Inhibition of Clostridium tyrobutyricum in cheese by Lactobacillus gasseri

A semi-hard cheese produced from milk artificially contaminated with Clostridium tyrobutyricum spores (2.5×10³ mL⁻¹) was used as a model for studying the ability of bacteriocin-producing Lactobacillus gasseri K7 (Rif^r) to inhibit clostridia. The added lactobacilli did not inhibit the primary starter culture (Streptococcus thermophilus), but inhibited non-starter mesophilic lactobacilli. Late blowing as a result of Cl. tyrobutyricum outgrowth and butyric acid fermentation occurred in all cheeses however it was reduced in cheeses with added Lb. gasseri. After 6 weeks, the average amount of butyric acid was significantly higher in cheeses without added lactobacilli (1.43 vs. 0.70 g kg⁻¹). At the end of 8-weeks ripening, 2.8×10⁷ cfu g⁻¹ of K7 (Rif^r) viable cells were detected. Using the total DNA from cheeses with added K7 (Rif^r) strain, PCR products were amplified with primers specific for Lactobacillus, Lb. gasseri and K7 bacteriocin gene.     

Novel bacteriocins produced by Lactobacillus fermentum strains with bacteriostatic effects in milk against selected indicator microorganisms

The aim of this work was to isolate and characterize bacteriocins produced by 2 Lactobacillus fermentum strains isolated from artisanal Mexican Cocido cheese. Fractions (F ≤3 kDa) obtained from cell-free supernatants of Lb. fermentum strains J23 and J32 were further fractionated by reversed-phase HPLC on a C18 column. Antimicrobial activities of F ≤3 kDa and bacteriocin-containing fractions (BCF), obtained from fractionation of F ≤3 kDa against 4 indicator microorganisms, were determined by the disk diffusion method and growth inhibition in milk. Subsequently, isolated BCF were analyzed by reversed-phase HPLC tandem mass spectrometry. Results showed that BCF presented antimicrobial activity against the 4 indicator microorganisms tested. For J23, one of the fractions (F3) presented the highest activity against Escherichia coli and was also inhibitory against Staphylococcus aureus, Listeria innocua, Salmonella Typhimurium, and Salmonella Choleraesuis. Similarly, fractions F3 and F4 produced by J32 presented antimicrobial activity against all indicator microorganisms. Furthermore, generation time and growth rate showed that F3 from J23 presented significantly higher antimicrobial activity against the 4 indicator microorganisms (2 gram-positive and 2 gram-negative) when inoculated in milk compared with F3 from J32. Interestingly, this fraction presented a broader antimicrobial spectrum in milk than nisin (control). Reversed-phase HPLC tandem mass spectrometry analysis revealed the presence of several peptides in BCF; however, F3 from J23 that was the most active fraction of all presented only 1 bacteriocin. The chemical characterization of this bacteriocin suggested that it was a novel peptide with 10 hydrophobic AA residues in its sequence and a molecular weight of 2,056 Da. This bacteriocin and its producing strain, J23, may find application as a biopreservative against these indicator microorganisms in dairy products.     

加氏乳杆菌的降脂活性研究

目的:探究加氏乳杆菌是否具有降脂活性,并验证其对HepG2细胞脂质堆积和高脂血症小鼠脂质代谢活性的影响.方法:利用油酸诱导建立HepG2细胞脂质堆积模型,通过多次油红O染色定量对166株加氏乳杆菌进行降脂活性筛选,选择活性最强的一株菌通过Bodipy染色、甘油三酯含量测定进行验证.利用C57BL/6J小鼠喂食高脂饲料建...     

嗜酸乳杆菌产细菌素培养基及培养条件的优化

为了提高嗜酸乳杆菌单位体积的活菌数及其产细菌素的产量,通过单因素试验和正交试验优化了培养基配方及培养条件。优化后培养基配方为葡萄糖10g/L,蛋白胨15g/L,牛肉膏15g/L,氯化钠3g/L,碳酸钙1g/L,MgSO4.7H2O 0.4g/L,柠檬酸三铵2g/L,K2HPO4.3H2O 4g/L,MnSO4.H2O 0.2g/L。优化后培养条件为37℃发酵22h,接种量为2%,初始pH值为6.8,摇床转速160r/min。在此条件下,细菌素效价为231.34AU/mL。     

乳酸菌细菌素抗菌潜力挖掘研究进展

乳酸菌产生的主要抗菌防腐物质是有机酸,但在乳酸菌代谢产物中发现了细菌素等其它抗菌物质,其中有的细菌素甚至具有强烈的抗真菌能力。世界范围内启动的乳酸菌基因组计划创造了巨大的生物信息流。乳酸菌基因组蕴含的生物信息将对研究乳酸菌细菌素的合成代谢途径,功能基因挖掘以及新型抗菌剂的开发搭建信息平台。结合乳酸菌细菌素的最新研究进展,讨论利用乳酸菌基因组探索乳酸菌的抗菌潜力及其作为生物防腐剂在食品保藏中的应用前景。     

Ability of Lactobacillus gasseri K 7 to inhibit Escherichia coli adhesion in vitro on Caco-2 cells and ex vivo on pigs' jejunal tissue

The ability of Lactobacillus (Lb.) gasseri K 7 to inhibit adhesion of Escherichia coli O8:K88 to intestinal mucosa was studied on cultured Caco-2 cells and ex vivo on pigs' small intestinal tissue. Lactobacilli were added simultaneously with E. coli, before E. coli and after E. coli for competition, exclusion and displacement assays. The concentration of lactobacilli on fully differentiated Caco-2 cells was 4.5+/-0.3 x 10(8) cfu/well, while the concentration of E. coli varied from 1.5 x 10(6) to 4.3 x 10(8) cfu/well. The number of E. coli adhered to Caco-2 monolayer (cfu/well) was lineary correlated (R(2)=0.97) to the concentration of added cells. In the assay simulating exclusion, E. coli adhesion was reduced by Lb. gasseri K 7 strain by 0.1 to 0.6 log cfu/well. The binding of E. coli was inhibited even more when incubated simultaneously with lactobacilli, particularly at the lowest concentration of E. coli (ratio E. coli/lactobacilli 1:248), where five-times reduction (or 0.7 log) was observed. When adhesion to tissue derived from pigs' jejunum was tested, concentration of E. coli was constant (6.9+/-0.14 x 10(7) cfu/ml), while the concentration of Lb. gasseri K 7 was 5.9 x 10(7) and 1.3 x 10(7) cfu/ml in two independent experiments, respectively. The adhesion of E. coli and Lb. gasseri K 7 cells to jejunal mucosa was similar (1.0+/-0.17 x 10(6) and 1.54+/-0.10 x 10(6) cfu/cm(2)) when the concentrations of single strains in suspensions were approximately the same. No significant competition, exclusion or displacement of E. coli by lactobacilli was observed on jejunal tissue. In conclusion, Lb. gasseri K 7 was found to be effective in reducing E. coli adhesion to Caco-2 enterocytes, but it was not able to do so in ex vivo conditions tested for pig jejunal tissue.     

响应面法优化双层包埋格氏乳杆菌微胶囊制备工艺

为提高格氏乳杆菌的包埋率,增强菌体在人体内的定殖能力。该文利用响应面试验对双层包埋格氏乳杆菌微胶囊的制备工艺进行优化。确定最佳工艺条件为成膜时间15 min、壳聚糖浓度0.6%、pH5.2。制得双层包埋格氏乳杆菌微胶囊包埋率76.52%。经工艺优化后制得的微胶囊对人体酸环境耐受性明显增大,在人工肠液中1.0 h～1.5 h内释放完毕,贮藏稳定性显著提高。     

Bile tolerance, taurocholate deconjugation, and binding of cholesterol by Lactobacillus gasseri strains

Bile tolerance, deconjugation of sodium taurocholate, and the cholesterol-binding ability of 28 strains of Lactobacillus gasseri were examined. There was significant variation among strains in growth in media containing bile and also variation in the ability to bind cholesterol. Cultures grown for 12 h at 37 degrees C bound significantly more cholesterol than did cells from a 48-h incubation. Variation among strains in the ability to deconjugate sodium taurocholate was not significantly different. Maximal deconjugation of sodium taurocholate was achieved with the cells during the stationary phase of growth (12 h). Statistical analysis showed no significant correlation between bile tolerance and sodium taurocholate deconjugation, bile tolerance and cholesterol-binding ability, or sodium taurocholate deconjugation and cholesterol-binding ability.     

Diversity of bacteriocins produced by lactic acid bacteria isolated from raw milk

Bacteriocin-producing lactic acid bacteria were isolated from 298 samples of raw ewes', goats’ or cows’ milk. Eighty-two bacteriocin producers were phenotypically and genotypically identified as L. lactis subsp. lactis (59 isolates), L. lactis subsp. cremoris (2 isolates), L. lactis subsp. lactis biovar diacetylactis (6 isolates), E. faecalis (7 isolates), E. faecium (1 isolate), L. paracasei subsp. paracasei (4 isolates), L. plantarum (1 isolate) and Leuconostoc spp. (2 isolates). By means of PCR-techniques, nisin was characterized in 39 of the 67 bacteriocin-producing lactococci and lacticin 481 in 23 isolates, some of which presented antilisterial activity. Enterocin AS-48 was produced by four enterococcal isolates. Four non-identified bacteriocins produced by 16 isolates showed a broad inhibitory spectrum. Nisin-producing lactic acid bacteria were the most abundant, but lacticin 481-producing lactococci and AS-48-producing enterococci were found at relatively high rates.     

乳酸菌Ⅱ类细菌素生物合成及其代谢调控策略

乳酸菌细菌素是乳酸菌经由核糖体合成的具有抑菌活性的多肽类物质,其中乳酸菌Ⅱ类细菌素具有很强的热稳定性和抑菌活性,但是产量较低尚不能实现商业化生产。因此,了解和掌握乳酸菌Ⅱ类细菌素合成及其代谢调控机制,对解决商业化生产过程的技术瓶颈具有重要的意义。该文综述了目前Ⅱ类细菌素生物合成和降解途径,并从代谢调控角度提出提高Ⅱ类细菌素产量的可行性策略,为提高Ⅱ类细菌素产量提供新的途径和思路,也为Ⅱ类细菌素工业化生产奠定基础。     

产细菌素乳酸菌在食品中的应用

食品在加工贮藏过程中容易被有害微生物侵染,从而导致其腐败变质。现常用的物理化学防腐保鲜方法虽然可以延长产品的货架期但却存在一定的安全隐患,而且目前消费者更倾向于选择绿色、天然的产品,因此生物抑菌剂的开发显得尤为重要。乳酸菌是公认的食品安全级微生物,是一种天然的防腐剂,安全、无毒、开发性能稳定,被广泛应用于食品中。乳酸菌细菌素是由乳酸菌在生长代谢过程中通过核糖体合成的一类具有抗菌活性的多肽或蛋白质,对一些有害微生物的生长繁殖有明显抑制作用。本文主要介绍了乳酸菌细菌素的分类、抑菌机制及乳酸菌作为天然抑菌剂在乳及乳制品、肉及肉制品、水产品、果蔬类产品及蛋制品中的应用,以期为产抑菌素乳酸菌的综合利用与进一步的研究提供一定的理论依据。     

Description of the bacteriocins produced by two strains of Enterococcus faecium isolated from Italian goat milk

In this study two strains of Enterococcus faecium, M241 and M249, isolated from goat milk, were studied for their capability to produce antibacterial compounds. It was determined that the bacteriocins produced by both strains were active towards Listeria monocytogenes and Clostridium butyricum, and they did not have any activity with respect to other species of lactic acid bacteria. Enterocins A and B were targeted by polymerase chain reaction (PCR) and sequenced, after cloning, in both strains. The bacteriocins contained in the cell free supernatants were stable when subjected to treatments at high and low temperatures or with lipase, catalase and alpha-amylase. Whereas, the activity was lost when proteinases were used. Lastly, a co-culture experiment with L. monocytogenes in skimmed milk was performed. In the presence of the E. faecium strains, the pathogen showed a delay in the growth of about 6h and it reached a maximum counts of about 10(6) colony forming unit, two orders of magnitude low with respect to the control. This result suggests the possibility to use the strains studied as starter cultures to enhance food safety of dairy products.     

山西老陈醋源萎缩芽孢杆菌细菌素对病原菌群体感应的抑制研究

该实验考察了不同理化因子、山西老陈醋源萎缩芽孢杆菌（Bacillus atrophaeus）SAV-CP 297细菌素对5种病原菌（大肠杆菌、金黄色葡萄球菌、沙门氏菌、志贺氏菌、单增李斯特菌）产信号分子二酮哌嗪（DKPs）能力的影响,并将该细菌素应用于草莓防腐中,考察其对群体感应的抑制作用。结果表明,5种病原菌均能产生cyclo-（L-Pro-L-Gly）、cyclo-（L-Pro-L-Leu）和cyclo-（L-Pro-L-Phe）3种类型的DKPs。随NaCl含量增加,DKPs的种类和含量减少;随pH、温度升高,DKPs含量均呈先升高后下降趋势,其种类在pH升高时增加,温度升高时不变。B. atrophaeus SAV-CP 297细菌素可以明显抑制病原菌产DKPs的能力,从而延缓贮藏期间草莓的腐败速率,达到良好的保鲜效果。     

植物乳杆菌JLA-9产细菌素的分离纯化

将来源于东北传统发酵酸菜中的植物乳杆菌JLA-9产的细菌素进行分离纯化,首先利用饱和度为80%的硫酸铵溶液对发酵上清液进行沉淀粗分离。复溶后利用Sephadex LH-20进行凝胶层析纯化,之后采用Hitrap QFF进一步进行离子交换层析纯化,利用反相高效液相色谱(Reversed-Phase High Performance Liquid Chromatography,RP-HPLC)C_(18)柱进行最终纯化,得到单一活性抑菌组分,说明细菌素得到基本纯化,经基质辅助激光解吸电离飞行时间质谱(Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry,MALDI-TOF/MS)确定该细菌素的分子质量约为0.9 kDa左右。采用琼脂扩散法测定了细菌素对常见的食源性致病菌和腐败菌的抑菌效果,结果显示该细菌素具有较好的抑制作用。     

Lactobacillus reuteri LA6 and Lactobacillus gasseri LA39 isolated from faeces of the same human infant produce identical cyclic bacteriocin

Reutericin 6, a bacteriocin produced by Lactobacillus reuteri LA6 that was isolated from the faeces of a human infant at 2 months of age, was purified to homogeneity from broth culture-supernatant by reverse-phase chromatography. Molecular weight (5652) by mass spectrometry and primary structure of reutericin 6 were identical to that of gassericin A produced by Lactobacillus gasseri LA39 which was isolated from the faeces of the same human infant at 4 months old. Reutericin 6 was shown to be a class II and cyclic bacteriocin. PCR amplification on the chromosome DNA of L. reuteri LA6 as a template with the primers based on the DNA sequences cloned (gassericin A and acidocin B fromLactobacillus acidophilus M46) and sequencing revealed that L. reuteri LA6 has the structural gene for gassericin A with no variations among those primers. These results indicate that a bacteriocin of the same structure has been produced by different lactobacilli species isolated from the same infant.     

Chemical and genetic characterization of bacteriocins: antimicrobial peptides for food safety

Antimicrobial peptides are produced across all domains of life. Among these diverse compounds, those produced by bacteria have been most successfully applied as agents of biocontrol in food and agriculture. Bacteriocins are ribosomally synthesized, proteinaceous compounds that inhibit the growth of closely related bacteria. Even within the subcategory of bacteriocins, the peptides vary significantly in terms of the gene cluster responsible for expression, and chemical and structural composition. The polycistronic gene cluster generally includes a structural gene and various combinations of immunity, secretion, and regulatory genes and modifying enzymes. Chemical variation can exist in amino acid identity, chain length, secondary and tertiary structural features, as well as specificity of active sites. This diversity posits bacteriocins as potential antimicrobial agents with a range of functions and applications. Those produced by food‐grade bacteria and applied in normally occurring concentrations can be used as GRAS‐status food additives. However, successful application requires thorough characterization. © 2013 Society of Chemical Industry     

Characterization of two bacteriocins produced by Pediococcus acidilactici isolated from "Alheira", a fermented sausage traditionally produced in Portugal

Lactic acid bacteria were isolated from "Alheira" sausages that have been sampled from different regions in Portugal. The sausages were produced according to different recipes and with traditional starter cultures. Two isolates (HA-6111-2 and HA-5692-3) from different sausages were identified as strains of Pediococcus acidilactici. Each strain produces a bacteriocin, designated as bacHA-6111-2 and bacHA-5692-3. Both bacteriocins are produced at low levels after 18 h of growth in MRS broth (3200 AU/ml against Enterococcus faecium HKLHS and 1600 AU/ml against Listeria innocua N27). BacHA-6111-2 and bacHA-5692-3 are between 3.5 kDa and 6.5 kDa in size, as determined by tricine-SDS-PAGE. Complete inactivation or significant reduction in antimicrobial activity was observed after treatment of cell-free supernatants with proteinase K, pronase and trypsin. No change in activity was recorded when treated with catalase. Both bacteriocins are sensitive to treatment with Triton X-114 and Triton X-100, but resistant to Tween 20, Tween 80, SDS, Oxbile, NaCl, urea and EDTA. The bacteriocins remained stable after 2 h at pH 6.0. A decrease in antibacterial activity was recorded after 60 min at 100 degrees C. After 60 min at 80 degrees C, 60 degrees C and 25 degrees C the antibacterial activity against L. innocua N27 decreased by 25%. Addition of bacHA-6111-2 and bacHA-5692-3 (1600 AU/ml) to a mid-log (5-h-old) culture of L. innocua N27 inhibited growth for 7 h. Addition of the bacteriocins (3200 AU/ml) to a mid-log (5-h-old) culture of E. faecium HKLHS repressed cell growth. The bacteriocins did not adhere to the surface of the producer cells. Both strains contain a 1044 bp DNA fragment corresponding in size to that recorded for pediocin PA-1. Sequencing of the fragments from both bacteriocins revealed homology to large sections of pedA (188 bp), pedB (338 bp) and pedC (524 bp) of pediocin PA-1 and the bacteriocins are considered similar to pediocin PA-1.     

A novel immunostimulating aspect of Lactobacillus gasseri: induction of "Gasserokine" as chemoattractants for macrophages

The chemotactic activity of the culture supernatants from 14 strains of Lactobacillus acidophilus and L. gasseri was examined for murine macrophages. Significant macrophage chemotactic activity was observed in three strains of L. acidophilus and all strains of L. gasseri. The highest activity was observed in the supernatant (1131-sup) from 24-h cultures of L. gasseri JCM1131T. The chemotactic factor from 1131-sup, designated as "Gasserokine", was purified by the C18 reverse phase and ion-exchange chromatography. The purity of Gasserokine was checked by HPLC with the reverse-phase mode. The chemotactic activity of Gasserokine was also observed for human monocytes. The macrophage chemotaxis induced by L. gasseri JCM1131T culture supernatants was discovered to be a new biological function exerted by probiotic lactic acid bacteria. Therefore, the activity is expected to be used for one of the functional parameters in the immunomodulating properties of probiotic lactic acid bacteria.     

Application of reuterin produced by Lactobacillus reuteri 12002 for meat decontamination and preservation

Lactobacillus reuteri strain 12002 was used for reuterin production in the two-step fermentation process. A batch culture fermentation was used to produce a maximum biomass of L. reuteri. Then cells were harvested, resuspended in a glycerol-water solution, and anaerobically incubated to produce reuterin. The lyophilized supernatants (approximately 4000 activity units (AU) of reuterin per ml) were diluted in distilled water for decontamination and preservation trials. The MIC values of reuterin for Escherichia coli O157:H7 and Listeria monocytogenes were 4 and 8 AU/ml, respectively. In meat decontamination experiments, the surface of cooked pork was inoculated with either L. monocytogenes or E. coli O157:H7 at a level of approximately log10 5 CFU/cm2, incubated for 30 min at 7 degrees C, and decontaminated by exposure to reuterin (500 AU/ml). The bactericidal effect of reuterin was analyzed 15 s and 24 h after exposure at 7 degrees C. After 15 s of exposure to reuterin, viable numbers decreased by 0.45 and 0.3 log10 CFU/cm2 for E. coli O157:H7 and L. monocytogenes, respectively. After 24 h the numbers decreased by 2.7 log10 CFU/cm2 for E. coli O157:H7 and by 0.63 log10 CFU/cm2 for L. monocytogenes. In the same experiment, the combined effect of reuterin and lactic acid was also investigated. Adding lactic acid (5%, vol/vol) to reuterin significantly enhanced (P < or = 0.05) the efficacy of reuterin. No additional effect (P < or = 0.05) was found when ethanol (40%) was added to the mixture of reuterin and lactic acid. To evaluate the preservative effect of reuterin during meat storage, reuterin was added to raw ground pork contaminated with E. coli O157:H7 or L. monocytogenes. Reuterin at a concentration of 100 AU/g resulted in a 5.0-log10 reduction of the viability of E. coli O157:H7 after 1 day of storage at 7 degrees C. Reuterin at a concentration of 250 AU/g reduced the number of the viable cells of L. monocytogenes by log10 3.0 cycles after 1 week of storage at 7 degrees C.     

嗜酸乳杆菌WS所产细菌素生物学特性的研究

LactobacliiusacidophilusWS在MRS培养基中经过37℃培养得到的细菌素经受70℃、121℃处理20min后,活性几乎不变,对以金黄色葡萄球菌为代表的革兰氏阳性菌有明显的抑制作用,而对大肠杆菌为代表的革兰氏阴性菌的抑制作用不明显,说明该细菌素是一种广谱抗菌的细菌素。细菌素对木瓜蛋白酶(papain)、蛋白酶K(proteinaseK)以及胰蛋白酶(trypsin)敏感,而对中性蛋白酶(subtilisin)和胃蛋白酶(pepsin)的敏感性差。在一定的范围内,细菌素的抑菌活性和环境的酸度呈正相关。