Purification and characterization of β-galactosidase from probiotic Pediococcus acidilactici and its use in milk lactose hydrolysis and galactooligosaccharide synthesis

β-galactosidase is a commercially important enzyme that was purified from probiotic Pediococcus acidilactici. The enzyme was extracted from cells using sonication and subsequently purified using ammonium sulphate fractionation and successive chromatographies on Sephadex G-100 and Q-Sepharose. The enzyme was purified 3.06-fold up to electrophoretic homogeneity with specific activity of 0.883 U/mg and yield of 28.26%. Molecular mass of β-galactosidase as estimated by SDS-PAGE and MALDI-TOF was 39.07 kDa. The enzyme is a heterodimer with subunit mass of 15.55 and 19.58 kDa. The purified enzyme was optimally active at pH 6.0 and stable in a pH range of 5.8–7.0 with more than 97% activity. Purified β-galactosidase was optimally active at 50 °C. Kinetic parameters K_m and V_max for purified enzyme were 400 µM and 1.22 × 10⁻¹ U respectively. Its inactivation by PMSF confirmed the presence of serine at the active site. The metal ions had different effects on enzyme. Ca²⁺, Mg²⁺ and Mn²⁺ slightly activated the enzyme whereas NH₄⁺, Co²⁺ and Fe³⁺ slightly decreased the enzyme activity. Thermodynamic parameters were calculated that suggested that β-galactosidase is less stable at higher temperature (60 °C). Purified enzyme effectively hydrolysed milk lactose with lactose hydrolysing rate of 0.047 min⁻¹ and t_1/2 of 14.74 min. This is better than other studied β-galactosidases. Both sonicated Pediococcus acidilactici cells and purified β-galactosidase synthesized galactooligosaccharides (GOSs) as studied by TLC at 30% and 50% of lactose concentration at 47.5 °C. These findings indicate the use of β-galactosidase from probiotic bacteria for producing delactosed milk for lactose intolerant population and prebiotic synthesis. pH and temperature optima and its activation by Ca²⁺ shows that it is suitable for milk processing.     

Mitochondria: Role of citrulline and arginine supplementation in MELAS syndrome

Mitochondria are found in all nucleated human cells and generate most of the cellular energy. Mitochondrial disorders result from dysfunctional mitochondria that are unable to generate sufficient ATP to meet the energy needs of various organs. Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a frequent maternally inherited mitochondrial disorder. There is growing evidence that nitric oxide (NO) deficiency occurs in MELAS syndrome and results in impaired blood perfusion that contributes significantly to several complications including stroke-like episodes, myopathy, and lactic acidosis. Both arginine and citrulline act as NO precursors and their administration results in increased NO production and hence can potentially have therapeutic utility in MELAS syndrome. Citrulline raises NO production to a greater extent than arginine, therefore, citrulline may have a better therapeutic effect. Controlled studies assessing the effects of arginine or citrulline supplementation on different clinical aspects of MELAS syndrome are needed.     

Low cell surface hydrophobicity is one of the key factors for high butanol tolerance of Lactic acid bacteria

Highly butanol‐tolerant strains have always been attractive because of their potential as microbial hosts for butanol production. However, due to the amphiphilic nature of 1‐butanol as a solvent, the relationship between the cell surface hydrophobicity and butanol resistance remained ambiguous to date. In this work, the quantitatively estimated cell surface hydrophobicity of 74 Lactic acid bacteria strains were juxtaposed to their tolerance to various butanol concentrations. The obtained results revealed that the strains’ hydrophobicity was inversely proportional to their butanol tolerance. All highly butanol‐resistant strains were hydrophilic (cell surface hydrophobicity<1%), whereas the more hydrophobic the strains were, the more sensitive to butanol they were. Furthermore, cultivation at increasing butanol concentrations showed a clear tendency to decrease the level of hydrophobicity in all tested organisms, thus suggesting possible adaptation mechanisms. Purposeful reduction of cell surface hydrophobicity (by removal of S‐layer proteins from the cell envelope) also led to an increase of butanol resistance. Since the results covered 23 different Lactic acid bacteria species of seven genera, it could be concluded that regardless of the species, the lower degree of cells’ hydrophobicity clearly correlates with the higher level of butanol tolerance.     

保元煎剂对疲劳大鼠自由基、乳酸、ATP的影响

目的:研究保元煎剂对疲劳大鼠力竭游泳时间以及ATP、羟自由基、乳酸含量的影响。方法:选用60只雄性SD大鼠,随机分为正常组、模型组、西洋参组、保元煎剂低、中、高剂量组。采用力竭游泳加睡眠剥夺建立复合疲劳大鼠模型,均予灌胃给药或蒸馏水(保元煎剂低、中、高剂量依次为1.04、2.08、4.15 g·kg-1·d-1;西洋参组给药量为0.27 g·kg-1·d-1),记录大鼠末次力竭游泳时间,检测疲劳大鼠血清ATP、羟自由基、乳酸含量。结果:与模型组比较,保元煎剂各组大鼠末次力竭游泳时间延长,ATP含量升高,羟自由基及乳酸含量下降,均有统计学意义,P0.05;与西洋参组比较,保元煎剂高剂量组末次力竭游泳时间延长、乳酸含量降低,保元煎剂高、中、低剂量组ATP升高,保元煎剂高、中剂量组自由基水平下降,均具有统计学意义,P0.05。结论:保元煎剂可提高疲劳大鼠力竭游泳时间,升高疲劳大鼠ATP含量,降低羟自由基、乳酸含量。     

Analysis of the Binding Moiety Mediating the Interaction between Monocarboxylate Transporters and Carbonic Anhydrase II

Proton-coupled monocarboxylate transporters (MCTs) mediate the exchange of high energy metabolites like lactate between different cells and tissues. We have reported previously that carbonic anhydrase II augments transport activity of MCT1 and MCT4 by a noncatalytic mechanism, while leaving transport activity of MCT2 unaltered. In the present study, we combined electrophysiological measurements in Xenopus oocytes and pulldown experiments to analyze the direct interaction between carbonic anhydrase II (CAII) and MCT1, MCT2, and MCT4, respectively. Transport activity of MCT2-WT, which lacks a putative CAII-binding site, is not augmented by CAII. However, introduction of a CAII-binding site into the C terminus of MCT2 resulted in CAII-mediated facilitation of MCT2 transport activity. Interestingly, introduction of three glutamic acid residues alone was not sufficient to establish a direct interaction between MCT2 and CAII, but the cluster had to be arranged in a fashion that allowed access to the binding moiety in CAII. We further demonstrate that functional interaction between MCT4 and CAII requires direct binding of the enzyme to the acidic cluster ⁴³¹EEE in the C terminus of MCT4 in a similar fashion as previously shown for binding of CAII to the cluster ⁴⁸⁹EEE in the C terminus of MCT1. In CAII, binding to MCT1 and MCT4 is mediated by a histidine residue at position 64. Taken together, our results suggest that facilitation of MCT transport activity by CAII requires direct binding between histidine 64 in CAII and a cluster of glutamic acid residues in the C terminus of the transporter that has to be positioned in surroundings that allow access to CAII.     

乳酸菌合成叶酸的研究进展

叶酸普遍存在于各类食物中,但由于叶酸的不稳定性以及饮食习惯的差异性,各国叶酸缺乏现象普遍存在。叶酸是参与核酸合成及细胞分裂分化的重要物质,叶酸缺乏引起机体功能的混乱,由此引发癌症等一系列的疾病。大部分乳酸菌是叶酸缺陷型菌株,但越来越多的研究表明其很多种类都具有叶酸合成能力。本文主要概述产叶酸乳酸菌的分类,乳酸菌生物合成叶酸的机制,以及利用乳酸菌进行的叶酸基因工程研究进展。     

发酵食品中乳酸菌的耐药性现状分析

乳酸菌中的许多种类都是食品工业中常用的发酵菌种,但其耐药性会给食品安全带来风险。本文就乳酸菌的耐药机制、敏感性检测方法及发酵食品中乳酸菌的耐药现状和耐药转移性进行分析,为同行研究者能准确找出问题切入点提供资料。     

乳酸菌对高脂小鼠降胆固醇作用的研究

目的探讨乳酸菌对高脂小鼠降胆固醇的作用。方法对昆明小鼠饲喂高脂饲料14 d建立高脂小鼠模型,确定模型建立成功后用1株经体外实验证实具有降胆固醇效果的乳酸菌对小鼠灌胃,测定小鼠经灌胃14、28 d的脏器指数、血清胆固醇含量和肝脏胆固醇含量。结果灌胃组小鼠脏器指数明显低于高脂组小鼠,且对于血清和肝脏胆固醇含量具有明显的降低作用。结论该菌株具有降低血清胆固醇,抑制肝脏胆固醇堆积的功能,为将来利用该菌株制作出降胆固醇功能的食品或药品提供实验基础。     

乳酸菌双组分信号转导系统研究进展

乳酸菌中存在着一种重要的调控机制--双组分信号转导系统,它可以通过调控乳酸菌的多种生理生化过程来适应外界环境的变化.就双组分信号转导系统的组成、作用机制以及乳酸菌中调控耐酸机制、细菌素的合成和黏性吸附等生理过程的双组分信号转导系统作一综述.