雪莲果粉对双歧杆菌的体外促生长作用研究

研究了雪莲果粉对3种双岐杆菌(长双歧杆菌、短双歧杆菌和两歧双歧杆菌)体外生长的影响。结果表明,雪莲果粉对3种双歧杆菌的体外生长均有显著的促进作用,优于葡萄糖作碳源时的生长效果,同时对不同的双歧杆菌的促进作用不完全一致。雪莲果粉浓度为1.6%～2.4%(m/v)时长双歧杆菌增殖效果最明显,0.8%～1.6%(m/v)时短双歧杆菌促生长效果明显,1.2%～2.4%(m/v)两歧双歧杆菌促生长效果明显。     

两歧双歧杆菌缓解Ⅱ型糖尿病的效果差异及机制分析

该文分析3株两歧双歧杆菌对高脂饮食结合链脲佐菌素诱导的Ⅱ型糖尿病小鼠症状的缓解作用及可能机制。将48只雄性C57BL/6J小鼠分为6组,菌处理组每周灌菌6次,灌胃15周。实验期间,测定小鼠的血糖指标和血脂四项,测定肝脏细胞因子和丙二醛含量,分析组织病理学损伤,测定小鼠粪便短链脂肪酸含量并分析小鼠的肠道菌群。3株菌在改善血糖血脂代谢和炎症等方面表现出明显差异。结果表明,与两歧双歧杆菌BB13和H35相比,BB1对Ⅱ型糖尿病小鼠的血糖血脂代谢异常、炎症反应均有明显改善,其可能机制是BB1缓解高脂饮食和STZ导致的肠道菌群失调,增加了肠道中Bacteroidales S24-7的丰度,从而使肠道内SCFAs含量升高,缓解了小鼠体内的低水平炎症,改善胰岛素抵抗,缓解了Ⅱ型糖尿病。     

中华人民共和国卫生部公告 2003年 第3号

根据《益生菌类保健食品评审规定》(卫法监发〔2 0 0 1〕84号附件 4 ) ,经审查 ,批准罗伊氏乳杆菌 (Ｌａｃｔｏｂａ ｃｉｌｌｕｓｒｅｕｔｅｒｉ)为可用于保健食品的益生菌菌种 ,列入卫法监发〔2 0 0 1〕84号附件 5“可用于保健食品的益生菌菌种名单”。目前 ,可用于保健食品的益生菌菌种名单如下 :两歧双歧杆菌　　　　　Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍｂｉｆｉｄｕｍ婴儿双歧杆菌　　　　　Ｂ .ｉｎｆａｎｔｉｓ长双歧杆菌　　　　　　Ｂ .ｌｏｎｇｕｍ短双歧杆菌　　　　　　Ｂ .ｂｒｅｖｅ青春双歧杆菌　　　　　Ｂ .ａｄｏｌｅｓｃｅ…     

青年人肠道中双歧杆菌的分离及其在发酵羊乳中的应用

在对湖北省襄阳地区青年人肠道中双歧杆菌进行分离鉴定的基础上,采用电子舌和电子鼻技术对其在羊乳中的发酵特性进行了评价。结果表明:分离出的20株双歧杆菌菌株,共鉴定为Bifidobacterium longum(长双歧杆菌)、B. breve(短双歧杆菌)、B. bifidum(两歧双歧杆菌)、B.pseudocatenulatum(假链状双歧杆菌)、B. adolescentis(青春双歧杆菌)和B. faecale(粪便双歧杆菌)6个种,其中11株为B. longum(长双歧杆菌)。通过电子舌分析发现,不同双歧杆菌制备的发酵羊乳在后味A(涩味的回味)上的差异最大,极差值为24.52。通过高效液相色谱法分析发现,乳酸和乙酸为双歧杆菌制备发酵羊乳中的主要有机酸。通过电子鼻分析发现,传感器W6S、W1W和W5S对不同双歧杆菌菌株制备发酵羊乳响应值差异较大,其变异值分别为35.56%、23.32%和17.20%。通过主成分分析发现,B. longum HBUAS55095和B. longum HBUAS55100具有良好的羊乳发酵特性。由此可见,B. longum(长双歧杆菌)为襄阳地区青年人肠道中双歧杆菌类群的优势种,且不同双歧杆菌制备的发酵羊乳品质差异主要体现在滋味上。     

基于宏基因测序及分箱技术的复合乳酸菌发酵乳菌种鉴定及定量

复合乳酸菌发酵乳产品的菌种组成与定量是产品质量的关键。以3款市售复合乳酸菌发酵乳产品为研究对象,利用宏基因组测序和分箱分析、平均核苷酸一致性(average nucleotide identity, ANI)分析等技术,研究复合乳酸菌发酵乳产品中菌种种水平鉴定及相对定量。结果表明,产品A和产品C分箱获得的3个bin通过ANI分析鉴定为嗜热链球菌(Streptococcus thermophilus)、德氏乳杆菌(Lactobacillus delbrueckii)和动物双歧杆菌(Bifidobacterium animalis),与产品声称添加菌种一致,产品A中3个物种的相对丰度分别为96.85%、0.12%和3.02%,产品C中3个物种的相对丰度分别为99.55%、0.18%和0.14%。产品B分箱获得的3个bin通过ANI分析鉴定为嗜热链球菌(Streptococcus thermophilus)、干酪乳酪杆菌(Lacticaseibacillus casei)和乳酸乳球菌(Lactococcus lactis),与产品声称添加菌种部分一致,3个物种的相对丰度分别为99.43%、0...     

蔓菁对小鼠肠道菌群的影响

目的:探究不同剂量蔓菁对小鼠粪样中肠道菌群和短链脂肪酸的影响。方法:选取BalB/C雄性小鼠(18~22 g)48只按体重随机分为对照组、低剂量组、中剂量组、高剂量组4组,三个剂量组分别饲喂不同剂量(1、2、4 g/kg·BW·d)的蔓菁粉,对照组正常喂养,连续干预14 d,分别在干预第0 d、第7 d、第14 d称重并无菌收集新鲜粪样。采用平板计数法检测肠道菌群数量,气相色谱-质谱联用法检测短链脂肪酸的含量,并用SPSS 21.0对结果进行数据分析。结果:蔓菁可增加肠道益生菌乳酸杆菌、双歧杆菌和中性菌肠杆菌的数量(p<0.05),减少中性菌肠球菌和致病菌产气荚膜梭菌的数量(p<0.05),中剂量(2 g/kg·BW·d)作用最为明显,且肠杆菌数量增加幅度低于乳酸杆菌和双歧杆菌增加幅度。蔓菁还可增加肠道乙酸、丙酸、正丁酸、异丁酸的含量(p<0.05),提高短链脂肪酸总含量,高剂量(4 g/kg·BW·d)作用最为明显。结论:蔓菁可促进小鼠肠道有益菌的增殖,抑制致病菌的增殖;蔓菁可促进小鼠肠道短链脂肪酸的产生。     

两歧双歧杆菌CCFM1167通过提升肠道中乙酸水平以抑制炎症从而缓解便秘

便秘(constipation)是一种临床常见疾病,发病率逐年上升。大量研究证明补充特定益生菌可以缓解便秘。因此该研究选取3株不同来源的双歧杆菌,研究其对便秘的缓解作用并进一步探究其可能的缓解途径。使用双歧杆菌干预小鼠4周,期间用盐酸洛哌丁胺处理3周构建便秘小鼠模型,通过对小鼠便秘相关指标的检测,确定两歧双歧杆菌CCFM1167对便秘具有较好的缓解作用,并进一步对小鼠便秘相关胃肠调节肽、炎症相关指标和短链脂肪酸进行检测以及相关性分析。结果表明两歧双歧杆菌CCFM1167提升便秘小鼠肠道中短链脂肪酸尤其是乙酸的含量,上调小鼠白细胞介素10及调节性T细胞含量,减轻肠道炎症。推测CCFM1167通过提高便秘小鼠肠道内乙酸的含量,缓解小鼠由洛哌丁胺导致的炎症,提高粪便含水量进而缓解便秘。     

双抗夹ELISA法检测双歧杆菌反应条件的优化

研究利用双抗夹心酶联免疫吸附法（Double-antibody Sandwich Enzyme-Linked Immunosorbent Assay,Double-antibody Sand-wich ELISA）快速检测双歧杆菌（Bifidobacterium）的最佳反应条件。使用实验室自制的兔抗两歧双歧杆菌免疫血清和鼠抗两歧双歧杆菌免疫血清,利用方阵法对两种血清抗体的反应浓度进行筛选,之后对包被液、封闭液、封闭时间及底物作用时间进行比较和选择,从而确定运用双夹心ELISA法快速检测双歧杆菌的最佳反应条件。最佳反应条件分别为：兔抗两歧双歧杆菌免疫血清和鼠抗两歧双歧杆菌免疫血清反应浓度分别为1︰1280,1︰320,包被液浓度0.05 mol/L（pH值为9.6）的Na2CO3-NaHCO3,封闭液为质量分数为5%的BSA-PBS,封闭30min,底物作用时间为30 min。优化了双抗夹心ELISA法检测双歧杆菌的反应条件,为双歧杆菌的血清学检测提供了参考。     

基于高通量测序技术分析霉变核桃仁真菌多样性

目的:研究核桃仁在适宜霉变条件(温度25 ℃、相对湿度85%)下的真菌侵染过程，揭示其霉变过程中真菌群落的结构与真菌群落样性变化。方法:采用Illumina Miseq高通量测序方法分析核桃仁霉变过程中的真菌群落组成和多样性。结果:子囊菌门是核桃样品霉变的主要真菌门类，相对丰度在27.41%~99.98%范围，其次是担子菌门，相对丰度在0.01%~32.56%范围。在真菌属水平上，霉变过程中的主要优势种群为镰孢菌属、链格孢属、赤霉属与曲霉属。1周内核桃仁的霉变过程可大致分为Ⅰ期(0~2 d)、Ⅱ期(3~4 d)、Ⅲ期(5~7 d)3个时期，Ⅱ期与Ⅰ期相比镰孢菌属出现明显的富集，Ⅲ期与Ⅱ期相比镰孢菌属、链格孢属与曲霉属出现明显的富集，Ⅲ期与Ⅰ期相比镰孢菌属、链格孢属与曲霉属出现明显的富集。结论:核桃仁霉变过程中真菌微生物群落的变化分为3个时期，真菌多样性呈下降趋势，镰孢菌属、链格孢属与曲霉菌属是其霉变的优势真菌种群。     

双歧杆菌的分离鉴定及保藏性能研究

为丰富发酵剂及益生菌菌种资源,从内蒙古阿拉善盟采集7份新生婴儿粪便样品,在传统细菌分离方法的基础上,通过添加莫匹罗星锂盐和X-Gal(5-溴-4-氯-3-吲哚-β-D-半乳糖苷)改良基础培养基,快速有效地分离获得双歧杆菌。结合全自动微生物分析系统和API20A试剂条鉴定菌种。结果共分离获得6株双歧杆菌,其中3株动物双歧杆菌,2株青春双歧杆菌,1株短双歧杆菌。试验验证甘油浓度为70%时冷冻保藏效果最佳。     

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。     

In vitro fermentability of human milk oligosaccharides by several strains of bifidobacteria

This study was conducted to investigate the catabolism and fermentation of human milk oligosaccharides (HMO) by individual strains of bifidobacteria. Oligosaccharides were isolated from a pooled sample of human milk using solid-phase extraction, and then added to a growth medium as the sole source of fermentable carbohydrate. Of five strains of bifidobacteria tested (Bifidobacterium longum biovar infantis, Bifidobacterium bifidum, Bifidobacterium longum biovar longum, Bifidobacterium breve, and Bifidobacterium adolescentis), B. longum bv. infantis grew better, achieving triple the cell density then the other strains. B. bifidum did not reach a high cell density, yet generated free sialic acid, fucose and N-acetylglucosamine in the media, suggesting some capacity for HMO degradation. Thin layer chromatography profiles of spent fermentation broth suggests substantial degradation of oligosaccharides by B. longum bv. infantis, moderate degradation by B. bifidum and little degradation by other strains. While all strains were able to individually ferment two monosaccharide constituents of HMO, glucose and galactose, only B. longum bv. infantis and B. breve were able to ferment glucosamine, fucose and sialic acid. These results suggest that as a potential prebiotic, HMO may selectively promote the growth of certain bifidobacteria strains, and their catabolism may result in free monosaccharides in the colonic lumen.     

Uncoupling of growth and acids production in Bifidobacterium ssp

Kinetics of batch cultivation of four species of Bifidobacterium in milk were examined in detail. Bifidobacteria could grow well in milk inoculated with cultures prepared in a synthetic medium. Cessation of growth occurred, however, in pH-controlled batch cultures, although incomplete utilization of lactose was observed. Lactate and acetate accumulation caused limitation on growth of bifidobacteria leading to an uncoupling of biomass and product formation. From 70 to 75% of both final lactate and acetate concentrations were produced during the stationary growth phase of Bifidobacterium bifidum, Bifidobacterium breve, and Bifidobacterium longum cultivated in milk, whereas Bifidobacterium infantis produced less acetate or lactate during this phase.     

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.     

双歧杆菌抗消化道逆环境特性的研究

应用模拟胃液pH、胃液、肠液、肠胆盐环境 ,研究青春双歧杆菌、婴儿双歧杆菌、长双歧杆菌和短双歧杆菌的抗消化道逆环境特性。在pH1 5酸性条件下作用 1 5h ,存活率分别为 0 14 %、17 86%、49 0 9%和 9 5 3 %。在加有胃蛋白酶的模拟胃液中作用 1 5h ,存活率分别为 :45 3 9%、97 2 7%、84 5 3 %和 85 5 1%。在含胰蛋白酶的模拟肠液中作用 12h ,存活率分别为 :2 9 17%、47 97%、87 70 %和 77 2 7%。青春双歧杆菌、长双歧杆菌和短双歧杆菌的活菌数随胆盐浓度升高而下降 ,2 %胆盐浓度条件下作用 12h ,三者的存活率分别为 5 92 %、13 96%和 61 2 4%。而婴儿双歧杆菌在各胆盐浓度条件下 ,均有生长趋势 ,这种趋势随胆盐浓度升高而下降。研究结果表明 ,消化道逆环境对双歧杆菌的存活有不同程度的影响 ,以婴儿双歧杆菌和长双歧杆菌的耐性最好。     

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.     

Production of oligosaccharides in yogurt containing bifidobacteria and yogurt cultures

Yogurts were prepared by using yogurt cultures combined to mixed cultures of bifidobacteria (Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium longum) and by adding a preincubation step (1.5 h at 50 degrees C) with bifidobacteria to the conventional method of manufacture in order to produce oligosaccharides. The survival of bifidobacteria was drastically affected during storage of yogurts, except for products containing B. animalis, in which viable counts remained at >10(6) cfu/g after 28 d of storage at 4 degrees C. Oligosaccharides with a degree of polymerization of 3 were produced during the preincubation step (0.31 to 0.68%), and the amount in the final products varied according to the species of bifidobacteria inoculated during the preincubation step or the concentration of bifidobacteria used as second inoculum during the fermentation process. In fact, the higher concentration of oligosaccharides measured at the end of the fermentation process (0.72%) and the 28 d-storage period (0.67%) was obtained for yogurts containing B. infantis. However, yogurts containing B. breve showed higher beta-galactosidase activities and had lower lactose concentrations after the fermentation process and the storage period than the other yogurts. The use of a mixed cultures of bifidobacteria (B. animalis, B. infantis, or B. breve) thus allows the production of yogurts in which bifidobacteria can survive in relatively high cell numbers and contain appreciable amount of oligosaccharides.     

Culture-dependent and culture-independent qualitative analysis of probiotic products claimed to contain bifidobacteria

A total of 58 probiotic products obtained worldwide, which were claimed to contain Bifidobacterium strains (including 22 yoghurts, 5 dairy fruit drinks, 28 food supplements and 3 pharmaceutical preparations) were investigated in parallel using a culture-dependent and a culture-independent approach. Three isolation media previously reported as selective for Bifidobacterium were evaluated for their suitability in the quality analysis of these products. Subsequently, possible bifidobacterial colonies were picked from the best medium and identified by means of rep-PCR fingerprinting using the BOX primer (BOX-PCR). Bifidobacterium animalis subsp. lactis, formerly classified as Bifidobacterium lactis, was most frequently found, but strains belonging to Bifidobacterium longum biotypes longum and infantis, Bifidobacterium bifidum and Bifidobacterium breve were recovered also. In parallel, all products were also subjected to culture-independent analysis which involved a nested-PCR step on total bacterial DNA extracted directly from the product, followed by separation of the amplicons by Denaturing Gradient Gel Electrophoresis (DGGE) and subsequent identification of species from the band patterns. By conventional cultivation, 70.7% of the products analysed were found to contain culturable bifidobacteria whereas by culture-independent DGGE analysis members of the genus Bifidobacterium could be detected in 96.5% of the analysed products. Genotypic characterization of a number of bifidobacterial isolates at the strain level by means of Pulsed-Field Gel Electrophoresis (PFGE) revealed a relatively high degree of genomic homogeneity among the Bifidobacterium strains currently used in the probiotic industry.     

Stability of bifidobacteria in powdered formula

Two stability tests of bifidobacteria in powdered formula were conducted. In a strain comparison test, three kinds of bifidobacterial powders; Bifidobacterium longum BB536, Bifidobacterium breve M-16V and Bifidobacterium infantis M-63 powders, admixed in follow-up formula were used. In a commercial product evaluation, powdered formulas for toddlers containing bifidobacteria sold in the Indonesian market were analysed. When the inactivation rate constant of each sample, which was used as an index of the loss rate, was determined from the stability tests, B. longum was the most stable strain. The mean inactivation rate constant of commercial products was significantly lower than those obtained in strain comparison, although the same strains ( B. longum BB536 and B. breve M-16V) were used. A possible reason was the lower water activity of commercial products compared to the follow-up formula. Also, higher storage temperature yielded lower stability in all strains or samples, which obeys the Arrhenius theory.     

鲟鱼肝酶解条件优化及酶解液对双歧杆菌增殖效果的研究

为提高鲟鱼肝脏的加工附加值，使用碱性蛋白酶，以水解度为评价指标，在单因素试验的基础上，通过Plackett-Burman试验、响应面分析法优化鲟鱼肝蛋白酶解条件。在此条件下，进一步探究了酶解液对双歧杆菌的促生长效果。结果表明，最优酶解条件为碱性蛋白酶添加量10 540 U/g鱼肝蛋白、体系初始pH值10.0、酶解时间8.6 h、酶解温度45 ℃、料液比1∶10（g∶mL），此条件下水解度（81.7%）是优化前（40.4%）的1.02倍。鲟鱼肝蛋白酶解液对青春双歧杆菌、两歧双歧杆菌、动物双歧杆菌、长双歧杆菌、短双歧杆菌和婴儿双歧杆菌6种均具有显著的促生长效果。研究结果为鲟鱼加工副产物利用提供了新途径，同时也为双歧杆菌促生长因子的研发提供新思路。