Ammonia production by ruminal microorganisms and enumeration, isolation, and characterization of bacteria capable of growth on peptides and amino acids from the sheep rumen
Ammonia production by ruminal microorganisms and enumeration, isolation, and characterization of bacteria capable of growth on peptides and amino acids from the sheep rumen
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DOI：
10.1128/aem.68.10.4925-4931.2002
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发表时间：
2002-10-01
影响因子：
4.4
通讯作者：
Wallace, RJ
中科院分区：
文献类型：
--
作者：
Eschenlauer, SCP;McKain, N;Wallace, RJ
瘤胃中过量的 NH3 产生是反刍动物营养效率低下的一个主要问题。进行实验以比较体外瘤胃液中不同底物产生 NH3 的速率，并评估解糖细菌在 NH3 产生中的作用。瘤胃液取自四只接受混合干草浓缩饲料的瘤胃瘘羊。胰蛋白酶产生 NH3 的计算速率在 1.8 至 19.7 nmol mg(-1) min(-1) 蛋白质之间变化，具体取决于底物、其浓度和所使用的方法。底物浓度为 2 mg/ml 时，莫能菌素 (5 muM) 抑制蛋白质、肽和氨基酸产生 NH3 的能力平均为 28%，而底物浓度为 20 mg/ml 时，抑制率平均为 48% (P = 0.011)。在总细菌种群中，1.4% 仅依靠胰蛋白酶而生长，其中 93% 被 5 mu 莫能菌素消除。仅靠氨基酸生长的细菌要少得多（占总数的 0.002%）。从四只羊身上获得了十九个能够在胰蛋白酶上生长的分离株。 16S核糖体DNA和传统鉴定方法表明细菌分为六类。所有细胞均对莫能菌素敏感，并且除一组（第 111 组，类似于 Atopobium minutum）外，所有细胞均以 >250 nmol min(-1) mg 蛋白质 (-1) 产生 NH3，具体取决于培养基（通过分批培养方法测定）。所有分离株均具有外肽酶活性，但仅第 III 组具有明显的二肽基肽酶 I 活性。第 1、11 和 IV 组与瘤胃和口腔梭状芽胞杆菌和真杆菌属最密切相关。 V 组包含一种分离株，类似于猪脱硫单胞菌（以前称为猪脱硫弧菌）。 VI 组与发酵氨基酸球菌有 95% 相似。糖促进了 Atopobium 和 Desulfomonas 样分离株的生长，而糖显着抑制了 I、II 和 V 组的生长。因此，本研究表明，不同的方法和不同的底物浓度为不同研究中报告的瘤胃 NH3 产生的不同表观速率提供了解释，并确定了绵羊瘤胃中多种高产氨细菌。
Excessive NH3 production in the rumen is a major nutritional inefficiency in ruminant animals. Experiments were undertaken to compare the rates of NH3 production from different substrates in ruminal fluid in vitro and to assess the role of asaccharolytic bacteria in NH3 production. Ruminal fluid was taken from four rumen-fistulated sheep receiving a mixed hay-concentrate diet. The calculated rate of NH3 production from Trypticase varied from 1.8 to 19.7 nmol mg of protein(-1) min(-1) depending on the substrate, its concentration, and the method used. Monensin (5 muM) inhibited NH3 production from proteins, peptides, and amino acids by an average of 28% with substrate at 2 mg/ml, compared to 48% with substrate at 20 mg/ml (P = 0.011). Of the total bacterial population, 1.4% grew on Trypticase alone, of which 93% was eliminated by 5 mu monensin. Many fewer bacteria (0.002% of the total) grew on amino acids alone. Nineteen isolates capable of growth on Trypticase were obtained from four sheep. 16S ribosomal DNA and traditional identification methods indicated the bacteria fell into six groups. All were sensitive to monensin, and all except one group (group 111, similar to Atopobium minutum), produced NH3 at >250 nmol min(-1) mg of protein(-1) depending on the medium, as determined by a batch culture method. All isolates had exopeptidase activity, but only group III had an apparent dipeptidyl peptidase I activity. Groups 1, 11, and IV were most closely related to asaccharolytic ruminal and oral Clostridium and Eubacterium spp. Group V comprised one isolate, similar to Desulfomonas piger (formerly Desulfovibrio pigra). Group VI was 95% similar to Acidaminococcus fermentans. Growth of the Atopobium- and Desulfomonas-like isolates was enhanced by sugars, while growth of groups I, II, and V was significantly depressed by sugars. This study therefore demonstrates that different methodologies and different substrate concentrations provide an explanation for different apparent rates of ruminal NH3 production reported in different studies and identifies a diverse range of hyper-ammonia-producing bacteria in the rumen of sheep.