Production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from Kimchi

Lactococcus lactis subsp. lactis A164 was isolated from Kimchi (Korean traditional fermented vegetables). The bacteriocin produced by strain A164 was active against closely related lactic acid bacteria and some food-borne pathogens including Staphylococcus aureus, Listeria monocytogenes and Salmonella typhimurium. The antimicrobial spectrum was nearly identical to that of nisin. Bacteriocin activity was not destroyed by exposure to elevated temperatures at low pH values, but the activity was lost at high pH values. This bacteriocin was inactivated by pronase E and alpha, beta-chymotrypsin, but not by trypsin, pepsin, and alpha-amylase. Cultures of L. lactis subsp. lactis A164 maintained at a constant pH of 6.0 exhibited maximum production of the bacteriocin. It was purified to homogeneity by ammonium sulphate precipitation, sequential ion exchange chromatography, and ultrafiltration. Tricine-SDS-PAGE of purified bacteriocin gave the same molecular weight of 3.5 kDa as that of nisin. The gene encoding this bacteriocin was amplified by PCR with nisin gene-specific primers and sequenced. It showed identical sequences to the nisin gene. These results indicate that bacteriocin produced by Lactococcus lactis A164 is a nisin-like bacteriocin.     

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.     

Note : Genetic and biochemical characterization of nisin Z produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719

The bacteriocin produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719 was purified and characterized. Two peaks exhibiting antimicrobial activity were obtained after purification. Primary structure of the peptide of major peak 2 was identical to that of nisin Z when determined by Edman degradation and confirmed by DNA sequence analysis. The molecular mass as determined by mass spectrometry was 3346·39 ± 0·40 Da for peak 1 and 3330·39 ± 0·27 Da for peak 2, which suggests that peak 1 may correspond to an oxidized form of nisin Z. The two purified peaks exhibiting xrantimicrobial activity appear to correspond with the oxidized and native forms of nisin Z.     

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.     

A comparison of the properties of bacteriocins formed by Lactococcus lactis subsp. lactis strains of diverse origin

Bacteriocins formed by four strains of Lactococcus lactis subsp. lactis have been studied and compared: 729 (a natural strain isolated from milk), 1605 (a mutant of strain 729), F-116 (a recombinant obtained by fusing of protoplasts of the two related strain 729 and 1605), and a nisin-forming strain obtained by adaptive selection at Moscow State University. Antimicrobial activity studies revealed differences between the strains in the effects on individual groups of microorganisms; the activities of the strains were also distinct from that of Nisaplin (a commercial preparation of the bacteriocin nisin). Methods for isolation and purification of bacteriocins have been developed, making it possible to obtain individual components of antibiotic complexes as chromatographically pure preparations. Bacteriocins formed by the strains of Lactococcus lactis subsp. lactis have been identified and differences in their biological and physicochemical properties, established. A novel potent broad-spectrum antibiotic substance distinct from nisin has been isolated from the recombinant strain F-116.     

Properties of nisin Z and distribution of its gene, nisZ, in Lactococcus lactis.

Two natural variants of the lantibiotic nisin that are produced by Lactococcus lactis are known. They have a similar structure but differ in a single amino acid residue at position 27; histidine in nisin A and asparagine in nisin Z (J.W.M. Mulders, I.J. Boerrigter, H.S. Rollema, R.J. Siezen, and W.M. de Vos, Eur. J. Biochem, 201:581-584, 1991). The nisin variants were purified to apparent homogeneity, and their biological activities were compared. Identical MICs of nisin A and nisin Z were found with all tested indicator strains of six different species of gram-positive bacteria. However, at concentrations above the MICs, with nisin Z the inhibition zones obtained in agar diffusion assays were invariably larger than those obtained with nisin A. This was observed with all tested indicator strains. These results suggest that nisin Z has better diffusion properties than nisin A in agar. The distribution of the nisin variants in various lactococcal strains was determined by amplification of the nisin structural gene by polymerase chain reaction followed by direct sequencing of the amplification product. In this way, it was established that the nisZ gene for nisin Z production is widely distributed, having been found in 14 of the 26 L. lactis strains analyzed.     

Identification and Characteristics of Nisin Z-Producing <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> Isolated from Kimchi

We isolated bacteriocin-producing Lactococcus lactis subsp. lactis from Kimchi. The bacteriocin inhibited strains of Clostridium perfringens, C. difficile, Listeria monocytogenes, vancomycin-resistant Enterococcus, and one out of four methicillin-resistant Staphylococcus aureus strains, as well as some closely related lactic acid bacteria. In tricine-SDS-PAGE, the bacteriocin migrated with an apparent molecular weight of about 4 kDa to the same location as nisin A and crude nisin Z. The gene encoding this bacteriocin was found to be identical to that of nisin Z with direct PCR sequence methods. The inhibitory activity was stable against heat and pH, but it was lost at 100°C for 1 h and at 121°C for 15 min. The bacteriocin was inactivated by proteolytic enzymes, but was not affected by lysozyme, lipase, catalase, or β-glucosidase. There were some differences in characteristics from those of nisins described previously. Received: 21 June 2002 / Accepted: 22 July 2002     

Conjugative 40-megadalton plasmid in Streptococcus lactis subsp. diacetylactis DRC3 is associated with resistance to nisin and bacteriophage

Streptococcus lactis subsp. diacetylactis DRC3 was examined for plasmid DNA and found to contain a previously unreported plasmid of 40 X 10(6) daltons. This plasmid, designated pNP40, was conjugally transferred to a plasmid-cured derivative of S. lactis C2. Transconjugants containing pNP40 acquired resistance to nisin produced by strains of S. lactis and to commercially available nisin when assay plates were incubated at 21, 32, and 37 degrees C. In addition, c2 phage growth was completely restricted in transconjugants containing pNP40 at 21 and 32 degrees C, but not at 37 degrees C. This result suggests that pNP40 may be coding for a temperature-sensitive enzyme that restricts phage growth at 21 and 32 degrees C, but not at 37 degrees C. Eight consecutive transfers of a transconjugant containing pNP40 in Elliker broth at 37 degrees C resulted in 100% loss of resistance to c2 phage when colonies were tested at 32 degrees C. These phage-sensitive isolates had lost pNP40 and had also become sensitive to nisin. This result suggests that pNP40 may also be thermosensitive in its replication. The finding of a phage resistance determinant located on a conjugative plasmid should prove useful in constructing phage-resistant variants for dairy fermentation processes.     

Conjugative 40-megadalton plasmid in Streptococcus lactis subsp. diacetylactis DRC3 is associated with resistance to nisin and bacteriophage.

Streptococcus lactis subsp. diacetylactis DRC3 was examined for plasmid DNA and found to contain a previously unreported plasmid of 40 X 10(6) daltons. This plasmid, designated pNP40, was conjugally transferred to a plasmid-cured derivative of S. lactis C2. Transconjugants containing pNP40 acquired resistance to nisin produced by strains of S. lactis and to commercially available nisin when assay plates were incubated at 21, 32, and 37 degrees C. In addition, c2 phage growth was completely restricted in transconjugants containing pNP40 at 21 and 32 degrees C, but not at 37 degrees C. This result suggests that pNP40 may be coding for a temperature-sensitive enzyme that restricts phage growth at 21 and 32 degrees C, but not at 37 degrees C. Eight consecutive transfers of a transconjugant containing pNP40 in Elliker broth at 37 degrees C resulted in 100% loss of resistance to c2 phage when colonies were tested at 32 degrees C. These phage-sensitive isolates had lost pNP40 and had also become sensitive to nisin. This result suggests that pNP40 may also be thermosensitive in its replication. The finding of a phage resistance determinant located on a conjugative plasmid should prove useful in constructing phage-resistant variants for dairy fermentation processes.     

Characterization of two nisin-producing Lactococcus lactis subsp. lactis strains isolated from a commercial sauerkraut fermentation.

Two Lactococcus lactis subsp. lactis strains, NCK400 and LJH80, isolated from a commercial sauerkraut fermentation were shown to produce nisin. LJH80 was morphologically unstable and gave rise to two stable, nisin-producing (Nip+) derivatives, NCK318-2 and NCK318-3. NCK400 and derivatives of LJH80 exhibited identical morphological and metabolic characteristics, but could be distinguished on the basis of plasmid profiles and genomic hybridization patterns to a DNA probe specific for the iso-ISS1 element, IS946. NCK318-2 and NCK318-3 harbored two and three plasmids, respectively, which hybridized with IS946. Plasmid DNA was not detected in NCK400, and DNA from this strain failed to hybridize with IS946. Despite the absence of detectable plasmid DNA in NCK400, nisin-negative derivatives (NCK402 and NCK403) were isolated after repeated transfer in broth at 37 degrees C. Nisin-negative derivatives concurrently lost the ability to ferment sucrose and became sensitive to nisin. A 4-kbp HindIII fragment containing the structural gene for nisin (spaN), cloned from L. lactis subsp. lactis ATCC 11454, was used to probe genomic DNA of NCK318-2, NCK318-3, NCK400, and NCK402 digested with EcoRI or HindIII. The spaN probe hybridized to an 8.8-kbp EcoRI fragment and a 10-kbp HindIII fragment in the Nip+ sauerkraut isolates, but did not hybridize to the Nip- derivative, NCK402. A different hybridization pattern was observed when the same probe was used against Nip+ L. lactis subsp. lactis ATCC 11454 and ATCC 7962. These phenotypic and genetic data confirmed that unique Nip+ L. lactis subsp. lactis strains were isolated from fermenting sauerkraut.     

Characterization of Lactic Acid Bacteria Coexisting with a Nisin Z Producer in <Emphasis Type="Italic">Tsuda</Emphasis>-Turnip Pickles

Bacteriocin-producing lactic acid bacteria (LAB) are believed to be associated with many types of fermented food. The present study reports the identification of lactic acid bacterium MS27 producing a bacteriocin isolated from the Tsuda-turnip pickle, which is a Japanese fermented food, and characterization of LAB coexisting with the bacteriocin producers in the Tsuda-turnip pickle. The strain MS27 was identified as Lactococcus lactis subsp. lactis based on a partial 16S rRNA gene sequence and sugar fermentation pattern analyses. Mass spectroscopy and genetic analysis revealed that it produces nisin Z. Microbial population analysis revealed that the LAB community in the Tsuda-turnip pickle comprises nisin Z-sensitive and nisin Z-insensitive LAB (nonbacteriocin producers) and nisin Z producers at population rates of 52.5%, 37.5%, and 10.0%, respectively. This revealed that Leuconostoc spp. (nisin Z insensitive) is the dominant species among LAB microflora and that nisin Z insensitivity of a bacterial strain is proportional to its ability to dominate the population in Tsuda-turnip pickles. Competitive growth assay revealed that Leuconostoc spp. considerably suppressed the bacteriocin production of L. lactis MS27. These results suggested that Leuconostoc spp. contributes to the formation of the LAB community with a wide variety of microorganisms in Tsuda-turnip pickles.     

Effects of Lactococcus lactis on composition of intestinal microbiota: role of nisin

This study examined the ability of (i) pure nisin, (ii) nisin-producing Lactococcus lactis strain CHCC5826, and (iii) the non-nisin-producing L. lactis strain CHCH2862 to affect the composition of the intestinal microbiota of human flora-associated rats. The presence of both the nisin-producing and the non-nisin-producing L. lactis strains significantly increased the number of Bifidobacterium cells in fecal samples during the first 8 days but decreased the number of enterococci/streptococci in duodenum, ileum, cecum, and colon samples as detected by selective cultivation. No significant changes in the rat fecal microbiota were observed after dosage with nisin. Pearson cluster analysis of denaturing gradient gel electrophoresis profiles of the 16S rRNA genes present in the fecal microbial population revealed that the microbiota of animals dosed with either of the two L. lactis strains were different from that of control animals dosed with saline. However, profiles of the microbiota from animals dosed with nisin did not differ from the controls. The concentrations of nisin estimated by competitive enzyme-linked immunosorbent assay (ELISA) were approximately 10-fold higher in the small intestine and 200-fold higher in feces than the corresponding concentrations estimated by a biological assay. This indicates that nisin was degraded or inactivated in the gastrointestinal tract, since fragments of this bacteriocin are detected by ELISA while an intact molecule is needed to retain biological activity.     

Characterization of two nisin-producing Lactococcus lactis subsp. lactis strains isolated from a commercial sauerkraut fermentation.

Two Lactococcus lactis subsp. lactis strains, NCK400 and LJH80, isolated from a commercial sauerkraut fermentation were shown to produce nisin. LJH80 was morphologically unstable and gave rise to two stable, nisin-producing (Nip+) derivatives, NCK318-2 and NCK318-3. NCK400 and derivatives of LJH80 exhibited identical morphological and metabolic characteristics, but could be distinguished on the basis of plasmid profiles and genomic hybridization patterns to a DNA probe specific for the iso-ISS1 element, IS946. NCK318-2 and NCK318-3 harbored two and three plasmids, respectively, which hybridized with IS946. Plasmid DNA was not detected in NCK400, and DNA from this strain failed to hybridize with IS946. Despite the absence of detectable plasmid DNA in NCK400, nisin-negative derivatives (NCK402 and NCK403) were isolated after repeated transfer in broth at 37 degrees C. Nisin-negative derivatives concurrently lost the ability to ferment sucrose and became sensitive to nisin. A 4-kbp HindIII fragment containing the structural gene for nisin (spaN), cloned from L. lactis subsp. lactis ATCC 11454, was used to probe genomic DNA of NCK318-2, NCK318-3, NCK400, and NCK402 digested with EcoRI or HindIII. The spaN probe hybridized to an 8.8-kbp EcoRI fragment and a 10-kbp HindIII fragment in the Nip+ sauerkraut isolates, but did not hybridize to the Nip- derivative, NCK402. A different hybridization pattern was observed when the same probe was used against Nip+ L. lactis subsp. lactis ATCC 11454 and ATCC 7962. These phenotypic and genetic data confirmed that unique Nip+ L. lactis subsp. lactis strains were isolated from fermenting sauerkraut.     

High nisin Z production by Lactococcus lactis UL719 in whey permeate with aeration

The influence of controlled pH (5.0–6.5) and initial dissolved oxygen level (0–90% air saturation) on nisin Z production in a yeast extract/Tween 80-supplemented whey permeate (SWP) was examined during batch fermentations with citrate positive Lactococcus lactis subsp. lactis UL719. The total activity corresponding to the sum of soluble and cell-bound activities, as measured by a critical dilution method, was more than 50% lower at pH 5.0 than in the range 5.5–6.5, although the specific production decreased as pH increased. A maximum nisin Z activity of 8200 AU/ml (4100IU/ml) was observed in the supernatant after 8h of culture for pH ranging from 5.5 to 6.5. Prolonging the culture beyond 12h decreased this activity at pH 6.0 and 6.5 but not at pH 5.5 or 5.0. A corresponding increase in cell-bound activity was probably due to adsorption of soluble bacteriocin to the cell wall. Aeration increased cell-bound and total activity to maximum values of 32800 and 41000 AU/ml (16400 and 20500IU/ml), respectively, with an initial level of 60% air saturation after 24h of incubation at pH 6.0. The specific production at 60% or 90% initial air saturation was eight-fold higher than at 0%.     

Antibacterial activities of nisin Z encapsulated in liposomes or produced in situ by mixed culture during cheddar cheese ripening

This study investigated both the activity of nisin Z, either encapsulated in liposomes or produced in situ by a mixed starter, against Listeria innocua, Lactococcus spp., and Lactobacillus casei subsp. casei and the distribution of nisin Z in a Cheddar cheese matrix. Nisin Z molecules were visualized using gold-labeled anti-nisin Z monoclonal antibodies and transmission electron microscopy (immune-TEM). Experimental Cheddar cheeses were made using a nisinogenic mixed starter culture, containing Lactococcus lactis subsp. lactis biovar diacetylactis UL 719 as the nisin producer and two nisin-tolerant lactococcal strains and L. casei subsp. casei as secondary flora, and ripened at 7 degrees C for 6 months. In some trials, L. innocua was added to cheese milk at 10(5) to 10(6) CFU/ml. In 6-month-old cheeses, 90% of the initial activity of encapsulated nisin (280 +/- 14 IU/g) was recovered, in contrast to only 12% for initial nisin activity produced in situ by the nisinogenic starter (300 +/- 15 IU/g). During ripening, immune-TEM observations showed that encapsulated nisin was located mainly at the fat/casein interface and/or embedded in whey pockets while nisin produced by biovar diacetylactis UL 719 was uniformly distributed in the fresh cheese matrix but concentrated in the fat area as the cheeses aged. Cell membrane in lactococci appeared to be the main nisin target, while in L. casei subsp. casei and L. innocua, nisin was more commonly observed in the cytoplasm. Cell wall disruption and digestion and lysis vesicle formation were common observations among strains exposed to nisin. Immune-TEM observations suggest several modes of action for nisin Z, which may be genus and/or species specific and may include intracellular target-specific activity. It was concluded that nisin-containing liposomes can provide a powerful tool to improve nisin stability and availability in the cheese matrix.     

Sensitivity of nisin-resistant Listeria monocytogenes to heat and the synergistic action of heat and nisin

Nisin, a bacteriocin produced by some strains of Lactococcus lactis, acts against foodborne pathogen Listeria monocytogenes. A single exposure of cells to nisin can generate nisin-resistant (Nisr) mutants, which may compromise the use of nisin in the food industry. The objective of this research was to compare the heat resistance of Nisr and wild type (WT) Listeria monocytogenes. The synergistic effect of heat-treatment (55 degrees C) and nisin (500 IU ml-1) on the Nisr cells and the WT L. monocytogenes Scott A was also studied. When the cells were grown in the absence of nisin, there was no significant (alpha = 0.05) difference in heat resistance between WT and Nisr cells of L. monocytogenes at 55, 60 and 65 degrees C. However, when the Nisr cells were grown in the presence of nisin, they were more sensitive to heat at 55 degrees C than the WT cells. The D-values at 55 degrees C were 2.88 and 2.77 min for Nisr ATCC 700301 and ATCC 700302, respectively, which was significantly (alpha = 0.05) lower than the D-value for WT, 3.72 min. When Nisr cells were subjected to a combined treatment of heat and nisin, there was approximately a four log reduction during the first 7 min of treatment.     

Components of fermentation medium regulate bacteriocin synthesis by the recombinant strain Lactococcus lactis subsp. lactis F-116

The regulation of the synthesis of bacteriocin produced by the recombinant strain Lactococcus lactis subsp. lactis F-116 has been studied. The synthesis is regulated by the components of the fermentation medium, the content of inorganic phosphate (KH2PO4), yeast autolysate (source of amine nitrogen), and changes in carbohydrates and amino acids. The strain was obtained by fusion of protoplasts derived from two related L. lactis subsp. lactis strains, both exhibiting a weak ability to synthesize the bacteriocin nisin. Decreasing the content of KH2PO4 from 2.0 to 1.0 or 0.5% caused bacteriocin production to go down from 4100 to 2800 or 1150 IU/ml, respectively; the base fermentation medium contained 1.0% glucose, 0.2% NaCl, 0.02% MgSO4, and yeast autolysate (an amount corresponding to 35 mg % ammonium nitrogen). The substitution of sucrose for glucose (as the source of carbon) increased the antibiotic activity by 26%, and the addition of isoleucine, by 28.5%. Elevation of the concentration of yeast autolysate in the low-phosphate fermentation medium stimulated both the growth of the lactococci and the synthesis of bacteriocin. Introduction of 1% KH2PO4, yeast autolysate (in an amount corresponding to 70 mg % ammonium nitrogen), 2.0% sucrose, and 0.1% isoleucine increased the bacteriocin-producing activity of the strain by 2.4 times.     

Thermosensitive plasmid replication, temperature-sensitive host growth, and chromosomal plasmid integration conferred by Lactococcus lactis subsp. cremoris lactose plasmids in Lactococcus lactis subsp. lactis.

Evidence is presented that lactose-fermenting ability (Lac+) in Lactococcus lactis subsp. cremoris AM1, SK11, and ML1 is associated with plasmid DNA, even though these strains are difficult to cure of Lac plasmids. When the Lac plasmids from these strains were introduced into L. lactis subsp. lactis LM0230, they appeared to replicate in a thermosensitive manner; inheritance of the plasmid was less efficient at 32 to 40 degrees C than at 22 degrees C. The stability of the L. lactis subsp. cremoris Lac plasmids in lactococci appeared to be a combination of both host and plasmid functions. Stabilized variants were isolated by growing the cultures at 32 to 40 degrees C; these variants contained the Lac plasmids integrated into the L. lactis subsp. lactis LM0230 chromosome. In addition, the presence of the L. lactis subsp. cremoris Lac plasmids in L. lactis subsp. lactis resulted in a temperature-sensitive growth response; growth of L. lactis subsp. lactis transformants was significantly inhibited at 38 to 40 degrees C, thereby resembling some L. lactis subsp. cremoris strains with respect to temperature sensitivity of growth.     

Characterization and structure analysis of a novel bacteriocin, lacticin Z, produced by Lactococcus lactis QU 14

A novel bacteriocin, lacticin Z, produced by Lactococcus lactis QU 14 isolated from a horse's intestinal tract was identified. Lacticin Z was purified through a three step procedure comprised of hydrophobic-interaction, cation-exchange chromatography, and reverse-phase HPLC. ESI-TOF MS determined the molecular mass of lacticin Z to be 5,968.9 Da. The primary structure of lacticin Z was found to consist of 53 amino acid residues without any leader sequence or signal peptide. Lacticin Z showed homology to lacticin Q from L. lactis QU 5, aureocin A53 from Staphylococcus aureus A53, and mutacin BHT-B from Streptococcus rattus strain BHT. It exhibited a nanomolar range of MICs against various Gram-positive bacteria, and the activity was completely stable up to 100 degrees C. Unlike many of other LAB bacteriocins, the stability of lacticin Z was emphasized under alkaline conditions rather than acidic conditions. All the results indicated that lacticin Z belongs to a novel type of bacteriocin.