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首页> 外文学位 >Occurrence and Characterization of Non-starter Lactic Acid Bacteria Producing Pediocin PA-1/AcH-like Bacteriocin in Retail Cheddar Cheese.
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Occurrence and Characterization of Non-starter Lactic Acid Bacteria Producing Pediocin PA-1/AcH-like Bacteriocin in Retail Cheddar Cheese.

机译：零售切达干酪中生产非发酵乳酸菌产生Pediocin PA-1 / AcH样细菌素的特性及表征。

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摘要

Lactic acid bacteria are known to produce antimicrobial compounds, including organic acids, hydrogen peroxide, and bacteriocins. However, bacteriocins have attracted strong interest as safe alternative food preservatives. In this study, bacteriocin producing non-starter lactic acid bacteria (NSLAB) were isolated from Cheddar cheese and characterized. A total of 300 NSLAB were isolated from retail Cheddar cheese. Of these, 130 isolates were found to have inhibitory activity. The isolates were grouped into five groups based on morphological and physiological characteristics and fifteen isolates were chosen as representatives for further studies. The representative isolates had antimicrobial activity against several foodborne pathogens: Listeria monocytogenes, Bacillus cereus, Salmonella typhi, Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa, and E. coli O157: H7. The antimicrobial activity of the supernatants was not affected by neutralization with NaOH, treatment with catalase, trypsin, lipase A and amylase. However, treatment with proteinase K resulted in partial inactivation of the supernatants suggesting that the compounds are bacteriocins. The mode of action of these bacteriocins was found to be bactericidal as revealed by Evos digital fluorescence microscopy. This was confirmed by the intra cellular detection of propidium iodide stain in most of the cells after exposure for 12 h, suggestive of a permeable membrane. Further characterization of the bacteriocins showed that they had high thermostability as activity was maintained after boiling for 6.5 h. Also autoclaving for 45 minutes had no significant effect on the activity. Similarly, storage at -20 ºC, 4 ºC, 25 ºC, 37 ºC, and 45 ºC for up to 30 days did not reduce the activity. Also, the activity was maintained at a wide pH range of one to eight. However, addition of sodium chloride 1-7% (w/w) enhanced the activity of the bacteriocins. Sonication at amplitude of 5 for 5 h (discontinuous mode) showed time dependent reduction in activity of the bacteriocins. Analysis of the fifteen representative isolates with API 50CH identified eight out of fifteen isolates to two Lactobacillus species: L. paracasei ssp. paracasei and L. brevis. The remaining isolates were identified to two genera: three Pediococcus sp. and four Lactococcus sp. 16S rRNA identified eight out of fifteen isolates to three Lactobacillus species: L. paracasei, L.=" L. paracasei, " and L. rhamnosus and the other seven isolates to Pediococcus sp. To assess species diversity, the isolates were analysed by pulse field gel electrophoresis using Not I restricted genomic DNA. Pediococccus sp. revealed only one banding pattern while Lactobacillus sp. had multiple banding patterns. However, cell wall protein analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed different banding patterns for each isolate. PCR analysis of the fifteen representative isolates using pediocin primers (PedF and PedR) identified a 322 bp fragment corresponding to pediocin in both the Lactobacillus and Pediococcus strains. Furthermore, PCR amplification with PEDRPO and PEDC1041 showed the presence of a 1044 bp DNA fragment corresponding in size to the structural gene of pediocin PA-1 in all fifteen selected isolates. Hind III digestion of pediocin PA-1 amplicons yielded two different profiles, 10 isolates showed 2 fragments while the other 5 showed one fragment suggesting possible difference in the genetic sequences of the pediocin PA-1 gene. The pediocin PA-1 structural gene seems to be located in the chromosome as no plasmid was detected under various growth conditions.
机译：已知乳酸菌会产生抗菌化合物，包括有机酸，过氧化氢和细菌素。然而，细菌素作为安全的替代食品防腐剂引起了人们的强烈兴趣。在这项研究中，从切达干酪中分离出产生细菌素的非发酵乳酸菌（NSLAB）并进行了表征。从零售切达干酪中分离出总共300个NSLAB。其中，发现130种分离物具有抑制活性。根据形态和生理特征将分离物分为五类，并选择了十五个分离物作为进一步研究的代表。代表性分离株对几种食源性病原体具有抗微生物活性：单核细胞增生性李斯特菌，蜡样芽孢杆菌，伤寒沙门氏菌，粪肠球菌，粪肠球菌，铜绿假单胞菌和大肠杆菌O157：H7。上清液的抗菌活性不受NaOH中和，过氧化氢酶，胰蛋白酶，脂肪酶A和淀粉酶处理的影响。然而，用蛋白酶K处理导致上清液部分失活，表明该化合物是细菌素。如Evos数字荧光显微镜所揭示的，发现这些细菌素的作用方式具有杀菌作用。暴露12 h后，大多数细胞的细胞内碘化丙啶染色检测结果证实了这一点，这表明存在可渗透膜。细菌素的进一步表征表明，它们具有高的热稳定性，因为煮沸6.5小时后仍能保持活性。同样高压灭菌45分钟对活性没有明显影响。同样，在-20ºC，4ºC，25ºC，37ºC和45ºC下保存最多30天不会降低活性。另外，将活性维持在1至8的宽pH范围内。但是，添加1％至7％（w / w）的氯化钠可增强细菌素的活性。在5振幅下超声处理5小时（不连续模式）显示出细菌素活性的时间依赖性降低。用API 50CH对15种代表性分离株进行分析后，从15种分离物中分离出8种分离到两种乳酸杆菌：副干酪乳杆菌。副干酪和短乳杆菌。其余的分离物被鉴定为两个属：三个Pediococcus sp。和四个乳球菌。 16S rRNA鉴定了15种细菌中的8种，分别属于三种乳酸杆菌：副干酪乳杆菌，副干酪乳杆菌和鼠李糖乳杆菌，以及其他7种细菌。为了评估物种多样性，使用Not I限制性基因组DNA通过脉冲场凝胶电泳分析了分离物。小球菌乳酸菌sp。揭示了仅一种条带模式。有多个条纹图案。但是，用十二烷基硫酸钠聚丙烯酰胺凝胶电泳进行的细胞壁蛋白分析显示每种分离物的条带模式不同。使用pediocin引物（PedF和PedR）对十五种代表性分离株进行PCR分析，鉴定出了在乳酸杆菌和Pecococcus菌株中对应于pediocin的322 bp片段。此外，用PEDRPO和PEDC1041进行的PCR扩增显示，在所有15个选定的分离物中，都存在一个大小与pediocin PA-1的结构基因相对应的1044 bp DNA片段。 pediocin PA-1扩增子的Hind III消化产生两个不同的图谱，其中10个分离物显示2个片段，而其他5个显示一个片段，表明pediocin PA-1基因的遗传序列可能存在差异。 pediocin PA-1结构基因似乎位于染色体中，因为在各种生长条件下均未检测到质粒。

著录项

  • 作者

    Mlalazi-Oyinloye, Mothomang.;

  • 作者单位

    Howard University.;

  • 授予单位 Howard University.;
  • 学科 Biology General.;Biology Microbiology.;Agriculture Food Science and Technology.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 122 p.
  • 总页数 122
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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