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.     

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。     

重组粪肠球菌gshF基因对副干酪乳杆菌(Lactobacillus paracasei)L14抗逆性能的影响

乳酸菌作为重要的食品工业微生物,其在工业生产应用过程中会受到多种非生物胁迫。已有研究表明部分乳酸菌可以吸收培养基中或是自身合成谷胱甘肽(Glutathione,GSH),提高对各种胁迫的抵抗作用。克隆了粪肠球菌(Enterococcus faecalis)中的谷胱甘肽合成酶基因gshF,通过构建乳杆菌重组表达质粒,实现了gshF在副干酪乳杆菌(Lactobacillus paracasei)L14菌株中的异源表达。通过对重组gshF-副干酪乳杆菌阳性菌株的抗逆性性测定,结果表明在过氧化氢、酸、冻干脱水和渗透等胁迫条件下,gshF重组菌株的存活率与对照菌株相比均有显著提高。     

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。     

五株乳酸菌和三株芽孢杆菌的生物学特性和功能

【背景】乳酸菌和芽孢杆菌是应用于生产最多的益生菌,但不同菌株间的生长特性均不相同,因此了解菌株的生物学特性具有重要意义。【目的】研究菌株的生物学特性,能合理地开发和利用菌株,以保证菌株生产应用的安全性。【方法】活化后鉴定5株乳酸菌和3株芽孢杆菌并对其形态进行观察,探究菌株的生长曲线、产酸能力及最适生长条件,测定菌株的抑菌活性和产酶性能,同时探究菌株的益生性和安全性。【结果】五株乳酸菌分别编号鉴定为干酪乳杆菌R1、副干酪乳杆菌R2、香肠乳杆菌R3、福莱乳杆菌R4和唾液乳杆菌R5;3株芽孢杆菌分别编号命名为贝莱斯芽孢杆菌Y1、枯草芽孢杆菌Y2和地衣芽孢杆菌Y3。八株菌形态结构均不相同但都为杆状,均在2–10 h为对数生长期,18–24 h为稳定期,培养24 h时乳酸菌和芽孢杆菌的活菌数均保持在10⁹和10⁸ CFU/mL,最适生长温度为37.0℃。乳酸菌具有较强的产酸能力和抑菌活性,芽孢杆菌有较强的产酶性,在人工胃液中都有较强的耐受性。八株菌都无溶血活性、无毒力基因、对抗生素都保持中度敏感以上;其中唾液乳杆菌有四环素耐药基因,但对四环素抗性为中度敏感。【结论】八株菌生长繁殖速度快,乳酸菌产酸能力和抑菌活性较强,芽孢杆菌具有较强的产酶性能,在体外具有较好的益生性和安全性,可应用于生产实践。     

乳酸菌表面蛋白对免疫细胞的诱导增殖及粘附抑制效应

目的研究表面蛋白在乳酸菌体外粘附和激活免疫细胞中的作用。方法应用5mol/L氯化锂结合盐酸胍提取植物乳杆菌LpYZU09、干酪乳杆菌LcYZU02、鼠李糖乳杆菌LrGG、发酵乳杆菌LfYZU15及戊糖片球菌PpYZU32的表面蛋白,并分析提取物对乳酸菌粘附鼠肠上皮细胞、巨噬细胞和脾细胞的抑制作用及诱导增殖效应。结果表面蛋白对3种细胞粘附菌体均具有显著抑制效应,抑制作用具有细胞和菌株差异性,其中菌株LrGG表面蛋白对巨噬细胞粘附5种菌体普遍显示了较强的抑制作用,抑制率为38.7%~76.0%。不同菌株表面蛋白对肠上皮细胞的诱导增殖指数为0.05~0.35,对巨噬细胞为0.05~0.42,对脾细胞为0.02~0.40,诱导效应具有菌株和剂量依赖性,菌株LrGG的诱导增殖指数显著高于其他四种。结论乳酸菌表面的蛋白类因子在粘附和激活免疫细胞中发挥了重要作用。     

酸胁迫下琥珀酸放线杆菌的生理及转录应答

【目的】通过琥珀酸放线杆菌Actinobacillus succinogenes CGMCC1593对酸胁迫的生理应答和转录组学分析,探究琥珀酸放线杆菌酸胁迫的机制。【方法】测定不同pH对细胞生长、H+-ATPase、细胞内pH的影响;测定酸胁迫前后细胞膜和谷氨酸脱氢酶的变化、谷氨酸对琥珀酸放线杆菌生长的影响;通过RNA-seq测序分析酸胁迫条件下的差异表达基因。【结果】随pH值的降低,细胞生长受抑制,H+-ATPase的活性下降。pH 4.7酸胁迫后,细胞膜受到严重损伤,谷氨酸对酸胁迫后的细胞有保护作用,GDH酶活响应酸胁迫后略有增加。酸胁迫后,39个基因差异表达较为显著,其中49%基因属于应激蛋白、转运蛋白,小部分基因与代谢相关。【结论】本文探究了琥珀酸放线杆菌酸胁迫下的生理及转录应答,研究结果可为寻找增强琥珀酸放线杆菌耐酸性策略提供参考。     

葡萄藤叶青贮中乳酸菌的分离与鉴定

为了筛选出性状优良的乳酸菌菌株,本试验以实验室纯培养方式从葡萄藤叶自然发酵的青贮中分离鉴定乳酸菌。经过菌落形态、细胞形态、生理生化鉴定和16S r DNA基因序列以及系统发育树的分析,从葡萄藤叶青贮中分离出5株乳酸菌,其中菌株P-1. 2和P-2. 2为戊糖乳杆菌(Lactobacillus pentosus),菌株P-1. 7为戊糖片球菌(Pediococcus pentosaceus),菌株P-2. 1为粪肠球菌(Enterococcus faecium),菌株P-2. 3为短乳杆菌(Lactobacillus brevis)。测定产酸速率和生长速率,发现菌株P-2. 1的产酸能力和生长性能最好。     

具有抗幽门螺杆菌能力的乳酸菌菌株筛选CSCD

目的 在8株乳酸菌中筛选能够抗幽门螺杆菌感染的乳酸菌菌株,为后续研发治疗幽门螺杆菌感染的益生菌制剂提供依据。方法 在8株乳酸菌菌株中,通过对幽门螺杆菌抑菌率的检测及构建人胃腺癌细胞感染模型检测8株乳酸菌对幽门螺杆菌的抑制作用,以及对尿素酶活性的检测判断8株菌的抗幽门螺杆菌的能力。结果 在抑菌试验中,植物乳植杆菌HCS03-001(t=2.938,P=0.008)和副干酪乳酪杆菌HCS17-040(t=3.864,P=0.006)上清液的抑菌作用较强;植物乳植杆菌HCS03-001、罗伊氏粘液乳杆菌RH02100、副干酪乳酪杆菌HCS17-040能够降低幽门螺杆菌对AGS细胞的黏附能力;通过幽门螺杆菌菌悬液与乳酸菌菌悬液共培养,发现植物乳植杆菌HCS03-001(t=6.257,P<0.001)、副干酪乳酪杆菌HCS17-040(t=5.873,P=0.005)抑制尿素酶活性的作用更强。结论 植物乳植杆菌HCS03-001和副干酪乳酪杆菌HCS17-040具有抗幽门螺杆菌感染的能力。     

鼠李糖乳杆菌菌毛SpaA亚基的表达、抗体制备及其种属特异性研究

【目的】制备鼠李糖乳杆菌菌毛亚基Spa A多克隆抗体,研究其种属特异性。【方法】应用PCR方法从鼠李糖乳杆菌GG的基因组扩增出spa A,并连接到质粒p ET-28α(+)中。将重组质粒转化大肠杆菌BL21(DE3),经IPTG诱导表达和镍柱纯化制备重组SpaA。通过免疫BALB/c小鼠获得多克隆抗体,利用全菌ELISA、Western和Dot-blot分析了SpaA在18株乳酸菌(12个种)中的分布特征。【结果】表达的重组SpaA分子量为36 k D,与预期大小一致;获得的Spa A抗体效价为1:12 800。Western结果显示抗体与天然Spa A具有良好的反应性。在测定的18株乳酸菌中,鼠李糖乳杆菌、干酪乳杆菌、副干酪乳杆菌3个种属菌株的spa A基因PCR和RT-PCR检测均为阳性。但全菌ELISA和Dot-blot结果显示,只有3株鼠李糖乳杆菌的全菌细胞与SpaA抗体呈特异性反应,而其它种属的菌株没有明显的交叉反应。【结论】尽管spa A基因在鼠李糖乳杆菌、干酪乳杆菌、副干酪乳杆菌中具有高度同源性,但SpaA蛋白只特异性地呈现在鼠李糖乳杆菌细胞表面。本研究中获得的Spa A抗体,为高黏附性鼠李糖乳杆菌的免疫磁珠分离及菌毛功能研究提供了工具。     

天冬氨酸提高乳酸乳球菌Lactococcus lactis NZ9000酸胁迫抗性的作用机制

【背景】乳酸菌作为重要的发酵微生物在应用过程中面临广泛存在的酸胁迫。【目的】确认天冬氨酸可有效提高乳酸乳球菌的酸胁迫抗性,通过解析天冬氨酸的作用机制,为进一步提高乳酸菌酸胁迫抗性提供可借鉴的思路。【方法】通过荧光定量PCR比较胁迫条件下天冬氨酸对L.lactisNZ9000产能和氨基酸代谢途径中关键基因转录水平的影响,并通过过量表达天冬酰胺酶增加胞内天冬氨酸的含量。【结果】天冬氨酸主要是在转氨酶的作用下生成草酰乙酸和谷氨酸。草酰乙酸参与三羧酸循环,为细胞提供更多的能量;谷氨酸经谷氨酸脱羧酶途径提高细胞的酸胁迫抗性。经pH4.0胁迫处理后,天冬氨酸使糖酵解和三羧酸循环产能途径中关键基因转录上调,胞内ATP含量为对照组的42倍;胞内谷氨酸含量为对照的1.99倍。通过过量表达天冬酰胺酶获得的重组菌株,在pH3.6条件下胁迫0.5h后,存活率约为对照组的11.11倍。【结论】在L. lactis NZ9000中探究了天冬氨酸提高酸胁迫抗性的作用机理,进一步完善了氨基酸代谢提高乳酸菌酸胁迫抗性的理论基础。     

耐乙酸乳杆菌的筛选及其产乳酸特性

【背景】耐受乙酸的乳酸菌是传统谷物醋醋酸发酵过程中产生乳酸及其风味衍生物的重要功能微生物。【目的】从镇江香醋醋醅中分离鉴定具有耐乙酸特性的乳酸菌,并评价不同条件下该菌株的产乳酸能力。【方法】利用4%(体积比)乙酸含量的MRS培养基分离耐乙酸乳酸菌;对其进行16S rRNA基因鉴定、基因组测序、形态观察以及生理生化特性研究;考察不同乙酸浓度、葡萄糖浓度、发酵温度和时间对菌株产乳酸能力的影响。【结果】分离得到一株可耐受6%乙酸的乳杆菌Lactobacillus sp. JN500903;在厌氧静置、接种量5%、乙酸浓度5%、葡萄糖浓度40 g/L、发酵温度37°C、发酵时间10 d条件下,该菌株乳酸产量为16.1 g/L。【结论】乳杆菌JN500903能够耐受6%乙酸浓度,具有在酸性环境下合成乳酸的能力,有一定的应用潜力。     

Degradation of cassava linamarin by lactic acid bacteria

Summary Six out of ten lactic acid bacteria strains tested displayed linamarase activity.Lactobacillus plantarum strain A6 displayed the greatest activity affecting 36U/g cells on MRS cellobiose. The strain also broke down in less than 2 hours the linamarin extracted from cassava juice. HPLC analysis of the products of the reaction showed that the bacteria converted the linamarin into lactic acid and acetone cyanohydrin.     

Display of alpha-amylase on the surface of Lactobacillus casei cells by use of the PgsA anchor protein, and production of lactic acid from starch

We developed a new cell surface engineering system based on the PgsA anchor protein from Bacillus subtilis. In this system, the N terminus of the target protein was fused to the PgsA protein and the resulting fusion protein was expressed on the cell surface. Using this new system, we constructed a novel starch-degrading strain of Lactobacillus casei by genetically displaying alpha-amylase from the Streptococcus bovis strain 148 with a FLAG peptide tag (AmyAF). Localization of the PgsA-AmyA-FLAG fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. The lactic acid bacteria which displayed AmyAF showed significantly elevated hydrolytic activity toward soluble starch. By fermentation using AmyAF-displaying L. casei cells, 50 g/liter of soluble starch was reduced to 13.7 g/liter, and 21.8 g/liter of lactic acid was produced within about 24 h. The yield in terms of grams of lactic acid produced per gram of carbohydrate utilized was 0.60 g per g of carbohydrate consumed at 24 h. Since AmyA was immobilized on the cells, cells were recovered after fermentation and used repeatedly. During repeated utilization of cells, the lactic acid yield was improved to 0.81 g per g of carbohydrate consumed at 72 h. These results indicate that efficient simultaneous saccharification and fermentation from soluble starch to lactic acid were carried out by recombinant L. casei cells with cell surface display of AmyA.     

大肠杆菌重组乳链菌肽抗性蛋白(NSR)的表达纯化及其功能分析

乳链菌肽(Nisin)是由某些乳酸菌产生的一种阳离子抗菌肽, 而Nisin抗性蛋白(Nisin resistance protein, NSR)的表达则使一些非 Nisin产生菌获得Nisin抗性。为深入探索NSR的作用机制, 本研究在大肠杆菌中表达了去除N末端38个氨基酸残基的NSR(NSRΔ38)与GST的融合蛋白GST-NSRΔ38。通过谷胱甘肽(GSH)亲和层析和GST标签的切除后, 得到纯化的NSRΔ38, 并测定了该蛋白可能的nisin降解活性。反应产物的抑菌活性测定结果表明被NSRΔ38作用     

Proteomic approach for characterization of hop-inducible proteins in Lactobacillus brevis

Resistance to hops is a prerequisite for the capability of lactic acid bacteria to grow in beer and thus cause beer spoilage. Bactericidal hop compounds, mainly iso-alpha-acids, are described as ionophores which exchange H+ for cellular divalent cations, e.g., Mn2+, and thus dissipate ion gradients across the cytoplasmic membrane. The acid stress response of Lactobacillus brevis TMW 1.465 and hop adaptation in its variant L. brevis TMW 1.465A caused changes at the level of metabolism, membrane physiology, and cell wall composition. To identify the basis for these changes, a proteomic approach was taken. The experimental design allowed the discrimination of acid stress and hop stress. A strategy for improved protein identification enabled the identification of 84% of the proteins investigated despite the lack of genome sequence data for this strain. Hop resistance in L. brevis TMW 1.465A implies mechanisms to cope with intracellular acidification, mechanisms for energy generation and economy, genetic information fidelity, and enzyme functionality. Interestingly, the majority of hop-regulated enzymes are described as manganese or divalent cation dependent. Regulation of the manganese level allows fine-tuning of the metabolism, which enables a rapid response to environmental (stress) conditions. The hop stress response indicates adaptations shifting the metabolism into an energy-saving mode by effective substrate conversion and prevention of exhaustive protein de novo synthesis. The findings further demonstrate that hop stress in bacteria not only is associated with proton motive force depletion but obviously implies divalent cation limitation.     

Effects of lactic acid fermentation and gamma irradiation of barley on antinutrient contents and nutrient digestibility in mink (Mustela vison) with and without dietary enzyme supplement

The experiment was conducted to study the effects of fermentation of barley, using two different strains of lactic acid bacteria, a Lactobacillus plantarum/pentosus strain isolated from spontaneously fermented rye sourdough (AD2) and a starch-degrading Lactobacillus plantarum (AM4), on contents of mixed-linked (1 --> 3) (1 --> 4)-beta-glucans, alpha-amylase inhibitor activity, inositol phosphates, and apparent digestibility of macronutrients in mink. Effects of fermentation were compared with effects of gamma irradiation (gamma-irradiation: 60Co gamma-rays at 25 kGy). The diets were fed to mink with and without a supplementary enzyme preparation. Both lactic acid fermentation and gamma-irradiation followed by soaking and incubation, reduced concentrations of soluble beta-glucans, phytate and alpha-amylase inhibitor activity. Dietary enzyme supplementation increased significantly digestibility of crude protein, fat, starch and crude carbohydrate (CHO). Fermentation of the barley increased digestibility of starch and CHO. Fermentation with lactic acid bacteria AD2 resulted in higher starch and CHO digestibility than strain AM4, and had greater effect than gamma-irradiation, soaking and incubation. The highest digestibility of starch and CHO was obtained after AD2 fermentation followed by enzyme supplementation. It is concluded that both lactic acid fermentation of barley and enzyme supplementation have positive nutritional implications in the mink by limiting the effects of antinutrients and improving digestibility and energy utilization.     

Strain improvement and metabolic flux analysis in the wild-type and a mutant Lactobacillus lactis strain for L(+)-lactic acid production

The effects of initial glucose concentration and calcium lactate concentration on the lactic acid production by the parent strain, Lactobacillus lactis BME5-18, were studied. The results of the experiments indicated that glucose and lactate repressed the cell growth and the lactic acid production by Lactobacillus lactis BME5-18. A L(+)-lactic acid overproducing strain, Lactobacillus lactis BME5-18M, was screened by mutagenizing the parent strain with ultraviolet (UV) light irradiation and selecting the high glucose and lactate calcium concentration repression resistant mutant. Starting with a concentration of 100g L(-1) glucose, the mutant produced 98.6 g L(-1) lactic acid after 60 h in flasks, 73.9% higher than that of the parent strain. The L(+)-lactic acid purity was 98.1% by weight based on the amount of total lactic acid. The culture of the parent strain could not be analyzed well by conventional metabolic flux analysis techniques, since some pyruvate were accumulated intracellularly. Therefore, a revised flux analysis method was proposed by introducing intracellular pyruvate pool. Further studies demonstrate that there is a high level of NADH oxidase activity (12.11 mmol mg(-1) min(-1)) in the parent strain. The molecular mechanisms of the strain improvement were proposed, i.e., the high level of NADH oxidase activity was eliminated and the uptake rate of glucose was increased from 82.1 C-mmol (g DW h)(-1) to 98.9 C-mmol (g DW h)(-1) by mutagenizing the parent strain with UV, and therefore the mutant strain converts mostly pyruvate to lactic acid with a higher productivity (1.76 g L(-1) h(-1)) than the parent strain (0.95 g L(-1) h(-1)).     

青海湖裸鲤肠道乳酸菌多样性与抑菌活性

【目的】通过生理生化特性,结合16S r RNA基因序列分析研究青海湖裸鲤肠道乳酸菌分离株的多样性,并对这些代表株的抑菌活性进行初步探讨,以期筛选具有高效抑菌活性的鱼源益生菌。【方法】对分离的47株乳酸菌代表株进行p H、温度生长范围、耐盐性等生理生化特征检测,结合16S r RNA基因序列对已分离到的乳酸菌进行基因分型和菌种鉴定,采用牛津杯双层平板法检测乳酸菌代表株的抑菌活性。【结果】鉴定结果显示:23株为Lactobacillus fuchuensis(48.94%),12株为Lactobacillus curvatus(25.53%),3株为Leuconostoc fallax(6.38%),2株为Lactobacillus sakei(4.26%),2株为Weissella ceti(4.26%);2株为Lactococcus cremoris(4.26%),1株为Leuconostoc lactis(2.13%),1株为Weissella minor(2.13%),1株为Enterococcus devriesei(2.13%)。qz1217、qz1196、qz1220所在的A、B、C三组乳酸菌在5-50°C的温度范围内生长良好,qz1196、qz1220所在的B、C组在pH 3.0-10.0的范围内生长良好,几乎所有乳酸菌都具有耐6.5%盐浓度特性。13株乳酸菌菌株对6种病原菌都具有抑制作用。通过排除酸、过氧化氢实验,发现上清液仍然具有抑菌活性。对qz1251发酵液进行蛋白酶处理,抑菌活性消失,确定其抑菌物质属于蛋白类物质,是一种细菌素。【结论】青海湖裸鲤肠道附着乳酸菌的多样性为益生性乳酸菌的筛选提供优质资源及数据参考。