Nisin Production Research Articles

Cascade pH Control in Antimicrobial Peptide, Nisin, Production Process, Utilizing a Mixed Culture System

In order to control pH in an antimicrobial peptide (nisin) production process by a lactic acid bacterium (LAB), Lactococcus lactis sbsp. lactis (ATCC11454), a novel method without alkaline addition and mechanistic separation system was developed, using interaction of L. lactis and another microorganism. A lactic acid assimilating yeast, Kulyveromyces marxianus was isolated from a fermentation food, kefir grains, and a mixed culture system with L. lactis and K. marxianus was developed. The kinetics of growth and production by both microorganisms were investigated in detail. The specific lactic acid consumption rate of K. marxianus was controlled by changing in the dissolved oxygen concentration (DO) and a cascade controller of pH and DO was developed. Lactic acid concentration was kept at low level and nisin was highly produced, comparing with the production processes without pH control and with pH control by alkaline addition.

Effects of gene disruptions in the nisin gene cluster of Lactococcus lactis on nisin production and producer immunity.

The lantibiotic nisin is produced by several strains of Lactococcus lactis subsp. lactis. The chromosomally located gene cluster nisABTCIPRKFEG is required for biosynthesis, development of immunity, and regulation of gene expression. Inframe deletions in the nisB and nisT genes, and disruption of nisC by plasmid integration, eliminated nisin production and resulted in a strongly reduced level of immunity of the strains. The transcription of two nisin operons was inactivated in these mutant strains, but could be restored by addition of small amounts of nisin to growing cultures. The immunity levels of the mutants were also raised by adding nisin to growing cultures, albeit not to wild-type level. A strain with an in-frame deletion in the nisI gene was still able to produce active nisin, but the production and immunity levels were markedly lower. By measuring immunity levels of the knock-out strains and determining mRNA levels, it is concluded that NisI has an important function for nisin immunity and must cooperate with nisFEG-encoded proteins to provide a high level of immunity. Maximal immunity could not be obtained in the mutant strains, probably because the wild-type transcription levels from nisA and nisF promoters are not reached when essential nis genes are disrupted. Using Southern hybridization with a consensus promoter probe, no other DNA sequences similar to the nisA and nisF promoters could be detected, indicating that these two elements are probably the only ones in the chromosome regulated by nisin and are thus the only ones involved in the regulation of producer immunity.

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.

Improving nisin production by increasing nisin immunity/resistance genes in the producer organism Lactococcus lactis

The effect on nisin production of increasing nisin immunity/resistance genes in Lactococcus lactis subsp. lactis MG1363 was investigated. The 60-kb nisin immunity/resistance plasmid pND300, which was isolated from a non-nisin-producing strain, encodes five genes involved in nisin immunity/resistance, which are very similar to those of the immunity/resistance system encoded by the nisin-production transposon. The introduction of pND300 into MG1363(TnNip) resulted in the construct being able to produce significantly more nisin than the parent MG1363(TnNip). The introduction of pND314, which contains the nisin immunity/resistance genes subcloned into pSA3, into MG1363(TnNip) allowed the strain to grow more rapidly than the parent MG1363(TnNip) with a concomitant increase in the rate of nisin production. This work illustrates that introduction of pND300 and a derivative containing the nisin immunity/resistance system of pND300 into MG1363 (TnNip) can result in significant alterations to the kinetics of nisin production. These observations indicate approaches that may be used successfully to improve the economics of nisin production.

Procedure for quantifiable assessment of nutritional parameters influencing Nisin production by Lactococcus lactis subsp. lactis

A modified rapid plate assay procedure was developed, that allowed quantifiable measurement of nisin production by Lactococcus lactis growing directly on agar media. Using this direct plate assay, several nutritional parameters were assessed for their influence on nisin production (as distinct from their influence on growth) by L. lactis subsp. lactis ATCC 11454 growing on standard M17 based media over 3 and 6 h incubation periods. Glucose was found to be the optimal carbon source tested, with glycerol having the greatest suppressive effect. The addition of salts suppressed nisin production on a per cell basis, except MnCl 2. This direct plate method proved to be a good pilot assay for rapidly and quantifiably investigating the initial effects of different parameters on nisin production by L. lactis, prior to conducting more intensive broth batch culture assays. The data obtained in this study indicate that certain nutritional parameters can impose a repressive effect on nisin production. Elucidation of how these parameters control the amount of nisin produced will provide further insight into the regulation of nisin biosynthesis in L. lactis.

Tn916 family conjugative transposons and dissemination of antimicrobial resistance determinants.

The widespread use of broad-spectrum antimicrobial agents poses a significant challenge to bacteria pathogenic for humans, a challenge that has been answered by the acquisition and spread of a variety of antimicrobial resistance determinants. Bacterial resistance to antibiotics results from the mutation of normal cellular genes, the acquisition of foreign resistance genes, or a combination of these two mechanisms. Resistance to some antimicrobials or classes of antimicrobials, notably, rifampin or the fluoroquinolones, occurs primarily through point mutations and is not transferable. Other forms of resistance, such as multiple antibiotic resistance efflux systems and inducible β-lactamase-mediated resistance, result from regulatory mutations in normal cellular processes and are also not transferable. For many bacteria-antibiotic combinations, however, such intrinsic mechanisms of resistance are not available, so resistance genes must be imported. To accomplish this end, bacteria use a complex array of mechanisms to share and disseminate useful resistance determinants. Mechanisms frequently cited as potentially important for the dissemination of antibacterial resistance determinants include natural transformation (penicillin resistance in Streptococcus pneumoniae and Neisseria spp.) (16, 60), bacteriophage-mediated transduction (β-lactamase-mediated penicillin resistance in staphylococci) (33), and plasmid- or transposon-mediated conjugation (ampicillin resistance in members of the family Enterobacteriaceae, tetracycline resistance in enterococci, and many others) (3, 20). With the widespread availability of techniques for analyzing the molecular genetics of bacterial pathogens, understanding of the role of transposons in the dissemination of antimicrobial resistance has expanded significantly. Among the more interesting and important classes of transposons are the conjugative transposons. As their name implies, conjugative transposons are mobile elements that possess the genetic machinery to facilitate their own transfer between bacterial cells. Several prototypes of conjugative transposons have been described. Conjugative transposons encoding the tet(Q) tetracycline resistance determinants are widespread in clinical Bacteroides isolates and have been the subject of several recent reviews (54, 55). Tn5276-like transposons are large (70- to 80-kb) elements that encode genes for nisin production (44, 45). These elements have not been shown to encode antimicrobial resistance determinants, appear to be restricted to lactococci, and will not be addressed further in this minireview. The present minireview focuses on the Tn916 and Tn1545 family of conjugative transposons that predominates in clinically important gram-positive bacteria. The basic mechanisms of movement of these transposons have recently been reviewed (57). The present work focuses on recent developments in the basic science of conjugative transposons and more specifically on the actual and potential role of these elements in the dissemination of important resistance determinants in pathogenic bacteria.

Lysis of Lactococcus lactis subsp. cremoris SK110 and its nisin-immune transconjugant in relation to flavor development in cheese.

To develop a nisin-producing cheese starter, Lactococcus lactis subsp. cremoris SK110 was conjugated with transposon Tn5276-NI, which codes for nisin immunity but not for nisin production. Cheese made with transconjugant SK110::Tn5276-NI as the starter was bitter. The muropeptide of the transconjugant contained a significantly greater amount of tetrapeptides than the muropeptide of strain SK110, which could have decreased the susceptibility of the cells to lysis and thereby the release of intracellular debittering enzymes.

Some Characteristics of Nisin Z, a Peptide Antibiotic Produced by Lactococcus lactis IO-1.

Lactococcus lactis IO-1 isolated in our laboratory produces a peptide antibiotic, a natural nisin variant, nisin Z. Nisin Z was heat stable at acidic pH and showed a bactericidal mode of action against an indicator strain, Bacillus subtilis C1. The effect of proteolytic enzyme treatment on the activity and the antimicrobial spectrum were tested. These results indicated nearly the same characteristics as those of nisin A. In this study, it was found that two nisin variants (nisin Z and nisin A) were practically inactivated by proteinase K and actinase E. Interestingly, nisin-producing strains, L. lactis IO-1 and L. lactis NCDO 497 (a nisin A producer) were sensitive to a high concentration of nisin variants including their own products. The growth of a Gram-negative bacterium, Escherichia coli, was also inhibited by a high concentration of nisin, although nisin producers and Gram-negative bacteria are generally resistant to nisin variants.

Characterization of the nisFEG operon of the nisin Z producing Lactococcus lactis subsp. lactis N8 strain.

Biosynthesis of the food additive nisin, a posttranslationally modified peptide antibiotic existing as two natural variants (A and Z), requires eleven genes (nisA/ZBTCIPRKFEG) involved in modification, secretion, regulation and self-immunity. The suggested self-immunity genes (nisFEG) of the nisin Z producer Lactococcus lactis subsp. lactis N8 were cloned and sequenced. Putative binding sites of the NisR transcription factor were recognized upstream of the nisF promoter. The hydrophilic NisF protein was expressed in Escherichia coli and shown to be associated with the membrane. Expression of the nisF gene from a plasmid in L. lactis MG1614, a strain lacking the nisin operons, did not increase the nisin resistance of the cells. This showed that NisF alone does not protect against nisin. Overexpression of the nisF gene in the N8 nisin producer did not affect the level of nisin immunity, indicating that the wild-type amount of NisF is not limiting the level of nisin immunity. Production of antisense-nisEG or antisense-nisG RNA in L. lactis N8 resulted in severe reduction in the level of nisFEG mRNA and a clearly reduced immunity showing that the nisFEG transcript is important for development of nisin self-immunity.

The effect of nisin concentration and nutrient depletion on nisin production of Lactococcuslactis

The kinetics of nisin production was studied in batch cultures using a construct of Lactococcus lactis subsp. lactis C2SmPrt-, containing a transposon (TnNip) that encodes nisin production. The introduction of TnNip into C2SmPrt- significantly lowered the specific growth rate and the maximum A620 reached was reduced from 15.2 to 11.0. The effect of nisin concentration and nutrient depletion on nisin production of the construct, C2SmPrt-(TnNip), was examined. Nisin production was found to be inhibited by high concentrations of nisin, when grown in excess nutrient, even though growth of the culture continued because nutrient limitation was not operating. However, in low nutrient concentrations nisin production was limited by nutrient depletion. The specific growth rate of C2SmPrt-(TnNip) was altered, by using different nutrient concentrations and different sugars, in order to examine the relationship between nisin production and growth. Nisin production was shown to be growth-associated for most of growth, but near the end of growth, when the specific growth rate was 0.05 h-1 or less, the production ceased.

Nisin production by Lactococcus lactis using two-phase batch culture.

Nisin production by Lactococcus lactis subsp. lactis C2SmPrt-(TnNip) was investigated using two-phase batch culture. A solvent (phenyl-methyl silicone oil) was introduced in addition to the aqueous phase. The partition coefficient of this solvent in aqueous nisin varied as the cultivation medium changed, with a minimum value being 1.04. The two-phase batch culture supported a 21% increase in growth and a 24% increase in the level of nisin produced compared to the single-phase batch culture.

Combined effect of bacteriocin-producing lactic acid bacteria and lactoperoxidase system activation on Listeria monocytogenes in refrigerated raw milk.

The bactericidal activity of three bacteriocin-producing lactic acid bacteria alone and in combination with milk lactoperoxidase (LP) system activation against Listeria monocytogenes in refrigerated raw milk was studied. After 4 d at 4 degrees C, the population of L. monocytogenes in milk inoculated with bacteriocin-producing Lactococcus lactis subsp. lactis ATCC 11454, L. lactis subsp. lactis ESI 515 or Enterococcus faecalis INIA 4 was reduced by 0.21-0.24 log units. Activation of the LP system did not enhance inhibition at this temperature. After 4 d at 8 degrees C, L. monocytogenes levels in the non-activated LP system milk inoculated with L. lactis subsp. lactis ATCC 11454, L. lactis subsp. lactis ESI 515 or Ent. faecalis INIA 4 were reduced by 1.87, 1.54 and 1.11 log units compared to control milk, whereas in the activated LP system milk, this reduction was 1.99, 2.10 and 1.06, respectively. The higher nisin production by L. lactis subsp. lactis ESI 515 in milk with activated LP system than in non-activated LP system milk was responsible for the more pronounced decrease of L. monocytogenes counts in the former.

Stabilization of the Lactococcus lactis nisin production transposon as a plasmid

A transposon (Tn Nip) encoding nisin production was visualized and stabilized as a plasmid of approximately 60 kb in the transconjugant, LM0230Fus(Tn Nip), formed from a conjugation between nisin-producing Lactococcus lactis subsp. lactis LL33-1 and a plasmid-free LM0230 derivative. The plasmid did not correspond in size to any of the plasmids resident in the donor. A plasmid-cured derivative of this construct was also able to stabilize the transposon, as a plasmid, from all donors used. Therefore, for the first time, a host has been identified that can maintain the Tn Nip transposons stably excised out of the chromosome. The plasmid DNA was isolated and characterized by restriction digestion and was used to successfully transform cells, thus linking the nisin production phenotype to the plasmid.

Neutralization/recovery of lactic acid from Lactococcus lactis: effects on biomass, lactic acid, and nisin production

Besides lactic acid, many lactic acid bacteria also produce proteinaceous metabolites (bacteriocins) such as nisin. As catabolite repression and end-product inhibition limit production of both products, we have investigated the use of alternative methods of supplying substrate and neutralizing or extracting lactic acid to increase yields. Fed-batch fermentation trials using a stillage-based medium with pH control by NH4OH resulted in improved lactic acid (83.4 g/l, 3.18 g/l/h, 95% yield) and nisin (1,260 IU/ml, 84,000 IU/l/h, 14,900 IU/g) production. Removing particulate matter from the stillage-based medium increased nisin production (1,590 IU/ml, 33,700 IU/g), but decreased lactic acid production (58.5 g/l, 1.40 g/l/h, 96% yield). Removing lactic acid by ion exchange resins stimulated higher lactic acid concentrations (60 to 65 g/l) and productivities (2.0 to 2.6 g/l/h) in the filtered stillage medium at the expense of nisin production (1,500 IU/ml, 25,800 IU/g).

Isolation of a Lactococcus lactis strain with high resistance to nisin and increased nisin production

Lactococcus lactis LAC48, a spontaneous erythromycin resistant mutant strain of N8, showed an increased nisin resistance from 1000 to 5000 IU ml -1 . The cells of LAC48 produced 10 times more nisin than the cells of the N8 strain. The results of Northern and Western analyses of LAC48 suggested that the majority of the exceptionally high level of nisin resistance in strain LAC48 was not due to changes in the expression level of the natural immunity genes. Strain LAC48 represents a good candidate to be used in industrial fermentation to achieve a high nisin titre.

Host specificity of nisin production by Lactococcus lactis

Kinetics of nisin production have been investigated in terms of endogenous features of the producer organism, Lactococcus lactis. Nisin-producing transposons (Tn Nip) were transferred to different hosts by conjugation. Constructs were cultivated in batch cultures and nisin produced was measured. The proteinase function of C2Prt (Tn Nip)-1 was eliminated by plasmid curing, resulting in the construct C2Prt - (Tn Nip)-1. C2Prt - (Tn Nip)-1 produced nisin to a higher concentration compared to C2Prt (Tn Nip)-1 and was able to maintain the maximum concentration till the end of cultivation. The final concentration of nisin produced was host-specific, because when different constructs carrying the same Tn Nip were cultivated they produced nisin to different concentrations. However, when the same host carried Tn Nip transposons derived from different donors the concentration of nisin produced was similar, suggesting that the two Tn Nip transposons may be similar.

Concentration and recovery of the bacteriocin nisin from Lactococcus lactis subsp. lactis.

This investigation compared various techniques to concentrate and recover nisin from Lactococcus lactis cells. Centrifugation, combined with pH manipulation, was initially investigated as it is known that nisin will adsorb to producer cells at pH 6.5 and desorb at pH < or = 3.0. Unfortunately, centrifugation stripped producer cells of nisin even at pH 6.5; therefore a milder separation process (microfiltration) was evaluated. Results using a medium (LTB) containing peptone, tryptone, yeast extract, NaCl, Na2HPO4 and glucose demonstrated that nisin could be at least partially concentrated with cells via microfiltration. However, when a filtered stillage-based medium was used, nisin production was boosted to levels which exceeded the holding capacity of producer cells, resulting in release of nisin from cells at pH 6.5. Since it appears unfeasible to use producer cells for nisin recovery, an alternative may be to separately immobilize cells/ fragments in a re-usable column to act as a "resin' to adsorb nisin. Microfiltration could then be used to release nisin from cells, with nisin recovered by passing the permeated material through the immobilized-cell columns at pH 6.5.

Presence of non-functional nisin genes in Lactococcus lactis subsp. lactis isolated from natural starters

Sixty-four lactococcal strains isolated from natural whey starters were screened for the presence of the nisin structural gene by polymerase chain reaction. Seven of them showed a specific PCR product of 320 bp; only two produced antagonistic activity and were resistant to nisin. Southern blots of SmaI-digested DNA from PCR-positive strains hybridized with a nis A probe displayed a location of the gene on different SmaI fragments. Among PCR-positive strains, nisin producers showed specific transcript after reverse transcriptase-PCR, as well as some non-nisin-producing strains. The RT-PCR product could not be shown in one non-nisin-producing PCR-positive strain.

Identification and characterization of a mobilizing plasmid, pND300, in Lactococcus lactis M189 and its encoded nisin resistance determinant.

A 60 kb conjugative plasmid, pND300, which encodes nisin resistance, was identified in Lactococcus lactis ssp. lactis (L. lactis) M189. pND300 was found to mobilize the transfer of some other plasmids as indicated by the mobilization of plasmids encoding lactose utilization. The nisin resistance determinant from pND300 was initially subcloned on a 12 kb DNA fragment and subsequently reduced to 10.4 kb. Restriction analysis, PCR, Southern hybridization and sequencing illustrated that the nisin resistance of pND300 is very similar to that encoded by the transposon involved in nisin production. pND300 encodes nisR as well as nisK and the recently reported nisF, nisE and nisG, but does not encode nisI. The DNA fragment encoding the nis genes is flanked by IS946 with a copy at each end in reverse orientation. The expression of these nis genes is probably controlled by a putative promoter upstream of nisR, which is composed of the TTGCAA hexanucleotide on the insertion sequence IS946 and the TATAAT sequence 21 bp downstream.

Presence of non-functional nisin genes in Lactococcus lactis subsp. lactis isolated from natural starters.

Sixty-four lactococcal strains isolated from natural whey starters were screened for the presence of the nisin structural gene by polymerase chain reaction. Seven of them showed a specific PCR product of 320 bp; only two produced antagonistic activity and were resistant to nisin. Southern blots of SmaI-digested DNA from PCR-positive strains hybridized with a nisA probe displayed a location of the gene on different SmaI fragments. Among PCR-positive strains, nisin producers showed specific transcript after reverse transcriptase-PCR, as well as some non-nisin-producing strains. The RT-PCR product could not be shown in one non-nisin-producing PCR-positive strain.