不同来源的植物乳杆菌基因组和抗生素抗性

为了研究不同分离源对植物乳杆菌基因组和功能的影响,选取了来自发酵酱、泡菜、粪便3种环境的33株植物乳杆菌,通过比较基因组学手段研究菌株的基因组基本特征、直系同源基因、系统进化关系,并结合功能基因注释结果与表型结果分析菌株对抗生素的耐药性。系统发育树揭示了分离源对植物乳杆菌的遗传进化具有较为显著的影响;同源基因结果表明粪便源的菌株其特殊基因数量要高于泡菜源和发酵酱源菌株的数量。33株植物乳杆菌均含有环丙沙星、四环素、氯霉素、甲氧苄氨嘧啶和万古霉素的抗性基因,菌株对这些抗生素也表现出抗性;绝大多数菌株对庆大霉素、链霉素、卡那霉素、新霉素、氨苄西林敏感,在基因组中也没有相关抗性基因;而红霉素和克林霉素的基因和表型并不对应。抗生素实验结果说明,分离源对菌株影响较小,大部分基因型与表型可以对应,基因组学对研究植物乳杆菌的生理特性起一定的指导作用。     

奶豆腐来源植物乳杆菌的益生特性评价

以奶豆腐来源的3株植物乳杆菌(Lactobacillus plantarum)为研究对象,分析了菌株的耐酸耐胆盐能力、表面疏水性和体外抗氧化能力,旨在评价菌株的益生特性。结果表明:植物乳杆菌对强酸和高胆盐具有较好的耐受能力,能保持较高的活性;供试菌株均对二甲苯的疏水性作用较强,而对三氯甲烷的疏水作用较弱;3株菌株均具有较好抗氧化活性。     

瑞士乳杆菌的益生特性

研究了瑞士乳杆菌作为发酵肉制品发酵剂的发酵特性;在模拟胃肠道环境中对酸和胆盐的耐受性;另以Caco-2细胞为体外肠道上皮细胞模型,研究了瑞士乳杆菌对小肠的黏附性和影响黏附的因素。实验结果表明,瑞士乳杆菌不产粘液,不产气,不产H2S,不产氨,不产H2O2,不具有氨基酸脱羧酶活性,不具有硝酸还原酶能力,具有抑制S.aureus和E.coli能力,能耐受一定量的NaCl和NaNO2,发酵葡萄糖产酸。瑞士乳杆菌同时还具有较好的耐酸和耐胆盐性能。通过瑞士乳杆菌的小肠黏附实验发现,生长稳定期的瑞士乳杆菌与Caco-2细胞有很好的黏附效果,当瑞士乳杆菌与Caco-2细胞共培养时间为2h时,黏附趋于饱和,且黏附环境pH值为7.0时黏附性最强。结果显示瑞士乳杆菌具备益生活性,满足发酵肉制品中益生菌制剂的筛选标准,可作为发酵菌种用于发酵肉制品的生产。     

植物乳杆菌天然质粒分类

为建立植物乳杆菌天然质粒分类方法,以质粒复制起始蛋白(replication initiation protein,Rep)作为分类标记,通过系统进化树分析方法,将植物乳杆菌53个编码Rep天然质粒划分为6个质粒类型,包括5个质粒家族和1个新的复制类型质粒,之后进一步提出了每个质粒家族特有的骨干序列,最终建立了一种简单、有效的植物乳杆菌天然质粒的分类方法和标准。与以往质粒功能和不相容性分类方法相比较,本方法具有较好的实用性和通用性。     

短乳杆菌天然质粒分类

为建立短乳杆菌天然质粒分类方法,通过质粒复制起始蛋白（replication initiation protein,Rep）进化树和基因组共线性分析,将短乳杆菌51 个天然质粒可以准确、有效地划分为6?个家族类型和11?个亚家族类型,并在家族4质粒中发现了一种新的复制子类型。研究结果表明,质粒Rep进化树和基因组共线性分析都是短乳杆菌天然质粒有效的分类方法,分类结果为今后短乳杆菌天然质粒科学分类和理性应用提供了理论研究依据。     

鼠李糖乳杆菌燕麦益生乳的研制

探讨了以燕麦和脱脂奶粉为主要原料,燕麦经双酶水解所得的糖化液,配以脱脂奶粉,经杀菌冷却,以鼠李糖乳杆菌为发酵剂,接种发酵而成的含有活性成分且具有保健功能的生物乳,它的活性多糖β-葡聚糖量为236mg/L,酸度为108°T,活菌数高达1010cuf/ml。      

泡菜中乳杆菌的快速检出和乳杆菌质粒资源的初步调查

乳杆菌(Lactobacillus)的质粒不仅决定了乳杆菌的某些性状,还应用于乳杆菌克隆表达载体的构建,是一种珍贵的遗传信息资源。为了调查中国泡菜中的乳杆菌质粒资源,采集了52份中国泡菜样品,采用乳杆菌PCR快速检出技术检出其中的乳杆菌,并用16s rDNA测序验证检出结果,然后提取检出乳杆菌菌株的质粒。结果检出1株清酒乳杆菌(Lactobacillus sakei)和18株植物乳杆菌(Lactobacillus plantarum),乳杆菌检出率为36.5%。检出含质粒的乳杆菌菌株11株,琼脂糖电泳显示有9种质粒谱型,乳杆菌质粒检出率为21.2%,质粒谱型检出率为17.3%。调查结果提示,中国泡菜中蕴藏丰富的乳杆菌质粒资源,值得进行更大规模的调查和研究。     

益生乳杆菌的筛选研究

以分离保存的15株乳杆菌为研究对象,从中筛选益生乳杆菌两株。通过检测乳杆菌的疏水性和自聚性,得到表面疏水性和自聚性均较高的菌株为Ind-3、CH10和M8。其中,乳杆菌CH10和M8在模拟胃肠环境下活菌数均达到106mL-1以上。通过灌胃乳杆菌CH10和M8,表明小鼠体重增加明显高与对照组,且实验组小鼠粪便中的β-半乳糖苷酶活性明显高于对照组。灌胃两株乳杆菌后,小鼠肠道内双歧杆菌与乳杆菌活菌数提高了,且肠杆菌、肠球菌和产气荚膜梭菌的生长繁殖受到不同程度的抑制,而对照组无明显变化。结果表明筛选出的两株乳杆菌可以促进小鼠对营养物质的吸收,提高肠道内有益菌的数量,抑制有害菌的生长繁殖,或许能够改善寄主肠道的微生态环境。     

乳杆菌质粒基因工具的发展概况

乳杆菌作为重要的益生菌已经被广泛地应用于食品及饮料加工业。利用分子生物学技术可改造乳杆菌的性状,改善其工业特性,满足生产需要。但相对于大肠杆菌,乳杆菌基因工程尚处于起步阶段。由于乳杆菌自身含有很多质粒,因此可以自身质粒为基础构建克隆载体或表达载体。主要介绍了乳杆菌质粒概况以及质粒基因工具的发展概况,并展望了乳杆菌质粒基因工具的发展前景。     

三株人源干酪乳杆菌的益生特性

评价3株人源干酪乳杆菌的益生性能,为其在食品和保健品等行业中的应用提供理论支持。通过体外测定3株干酪乳杆菌的对模拟肠道的耐受性、黏附性、抑菌性及抗生素敏感性来评价其益生性能。3株干酪乳杆菌,特别是NCU011056,对高盐、酸和胆盐均具有高耐受性,NCU011056在100 g/L NaCl、pH 2. 0和3 g/L胆盐环境下存活率分别为95. 7%、87. 2%和80%; 3株干酪乳杆菌对Caco-2细胞系黏附能力各异,其中NCU011056黏附能力较强,黏附指数为60. 08%; 3株干酪乳杆菌对4种常见革兰氏阳性和革兰氏阴性致病菌均具有较强的抑菌能力; 3株干酪乳杆菌对青霉素、阿莫西林、头孢曲松、四环素、氯霉素、红霉素、利福平、环丙沙星均敏感。3株人源干酪乳杆菌,特别是NCU011056,具有良好的益生潜力,可考虑后期应用于食品、保健品等行业中。     

一株植物乳杆菌体外抗氧化和耐药性的研究

目的:研究了植物乳杆菌KLDS 1.0386的体外抗氧化能力和10种抗生素的敏感性。方法:以过氧化氢耐受性、DPPH自由基清除力、羟自由基清除力、超氧阴离子清除能力、抗脂质过氧化能力、还原能力和螯合金属离子能力为指标进行体外抗氧化评定;检测KLDS 1.0386对β-内酰胺类、碳青霉烯类、糖肽类、氨基糖苷类、大环内酯类、林可酰胺类和酰胺醇类抗生素的敏感性。结果:KLDS 1.0386菌悬液、菌体菌体细胞破碎液和发酵上清液三者对比,菌悬液的抗氧化能力最佳;在抗生素敏感性上,植物乳杆菌KLDS 1.0386对氨苄西林、青霉素、亚胺培南、庆大霉素、红霉素和四环素表现为敏感;对万古霉素、卡那霉素、克林霉素和氯霉素表现为耐受。结论:实验获得一株具有潜在抗氧化能力和耐药性的植物乳杆菌。      

Evaluation of the possible use of potentially probiotic Lactobacillus strains in dairy products

In the study, the ability of two potentially probiotic strains Lactobacillus plantarum 14 and Lactobacillus fermentum 4a to milk fermentation and the possibility to use them in yogurt production were investigated. The strains did not acidify milk during 24 h and 72 h fermentation at 37C, but grew well and remained at the level of 10⁸ colony-forming units (CFU)/mL during 21 days of cold storage. Their application to yogurt production along with commercial starter culture consisted on L. delbrueckii ssp. bulgaricus and S. thermophilus allowed to obtain products with typical sensory properties, pH values and numbers of potentially probiotic bacteria at desired level 10⁷ CFU/mL.     

Species-specific identification of commercial probiotic strains

Products containing probiotic bacteria are gaining popularity, increasing the importance of their accurate speciation. Unfortunately, studies have suggested that improper labeling of probiotic species is common in commercial products. Species identification of a bank of commercial probiotic strains was attempted using partial 16S rDNA sequencing, carbohydrate fermentation analysis, and cellular fatty acid methyl ester analysis. Results from partial 16S rDNA sequencing indicated discrepancies between species designations for 26 out of 58 strains tested, including two ATCC Lactobacillus strains. When considering only the commercial strains obtained directly from the manufacturers, 14 of 29 strains carried species designations different from those obtained by partial 16S rDNA sequencing. Strains from six commercial products were species not listed on the label. The discrepancies mainly occurred in Lactobacillus acidophilus and Lactobacillus casei groups. Carbohydrate fermentation analysis was not sensitive enough to identify species within the L. acidophilus group. Fatty acid methyl ester analysis was found to be variable and inaccurate and is not recommended to identify probiotic lactobacilli.     

Adhesive and chemokine stimulatory properties of potentially probiotic Lactobacillus strains

Five Lactobacillus plantarum strains and two Lactobacillus johnsonii strains, stemming either from African traditionally fermented milk products or children's feces, were investigated for probiotic properties in vitro. The relationship between the hydrophobic-hydrophilic cell surface and adhesion ability to HT29 intestinal epithelial cells was investigated, and results indicated that especially the L. johnsonii strains, which exhibited both hydrophobic and hydrophilic surface characteristics, adhered well to HT29 cells. Four L. plantarum and two L. johnsonii strains showed high adherence to HT29 cells, generally higher than that of the probiotic control strain Lactobacillus rhamnosus GG. Most strains with high adhesion ability also showed high autoaggregation ability. The two L. johnsonii strains coaggregated well with the intestinal pathogens Listeria monocytogenes Scott A, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Salmonella enterica serovar Typhimurium ATCC 14028. The L. plantarum BFE 1685 and L. johnsonii 6128 strains furthermore inhibited the adhesion of at least two of these intestinal pathogens in coculture with HT29 cells in a strain-dependent way. These two potential probiotic strains also significantly increased interleukin-8 (IL-8) chemokine production by HT29 cells, although modulation of other cytokines, such as IL-1, IL-6, IL-10, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor alpha (TNF-alpha), and transforming growth factor beta (TGF-beta), did not occur. Altogether, our results suggested that L. plantarum BFE 1685 and L. johnsonii BFE 6128 showed good adherence, coaggregated with pathogens, and stimulated chemokine production of intestinal epithelial cells, traits that may be considered promising for their development as probiotic strains.     

Edible table (bio)spread containing potentially probiotic Lactobacillus and Bifidobacterium species

A prototype of a reduced fat (60% w/w) edible table biospread, with an added viable, nongrowing, mixed-strain and potentially probiotic culture was developed. Conventional commercial aqueous-phase ingredient and reduced fat spread processing technologies were modified to achieve acceptable strain viability ( ≥   10⁵ cfu/mL) during scraped-surface heat exchange emulsion processing and biospread shelf life. The modifications consisted of:

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   spilt-stream pasteurization of the aqueous and lipid phases (thereby obviating the need for in-line pasteurization of the water-in-oil emulsion during processing);

       
  
  

乳杆菌和双歧杆菌的生理代谢多样性研究

比较了拥有潜在益生特性的28株乳杆菌(Lactobacillus)和26株双歧杆菌(Bifidobacterium)的生理代谢差异，这些菌株多数来源于波兰。筛选过程当中，首要的指标是检测菌株对低pH值和对胆盐的抗性以及在发酵或非发酵乳制品当中的存活能力。考察了这些候选菌株是否产生某种拮抗物质，同时评价这些物质对来自于食物或肠道内的病原微生物或生产当中不渴望微生物的抑制效果。研究了混合发酵剂中各菌株之间的共生能力，并对它们在生产加工过程中表现出的对诸如冷冻、冻干或储藏等处理的耐受能力进行评价。采用高效液相色谱(HPLC)的方法对菌株利用各种碳源所生成的代谢产物的类型进行了测试。结果表明，所有供试菌株存在很大的生理代谢差异，因而，应选择那些具有科学测定基础的菌株作为益生菌菌株。     

内蒙古牧区传统乳制品中潜在益生植物乳杆菌的药物敏感性研究

采用纸片琼脂扩散法(K-B法),对从内蒙古牧区传统乳制品中分离到的16株对pH3及0.3%牛胆酸钠有一定耐受性的植物乳杆菌的药物敏感性进行了测定。结果显示,所测试的16株植物乳杆菌均对青霉素G、万古霉素、诺氟沙星、氧氟沙星、环丙沙星不敏感;对头孢哌酮、乙酰螺旋霉素中度敏感;对哌拉西林、阿莫西林、头孢唑啉、克林霉素、磺胺甲恶唑、利福平、呋喃妥因敏感;对其他抗生素的敏感性因菌株不同而有差异。所有菌株对阿奇霉素、新霉素表现为敏感或中度敏感;多数菌株对氨苄青霉素敏感(11/16),对头孢噻肟敏感(8/16)或中度敏感(6/16),对多黏菌素B不敏感(10/16)。     

5株植物乳杆菌生物学特性及安全性评价

通过对不同分离源的5株植物乳杆菌进行产酸、抑菌、耐酸耐胆盐、耐药性、溶血性、产生物胺能力测定,结果显示:5株菌的产酸及抑菌能力较强,差异显著（p<0.05）;耐酸性能较强,2 h的存活率在85.67%⁹8.00%,PBIL1-002与其他各菌株之间差异显著（p<0.05）;耐胆盐能力相对较弱,2 h的存活率在21.17%⁵1.20%,除PBIL1-002与PBIL1-006外,其余各菌株间差异显著（p<0.05）;5株菌均对链霉素和万古霉素耐药,PBIL1-009和PBIL1-018对复方新诺明耐药;PBIL1-006和PBIL1-009对庆大霉素耐药;菌株不具溶血性能;定性检测菌株产生物胺能力显示,PBIL1-009代谢产生精胺,PBIL1-018代谢产生精胺和酪胺。说明不同分离源的菌株特性及安全性有一定差异,生产应用时需选择生物学特性佳且无安全隐患的菌株。