中草药添加剂的抗小鼠腹泻效果及其对小鼠肠道微生物菌群的影响

为研究中草药提取液的抗小鼠腹泻效果及其对小鼠肠道菌群的影响,本实验选用50只体重为18～22 g的清洁级ICR(Institute of Cancer Researceh,USA)小鼠(Mus musculus),随机分为5个处理组:空白对照组、复方提取液组、黄柏提取液组、白头翁提取液组和白头翁:黄柏(1:1)提取液组.每天按照4 g生药/kg体重剂量定时灌胃,空白对照组给予等剂量的蒸馏水.第10天每只小鼠按0.02 mL/g体重剂量腹腔注射108 cfu/mL大肠杆菌(Escherichia coli)菌液,建立小鼠细菌性致腹泻病理模型,观察各提取液对小鼠的腹泻的影响,第13天收集粪样,变性梯度凝胶电泳(DGGE)研究肠道微生物区系的变化.结果显示,造模当天对照组的体重变化最为明显,腹泻最为严重,与其他各给药组相比均差异显著(P＜0.05);4个中草药提取液添加组小鼠的腹泻次数比对照组分别降低了51％、26％、33％和35％,其中复方组抑制小鼠的腹泻效果最为显著(P＜0.05);小鼠粪便微生物DGGE图谱分析显示,复方组的条带数和多样性指数均显著高于其他处理组(P＜0.05).研究结果表明,中草药添加剂能够显著地抑制小鼠腹泻,有效地改善肠道微生物菌群结构,其中复方组的效果最优.     

螺旋藻对抗生素相关性腹泻小鼠大便中菌群的影响

通过对腹泻小鼠灌食螺旋藻,对比给药组、模型组和正常组小鼠大便中菌种的变化,探索螺旋藻对抗生素相关腹泻小鼠肠道菌群的影响。结果表明:抗生素相关性腹泻导致小鼠的大便菌群异常,异常菌群有一定的自我恢复能力,生理盐水对其无明显治疗作用,螺旋藻灌胃可以明显抑制大便菌群的异常增殖,并促进其中双歧杆菌和乳杆菌菌群数量回升,对菌群异常的恢复有显著效果。     

秋葵微粉对小鼠肠道内环境的影响

为研究不同剂量秋葵微粉对小鼠肠道内环境的影响,将小鼠随机分为4组,即对照组、高剂量组(1 800 mg·kg^-1·d^-1)、中剂量组(900 mg·kg^-1d^-1)以及低剂量组(450 mg·kg^-1d^-1),灌胃6周后,检测其粪便、结肠、盲肠相关的肠道指标。结果表明,与对照组相比,各剂量组小鼠粪便含水量均增加,且高剂量组粪便含水量显著高于对照组(P<0.05);各剂量组粪便pH值均极显著降低(P<0.01);各剂量组盲肠内容物 pH 值和结肠内容物pH值均降低。各剂量组盲肠内容物菌群OTU数量均极显著高于对照组(P<0.01),中剂量组Chaol值显著高于对照组(P<0.05);各剂量组结肠内容物菌群的OTU数量较对照组均有增加,中剂量组和低剂量组Chaol值极显著(P<0.01)和显著(P<0.05)高于对照组。各剂量组盲肠、结肠内容物中短链胎肪酸(SCFA)含量均较对照组增加。秋葵微粉可以提高小鼠肠道中的益生菌比例,降低有害菌比例,促进肠道乙酸、丙酸和丁酸等SCFA含量明显升高。本研究结果为秋葵微粉用于改善动物肠道健康提供了理论依据。     

番石榴结合态多酚对小鼠肠道菌群结构及多样性的影响研究

为探究番石榴结合态多酚对小鼠肠道菌群的影响,本试验将CD-1小鼠分为空白组、高浓度组(500 mg·kg^-1)和低浓度组(100 mg·kg^-1)3组,在灌胃5周后,收集小鼠的无菌粪便,扩增肠道菌群16S rRNA基因高变域V3-V4,通过16S rRNA基因扩增子测序技术检测小鼠肠道菌群的α多样性、β多样性,以及门、属水平的物种组成。结果表明,与空白组相比,低浓度组中菌群的均匀性和丰富度显著提高(P<0.05);基于门水平分析,厚壁菌门、拟杆菌门和疣微菌门3个菌门的相对丰度值较大;在属水平中,高低浓度组均能降低拟杆菌属相对丰度,低浓度组能提高乳杆菌相对丰度,并且高浓度组中艾克曼菌属的相对丰度大幅提高。综上所述,番石榴结合态多酚能提高小鼠肠道菌群的均匀性和丰富度,且对菌群结构具有调节作用,既能提高益生菌的相对丰度,也能降低有害菌的相对丰度。本研究结果可为番石榴结合态多酚的活性研究和实际应用提供理论参考。     

双Bt基因提高青花菜对菜粉蝶和小菜蛾抗性

以青花菜（Brassica oleracea L.ssp.italica）下胚轴为外植体,通过农杆菌介导的遗传转化方法将苏云金芽胞杆菌（Bacillus thuringiensis）晶体毒蛋白基因Cry1Ac和Cry1C分别导入青花菜栽培品种绿彗星中,各获得35株卡那霉素抗性植株。分别以Cry1Ac和Cry1C特异引物对转基因植株进行PCR扩增,结果得到27株Cry1Ac阳性植株和13株Cry1C阳性植株。以目的基因Cry1Ac和Cry1C为探针,进行Southernblot分析,证明多数植株目的基因已经整合到青花菜基因组中。对含有单拷贝目的基因植株自交纯合后通过有性杂交获得带有Cry1Ac和Cry1C双价的转基因青花菜。利用RT-PCR检测Bt基因在转基因植株中的表达情况,发现不同株系间在RNA水平上存在显著的表达差异,部分株系中双价Bt同时得到表达。ELISA检测转基因植株Bt毒蛋白含量为0.12～0.82μg/gFW。结合室内和田间饲虫试验,证明抗虫效果与Bt毒蛋白表达水平正相关。通过多世代选择,获得高抗菜粉蝶（Pieris rapae L.）和小菜蛾（Plutella xylostella L.）的纯合转基因青花菜材料。双价抗虫基因共同作用,提高了青花菜的抗虫性。     

苏云金芽胞杆菌杀虫晶体蛋白Cry7Ba1溶解性改良

苏云金芽胞杆菌杀虫晶体蛋白Cry7Ba1需要在强碱性缓冲液中(pH12.5)才能溶解,而且只有溶解后才能表现出毒力。分别用Cry1Ac和Cry1C晶体蛋白的C-半段替换Cry7Ba1的C-半段,结果发现当Cry7Ba1的C-半段被替换后,重组晶体蛋白与Cry1Ac和Cry1C等其它晶体蛋白一样在弱碱性条件下(pH9.5)能有效溶解,而且其杀虫活性没有发生明显变化。本研究结果表明Cry7Ba1晶体蛋白难溶的特性是由其C-半段结构决定的,通过改变其C-半段结构改善溶解性,为该晶体蛋白的应用提供了可能。     

不同脆化阶段草鱼肠道菌群动态变化、血清酶指标及生长性能

本研究旨在探讨草鱼(Ctenopharyngodon idellus)不同脆化阶段的肠道菌群组成结构、血清酶活性及生长性能,进而衡量脆肉鲩形成过程中的生理生化变化,以期为脆肉鲩的健康养殖提供科学数据.脆化不同阶段(30、60、90和120 d)的草鱼,在脆化结束后转投喂人工配合饲料30 d(第150天),鉴定整个过程中草鱼肠道菌群组成、血清酶活性和生长性能的变化.结果表明,60～150 d蚕豆组草鱼体重均低于对照组(P＜0.05,P＜0.01),90～120 d蚕豆组草鱼头肾体指数(head kidney somatic index,HKBI)和肝体指数(hepatosomatic index,HSI)均低于对照组(P＜0.05或P＜0.01),150 d时蚕豆组草鱼肝体指数与对照组无显著差异(P＞0.05);蚕豆组草鱼血清碱性磷酸酶、溶菌酶和补体C3(第30天除外)活性均低于对照组的,在转投喂饲料后,3种血清酶活性均出现回升趋势;与对照组相比,蚕豆组草鱼肠道菌群多样性降低,芽胞杆菌属(Bacillus)和鲸杆菌属(Cetobacterium)等有益菌数量减少,维氏气单胞菌(Aeromonas veronii)、温和气单胞菌(Aeromonas sobria)和鳗弧菌(Vibrio anguillarum)等条件致病菌数量增多.两组草鱼肠道核心菌群主要为变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)和梭杆菌门(Fusobacteria).本研究结果提示,随着脆化时间的增加,摄食蚕豆改变草鱼肠道菌群组成、抑制血清酶活性和生长性能,在转投饲料后其肠道菌群、血清酶活性及生长性能均有所改善.本研究结果对脆肉鲩的健康养殖具有重要的科学指导意义.     

梯度有机负荷下农业废弃物厌氧发酵特性及微生物群落

为了解梯度负荷对不同C/N农业废弃物厌氧发酵特性及微生物群落的影响,该试验以猪粪、金针菇菌包、稻秆和甘蔗叶等C/N差异较大的废弃物作为原料,通过逐渐提高全混合厌氧反应器(continuous stirred tank reactor,CSTR)的有机负荷率(organic loading rate,OLR),研究物料在4个OLR(1.11、1.67、2.22和2.78g/(L·d),以可挥发性固体计)下的产甲烷特性和微生物群落结构变化.结果表明:4种物料的日甲烷产量均随OLR递增而增加,但单位物料甲烷产率与微生物菌群结构则因物料C/N差异而表现出不同的变化趋势.其中:猪粪的日甲烷产量最高,细菌和古菌的菌群结构与对照组相比变化不大;多样性指数先增后减,甲烷产率也在OLR达到1.67g/(L·d)后逐渐下降.金针菇菌包甲烷产率相对稳定,细菌和古菌的多样性指数随OLR递增而增长.稻秆和甘蔗叶同属于碳质量分数高的秸秆类物料,二者的细菌和古菌的菌群结构变化明显;但在OLR达到2.78g/(L·d)时,稻秆受系统酸化(VFA/TIC>0.8)影响,甲烷产率下降明显,细菌和古菌的多样性指数也出现下降;而甘蔗叶则因VFA/TIC<0.8,其甲烷产率在发酵过程中未出现明显下降.此外,不同C/N物料对优势菌的形成存在影响.其中:Methanolobus zinderi为猪粪物料特有的优势古菌;Proteiniphilum acetatigenes,Acetivibrio cellulolyticus为金针菇菌包、甘蔗叶特有的优势细菌;Methanospirillum hungatei为稻秆物料独有的优势古菌.     

主养草鱼池塘三种混养模式下鱼类肠道菌群PCR-DGGE比较

消化道微生物在宿主生长、营养和健康等方面起到重要的作用.本实验采用基于PCR扩增的变性梯度凝胶电泳(PCR-DGGE)技术比较研究了主养草鱼(Ctenopharyngodon idellus)池塘三种不同混养模式下的鱼类肠道菌群差异.结果表明,三种混养模式下同种鱼的生长率出现显著性差异(P＜0.05),而肠道细菌16SrDNA V3区特征片段PCR-DGGE指纹分析显示草鱼的肠道菌群相似性较高(＞42.2％);投喂配合饲料的草鱼与摄食浮游生物的鲢(Hypophthalmichthys molitrix)、鳙(Aristichthys nobilis和匙吻鲟(Polyodon spathula)肠道菌群结构相差最大(＜19％),鲢和鳙肠道菌群相似性较高(＞41.6％),除模式Ⅱ鳙的肠道和匙吻鲟的胃菌群具有较高的相似性(＞50.3％)外,匙吻鲟的消化道菌群和鲢鳙的相似性低.实验共回收测序了14条特定DGGE条带中的DNA片段,并进行系统进化分析,结果显示,这14条条带分别归属于4个细菌类群:变形细菌门(Proteobacteria),拟杆菌门(Bacteroidetes),厚壁菌门(Firmicutes)和梭杆菌门(Fusobacteria).研究结果为鱼类混养模式的优化,饲料研发和鱼病防治提供了基础参考资料.     

转Bt基因棉花根际细菌与古菌群落结构分析

在一年内棉花的四个生长时期（苗期,蕾期,花铃期,吐絮期）分别采集转Bt基因抗虫棉GK12和非转基因亲本棉花泗棉3号根际土壤,以及未种植棉花的背景土壤,利用末端标记限制性片段长度多态性（T-RFLP）分析技术,分析三种土壤中细菌和古菌的16S rRNA基因片段多态性,结合克隆文库建立和测序,研究了土壤中细菌和古菌群落结构的变化.结果表明：在棉花生长的各个时期,背景土壤中细菌群落结构发生了明显的变化,生物多样性指数明显降低,古菌群落结构也有一定的变化,说明季节性变化对土壤中微生物群落产生了明显的影响.与背景土壤相比,棉花种植后根际土壤中细菌和古菌群落发生显著的变化.转基因棉花与非转基因棉花相比,根际土壤细菌和古菌的种类和种群大小的分布也发生了明显的改变.克隆文库和测序结果表明土壤中主体微生物为目前未培养的、功能特性未知的细菌和古菌,转基因棉花种植对这些细菌和古菌影响的原因、环境危害和生态风险目前尚不清楚.与古菌群落相比,棉花种植对细菌群落结构的影响较小.     

Effect of Lactobacillus pentosus ONRIC b0240 on intestinal IgA production in mice fed differing levels of protein

Protein-energy malnutrition (PEM) is frequently associated with the occurrence of infection due to a decline in immune function. Here, an experiment was conducted with the objective of enhancing mucosal immunity by administration of Lactobacillus pentosus ONRIC b0240 (b240) in PEM model mice. Three groups of male C3H/HeN mice aged approximately 12 weeks were caged in groups of five or six and received various treatments. The mice were fed 4 (low-protein diet; PEM model), 20 (standard-protein diet), or 40% (high-protein diet) ovalbumin (OVA) with or without 0.05% b240. Five weeks later, all mice were sacrificed, and the organs were extracted for analysis of the immune response. Acute toxicity was not observed in this study. The addition of b240 showed no influence on body weight; however, body weight decreased with increasing protein level. Interestingly, intestinal total IgA was significantly increased (p<0.05) in all test diets with b240. The in vitro study showed that the number of B cells and type 2 helper T (Th2) cells were significantly increased in mouse spleen cells with b240 treatment, whereas no differences were found in the number of Th1 cells. b240 also has the ability to augment IgA and IgG production in mouse Peyer's patch cells. These results suggest that b240 enhances IgA production and helps recover the intestinal immune system in PEM model mice via augmentation of humoral immunity.     

Liming induces growth of a diverse flora of ammonia-oxidising bacteria in acid spruce forest soil as determined by SSCP and DGGE

The autotrophic ammonia-oxidising bacterial (AOB) community composition was studied in acid coniferous forest soil profiles at a site in southwestern Sweden 6 years after liming. Liming caused a significant increase in pH in the organic horizons, while the mineral soil was unaffected. The AOB communities were studied by single-strand conformation polymorphism (SSCP) in parallel with denaturing gradient gel electrophoresis (DGGE) analysis of partial 16S rRNA genes amplified by PCR using primers reported to be specific for β-Proteobacteria AOB, followed by nucleotide sequencing. High genetic diversity of Nitrosospira-like sequences was found in the limed soil profiles, whereas no AOB-like sequences were detected in the control soil at any depth, according to both the SSCP and DGGE analyses. This clearly showed that liming induced growth of a diverse flora of AOB at this forest site. Both Nitrosospira cluster 2 and cluster 4 sequences were present in the limed soil profiles, regardless of soil pH, but we found a higher number of sequences affiliated with cluster 4. The high lime dose seemed to affect the AOB community more than the low dose, and its effects reached deeper into the soil profile. Seven different Nitrosospira-like sequences were found 10 cm under the litter layer in the soil limed with the high dose, but only two in the soil amended with the low lime dose.     

植物乳杆菌对苏尼特羊肠道菌群、血浆代谢物及肉品质的影响

为了研究植物乳杆菌对苏尼特羊肠道菌群、血浆代谢物和肉品质的影响及其作用机理,选取三月龄苏尼特羊为试验对象,随机分为2组:对照组(C组,基础日粮)和植物乳杆菌组(R组,基础日粮、活菌数为3×1010cfu/g植物乳杆菌),进行为期90 d的饲喂试验.屠宰后取其肠道内容物及背最长肌,利用高通量测序技术、代谢组学液相-色谱联...     

PCR-DGGE技术用于猪场沼气池细菌群落分析的条件优化

本研究采用变性梯度凝胶电泳（PCR-DGGE）技术研究猪场沼气池细菌群落,对基于16S rDNAV3区的PCR-DGGE电泳的最佳变性剂梯度范围、电泳时间、染色时间进行优化。研究结果表明,最佳变性剂梯度范围为35%～60%,电泳时间为12h,SYBR Green Fluorescent Dye染料的染色时间是30min,优化后的PCR-DGGE确保了实验的准确性、灵敏度和可重复性。运用此优化后的PCR-DGGE技术对5个猪场沼液细菌群落进行了研究,获得了较丰富的多样性。该实验为深入研究畜禽养殖粪污治理的菌种筛选奠定了基础。     

Bacterial communities in manganese nodules in rice field subsoils: Estimation using PCR-DGGE and sequencing analyses

Abstract

The phylogenetic positions of bacterial communities in manganese (Mn) nodules from subsoils of two Japanese rice fields were estimated using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis followed by sequencing of 16S rDNA. The DGGE band patterns and sequencing analysis of characteristic DGGE bands revealed that the bacterial communities in Mn nodules were markedly different from those in the plow layer and subsoils. Three out of four common bands found in Mn nodules from two sites corresponded to Deltaproteobacteria and were characterized as sulfate-reducing and iron-reducing bacteria. The other DGGE bands of Mn nodules corresponded to sulfate and iron reducers (Deltaproteobacteria), methane-oxidizing bacteria (Gamma and Alphaproteobacteria), nitrite-oxidizing bacteria (Nitrospirae) and Actinobacteria. In addition, some DGGE bands of Mn nodules showed no clear affiliation to any known bacteria. The present study indicates that members involved in the reduction of Mn nodules dominate the bacterial communities in Mn nodules in rice field subsoils.     

DGGE fingerprinting of culturable soil bacterial communities complements culture-independent analyses

Culture-dependent DGGE (CD DGGE) fingerprinting of the 16S rRNA gene was used to characterize mixed bacterial communities recovered on agar plates. Using R2A Agar as a growth medium, CD DGGE analysis resulted in clear banding patterns of sufficient complexity (16-32 major bands) and reproducibility to investigate differences in bacterial communities in a silt loam soil. Replicate CD DGGE profiles from plates inoculated with less-dilute samples (10⁻³) had a higher band count and were more similar (72-77%) than profiles from more-dilute samples (51-61%). Different culture media and incubation conditions resulted in distinct community fingerprints and increased the cumulative number of unique bands detected. When CD DGGE fingerprints were compared to profiles constructed from 16S rRNA genes obtained from culture-independent clone libraries (CB DGGE profiles) 34% of the bands were unique to the culture-dependent profiles, 32% were unique to the culture-independent profiles and 34% were found in both communities. These data demonstrate that culture-independent DGGE profiles are supplemented by the distinct bands detected in culture-dependent profiles. CD DGGE can be a useful technique to follow the dynamics of distinct culturable fractions of the soil bacterial community.     

Iberian pig as a model to clarify obscure points in the bioavailability and metabolism of ellagitannins in humans

Ellagitannin-containing foods (strawberries, walnuts, pomegranate, raspberries, oak-aged wine, etc.) have attracted attention due to their cancer chemopreventive, cardioprotective, and antioxidant effects. Ellagitannins (ETs) are not absorbed as such but are metabolized by the intestinal flora to yield urolithins (hydroxydibenzopyran-6-one derivatives). In this study, Iberian pig is used as a model to clarify human ET metabolism. Pigs were fed either cereal fodder or acorns, a rich source of ETs. Plasma, urine, bile, lumen and intestinal tissues (jejunum and colon), feces, liver, kidney, heart, brain, lung, muscle, and subcutaneous fat tissue were analyzed. The results demonstrate that acorn ETs release ellagic acid (EA) in the jejunum, then the intestinal flora metabolizes EA sequentially to yield tetrahydroxy- (urolithin D), trihydroxy- (urolithin C), dihydroxy- (urolithin A), and monohydroxy- (urolithin B) dibenzopyran-6-one metabolites, which were absorbed preferentially when their lipophilicity increased. Thirty-one ET-derived metabolites were detected, including 25 urolithin and 6 EA derivatives. Twenty-six extensively conjugated metabolites were detected in bile, glucuronides and methyl glucuronides of EA and particularly urolithin A, C, and D derivatives, confirming a very active enterohepatic circulation. Urolithins A and B as well as dimethyl-EA-glucuronide were detected in peripheral plasma. The presence of EA metabolites in bile and in urine and its absence in intestinal tissues suggested its absorption in the stomach. Urolithin A was the only metabolite detected in feces and together with its glucuronide was the most abundant metabolite in urine. No metabolites accumulated in any organ analyzed. The whole metabolism of ETs is shown for the first time, confirming previous studies in humans and explaining the long persistency of urolithin metabolites in the body mediated by an active enterohepatic circulation.     

芦笋对高脂饮食小鼠肠道内容物细菌多样性的影响

为探讨芦笋对高脂饮食小鼠肠道内容物细菌多样性的影响,设计动物试验,试验分成4组,其中,正常对照组饲喂基础饲料,高脂饮食组、高脂饮食芦笋组、高脂饮食降脂理肝汤组均喂食高脂饲料;每只小鼠每天灌胃2次,每次0.35 mL,正常组与高脂饮食组为蒸馏水,高脂饮食芦笋组为2.10 g·kg^-1芦笋液,高脂饮食降脂理肝汤组为1.19 g·kg^-1降脂理肝汤。试验结束后提取肠道内容物微生物总DNA,进行16S rDNA V4区基因组测序。结果表明,从操作分类单元(OTU)总数来看,正常对照组OTU总数有 1 264 种,高脂饮食组OUT总数为1 564 种,高脂饮食降脂理肝汤组和高脂饮食芦笋组OUT总数分别为 1 354 和1 654 种,表明芦笋液抑制、降脂理肝汤能促进肠道细菌OUT总数恢复至正常水平。样本群落(Alpha)多样性分析表明,高脂饮食芦笋组、高脂饮食降脂理肝汤组和正常对照组丰富度估计指数(Chao)、多样性指数(Shannon)、丰富度估计指数(ACE)及多样性指数(Simpson)均低于高脂饮食组,且高脂饮食芦笋组Shannon指数显著低于高脂饮食组。细菌门分类分析表明,高脂饮食芦笋组变形菌门(Proteobacteria)、放线菌门(Actinobacteria)相对丰度介于高脂饮食组与正常对照组之间,高脂饮食降脂理肝汤组厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)相对丰度介于高脂饮食组与正常对照组之间,蓝细菌(Cyanobacteria)相对丰度在各组间有差别,但差异不显著。本研究结果表明,芦笋通过改变肠道菌群结构达到了降脂的效果,为芦笋产品的进一步开发研究提供了理论依据。     

Community structure of methanogenic archaea in paddy field soil under double cropping (rice-wheat)

Community structure of methanogenic archaea in paddy field soil under double cropping (rice [Oryza sativa L.] and wheat [Triticum aestivum L.]) was studied by the denaturing gradient gel electrophoresis (DGGE) method. Soil samples under flooded and upland conditions were collected 7 and 6 times, respectively, from two paddy fields throughout a year, and two primer sets, 0357F-GC/0691R and newly designed 1106F-GC/1378R, were used for DGGE analysis. The 25 and 29 different bands were observed on the DGGE gels with the primers 0357F-GC/0691R and 1106F-GC/1378R, respectively. DGGE band patterns of the methanogenic archaeal community were stable throughout a year including the cultivation periods of rice under flooded conditions and of wheat under upland conditions. Cluster analysis and principal component analysis suggested that the difference in the soil type (sampling region) largely influenced the community structures of methanogenic archaea in paddy field soil, while the effects of sampling period and different fertilizer treatments on them were small. Most of the sequences obtained from the DGGE bands were closely related to Methanomicrobiales, Methanosarcinaceae, Methanosaetaceae and Rice cluster-I.