双歧杆菌中胆盐水解酶的研究进展

胆盐水解酶的特性及生理功能已成为研究热点。我们探讨了国内外关于双歧杆菌中胆盐水解酶的生化特性、降胆固醇机理及其生理作用几个方面的研究进展,以期对研究双歧杆菌胆盐水解酶,以及双歧杆菌及其制品的开发利用有一定的指导意义。     

从人胃肠道源乳杆菌株中初步筛选耐酸耐胆盐优良株

目的筛选对酸和胆盐耐受性均较强的优良乳杆菌株。方法模拟人体胃酸环境（pH=3）和十二指肠高胆盐环境（胆盐含量=3‰）测定104株人胃肠道源乳杆菌在酸性及高胆盐环境下作用4h后的存活菌数,并与对照相比较。对结果进行统计学分析。结果16株菌在酸性环境及高胆盐环境下4h后的活菌下降对数值均小于2。筛选出3株具有较强耐酸耐胆盐能力的乳杆菌株。结论大多数人胃肠道源乳杆菌对酸和胆盐的耐受能力均不强,对酸的耐受性普遍强于对胆盐的耐受性。     

类植物乳杆菌的耐酸、耐胆盐及降胆固醇特性

摘要：【目的】研究了类植物乳杆菌Ⅱ32的酸、胆盐耐受性和胆固醇去除能力,并通过菌株生长和胆固醇去除的关系探讨可能的机理。【方法】菌株Ⅱ32生长在高胆固醇MRS培养基中,胆固醇的检测通过气相色谱法。检测菌株在不同生长阶段对胆固醇的去除情况。【结果】菌株Ⅱ32具有酸耐受性、胆盐耐受性和一定的胆固醇清除能力。菌株在pH2.0培养2 h后仍能达到104 cfu/mL;加胆盐（0.3%～0.4%）对菌株生长量达到OD值0.6的时间延迟在0.5 h以内;热杀死的和休眠的细胞能去除很少的胆固醇,分别是5.64、5.90 mg/g细胞干重,而生长的细胞去除的胆固醇达到16.98 mg/g细胞干重。另外,研究表明胆固醇去除与菌体的生长有一定的相关性。【结论】菌株Ⅱ32去除胆固醇可能的机理是菌体对胆固醇的吸附及菌体在生长过程中对胆固醇的吸收利用,为此该菌株具有添加到食品中来降低血液胆固醇的潜能。     

罗伊乳杆菌LE16益生特性的研究

目的研究罗伊乳杆菌LE16的益生特性。方法通过体外和动物实验,评价罗伊乳杆菌LE16的益生特性。结果该菌株对p H 2.5的强酸和0.3%高胆盐环境均有良好的耐受性。对引起肠道感染的几种病原菌均有不同程度的拮抗,尤其对枯草芽胞杆菌、伤寒沙门菌、单核细胞增生李斯特菌及蜡样芽胞杆菌的抑制作用最强。对肠道菌群正常和失调的小鼠均具有调节功能。结论罗伊乳杆菌LE16具有良好的益生性能,可作为益生菌菌株开发应用于食品和保健食品中。     

一种益生菌组合的降胆固醇能力研究

目的研究一种由乳双歧杆菌HN019和鼠李糖乳杆菌HN001构成的益生菌组合的降胆固醇能力。方法分别采用体外测定胆固醇脱除率和灌胃高脂模型大鼠的方法,考察该益生菌组合物的降胆固醇能力。结果该益生菌组合物体外的胆固醇脱除率达到78.82%,能够显著降低高脂模型大鼠的血清总胆固醇(TC)、甘油三酯(TG)和低密度脂蛋白胆固醇(LDL-C),且差异有统计学意义(P0.01)。该组合物的降胆固醇能力均优于单一菌株。结论该益生菌组合物具有较强的降胆固醇能力,可考虑进一步开发为降胆固醇微生态制剂或保健食品。     

模拟人体胃肠道环境筛选益生乳杆菌

【目的】筛选具有益生特性的乳杆菌作为保健型酸奶的候选菌株。【方法】从健康人肠道和奶豆腐中分离筛选出耐受人工胃液的乳杆菌,对其进行体外益生特性(人工胃肠液耐受性、胆盐耐受性、抑菌活性及胆固醇降解能力)研究。【结果】从在乳杆菌分离培养基上有溶钙圈的41株菌株中筛选出5株耐酸、耐人工胃液较强的菌株,经16S rR NA基因测序鉴定,其中3株为乳杆菌,分别命名为植物乳杆菌Lp MT-3、植物乳杆菌Lp MT-5和唾液乳杆菌LsA F-7。在人工胃液中3株菌的耐受力均强于商品化的对照菌株LGG(鼠李糖乳杆菌GG);转入肠液4 h后直至26 h,Lp MT-5存活率基本稳定在45%左右,仅次于LGG。胆盐浓度为0.10%时,3株乳杆菌的耐胆盐能力均强于LGG;胆盐浓度为0.20%时,Lp MT-3和LsA F-7仍能存活。3株乳杆菌均具有抑菌活性,对粪肠球菌的抑制最明显,其次是金黄色葡萄球菌,对大肠杆菌、沙门氏菌的抑制作用较差。3株乳杆菌对胆固醇的清除效力依次为Lp MT-3LpM T-5Ls AF-7;清除率依次为Ls AF-7Lp MT-3LpM T-5。【结论】筛选出3株适应人体胃肠液环境、耐胆盐、抑菌及降胆固醇活力强的乳杆菌,可作为进一步开发新的益生菌产品和保健型酸奶的菌株。     

长寿老人源双歧杆菌优良菌株的筛选*

以人结肠腺癌细胞系HT-29细胞为试材,对来源于广西巴马百岁以上长寿老人肠道的24株双歧杆菌进行了体外黏附试验。结果发现,双歧杆菌均具有一定的黏附能力,其中TTF、Z2、TZ5和J-1菌株具有较高的黏附能力。进一步对4株初筛双歧杆菌耐胃酸、胆汁酸和合成B族维生素能力的试验发现,双歧杆菌TTF菌株不仅能合成较高的B1、B2、B6、B12等多种B族维生素,而且在pH 3.0的条件下处理120m in存活率达93.11%,同时在2%胆盐浓度下处理24h有较好的存活,具有显著的综合优势。     

降胆固醇嗜酸乳杆菌的诱变选育

通过诱变获得具有降胆固醇功能的优良嗜酸乳杆菌新菌株。利用亚硝基胍(作用浓度为1 g/L)对嗜酸乳杆菌进行诱变。突变菌株测定其耐渗透压和抗胆盐能力后,在含有0.3 g/L胆固醇的培养基中培养48 h,测定降胆固醇率。挑选优良突变菌株制成酸奶并喂养高脂大鼠模型,28 d后测定血清及粪便胆固醇指标。嗜酸乳杆菌突变后获得的60个突变菌株中有8株具有良好的耐渗透压和抗胆盐能力,其中突变株Y48的清除胆固醇能力最高,清除率达到(61.44±1.8)%。Y48发酵酸奶喂养高脂大鼠模型28 d后与对照大鼠模型相比,血清中TC、TG明显减少(P<0.05),粪便TC明显增加。通过诱变获得了优良的嗜酸乳杆菌突变菌株,为今后获得优质降胆固醇乳酸制品提供良好的候选菌种。     

不同物质对双歧杆菌胆盐耐受性的影响研究

初步研究不同物质对双歧杆菌的胆盐耐受性的影响。结果表明,脱氧胆酸钠盐对双歧杆菌Blm和Bbm有毒性作用;在培养基中补充一定量的葡萄糖和果糖可以改善Bbm的胆盐耐受性,而菊糖和菊芋粉对提高Blm和Bbm的胆盐耐受性没有明显作用。     

人体肠道益生菌体外降胆固醇活性研究

从健康儿童和青年人肠道分离并鉴定了21株乳杆菌和双歧杆菌,连同6株实验室保藏菌株进行了体外降胆固醇、耐酸及耐胆汁盐实验。结果表明,所有实验菌株都能从培养基中去除胆固醇,5株去除率可达40%以上,同时去除效力也较高。胆汁盐耐受性和耐酸性具有菌株特异性。菌株Bm26同时具有较高降胆固醇能力和耐胆汁盐及耐酸性能。     

蒙古族儿童源益生特性双歧杆菌的筛选及鉴定

【目的】双歧杆菌在人和动物胃肠道中发挥着重要的生理作用,然而双歧杆菌能否耐受胃酸、肠液及胆汁酸是影响活菌制剂益生效果的关键因素,本研究旨在从蒙古族儿童粪便中分离筛选出具有良好益生特性的双岐杆菌。【方法】本文采用双岐杆菌选择性培养基对样品进行分离纯化,并对菌株进行生理生化鉴定,以耐受人工胃肠液、耐受牛胆盐为手段对各菌株的益生特性进行评价,并且对B. animalisV9进行了16S rDNA分子生物学鉴定。【结果】本文从12位健康蒙古族儿童粪便中分离得到11株双歧杆菌,经传统生理生化试验鉴定为5株B. adolescentis：A1、H3、G4、A8、V10;3株B. longum：C6、C7、D11;1株B. pseudocatenlatum：B2;1株B. bifidum：G5;1株B. animalis：V9。B. animalis V9具有较强的耐酸性,在pH2.0的人工胃液中厌氧培养3h后存活率为92.4%,而其它10株双歧杆菌在此条件下的存活率均小于31.25%;B. animalis V9在pH2.0的人工胃液中厌氧培养3h后接入pH8.0的人工肠液中消化8h,存活率为99.7%,并且可以耐受0.3%的牛胆盐。进一步对V9菌株进行16S rDNA分子生物学鉴定,发现与Bifidobacterium animalis subsp. lactic BB12的同源性为99%。【结论】本研究结果显示B. animalis V9来源安全,并且具有良好耐酸、耐胆盐益生特性,有望在乳制品及保健类产品中得到广泛的应用。     

Molecular characterization of bile salt hydrolase from Bifidobacterium animalis subsp. lactis Bi30

The present work describes the identification, purification, and characterization of bile salt hydrolase (BSH) from Bifidobacterium animalis subsp. lactis. The enzyme was purified to electrophoretic homogeneity by hydrophobic chromatography, ion-exchange chromatography and ultrafiltration. SDS-PAGE analysis of putative BSH and gel filtration revealed that the analyzed protein is presumably a tetramer composed of four monomers each of about 35 kDa. The purified enzyme was analyzed by liquid chromatography coupled to LTQ FT ICR mass spectrometry and unambiguously identified as a bile salt hydrolase from B. animalis. The isoelectric point of the studied protein was estimated to be around pH 4.9. The pH optimum of the purified BSH is between 4.7 to 6.5, and the temperature optimum is around 50 degrees C. The BSH of B. animalis could deconjugate all tested bile salts, with clear preference for glycine-conjugated bile salts over taurine-conjugated forms. Genetic analysis of the bsh showed high similarity to the previously sequenced bsh gene from B. animalis and confirmed the usefulness of bile salt hydrolase as a genetic marker for B. animalis identification.     

Effects of fructooligosaccharides and their monomeric components on bile salt resistance in three species of bifidobacteria

The influence of fructooligosaccharides (FOS) and their monomeric components on bile salt resistance of Bifidobacterium breve ATCC 15700, Bif. longum ATCC 15707 and Bif. animalis ATCC 25527 was examined. The neosugars induced fructofuranosidase activities for the degradation of these saccharides. For the three strains tested the growth was identical and bile salts had the same inhibitory effect on growth whatever the carbohydrate used. The survival of Bif. breve and Bif. longum, in the presence of glycodeoxycholic acid depended, however, on carbohydrates: the toxic effects of the bile salt could be partly alleviated by the addition of a metabolizable C-source. For Bif. animalis, the presence of any carbohydrate in the incubation medium did not enhance the viability of the strain. But in the three deconjugating strains of bifidobacteria studied, the presence of neosugar during the growth led to improved resistance to the bactericidal effect of the bile salt compared with the monomeric components of these neosugars (glucose and fructose).     

Adaptation and response of Bifidobacterium animalis subsp. lactis to bile: a proteomic and physiological approach

Bile salts are natural detergents that facilitate the digestion and absorption of the hydrophobic components of the diet. However, their amphiphilic nature makes them very inhibitory for bacteria and strongly influences bacterial survival in the gastrointestinal tract. Adaptation to and tolerance of bile stress is therefore crucial for the persistence of bacteria in the human colonic niche. Bifidobacterium animalis subsp. lactis, a probiotic bacterium with documented health benefits, is applied largely in fermented dairy products. In this study, the effect of bile salts on proteomes of B. animalis subsp. lactis IPLA 4549 and its bile-resistant derivative B. animalis subsp. lactis 4549dOx was analyzed, leading to the identification of proteins which may represent the targets of bile salt response and adaptation in B. animalis subsp. lactis. The comparison of the wild-type and the bile-resistant strain responses allowed us to hypothesize about the resistance mechanisms acquired by the derivative resistant strain and about the bile salt response in B. animalis subsp. lactis. In addition, significant differences in the levels of metabolic end products of the bifid shunt and in the redox status of the cells were also detected, which correlate with some differences observed between the proteomes. These results indicate that adaptation and response to bile in B. animalis subsp. lactis involve several physiological mechanisms that are jointly dedicated to reduce the deleterious impact of bile on the cell's physiology.     

Screening of bifidobacteria with acquired tolerance to human gastrointestinal tract

Tolerance capabilities of 38 Bifidobacterium strains were achieved by simulating the micro-environment of human gastrointestinal tract using modified MRSC broth (pH 3.0). Fourteen strains of them with high viability were obtained in MRSC with 0.3% bile salts. Sequently, six strains of bifidobacteria with higher survivability were picked in MRSC broth (pH 3.0) supplemented with 1-20mg/ml bile salt. Finally, strain A04 with the optimal ability was chosen for further studies. It had been seen that Bifidobacterium breve A04 had better survival capability to 0.5% pepsin (w/v) or 1% pancreatin (w/v) than other bifidobacteria, and viable bacteria were above 8.00 log cfu/ml after incubation for 24h. Meanwhile, it had higher adhesive capability to HT-29 cells in vitro and average adhesive bacteria numbers reached 12.8+/-0.9 for each HT-29 cell. The results indicated that the ability to tolerate gastroenteric environment and the adhesive capacity to HT-29 cells among Bifidobacterium strains was different. B. breve A04 has several aspects of advantages and may be regarded as potential probiotics.     

The F1F0-ATPase of Bifidobacterium animalis is involved in bile tolerance

Adaptation and tolerance to bile stress are important factors for the survival of bifidobacteria in the intestinal tract. Bifidobacterium animalis is a probiotic microorganism which has been largely applied in fermented dairy foods due to its technological properties and its health-promoting effects for humans. The effect of the presence of bile on the activity and expression of F₁F₀-ATPase, the pool of ATP and the intracellular pH of B. animalis IPLA 4549 and its mutant with acquired resistance to bile B. animalis 4549dOx was determined. The bile-resistant mutant tolerated the acid pH better than its parent strain. Bile induced the expression of the F₁F₀-ATPase and increased the membrane-bound H⁺-ATPase activity, in both parent and mutant strains. In acidic conditions (pH 5.0), the expression and the activity of this enzyme were higher in the mutant than in the parent strain when cells were grown in the absence of bile. Total ATP content was higher for the mutant in the absence of bile, whereas the presence of bile induced a decrease of intracellular ATP levels, which was much more pronounced for the parent strain. At pH 4.0, and independently on the presence or absence of bile, the mutant showed a higher intracellular pH than its parent strain. These findings suggest that the bile-adapted B. animalis strain is able to tolerate bile by increasing the intracellular ATP reserve, and by inducing proton pumping by the F₁F₀-ATPase, therefore tightly regulating the internal pH, and provide a link between the physiological state of the cell and the response to bile.     

A bile salt-resistant derivative of Bifidobacterium animalis has an altered fermentation pattern when grown on glucose and maltose

The growth of Bifidobacterium animalis subsp. lactis IPLA 4549 and its derivative with acquired resistance to bile, B. animalis subsp. lactis 4549dOx, was evaluated in batch cultures with glucose or the glucose disaccharide maltose as the main carbon source. The acquisition of bile salt resistance caused a change in growth pattern for both sugars, which mainly resulted in a preferential use of maltose compared to glucose, whereas the mother strain used both carbohydrates in a similar way. High-performance liquid chromatography and gas chromatography-mass spectrometry analyses were performed to determine the amounts of glucose consumption and organic acid and ethanol formation from glucose by buffered resting cells taken at different points during growth. Resting cells of the bile-adapted strain generally consumed less glucose than those of the nonadapted one but showed an enhanced production of ethanol and higher acetic acid-to-lactic acid as well as formic acid-to-lactic acid ratios. These findings suggest a shift in the catabolism of carbohydrates promoted by the acquisition of bile resistance that may cause changes in the redox potential and improvements in the cellular ATP yield.     

一株降血压功能乳酸菌在模拟胃肠环境中抗性的研究

目的研究具有降血压功能乳酸菌DM9057株对pH、胆汁盐、胃蛋白酶及胰蛋白酶的耐受性。方法以MRS为基础培养基,模拟胃肠环境。结果 DM9057在pH2~3、胆汁盐浓度0.1%~0.3%、胃蛋白酶0.592~1.482μg/mL和胰蛋白酶14.24~72.32 U/g的条件下,37℃培养4 h,活菌数均能达到107CFU/mL以上。结论乳酸菌DM9057能顺利通过胃肠道,达到促进健康的作用。