The effects on vegetative cells and spores of three bacteriocins from lactic acid bacteria

The sensitivities of vegetative cells of strains ofListeria, Clostridium, Staphylococcus, Lactococcus, Lactobacillus, MicrococcusandPediococcus, and of spores ofClostridiumandBacillusto three broad spectrum bacteriocins (nisin A, nisin Z and pediocin) from lactic acid bacteria were determined by a critical dilution micro-assay. The minimal inhibitory concentrations (MIC) of partially purified bacteriocins, prepared by a pH-dependent adsorption/desorption process, were determined and expressed in arbitrary units ml⁻¹and in μ g ml⁻¹of pure bacteriocin. The MICs of bacteriocins varied considerably between species and even between strains of the same species, as clearly shown for nine strains ofListeria monocytogenes. When bacteriocin activity was expressed in μ g ml⁻¹, pediocin was more effective againstListeria monocytogenesthan nisin A or nisin Z. The latter bacteriocins, in concentrations between 23 and 69 μ g ml⁻¹, prevented outgrowth ofClostridiumandBacillusspores for at least 10 days. Although pediocin at 17 μ g ml⁻¹prevented outgrowth ofB. stearothermophilusandC. butyricumspores for up to 7 days, it apparently activated the germination ofB. subtilisspores.     

Characterisation of an antiviral pediocin-like bacteriocin produced by Enterococcus faecium

The bacteriocin-producing strain Enterococcus faecium ST5Ha was isolated from smoked salmon and identified by biomolecular techniques. Ent. faecium ST5Ha produces a pediocin-like bacteriocin with activity against several lactic acid bacteria, Listeria spp. and some other human and food pathogens, and remarkably against HSV-1 virus. Bacteriocin ST5Ha was produced at high levels in MRS broth at 30 °C and 37 °C, reaching a maximum production of 1.0 × 10⁹ AU/ml, checked against Listeria ivanovii ATCC19119 as target strain and surrogate of pathogenic strain Listeria monocytogenes. The molecular weight of bacteriocin ST5Ha was estimated to be 4.5 kDa according to tricine-SDS-PAGE data. Ent. faecium ST5Ha harbors a 1.044 kb chromosomal DNA fragment fitting in size to that of pediocin PA-1/AcH. In addition, the sequencing of bacteriocin ST5Ha gene indicated 99% of DNA homology to pediocin PA-1/AcH. The combined application of low levels (below MIC) of ciprofloxacin and bacteriocin ST5Ha resulted in a synergetic effect in the inhibition of target strain L. ivanovii ATCC19119. Bacteriocin ST5Ha displayed antiviral activity against HSV-1, an important human pathogen, with a selectivity index of 173. To the best of our knowledge, this is the first report on Ent. faecium as a potential producer of pediocin-like bacteriocin with antiviral activity.     

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.     

Antilisterial and amylase-sensitive bacteriocin producing Enterococcus faecium SH01 from Mukeunji,a Korean over-ripened kimchi

A bacteriocinogenic strain identified as Enterococcus faecium SH01 was isolated from mukeunji, a Korean traditional over-ripened kimchi with antimicrobial activities against Listeria monocytogenes KCTC 3569 and Lactobacillus curvatus KFRI 166. The maximum bacteriocin titer (1,280 AU/mL) was detected at the early stationary phase and was maintained for 28 h with no activity loss. The bacteriocin activity, which disappeared after treatment with the proteolytic enzymes α-chymotrypsin, pronase E, proteinase K, and trypsin, was partially inactivated using α-amylase. The activity of bacteriocin SH01 remained after heat treatment (121°C, 15 min) and exposure to pH values from 2–12. The molecular weight of crude bacteriocin SH01 was 3 kDa. The bacteriocin production phenotype (Bac⁺) was linked to a 6 kb plasmid. Bacteriocin SH1 production was not induced by the co-presence of viable cells, heat treatment, or a cell-free indicator supernatant. The mode of action of bacteriocin SH1 was bactericidal.     

Immunity gene pedB enhances production of pediocin PA-1 in naturally resistant Lactococcus lactis strains

Heterologous production of the antilisterial bacteriocin pediocin PA-1 in lactococci is an attractive objective to increase the safety of dairy products. In a previous paper, we developed a system for the heterologous production of the bacteriocin pediocin PA-1 in pediocin-resistant lactococcal hosts through a leader exchange strategy. The system was based on 3 genes, 1 encoding the fusion between the lactococcin A leader and propediocin PA-1, and the other 2 encoding the lactococcin A secretion machinery. In this study, we investigated whether the addition of the pediocin PA-1 immunity gene (pedB) to this system has any effect on pediocin production. Introduction of the plasmid(s) carrying the genes described above into nisinproducing and non-nisinproducing lactococcal hosts led to a significant increase in the production of pediocin compared with the equivalent pedB-devoid systems. In addition, we obtained a nisin-producing strain with the ability to secrete pediocin PA-1 at a level equivalent to that of the parental strain Pediococcus acidilactici 347, which represents a notable improvement over our previous systems.     

Inhibition ofListeria monocytogenesandStaphylococcus aureusby enterocin CCM 4231 in milk products

The inhibitory effect of purified enterocin CCM 4231 (concentration 3200 AU/ml) on the growth ofStaphylococcus aureusSA1 strain in skimmed milk as well as in the yoghurt-making procedure was investigated. The decrease in viable cells of SA1 strain in skimmed milk from 10¹⁰cfu ml^–1in the control sample (CS) up to 10²cfu ml^–1in the experimental sample (ES) was detected 24 h after enterocin addition. In the case of the yoghurt-making procedure, significant inhibition of SA1 strain was noted after 3·5 h from enterocin CCM 4231 addition (10³cfu ml^–1in ES, 10⁶cfu ml^–1in CS). The lowest pH value of final product was determined in yoghurt containing enterocin. In addition, the antilisterial effect of enterocin CCM 4231 in yoghurt milk was analysed, and an expressive inhibitory effect of enterocin CCM 4231 added toListeria monocytogenesOHIO culture in yoghurt was found. The decrease of viable bacterial cells by 3.77 log cfu ml^–1in ES was detected in comparison with CS. Although the inhibitory effect of enterocin added was confirmed, no bacteriocin activity could be detected analytically in the samples. Results presented indicate that the enterocin CCM 4231 strain has possibilities for further application as a protective culture in some milk products vsListeriaandStaphlococcus. However, major obstacles preventing this application at present are legislation and lack of experimental data.     

Bacteriocinogenic Lactobacillus plantarum ST16Pa isolated from papaya (Carica papaya) — From isolation to application: Characterization of a bacteriocin

Strain ST16PA, isolated from papaya was identified as Lactobacillus plantarum based on biochemical tests, PCR with species-specific primers and 16S rDNA sequencing. L. plantarum ST16PA produces a 6.5 kDa bacteriocin, active against different species from genera Enterobacter, Enterococcus, Lactobacillus, Pseudomonas, Streptococcus and Staphylococcus and different serotypes of Listeria spp. The peptide is inactivated by proteolytic enzymes, but not when treated with ??-amylase, catalase, lipase, Triton X-100, SDS, Tween 20, Tween 80, urea, NaCl and EDTA. However, presence of 1% Triton X-114 deactivates the bacteriocin. No change in activity was recorded after 2 h at pH values between 2.0 and 12.0, and after treatment at 100 °C for 120 min or 121 °C for 20 min. The mode of activity against Lactobacillus sakei ATCC 15521, Enterococcus faecalis ATCC 19443 and Listeria innocua 2030C was bactericidal, resulting in cell lysis and enzyme-leakage. No significant differences in cell growth and bacteriocin production were observed when strain ST16Pa was cultured in MRS broth at 26 °C and 30 °C for 24 h (25 600 AU/ml). However, even though strain ST16PA grows well in MRS broth at 15 °C and 37 °C, a reduction of bacteriocin production was observed (400 AU/ml and 1600 AU/ml, respectively). In addition, effect of MRS medium components, different initial pH and additions of glycerol or vitamins to the media on bacteriocin ST16Pa production was studied.Peptide ST16PA adsorbs (400 AU/ml) to producer cells. However, bacteriocin ST16Pa was adsorbed at 50% to cells of L. innocua 2030C and at 75% to L. sakei ATCC 15521 and E. faecalis ATCC 19433 when experiments were conducted at 30 °C and pH 6.5. Adsorption of bacteriocin ST16Pa to target cells at different temperatures, pH and in presence of potassium sorbate, sodium nitrate, sodium chloride, ascorbic acid, Tween 80 and Tween 20 were also studied. To the best of our knowledge, this is the first report on detection of L. plantarum in papaya.     

Production of β-galactosidase for lactose hydrolysis in milk and dairy products using thermophilic lactic acid bacteria

The aim of this work was to establish optimal conditions for the maximum production of β-galactosidase using an industrially suitable medium. Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was cultivated in skim milk, whey and whey permeate basal media, supplemented with whey protein products, yeast extract or De Man–Rogosa–Sharpe (MRS) broth, at pH 5.6 and 43°C. All supplementations of the whey and whey permeate basal media resulted in the enhancement of the specific growth rates, rate of lactic acid production and β-galactosidase activity. However, unsupplemented skim milk gave the greatest rate of lactic acid production (3.50±0.269 mg lactic acid ml⁻¹ media h⁻¹) and the highest β-galactosidase activity (5.491±0.116 U activity ml⁻¹ media); far superior to the best whey-based medium supplemented with MRS (2.71±0.176 mg lactic acid ml⁻¹ media h⁻¹ and 3.091±0.089 U activity ml⁻¹ media, respectively). A technologically feasible approach for the reprocessing of the spent skim milk was tested and a conceptual process scheme is proposed.     

Isolation and characterisation of the bacteriocin-producing Leuconostoc citreum HW02 from malts

Herein, a lactic acid bacterium which produces bacteriocin was isolated from malted barley. The isolated strain was identified by 16 S rRNA analysis and designated Leuconostoc citreum HW02. Bacteriocin HW02 was shown to have growth inhibition against health-threatening Gram-positive microorganisms such as Staphylococcus aureus, Listeria monocytogenes, and Latilactobacillus curvatus. Bacteriocin HW02 was reported to be incapable of inhibiting the growth of health-threatening microorganisms after treatment with proteolytic enzymes; thus, it was indicated that bacteriocin is proteinaceous in nature. Bacteriocin HW02 showed stability against a broad range of temperatures (60–121 °C), pH (2–10), various detergents, or organic solvents. Its antimicrobial activity against the indicator strain, Lactiplantibacillus plantarum NCDO 955, was maximised (320 AU mL⁻¹) midway through the stationary phase at 12 h. The crude bacteriocin HW02 molecular weight was estimated to be about 7.7 kDa. After the treatment of the indicator strain with bacteriocin, the maintenance of the optical density of the culture, the morphological analysis of cell-structure abnormalities, and cell shrinkage implied that bacteriocin showed bactericidal activity.     

Growth of Carnobacterium divergens M35 and production of Divergicin M35 in snow crab by-product, a natural-grade medium

This work aimed to study the factors influencing the growth of Carnobacterium divergens strain M35 and its ability to produce a new class IIa bacteriocin (divergicin M35) in various synthetic media and in medium supplemented with snow crab hepatopancreas (SCH), a natural-grade by-product of crustacean processing. C. divergens M35 growth and bacteriocin production in SCH-supplemented medium was evaluated at different temperatures in batch fermentations and under controlled pH. C. divergens M35 was shown to grow well in SCH medium at tested temperatures between 4 and 30 °C, except at 37 °C. Maximum divergicin M35 production was obtained after 10 h of growth in SCH medium at 25 and 30 °C with a total activity of 3.7 × 10⁴ AU mL⁻¹. Less growth was observed at 37 °C, for which a total bacteriocin activity of only 256 AU mL⁻¹ was obtained. The production of divergicin M35 was greatly influenced by the medium composition, especially by the type of added carbon source. The best production of divergicin M35 in SCH medium was observed at a controlled pH of 7.0. This study describes the optimal parameters for the growth of C. divergens M35 and production of divergicin M35 and demonstrates the effectiveness of the marine by-product as a medium supplement for this culture.     

The conjugal plasmid pLL10236 encodes lactose fermentation ability,restriction/modification activity,bacteriocin production and immunity in Lactococcus lactis subsp. lactis LL102

A 36 kb plasmid, designed as pLL 10236, was determined in Lactococcus lactis subsp.Lactis LL102. This plasmid mediates lactose fermentation ability (Lac⁺), bacteriocin production (Bac⁺) and immunity (Imm⁺), and restriction/modification (R⁺M⁺) activity against bacteriophage Ø1120 in strain LL102. The conjugal ability of pLL 10236 and expression of pLL 10236 encoding traits were determined by using a Lac⁻, Bac⁻, Imm⁻and (R⁻M⁻) recipient strain L. lactis subsp. lactis P81-1.     

Biochemical,antimicrobial and molecular characterization of a noncytotoxic bacteriocin produced by Lactobacillus plantarum ST71KS

Lactobacillus (Lb.) plantarum ST71KS was isolated from homemade goat feta cheese and identified using biochemical and molecular biology techniques. As shown by Tricine-SDS-PAGE, this lactic acid bacterium produces a bacteriocin (ST71KS) with an estimated molecular weight of 5.0 kDa. Bacteriocin ST71KS was not affected by the presence of α-amylase, catalase and remained stable in a wide range of pH and after treatment with Triton X-100, Triton X-114, Tween 20, Tween 80, NaCl, SDS, urea and EDTA. This bacteriocin also remained active after being heated at 100 °C for 2 h and even after 20 min at 121 °C; however, it was inactivated by proteolitic enzymes. Production of bacteriocin ST71KS reached 6400 AU/mL during stationary growth phase of Lb. plantarum cultivated in MRS at 30 °C and 37 °C. Bacteriocin ST71KS displayed a bactericidal effect against Listeria monocytogenes strains 603 and 607 and did not adsorb to the producer cells. Lb. plantarum ST71KS harbors two bacteriocin genes with homology to plantaricin S and pediocin PA-1. These characteristics indicate that bacteriocin ST71KS is a class IIa bacteriocin. The peptide presented no toxic effect when tested in vitro with kidney Vero cells, indicating safe technological application to control L. monocytogenes in foods.     

Temperature effect on the biological activity of Bifidobacterium longum CRL 849 and Lactobacillus fermentum CRL 251 in pure and mixed cultures grown in soymilk

The influence of temperature on the growth and biological activity of two probiotic strains (Bifidobacterium longum CRL 849 and Lactobacillus fermentum CRL 251) as pure and mixed cultures in soymilk (SM) were evaluated. Maximum growth was observed at 37°C in both mixed and pure cultures. In a product prepared with the mixed culture (1:1) at 37°C, the amount of lactic acid produced was approximately 55 mmol l⁻¹ after 24 h with a slow production rate (2.8 mmol l⁻¹ h⁻¹); the formation of acetic acid was higher with respect to pure cultures (82.01 mmol l⁻¹ after 24 h), and final pH (24 h) was 5.0. About 85% of the total amount of sugars in SM was reduced, mainly sucrose. Stachyose was reduced (71%) after 4 h of incubation. Maximum activity of alpha-galactosidase (alpha-gal) (13.2 U ml⁻¹) was observed after 6 h. At 37°C the bifidobacterium strain was viable in mixed culture throughout the period assayed. At lower (30°C) or higher (42°C) temperatures, mixed culture showed slower growth and lower acid production in SM but the alpha-gal activity was stimulated at 30°C.     

Detection of pediocin PA-1-producing pediococci by rapid molecular biology techniques

Five bacteriocinogenic lactic acid bacteria strains (347, X13, Z102, A172 and P20) independently isolated from fermented sausages were identified asPediococcus acidilacticiby carbohydrate fermentation patterns and other biochemical characteristics. This fact, together with their activity againstListeria monocytogenes, suggested that they could be pediocin PA-1 producers. Rapid molecular biology techniques were used to detect the pediocin PA-1 operon in these strains. PCR, dot-blot and Southern hybridization, and DNA sequencing results confirmed that all of them had the genetic determinants for pediocin PA-1 biosynthesis encoded in a 9.4 kb plasmid. The bacteriocin produced byP. acidilactici347 has been purified by a procedure that included ammonium sulphate precipitation and cation exchange, hydrophobic-interaction and reverse-phase chromatography. The amino acid sequence of this bacteriocin was identical to pediocin PA-1, confirming the expression of the pediocin PA-1 genes.     

Bactericidal synergism through bacteriocins and high pressure in a meat model system during storage

High hydrostatic pressure represents an attractive non-thermal process for meat products to avoid post-processing contamination. When combined with antimicrobials, like bacteriocins, the death rate may be increased because of sub-lethal injuries to living cells. The behaviour of several foodborne bacteria inoculated in a meat model system with added bacteriocins (enterocins A and B, sakacin K, pediocin AcH or nisin) after pressurization (400 MPa, 10 min, 17°C) and during chilled storage was investigated. Although Staphylococcus was the genus least sensitive to pressurization, the samples including nisin displayed lower and significantly different counts during the 4°C storage than the rest of the treatments. A greater inactivation of Escherichia coli (>6 log₁₀) in the presence of nisin was recorded, the number of survivors remained unchanged during storage at 4°C for 61 days. Nisin was also the bacteriocin capable of maintaining slime-producing lactic acid bacteria below the detection limit (<10² cfu g⁻¹ ). Listeria monocytogenes in treatments with sakacin, enterocins or pediocin was kept <10² cfu g⁻¹ till the end of storage (61 days). Salmonella enterica subsp. enterica ser London and Salmonella enterica subsp.enterica ser Schwarzengrund counts in every treatment were kept at the level obtained after pressurization, with no significant differences between treatments during the chilled storage.     

Production of pediocin PA-1, and coproduction of nisin A and pediocin PA-1, by wild Lactococcus lactis strains of dairy origin

Heterologous production of pediocin PA-1 in nisin and non-nisin-producing Lactococcus lactis strains, which had been previously selected because of their technological properties for cheese making, was investigated. Plasmid pFI2160, which contains a hybrid gene (L-pedA) encoding the fusion between the lactococcin A leader and propediocin PA-1, and also the genes lcnC and lcnD, that encode the lactococcin A secretion apparatus, was introduced into L. lactis ESI 153 and L. lactis ESI 515 (Nis⁺). The pediocin production level of their respective transformants, L. lactis CL1 and L. lactis CL2 (Nis⁺), was approximately 600 and 400 ng mL⁻¹, respectively, which represents a 30% and a 20% of the quantity produced by the natural pediocin PA-1 producer Pediococcus acidilactici 347. Transformation of L. lactis ESI 515 with pFI2160 did not affect its ability to produce nisin. Pediocin bioassays showed the stability of pFI2160 in both heterologous hosts under selective and non-selective conditions.     

Nisin-curvaticin 13 combinations for avoiding the regrowth of bacteriocin resistant cells of Listeria monocytogenes ATCC 15313

Nisin (25-100 IU/ml) and curvaticin 13 (160 AU/ml), a bacteriocin produced by Lactobacillus curvatus SB13, were shown to have a bactericidal effect against Listeria monocytogenes ATCC 15313 in TSB-YE broth (pH 6.5), but it was only transitory. Regrowth was not due to the loss of bacteriocin activity. Cells surviving nisin or curvaticin 13 were more resistant to the respective bacteriocin than the parental strain. Survivors to curvaticin 13 were resistant to the class IIa bacteriocins (camocin CP5, pediocin AcH) but remained sensitive to nisin. The frequencies of spontaneous nisin resistants decreased with increasing bacteriocin concentration and the presence of salts (NaCl, K2HPO4). The behaviour of nisin (1000 IU/ml) or curvaticin 13 (640 AU/ml) resistant variants (Nis1000, Curv645) was investigated in the presence of nisin (100 IU/ml) or curvaticin 13 (320 AU/ml) at 22 and 37 degrees C, and compared with that of the parental strain. The effectiveness of nisin was the same at both temperatures, whereas curvaticin 13 displayed a faster bactericidal action at 37 degrees C. Nis1000 cells were less sensitive to curvaticin 13 than the parental strain, whereas Curv640 cells were more sensitive to nisin than the parental strain. Simultaneous or sequential additions of nisin (50 IU/ml) and curvaticin 13 (160 AU/ml) were performed at 22 degrees C in broth inoculated with the parental strain. All combinations induced a greater inhibitory effect than the use of a single bacteriocin. Simultaneous addition of bacteriocins at t0 led to the absence of viable cells in the broth after 48 h.     

Combined use of bacteriocin‐producing strains to control Listeria monocytogenes regrowth in raw pork meat

Avoiding the presence of Listeria in meat and dairy products is a major challenge for the food industry. In this work, a Lactobacillus curvatus strain producing the bacteriocin sakacin P and a Pediococcus acidilactici strain producing another bacteriocin, pediocin AcH, were used as starter cultures under laboratory conditions in a Listeria‐seeded raw‐pork‐meat matrix, which was then stored for 6 weeks at 4 °C. At 1 week intervals during the storage period, the antilisterial activity was evaluated. When either strain was added alone, the Listeria monocytogenes cfu count initially dropped from 10² cfu g^?1 to an undetectable level by the end of week 1 or 2, but this was followed by a rebound (regrowth) 1 week later. When both strains were added together to the meat matrix, rebound was delayed, Listeria remaining undetected from the end of week 1 to the end of week 5. A rebound was observed 6 weeks post‐inoculation, but fewer than 10 cfu g^?1 were counted. The use of more than one bacteriocin‐producing strain may thus overcome some of the problems limiting the effectiveness of bacteriocins in food systems.     

Antimicrobial activity of pediocin PA-1 against Oenococcus oeni and other wine bacteria

Pediocin PA-1 is an antimicrobial peptide produced by lactic acid bacteria (LAB) that has been sufficiently well characterised to be used in food industry as a biopreservative. Sulphur dioxide is the traditional antimicrobial agent used during the winemaking process to control bacterial growth and wine spoilage. In this study, we describe the effect of pediocin PA-1 alone and in combination with sulphur dioxide and ethanol on the growth of a collection of 53 oenological LAB, 18 acetic acid bacteria and 16 yeast strains; in addition, production of pediocin PA-1 by Pediococcus acidilactici J347-29 in presence of ethanol and grape must is also reported. Inhibitory concentrations (IC) and minimal bactericide concentrations of pediocin PA-1 were determined against LAB, and revealed a bacteriostatic effect. Oenococcus oeni resulted more sensitive to pediocin PA-1 (IC₅₀ = 19 ng/ml) than the other LAB species (IC₅₀ = 312 ng/ml). Cooperative inhibitory effects of pediocin PA-1 and either sulphur dioxide or ethanol were observed on LAB growth. Moreover, the pediocin PA-1 producing P. acidilactici strain J347-29 was able to grow and produce the bacteriocin in presence of ethanol (up to 4% ethanol in the fermentation broth) and grape must (up to 80%), which indicated that pediocin PA-1 can be considered as a potential biopreservative in winemaking.