Isolation of nisin-producing Lactococcus lactis WNC 20 strain from nham,a traditional Thai fermented sausage

A total of 14,020 lactic acid bacteria (LAB) were isolated from nham and screened for bacteriocin production. One Lactococcus lactis strain WNC 20 produced a bacteriocin that not only inhibited closely related LAB, but also some food-borne pathogens including Listeria monocytogenes, Clostridium perfringens, Bacillus cereus and Staphylococcus aureus. Biochemical studies revealed that the bacteriocin was heat-stable even at autoclaving temperature (121 degrees C for 15 min) and was active over a wide pH range (2-10). The bacteriocin was inactivated by alpha-chymotrypsin and proteinase K but not other proteases. The antimicrobial spectrum and some characteristics of this bacteriocin were nearly identical to that of nisin. The gene encoding this bacteriocin was amplified by polymerase chain reaction (PCR) with nisin gene-specific primer. Sequencing of this gene showed identical sequences to nisin Z as indicated by the substitution of asparagine residue instead of histidine at position 27. The ability of the bacteriocin produced by Lc. lactis WNC 20 may be useful in improving the food safety of the fermented product.     

Efficacy of nisin in combination with protective cultures against Listeria monocytogenes Scott A in tofu.

Nisin can be used as a biopreservative to control growth of Listeria monocytogenes in various minimally processed foods. Tofu is an example of a non-fermented soybean product, which may allow growth of Listeria at refrigeration temperatures and in which nisin may be applied to prevent multiplication of Listeria. The efficacy of nisin against Listeria may be compromised by the emergence of spontaneous nisin-resistant mutants. Exposure of L. monocytogenes Scott A to nisin in a culture medium or in a food product results in an initial reduction of Listeria population which is followed by regrowth of survivors to nisin during further incubation. In vitro studies using Standard I Nutrient broth showed that Enterococcus faecium BFE 900-6a and Lactobacillus sakei Lb 706-1a used as protective cultures in combination with nisin were able to suppress proliferation of Listeria cells not killed by nisin at 10 degrees C. Growth and bacteriocin production of these two strains and a third protective culture, Lactococcus lactis BFE 902 was also observed in soymilk and tofu at 10 degrees C. Inoculation studies with tofu prepared with nisin and protective cultures showed that lower amounts of nisin are required for an effective inhibition of L. monocytogenes Scott A when either E. faecium BFE 900-6a or Lc. lactis BFE 902 are used in addition. The combination of nisin with these bacteriocinogenic lactic acid bacteria (LAB) resulted in a complete suppression of listerial growth in homemade tofu stored at 10 degrees C for 1 week. Lb. sakei Lb 706-1a was less effective and did not prevent a slight increase of L. monocytogenes Scott A numbers during storage.     

Technological characterization of bacteriocin producing Lactococcus lactis strains employed to control Listeria monocytogenes in cottage cheese

In recent years, there has been a particular focus on the application of antimicrobial compounds produced by lactic acid bacteria (LAB) as natural preservatives to control the growth of spoilage and pathogenic bacteria in food. Bacteriocins are antimicrobial peptides which can be added to foods in concentrated forms as food preservatives, e.g. additives, or they can be produced in situ by starters or protective cultures. In this study, twenty Lactococcus lactis bacteriocin producers previously isolated from Italian fermented foods were subjected to a variety of physical and biochemical tests in order to identify those with the greatest potential as starter cultures in cheese production. Of these, four strains isolated from cheese (one nisin Z producer, one nisin A producer and two lacticin 481 producers) which fulfilled the desired technological criteria were assessed for their ability to control Listeria monocytogenes. The subsequent application of these bacteriocinogenic strains as starter cultures in Cottage cheese established that the nisin A producing Lact. lactis 40FEL3, and to a lesser extent the lacticin 481 producers 32FL1 and 32FL3, successfully controlled the growth of the pathogen. This is the first study to directly compare the ability of nisin A, nisin Z and lacticin 481 producing strains to control listerial growth during the manufacture and storage of Cottage cheese.     

抗食源性病原菌细菌素的筛选及特性研究

目的 筛选一种抗食源性病原菌的细菌素,并对其稳定性进行研究.方法 以食源性病原菌Bacillus cereus ATCC 14579、Listeria monocytogenes LM201、Listeria monocytogenes LM605为指示菌,采用琼脂扩散法筛选细菌素产生菌,通过离子交换树脂法和高效液相色...     

An α-amylase sensitive bacteriocin of Leuconostoc carnosum

Leuconostoc carnosum LA54A isolated from processed meat produces an antimicrobial substance that inhibits the growth of strains of lactic acid bacteria, Listeria monocytogenes and Enterococcus faecalis. The antibacterial substance was sensitive to proteases suggesting that Lc. carnosum is producing a bacteriocin. The bacteriocin was in addition sensitive against the activity of different α-amylases indicating that the molecule consists of a protein and a carbohydrate moiety. The bacteriocin was active over a wide pH range with an optimum between pH 3 and 5. It was stable after heat treatment at 100°C for 15 min. Optimum production of the bacteriocin by Lc. carnosum was during the late logarithmic phase. The purified bacteriocin showed a molecular weight of about 4000 Da when analyzed by SDS-polyacrylamide gel electrophoresis.     

Preservation and fermentation: past,present and future

Preservation of food and beverages resulting from fermentation has been an effective form of extending the shelf-life of foods for millennia. Traditionally, foods were preserved through naturally occurring fermentations, however, modern large scale production generally now exploits the use of defined strain starter systems to ensure consistency and quality in the final product. This review will mainly focus on the use of lactic acid bacteria (LAB) for food improvement, given their extensive application in a wide range of fermented foods. These microorganisms can produce a wide variety of antagonistic primary and secondary metabolites including organic acids, diacetyl, CO2 and even antibiotics such as reuterocyclin produced by Lactobacillus reuteri. In addition, members of the group can also produce a wide range of bacteriocins, some of which have activity against food pathogens such as Listeria monocytogenes and Clostridium botulinum. Indeed, the bacteriocin nisin has been used as an effective biopreservative in some dairy products for decades, while a number of more recently discovered bacteriocins, such as lacticin 3147, demonstrate increasing potential in a number of food applications. Both of these lactococcal bacteriocins belong to the lantibiotic family of posttranslationally modified bacteriocins that contain lanthionine, beta-methyllanthionine and dehydrated amino acids. The exploitation of such naturally produced antagonists holds tremendous potential for extension of shelf-life and improvement of safety of a variety of foods.     

Potential of lactic acid bacteria isolated from specific natural niches in food production and preservation

Autochthonous strains of lactic acid bacteria (LAB) have been isolated from traditionally homemade cheeses collected from specific ecological localities across Serbia and Montenegro. Genetic and biochemical analysis of this LAB revealed that they produce bacteriocins, proteinases and exopolysaccharides. LAB produces a variety of antimicrobial substances with potential importance for food fermentation and preservation. Apart from the metabolic end products, some strains also secrete antimicrobial substances known as bacteriocins. Among the natural isolates of LAB from homemade cheeses, bacteriocin producers were found in both lactococci and lactobacilli. Lactococcus lactis subsp. lactis BGMN1-5 was found to produce three narrow spectrum class II heat-stable bacteriocins. In addition to bacteriocin production, BGMN1-5 synthesized a cell envelope-associated proteinase (CEP) and shows an aggregation phenotype. Another isolate, L. lactis subsp. lactis BGSM1-19 produces low molecular mass (7 kDa) bacteriocin SM19 that showed antimicrobial activity against Staphylococcus aureus, Micrococcus flavus and partially against Salmonella paratyphi. Production of bacteriocin reaches a plateau after 8 h of BGSM1-19 growth. Bacteriocin SM19 retained activity within the wide pH range from 1 to 12 and after the treatment at 100 degrees C for 15 min. Among collection of lactobacilli, the isolate Lactobacillus paracasei subsp. paracasei BGSJ2-8 produces heat-stable bacteriocin SJ (approx. 5 kDa) polypeptide. It retained activity after treatment for 1 h at 100 degrees C, and in the pH range from 2 to 11. In addition to isolates from cheeses, bacteriocin-producing human oral lactobacilli were detected. Most of them showed antimicrobial activity against streptococci, staphylococci and micrococci, but not against Candida. Isolate BGHO1 that showed the highest antimicrobial activity was determined as L. paracasei. Interestingly, Lactobacillus helveticus BGRA43, which was isolated from the human intestine showed strong activity against Clostridium sporogenes, but it was not possible to detect any bacteriocin production in this isolate by using standard procedures. Further analysis of antimicrobial activity revealed that BGRA43 has a relatively broad spectrum. Lactobacilli resistant to nisin were also detected among natural isolates. They produce bacteriocins, which have no activity against nisin producing lactococci.     

Incidence and antibiotic susceptibility of bacteriocin‐producing lactic acid bacteria from dairy products

Screening for bacteriocin production by strains of lactic acid bacteria (LAB) from local dairy products in Iran resulted in the detection of 10 bacteriocin‐producing strains. Among 105 isolated, 10 bacteriocin producers were phenotypically and genotypically identified as Enterococcus spp. The antimicrobial compounds produced by these novel strains were inactivated by trypsin, proteinase k. These bacteriocins also were active in a wide range of pH and temperature values, and inhibited not only the closely related LAB, but also Listeria monocytogenes.     

Isolation of lactic acid bacteria with inhibitory activity against pathogens and spoilage organisms associated with fresh meat

The use of lactic acid bacteria (LAB) as protective cultures in vacuum-packed chill-stored meat has potential application for assuring and improving food quality, safety and market access. In a study to identify candidate strains suitable for evaluation in a meat model, agar-based methods were employed to screen 181 chilled meat and meat process-related LAB for strains inhibitory to pathogens and spoilage organisms of importance to the meat industry. Six meat-derived strains, including Lactobacillus sakei and Lactococcus lactis, were found to be inhibitory to one or more of the target strains Listeria monocytogenes, Brochothrix thermosphacta, Campylobacter jejuni and Clostridium estertheticum. The inhibitory agents appeared to be either cell-associated or molecules released extracellularly with bacteriocin-like properties. Variations detected in the antimicrobial activity of LAB associated with changes to test parameters such as substrate composition underlined the importance of further in situ evaluation of the inhibitory strains in stored meat trials.     

天然食品防腐剂——乳链菌肽（Nisin）

乳链菌肽是某些乳酸链球菌产生的一种天然食品防腐剂，它强烈抑制许多引起食品腐败的革兰氏阳性菌的生长，繁殖，并对人体安全无毒。已被广泛应用于热加工食品，尤其是罐头食品，奶制品，高蛋白食品和乙醇饲料的防腐保鲜。此外，加入乳链菌肽还可降低食品加工过程中的能源消耗，改进食品的营养价值，外观，风味和质地，延长货架寿命。对乳链菌肽的应用和开发将具有重大的经济效益和社会效益。     

EFFECT OF A BACTERIOCIN-LIKE SUBSTANCE (BLS) PRODUCED BY STREPTOCOCCUS THERMOPHILUS STRAINS ON LISTERIA SPP. STRAINS

In this study, fifty-two strains of Streptococcus, isolated from Turkish yogurt were tested for their ability to inhibit Listeria strains. Only two isolates exhibited antimicrobial activity (CFF; cell-free filtrate) against eight strains of Listeria innocua, four strains of Listeria monocytogenes and one strain of Listeria welshimeri by agar diffusion. However, bacteriocin-like substance (BLS) producing strains were determined under conditions with the effects of organic acids and hydrogen peroxide eliminated. Inhibitory activities of Streptococcus thermophilus ET5 and S. thermophilus H22 were due to BLS. BLS of S. thermophilus ET5 was observed to exhibit more significant inhibition to the Listeria strains than BLS of S. thermophilus H22.
Inhibitory effect of nisin in 50, 100, 150, 200, 250 and 300 ppm concentrations was compared with BLS producing S. thermophilus strains against Listeria spp. strains. While all Listeria strains were resistant to 50 and 100 ppm concentrations of nisin, some strains of Listeria spp. were only sensitive to 150, 200, 250 and 300 ppm concentrations. The inhibitory activity of S. thermophilus ET5 was more effective against Listeria strains than the inhibitory activity of nisin. It appears that BLS of S. thermophilus ET5 could provide greater potential and usefulness as bacteriocin than nisin. These results indicate that the potential application of antimicrobial-production of S. thermophilus ET5 and H22 as bio-preservatives against Listeria contamination on dairy products could be suggested.     

Preliminary characterization of bacteriocin-like substances from lactic acid bacteria isolated from organic leafy vegetables

This study presents the characterization of new strains of lactic acid bacteria (LAB) from organic vegetables. Forty-five strains of LAB isolated from vegetables were investigated by its antimicrobial activity against taxonomically related microorganisms. Genetic identification of selected LAB was performed by means of PCR method. These strains were Enterococcus faecium, Lactococcus lactis, Enterococcus hirae and Enterococcus canis. Bacteriocin-like substances were active against Gram-positive bacteria and Gram-negative foodborne pathogens (Listeria monocytogenes and Escherichia coli, respectively). The antimicrobial activity of LAB strains was inactivated by the addition of proteases, thus confirming the proteinaceous nature of the inhibition. In all four strains the bacteriocin activity was stable after extended refrigerated storage and freezing-thawing cycles. This fact suggests that bacteriocin produced by the four LAB strains may find application as biopreservatives in minimally processed vegetables.     

从酸白菜中分离出一株产细菌素的乳酸片球菌

本试验从酸白菜中分离出一株有抑制单核细胞增多症李氏杆菌作用的菌株,经鉴定为乳酸片球菌,还对植物乳杆菌、金黄色葡萄球菌、蜡样芽胞杆菌等革兰氏阳性细菌有抗菌作用。该抗菌物质对蛋白水解酶敏感、对过氧化氢酶不敏感、耐热、在较宽的pH范围内保持活性。在排除是有机酸和过氧化氢后,初步认为该菌产生的抗菌物质为抗菌肽,即细菌素。     

Growth and bacteriocin production by lactic acid bacteria in vegetable broth and their effectiveness at reducing Listeria monocytogenes in vitro and in fresh-cut lettuce

The fresh-cut fruit and vegetable industry is searching for alternatives to replace chemical treatments with biopreservative approaches that ensure the safety of the product and fulfil consumer preferences for minimally processed foods. In this study, the use of bacteriocins produced by lactic acid bacteria has been tested as a substitute for chemical disinfection of fresh-cut iceberg lettuce. First, the ability of several non-plant origin bacteriocinogenic strains (nisin Z(+), plantaricin C(+), lacticin 481(+), coagulin(+) or pediocin PA-1(+)) to grow in a lettuce extract at 4 degrees C, 10 degrees C and 32 degrees C was tested. All strains were able to grow, but bacteriocin production was predominantly detected at 32 degrees C. Addition of bacteriocinogenic supernatants (nisin(+), coagulin(+) and a nisin-coagulin(+) cocktail) to tryptic-soy agar plates inoculated with Listeria monocytogenes reduced Listeria counts by approximately 1-1.5 log units compared with the control plates without bacteriocin, after 48 h of storage at 4 degrees C. The effect of washing with bacteriocin-containing solutions on survival and proliferation of Listeria monocytogenes was also evaluated in fresh-cut lettuce packaged in macro-perforated polypropylene bags and stored for 7 days at 4 degrees C. Washing fresh-cut lettuce with these solutions decreased the viability of Listeria monocytogenes by 1.2-1.6 log units immediately after treatment, but, during storage at 4 degrees C, bacteriocin treatments only exerted minimal control over the growth of the pathogen. Natural microbiota were little affected by bacteriocins during storage.     

Nisin的生产、提纯和检测

Nisin是一种由乳酸乳球菌产生的羊毛硫氨酸类细菌素 ,在许多国家被许可作为生物防腐剂。Nisin的产量受许多因素的制约 ,如产生菌性能、培养基组成 (碳源、氮源、磷源和阳离子 )、发酵条件 (pH、温度、搅拌、通风 )、发酵类型 (分批发酵、连续发酵、自由细胞、固定化细胞 )等 ;大规模回收和纯化Nisin主要采用一些基于吸附-解析或者相分配原理的方法 ;最常用的定量检测Nisin的方法主要有生物分析法和免疫检测法 ,采用各种特定nisin抗体的免疫检测方法具有迅速、灵敏、准确等特性并能实现Nisin的在线检测。     

Microbial ecology of artisanal products from North West of Italy and antimicrobial activity of the autochthonous populations

A total of about 1000 autochthonous strains were isolated from fresh and fermented artisanal products (cheese and meat) of the Piedmont area (Northwest of Italy) and screened for their antimicrobial activity. Among them, 98 bacteriocin-producing strains were detected and identified by molecular methods and genetic determinants encoding the antimicrobial proteins were targeted by PCR. The bacteriocins produced by isolates showed antimicrobial activity towards different spoilage and pathogenic microorganisms such as Listeria monocytogenes, Staphylococcus aureus, Clostridium tyrobutyricum and Brochothrix thermosphacta. Identification of the bacteriocin producing strains resulted in a large number of Lactococcus and Enterococcus strains with a broad spectrum of activity towards more that one spoilage and/or pathogenic microorganisms tested. In several strains it was possible to detect a combination of different genes responsible for the bacteriocin activity. These newly isolated bacteriocin-producing strains represent potential starters or co-starters to be used in food stabilization using the approach of the biopreservation.     

In vitro fermentation studies for selection and evaluation of Bacillus strains as starter cultures for the production of okpehe, a traditional African fermented condiment

Selected Bacillus and Enterococcus strains, isolated from traditional okpehe fermentations, were studied for their suitability as starter cultures in laboratory-scale fermentations of Prosopis africana seeds for the production of okpehe, a traditional fermented vegetable product of Nigeria. The strains were selected on the basis of highest proteolytic activity, as determined with the APIZYM (BioMerieux) test. The choice of starter strains was narrowed to Bacillus subtilis strains BFE 5301 and BFE 5372. These were determined as the best starter combination because of rapid growth, high amylolytic and proteolytic activities, high levels of polyglutamic acid production by strain BFE 5372, as well as bacteriocin production by strain BFE 5301. Other mixed culture fermentations did not yield sensorically acceptable products. Although a monoculture fermentation, using only B. subtilis strain BFE 5372, produced okpehe with very good sensory characteristics, the growth of B. cereus could be detected after 48 h fermentation, indicating that this starter did not sufficiently contribute to product safety. Mixed culture fermentation with the combination of bacteriocin-producing starter B. subtilis BFE 5301 and the non-bacteriocin-producing B. subtilis BFE 5372, produced a product with good sensory characteristics, in which growth of B. cereus was delayed. The bacteriocin produced by B. subtilis strain BFE 5301 was identified as subtilisin, using subtilisin-specific primers and PCR amplification of the subtilisin gene. The bacteriocin was heat-stable at 100 degrees C for 10 min and exhibited highest activity at pH values lower or equal to pH 6.0. The bacteriocin was sensitive to the proteolytic enzymes trypsin and alpha-chymotrypsin at concentrations of 10 mg/ml.     

乳链菌肽生物合成及其调控

乳链菌肽是由乳酸链球菌的某些菌株代谢所产生的一种羊毛硫抗生素,对芽孢杆菌、链球菌、李斯特菌等许多革兰氏阳性菌有较强的抑制作用,是一种应用广泛的天然食品防腐剂。研究发现,乳链菌肽生物合成基因簇包括nisABTCIPRKFEG,与蔗糖发酵基因以及1个或多个噬菌体抗性系统和N5-(羧乙基)鸟氨酸酶的基因共同定位于染色体上的1个大的接合性转座子上,通过3个操纵子转录,并通过两组分调控体系的信号传导调控自身结构基因的表达。文章综述了乳链菌肽的分子结构、基因簇及生物合成调控机制。     

牦牛奶酪中产细菌素乳酸菌菌株的筛选

本研究用分离自西藏、新疆、云南地区牦牛奶酪的39 株乳酸菌作为供试菌株,制备细菌素粗提液,用琼脂井法分析粗提液对9 株致病菌（蜡样芽孢杆菌、金黄色葡萄球菌、埃希氏大肠杆菌O157∶H7、单增李斯特菌、费氏柠檬酸杆菌、鼠伤寒沙门氏菌、阴沟肠杆菌、产气荚膜梭菌、宋内志贺菌）和2 株非致病菌（无害李斯特菌、嗜热链球菌）指示菌株的抑菌作用。测定结果表明有30 株菌的细菌素粗提液对指示菌株有抑菌作用,筛选出Y13、X29和X30这3 株抑菌谱较宽的细菌素产生菌。     

Production,mode of action and sequencing of the corresponding gene of a bacteriocin produced by Lactococcus lactis 19.3

Lactococcus lactis 19.3 produces a bacteriocin with a wide inhibitory spectrum, including the food pathogen Listeria monocytogenes. The producing strain was able to grow and produce similar amounts of bacteriocin in MRS medium, cow's milk and soya milk, respectively. The mode of action of this bacteriocin was further investigated using two indicator strains, Lactobacillus delbrueckii subsp. bulgaricus LMG 6901^T and Listeria monocytogenes ATCC 1911‐1. The bacteriocin displayed a bactericidal effect, causing a rapid decrease of the cell viability. Bacteriocin treatment of the sensitive strains resulted in major morphological changes, including pore formation, as shown by scanning electron microscopy. Finally, the bacteriocin‐encoding gene was identified by sequencing. The presence of nisin gene was confirmed. Based on all our data gathered so far, L. lactis 19.3 is a good candidate for a starter or protective culture in the manufacturing of both fermented dairy and vegetarian food products.