双歧杆菌RH 菌株不同处理物修复肠道菌群平衡失调的研究

为探讨双歧杆菌RH 菌株对肠道菌群平衡失调的修复功能,本研究通过动物实验,考察双歧杆菌活菌(LB)、菌体破碎物(CE)及发酵上清液(FS)3 种处理物灌胃由抗生素造成的菌群失调模型小鼠后对其肠道内双歧杆菌、乳杆菌、产气荚膜梭菌、肠球菌、肠杆菌和拟杆菌6 大类主要菌群的影响。结果显示：3 种处理物均可使小鼠肠道内双歧杆菌和乳杆菌数量极显著增加(P ＜ 0.01),产气荚膜梭菌和肠球菌数量显著降低(P ＜ 0.05 和P ＜ 0.01),并能使肠杆菌和拟杆菌数量恢复至正常水平,说明该双歧杆菌LB、CE 和FS 均可起到修复、调节小鼠肠道微生态平衡的作用。     

大豆低聚糖对肠道菌群的调节作用

通过动物实验及人体实验研究大豆低聚糖对肠道菌群的影响.实验小鼠灌胃不同剂量的大豆低聚糖,观察其肠道内肠杆菌、肠球菌、产气荚膜梭菌、拟杆菌、乳酸杆菌、双歧杆菌的变化.受试人群每日服用10mL大豆低聚糖,同样观察以上菌群变化 . 结果表明,大豆低聚糖具有调节肠道菌群的作用.     

苦荞粉对小鼠肠道菌群的影响

为了探索苦荞粉对小鼠肠道菌群的影响,给予小鼠不同剂量的苦荞粉,35d后考察其对小鼠结肠中大肠杆菌、乳酸杆菌以及双歧杆菌的影响;并取小鼠空肠组织,研究苦荞粉对小鼠肠道组织形态的影响。结果表明,与对照组相比,苦荞粉的灌胃剂量大于3.250g/(kg.d)时,小鼠肠道中乳酸杆菌和双歧杆菌数量均显著增加,同时大肠杆菌的数量显著下降(P<0.05)。灌胃苦荞粉改变了小鼠空肠组织结构形态。研究证实苦荞粉可以作为益生元。     

益生菌对抗生素性肠道菌群失衡的调节作用

以小鼠粪便中的双歧杆菌、肠球菌、肠杆菌、厌氧总菌数量变化为检测指标,采用随机饮用盐酸克林霉素水溶液(1.2 g/L)6 d的方法造成抗生素性肠道菌群失衡小鼠模型.之后,灌胃自筛嗜酸乳杆菌和植物乳杆菌的混合液,以生理盐水为对照,评价了混合菌液对小鼠模型的影响.结果表明,与对照组相比,混合菌液可迅速恢复肠道菌群平衡,停止灌胃/3～7 d内,仍可发挥其调节作用,产生作用的效应剂量是107mL-1.     

植物乳杆菌ST-Ⅲ对小鼠肠道菌群的调节

以小鼠粪便中的双歧杆菌、乳杆菌、肠杆菌、肠球菌、产气荚膜梭菌数量为检测指标,比较了灌胃含有植物乳杆菌ST-Ⅲ活菌量6×107,6×108,6×109mL-1的脱脂奶液对小鼠肠道菌群的影响。结果表明,植物乳杆菌ST-Ⅲ对小鼠肠道菌群具有一定的调节效果,有效剂量为6×108mL-1。灌胃108mL-1活菌15d,停止灌胃3～5d后仍具有显著效果,5～7d后各项指标菌的量回复到灌胃前水平。     

乳双歧杆菌V9对头孢曲松钠作用小鼠肠道菌群的变化

该研究主要探讨了乳双歧杆菌V9对头孢曲松钠作用的小鼠肠道菌群的变化。头孢曲松钠连续灌胃5 d建立小鼠肠道菌群失调的模型,然后随机分为4组,分别为模型组及低、中和高剂量组。低、中和高剂量组灌胃不同剂量乳双歧杆菌V9溶液,另设正常对照组,与模型组灌胃等体积生理盐水,连续灌胃23 d。灌胃结束后,采集小鼠的粪便进行活菌计数和16S rDNA测序,检测粪便中微生物组成及分布,测定血清中IL-1β、IL-6、TNF-α以及IL-2的含量,测定小肠及肝脏组织中SOD、MDA、GSH、GSH-Px的含量,HE染色观察小鼠小肠组织病理学变化。结果表明,灌胃乳双歧杆菌V9溶液后,中、高剂量组IL-1β、IL-6、TNF-α及IL-2的含量分别显著降低31.73、17.04、12.57及31.71 pg/mL;高剂量组小肠及肝脏组织中MDA含量显著降低（p<0.01）,中、高剂量组SOD、GSH及GSH-Px均极显著升高（p<0.01）。同时,乳双歧杆菌V9使得头孢曲松钠导致的小鼠肠道菌群失调得到明显改善,粪便活菌计数显示高剂量组肠杆菌数量显著降低0.34 lg cfu/g,乳杆菌及双歧杆菌数量显著增加,分别增加0.40 lg cfu/g和0.26 lg cfu/g。拟普雷沃菌属和魏斯氏菌丰度显著降低（p<0.01）。说明乳双歧杆菌V9对由头孢曲松钠引起的肠道菌群失衡具有一定的调节作用,并调节菌群多样性。     

木糖调节小鼠肠道菌群作用的研究

本实验研究木糖对小鼠肠道菌群的影响。实验小鼠灌胃不同剂量的木糖,观察双歧杆菌、乳杆菌、肠杆菌和肠球菌等4 种肠道菌群的变化。结果表明,木糖具有调节小鼠肠道菌群及促进双歧杆菌增殖功能。     

短双歧杆菌对Aβ;导致的阿尔兹海默症小鼠肠道菌群及代谢物的影响

为分析2株不同来源的短双歧杆菌对脑部微注射Aβ;蛋白导致的阿尔兹海默症小鼠肠道菌群及其代谢物的影响,给小鼠海马区注射Aβ;蛋白建立阿尔兹海默症小鼠模型,连续灌胃6周2株不同来源的短双歧杆菌,收集小鼠粪便,采用Illumina MiSeq高通量测序技术分析菌群的多样性及物种组成,并采用GC-MS技术检测小鼠粪便中短链脂肪酸的含量。菌群多样性分析发现,Aβ;蛋白注射改变了小鼠的菌群多样性及物种结构,灌胃短双歧杆菌可一定程度上改善菌群紊乱。进一步在属水平和种水平分析各组菌群差异,发现灌胃短双歧杆菌MY显著提高了大鼠肠道中产短链脂肪酸菌Coprococcus spp.、Lactobacillus reuteri和Akkermansia muciniphila的相对丰度,且有效调控了肠道内3种短链脂肪酸的水平。短双歧杆菌MY可能通过调节肠道菌群及其代谢物短链脂肪酸的水平缓解小鼠的认知障碍。     

酪蛋白糖巨肽(CGMP)对小鼠肠道菌群消长规律的影响

目的:研究了不同剂量的CGMP对小鼠肠道菌群的影响。方法:选用60只BALB/c小鼠(25～30g),随机分成十组(对照组和4～10七个试验组),每组6只;对照1组(日食组)既不灌胃CGMP也不灌胃安慰剂,对照2组灌胃甜乳清粉溶液(0.2ml),对照3组灌胃生理盐水(0.2ml),试验4～10组灌胃等体积(0.2ml)不同浓度的CGMP溶液,灌胃期为15d。分别于灌胃前以及试验的第3、5、7、10、15d和停止灌胃后一周(第22d)用逼迫法采集小鼠新鲜粪便进行菌群测定,观察肠道内菌群的消长规律。结果表明:与对照组相比,随着灌胃时间的延长,各试验组均可促进小鼠肠道和粪便中的双歧杆菌和乳酸杆菌增殖(p<0.01),使致病菌数量明显减少(p<0.05),对肠杆菌、肠球菌数量的影响不明显。CGMP对小鼠肠道菌群的影响作用存在最适剂量(100μg/d),最适剂量组使肠杆菌、肠球菌、致病菌数量明显减少。说明CGMP具有有效地促进和调控小鼠肠道生理性细菌、构筑膜菌群的作用。     

牛蒡寡糖的提取及对小鼠肠道菌群的调节作用

目的：研究牛蒡多糖的提取条件和对小鼠肠道菌群的调节作用。方法：采用单因素试验和正交试验优化牛蒡多糖提取工艺。15 只小鼠随机分为空白对照组和牛蒡多糖低、高剂量组,每组5 只,经口灌胃给药,灌胃容积为10ml/kg,1 次/d,连续14d。给药结束后,分别采集小鼠粪便,检查肠道肠杆菌,乳杆菌和双歧杆菌。结果：牛蒡多糖优化提取条件为：温度80℃,固液比1:10(m/V),时间60min,提取两次。与空白对照组相比,牛蒡多糖组乳杆菌与双歧杆菌数量均显著增加(P ＜ 0.05),肠杆菌数量变化不明显(P ＞ 0.05)。结论：牛蒡多糖能有效调节小鼠肠道菌群,增殖乳杆菌和双歧杆菌数量。     

魔芋葡甘低聚糖对大鼠肠道环境的影响

目的：研究以半干法酶解制备的魔芋葡甘低聚糖（konjac oligosaccharides,KOS）对大鼠肠道环境的影响。方法：将KOS掺入到饲料中饲喂Sprague-Dawley（SD）大鼠,30 d后测定大鼠盲肠内容物各项指标和盲肠、小肠的理化指标。结果：KOS可以明显增加大鼠粪便含水率及盲肠壁表面积、小肠的拉伸性能、盲肠内容物中双歧杆菌和乳酸菌的数量;降低盲肠内容物pH值及含水率、游离氨及挥发性醛类和含氮类物质含量;抑制大肠杆菌和梭状芽孢杆菌的生长,增加短链脂肪酸的产生量。结论：KOS可以调节大鼠肠道菌群组成,改善肠道环境,促进肠道健康,是一种优良的益生元。     

A Bifidobacterium-based synbiotic product to reduce the transmission of C. jejuni along the poultry food chain

With the ban of dietary antimicrobial agents, the use of probiotics, prebiotics and synbiotics has attracted a great deal of attention in order to improve intestinal health and control food-borne pathogens, which is an important concern for the production of safe meat and meat products. Recently, Campylobacter jejuni has emerged as a leading bacterial cause of food-borne gastroenteritis in humans, and epidemiological evidences indicate poultry and poultry products as the main source of human infection. This work aimed at the development of a synbiotic mixture capable of modulating the gut microbiota of broiler chickens to obtain an increase of the beneficial bacteria (i.e. bifidobacteria, lactobacilli) and a competitive reduction of C. jejuni. The prebiotic compound used in the mixture was chosen after an in vivo trial: a fructooligosaccharide and a galactooligosaccharide were separately administered to broilers mixed with normal feed at a concentration of 0.5% and 3%, respectively. Quantitative PCR on DNA extracted from fecal samples revealed a significant (p<0.05) increase of Bifidobacterium spp. in broilers treated with the galactooligosaccharide, coupled to a decrease (p<0.05) of Campylobacter spp. The galactooligosaccharide was then combined with a probiotic Bifidobacterium strain (B. longum subsp. longum PCB133), possessing in vitro antimicrobial activity against C. jejuni. The strain was microencapsulated in a lipid matrix to ensure viability into the feed and resistance to stomach transit. Finally, the synbiotic mixture was administered to broiler chickens for 14 days mixed with normal feed in order to have an intake of 10(9)CFU of PCB133/day. Bifidobacterium spp., Lactobacillus spp., Campylobacter spp., B. longum subsp. longum and C. jejuni were quantified in fecal samples. PCB133 was recovered in feces of all animals. C. jejuni concentration in poultry feces was significantly (p<0.05) reduced in chickens administered with the synbiotic mixture. This study allowed to highlight the positive effect of the synbiotic approach for C. jejuni reduction in broiler chickens, which is of fundamental importance for the safety of poultry meat consumers.     

Effect of honey on the growth of and acid production by human intestinal Bifidobacterium spp.: an in vitro comparison with commercial oligosaccharides and inulin.

Five human intestinal Bifidobacterium spp., B. longum, B. adolescentis, B. breve, B. bifidum, and B. infantis, were cultured in reinforced clostridial medium (control) and in reinforced clostridial medium supplemented with 5% (wt/vol) honey, fructooligosaccharide (FOS), galactooligosaccharide (GOS), and inulin. Inoculated samples were incubated anaerobically at 37degrees C for 48 h. Samples were collected at 12-h intervals and examined for specific growth rate. Levels of fermentation end products (lactic and acetic acids) were measured by high-pressure liquid chromatography. Honey enhanced the growth of the five cultures much like FOS, GOS, and inulin did. Honey, FOS, GOS, and inulin were especially effective (P < 0.05) in sustaining the growth of these cultures after 24 h of incubation as compared with the control treatment. Overall, the effects of honey on lactic and acetic acid production by intestinal Bifidobacterium spp. were similar to those of FOS, GOS, and inulin.     

婴儿源益生性双歧杆菌的筛选及肠道定殖性研究

本研究旨在从婴儿粪便中筛选出具有潜在益生特性的双歧杆菌,并探究其肠道定殖情况,为双歧杆菌的产品开发提供优良的菌株。采用MRS培养基对样品进行分离纯化,菌株经F6PPK检测及16S r DNA测序鉴定,之后进行模拟胃肠液、胆盐耐受性、对食源性致病菌(大肠杆菌、沙门氏菌、单增李斯特菌等)的抑制及对HT-29细胞的粘附能力测定,将筛选出的菌株进行动物实验,测定其肠道定殖能力。分离到的27株双歧杆菌,经分子生物学鉴定为7个不同的种:Bifidobacterium longum、Bifidobacterium breve、Bifidobacterium bifidum、Bifidobacterium pseudocatenulatum、Bifidobacterium infantis、Bifidobacterium animalis和Bifidobacterium adolescentis。体外实验表明,B.longum A9、B.breve A4、B.bifidum B6、B.longum C6、B.adolescentis F8和B.infantis H6等具有较强的潜在益生特性;动物实验表明,B.infantis H6和B.longum C6具有较强的肠道定殖能力。B.longum C6和B.infantis H6有望作为优良的益生性菌株,应用于双歧杆菌的产品开发。     

Use of tuf gene-based primers for the PCR detection of probiotic Bifidobacterium species and enumeration of bifidobacteria in fermented milk by cultural and quantitative real-time PCR methods

Due to the increasing use of bifidobacteria in probiotic products, it is essential to establish a rapid method for the qualitative and quantitative assay of the bifidobacteria in commercial products. In this study, partial sequences of the tuf gene for 18 Bifidobacterium strains belonging to 14 species were determined. Alignment of these sequences showed that the similarities among these Bifidobacterium species were 82.24% to 99.72%. Based on these tuf gene sequences, 6 primer sets were designed for the polymerase chain reaction (PCR) assay of B. animalis subsp. animalis, B. animalis subsp. lactis, B. bifidum, B. breve, B. longum subsp. infantis, B. longum subsp. longum, and the genus of Bifidobacterium, respectively. These Bifidobacterium species are common probiotic species present in dairy and probiotic products. When each target Bifidobacterium spp. was assayed with the designed primers, PCR product with expected size was generated. In addition, for each target species, more than 70 bacterial strains other than the target species, including strains of other Bifidobacterium species, strains of Lactobacillus spp., Enterococcus spp., and other bacterial species, all generated negative results. PCR assay with primers specific to B. animalis subsp. lactis and B. longum subsp. longum confirmed the presence of these Bifidobacterium species in commercial yogurt products. In addition, for each product, enumeration of the bifidobacteria cells by culture method with BIM-25 agar and the quantitative real-time PCR showed similar cell counts. Such results indicated that within 15-d storage (4 °C) after manufacture, all the bifidobacteria cells originally present in yogurt products were viable and culturable during the storage.     

益生菌混合菌种在发酵豆乳中的优化

采用益生菌混合物AST和TLB分别在42℃进行豆乳的发酵,在发酵豆乳的发酵和贮存过程中(4℃下28d),观察pH值和活菌数的变化情况。在AST益生菌混合物(嗜酸性乳杆菌,双歧杆菌和嗜热链球菌)的培育下,42℃下发酵时间减少到8h。但嗜酸性乳杆菌在冷藏的过程中生存状态较差,其活菌数在冷藏后一周后未达到标准。将豆乳在42℃下用TLB益生菌(嗜热链球菌,保加利亚乳杆菌和动物双歧杆菌乳酸亚种Bb12)进行发酵,结果发酵时间缩短到4h,双歧杆菌活菌数目的对数增加了约一半,而且经过28d的冷藏,细菌数仍然维持在107 CFU/mL以上。     

食品检验中乳酸菌鉴定方法的探讨

目的评价GB 4789《食品安全国家标准食品微生物学检验》中乳酸菌的鉴定方法。方法选择5种双歧杆菌、4种乳酸杆菌和1种链球菌共10株乳酸菌,分别采用GB 4789标准中的方法、API生化鉴定系统、16S rRNA基因序列分析法和RiboPrinter全自动基因指纹图谱分析法进行鉴定。结果 GB 4789对乳酸杆菌和嗜热链球菌鉴定效果较好,对双歧杆菌的鉴定能力较弱;API鉴定乳酸菌一般至"属"水平;16S rRNA序列分析和RiboPrinter系统可鉴定乳酸菌至"种"水平,婴儿双歧杆菌的鉴定结果为长双歧杆菌婴儿亚种。结论建议GB 4789标准中增加分子生物学方法作为乳酸菌鉴定方法的补充,同时建议及时更新标准中菌株的分类和名称。     

富硒长双歧杆菌DD98菌株对伊立替康所致小鼠腹泻及肠道菌群的影响

本研究建立伊立替康所致腹泻小鼠模型,通过长期灌胃富硒DD98(Selenium-enriched Bifidobacterium longum Se-DD98)菌株(一株新的长双歧杆菌菌株),评价其对伊立替康所致腹泻及肠道菌群的影响。正常对照组和伊立替康组小鼠每天灌胃无菌蒸馏水,实验组小鼠每天灌胃低剂量(活菌数1×108 CFU/kg)、高剂量(活菌数1×109 CFU/kg)Se-DD98菌液,除正常对照组外其他各组在第17 d腹腔注射伊立替康,实验周期为24 d。测定小鼠体重,小鼠腹泻等级,免疫指标,肠道菌群以及回肠组织结构。结果表明:与伊立替康组比较,Se-DD98低、高剂量组小鼠体重显著增加(P<0.05);腹泻等级显著降低(P<0.05);高剂量组白细胞数目显著增加(P<0.05),低、高剂量组脾脏指数和中性粒细胞数目均显著增加(P<0.05),说明Se-DD98具有调节免疫功能的作用。同时,低、高剂量组大肠杆菌、梭菌和拟杆菌含量均比正常组和伊立替康组显著减少(P<0.05),而双歧杆菌含量显著增加(P<0.01)。此外Se-DD98低、高剂量均能减轻伊立替康导致的肠道粘膜、细胞和腺体损伤。上述结果表明Se-DD98可能通过调节肠道菌群和保护肠道粘膜发挥缓解伊立替康导致的腹泻的作用。提示Se-DD98在辅助治疗伊立替康所致的腹泻等方面具有潜在应用前景。     

茯苓多糖对双岐杆菌BB-12关键代谢产物的影响

为探究动物双岐杆菌乳双歧亚种BB-12代谢茯苓多糖的关键代谢产物,采用基于液质联用技术（LC-MS/MS）的非靶向代谢组学手段,以葡萄糖作为对照,分析茯苓多糖对动物双岐杆菌乳双歧亚种BB-12关键代谢产物的影响。结果表明,茯苓多糖可显著促进双歧杆菌BB-12的生长。结合主成分分析（Principal Component Analysis, PCA）和正交偏最小二乘判别模型（Orthogonal Partial Least Squares Discriminant Analysis, OPLS-DA）等分析手段,筛选出丹芝酸、黄藤素等具有明确生理功能的差异代谢产物共12种,并显著富集到ABC转运蛋白通路、亚油酸代谢通路、半乳糖代谢通路等17条差异代谢通路。这些代谢物和代谢通路均可证明双歧杆菌BB-12在发酵茯苓多糖的情况下表现出更优异的生长性能和益生功效。该实验从小分子代谢物水平分析了动物双歧杆菌乳双歧亚种BB-12代谢茯苓多糖后产生的代谢产物,为进一步研究茯苓及动物双岐杆菌乳双歧亚种BB-12在人体内发挥益生功效的相关机制提供理论依据和指导。