马乳酒样乳杆菌马乳酒样亚种及其研究进展

马乳酒样乳杆菌(Lactobacillus kefiranofaciens)是一类革兰氏阳性乳杆菌,其来源广泛,功能多样,安全性好.2020年12月国家卫健委批准马乳酒样乳杆菌马乳酒样亚种作为我国新食品原料.本文主要综述国内外马乳酒样乳杆菌和马乳酒样乳杆菌马乳酒样亚种的菌种形态学和基因组特性、益生功能、主要价值和发展前...     

短乳杆菌与植物乳杆菌的发酵特性

【背景】目前对于酸菜发酵的研究主要关注点是植物乳杆菌(Lactobacillus plantarum),有关短乳杆菌(Lactobacillus brevis)在酸菜方面的研究报道很少。【目的】为了挖掘短乳杆菌的发酵性能并开发酸菜发酵剂,将2株短乳杆菌分别与1株植物乳杆菌进行组合并发酵酸菜,分析短乳杆菌对酸菜发酵品质的影响。【方法】分别测定短乳杆菌与植物乳杆菌的单菌株生长产酸性能、耐酸性及亚硝酸盐降解力,并将两菌种组合后发酵酸菜,分析1-7d内酸度、乳酸菌活菌数、亚硝酸盐含量及酸菜质构特性的变化趋势。【结果】相较于短乳杆菌Lb-9-2,短乳杆菌Lb-5-3的生长和产酸速率较慢、酸耐受力较弱,但其亚硝酸盐降解力较强。两株短乳杆菌分别与植物乳杆菌Lp-9-1组合后产酸力显著增强,并在3 d时达到最低pH值(约3.10);植物乳杆菌Lp-9-1的添加使酸菜中总体乳酸菌生长延迟,在5 d时达到最高活菌数;组合菌种的样品中亚硝酸盐含量在1-7 d内变化较为平缓,前5天内两个组合之间差异不显著;接种乳酸菌会降低酸菜硬度和弹性,发酵3d时Lb-5-3/Lp-9-1组合的硬度最大,感官评价得分最高。【...     

Isolation, characterization and transposition of an (IS2)2 intermediate

We have isolated and characterized a dimer derivative of the extensively studiedEscherichia coli insertion sequence IS2. The dimer structure — called (IS2)₂ — consists of two IS2 elements arranged as a direct repeat, separated by 1 bp. The junction between the (IS2)₂ dimer and target sequences is located at various positions in independent isolates; however, one position was preferred. The transposition of (IS2)₂ into a target plasmid resulted in cointegrate-type structures. The transposition frequency of the (IS2)₂ dimer itself was significantly higher than that of the isogenic monomer IS2 insertion. The poor stability and high activity of (IS2)₂ indicates that this is an active transposition intermediate. The mode of transposition of (IS2)₂ is analogous to the joined dimer model described in the case of (IS21)₂ and (IS30)₂.     

Comparative Genomic Sequence Analysis of the FXR Gene Family: FMR1, FXR1, and FXR2

Mutations in the X-linked gene FMR1 cause fragile X syndrome, the leading cause of inherited mental retardation. Two autosomal paralogs of FMR1 have been identified, and are known as FXR1 and FXR2. Here we describe and compare the genomic structures of the mouse and human genes FMR1, FXR1, and FXR2. All three genes are very well conserved from mouse to human, with identical exon sizes for all but two FXR2 exons. In addition, the three genes share a conserved gene structure, suggesting they are derived from a common ancestral gene. As a first step towards exploring this hypothesis, we reexamined the Drosophila melanogaster gene Fmr1, and found it to have several of the same intron/exon junctions as the mammalian FXRs. Finally, we noted several regions of mouse/human homology in the noncoding portions of FMR1 and FXR1. Knowledge of the genomic structure and sequence of the FXR family of genes will facilitate further studies into the function of these proteins.     

灌喂植物乳杆菌和干酪乳杆菌增加仔猪肠道菌群多样性及短链脂肪酸生成

【目的】研究断奶前给仔猪饲喂植物乳杆菌和干酪乳杆菌对断奶前、后肠道菌群组成、数量和短链脂肪酸(SCFA)浓度的影响,分析仔猪生长性能与肠道形态、微生物菌群及SCFAs的相关性,探讨测试菌株缓解仔猪断奶应激的可能机制。【方法】选取15窝7 d龄杜长大仔猪,随机分为3组,分别灌喂2 mL去离子水(对照组)、0.5×10~9 CFU/mL植物乳杆菌(LP组)或干酪乳杆菌(LC组)的菌液,每组以窝为单位5个重复,于21 d(断奶)、24 d和35 d屠宰,采集回肠和结肠食糜,分析菌群组成和数量的变化,测定SCFAs浓度。【结果】测试菌株均能显著提高断奶2周后回肠、结肠菌群多样性(P0.05),促进乳酸杆菌和双歧杆菌增殖;显著促进断奶前回肠和结肠中乙酸、丙酸、丁酸和总SCFA生成,促进断奶后乙酸和总SCFA产生;相关分析显示,测试菌株组仔猪腹泻率下降与SCFAs浓度上升、回肠绒毛高度增加和总菌数量上升显著相关,日增重提高与结肠乙酸和TSCFA浓度增加显著相关。【结论】测试菌株促进乳酸杆菌、双歧杆菌等有益菌增殖,增加肠道菌群多样性,促进肠道SCFAs生成。     

低H~+_-ATPase活性植物乳杆菌突变菌筛选及基因表达的相对定量分析

【目的】筛选H~+_-ATPase活性降低的植物乳杆菌突变菌,比较其与亲本菌基因表达水平的差异,进一步探索H~+_-ATPase的调控机制。【方法】利用硫酸新霉素诱变、筛选突变菌,并对亲本菌(ZUST)和突变菌(ZUST-1、ZUST-2)进行生长、产酸能力及H~+_-ATPase活性的测定。分别提取亲本菌和突变菌的基因组DNA,扩增H~+_-ATPase全部编码基因并测序。通过荧光定量PCR对H~+_-ATPase全部编码基因进行相对定量分析。【结果】突变菌的生长和产酸能力均低于亲本菌,突变菌ZUST-1和ZUST-2的H~+_-ATPase活性比亲本菌分别降低了10.1%和28.8%。突变菌ZUST-1和ZUST-2的atp A基因均有22个位点发生突变,而ZUST-2的atp C基因有6个位点发生突变。突变菌ZUST-1和ZUST-2的atp A在对数期基因表达水平分别比亲本菌ZUST下调了41.1%和35.7%,在稳定期分别下调了43.6%和14.2%;ZUST-1的atp C基因在对数期的表达水平比ZUST略高,在稳定期比ZUST上调了30%,而ZUST-2的atp C基因未表达。【结论】突变菌H~+_-ATPase活性减弱会导致其全部编码基因在稳定期表达水平上调(除ZUST-2的atp C不表达外),而且atp A和atp C基因突变导致的基因表达水平的差异是影响H~+_-ATPase活性的主要因素,此研究结果为进一步研究植物乳杆菌中H~+_-ATPase的调控机制奠定了基础。     

Isolation and Characterization of IS1181, an Insertion Sequence from Staphylococcus aureus

The repeated nucleotide sequence isolated from a methicillin-resistant Staphylococcus aureus isolate displays the characteristic features of an insertion sequence and was named IS1181. It has a size of 1512 bp and consists of a 1359-bp open reading frame that encodes a 439-amino-acid protein which is predicted to be highly basic and 23-bp terminal inverted complementary repeated sequences exhibiting six mismatches. The three copies of IS1181 isolated from distinct parts of the chromosome of S. aureus, BM3121, are flanked at their ends by 8-bp direct repeats, suggesting a duplication of the target sequence. IS1181 exhibits similarities with IS1165 from Leuconostoc mesenteroides and IS1001 from Bordetella parapertusis. IS1181 was detected in at least two to eight copies in 41 of the 52 S. aureus isolates tested, whereas none of the 26 coagulase-negative staphylococci, 24 streptococci, or 11 enterococci analyzed carried nucleotide sequences hybridizing with IS1181.     

香坊肠球菌和罗伊氏乳杆菌在无菌猪肠道中的定殖

【背景】已有研究表明,微生物在宿主肠道中的定殖受宿主、肠道环境、微生物物种特性和菌株来源等多个因素的影响。一般认为,来源于同类宿主的微生物菌株,在该类宿主肠道中具有定殖优势,但缺乏在物种和菌株水平上研究微生物自身特性在宿主肠道中定殖的研究报道。【目的】将不同来源(同类宿主肠道、非同类宿主肠道和非肠道环境)、具有不同生物学特性的3株香坊肠球菌(Enterococcus xiangfangensis)和4株罗伊氏乳杆菌(Lactobacillusreuteri)对无菌猪肠道进行定殖,在物种和菌株2个水平上探究物种特性和菌株来源对宿主肠道定殖的偏好性,揭示影响微生物定殖效率的关键因素。【方法】在本项研究中,将从藏猪(Tibetan pigs)、小鼠(ob/ob mice)、食蟹猴(Macaca fascicularis)和发酵食品中分离得到的多株香坊肠球菌和罗伊氏乳杆菌,制成混合菌剂对无菌巴马香猪(Bama miniature pig)进行为期4周的饲喂,并通过实时荧光定量PCR方法检测这7株菌在无菌猪肠道中的定殖情况。【结果】在物种水平上,香坊肠球菌和罗伊氏乳杆菌在无菌猪体内具有相近的定殖...     

不同来源植物乳杆菌基因组结构和功能差异的比较研究

[目的] 本试验研究不同来源植物乳杆菌（Lactobacillus plantarum）基因特点以及在不同环境下其基因多样性,探究2株L.plantarum A8和P9在肠道生境及植物表面适应性的异同,为优良菌株的开发提供理论基础。[方法] 本研究对从动物肠道和植物表面分离获得的L.plantarum A8和L.plantarum P9的基因组进行分析,利用第二代测序技术（NextGeneration Sequencing,NGS）,基于Illumina NovaSeq测序平台,同时利用第三代单分子测序技术,基于PacBio Sequel测序平台,对L.plantarum A8和L.plantarum P9进行测序。采用Carbohydrate-active enzymes（CAZy）、Koyto encyclopedia of genes and genomes（KEGG）和Clusters of orthologous genes（COG）数据库对基因组进行功能注释;采用CGView软件绘制菌株的基因组环形图谱。应用比较基因组学与已经公开发表的其他L.plantarum基因组进行比较分析。[结果] 由研究可知L.plantarum A8和L.plantarum P9基因组大小存在差异,通过构建系统发育树发现2株菌与其他来源的L.plantarum分在同一分支,并且L.plantarum P9与母乳来源的L.plantarum WLPL04菌株距离最近,而L.plantarum A8与L.paraplantarum DSM10667距离最近。通过基因家族分析可知,2株菌共有基因为2643个,其中包括一些抗应激蛋白如热休克蛋白、冷休克蛋白。L.plantarum A8和P9独特基因分别为321和336个,L.plantarum A8中独特基因主要参与DNA复制、ABC转运系统（ABC transfer system）、PTS系统（phosphotransferase system）、磺酸盐转运系统、氨基酸生物合成等代谢通路;L.plantarum P9的独特基因以参与碳水化合物的运输和代谢基因居多,例如rpiA基因、lacZ基因、FruA基因等。[结论] 通过比较基因组学方法解析L.plantarum的基因组信息,发现动物肠道来源的L.plantarum具有较好的氨基酸转运能力,植物表面附着的L.plantarum菌株具有较好碳水化合物利用能力,从而为益生菌的开发与利用提供理论依据。     

罗伊氏乳杆菌肠道分离株的遗传多样性研究

动物体内定殖着丰富且多样的细菌,其对宿主的健康发挥着举足轻重的作用。罗伊氏乳杆菌(Lactobacillus reuteri)是存在于动物肠道中的益生菌,是研究肠道菌群与宿主进化关系的模式菌种。【目的】以下载自NCBI的分离自猪、家禽类、人类、啮齿类动物的116株L.reuteri和分离自内蒙古锡林郭勒牛、羊和马肠道中的16株L. reuteri为研究对象,解析L. reuteri不同分离株的遗传多样性和宿主特异性,为L. reuteri的开发利用提供理论依据。【方法】利用MLST技术,以ddl、pkt、leu S、gyr B、dlt A、rpo A、rec A共7个看家基因为研究靶点,对L.reuteri分离株遗传多样性进行研究,推演分离株与宿主生境的进化关系。【结果】132株L. reuteri共划分为63个序列型,6个克隆复合体。等位基因序列重组分析发现,在L.reuteri的进化中发生了个别的重组事件,eBURST、MSTree分析表明不同分离源的L. reuteri分离株经历了不同的进化过程,系统发育分析表明132株L. reuteri来自5个Clusters且与分离源表现出较强的相关性。【结论】本研究利用MLST技术完成了132株L. reuteri肠道分离株的遗传多样性分析,利用MSTree、系统发育等群体结构分析,发现不同分离源菌株有着高度的宿主特异性,表明L. reuteri为适应不同生存环境经历了不同的进化过程。     

马乳酒样乳杆菌1207对CUMS小鼠焦虑及抑郁样行为的缓解作用

【目的】越来越多的研究表明,益生菌能够通过“微生物-肠-脑”轴来调节中枢神经系统,影响精神障碍疾病的发生和发展。因此,精神健康相关的益生菌资源的开发具有重要的意义。本文利用慢性不可预知温和应激小鼠模型(chronic unpredictable mild stress,CUMS),研究了一株分离自西藏Kefir粒的马乳酒样乳杆菌1207对小鼠焦虑样和抑郁样行为的缓解作用。【方法】本研究构建了CUMS模型,并利用马乳酒样乳杆菌1207 (109 CFU/d)灌胃进行2周的干预。随后,分别利用旷场实验、悬尾实验和强迫游泳评估了该菌株对小鼠的焦虑样和抑郁样行为的影响;通过测定下丘脑、血清和脾脏中生物标记物的含量变化,研究了该菌株对色氨酸代谢、下丘脑-垂体-肾上腺(hypothalamic-pituitary-adrena,HPA)轴和炎症因子的调节作用。【结果】与对照小鼠相比,灌胃马乳酒样乳杆菌1207可显著增加小鼠在旷场实验中中央区域的滞留时间(P<0.05);降低小鼠在悬尾实验(P<0.01)和强迫游泳实验(P<0.05)中的不动时间;可通过降低小鼠下丘脑中5-羟基吲哚...     

副干酪乳杆菌PC-01全基因组测序及不同副干酪乳杆菌菌株比较基因组学分析

【背景】副干酪乳杆菌(Lactobacillus paracasei)作为乳酸菌中重要菌种之一,常被认为是优良益生菌开发的潜在资源。【目的】以L.paracasei PC-01和L.paracasei Zhang为例,分析不同L.paracasei的基因组差异和遗传背景,为菌株的鉴定和开发奠定基础。【方法】采用PacBioSMRT三代测序技术对L.paracasei PC-01进行全基因组测序,结合2株L.paracasei模式菌株和公开的36株全基因组数据,通过比较基因组学方法揭示39株L.paracasei菌株之间的差异。【结果】L.paracasei PC-01基因组不包含质粒,染色体大小为2 829 251 bp,GC含量为46.64%;L.paracasei Zhang包含一个质粒基因组大小为2 898 456 bp,GC含量为46.51%;不同L.paracasei菌株基因组大小、质粒数及GC含量均存在一定差异。L.paracasei群体为开放式基因组,基因组具有高度多样性。基于核心基因构建系统发育树对于L.paracasei种内区分效果最好,L.paracasei PC-...     

ISL2, a new mobile genetic element in Lactobacillus helveticus

Spontaneous, phenotypically stable mutations at the -galactosidase locus (lacL-lacM) in Lactobacillus helveticus were identified and analyzed. We found that a significant number of mutations were caused by integration of a new IS element, ISL2, into these lac genes. ISL2 is 858 by long, flanked by 16-bp perfect inverted repeats and generates 3-bp target duplications upon insertion. It contains one open reading frame, which shows significant homology (40.1 % identity) to the putative transposase of IS702 from Cyanobacterium calothrix. ISL2 is present in 4–21 copies in the L. helveticus genome, but it is not found in other lactic acid bacteria. Its divergence in copy number and genomic locations in different L. helveticus strains makes it useful as a tool for strain identification by genetic fingerprinting.     

Complete Sequence of Plasmid pLH1 from Lactobacillus helveticus ATCC15009: Analysis Reveals the Presence of Regions Homologous to Other Native Plasmids from the Host Strain

The complete sequence for plasmid pLH1 from Lactobacillus helveticus ATCC15009 has been determined. Analysis of the 19,360-bp primary sequence revealed a putative replication origin and initiation protein, information that could provide the basis for the construction of cloning vectors for L. helveticus. Evidence that pLH1 is theta-replicating could be deduced from the plasmid size, from the homology to the replication protein of the Bacillus natto theta-replicating plasmid pLS32, and from the identification of a putative resolvase gene (orf-195). Although 14 open reading frames capable of encoding polypeptides longer than 100 amino acids were identified, none, on the basis of homology with known sequences, appeared to encode a well-characterized trait relevant to milk fermentation. Plasmid pLH1 revealed regions of identity with the smaller cryptic plasmids (pLH2 and pLH3) from the same strain and with other tracts of DNA, including insertion sequence elements, from a variety of other lactic acid bacteria. The presence of such regions provides a basis for developing an explanation of the phenotypic variability observed in these bacteria. The plasmid also appears to possess a number of genetic elements present in other lactic acid bacterial plasmids, conservation of which would be consistent with an important functional or evolutionary role. It could be argued that the plasmid complement of L. helveticus ATCC15009 consists of parasitic entities concerned only with their own replication and survival.     

仿刺参(Apostichopus japonicus)肠道源植物乳杆菌(Lactobacillus plantarum) HY21的生长及益生特性分析

【目的】在仿刺参(Apostichopus japonicus)健康养殖中为了寻找具有良好耐受性、益生性、安全性的益生菌,本研究从仿刺参肠道内容物中分离获得一株植物乳杆菌(Lactobacillus plantarum) HY21,对其益生潜能进行评价。【方法】利用摇瓶发酵培养、应用环境因子模拟试验、抗生素药敏测试等方法,分析L. plantarum HY21的生长特性、产酸性能、发酵液的抗氧化性质和对水产致病菌的抑制作用;检测菌体的疏水性、自聚性和共聚性、黏附力和药物敏感性,以及对应用环境因子的耐受性。【结果】L. plantarum HY21摇瓶发酵培养温度30℃、初始pH 8.0,于2-10 h为对数生长期,发酵18 h后pH达到最低3.6;发酵液对溶藻弧菌的抑菌直径达到(13.96±0.30) mm,对1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-trinitrophenylhydrazine, DPPH)自由基清除率达到95.45%±1.56%;L. plantarum HY21的疏水率为60.42%±2.78%、自聚率为22.69%±1.36%,与溶藻弧菌共聚率为27.98%±1.45%;对体表和肠道黏液蛋白的黏附量分别达到(1.66±0.01)×10⁶ CFU/mL和(1.23±0.15)×10⁶ CFU/mL;对13种常见的抗菌药物均表现敏感;菌体在模拟胃肠液(pH 3.0, pH 6.8)中孵育3 h存活率均可达99%以上,在10%鱼胆汁溶液中孵育3 h存活率达到68.11%±7.98%,在3%海盐溶液中孵育16 h存活率达到97.58%±7.14%。【结论】L. plantarum HY21具有良好的定殖作用、抑菌作用、抗氧化作用等益生特性,而且在应用环境中具有高存活率和高安全性,可以作为一株新的水产益生菌,为开发水产益生菌制剂提供科学理论依据。