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.     

A nisin bioassay based on bioluminescence.

A Lactococcus lactis subsp. lactis strain that can sense the bacteriocin nisin and transduce the signal into bioluminescence was constructed. By using this strain, a bioassay based on bioluminescence was developed for quantification of nisin, for detection of nisin in milk, and for identification of nisin-producing strains. As little as 0.0125 ng of nisin per ml was detected within 3 h by this bioluminescence assay. This detection limit was lower than in previously described methods.     

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.     

Requirement of autolytic activity for bacteriocin-induced lysis

The bacteriocin produced by Lactococcus lactis IFPL105 is bactericidal against several Lactococcus and Lactobacillus strains. Addition of the bacteriocin to exponential-growth-phase cells resulted in all cases in bacteriolysis. The bacteriolytic response of the strains was not related to differences in sensitivity to the bacteriocin and was strongly reduced in the presence of autolysin inhibitors (Co(2+) and sodium dodecyl sulfate). When L. lactis MG1363 and its derivative deficient in the production of the major autolysin AcmA (MG1363acmADelta1) were incubated with the bacteriocin, the latter did not lyse and no intracellular proteins were released into the medium. Incubation of cell wall fragments of L. lactis MG1363, or of L. lactis MG1363acmADelta1 to which extracellular AcmA was added, in the presence or absence of the bacteriocin had no effect on the speed of cell wall degradation. This result indicates that the bacteriocin does not degrade cell walls, nor does it directly activate the autolysin AcmA. The autolysin was also responsible for the observed lysis of L. lactis MG1363 cells during incubation with nisin or the mixture of lactococcins A, B, and M. The results presented here show that lysis of L. lactis after addition of the bacteriocins is caused by the resulting cell damage, which promotes uncontrolled degradation of the cell walls by AcmA.     

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

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

Identification and characteristics of nisin Z-producing Lactococcus lactis subsp. lactis 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 degrees C for 1 h and at 121 degrees C for 15 min. The bacteriocin was inactivated by proteolytic enzymes, but was not affected by lysozyme, lipase, catalase, or beta-glucosidase. There were some differences in characteristics from those of nisins described previously.     

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.     

Antimicrobial activity of lactic acid bacteria isolated from goat's milk and artisanal cheeses: characteristics of a bacteriocin produced by Lactobacillus curvatus IFPL105

A total of 203 lactic acid bacteria isolated from raw goat's milk and artisanal cheese were tested for antibacterial activity. Only two strains of Lactococcus lactis , one strain of Enterococcus faecalis and one strain of Lactobacillus curvatus were shown to produce a bacteriocin-like substance. Lactobacillus curvatus IFPL105 produced a heat-stable bacteriocin, which was hydrolysed by α-chymotrypsin, proteinase K and pancreatin and exhibited a broad spectrum of inhibitory activity. The bactericidal activity of the bacteriocin was more potent when sensitive strains were in the logarithmic growth phase, inducing cell lysis, as observed by decreases in optical density and release of intracellular marker enzymes. Curing experiments resulted in variants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of the parental strain and the bacteriocin-negative variants indicated that a plasmid of about 46 kbp may be involved in bacteriocin production and immunity to this antibacterial compound.     

Inhibition of Listeria innocua in cheddar cheese by addition of nisin Z in liposomes or by in situ production in mixed culture

The effect of addition of purified nisin Z in liposomes to cheese milk and of in situ production of nisin Z by Lactococcus lactis subsp. lactis biovar diacetylactis UL719 in the mixed starter on the inhibition of Listeria innocua in cheddar cheese was evaluated during 6 months of ripening. A cheese mixed starter culture containing Lactococcus lactis subsp. lactis biovar diacetylactis UL719 was selected for high-level nisin Z and acid production. Experimental cheddar cheeses were produced on a pilot scale, using the selected starter culture, from milk with added L. innocua (10(5) to 10(6) CFU/ml). Liposomes with purified nisin Z were prepared from proliposome H and added to cheese milk prior to renneting to give a final concentration of 300 IU/g of cheese. The nisin Z-producing strain and nisin Z-containing liposomes did not significantly affect cheese production and gross chemical composition of the cheeses. Immediately after cheese production, 3- and 1.5-log-unit reductions in viable counts of L. innocua were obtained in cheeses with encapsulated nisin and the nisinogenic starter, respectively. After 6 months, cheeses made with encapsulated nisin contained less than 10 CFU of L. innocua per g and 90% of the initial nisin activity, compared with 10(4) CFU/g and only 12% of initial activity in cheeses made with the nisinogenic starter. This study showed that encapsulation of nisin Z in liposomes can provide a powerful tool to improve nisin stability and inhibitory action in the cheese matrix while protecting the cheese starter from the detrimental action of nisin during cheese production.     

一个含有乳链菌肽抗性基因的乳酸乳球菌质粒pTS50的鉴定

在添加乳链菌肽、乳糖及溴甲酚紫的M1 7选择培养基上 ,从 1 97个新鲜牛奶样品中筛选到 3株乳链菌肽抗性菌株 ,PCR扩增证实它们都含有乳链菌肽抗性基因。菌种生理生化特性鉴定及特异性 1 6SrDNAPCR扩增产物的序列测定结果表明这 3株菌都属于乳酸乳球菌乳酸亚种。质粒转化实验发现乳酸乳球菌乳酸亚种TS 1 640中的乳链菌肽抗性基因位于一个约47kb的大质粒pTS50上。BamHI、EcoRI、HindⅢ、NcoI、PstⅠ酶切分析和Southern杂交 ,进一步将乳链菌肽抗性基因定位于pTS50的一个约 1 9kbEcoRI酶切片段中     

Intra-specific variation of Lactobacillus plantarum and Lactobacillus pentosus in sensitivity towards various bacteriocins

Fifty-two strains belonging to the Lactobacillus plantarum species group were identified and typed. They represented 32 clones of Lactobacillus plantarum and 7 clones of Lactobacillus pentosus. Sensitivity of all strains towards bacteriocins of four different producer strains was investigated using a deferred inhibition test (DIT). Substantial intra-specific variation in sensitivity of clones was observed towards bacteriocinogenic lactic acid bacteria producing nisin ( Lactococcus lactis ATCC 11454) or pediocin PA-1 ( Pediococcus acidilactici PAC-1.0), while none of the strains were sensitive towards the two remaining bacteriocin producers. The minimum inhibitory concentration (MIC) of nisin towards selected strains confirmed the DIT results. No correlation between the susceptibility of fourteen selected strains towards nisin and an array of antibiotics was found. The present study indicates that the variation in bacteriocin-sensitivity within target species might be a potential limitation for the application of bacteriocins as biopreservatives.     

Influence of the phosphorus and nitrogen source on nisin production in Lactococcus lactis subsp. lactis batch fermentations using a complex medium

The influence of different phosphorus and nitrogen sources on Lactococcus lactis subsp. lactis NIZO 22186 growth and nisin production was studied in batch fermentations using a complex medium. KH₂PO₄ was found to be the best phosphorus source for nisin production. Increasing initial phosphate concentrations from 0 to 5% KH₂PO₄ exerted a double effect, creating favourable pH conditions and particularly stimulating the nisin production levels, which were highest at 5% KH₂PO₄. Up to now, no such high initial phosphate concentrations have been reported for the production of other antibiotics or bacteriocins. Nisin, a lanthionine-containing peptide antibiotic with bacteriocin properties, clearly behaved as a primary metabolite, since its formation was linked with active growth and was not suppressed by phosphate concentrations up to 5%. A complex medium supplemented with cotton seed meal as nitrogen source also gave very high nisin yields. Correspondence to: L. De Vuyst     

Prevention of Histamine Formation in Cheese by Bacteriocin-Producing Lactic Acid Bacteria

The susceptibility of 13 amine-forming lactobacilli to several bacteriocins was investigated by an agar diffusion assay. All strains were susceptible to nisin and to five bacteriocins of enterococcal origin. Pediocin PA-1, bavaricin A, lactococcin A, and a bacteriocin from Enterococcus faecalis 1061 did not show inhibitory activity. Two bacteriocin-producing enterococci and a nisin-producing Lactococcus lactis strain were employed as starters in separate cheese-making experiments. Outgrowth of histamine producer Lactobacillus buchneri St2A, which was added to the milk at levels of up to 190 CFU/ml, was almost completely inhibited. No histamine formation was detected in the cheeses made with bacteriocin-producing starters. In the control cheese without bacteriocin, St2A reached levels of 1.1 x 10(sup8) CFU/g, and 200 mg of histamine per kg was found after 4 months of ripening. To our knowledge, this is the first report of bacteriocin-mediated inhibition of histamine formation in foods.     

Immunodot detection of nisin Z in milk and whey using enhanced chemiluminescence

A highly specific antisera was produced in New Zealand white rabbits against nisin Z, a 3400 Da bacteriocin produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719. A dot immunoblot assay was then developed to detect nisin Z in milk and whey. As few as 1·5 10⁻¹ international units per ml (IU ml⁻¹), corresponding to 0·003 μg ml⁻¹ of pure nisin Z, were detected in carbonate-bicarbonate buffer within 6 h using chemiluminescence. When milk and whey samples were tested, approximately 0·155 μg ml⁻¹ (7·9 IU ml⁻¹) of nisin Z was detected. The detection limit obtained was lower than that of traditional methods including microtitration and agar diffusion.     

Nisin A depletes intracellular ATP and acts in bactericidal manner against Mycobacterium smegmatis

Nisin is a bacteriocin produced by many strains of Lactococcus lactis. This study examined the effect of nisin on Mycobacterium smegmatis, a non-pathogenic species of Mycobacterium. Nisin had a minimum inhibitory concentration of 8.0 micrograms ml-1 and a minimum inhibitory dose of 7.5 micrograms ml-1 against Myco. smegmatis. Treatment with 25.0 micrograms ml-1 nisin caused partial inhibition of Myco smegmatis; the survivors were nisin-sensitive when tested in a separate experiment. Mycobacterium smegmatis cells exposed to 50.0 micrograms ml-1 of nisin, lost their viability. the effect of nisin on the growth of Myco. smegmatis was both time- and concentration-dependent. Nisin (10.0 micrograms ml-1) caused 97.7 +/- 2.0% reduction in internal ATP and leakage of intracellular ATP out of Myco. smegmatis cells after several hours of treatment. These data suggest that nisin inhibits Myco. smegmatis by the same mechanism by which it inhibits other bacteria and warrants further investigation as a possible antitubercular agent.     

Nisin Production by Enterococcus hirae DF105Mi Isolated from Brazilian Goat Milk

The purpose of this study was to select the promising biopreservation bacteriocin producer strain from goat milk and characterize the expressed bacteriocin, related to its physiological and biochemical properties and specificity of operon encoding production and expression of antimicrobial peptide. Brazilian goat milk was used as the source for the selection of bacteriocin-producing lactic acid bacteria. One strain (DF105Mi) stood out for its strong activity against several Listeria monocytogenes strains. Selected strain was identified based on the biochemical and physiological characteristics and 16s rRNA analysis. The bacteriocin production and inhibitory spectrum of strain DF105Mi were studied, together with the evaluation of the effect of temperature, pH, and chemicals on bacteriocin stability and production, activity, and adsorption to target cells as well as to the cell surface of bacteriocin producers. Physiological and bio-molecular analyses based on targeting of different genes, parts of nisin operon were performed in order to investigate the hypothesis that the studied strain can produce and express nisin. Based on biochemical, physiological, and 16s rRNA analysis, the strain DF105Mi was classified as Enterococcus hirae. The selected strain produces a bacteriocin which is stable in a wide range of pH (2.0–12.0), temperature (up to 120 °C), presence of selected chemicals and presents adsorption affinity to different test organisms, process influenced by environmental conditions. Higher bacteriocin production by Ent. hirae DF105Mi was recorded during stationary growth phase, but only when the strain was cultured at 37 °C. The strain’s genetic analysis indicated presence of the genes coding for the production of the bacteriocin nisin. This result was confirmed by cross-checking the sensitivity of the produced strain to commercial nisin A. The strong anti-Listeria activity, bacteriocin adsorption, and stability of produced bacteriocin indicate that Ent. hirae DF105Mi presents a differentiated potential application for biopreservation of fermented dairy products.

     

Influence of growth conditions on the production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from kimchi

The influence of growth parameters on the fermentative production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from kimchi was studied. The bacteriocin production was greatly affected by carbon and nitrogen sources. Strain A164 produced at least 4-fold greater bacteriocin in M17 broth supplemented with lactose than other carbon sources. The amount of 3% yeast extract was found to be the optimal organic nitrogen source. While the maximum biomass was obtained at 37 degrees C, the optimal temperature for the bacteriocin production was 30 degrees C. The bacteriocin production was also affected by pH of the culture broth. The optimal pH for growth and bacteriocin production was 6.0. Although the cell growth at pH 6.0 was nearly the same level at pH 5.5 and 6.5, the greater bacteriocin activity was observed at pH 6.0. Exponential growth took place only during an initial period of the cultivation, and then linear growth was observed. Linear growth rates increased from 0.160 g(DCW) x l(-1) x h(-1) to 0.245 g(DCW) x l(-1) x h(-1) with increases in lactose concentrations from 0.5 to 3.0%. Maximum biomass was also increased from 1.88 g(DCW) x l(-1) to 4.29 g(DCW) x l(-1). However, increase in lactose concentration did not prolong the active growth phase. After 20 h cultivation, cell growth stopped regardless of lactose concentration. Production of the bacteriocin showed primary metabolic kinetics. However, bacteriocin yield based on cell mass increased greatly during the late growth phase. A maximum activity of 131x10(3) AU x ml(-1) was obtained at early stationary growth phase (20 h) during the batch fermentation in M17L broth (3.0% lactose) at 30 degrees C and pH 6.0.     

Nutritional factors affecting nisin production by Lactococcus lactis subsp. actis NIZO 22186 in a synthetic medium

L. DE VUYST. 1995 A minimal synthetic medium (SM8) for nisin-producing Lactococcus lactis subsp. lactis strains has been designed; it consists of eight growth-stimulating amino acids (glutamic acid, methionine, valine, leucine, threonine, arginine, isoleucine and histidine), five vitamins (biotin, calcium pantothenate, nicotinic acid, pyridoxine and riboflavin) and the mineral salts dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, magnesium sulphate and trisodium citrate. Nisin biosynthesis is strongly dependent on the presence of a sulphur source, either an inorganic salt (magnesium sulphate or sodium thiosulphate) or the amino acids methionine, cysteine or cystathionine. The amino acids serine, threonine and cysteine highly stimulate nisin production without affecting the final cell yield, indicating their precursor role during nisin biosynthesis.     

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

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