Identification and Characteristics of Nisin Z-Producing <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> Isolated from Kimchi

We isolated bacteriocin-producing Lactococcus lactis subsp. lactis from Kimchi. The bacteriocin inhibited strains of Clostridium perfringens, C. difficile, Listeria monocytogenes, vancomycin-resistant Enterococcus, and one out of four methicillin-resistant Staphylococcus aureus strains, as well as some closely related lactic acid bacteria. In tricine-SDS-PAGE, the bacteriocin migrated with an apparent molecular weight of about 4 kDa to the same location as nisin A and crude nisin Z. The gene encoding this bacteriocin was found to be identical to that of nisin Z with direct PCR sequence methods. The inhibitory activity was stable against heat and pH, but it was lost at 100°C for 1 h and at 121°C for 15 min. The bacteriocin was inactivated by proteolytic enzymes, but was not affected by lysozyme, lipase, catalase, or β-glucosidase. There were some differences in characteristics from those of nisins described previously. Received: 21 June 2002 / Accepted: 22 July 2002     

In Vitro Evaluation of Beneficial Properties of Bacteriocinogenic <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> ST8Sh

Lactobacillus plantarum ST8Sh, isolated from Bulgarian salami “shpek” and previously characterized as bacteriocin producer, was evaluated for its beneficial properties. Based on the PCR analysis, Lb. plantarum ST8Sh was shown to host a gene related to the production of adhesion proteins such as Mab, Mub, EF, and PrgB. Genetic and physiological tests suggest Lb. plantarum ST8Sh to represent a potential probiotic candidate, including survival in the presence of low levels of pH and high levels of ox bile, production of β-galactosidase, bile salt deconjugation, high level of hydrophobicity, functional auto- and co-aggregation properties, and adhesion to cell lines. Application of semi-purified bacteriocin produced by Lb. plantarum ST8Sh in combination with ciprofloxacin presented synergistic effect on inhibition of Listeria monocytogenes Scott A. Based on observed properties, Lb. plantarum ST8Sh can be considered as a potential probiotic candidate with additional bacteriocinogenic properties.     

Genetic modification of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> by heterologous gene integration in a not functional region of the chromosome

This report describes the vector-free engineering of Lactobacillus plantarum by chromosomal integration of an exogenous gene without inactivation of physiological traits. The integrative plasmid vector pP7B6 was derived from pGIP73 by replacing the cbh site, encoding the L. plantarum conjugated bile salt hydrolase, with the prophage fragment P7B6, from L. plantarum Lp80 (DSM 4229). Plasmid pP7B6NI was obtained by inserting the nisin immunity gene nisI of Lactococcus lactis subsp. lactis DSM 20729, preceded by the constitutive promoter P32 from the same strain, in a unique XbaI site of fragment P7B6 and was used to electrotransform L. plantarum Lp80. A food grade recombinant L. plantarum Lp80NI, with 480-fold higher immunity to nisin than the wild type, was derived by integration of pP7B6NI followed by the excision of pP7B6. Polymerase chain reaction tests demonstrated that the integration of nisI in the prophage region had occurred and that the erythromycin resistance marker from pP7B6 was lost. Fifteen among 31 L. plantarum strains tested hybridized with P7B6, indicating that the integration of pP7B6-derived vectors might occur in some other L. plantarum strains. This was experimentally confirmed by constructing the recombinant strain L. plantarum LZNI from the dairy isolate L. plantarum LZ (LMG 24600).     

Enhancement of Nisin Production by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis>

Lactococcus lactis subsp lactis BSA (L. lactis BSA) was isolated from a commercial fermented product (BSA Food Ingredients, Montreal, Canada) containing mixed bacteria that are used as starter for food fermentation. In order to increase the bacteriocin production by L. lactis BSA, different fermentation conditions were conducted. They included different volumetric combinations of two culture media (the Man, Rogosa and Sharpe (MRS) broth and skim milk), agitation level (0 and 100 rpm) and concentration of commercial nisin (0, 0.15, and 0.30 µg/ml) added into culture media as stimulant agent for nisin production. During fermentation, samples were collected and used for antibacterial evaluation against Lactobacillus sakei using agar diffusion assay. Results showed that medium containing 50 % MRS broth and 50 % skim milk gave better antibacterial activity as compared to other medium formulations. Agitation (100 rpm) did not improve nisin production by L. lactis BSA. Adding 0.15 µg/ml of nisin into the medium-containing 50 % MRS broth and 50 % skim milk caused the highest nisin activity of 18,820 AU/ml as compared to other medium formulations. This activity was 4 and ~3 times higher than medium containing 100 % MRS broth without added nisin (~4700 AU/ml) and 100 % MRS broth with 0.15 µg/ml of added nisin (~6650 AU/ml), respectively.     

Bacteriocin production by strain <Emphasis Type="Italic">Lactobacillus delbruecki</Emphasis>i ssp. <Emphasis Type="Italic">bulgaricus</Emphasis> BB18 during continuous prefermentation of yogurt starter culture and subsequent batch coagulation of milk

By screening for bacteriocin-producing lactic acid bacteria of 1,428 strains isolated from authentic Bulgarian dairy products, Lb. bulgaricus BB18 strain obtained from kefir grain was selected. Out of 11 yogurt starters containing Lb. bulgaricus BB18 and S. thermophilus strains resistant to bacteriocin secreted by Lb. bulgaricus BB18 a yogurt culture (S. thermophilus 11A+Lb. bulgaricus BB18) with high growth and bacteriocinogenic activity in milk was selected. Continuous (pH-stat 5.7) prefermentation processes were carried out in milk at 37 degrees C in a 2l MBR bioreactor (MBR AG, Zurich, Switzerland) with an IMCS controller for agitation speed, temperature, dissolved oxygen, CO2 and pH. Prefermented milk with pH 5.7 coagulated in a thermostat at 37 degrees C until pH 4.8-4.9. S. thermophilus 11A and Lb. bulgaricus BB18 grew independently in a continuous mode at similar and sufficiently high-dilution rates (D=1.83 h(-1)-S. thermophilus 11A; D=1.80 h(-1)-Lb. bulgaricus BB18). The yogurt cultures developed in a stream at a high-dilution rate (D=2.03-2.28 h(-1)). The progress of both processes (growth and bacteriocin production) depended on the initial ratio between the two microorganisms. The continuous prefermentation process promoted conditions for efficient fermentation and bacteriocinogenesis of the starter culture during the batch process: strong reduction of the times for bacteriocin production and coagulation of milk (to 4.5-5.0 h); high cell productivity (lactobacilli-4x10(12) CFU ml(-1), streptococci-6x10(12) CFU ml(-1)); high productivity of bacteriocins (4,500 BU ml(-1))-1.7 times higher than the bacteriocinogenic activity of the batch starter culture.     

Parameters affecting the adsorption of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum 423 isolated from sorghum beer

Plantaricin 423 is bactericidal to logarithmic and stationary-phase cells of Enterococcus sp. HKLHS and L. sakei DSM 20017. Detection of extracellular DNA and beta-galactosidase suggests that the mode of action is most probably by destabilizing of the cell membrane. Adsorption of plantaricin 423 to target cells ranged from 17% for Streptococcus caprinus ATCC 700066 to 67% for Lactobacillus plantarum LMG 13556, Lactobacillus curvatus DF38, Listeria innocua LMG 13568 and Lactobacillus sakei DSM 20017. Treatment of Enterococcus sp. HKLHS and L. sakei DSM 20017 with Triton X-100, Triton X-114 and chloroform increased the adsorption of plantaricin.     

Purification and characterization of plantaricin LR14: a novel bacteriocin produced by <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> LR/14

Bacteriocin produced by Lactobacillus plantarum strain LR/14 was purified to homogeneity by a multi-step protocol consisting of ammonium sulfate precipitation, cation-exchange chromatography, gel-filtration, and reverse-phase FPLC. L. plantarum LR/14 secreted a low-molecular-weight bacteriocin consisting of two peptides designated as plantaricin LR14alpha and -beta with molecular mass of 3,012.46 and 5,605.74 Da, respectively. The purified peptides were characterized to be highly thermostable and active in acidic pH range, with a pI of >10.0. Both alpha and beta peptides showed bactericidal mode of action against indicator strain, Micrococcus luteus and together showed a synergistic action. These peptides were differentially sensitive to a range of proteolytic enzymes, indicating differences in their composition. Amino acid sequencing revealed that the N-terminus in both the cases is blocked; thus, only a partial sequence could be obtained after CNBr digestion. These sequences, when compared with those available in the database, showed no homology with known bacteriocins, indicating it to be a novel compound.     

<Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> isolated from cheese: production and partial characterization of bacteriocin B391

Lactobacillus plantarum B391, a strain isolated from an artisanal French cheese, is a producer of a bacteriocin, expressing activity against Enterococcus faecalis NCTC 775, Clostridium perfringens NCTC 13170 and several Listeria monocytogenes strains. High stability was recorded after heat treatment at 121 °C for 20 min and when stored at 4 °C for more than 40 days. A challenge test performed in milk for 11 days showed potential for the control of L. monocytogenes. In the presence of the lytic bacteriocin B391, L. monocytogenes cells present numerous morphology modifications of cell shape and surface structure as well as in the cell division pattern, resulting ultimately in lysis. The high level of Listeria growth inhibition obtained in the presence of Lb. plantarum B391, and the stability of B391 bacteriocin for a long period of time, make this strain potentially interesting to use in milk products to increase food safety.     

In Vitro Antibacterial Activity of Phlorotannins from Edible Brown Algae, <Emphasis Type="Italic">Eisenia bicyclis</Emphasis> Against Streptomycin-Resistant <Emphasis Type="Italic">Listeria monocytogenes</Emphasis>

Listeria monocytogenes (LM) is an important food borne pathogen responsible for listeriosis. Further, LM is an etiological agent associated with life threatening conditions like meningitis and encephalitis. Biofilm forming and drug resistant LM may potentially become difficult to treat infections and hence effective controlling measures are required to prevent LM infections. In view of this, the present study evaluated an anti-listerial potential of edible brown seaweed, Eisenia bicyclis, by disc diffusion and micro-dilution methods. The results of the present study suggested that the anti-listerial activity of various phlorotannins isolated form E. bicyclis were in the range of 16–256 µg/ml. Among the phlorotannins isolated, fucofuroeckol-A (FAA) exhibited the highest anti-listerial potential (MIC range 16–32 µg/ml) against LM strains tested. Further, in checker board synergy assays, FFA-streptomycin combination exhibited significant synergy (fractional inhibitory concentration index, ∑FIC < 0.5) against aminoglycoside resistant clinical strains of LM. The results of the present study suggested the potential use of edible seaweed E. bicyclis as a source of natural phlorotannins to control food borne pathogenic infections.     

Nisin Z produced by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> from bullfrog hatchery is active against <Emphasis Type="Italic">Citrobacter freundii</Emphasis>, a red-leg syndrome related pathogen

Lactococcus lactis subsp. lactis CRL 1584 isolated from a bullfrog hatchery produces a bacteriocin that inhibits both indigenous Citrobacter freundii (a Red-Leg Syndrome related pathogen) and Lactobacillus plantarum, and Listeria monocytogenes as well. Considering that probiotics requires high cell densities and/or bacteriocin concentrations, the effect of the temperature on L. lactis growth and bacteriocin production was evaluated to find the optimal conditions. Thus, the growth rate was maximal at 36 °C, whereas the highest biomass and bacteriocin activity was achieved between 20 and 30 °C and 20–25 °C, respectively. The bacteriocin synthesis was closely growth associated reaching the maximal values at the end of the exponential phase. Since bacteriocins co-production has been evidenced in bacterial genera, a purification of the bacteriocin/s from L. lactis culture supernatants was carried out. The active fraction was purified by cationic-exchange chromatography and then, a RP-HPLC was carried out. The purified sample was a peptide with a 3353.05 Da, a molecular mass that matches nisin Z, which turned out to be the only bacteriocin produced by L. lactis CRL 1584. Nisin Z showed bactericidal effect on C. freundii and L. monocytogenes, which increased in the presence l-lactic acid?+?H₂O₂. This is the first report on nisin Z production by L. lactis from a bullfrog hatchery that resulted active on a Gram-negative pathogen. This peptide has potential probiotic for raniculture and as food biopreservative for bullfrog meat.     

Actinomycetemcomitin: a new bacteriocin produced by <Emphasis Type="Italic">Aggregatibacter</Emphasis> (<Emphasis Type="Italic">Actinobacillus</Emphasis>) <Emphasis Type="Italic">actinomycetemcomitans</Emphasis>

Aggregatibacter (Actinobacillus) actinomycetemcomitans P_7–20 strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30–60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45°C, and after treatment with proteolytic enzymes such as trypsin, α-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.     

Autohydrolysed <Emphasis Type="Italic">Tilapia nilotica</Emphasis> Fish Viscera as a Peptone Source in Bacteriocin Production

Fish processing generates large amounts of solid and liquid wastes. Many different by-products have been produced from fish processing wastes. Studies on solubilization of Bolti fish (Tilapia nilotica) viscera by endogenous enzymes at different pHs are described. Hydrolysis reactions were conducted with freshly thawed viscera utilizing an initial temperature gradient and terminated at various time points by heat inactivation of the enzymes. Various peptones obtained from hydrolysed visceral homogenates of Bolti fish residues showed their suitability for promoting the growth of lactic acid bacteria (mainly Lactobacillus sake Lb 706), microorganisms with particularly complex nutritional requirements especially peptidic sources. The assay of several treatments with L. sakei Lb 706, producer of the bacteriocin sakacin A, demonstrated that optimum conditions for biomass and bacteriocin production only imply a brief autohydrolysis at room temperature. The results showed that the Bolti fish hydrolysates gave remarkable results to those found in costly commercial media, specifically recommended for culturing and large-scale production of lactic acid bacteria.     

Cloning and heterologous expression of a bacteriocin sakacin P from <Emphasis Type="Italic">Lactobacillus sakei</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Sakacin P, a bacteriocin from Lactobacillus sakei, shows strong activity against food-borne pathogens such as Listeria monocytogenes. In L. sakei, the structural gene (sppA) encoding sakacin P is controlled by a strict regulatory mechanism, and the quantity of secreted sakacin P is limited. In this study, the sppA gene was synthesized by splicing overlap extension PCR and cloned into Escherichia coli. After the induction with isopropyl-β-d-thiogalactopyranoside, the recombinant sakacin P was successfully expressed. The collected cells were sonicated, and the activity was detected by agar diffusion method. The results also showed that the low-temperature induction can improve the activity of sakacin P.     

Properties of the strains<Emphasis Type="Italic">Enterococcus haemoperoxidus</Emphasis> and<Emphasis Type="Italic">E. moraviensis</Emphasis>, new species among enterococci

Antibiotic susceptibility or resistance, urease activity, detection of the structural genes for bacteriocin production, bacteriocin activity as well as sensitivity of the isolates to enterocins (Ent) A and M were determined in 23 isolates of new species Enterococcus haemoperoxidus and E. moraviensis. The majority of the strains were antibiotic sensitive and exhibited low urease activity (< 10 nkat/mL). The most frequently detected genes for Ent were entA and entP. However, only the strain 466 of E. haemoperoxidus produced an antibacterial substance with inhibitory activity against 21 G+ indicators. It was partially purified reaching an activity of up to 12 800 AU/mL. This bacteriocin active strain also possessed the genes for EntA and EntP. The other strains did not inhibit the indicator strains. The substance produced by the 466 strain was active even after a 5-months storage at +4 and -20 degrees C. This substance has proteolytic and hydrophilic character, pH optimum of bacteriocin production by this strain being between 4 and 7. While E. moraviensis strains showed sensitivity to EntA (produced by E. faecium EK13) and to EntM (produced by E. faecium AL41), E. haemoperoxidus strains were sensitive to EntA (except strain 382) but less sensitive to the treatment by EntM.     

Characterization of bacteriocins produced by strains of <Emphasis Type="Italic">Pediococcus pentosaceus</Emphasis> isolated from Minas cheese

Interest in obtaining bacteriocin-producing strains of lactic acid bacteria (LAB) from different sources has been increasing in recent years due to their multiple applications in health and food industries. This study focused on the isolation and characterization of metabolically active populations of bacteriocinogenic LAB and the evaluation of their antimicrobial substances as well as of some nutritional requirements of them. One hundred and fifty colonies of LAB from artisanal cheeses produced in Minas Gerais state (Brazil) were isolated and screened for their antimicrobial activity. According to their activity against Listeria monocytogenes, ten strains were selected and subsequently identified using biochemical and molecular techniques including 16s rRNA amplification and sequencing as Enterococcus faecalis, Lactobacillus spp., and Pediococcus pentosaceus. Antimicrobial substances produced by four of the selected strains, P. pentosaceus 63, P. pentosaceus 145, P. pentosaceus 146, and P. pentosaceus 147, were biochemically characterized, and presented sensitivity to proteolytic enzymes (suggesting their proteinaceous nature) and to extreme pH. Antimicrobial activity showed stability after treatment with lipase, catalase, α-amylase, and chemicals. Growth kinetics of the P. pentosaceus selected showed maximal bacteriocin production at 37 °C during the end of the exponential growth phase (25,600 AU/mL) and stable production during 24 h of incubation. Dextrose, maltose, and a mixture of peptone, meat extract, and yeast extract increased bacteriocin production. This study demonstrated that dairy products provide a good alternative for obtaining LAB, with the ability to produce antimicrobial substances such as bacteriocins that have potential use as biopreservatives in food.     

Adhesion of the probiotic strains <Emphasis Type="Italic">Enterococcus mundtii</Emphasis> ST4SA and <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> 423 to Caco-2 cells under conditions simulating the intestinal tract,and in the presence of antibiotics and anti-inflammatory medicaments

Adhesion of Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 to Caco-2 (human carcinoma epithelial) cells was visualized by fluorescent staining. Both strains showed good adhesion compared to L. casei MB1, L. casei Shirota, L. johnsonii La1 and L. rhamnosus GG. No correlation was found between hydrophobicity, aggregation and adhesion to Caco-2 cells. Presence of antibiotics and anti-inflammatory medicaments reduced adhesion of bacterial strains to Caco-2 cells. Proteins sensitive to pepsin, trypsin and pronase are involved in the adhesion of E. mundtii ST4SA and L. plantarum 423 to Caco-2 cells. Adhesion of Listeria monocytogenes ScottA to Caco-2 cells was not prevented by E. mundtii ST4SA and L. plantarum 423. Cell-free culture supernatants of strains ST4SA and 423, containing the antimicrobial peptides plantaricin 423 and peptide ST4SA, prevented the invasion of L. monocytogenes ScottA into Caco-2 cells.     

Mode of action of leucocin K7 produced by <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> K7 against <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and its potential in milk preservation

Objectives

To investigate the mode of action of leucocin K7 against Listeria monocytogenes and to assess its inhibitory effect on Lis. monocytogenes in refrigerated milk.

Results

A bacteriocin-producing strain, Leuconostoc mesenteroides K7, was isolated from a fermented pickle. The bacteriocin, leucocin K7, exhibited antagonistic activity against Lis. monocytogenes with an MIC of 28 µg/ml. It was sensitive to proteaseS and displayed good thermal stability and broad active pH range. Leucocin K7 had no effect on the efflux of ATP from Lis. monocytogenes but triggered the efflux of K⁺ and the intracellular hydrolysis of ATP. It also dissipated the transmembrane electrical potential completely and transmembrane pH gradient partially. It 80 AU/ml inhibited the growth of Lis. monocytogenes by 2.3–3.9 log units in milk; when combined with glycine (5 mg/ml), it completely eliminated viable Lis. monocytogenes over 7 days

Conclusion

Leucocin K7 shows different mode of action from nisin and may have potential application in milk preservation.

     

A Novel Bacteriocin-Like Substance Produced by <Emphasis Type="Italic">Enterococcus faecium</Emphasis> 3587

Secretion of a novel bacteriocin-like inhibitory substance from Enterococcus faecium 3587 is described herein for the first time. Whereas some bacteriocins receive their denomination based on the species or genus name of the producer microorganism, the newly discovered bacteriocin-like substance was named “enterocin 3587.” The growth characteristics of the producer strain, as well as the type of production and the primary characteristic of the peptide, were investigated. It was found by sodium dodecyl sulfate–polyacrylamide gel electrophoresis that the molecule possesses a molecular weight <6.5 kDa; its secretion is growth-phase dependent; and it shows activities only against other closely related enterococci but not against other Gram-positive bacteria, such as L. innocua, S. aureus 209, and Lactobacillus delbrueckii B2, nor against some Gram-negative species, such as Escherichia coli HB101.     

Development of<Emphasis Type="Italic"> Lactobacillus plantarum</Emphasis> LL441 and its plasmid-cured derivatives in cheese

A wild Lactobacillus plantarum strain and two of its plasmid-cured derivatives were separately used as adjunct cultures in the manufacture of a Gouda-like traditional Spanish cheese. The wild strain, LL441, harbours seven plasmids and produces a lantibiotic-like bacteriocin. The LL441-B2 derivative has lost plasmids of 40 and 80 kb and the bacteriocin-producing capability. The LL441-B11 derivative has lost in addition a 70 kb plasmid encoding active α- and β-galactosidases. All three strains could be used as adjunct cultures as none of the technological and biochemical parameters of the cheeses was affected. Both the wild-type and the two derivatives were recovered from experimental cheeses up to 30 days after manufacture at similar rates of nearly 20%. Thus, the phenotypic traits under examination were not essential for L. plantarum to grow into the cheese matrix. Electronic Publication