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.     

一株产胆盐水解酶植物乳杆菌的发酵培养基的优化

为了提高植物乳杆菌Lactobacillus plantarum KLDS1.0386的胆盐水解酶产量,本实验通过响应面法对其发酵培养基进行优化。通过单因素实验、Plackett-Burman实验、最陡爬坡实验和Box-Behnken实验,最终获得最优培养基为:葡萄糖18.2 g/L、蛋白胨15.03 g/L、酵母粉9.97 g/L、乙酸钠3.13 g/L、柠檬酸氢二铵2.00 g/L、磷酸氢二钾2.00 g/L、硫酸锰0.25 g/L、硫酸镁0.58 g/L。优化前植物乳杆菌KLDS1.0386产胆盐水解酶的比酶活为1.04 U/mg,经过培养基的优化后,胆盐水解酶的比酶活为3.37 U/mg,比优化前提高了3.24倍。且实验结果与模型预测结果误差在允许范围内,说明该模型可以投入使用。      

Measurement of bile salt hydrolase activity from Lactobacillus acidophilus based on disappearance of conjugated bile salts

Bile salt hydrolase activity of Lactobacillus acidophilus was measured based on the disappearance of sodium glycocholate and sodium taurocholate from the reaction mixture using HPLC. The amount of sodium glycocholate and sodium taurocholate that disappeared was proportional to the amount of sodium cholate that appeared in the mixture as detected by HPLC. Sodium glycocholate did not precipitate at the enzyme reaction conditions (37 degrees C and pH 5.4) for determining bile salt hydrolase activity. The bile salt hydrolase assay was insensitive to low oxidation-reduction potential when measuring bile salt hydrolase from L. acidophilus, an intestinal microorganism. However, EDTA and freezing temperatures were necessary to maintain stability of the partially purified enzyme during storage.     

响应面法优化植物乳杆菌发酵产胆盐水解酶的培养基成分

以胆盐水解酶的活性为响应值,采用响应面设计分析法对植物乳杆菌发酵产胆盐水解酶培养基的成分进行优化.获得最佳培养基组成(g/L):葡萄糖19.40、蛋白胨17.05、大豆蛋白胨14.00、乙酸钠1.82、K2HPO4 2.00、酵母浸粉6.00、柠檬酸氢二铵1.00、MgSO40.87、MnSO40.45、L-半胱氨酸0.5、吐温-80 2.2mL/L.在该优化条件下,胆盐水解酶产量为(7.02±0.28) U/mL,较单因素优化前提高了6.44倍.所得数学模型的预测值与实验观察值相符,能够预测不同培养条件下植物乳杆菌发酵产胆盐水解酶产量的变化.     

植物乳杆菌中胆盐水解酶的研究现状及展望

益生菌的降胆固醇作用与它们所产的胆盐水解酶(bile salt hydrolase,BSH)有关,这使得胆盐水解酶成为近年来研究的热点之一。文中主要对植物乳杆菌中胆盐水解酶的克隆表达、生化特性以及降胆固醇作用等方面的研究现状进行了综述,并展望了它未来的发展方向。以期对植物乳杆菌胆盐水解酶的研究及其相关制品的开发利用提供一定的指导意义。      

Hypocholesteremic effect of bile salt hydrolase from Lactobacillus buchneri ATCC 4005

The effect of oral administration of the immobilized bile salt hydrolase enzyme on serum cholesterol, triglyceride, high density lipoprotein levels and its application in the therapeutic treatment of hypercholesteremia was assessed. Culture conditions were optimized for the production of bile salt hydrolase, which resulted in 2.9-fold enhancement in activity. Bile salt hydrolase (BSH; E.C.3.5.1.24) was isolated from Lactobacillus buchneri ATCC 4005 and immobilized in 0.5% gellan gum gel. The immobilized enzyme was orally delivered in wistar rats, induced with hypercholesteremia by triton X-100. The serum cholesterol and triglycerides were reduced by 50% and 15%, respectively, in the group fed with immobilized enzyme 10 IU/kg dose whereas administration of 20 IU/kg immobilized enzyme resulted in reduction of serum cholesterol by 58% and triglycerides by 45%, respectively. The results indicate that bile salt hydrolase has potential cholesterol lowering property and oral administration of the immobilized enzyme is an alternative pharmacological approach to reduce serum cholesterol levels.     

Bile salt hydrolase activity of three strains of Lactobacillus acidophilus

Three strains of Lactobacillus acidophilus, two from human intestinal origin (016 and L1) and one from porcine intestinal origin (ATCC 43121), were tested for their bile salt deconjugation activity. The L. acidophilus ATCC 43121 had more deconjugating activity of both sodium glycocholate and sodium taurocholate at pH 6.5 than did either L. acidophilus 016 or L1. The activity of intracellular bile salt hydrolase found in strain ATCC 43121 was 14-fold higher than that in either of the other two strains. The optimum pH for deconjugation of sodium glycocholate was between 4 and 5.5 for all three strains. For deconjugation of sodium taurocholate, the optimum pH was between 3.5 and 4.5 for strains L1 and ATCC 43121 and was between pH 5 and 6 for strain O16. The molecular mass of the enzyme in all three strains of L. acidophilus was estimated to be 126 kDa by Sephadex G-200 gel filtration. All three strains exhibited more bile salt hydrolase activity towards sodium glycocholate than towards sodium taurocholate.     

Screening of lactic acid bacteria for bile salt hydrolase activity

Bile salt hydrolysis is an important metabolic reaction in the bile salt metabolism of mammals. This reaction has a facilitating effect for bile salt excretion but can also be involved in various illnesses. In recent years interest has increased to use bile salt hydrolysis to influence the cholesterol metabolism of humans and farm animals. To understand the distribution and range of bile salt hydrolase activity in lactic acid bacteria, we screened more than 300 strains of the genera Bifidobacterium and Lactobacillus and the species Lactococcus lactis, Leuconostoc mesenteroides, and Streptococcus thermophilus. Results obtained for 273 strains showed that bile salt hydrolase activity is common in Bifidobacterium and Lactobacillus but absent in L. lactis, Leu. mesenteroides, and S. thermophilus. Nearly all bifidobacteria species and strains have bile salt hydrolase activity, whereas this activity can only be found in selected species of lactobacilli. A strong correlation can be observed between the habitat of a genus or species and the presence of bile salt hydrolase activity. Most often bile salt hydrolase activity is found in strains that have been isolated from the intestines or from feces from mammals--an environment rich in conjugated and unconjugated bile acids. Strains and species from other habitats like milk or vegetables--environments from which bile salts are absent--do normally not have bile salt hydrolase activity. In two independent assays, we established that bile salt hydrolase activity in bifidobacteria is, in general, much higher than in lactobacilli.     

胆盐水解酶的研究现状

阐述了胆盐水解酶(BSH)的功能、特性以及对宿主微生物产生的影响;介绍了胆盐水解酶基因、基因调节及同系物;讨论了胆盐水解酶未来研究方向和应用的前景。     

共固定甘氨酸甜菜碱与植物乳杆菌在高盐稀态酱油酿造中的应用

报道了甘氨酸甜菜碱对高渗条件下植物乳杆菌(Lactobacillus plantarum YSQ18)形态的影响,并研究了甜菜碱与乳杆菌共固定化技术对提高高盐稀态酿造酱油蛋白质转化率、缩短酱油发酵周期等的作用。利用包埋法将甜菜碱与乳杆菌制成共固定化颗粒,加入酱醪自然发酵。电镜观察显示,外源添加甜菜碱时,处在高盐环境的植物乳杆菌YSQ18生长良好,细胞形态与正常培养相近。发酵结果表明,共固定化组氨基酸态氮、全氮相比空白组分别增加0.234 g/100 mL和0.358 g/100 mL。氨基酸态氮生成率和蛋白质转化率比空白组分别提高1.7%和6.7%,比单独固定乳杆菌和添加游离乳杆菌与甜菜碱的实验组平均提前约1周时间达到发酵终点,与空白组相比提前幅度更为明显。因此,该共固定化技术在酱油酿造中具有较好的应用价值。     

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.     

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.     

胆盐水解酶合成条件的优化

以MRS，MRS肉汤，改良MRS为基础培养基，考察了培养基对菌株产胆盐水解酶活力的影响，确定了MRS肉汤为基础培养。对KTx在MRS肉汤中最适碳源、氮源、培养温度、培养基起始pH值、刺激因子作了单因素试验，得出最适碳源为葡萄糖，最适氮源为大豆蛋白胨，最适培养温度37℃，最适培养基起始pH值为5．5～6．0，最适刺激因子吐温80。在此基础上选择葡萄糖、大豆蛋白胨、培养温度、接种量进行L16（4^4）四因素四水平正交试验。结果表明，对胆盐水解酶活力影响因子水平：葡萄糖〉大豆蛋白胨〉培齐温度〉接种量：最佳产胆盐水解酶条件：葡萄糖4％，大豆蛋白胨2％，接种量2％（均为质量分数），培养温度为37℃。优化后菌株的产酶活力可提高到优化前的13．4倍。     

Factors affecting the caseinolytic activity of Lactobacillus casei and Lactobacillus plantarum

Whole cell suspensions of some strains of each Lactobacillus casei and Lactobacillus plantarum were assayed for their caseinolytic activity in 0.1 M NaH₂PO₄ buffer, pH 6.5, at 30 °C, using different assay methods. Azocasein was not as sensitive as casein (Hammarsten) as a substrate. Inclusion of glucose in the assay mixture reduced the released α-amino groups as evidenced by fluorescent labelling, but generally increased the amounts of excreted amino acids. Divalent cations, including calcium ions, played only a minor role in the activation of the cell-bound proteinase, whereas NaCl inhibited it markedly. Inhibitor studies suggest that the enzyme is a serine proteinase. The different assay methods used did not give identical results. Fluorescent labelling of the free α-amino groups at pH 6.0 appears, on the contrary, to be a more reliable method.     

具有胆固醇去除能力和胆盐水解酶活性的乳酸菌的体外筛选

对分离自内蒙古传统稀奶油或酸化稀奶油中的10株乳酸菌进行了胆固醇去除能力和胆盐水解酶活性的研究(定性和定量),筛选出了3株具有较高胆固醇去除能力和胆盐水解酶活力的乳酸菌菌株,分别为KLDS6.0330、KLDS6.0333和KLDS6.0335,并对它们进行16SrDNA分子生物学鉴定、酸耐受性和胆盐耐受性的测定。结果表明:这3株菌均为植物乳杆菌(L.Plantarum),并且表现出了较好的酸耐受性和胆盐耐受性。     

益生菌中胆盐水解酶作用机理研究现状

对乳酸菌中胆盐水解酶研究现状进行分析,从编码基因、蛋白氨基酸结构以及酶与肠道间密切关系等方面入手,阐述了肠道益生菌通过胆盐水解酶对胆盐的水解,可以达到耐受肠道胆盐压力,增强细菌表面对体内免疫系统的抵抗能力建立长期感染,促进在高等动物肠道内细菌的定殖,降低血清胆固醇水平等作用,为最终治愈高胆固醇血症提供了良好的解决方案。     

具有拮抗幽门螺杆菌作用的植物乳杆菌特性研究

幽门螺杆菌是螺旋形或是S形的微需氧革兰氏阴性菌,是慢性活动性胃炎、消化性溃疡发病的主要病因,并与胃恶性肿瘤(胃癌、胃粘膜相关淋巴样组织淋巴瘤)关系密切。本文探讨了具有拮抗幽门螺杆菌作用的植物乳杆菌的生物特性,结果表明L.plantarum 18在低酸条件下可以生长,耐受pH2.5的人工胃液,能够抑制多种幽门螺杆菌的生长。该菌株生长比较快,4h进入生长对数期,14h后进入稳定期;最适生长pH为5～7,最适生长温度为30～37℃,最适接种量为1%～2%(V/V),可在0.2%的胆盐中生长。     

克鲁维酵母菌产胆盐水解酶发酵条件的优化研究

利用从藏灵菇中筛选的产胆盐水解酶(bile salt hydrolase,BSH)的马克斯克鲁维酵母菌K1(Kluyveromyces marxianus K1)研究提高酶活力的环境因素。针对主要影响胆盐水解酶合成的4个因素,采用4因素3水平L9(34)正交试验确定了高产胆盐水解酶优化发酵条件:发酵温度为32℃,发酵时间为24h,培养基的初始pH值为6.0,接种量为2%;在优化发酵条件下,K1菌株产BSH活力比优化前提高了52.4%,为开发研制降胆固醇功能食品及微生态制剂提供了试验基础。     

Proteolytic activity of crude cell-free extract of Lactobacillus casei and Lactobacillus plantarum

Crude cell-free extracts of some strains of each L. casei and L. plantarum were assayed for their amino-, imino- and endopeptidase as well as the caseinolytic activity. L-alanine-, L-phenylalanine- and L-leucine-p-nitroanilide but not L-glutamic acid-p-nitroanilide, were hydrolyzed by all the strains indicating an amino-peptidase activity. The activity of proline iminopeptidase was very low compared to that of the aminopeptidase. L. casei could be distinguished from L. plantarum by its high endopeptidase activity against succinyl-phenylalanine- and glutaryl-phenylalanine-p-nitroanilide. The caseinolytic activity of cell-free extract of L. casei ATCC 393 was about one seventh the caseinolytic activity of intact cells, suggesting that the bulk of the cellular proteinase activity is located in the cell wall. It appears that a metallo aminopeptidase and a probable cysteine one are involved in the hydrolysis of amino acid-p-nitroanilide, whereas the endopeptidase activity appears to be related to a cysteine enzyme. Incubation of gels with L-leucine-p-nitroanilide after electrophoresis allowed the revealing of 2 zones of aminopeptidase activity in a strain of L. casei and only one in two others, but in L. plantarum it did not allow the revealing of any. The high proteolytic activities of L. casei compared to those of L. plantarum may explain its more frequent occurence in cheese and its probable role in the ripening of many cheese varities.     

植物乳杆菌高密度培养通气条件的研究

为研究通气条件对植物乳杆菌高密度生理活性、高密度培养及存活率的影响,在FUS-50-L (A)发酵罐中,分别采用4种通气策略进行发酵培养.结果表明,当通入空气时,植物乳杆菌生长速率较小,而通入氮气时,菌体的生理活性、比生长速率、营养物质消耗速率和细胞存活率最高,最大活菌数可达到1.8× 1010CFU/mL.说明植物乳杆菌在微量氧气短暂存在时能存活,欲实现发植物乳杆菌的高密度培养,必须通过通入氮气等惰性气体来保持发酵过程的厌氧状态.