应用16S rRNA基因技术研究仔猪结肠梭菌Ⅳ群变化

【目的】研究从7日龄到35日龄(断奶后两周)仔猪结肠中梭菌Ⅳ群(柔嫩梭菌群,Clostridium Leptumgroup)结构以及数量的变化,探究该菌群与结肠中丁酸浓度的关系。【方法】选取3窝新生仔猪,分别在7、14、21(断奶)、24和35日龄每窝随机屠宰1头,收集结肠内容物,利用气相色谱测定挥发性脂肪酸(Volatilefatty acid,VFA);提取结肠内容物总细菌DNA,利用基于16S rRNA基因的变性梯度凝胶电泳(Denaturinggradient gel electrophoresis,DGGE)和Real-time PCR技术定性、定量分析梭菌Ⅳ群。【结果】对仔猪结肠中梭菌Ⅳ群DGGE图谱进行相似性分析显示,7日龄仔猪样品归于一簇,数值达90%以上,而与其他日龄的样品间相似性较低,断奶前(21日龄)和断奶后3天菌群则没有显著变化。Real-time PCR定量分析显示,仔猪结肠总细菌拷贝数在断奶后3天显著降低,这一趋势与结肠中的总挥发性脂肪酸和丁酸浓度变化相似,而梭菌Ⅳ群拷贝数变化则不显著。克隆和测序分析表明,仔猪结肠梭菌Ⅳ群中的优势菌为Faecalibacteriumprausnitzii,Subdoligranulum variabile以及一些未培养细菌。【结论】7日龄至14日龄仔猪结肠中梭菌Ⅳ群发生改变,但断奶对其影响不大;该菌群和结肠丁酸浓度关系还需进一步研究。     

变性梯度凝胶电泳法研究断奶仔猪粪样细菌区系变化

利用PCR和DGGE技术分析了12头仔猪在断奶后其粪样细菌区系的变化。粪样细菌16S rDNA的V6～V8可变区经PCR扩增,扩增产物经DGGE电泳后再进行相似性分析。结果表明,仔猪断奶当天粪样 DGGE谱带少,同窝仔猪间图谱相似。断奶后,随着断奶时间的推移,每头仔猪的DGGE图谱带逐渐增多,变得复杂和多样,仔猪个体间DGGE图谱差异逐渐增大。仔猪是否同窝以及所采食日粮类型对DGGE图谱没有明显影响。相似性分析还表明,日粮中添加寡果糖的仔猪在断奶后第1周,其粪样微生物区系变化迅速,而后缓慢。     

16S rDNA技术研究新生腹泻仔猪粪样细菌区系的多样性变化

用PCR/DGGE技术跟踪一窝5头新生腹泻仔猪自然康复、补饲、断奶过程中粪样细菌区系的演变,构建3头仔猪42日龄粪样的16S rDNA克隆库,分析匹配于DGGE优势谱带23个克隆的16S rDNA序列。结果表明,DGGE图谱由简单(2日龄)到复杂(10日龄),再回复简单(16日龄)到复杂(断奶),最后趋于稳定。2、16日龄DGGE图谱最简单、相似,最优势谱带为大肠杆菌;10日龄(补饲后3天)图谱复杂,大肠杆菌存在但不是最优势谱带,补饲前后图谱的相似性低,补饲导致了粪样细菌区系结构的显著变化;断奶前(27日龄)和后(35、42日龄)图谱复杂,优势谱带、图谱相似性均趋向稳定。序列分析表明,23个克隆中除5个与未知细菌最相似外,其余最相似菌分属于肠球菌(Enterococcus),链球菌(Streptococcus),梭菌(Clostridium),消化链球菌(Peptostreptococcus)和乳酸杆菌(Lactobacillus)。     

高通量测序和DGGE分析土壤微生物群落的技术评价

【目的】比较新一代高通量测序与传统的变性梯度凝胶电泳(Denaturing Gradient Gel Electrophoresis,DGGE)指纹图谱技术,评价两种技术研究土壤微生物群落结构的优缺点。【方法】针对新西兰典型草地和森林土壤,以16S rRNA基因为标靶,通过高通量测序和DGGE技术分析土壤微生物群落的组成、丰度和多样性,比较两种方法在土壤微生物研究中的适用性。【结果】在不同的微生物分类水平,高通量测序草地土壤检测到22门,54纲,60目,131科,350属;而DGGE仅检测到6门,9纲,8目,10科,10属,表明DGGE显著低估了土壤微生物的群落组成。森林土壤也得到了类似规律,高通量测序的检测灵敏度是DGGE的3.8、6.7、6.4、19.2及39.4倍。进一步分析土壤中主要微生物类群的相对丰度,发现分类水平越低,高通量测序与DGGE的结果差异越大,尤其在科和属的水平上差异最大。以高通量测序结果为标准,DGGE明显高估了土壤中大多数微生物类群的相对丰度,最高可达2000倍。两种方法都表明草地土壤的多样性指数高于森林土壤,但DGGE多样性指数的绝对值远低于高通量测序结果。【结论】高通量测序能够较为全面和准确的反映土壤微生物群落结构,而DGGE仅能够反映有限的优势微生物类群,在很大程度上极可能低估土壤微生物的物种组成并高估其丰度。     

转Bar基因稻谷对小鼠(Mus musculus)肠道菌群的作用研究

选取6周龄、雌雄各半、体重18~22 g的SPF级昆明小鼠(Mus musculus)100只,随机分成5组,每组4个重复,每个重复5只小鼠。分别用含量为40%和60%的转Bar基因稻谷Bar68-1和非转基因稻谷D68日粮喂养小鼠,亲代小鼠饲养180 d后开始繁殖子一代(F1),每代小鼠饲养180 d。在180 d后分别从亲代和子一代每组随机抽取5只小鼠,提取肠道内容物基因组DNA,利用细菌通用引物对细菌16S r DNA的V3区序列进行PCR扩增,并将扩增产物用变性梯度凝胶电泳(denaturing gradient gel electrophoresis DGGE)。研究显示,各组小鼠肠道菌群相似性系数无显著差异(P0.05);肠道主要优势菌群相同。结果表明,转Bar基因稻谷对小鼠肠道菌群的作用不明显。     

应用变性梯度凝胶电泳和16S rDNA序列分析对kefir粒中细菌多样性的研究

以开菲尔(Kefir)粒为材料,经过DNA抽提和16S rDNA V3区PCR扩增,扩增产物经变性梯度凝胶电泳(DGGE)分离并切割电泳条带进行序列测定,并与现有的数据库进行了比较,对Kefir粒的细菌多样性进行分析。结果表明,DGGE图谱中可检测到的8条带的16S rDNA基因序列中有7个基因序列与GenBank数据库登录的相关序列的相似性大于98%,余下的1个基因序列的相似性也大于96%。相似性大于98%的7个克隆中,有3个属于鞘氨醇杆菌属(Sphingobacterium),2个属于乳杆菌属(Lactobacillus),其它2个分别属于肠杆菌属(Enterobacter)和不动杆菌属(Acinetobacter)。首次报道了鞘氨醇杆菌作为优势菌群存在开菲尔Kefir粒中。     

接种微生物菌剂对猪粪堆肥过程中细菌群落多样性的影响

利用PCR-DGGE方法研究了接种外源微生物菌剂对鲜猪粪高温好氧堆肥过程中细菌群落多样性的影响.结果表明：接种外源微生物菌剂可以促进堆肥的顺利进行,比不接种处理的高温期提前2 d.DGGE图谱分析表明,堆肥中优势细菌群落组成发生了明显的更迭现象,不同堆肥时期细菌群落的Shannon-Wiener指数呈显著差异.目的条带克隆测序结果表明,整个堆肥过程Clostridium stercorarium subsp. thermolacticum sp.一直是优势菌属,不经培养细菌、Bacillus coagulans sp.、Clostridium thermocellum sp.在接种外源微生物菌剂处理的第10、16天成为优势菌属,不经培养的Firmicutes sp.和不经培养的 delta proteobacterium分别在未接种外源微生物菌剂处理堆肥发酵的第5天和第16天成为优势菌属.非优势菌属Ureibacillus thermosphaericus、不经培养的Silvimonas sp.出现在堆肥腐熟后期,不经培养的土壤细菌主要出现在堆肥初期和高温初期.UPGMC聚类分析表明,接种外源微生物菌剂明显影响了堆肥不同时期的细菌群落结构组成.堆肥化过程中细菌DGGE图谱主成分分析表明,细菌群落变化主要受外源接种微生物菌剂的影响.     

Sugary vs salty food industry leftovers in postweaning piglets: effects on gut microbiota and intestinal volatile fatty acid production

Awareness of the need to improve the sustainability of livestock by reducing the loss of natural resources has increased significantly. This study investigated the effects of two categories of food industry leftovers, also referred to as former foodstuff products (FFPs), on pig gut microbiota and intestinal volatile fatty acid (VFA) production. Thirty-six female postweaning piglets (28 days old, Large White × Landrace, 6.5 ± 1.1 kg) were separated into three groups and fed a conventional diet (CTR), and diets in which cereals were partially replaced (30% w/w) by sugary confectionery products (FFPs-C) or salty bakery products (FFPs-B), respectively. After 42 days of dietary treatments, faeces were collected from the rectal ampulla, snap-frozen, and used for next-generation sequencing to analyse the composition and the alpha and beta diversity indexes of the microbial population. The concentration of VFAs in the intestinal content collected at the slaughterhouse was also analysed. The study demonstrated that balanced diets can be obtained by the inclusion of both FFPs-C and FFPs-B, with a similar chemical composition compared to traditional diets. Neither the FFPs-C nor FFPs-B diets affected the abundance and biodiversity indexes of the microbial community. Only a few taxa, normally attributed to a healthy gut, increased with FFPs-C and FFPs-B compared to the CTR. The experimental diets had no impact on the production of the VFAs in the faeces. Lastly, the inclusion at 30% (w/w) of both categories of FFP diets slightly affected the faecal microbiota. FFPs could thus be used as a promising alternative to traditional ingredients in pig diets; however, additional analyses are needed to further investigate the presence of potentially pathogenic bacteria. The effects of such ingredients on other markers of gut health, and on product quality when used in the fattening period also need to be investigated.     

Comparison of fecal microbiota and polyamine concentration in adult patients with intractable atopic dermatitis and healthy adults

Fecal microbiota and polyamine concentration obtained from eleven intractable adult-type atopic dermatitis (AD) patients and thirteen healthy adults were compared. Fecal microbiota were analyzed using terminal-restriction fragment length polymorphism. The fecal microbiota of volunteers were divided into two clusters, A (n=16) and B (n=8), and the number of AD patients was found to be higher in Cluster B than Cluster A, suggesting that there are relationships between the obstinacy of intractable adult-type AD and intestinal microbiota in Cluster B. Fecal spermidine concentration in Cluster B were lower than that in Cluster A significantly (P<0.05). Fecal putrescine concentration in Cluster B also tended to be lower than that in Cluster A. Terminal-restriction fragment (T-RF) of 122 bp generated by digestion with Hha I, which were predicted as unknown bacteria, were detected characteristically in Cluster A. In contrast, T-RFs of 368/9 bp generated by digestion with Hha I, which were predicted as Enterobacteriaceae, were detected characteristically in Cluster B. These bacteria are closely associated with intestinal polyamine concentration. These findings raise the possibility that a low concentration of intestinal polyamines produced by intestinal microbiota is one of the important factors in the onset of intractable adult-type AD.     

Effects of live yeast on the performance, nutrient digestibility, gastrointestinal microbiota and concentration of volatile fatty acids in weanling pigs

Two experiments were conducted to evaluate the effects of live yeast supplementation on performance, nutrient digestibility, enteric microbial populations and volatile fatty acid (VFA) concentration of weanling pigs, receiving diets supplemented with aureomycin and elevated doses of CuSO4. In experiment 1, 90 crossbred pigs (7.20 +/- 0.44 kg, 28 d of age) were randomly allotted to one of five dietary treatments containing either 0, 4.0 x 10(6), 9.0 x 10(6), 2.6 x 10(7), or 5.1 x 10(7) cfu Saccharomyces cerevisiae per gram with six replicate pens per treatment and three pigs per pen. BWG and feed intake increased quadratically during days 1-14 and days 1-28 as live yeast levels increased (p < 0.01). Pigs fed the diet containing 2.6 x 10(7) cfu yeast per gram had the highest BWG and feed intake among the treatments. In experiment 2, 48 crossbred pigs (7.64 +/- 0.72 kg, 28 d of age) were fed diets containing live yeast at 0 or 3.2 x 10(7) cfu of S. cerevisiae per g with six replicate pens per treatment and four pigs per pen. The yeast supplementation improved BWG and feed intake during days 1-14 (p < 0.01) and days 1-28 (p < 0.05). Treatment differences were not observed in any of the bacterial populations, yeast numbers or VFA concentrations, at any of the sites of the gastrointestinal tract tested. Total tract nutrient digestibility was also not different between treatments. Overall, dietary supplementation of live yeast had a positive effect on BWG and feed intake of weanling pigs, receiving diets supplemented with aureomycin and elevated doses of CuSO4. The improvement in BWG appears to be partly related to an increase in feed intake. The mechanism of yeast improving feed intake of piglets needs to be explored.     

Effect of inulin supplementation on selected gastric, duodenal, and caecal microbiota and short chain fatty acid pattern in growing piglets

We explored whether bifidobacteria and lactobacilli numbers and other selected bacteria in the upper intestine and the caecum of growing pigs were affected by diet and intake of inulin. Starting at two weeks after weaning (28 d) 72 pigs were fed two types of diets (wheat/barley (WB) or maize/gluten (MG)), without or with 3% inulin (WB + I, MG + I) for three and six weeks. Intestinal bacteria were quantified by fluorescence-in-situ-hybridization (n = 8/group). Duration of feeding had no effect on the variables tested, so data for both periods were pooled. Gastric total bacteria amounted to log(10) 7.4/g digesta. Bifidobacteria were detected in stomach and duodenum two weeks after weaning and disappeared thereafter. In jejunum and caecum bifidobacteria were present at a level of log(10) 7.0/g digesta. Inulin did not alter numbers of lactobacilli, bifidobacteria, enterococci, enterobacteria and bacteria of the Clostridium coccoides/Eubacterium rectale-group. Inulin disappearance in stomach plus jejunum was higher with the MG diet (73.7 vs. 60.7%, p = 0.013). Caecal acetate was lower in inulin-supplemented diets (p < 0.05) whereas propionate and butyrate were higher in pigs fed the WB diets (p < 0.05). With the WB diet total caecal short chain fatty acids concentration was higher which resulted in a lower pH value (p < 0.05).     

Biomass-derived volatile fatty acid platform for fuels and chemicals

The typical biorefinery platforms are sugar, thermochemical (syngas), carbon-rich chains, and biogas platforms, each offering unique advantages and disadvantages. The sugar platform uses hexose and pentose sugars extracted or converted from plant body mass. The thermochemical (syngas) platform entails a chemical or biological conversion process using pyrolysis or gasification of plants to produce biofuels. The carbon-rich chains platform is used to produce biodiesel from long-chain fatty acids or glycerides. In the present work, we suggest a new platform using volatile fatty acids (VFAs) for the production of biofuels and biochemicals production. The VFAs are short-chain fatty acids composed mainly of acetate and butyrate in the anaerobic digestion (AD) process, which does not need sterilization, additional hydrolysis enzymes (cellulose or xylanase), or a high cost pretreatment step. VFAs are easily produced from almost all kinds of biomass with low lignin content (terrestrial, aquatic, and marine biomass) by the AD process. Considering that raw material alone constitutes 40∼80% of biofuel production costs, biofuels made from VFAs derived from waste organic biomass potentially offer significant economical advantage.     

Relations between fatty acid synthesis,pyruvate concentration and cell concentration of suspensions of isolated rat hepatocytes

• 1.1. The cell concentration of suspensions of isolated rat hepatocytes affects both the rate of pyruvate accumulation in the incubation medium and the rate of fatty acid synthesis.
• 2.2. At low cell concentrations pyruvate accumulation is directly related to the cell concentration but levels off at higher concentrations even when maximum pyruvate concentrations in the medium are not yet reached.
• 3.3. The rate of fatty acid synthesis in the 30–60-min incubation interval is proportional to the cell concentration. In contrast, the rate of fatty acid synthesis during the 0–30-min incubation period decreases with increasing cell concentrations and subsequently becomes independent of the cell concentration.

     

Differences between the fecal microbiota of coeliac infants and healthy controls

Coeliac disease (CD) is an immune-mediated enteropathy with a multifactorial aetiology, characterized by chronic inflammation of the small intestinal mucosa. Although evidence suggests that the gut microbiota contributes to other chronic inflammatory disorders, its possible role in CD has not been determined. In this study, the composition of the fecal microbiota of coeliac children and age-matched controls was investigated by culture-dependent and -independent methodologies, using fluorescent in situ hybridization (FISH). The levels of Bacteroides, Clostridium and Staphylococcus were significantly higher (p < 0.05) in fecal samples from coeliac patients than in healthy subjects when analysed by culture methods. The numbers of Bacteroides-Prevotella, Clostridium histolyticum, Eubacterium rectale-C. coccoides, Atopobium, and sulfate reducing bacterial groups were also significantly higher (p < 0.05) in fecal samples from coeliac infants when analysed by FISH. The counts of Bifidobacterium tended to be higher in healthy controls by the two type of analysis but the differences were not significant. This is the first report on the identification of the specific bacterial groups responsible for alterations in the intestinal microecology of children with active CD. The bacterial pattern detected in coeliac patients, correlates with the epidemiological data and metabolic deviations associated with CD, and involve bacterial groups link to other chronic inflammatory disorders.     

肠道菌群与胆汁酸代谢的互相作用

肠道菌群是胃肠道的多种共生细菌和其他微生物的统称,是一个复杂而动态的微生物生态系统,有数十万亿个微生物.胆汁酸是胆汁的主要成分,由肝脏中的胆固醇合成并释放到肠道中以帮助消化吸收膳食脂肪.肠道菌群在胆汁酸代谢中发挥着重要作用,它借助胆盐水解酶和类固醇脱氢酶等通过脱氢、脱羟基和脱硫等作用改变胆汁酸池的组成;随后通过影响胆汁...     

Artifacts in ultracentrifugal estimation of aqueous fatty acid concentration

Ultracentrifugation for determination of isotropic concentrations of fatty acids is widely used. However, several artifacts, which would affect the isotropic concentration, could occur if care is not taken. These include sedimentation of micelles, incomplete flotation of unsolubilized oil, and uptake of labeled fatty acid by the walls of centrifuge tubes. The first artifact can be overcome by sampling large volumes from the ultracentrifuged sample, and the second by ultracentrifugation for long periods; a force-duration of 7.2 X 10(7) g-min is suitable. The third artifact cannot be eliminated but may be made constant if the duration of exposure of lipid mixtures to centrifuge tubes is kept constant.