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.     

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.     

Isolation and characterization of nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts

Bacterial isolates from bean-sprouts were screened for anti- Listeria monocytogenes bacteriocins using a well diffusion method. Thirty-four of 72 isolates inhibited the growth of L.monocytogenes Scott A. One, HPB 1688, which had the biggest inhibition zone against L.monocytogenes Scott A, was selected for subsequent analysis. Both ribotyping and DNAsequencing of 16S ribosomal RNA gene demonstrated that the isolate was Lactococcus lactis subsp. lactis . Polymerase chain reaction and nucleotide sequencing revealed that thegenomic DNA of the bean-sprout isolates contained a nisin Z structural gene. In MRS broth,bean-sprout isolate HPB 1688 survived at 3–4·5°C for at least 20 d, grew at 4°Cand produced anti-listerial compoundsat 5°C. When co-cultured with L. monocytogenes in MRS broth, the isolate inhibited thegrowth of L. monocytogenes at 4°C after 14d and at 10°C after 2 d. When co-inoculatedwith 10²cells g⁻¹ of L.monocytogenes on fresh-cut ready-to-eat Caesar salad, L. lactis subsp. lactis (10⁸cells g⁻¹) was able to reduce the number of L. monocytogenes by 1–1·4 logs after storage for 10 d at 7° and 10°C. A bacteriocin-producing Enterococcusfaecium was also able to reduce the numbers of L. monocytogenes onCaesar salad, butdid not act synergistically when co-inoculated with L. lactis subsp. lactis .     

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.     

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.     

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.     

Glucose metabolism and internal pH of Lactococcus lactis subsp. lactis cells utilizing NMR spectroscopy

The metabolism of glucose was studied in Lactococcus lactis subsp. lactis CNRZ 125 by ¹³C NMR. The initial rate of glucose utilization was higher for exponential phase cells than for stationary phase cells [150 vs 85 nmol g (dry wt)^-1 s^-1]. ³¹P NMR was used to determine changes in glycolytic phosphorylated intermediates (fructose-1,6-diphosphate, dihydroxyacetone phosphate and phosphoglycerate). The internal pHs of L. lactis subsp. lactis CNRZ 141 and CNRZ 125 were also measured by ³¹P NMR as a function of the external pH during growth. When the external pH was 6·8, the internal pHs of strain CNRZ 141 and CNRZ 125 were similar, 7·4. After the external pH had decreased to 5·5, the internal pH of strain CNRZ 141 had declined by 0·6 unit, whereas that of strain CNRZ 125 had decreased by only 0·2 unit of pH.     

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°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°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.     

乳链菌肽前体基因（nisZ）在乳酸乳球菌中的克隆和表达

用PCR技术从克隆有完整乳链菌肽生物合成基因簇（来自于乳链菌肽高产菌株L.lactis AL2)的重组噬菌体λHJ-3中扩增了编码乳链菌肽的前体基因,与pMG36e连接得到重组质粒pHJ201,用电击转化法将pHJ201转化到L.lactis NZ9800中,经活性测定和Tricine－SDS－PAGE电泳证实乳链菌肽前体基因获得了功能表达。DNA序列分析表明乳链菌肽高产菌株L.lactis AL2产生的是NisinZ。发现pHJ201d L.lactis NZ9800 中有良好的稳定性。     

Production and utilization of polyclonal antibodies against nisin in an ELISA and for immuno-location of nisin in producing and sensitive bacterial strains

Specific nisin polyclonal antibodies (PAb) were produced in rabbits using nisin Z produced by Lactococcus lactis subsp. lactis biovar diacetylactis UL 719. Antisera were obtained from white female New Zealand rabbits that were first immunized with a nisin Z-keyhole limpet haemocyanin conjugate and boosted with free nisin Z. Nisin-specific PAb were purified by affinity chromatography with a yield of 15 mg specific antinisin 100 ml-1 serum. The detection limit of the ELISA test for nisin Z was 0.75 ng ml-1 in buffer but was 1.7 and 3.5 ng ml-1 in milk and complex media broth spiked (5, 10, 20 microg ml-1) with nisin Z, respectively. In nisin Z-spiked samples, the average concentration was between 90 and 107% of actual added amount. In contrast, when the bioassay (microtitration method) was used, only 50-63% of nisin Z biological activity could be detected. In addition, the affinity-purified nisin PAb, antirabbit IgG gold conjugate and transmission electron microscopy were successfully used to locate nisin Z on producing cells and to observe its bactericidal effects against sensitive cells.     

Detection and characterization of a bacteriocin produced by Lactococcus lactis subsp. cremoris R isolated from radish

Bacteria isolated from radish were identified as Lactococcus lactis subsp. cremoris R and their bacteriocin was designated lactococcin R. Lactococcin R was sensitive to some proteolytic enzymes (proteinase-K, pronase-E, proteases, pepsin, α-chymotrypsin) but was resistant to trypsin, papain, catalase, lysozyme and lipase, organic solvents, or heating at 90 °C for 15, 30 and 60 min, or 121 °C for 15 min. Lactococcin R remained active after storage at −20 and −70 °C for 3 months and after exposure to a pH of 2–9. The molecular weight of lactococcin R was about 2·5 kDa. Lactococcin R was active against many food-borne pathogenic and food spoilage bacteria such as Clostridium, Staphylococcus, Listeria, Bacillus, Micrococcus, Enterococcus, Lactobacillus, Leuconostoc, Streptococcus and Pediococcus spp., but was not active against any Gram-negative bacteria. Lactococcin R was produced during log phase and reached a maximum activity (1600 AU ml⁻¹) at early stationary phase. The highest lactococcin R production was obtained in MRS broth with 0·5% glucose, at 6·5–7·0 initial pH values, 30 °C temperature and 18–24-h incubation times. Lactococcin R adsorbed maximally to its heat-killed producing cells at pH 6–7 (95%). Crude lactococcin R at 1280 AU ml⁻¹ was bactericidal, reducing colony counts of Listeria monocytogenes by 99·98% in 3 h. Lactococcin R should be useful as a biopreservative to prevent growth of food-borne pathogenic and food spoilage bacteria in ready-to-eat, dairy, meat, poultry and other food products. Lactococcin R differs from nisin in having a lower molecular weight, 2·5 kDa vs 3·4 kDa, and in being sensitive to pepsin and α-chymotrypsin to which nisin is resistant.     

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.     

Bacteriocin-Producing Lactic Acid Bacteria Isolated from Mangrove Forests in Southern Thailand as Potential Bio-Control Agents: Purification and Characterization of Bacteriocin Produced by Lactococcus lactis subsp. lactis KT2W2L

The aim of this work was to purify and characterize the bacteriocin produced by Lactococcus lactis subsp. lactis KT2W2L previously isolated from mangrove forests in southern Thailand, in order to evaluate its potential as new food protective agent. The active peptide from the cell-free supernatant of this strain was purified in 4 steps: (1) precipitation with 70 % saturated ammonium sulfate, (2) elution on a reversed-phase cartridge using different concentrations of acetonitrile, (3) cation-exchange chromatography and (4) final purification by reversed-phase HPLC on a C₈ column. The molecular mass of 3,329.5254 Da of the purified bacteriocin, determined by mass spectrometry, is nearly identical to that of peptide nisin Z. The activity of the purified bacteriocin was unaffected by pH (2.0–10.0), thermostable but was sensitive to proteolytic enzymes. The bacteriocin activity was stable after 8 weeks of storage at ?20 °C and 7 weeks of storage at 4 °C, but decreased after 3 weeks of storage at 37 °C. It was stable when incubated for 1 month at 4 °C in 0–30 % NaCl. Inhibitory spectrum of this bacteriocin showed a wide range of activity against similar bacterial strains, food-spoilage and food-borne pathogens. L. lactis subsp. lactis KT2W2L was sensitive to kanamycin, penicillin and tetracycline but resistant to ampicillin, gentamicin and vancomycin. The fragment obtained after amplification of genomic DNA from L. lactis subsp. lactis KT2W2L, with specific primers for bacteriocin genes, presented 99 % homology to the nisin Z gene. PCR amplification demonstrated that L. lactis subsp. lactis KT2W2L does not harbor virulence genes cylA, cylB, efaAfs and esp. The bacteriocin and its producing strain may find application as bio-preservatives for reduction in food-spoilage and food-borne pathogens in food products.     

Antilisterial activity of lactic acid bacteria isolated from rigouta, a traditional Tunisian cheese

AIMS: Screening for lactic acid bacteria (LAB) producing bacteriocins and other antimicrobial compounds is of a great significance for the dairy industry to improve food safety. METHODS AND RESULTS: Six-hundred strains of LAB isolated from 'rigouta', a Tunisian fermented cheese, were tested for antilisterial activity. Eight bacteriocinogenic strains were selected and analysed. Seven of these strains were identified as Lactococcus lactis and produced nisin Z as demonstrated by mass spectrometry analysis of the purified antibacterial compound. Polymerase chain reaction experiments using nisin gene-specific primers confirmed the presence of nisin operon. Plasmid profiles analysis suggests the presence of, at least, three different strains in this group. MMT05, the eighth strain of this antilisterial collection was identified, at molecular level, as Enterococcus faecalis. The purified bacteriocin produced by this strain showed a molecular mass of 10 201.33 +/- 0.85 Da. This new member of class III bacteriocins was termed enterocin MMT05. CONCLUSIONS: Seven lactococcal strains producing nisin Z were selected and could be useful as bio-preservative starter cultures. Additional experiments are needed to evaluate the promising strain MMT05 as bio-preservative as Enterococci could exert detrimental or beneficial role in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: Only a few antibacterial strains isolated from traditional African dairy products were described. The new eight strains described herein contribute to the knowledge of this poorly studied environment and constitute promising strains for fermented food safety.     

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%.     

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.     

Production and characterization of anti-nisin Z monoclonal antibodies: suitability for distinguishing active from inactive forms through a competitive enzyme immunoassay

As a pre-requisite to monoclonal antibody development, an efficient purification strategy was devised that yielded 72 mg of nisin Z from 14.5 1 of Lactococcus lactis subsp. lactis biovar. diacetylactis UL 719 (L. diacetylactis UL719) culture in supplemented whey permeate. Specific monoclonal antibodies (mAbs) were produced in mice against the purified nisin Z using keyhole limpet hemocyanin as a carrier protein. These antibodies did not recognize nisin A, suggesting that the asparagine residue at position 27 is involved in antibody recognition to nisin Z. However, the high reactivity of mAbs against biologically inactive nisin Z degradation products, produced during storage of freeze-dried pure nisin Z at -70 degrees C, indicated that the dehydroalanine residue at position 5 (Dha5), required for biological activity, is not necessary in nisin Z recognition by the mAb. A competitive enzyme immunoassay (cEIA) using the specific anti-nisin Z mAb was developed and used for rapid and sensitive detection and quantification of nisin Z in fresh culture supernatant, milk and whey. Detection limits of 78 ng/ml in phosphate-buffered saline, 87 ng/ml in culture supernatant, 106 ng/ml in milk and 90.5 ng/ml in whey were obtained for this assay. The cEIA using specific mAbs can be used to quantify nisin Z in food products.     

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.     

Fluorescent labeling of nisin Z and assessment of anti-listerial action

Biomolecule labeling by fluorescent markers has emerged as an innovative methodology for bio-analytical purposes in food microbiology, medicine and pharmaceutics due to the great advantages of this method such as precision, wide detection limits, and in vivo recognition. Fluorescent nisin Z was synthesized by linking the carboxyl group and amino group of nisin Z and 5-aminoacetamido fluorescein (AAA-flu). This new structure was fully characterized by mass spectrometry with a molecular weight of 3717.3 Da. Intracellular K⁺ leakage and transmembrane electrical potential (Δψ) were used to evaluate the antibacterial action of the labeled molecule against three listerial strains and demonstrated that nisin Z endured the labeling process without any activity loss. In vivo activity of labeled nisin was observed by confocal laser microscope which revealed its localization at the septum of listerial cell division site where the membrane-bound cell wall precursor lipid II is maximal. Fluorescent nisin Z showed its great potential as a tool to study antibacterial mechanism of action of nisin in biological systems.