不同来源胆盐水解酶基因在大肠杆菌中的表达

根据GenBank上报道的两歧双歧杆菌ATCC 29521的胆盐水解酶基因(BSH)序列和嗜酸乳杆菌NCFM的胆盐水解酶基因(BSHA和BSHB)序列,设计引物通过PCR扩增获得BSH基因,将其连接到表达载体pET28a(+),构建pETBSH表达质粒,经IPTG诱导表达后,聚丙烯酰胺凝胶电泳(SDS-PAGE)检测表明在分子质量大小约40、43、45kD处有预期条带出现,初步表明蛋白表达成功。重组菌产生的胆盐水解酶水解甘氨胆酸钠产生的甘氨酸经茚三酮比色法分析,表明该重组的胆盐水解酶具有水解活性。     

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。     

Novel bifidobacterial glycosidases acting on sugar chains of mucin glycoproteins

Bifidobacterium bifidum was found to produce a specific 1,2-alpha-L-fucosidase. Its gene (afc A) has been cloned and the DNA sequence was determined. The Afc A protein consisting of 1959 amino acid residues with a predicted molecular mass of 205 kDa can be divided into three domains; the N-terminal function-unknown domain (576 aa), the catalytic domain (898 aa), and the C-terminal bacterial Ig-like domain (485 aa). The recombinant catalytic domain specifically hydrolyzed the terminal alpha-(1-->2)-fucosidic linkages of various oligosaccharides and sugar chains of glycoproteins. The primary structure of the catalytic domain exhibited no similarity to those of any glycoside hydrolases but showed similarity to those of several hypothetical proteins in a database, which resulted in establishment of a novel glycoside hydrolase family (GH family 95). Several bifidobacteria were found to produce a specific endo-alpha-N-acetylgalactosaminidase, which is the endoglycosidase liberating the O-glycosidically linked galactosyl beta1-->3 N-acetylgalactosamine disaccharide from mucin glycoprotein. The molecular cloning of endo-alpha-N-acetylgalactosaminidase was carried out on Bifidobacterium longum based on the information in the database. The gene was found to comprise 1966 amino acid residues with a predicted molecular mass of 210 kDa. The recombinant protein released galactosyl beta1-->3 N-acetylgalactosamine disaccharide from natural glycoproteins. This enzyme of B. longum is believed to be involved in the catabolism of oligosaccharide of intestinal mucin glycoproteins. Both 1,2-alpha-L-fucosidase and endo-alpha-N-acetylgalactosaminidase are novel and specific enzymes acting on oligosaccharides that exist mainly in mucin glycoproteins. Thus, it is reasonable to conclude that bifidobacteria produce these enzymes to preferentially utilize the oligosaccharides present in the intestinal ecosystem.     

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.     

Myo-inositol hexakisphosphate degradation by Bifidobacterium infantis ATCC 15697

The ability of five strains of different bifidobacterial species (Bifidobacterium animalis, B. bifidum, B. infantis, B. longum, B. pseudolongum) isolated from human and animal gut to degrade myo-inositol hexaphosphate or phytic acid (InsP(6)) has been evaluated. The disappearance of phytate and the generation of lower myo-inositol phosphates were determined in a complex medium in which phytic acid was the only source of phosphorus. Bifidobacterium infantis ATCC 15697 showed the highest level of phytate-degrading activity. This strain displayed optimal activity at slight acid pH (6.0-6.5) and 50 degrees C, but also retained high activity levels at neutral pH and 37 degrees C. B. infantis ATCC 15697 produced maximum activity during the stationary phase of growth and when 1% lactose was used as carbon source. In contrast, the presence of inorganic phosphate in the growth medium inhibited phytase activity. The ability of B. infantis ATCC 15697 to generate lower myo-inositol phosphates and, particularly, accumulate myo-inositol tri-phosphates (InsP(3)) from partial hydrolysis of InsP(6) could contribute to the reduction of the anti-nutritional properties of InsP(6) and the generation of intermediate compounds with beneficial properties during food processing and gastrointestinal transit.     

Bile salt deconjugation and BSH activity of five bifidobacterial strains and their cholesterol co-precipitating properties

Five strains of bifidobacteria were screened for their bile salt deconjugation ability, bile salt hydrolase (BSH) activity and co-precipitation of cholesterol with deconjugated bile. Bile salt deconjugation was determined by the release of cholic acid. All strains exhibited deconjugation of both sodium glycocholate and sodium taurocholate. More cholic acid was liberated from the deconjugation of sodium glycocholate than sodium taurocholate. BSH activity was quantified by determining the amount of glycine or taurine liberated from conjugated bile salts by bifidobacteria strains. There was higher substrate specificity for glycine-conjugated bile compared to taurine-conjugated bile. Co-precipitation of cholesterol with cholic acid was observed from deconjugation of both sodium glycocholate and sodium taurocholate, and by all bifidobacteria strains studied. More cholesterol was precipitated with cholic acid when sodium glycocholate was used compared to sodium taurocholate. Increased cholesterol co-precipitation with deconjugated bile was observed with decreasing pH levels. Bifidobacterium infantis 17930 showed highest deconjugation ability and BSH activity towards bile mixtures that resemble the human bile, and may be a promising candidate to exert beneficial bile deconjugation activity in vivo.     

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.     

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。     

Short communication: Identification and differentiation of bifidobacteria obtained from Ukraine

Ten freeze-dried bifidobacterial strains used as probiotics in Ukrainian dairy foods, identified by the supplier as Bifidobacterium adolescentis (2), Bifidobacterium bifidum (2), Bifidobacterium longum (4), Bifidobacterium animalis (1), and Bifidobacterium infantis (1), were characterized. Following rehydration and anaerobic growth on de Man, Rogosa, and Sharpe-cysteine medium at 37°C for 72 h, single-colony isolates were picked and evaluated using PCR primers specific for the Bifidobacterium genus, for the supplier-identified species, and for B. animalis ssp. lactis. All isolates were identified as members of the genus Bifidobacterium; however, species-specific PCR revealed all 10 isolates were actually strains of B. animalis ssp. lactis. Further evaluation using pulsed-field gel electrophoresis was only able to separate a single strain (RT 09) from the other 9 strains evaluated. Application of genome-wide allelic profiling to the Ukrainian bifidobacterial strains revealed 4 distinct groups. Interestingly, 6 (60%) of the isolates fell into the same cluster as that containing the common commercial probiotic strain BB-12. Two of the strains (RT 02 and RT 09) were found to be in the same group as ATCC 27536 and one strain (RT 08) was in the same group as the RB 7239 (a previously evaluated commercial strain). One strain, RT 04, was placed on a unique branch. These results highlight the importance of employing routine typing of bifidobacterial isolates, demonstrate the utility of single nucleotide polymorphism/insertion-deletion polymorphism-based allelic typing in B. animalis ssp. lactis strain differentiation and further point to the limited genetic variability of B. animalis ssp. lactis strains and the worldwide distribution of a small number of commercial strains.     

Bile salt deconjugation ability,bile salt hydrolase activity and cholesterol co-precipitation ability of lactobacilli strains

Eleven strains of lactobacilli were screened for their bile salt deconjugation ability, bile salt hydrolase activity (BSH) and co-precipitation of cholesterol with deconjugated bile. Bile salt deconjugation as determined by the release of cholic acid showed that more cholic acid was liberated from the deconjugation of sodium glycocholate than sodium taurocholate, and Lactobacillus acidophilus strains had higher deconjugation ability than L. casei strains. BSH activity, as quantified by the amount of taurine or glycine liberated from conjugated bile salts, indicated that substrate specificity was more towards glycine-conjugated bile compared to taurine-conjugated bile. Co-precipitation of cholesterol with cholic acid was observed from deconjugation of both conjugated bile, with more cholesterol being precipitated upon deconjugation of sodium glycocholate than upon that of sodium taurocholate. Cholesterol co-precipitation with deconjugated bile increased with decreasing pH. L. acidophilus ATCC 33200, 4356 and 4962 and L. casei ASCC 1521 showed highest deconjugation ability and BSH activity towards bile mixtures that resemble the human bile, and may be promising candidates to exert beneficial bile deconjugation activity in vivo.     

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。     

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.     

Protection of bifidobacteria encapsulated in polysaccharide-protein gel beads against gastric juice and bile

Bifidobacterium cells were encapsulated in a mixed gel composed of alginate, pectin, and whey proteins. Two kinds of capsules were obtained: gel beads without membranes and gel beads with two membranes formed by the transacylation reaction. In vitro studies were carried out to determine the effects of simulated gastric pH and bile salts on the survival of free and encapsulated Bifidobacterium bifidum. The protective effects of gel beads without membranes and gel beads coated with two membranes formed by the transacylation reaction were evaluated. After 1 h in an acidic solution (pH 2.5), the free-cell counts decreased by 4.75 log units, compared with a <1-log decrease for entrapped cells. The free cells did not survive after 2 h of incubation at pH 2.5, while immobilized-cell counts decreased by about 2 log units. After incubation (1 or 3 h) in 2 and 4% bile salt solutions, the bifidobacterium mortality level for membrane-free gel beads (4 to 7 log units) was higher than that for free cells (2 to 3 log units). However, counts of bifidobacteria immobilized in membrane-coated gel beads decreased by <2 log units. Cell encapsulation in membrane-coated protein-polysaccharide gel beads could be used to increase the survival of healthy probiotic bacteria during their transit through the gastrointestinal tract.     

Characterisation of a Bifidobacterium strain with acquired resistance to cholate--a preliminary study

The susceptibility levels against bile salts (ox gall) and sodium cholate of 19 Bifidobacterium strains from different origins ranged between 0.125% and 2.0% and 0.05% and 0.4%, respectively. Seven of these strains were subsequently selected for the isolation of sodium cholate resistant derivatives by exposure to gradually increasing concentrations of this compound. Derivatives resistant to at least 1.2% of cholate were obtained from Bifidobacterium bifidum CECT 4549 and B. bifidum M6. Further analysis of the CECT 4549 derivative indicated that the phenotype induced was stable; the enzymatic activities analysed remained unchanged and no major rearrangements of chromosomal DNA were produced. However, the cells of the resistant derivative were smaller and more regular in average size than those of the original strain. In addition, the derivative had lost the ability to use lactose, fructose and galactose, and showed reduced expression levels of two membrane proteins of 78 and 114 kDa. Finally, the resistant strain displayed higher survival and lower growth inhibition in the presence of cholate than the original strain.     

乳清蛋白在转谷氨酰胺酶作用下包埋益生菌的研究

为了提高双歧杆菌在人体胃肠道中的存活率,以乳清蛋白为壁材,转谷氨酰胺酶为交联剂,通过乳化凝胶的方法制备包埋有两歧双歧杆菌的蛋白质微球。实验表明:以此工艺制备的微球成球性较好,粒径为(308.2±16.2)μm,益生菌包埋率为87.8%±10.0%,与未包埋的两歧双歧杆菌比较,经过包埋后的两歧双歧杆菌在模拟胃液和高胆盐溶液中的存活率分别提高了5个和2个对数值。     

All 4 bile salt hydrolase proteins are responsible for the hydrolysis activity in Lactobacillus plantarum ST-III

In vertebrates, bile salt hydrolysis plays an essential role in fat metabolism. Bile salts are synthesized in the liver. And in the small intestine glycine and taurine are de-conjugated from bile salts by the enzyme bile salt hydrolase (BSH) from intestinal microbes. However, the mechanism of bile salt hydrolysis in Lactobacillus plantarum is still ambiguous. Four predicted bile salt hydrolase (bsh) genes from L. plantarum ST-III were cloned into Escherichia coli. The function of these genes was explored by overexpression. All 4 proteins that were studied showed activity against glycine- or taurine-conjugated bile salts. Substrate preference was also observed in BSH proteins, especially for the enzyme BSH1, which had high hydrolysis activity for glycodeoxycholic acid. These results suggest that all 4 bile salt hydrolases may be responsible for the bile salt hydrolysis activity in L. plantarum ST-III. PRACTICAL APPLICATION: Hypercholesterolemia is considered one of the major risk factors for coronary heart disease. More interest has focused on intestinal microbes because of their role in the decrease of serum cholesterol. BSH proteins play an important role in the reduction of cholesterol. This paper adds to a better understanding of BSH proteins of intestinal microbes. It gives a great hint that probiotics can be used to solve hypercholesterolemia one day.     

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。     

Competitive exclusion of enteropathogens from human intestinal mucus by Bifidobacterium strains with acquired resistance to bile--a preliminary study

The ability to inhibit the adhesion and to displace selected pathogens from human intestinal mucus of two Bifidobacterium strains with acquired resistance to bile, were assessed and compared with those of their bile sensitive original strains. A preliminary characterization of the macromolecules involved in the adhesion was also carried out. The inhibition of adhesion and the displacement of enteropathogens previously adhered were found to be specific, depending on the strains used. The cholate-resistant strain Bifidobacterium bifidum M6dCo, that adhered more to mucus than its original, was able to inhibit the adhesion and to displace pathogens from mucus significantly more than its original cholate-sensitive strain B. bifidum M6. Contrary to this, two strains showing similar adhesion levels, B. bifidum A1 and its bile resistant derivative B. bifidum A1dOx, did not display any differences. Different molecules appear to be involved in the adhesion of the strains B. bifidum M6 and B. bifidum M6dCo. These differences in the cellular surface may explain the differences in competitive exclusion observed between both strains.     

Short communication: Improving the activity of bile salt hydrolases in Lactobacillus casei based on in silico molecular docking and heterologous expression

Bile salt hydrolase (BSH) plays an essential role in the cholesterol-removing effect of lactic acid bacteria, which hydrolyze conjugated bile salts to amino acid and deconjugated bile salts. However, Lactobacillus casei lacks the bsh gene, which may make it highly sensitive to bile salt stress. We wanted to improve the BSH activity of L. casei for various food-industry applications (e.g., milk fermentation). Plate assay testing indicated that Lactobacillus plantarum AR113 has the highest BSH activity. We cloned and sequenced 4 bsh genes from the genome of L. plantarum AR113. Structure modeling and molecular docking of BSH indicated that BSH1 and BSH3 could react efficiently with bile salts, so we selected BSH1 and BSH3 for heterologous expression in L. casei. Compared with single expression of BSH1 or BSH3, co-expression of both protein sequences showed the highest hydrolysis activity by HPLC analysis. Our results suggested that heterologous expression of BSH in L. casei can significantly improve host activity against bile salts, and in silico molecular docking could be an efficient method of rapid screening for BSH with high activity.     

Molecular cloning,expression and characterization of bile salt hydrolase from Lactobacillus rhamnosus E9 strain

Bile salt hydrolase (BSH) enzyme, commonly found in probiotic bacteria of gut origin, catalyzes the hydrolysis of glycine and/or taurine-conjugated bile salts allowing for colonization of the bacteria in the gut and contributing to a decrease in levels of cholesterol. However an excessive deconjugation of tauro-conjugated bile salts and production of secondary bile acid can have harmful side-effects. The aim of this study was to characterize the activity of BSH enzymes from Lactobacillus rhamnosus E9, a popular probiotic strain. The bsh gene was cloned, expressed, purified and characterized in Escherichia coli BLR(DE3) strain. The hydrolysis activities and substrate specificities of the recombinant BSH (rBSH) enzyme were examined using six different bile acids. Nucleotide sequence analysis results indicated that the bsh of E9 contained an open reading frame (ORF) of 1014 and nucleotides encoding a 338-amino acid protein with a molecular weight of 37 kDa. Five catalytically important amino acids and the amino acid motifs located around the active site were highly conserved. The rBSH showed a slight preference towards glycine-conjugated to tauro-conjugated bile salts. This confirms that it is a safe strain for probiotics and its preference for glycine-conjugated bile salts should be further investigated.