Characterization of a two-peptide plantaricin produced by Lactobacillus plantarum MBSa4 isolated from Brazilian salami

The aim of this study was to explore the biochemical and genetic features of the two-peptide bacteriocin produced by a Lactobacillus plantarum strain isolated from Italian type salami produced in Brazil (Lb. plantarum MBSa4). Identification of bacteriocinogenic Lb. plantarum MBSa4 was performed by 16S rRNA sequencing. Expressed bacteriocin was evaluated for spectrum of activity, heat and pH stability, mechanism of action, and molecular mass. Partial purification was achieved by cation-exchange, and reversed phase - HPLC. Total DNA of Lb. plantarum MBSa4 was extracted and tested for presence of previously described bacteriocin genes. Bacteriocin MBSa4 was heat-stable, unaffected by pH 2.0 to 6.0 and active against all tested Listeria monocytogenes strains and most of tested fungi. Maximal production (1600 AU/ml) in MRS broth occurred after 22 h at 25 °C, presenting bacteriostatic activity as result of combined action of two components. The molecular mass determined by SDS-PAGE was 2.3 kDa. PCR-amplified DNA indicated the same nucleotide sequence of plantaricin W. Results indicate that Lb. plantarum MBSa4 produces plantaricin W, a two-peptide lantibiotic with remarkable anti-Listeria activity.     

Antimicrobial Effect of Bacteriocin KU24 Produced by Lactococcus lactis KU24 against Methicillin‐Resistant Staphylococcus aureus

Bacteriocin KU24 produced by Lactococcus lactis KU24 exhibited an inhibitory effect against methicillin‐resistant Staphylococcus aureus (MRSA). Bacteriocin KU24 was inactivated by protease XIV, showing that it has a proteinaceous nature on S. aureus ATCC 33591. Also, bacteriocin KU24 exhibited a strong heat stability (121 °C for 15 min) and pH stability (pH 3 to 9). The mode of inhibition was determined for S. aureus ATCC 33591 by treatment of 0, 250, and 500 AU/mL of bacteriocin KU24. S. aureus ATCC 33591 was inhibited by added bacteriocin KU24, while control was increased. The cell membranes of S. aureus ATCC 33591 were damaged with treatment of 500 AU/mL of bacteriocin KU24. Also, bacteriocin KU24 inhibited the occurrence of mecA gene, the methicillin resistance gene in S. aureus ATCC 33591. Bacteriocin KU24 was purified by C₁₈ Sep‐Pack column, cation exchange chromatography, and gel filtration chromatography, and molecular mass is approximately 6.5 kDa by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. These results demonstrate that bacteriocin KU24 can be used as an alternative antimicrobial agent for the treatment of infection of MRSA in the food industry.     

细菌素在食品贮藏保鲜中的应用

由微生物污染所引起的食品腐败变质问题是食品工业始终面临的巨大挑战之一。而传统的理化保鲜技术一方面会导致营养成分流失,另一方面对人体健康会造成潜在的威胁。人们一直在寻找一种安全、高效、天然的食品保鲜剂。现代生物技术的发展使得微生物保鲜剂逐渐进入人们的视野并不断地被开发、研究和应用。细菌素是由某些细菌分泌的一种具有抑菌活性的代谢产物。它无毒无抗性,能够在杀死微生物的同时最大程度地保留食品的鲜度和营养价值,可以替代化学防腐剂应用于食品保鲜,作为天然微生物源保鲜剂的一种,近些年受到了广泛关注。因此,本文简要介绍了细菌素的分类、特点,概述了各种类型的细菌素在不同种类食品保鲜方面的应用和研究进展,总结了在保鲜领域中存在的问题和挑战,并对其未来发展趋势提出展望,为细菌素更好地应用于食品工业提供一定的理论依据。     

细菌素的生物学特性及作为防腐剂在熟肉制品中的应用

本对选育出的三株具有广谱、高效抑菌作用乳酸菌所产细菌素的理化特性以及作为防腐剂在肉品中的应用进行了研究。结果表明：三株产细菌素菌株产生的细菌素均表现出良好的热稳定性，在80℃15min的热处理条件下活性基本不变；能被胃蛋白酶、胰蛋白酶、蛋白酶K、木瓜蛋白酶、中性蛋白酶降解而完全失活，表明该细菌素是一种蛋白类物质；对酸具有较好的稳定性；具有较强的防腐能力。     

Nisin‐producing microorganisms and their implementation in brewers' wort

Nisin is a bacteriocin, which is capable of eliminating more than 90% of all potential beer spoilage Gram‐positive bacteria. Hence, the implementation of nisin‐producing cultures into the brewing process needs to be evaluated systematically. In this work, the genetic relationships and properties, as well as the reactions of four strains of Lactococcus lactis ssp. lactis, known to be capable of producing nisin (NCIMB 8780, 8586, 701402 and 701403), and seven isolated strains of the same species found in the beverage environment (TUM 575, 8947, 8127, 8446, 8673, 8973 and 8872), were tested under typical brewery conditions. As in previous work, it was found, that all of the tested strains could be genetically differentiated via PCR‐(GTG)₅. In addition, the absence of the genes HorA, HorC and ORF5 indicated that all strains were sensitive to hop components. The agar diffusion assay test proved to be the most reliable method to precisely determine different nisin concentrations. Nisin formation, acid formation and the reproduction rates of the organisms were tested subsequently in various brewery relevant culture media such as MRS, first wort (unhopped) and wort (hopped). MRS provided the best environment for bacterial growth and hence acid and nisin production. The four NCIMB strains, which were the only ones capable of producing nisin under the named conditions, were chosen for study with regards to their tolerance to specific compounds found in the brewery environment. The strains NCIMB 8780 and 8586 produced comparatively higher amounts of nisin and were more tolerant to hop bitterness, ethanol, high gravity and low extract conditions. The growth rates, acid production and nisin production of all strains decreased with increasing bitter units and ethanol content. The optimum extract concentration was 5–10°P. Copyright © 2015 The Institute of Brewing & Distilling     

乳酸菌细菌素Durancin GL对冷鲜鸡肉中单增李斯特菌抑制效果研究

以乳酸菌细菌素Durancin GL处理冷鲜鸡肉,进行储藏试验。从感官评定、品质分析以及有害微生物抑制情况考察该细菌素对冷鲜鸡肉的防腐保鲜效果。试验结果显示:200AU/mL的细菌素保鲜液处理冷鲜鸡肉,可抑制单增李斯特菌的生长,减缓挥发性盐基氮(TVB-N)含量增加以及pH上升,减缓肉质劣变;在4℃储藏条件下,一级冷鲜肉存储期由4d延长至8d。表明该乳酸菌细菌素对冷鲜鸡肉延长储藏期具有积极效果。     

Development of a New Paenibacillin‐Producing Strain and Testing its Usability in Improving Food Safety

A new bacterial strain that produces a bacteriocin (paenibacillin) without polymyxin was developed from Paenibacillus polymyxa that co‐produces the 2 antimicrobial agents. Gamma radiation was used successfully to develop the new strain, P. polymyxa OSY‐HG. Subsequently, we explored the feasibility of using food or food ingredients as growth media for the new strain. Milk supported the growth of P. polymyxa OSY‐HG which produced up to 32 mg paenibacillin/L milk without polymyxin. Fermentation crude extract was applied in a model food (Vienna sausage) to control Listeria innocua, a Listeria monocytogenes surrogate. The treatment increased Listeria lag time by 2 d at 7 °C and at least 6 h at 37 °C. In conclusion, a new paenibacillin‐producing P. polymyxa strain has been developed for potential industrial use. Using the new strain in applications that enhance food safety is feasible.     

Short communication: Naturally sensitive Bacillus thuringiensis EG10368 produces thurincin H and acquires immunity after heterologous expression of the one-step-amplified thurincin H gene cluster

Heterologous expression of bacteriocin genetic determinants (or operons) has long been a research interest for the functional analysis of genes involved in bacteriocin biosynthesis, regulation, modification, and immunity. Previously, construction of genomic libraries of the bacteriocin producer strains was usually required to identify new bacteriocin operons, a method that is tedious and time consuming. For the first time, we directly amplified an 8.14-kb bioinformatically identified thurincin H gene cluster using a one-step PCR method with 100% accuracy. This amplified gene cluster was cloned into plasmid pHT315, resulting in plasmid pGW139, and subsequently transformed to Bacillus thuringiensis EG10368, a strain naturally sensitive to thurincin H. Heterologous expression of the gene cluster makes the sensitive B. thuringiensis EG10368 produce thurincin H at a higher level compared with the wild-type producer, B. thuringiensis SF361. Moreover, B. thuringiensis EG10368pGW139 acquired complete immunity to thurincin H. The results indicated that one-step PCR is a promising tool to accurately amplify long bacteriocin gene clusters used in bacteriocin functional analysis studies and it is an effective way to produce bacteriocins at a higher level, without the need to clone large chromosomal fragments.     

植物乳杆菌CGMCC.5297产细菌素条件的优化及应用

用响应面法对植物乳杆菌CGMCC.5297生产细菌素的培养基进行了优化。通过Plackett-Burman设计和中心组合试验设计,植物乳杆菌CGMCC.5297代谢产细菌素的最佳培养条件为酵母粉5.09g/L,牛肉膏10.85g/L,葡萄糖55.34g/L。此时的细菌素上清与指示菌单核细胞增多性李斯特菌CVCC1595共培养4h后,指示菌OD600nm为0.001 73,效价为4499 IU/mL,提高了1.4倍。在最优发酵条件下获得的试验结果与模型预测值吻合,说明所建立的模型是切实可行的。将优化后的植物乳杆菌上清加入到自制酸奶中,发现此细菌素对酸奶具有良好的保鲜效果。