Nisin Production Research Articles

Effects of pH profiles on nisin production in biofilm reactor

Apart from its widely accepted commercial applications as a food preservative, nisin emerges as a promising alternative in medical applications for bacterial infection in both humans and livestock. Improving nisin production through optimization of fermentation parameters would make nisin more cost-effective for various applications. Since nisin production by Lactococcus lactis NIZO 22186 was highly influenced by the pH profile employed during fermentation, three different pH profiles were evaluated in this study: (1) a constant pH profile at 6.8 (profile 1), (2) a constant pH profile with autoacidification at 4 h (profile 2), and (3) a stepwise pH profile with pH adjustment every 2 h (profile 3). The results demonstrated that the low-pH stress exerted during the first 4 h of fermentation in profile 3 detrimentally affected nisin production, resulting in a very low maximum nisin concentration (593 IU ml(-1)). On the other hand, growth and lactic acid production were only slightly delayed, indicating that the loss in nisin production was not a result of lower growth or shifting of metabolic activity toward lactic acid production. Profile 2, in which pH was allowed to drop freely via autoacidification after 4 h of fermentation, was found to yield almost 1.9 times higher nisin (3,553 IU ml(-1)) than profile 1 (1,898 IU ml(-1)), possibly as a result of less adsorption of nisin onto producer cells. Therefore, a combination of constant pH and autoacidification period (profile 2) was recommended as the pH profile during nisin production in a biofilm reactor.

Increase of nisin production by Lactococcus lactis in different media

Nisin production related to the growth conditions of Lactococcus lactis subsp . lactis ATCC 11454, the effects of various media components and concomitant release of nisin into the media, were studied through transfers (five times). Nisin production was assayed by agar diffusion using Lactobacillus sake ATCC 15521 as the sensitive test organism. The expression of nisin was strongly influenced by the addition of skimmed milk to both MRS and M17 broth, with the highest production obtained after the second and the fifth transfers, respectively, with maximum expression after 36 h of incubation. African Journal of Biotechnology Vol. 4 (3), pp. 262-265, 2005

Preliminary tests on nisin and pediocin production using waste protein sources: Factorial and kinetic studies

Lactic acid bacteria, the object of current interest as bacteriocin producers, are microorganisms with complex requirements for peptidic sources, making them appropriate indicators for testing the suitability of formulations based on proteinaceous wastes for use as microbiological media. Different peptones obtained from visceral and fish muscle residues promoted growth of lactic acid bacteria when applied individually or in combination. Kinetic parameters and bacteriocin production were similar and, in some cases (pediocin), far superior (>500%) to those obtained with bactopeptones and commercial media specifically recommended for lactic acid bacteria growth. Visceral residues, especially when subjected to a brief process of autohydrolysis at 20 °C, were more efficient for bacterial growth than muscle, even when muscle was treated with pepsin.

Modelling the production of nisin by Lactococcus lactis in fed-batch culture

Nisin production in batch culture and fed-batch cultures (sucrose feeding rates were 6, 7, 8, and 10 g l(-1) h(-1), respectively) by Lactococcus lactis subsp. lactis ATCC 11454 was investigated. Nisin production showed primary metabolite kinetics, and could be improved apparently by altering the feeding strategy. The nisin titer reached its maximum, 4,185 IU ml(-1), by constant addition of sucrose at a feeding rate of 7 g l(-1) h(-1); an increase in 58% over that of the batch culture (2,658 IU ml(-1)). Nisin biosynthesis was affected strongly by the residual sucrose concentration during the feeding. Finally, a mathematical model was developed to simulate the cell growth, sucrose consumption, lactic acid production and nisin production. The model was able to describe the fermentation process in all cases.

Enhanced nisin production by increasing genes involved in nisin Z biosynthesis in Lactococcus lactis subsp. lactis A164

Nisin Z production in Lactococcus lactis subsp. lactis A164 was improved by introducing multicopy genes, nisZ, nisRK, or nisFEG, involved in nisin biosynthesis into A164 strain. A similar growth profile was obtained from all strains tested. However, the cells expressing nisRK produced 25,000 AU nisin Z ml(-1) compared to 16,000 AU ml(-1) by the control strain. Northern blot analysis revealed that over-expression of nisRK promoted the transcription of the nisZ gene. The A164 strain expressing multicopy nisFEG also had an increased nisin Z production (25,000 AU ml (-1)) but produced the nisin more slowly than the cells expressing multicopy nisRK.

Effect of sucrose on nisin production in batch and fed-batch culture byLactococcus lactis

The effects of sucrose on cell growth and nisin production by Lactococcus lactis were investigated in batch and pH feed-back controlled fed-batch cultures. In batch cultures, nisin titer reached its maximum, 2658 IU cm−3, at the initial sucrose concentration of 30 g dm−3. With sucrose concentrations higher than 30 g dm−3, nisin production decreased while the biomass was not influenced significantly. By using the pH feed-back controlled method, residual sucrose concentration could be controlled well in fed-batch cultures and three conditions (sucrose maintained at 2, 16, 20 g dm−3, respectively) were evaluated. Maintaining a low sucrose concentration at 2 g dm−3 during feeding favored nisin biosynthesis, and the maximum nisin titer obtained was 4961 IU cm−3 compared with 3370 IU cm−3 (16 g sucrose dm−3)and 3498 IU cm−3 (20 g sucrose dm−3), respectively. Copyright © 2005 Society of Chemical Industry

Production of Nisin by <I>Lactococcus lactis </I>in Media with Skimmed Milk

Nisin is a bacteriocin that inhibits the germination and growth of Gram-positive bacteria. With nisin expression related to growth conditions of Lactococcus lactis subsp. lactis, the effects of growth parameters, media components, and incubation time were studied to optimize expression. L. lactis ATCC 11454 was grown (100 rpm at 30 degrees C for 36 h) in both M17 and MRS standard broth media (pH 6.0-7.0) supplemented with sucrose (1.0-12.5 g/L), potassium phosphate (0.13 g/L), asparagine (0.5 g/L), and sucrose (0.24 g/L), and diluted 1:1 with liquid nonfat milk. Liquid nonfat milk, undiluted, was also used as another medium (9% total solids, pH 6.5). Nisin production was assayed by agar diffusion using Lactobacillus sake ATCC 15521 (30 degrees C for 24 h) as the sensitive test organism. The titers of nisin expressed and released in culture media were quantified and expressed in arbitrary units (AU/L of medium) and converted into known concentrations of "standard nisin" (Nisaplin, g/L). The detection of nisin activity was <0.01 AU/L in M17 and MRS broths, and 7.5 AU/L in M17 with 0.14% sucrose or 0.13% other supplements, and the activity increased to 142.5 AU/L in M17 diluted with liquid nonfat milk (1:1). The 25% milk added to either 25% M17 or 25% MRS provided the highest levels of nisin assayed.

Effects of Nutrient Supplements on Simultaneous Fermentation of Nisin and Lactic Acid from Cull Potatoes

The feasibility of using cull potatoes as substrate for the simultaneous production of nisin, a natural food preservative, and lactic acid, a raw material for biopolymer production, was studied. Cull potatoes are potato tubers unacceptable for food processing because of size or damage caused by bruising or disease. Although cull potatoes are enriched in various nutrients including starch, minerals, and proteins, they alone still cannot provide enough essential nutrients for the growth and metabolism of Lactococcus lactis subsp. lactis (ATCC 11454). Stimulation of bacterial growth, nisin biosynthesis, as well as lactic acid production was observed when additional nutrients such as yeast extract, peptone from meat, peptone from soy (PS), corn steep solid (CSS), and distillers' dried grains with solubles were provided. Considering the cost and availability, PS and CSS were selected as nutrient supplements for nisin and lactic acid coproduction. The conditions for nisin biosynthesis and lactic acid coproduction by L. lactis subsp. lactis in a cull potato-based medium were subsequently optimized using a statistically based experimental design.

Improvement of nisin production in pH feed-back controlled, fed-batch culture by Lactococcus lactissubsp. lactis

Nisin production by Lactococcus lactis subsp. lactis in fed-batch culture was doubled by using a pH feed-back controlled method. Sucrose concentration was controlled at 10 g l(-1) giving 5010 IU nisin ml(-1) compared to 2660 IU nisin ml(-1) in batch culture.

Nisin-controlled production of pediocin PA-1 and colicin V in nisin- and non-nisin-producing Lactococcus lactis strains.

The introduction of chimeric genes encoding the fusion leader of lactococcin A-propediocin PA-1 or procolicin V under the control of the inducible nisA promoter and the lactococcin A-dedicated secretion genes (lcnCD) into Lactococcus lactis strains, including a nisin producer, expressing the two component regulator NisRK led to the production or pediocin PA-1 or colicin V, respectively.

Peptones from autohydrolysed fish viscera for nisin and pediocin production

Various peptones obtained from hydrolysed visceral homogenates of four fishery residues showed their suitability for promoting the growth of lactic acid bacteria, micro-organisms with particularly complex requirements regarding peptidic nutrients. The assay of several treatments with two bacterial species, producers of the two main bacteriocins (nisin and pediocin) demostrated that optimum conditions only imply a brief autohydrolysis at natural pH and room temperature, with subsequent steam-flow stabilisation. Later kinetic analysis of the cultures of both bacteria in the best media provided parameters which, for production of both biomass and bacteriocins (the latter behaved in the majority of cases as a mixed metabolite), indicate comparable or superior results to those found in costly commercial media, specifically recommended for culture of lactic acid bacteria.

Inhibition of a Methicillin-Resistant Staphylococcus aureus Strain in Afuega'l Pitu Cheese by the Nisin Z–Producing Strain Lactococcus lactis subsp. lactis IPLA 729

Methicillin-resistant Staphylococcus aureus strains are a potential threat for food safety because foodborne illness caused by methicillin-resistant Staphylococcus aureus has been reported even though these strains were only associated with nosocomial infections until recently. This article focuses on the inhibitory effect of the nisin Z–producing strain Lactococcus lactis subsp. lactis IPLA 729 on the growth of Staphylococcus aureus CECT 4013, a methicillin-resistant strain. S. aureus was inhibited by the presence of the nisin producer IPLA 729 in buffered Trypticase soy broth, milk, and Afuega'l Pitu cheese, an acid-coagulated cheese manufactured in Asturias, Northern Spain. A reduction of 3.66 log units was observed in Trypticase soy broth at the end of the incubation period. In milk, viable counts of S. aureus were undetectable or were reduced by 2.16 log units in 24 h depending on the initial inoculum (1.8 × 104 and 7.2 × 106 CFU/ml). The staphylococcal strain was also undetected in test cheeses in which the nisin Z producer was present whereas 2 log units were detected in control cheeses at the end of ripening.

The role of amino acids in nisin and pediocin production by two lactic acid bacteria: A factorial study

In conventional and repeatedly re-alkalized cultures of two lactic acid bacteria, the effects of five amino acids and their interactions on biomass and bacteriocin (nisin and pediocin) production were studied. Nisin production was stimulated by cysteine and tryptophan, and depressed by proline. Equations describing biomass production in Lactococcus lactis, and biomass and pediocin productions in Pediococcus acidilactici, contain numerous terms with three and four variables, indicating that none of the amino acids seems to play a relevant individual role. Stimulation due to the pH-stepwise profiles exceed significantly those of the most effective amino acid combinations and promote much faster consumption of some amino acids. Since a pH-stepwise culture can be considered as a series of successive incubations, each starting up in presence of the metabolic products from the preceding step, these results are more compatible with an auto-stimulatory mechanism rather than a nutritional phenomenon.

Continuous nisin production in laboratory media and whey permeate by immobilized Lactococcus lactis

Continuous production of nisin in laboratory media and whey permeate was investigated using a packed-bed bioreactor. Lactococcus lactis subsp. lactis ATCC 11454 was immobilized by natural attachment to fibre surfaces and entrapment in the void volume within spiral wound fibrous matrix. The inoculated bioreactor was fed M17 medium and effect of pH, temperature, dilution rate, and substrate concentrations on nisin production was examined. Bioreactor performance was monitored by checking cell density, lactic acid production, lactose consumption, and nisin activity. Optimal nisin activity was observed at pH 5.5, 31 °C, 0.2–0.3/h dilution rate, and 30 g/l lactose in M17. The maximum nisin titre was 2.6×10 4 AU/ml against a nisin sensitive strain, Lactobacillus leichmannii ATCC 4797. The bioreactor was fed whey permeate, supplemented with casein hydrolysate, and growth of L. lactis and associated nisin production were monitored. Optimal conditions for continuous nisin production in whey permeate were pH 5.5, 31 °C, 10–20 g/l casein hydrolysate, and 0.2/h dilution rate. Under these conditions, a maximum nisin titre of 5.1×10 4 AU/ml was observed. The bioreactor was operated continuously for 6 months without encountering any clogging, degeneration, or contamination problems. The cell density in the bioreactor was 52.0 g/l with 96.4% of the cells immobilized. This study illustrates the possibility of continuous production of high concentration of bacteriocins by lactic acid bacteria for use as food biopreservatives.

Simultaneous production of nisin and lactic acid from cheese whey: optimization of fermentation conditions through statistically based experimental designs.

A biorefinery process that utilizes cheese whey as substrate to simultaneously produce nisin, a natural food preservative, and lactic acid, a raw material for biopolymer production, was studied. The conditions for nisin biosynthesis and lactic acid coproduction by Lactococcus lactis subsp. lactis (ATCC 11454) in a whey-based medium were optimized using statistically based experimental designs. A Plackett-Burman design was applied to screen seven parameters for significant factors for the production of nisin and lactic acid. Nutrient supplements, including yeast extract, MgSO4, and KH2PO4, were found to be the significant factors affecting nisin and lactic acid formation. As a follow-up, a central-composite design was applied to optimize these factors. Second-order polynomial models were developed to quantify the relationship between nisin and lactic acid production and the variables. The optimal values of these variables were also determined. Finally, a verification experiment was performed to confirm the optimal values that were predicted by the models. The experimented results agreed well with the model prediction, giving a similar production of 19.3 g/L of lactic acid and 92.9 mg/L of nisin.

Impact of Autolytic and Proteolytic Lactobacilli and Nisin‐Producing Culture on Proteolysis and Sensory Characteristics in Cheddar Cheese

ABSTRACT: Cheddar cheeses were made using a nisin‐tolerant starter culture with either Lactobacillus delbrueckii subsp. bulgaricus UL12 (autolytic strain), Lactobacillus casei subsp. casei L2A (proteolytic strain), Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 (nisin producer), or of Lb. bulgaricus UL12 and Lc. diacetylactis UL719. Lb. bulgaricus UL12 produced more trichloroacetic acid‐soluble nitrogen than did Lb. casei L2A, which produced more phosphotungstic acid‐soluble nitrogen than did Lc. diacetylactis UL719. High‐performance liquid chromatography analyses showed that either lactobacilli or Lc. diacetylactis UL719 increased the hydrophilic and hydrophobic peptide contents. Cheeses containing both Lb. bulgaricus UL12 and Lc. diacetylactis UL719 had the most intense old Cheddar cheese flavor after 6 mo of ripening.

Construction of vectors for inducible gene expression in Lactobacillus sakei and L. plantarum

We have constructed vectors for inducible expression of genes in Lactobacillus sakei and Lactobacillus plantarum. The key elements of these vectors are a regulatable promoter involved in the production of the bacteriocins sakacin A and sakacin P and the genes encoding the cognate histidine protein kinase and response regulator that are necessary to activate this promoter upon induction by a peptide pheromone. The vectors are built up of cassettes that permit easy exchange of all parts through restriction enzyme digestion and ligation. Using β-glucuronidase as a reporter enzyme, variants of these vectors were compared with each other, and with a corresponding system based on genes involved in the production of nisin. Several of the new vectors permitted tightly controlled and efficient expression of β-glucuronidase in both L. sakei and L. plantarum.

Production of nisin with continuous adsorption to Amberlite XAD-4 resin using Lactococcus lactis N8 and L. lactis LAC48.

The production of nisin, biomass and lactic acid in pH-controlled and uncontrolled batch fermentation and batch fermentation (pH 5.5) with continuous removal of nisin was examined in the parent strain Lactococcus lactis N8 and LAC48. Strain LAC48 in batch fermentor (pH not controlled) gave a maximum nisin concentration of 2.5 x 10(6) IU g dcw(-1). The nisin concentration remained high (2.0 x 10(6) IU g dcw(-1)) after the logarithmic growth phase (10-22 h), whereas nisin production of strain N8 decreased after the logarithmic growth phase. The maximum nisin production of strain LAC48 was not directly related to the biomass formation and not associated with growth. In order to study end product inhibition in nisin production, a system was built for adsorption of nisin during fermentation. The adsorbent Amberlite XAD-4 was found to have an effective binding capacity for nisin. Cells of LAC48 and N8 compensated for the removal of nisin, indicating that nisin production also occurs in the stationary phase.

Influence of pH drop on both nisin and pediocin production by Lactococcus lactis and Pediococcus acidilactici.

To develop a kinetic model for describing the specific effect of pH drop on nisin and pediocin production in whey. The effect of pH drop on both bacteriocin productions was tested in non-buffered whey and whey buffered at initial pH 6.3 with 0.03, 0.10 and 0.25 mol l-1 of potassium hydrogen phthalate-NaOH. An accurate description of the experimental data of nisin and pediocin obtained at different pH drops is obtained with the proposed model. The proposed model was able to typify both bacteriocins as pH-dependent primary metabolites. The decisive role of pH drop for bacteriocin production on whey was demonstrated and modelled. This study contributes to a better understanding of underlying metabolic regulatory mechanisms, which could facilitate the optimization of bacteriocin production for upscaling.

Application of statistically-based experimental designs for the optimization of nisin production from whey.

Statistically-based experimental designs were applied for the optimization of nisin production by Lactococcus lactis in a whey-based medium. Yeast extract, KH2PO4, and MgSO4 were identified to have significant effects on nisin biosynthesis by a Plackett-Burman design. These three significant factors were subsequently optimized using central composite design, and the optimal conditions were determined to be 12.067 g l(-1) for yeast extract, 0.569 g l(-1) for KH2PO4, and 0.572 g l(-1) for MgSO4. The validity of the optimal conditions was verified by a separate experiment.