山羊瘤胃中主要乳酸代谢菌代谢机制及其调控瘤胃酸中毒的应用研究
发布时间：2018-07-31 06:13
【摘要】：有关瘤胃酸中毒发生机制研究表明,瘤胃乳酸的累积可能对酸中毒诱导起重要作用,而饲喂高精料日粮下诱发瘤胃乳酸累积主要取决于瘤胃乳酸产生菌和乳酸利用菌间的平衡程度。本论文即研究瘤胃中主要乳酸代谢菌代谢机制与调控方法,旨在为反刍动物瘤胃酸中毒的乳酸中毒机制深入解析提供参考。试验一建立山羊体外、内瘤胃酸中毒模型。将5种主要乳酸代谢菌(Streptococcus bovis,Lactobacilli fermentum,Butyrivibrio fibrisolvens,Megasphaera elsdenii 和Selenomonasruminantium)体外共培养并通过底物淀粉浓度(1,3和9g/L)诱导建立体外多菌共培养瘤胃酸中毒模型。结果表明:1)乳酸产生菌(S.bovis,L.fermentum和B.fibrisolvens)数量随底物淀粉浓度提高而提高(0.001),乳酸利用菌(M.elsenii和S.ruminantium)数量在急性酸中毒(ARA)组中显著低于亚急性酸中毒(SARA)组(P0.001)。2)ARA组中乳酸、乙酸、甲酸及丙酸浓度显著高于SARA组(P0.001),但丁酸浓度显著低于SARA组(P0.001)。3)乳酸脱氢酶(LDH)活性随底物淀粉浓度提高而显著提高(P0.001),ARA组中α-淀粉酶(α-AMY)活性显著低于SARA组(P0.001)。4)ARA组中脂多糖(LPS)浓度显著高于SARA组和对照组(P0.001),但对照组和SARA组无显著差异(P=0.659);选择8只干奶期萨能奶山羊,采用自身对照试验设计,通过高淀粉饲粮(50%玉米粉)诱导建立山羊SARA模型。结果表明:1)SARA 组 pH显著低于对照组(P0.001)。2)SARA 组 S.bovis,M.elsenii和S.ruminantium数量显著高于对照组(P0.001),Lactobacill 数量显著低于对照组(P0.001),B.fibrisolvens数量组间无显著差异(P = 0.800)。3)SARA组乳酸、甲酸、丙酸及丁酸浓度显著高于对照组(P0.01),但乙酸浓度无显著差异(P = 0.209)。4)SARA组LDH和α-AMY酶活及LPS浓度显著高于对照组(P0.001)。本试验结果表明:可以通过主要乳酸代谢菌共培养建立体外酸中毒模型及通过高淀粉饲粮诱导山羊瘤胃酸中毒发生。与此同时,S.bovis和M.elsdenii是瘤胃酸中毒进程中主要的乳酸产生菌和分解菌。试验二研究不同底物(淀粉和葡萄糖)、底物浓度(1,3和9g/L)和pH(6.5和5.5)条件下S.bovis有机酸产生通路及调控。结果表明:1)乳酸产量随底物浓度的提高显著提高(P0.001),且以淀粉为底物时pH为6.5下乳酸产量显著高于pH为5.5(P0.001)。甲酸和乙酸产量随底物浓度提高波动,但占总有机酸比例较小。2)LDH和α-AMY酶活随底物淀粉浓度提高而显著提高(P0.05),且pH为5.5时显著高于pH为6.5(P0.001),但以葡萄糖为底物时各组无显著差异(P0.05)。3)LDH、α-AMY、甲酸裂解酶(PFL)及分解控制蛋白A(CcpA)编码基因表达与底物浓度总体上呈正相关。4)果糖-1,6-二磷酸(FDP)浓度随底物浓度提高而显著提高(P0.05),且pH为6.5下FDP浓度显著高于pH为5.5(P = 0.032)。5)相较底物淀粉浓度的影响,S.bovis产乳酸对pH变化更敏感。本试验结果表明:底物类型、浓度及pH可在转录水平调控S.bovis有机酸产生模式。试验三研究底物乳酸浓度(15,30和90 mM)和pH(6.5和5.5)条件下M.elsdenii乳酸分解通路及调控。结果表明:1)M.elsdenii分解乳酸主要产生乙酸,其次丙酸和丁酸。2)pH为6.5时乙酸、丙酸和丁酸产量均显著高于pH为5.5(P0.05)。pH为6.5时,产乙酸比例显著低于pH为5.5(P0.001),而产丙酸和丁酸比例显著高于pH为5.5(P0.001)。3)底物乳酸浓度为90 m时,乙酸产量和比例显著高于其他两组(P0.05)。底物乳酸浓度为30 m组,丙酸和丁酸产量和比例显著高于底物乳酸浓度为90 mM组(P0.05),且90 mM组显著高于15 mM组(P0.05)。4)涉及乳酸分解相关关键酶编码基因随底物乳酸浓度及pH变化出现不出程度的显著差异表达(P0.05)。本试验结果表明:底物浓度及pH可以在转录水平调控M.elsenii乳酸分解通路。试验四研究山羊瘤胃灌注低营养富集诱导M.elsdenii对高淀粉饲粮诱导SARA缓解作用。结果表明:1)灌菌组pH显著高于SARA组,同时显著低于对照组(P0.05)。2)SARA组乳酸和丙酸浓度显著高于灌菌组及对照组(P0.05),但对照组与灌菌组差异不显著(P0.05)。SARA组和灌菌组丁酸浓度显著高于对照组(P0.05)。对照组乙酸浓度显著高于灌菌组(P0.05),但SARA组与对照组和灌菌组差异不显著(P0.05)。3)SARA组S.bovi 数量显著高于灌菌组和对照组(P0.05)。灌菌组Lactobacilli和B.fibrisolvens数量显著高于SARA组和对照组(P0.05)。SARA组和灌菌组M.elsdenii和S.ruminantium数量显著高于对照组(P0.05)。4)SARA组α-AMY和LDH酶活显著高于灌菌组(P0.05),且灌菌组α-AMY酶活显著高于对照组(P0.05)。5)SARA组LPS浓度显著高于灌菌组,且灌菌组显著高于对照组。本试验结果表明:通过瘤胃灌注低营养富集诱导M.elsdenii菌液可以改善高淀粉饲粮诱导SARA山羊瘤胃发酵,在一定程度上缓解酸中毒的发生。
[Abstract]:The study of the pathogenesis of gastric acidosis shows that the accumulation of lactic acid in the rumen may play an important role in the induction of acidosis, while feeding the rumen lactic acid accumulation under the diet of high concentrate depends mainly on the balance between the lactic acid producing bacteria in the rumen and the lactic acid use bacteria. Control methods are designed to provide a reference for the mechanism of lactic acidosis in ruminant gastric acidosis. A model of goats in vitro, internal tumor gastric acidosis was established. 5 major lactic acid metabolites (Streptococcus bovis, Lactobacilli fermentum, Butyrivibrio fibrisolvens, Megasphaera elsdenii, and Selenomonasruminantium) were established in vitro. The results showed that the number of lactic acid producing bacteria (S.bovis, L.fermentum and B.fibrisolvens) increased with the increase of substrate starch concentration (0.001), and the number of lactic acid bacteria (M.elsenii and S.ruminantium) in acute acidosis (ARA) group was increased by 1,3 and 9g/L induced by substrate starch concentration (1,3 and B.fibrisolvens). The concentration of lactic acid, acetic acid, formic acid and propionic acid in group ARA was significantly higher than that in group SARA (P0.001), but the concentration of butyric acid was significantly lower than that of SARA group (P0.001).3), but the activity of lactate dehydrogenase (LDH) in ARA group was significantly higher than that of SARA group (P0.001), but the activity of alpha amylase (alpha.2) in the ARA group was significantly lower than that of the SARA group (P0.001). 001).4) the concentration of lipopolysaccharide (LPS) in the ARA group was significantly higher than that in the SARA group and the control group (P0.001), but there was no significant difference between the control group and the SARA group (P=0.659); the selected 8 dried milk goats were designed by self control experiment, and the goat SARA model was established through the high starch diet (50% corn flour). The results showed that 1) pH of the SARA group was significantly lower than the control. Group (P0.001).2) the number of S.bovis, M.elsenii and S.ruminantium in group SARA was significantly higher than that of the control group (P0.001), the number of Lactobacill was significantly lower than that of the control group (P0.001), there was no significant difference between the B.fibrisolvens quantity groups (P = 0.800) and the concentration of lactic acid, propionic acid and butyric acid was significantly higher than that of the control group, but there was no significant difference in the concentration of acetic acid. LDH and alpha -AMY enzyme activity and LPS concentration in group SARA were significantly higher than those of control group (P0.001). The results of this experiment showed that the model of stereotactic acid poisoning and goaltumor gastric acidosis could be induced by major lactic acid metabolites and induced by high starch diet. At the same time, S.bovis and M.elsdenii were the main milk in the process of tumor gastric acidosis. Acid producing bacteria and decomposing bacteria. Experiment two studied the pathway and regulation of S.bovis organic acids in different substrates (starch and glucose), substrate concentration (1,3 and 9g/L) and pH (6.5 and 5.5). The results showed that 1) the production of lactic acid increased significantly with the increase of substrate concentration (P0.001), and the yield of lactic acid under the pH of 6.5 was significantly higher than that of pH at 5.5 when starch was the substrate. (P0.001). The yield of formic acid and acetic acid fluctuated with the concentration of substrate, but the proportion of the total organic acids was smaller.2). The activity of LDH and alpha -AMY increased significantly with the increase of substrate starch concentration (P0.05), and the pH was 5.5 higher than pH was 6.5 (P0.001), but there was no significant difference in each group (P0.05).3) LDH, alpha -AMY, formate lyase and fractions when the glucose was the substrate. The expression of A (CcpA) encoding gene was positively correlated with substrate concentration (.4). The concentration of fructose -1,6- two phosphoric acid (FDP) increased significantly with the increase of substrate concentration (P0.05), and the concentration of pH to 6.5 FDP was significantly higher than pH (P = 0.032).5) compared with the concentration of substrate starch. The substrate type, concentration and pH can regulate the production mode of S.bovis organic acid at the transcriptional level. Experiment three studies the pathway and regulation of the M.elsdenii lactic acid decomposition pathway under the conditions of substrate lactic acid (15,30 and 90 mM) and pH (6.5 and 5.5). The results show that 1) M.elsdenii decomposition lactic acid mainly produces acetic acid, and then propionic acid and butyric acid.2) pH is 6.5, acetic acid, propionic acid And the yield of butyric acid was significantly higher than that of pH 5.5 (P0.05).PH 6.5, the proportion of acetic acid was significantly lower than that of pH 5.5 (P0.001), while the ratio of propionic acid and butyric acid was significantly higher than that of pH 5.5 (P0.001).3). When the substrate lactic acid concentration was 90 m, the yield and proportion of acetic acid were significantly higher than those of the other two groups (P0.05). The substrate lactic acid concentration was 30 m group, propionic acid and butyric acid yield and ratio. The sample was significantly higher than the substrate lactate concentration of 90 mM (P0.05), and the 90 mM group was significantly higher than the 15 mM group (P0.05).4) involved in the significant difference expression of the lactic acid decomposition related key enzyme encoding genes with the substrate lactic acid concentration and pH changes (P0.05). The results showed that the substrate concentration and pH could regulate the M.elsenii lactic acid at the transcriptional level. Decomposition pathway. Experiment four study the effect of M.elsdenii induced SARA on high starch diet induced by rumen perfusion in goats. The results showed that: 1) the pH of the group was significantly higher than that of the SARA group, and the concentration of lactic acid and propionic acid in the SARA group was significantly higher than that of the control group and the control group (P0.05), but the difference between the control group and the perfusion group was worse than the control group (P0.05). The concentration of butyric acid in group P0.05.SARA and perfusion group was significantly higher than that of control group (P0.05). The concentration of acetic acid in the control group was significantly higher than that of the perfusion group (P0.05), but there was no significant difference between the SARA group and the control group and the control group (P0.05).3). The S.bovi number of the SARA group was significantly higher than that of the perfusion group and the opposite group (P0.05). The number of M.elsdenii and S.ruminantium in the group.SARA and the control group (P0.05) and the control group was significantly higher than that of the control group (P0.05).4). The enzyme activity of alpha -AMY and LDH in the SARA group was significantly higher than that of the group (P0.05), and the alpha -AMY enzyme activity of the group was significantly higher than that of the control group (P0.05), and the concentration of SARA was significantly higher than that of the control group, and the perfusion group was significantly higher than the control group. The results showed that the induced M.elsdenii bacteria could improve the rumen fermentation of SARA goats induced by high starch diet, and alleviated the occurrence of acidosis to a certain extent.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.27
本文编号：2154680
[Abstract]:The study of the pathogenesis of gastric acidosis shows that the accumulation of lactic acid in the rumen may play an important role in the induction of acidosis, while feeding the rumen lactic acid accumulation under the diet of high concentrate depends mainly on the balance between the lactic acid producing bacteria in the rumen and the lactic acid use bacteria. Control methods are designed to provide a reference for the mechanism of lactic acidosis in ruminant gastric acidosis. A model of goats in vitro, internal tumor gastric acidosis was established. 5 major lactic acid metabolites (Streptococcus bovis, Lactobacilli fermentum, Butyrivibrio fibrisolvens, Megasphaera elsdenii, and Selenomonasruminantium) were established in vitro. The results showed that the number of lactic acid producing bacteria (S.bovis, L.fermentum and B.fibrisolvens) increased with the increase of substrate starch concentration (0.001), and the number of lactic acid bacteria (M.elsenii and S.ruminantium) in acute acidosis (ARA) group was increased by 1,3 and 9g/L induced by substrate starch concentration (1,3 and B.fibrisolvens). The concentration of lactic acid, acetic acid, formic acid and propionic acid in group ARA was significantly higher than that in group SARA (P0.001), but the concentration of butyric acid was significantly lower than that of SARA group (P0.001).3), but the activity of lactate dehydrogenase (LDH) in ARA group was significantly higher than that of SARA group (P0.001), but the activity of alpha amylase (alpha.2) in the ARA group was significantly lower than that of the SARA group (P0.001). 001).4) the concentration of lipopolysaccharide (LPS) in the ARA group was significantly higher than that in the SARA group and the control group (P0.001), but there was no significant difference between the control group and the SARA group (P=0.659); the selected 8 dried milk goats were designed by self control experiment, and the goat SARA model was established through the high starch diet (50% corn flour). The results showed that 1) pH of the SARA group was significantly lower than the control. Group (P0.001).2) the number of S.bovis, M.elsenii and S.ruminantium in group SARA was significantly higher than that of the control group (P0.001), the number of Lactobacill was significantly lower than that of the control group (P0.001), there was no significant difference between the B.fibrisolvens quantity groups (P = 0.800) and the concentration of lactic acid, propionic acid and butyric acid was significantly higher than that of the control group, but there was no significant difference in the concentration of acetic acid. LDH and alpha -AMY enzyme activity and LPS concentration in group SARA were significantly higher than those of control group (P0.001). The results of this experiment showed that the model of stereotactic acid poisoning and goaltumor gastric acidosis could be induced by major lactic acid metabolites and induced by high starch diet. At the same time, S.bovis and M.elsdenii were the main milk in the process of tumor gastric acidosis. Acid producing bacteria and decomposing bacteria. Experiment two studied the pathway and regulation of S.bovis organic acids in different substrates (starch and glucose), substrate concentration (1,3 and 9g/L) and pH (6.5 and 5.5). The results showed that 1) the production of lactic acid increased significantly with the increase of substrate concentration (P0.001), and the yield of lactic acid under the pH of 6.5 was significantly higher than that of pH at 5.5 when starch was the substrate. (P0.001). The yield of formic acid and acetic acid fluctuated with the concentration of substrate, but the proportion of the total organic acids was smaller.2). The activity of LDH and alpha -AMY increased significantly with the increase of substrate starch concentration (P0.05), and the pH was 5.5 higher than pH was 6.5 (P0.001), but there was no significant difference in each group (P0.05).3) LDH, alpha -AMY, formate lyase and fractions when the glucose was the substrate. The expression of A (CcpA) encoding gene was positively correlated with substrate concentration (.4). The concentration of fructose -1,6- two phosphoric acid (FDP) increased significantly with the increase of substrate concentration (P0.05), and the concentration of pH to 6.5 FDP was significantly higher than pH (P = 0.032).5) compared with the concentration of substrate starch. The substrate type, concentration and pH can regulate the production mode of S.bovis organic acid at the transcriptional level. Experiment three studies the pathway and regulation of the M.elsdenii lactic acid decomposition pathway under the conditions of substrate lactic acid (15,30 and 90 mM) and pH (6.5 and 5.5). The results show that 1) M.elsdenii decomposition lactic acid mainly produces acetic acid, and then propionic acid and butyric acid.2) pH is 6.5, acetic acid, propionic acid And the yield of butyric acid was significantly higher than that of pH 5.5 (P0.05).PH 6.5, the proportion of acetic acid was significantly lower than that of pH 5.5 (P0.001), while the ratio of propionic acid and butyric acid was significantly higher than that of pH 5.5 (P0.001).3). When the substrate lactic acid concentration was 90 m, the yield and proportion of acetic acid were significantly higher than those of the other two groups (P0.05). The substrate lactic acid concentration was 30 m group, propionic acid and butyric acid yield and ratio. The sample was significantly higher than the substrate lactate concentration of 90 mM (P0.05), and the 90 mM group was significantly higher than the 15 mM group (P0.05).4) involved in the significant difference expression of the lactic acid decomposition related key enzyme encoding genes with the substrate lactic acid concentration and pH changes (P0.05). The results showed that the substrate concentration and pH could regulate the M.elsenii lactic acid at the transcriptional level. Decomposition pathway. Experiment four study the effect of M.elsdenii induced SARA on high starch diet induced by rumen perfusion in goats. The results showed that: 1) the pH of the group was significantly higher than that of the SARA group, and the concentration of lactic acid and propionic acid in the SARA group was significantly higher than that of the control group and the control group (P0.05), but the difference between the control group and the perfusion group was worse than the control group (P0.05). The concentration of butyric acid in group P0.05.SARA and perfusion group was significantly higher than that of control group (P0.05). The concentration of acetic acid in the control group was significantly higher than that of the perfusion group (P0.05), but there was no significant difference between the SARA group and the control group and the control group (P0.05).3). The S.bovi number of the SARA group was significantly higher than that of the perfusion group and the opposite group (P0.05). The number of M.elsdenii and S.ruminantium in the group.SARA and the control group (P0.05) and the control group was significantly higher than that of the control group (P0.05).4). The enzyme activity of alpha -AMY and LDH in the SARA group was significantly higher than that of the group (P0.05), and the alpha -AMY enzyme activity of the group was significantly higher than that of the control group (P0.05), and the concentration of SARA was significantly higher than that of the control group, and the perfusion group was significantly higher than the control group. The results showed that the induced M.elsdenii bacteria could improve the rumen fermentation of SARA goats induced by high starch diet, and alleviated the occurrence of acidosis to a certain extent.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.27
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