Nisin Production Research Articles

Effects of pH profiles on nisin fermentation coupling with foam separation

Online foam separation was proposed to recover nisin during fermentation of Lactococcus lactis subsp. lactis ATCC 11454. Firstly, the optimal pH profile of nisin fermentation was investigated including different realkalization set values and pH drop gradients. Then the selected pH profiles of 5.75+/-0.05 and 6.25-5.75 (+/-0.02) were used to perform nisin fermentation coupling with foam separation. The results showed that pH profile of 5.75+/-0.05 was better than that of 6.25-05.75 (+/-0.02) for online foam separation. With the optimal pH profile, an aeration of 20 ml min(-1) that started at 8 h of incubation and lasted for 2 h resulted in 6.6 times higher specific productivity than that of the fermentation without aeration. Nisin synthesis was therefore prolonged with low sucrose concentration in the culture broth, which indicated that the feedback inhibition of nisin is more influential than the substrate limitation of sucrose in the late phase of nisin fermentation. Total nisin production (4,870+/-180 IU ml(-1)) was increased by 30.3% with online foam separation. This effective online recovery method for nisin production could be easily scaled up due to the facile operation of foaming process.

Production and nano-formulation of nisin in liposome as a slow release preservative against important food-born pathogens in UF cheese

Production and nano-formulation of nisin in liposome as a slow release preservative against important food-born pathogens in UF cheese

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.

Continuous nisin production with bioengineered Lactococcus lactis strains

Nisin production in continuous cultures of bioengineered Lactococcus lactis strains that incorporate additional immunity and regulation genes was studied. Highest nisin activities were observed at 0.2 h(-1) dilution rate and 12.5 g l(-1) fructose concentration for all strains. Recombinant strains were able to produce greater amounts of nisin at dilution rates below 0.3 h(-1) compared to the control strain. However, this significant difference disappeared at dilution rates of 0.4 and 0.5 h(-1). For the strains LL27, LAC338, LAC339, and LAC340, optimum conditions for nisin production were determined to be at 0.29, 0.26, 0.27, and 0.27 h(-1) dilution rates and 11.95, 12.01, 11.63, and 12.50 g l(-1) fructose concentrations, respectively. The highest nisin productivity, 496 IU ml(-1) h(-1), was achieved with LAC339. The results of this study suggest that low dilution rates stabilize the high specific nisin productivity of the bioengineered strains in continuous fermentation. Moreover, response surface methodology analysis showed that regulation genes yielded high nisin productivity at wide ranges of dilution rates and fructose concentrations.

Identification of Nisin-Producing Strains by Nisin-Controlled Gene Expression System

A specific method to identify nisin-producing strains was developed based on Nisin-Controlled gene Expression (NICE) vector pSec:Nuc. The plasmid pSec:Nuc was transformed into non-nisin-producing strain Lactococcus lactis NZ9000, a host commonly used for the NICE system. The generating strain L. lactis NZ9000/pSec:Nuc could sense extracellular inducer nisin and efficiently secrete a reporter protein Nuc, the staphylococcal nuclease (Nuc) into the medium. Instead of using purified nisin, the culture supernatants of nisin-producing strains were also used as inducers. Therefore, the NICE system could be used to identify nisin-producing strains. With this principle, 4 among 56 lactococci strains isolated from raw milk were identified as nisin producers. The results were further confirmed by polymerase chain reaction amplification with their genomic DNA as templates, and nucleotide sequencing revealed that three of them produced nisin A, and the others produced nisin Z. Those results made it possible to isolate and identify nisin-producing strains specifically and rapidly using NICE system.

Production of nisin Z using Lactococcus lactis IO-1 from hydrolyzed sago starch

A membrane bioreactor for production of nisin Z was constructed using Lactococcus lactis IO-1 in continuous culture using hydrolyzed sago starch as carbon source. A strategy used to enhance the productivity of nisin Z was to maintain the cells in a continuous growth at high cell concentration. This resulted in a volumetric productivity of nisin Z, as 50,000 IU l(-1) h(-1) using a cell concentration of 15 g l(-1), 30( degrees )C, pH 5.5 and a dilution rate of 1.24 h(-1). Adding 10 g l(-1) YE and 2 g l(-1) polypeptone, other inducers were unnecessary to maintain production of nisin. The operating conditions of the reactor removed nisin and lactate, thus minimizing their effects which allowed the maintenance of cells in continuous exponential growth phase mode with high metabolic activity.

Influence of growth conditions on the nisin production of bioengineered Lactococcus lactis strains

Nisin production of three bioengineered strains, (LAC338, LAC339 and LAC340) with immunity (nisFEG) and/or regulation (nisRK) genes of nisin biosynthesis on plasmids in the Lactococcus lactis LL27 nisin producer, was evaluated under pH-controlled and pH-uncontrolled batch fermentations. Optimization studies showed that fructose and yeast extract yielded the highest nisin activity. The strains LAC338, LAC339, and LAC340 produced 24, 45, and 44% more nisin, respectively, than wild-type L. lactis LL27 after 12-h incubation. However, sharp decreases in the yield of nisin were observed at the late phase of fermentation with LAC339 and LL27 in contrast to LAC340 and LAC338 strains for which the high level of nisin could be maintained longer. Obviously, increasing the copy number of the regulation genes together with immunity genes in the nisin producers retarded the loss of nisin in the late phase of the fermentation.

Inhibition of toxicogenic Bacillus licheniformis 553/1 in Nigerian Wara soft cheese by nisin‐producing Lactococcus lactis LAC309

SummaryWara soft cheese is a traditionally produced cheese in Nigeria. The production of this cheese includes a heating step for killing vegetative cells. Therefore, mainly spore‐forming bacteria surviving the process may function as the first spoilers resulting in lowered shelf‐life and safety of the product. In this study, we investigated if the addition of a nisin‐producing Lactococcus lactis LAC309 starter after the high temperature treatment could result in nisin production and inhibition of the toxicogenic Bacillus licheniformis 553/1 strain spiked into the cheese. The results showed that L. lactis LAC309 could produce nisin in Wara cheese and that the strain inhibited (3 log reduction) B. licheniformis 553/1 in Wara cheese. Food technologists developing industrialised Wara cheese should therefore consider including a nisin‐producing starter strain in the cheese‐making process.

Phenotypic and Genotypic Characterization of Nisin-Producing Lactococcus Lactis Subsp. Lactis YB23 Isolated from Raw Milk in Turkey

ABSTRACTA total of 142 presumptive lactococci were isolated from 40 raw milk samples and screened for bacteriocin production. Four presumptive lactococci isolates (YB23, YB42, YB98 and YB112) exhibited antimicrobial activity against indicator strains. Antimicrobial activity and protease sensitivity assays showed that a strain, YB23, produced bacteriocin that inhibited a broad spectrum of Gram-positive bacteria, and was selected for further analyses. It was identified as Lactococcus lactis subsp. lactis, phenotypically by the API system and genotypically by 16S rDNA homology. YB23 was tested for determination of antimicrobial activity against indicator strains including known bacteriocin producing lactococci and it showed no activity against nisin producer Lactococcus lactis SIK83. Different pH and heat treatments showed that the bacteriocin produced by L. lactis subsp. lactis YB23 exhibited a similar behavior with nisin produced by L. lactis SIK83. The bacteriocin produced by YB23 strain was characterized as nisin by PCR analysis of its genomic DNA. Lactic acid production and proteolytic activity in strain YB23 were determined as 0.38% and 25.3 μg Tyr/mL, respectively.

Mu transposition complex mutagenesis inLactococcus lactis- identification of genes affecting nisin production

This paper describes optimization of electrotransformation of Mu transposition complexes into Lactococcus lactis cells and identification of genes affecting nisin production. The highest transformation efficiency, 1.1 x 10(2) transformants microg(-1) of input transposon DNA, was achieved when cells were grown to an OD(600) of 0.5 in the presence of 1.5% of glycine and treated with 20 microg ml(-1) ampicillin for 60 min. Three insertions affecting nisin production, which were identified at nisB, fhuR, and rpiA genes, were screened from a library of approximately 2000 erythromycin-resistant transformants using a nisin bioassay method. NisB is part of the nisin biosynthetic machinery, explaining the loss of nisin production in nisB mutant. FhuR is a transcription regulator involved in sulphur acquisition. Inactivation of fhuR presumably results in a low cellular cystein level, which affects nisin biosynthesis that involves utilization of cystein. RpiA is involved in pentose phosphate pathway and carbon fixation. The rpiA mutant showed reduction in nisin production and slow growth rate. The results showed that Mu transposition complex mutagenesis can be used to identify genes in L. lactis. Three genes involved in nisin production were identified. Expanding the Mu transposition-based mutagenesis to Lactococci adds a new tool for studies of industrially important bacteria.

Cycle changing the medium results in increased nisin productivity per cell in Lactococcus lactis

High specific cellular nisin production was aimed by cycle changing the medium of Lactococcus lactis N8 and LAC48. The highest level of nisin production was reached with the 120 min cycles but maximal production was unstable. In shorter cycles (30 and 60 min) cells could be maintained in a high production state up to the end of the fermentation (28 and 14 cycles). N8 produced 19-fold and LAC48 15-fold more nisin with cycle changing the medium than without cycle incubation.

Biosynthetic characterization and biochemical features of the third natural nisin variant, nisin Q, produced byLactococcus lactis61-14

To characterize the genetic and biochemical features of nisin Q. The nisin Q gene cluster was sequenced, and 11 putative orfs having 82% homology with the nisin A biosynthesis gene cluster were identified. Nisin Q production was confirmed from the nisQ-introduced nisin Z producer. In the reporter assay, nisin Q exhibited an induction level that was threefold lower than that of nisin A. Nisin Q demonstrated an antimicrobial spectrum similar to those of the other nisins. Under oxidizing conditions, nisin Q retained a higher level of activity than nisin A. This higher oxidative tolerance could be attributed to the presence of only one methionine residue in nisin Q, in contrast to other nisins that contain two. The 11 orfs of the nisin producers were identical with regard to their functions. The antimicrobial spectra of the three natural nisins were similar. Nisin Q demonstrated higher oxidative tolerance than nisin A. Genetic and biochemical features of nisin Q are similar to those of other variants. Moreover, owing to its higher oxidative tolerance, nisin Q is a potential alternative for nisin A.

Influence of complex nutrient sources: Dates syrup and dates pits on Lactococcus lactis growth and nisin production

Influence of complex nutrient sources: Dates syrup and dates pits on Lactococcus lactis growth and nisin production

Utilization of Fermented Barley Extract Obtained from a By-product of Barley Shochu for Nisin Production

Fermented barley extract (FBE) obtained from a barley shochu by-product (shochu kasu) and its ethanol fractions were evaluated as a medium and supplement, respectively, for nisin A production by Lactococcus lactis subsp. lactis ATCC 11454. A Brix 2.5 FBE medium supplemented with glucose provided a high level of nisin A production with a nisin yield comparable to that from a nutritionally rich laboratory medium (basal medium). By adding the ethanol-insoluble (EI) fraction of FBE to the basal medium, nisin A production was enhanced concomitant with an increase in bacterial cell growth, while the ethanol-soluble (ES) fraction had a negative effect on nisin A production. These findings indicate that FBE obtained from shochu kasu can be utilized as a preferable medium for nisin A production and could be converted into a value-added food product having preservative functions. The procedure developed in this study would promote recycling of shochu kasu.

Stress Response Kinetics of Two Nisin Producer Strains of Lactococcus lactis spp. lactis

The purpose of this study is to determine the survival and nisin production behaviors of two strains of Lactococcus lactis under different stress conditions that represent the food ecosystem. In this respect, the survival ratios of two nisin producers were determined under different pH, temperature, NaCl, and bile salt concentrations. Then, nisin production levels of the strains were determined at each stress conditions. Both strains had similar growth or inactivation patterns under the same stress conditions. NaCl and bile salt stresses on the survival ratio of the strains could be successfully described by the exponential decay function, whereas Gaussian function produced good fits for temperature and pH stresses. The nisin activity of two nisin producers (in their mid-exponential and/or early stationary phase) decreased dramatically under all stress conditions, except osmotic (NaCl) and low temperature applications. The results of this study showed that two nisin producers had similar adaptive responses under severe stress conditions, which could be described by appropriate mathematical equations. Moreover, the effect of harsh environment on the nisin activity of L. lactis strains depends on the stress factors applied.

Variable Volume Fed-Batch Fermentation for Nisin Production by Lactococcus lactis subsp. lactis W28

A feeding technology that was suitable for improving the nisin production by Lactococcus lactis subsp. lactis W28 was established. The effects of initial sucrose concentration (ISC) in the fermentation broth, feeding time, and feeding rate on the fermentation were studied. It was observed that a fed-batch culture (ISC = 10 g l(-1)) with 100 ml sucrose solution (190 g l(-1)) being evenly fed (9-10 ml h(-1)) into the fermenter after 3-h fermentation gave the best performance in terms of biomass and nisin yield. Under these conditions, the total biomass and the total nisin yield were approximately 23% and 51% higher than those in batch fermentation, respectively. When the sucrose concentration was controlled at 5-10 g l(-1) in variable volume intermittent fed-batch fermentation (VVIF) with ISC = 10 g l(-1), the total biomass and the total nisin yield were 29% and 60% above those in batch fermentation, respectively. The VVIF proved to be effective to eliminate the substrate inhibition by maintaining sucrose at appropriate levels. It is also easy to be scaled up, since various parameters involved in industrial production were taken into account.

Fermentative Production, Purification and Characterization of Nisin

Bacteriocins are bactericidal or bacteriostatic in action and active against closely related species. Nisin is the bacteriocin produced by the lactic acid bacteria (LAB). Nisin is a small (3353 Da), cationic, hydrophobic, and 34-amino acid peptide. It is used in products such as pasteurized processed cheese, salad dressing, and liquid whole eggs to inhibit the growth of Gram-positive microorganisms including Listeria monocytogenes. The objective of the present work was to study production, purification and characterization of nisin. The production of nisin was carried out using Lactococcus lactis subsp lactis MTCC 440 by one-factor-at-a-time method and statistical design (Orthogonal array). Purification was carried out using ammonium sulphate precipitation followed by hydrophobic interaction and gel chromatography. Characterization was done for pH and temperature stability. The activity of nisin after one factor at a time optimization was found to be 5120 AU/ml. The activity of the nisin increased to 6800 AU/ml after optimization by Orthogonal Array design. Ammonium sulphate precipitation gives good yield of nisin with 60 to 80% saturation with 2.52 fold purity. The overall purification by hydrophobic interaction and gel filtration chromatography was 10.87 fold with 50.84% yield and 8.8 fold with 49.65% yield as compared to crude broth respectively.

Optimization of Medium Composition for Nisin Fermentation with Response Surface Methodology

Nisin is an effective food biopreservative widely used in food industry. However, 1 problem of concern is limited production rate and final nisin concentration. A nisin-producing strain, L. lactis Lac2, a mutant strain with high yield of nisin, was obtained in our laboratory recently. In the present study, a fractional factorial design was applied to investigate the main factors that affect the yield of L. lactis Lac2. Central composite experimental design and response surface methodology were adopted to derive a statistical model for optimizing the composition of the medium. The results showed that the optimum medium for nisin production of L. lactis Lac2 was composed of 2.68% sucrose (w/v), 0.5% tryptone (w/v), 1% yeast extract (w/v), 0.3% Tween-80 (w/v), 0.02% MgSO4.7H2O (w/v), 0.81% NaCl (w/v), 1.91% K2HPO4 (w/v), 0.05% ascorbic acid (w/v), and 2% agar (w/v) (if necessary) at pH 6.5. When cultured in the optimum medium, the nisin yield is an average of 3381.81 IU/mL, which nearly doubled the yield when incubated in the initial medium. Also, the concentration of tryptone was decreased while that of the sucrose was increased when compared with CM broth, which means a reduction of the fermentation cost.

Distinct Contributions of the Nisin Biosynthesis Enzymes NisB and NisC and Transporter NisT to Prenisin Production by Lactococcus lactis

Several Lactococcus lactis strains produce the lantibiotic nisin. The dedicated enzymes NisB and NisC and the transporter NisT modify and secrete the ribosomally synthesized nisin precursor peptide. NisB can function in the absence of the cyclase NisC, yielding the dehydrated prenisin that lacks the thioether rings. A kinetic analysis of nisin production by L. lactis NZ9700 demonstrated that the prenisin was released from the cell into the medium before the processing of the leader sequence occurred. Upon the deletion of nisC, the production of prenisin was reduced by 70%, while in the absence of nisB, the production of prenisin was nearly completely abolished. In cells lacking nisT, no secretion was observed, while the expression of nisABC in these cells resulted in considerable growth rate inhibition caused by the intracellular accumulation of active nisin. Overall, these data indicate that the efficiency of prenisin transport by NisT is markedly enhanced by NisB, suggesting a channeling mechanism of prenisin transfer between the nisin modification enzymes and the transporter.

Nisin expression production from Lactococcus lactis in milk whey medium

AbstractBACKGROUND: Nisin is a commercially available bacteriocin produced by Lactococcus lactis ATCC 11454 and used as a natural agent in the biopreservation of food. In the current investigation, milk whey, a byproduct from dairy industries was used as a fermentation substrate for the production of nisin. Lactococcus lactis ATCC 11454 was developed in a rotary shaker (30 °C/36 h/100 rpm) using two different media with milk whey (i) without filtration, pH 6.8, adjusted with NaOH 2 mol L−1 and without pH adjustment, both autoclaved at 121 °C for 30 min, and (ii) filtrated (1.20 µm and 0.22 µm membrane filter). These cultures were transferred five times using 5 mL aliquots of broth culture for every new volume of the respective media.RESULTS: The results showed that culture media composed of milk whey without filtration supplied L. lactis its adaptation needs better than filtrated milk whey. Nisin titers, in milk whey without filtration (pH adjusted), was 11120.13 mg L−1 in the second transfer, and up to 1628‐fold higher than the filtrated milk whey, 6.83 mg.L−1 obtained in the firstt transfer.CONCLUSIONS: Biological processing of milk byproducts (milk whey) can be considered a profitable alternative, generating high‐value bioproducts and contributing to decreasing river disposals by dairy industries. Copyright © 2008 Society of Chemical Industry