婴儿源双歧杆菌对人胎结肠上皮细胞的增殖作用及机制研究

分析12株婴儿源双歧杆菌的发酵上清液和破碎物对人胎结肠上皮细胞(CCD841 CoN)的增殖效果。筛选出对细胞增殖促进效果最好的菌株,提取其表面蛋白和分泌蛋白,检测这两类蛋白对细胞增殖、周期及增殖相关基因表达的影响,探讨其对CCD841 CoN细胞增殖促进的作用。结果表明:筛选出双歧杆菌H3-R2,其浓度为50 μg/mL表面蛋白和10 μg/mL分泌蛋白与CCD841 CoN细胞共作24 h后,对细胞有显著增殖促进作用(P<0.05),提高细胞S期比例,降低G0/G1期比例(P<0.05),提高细胞内 β-catenin、c-Myc和Cyclin D1基因mRNA的转录水平(P<0.05),降低 Axin2、GSK-3β 基因mRNA的转录水平(P<0.05)。综上,婴儿源双歧杆菌可在mRNA转录水平上对人胎结肠上皮细胞有增殖促进作用,具有促进新生儿肠道发育的潜在应用价值。     

婴儿源动物双歧杆菌消化道胁迫抗性及体外对免疫细胞活性影响的比较

以4 株婴儿源动物双歧杆菌乳亚种（Bifidobacterium animalis subsp. lactis）为研究对象,分析菌株对酸、胆盐、模拟消化道环境的耐受能力及对致病菌的黏附抑制作用,并评价体外对Caco-2细胞的黏附性。此外,测定菌株合成胞外多糖的能力,并探讨其与耐受性及黏附性的关联。经上述实验获得黏附性及抗性双优的菌株H15-2,再经脾淋巴细胞增殖实验、巨噬细胞能量代谢水平及巨噬细胞吞噬中性红能力3 个方面评价其免疫调节能力。结果表明：动物双歧杆菌H15-2对模拟胃肠道环境有较强的耐受性,对Caco-2细胞有较高的黏附能力,并对免疫细胞活性具有较好的调节作用,有应用开发潜力。     

新疆喀什地区维吾尔族母乳源双歧杆菌分离筛选及其益生特性分析

采用多重聚合酶链式反应指纹图谱结合16S rRNA基因测序技术对新疆喀什地区维吾尔族母乳分离的双歧杆菌进行鉴定和遗传差异分析,并检测常规生理生化和糖代谢表型特征,同时测试菌株对6 种常见病原菌和3 种母乳源条件致病菌的抑菌性能和对胃肠液的耐受性。结果显示,15 份母乳样品中共分离15 株双歧杆菌,测序结果将菌株归属于3 个种以及2 个亚种,包括8 株假小链双歧杆菌（Bifidobacterium pseudocatenulatum）、2 株短双歧杆菌（B. breve）、2 株长双歧杆菌长亚种（B. longum subsp. longum）和3 株长双歧杆菌婴儿亚种（B. longum subsp. infantis）。抑菌实验表明,15 株测试菌株中,隶属于B. pseudocatenulatum的4 株菌MY92、MY75-1、MY72、MY81的抑菌谱更广,抑菌能力更强;胃肠液耐受性实验表明菌株MY92无论在模拟胃液还是模拟肠液中存活率均最高,分别达到20.37%和0.302%。基于以上描述特性,MY92作为一株有效的益生菌株,具有潜在的利用价值,为后期进一步作为防止婴幼儿腹泻辅助制剂的开发提供参考。     

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。     

新疆维吾尔族肠道中高产胞外多糖双歧杆菌的筛选及其抗氧化活性

使用Wilkins-Chalgren厌氧菌琼脂和BSM培养基作为筛选平板,结合重复基因外回文序列-聚合酶链式反应和16S rRNA序列分析,对新疆维吾尔族婴儿及其母亲粪便中的双歧杆菌(Bifidobacterium)进行分离鉴定,并筛选出高产胞外多糖的双歧杆菌,测定其多糖的抗氧化活性以及菌株的耐受性和黏附性。结果显示,20份粪便样品中共分离出52株双歧杆菌,其中假小链双歧杆菌(B. pseudocatenulatum)14株,假长双歧杆菌(B. pseudolongum)8株,两歧双歧杆菌(B.bifidum)9株,短双歧杆菌(B.breve)7株,长双歧杆菌婴儿亚种(B.longumsubsp.infantis)5株,动物双歧杆菌乳亚种(B. animal subsp. lactis)6株以及长双歧杆菌(B. longum)3株。经过表型初筛和苯酚-硫酸法复筛,共筛选出7株高产胞外多糖的双歧杆菌,37℃发酵36 h后胞外多糖产量均可达400 mg/L以上。抗氧化活性实验结果表明7株双歧杆菌所产的胞外多糖对过氧化氢自由基、超氧阴离子自由基、羟自由基和1,1-二苯基-2-三硝基苯肼自由基均有一定的清除能力。此外,菌株BF66-16相较于其他几株双歧杆菌具有较强的胃肠液耐受性以及黏附性,因此来源于婴儿粪便的BF66-16可以作为潜在的抗氧化菌株应用于制药和食品工业中。     

新疆喀什地区维吾尔族婴幼儿肠道双歧杆菌遗传差异及益生特性分析

分析新疆喀什地区维吾尔族婴幼儿肠道双歧杆菌的遗传差异,以期为少数民族婴幼儿肠道益生双歧杆菌的开发奠定一定基础。本实验采用16S rRNA基因序列结合基因外重复回文序列聚合酶链式反应指纹分型技术,对菌株遗传结构差异进行分析,根据遗传指纹挑选部分代表性双歧杆菌,检测它们对常见6种致病菌的抑菌活性并对抑菌性能较好的菌株进行耐酸耐胆盐、碳源代谢及抗生素药敏实验。研究发现有75株为双歧杆菌,隶属于4个种及2个亚种,分别为Bifidobacterium bifidum、B. pseudocatenulatum、B. adolescentis、B. longum subsp. infantis和B. longum subsp. suis。抑菌实验结果表明,有16株双歧杆菌具有广谱抑菌性能;药敏实验显示这些菌株除对阿米卡星及万古霉素表现耐药外,对其他抗生素均表现敏感或中度敏感;耐酸耐胆盐实验表明抑菌菌株中B. longum subsp.infantis f65-26和B. pseudocatenulatum f115-8为耐受性最优菌株,具有潜在开发利用价值。     

嗜酸乳杆菌和双歧杆菌遗传性对其益生特性及货架期菌数的影响

以1株嗜酸乳杆菌和6株双歧杆菌为研究对象,鼠李糖乳杆菌LGG作为对照,对经传代4、5、6次过程中菌株益生特性和货架期菌数稳定性进行测试,结果表明：嗜酸乳杆菌Z-43在遗传稳定性、细胞黏附性和货架期菌数方面表现最好,连续传代6次对氯仿静电作用率约为100%,在25和-20℃条件下90 d菌数存活率在60%～100%。乳双歧杆菌Z-1传代4次表现为最佳益生特性,在模拟胃液孵育60 min和模拟肠液中孵育5 h存活率分别约为93.07%和106.5%,其静电作用率可达97.35%,25和-20℃货架期90 d菌数存活率分别在30.10%和92%,为最佳抗性菌株。     

鼠李糖乳杆菌LR-ZB1107-01的安全性评价及其益生特性初步研究

从广州1月龄婴儿粪便中分离得一株乳酸菌,经鉴定为鼠李糖乳杆菌(Lactobacillus rhamnosus ZB1107-01),通过对该菌的溶血性、抗生素敏感性和动物经口急性毒理等安全性评价,并研究标准菌株与其对致病菌抑菌性、胃肠道液耐受性、肠道黏附性、疏水性和自聚力。结果表明:此菌无溶血性;对四环素、红霉素、氨基西林和卡那霉素等4种抗生素表现高敏感;小鼠经口急性毒理无毒,确定该菌株为食用安全菌。该菌可抑制金黄色葡萄球菌、大肠杆菌、单增李斯特菌的生长,抑菌效果与标准菌株ATCC7469相当。对人工模拟的胃肠道液有很好的耐受性,对pH2的人工胃液耐受性略强于标准菌株。黏附Caco-2细胞数与标准菌株相当。疏水性(48.6%)高于标准菌株(27.2%),自聚性与标准菌株相当。研究为鼠李糖乳杆菌LR-ZB1107-01在食品领域进行商业化应用提供理论依据。     

动物双歧杆菌冻干保护剂配方优化及酸对菌粉存活率的影响

目的 筛选动物双歧杆菌冻干菌粉保护剂,优化冻干保护剂配方,探究菌悬液制备过程中有机酸积累对菌粉存活率的影响。方法 以菌粉中动物双歧杆菌存活率为指标,通过发酵培养、离心收集菌泥、制备菌悬液、预冻和冷冻干燥的菌粉制备工艺,通过单因素实验和正交实验优化冻干保护剂。根据单因素实验探究冻干前菌悬液制备条件和最适pH。结果 最佳保护剂组合为5.00%麦芽糊精、6.00%海藻糖、0.15%抗坏血酸、1.50%谷氨酸钠、1.00%甘油。通过对菌悬液制备过程中菌粉活菌数的研究确定菌悬液制备和冻干条件,菌悬液pH 6.5,无菌水洗涤2次菌, 4℃菌悬液融合30 min,在-80℃预冻2 h,-40℃下干燥24 h,获得的冻干菌粉活菌数为1.38×10¹² CFU/g,菌粉最高存活率可达98.60%。结论 本研究优化后的保护剂组合可以制备高活性动物双歧杆菌菌粉,提高经济效益。     

几株乳酸菌与降血糖相关的性能研究

选用从陕、甘、宁等地区传统乳酸菌发酵食品中筛选的18株乳酸菌,通过对其与降血糖相关的性能,即:对α-葡萄糖苷酶和α-淀粉酶抑制性、对自由基DPPH和O~(2-)的清除能力、对菌株还原能力等五个方面的研究,结果表明:18株乳酸菌的菌悬液对α-葡萄糖苷酶的抑制率总体高于发酵上清液和细胞破碎上清,其中乳双歧杆菌1号菌株的菌悬液对α-葡萄糖苷酶抑制率在所有菌株中最高为37.48%。其余四个方面均是发酵上清液作用较强,乳双歧杆菌1号发酵上清液对α-淀粉酶抑制抑制率、还原能力和对O~(2-)清除率在所有菌株中均最高,其中抑制率为33.85%,还原浓度为105.25μmol/L,清除率为34.18%。保加利亚乳杆菌1号发酵上清液对DPPH清除率在所有菌株中最高为82.92%。     

高产γ-氨基丁酸的双歧杆菌的筛选及对抑郁细胞模型的改善作用

为了筛选出具有缓解抑郁作用的菌株,本研究以94株双歧杆菌为研究对象,以胆盐水解酶活性、抗生素耐性、抗逆性、粘附性作为益生菌评价的基本指标;以高产γ-氨基丁酸(gamma-aminobutyric acid,GABA)作为潜在抗抑郁症菌株的筛选指标;最后通过体外抑郁细胞模型试验,研究菌株对抑郁细胞模型的改善作用,进而评价菌株改善抑郁的潜力。结果显示:13株双歧杆菌的胆盐水解酶活性较高,且都不具有种属特异性耐性之外的抗生素耐性。耐酸耐胆盐实验证明:双歧杆菌ZT3、84和95都具有较高的抗逆性能;粘附结果表明:双歧杆菌91、92、95和98具有较强的粘附能力,粘附能力分别为:19.34、33.77、23.23、20.46 CFU/Cell。13株双歧杆菌产GABA能力检测结果显示,双歧杆菌98和95的GABA的产量较高,分别为285.0和232.8 mg/L。抑郁细胞模型试验结果显示:高、中、低剂量高产GABA的双歧杆菌98都具有缓解CORT诱导的细胞损伤的能力,而只有高剂量的双岐杆菌95可以缓解CORT诱导的细胞损伤。由于双歧杆菌98、95具有安全的抗生素耐受范围,较高的酸、胆盐耐受性和粘附能力以及高产GABA和缓解细胞模型损伤的能力,因此,其可以作为潜在的改善抑郁症状的菌株。     

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.     

Interaction of probiotic Lactobacillus and Bifidobacterium strains with human intestinal epithelial cells: adhesion properties, competition against enteropathogens and modulation of IL-8 production

The human intestinal microbiota plays a pivotal role in human nutrition and health by promoting the supply of nutrients, preventing pathogen colonization and shaping and maintaining normal mucosal immunity. The depletion of the individual microbiota can result in a higher susceptibility to enteropathogenic bacteria infection. In order to reduce this risk, the use of food supplements containing probiotic bacteria has been recently addressed. In this paper, we investigate the protective role toward enteropathogen infection of probiotic strains belonging to Lactobacillus and Bifidobacterium. According to our experimental data, Lactobacillus acidophilus Bar13, L. plantarum Bar10, Bifidobacterium longum Bar33 and B. lactis Bar30 were effective in displacing the enteropathogens Salmonella typhimurium and Escherichia coli H10407 from a Caco-2 cell layer. Moreover, L. acidophilus Bar13 and B. longum Bar33 have been assessed for their immunomodulatory activity on IL-8 production by HT29 cells. Both strains showed the potential to protect enterocytes from an acute inflammatory response. These probiotic strains are potential candidates for the development of new functional foods helpful in counteracting enteropathogen infections.     

Bifidobacterium longum Survival During Frozen and Refrigerated Storage as Related to pH during Growth

Four strains of Bifidobacterium longum were grown at pH 5.5, 6.0, 6.5 and 7.0 and evaluated for survival and bile tolerance during frozen and subsequent refrigerated storage in milk. There were no reductions in cell numbers following initial freezing. There were effects for strain, pH and storage for three of the four strains of B. longum du ring storage at 5°C in milk. Bifidobacterium longum S9 was more stable than other strains in that no losses occurred, regardless of pH during growth. Results were variable for strains II, III, and ATCC 15707 grown at the various pH levels. Bifidobacterium longum S9 did not lose bile resistance during refrigerated storage as the other three strains did.     

Production of beta-galactosidase by Bifidobacteria as influenced by various culture conditions

Beta-Galactosidase production by Bifidobacterium longum CCRC 15708, Bifidobacterium longum B6 and Bifidobacterium infantis CCRC 14633 was first examined with B. longum CCRC 15708 showing the highest production of beta-galactosidase and the highest specific activity. Further study with B. longum CCRC 15708 revealed that the highest level of beta-galactosidase was produced with lactose and yeast extract as carbon and nitrogen sources, respectively. Optimal enzyme production occurred at an initial pH of 6.5 and at 37 degrees C. Under these optimum culture conditions, a maximumbeta-galactosidase activity of 18.6 U/ml could be obtained after 16 h of fermentation in a medium contain 4% lactose, 3.5% yeast extract, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO4.7H2O and 0.03% L-cysteine. The highest transgalactosylation activity was also detected in this culture after 14-16 h of fermentation.     

成人粪便中长双歧杆菌长亚种的分离鉴定及多位点序列分型分析

为了对成人粪便中分离的长双歧杆菌长亚种进行多位点序列分型分析,采用改良MRS培养基从健康成人粪便中分离的长双歧杆菌长亚种,通过生理生化试验结合16S rDNA和热应激蛋白60(heat-shock protein,hsp60)同源性分析鉴定分离株,利用同源性分析及多位点序列分型(multilocus sequence typing,MLST)技术进一步分析长双歧杆菌长亚种的基因多态性。从12个健康成人体内分离得到24株厌氧的细菌菌株,经形态学观察、生理生化试验、16S rDNA及hsp60同源性分析发现,其中14株分离株为长双歧杆菌长亚种(Bifidobacterium longum subsp. longum)。MLST结果表明:14株长双歧杆菌长亚种可分为10种基因型,且均与Bifidobacterium longum subsp. longum ATCC 15707为不同基因型。健康成人粪便中分离的长双歧杆菌长亚种具有较大的基因多态性。     

长双歧杆菌多因素连续驯化的研究

双歧杆菌因对外界环境的耐受性较差,限制了其规模化生产和实际应用。通过对长双歧杆菌进行连续的耐氧、耐酸以及耐胆盐的驯化,使长双歧杆菌对氧、酸以及胆盐的耐受性有了一定的提高,活菌数和产酸性能都有所提高。耐氧驯化时,通过逐渐增加培养基中的氧分压的方法进行;耐酸驯化时,通过逐渐降低培养基的初始pH的方法进行;耐胆盐驯化时,通过逐渐增加培养基初始胆盐含量的方法进行。实验结果表明,耐氧耐酸驯化后的菌株活菌数能达到9.4×108 cfu/mL,为初始菌的1.92倍;耐胆盐驯化后的菌株在胆盐浓度为0.3%时培养也能保持一定的活菌数,而初始菌株不能存活。