Bacteriocin-producing Bacteria Research Articles

Isolation and Molecular Identification of Bacteriocin-producing Enterococci with Broad Antibacterial Activity from Traditional Dairy Products in Kerman Province of Iran.

One of the critical limitations to use of bacteriocins produced by lactic acid bacteria as a substitute for chemical antibiotics is the narrow spectrum of their antibacterial activity. The aim of present study was isolation and molecular identification of bacteriocin-producing enterococci with broad antibacterial spectrum. Bacteriocin-producing bacteria were isolated from native dairies in Kerman. Bacteriocins were purified by ammonium sulfate method and the effects of them were investigated on different strains of bacteria. Also, the effects of pH and heat on produced bacteriocins were investigated. High level bacteriocin-producing isolates were identified based on molecular tests. A total of 15 strains of bacteriocin-producing Enterococcus were isolated initially. Enterococcus faecium C-2 and Y-1 strains produced bacteriocins with the highest antibacterial effect. The bacteriocins were stable in pH ranges from 2 to 12 and their antibacterial activity was maintained after autoclave treatment. The maximum bactericidal effect was observed against Listeria monocytogenes and Pseudomonas aeruginosa. In conclusion, use of these bacteriocins as a substitute for chemical antibiotics is recommended.

D-Ribose Interferes with Quorum Sensing to Inhibit Biofilm Formation of Lactobacillus paraplantarum L-ZS9.

Biofilms help bacteria survive under adverse conditions, and the quorum sensing (QS) system plays an important role in regulating their activities. Quorum sensing inhibitors (QSIs) have great potential to inhibit pathogenic biofilm formation and are considered possible replacements for antibiotics; however, further investigation is required to understand the mechanisms of action of QSIs and to avoid inhibitory effects on beneficial bacteria. Lactobacillus paraplantarum L-ZS9, isolated from fermented sausage, is a bacteriocin-producing bacteria that shows potential to be a probiotic starter. Since exogenous autoinducer-2 (AI-2) promoted biofilm formation of the strain, expression of genes involved in AI-2 production was determined in L. paraplantarum L-ZS9, especially the key gene luxS. D-Ribose was used to inhibit biofilm formation because of its AI-2 inhibitory activity. Twenty-seven differentially expressed proteins were identified by comparative proteomic analysis following D-ribose treatment and were functionally classified into six groups. Real-time quantitative PCR showed that AI-2 had a counteractive effect on transcription of the genes tuf, fba, gap, pgm, nfo, rib, and rpoN. Over-expression of the tuf, fba, gap, pgm, and rpoN genes promoted biofilm formation of L. paraplantarum L-ZS9, while over-expression of the nfo and rib genes inhibited biofilm formation. In conclusion, D-ribose inhibited biofilm formation of L. paraplantarum L-ZS9 by regulating multiple genes involved in the glycolytic pathway, extracellular DNA degradation and transcription, and translation. This research provides a new mechanism of QSI regulation of biofilm formation of Lactobacillus and offers a valuable reference for QSI application in the future.

이상발효유산균과 내산성 효모와의 혼합배양이 사워도우의 저장성에 미치는 영향

본 연구에서는 묵은지로부터 분리된 박테리오신을 생산하는 이상발효 유산균 및 내산성 효모가 사워도우의 저장 기간 연장과 품질 개선에 미치는 영향을 조사하였다. 유전자 염기서열 분석 결과 빵 부패세균인 Bacillus 속에 대한 항균활성을 나타낸 이상발효 유산균은 Leuconostoc mesenteroides LAS112, Lactobacillus brevis LAS129 및 L. mesenteroides subsp. dextranicum LAB137으로 동정되었고, 산성 pH 하에서 증식 가능한 효모는 Pichia membranifaciens YS05, Pichia fermentans YS19 및 Pichia anomala YS26으로 확인되었다. 사워도우 발효에 사용된 L. brevis LAS129는 L. mesenteroides LAS112 및 L. mesenteroides subsp. dextranicum LAS 137 보다 더많은 양의 초산과 박테리오신 활성을 나타내었으나, LAS112는 가장 많은 양의 유산을 생산하였다. Bacillus subtilis ATCC 35421에 대한 최대의 박테리오신 활성(640 AU/g)은 L. brevis LAS129와 P. membranifaciens YS05 혹은 P. anomala YS26으로 혼합 발효시킨 사워도우 내에서 관찰되었다. <TEX>$30^{\circ}C$</TEX>에서 24시간 발효 후 사워도우 내 LAS129의 균수(<TEX>$10^9CFU/g$</TEX>)는 YS05 혹은 YS26의 효모 균수(<TEX>$10^7CFU/g$</TEX>)보다 높게 검출되었다. 한편, 이들 균주들을 이용하여 발효시킨 사워도우 내에 존재하는 빵 부패균의 균수는 대조구 보다 유의하게(P < 0.05) 낮은 수준으로 나타났다. The primary objective of this study was to investigate the effects of the bacteriocin-producing heterofermentative lactic acid bacteria (LAB) and acid-resistant yeast isolated from Mukeunji, a Korean ripened kimchi on shelf-life extension and quality improvement of sourdough. According to gene sequence analysis the heterofermentative LAB that showed the antimicrobial activity against bread-spoilage Bacillus strains were identified as Leuconostoc mesenteroides LAS112, Lactobacillus brevis LAS129, and L. mesenteroides subsp. dextranicum LAB137. In addition, the yeasts that were able to grow at acidic pH were identified as Pichia membranifaciens YS05, Pichia fermentans YS19, and Pichia anomala YS26. During sourdough fermentation the levels of acetic acid and bacteriocin produced by L. brevis LAS129 strain were higher than those of L. mesenteroides LAS112 and L. mesenteroides subsp. dextranicum LAS 137 strains, whereas LAS112 strain produced the highest levels of lactic acid. The maximum bacteriocin activity (640 AU/g) against Bacillus subtilis ATCC 35421 was obtained in sourdough fermented by mixed culture of L. brevis LAS129 and P. membranifaciens YS05 or P. anomala YS26. After 24 h of fermentation at <TEX>$30^{\circ}C$</TEX>, the viable cell counts of LAS129 (<TEX>$10^9CFU/g$</TEX>) in sourdough were higher than those of the YS05 or YS26 (<TEX>$10^7CFU/g$</TEX>). Meanwhile, the viable cells of bread-spoilage strain in sourdough fermented with these strains were significantly (P < 0.05) lower than the control group.

Inhibition of Listeria monocytogenes biofilms by bacteriocin-producing bacteria isolated from mushroom substrate.

This study was designed to investigate the ability of naturally occurring bacteria isolated from mushroom substrate to prevent biofilm formation by Listeria monocytogenes or to remove existing biofilms in mushroom production facilities. It is generally recognized that L. monocytogenes forms biofilms that can facilitate its survival in food-processing environments. Eleven bacteriocin-producing isolates were identified and the bacteriocins characterized based on heat and enzyme inactivation studies. Further characterization was undertaken by MALDI-TOF mass spectrometry, PCR and sequencing. Production of nisin Z (by Lactococcus lactis isolates), subtilomycin (by Bacillus subtilis isolates) and lichenicidin (by Bacillus licheniformis and Bacillus sonorensis isolates) was detected. In co-culture with L. monocytogenes, the bacteriocin-producing strains could prevent biofilm formation and reduce pre-formed biofilms. Mushroom substrate can be a source of bacteriocin-producing bacteria that can antagonize L. monocytogenes. The results highlight the potential of bacteriocin-producing strains from mushroom substrate to reduce L. monocytogenes biofilm in food production environments, contributing to a reduction in the risk of food contamination from the environment.

Two-Component Systems Involved in Susceptibility to Nisin A in Streptococcus pyogenes.

Two-component systems (TCSs) are regulatory systems in bacteria that play important roles in sensing and adapting to the environment. In this study, we systematically evaluated the roles of TCSs in the susceptibility of the group A Streptococcus (GAS; Streptococcus pyogenes) SF370 strain to several types of lantibiotics. Using individual TCS deletion mutants, we found that the deletion of srtRK (spy_1081-spy_1082) in SF370 increased the susceptibility to nisin A, which is produced by Lactococcus lactis ATCC 11454, but susceptibility to other types of lantibiotics (nukacin ISK-1, produced by Staphylococcus warneri, and staphylococcin C55, produced by Staphylococcus aureus) was not altered in the TCS mutants tested. The expression of srtFEG (spy_1085 to spy_1087), which is located downstream of srtRK and is homologous to ABC transporters, was increased in response to nisin A. However, srtEFG expression was not induced by nisin A in the srtRK mutant. The inactivation of srtFEG increased the susceptibility to nisin A. These results suggest that SrtRK controls SrtFEG expression to alter the susceptibility to nisin A. Further experiments showed that SrtRK is required for coexistence with L. lactis ATCC 11454, which produces nisin A. Our results elucidate the important roles of S. pyogenes TCSs in the interactions between different bacterial species, including bacteriocin-producing bacteria. In this study, we focused on the association of TCSs with susceptibility to bacteriocins in S. pyogenes SF370, which has no ability to produce bacteriocins, and reported two major new findings. We demonstrated that the SrtRK TCS is related to susceptibility to nisin A by controlling the ABC transporter SrtFEG. We also showed that S. pyogenes SrtRK is important for survival when the bacteria are cocultured with nisin A-producing Lactococcus lactis This report highlights the roles of TCSs in the colocalization of bacteriocin-producing bacteria and non-bacteriocin-producing bacteria. Our findings provide new insights into the function of TCSs in S. pyogenes.

Utilization of bacteriocin-producing bacteria in dairy products

Utilization of bacteriocin-producing bacteria in dairy products

Antibiotic resistance in potentially bacteriocinogenic probiotic bacteria in aquaculture environments

Bacteriocin-producing bacteria in aquaculture may prevent spreading of potentially pathogenic microorganisms, and could be used as an alternative to the empirical use of antimicrobial drugs, especially for prophylaxis. Bacteriocinogenic bacteria inoculated as probiotics should not carry undesirable traits, such as antibiotic resistance. This study aimed to find potentially bacteriocinogenic bacteria in an aquaculture system and evaluate their antimicrobial susceptibility patterns. Selective cultures for enterobacteria, non-fermenting Gram-negative rods and Gram-positive cocci were obtained from water samples before bacterial isolation and biochemical identification. Overall, 160 representative strains were recovered and for 57 antagonism was observed against selected strains such as Staphylococcus aureus, with antagonism being expressed better on Brain Heart Infusion medium. After exclusion of interfering factors, bacteriocin or bacteriocin-like substances were suggested to be related to the antagonism observed. Higher drug-resistant rates were observed among potentially bacteriocinogenic bacteria for different antimicrobials of clinical relevance. Although antibiotic resistance is a global health problem and bacteriocins are attractive alternatives to classical antibiotic even to multiresistant bacteria, the data obtained suggest that bacteriocin-producing bacteria may harbour resistance genes available for transference in different environments. From the ecological and biotechnological perspective, antimicrobial susceptibility tests must be always performed when prospecting potentially bacteriocinogenic bacteria as probiotic candidates in the environment.

DIFFERENCES OF ANTIBACTERIAL ACTIVITY SPECTRA AND PROPERTIES OF BACTERIOCINS, PRODUCED BY GEOBACILLUS SP. BACTERIA ISOLATED FROM DIFFERENT ENVIRONMENTS

Ability of bacteriocins to inhibit pathogenic bacteria may depend on the environment inhabited by the producing strain. However, there are just few reports on bacteriocin-producing thermophilic bacteria from unconventional environments. Therefore, in this work we investigated differences in antibacterial activity spectra of microorganisms isolated from two different environments: the oil wells (first collection) and surface soil above the oil pools (second collection). All strains were Gram-positive, rod-shaped, thermophilic and endospore forming bacteria. Using spot-on-lawn method we have demonstrated that strains from the second collection possess higher and broader antibacterial activity spectra than first collection strains. Furthermore, all strains were active against at least 1 of 19 tested pathogenic bacteria. Analysis of antibacterial compounds has shown that all of them were stable in broad temperature and pH ranges, sensitive to proteolytic enzymes, proving their proteonaceus nature. They were all secreted during the exponential growth phase and active against closely related thermophilic bacteria, which suggest that these substances are most likely bacteriocins. Most of the bacteriocins had molecular masses under 20 kDa. Plasmid isolation and elimination experiments suggest that bacteriocins are most likely chromosome-encoded. Our results confirm the differences between antibacterial spectra of bacteriocins produced by bacterial strains isolated from different environmental niches.

Study of Lactic Acid Bacteria Community From Raw Milk of Iranian One Humped Camel and Evaluation of Their Probiotic Properties.

Background:Camel milk is amongst valuable food sources in Iran. On the other hand, due to the presence of probiotic bacteria and bacteriocin producers in camel milk, probiotic bacteria can be isolated and identified from this food product.Objectives:The objectives of the present research were the isolation and molecular identification of lactic acid bacteria from camel milk and evaluation of their probiotic properties.Materials and Methods:A total of ten samples of camel milk were collected from the Golestan province of Iran under aseptic conditions. Bacteria were isolated by culturing the samples on selective medium. Isolates were identified by amplification of the 16S rDNA and Internal Transcribed Spacer (ITS) region between the 16S and 23S rRNA genes by Polymerase Chain Reaction (PCR) and were then screened and grouped by the Amplified Ribosomal DNA Restriction Analysis (ARDRA) method. To evaluate probiotic properties, representative isolates of different ARDRA profiles were analyzed. The antimicrobial activity of Lactic Acid Bacteria (LAB) against Pediococcus pentosaceus, Escherichia coli and Bacillus cereus was examined by the agar diffusion assay. Acid and bile tolerance of isolates were evaluated.Results:A total of 64 isolates were analyzed based on biochemical tests and morphological characteristics. The most frequently isolated LAB was Enterococci. Weissella, Leuconostoc, Lactobacilli and Pediococci were less frequently found. Based on restriction analysis of the ITS, the isolates were grouped into nine different ARDRA patterns that were identified by ribosomal DNA sequencing as P. pentosaceus, Enterococcus faecium strain Y-2, E. faecium strain JZ1-1, E. faecium strain E6, E. durans, E. lactis, Leuconostoc mesenteroides, Lactobacillus casei and Weissella cibaria. The results showed that antimicrobial activity of the tested isolates was remarkable and P. pentosaceus showed the most antibacterial activity. In addition, E. durans, E. lactis, L. casei and P. pentosaceus were selected as probiotic bacteria.Conclusions:This study revealed the presence of bacteriocin-producing bacteria and probiotic bacteria in camel milk from the Golestan province of Iran.

Isolation and Characterization of Bacteriocin Producing Lactic Acid Bacteria from Diary Effluent

Bacteriocins are antimicrobial peptides produced by bacteria and Achaea. This study was focused to isolate bacteriocin producing bacteria from dairy effluent and the species were screened for antimicrobial and probiotic characteristics. The strains 3 and 5 confirmed highest antimicrobial activity against multidrug resistant pathogens such as Escherichia coli, Staphylococcus aureus, Streptococcus sp, Klebsiella pneumoniae, Enterobacter aerogenes, and Proteus vulgaris and negative activity was found against Bacillus subtilis. The strains showed high antimicrobial activity at neutral pH and temperatures in the range of 30–50oC. The strains were also tested for antibiotic resistance as well as acid and bile salt tolerance tests.

Screening of Bacteriocin-producing Enterococcus faecalis Strains for Antagonistic Activities against Clostridium perfringens.

This study was conducted to isolate and characterize bacteriocin-producing bacteria against Clostridium perfringens (C. perfringens) from domestic animals to determine their usefulness as probiotics. Bacteriocin-producing bacteria were isolated from pig feces by the spot-on-lawn method. A total of 1,370 bacterial stains were isolated, and six were tentatively selected after identifying the inhibitory activity against the pathogenic indicator C. perfringens KCTC 3269 and KCTC 5100. The selected strains were identified as Enterococcus faecalis (E. faecalis) by 16s rRNA sequencing. Most of the isolated bacterial strains were resistant to 0.5% bile salts for 48 h and remained viable after 2 h at pH 3.0. Some E. faecalis also showed strong inhibitory activity against Listeria monocytogenes KCTC 3569, KCTC 3586 and KCTC 3710. In the present study, we finally selected E. faecalis AP 216 and AP 45 strain based on probiotic selection criteria such as antimicrobial activity against C. perfringens and tolerance to acid and bile salts. The bacteriocins of E. faecalis AP 216 and AP 45 strains were highly thermostable, showing anticlostridial activities even after incubation at 121℃ for 15 min. These bacteriocinproducing bacteria and/or bacteriocins could be used in feed manufacturing as probiotics as an alternative to antibiotics in the livestock industry.

Isolation and characterization of bacteriocin-producing bacteria from the intestinal microbiota of elderly Irish subjects

To isolate and characterize bacteriocins produced by predominant species of lactic acid bacteria from faeces of elderly subjects. Screening over 70,000 colonies, from faecal samples collected from 266 subjects, using the indicator organisms Lactobacillus bulgaricus LMG 6901 and Listeria innocua DPC 3572, identified 55 antimicrobial-producing bacteria. Genomic fingerprinting following ApaI digestion revealed 15 distinct strains. The antimicrobial activities associated with 13 of the 15 strains were sensitive to protease treatment. The predominant antimicrobial-producing species were identified as Lactobacillus salivarius, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus crispatus and Enterococcus spp. A number of previously characterized bacteriocins, including ABP-118 and salivaricin B (from Lact. salivarius), enterocin B (Enterococcus faecium), lactacin B (Lact. acidophilus), gassericin T and a variant of gassericin A (Lact. gasseri), were identified. Interestingly, two antimicrobial-producing species, not generally associated with intestinally derived microorganisms were also isolated: Lactococcus lactis producing nisin Z and Streptococcus mutans producing mutacin II. These data suggest that bacteriocin production by intestinal isolates against our chosen targets under the screening conditions used was not frequent (0.08%). The results presented are important due to growing evidence indicating bacteriocin production as a potential probiotic trait by virtue of strain dominance and/or pathogen inhibition in the mammalian intestine.

Effects of the Oral Administration of Viable and Heat-Killed Streptococcus bovis HC5 Cells to Pre-Sensitized BALB/c Mice

Antimicrobial peptides have been suggested as an alternative to classical antibiotics in livestock production and bacteriocin-producing bacteria could be added to animal feeds to deliver bacteriocins in the gastrointestinal (GI) tract of ruminant and monogastric animals. In this study, viable (V) and heat-killed (HK) Streptococcus bovis HC5 cells were orally administered to pre-sensitized mice in order to assess the effects of a bacteriocin-producing bacteria on histological parameters and the immune response of the GI tract of monogastric animals. The administration of V and HK S. bovis HC5 cells during 58 days to BALB/c mice did not affect weight gain, but an increase in gut permeability was detected in animals receiving the HK cells. Viable and heat killed cells caused similar morphological alterations in the GI tract of the animals, but the most prominent effects were detected in the small intestine. The oral administration of S. bovis HC5 also influenced cytokine production in the small intestine, and the immune-mediated activity differed between V and HK cells. The relative expression of IL-12 and INF-γ was significantly higher in the small intestine of mice treated with V cells, while an increase in IL-5, IL-13 and TNF-α expression was only detected in mice treated with HK cells. Considering that even under a condition of severe challenge (pre-sensitization followed by daily exposure to the same bacterial immunogen) the general health of the animals was maintained, it appears that oral administration of S. bovis HC5 cells could be a useful route to deliver bacteriocin in the GI tract of livestock animals.

Evaluation of two bacteriocin-producing probiotic lactic acid bacteria as inoculants for controlling Listeria monocytogenes in grass and maize silages

This study evaluated the effectiveness as silage inoculants of two bacteriocin-producing lactic acid bacteria (LAB) with proven probiotic activity in post-weaned piglets, Lactococcus lactis CECT 539 and Pediococcus acidilactici NRRL B-5627, in combination with the ensiling strain Lactobacillus plantarum CECT 220. Four combinations of LAB were tested in grass and maize silages: L. plantarum (Lb), L. plantarum + L. lactis (LbL), L. plantarum + P. acidilactici (LbP), and the three species together (LbLP). Untreated silages and silages inoculated with commercial starters (L. plantarum + Lactobacillus buchneri + Enterococcus faecium) were prepared and used as controls. Since both bacteriocinogenic strains have shown antilisterial activity in vitro, this study also aimed to determinate their effectiveness for controlling Listeria monocytogenes in the silo. Therefore, silages were inoculated with the strain L. monocytogenes CECT 4032 (1.0×105cfu/g), the evolution of which was monitored throughout fermentation using microbiological and DNA based methods. Each treatment was assayed in triplicate in lab-scale silos and sampled after 1, 2, 8, 16 and 30 days to extract DNA and determination of the pH, volume, organic acids (lactic, acetic, propionic and butyric), alcohols (ethanol and propane-1, 2-diol), water soluble reducing sugars (WSRS) and make LAB and L. monocytogenes counts. Our results showed that the silages treated with bacteriocin-producing LAB had a higher rate of pH decline (P<0.01) and lactic acid production (P<0.01). After 30 days of fermentation, we observed low acetic acid (P<0.01) and ethanol (P<0.01), lower LAB counts (P<0.01) and higher (P<0.01) residual sugar concentrations in the Lb, LbL, LbP and LbLP silages. No L. monocytogenes colonies were found during ensilage; however, its DNA was present, which suggests that the adverse silage conditions (pH, anaerobiosis) might favour a viable but non-culturable state in the cells. In both forages, all treatments showed the anticipated disappearance of L. monocytogenes, unlike the control. It can be concluded that the bacteriocinogenic LAB tested in this study can be used as silage inoculants for controlling L. monocytogenes in the silo and also in the rumen of cattle due to their reported tolerance to acid.

Selection and Characterization of Bacteriocin-Producing Lactobacillus sp. AP 116 from the Intestine of Pig for Potential Probiotics

The purpose of this study was to isolate bacteriocin-producing bacteria with antagonistic activities against pathogens from the intestines of pigs for probiotic use. Lactobacillus sp. AP 116 possessing antimicrobial property was selected from a total of 500 isolates. The AP 116 strain showed a relatively broad spectrum of inhibitory activity against Listeria monocytogenes, Clostridium perfringens, Pediococcus dextrinicus, and Enterococcus strains using the spot-on-lawn method. Bacteriocin activity remained unchanged after 15 min of heat treatment at <TEX>$121^{\circ}C$</TEX> and exposure to organic solvents; however, it diminished after treatment with proteolytic enzymes. Maximum production of bacteriocin occurred at <TEX>$34^{\circ}C$</TEX> when a pH of 6.0 was maintained throughout the culture during fermentation. According to a tricine SDS-PAGE analysis, the molecular weight of the bacteriocin was approximately 5 kDa. The isolate tolerated bile salts and low pH, and also induced nitric oxide (NO) in mouse peritoneal macrophages. Bacteriocin and bacteriocin-producing bacteria, such as Lactobacillus sp. AP 116, could be potential candidates for use as probiotics as an alternative to antibiotics in the pig industry.

Anti-Listeria activity of lactic acid bacteria isolated from golka, a regional cheese produced in Poland

Eight hundred lactic acid bacteria (LAB) strains isolated from golka, one of the oldest and most popular regional cheese produced in Poland, were screened for anti-Listeria activity.Nearly one third of isolates showed antagonistic activity against Listeria monocytogenes. Seven strains produced other substances active against L. monocytogenes. These substances were detected in neutralized cell-free supernatant fluids and were classified as bacteriocins. 16S rDNA identification of the selected bacteriocin-producing bacteria was performed. By using primers specific to a fragment of class IIa bacteriocin-coding genes, developed by the authors, presence of the bacteriocin structural gene was confirmed for all Lactococcus garvieae strains, Leuconostoc mesenteroides 453Lab and Lactobacillus plantarum Lab572. Presence of the class IIa bacteriocin-coding genes within genomes of non-bacteriocin-producing strains Lactococcus lactis subsp. lactis was also detected. Bacteriocins with the highest activity against Listeria, i.e. bacteriocins of L. garvieae Lab428, Lc. mesenteroides Lab25 and Lb. plantarum Lab572 strains, were partially characterized. The bacteriocins did not adhere to surface of the producer cells and exhibit a bactericidal mode of action. Bacteriocin of L. garvieae 428Lab was produced at much higher levels (12.800 AU/ml), as compared to the other bacteriocins (3.200 AU/ml).Our results indicate that golka cheese is a rich source of LAB active against L. monocytogenes. The bacteriocinogenic isolates from golka described in this work may potentially find application as starter or protective cultures and could constitute a superb genetic pool for production of new bacteriocins.

Construction of a food-grade cloning vector for Lactobacillus plantarum and its utilization in a food model

The development of Lactobacillus plantarum to be used in starter cultures in the food industry has been limited because of the lack of a food-grade cloning vector for the bacterium. In this study, the plasmid pFLP1 was constructed by joining 2 DNA fragments derived from food-approved organisms. The 5.2-kb BamHI/KpnI DNA fragment of pRV566 containing the theta-type replicon of Lactobacillus sakei was ligated to the BamHI/KpnI DNA fragment of a 2.9-kb lactococcal cadmium resistance determinant amplified from pND918. The 8.1-kb newly constructed plasmid could transform L. plantarum N014, a bacteriocin-producing bacteria originally isolated from nham, a traditional Thai fermented sausage. The resulting transformant, L. plantarum N014-FLP, and its parent strain were shown to be very similar in growth rate and bacteriocin activity. In addition, the plasmid was very stable in its host bacteria under nonselective pressure for 100 generations in MRS medium and for 5 days in a nham model. These results suggest that pFLP1 is a potential food-grade cloning vector for L. plantarum.

Screening of Bacteriocin-producing Bacillus Strains Isolated from Domestic Animal Feces for Antagonistic Activities against Clostridium perfringens

The purpose of this study was to isolate and characterize bacteriocin-producing bacteria against Clostridium perfringens from domestic animals to determine their usefulness as probiotics. The feces of cattle and chicken were used as sources to isolate bacteriocin-producing bacteria using the spot-on-lawn method. In total, 900 bacterial stains were isolated from domestic animal feces, and 19 strains were finally selected after determining the inhibitory activity against the pathogenic indicator C. perfringens KCTC 3269. Eighteen strains of Bacillus subtilis and one strain of Brevibacillus parabrevis were identified by 16s rRNA sequencing. Most of the bacterial strains isolated were resistant to 0.5% bile salts and remained viable after 2 h at pH 3.0. Additionally, some B. subtilis strains showed strong inhibitory activity against Listeria monocytogenes. We isolated and screened B. subtilis strains CB 153 and CB 189 from cattle and B. subtilis MSC 156 and B. parabrevis MSC 164 from chickens using probiotic selection criteria such as inhibition activity against C. perfringens and tolerance to acid and bile salts. The isolated bacteriocin-producing bacteria and/or bacteriocin have the potential to be used as probiotics in the livestock industry.

Characterization of the Bacteriocin J4 Produced by Bacillus amyloliquefaciens J4 Isolated from Korean Traditional Fermented Soybean Paste

In order to isolate the antibacterial bacteriocin-producing bacteria against Listeria monocytogenes, one of noxious and prevalent food-poisoning bacterium, 117 strains of indigenous ferment bacteria were isolated from Korean traditional fermented soybean paste (Doenjang) in Korea. Among the isolated strains, 4 isolates inhibited the growth of L. Monocytogenes. Strain J4, showing the strongest inhibition activity against L. Monocytogenes, was identified as a Bacillus amyloliquefaciens by 16S rDNA sequencing. The strain had a broad spectrum of antibacterial activity against various food-borne pathogens such as Micrococcus luteus, Vibrio parahaemolyticus, and Staphylococcus aureus besides L. monocytogenes. The bacteriocin J4 was purified by 60% ammonium sulfate precipitation, carboxymethyl (CM)-Sephadex column chromatography, and Sephadex G-100 gel filtration. The purified bacteriocin J4 was estimated to be 39 kDa molecular weight by tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Bacterocin J4 had antimicrobial activity in a wide pH range (2.0-12.0) and was heat-stable at 80oC for 20 min, but the activity of bacterocin was inactivated by heating at 100oC for 15 min. The antimicrobial activity of the bacteriocin J4 was completely abolished by treatment with proteolytic enzymes such as protease (type I and IX), papain, trypsin, proteinase K, and pepsin, but not when treated with α-amylase, α-glucosidase, and β-glucosidase. The encoding gene of the bacteriocin J4 was included in chromosomal DNA, not plasmid. The bacteriocin J4 had a bacteriolytic mechanism, resulting in cell wall degradation of L. Monocytogenes.

Anti-Listeria bacteriocin-producing bacteria from raw ewe’s milk in northern Greece

The isolation and partial characterization of anti-Listeria bacteriocin producing strains present in milk from areas of northern Greece in view of their potential use as protective cultures in food fermentations. Three hundred and thirty-two isolates were obtained from milk samples intended for Feta cheese production and gathered from 40 individual producers in Northern Greece. Isolates with anti-Listeria activity were identified by multiplex PCR as Enterococcus faecium and grouped by (GTG)(5) -PCR. The genomes of the anti-Listeria isolates were examined for the presence of known enterocin genes and major virulence genes by means of specific PCR. At least three known enterocin encoding genes were present in the genome of each of the 17 isolates. None of the 17 isolates harboured any of the virulence genes tested for or exhibited haemolytic activity. Enterococcus faecium was the dominant anti-Listeria species in the milk samples. The isolates had the potential of multiple bacteriocin production and did not exhibit some important elements of virulence. Enterococci present in milk of this area of northern Greece may be partly responsible for the safety of Feta cheese and could be useful for the production of anti-Listeria protective cultures.