乳酸菌微进化的研究进展

摘要:乳酸菌是食品工业中重要的微生物,乳酸菌微进化研究有助于深入解析其生物学功能与机制。随着分子生物学的发展,多位点序列分型(Multi-locus Sequence Typing,MLST)及基因组重测序(Whole-genome resequencing)等技术手段应运而生,使得从分子水平上阐述乳酸菌的系统发育和种群进化关系成为可能。MLST已被广泛用于乳酸菌遗传多样性和种群结构等微进化研究中,近期,测序成本的锐减使全基因组测序技术在乳酸菌微进化研究中的优势日益突显。本文对乳酸菌微进化的理论基础、研究方法和意义进行了阐述,并介绍了全基因组测序技术在乳酸菌微进化方面的应用,旨在为乳酸菌微进化分析研究提供新思路。     

内蒙古原始森林桦树皮内生和表生乳酸菌分离鉴定及其特性

[背景]随着乳酸菌产业的快速发展,不同环境乳酸菌群落结构的分析和菌种资源的开发利用已经成为该领域的研究热点.从不同环境、不同来源分离、保藏乳酸菌对于优良菌种的筛选和开发利用具有重要意义.[目的]阐明内蒙古原始森林桦树皮中乳酸菌的组成,并获得丰富的野生型乳酸菌资源,为优良菌株的筛选和工业化应用提供核心菌株,从原始森林桦树...     

一株产共轭亚油酸乳酸菌的鉴定及其特性

从酸菜汁中分离筛选到一株产共轭亚油酸(CLA)能力较高的乳酸菌。经鉴定,确定为植物乳杆菌Lactobacliius plantarum。微氧条件可提高CLA的产量,催化亚油酸(LA)生成CLA的酶受着LA的诱导。37℃对细胞生长和CLA生成最为有利。对数生长期为6～12h,18h后进入稳定期。在14～22h,CLA生成量快速增加,24h时达到最高值。该菌的培养物经萃取、甲酯化后,进行了气相色谱分离,生成的CLA产物为c9/t9,c11-CLA和t10,c12-CLA异构体的混合物。     

乳酸菌对真菌毒素的脱毒作用研究进展

真菌毒素广泛存在于农业产品中,对人和动物的健康构成巨大威胁。乳酸菌作为一种公认安全的微生物,在食品生物减毒方面具有巨大的应用潜力,成本低廉且不会对食品品质及生态环境造成不良影响。文章主要根据近年来国内外研究进展,阐述乳酸菌对食品和饲料中几种常见真菌毒素的脱毒作用(抑制真菌生长、毒素的吸附和降解),关注乳酸菌在生物脱毒方面的实际应用,为乳酸菌在食品保鲜领域的应用提供理论指导。     

一株屎肠球菌烈性噬菌体的分离鉴定及基因组测序分析

【背景】屎肠球菌为ESKAPE(由屎肠球菌、金黄色葡萄球菌、肺炎克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌和肠杆菌属六大超级细菌的拉丁学名首字母组成)病原体之一,对多种抗菌药物具有耐药性,严重威胁全球人类健康,被世界卫生组织列入亟须研发新抗菌药的病原体名单。【目的】分离针对屎肠球菌的烈性噬菌体,测定其基本生物学特性并进行基因组测序分析,为屎肠球菌噬菌体疗法提供原料。【方法】从牧场污水中分离筛选出一株烈性屎肠球菌噬菌体,命名为Enterococcus phage 1A11,通过透射电镜观察噬菌体的形态,测定其最佳感染复数、一步生长曲线和裂解谱,并进行全基因组的测序和分析,以阐释该噬菌体的基本生物学特性。【结果】电镜下可观察到屎肠球菌噬菌体1A11具有典型的正二十面体头部结构和较长的尾部结构,属于有尾病毒目长尾病毒科,而且测得其最佳感染复数为0.01,裂解周期为70 min,潜伏期为30 min,暴发期为40 min,并特异性地对部分屎肠球菌产生裂解作用。噬菌体1A11的基因组大小为42 750 bp,GC含量为34.71%,含有70个推定的开放阅读框(open reading frame, O...     

一株具有促生作用的生防细菌YN-2A的分离、鉴定及全基因组测序分析

【背景】根际土壤中分布着丰富的微生物类群,其中不少微生物具有拮抗病原菌的能力,以此保护植株免受病害侵袭。【目的】分离并鉴定出一株具有促生作用的生防细菌,从基因组学与生物信息学角度探究生防菌的抑菌机制。【方法】采用平板稀释涂布法筛选出具有拮抗黄瓜炭疽病作用的细菌。通过16S rRNA基因和基因组序列比对来确定生防细菌的生物学分类。以500倍生防菌稀释液(5×10⁸cfu/mL)为材料,测定其对番茄种子、幼苗的促生效果。通过平板对峙试验测定生防细菌的广谱抑菌性。利用三代(PacBio、Nanopore)结合二代测序技术解析菌株的基因组信息、基因功能注释等。【结果】从鹅掌柴根际土壤分离出一株生防细菌YN-2A,结合16S rRNA基因与核酸序列数据库(nucleotide sequence database, NT)比对结果,鉴定出菌株YN-2A为贝莱斯芽孢杆菌(Bacillus velezensis)。菌株YN-2A是一株好氧型的革兰氏阳性菌,具有产生生物膜的能力。菌株YN-2A的500倍稀释液(5×10⁸ cfu/mL)能够有效促进番茄种子发芽率和幼苗的生长,同时提高叶片的叶绿素荧光参数。在72、96和120 h的发芽率分别比CK组提高1.45倍、1.42倍和1.09倍;在幼苗形态指标上,株高提升5.90 cm。光化学猝灭指数(photochemical quenching, qP)在荧光参数中提升最为显著,处理后番茄叶片qP指数同CK组相比提高1.27倍。平板对峙试验中菌株YN-2A能够拮抗多种真菌病原。全基因组测序结果表明,菌株YN-2A全基因组长度为4 046 066 bp,G+C含量为46%,包含4 090个编码基因。将全基因组测序数据提交至NCBI获得GenBank登录号为CP139086。基本功能注释中大部分基因注释到氨基酸与碳水化合物代谢途径。另外,在病原与宿主互作(pathogen host interactions, PHI)数据库中毒力降低(reduced virulence)分类涉及基因数量高达905个,并有69个基因注释到病原菌失去致病力(loss of pathogenicity)分类。初步推断生防细菌YN-2A因其含有大量抗病、降低毒力的基因来发挥抑菌作用。【结论】分离并鉴定出一株贝莱斯芽孢杆菌(B. velezensis) YN-2A,该菌株对番茄的种子和幼苗具有良好的促生效果,同时该菌株具备广谱抑菌性。通过测序技术,获得了菌株YN-2A的全基因组信息,为深入挖掘菌株YN-2A的抑菌机制提供参考信息。     

Alcoholic fermentation and its prevention by Lactobacillus buchneri in whole crop rice silage

AIMS: To evaluate the fermentation characteristics and the effects of Lactobacillus buchneri inoculation in ensiling whole crop rice. METHODS AND RESULTS: Laboratory-scale silages were prepared from whole crop rice harvested at yellow-ripe stage. The crop was ensiled for 2 months with and without inoculation of L. buchneri at 10(4), 10(5) and 10(6) CFU g(-1). The effect of prolonged ensiling was also studied by using the same crop; the silos were opened at 1, 3, 6 and 12 months, while the inoculation was made at 10(5) CFU g(-1). Enhanced alcoholic fermentation was found in untreated silage; the sum of ethanol and 2,3-butanediol were seven times higher at 2 months than those of lactic and volatile fatty acids, while the differences were diminished at 12 months owing to the reduction of ethanol in the late ensiling period. Inoculation of L. buchneri inhibited the alcohols; however, ethanol yet prevailed over the fermentation until 6 months, after which acetic acid became the main product in the inoculated silage. Regardless of inoculation and ensiling period, yeasts were not found in whole crop rice silage. CONCLUSIONS: Substantial amounts of ethanol and 2,3-butanediol would be produced in silage prepared from whole crop rice. The alcoholic fermentation can be suppressed when inoculated with L. buchneri. SIGNIFICANCE AND IMPACT OF THE STUDY: Inoculation of L. buchneri could be an option to prevent ethanol fermentation in silage.     

The Lactic Acid Enterococci Enterococcus faecium and Enterococcus durans: Nucleotide Sequence Diversity in 16S rRNA Genes

Among the strains used as starters for making sour milk products on the territory of the CIS, the bacteria Enterococcus faecium and Enterococcus durans are frequently found. In this work, we studied a new collection of lactic acid enterococci and also obtained more complete data on the nucleotide sequences of 16S rRNA genes in some strains studied earlier and found that most strains had certain distinctions in their 16S rRNA genes as compared with the E. durans and E. faecium genes available in the NCBI database. Based on these data, it is suggested that the strains of lactic acid enterococci represent new, earlier unknown taxa of enterococci that use milk as an ecological niche.     

Elucidation of the Taxonomic Status of Industrial Strains of Thermophilic Lactic Acid Bacteria by Sequencing of 16S rRNA Genes

Both phenotypic characteristics and results of PCR tests for the presence of species-specific genes indicate that a number of strains of thermophilic lactic acid bacteria previously considered as belonging to Streptococcus thermophilus are actually closely related to enterococci. In the present study, partial (over 500 nucleotides) sequencing of 16S rRNA genes from 12 strains of thermophilic lactic acid bacteria used as starters for manufacturing sour milk products on the territory of the Commonwealth of Independent States (CIS) has been performed. According to the results of the sequencing, seven of the strains have been classified with Enterococcus durans. The earlier classification (based on PCR tests) of two of the strains as S. thermophilus and three of the strains as E. faecium has been confirmed. The data obtained demonstrate that the enterococci E. durans and E. faecium are widely used as thermophilic starters for manufacturing sour milk products on the territory of the CIS.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 520–525.Original Russian Text Copyright © 2005 by Botina, Lysenko, Sukhodolets.     

解磷菌PSB-R全基因组测序鉴定及其解磷特性分析

采用生理生化指标结合分子生物学技术对解磷菌PSB-R进行分类学鉴定,确定为粘质沙雷氏菌（Serratia marcescens）,通过二代测序平台Illumina NovaSeq PE150对PSB-R进行全基因组测序,分析预测了与解磷能力相关基因及其他植物促生基因组成情况。通过响应面优化试验检测了PSB-R最大解磷能力为805.199 mg/L,连续培养10代后解磷能力稳定且对多种难溶性磷酸盐均具有溶解能力。本研究为解磷菌解磷机制的进一步研究提供了基因组数据基础,同时证实PSB-R具有应用于菌肥的潜力,为后续解磷菌肥的研制提供了研究基础。     

老面中玉米赤霉烯酮消减乳酸菌的筛选、鉴定及机理解析

【背景】玉米赤霉烯酮(zearalenone,ZEN)是一种由镰刀菌属真菌产生的具有雌激素效应的真菌毒素,是世界范围内严重危害人类健康和农业安全的污染物之一。乳酸菌作为一类被公认安全(generally recognized as safe,GRAS)的食品级非致病微生物,近年研究显示其具有良好的真菌毒素消减能力,为其在保障食品安全方面带来潜在应用前景。【目的】以来自山东、河南和甘肃等7份老面样品作为研究对象,从中筛选具有消减ZEN活性的乳酸菌,探究该菌对ZEN的吸附机理。【方法】利用稀释平板涂布法获得菌株;超高效液相色谱检测具有消减ZEN活性的菌株,质谱(mass spectrometry)确定代谢产物;16s rRNA基因序列分析比对确定菌株属种,透射电镜(transmission electron microscopy,SEM)观察菌株形态;分析不同初始毒素浓度及菌体浓度下的吸附效果,对其进行动力学模型拟合;定位吸附位点并通过傅里叶变换红外光谱(Fourier transform infrared spectroscopy,FTIR)分析确定菌株中参与吸附ZEN的官能团,阐明对ZEN的吸附机理。【结果】初步筛选获得63株乳酸菌,从中复筛获得3株具有消减ZEN活性的乳酸菌,经鉴定：菌株6-8和菌株18-2为短促生乳杆菌(Levilactobacillus brevis),菌株12-6为热带醋杆菌(Acetobacter tropicalis)。两株短促生乳杆菌具有降解效果,在ZEN浓度为10 mg/L的条件下,48 h内降解率分别达到85.5%和87.3%,质谱结果显示降解产物为α-ZEL;另一株具有吸附效果,在菌株浓度为4.26×10¹⁰ CFU/mL、ZEN浓度为10 mg/L条件下,20 min内吸附率达到62.9%,灭活后吸附率上升20%。吸附过程同时符合准一级(pseudo-first-order)动力学及准二级(pseudo-second-order)动力学模型。傅里叶变换红外光谱结果显示菌株12-6的主要吸附位点为细胞壁上的肽聚糖与磷壁酸分子,羟基、次甲基、羧基和酰胺基等作为主要官能团参与吸附。【结论】菌株6-8和菌株18-2对ZEN有较强的降解能力;菌株12-6对ZEN有较强的吸附能力。其吸附动力学符合准一级及准二级动力学模型,吸附位点为细胞壁上的磷壁酸及肽聚糖分子。本研究为乳酸菌清除食品和饲料中有害物质的应用提供了理论基础。     

Bacterial population in traditional sourdough evaluated by molecular methods

AIMS: To study the microbial communities in artisanal sourdoughs, manufactured by traditional procedure in different areas of Sicily, and to evaluate the lactic acid bacteria (LAB) population by classical and culture-independent approaches. METHODS AND RESULTS: Forty-five LAB isolates were identified both by phenotypic and molecular methods. The restriction fragment length polymorphism and 16S ribosomal DNA gene sequencing gave evidence of a variety of species with the dominance of Lactobacillus sanfranciscensis and Lactobacillus pentosus, in all sourdoughs tested. Culture-independent method, such as denaturing gradient gel electrophoresis (DGGE) of the V6-V8 regions of the 16S rDNA, was applied for microbial community fingerprint. The DGGE profiles revealed the dominance of L. sanfranciscensis species. In addition, Lactobacillus-specific primers were used to amplify the V1-V3 regions of the 16S rDNA. DGGE profiles flourished the dominance of L. sanfranciscensis and Lactobacillus fermentum in the traditional sourdoughs, and revealed that the closely related species Lactobacillus kimchii and Lactobacillus alimentarius were not discriminated. CONCLUSIONS: Lactobacillus-specific PCR-DGGE analysis is a rapid tool for rapid detection of Lactobacillus species in artisanal sourdough. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports a characterization of Lactobacillus isolates from artisanal sourdoughs and highlights the value of DGGE approach to detect uncultivable Lactobacillus species.     

利用内源CRISPR-Cas系统开展乳酸菌基因编辑的研究进展

CRISPR-Cas9技术是一种高效、精准的基因编辑工具,该技术的建立推动基因组编辑进入快速发展阶段。目前应用最广泛的Cas9蛋白是来源于酿脓链球菌(Streptococcuspyogenes)的SpyCas9,该蛋白作为“基因剪刀”在哺乳动物、植物等真核生物中应用较为广泛且成熟,但是该蛋白在一些乳酸菌中的应用仍然受到多种因素的限制。乳酸菌基因组上已发现多种类型的CRISPR系统,也蕴含着多种未表征的Cas蛋白,利用乳酸菌内源CRISPR-Cas系统,结合外源导入的向导RNA和同源修复模板,也可实现对乳酸菌基因组的编辑。这种基于内源CRISPR-Cas系统实现基因编辑的方式,具有打靶载体相对较小易转化、无外源Cas9蛋白对宿主细胞产生毒性等优势,相比于CRISPR-SpyCas9更适合于乳酸菌基因组进行编辑,可能是一些乳酸菌未来开展基因组编辑的主要手段,本文重点对此部分内容进行了综述。     

泡菜中优良乳酸菌的分离、鉴定及发酵特性

从几种泡菜中分离出86株菌,对其在适温和低温下产酸速率及硝酸盐降解能力进行测定,筛选出5株产酸速率快、硝酸盐降解能力强的菌株。经形态学鉴定及生理生化反应试验,初步鉴定为：植物乳杆菌2株,短乳杆菌1株,戊糖乳杆菌1株,肠膜明串珠菌葡聚糖亚种1株,并对5株菌的发酵性能进行了测定。     

微生物全基因组鸟枪法测序

全基因组测序主要有二种策略,一种是分级鸟枪法测序,另一种是全基因组鸟枪法测序。微生物是一种十分重要的遗传资源,运用全基因组鸟枪法可以方便、快捷地完成其基因组的测序任务。本文对微生物全基因组鸟枪法测序中文库构建、插入片段的长短比例、反应投入量、拼接以及补洞等问题作了较细致的描述,有些步骤作了举例说明。Abstract:Two strategies introduced for whole genome sequencing,one is clone by clone method,the other is whole genome shotgun sequencing,for microbes which are very important to us,whole genome shotgun sequencing method is very convenient.In this article we discussed the library construction、long-to-short-ratio of insert,、total number of reads should be sequenced、assembly and gap filling technologies of the whole microbial genome shotgun sequencing method while some examples presented.     

产黏乳酸菌的发酵特性研究

通过对2株产黏乳酸球菌的发酵特性比较研究,以感观、酸度、活菌数对数值、培养时间、粘度及乳清析出率为考察指标,结果表明:乳酸球菌Q26具备较好的产黏、产香特性,遗传性质较稳定.通过与乳酸杆菌G18进行发酵应用试验,确定G18和Q26存在共生关系,混合发酵的酸牛乳乳清析出率为0.1％,粘度为4 528mPa·s.通过保质期试验,产品在28、20、4℃下保质期分别为3、6、15d.确定Q26和G18为最佳酸牛乳生产用菌种.