乳酸菌S-层蛋白的功能特性及在微生态制剂中的应用

S-层蛋白(surface layer protein,slp)是一些乳酸菌,细菌及古细菌细胞壁表面所包被的生物活性大分子。这些蛋白能够形成周期性的有序结构,如倾斜形,正方形或是匀称的六边形。最新研究表明,这些结构是通过非共价键相互作用,在熵驱动下自我组装而成。与其他S-层蛋白相比,乳酸菌S-层蛋白的显著特点是分子质量小、等电点高。一些乳酸菌含有多种S-层蛋白编码基因,他们能够分别或同时表达。随后,文中介绍了3种乳酸菌S-层蛋白的分离方法。最后简单介绍了乳酸菌S-层蛋白在益生菌微生态制剂中的的应用。     

嗜酸乳杆菌S-层蛋白对感染大肠杆菌小鼠肠黏膜免疫功能的影响

研究嗜酸乳杆菌S-层蛋白对感染致病性大肠杆菌小鼠肠黏膜免疫的影响。用5 M的氯化锂溶液对嗜酸乳杆菌的S-层蛋白进行提取,获得脱除S-层蛋白的嗜酸乳杆菌菌体。将SPF级BALB/c小鼠随机分为对照组、致病性大肠杆菌感染组、嗜酸乳杆菌预防组治疗组,脱除S-层蛋白的嗜酸乳杆菌预防组和治疗组六组,实验进行14 d。小鼠处死后取眼球血、小肠及肠黏膜,观察各组小鼠肠黏膜形态变化、肠黏膜功能因子表达的差异。致病性大肠杆菌感染组与对照组相比,肠黏膜形态明显改变,感染大肠杆菌的小鼠在灌胃嗜酸乳杆菌后各黏膜SIg A和IL-10含量明显升高,同时血清IFN-γ、TNF-α和IL-1β含量对比感染组有了显著下降,说明可以减缓炎症发生,并且比脱除S-层蛋白的嗜酸乳杆菌治疗组对肠黏膜免疫保护效果好。嗜酸乳杆菌S-层蛋白对于嗜酸乳杆菌定植于肠黏膜并形成生物屏障保护其免疫功能有着重要影响。     

乳酸菌S-层蛋白的多样性及其研究方法

乳酸菌是人体和许多动物肠道的正常菌群,在预防肠道感染、促进人体健康方面,具有不可或缺的作用.乳酸茵通过竞争性黏附来抑制致病茵的繁殖.研究表明,乳酸菌的黏附作用与细胞表层蛋白密切相关.表层蛋白又名S-层蛋白(surface layer protein,Sip),是许多细菌及古生菌细胞壁表面所包被的生物活性大分子,在一些乳酸茵中也发现了S-层蛋白的存在.S-层蛋白的相对分子质量在40 000～200 000间,而在已发现的乳酸菌中,S-层蛋白相对分子质量在25 000～70 000间.作者讨论了乳酸菌中S-层蛋白的多样性及主要特性,对乳酸菌S-层蛋白的研究方法进行了初步探索.     

2种山葡萄白藜芦醇合酶基因的克隆与序列分析

目的:白藜芦醇合酶(resveratrol synthetic enzyme,RS)是植物白藜芦醇合成途径中的关键酶,为获得高效表达的白藜芦醇合酶基因,方法:利用已知的葡萄RS基因(AF274281)序列设计并合成一对引物,以2种不同来源的山葡萄叶片基因组DNA为模板,PCR扩增包含RS完整基因在内的一段序列;在此基础上采用悬挂延伸PCR法分别克隆目的基因。结果:测序与序列分析表明,RS全长基因片段大小均为1 536bp,其中包含一个内含子及两个外显子,同源性比较,其内含子碱基变化较大。悬挂延伸PCR法克隆获得的目的基因,开放读码框分别为1 170 bp和1 182bp,编码389/392个氨基酸残基,命名为CNRS1、CNRS3。同源性比较发现,CNRS1、CNRS3与已知葡萄RS基因序列的同源性分别为98%、97.5%。结论:以上结果证实CNRS1、CNRS3属葡萄RS基因家族成员,为今后进一步对该基因的研究与利用打下了基础。     

乳酸菌S-层蛋白及其黏附性相关研究技术

乳酸菌为人体的重要生理性细菌,在维持人体肠道生态环境、提高机体免疫力等方面起到重要的作用。然而,乳酸菌发挥这些生理功能的重要前提是黏附到肠细胞的表面。随着研究的深入,越来越多的研究表明乳酸菌S-层蛋白对宿主细胞具有黏附性,如罗伊氏乳酸菌S-层蛋白可与黏蛋白及人结肠癌细胞HT-29进行黏附。在研究乳酸菌S-层蛋白对宿主细胞的黏附时,人们采用了不同的方法。一些常用的基本方法有SDS-PAGE、层析纯化技术、显微镜观察技术等。随着新技术的应用,表面等离子共振技术、免疫学技术等也用于研究S-层蛋白的黏附性。本文将着重介绍S-层蛋白及S-层蛋白黏附性研究的相关技术。     

富含S-层蛋白乳酸菌的鉴定及其消化酶耐受性研究

通过平板划线分离法从新鲜牛奶中筛选获得一株乳酸菌菌株,命名为M8,SDS-PAGE分析该菌株富含S-层蛋白。结合表型特征、生理生化特征与16S rRNA基因序列分析法,鉴定该菌株为短乳杆菌(Lactobacillus brevis)。对其人工消化液耐受性分析,结果显示:其对人工胃液(3.0g/L,pH2.5)具有良好耐受能力,能在人工肠液(10g/L,pH8.0)中增殖,且S-层蛋白具有耐消化酶酶解能力。提示S-层蛋白作为保护屏障,可抵抗消化酶的作用。     

枯草芽孢杆菌BJ3-2 S-腺苷甲硫氨酸脱羧酶基因speD的克隆与序列分析

S-腺苷甲硫氨酸脱羧酶（SAMDC）是多胺合成过程中的限速酶。根据GenBank中枯草芽孢杆菌168菌株的S-腺苷甲硫氨酸脱羧酶基因（speD）序列设计同源引物,PCR扩增Bacillus subtilis BJ3-2gDNA,并克隆至pGEM-T载体后测序。序列分析结果表明：B. subtilis BJ3-2 speD基因ORF为381 bp,可编码126个氨基酸,分子质量为13.87 ku,基因登录号为KJ561347,Blast比对,SAMDC基因和蛋白序列的同源性都达到90%以上。B. subtilis BJ3-2的SAMDC具有丙酮酸依赖型脱羧酶的酶原剪切结构域[GVSGVVIISESHLTIH]和保守结构域[TCG],属于典型的I类B型SAMDC家族。SAMDC在多胺生物合成过程中扮演重要的角色,是多胺合成途径中的关键酶。研究为控制发酵豆豉中生物胺含量奠定了基础。     

嗜热链球菌胸苷酸合成酶基因的克隆与序列分析

本研究以嗜热链球菌(S．thermophilus)染色体DNA为模板,利用PCR技术扩增出胸苷酸合成酶(thymidylate synthase,thyA)基因,将其克隆入T载体中,转化DH5α,筛选阳性克隆,提取质粒,进行酶切鉴定,PCR扩增鉴定,进行测序,与已知序列进行同源性比较,结果表明成功克隆了thyA基因,全长900bp,与国外报道的S．thermophilus ATCC19258 thyA基因同源性达99．9%。这为构建以thyA基因为选择压力的非抗生素抗性载体提供了理论依据。     

苦荞麦主要过敏蛋白N端基因片段的克隆及序列分析

为了获得苦荞麦主要过敏蛋白全长基因并深入开展苦荞过敏蛋白的研究,本实验在先前获得的苦荞麦主要过敏蛋白C端(TBa)基因序列的基础上,设计不同的引物,以开花20d左右的苦荞麦果实为材料提取总RNA,并以此RNA为模板,通过RT-PCR和5’-RACE等方法,扩增,克隆获得苦荞麦主要过敏蛋白N端(命名为TBb)cDNA序列。序列分析结果表明,该序列由1005bp组成,开放阅读框为960bp,可编码一个由320个氨基酸残基组成的功能蛋白。同源性分析显示,此核苷酸序列与甜荞麦过敏性贮藏蛋白及豆球类蛋白的同源性达到90%。由此核苷酸序列推导出的氨基酸序列与甜荞麦13S贮藏蛋白有84%的同源性,与甜荞麦主要过敏蛋白有76%的同源性。苦荞麦主要过敏蛋白N端基因的获得为该蛋白的重组表达及其生物学活性等研究建立了前期基础。     

含金属硫蛋白鸡蛋水解物对两种乳酸菌体外生长的影响

以乳酸菌生长过程中的pH、OD值和菌落总数为评价指标,研究了普通鸡蛋、含金属硫蛋白鸡蛋、普通鸡蛋水解物以及含金属硫蛋白的鸡蛋水解物对动物双歧杆菌、嗜酸乳杆菌以及两种混合菌的影响。结果表明,鸡蛋水解物作为氮源能够促进乳酸菌的增殖,经比较,最佳氮源为含金属硫蛋白鸡蛋水解物,其动物双歧杆菌、嗜酸乳杆菌和混合菌发酵液OD值为普通鸡蛋水解物的1.05倍、1.08倍和1.09倍。含金属硫蛋白的鸡蛋水解物在动物双歧杆菌、嗜酸乳杆菌以及两种混合菌发酵液的最佳添加量分别为2.0%、1.5%和2.0%,活菌数分别可达7.5×109、3.21×109和2.33×1010 CFU/mL。本研究为开发乳酸菌发酵金属硫蛋白鸡蛋肽产品提供指导。     

Molecular cloning and deoxyribonucleic acid polymorphisms in Lactobacillus acidophilus and Lactobacillus gasseri.

Lactobacillus strain ADH is a bile-resistant, bacteriocin-producing human isolate that was phenotypically classified within the Lactobacillus acidophilus group. Total DNA and phage DNA extracted from strain ADH were separately digested with BclI and ligated with BclI-digested pGK12. Following electroporation of these ligation mixtures directly into strain ADH, electrotransformants were recovered at frequencies of 1.5 x 10(3) and 2.0 x 10(4)/micrograms of pGK12 for preparations of pGK12::phage DNA and pGK12::total DNA, respectively. Among the electrotransformants screened, 6 and 22% contained passenger DNA of either phage DNA or chromosomal origin, respectively, as determined by restriction-enzyme analyses and hybridization assays. Derivatives of pGK12 containing passenger DNA of chromosomal (pTRK120) or phage (pTRK121) origin and pTRK15 (native cryptic plasmid) were evaluated for use as species-specific probes. The strain ADH-derived probes hybridized primarily to members of the B-1 and B-2 lactobacilli homology groups and demonstrated strain-specific polymorphisms within these groups. Identical hybridization patterns were established for strain ADH and Lactobacillus gasseri VPI 6033 (ATCC 19992). Identification of DNA probes and establishment of a host-vector cloning system have facilitated our efforts to characterize the Lactobacillus chromosome and to distinguish between closely related species thought to be important inhabitants of the gastrointestinal tract.     

嗜酸乳杆菌S-层蛋白对肠道细胞黏附及巨噬细胞增殖的影响

比较去除S-层蛋白前后嗜酸乳杆菌对肠道细胞黏附及菌体自我凝集率的变化,并探究S-层蛋白对巨噬细胞增殖及溶酶体分泌的影响。采用4M氯化锂提取嗜酸乳杆菌ATCC 4356的表层蛋白,经透析及微孔过滤等步骤得到S-层蛋白,SDS-PAGE确定其分子量。利用酶标仪测定嗜酸乳杆菌在37℃孵育过程中(0 h~5.5 h)自我凝集率的变化情况,光学显微镜观察去除S-层蛋白前后嗜酸乳杆菌对结肠癌细胞HT-29的黏附情况变化。将S-层蛋白与巨噬细胞RAW264.7孵育2 h后,MTS法测定细胞增殖情况,并采用溶酶体荧光探针研究溶酶体分泌量的变化。结果表明:所提蛋白为嗜酸乳杆菌S-层蛋白(分子量M≈46ku)。嗜酸乳杆菌自我凝集率随孵育时间的延长而呈现出上升趋势,去除S-层蛋白后,菌体自我凝集率降低,嗜酸乳杆菌对HT-29细胞的黏附量也明显减少,且S-层蛋白能促进巨噬细胞增殖及其溶酶体分泌。     

嗜酸乳杆菌zrx02的plnD基因克隆及生物信息学分析

目的:对zrx02嗜酸乳杆菌细菌素编码基因进行克隆与序列分析。方法:对分离出的菌株进行16S rRNA鉴定,并根据已报道的plnD编码产物基因设计引物,以该菌基因组DNA为模板进行PCR扩增和测序,利用Pro Param tool,TMHMM等在线软件对编码蛋白进行生物信息学分析。结果:该菌为嗜酸乳杆菌,命名为嗜酸乳杆菌zrx02,该菌含有plnD基因,通过与Gen Bank中相关的抗菌素基因进行比对,发现其基因序列与Gen Bank库中的Lactobacillus plantarum strain（WP 016526896.1）plnD gene的同源性达到100%。生物信息学分析显示该基因全长355 bp,编码92个氨基酸,相对分子量10.54 ku,理论等电点9.26,该基因编码蛋白是稳定蛋白,不具有明显的跨膜结构,二级结构主要有α-螺旋、无规则卷曲和延伸链组成,三级结构结果显示plnD蛋白模型与Streptococcus pneumoniae中的ComE同源性最高。结论:对嗜酸乳杆菌zrx02 plnD基因的克隆与分析,为进一步探究细菌素的形成机制奠定理论基础。      

Expression of the sweet-tasting protein brazzein in Lactobacillus spp.

As sucrose has been implicated in healthrelated problems such as obesity and diabetes, awareness of a safe natural non-sugar sweetener has increased. Brazzein is the intensely sweet-tasting plant protein which consists of 54 amino acids. In this study, brazzein was expressed in Lactobacillus spp., probiotics, which are widely used in food industry. Three Lactobacillus spp. (Lb. paracasei KLB58, Lb. plantarum KLB213, and Lb. reuteri KCTC3594) were electrotransformed with pMSP3545::Braht which contains brazzein gene along with nisin-controlled expression (NICE) system. Brazzein expression in Lactobacillus spp. was confirmed by Western blot analysis. Overall, higher expression rate was obtained with nisin concentration of 25–40 ng/mL.     

Immune enhancing effects of Lactobacillus acidophilus LAFTI L10 and Lactobacillus paracasei LAFTI L26 in mice

The immune enhancing properties of Lactobacillus acidophilus LAFTI L10 and Lactobacillus paracasei LAFTI L26 in mice were investigated. Each mouse (BALB/c) was orally fed with cultures of either L. acidophilus or L. paracasei at 10(8) CFU/50 mul per day for 14 days. The effect of these strains on immunoglobulin A, interleukin-10 and interferon gamma producing cells in the gut immune system was determined by immunofluorescence assays. Systemic immune responses were analysed in mice serum upon euthanising after a 14-day feeding trial to estimate cytokines such as IL-10 and IFN-gamma by enzyme-linked immunosorbent assays. L. acidophilus and L. paracasei strains demonstrated an increase in the number of IgA producing cells, IL-10 and IFN-gamma cytokine producing cells in the small intestine. In the systemic immune response, mice fed with L. acidophilus or L. paracasei also enhanced the secretion of anti-inflammatory cytokine (IL-10) and pro-inflammatory cytokine (IFN-gamma). The results of this study suggest that L. acidophilus and L. paracasei were able to enhance specific gut and systemic immune responses in mice.     

Selective and differential enumerations of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp. in yoghurt--a review

Yoghurt is increasingly being used as a carrier of probiotic bacteria for their potential health benefits. To meet with a recommended level of ≥ 10⁶ viable cells/g of a product, assessment of viability of probiotic bacteria in market preparations is crucial. This requires a working method for selective enumeration of these probiotic bacteria and lactic acid bacteria in yoghurt such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lb. acidophilus, Lb. casei and Bifidobacterium. This chapter presents an overview of media that could be used for differential and selective enumerations of yoghurt bacteria. De Man Rogosa Sharpe agar containing fructose (MRSF), MRS agar pH 5.2 (MRS 5.2), reinforced clostridial prussian blue agar at pH 5.0 (RCPB 5.0) or reinforced clostridial agar at pH 5.3 (RCA 5.3) are suitable for enumeration of Lb. delbrueckii subsp. bulgaricus when the incubation is carried out at 45 °C for 72 h. S. thermophilus (ST) agar and M17 are recommended for selective enumeration of S. thermophilus. Selective enumeration of Lb. acidophilus in mixed culture could be made in Rogosa agar added with 5-bromo-4-chloro-3-indolyl-β-d-glucopyranoside (X-Glu) or MRS containing maltose (MRSM) and incubation in a 20% CO₂ atmosphere. Lb. casei could be selectively enumerated on specially formulated Lb. casei (LC) agar from products containing yoghurt starter bacteria (S. thermophilus and Lb. delbrueckii subsp. bulgaricus), Lb. acidophilus, Bifidobacterium spp. and Lb. casei. Bifidobacterium could be enumerated on MRS agar supplemented with nalidixic acid, paromomycin, neomycin sulphate and lithium chloride (MRS-NPNL) under anaerobic incubation at 37 °C for 72 h.     

乳酸菌发酵荠菜－番茄复合蔬菜汁的动力学研究

以荠菜和番茄为基料,用嗜酸乳杆菌和植物乳杆菌为试验菌种进行乳酸发酵,在发酵工艺优化的基础上,对5L 罐乳酸菌分批发酵过程进行研究,建立了菌体生长、产物生成和底物消耗动力学模型,经验证,模型的理论值和实验值拟和较好。所建动力学模型对乳酸菌发酵荠菜- 番茄复合蔬菜汁的反应器设计、工艺原理以及有效的控制发酵过程具有指导意义。     

乳酸菌饮料中嗜酸乳杆菌的实时荧光定量PCR检测方法

目的：为快速准确检测乳酸菌饮料中的嗜酸乳杆菌,建立实时荧光定量聚合酶链式反应（polymerase chain reaction,PCR）检测方法。方法：根据嗜酸乳杆菌NCFM的SPIDR保守区域设计特异性引物与探针,借助建立的实时荧光定量PCR方法进行特异性、灵敏度、重复性以及抗干扰能力验证,并利用模拟样品对方法进行检验,最后对市售的实际样品进行检测。结果：该方法的特异性较好;方法的绝对灵敏度达到3pg,相对灵敏度达103 CFU/mL;重复性检测表明相对标准偏差在2.6%以下。同时进行杂菌干扰实验,在纯基因组水平和培养物水平混合大肠杆菌,扩增均无明显影响,表明建立的方法抗干扰能力较好。利用建立的实时荧光定量PCR方法对模拟样品进行检测并建立标准曲线,得出R2为0.987,线性较好,可进行实际样品的检测。对市售的11 份样品进行检测,其中6 份含有嗜酸乳杆菌菌株,5份不含嗜酸乳杆菌菌株,标记嗜酸乳杆菌的样品全部检测出嗜酸乳杆菌且含量在5.83×102～3.68×104 CFU/mL之间。结论：建立的实时荧光定量PCR方法可快速、准确地检测出乳酸菌饮料中嗜酸乳杆菌。     

Bifidobacterium spp. and Lactobacillus acidophilus: biological,biochemical, technological and therapeutical properties relevant for use as probiotics

This review focuses on the biological properties and consequent technological roles of intestinal bacteria with potential health-promoting capacities, and provides selected examples available in the literature that are pertinent to the aforementioned concepts. A comprehensive overview pertaining to the taxonomy and ecology, as well as nutritional and health effects of Bifidobacterium spp. and Lactobacillus acidophilus, is provided; particular attention is paid to their incorporation, and growth and acidification features in fermented dairy products. The typical poor growth of these species in milk is highlighted, and the use of bifidogenic and growth factors, including their nature and function, is discussed. Efforts to establish optimum environmental conditions for their growth are critically reviewed, in addition to the effects of the food and storage conditions on microbial survival. Criteria for selection of effective microbial strains for their probiotic effect are listed, and modifications to improve fermentation efficiency and shelf-life of final dairy products are suggested; among these, the incorporation of Bifidobacterium spp. and L. acidophilus into a solid matrix, such as cheese, is emphasized.