The control of Listeria innocua and Lactobacillus sakei in broth and meat slurry with the bacteriocinogenic strain Lactobacillus casei CRL705

The effect of the bacteriocin-producing strain, Lactobacillus casei CRL705, in the control of Listeria innocua 7 and Lactobacillus sakei CRL1424 in MRS medium and meat slurry during the storage under vacuum at chill temperatures was evaluated. L. sakei CRL 1424 isolated from vacuum-packaged contaminated raw meat was identified as the predominant indigenous lactic acid bacterial flora. Co-inoculation of MRS broth at 8°C with L. casei CRL705 caused growth inhibition of L. sakei CRL1424 and L. innocua 7 after 10 and 4 days of storage, respectively. At 4°C the complete inhibition of both strains occurred within 14 and 8 days for L. sakei CRL1424 and L. innocua 7, respectively. Bacteriocin activity in broth was observed to be maximal at 8°C reaching 2130 AU ml⁻¹ after10 days and 400 AU m1⁻¹ after 8 days of storage, while at 4°C maximal activities, 690 and 30 AU ml⁻¹ were obtained at 14 and 10 days, respectively. Addition of bacteriocinogenic strain to the meat slurry did not allow the growth of L. sakei and L. innocua, showing a bacteriostatic effect during 21 days of storage at 4°C. In addition, L. casei CRL705, as a protective culture did not change significantly the pH of the meat slurry in the assayed storage conditions. The results presented here confirm that the bacteriocinogenic strain L. casei CRL705 can be used as a useful tool to improve the microbial stability and safety in the commercial meat preservation.     

Characterization of a multilayer film activated with Lactobacillus curvatus CRL705 bacteriocins

BACKGROUND: Bacteriocins produced by lactic acid bacteria offer enormous promise for food safety preservation. In this study an active multilayer film obtained by the incorporation of lactocin 705 and lactocin AL705, two bacteriocins produced by Lactobacillus curvatus CRL705 with antimicrobial activity against Lactobacillus plantarum CRL691 and Listeria innocua 7, respectively, was characterized for its potential application in active packaging technology. Film activity performance at different storage conditions, bacteriocins transfer into water and sunflower oil, and film surface properties were evaluated. RESULTS: Film activity against L. innocua 7 was maintained during 2, 4 and 6 weeks at 30, 10 and 5 °C respectively. At 30 and 10 °C, activity loss against L. plantarum CRL691 was observed on the second week of storage and after the fourth week at 5 °C. Results showed no significant difference for active multilayer film contact angle and seal properties compared to the control (without bacteriocins). A decrease in lactocin 705 inhibitory activity after sunflower oil contact was observed, while lactocin AL705 remained unaffected. After water contact, film activity was retained for both bacteriocins. CONCLUSIONS: As demonstrated by antimicrobial activity and physico‐mechanical properties retention, lactocin 705 and AL705 active multilayer film present potential for application in active packaging technology. Copyright © 2011 Society of Chemical Industry     

Lactobacillus casei CRL 431 and Lactobacillus rhamnosus CRL 1224 as biological controls for Aspergillus flavus strains

The effect of two species of lactobacilli, Lactobacillus casei CRL 431 and Lactobacillus rhamnosus CRL 1224, on growth of different Aspergillus flavus strains was determined. A. flavus strains (Ap, TR2, or CF80) were grown in LAPTg broth at 37 degrees C for 7 days as a single culture and in association with L. casei CRL 431 or L. rhamnosus CRL 1224 at initial inoculum ratios of 1:1, 1:10, and 1:100. In most cases, the mixed cultures had a lower fungal growth and a lower pH than the control cultures. Mycelial dry weight was reduced to 73 and 85% using L. casei CRL 431 and L. rhamnosus CRL 1224, respectively. The pH decrease in mixed cultures when the fungal mycelial dry weight is reduced may play an important role in inhibition. The number of viable bacteria was variably affected by fungal growth. These results indicate that L. casei CRL 431 and L. rhamnosus CRL 1224 may be useful as potential biocontrol agent against A. flavus.     

Development and characterization of an active polyethylene film containing Lactobacillus curvatus CRL705 bacteriocins

The development and characterization of a bacteriocin-containing polyethylene-based film is described, incorporating lactocin 705 and lactocin AL705, produced by Lactobacillus curvatus CRL705, and nisin. Three different procedures to obtain lactocin 705 and AL705 solution were evaluated, with the partially purified aqueous bacteriocin solution showing the highest inhibitory activity against indicator strains (Lactobacillus plantarum CRL691 and Listeria innocua 7). Pouch contact, soaking and a contact method were compared for incorporating bacteriocins onto PE-based films. Contact between the PE film and bacteriocin solution was the most effective, resulting in a more uniform distribution of bacteriocins on the film surface and using less active solution. The minimal inhibitory concentration of bacteriocin solution was 267 AU cm^?3 (lactocin 705) and 2133 AU cm^?3 (lactocin AL705), while the minimal contact time was 1 h. When relative inhibition area for antilisterial activity of the active films was compared, those treated with L. curvatus CRL705 bacteriocins displayed higher inhibitory activity than nisin-treated films. Functional properties of active PE-films containing lactocin 705 and AL705 showed no differences compared with non-active control films. Bacteriocin-active PE-based films are shown to be highly effective in inhibiting growth of Listeria. The potential use of commercially available packaging films as bacteriocins carriers may benefit active-packaging systems.     

Study of adhesion of Lactobacillus casei CRL 431 to ileal intestinal cells of mice

It is well known that the cell wall of Lactobacillus casei CRL 431, a strain present in probiotics, presents lectinlike surface molecules. Presence of these molecules stimulates the immune system. Given the role that lectins and lectinlike substances play in the adhesion phenomenon, it is probable that this is an initial stage in the immunostimulation produced by this bacterium. To confirm this, adhesion of this microorganism to exfoliated mouse ileal epithelial cells was studied in vitro. Other L. casei strains isolated from adult human intestines and one of dairy origin were also examined for their ability to adhere to ileal epithelial cells. Another strain, which was included in the present study, was Lactobacillus acidophilus CRL 730. L. casei strains isolated from humans showed good ability to adhere to ileal epithelial cells, whereas L. casei isolated from dairy origin did not. Adhesion was only observed at 37 degrees C and at a pH between 6 and 7.5. The exposure time needed for highest adhesion was 30 min. Presence of lectinlike substances on the surface of L. casei CRL 431 is important to this adhesion phenomenon, since adherence capacity was lost after removal of these substances.     

干酪乳杆菌细菌素的抗菌机制分析

目的：分析和预测干酪乳杆菌细菌素基因及其编码蛋白的分子结构和理化性质,明确细菌素的抗菌机制。方法：利用生物信息学软件进行细菌素编码蛋白的结构预测和功能分析,采用氨基酸定点突变技术对预测的氨基酸位点进行点突变,检测突变体的抗菌活性。结果：干酪乳杆菌细菌素（LacA和LacB）属于热稳定、分泌信号肽的跨膜蛋白,LacA蛋白存在典型的抗菌结构域GxxxG,构建LacA蛋白中第40位V和第57位I的突变载体,两个位点中任何一个发生突变都明显影响细菌素的抗菌活性。结论：推测V40和I57是干酪乳杆菌细菌素发挥抑菌活性的关键氨基酸位点,为今后探索干酪乳杆菌细菌素（class IIb）抗菌机制提供理论依据。     

Development of an active wheat gluten film with Lactobacillus curvatus CRL705 bacteriocins and a study of its antimicrobial performance during ageing

Antimicrobial wheat gluten film was obtained at pilot scale by Lactobacillus curvatus CRL705 bacteriocins inclusion in the film-forming solution. Bacteriocins’ minimum inhibitory concentration for the film activation was 2133 AU cm^?3 (lactocin AL705) and 267 AU cm^?3 (lactocin 705). Mechanical and barrier properties as well as film ageing kinetics were not significantly affected by the addition of bacteriocins. The antimicrobial film performance during ageing was assessed. Film activity against Listeria innocua 7 and Lactobacillus plantarum CRL691 was observed over 50 days of ageing. Even when the release of bacteriocins from the film upon water contact was observed for both bacteriocins at the beginning of the ageing period, and anti-Listeria activity was delivered to the simulant up to the 15th day of ageing, film residual activity for both bacteriocins was observed over 50 days. The results confirm the potential of a gluten film doped with L. curvatus CRL705 bacteriocins as a carrier of bacteriocins to avoid Listeria and lactic acid bacterial growth, thus enhancing quality and safety in foods.     

Mode of action, purification and amino acid sequence of plantaricin C19, an anti-Listeria bacteriocin produced by Lactobacillus plantarum C19.

Plantaricin C19, an anti-Listeria bacteriocin, was successfully purified by adsorption to and release from producing cells at low pH combined with reverse phase high-performance liquid chromatography (HPLC). The purification resulted in a 900-fold increase in specific activity with a yield of 15% of the original activity. Mass spectrometry analysis gave a molecular weight of 3845.3. Protein microsequencing identified 36 amino acids. Plantaricin C19 is rich in both hydrophobic and basic amino acids in good accordance with its basic and hydrophobic character. Comparison of the amino acid sequence of plantaricin C19, with the sequence of some other anti-Listeria bacteriocins produced with lactic acid bacteria, revealed that plantaricin C19 has in its N-terminal region the consensus sequence--YYGNGL--(uniquely with Valine instead of Leucine as found in all other bacteriocins), identifying plantaricin C19 as a pediocin-like bacteriocin. Plantaricin C19 exerted a bacteriostatic action on sensitive cells of Listeria grayi IP 6818 in BHI broth. No loss of intracellular K+, Mg2+ or UV-absorbing materials was observed. Adsorption of plantaricin C19 on L. grayi CIP 6818 decreased in the presence of salts.     

Effects of curing additives on the control ofListeria monocytogenesby lactocin 705 in meat slurry

Meat slurry inoculated withListeria monocytogenes(4.00 cfu g⁻¹) was mixed with different levels of curing additives and their influence on the inhibitory effect of lactocin 705 (17,000 AU ml⁻¹) was evaluated at 20°C. Inhibition ofL. monocytogeneswas 1.90 and 1.00 log less in meat slurry with 5 and 7% NaCl than in meat slurry without added sodium chloride. When nitrite and bacteriocin were added together, less nitrite (200 μg g⁻¹) was required to obtain the sameListeriapopulation (3.00 log cfu g⁻¹) as when 800 μg g⁻¹NaNO₂was used. However, when compared with lactocin 705 alone, lessListeriainhibition was observed showing also a protective effect of NaNO₂. When ascorbic acid and alginate meat binder were assayed in the presence of the bacteriocin, the inhibition ofL. monocytogeneswas less effective, but when sodium lactate (2%) was added to the meat slurry, almost no protective effect was observed. These results indicated that the use of lactocin 705 to controlL. monocytogeneswas less effective in the presence of curing ingredients.     

Purification and characterization of intracellular proteinase from Lactobacillus casei ssp. casei LLG

The intracellular proteinase of Lactobacillus casei ssp. casei LLG was isolated in the cytoplasmic fraction with 0.05 M Tris-HCl buffer (pH 7.5). The enzyme was purified by the fast protein liquid chromatography system equipped with ion-exchange and gel filtration chromatographies. This proteinase comprised a single monomeric form and had a molecular weight of about 55 kDa and an isoelectric point near pH 4.9. The optimum pH and temperature for the enzyme activity were determined to be pH 6.5 and 37 degrees C, respectively. The enzyme was inactivated by metal-chelating compounds (EDTA, 1,10-phenanthroline) and less affected by serine proteinase inhibitors (diisopropylfluorophosphate, phenylmethylsulfonyl fluoride). Proteinase activity was increased by Ca++, Mn++, and Co++, and inhibited by Cu++, Mg++, and Zn++. The activity of this enzyme to hydrolyze casein appeared to be more active on beta-casein than alphas1-casein and kappa-casein as monitored by polyacrylamide gel electrophoresis.     

植物乳杆菌(Lactobacillus plantarum)CW5的筛选及细菌素抑菌活性的研究

为了提高抑菌活性,对植物乳杆菌(Lactobacillus plantarum)CW5产细菌素的发酵条件进行了优化,分别研究了培养时间、温度、接种量、培养基起始pH、培养基碳源、氮源等因素对细菌素产生的影响,通过单因素水平实验和正交实验,确定产细菌素的最佳培养基组合和最佳发酵条件为:葡萄糖3%,胰蛋白胨1.5%,蛋白胨1.5%,酵母膏1%,硫酸镁0.058%,吐温80 0.2%,30℃培养24 h,培养基起始pH为6.5,接种量2%。CW5在优化前效价为367.82 IU/mL,优化后效价为1619.85 IU/mL,提高了340.39%。     

西藏干酪乳酸菌降胆固醇特性研究

从西藏传统发酵乳(西藏灵菇)中分离筛选到1株高效降胆固醇乳酸菌KM-16。应用VITEK-32全自动微生物分析系统鉴定该菌为干酪乳杆菌(Lactobacillus casei)。在液体培养基中KM-16乳酸菌对降胆固醇达51.8%。用KM-166灌胃高脂血症模型小鼠研究其对血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白(HDL-C)的影响。结果表明灌胃14d后,实验组小鼠血清中TC、TG浓度较对照组显著降低(p<0.01),同时HDL-C浓度有所增加,动脉硬化指数(AI)低于对照的水平。     

Production and evaluation of a probiotic yogurt using Lactobacillus casei ssp. casei

Skimmed milk was inoculated with the commercial starter and Lactobacillus casei ssp. casei. pH changes, viable counts, and organoleptic properties of the produced control and probiotic yogurts were analysed. The pH decrease during the fermentation period was faster in the milk inoculated with L. casei plus starter. The growth of both starters in probiotic yogurt was significantly lower than their growth in control yogurt during the fermentation period. The viable count of the probiotic bacterium remained higher than the standard limit for probiotic products. There was no significant difference between the organoleptic properties of the control and the probiotic yogurts.     

Protective action of Lactobacillus curvatus CRL705 on vacuum-packaged raw beef. Effect on sensory and structural characteristics

Lactobacillus curvatus CRL705 was examined for its effectiveness as protective culture in the biopreservation of vacuum-packaged fresh beef stored during 60 days at 2 °C. For this purpose, L. curvatus CRL705, producer of lactocin 705 and lactocin AL705, was inoculated on the meat surface (10⁶ cfu g⁻¹). This microorganism became the dominating population throughout the storage period controlling the growth of Brochothrix thermosphacta and spoilage lactic acid bacteria naturally present on the meat. When the microstructural characteristics of the meat were evaluated using light microscopy, beef samples inoculated with the bioprotective culture showed a 10 days delay for the appearance of tissue degradation signs. Sensory analysis demonstrated that beef samples treated with L. curvatus CRL705 only developed an “acid” off-flavor after 60 days of refrigerated storage, and no undesirable off-odors were found. Therefore, inoculation with this bacteriocinogenic strain would provide an additional hurdle to improve storage life of refrigerated vacuum-packaged beef without affecting its sensory and structural characteristics.     

益生乳酸菌Lactobacillus casei Zhang从基础研究到产业化开发

2001年-2009年,内蒙古农业大学乳品生物技术与工程教育部重点实验室从采集自中国西部7个省市及蒙古国14个省市不同少数民族地区951份自然发酵乳制品等样品中分离鉴定出3 388株乳酸菌,建立了中国首个原创性乳酸菌菌种资源库;从菌种库中分离自传统发酵酸马奶(koumiss)的243株乳杆菌中筛选获得1株性能优良的乳酸菌—Lactobacillus casei Zhang(L.casei Zhang)。采用5种动物模型和人体试验进行了功能评价,并利用基因组学和蛋白质组学研究技术对L.casei Zhang益生分子机制进行了深入剖析。经过多年系统的基础研究,自主解决了其产业化的关键技术问题,包括其直投式发酵剂、发酵乳制品和发酵豆乳益生菌饮料的研发并实现了产业化。     

Intracellular esterase from Lactobacillus casei LILA: nucleotide sequencing,purification, and characterization

An esterase gene (estC) was isolated from a genomic library of Lactobacillus casei LILA. The estC gene consisted of a 777 bp open reading frame encoding a putative peptide of 28.9 kDa. A recombinant EstC fusion protein containing a C-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of EstC revealed that it was a serine-dependent dimeric enzyme. Optimum temperature, NaCl concentration, and pH for EstC were determined to be 30 degrees C, 0% NaCl, and pH 5.5, respectively. EstC had significant activity under conditions simulating those of ripening cheese (10 degrees C, 4% NaCl, and pH 5.1). Kinetic constants (KM and Vmax) were determined for EstC action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, the previously studied EstA from Lactococcus lactis MG1363 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstC and EstA.     

干酪乳杆菌6033蛋白酶降解肌肉蛋白质的作用研究

本试验将干酪乳杆菌(Lactobacillus casei，Lc)6033蛋白酶液按5％的比例分别加入肌浆蛋白和肌原纤维蛋白提取液，然后设定不同的pH值，于15℃培养7d，定时取样，测定反应后的pH值、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测蛋白质的变化。结果发现：不同设定的肌浆蛋白和肌原纤维蛋白试验组在反应初期pH值均稍稍降低，随着反应进程，各试验组的pH值呈现出逐步升高的趋势；其中，肌浆蛋白以pH5．0、4．5组的变化较为明显，肌原纤维蛋白则以pH6．0、5．0、4．5组的变化较为明显。经7d振荡培养后，电泳检测发现，各试验组肌浆蛋白均表现出分解现象，尤其以pH5．0组和pH4．5组更为明显；而肌原纤维蛋白也都表现出了明显的分解现象，尤以pH5．0组肌原纤维蛋白分解最明显。     

Oat-based Symbiotic Beverage Fermented by Lactobacillus plantarum, Lactobacillus paracasei ssp. casei, and Lactobacillus acidophilus

ABSTRACT: Oats and probiotics have long been recognized for their health benefits. The objectives of this study were (1) to study the ability of Lactobacillus plantarum (B-28), Lactobacillus paracasei ssp. casei (B-29) isolated from a traditional Bulgarian cereal-based fermented beverage, and Lactobacillus acidophilus from Chr. Hansen, Milwaukee, Wis., U.S.A., to remove cholesterol from the media and to adhere to the Caco-2 cell line, (2) to optimize the fermentation conditions to develop a beverage using these probiotics and oats with acceptable sensory and nutritional qualities, and (3) to assess the quality and shelf-life of this beverage and survivability of probiotics in the beverage. Lactobacillus acidophilus , B-28, and B-29 were found to remove 70.67%± 2.35%, 20.26%± 2.63%, and 16.75%± 3.83% of cholesterol from media and the percentage of adhesion was 4.69%± 0.78%, 1.92%± 0.78%, and 8.36%± 0.78%, respectively. The blend of oat flour, sugar, inulin, and whey protein concentrate was cooked in water and fermented for 12 h at 37°C by 2 ± 10⁶ colony-forming units (CFU) /mL each of B-28 and B-29 and 2 ± 10⁸ CFU/mL of L. acidophilus . The beverage had 0.87%± 0.03% of dietary fiber and had better sensory qualities compared with the commercially available similar product. The probiotics survived for 10 wk of storage at 4°C, except for L. acidophilus , which survived for about 4 wk. The population of B-28 was 1.77 ± 10⁶ to 1.29 ± 10⁷ CFU/mL and that of B-29 was 7.39 ± 10⁷ to 4.49 ± 10⁸ CFU/mL throughout the storage period. The oat-based symbiotic beverage is a functional drink providing both probiotics and prebiotics at the same time.     

干酪乳杆菌蛋白酶的性质研究

干酪乳杆菌(Lactobacilluscasei,Lc)6033经增菌培养、超声波破碎、高速冷冻离心后获得粗酶液,并经70%～80%饱和度范围的硫酸铵分级沉淀初步提纯,酶活力回收率(OD275)为9.71%,酶液最适pH为6.5左右、最适温度为40℃,Co2 、Mn2 能显著增强酶液活力;Cu2 、Fe3 、乙二胺四乙酸(EDTA)和苯甲基磺酰氟(PMSF)对酶液有较强的抑制作用;而Zn2 、碘乙酸和β-巯基乙醇的抑制作用较轻微。可见,酶液中可能含金属酶、丝氨酸酶及巯基酶等多种蛋白酶。