云南传统发酵豆豉中高产豆豉纤溶酶菌株的筛选及其酶谱分析

从云南昆明、元阳、玉溪、昭通地区采集豆豉样品26个,通过琼脂-纤维蛋白平板试验,SDS-fibrin-PAGE蛋白电泳结合Zymography活性染色对相应酶谱进行分析;同时采用茚三酮法测定其豆豉纤溶酶活性,筛选得到5株豆豉纤溶酶活性较高的菌株,其中尤以ZT-13茵株纤溶酶活性达到97.09 U/mL,是工业化生产豆豉纤溶酶的潜在优良菌株.     

产纤溶酶少根根霉菌株的诱变筛选

目的:通过对自南方小酒药中筛选得到的1株产纤溶酶的少根根霉Or株的诱变筛选,提高原有菌株的产酶能力。方法:以Or为出发菌株,进行亚硝基胍、紫外线、Co60诱变,以血纤维蛋白平板法为检测方法,筛选高产酶突变株。结果:经诱变传代后得到高产突变株8B,其产酶活性稳定为291.05U/mL,为原菌株产酶活力的6.34倍。该菌在血琼脂平板上不产生溶圈,诱变后孢子成熟提前8h。结论:物理诱变和化学诱变交替应用,能显著提高少根根霉Or株单位体积发酵液的产酶量,并能缩短孢子的成熟周期。该菌不具有溶血性,与已有报道的产纤溶酶的菌株不同。     

一株产纤溶酶凝结芽孢杆菌的筛选及其生物学特性

为了筛选性能良好的产纤溶酶菌株,本研究通过脱脂乳平板及纤维蛋白平板双层筛选从传统发酵食品豆豉中,分离到1株具有溶解纤维蛋白能力的凝结芽孢杆菌(Bacillus coagulans) HQ-1。分析其发酵上清液中纤溶酶的纤溶能力、组成特征和作用方式,并研究菌株HQ-1生物学特性。结果表明:菌株HQ-1发酵上清液中的纤溶酶活力为383.1 U/mL;Native-PAGE实验表明HQ-1发酵上清液中能溶解纤维蛋白的纤溶酶组分仅有一种,并且通过激活纤溶酶原实现HQ-1降解纤维蛋白。HQ-1生物学特性实验表明:菌株HQ-1对阿莫西林等11种常见抗生素敏感;对沙门氏菌、大肠杆菌和金黄色葡萄球菌的抑制作用显著。菌株HQ-1产纤溶酶活性及稳定性较好,具有潜在开发价值,本研究结果为产纤溶酶微生物资源的开发提供理论依据。     

一株产纤溶酶菌株的分离鉴定及其纤溶组分分析

【目的】筛选性能良好的产纤溶酶菌株,对菌株进行多项分类鉴定,分析其纤溶酶系的组成特征及纤溶能力。【方法】通过酪蛋白培养基初筛,琼脂-纤维蛋白双层平板复筛,从海泥、土壤等环境中筛选纤维蛋白降解菌,以尿激酶为标准测定纤溶酶活性。通过形态学、生理生化特征研究,结合16S rDNA基因序列分析菌株种类及系统分类地位。通过SDS-PAGE和纤维蛋白酶谱法分析胞外纤溶酶系的组成特征。【结果】筛选到一株能降解纤维蛋白的细菌CNY16,鉴定其为沙福芽孢杆菌(Bacillus safensis)。该酶为胞外酶,SDS-PAGE和纤维蛋白酶谱结果表明该纤溶酶系有至少两种分子量大小不同的纤溶酶,分别约33 kD和23 kD。能有效溶解血块中纤维蛋白,并且对红细胞无降解作用。【结论】细菌CNY16是一株新的纤溶酶产生菌,纤溶酶活性及稳定性较好,具有潜在开发价值。为获取新型纤溶酶提供了一种新的菌源。     

豆豉纤溶酶产生菌的筛选及诱变

从广泛收集的豆豉成品及半成品中筛选到数株菌落形态各异且具有纤溶酶活性的菌株.分别采用亚硝酸和紫外线对豆豉纤溶酶产生菌DC-12进行诱变育种,成功筛选到了3株突变株,其纤溶酶产量较出发菌株分别提高了3.6、3.7和4.75倍.     

豆豉纤溶酶产生菌的筛选及菌种鉴定

在收集的全国 2 1个豆豉产品中分离到 36 9株革兰氏阳性芽孢杆菌 ,对其进行产纤溶酶活性筛选 ,得到 1株高产纤溶酶菌株HGD10 7。对菌株HGD10 7进行形态、生理、生化鉴定 ,初步鉴定为枯草芽孢杆菌。     

一株产纤溶酶菌株的分离、鉴定及其酶活特征的初步研究

[目的]分离筛选并鉴定产纤溶酶的菌株.[方法]采用血粉培养基富集,琼脂糖-纤维蛋白平板筛选,从自然界中分离筛选出一株产纤溶活性物质的菌株.通过形态学特征、生理生化特征研究,并结合16S rRNA基因序列分析及分子系统发育树的构建结果,确定菌株的种类.[结果]从自然界分离筛到一株产纤溶酶的菌株EF608,经鉴定该菌株为粪肠球菌(Enterococcus faecalis). SDS-PAGE和纤维蛋白自显影表明该纤溶酶的分子量为37 kD,最适反应温度和pH分别为35℃和7.5,EDTA能完全抑制其纤溶活性,而PMSF对其活性无抑制作用.菌株EF608发酵液不仅可以直接水解纤维蛋白,而且具有体外溶栓的作用,对血红细胞没有溶解作用.[结论]筛选到一株具有纤溶活性的粪肠球菌——EF608,为获取新型纤溶酶提供了一种的新的菌源.     

豆豉纤溶酶高产菌株的筛选研究

以中国豆豉为材料,取6个不同来源的样品经富集培养后,用自制血纤维蛋白平板进行初筛;利用LB纤维蛋白平板复筛与血琼脂平板进行体外溶血试验相结合的方法,筛选出安全性良好并有一定纤溶活性的菌株。再对复筛得到的菌株进行摇瓶培养,用纤维蛋白平板法测定并比较不同菌株发酵液的纤溶活性。结果成功地筛选出5株纤溶活性高和通过溶血性实验的芽孢杆菌菌株,其中菌株DC-C4粗酶液的酶活稍高,达到280IU/mL。采用16S-23S rDNA序列分析法及传统的生理生化特征鉴定法对该菌株进行鉴定,确定菌株DC-C4为蜡状芽孢杆菌(Bacillus cereus)。本实验为开发新型溶栓药物及保健食品提供了实验参考依据。     

一株产纤溶酶放线菌的分离和鉴定

目的：从黄精根际土壤筛选产纤溶酶的放线菌,并对其进行鉴定。方法：采用稀释法分离放线菌,利用纤维蛋白平板法筛选能产生纤溶酶的菌株,根据16SrDNA序列、形态学观察和生理生化特征对纤溶活性高的菌株进行鉴定。结果：菌株XZNUM 00004代谢产物的纤溶酶活性为69U/ml,该酶加热到65℃仍能保留85%的活性。经形态学观察,培养特征、生理生化和分子分类学分析鉴定,与胶样链霉菌（Streptomyces gelaticus）相似性为99%。结论：初步推断XZNUM 00004属于链霉菌属（Streptomyces）。     

来自光裸方格星虫产纤溶酶蜡状芽孢杆菌的鉴定

对广西北部湾光裸方格星虫中产纤溶酶菌株进行筛选和鉴定,结合脱脂乳平板与纤维蛋白板筛选出产纤溶酶菌株,并对其粗酶液的血栓溶解机制进行了体外初步研究。结果显示,从光裸方格星虫获得5株产纤溶酶菌株,其中GXUSP-1菌株纤溶酶粗酶活力(相当尿激酶酶活力单位)为303.2 U/mL,经初步鉴定该菌为蜡状芽孢杆菌属,属于海洋共生菌,该菌株粗酶液能通过直接和间接两种方式溶解血栓,且体外溶解血栓作用显著。GXUSP-1具有良好的潜在工业应用价值。     

瘤胃木质纤维素降解菌及降解酶基因的研究进展

反刍动物瘤胃是公认的木质纤维素高效降解的天然反应器,对瘤胃微生物的研究已成为开发生物能源的热点领域之一。目前的研究手段已经从传统的依赖分离培养从瘤胃中获得木质纤维素降解菌,并对降解菌中的木质纤维素降解酶逐一分析,发展到通过基因组/元基因组学技术,直接从瘤胃中发现并获得大量新的木质纤维素降解酶基因/基因簇,进而探讨其降解的分子机理。已有的研究结果表明,瘤胃微生物降解木质纤维素的过程非常复杂,涉及大量不同种类的微生物及基因/基因簇,随着新分析技术的建立和完善,对这些微生物和基因的研究已取得了诸多进展。本论文综述了近期有关该方向的研究进展。     

瘤胃中木质纤维素降解菌及降解酶基因的研究进展

摘要：反刍动物瘤胃是公认的木质纤维素高效降解的天然反应器,对瘤胃微生物的研究成为开发生物能源的热点领域之一。其研究手段已经从传统的依赖分离培养从瘤胃中获得木质纤维素降解菌,并对降解菌中的木质纤维素降解酶逐一分析,发展到通过基因组/元基因组技术,直接从瘤胃中发现获得大量新的木质纤维素降解酶基因/基因簇,进而探讨其降解的分子机理。已有的研究结果表明,瘤胃微生物降解木质纤维素的过程非常复杂,其中涉及到大量不同种类的微生物、酶及基因/基因簇,随着新分析技术的建立和完善,对这些微生物、酶和基因的研究已取得了诸多进展。本论文综述报道了近期有关该方向的研究进展。     

Metagenomic insights into the fibrolytic microbiome in yak rumen

The rumen hosts one of the most efficient microbial systems for degrading plant cell walls, yet the predominant cellulolytic proteins and fibrolytic mechanism(s) remain elusive. Here we investigated the cellulolytic microbiome of the yak rumen by using a combination of metagenome-based and bacterial artificial chromosome (BAC)-based functional screening approaches. Totally 223 fibrolytic BAC clones were pyrosequenced and 10,070 ORFs were identified. Among them 150 were annotated as the glycoside hydrolase (GH) genes for fibrolytic proteins, and the majority (69%) of them were clustered or linked with genes encoding related functions. Among the 35 fibrolytic contigs of >10 Kb in length, 25 were derived from Bacteroidetes and four from Firmicutes. Coverage analysis indicated that the fibrolytic genes on most Bacteroidetes-contigs were abundantly represented in the metagenomic sequences, and they were frequently linked with genes encoding SusC/SusD-type outer-membrane proteins. GH5, GH9, and GH10 cellulase/hemicellulase genes were predominant, but no GH48 exocellulase gene was found. Most (85%) of the cellulase and hemicellulase proteins possessed a signal peptide; only a few carried carbohydrate-binding modules, and no cellulosomal domains were detected. These findings suggest that the SucC/SucD-involving mechanism, instead of one based on cellulosomes or the free-enzyme system, serves a major role in lignocellulose degradation in yak rumen. Genes encoding an endoglucanase of a novel GH5 subfamily occurred frequently in the metagenome, and the recombinant proteins encoded by the genes displayed moderate Avicelase in addition to endoglucanase activities, suggesting their important contribution to lignocellulose degradation in the exocellulase-scarce rumen.     

Effects of exogenous fibrolytic enzymes on ruminal fermentation and digestibility of total mixed rations fed to lambs

This experiment was designed to study effects of exogenous fibrolytic enzymes on digestibility and ruminal fermentation of total mixed rations (TMR) with different forage:concentrate (F:C) ratios. The experimental design was a 6×6 Latin square with six ruminally cannulated Ramboulliet lambs (58 ± 7.4 kg body weight) used in a 3×2 factorial arrangement of treatments using three forage:concentrate (F:C) ratios (400:600, 500:500, 600:400 kg/kg) and two (0 or 2 g) levels of fibrolytic enzymes/kg TMR dry matter (DM). Exogenous fibrolytic enzymes increased the soluble fraction of DM (P<0.01) and ruminal in situ disappearance rate of DM and neutral detergent fibre (aNDF; P<0.001). However, there were no differences among treatments in DM and aNDF in vivo digestibility due to enzyme addition to the diet. Feed intake and N balance were also not affected by enzymes, and ruminal fermentation patterns were unchanged. Exogenous fibrolytic enzymes improved ruminal disappearance rates of DM and aNDF, but had no impact on lamb performance or ruminal fermentation.     

Effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes and fibre breakdown in the rumen ecosystem.

The effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents was examined after the refaunation of ciliate-free sheep with an A-type rumen protozoal population. Although the culturable rumen bacterial population was reduced after refaunation the number of fibrolytic micro-organisms detected was higher; the xylanolytic bacterial population and numbers of fungal zoospores were increased after refaunation. The proportion of propionic acid was lower in the refaunated animals, whereas the concentration of ammonia and the acidic metabolites acetate, butyrate and valerate were all increased. The range of enzyme activities present in the digesta subpopulations were the same in defaunated and refaunated animals. The activities of the polysaccharide-degrading enzymes, however, were increased in the microbial populations associated with the digesta solids after refaunation, and at 16 h after feeding the activities were 4-8 times (beta-D-xylosidase 20 times) higher than the levels detected in the adherent population from defaunated sheep. The protozoa, either directly through their own enzymes or indirectly as a consequence of their effects on the population size and activity of the other fibrolytic micro-organisms present, have an important role in determining the level of activity of polysaccharide-degrading enzymes in the rumen ecosystem. Although the extent of ryegrass (Lolium perenne) hay digestion was similar after 24 h in the absence or presence of protozoa, the initial ruminal degradation was higher in refaunated sheep.     

Effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes and fibre breakdown in the rumen ecosystem

The effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents was examined after the refaunation of ciliate-free sheep with an A-type rumen protozoal population. Although the culturable rumen bacterial population was reduced after refaunation the number of fibrolytic micro-organisms detected was higher; the xylanolytic bacterial population and numbers of fungal zoospores were increased after refaunation. The proportion of propionic acid was lower in the refaunated animals, whereas the concentration of ammonia and the acidic metabolites acetate, butyrate and valerate were all increased. The range of enzyme activities present in the digesta subpopulations were the same in defaunated and refaunated animals. The activities of the polysaccharide-degrading enzymes, however, were increased in the microbial populations associated with the digesta solids after refaunation, and at 16 h after feeding the activities were 4–8 times (β-d-xylosidase 20 times) higher than the levels detected in the adherent population from defaunated sheep. The protozoa, either directly through their own enzymes or indirectly as a consequence of their effects on the population size and activity of the other fibrolytic micro-organisms present, have an important role in determining the level of activity of polysaccharide-degrading enzymes in the rumen ecosystem. Although the extent of ryegrass ( Lolium perenne ) hay digestion was similar after 24 h in the absence or presence of protozoa, the initial ruminal degradation was higher in refaunated sheep.     

A xylanase produced by the rumen anaerobic protozoan Polyplastron multivesiculatum shows close sequence similarity to family 11 xylanases from gram-positive bacteria

We report for the first time the cloning and characterisation of a protozoal enzyme involved in plant cell wall polysaccharide degradation. A cDNA library was constructed from the ruminal protozoan Polyplastron multivesiculatum and a stable clone expressing xylanase activity was isolated. The encoded enzyme belongs to the glycoside hydrolase family 11, and phylogenetic analysis indicates a closer relationship with catalytic domains from Gram-positive bacteria than the other fibrolytic eukaryotes from the rumen, the anaerobic fungi.     

Fiber-degrading systems of different strains of the genus Fibrobacter

The S85 type strain of Fibrobacter succinogenes, a major ruminal fibrolytic species, was isolated 49 years ago from a bovine rumen and has been used since then as a model for extensive studies. To assess the validity of this model, we compared the cellulase- and xylanase-degrading activities of several other F. succinogenes strains originating from different ruminants, including recently isolated strains, and looked for the presence of 10 glycoside hydrolase genes previously identified in S85. The NR9 F. intestinalis type strain, representative of the second species of the genus, was also included in this study. DNA-DNA hybridization and 16S rRNA gene sequencing first classified the strains and provided the phylogenetic positions of isolates of both species. Cellulase and xylanase activity analyses revealed similar activity profiles for all F. succinogenes strains. However, the F(E) strain, phylogenetically close to S85, presented a poor xylanolytic system and weak specific activities. Furthermore, the HM2 strain, genetically distant from the other F. succinogenes isolates, displayed a larger cellulolytic profile on zymograms and higher cellulolytic specific activity. F. intestinalis NR9 presented a higher cellulolytic specific activity and a stronger extracellular xylanolytic activity. Almost all glycoside hydrolase genes studied were found in the F. succinogenes isolates by PCR, except in the HM2 strain, and few of them were detected in F. intestinalis NR9. As expected, the fibrolytic genes of strains of the genus Fibrobacter as well as the cellulase and xylanase activities are better conserved in closely related phylogenetic isolates.     

Cereal supplementation modified the fibrolytic activity but not the structure of the cellulolytic bacterial community associated with rumen solid digesta

4 ruminally cannulated cows were fed a forage diet (93% hay + 7% straw) and a mixed diet (33 % hay + 7% straw + 40% barley) in a 2 x 2 crossover experimental design. In sacco degradation of forage, fibrolytic activities (polysaccharidases and glycosidases) of the solid-associated bacteria (SAB), and distribution of the 3 main cellulolytic bacterial species (Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens) were determined for both diets. Barley supplementation decreased the hay degradation rate and mainly the polysaccharidase activities of the SAB (30% on average). The sum of rRNA of the 3 cellulolytic bacterial species represented on average 17% of the total bacterial signal and R. albus was the dominant cellulolytic bacterial species of the 3 studied. Barley supplementation did not modify the proportion of the 3 cellulolytic bacteria attached to plant particles. The negative effect of barley on the ruminal hay degradation rate is due to a decrease in fibrolytic activity of the SAB, and not to a modification of the balance of the three cellulolytic bacterial species examined.     

The dynamics of major fibrolytic microbes and enzyme activity in the rumen in response to short- and long-term feeding of Sapindus rarak saponins

AIMS: To investigate the short- and long-term effects of an extract of Sapindus rarak saponins (SE) on the rumen fibrolytic enzyme activity and the major fibrolytic micro-organisms. METHODS AND RESULTS: Two feeding trials were conducted. In the short-term trial, four fistulated goats were fed a basal diet containing sugar cane tops and wheat pollard (65:35, w/w) and were supplemented for 7 days with SE at a level of 0.6 g kg(-1) body weight. Rumen liquor was taken before, during and after SE feeding. In the long-term trial, 28 sheep were fed the same basal diet as the goats and were supplemented for 105 days with 0.24, 0.48 and 0.72 g kg(-1) body mass of the extract. Rumen liquor was taken on days 98 and 100. Protozoal numbers were counted under the microscope. Cell wall degradation was determined by enzyme assays and the major fibrolytic micro-organisms were quantified by dot blot hybridization. Sapindus extract significantly depressed rumen xylanase activity in both trials and carboxymethylcellulase activity in the long-term trial (P < 0.01). Fibrobacter sp. were not affected by the SE in both trials, while ruminococci and the anaerobic fungi showed a short-term response to the application of saponins. Protozoal counts were decreased only in the long-term trial with sheep. CONCLUSION: These data suggest that there is an adaptation of Ruminococcus albus, Ruminococcus flavefaciens and Chytridiomycetes (fungi) to saponin when fed over a long period. The fact that no correlation between the cell wall degrading enzyme activities and the cell wall degrading micro-organisms was observed suggests that the organisms tracked in this experiment are not the only key players in ruminal cell wall degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: Sapindus rarak saponins partially defaunate the rumen flora. Their negative effect on cell wall degradation, however, is not related to rumen organisms currently recognized as the major cell wall degrading species. The adaptation of microbes in the long-term feeding experiment suggests that the results from short-term trial on the ruminal microbial community have to be interpreted carefully.