Nisin and pediocin production on mussel-processing waste supplemented with glucose and five nitrogen sources

AIMS: Optimization of bacteriocin production by L. lactis subsp. lactis CECT 539 and Ped. acidilactici NRRL B-5627 on mussel-processing wastes. METHODS AND RESULTS: The concentrations of glucose and five nitrogen sources that optimize nisin and pediocin production were determined using a second order orthogonal factorial design. NH4Cl, glycine and glutamic acid were poor nitrogen sources. Enhanced pediocin and nisin productions were achieved in a medium supplemented with yeast extract or Bacto casitone. CONCLUSIONS: Mussel-processing wastes could be successfully used as a culture medium for bacteriocin production at low production cost. SIGNIFICANCE AND IMPACT OF THE STUDY: Average optimization led to a threefold increase in pediocin production (from 322 to 934 BU ml(-1)) and in nisin production (from 32 to 100 BU ml(-1)) when compared with the unsupplemented medium. For this reason, mussel-processing waste warrants further investigation due to its potential use as a cheap culture medium for upscaling bacteriocin production.     

Production of a nisin Z/pediocin mixture by pH-controlled mixed-strain batch cultures in supplemented whey permeate

The conditions for high production of nisin Z and pediocin during pH-controlled, mixed-strain batch cultures in a supplemented whey permeate medium with Lactococcus lactis subsp. lactis biovar. diacetylactis UL719, a nisin Z producer strain, and variant T5 of Pediococcus acidilactici UL5, a pediocin-producing strain resistant to high concentrations of nisin, were studied. Mixed cultures were performed at 37 °C and pH 5·5 by first inoculating with variant T5 and then with L. diacetylactis UL719 after 8 h incubation, and were compared with single-strain batch cultures. High productions of both nisin Z and pediocin were obtained after 18 or 16 h incubation during mixed cultures, with titres of 3000 and 730 AU ml⁻¹, or 1060 and 1360 AU ml⁻¹, respectively, corresponding to approximately 75 and 55, or 25 and 100 mg l⁻¹ of pure nisin Z and pediocin, respectively. In pure cultures, nisin Z and pediocin productions were higher than in mixed cultures, and maximum activities were obtained after 10 h incubation, with approximately 10 000 AU ml⁻¹ (250 mg l⁻¹ pure nisin Z) and 2500 AU ml⁻¹ (190 mg l⁻¹ pure pediocin). During mixed cultures, significant pediocin degradation was observed in the culture supernatant fluid after 16 h incubation, together with a sharp drop in Ped. acidilactici UL5 cell viability. In the test conditions, the feasibility of producing a nisin/pediocin mixture by mixed-strain fermentation was demonstrated. The bacteriocin mixture produced in a supplemented whey permeate medium could be used as a natural food-grade biopreservative with a broad activity spectrum.     

Modeling the batch bacteriocin production system by lactic acid bacteria by using modified three-dimensional Lotka–Volterra equations

Different batch cultures of Lactococcus lactis CECT 539, a nisin-producing strain, were carried out in culture media prepared with whey and mussel processing wastes. From these cultures, a reasonable system of differential equations, similar to the three-dimensional Lotka–Volterra two predators-one prey model, was set up to describe, for the first time, the relationship between the absolute rates of growth, pH drop and nisin production.Thus, the nisin production system was described as a three-species (pH, biomass and nisin) ecosystem. In this case, both nisin and biomass production were considered as two pH-dependent species that compete for the nitrogen source. Excellent agreement (R² values ≥0.9885) resulted between model predictions and the experimental data, and significant values for all the model parameters were obtained. The developed model was demonstrated (R² values ≥0.9874) for five batch cultivations of the strains L. lactis CECT 539 in MRS broth and Lactobacillus sakei LB 706 (sakacin A producer), Pediococcus acidilactici LB42-923 (pediocin AcH producer), L. lactis ATCC 11454 (nisin producer) and Leuconostoc carnosum Lm1 (leuconocin Lcm1 producer) in TGE broth. These results suggest that the batch bacteriocin production system in these culture media can be successfully described by using the Lotka–Volterra approach.     

Influence of Environmental Factors on Bacteriocin Production by Human Isolates of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> MM19 and <Emphasis Type="Italic">Pediococcus acidilactici</Emphasis> MM33

The influence of temperature, initial pH, and carbon and nitrogen sources on bacteriocin secreted by Lactococcus lactis MM19 (MM19) and Pediococcus acidilactici MM33 (MM33) was evaluated. It was found that 30 and 45 °C were the growth temperatures for higher nisin and pediocin production by MM19 and MM33, respectively. The initial pH values for higher production of nisin and pediocin were 9 and 6, respectively. Glucose and wheat peptone E430 were found as suitable carbon and nitrogen sources, respectively, for highest nisin production by MM19 at 30 °C and initial pH of 9. In these conditions, nisin production could be increased by 6.7 times as compared to the control medium (de Man, Rogosa, and Sharpe—MRS broth). Similarly, fructose and pea peptone were suitable carbon and nitrogen sources, respectively, for highest production of pediocin by MM33 at 45 °C and initial pH of 6. In these conditions, pediocin production by MM33 was increased by three times as compared to the control medium (tryptone-glucose-yeast extract—TGE broth).     

Influence of pH,temperature and culture media on the growth and bacteriocin production by vaginal Lactobacillus salivarius CRL 1328

AIMS: To study the influence of pH, temperature and culture medium on the growth and bacteriocin production by vaginal Lactobacillus salivarius subsp. salivarius CRL 1328. METHODS AND RESULTS: The study was performed using a complete factorial experimental design. Lactobacillus salivarius was cultivated in LAPTg and MRS broths, adjusted to specific initial pH, and at different temperatures of incubation. The growth, which was evaluated by the Gompertz model, was higher in MRS broth than in LAPTg broth. The initial pH of the culture medium and the temperature had a dramatic effect on the production of bacteriocin. The optimal conditions for bacteriocin production were different to those for optimal growth. The decrease in the pH of the culture medium was parallel to the growth; pH had similar final values in both the MRS and the LAPTg broths. CONCLUSIONS: The optimal growth conditions were recorded in MRS broth, with an initial pH of 6.5 and a temperature of 37 degrees C. The maximum bacteriocin activity was obtained in LAPTg after 6 h at 37 degrees C, and at an initial pH of 6.5 or 8.0. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of a complete factorial design, and the evaluation of the growth parameters through the Gompertz model, enabled a rapid and simultaneous exploration of the influence of pH, temperature and growth medium on both growth and bacteriocin production by vaginal Lact. salivarius CRL 1328.     

Enhanced production of pediocin PA-1 and coproduction of nisin and pediocin PA-1 by Lactococcus lactis.

The production and secretion of class II bacteriocins share a number of features that allow the interchange of genetic determinants between certain members of this group of antimicrobial peptides. Lactococcus lactis IL1403 encodes translocatory functions able to recognize and mediate secretion of lactococcin A. The ability of this strain to also produce the pediococcal bacteriocin pediocin PA-1, has been demonstrated previously by the introduction of a chimeric gene, composed of sequences encoding the leader of lactococcin A and the mature part of pediocin PA-1 (N. Horn, M. I. Martínez, J. M. Martínez, P. E. Hernández, M. J. Gasson, J. M. Rodríguez, and H. M. Dodd, Appl. Environ. Microbiol. 64:818-823, 1998). This heterologous expression system has been developed further with the introduction of the lactococcin A-dedicated translocatory function genes, lcnC and lcnD, and their effect on bacteriocin yields in various lactococcal hosts was assessed. The copy number of lcnC and lcnD influenced production levels, as did the particular strain employed as host. Highest yields were achieved with L. lactis IL1403, which generated pediocin PA-1 at a level similar to that for the parental strain, Pediococcus acidilactici 347, representing a significant improvement over previous systems. The genetic determinants required for production of pediocin PA-1 were introduced into the nisin-producing strain L. lactis FI5876, where both pediocin PA-1 and nisin A were simultaneously produced. The implications of coproduction of these two industrially relevant antimicrobial agents by a food-grade organism are discussed.     

Interaction of the bacteriocin pediocin SJ-1 with the cytoplasmic membrane of sensitive bacterial cells as detected by ANS fluorescence

The l-anilino-8-naphthalenesulphonic acid (ANS) fluorescent probe was used to monitor alterations in the cytoplasmic membrane of sensitive Lactobacillus plantarurm cells, caused by pediocin SJ-1, a bacteriocin produced by Pediococcus acidilactici. The addition of pediocin SJ-1 to the sensitive cells caused an increase in fluorescence intensity of ANS and a blue shift in its emission maximum from 520 to 475 nm. None of these spectral changes could be detected when pediocin SJ-1 was added to cells of a Lact. plantarum variant resistant to pediocin SJ-1. Upon the addition of pediocin SJ-1, dose-dependent energy transfer took place between tryptophanyl residues in the cytoplasmic membrane of sensitive cells and ANS. Similar ANS-fluorescence changes were observed with the bacteriocin nisin. The concentrations of pediocin SJ-1 needed to effect changes in the fluorescence spectrum of ANS were of the same magnitude as those required for a bactericidal effect and the release of u.v.-absorbing material. A hypothesis on the mode of action of pediocin SJ-1 is proposed.     

Growth optimization of Pediococcus damnosus NCFB 1832 and the influence of pH and nutrients on the production of pediocin PD-1

AIMS: Optimization of the growth of Pediococcus damnosus NCFB 1832 and the production of pediocin PD-1 by traditional fermentation methods. METHODS AND RESULTS: Fermentation studies were conducted in De Man Rogosa and Sharpe (MRS) broth (Oxoid), preadjusted to specific pH values, and in MRS broth supplemented with various nitrogen sources, MnSO4, MgSO4 and Tween 80. The production of pediocin PD-1 closely followed the growth curve of Ped. damnosus NCFB 1832. Maximum levels of bacteriocin activity (3249 AU ml(-1)/O.D.max) were recorded in MRS broth with an initial pH of 6.7. In media with an initial pH of 4.5 bacteriocin activity as low as 222 AU ml(-1)/O.D.max was recorded. The highest bacteriocin activity was recorded in growth conditions allowing the greatest pH variation (highest DeltapH). The addition of bacteriological peptone (1.7%, w/v), MnSO4 (0.014%, w/v) and Tween 80 (3%, v/v) to MRS and adjustment of the medium pH to 6.7 resulted in a further increase in activity (from 3249 to 5078 AU ml(-1)/O.D.max). The same medium, but with an initial pH of 6.2, resulted in an 82.5% decrease in bacteriocin activity. CONCLUSIONS: Pediocin PD-1 production is not only stimulated by the presence of specific growth factors (e.g., bacteriological peptone, MnSO4 or Tween 80), but may also be stimulated by the lowering in pH during growth (highest DeltapH), and thus also the amount of organic acids produced. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of pediocin PD-1 by the wild-type producer strain was significantly improved by using a defined medium and traditional fermentation methods.     

Comparative effectiveness of nisin and bacteriocin J46 at different pH values

E. HUOT, C. BARRENA-GONZALEZ AND H. PETITDEMANGE. 1996. A Comparative study of the inhibitory activity of nisin, the well-known lantibiotic produced by certain strains of Lactococcus lactis subsp. lactis , and of the bacteriocin produced by L. lactis subsp. cremoris J46, a strain previously isolated from fermented milk, was conducted. For both bacteriocins, the activity against L. lactis subsp. cremoris decreased with increasing pH. In addition, the bacteriocin preparations were more stable at 4 than at 20°C. The influence of the storage temperature was more crucial for nisin. Essentially the same activity was observed for bacteriocin J46 stored for 3 h at 4 or 20°C. More interesting was the observed stability of bacteriocin J46 at pH values between 5.8 and 6.8. For example, about 23% of nisin activity was lost at pH 6.4 whereas no loss of bacteriocin J46 activity was observed.     

Development of a low-cost medium for production of nisin from Lactococcus lactis subsp. lactis

The goal of this project was to develop a lower-cost medium for nisin production, so this bacteriocin could be used in a broader range of industrial fermentation processes. The objectives included: (1) evaluating methods for controlling the inhibitory effect of lactic acid produced during fermentation, and (2) comparing two inexpensive complex media for nisin production. Lactococcus lactis subsp. lactis was cultured in shake flasks on Laurel–Tryptose broth to evaluate a range of buffers for pH control. NaHCO₃ proved to be an effective buffer for increasing nisin production. Subsequent trials then evaluated condensed corn soluble (CCS, a fuel ethanol production byproduct) and cheese whey as inexpensive growth media. CCS was shown to be an efficient, low-cost medium for high nisin titers and yields. These modifications reduced the medium costs for nisin production from $600/kg nisin (based on Laurel–Tryptose broth medium) to $35–40/kg nisin for the corn solubles medium.     

Frequency of bacteriocin resistance development and associated fitness costs in Listeria monocytogenes

Bacteriocin-producing starter cultures have been suggested as natural food preservatives; however, development of resistance in the target organism is a major concern. We investigated the development of resistance in Listeria monocytogenes to the two major bacteriocins pediocin PA-1 and nisin A, with a focus on the variations between strains and the influence of environmental conditions. While considerable strain-specific variations in the frequency of resistance development and associated fitness costs were observed, the influence of environmental stress seemed to be bacteriocin specific. Pediocin resistance frequencies were determined for 20 strains and were in most cases ca. 10(-6). However, two strains with intermediate pediocin sensitivity had 100-fold-higher pediocin resistance frequencies. Nisin resistance frequencies (14 strains) were in the range of 10(-7) to 10(-2). Strains with intermediate nisin sensitivity were among those with the highest frequencies. Environmental stress in the form of low temperature (10 degrees C), reduced pH (5.5), or the presence of NaCl (6.5%) did not influence the frequency of pediocin resistance development; in contrast, the nisin resistance frequency was considerably reduced (<5 x 10(-8)). Pediocin resistance in all spontaneous mutants was very stable, but the stability of nisin resistance varied. Pediocin-resistant mutants had fitness costs in the form of reduction down to 44% of the maximum specific growth rate of the wild-type strain. Nisin-resistant mutants had fewer and less-pronounced growth rate reductions. The fitness costs were not increased upon applying environmental stress (5 degrees C, 6.5% NaCl, or pH 5.5), indicating that the bacteriocin-resistant mutants were not more stress sensitive than the wild-type strains. In a saveloy-type meat model at 5 degrees C, however, the growth differences seemed to be negligible. The applicational perspectives of the results are discussed.     

Industrial Whey Utilization as a Medium Supplement for Biphasic Growth and Bacteriocin Production by Probiotic Lactobacillus casei LA-1

The ability of probiotic Lactobacillus casei LA-1 for bacteriocin production using industrial by-products, such as whey, as supplement in growth medium has been demonstrated for the first time. Whey was investigated as a sole carbon source in cooperation with other components to substitute expensive nutrients as MRS for economical bacteriocin production. Industrial whey-supplemented MRS medium was then selected as to determine the effect of four variables (temperature, initial pH, incubation time, and whey concentration) by response surface methodology on bacteriocin production. Statistical analysis of results showed that two variables have a significant effect on bacteriocin production. Response surface data showed maximum bacteriocin production of 6,132.33?AU/mL at an initial pH of 7.12, temperature 34.29?°C, and whey concentration 13.74?g/L. The production of bacteriocin started during the exponential growth phase, reaching maximum values at stationary phase, and a biphasic growth and production pattern was observed. Our current work demonstrates that this approach of utilization of whey as substitution in costly medium as MRS has great promise for cost reduction in industry for the production of novel biological metabolic product that can be utilized as a food preservative.     

Collapse of the proton motive force in Listeria monocytogenes caused by a bacteriocin produced by Pediococcus acidilactici.

The effect of pediocin JD, a bacteriocin produced by Pediococcus acidilactici JD1-23, on the proton motive force and proton permeability of resting whole cells of Listeria monocytogenes Scott A was determined. Control cells, treated with trypsin-inactivated bacteriocin at a pH of 5.3 to 6.1, maintained a pH gradient and a membrane potential of approximately 0.65 pH unit and 75 mV, respectively. However, these gradients were rapidly dissipated in cells after exposure to pediocin JD, even though no cell lysis had occurred. The pH gradient and membrane potential of the producer cells were also unaffected by the bacteriocin. Whole cells treated with bacteriocin were twice as permeable to protons as control cells were. The results suggest that the inhibitory action of pediocin JD against L. monocytogenes is directed at the cytoplasmic membrane and that inhibition of L. monocytogenes may be caused by the collapse of one or both of the individual components of the proton motive force.     

Collapse of the proton motive force in Listeria monocytogenes caused by a bacteriocin produced by Pediococcus acidilactici.

The effect of pediocin JD, a bacteriocin produced by Pediococcus acidilactici JD1-23, on the proton motive force and proton permeability of resting whole cells of Listeria monocytogenes Scott A was determined. Control cells, treated with trypsin-inactivated bacteriocin at a pH of 5.3 to 6.1, maintained a pH gradient and a membrane potential of approximately 0.65 pH unit and 75 mV, respectively. However, these gradients were rapidly dissipated in cells after exposure to pediocin JD, even though no cell lysis had occurred. The pH gradient and membrane potential of the producer cells were also unaffected by the bacteriocin. Whole cells treated with bacteriocin were twice as permeable to protons as control cells were. The results suggest that the inhibitory action of pediocin JD against L. monocytogenes is directed at the cytoplasmic membrane and that inhibition of L. monocytogenes may be caused by the collapse of one or both of the individual components of the proton motive force.     

Substrate inhibition of Pediococcus acidilactici by glucose on a waste medium. Simulations and experimental results

AIMS: The possibility of substrate inhibition by glucose on biomass and pediocin production was studied in cultures of Pediococcus acidilactici on a residual medium. METHODS AND RESULTS: Calculation of the substrate inhibition coefficient in the context of microbial growth is generally laborious, and very prone to experimental error. However, a simulation combining logistic and Monod kinetics equations demonstrates that quantitative evidence for this type of inhibition, without the possibility of misinterpretation, can be obtained through the comparison of punctual preasymptotic productions as a function of substrate concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: It was concluded that glucose had an inhibitory effect on growth, but not on bacteriocin production.     

Mechanism of Nisin, Pediocin 34, and Enterocin FH99 Resistance in Listeria monocytogenes

Nisin-, pediocin 34-, and enterocin FH99-resistant variants of Listeria monocytogenes ATCC 53135 were developed. In an attempt to clarify the possible mechanisms underlying bacteriocin resistance in L. monocytogenes ATCC 53135, sensitivity of the resistant strains of L. monocytogenes ATCC 53135 to nisin, pediocin 34, and enterocin FH99 in the absence and presence of different divalent cations was assessed, and the results showed that the addition of divalent cations significantly reduced the inhibitory activity of nisin, pediocin 34, and enterocin FH99 against resistant variants of L. monocytogenes ATCC 53135. The addition of EDTA, however, restored this activity suggesting that the divalent cations seem to affect the initial electrostatic interaction between the positively charged bacteriocin and the negatively charged phospholipids of the membrane. Nisin-, pediocin 34-, and enterocin-resistant variants of L. monocytogenes ATCC 53135 were more resistant to lysozyme as compared to the wild-type strain both in the presence as well as absence of nisin, pediocin 34, and enterocin FH99. Ultra structural profiles of bacteriocin-sensitive L. monocytogenes and its bacteriocin-resistant counterparts revealed that the cells of wild-type strain of L. monocytogenes were maximally in pairs or short chains, whereas, its nisin-, pediocin 34-, and enterocin FH99-resistant variants tend to form aggregates. Results indicated that without a cell wall, the acquired nisin, pediocin 34, and enterocin FH99 resistance of the variants was lost. Although the bacteriocin-resistant variants appeared to lose their acquired resistance toward nisin, pediocin 34, and enterocin FH99, the protoplasts of the resistant variants appeared to be more resistant to bacteriocins than the protoplasts of their wild-type counterparts.     

Lantibiotic nisin Z fermentative production by Lactococcus lactis IO-1: relationship between production of the lantibiotic and lactate and cell growth

 The influence of several parameters on the fermentative production of nisin Z by Lactococcus lactis IO-1 was studied. Considerable attention has been focused on the relationship between the primary metabolite production of bacteriocin and lactate and cell growth, which has so far not been clarified in detail. Production of nisin Z was optimal at 30°C and in the pH range 5.0–5.5. The addition of Ca²⁺ to the medium showed a stimulating effect on the production of nisin Z. A maximum activity of 3150 IU/ml was obtained during pH-controlled batch fermentation in the medium supplemented with 0.1 M CaCl₂. It was about three times higher than that obtained under the optimal conditions for cell growth and lactic acid production. Received: 12 July 1995/Received revision: 11 September 1995/Accepted: 4 October 1995     

Isolation, screening and characterization of bacteriocin-producing lactic acid bacteria isolated from traditional fermented food

100 lactic acid bacterial strains isolated from traditional fermented foods (yoghurt, milk cream, sour dough and milk) were screened for bacteriocin production. Twenty six strains producing a nisin-like bacteriocin were selected. Most of these isolates gave only a narrow inhibitory spectrum, although one showed a broad inhibitory spectrum against the indicator strains tested, this strain was determined as Lactococcus lactis. The influence of several parameters on the fermentative production of nisin by Lactococcus lactis was studied. Production of nisin was optimal at 30 degrees C and in the pH range 5.5-6.3. The effect of different sulphur and nitrogen sources on Lactococcus lactis growth and nisin production was studied. Magnesium sulfate and manganese sulfate were found to be the best sulphur sources while triammonium citrate was the best inorganic nitrogen source and meat extract, peptone and yeast extract were the best organic nitrogen source for nisin production.     

Production, recovery and purification of bacteriocins from lactic acid bacteria

Bacteriocins produced by lactic acid bacteria are a heterogeneous group of peptide inhibitors which include lantibiotics (class I, e.g. nisin), small heat-stable peptides (class II, e.g. pediocin AcH/PA1) and large heat-labile proteins (class III, e.g. helveticin J). Many bacteriocins belonging to the first two groups can be successfully used to inhibit undesirable microorganisms in foods, but only nisin is produced industrially and is licensed for use as a food preservative in a partially purified form. This review focuses on the production and purification of class I and class II bacteriocins from lactic acid bacteria. Bacteriocin production is growth associated but the yield of bacteriocin per unit biomass is affected by several factors, including the producing strain, media (carbohydrate and nitrogen sources, cations, etc.) and fermentation conditions (pH, temperature, agitation, aeration and dilution rate in continuous fermentations). Continuous fermentation processes with cell recycle or immobilized cells can result in a dramatic improvement in productivity over batch fermentations. Several simple recovery processes, based on adsorbing bacteriocin on resins or silica compounds, have been developed and can be used to build integrated production processes. Received: 29 December 1998 / Received revision: 23 April 1999 / Accepted: 23 April 1999     

Conjugal transfer of bacteriocin plasmids from different genera of lactic acid bacteria into Enterococcus faecalis JH2-2

The lactic acid bacteria (LAB) are of great interest because of their food grade quality and industrial importance. In the recent past, the pediocin PA-1 like bacteriocin was found to be synthesized in cross-species and cross-genera. Hence, the present work was carried out in order to determine the transfer of plasmid encoded pediocin PA-1 like bacteriocin among LAB. The objective of this study is to demonstrate the dissemination of bacteriocin-encoded plasmid from Pediococcus acidilactici NCIM 5424, Enterococcus faecium NCIM 5423 and Lactobacillus plantarum Acr2 to Enterococcus faecalis JH2-2 under in vitro (filter mating method) and in situ (soymilk model) conditions. The fermentation of the soymilk was determined by the selected pediocin producers. E. faecium NCIM 5423 was able to transfer the bacteriocin only under in situ conditions, whereas the native pediocin producer P. acidilactici NCIM 5424 transferred the bacteriocin under both the methods used. The in situ method gave more transfer frequency, ranging from 10^?7 to 10^?4 transconjugants per recipient cell. No conjugal transfer by L. plantarum Acr2 was observed. The physiological conditions like pH and temperature were found to influence the production of bacteriocin in the obtained transconjugants. The results suggest the horizontal gene transfer (HGT) and the natural spread of pediocin PA-1-like bacteriocin among LAB present in their close vicinity by means of conjugation. The dissemination of pediocin PA-1-like bacteriocin under in situ conditions is noteworthy, and such bacteriocin producers can be useful in the fermentation of dairy products and construction of novel cultures.