交互保护对干酪乳杆菌ATCC 393~(TM)存活的影响

【目的】以干酪乳杆菌典型株ATCC 393TM(Lactobacillus casei ATCC 393TM)为实验菌株,研究其在多重胁迫环境下的交互保护应答机制。【方法】比较不同亚适应条件(热、H2O2、酸、胆盐)处理后菌体细胞在热致死条件(60℃)及氧致死条件H2O2(5mmol/L)下的存活率变化,并集中考察了最佳亚适应条件-酸适应的不同处理方式对细胞交互保护存活率、胞内pH以及脂肪酸含量的影响。【结果】交互保护对干酪乳杆菌ATCC393生理活性的影响因亚适应及致死条件而异:酸胁迫预适应能够显著提高细胞的交互胁迫抗性,其中,盐酸预适应的交互保护效果优于乳酸,其预适应引发的生理应答效应使细胞在应对热致死和氧致死胁迫时存活率分别提高了305倍和173倍;进一步的研究表明,酸预适应提高细胞存活率的作用机制可能与其能够显著改善胁迫环境下的胞内pH和细胞膜脂肪酸不饱和度相关。【结论】盐酸预适应对干酪乳杆菌典型株ATCC393的交互保护作用最为显著,并能够维持胁迫条件下细胞生理状态的相对稳定,本研究将有助于进一步解析干酪乳杆菌在对抗不同胁迫环境的过程中生理应答机制间的相互作用关系。     

Distinct adhesion of probiotic strain Lactobacillus casei ATCC 393 to rat intestinal mucosa

Adhesion to the intestine represents a critical parameter for probiotic action. In this study, the adhesion ability of Lactobacillus casei ATCC 393 to the gastrointestinal tract of Wistar rats was examined after single and daily administration of fermented milk containing either free or immobilized cells on apple pieces. The adhesion of the probiotic cells at the large intestine (cecum and colon) was recorded at levels ≥6 logCFU/g (suggested minimum levels for conferring a probiotic effect) following daily administration for 7 days by combining microbiological and strain-specific multiplex PCR analysis. Single dose administration resulted in slightly reduced counts (5 logCFU/g), while they were lower at the small intestine (duodenum, jejunum, ileum) (≤3 logCFU/g), indicating that adhesion was a targeted process. Of note, the levels of L. casei ATCC 393 were enhanced in the cecal and colon fluids both at single and daily administration of immobilized cells (6 and 7 logCFU/g, respectively). The adhesion of the GI tract was transient and thus daily consumption of probiotic products containing the specific strain is suggested as an important prerequisite for retaining its levels at an effective concentration.     

重组干酪乳杆菌在模拟消化环境中生存性能的研究

目的 探讨重组干酪乳杆菌Lactobacillus casei 393在模拟胃肠道环境中的存活能力.方法 人工模拟胃肠道环境,即人工胃液（pH=1.5～4.5）、人工肠液、胆汁（质量浓度0.3～3.0 g/L）和高盐（质量浓度40～90g/L）.结果 重组干酪乳杆菌在pH为2.5～4.5的人工胃液中具有较强的生存能力,3 h活菌数仍达108/ml;在人工肠液中经过不同时间的作用后,重组干酪乳杆菌显出生长趋势;在0.3%的胆汁环境作用8 h仍有存活,且能耐受7%NaCl浓度的高渗环境.结论 实验为干酪乳杆菌Lactobacillus casei 393能否作为益生菌制剂在胃肠道中发挥作用提供了理论基础.     

基于蛋白表达分析的干酪乳杆菌ATCC393交互胁迫保护机制

为考察干酪乳杆菌典型株ATCC393在交互胁迫环境下的生理应答机制,应用二维电泳和iTRAQ技术在蛋白水平上比较了交互胁迫前后干酪乳杆菌蛋白质组的变化情况。在对不同处理条件下细胞全蛋白的二维电泳分析中发现,干酪乳杆菌的主要蛋白分布在等电点pI4～7的范围,经酸预适应处理后细胞的蛋白表达产生了较大的变化。通过iTRAQ技术对细胞在酸适应前后以及相应致死条件下蛋白表达的定性及相对定量分析得知,酸诱导所产生的热胁迫应激蛋白（dnaK,dnaJ等）、氧胁迫应激蛋白（mutS,YeaO）以及与代谢相关的酶类上调可能是提高细胞对交互胁迫耐受能力的主要原因,而酸适应后GTP环化水解酶I和GMP合成酶的高表达可能与这一过程的诱导有关。上述研究结果为提高工业生产菌株在发酵生产及加工过程中对外界不利环境的抵御能力,进而通过调控与微生物生理应答机制密切相关的功能元器件实现生产菌株的性能强化提供了重要的生物信息和可借鉴的研究思路。     

Protective mechanism of Lactobacillus casei ATCC393 against multiple stresses by proteomics analysis

为考察干酪乳杆菌典型株ATCC 393在交互胁迫环境下的生理应答机制,应用二维电泳和iTRAQ技术在蛋白水平上比较了交互胁迫前后干酪乳杆菌蛋白质组的变化情况.在对不同处理条件下细胞全蛋白的二维电泳分析中发现,干酪乳杆菌的主要蛋白分布在等电点pI 4～7的范围,经酸预适应处理后细胞的蛋白表达产生了较大的变化.通过iTRAQ技术对细胞在酸适应前后以及相应致死条件下蛋白表达的定性及相对定量分析得知,酸诱导所产生的热胁迫应激蛋白( dna K,dna J等)、氧胁迫应激蛋白(mut S,rec O)以及与代谢相关的酶类上调可能是提高细胞对交互胁迫耐受能力的主要原因,而酸适应后GTP环化水解酶Ⅰ和GMP合成酶的高表达可能与这一过程的诱导有关.上述研究结果为提高工业生产菌株在发酵生产及加工过程中对外界不利环境的抵御能力,进而通过调控与微生物生理应答机制密切相关的功能元器件实现生产菌株的性能强化提供了重要的生物信息和可借鉴的研究思路.     

Osmotic response in Lactobacillus casei ATCC 393: biochemical and biophysical characteristics of membrane

The biochemical and biophysical properties of the membrane and some general characteristics of the response of Lactobacillus casei ATCC 393 (reclassified Lactobacillus zeae) to hyperosmotic conditions were studied. Under hypertonic conditions, the hydrophobicity and the bile salt sensitivity of the cultures were increased. The glycolipid AcylH3DG is only present in membranes of NaCl containing medium, whereas, H4DG undergoes a significant increment and H2DG a significant decrease. The fluidity of both the purified membranes and the total lipid vesicles, as determined with the fluorescent probe DPH, did not change in conditions of high salinity. This was coincident with changes in the fatty acid (FA) composition where an increase in the saturated/unsaturated FA ratio was compensated by a rise in the fluidifying 11,12-methyleneoctadecanoic FA (cyc 19:0). Under osmotic stress conditions, Laurdan and acridine orange in total lipid vesicles showed increased lateral lipid packing and proton permeability, respectively.     

Expression of cbsA encoding the collagen-binding S-protein of Lactobacillus crispatus JCM5810 in Lactobacillus casei ATCC 393(T)

The cbsA gene encoding the collagen-binding S-layer protein of Lactobacillus crispatus JCM5810 was expressed in L. casei ATCC 393^T. The S-protein was not retained on the surface of the recombinant bacteria but was secreted into the medium. By translational fusion of CbsA to the cell wall sorting signal of the proteinase, PrtP, of L. casei, CbsA was presented at the surface, rendering the transformants able to bind to immobilized collagens.     

Homology modeling of the central catalytic domain of insertion sequence ISLC3 isolated from Lactobacillus casei ATCC 393

The tertiary structure of the central catalytic domain of insertion sequence ISLC3 isolated from Lactobacillus casei ATCC 393 was predicted using the homology modeling approach. The novel insertion sequence was isolated by us from the template bacteriophage phiA3 of L.casei ATCC 393. The number of amino acid residues of the ISLC3 central catalytic domain was 116 and was treated as the query sequence. There were five Web-available threading methods used to find some primary structure templates for the query sequence. These primary templates were further screened using the SWISS-MODEL Protein Modeling Server and the default parameter settings therein to give six final structure templates. All of these final structure templates were the integrase (IN) protein of retroviruses. Multiple sequence alignment using these IN sequences against the query one revealed the signature DDE motif. Based on the structures of these final templates, the structure of the query sequence was constructed using the InsightII/Discover/Homology programs. A metal ion, Mg(2+), was inserted into the center of the putative catalytic pocket formed by the DDE residues of the predicted structure in the final rounds of refinement by molecular dynamics (MD) simulations. The structure with a metal ion included was designated with Mg and that without a metal ion was designated free Mg. The average exposed surface area of some hydrophobic residues of both the predicted free Mg and with Mg structures were computed and compared with those computed for the six structure templates. Whereas the predicted with Mg structure was slightly more exposed than the predicted free Mg structure, the former appeared to be more stable than the latter, as revealed by the lower conformation energy recorded for the former during the structure refinement by MD simulations. To verify further the predicted structures, the coordinates of both predicted structures were fed into the ERRAT Protein Verification Server. It was found that the quality of the predicted with Mg structure was much better than that of the free Mg structure. The validation results also indicated that regions of the predicted with Mg structure that can be rejected at the 95% confidence level were approximately 20% whereas those which can be rejected at the same level for the six structure templates were approximately 10%. The predicted with Mg structure was also docked into a short oligonucleotide representing the substrate of the ISLC3 transposase using the DOCK_4.0.2 program. It was found that both Glu140 and Asp68 residues of the DDE motif of the predicted with Mg structure were able to form hydrogen bonds with the DNA substrate, which was similar to what was observed in a docking study using the retrovirus IN 1asu and its DNA substrate.     

Expression of Lactobacillus casei ATCC 393 β-galactosidase encoded by plasmid pLZ15 in Lactococcus lactis CNRZ 1123

Lactococcus lactis subsp. lactis CNRZ 1123, a Lac^- derivative of CNRZ 1122 was transformed by electroporation with the Lactobacillus casei ATCC 393 plasmid pLZ15, which bears a β-galactosidase gene. The transformants expressed a constitutive β-galactosidase activity at a higher level than in Lact. casei , and in the cell-free extract two additional protein bands were detected by SDS-PAGE which could correspond to lactose metabolism enzymes. Both plasmid and β-gal activity were stable in Lactococcus after 100 generations in glucose-containing medium.     

Selective enumeration of Lactobacillus casei from yogurts and fermented milk drinks

A selective medium (LC agar) was developed for enumeration of Lactobacillus casei populations from commercial yogurts and fermented milk drinks that may contain strains of yogurt bacteria (Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus), probiotic bacteria (Lactobacillus acidophilus and bifidobacteria) and L. casei. Appropriate dilutions were pour-plated in specially formulated LC agar acidified to pH 5.1 and the plates incubated at 27°C for 72 to 96 h under anaerobic conditions. Growth of S. thermophilus was prevented by adjusting pH to 5.1. L. delbrueckii ssp. bulgaricus did not ferment ribose as the carbon source, as a result the organisms did not form colonies. L. acidophilus formed colonies on MRS-ribose agar; however, this organism did not grow in the specially formulated LC agar containing ribose. Similarly, Bifidobacterium spp. did not form colonies in LC agar. L. casei formed colonies on LC agar. © Rapid Science Ltd. 1998     

活性乳酸菌饮料对人体肠道菌群影响的研究

目的 探讨一种活性乳酸菌饮料对人体肠道菌群的影响作用.方法 采用卫生部《保健食品检验与评价技术规范》调节肠道菌群功能检验方法进行检验.结果 试食组试验前后自身比较：肠道内双歧杆菌、乳杆菌数量明显增加（P＜0.01）,肠球菌数量增加（P＜0.05）,产气荚膜梭菌数量明显减少（P＜0.01）,肠杆菌和拟杆菌无明显变化;试验后对照组与试食组组间比较：双歧杆菌数量明显增加（P＜0.01）,乳杆菌的数量增加（P＜0.05）,肠杆菌、肠球菌、产气荚膜梭菌和拟杆菌无明显变化.结论 活性乳酸菌饮料具有调节人体肠道菌群、改善肠道内环境的作用.