芝麻香型白酒微生物多样性及其与胁迫因子相关性

【背景】近年来芝麻香型白酒的生产工艺日臻成熟,然而相应的科学研究却没有同步发展起来。高通量测序技术越来越多地应用于物种多样性的研究,但偏重于研究物种的相对丰度,没有关注物种的生物数量。【目的】深度解析芝麻香型白酒发酵过程微生物群落结构变化及其与胁迫因子相关性,并研究主要酵母菌与细菌的相关性,为揭示芝麻香型白酒发酵机理和控制发酵质量提供理论支撑。【方法】使用Thermofisher的Ion S5~(TM)XL测序平台进行16S rDNA和ITS rDNA扩增子高通量测序,结合微生物传统的定量方法,测定芝麻香型白酒发酵过程微生物群落结构的变化,同时监测发酵过程乳酸、乙酸、乙醇的含量变化,通过样品复杂度分析、多样品比较分析、环境因子关联性分析探究发酵过程微生物群落及其与胁迫因子的关系。通过Pearson相关性分析酵母菌与细菌的相关性。【结果】发酵前期纤维素菌、魏斯氏菌和芽孢杆菌占主要优势,发酵中后期乳杆菌占绝对优势,其次是纤维素菌、魏斯氏菌和芽孢杆菌。整个发酵过程伊萨酵母占绝对优势,其次是维克霉菌、酿酒酵母、假丝酵母。大部分微生物与胁迫因子呈负相关,只有乳杆菌与乙酸呈极显著性正相关。酵母菌与部分细菌呈正相关性。【结论】白酒发酵过程胁迫因子和微生物间的相互作用促进了群落演替过程,发酵后期乳杆菌和芽孢杆菌发酵产酸抑制了大部分不耐酸菌,有机酸是影响群落结构变化的主要胁迫因子。微生物数量结合相对丰度揭示了发酵过程群落结构演替及其与环境因子相关性的更多信息。     

海洋沉积物中铁还原细菌组成及异化铁还原与产氢性质分析

【背景】一些铁还原细菌具有异化铁还原与产氢的能力,该类细菌在环境污染修复的同时能够解决能源问题。【目的】从海洋沉积物中富集获得异化铁还原菌群,明确混合菌群组成、异化铁还原及产氢性质。获得海洋沉积物中异化铁还原混合菌群组成,分析菌群异化铁还原和产氢性质。【方法】利用高通量测序技术分析异化铁还原菌群的优势菌组成,在此基础上,分析异化铁还原混合菌群在不同电子供体培养条件下异化铁还原能力和产氢性质。【结果】高通量数据表明,在不溶性氢氧化铁为电子受体和葡萄糖为电子供体厌氧培养条件下,混合菌群的优势菌属主要是梭菌(Clostridium),属于发酵型异化铁还原细菌。混合菌群能够利用电子供体蔗糖、葡萄糖以及丙酮酸钠进行异化铁还原及发酵产氢。葡萄糖为电子供体时,菌群累积产生Fe(Ⅱ)浓度和产氢量最高,分别是59.34±6.73 mg/L和629.70±11.42 mL/L。【结论】异化铁还原混合菌群同时具有异化铁还原和产氢能力,拓宽了发酵型异化铁还原细菌的种质资源,探索异化铁还原细菌在生物能源方面的应用。     

库木塔格沙漠土壤细菌多样性及具有群体感应抑制活性放线菌筛选

【背景】群体感应抑制剂(quorum sensing inhibitor,QSI)作为抗生素潜在替代品,可有效降低致病菌传染性和毒性。沙漠土壤蕴藏着丰富的放线菌资源,是挖掘群体感应抑制剂的重要来源。【目的】解析库木塔格沙漠土壤细菌群落多样性,筛选并挖掘群体感应抑制活性放线菌资源。【方法】采用Illumina Nova Seq高通量测序技术揭示库木塔格沙漠土壤细菌群落组成,利用可培养方法进行土壤放线菌分离和鉴定;选用紫色杆菌CV026模型筛选群体感应抑制活性放线菌,并对其功能特性进行初步评价。【结果】Illumina Nova Seq高通量测序结果显示,样品土壤细菌涉及23门96目150属,优势菌门为变形菌门(Proteobacteria,61%)、放线菌门(Actinobacteria,28%),其中分枝杆菌属(Mycobacterium)为放线菌门最优势菌属(87.3%),其次为红球菌属(Rhodococcus,6.8%)和丙酸杆菌属(Cutibacterium,0.9%)。可培养方法共分离到108株放线菌,归属9科10属,其中优势菌属为链霉菌属(Streptomyces),占65....     

渤海沉积物产脂肪酶细菌的筛选及其多样性分析

【目的】从渤海沉积物中分离筛选产脂肪酶细菌,分析其物种多样性,增加人们对渤海生态系统中产脂肪酶菌多样性的认识,获取高效产脂肪酶菌株,为海洋产脂肪酶微生物的挖掘提供菌群资源。【方法】分别将8个渤海沉积物样品梯度稀释涂布至吐温-80筛选平板和三丁酸甘油酯筛选平板,选择性分离产脂肪酶细菌;分析基于16SrRNA基因序列的系统发育关系,揭示这些细菌的分类地位和遗传多样性;利用对硝基苯酚法测定胞外脂肪酶活性,筛选出高效产脂肪酶菌株。【结果】从8个渤海沉积物样品中分离获得51株产脂肪酶细菌,这些菌株隶属于Bacteroidetes、Proteobacteria和Firmicutes三个门的8个属,其中Pseudoalteromonas(35.2%)、Marinobacter(23.5%)和Sulfitobacter(17.6%)是优势菌群;脂肪酶酶活性实验表明所有测定菌株都能够分泌脂肪酶,菌株70623分泌的脂肪酶酶活最高,为42.4 U/m L。【结论】渤海沉积物中可培养产脂肪酶细菌类群较为丰富,Pseudoalteromonas、Marinobacter和Sulfitobacter菌株是优势菌群,测定菌株所产胞外脂肪酶能力不同,获得了一株高效产脂肪酶菌株Marinobacter sp.70623。     

两株母乳源植物乳杆菌的全基因组测序分析

【背景】母乳是一个重要的益生菌筛选库,其中植物乳杆菌是一种用途广泛、适应性强的益生菌。然而不同菌株具有不同的功能,现有的生理生化方法对其潜在益生特性研究十分有限,有必要采用高通量的方法寻找具有种群特异性的优质益生菌。【目的】结合菌株生化特征在全基因组的测序与分析的基础上对两株植物乳杆菌的潜在功能进行预测,并重点找寻与肠液耐受性及细菌素的合成相关的基因,即在基因组的结构上对菌株的表型进行探究。【方法】分离筛选出两株母乳源植物乳杆菌MP55、MP37,并利用Illumina genome analyzer对菌株的全基因组进行测序,采用Prokka软件对细菌基因组进行注释,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;同时采用Prodigal、RNAmmer等工具对编码序列、核糖体RNA进行预测,并用CGView软件绘制菌株的基因组环形图谱。【结果】通过基因组装得到了两株植物乳杆菌的全基因组信息,植物乳杆菌MP37、MP55基因组大小分别为3 204 421 bp和3 299 180 bp;(G+C)mol%含量分别为44.36%和44.46%;分别包含3 012个和3 101个DNA编码序列,结合菌株生化特征在基因组上找到4个与肠液耐受相关的基因及一段细菌素合成相关基因簇。基因组序列原始数据和拼接结果已提交至"gcMeta"平台。【结论】通过高通量测序分析在基因组水平上揭示了植物乳杆菌MP55、MP37在肠道存活性与抑制病原菌相关的可能机理。植物乳杆菌MP55、MP37是两株潜在的益生菌候选菌株,实验结果为进一步阐明其益生菌特性的功能机制提供了遗传学基础。     

多糖类益生元的筛选及凝结芽孢杆菌体外发酵特性北大核心

【目的】探究9种多糖对凝结芽孢杆菌(Bacillus coagulans)的增殖、产酶特性的影响。【方法】将凝结芽孢杆菌分别添加至菊粉多糖(inulin polysaccharide)、刺五加多糖(Eleutherococcus senticosus polysaccharide)、壳寡糖(chitosan oligosaccharide)、防风多糖(Saposhnikovia divaricata polysaccharide)、低聚木糖(xylo-oligosaccharide)、黄芪多糖(Astragalus polysaccharide)、甘露糖(D-mannose)、白术多糖(Atractylodes macrocephala Koidz polysaccharide)和玉屏风多糖(Yu Ping Feng polysaccharide)为唯一碳源的培养基中,通过菌株生长、酶活性及其体外厌氧发酵等作为指标,筛选出最优益生元。【结果】凝结芽孢杆菌能很好地利用防风多糖、黄芪多糖、白术多糖和玉屏风多糖;添加量为4%的防风多糖和白术多糖,pH差值差异最大,蛋白酶活性差异显著(P<0.05)。体外发酵乳酸活性和总蛋白酶活性均提高,4%白术多糖的乳酸和总蛋白酶活性差异显著(P<0.05);肠道内容物发酵液16S rRNA基因高通量测序结果表明,与对照组比较,添加黄芪多糖、防风多糖、甘露糖3种益生元发酵凝结芽孢杆菌显著降低了气单胞菌(Aeromonas)、α-变形菌(α-Proteobacteria)、链球菌(Streptococcus)、志贺氏杆菌属(Shigella)等致病菌的相对丰度,提高了乳杆菌(Lactobacillus)、厚壁菌门(Firmicutes)、乳酸乳球菌(Lactococcus lactis)、产酸杆菌(Acidobacteria)的相对丰度。【结论】凝结芽孢杆菌发酵4%白术多糖具有较好的产酶性能与益生特性,二者协同发酵添加至饲料中具有较好的发展潜力。     

印度洋红树林沉积物可培养海洋放线菌多样性及其活性

【目的】本研究旨在了解印度洋红树林沉积物可培养海洋放线菌多样性、抗菌活性及产酶活性。【方法】选用24种碳源为唯一能源培养基,利用稀释平板涂布方法对8个印度洋红树林沉积物样品进行分离,并基于16S rRNA基因系统发育分析的方法研究样品中海洋放线菌多样性;对分离得到的菌株进行抗菌活性和产酶活性检测。【结果】24种唯一碳源分离培养基中,非糖类碳源特别是甘油、丙氨酸分离效果最好,其次是多糖物质,最后是单糖。共分离得到521株海洋放线菌,经并菌后选取其中的139株代表性菌株测序,结果发现它们主要分布在放线菌纲7个亚目10个科的16个属,其中35个为潜在新种。有43.1%、33.3%、26.9%、25.5%、15.7%的实验菌株分别对枯草芽孢杆菌、白色念珠菌、大肠埃希氏菌、金黄色葡萄球菌、黑曲霉具有抑制作用;有36.5%、26.5%、22.4%、15.9%的实验菌株分别具有蛋白酶活性、纤维素酶活、淀粉酶活性、酯酶活性。【结论】印度洋红树林沉积物蕴藏着丰富的海洋放线菌资源,并具有较高生物活性,为后续工作提供良好的实验材料。     

具有群体感应抑制活性的海洋细菌的筛选

【目的】从海洋环境中筛选出能有效抑制细菌群体感应的活性菌株,为以致病菌群体感应为靶点的新型疗法提供新的天然产物资源。【方法】以紫色杆菌(Chromobacteriumviolaceum)为报告菌,采用滤纸片法和双层软琼脂法相结合的筛选模型进行群体感应抑制活性菌的筛选。【结果】通过对美国圣璜岛海域海绵中分离出来的272株海洋细菌群体感应抑制活性的筛选,得到了具有抑制紫色杆菌素产生的细菌51株,其中74号菌株抑制效果最好,具有进一步研究的价值。【结论】海洋细菌中有很多具有抑制细菌群体感应效应的菌株,是天然群体感应抑制剂的潜在来源。     

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。     

锡林郭勒地区鲜马奶中细菌多样性分析

【背景】传统的酸马奶是由鲜马奶自然发酵而成,鲜马奶中的微生物在很大程度上决定了酸马奶的品质。然而,鲜马奶中的微生物组成却鲜有报道。【目的】解析鲜马奶中细菌多样性,比较分析鲜马奶和酸马奶中微生物的差异,探究马奶发酵过程中微生物的变化。【方法】采用PacBio SMRT测序技术,对6份采集自内蒙古锡林浩特牧区的鲜马奶中细菌16SrRNA基因序列全长进行测序,在种水平解析鲜马奶样品的细菌组成,并与前期同一地区的酸马奶菌群组成作对比。【结果】鲜马奶中包含大量的乳酸菌,主要为乳酸乳球菌(12.43%)、格氏乳球菌(3.53%)、肠膜明串珠菌(1.77%)等。此外还有部分板崎肠杆菌等条件致病菌的存在。马奶发酵前后菌群变化较大,鲜马奶中微生物多样性显著高于酸马奶,其中乳酸乳球菌和瑞士乳杆菌为鲜马奶和酸马奶的共有细菌。鲜马奶中的肠膜明串珠菌与乳酸乳球菌、格氏乳球菌呈正相关关系,与嗜水气单胞菌等有害菌为负相关关系,表明在马奶发酵过程中乳酸菌可能会抑制有害菌的生长。【结论】在马奶的发酵过程中,乳酸菌的大量生长抑制了马奶中有害菌生长,降低了马奶中微生物的多样性,提升了酸马奶的品质。为酸马奶的工业化生产提供了参考。     

Complete genome sequence of Lactobacillus helveticus H10

Lactobacillus helveticus strain H10 was isolated from traditional fermented milk in Tibet, China. We sequenced the whole genome of strain H10 and compared it to the published genome sequence of Lactobacillus helveticus DPC4571.     

Cardiovascular effects of fermented milk containing angiotensin-converting enzyme inhibitors evaluated in permanently catheterized,spontaneously hypertensive rats

In this study, two strains of Lactobacillus helveticus were used to produce fermented milk rich in angiotensin-converting enzyme (ACE) inhibitors. In vitro tests revealed that the two milks contained competitive inhibitors of ACE in amounts comparable to what has been obtained in previously reported studies. The two milks were administered by gavage to spontaneously hypertensive rats that had had a permanent aortic catheter inserted through the left arteria carotis, and mean arterial blood pressure and heart rate were monitored from 4 to 8 h after administration. Unfermented milk and milk fermented with a lactococcal strain that does not produce inhibitors were used as controls. Highly significant blood pressure effects were observed; i.e., milk fermented with the two strains of L. helveticus gave a more pronounced drop in blood pressure than the controls. Significant differences in heart rate effects were detected with one of the strains.     

Doxycycline-Dependent Inducible and Reversible RNA Interference Mediated by a Single Lentivirus Vector

Most fermented milk prepared by strains of Lactobacillus helveticus showed significant antihypertensive effect in spontaneously hypertensive rats (SHR) by oral administration. However, milk fermented by other species of lactic acid bacteria did not show significant antihypertensive effects. Most of the whey fractions of the milk fermented by L. helveticus or Lactobacillus delbrueckii subsp. bulgaricus showed higher angiotensin I-converting enzyme (ACE) inhibitory activity than the activity of milk fermented by other species. Proteolytic activity in cell wall and peptide content of the fermented milk were higher in L. helveticus strains than other species.     

蒙古戈壁地区自然发酵乳中乳酸菌的分离鉴定

从采集自蒙古国戈壁地区的6份自然发酵乳中分离到14株乳酸菌,经过形态特征,生理生化特性,糖发酵试验和乳酸旋光性的测定,鉴定结果:乳酸球菌5株,包括Lactococcus lactissubsp.cremoris 1株,Pedio-coccus.(后缩写为Ped.).urinaeequi3株,Pediococcus.pentosaceus1株;乳杆菌9株,包括Lactobacillus.(后缩写为L.)helveticus8株,Lactobacillus.delbrueckii.subsp.bulgaricus1株。蒙古国戈壁地区自然发酵乳中的优势菌为Lactobacillus.helveticus,其次为Pediococcus.urinaeequi。     

Production of active angiotensin-I converting enzyme inhibitory peptides derived from bovine beta-casein by recombinant DNA technologies

A wide range of anti-hypertensive peptides potentially able to lower blood pressure through the inhibition of vasoactive enzymes such as angiotensin-I converting enzyme (ACE) are known. Currently, ACE-inhibitory peptides can be produced from precursor proteins via enzymatic hydrolysis by proteolytic enzymes, or food fermentation with proteolytic starter cultures. These approaches are neither selective nor easy. In this study a novel procedure has been developed, based on recombinant DNA technologies, for the production of highly purified fractions of three polypeptides derived from bovine beta-casein active as ACE inhibitors in vitro. The procedure includes peptide expression in Escherichia coli cells as recombinant fusion proteins, purification by affinity chromatography, cleavage by proteinase from a selected strain of Lactobacillus helveticus and isolation of bioactive peptides (BPs). The reported concentration of inhibitor needed to reduce at 50% ACE activity (IC(50)) values for single BP calculated in inhibiting the ACE enzyme gave results in agreement with the same parameters available in literature for other milk-derived BPs. This procedure could be used to obtain quantities of pure peptides to determine their interactions with ACE, with the aim of designing peptides that have stronger inhibitory properties and exhibit new pharmacological profiles. Moreover, its scale up would be of commercial interest for the production of functional foods, e.g., milk beverages with blood pressure-lowering effects.     

蒙古国地区酸乳中乳酸菌的鉴定及耐酸菌株筛选

本研究对采集自蒙古国地区牧民家庭中17份发酵乳样品中的乳杆菌进行了分离、鉴定、生物学特性和耐酸性研究。共分离出45株乳杆菌。通过形态观察、生理生化试验、糖发酵试验及16S rDNA序列分析等研究将这些菌株鉴定为Lactobacillum fermentum(L. fermentum)31 株, L. helveticus 12株, L. plantarum 1株 和L. casei 1株, 所以认为L. fermentum是蒙古国地区传统酸乳中的优势菌群。经pH 为3.0 的人工胃液耐受性试验复筛后发现, 存活率在80%以上的仅1株, IMAU20085的存活率高达81.44%。菌株的分离鉴定以及高耐酸性菌株的筛选, 对我国益生菌资源的保藏和开发有重要的意义, 对我国未来益生菌的开发具有重要价值。     

Lactic acid bacteria: inhibition of angiotensin converting enzyme in vitro and in vivo

A total of 26 strains of wild-type lactic acid bacteria, mainly belonging to Lactococcus lactis and Lactobacillus helveticus, were assayed in vitro for their ability to produce a milk fermentate with inhibitory activity towards angiotensin converting enzyme (ACE). It was clear that the test strains in this study, in general, produce inhibitory substances in varying amounts. Using a spectrophotometric assay based on amino group derivatization with ortho-phthaldialdehyde as a measure of relative peptide content, it was shown that there is a significant correlation between peptide formation and ACE inhibition, indicating that peptide measurement constitutes a convenient selection method. The effect of active fermentates on in vivo ACE activity was demonstrated in normotensive rats. The pressor effect of angiotensin I (0.3 μg/kg) upon intravenous injection was significantly lower when rats were pre-fed with milks fermented using two strains of Lactobacillus helveticus. An increased response to bradykinin (10 μg/kg, intravenously injected) was observed using one of these fermented milks. It is concluded that Lactobacillus helveticus produces substances which in vivo can give rise to an inhibition of ACE. The inhibition in vivo was low compared to what can be achieved with classical ACE inhibitors. The clinical relevance of this finding is discussed. This work is the first in which an effect of fermented milk on ACE in vivo has been demonstrated, measured as decreased ability to convert angiotensin I to angiotensin II. This revised version was published online in June 2006 with corrections to the Cover Date.     

Role of Bioactive Peptides in Reducing the Severity of Hypertension with the Inhibition of ACE

International Journal of Peptide Research and Therapeutics - Bovine milk protein, fermented with LC (Lactobacillus casei) was used to evaluate its ACE inhibitory activity in-vitro. After evaluating...     

藏北地区传统发酵乳中乳杆菌的多样性分析

摘要：【目的】针对藏北地区的乳杆菌来源及种属,对其多态性进行研究。【方法】采用ERIC-PCR技术和NTSYS-pc2.1软件对从藏北地区牧民家庭制作的发酵乳制品中分离出的77株乳杆菌进行多样性分析。【结果】ERIC-PCR扩增出的条带清晰,重复性好,多态性高。聚类分析表明,在0.73的水平上,77株乳杆菌共分为4大类群：干酪乳杆菌群A1、发酵乳杆菌群A2,瑞士乳杆菌群A3和植物乳杆菌群A4。其中,干酪乳杆菌群A1和发酵乳杆菌群A2分别占供试乳杆菌的35.06%和61.04%,为优势菌群。进一步对优势菌群