A comparative study on the use of flow cytometry and colony forming units for assessment of the antibacterial effect of bacteriocins

Flow cytometry was investigated as a rapid method to determine the antibacterial effect of the bacteriocins nisin, pediocin PA-1, and sakacin A on the indicator organisms Lactobacillus reuteri DSM 12246, Lactobacillus sakei NCFB 2714 and Lactobacillus sakei DSM 20017, respectively. Fluorescence intensities of the cells were measured by flow cytometry upon exposure to bacteriocins after staining with carboxyfluorescein diacetate (cFDA) and were compared to the number of colony forming units (CFU). The fluorescence index (FI) of the bacterial populations decreased when exposed to the bacteriocins. For the different bacteriocins the pattern of decreases in FI and colony forming units differed at equal bacteriostatic concentrations. FI was the most sensitive measure of bacteriocin activity, i.e. the decrease in FI was observed at lower bacteriocin concentrations than decrease in CFU. It was demonstrated that the decrease in FI was caused by rapid leakage of carboxyfluorescein from cells exposed to pediocin. Cells showing severe leakage after pediocin treatment could be detected as CFU when transferred to a rich medium. Such a repair was less pronounced for cells exposed to sakacin and very limited for cells exposed to nisin. The influence of temperature and NaCl in combination with pediocin on FI and CFU of Lactobacillus sakei NCFB 2714 was examined at conditions relevant to foods. At all temperatures (5, 10, 20 and 37 degrees C) and NaCl concentrations (0, 2 and 4% w/v) investigated the flow cytometric measurements were significantly more sensitive compared to CFU. Both methods showed that the inhibitory effect of pediocin increased with increasing temperatures and decreased with increasing NaCl concentrations.     

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.     

Comparison of the sensitivity of commercial strains and infant isolates of bifidobacteria to antibiotics and bacteriocins

Eighteen bifidobacteria, six isolated from infant feces and 12 commercial strains (of which 10 were purchased from the American Type Culture Collection), were tested for sensitivity to 14 antibiotics and four bacteriocins including nisin A, nisin Z, pediocin PA-1 and mutacin B-Ny266. All bacteria were resistant to vancomycin, kanamycin, neomycin and streptomycin. Infant isolates were more sensitive than commercial strains to cloxacillin, ampicillin, chloramphenicol, tetracycline, rifampicin and novobiocin. Sensitivity to bacteriocins determined by a microplate assay varied widely. Generally, nisin A was the most effective bacteriocin followed by nisin Z and mutacin B-Ny266. Pediocin PA-1 appeared to have no inhibition at 70 μg mL⁻¹. Commercial strains showed relatively variable sensitivity to bacteriocins compared to infant isolates, which were inhibited within a narrow range of bacteriocin concentration. Bacteriocin tolerance could be easily gained by prolonging the exposure time. Death–time curves and transmission electron microscopy (TEM) of logarithmic and stationary cells of two strains (Bifidobacterium adolescentis ATCC 15704 and infant isolate Bifidobacterium sp. RBL67) incubated with twice the minimum inhibitory concentration of nisin A and nisin Z for 3 h revealed that log-phase cells were more sensitive than stationary-phase cells. TEM showed that the cell membrane is the most likely site of bactericidal effects of nisin.     

Effects of combinations of lactoperoxidase system and nisin on the behaviour of Listeria monocytogenes ATCC 15313 in skim milk

Individual or combined effects of nisin (100 or 200 IU/ml) and the lactoperoxidase system (LPS) were analysed against 1 x 10(4) cfu/ml Listeria monocytogenes ATCC 15313 cells in skim milk, at 25 degrees C for 15 days. Nisin induced an immediate bactericidal effect and LPS a 48 h bacteriostatic phase which in both cases was followed by re-growth of L. monocytogenes. LPS and nisin added together at t0 showed a synergistic and lasting bactericidal effect which after 8 days and until 15 days resulted in no detectable cells in 1 ml of milk. When LPS was added to cells already in contact with 100 or 200 IU/ml nisin for a period of 4 h, the inhibitory activity was enhanced with no L. monocytogenes detectable after 72 or 48 h, respectively, and until 15 days. When LPS was added after 12 h, the nisin bactericidal phase was followed by re-growth. When nisin, 100 or 200 UI/ml, was added to cells already in contact with LPS over 24 h, L. monocytogenes was not detectable after 196 and 244 h, respectively, without any re-growth. For nisin addition after 72 h, cell counts were 8 log10 cycles lower than in the control milk after 196 h, but population levels were similar to the control within 15 days. The best combination to inhibit L. monocytogenes ATCC 15313 was nisin present at t0 followed by the LPS addition 4 h later, when the maximum inhibitory effect of nisin was reached.     

Inhibition of Listeria monocytogenes by in situ produced and semipurified bacteriocins of Carnobacterium spp. on vacuum-packed, refrigerated cold-smoked salmon

Listeria monocytogenes inhibition by Carnobacterium strains and crude bacteriocins on sterile and commercial vacuum-packed cold-smoked salmon stored at 4 degrees C and 8 degrees C was investigated. Carnobacterium piscicola V1 was bactericidal against L. monocytogenes at the two temperatures, whereas Carnobacterium divergens V41 presented a bacteriostatic effect. C. piscicola SF668 delayed L. monocytogenes growth at 8 degrees C and had a bacteriostatic effect at 4 degrees C. Listeria growth was not affected by a non-bacteriocin-producing C. piscicola. Crude extracts of piscicocins were bactericidal at 4 degrees C and 8 degrees C. Listeria growth was delayed by divercin V41 at 8 degrees C and was inhibited at 4 degrees C. Nisin delayed Listeria growth at 8 degrees C and was bacteriostatic at 4 degrees C. The present study demonstrates that L. monocytogenes growth could be prevented on vacuum-packed cold-smoked salmon by Carnobacterium and associated bacteriocins at chilled temperatures. Moreover, no product spoilage could be observed with the use of such bacteriocin-producing strains as demonstrated by good sensorial analyses and low biogenic amine production.     

Class I/Class IIa bacteriocin cross-resistance phenomenon in Listeria monocytogenes

Variants resistant to nisin A (vA), nisin Z (vZ), pediocin PA-1 (vP), divergicin M35 (vD) and to bacteriocin-like compounds produced by Bifidobacterium thermophilum subsp. infantis RBL67 (vB) were developed from Listeria monocytogenes LSD530. Lactic acid production, specific growth rate, potassium ion efflux, susceptibility to 13 antibiotics, cell-envelope fatty acid composition and bacteriocin cross-resistance were evaluated. Lactic acid production decreased to 75% or less of that by strain LSD530 for vP, vD and vB and to 20% or less for vA and vZ. Specific growth rates also decreased for all five variants. Acquired resistance to nisin A or Z increased resistance to pediocin and divergicin while vD showed increased resistance to nisin Z but decreased resistance to nisin A and vP exhibited increased resistance to nisin Z, pediocin and divergicin but decreased resistance to nisin A. Acquired bacteriocin resistance generally decreased antibiotic sensitivity, particularly to ampicillin, chloramphenicol, erythromycin and tetracycline. Palmitic acid (C(16:0)) in the cell wall fraction of all variants was significantly higher than in strain LSD530, accounting for 18%, 43%, 32%, 26%, 53% and 44% of the total fatty acids for LSD530, vP, vD, vB, vA, and vZ, respectively. The relationship between the acquisition of bacteriocin resistance, cross-resistance and pathogenicity of Listeria monocytogenes should be studied.     

Comparative antimicrobial activity of enterocin L50, pediocin PA-1, nisin A and lactocin S against spoilage and foodborne pathogenic bacteria

The present work compares, under the same stated experimental conditions, the antimicrobial activity of crude and purified enterocin L50, pediocin PA-1, nisin A and lactocin S, produced by lactic acid bacteria (LAB) isolated from Spanish dry-fermented sausages. The bacteriocins were purified to homogeneity by ammonium sulphate precipitation, gel filtration (for lactocin S), and cation-exchange, hydrophobic-interaction, and reverse-phase-chromatography; high yields of pure bacteriocins were obtained. Minimal inhibitory concentration (MIC) of pure enterocin L50, pediocin PA-1, nisin A and lactocin S was determined against a broad spectrum of Gram-positive bacteria, including spoilage and foodborne pathogenic bacteria. The purified bacteriocins showed a broad antimicrobial spectrum similar to that exerted by crude bacteriocins. Enterocin L50 and pediocin PA-1 were very active againstListeria monocytogenes, which was quite resistant to nisin A and lactocin S. Enterocin L50 also displayed antimicrobial activity againstStaphylococcus aureus,Clostridium perfringensandClostridium botulinum. However, these pathogens were weakly inhibited, or not at all, by the other pure bacteriocins.     

Nutritional factors affecting the production of two bacteriocins from lactic acid bacteria on whey

The ability of Lactococcus lactis subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 to produce bacteriocins on both diluted and concentrated whey was investigated in batch fermentations. Both strains produced the higher amounts of biomass and bacteriocin titres on diluted whey. Luedeking and Piret expression was able to model the production of nisin, which was produced as a primary metabolite on both culture media. However, the pediocin production could not be typified in any case due to the negligible growth of P. acidilactici. Although the whey supported the growth and bacteriocin production by the two strains, both biomass and bacteriocin productions were lower than those obtained on MRS broth. The effect of total sugar, nitrogen, phosphorous and buffer concentrations on the production of nisin and pediocin was studied in diluted whey using factorial experiments and empirical modelling. The production of nisin was greatly inhibited by the increase in nitrogen, buffer, and to a lesser extent, sugar concentration in the medium, nevertheless, the used phosphorous source produced a light stimulatory effect on bacteriocin synthesis. In addition, the growth of Lc 1.04 was mainly affected by the nitrogen source used. On the other hand, pediocin was inhibited by the increase in buffer, phosphorous, and to a lesser degree, by the sugar and nitrogen concentration.

The inhibitory activity of pediocin disappeared almost totally after 15 min of treatment with trypsin, papain, subtilisin and pepsin. The activity of nisin was drastically reduced by treatment with trypsin, subtilisin and pepsin. Nevertheless, 50% of the initial activity was retained when nisin was treated with papain. Both bacteriocins showed the highest heat stability at acidic pH and short incubation times.     

INABILITY OF HEAT STRESS TO AFFECT SENSITIVITY OF LISTERIA MONOCYTOGENES TO PEDIOCIN IN PORK

The effect of heat-shock (48C for 20 min) on sensitivity of Listeria monocytogenes Scott A to bacteriocin produced by Pediococcus acidilactici was evaluated. Exposure of Listeria to pediocin (68, 267 AU/ mL) resulted in lower number of survivors in the first 2 h of incubation in Trypticase soy broth at 37C compared with samples without pediocin, regardless of whether the cells were heat-shocked or not. Further incubation for 24 h resulted in resumption of growth by L. monocytogenes, with counts reaching the same level as samples without pediocin. Cold storage of Listeria in pork in the presence of pediocin (8,192 AU/g) resulted in a 2.0 log₁₀ reduction after 24 h, when compared with controls not exposed to pediocin, regardless of whether the samples were stored under air, vacuum, or a modified atmosphere. Prior heat-shocking did not have an effect on sensitivity of Listeria to pediocin during storage. Thus, storage atmosphere and exposure to heat shock, which are known to induce thermotolerance in L. monocytogenes, had no effect in sensitivity of this organism to pediocin .     

Antimicrobial activity of reuterin in combination with nisin against food-borne pathogens

Antimicrobial activity of reuterin individually or in combination with nisin against different food-borne Gram-positive and Gram-negative pathogens in milk was investigated. Reuterin (8 AU/ml) exhibited bacteriostatic activity against Listeria monocytogenes, whereas its activity was slightly bactericidal against Staphylococcus aureus at 37 degrees C. Higher bactericidal activity was detected against Escherichia coli O157:H7, Salmonella choleraesuis subsp. choleraesuis, Yersinia enterocolitica, Aeromonas hydrophila subsp. hydrophila and Campylobacter jejuni. A significant synergistic effect on L. monocytogenes and a slight additive effect on S. aureus after 24 h at 37 degrees C were observed when reuterin was combined with nisin (100 IU/ml). The combination of reuterin with nisin did not enhance the antimicrobial effect of reuterin against Gram-negative pathogens.     

Viability loss and morphology change of foodborne pathogens following exposure to hydrostatic pressures in the presence and absence of bacteriocins

Cell suspensions of three pathogens were exposed to hydrostatic pressure (HP), bacteriocin mixture (nisin and pediocin) or a combination of HP+bacteriocins and changes in colony forming units (cfu) and cell-morphology by scanning electron microscopy (SEM) were studied. Cell viability loss, as determined from the reduction in cfu before and after a treatment, occurred in Listeria monocytogenes by all three treatments and in Salmonella typhimurium and Escherichia coli O157:H7 by HP and HP+bacteriocin combination. Cell wall and cell membrane collapse and cell lysis was indicated in L. monocytogenes exposed to bacteriocin or HP+bacteriocin and in Salmonella and E. coli exposed to HP or HP+bacteriocin.     

In vitro assessment of the cytotoxicity of nisin,pediocin, and selected colicins on simian virus 40-transfected human colon and Vero monkey kidney cells with trypan blue staining viability assays

Gram-positive bacterial bacteriocins (nisin and pediocin) and gram-negative bacterial bacteriocins (colicins [Col] E1, E3, E6, E7, and K) were evaluated for cytotoxicity against cultured simian virus 40-transfected human colon (SV40-HC) and Vero monkey kidney (Vero) cells. Bacteriocin-treated cells were assessed for viability by trypan blue staining. Monolayers of SV40-HC and Vero cells were cultured in tissue culture plates (35 degrees C, 10% CO2 in humidified air) with the use of Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) calf serum. Actively growing cells in the log phase (ca. 10(4) cells per ml) were treated with individual partially purified bacteriocin preparations at 170, 350, and 700 activity units per ml. Duplicate culture plates for each bacteriocin treatment and untreated controls were withdrawn after 16, 32, and 48 h of incubation. Cells were dissociated with trypsin and treated with trypan blue and were then counted in a hemocytometer with the use of a phase-contrast microscope. Viability assays indicated dose-dependent toxicity for some bacteriocins. Nisin, pediocin, and Col E6 were the most cytotoxic bacteriocins; SV40-HC cells demonstrated greater sensitivity than Vero cells did. Some bacteriocins can be toxic to mammalian cells; therefore, bacteriocins intended for use as biopreservatives must be evaluated for toxicity to mammalian cells and for other toxicities. Col E1, Col E3, Col E7, and Col K demonstrated little toxicity at the activities tested, indicating that they are safe and thus have potential for use as food biopreservatives.     

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.     

Acid-adapted Listeria monocytogenes displays enhanced tolerance against the lantibiotics nisin and lacticin 3147.

Log-phase Listeria monocytogenes cells become tolerant to a variety of environmental stresses following acid adaptation at pH 5.5. We demonstrated that adapted cells also exhibit increased tolerance to nisin and, to a lesser extent, lacticin 3147. At nisin concentrations of 100 and 200 IU/ml the survival of acid-adapted cells was approximately 10-fold greater than nonadapted cells. However, acid adaptation had only a moderate effect on the tolerance of L. monocytogenes to lacticin 3147, a phenomenon that possibly reflects the distinct mode of action of this bacteriocin. Analysis of the fatty acid composition of the bacterial membrane indicated that straight-chain fatty acids C14:0 and C16:0 were significantly increased in acid-adapted cells while levels of C18:0 decreased. The results indicate that stress mechanisms that are induced in mildly acidic conditions provide protection against the antimicrobial action of bacteriocins. This increased resistance of acid-adapted L. monocytogenes could cause increased survival of this pathogen in food products in which nisin or other bacteriocins are used as preservatives.     

A rapid turbidometric microplate bioassay for accurate quantification of lactic acid bacteria bacteriocins

A 1 day turbidometric microplate bioassay (TMB) was developed for the rapid, accurate and precise quantification of lactic acid bacteria (LAB) bacteriocins (nisin Z and pediocin PA-1). Parameters such as the concentration of the indicator strains and the incubation time were optimized for each bacteriocin. A high correlation coefficient (r²=0.992±0.004) was obtained for the exponential regression in the nisin Z concentration range of 20–120 ng/ml with 1×10⁷ CFU indicator strain (Pediococcus acidilactici UL5) and an incubation time of 3 h. Using these parameters, the detection limit was estimated at 80 ng/ml (3.2 IU/ml), compared to 300 ng/ml for the agar diffusion assay (ADA). High precision (<7%) and accuracy (10%) were obtained for all nisin Z concentrations tested. Similar results were obtained with pediocin PA-1 with r²=0.993±0.005, a precision (8.2%) and an accuracy lower than 15%.     

Effectiveness of Some Natural Antimicrobial Compounds in Controlling Pathogen or Spoilage Bacteria in Lightly Fermented Chinese Cabbage

ABSTRACT: This study was designed to evaluate the bactericidal or bacteriostatic effect of chitosan, an allyl isothiocyanate (AIT) product, and nisin for the artificially inoculated pathogenic bacteria ( Escherichia coli , Salmonella Enteritidis, Staphylococcus aureus , and Listeria monocytogenes ) or natural microflora of fermented Chinese cabbage. Addition of 0.1% chitosan decreased the population of pathogens from 0.7 to 1.7 log colony-forming units (CFU)/g after 4 d of storage at 10 °C. The bactericidal activity of chitosan was found to be stronger than that of nisin (0.05 mg/g). Addition of 0.2% of the AIT product (containing AIT and hop extract) exhibited a bacteriostatic effect. However, a combination of AIT product and chitosan enhanced bactericidal efficacy against L. monocytogenes . The addition of chitosan or AIT product was observed to suppress the populations of mesophilic and coliform bacteria during storage at 10 °C for 4 d. Moreover, the use of chitosan or the AIT product did not change the sensory quality of the lightly fermented vegetable. Therefore, these results suggest that chitosan or the AIT product could be useful to improve the microbial safety and quality of lightly fermented vegetable.     

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.     

Factors influencing death and injury of foodborne pathogens by hydrostatic pressure-pasteurization

The key objective of hydrostatic pressure-pasteurization of food is the destruction of pathogenic bacterial cells at a high level (8log cycles) without adversely affecting the acceptance characteristics of a food. To achieve these goals, hydrostatic pressure-pasteurization may best be conducted at a moderate pressure, which alone will not kill the desired level of pathogens. However, along with hydrostatic pressure other parameters, namely pressurization-time, temperature and bactericidal compounds, such as bacteriocins, can be used to enhance bactericidal effects of pressurization.Our results with four pathogens indicated that viability loss at 25°C were minimal up to 207MPa, then increased at a rapid rate. However, even above 483MPa, not all species had 8log cycles viability loss. At and above 276MPa a large number of survivors were injured. At 207MPa and 25°C, pressurization for 30min did not greatly enhance viability loss and injury of the four pathogens. However, pressurization temperatures beyond 35°C greatly increased cell death and injury. By pressurizing the cells in the presence of a mixture of pediocin AcH and nisin viability loss of the pathogens was increased by an additional 1.3 to 5.1log cycles. This increase was due to the bactericidal effect of the two bacteriocins on sensitive and injured cells. These results indicated that at moderate pressure, a high level of destruction of pathogens can be induced within a short time by using moderate temperature in combination with bactericidal preservative(s).     

INDIVIDUAL AND COMBINED LISTERICIDAL EFFECTS OF SODIUM LACTATE, POTASSIUM SORBATE, NISIN AND CURING SALTS AT REFRIGERATION TEMPERATURE

The individual and combined antilisterial activity of the preservatives sodium lactate (4%), potassium sorbate (0.3%) and nisin (400 IU/ml), in either the presence or absence of the curing salts nitrite (125 ppm) and polyphosphate (0.5%), was assessed in buffered BHI broth (pH 5.5) during incubation at 4C. A cocktail of the strain Listeria monocytogenes NCTC 7973 and two food derived strains was used as the inoculum in challenge studies. In the absence of the preservatives , L. monocytogenes grew in the presence of polyphosphate but not in the presence of nitrite or a combination of nitrite and polyphosphate. The antilisterial action of nitrite was predominantly bacteriostatic in nature. Lactate acting alone, or in the presence of curing salts, produced a bacteriostatic effect. Sorbate acting alone had a bacteriostatic effect. Sorbate acting in the presence of nitrite or nitrite and polyphosphate produced a marked listericidal effect, reducing the population by 6.7 logs and 5.4 logs, respectively. Nisin acting alone produced an almost immediate 90% listericidal response (1 log population decrease), but initial numbers were restored within 14 days by regrowth. Regrowth was eliminated when nisin was used in combination with lactate, nitrite or nitrite and polyphosphate. The combination of lactate and sorbate offered no advantage over the use of sorbate alone. Sorbate and nisin acting in combination produced an enhanced listericidal effect, which was also seen in the presence of curing salts, but was delayed. No listericidal advantage over sorbate-nisin combination was achieved through the use of the lactate-sorbate-nisin combination. The combined use of sorbate and nisin offers promise as a means of eliminating L. monocytogenes from low pH cured meat products. The efficacy of this preservative combination remains to be evaluated in a meat system before its practical application can be considered .     

Enhanced antimicrobial activity of nisin-loaded liposomal nanoparticles against foodborne pathogens

This study was designed to evaluate the prolonged antimicrobial stability of nisin-loaded liposome (LipoN) nanoparticles against Listeria monocytogenes and Staphylococcus aureus. The sizes of bare liposomes and LipoN were uniformly distributed between 114 and 125 nm. The nanoparticles were homogeneously dispersed in water with less than 0.2 of polydispersity index. The zeta potential value of LipoN was +17.1 mV due to the positive charged nisin, attaining 70% of loading efficiency. The minimum inhibitory concentration of LipoN against L. monocytogenes and S. aureus was 320 international unit/mL. The LipoN significantly enhanced the antimicrobial stability in brain heart infusion agar compared to free nisin. The numbers of L. monocytogenes and S. aureus exposed to LipoN were effectively reduced by more than 6 log colony-forming unit/mL after 48 and 72 h of incubation, respectively. These results provide useful information for the development of antimicrobial delivery system to improve food safety.