CRISPR/Cas基因组编辑技术在乳酸菌中的应用及研究展望

乳酸菌是一类重要的食品工业微生物,目前对其功能基因鉴定和挖掘优良功能基因主要依赖于传统的基因同源重组技术,该技术尽管有较高的可靠性,但是存在操作繁琐、效率低下等不足,严重制约了乳酸菌优良菌株的遗传选育。CRISPR/Cas基因编辑技术极大提升了对多物种基因组的编辑效率,这为乳酸菌功能基因的快速鉴定及遗传改良提供了可能,但是现有的CRISPR/Cas基因编辑技术在乳酸菌的应用还存在诸多限制。本文综述了CRISPR/Cas基因编辑技术在乳酸菌基因组上的应用现状及亟待解决的问题,并展望了乳酸菌基因组编辑技术的未来发展趋势,为乳酸菌功能基因鉴定及遗传改良提供参考。     

Gene expression systems for lactic acid bacteria.

Considerable advances have been made in the genetics and molecular biology of lactic acid bacteria, including Lactococcus, Lactobacillus, Leuconostoc, Pediococcus and Streptococcus spp. These have resulted in the construction of constitutive gene expression cassettes, inducible gene expression systems, and specific protein targeting systems for these bacteria. These developments are important in the food industry where lactic acid bacteria can be exploited as food-grade cell factories.     

新疆喀什地区母乳中乳酸菌的多样性分析

【目的】对新疆喀什地区母乳中乳酸菌多样性进行分析。【方法】采用菌落培养、显微镜观察、Repetitive genomic fingerprinting(Rep-PCR)指纹图谱和16S r RNA基因序列分析相结合的方法研究母乳中乳酸菌的菌群分布。【结果】从11份母乳中共分离出乳酸菌193株,利用16S r RNA基因序列同源分析和系统发育树对代表菌株进行了分子鉴定,193株乳酸菌隶属于4个属,分别为Lactobacillus(22株)、Streptococcus(42株)、Lactococcus(40株)、Enterococcus(89株)。其中,Enterococcus所占比例最大,达到46%。【结论】新疆喀什地区母乳中乳酸菌多样性丰富,极具开发潜力,将为开发母乳中的益生菌以及安全可靠的微生态制剂提供理论依据。     

乳酸菌的安全性研究

乳酸菌通常被认为是安全的,但在机体免疫抑制、肠道发育未成熟、肠上皮受损等情况下,可以导致心内膜炎、菌血症、局部性感染等临床症状.本文综述了国外有关乳酸菌的致病性、抗药性基因转移方面研究的一些新进展,以期引起国内对乳酸菌安全性问题的重视.     

新疆伊犁地区原牛乳中乳酸菌的多样性分析

【目的】对新疆伊犁地区原牛奶中乳酸细菌的遗传多样性进行分析。【方法】采用菌落培养、Rep-PCR(Repetitive genomic fingerprinting)指纹图谱和16S r RNA基因序列分析相结合的方法研究牛乳内乳酸菌的遗传多样性。【结果】从5份原牛乳中分离出乳酸菌29株,基因序列分析和系统进化分析显示29株乳酸菌隶属于5个属,分别为:Lactococcus、Lactobacillus、Leuconostoc、Pediococcus和Enterococcus。优势属为Leuconostoc(27.6%),其次为Lactococcus(24.0%)。【结论】新疆伊犁地区原牛乳中乳酸菌多样性丰富,为开发新疆地区益生乳酸菌提供了丰富的活性资源。     

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。     

饲料乳酸菌的分离鉴定及优良菌株筛选

对从饲料玉米、高粱、麦秆及棉花中筛选出的乳酸菌进行分类鉴定和综合性分析。用MRS+CaCO3固体培养基从棉花中分离出乳酸菌18株、高粱中30株、饲料玉米中18株、麦秆中18株。经形态学、生理生化试验进行初步鉴定并按产酸试验,耐盐及耐酸试验挑选出32株产酸率强的乳酸菌对其进行16S rDNA分子鉴定。结果显示,32株菌都具有良好的耐盐、耐酸能力;经生理生化和16S rDNA基因序列鉴定可知32株乳酸菌分属于两个属,即乳杆菌属、肠球菌属,4个种,即干酪乳杆菌(Lactobacilluscasei)、肠道球菌(Entercoccus faecium)、植物乳杆菌(Lactobacillus plantarum)、海氏肠球菌(Entercoccus hirae)。4种饲料原料中肠道球菌普遍存在。除了这种乳酸菌以外,棉花有干酪乳杆菌、植物乳杆菌、海氏肠球菌,玉米和麦秆内有植物乳杆菌。从饲料中筛选出4株具有较强产酸能力的乳酸菌,可进一步研发成青贮饲料添加剂。     

mCherry红色荧光标记乳酸菌的融合表达系统构建及应用

为开发新型荧光蛋白标记乳酸菌以填补国内研究空白,利用pSIP载体,构建了以红色荧光蛋白mCherry为标记,并以乳酸菌胆盐水解酶基因bsh为报告基因的乳酸菌融合表达系统。在4种不同启动子(P_(sppA)、P_(ldhL)、P_(32)和P_(slpA))调节下,相继实现了融合基因的诱导型和组成型表达,表达的融合重组蛋白mCherry-BSH同时检测出红色荧光活性和胆盐水解酶BSH活性。mCherry红色荧光标记的乳酸菌融合表达系统的成功构建不仅为研究乳酸菌在生物体内的分布、定植及存活情况从而揭示其益生功能的作用机理提供有利条件,也为更多活性蛋白在乳酸菌宿主中的表达、细胞定位、功能鉴定的研究奠定基础。     

青藏高原垂穗披碱草青贮饲料中乳酸菌的多样性及低温发酵菌株的筛选

【目的】探究青藏高原垂穗披碱草青贮饲料中乳酸菌的多样性,筛选在低温条件下(10、15和25°C)生长性能较好的优良菌株。【方法】将垂穗披碱草青贮饲料中分离纯化的乳酸菌进行形态特征观察及16S r RNA基因测序鉴定;用MRS液体培养基在10、15和25°C条件下分离、初筛乳酸菌,选取高吸光度值的菌株作为优势菌株。用绿汁发酵液在10、15和25°C条件下培养测定其p H值,选取低p H值菌株作为优势菌株,并综合MRS培养基筛选结果确定优良菌株。【结果】从不同温度和发酵阶段的垂穗披碱草青贮饲料中共分离得到108个乳酸菌菌株,它们分属于6个属、18个种。其中,清酒乳杆菌LS-24在15°C条件下发酵液p H值显著降低(P0.05),戊糖片球菌PP-63在发酵初期生长速度较快,植物乳杆菌LP-21在15°C条件下发酵液p H值降至3.9且有最大活菌数。【结论】在青藏高原垂穗披碱草青贮饲料中发现的乳酸菌属基本涵盖了前人在常温青贮饲料中发现的所有属,但种数略少;在108株菌中,清酒乳杆菌LS-24、戊糖片球菌PP-63和植物乳杆菌LP-21在低温条件下均表现出较好的繁殖和发酵特性,可作为青贮饲料低温发酵的备选菌株。     

芝麻香型白酒发酵过程中乳酸菌多样性及其演替规律

【背景】乳酸菌是白酒发酵过程中一类非常重要的微生物,其种类及动态变化对于白酒品质具有重要影响。然而,目前对于芝麻香型白酒发酵过程中乳酸菌群落结构及其演替规律的认识并不全面。【目的】揭示芝麻香型白酒发酵过程中乳酸菌的多样性及菌群的演替规律,为更好地探索白酒酿造机理和控制白酒品质提供生物学依据。【方法】利用高通量测序技术对芝麻香型白酒发酵过程中乳酸菌菌群演替进行跟踪分析,同时采用实时荧光定量PCR对发酵过程中乳酸菌的生物量进行定量分析。【结果】高通量测序结果显示,芝麻香型白酒发酵过程涉及5个属的乳酸菌:魏斯氏菌属(Weissella)、片球菌属(Pediococcus)、乳杆菌属(Lactobacillus)、明串珠菌属(Leuconostoc)和乳球菌属(Lactococcus),共计43种乳酸菌。其中,在发酵过程中平均相对丰度大于0.5%的乳酸菌有10种,分别是类肠膜魏斯氏菌(Weissella paramesenteroides)、食窦魏斯氏菌(Weissella cibaria)、融合魏斯氏菌(Weissella confusa)、戊糖片球菌(Pediococcus pentosaceus)、假肠膜明串珠菌(Leuconostoc pseudomesenteroides)、发酵乳杆菌(Lactobacillus fermentum)、植物乳杆菌(Lactobacillus plantarum)、副干酪乳杆菌(Lactobacillus paracasei)、耐酸乳杆菌(Lactobacillus acetotolerans)和Lactobacillus sp.。在堆积发酵过程中,Weissella属占细菌总量的50%以上,其次是Pediococcus属和Lactobacillus属,而Leuconostoc属和Lactococcus属相对较少。在窖池发酵过程中Lactobacillus属的乳酸菌逐渐成为优势细菌,尤其是Lactobacillus sp.在窖池发酵中后期相对丰度达到80%以上。实时荧光定量PCR结果显示,在堆积发酵和窖池发酵前期乳酸菌总量变化不大;从窖池发酵5 d开始,乳酸菌总量迅速上升,30 d时达到最大值。【结论】对白酒发酵过程中乳酸菌种类及动态变化的研究有助于探究白酒酿造过程中乳酸菌功能,进而解析白酒酿造机理,最终达到控制白酒品质的目的。     

乳酸菌的系统分类概况

乳酸菌是重要的益生菌资源,在食品、农业、化工业、医学等领域具有广阔的应用前景。目前,人们熟知的乳酸菌主要集中在乳杆菌属、双歧杆菌属、链球菌属、肠球菌属、乳球菌属、片球菌属和明串珠菌属等少数属种。为了拓宽人们对乳酸菌的认知,本文就乳酸菌的系统分类学进行阐述。在系统分类学上,乳酸菌分别隶属于厚壁菌门4纲7目18科39属653种和放线菌门2纲2目3科12属88种。最后,对乳酸菌的益生作用、安全性与有效来源、益生潜能的体外评价指标等进行简要的讨论。     

Identification of industrial strains of lactic acid bacteria by methods of molecular genetic typing

Various methods currently used in microbiology for determining taxonomic state of bacteria are discussed. The main focus is aimed at identifying and gene typing of lactic acid bacteria, used as starter cultures for industrial process of production of sour milk products, meat products, and probiotics.     

The molecular and phenotypic characterization of fructophilic lactic acid bacteria isolated from the guts of <Emphasis Type="Italic">Apis mellifera</Emphasis> L. derived from a Polish apiary

This paper describes taxonomic position, phylogeny, and phenotypic properties of 14 lactic acid bacteria (LAB) originating from an Apis mellifera guts. Based on the 16S rDNA and recA gene sequence analyses, 12 lactic acid bacteria were assigned to Lactobacillus kunkeei and two others were classified as Fructobacillus fructosus. Biochemically, all isolated lactic acid bacteria showed typical fructophilic features and under anaerobic conditions grew well on fructose, but poorly on glucose. Fast growth of bacteria on glucose was noted in the presence of oxygen or fructose as external electron acceptors. The residents of honeybee guts were classified as heterofermentative lactic acid bacteria. From glucose, they produced almost equimolar amounts of lactic acid, acetic acid, and trace amounts of ethanol. Furthermore, they inhibited the growth of the major honeybee pathogen, Paenibacillus larvae, meaning that the LAB studied may have the health-conferring properties of probiotics.     

Lactic acid bacterial cell factories for gamma-aminobutyric acid

Gamma-aminobutyric acid is a non-protein amino acid that is widely present in organisms. Several important physiological functions of gamma-aminobutyric acid have been characterized, such as neurotransmission, induction of hypotension, diuretic effects, and tranquilizer effects. Many microorganisms can produce gamma-aminobutyric acid including bacteria, fungi and yeasts. Among them, gamma-aminobutyric acid-producing lactic acid bacteria have been a focus of research in recent years, because lactic acid bacteria possess special physiological activities and are generally regarded as safe. They have been extensively used in food industry. The production of lactic acid bacterial gamma-aminobutyric acid is safe and eco-friendly, and this provides the possibility of production of new naturally fermented health-oriented products enriched in gamma-aminobutyric acid. The gamma-aminobutyric acid-producing species of lactic acid bacteria and their isolation sources, the methods for screening of the strains and increasing their production, the enzymatic properties of glutamate decarboxylases and the relative fundamental research are reviewed in this article. And the potential applications of gamma-aminobutyric acid-producing lactic acid bacteria were also referred to.     

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。     

Species diversity and relative abundance of vaginal lactic acid bacteria from women in Uganda and Korea

AIMS: Lactobacilli play an important role in maintaining vaginal health of women. The aim of this study was to compare the species richness and relative abundance of Lactobacillus and other lactic acid bacteria in women of two geographically distant countries, Uganda and Korea. METHODS AND RESULTS: Vaginal samples were obtained from two women populations in Uganda and Korea. The Lactobacillus Rogosa SL agar was used for initial isolation of lactic acid bacteria. After phenotypic analyses, the 16S rRNA gene was amplified by polymerase-chain reaction and analysed by the BLAST program and phylogenetic tree construction. A total of 338 (128 Korean and 210 Ugandan) vaginal lactic acid bacterial strains were isolated, including five genera: Lactobacillus, Leuconostoc, Pediococcus, Streptococcus and Weissella. While Lactobacillus crispatus was common in both populations, Lactobacillus fermentum was common only in Korean women, and Lactobacillus gasseri, Lactobacillus reuteri and Lactobacillus vaginalis only in Ugandan women. Among other lactic acid bacteria, Weissella was more common in Ugandan, and Pediococcus in Korean women. All Weissella strains produced hydrogen peroxide, and all Pediococcus strains inhibited Candida species. CONCLUSION: Although many lactic acid bacteria colonize women, their species distributions may be different in women of geographically separated communities. SIGNIFICANCE AND IMPACT OF THE STUDY: The knowledge of species richness and relative abundance of vaginal lactic acid bacteria, including Lactobacillus, Pediococcus and Weissella, may lead to the design of better probiotic products as bacterial replacement therapy.     

Identification of industrial strains of lactic acid bacteria by methods of molecular genetic typing

Various methods currently used in microbiology for determining taxonomic state of bacteria are discussed. The main focus is aimed at identifying and gene typing of lactic acid bacteria, used as starter cultures for industrial process of production of sour milk products, meat products, and probiotics.     

解淀粉乳酸细菌在L-乳酸发酵生产中的应用

对解淀粉乳酸细菌及其产生的淀粉酶和发酵工艺等方面的国内外研究现状进行了综述。解淀粉乳酸细菌具有分泌淀粉酶的能力,可免去原料水解处理工序直接发酵淀粉质原料生产乳酸,可以简化生产工艺,并可节约设备投资,进而降低生产成本。解淀粉乳酸细菌主要分离自传统发酵食品,也可从有机废弃物和厨余垃圾中分离得到。介绍了解淀粉乳酸细菌直接利用淀粉质原料的机理,比较了解淀粉乳酸菌发酵生产L-乳酸的工艺。提出通过诱变育种和基因工程育种等方法获得更加高效的解淀粉乳酸细菌,并结合先进的发酵、分离技术来提高乳酸生产效率。     

低温环境中乳酸菌的开发利用

乳酸菌作为一种益生资源,越来越为人们所重视.在自然界存在的乳酸菌中,有一类虽然人们一直在利用,但是还没有充分的研究和开发,这类乳酸菌就是低温环境中生长的乳酸菌.国外目前所见有关低温乳酸菌的报道多集中于低温冷藏肉、鱼制品及泡菜中的乳酸菌,集中研究和应用的菌属主要包括Leuconostoc和Lactobacillus,而国内这方面的研究并不多见.笔者根据目前国内外低温乳酸菌的研究现状,阐述了其存在环境、种类及相应作用,并对其研究涉及的领域、趋势及应用前景进行了探讨和展望,以期为国内这类乳酸菌资源的研究开发提供参考和依据.