瘤胃微生物氨基酸代谢及甲硫氨酸降解菌的研究
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摘要
甲硫氨酸和赖氨酸是反刍动物玉米-豆粕型日粮的限制性氨基酸,改善其利用率对于提高反刍动物生产性能具有重要意义。但由于反刍动物瘤胃微生物对进入瘤胃的部分饲料蛋白质、氨基酸的发酵降解,导致大量的蛋白质和氨基酸在瘤胃截流。为了提高氨基酸到达小肠的量,前人研究出了很多物理、化学的有效方法,但这些方法都不是从微生物的代谢途径入手,仅是治标的办法,且处理成本较高。因此我们期望通过对瘤胃微生物代谢规律的研究和甲硫氨酸利用菌的分离,来调控瘤胃中参与氨基酸代谢的重要酶的活性和瘤胃微生物中甲硫氨酸利用菌的数量,从而达到降低甲硫氨酸在瘤胃中的降解率,以及增加动物对限制性氨基酸的利用率。
1、采样和体外培养时间对瘤胃液氨基酸含量变化规律的影响,本试验旨在研究不同采样时间和不同培养时间对瘤胃微生物的氨基酸代谢的影响,试验一采用3只装有永久性瘤胃瘘管的萨能母羊为试验动物,不同采样时间和不同体外培养时间二因素下分别于饲前和饲后的不同时间点采集瘤胃液,经分别处理后分装到18个血清瓶中,分成两组,体外培养0、8、16h后抽取样本,测定样本中氨基酸的含量。试验结果表明:不同采样时间对于部分氨基酸的变化规律影响较显著(P＜0.05);不同的体外培养时间对于谷氨酸(Glu)、缬氨酸(Val)、苯丙氨酸(Phe)、赖氨酸(Lys)以及氨基酸总量的变化规律都有相当显著的影响(P＜0.05)。
2、混合瘤胃微生物体外利用甲硫氨酸、赖氨酸的研究,本试验旨在研究瘤胃液中的瘤胃微生物对添加的甲硫氨酸、赖氨酸利用情况,并进一步探讨添加氨基酸对瘤胃液中尿素氮、部分酶活的影响。试验二将采集到的来自3头装有永久性瘘管的成年萨能山羊的瘤胃液混合后,平均分装成18个瓶,随机分成3个处理组,每组6个重复。采用6重复随机因子试验设计。第一组中每瓶添加甲硫氨酸2mM(0.25mol/L Met溶液8ml),第二组中每瓶添加赖氨酸2mM(0.25mol/L Lys溶液8ml),第三组中每瓶添加等体积蒸馏水作为空白对照组,体外培养16小时。试验结果表明:添加氨基酸后混合瘤胃液中的尿素氮(UN)含量显著高于对照组(P＜0.05);谷氨酸脱氢酶(GLDH)活性随着体外培养时间的延长而迅速增加,并且添加甲硫氨酸组的GLDH的活性明显高于其他两组(P＜0.01),而添加赖氨酸组的GLDH活性则极显著低于甲硫氨酸组和对照组(P＜0.01);体外培养时间的长短对于γ-谷氨酰转移酶(γ-GT或GGT)的活性基本没影响,添加氨基酸后,赖氨酸有助于GGT活性的提高(P＜0.05),而甲硫氨酸对其基本不影响;添加的氨基酸和体外培养时间对于谷丙转氨酶(GPT)活性没有显著的影响(P＞0.05);添加氨基酸组的谷草转氨酶(GOT)的活性显著低于对照组(P＜0.05),但体外培养时间对于各组中GOT的活性没有显著的影响。
3、瘤胃甲硫氨酸利用菌的分离及鉴定,为了研究分析山羊瘤胃液中甲硫氨酸降解菌群的物种资源,以甲硫氨酸为唯一氮、碳源,对瘤胃微生物进行严格的厌氧分离、纯化,筛选出具有甲硫氨酸降解活性的三株细菌MB6-1、MB6-2、MB7-1,采用PCR方法扩增出16S rDNA基因,测定其基因的核苷酸全序列,并做部分的生理生化鉴定。菌株MB6-1、MB6-2以及MB7-1的16S rDNA序列已经被GenBank数据库收录,其序列号分别为DQ436917、EF581856、DQ453797;基于16S rDNA序列的同源性比较和系统发育学分析,发现MB6-1与普罗威登斯菌属的同源性高达96.7%,所以它可能是普罗威登斯菌属(Providencia)中的一个新种;而MB6-2与类芽孢杆菌属Paenibacillus cookii种的同源性达99.1%,故它可能是Paenibacillus cookii的一个亚种;菌株MB7-1与其他菌株的同源性都比较低,因此还不能断定其种属类别。
采样时间和培养时间对于部分的氨基酸浓度和变化趋势的影响都比较大,但Met和Lys的浓度始终呈现下降趋势,瘤胃微生物更优先利用赖氨酸,同时微生物降解这两种氨基酸的速度要大于合成他们的速度;添加氨基酸和体外培养时间的延长对瘤胃液中UN的含量和GGT、GLDH的活性影响较大(P＜0.05),但对GPT、GOT无显著影响(P＞0.05),说明瘤胃微生物发酵过程中氧化酶的活性变化范围比较大,从而氧化脱氨基反应发生的几率波动性较大,而转氨酶的活性相对比较稳定,所以发生转氨基反应相对比较稳定。以甲硫氨酸为唯一氮、碳源,经过严格筛选得到的菌株MB6-1、MB6-2和MB7-1,有利于我们今后研究分析山羊瘤胃液中甲硫氨酸降解菌群的更多物种资源,并期望通过对瘤胃中甲硫氨酸降解菌群数量的调控,来增加甲硫氨酸的过瘤胃率。
Methionine and lysine are the limited amino acids in corn and soybean-meal diets of ruminant,and improving their utilization rate is very important to improve the performance of ruminant.But owing to the parts of feed protein and amino acids what had been in rumen were fermented and degraded by rumen microorganism,most of protein and amino acids were intercepted in rumen.A lot of predecessors had been investigated many modus operandies for improving amino acids to get into small intestine,but these methods didn't begin with metabolic pathway of microorganism,and they were only the palliative methods and the cost price were much higher.So it was expected to control the activities of the main enzymes what participated the amino acid metabolism in rumen and the quantities of the methionine degradable bacterium in rumen microorganism by studying of the metabolism regularity of amino acid by rumen microorganism and isolating of the methionine degradable bacterium,accordingly lowed the degradation rate of the methionine in rumen and increased the utilization rate of limit amino acid for animals.
1、Effect of the sampling and culturing time in vitro on amino acid concentration of ruminal fluid.The aim of this study was to investigate the influence of the different sampling and culturing time on amino acid concentration of ruminal fluid by mixed rumen microorganisms in vitro.Three fistulated Salon goats used as the experimental animals in this experiment,and two different sampling times(before feeding and after feeding)were designed.This aim was to determine the effect of the different culturing time on metabolism of the amino acid in vitro.Rumen fluids from three perpetually fistulated Salon goats were collected on different time points(before feeding and after feeding),then divided into 18 bottles and allocated into two treatments,sampled after culturing 0、8、16h in vitro.The result indicated:there was significant influence between the different sampling time on the change regulation of some amino acid (P＜0.05);but there were great effect on the glutaminic acid(Glu)、valine(Val)、phenylalanine(Phe)、lysine(Lys)and the total amino acid(P＜0.05)between different culturing time in vitro.
2、Studies on the Utilization of Methionine and Lysine by Mixed Rumen Microorganisms in vitro.The aim of this study was to investigate the utilization of supplementary methionine and lysine by mixed rumen microorganisms in vitro,and further effects of adding methionine and lysine on urea nitrogen(UN)and enzyme activities(γ-glutamyltransferaseγ-GT or GGT,glutamic pyruvic transaminase GPT, glutamic oxalacetic transaminase GOT and glutamic acid dehydrogenase GLDH)were studied.Mixed rumen fluids from three perpetually fistulated Salon goats were collected, then divided into 18 bottles and allocated into three treatments arranged in a randomized complete block design with 6 which were added methionine(2mM),or lysine(2mM) and then added double distilled water as control groups,at last all treatments incubated for 16 hours at 39℃in vitro.Results indicated that the content of the UN in mixed rumen fluid increased when added with methionine or lysine compared with that in control group(P＜0.05).The activities of the GLDH increased rapidly with culture time getting longer.GLDH activity in methionine group was highly significant higher than that in other two groups(P＜0.01),however,GLDH activity in lysine group was highly significant lower than that in other two groups(P＜0.01).Culture time and added amino acid had no effect on GPT activity.GOT activities in both methionine and lysine groups were significantly lower than that in control.(P＜0.05),and culture time had no significant influence on them.Lysine was degraded more quickly than the methionine by rumen microorganisms during culturing in vitro.The UN was increased after adding amino acids,however activities of GOT and GPT were inhibited,and activities of GLDH was increased after adding methionine.
3、Isolation and identification of Methionine utilization bacterium from rumen fluid. In order to research and analyze species resource of the methionine utilization bacterium from goats rumen fluid,after strict anaerobe isolation and purity for rumen microorganism using methionine as the sole sources of nitrogen and carbon,the bacterium which can degrade the methionine were screened,and the 16S rDNA encoding gene(16S rDNA)from the three strains MB6-1、MB6-2、MB7-1 of methionine utilization bacterium were amplified by PCR,and their nucleotide sequences were determined,and identified the characters by partial physiology and biochemistry experiments.The 16S rDNA gene sequences of the strains MB6-1、MB6-2、MB7-1 had been accepted by GenBank,and the GenBank accession numbers were DQ436917、EF581856、DQ453797 respectively;On the basis of the homology and phylogenetic analysis of 16S rDNA,the homology of the strains MB6-1 and providencia rettgeri was up to 96.7%,so it was considered that strain MB6-1 may be a novel species of the providencia family;The homology of the strains MB6-2 and the Paenibacillus cookii was up to 99.1%,so it was considered that strain MB6-2 just may be a subspecies of the Paenibacillus cookii;The homology of the strains MB7-1 and other strains was low,so it can not be confirmed the genus categorization.
The influences of sampling and culturing time on the partial amino acid concentration and the change tendency were very significant,but the concentrations of methionine and lysine presented to descend tendency all the time,rumen microorganism were priority to use lysine,and the speed of the rumen microorganism degraded the two amino acids was quicker than the speed of the rumen microorganism synthesized the two amino acids;The effect of adding amino acids and extending the culture time in vitro on the content of UN and the activities of GGT、GLDH were significant,but there were no significant influences on GPT、GOT,it means that oxydasic activities had an extensive changing scope during the course of rumen microorganism fermentation,so the genetic probability of the oxidative deamination reaction was fluctuated,but transaminase activities were stable corresponding to oxydasic activities,so the genetic probability of the transamination was low.We got the strains MB6-1、MB6-2、MB7-1 by strict screening and adding the methionine as sole sources of nitrogen and carbon;It was profit to research and analyze more species resource of the methionine utilization bacterium from rumen fluid of goat in the future,and it was expected to increase the utilization rate of the Methionine in rumen by regulating the quantities of the Methionine degradable bacterium in rumen microorganism.
1、采样和体外培养时间对瘤胃液氨基酸含量变化规律的影响,本试验旨在研究不同采样时间和不同培养时间对瘤胃微生物的氨基酸代谢的影响,试验一采用3只装有永久性瘤胃瘘管的萨能母羊为试验动物,不同采样时间和不同体外培养时间二因素下分别于饲前和饲后的不同时间点采集瘤胃液,经分别处理后分装到18个血清瓶中,分成两组,体外培养0、8、16h后抽取样本,测定样本中氨基酸的含量。试验结果表明:不同采样时间对于部分氨基酸的变化规律影响较显著(P＜0.05);不同的体外培养时间对于谷氨酸(Glu)、缬氨酸(Val)、苯丙氨酸(Phe)、赖氨酸(Lys)以及氨基酸总量的变化规律都有相当显著的影响(P＜0.05)。
2、混合瘤胃微生物体外利用甲硫氨酸、赖氨酸的研究,本试验旨在研究瘤胃液中的瘤胃微生物对添加的甲硫氨酸、赖氨酸利用情况,并进一步探讨添加氨基酸对瘤胃液中尿素氮、部分酶活的影响。试验二将采集到的来自3头装有永久性瘘管的成年萨能山羊的瘤胃液混合后,平均分装成18个瓶,随机分成3个处理组,每组6个重复。采用6重复随机因子试验设计。第一组中每瓶添加甲硫氨酸2mM(0.25mol/L Met溶液8ml),第二组中每瓶添加赖氨酸2mM(0.25mol/L Lys溶液8ml),第三组中每瓶添加等体积蒸馏水作为空白对照组,体外培养16小时。试验结果表明:添加氨基酸后混合瘤胃液中的尿素氮(UN)含量显著高于对照组(P＜0.05);谷氨酸脱氢酶(GLDH)活性随着体外培养时间的延长而迅速增加,并且添加甲硫氨酸组的GLDH的活性明显高于其他两组(P＜0.01),而添加赖氨酸组的GLDH活性则极显著低于甲硫氨酸组和对照组(P＜0.01);体外培养时间的长短对于γ-谷氨酰转移酶(γ-GT或GGT)的活性基本没影响,添加氨基酸后,赖氨酸有助于GGT活性的提高(P＜0.05),而甲硫氨酸对其基本不影响;添加的氨基酸和体外培养时间对于谷丙转氨酶(GPT)活性没有显著的影响(P＞0.05);添加氨基酸组的谷草转氨酶(GOT)的活性显著低于对照组(P＜0.05),但体外培养时间对于各组中GOT的活性没有显著的影响。
3、瘤胃甲硫氨酸利用菌的分离及鉴定,为了研究分析山羊瘤胃液中甲硫氨酸降解菌群的物种资源,以甲硫氨酸为唯一氮、碳源,对瘤胃微生物进行严格的厌氧分离、纯化,筛选出具有甲硫氨酸降解活性的三株细菌MB6-1、MB6-2、MB7-1,采用PCR方法扩增出16S rDNA基因,测定其基因的核苷酸全序列,并做部分的生理生化鉴定。菌株MB6-1、MB6-2以及MB7-1的16S rDNA序列已经被GenBank数据库收录,其序列号分别为DQ436917、EF581856、DQ453797;基于16S rDNA序列的同源性比较和系统发育学分析,发现MB6-1与普罗威登斯菌属的同源性高达96.7%,所以它可能是普罗威登斯菌属(Providencia)中的一个新种;而MB6-2与类芽孢杆菌属Paenibacillus cookii种的同源性达99.1%,故它可能是Paenibacillus cookii的一个亚种;菌株MB7-1与其他菌株的同源性都比较低,因此还不能断定其种属类别。
采样时间和培养时间对于部分的氨基酸浓度和变化趋势的影响都比较大,但Met和Lys的浓度始终呈现下降趋势,瘤胃微生物更优先利用赖氨酸,同时微生物降解这两种氨基酸的速度要大于合成他们的速度;添加氨基酸和体外培养时间的延长对瘤胃液中UN的含量和GGT、GLDH的活性影响较大(P＜0.05),但对GPT、GOT无显著影响(P＞0.05),说明瘤胃微生物发酵过程中氧化酶的活性变化范围比较大,从而氧化脱氨基反应发生的几率波动性较大,而转氨酶的活性相对比较稳定,所以发生转氨基反应相对比较稳定。以甲硫氨酸为唯一氮、碳源,经过严格筛选得到的菌株MB6-1、MB6-2和MB7-1,有利于我们今后研究分析山羊瘤胃液中甲硫氨酸降解菌群的更多物种资源,并期望通过对瘤胃中甲硫氨酸降解菌群数量的调控,来增加甲硫氨酸的过瘤胃率。
Methionine and lysine are the limited amino acids in corn and soybean-meal diets of ruminant,and improving their utilization rate is very important to improve the performance of ruminant.But owing to the parts of feed protein and amino acids what had been in rumen were fermented and degraded by rumen microorganism,most of protein and amino acids were intercepted in rumen.A lot of predecessors had been investigated many modus operandies for improving amino acids to get into small intestine,but these methods didn't begin with metabolic pathway of microorganism,and they were only the palliative methods and the cost price were much higher.So it was expected to control the activities of the main enzymes what participated the amino acid metabolism in rumen and the quantities of the methionine degradable bacterium in rumen microorganism by studying of the metabolism regularity of amino acid by rumen microorganism and isolating of the methionine degradable bacterium,accordingly lowed the degradation rate of the methionine in rumen and increased the utilization rate of limit amino acid for animals.
1、Effect of the sampling and culturing time in vitro on amino acid concentration of ruminal fluid.The aim of this study was to investigate the influence of the different sampling and culturing time on amino acid concentration of ruminal fluid by mixed rumen microorganisms in vitro.Three fistulated Salon goats used as the experimental animals in this experiment,and two different sampling times(before feeding and after feeding)were designed.This aim was to determine the effect of the different culturing time on metabolism of the amino acid in vitro.Rumen fluids from three perpetually fistulated Salon goats were collected on different time points(before feeding and after feeding),then divided into 18 bottles and allocated into two treatments,sampled after culturing 0、8、16h in vitro.The result indicated:there was significant influence between the different sampling time on the change regulation of some amino acid (P＜0.05);but there were great effect on the glutaminic acid(Glu)、valine(Val)、phenylalanine(Phe)、lysine(Lys)and the total amino acid(P＜0.05)between different culturing time in vitro.
2、Studies on the Utilization of Methionine and Lysine by Mixed Rumen Microorganisms in vitro.The aim of this study was to investigate the utilization of supplementary methionine and lysine by mixed rumen microorganisms in vitro,and further effects of adding methionine and lysine on urea nitrogen(UN)and enzyme activities(γ-glutamyltransferaseγ-GT or GGT,glutamic pyruvic transaminase GPT, glutamic oxalacetic transaminase GOT and glutamic acid dehydrogenase GLDH)were studied.Mixed rumen fluids from three perpetually fistulated Salon goats were collected, then divided into 18 bottles and allocated into three treatments arranged in a randomized complete block design with 6 which were added methionine(2mM),or lysine(2mM) and then added double distilled water as control groups,at last all treatments incubated for 16 hours at 39℃in vitro.Results indicated that the content of the UN in mixed rumen fluid increased when added with methionine or lysine compared with that in control group(P＜0.05).The activities of the GLDH increased rapidly with culture time getting longer.GLDH activity in methionine group was highly significant higher than that in other two groups(P＜0.01),however,GLDH activity in lysine group was highly significant lower than that in other two groups(P＜0.01).Culture time and added amino acid had no effect on GPT activity.GOT activities in both methionine and lysine groups were significantly lower than that in control.(P＜0.05),and culture time had no significant influence on them.Lysine was degraded more quickly than the methionine by rumen microorganisms during culturing in vitro.The UN was increased after adding amino acids,however activities of GOT and GPT were inhibited,and activities of GLDH was increased after adding methionine.
3、Isolation and identification of Methionine utilization bacterium from rumen fluid. In order to research and analyze species resource of the methionine utilization bacterium from goats rumen fluid,after strict anaerobe isolation and purity for rumen microorganism using methionine as the sole sources of nitrogen and carbon,the bacterium which can degrade the methionine were screened,and the 16S rDNA encoding gene(16S rDNA)from the three strains MB6-1、MB6-2、MB7-1 of methionine utilization bacterium were amplified by PCR,and their nucleotide sequences were determined,and identified the characters by partial physiology and biochemistry experiments.The 16S rDNA gene sequences of the strains MB6-1、MB6-2、MB7-1 had been accepted by GenBank,and the GenBank accession numbers were DQ436917、EF581856、DQ453797 respectively;On the basis of the homology and phylogenetic analysis of 16S rDNA,the homology of the strains MB6-1 and providencia rettgeri was up to 96.7%,so it was considered that strain MB6-1 may be a novel species of the providencia family;The homology of the strains MB6-2 and the Paenibacillus cookii was up to 99.1%,so it was considered that strain MB6-2 just may be a subspecies of the Paenibacillus cookii;The homology of the strains MB7-1 and other strains was low,so it can not be confirmed the genus categorization.
The influences of sampling and culturing time on the partial amino acid concentration and the change tendency were very significant,but the concentrations of methionine and lysine presented to descend tendency all the time,rumen microorganism were priority to use lysine,and the speed of the rumen microorganism degraded the two amino acids was quicker than the speed of the rumen microorganism synthesized the two amino acids;The effect of adding amino acids and extending the culture time in vitro on the content of UN and the activities of GGT、GLDH were significant,but there were no significant influences on GPT、GOT,it means that oxydasic activities had an extensive changing scope during the course of rumen microorganism fermentation,so the genetic probability of the oxidative deamination reaction was fluctuated,but transaminase activities were stable corresponding to oxydasic activities,so the genetic probability of the transamination was low.We got the strains MB6-1、MB6-2、MB7-1 by strict screening and adding the methionine as sole sources of nitrogen and carbon;It was profit to research and analyze more species resource of the methionine utilization bacterium from rumen fluid of goat in the future,and it was expected to increase the utilization rate of the Methionine in rumen by regulating the quantities of the Methionine degradable bacterium in rumen microorganism.
引文
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