquit, the results showed that three bacterial strains produced protease and cellulase, out of producing amylase, lipase andβ-mannanase. After 72 h of fermentation, the activity of protease which produced by SC, SL1 and NT reached to 1426.292, 1449.29 and 1439.326 U/mL, the cellulase activity reached to 76.2, 66.4 and 45.2 U/mL. By measuring the pH value of fermentation broth, the acid content produced by three bacterial strains came to balance after fermenting 12 h and maintained 36 h. Along with fermentation, the spore formation rate of three bacterial strains increased gradually.
The bacteriostatic activity in the metabolite of three bacterial strains was detected by way of Oxford cup double layer flat quantitative diffusion detection , the results showed that metabolite had strong bacterioststic activity against Staphylococcus aureus and light inhibit to E. Coli. According to analyse three bacterias metabolite by GC/MS, the test results indicated that metabolite contained antibacterial substances and aromatic substances. The antibacterial substances included:Benzaldehyde,2,5-dimethyl-, N,N-Diethyl-1-oxaspiro[4.4]nonan-2-one-4-carboxamide and Thiazole, 2-butyl-4-methyl-. The aromatic substances were: Phenol,2,4-bis(1,1-dimethylethyl)- and Ethanone.1-[4-(1,1-dimethylethyl)-2,6-dimethyl-3,5-dinitrophenyl]-.
The various liquid fermentation medium was preparated by using soybean meal, bean juice, corn flour, wheat bran, white sugar, K2HPO4, MgSO4, MnSO4 and NaCl, the suitable liquid fermentation medium was selected by measuring protease, cellulase, pH and spore formation rate in the symbiotic fermentation liquid of three bacterial strains, the result showed that the better medium compositions were 10 % bean juice 100 mL/L, white sugar 60 g/L, K2HPO4 1.5 g/L, MgSO4 0.6 g/L, MnSO4 0.1 g/L, NaCl 5 g/L, pH was initial.
The semi-solid fermentation medium was preparated by using different amounts of soybean meal, wheat bran, fish meal, white sugar and water, the suitable semi-solid fermentation medium was selected by measuring protease, cellulase, pH and total bacteria in the symbiotic fermentation product of three bacterial strains. The result indicated that the better medium compositions were soybean meal 600 g/kg, wheat bran 200 g/kg, fish meal 100 g/kg, white sugar 100 g/kg, the ratio of solid and water was 1:1.2, pH was initial. Supplemented with 300 g/kg wheat bran in semi-solid fermentation, stir uniformity, dried in 45℃for 48 h, crushed which measured the number of spores, namely lactate bacillus probiotic.
Forty-five crossbred piglets (Dures×Landrace×Large white) were randomly assigned to 5 treatment groups by a randomized complete block design. The control group was provided with the basal diet, the antibiotic group was fed with basal diet supplemented with amoxicillin at the level of 2 g/kg. The three experiment groups was provided with basal diet supplemented with Lactate Bacillus probiotics at the level of 5, 10, 20 g/kg. The whole experiment period was 28 d. The results showed that: Compared with control group, 10 g/kg addition group increased 11.54 % (p<0.05) on average daily gain which had largest increased; 20 g/kg addition group decreased 3.95 %(p<0.01) on feed weight ratio which had largest decreased. Bacillus Lactate probiotic could increased or decreased serum glucose and total cholesterol. Serum albumin and total protein had significantly increased in the 56 days of age. Serum IgG decreased, serum IgA maintain at the same level. The number of fecal E. Coli reduced.
Based on the results above, preparation of the Lactate Bacillus probiotic was suitable to weaned piglets. The feasible supplement of probiotics addition was 1 %~2%.
用不同的产酶发酵培养基培养乳酸芽孢杆菌SC、SL1和NT株,通过测定待测粗酶液的酶活性表明:3株菌均产生蛋白酶和纤维素酶,不产淀粉酶、脂肪酶和β-甘露聚糖酶;在发酵72 h后乳酸芽孢杆菌SC、SL1和NT株的蛋白酶活力分别为1426.292、1449.29、1439.326 U/mL,纤维素酶活力分别为76.2、66.4、45.2 U/mL。通过测定发酵液pH值,确定3株菌在发酵12 h后产酸量达到平衡状态,并维持36 h。通过测定芽孢形成率,确定3株菌随着发酵时间的延长,芽孢形成率逐渐提高。
用牛津杯双层平板定量扩散法检测3株菌代谢产物的抑菌活性,结果表明:代谢产物有抑菌活性,对金黄色葡萄球菌有极强的抑制作用,对大肠埃希菌抑制作用较弱。通过GC/MS分析,表明3株菌胞外代谢产物中含有抑菌物质和芳香类物质,初步确定抑菌物质为:烯丙基苯酚、沙丁胺醇、2-异丁基-4-甲基噻唑;芳香类物质为:1-[4-(1,1-二甲基乙基)-2,6-二甲基-3,5-二硝基苯基]乙酮和开米司酮。
用豆粕、豆汁、玉米粉、麸皮、白糖、K2HPO4、MgSO4、MnSO4、NaCl等成分配制不同的液体发酵培养基,通过测定3株菌共生发酵液中的蛋白酶、纤维素酶、pH值和芽孢形成率筛选出适宜的液体发酵培养基配方为:10 %豆汁100 mL/L,白糖60 g/L,K2HPO4 1.5 g/L,MgSO4 0.6 g/L,MnSO4 0.1 g/L,NaCl 5 g/L,pH值自然。
用不同添加量的豆粕、麸皮、鱼粉、白糖和水分配制半固体发酵培养基,通过测定3株菌共生发酵物中的蛋白酶、纤维素酶、pH值和细菌总数筛选出适宜的半固体发酵培养基配方为:按豆粕600 g/kg、麸皮200 g/kg、鱼粉100 g/kg、白糖100 g/kg配制成固形物,按固形物与水的比例为1:1.2(g/mL)添加自来水,pH值自然。在3株乳酸芽孢杆菌共生发酵后的半固体发酵培养基中添加300 g/kg麸皮,搅拌均匀,45℃烘干,粉碎,测定其中的芽孢数,即为乳酸芽孢杆菌益生素。
选用45头28±2日龄杜长大三元杂交断奶仔猪,分为5组,每组设3个重复,每重复3头仔猪;对照组饲喂基础日粮;抗生素组在基础日粮中添加2 g/kg阿莫西林;3个试验组在基础日粮中添加乳酸芽孢杆菌益生素,添加量分别为5、10、20 g/kg;试验期为28 d。试验结果表明:10 g/kg益生素添加量的仔猪日增质量提高最多,比对照组增加11.54 %(P<0.05);20 g/kg益生素添加量的料质量比降低最多,比对照组减少3.95 %(P<0.01);乳酸芽孢杆菌益生素可使断奶仔猪血清葡萄糖和总胆固醇含量升高或降低,血清白蛋白和总蛋白含量在56日龄时极显著升高,血清IgG含量降低,血清IgA含量基本不变,粪便中大肠埃希菌数量降低。
本研究制备出了断奶仔猪使用的乳酸芽孢杆菌益生素,在断奶仔猪日粮中的适宜添加量为10 ~ 20 g/kg。
The experiment was conducted to study some performance of three lactate Bacillus strains, included its ability of producing enzyme and spore formation rate, the composition of liquid medium and semi-solid medium by three bacterial strains symbiotic fermentation, activity of bacteriostasis, components of extracellular metabolites and breeding experiment in weaned piglets. According to those studies that aim to establish base of the development and application about lactate Bacillus probiotics.
Lactate Bacillus strain SC, SL1 and NT were cultured by various ferment medium producing enzyme. By testing the activity of crude enzyme liquit, the results showed that three bacterial strains produced protease and cellulase, out of producing amylase, lipase andβ-mannanase. After 72 h of fermentation, the activity of protease which produced by SC, SL1 and NT reached to 1426.292, 1449.29 and 1439.326 U/mL, the cellulase activity reached to 76.2, 66.4 and 45.2 U/mL. By measuring the pH value of fermentation broth, the acid content produced by three bacterial strains came to balance after fermenting 12 h and maintained 36 h. Along with fermentation, the spore formation rate of three bacterial strains increased gradually.
The bacteriostatic activity in the metabolite of three bacterial strains was detected by way of Oxford cup double layer flat quantitative diffusion detection , the results showed that metabolite had strong bacterioststic activity against Staphylococcus aureus and light inhibit to E. Coli. According to analyse three bacterias metabolite by GC/MS, the test results indicated that metabolite contained antibacterial substances and aromatic substances. The antibacterial substances included:Benzaldehyde,2,5-dimethyl-, N,N-Diethyl-1-oxaspiro[4.4]nonan-2-one-4-carboxamide and Thiazole, 2-butyl-4-methyl-. The aromatic substances were: Phenol,2,4-bis(1,1-dimethylethyl)- and Ethanone.1-[4-(1,1-dimethylethyl)-2,6-dimethyl-3,5-dinitrophenyl]-.
The various liquid fermentation medium was preparated by using soybean meal, bean juice, corn flour, wheat bran, white sugar, K2HPO4, MgSO4, MnSO4 and NaCl, the suitable liquid fermentation medium was selected by measuring protease, cellulase, pH and spore formation rate in the symbiotic fermentation liquid of three bacterial strains, the result showed that the better medium compositions were 10 % bean juice 100 mL/L, white sugar 60 g/L, K2HPO4 1.5 g/L, MgSO4 0.6 g/L, MnSO4 0.1 g/L, NaCl 5 g/L, pH was initial.
The semi-solid fermentation medium was preparated by using different amounts of soybean meal, wheat bran, fish meal, white sugar and water, the suitable semi-solid fermentation medium was selected by measuring protease, cellulase, pH and total bacteria in the symbiotic fermentation product of three bacterial strains. The result indicated that the better medium compositions were soybean meal 600 g/kg, wheat bran 200 g/kg, fish meal 100 g/kg, white sugar 100 g/kg, the ratio of solid and water was 1:1.2, pH was initial. Supplemented with 300 g/kg wheat bran in semi-solid fermentation, stir uniformity, dried in 45℃for 48 h, crushed which measured the number of spores, namely lactate bacillus probiotic.
Forty-five crossbred piglets (Dures×Landrace×Large white) were randomly assigned to 5 treatment groups by a randomized complete block design. The control group was provided with the basal diet, the antibiotic group was fed with basal diet supplemented with amoxicillin at the level of 2 g/kg. The three experiment groups was provided with basal diet supplemented with Lactate Bacillus probiotics at the level of 5, 10, 20 g/kg. The whole experiment period was 28 d. The results showed that: Compared with control group, 10 g/kg addition group increased 11.54 % (p<0.05) on average daily gain which had largest increased; 20 g/kg addition group decreased 3.95 %(p<0.01) on feed weight ratio which had largest decreased. Bacillus Lactate probiotic could increased or decreased serum glucose and total cholesterol. Serum albumin and total protein had significantly increased in the 56 days of age. Serum IgG decreased, serum IgA maintain at the same level. The number of fecal E. Coli reduced.
Based on the results above, preparation of the Lactate Bacillus probiotic was suitable to weaned piglets. The feasible supplement of probiotics addition was 1 %~2%.
引文
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