一株产γ-氨基丁酸的戊糖片球菌分离鉴定及药敏分析

目的 从健康人肠道微生物群中分离功能菌株。方法 从健康人粪便中分离肠道菌株,进行形态学观察、生理生化鉴定和16S rDNA测序分析,并对分离的菌株进行耐药特性评估,利用ELISA试剂盒测定发酵液中γ-氨基丁酸的含量。结果 本研究分离得到菌株为革兰阳性球菌,生理生化反应初步判断为片球菌属,16S rDNA测序鉴定为戊糖片球菌（Pediococcus pentosaceus）,该菌株命名为P. pentosaceus WMU002。该菌株对氨苄西林、亚胺培南、头孢曲松、头孢噻肟、大环内酯类红霉素、克林霉素和哌拉西林敏感,对丁胺卡那、新生霉素、头孢西丁和甲氧嘧啶耐药。24 h发酵液γ-氨基丁酸含量为4.12μmol/L。结论 从健康人肠道中分离获得的1株产γ-氨基丁酸的P. pentosaceus WMU002菌株可作为人体益生菌制剂候选菌株。     

戊糖片球菌热胁迫应答机制探究

目的 以发酵柚子皮中分离到的戊糖片球菌WPPE03、WPPE04作为研究对象,用标准株CGMCC1.2695作为对照,探索其热胁迫相关机制。方法 通过平板计数法,确定戊糖片球菌的热适应条件和热致死条件;采用扫描电子显微镜观察细胞热应激前后形态学变化;应用RT-qPCR分析热休克蛋白及细胞膜脂肪酸合成相关基因转录水平变化,阐明热胁迫机制。结果 确定了三株戊糖片球菌热适应条件（47℃,60 min）和热致死评价条件（62℃,15 min）;发现了热致死条件处理菌体,膜表面会出现凹陷、皱褶,部分菌体出现破裂、胞内物质外溢等现象,而热致死条件处理前先经过热适应处理,菌体破裂现象明显减少;同时,证明了热应激处理过程中,相关热休克蛋白基因表达显著上调（P<0.05）,脂肪酸合成相关基因表达发生了变化。结论 研究戊糖片球菌的热应答相关机制,可为合理开发、利用具有发酵性能的戊糖片球菌提供借鉴。     

柚子皮黑曲霉的分离鉴定及产酶特性研究

对柚子皮上自然生长的黑曲霉进行分离鉴定,并探讨其产酶特性。以平板稀释法从柚子皮上分离出一株霉菌菌株,通过观察其形态特征和培养特征,对照《真菌鉴定手册》判定该菌株的种属;采用鉴定培养基法对其产酶特性进行分析。根据柚子皮的成分特性,以干柚子皮为主要原料,该菌为生产菌株,采用固态发酵法探究培养基的成分、柚子皮含量、培养基初始含水量及发酵时间4个因素对纤维素酶活力的影响。结果表明,该菌株为黑曲霉(Aspergillus nige),可产淀粉酶、蛋白酶、纤维素酶、果胶酶;固态发酵培养基中添加柚子皮12g,麸皮0.5 g和(NH_4)_2SO_40.5 g,培养基初始含水量保持在68.5 mL/100 g,培养时间控制在60 h左右时纤维素酶产量较高。     

戊糖片球菌益生效能及其在动物中的应用前景

动物肠道菌群紊乱是引起其消化系统疾病的重要原因。与抗生素相比,益生菌更安全,有良好的发展前景,而且戊糖片球菌本身就是乳酸菌,因此其具体应用过程备受业界关注。为此,文章对戊糖片球菌的作用机制、应用功能等展开了研究,以期为今后的动物生产应用提供参考价值。     

片球菌素PediocinPA-1抑菌活性检测

目的检测通过基因工程获得的片球菌素Pediocin PA-1抑菌活性。方法采用琼脂扩散法检测片球菌素Pediocin PA-1对单核细胞增生李斯特杆菌、金黄色葡萄球菌、铜绿假单胞菌、沙门菌和大肠埃希菌O157的抑菌活性。结果片球菌素Pediocin PA-1对单核细胞增生李斯特杆菌、金黄色葡萄球菌、沙门菌、铜绿假单胞菌和大肠埃希菌O157等均有抑制作用。其中对单核细胞增生李斯特杆菌、沙门菌、大肠埃希菌和金黄色葡萄球菌的抑制作用效果明显,对铜绿假单胞菌有微弱的抑制作用。结论通过基因工程获得的片球菌素Pediocin PA-1具有抑菌活性。     

嗜热网球菌纤维二糖差向异构酶在枯草芽孢杆菌中的表达及发酵优化

通过化学方法合成嗜热网球菌(Dictyoglomus thermophilum)来源的纤维二糖差向异构酶基因ce,将其引入到载体pBSuL3-ce,构建重组质粒pBSuL3-ce并转化进枯草芽孢杆菌,发酵48h后测定胞内酶活为7. 5U/ml。酶学性质结果表明:该酶的最适pH为8. 5;最适温度为85℃,85℃的半衰期为120min。为降低发酵成本,对发酵培养基进行优化:以35g/L豆粕粉为氮源、5g/L甘油为碳源时,酶活力最高可达12. 3U/ml。依据摇瓶优化的条件在3L发酵罐中扩大培养,胞内酶活达到56U/ml,比摇瓶培养酶活提高了8倍。利用发酵所得酶制备乳果糖,在乳糖浓度为400g/L、反应温度为85℃、初始pH 8. 5、加酶量为20U/ml的条件下,乳果糖转化率可达51%。     

戊糖片球菌368对猪生长性能、粪菌结构和代谢产物的影响

[背景]随着"禁抗令"的推行,如何寻找安全的抗生素替代品迫在眉睫.[目的]研究戊糖片球菌368对育肥猪的影响.[方法]选用20头育肥猪,随机分为2组,每组2个重复,每个重复5头.对照组饲喂基础饲粮,试验组饲喂基础饲粮+戊糖片球菌368(1010CFU/kg),试验期28 d.[结果]试验期内,试验组和对照组的平均日增重...     

具有优良抑菌特性乳酸菌的筛选鉴定及活性物质检测

【背景】有益性乳酸菌在人体和动物体内分布极为广泛,是维持胃肠道菌群平衡、提高机体免疫力的主力军。近年来,为了解决禁用抗生素而导致动物发病率不断增高的问题,分析和研究乳酸菌及其所产活性物质的益生特性并开发新型饲料添加剂成为一个重要手段。【目的】本实验旨在从土壤中分离筛选出具有优良抑菌特性的乳酸菌,并对其所产活性物质的特性进行分析评价。【方法】采用溴甲酚紫平板法筛选并结合抑菌能力检测,得到2株具有优良抑菌特性的产酸菌株,分别命名为H-3和H-4。经形态学鉴定及16S rRNA基因序列测定后,对2株菌分别进行生长曲线和产酸量检测;通过排除酸处理、蛋白酶处理和热处理的方法分析2株菌所产抑菌物质的有效成分。【结果】H-3和H-4菌株经初步鉴定为乳酸片球菌(Pediococcus acidilactici),2株菌均具有良好的生长性能及产酸性能。菌株发酵上清液对大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)、猪霍乱沙门氏菌(Salmonella choleraesuis)、福氏志贺氏菌(Shigella flexneri)均表现出明显的抑...     

淋球菌的快速糖发酵鉴定法

应用带有ｐＨ指示剂的缓冲平衡盐溶液建立了快速糖发酵鉴定淋球菌（Ｎｅｉｓｓｅｒｉａｇｏｎｏｒｒｈｏｅａｃ）的实验方法。０．５—４小时内可获得实验结果。将本法与常规的糖发酵方法进行了比较，本法敏感、快速，明显优于常规法，此法是快速鉴定的可靠方法。     

Autolytic activity and pediocin-induced lysis in Pediococcus acidilactici and Pediococcus pentosaceus strains

AIMS: To evaluate the autolytic phenotype of Pediococcus acidilactici and P. pentosaceus, the peptidoglycan hydrolases content and the effect of pediocin AcH/PA-1 and autolysins on cell lysis. METHODS AND RESULTS: The autolytic phenotype of Pediococcus strains was evaluated under starvation conditions in potassium phosphate buffer. The strains tested showed an extent of autolysis ranging between 40 and 90% after 48 h of starvation at 37 degrees C. Peptidoglycan hydrolase content was evaluated by renaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using cells of Micrococcus lysodeikticus as a target for the enzymatic activity and a major activity band migrating at about 116 kDa was detected. Additional secondary lytic bands migrating in a range of molecular weight between 45 and 110 kDa were also detected. The lytic activity, evaluated in the presence of different chemicals, was retained in 15 mM CaCl2 and in a range of pH between 5 and 9.5 but was strongly reduced in presence of 8% NaCl and in the presence of protease inhibitors. The substrate specificity of peptidoglycan hydrolases of Pediococcus strains was evaluated in renaturing SDS-PAGE incorporating cells of different bacterial species. Lytic activity was detected against cells of Lactococcus lactis subsp. lactis, L. delbrueckii subsp. bulgaricus, Lactobacillus helveticus and Listeria monocytogenes. The interaction between pediocin AcH/PA-1 and autolysis was evaluated and a relevant effect of bacteriocin in cell-induced lysis was observed. CONCLUSIONS: The autolytic phenotype is widely distributed among P. acidilactici and P. pentosaceus and the rate of autolysis is high in the majority of the analysed strains. Several autolytic bands, detected by renaturing SDS-PAGE, retained their activity against several lactic acid bacteria and L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization of the autolytic phenotype of Pediococcus acidilactici and P. pentosaceus strains should expand the knowledge of their role in fermentation processes where these species occur as primary or secondary bacterial population.     

Isolation and partial characterization of a bacteriocin produced by Pediococcus pentosaceus K23-2 isolated from Kimchi

Aims:  Screening and partial characterization of a bacteriocin produced by Pediococcus pentosaceus K23-2 isolated from Kimchi, a traditional Korean fermented vegetable.
Methods and Results:  A total of 1000 lactic acid bacteria were isolated from various Kimchi samples and screened for the production of bacteriocin. Pediocin K23-2, a bacteriocin produced by the Pediococcus pentosaceus K23-2 strain, showed strong inhibitory activity against Listeria monocytogenes . The bacteriocin activity remained unchanged after 15 min of heat treatment at 121°C or exposure to organic solvents; however, it diminished after treatment with proteolytic enzymes. The bacteriocin was maximally produced at 37°C, when the pH of the culture broth was maintained at 5·0 during the fermentation, although the optimum pH for growth was 7·0. The molecular weight of the bacteriocin was about 5 kDa according to a tricine SDS-PAGE analysis.
Conclusions:  Pediococcus pentosaceus K23-2 isolated from Kimchi produces a bacteriocin, which shares similar characteristics to the Class IIa bacteriocins. The bacteriocin is heat stable and shows wide antimicrobial activity against Gram-positive bacteria, especially L. monocytogenes .
Significance and Impact of the Study:  Pediocin K23-2 and pediocin K23-2-producing P. pentosaceus K23-2 could potentially be used in the food and feed industries as natural biopreservatives, and for probiotic application to humans or livestock.     

Identification of Pediococcus acidilactici and Pediococcus pentosaceus based on 16S rRNA and ldhD gene-targeted multiplex PCR analysis

A multiplex PCR assay that can readily and unambiguously identify Pediococcus acidilactici and Pediococcus pentosaceus strains was developed to give an easy-to-read profile based on the amplification of a 16S rRNA gene fragment, specific for each species, and a d-lactate dehydrogenase gene fragment specific for Pediococcus acidilactici strains.     

Physiological studies of the Pediococcus pentosaceus biofilm

Pediococcus pentosaceus, a bacterium recently used in human and animal probiotics, was used in combination with supports made from polylactic acid composite soybean meal was used to study biofilm formation, and it was found that dense biofilms developed by Day 1. Proteomic comparison between planktonic and biofilm cultures of P. pentosaceus showed distinct expression patterns of intracellular and extracellular proteins. Type I glyceraldehyde-3-phosphate dehydrogenase was upregulated in biofilm cultures and mediated cell adhesion and encouraged biofilm production. GMP synthase, which regulates GMP synthesis and acts as an intracellular signal molecule to control cell mechanisms and has been exploited in the development of new therapeutic agents, was also upregulated in the biofilm mode of growth. The present work serves as a basis for future studies examining the complex network of systems that regulate lactic acid bacterial (LAB) biofilm formation and can serve as a framework for studies of production of therapeutic agents from LAB.     

Prediction of dynamic metabolic behavior of Pediococcus pentosaceus producing lactic acid from lignocellulosic sugars

A dynamic metabolic model is presented for Pediococcus pentosaceus producing lactic acid from lignocellulose-derived mixed sugars including glucose, mannose, galactose, arabinose, and xylose. Depending on the pairs of mixed sugars, P. pentosaceus exhibits diverse (i.e., sequential, simultaneous or mixed) consumption patterns. This regulatory behavior of P. pentosaceus is portrayed using the hybrid cybernetic model (HCM) framework which views elementary modes of the network as metabolic options dynamically modulated. Comprehensive data are collected for model identification and validation through fermentation experiments involving single substrates and various combinations of mixed sugars. Most sugars are metabolized rather sequentially while co-consumption of galactose and arabinose is observed. It is demonstrated that the developed HCM successfully predicts mixed sugar data based on the parameters identified mostly from single substrate data only. Further, we discuss the potential of HCMs as a tool for predicting intracellular flux distribution with comparison with flux balance analysis.     

Chemostat production of pediocin SM‐1 by Pediococcus pentosaceus Mees 1934

Production of the bacteriocin pediocin SM‐1 by Pediococcus pentosaceus Mees 1934 was investigated in pH‐controlled batch and chemostat cultures using a complex medium containing glucose, sucrose or fructose. In chemostat cultures operated at 150 rpm, 30°C, 60% dissolved oxygen tension, pH 6.5, and D = 0.148 h^?1, the pediocin titer reached 185 AU/mL representing an increase of 32% compared with batch cultures in which glucose was used as the carbon source. Pediocin biosynthesis was markedly affected by the growth rate of the producer microorganism. For all carbon sources tested, pediocin production appeared to take place only at dilution rates lower than μ_max. However, only glucose supported production at the very low dilution rate of 0.05 h^?1 indicating a direct regulation of pediocin biosynthesis by the carbon source. Glucose supported higher biomass productivity and higher pediocin titers and yields compared with the other sugars used. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1481–1486, 2015     

Molecular characterization of the replicon of the Pediococcus pentosaceus 43200 pediocin A plasmid pMD136

The pediocin A-encoding plasmid of Pediococcus pentosaceus 43200, pMD136, was characterized by restriction enzyme analysis. Analysis of its replicon was facilitated by the construction of a probe vector consisting of the Escherichia coli plasmid pSP72 and the cat gene from Staphylococcus aureus plasmid pC194. The replication region of pMD136 was localized on a 1.6-kb EcoRI/BglII fragment. Sequencing analysis revealed a non-coding region, repA, spanning the first 440 bp, followed by an open reading frame, repB, encoding a putative protein of 390 amino acids. The non-coding region contained two sets of 6-bp and two sets of 22-bp direct repeats and two sets of inverted repeats upstream of the open reading frame. Strong homology of the isolated replicon was found to theta-type replicons of Lactococcus lactis plasmids. Segregational stability assay suggested at least two regions as potentially involved in the stabilization of pMD136. The plasmid's strong homology to other theta-type replicons and its relatively high stability suggest that pMD136 belongs to the widespread family of theta-replication plasmids.