枯草芽孢杆菌细菌素的分离、表达及稳定性分析

旨在分离枯草芽孢杆菌及其所产细菌素,并分析重组表达后的细菌素的抑菌活性和稳定性。通过牛津杯扩散法筛选羊驼粪便中高产细菌素的芽孢杆菌;借助16S rRNA鉴定分离菌;采用硫酸铵沉淀、氯仿抽提、SDS-PAGE、质谱分析等技术获得细菌素的氨基酸序列;利用大肠杆菌表达系统对细菌素进行体外表达,并利用牛津杯扩散法测定其抑菌活性和稳定性。结果显示,分离菌是一种枯草芽孢杆菌,命名为枯草芽孢杆菌SXAU18,其可产生抑制金黄色葡萄球菌、表皮葡萄球菌、藤黄微球菌和单增李斯特菌生长的细菌素;枯草芽孢杆菌SXAU18所产细菌素可能是分子量为10~20 ku的DarA蛋白和两种未知蛋白,属于类细菌素的范畴,将未知蛋白分别命名为BLIS SXAU181和182;经原核表达的BLIS SXAU181和182主要以可溶性上清蛋白形式表达,纯化后为单一条带;重组BLIS SXAU181蛋白没有抑菌活性,重组BLIS SXAU182蛋白具有良好的抑菌活性,且具有耐高温、耐酸碱、耐人工胃液和肠液的的特性。综上,本研究从枯草芽孢杆菌SXAU18分离到具有抑制革兰阳性菌生长活性的细菌素,且重组表达后的BLIS SXAU182具有良好的抑菌活性和和稳定性。

传统发酵乳品中产广谱细菌素乳酸菌选育

为选育产广谱细菌素乳酸菌,采用琼脂扩散法对甘肃牧区传统发酵乳品中分离纯化的96株乳酸菌进行了筛选,并确定产细菌素菌株,具有最佳抑菌效果的菌株经重离子束12C6+辐照诱变,再采用培养皿分区法进行6种指示菌广谱抑菌性能比较。结果表明,琼脂扩散法筛选出抑菌效果最佳的产细菌素菌株Lp1,经辐照诱变得到98株突变菌,选育出对大肠埃希氏菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)、单核细胞增生李斯特氏菌(Listeria monocytogenes)、沙门氏菌(Salmonella)、志贺氏菌(Shigella)和克罗诺杆菌(Cronobacter)抑菌性能均明显优于出发菌株Lp1的5株突变株L088、L087、L063、L049、L010进行广谱抑菌试验,其中菌株L088和L063为广谱抑菌性能较好菌株,突变株L088对上述6种指示菌的抑菌扇环半径比出发菌株Lp1抑菌扇环半径分别提高47.81%、33.28%、22.97%、45.25%、21.39%、40.21%,突变株L063对上述6种指示菌的抑菌扇环半径比出发菌株Lp1抑菌扇环半径分别提高54.27%、29.18%、25.37%、58.03%、17.85%、42.13%。采用培养皿分区法可同时在1个培养皿中完成检测6种指示菌的抑菌试验,可高效筛选产广谱细菌素乳酸菌。

LuxS/AI-2群体感应系统在乳酸菌细菌素合成中的作用

乳酸菌细菌素具有抑菌谱广、稳定性高、安全性高等优势,作为新型生物防腐剂备受青睐,但目前工业化应用的细菌素数量有限,合成量低是其应用受限的重要原因。国内外研究显示与特定的微生物共培养是提高乳酸菌细菌素合成量的有效方法,该过程受到LuxS/AI-2群体感应系统的调控。文章从乳酸菌LuxS/AI-2群体感应系统的组成及LuxS/AI-2群体感应系统在乳酸菌细菌素合成中的作用两个方面进行论述,为乳酸菌细菌素合成量的提高和工业化应用的实现提供了一定的理论依据。

枯草芽孢杆菌SNBS-3全基因组测序及其抑菌物质预测分析

为了详细解析枯草芽孢杆菌SNBS-3的基因组特征,本研究在前期研究基础上,采用Illumina二代测序技术和第三代高通量Pacbio测序平台,对从传统豆酱中筛选得到的枯草芽孢杆菌SNBS-3进行全基因组测序,获得菌株基因组特征信息、基因功能注释及分类、系统发育进化和次级代谢产物等关键信息。结果表明:SNBS-3的基因组为一条环状闭合DNA,大小为4076387 bp,共有4000个蛋白质编码基因,在直系同源集、基因本体论、京都基因与基因组百科全书、碳水化合物活性酶、抗生素耐药性数据库和致病毒力因子数据库分别注释到3209、2824、2560、147、12个和4个功能基因。应用在线软件AntiSMASH和Bagel4发现其除具有合成Surfactin、Mycosubtilin、Plipastatin、Bacilysin和Bacillaene等多种抑菌物质的相关基因外,还具有一条完整的细菌素Subtilosin A合成基因簇,结合抑菌实验和蛋白酶K实验结果推测枯草芽孢杆菌SNBS-3具有合成细菌素SubtilosinA的能力。综上,枯草芽孢杆菌SNBS-3全基因组测序结果表明其本身可产生多种抑菌物质,是1株具有生防潜力的微生物,相关分析结果可为包括细菌素SubtilosinA在内的多种抑菌物质的进一步开发应用提供理论基础。

细菌素Bacteriocin E50-52(H)基因设计及其毕赤酵母表达载体构建

设计及合成细菌素Bacteriocin E50-52(H)基因,克隆到组成型分泌表达质粒pGAPZαA中构建重组质粒pGAPZα-Bacteriocin E(H),经PCR、测序验证正确后,电转化整合到毕赤酵母基因组,基因组PCR、测序验证结果表明成功构建了细菌素组成型重组表达载体,为在毕赤酵母中表达奠定了基础。

基于文献计量的乳酸菌细菌素研究进展分析

乳酸菌细菌素是乳酸菌在代谢过程中产生的一类具有抑菌活性的天然蛋白质或多肽类物质,具有无抗药性、可生物降解、抑菌效果好等优点,在食品、医药、饲料领域应用前景广阔。本文基于文献计量学方法,检索2000~2023年CNKI数据库和Web of Science(WOS)核心合集有关乳酸菌细菌素领域的文献,得到627篇中文文献和2543篇英文文献。使用CiteSpace软件从年度发文量,发文国家、作者、机构和期刊,高被引文献,关键词共现聚类等角度分析该领域的研究现状和热点。结果显示:2000~2022年发文量总体呈上升的趋势。西班牙、印度、中国的发文量在国家排行前三。CNKI数据库与WOS数据库发文量最高的作者分别是中国农业大学李平兰和巴西圣保罗大学Todorov SD。发文量最高的期刊分别是《食品工业科技》与《Journal of Appled Micbiogy》。CNKI数据库中关键词分析可以看出,文章多是从不同食品样本中分离、筛选、鉴定获得产细菌素的乳酸菌,进一步评价细菌素的抑菌活性,并探讨其在食品行业中的应用;突现词分析表明细菌素抑菌机制、细菌素与益生菌的联系是目前研究热点。WOS数据库中关键词“胃肠道”、“基因特征”、“单增核李斯特菌”、“抑菌活性”出现频次较高,主要热点是抑制抗生素耐药细菌的乳酸菌细菌素的挖掘及其基因特征的解析。通过文献综合分析,为我国科研人员从事相关研究和预测行业未来发展趋势提供参考和帮助。

豆酱源枯草芽孢杆菌S1-2产细菌素分离鉴定及其稳定性研究

本研究首先从东北传统豆酱中分离筛选出一株具有良好抑菌作用的菌株,结合16S rDNA与持家基因gyrA测序分析等方法对菌株鉴定。其次,利用乙酸乙酯萃取法对其所产细菌素进行粗提,使用葡聚糖凝胶Sephadex-G50和高效液相色谱(HPLC)技术进一步纯化,结合SDS-PAGE和液相色谱-质谱联用(LC-MS/MS)技术进行鉴定。最后通过最小浓度实验与生长抑制曲线分析了细菌素对金黄色葡萄球菌的抑制效果,并探究了细菌素对热、酸碱和蛋白酶的稳定性。结果表明,枯草芽孢杆菌(Bacillus subtilis)菌株S1-2对金黄色葡萄球菌和单增李斯特氏菌抑菌效果最好,该菌株产生的细菌素经分离纯化鉴定为subtilosin A,分子量约为3 kDa,对金黄色葡萄球菌和单增李斯特氏菌有明显的抑制效果,对金黄色葡萄球菌的最小抑制浓度为0.625 mg/mL。该细菌素在20~80℃抑菌活性稳定,且在100~120℃仍表现出抑菌活性;对酸碱耐受性较高,经过pH为1、10的强酸强碱处理后仍表现出抑菌活性,在pH6.0~8.0的范围内表现出最高的抑菌活性;能够被蛋白酶K、胃蛋白酶、胰蛋白酶水解。综上,枯草芽孢杆菌S1-2与其产生的细菌素subtilosin A在食品生物防腐中具有较强的研究和应用潜力。

细菌素的特点及其在反刍动物中的应用

细菌素是细菌通过核糖体产生的小肽类物质,对很多革兰氏阳性病原菌和耐药菌具有良好的抗菌活性,并广泛存在于反刍动物瘤胃中。根据结构特征,细菌素可分为翻译后修饰细菌素和非翻译后修饰细菌素两大类,通常具有双亲性结构并带有正电荷。细菌素可通过在靶细胞膜上形成孔洞导致内容物外流、抑制靶细胞壁、蛋白质或脱氧核糖核酸(DNA)的合成。细菌素能够促进反刍动物瘤胃发育,参与瘤胃发酵和微生态系统调控,减少甲烷和氨气产生,并具有预防和治疗乳房炎等作用。文章综述了细菌素的分类、生物合成、作用机理及其在反刍动物养殖中的应用研究,为更好地开发细菌素类产品提供参考。

发酵蔬菜来源具抑菌活性明串珠菌的筛选及其细菌素基因簇挖掘

为提高发酵蔬菜的安全性和保藏性,从来自云南传统发酵蔬菜的8株明串珠菌中筛选出1株对食源性致病菌和引起泡菜过酸化的细菌抑制效果好的肠膜明串珠菌AP7。通过排除酸和H2O2影响及蛋白酶敏感性确定AP7的主要抑菌物质,分析其酸稳定性和热稳定性,根据AP7的全基因组序列挖掘潜在的细菌素基因簇。结果表明:排除酸及H2O2的影响后,菌株的发酵上清液仍具有明显抑菌活性,经蛋白酶处理后,抑菌效果明显下降,推测AP7发酵上清液浓缩液中的抑菌物质为细菌素;该细菌素对pH变化敏感,热稳定性高,分子质量在6.51~14.4 kDa;全基因组测序表明,菌株AP7全基因组包含1条染色体(1948310 bp)和2个质粒(37366和20698 bp),GC含量37.7%;存在1个以Enterocin_X_chain_beta细菌素为核心的基因簇,其编码产物预测为带正电的亲水性稳定蛋白,二级结构以α-螺旋为主,三级结构主要由两端松散肽链和中间α-螺旋构成。综上,产细菌素的肠膜明串珠菌AP7具有优良抑菌性能,有潜力应用于酸性食品的发酵和防腐。

海洋源乳酸菌细菌素及其在海产品中的应用研究进展

海鲜在加工和储存过程中因酶、微生物和化学反应等因素极易发生腐烂变质。海洋源乳酸菌细菌素是由海洋乳酸菌核糖体合成的一种天然抗菌肽,能有效抑制多种食源性病原菌,将其用于海产品保鲜中可以减少化学防腐剂的添加以及物理处理的强度,有效延长食品的保质期。该文综述海洋源乳酸菌细菌素来源、海产品中影响乳酸菌细菌素抗菌效果的因素及其在海产品中的应用研究进展,为海产品保鲜中乳酸菌细菌素的选择和应用提供参考。

产细菌素海洋乳酸菌的筛选及细菌素抑菌机制

【目的】筛选海洋源产细菌素乳酸菌,探讨细菌素抑菌机制。【方法】从市售海鱼的肠道中筛选出一株对金黄色葡萄球菌(Staphylococcus aureus)和大肠埃希菌(Escherichia coli)有较强抑菌效果的菌株GOFEL226,通过16S rDNA序列鉴定乳酸菌,用乙酸乙酯提取细菌素,利用分光光度法、电导率法、扫描电镜技术探究细菌素的抑菌机制。【结果】菌株GOFEL226为戊糖乳植物杆菌(Lactiplantibacillus pentosus),GFEB226细菌素对金黄色葡萄球菌和大肠埃希菌的最小抑菌质量浓度为0.883 mg/mL;其对指示菌的生长有明显的抑制作用,可导致细胞的蛋白质、核苷酸、带电离子泄漏到细胞外,对细胞膜具有破坏作用。【结论】GFEB226细菌素可通过破坏革兰阳性和革兰阴性菌的细胞膜,使其胞内物质泄漏到胞外,进而导致细胞的死亡。

基于蛋白质组学技术对植物乳杆菌YP36细菌素的抑菌机制研究

为深入了解植物乳杆菌(Lactobacillus plantarum)YP36细菌素的抑菌机理,该研究利用高效液相色谱-串联质谱(HPLC-MS/MS)联用非标记定量蛋白质组学技术对细菌素处理前后植物乳杆菌SL32-2差异表达蛋白进行鉴定,并对差异蛋白进行代谢功能分析。结果表明,细菌素处理前后植物乳杆菌SL32-2发酵液中鉴定到的蛋白分别为1999个、2005个,显著上调和下调的蛋白质分别有15个和59个,另外有26个蛋白消失,33个蛋白诱导表达。所鉴定蛋白质中酶占比最大,其次为转运蛋白。基因本体论(GO)富集分析表明,显著性差异表达蛋白的功能条目共99条,主要集中在肽聚糖分解代谢和细胞壁大分子分解代谢、碳水化合物代谢以及嘧啶核苷酸代谢和生物合成等方面。京都基因与基因组百科全书(KEGG)富集分析表明,差异蛋白共涉及淀粉和蔗糖代谢途径、丙氨酸、天冬氨酸和谷氨酸代谢途径、辅因子生物合成途径和嘧啶代谢途径等53条通路。

Physical chemical and biological characterization of a new bacteriocin produced by Bacillus cereus NS02

Objective:To screen the bacteriocinogenic isolate from buffalo milk and to characterize it on physical,chemical and biological aspects for the application in biopreservation.Methods:Bacillus cereus(B.cereus)was isolated and assessed for its baceteriocinogenic activity.Bacteriocin was produced and purified by ammonium sulphate precipitation,dialysis and gel filtration chromatography.Purified bacteriocin was used to clieck its antimicrobial activity against food borne bacteria.Effect and stability of bacteripcin was determined with the respect to temperature,pH,enzymes,organic solvents and chemicals.Bacteriocin was also subjected to SDS PAGE analysis to determine its molecular weight.In addition,functional groups exist in the bacteriocin was determined by FTIR analysis.Results:B.cereus was identified by 16S rRNA sequence analysis.Bacteriocin showed increased activity against all the bacteria used and its activity unit was found to be 51,200 AU/mL.It was stable to high temperature(100℃)and wide range of pH(3-10),sensitive to proteolytic enzymes and resistant to nonprotcolytic enzymes.It was low molecular weight(3.5-6 kDa)protein and FTIR study revealed the presence of amide group and NH stretching,Conclusions:Bacteriocin produced in this study possesses the highest antimicrobial activity against both gram positive and gram negative bacteria thereby it has immense application as biopreservative agent.FTIR proved its peptide nature.

Potential applications of lactic acid bacteria and bacteriocins in antimycobacterial therapy

Tuberculosis(TB) is a communicable disease caused by Mycobacterium tuberculosis(M. tuberculosis). WHO estimated that 10.4 million new(incident) TB cases worldwide in year 2016. The increased prevalence of drug resistant strains and side effects associated with the current anti-tubercular drugs make the treatment options more complicated. Hence, there are necessities to identify new drug candidates to fight against various sub-populations of M. tuberculosis with less or no toxicity/side effects and shorter treatment duration. Bacteriocins produced by lactic acid bacteria(LAB) attract attention of researchers because of its "Generally recognized as safe" status. LAB and its bacteriocins possess an effective antimicrobial activity against various bacteria and fungi. Interestingly bacteriocins such as nisin and lacticin 3147 have shown antimycobacterial activity in vitro. As probiotics, LAB plays a vital role in promoting various health benefits including ability to modulate immune response against various infectious diseases. LAB and its metabolic products activate immune system and thereby limiting the M. tuberculosis pathogenesis. The protein and peptide engineering techniques paved the ways to obtain hybrid bacteriocin derivatives from the known peptide sequence of existing bacteriocin. In this review, we focus on the antimycobacterial property and immunomodulatory role of LAB and its metabolic products. Techniques for large scale synthesis of potential bacteriocin with multifunctional activity and enhanced stability are also discussed.

Antibacterial Activity of Lactic Acid Bacteria and Extraction of Bacteriocin Protein

Biopreservation systems in foods are of increasing interest for industry and consumers. Bacteriocin producing Lactobacillus spp. is considered Generally Recognize as Safe (GRAS), useful to control the fast development of pathogens and spoiling microbes in food and feed. Lactobacillus spp. was isolated from traditional winter fermented vegetable cucumber & carrot by the use of selective media. Especially De Man, Rogosa and Sharpe (MRS) Agar media were used to isolate the Lactobacillus species. Morphologically identified by gram staining & colony morphology. Biochemically recognized by catalase, oxidase, MRVP & carbohydrate fermentation test. Antimicrobial activity of Lactobacillus spp. was confirmed by Well Diffusion Method. Molecular characterization of bacteriocin protein and molecular weight determined by SDS PAGE method. The isolate was found to be facultative anaerobic, Gram positive, and catalase negative. The result of antimicrobial activity measured by the Arbitrary Unit (AU/ml) of zone of the inhibition. Six isolates found from the sample but most activities exhibited isolate 4 against Bacillus megaterium (55 mm) zone of diameter. The molecular weight of the washed bacteriocin was calculated to be about 40 kDa (Isolate 1) and 15 kDa & 30 kDa (Isolate 5). Bacteriocin protein reduces chemical preservatives and uses in future as biopreservative in food industry.

Study Bacteriocin Production and Optimization Using New Isolates of <i>Lactobacillus</i>spp. Isolated from Some Dairy Products under Different Culture Conditions

Lactobacilli belong to the group of lactic acid bacteria (LAB), that have several distinguished abilities such as production of lactic acid, enzymes such as β-Galactosidase and natural antimicrobial substances called bacteriocins. Bacteriocin is a biopreservative agent potential of suppressing growth of some contaminant bacteria in food industry but its commercial availability is limited and costly. The study aimed to select isolates of Lactobacillus spp. potential for producing bacteriocins to suppress the growth of Escherichia coli ATCC 25922 and Bacillus subtilis NCIB3610, and to optimize the process of bacteriocin production. Results obtained in this study showed that L. acidophilus isolate CH1 was selected as the best candidate for bacteriocin among the four isolates that tested. The largest amounts of the bacteriocins were synthesized only in MRS medium was supplemented with K2HPO4 (1.0%), Tween 80 (1%), Beef extract (1%), glucose, cyctein and peptone extract (1%). The optimization of culture conditions for bacteriocin production areas showed that corn steep liquor medium was the best medium for all isolates against Bacillus subtilis while no effect was observed on Escherichia coli ATCC 25922 except when used MRS medium. The optimum conditions for bacteriocin production were pH 6.0, temperature 34?C with 4% Phenyl acetamide showing the greatest growth inhibition areas.

Isolation and Characterization of Enterococci Bacteriocinic Strains from Tunisian Fish Viscera

A collection of lactic acid bacteria isolates from fish viscera was studied and investigated regarding to their functional properties and safety aspects. From these, three isolates GM1, GM2 and GM3 were identified as Enterococcus feacium species using molecular methods. Partial Amplified rDNA Restriction Analysis (partial ARDRA) with restriction enzyme HaeIII separated these isolates into distinctive group which suggest genotypic variability within enterococci strains isolated from fish viscera. The three strains GM1, GM2 and GM3 exhibited antimicrobial activity. Indeed strains have been shown to produce bacteriocins with inhibitory effect against food spoilage bacteria and pathogenic fish including Carnobacterium maltaromaticum. The molecular mass of bacteriocin, as calculated by tricine-SDS-PAGE, was found to be 4.5 kDa. All isolates were tested positive upon PCR amplification of enterocin A structural gene. Investigations of antibiotic resistance show that the isolates were mostly sensitive to several antibiotics (ampicillin, penicillin, tetracycline, gentamycine) and resistance to rifampicin. All isolates grow in esculin azide agar as a selective medium for enumeration of probiotic enterococci. This study suggests that our strains can be employed as probiotic or to improve the safety of food products.

Use of Bacteriocin Producing <i>Lactococcus lactis</i>subsp. <i>lactis</i>LABW4 to Prevent <i>Listeria monocytogenes</i>Induced Spoilage of Meat

A bacteriocin producing strain of Lactococcus lactis subsp. lactis LABW4 was isolated from naturally fermented milk product which exhibited strong antibacterial activity against Listeria monocytogenes MTCC657, a food spoilage psychrophilic organism. Both cell free and heat killed supernatants of LABW4 were effective to produce zones of inhibition against L. monocytogenes in vitro. The antibacterial metabolite(s) of LABW4 showed strong cidal effect on the growth of L. monocytogenes. Meat samples, mixed with heat killed supernatant of LABW4 when inoculated with Listeria, remain fresh up to 25 days in refrigerated condition whereas spoilage started immediately after 24 hours of inoculation for control sets. Enhancement of Lactate dehydrogenase of L. monocytogenes upon treatment with LABW4 cell free supernatant suggested its lytic mode of action. Cell lysis or degradations were also supported by scanning electron micrograph of treated cells.

Partial Characterization of Bacteriocins from Two <i>Pediococcus acidilactici</i>Strains Isolated during Traditional Sorghum Beer Processing in Côte d’Ivoire

Two lactic acid bacteria strains (At1BEAE22 and At344E21) isolated during tchapalo production were identified on the basis of phenotypic analyses. Bacteriocins produced by these strains were tested for their antimicrobial activities using well diffusion agar method. Heat resistance, pH sensitivity and enzyme treatments were also analyzed. Results showed that both lactic acid bacteria strains were identified as Pediococcus acidilactici. Their bacteriocins inhibited growth of Lactobacillus delbrueckii F/31, Listeria innocua ATCC 33090, Enterococcus faecalis, Enterococcus faecalis ATCC 29212, Streptococcus sp, Enterococcus faecalis CIP 105042 and Enterococcus faecium ATCC 51558. These bacteriocins were heat stable at 60&degC for 30 min for all indicator bacteria. However, they remained active only against Lactobacillus delbrueckii and Listeria innocua at 121&degC for 60 min. Moreover, they were active in a wide range of pH (3 to 9) with a maximum activity observed at pH 5 and 6 on all indicator bacteria. But, bacteriocin from Pediococcus acidilactici At34E21 was more stable at acidic pH than basic one. The fact that the bacteriocin was inactivated by proteinase K and α-chymotrypsin indicated its proteinaceous nature, a general characteristics of bacteriocins.

Obtaining Bacteriocins by Chromatographic Methods

Bacteriocins are a large group of chromosome or plasmid-encoded and ribosomally synthesized low-molecular-weight (2 to 6 kDa) antimicrobial and amphiphilous peptides produced by Gr+ or Gr- bacteria [1]. Their low toxicity as well as the absence of allergenicity and reactogenicity is confirmed by testing selected bacteriocins [2] [3]. Bacteriocins can be widely used as preservatives and antibiotic alternatives in medicine. Nisin, a Streptococcus lactis-derived bacteriocin, has been in practice in food industry for a long time. A relevant product contains about 2.5% of nisin. For medical use (e.g., when injected into the blood stream), highly purified drugs are required. However, the yield of bacteriocins accounts for no more than a few percents from the total activity in the culture liquid. In this paper, we propose methods (by example of two B. subtilis strains), allowing to increase the yield up to ~80%. It is believed that other bacteriocins may be purified by these methods and with the same yield.