Enhancement of Nisin Production by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis>

Lactococcus lactis subsp lactis BSA (L. lactis BSA) was isolated from a commercial fermented product (BSA Food Ingredients, Montreal, Canada) containing mixed bacteria that are used as starter for food fermentation. In order to increase the bacteriocin production by L. lactis BSA, different fermentation conditions were conducted. They included different volumetric combinations of two culture media (the Man, Rogosa and Sharpe (MRS) broth and skim milk), agitation level (0 and 100 rpm) and concentration of commercial nisin (0, 0.15, and 0.30 µg/ml) added into culture media as stimulant agent for nisin production. During fermentation, samples were collected and used for antibacterial evaluation against Lactobacillus sakei using agar diffusion assay. Results showed that medium containing 50 % MRS broth and 50 % skim milk gave better antibacterial activity as compared to other medium formulations. Agitation (100 rpm) did not improve nisin production by L. lactis BSA. Adding 0.15 µg/ml of nisin into the medium-containing 50 % MRS broth and 50 % skim milk caused the highest nisin activity of 18,820 AU/ml as compared to other medium formulations. This activity was 4 and ~3 times higher than medium containing 100 % MRS broth without added nisin (~4700 AU/ml) and 100 % MRS broth with 0.15 µg/ml of added nisin (~6650 AU/ml), respectively.     

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.     

Bioassay for Nisin in Milk, Processed Cheese, Salad Dressings, Canned Tomatoes, and Liquid Egg Products

A sensitive nisin quantification bioassay was constructed, based on Lactococcus lactis chromosomally encoding the nisin regulatory proteins NisK and NisR and a plasmid with a green fluorescent protein (GFP) variant gfp_uv gene under the control of the nisin-inducible nisA promoter. This strain, LAC275, was capable of transducing the signal from extracellular nisin into measurable GFPuv fluorescence through the NisRK signal transduction system. The LAC275 cells detected nisin concentrations of 10 pg/ml in culture supernatant, 0.2 ng/ml in milk, 3.6 ng/g in processed cheese, 1 ng/g in salad dressings and crushed, canned tomatoes, and 2 ng/g in liquid egg. This method was up to 1,000 times more sensitive than a previously described GFP-based nisin bioassay. This new assay made it possible to detect significantly smaller amounts of nisin than the presently most sensitive published nisin bioassay based on nisin-induced bioluminescence. The major advantage of this sensitivity was that foods could be extensively diluted prior to the assay, avoiding potential inhibitory and interfering substances present in most food products.     

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.     

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.     

Directionality and Coordination of Dehydration and Ring Formation during Biosynthesis of the Lantibiotic Nisin

The lantibiotic nisin is a potent antimicrobial substance, which contains unusual lanthionine rings and dehydrated amino acid residues and is produced by Lactococcus lactis. Recently, the nisin biosynthetic machinery has been applied to introduce lanthionine rings in peptides other than nisin with potential therapeutic use. Due to difficulties in the isolation of the proposed synthetase complex (NisBTC), mechanistic information concerning the enzymatic biosynthesis of nisin is scarce. Here, we present the molecular characterization of a number of nisin mutants that affect ring formation. We have investigated in a systematic manner how these mutations influence dehydration events, which are performed enzymatically by the dehydratase NisB. Specific mutations that hampered ring formation allowed for the dehydration of serine residues that directly follow the rings and are normally unmodified. The combined information leads to the conclusion that 1) nisin biosynthesis is an organized directional process that starts at the N terminus of the molecule and continues toward the C terminus, and 2) NisB and NisC are alternating enzymes, whose activities follow one after another in a repetitive way. Thus, the dehydration and cyclization processes are not separated in time and space. On the basis of these results and previous knowledge, a working model for the sequence of events in the maturation of nisin is proposed.Nisin is a lantibiotic produced by Lactococcus lactis, which has been known since 1928 (1, 2). This antimicrobial peptide is active against various Gram-positive bacteria and has attained commercial success as a food preservative (3). In addition to the wide industrial applications of nisin, it became also a model system to study various aspects of lantibiotic biosynthesis, regulation, and mode of action (2). Furthermore, recently, other applications of nisin have emerged. Its biosynthetic machinery can be successfully used to install dehydrated amino acids and lanthionine rings in peptides, which are either related or totally unrelated to nisin (4–11). This offers great opportunities to modulate the stability and activity of peptides that are used as therapeutics (8).The post-translational modified nisin molecule is classified as a member of the Group A lantibiotics (12). Mature nisin contains 34 amino acids, three of which are posttranslationally modified, and five thioether rings that are enzymatically formed upon cyclization of five free cysteines and five dehydroamino acid residues (Fig. 1). These peculiar modifications, which are very rare in nature, give nisin its exceptional stability against proteolysis and contribute greatly to its antimicrobial activity.Open in a separate windowFIGURE 1.Primary structure of prenisin and generated mutants. Dehydrated residues are shaded gray; serine 33 sometimes escapes dehydration and is shaded light gray. Serine at position 29 is never dehydrated in wild type prenisin. The impact of mutations on the dehydration pattern of new prenisin species is schematically depicted. Mutated residues are indicated by filled red circles. Newly formed dehydrated residues are pointed to by a black arrow. Letters A–E correspond to the five consecutive lanthionine rings in nisin.Nisin is synthesized ribosomally as a 57-amino acid residue-long polypeptide. Subsequently, it is directed to a putative synthetase complex that probably consists of three different proteins that include the dehydratase NisB, responsible for dehydration of serines and threonines to dehydroalanines and dehydrobutyrines, respectively; the cyclase NisC, which forms (methyl) lanthionine bridges between cysteines and dehydroamino acids; and the ABC transporter NisT, which performs transport across the lipid bilayer by consuming ATP. Newly synthesized and modified prenisin is still antimicrobially inactive. Only upon cleavage of the leader sequence that encompasses the first 23 amino acids by the dedicated protease NisP, an active molecule is liberated.Although there are data pointing to the existence of a synthetase complex that modifies nisin, such a complex has not been isolated so far. However, both NisB (13, 14) and NisC (13) were shown by specific antibody detection to localize at the cytoplasmic membrane, although some soluble signal was also detected. This localization gives NisBC the opportunity to interact with the transporter NisT, which is an integral membrane protein. Furthermore, co-immunoprecipitation and yeast two-hybrid studies suggested an interaction between members of the nisin modification machinery and nisin itself (13). The function of each member of the putative multimeric synthetase has been investigated in vivo by knock-out studies. It also has been demonstrated that subsequent steps in nisin biosynthesis can be performed separately. Dehydration, cyclization, and transport of the modified product were dissected in vivo, and also the dehydratase has been shown to perform enzymatic reactions without the presence of other members of the complex in vivo (7) although with very low efficiency. The cyclization activity of NisC was demonstrated in vitro (15), and the ABC transporter NisT was shown to be capable of transport of unmodified prenisin in vivo (10). Based on the available data, it is difficult to assess whether multimeric lanthionine complexes are indispensable for efficient nisin production and modification. However, in vivo localization studies and interaction experiments suggest that these proteins work in a concerted manner.Here, we present data that indicates a strong coordination between members of the nisin modification machinery. The analysis of sets of nisin mutants, where key residues that take part in ring formation as well as substitutions of residues that directly follow lanthionine structures, suggests a strong interdependency of dehydratase and cyclase activity. Moreover, the data indicate that these enzymes alternate during catalysis and that they are intertwined in time and space. Our data also suggest that nisin modification is an ordered process that proceeds consecutively from the N terminus of prenisin toward its C terminus. Based on the available literature data and the data presented here, we propose a model wherein nisin is being posttranslationally modified in consecutive steps from its N terminus toward its C terminus.     

Bioassay for nisin in milk, processed cheese, salad dressings, canned tomatoes, and liquid egg products

A sensitive nisin quantification bioassay was constructed, based on Lactococcus lactis chromosomally encoding the nisin regulatory proteins NisK and NisR and a plasmid with a green fluorescent protein (GFP) variant gfp(uv) gene under the control of the nisin-inducible nisA promoter. This strain, LAC275, was capable of transducing the signal from extracellular nisin into measurable GFPuv fluorescence through the NisRK signal transduction system. The LAC275 cells detected nisin concentrations of 10 pg/ml in culture supernatant, 0.2 ng/ml in milk, 3.6 ng/g in processed cheese, 1 ng/g in salad dressings and crushed, canned tomatoes, and 2 ng/g in liquid egg. This method was up to 1,000 times more sensitive than a previously described GFP-based nisin bioassay. This new assay made it possible to detect significantly smaller amounts of nisin than the presently most sensitive published nisin bioassay based on nisin-induced bioluminescence. The major advantage of this sensitivity was that foods could be extensively diluted prior to the assay, avoiding potential inhibitory and interfering substances present in most food products.     

The combined effects of high pressure and nisin on germination and inactivation of Bacillus spores in milk

Aims:  The aim of this work was to investigate the germination and inactivation of spores of Bacillus species in buffer and milk subjected to high pressure (HP) and nisin.
Methods and Results:  Spores of Bacillus subtilis and Bacillus cereus suspended in milk or buffer were treated at 100 or 500 MPa at 40°C with or without 500 IU ml⁻¹ of nisin. Treatment at 500 MPa resulted in high levels of germination (4 log units) of B. subtilis spores in both milk and buffer; this increased to >6 logs by applying a second cycle of pressure. Viability of B. subtilis spores in milk and buffer was reduced by 2·5 logs by cycled HP, while the addition of nisin (500 IU ml⁻¹) prior to HP treatment resulted in log reductions of 5·7 and 5·9 in phosphate buffered saline and milk, respectively. Physical damage of spores of B. subtilis following HP was apparent using scanning electron microscopy. Treating four strains of B. cereus at 500 MPa for 5 min twice at 40°C in the presence of 500 IU ml⁻¹ nisin proved less effective at inactivating the spores of these isolates compared with B. subtilis and some strain-to-strain variability was observed.
Conclusions:  Although high levels of germination of Bacillus spores could be achieved by combining HP and nisin, complete inactivation was not achieved using the aforementioned treatments.
Significance and Impact of the Study:  Combinations of HP treatment and nisin may be an appealing alternative to heat pasteurization of milk.     

Promoting acid resistance and nisin yield of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> F44 by genetically increasing D-Asp amidation level inside cell wall

Nisin fermentation by Lactococcus lactis requires a low pH to maintain a relatively higher nisin activity. However, the acidic environment will result in cell arrest, and eventually decrease the relative nisin production. Hence, constructing an acid-resistant L. lactis is crucial for nisin harvest in acidic nisin fermentation. In this paper, the first discovery of the relationship between D-Asp amidation-associated gene (asnH) and acid resistance was reported. Overexpression of asnH in L. lactis F44 (F44A) resulted in a sevenfold increase in survival capacity during acid shift (pH 3) and enhanced nisin desorption capacity compared to F44 (wild type), which subsequently contributed to higher nisin production, reaching 5346 IU/mL, 57.0% more than that of F44 in the fed-batch fermentation. Furthermore, the engineered F44A showed a moderate increase in D-Asp amidation level (from 82 to 92%) compared to F44. The concomitant decrease of the negative charge inside the cell wall was detected by a newly developed method based on the nisin adsorption amount onto cell surface. Meanwhile, peptidoglycan cross-linkage increased from 36.8% (F44) to 41.9% (F44A), and intracellular pH can be better maintained by blocking extracellular H⁺ due to the maintenance of peptidoglycan integrity, which probably resulted from the action of inhibiting hydrolases activity. The inference was further supported by the acmC-overexpression strain F44C, which was characterized by uncontrolled peptidoglycan hydrolase activity. Our results provided a novel strategy for enhancing nisin yield through cell wall remodeling, which contributed to both continuous nisin synthesis and less nisin adsorption in acidic fermentation (dual enhancement).     

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.     

A Comparative Study Between the Antibacterial Effect of Nisin and Nisin-Loaded Chitosan/Alginate Nanoparticles on the Growth of Staphylococcus aureus in Raw and Pasteurized Milk Samples

The aim of this study was to evaluate the antibacterial effect of nisin-loaded chitosan/alginate nanoparticles as a novel antibacterial delivery vehicle. The nisin-loaded nanoparticles were prepared using colloidal dispersion of the chitosan/alginate polymers in the presence of nisin. After the preparation of the nisin-loaded nanoparticles, their physicochemical properties such as size, shape, and zeta potential of the formulations were studied using scanning electron microscope and nanosizer instruments, consecutively. FTIR and differential scanning calorimetery studies were performed to investigate polymer–polymer or polymer–protein interactions. Next, the release kinetics and entrapment efficiency of the nisin-loaded nanoparticles were examined to assess the application potential of these formulations as a candidate vector. For measuring the antibacterial activity of the nisin-loaded nanoparticles, agar diffusion and MIC methods were employed. The samples under investigation for total microbial counts were pasteurized and raw milks each of which contained the nisin-loaded nanoparticles and inoculated Staphylococcus aureus (ATCC 19117 at 10⁶ CFU/mL), pasteurized and raw milks each included free nisin and S. aureus (10⁶ CFU/mL), and pasteurized and raw milks each had S. aureus (10⁶ CFU/mL) in as control. Total counts of S. aureus were measured after 24 and 48 h for the pasteurized milk samples and after the time intervals of 0, 6, 10, 14, 18, and 24 h for the raw milk samples, respectively. According to the results, entrapment efficiency of nisin inside of the nanoparticles was about 90–95%. The average size of the nanoparticles was 205 nm, and the average zeta potential of them was ?47 mV. In agar diffusion assay, an antibacterial activity (inhibition zone diameter, at 450 IU/mL) about 2 times higher than that of free nisin was observed for the nisin-loaded nanoparticles. MIC of the nisin-loaded nanoparticles (0.5 mg/mL) was about four times less than that of free nisin (2 mg/mL). Evaluation of the kinetic of the growth of S. aureus based on the total counts in the raw and pasteurized milks revealed that the nisin-loaded nanoparticles were able to inhibit more effectively the growth of S. aureus than free nisin during longer incubation periods. In other words, the decrease in the population of S. aureus for free nisin and the nisin-loaded nanoparticles in pasteurized milk was the same after 24 h of incubation while lessening in the growth of S. aureus was more marked for the nisin-loaded nanoparticles than the samples containing only free nisin after 48 h of incubation. Although the same growth reduction profile in S. aureus was noticed for free nisin and the nisin-loaded nanoparticles in the raw milk up to 14 h of incubation, after this time the nisin-loaded nanoparticles showed higher growth inhibition than free nisin. Since, generally, naked nisin has greater interactions with the ingredients present in milk samples in comparison with the protected nisin. Therefore, it is concluded that the antibacterial activity of nisin naturally decreases more during longer times of incubation than the protected nisin with the chitosan/alginate nanoparticles. Consequently, this protection increases and keeps antibacterial efficiency of nisin in comparison with free nisin during longer times of storage. These results can pave the way for further research and use of these nanoparticles as new antimicrobial agents in various realms of dairy products.     

The increase of O-acetylation and N-deacetylation in cell wall promotes acid resistance and nisin production through improving cell wall integrity in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Cell wall is closely related to bacterial robustness and adsorption capacity, playing crucial roles in nisin production in Lactococcus lactis. Peptidoglycan (PG), the essential component of cell wall, is usually modified with MurNAc O-acetylation and GlcNAc N-deacetylation, catalyzed by YvhB and XynD, respectively. In this study, increasing the two modifications in L. lactis F44 improved autolysis resistance by decreasing the susceptibility to PG hydrolases. Furthermore, both modifications were positively associated with overall cross-linkage, contributing to cell wall integrity. The robust cell wall rendered the yvhB/xynD-overexpression strains more acid resistant, leading to the increase of nisin production in fed-batch fermentations by 63.7 and 62.9%, respectively. Importantly, the structural alterations also reduced nisin adsorption capacity, resulting in reduction of nisin loss. More strikingly, the co-overexpression strain displayed the highest nisin production (76.3% higher than F44). Our work provides a novel approach for achieving nisin overproduction via extensive cell wall remodeling.     

Influences on the antimicrobial activity of surface-adsorbed nisin

The efficacy of the antimicrobial peptide nisin was examined after adsorption to silica surfaces. Three protocols were used to evaluate nisin's activity against adhered cells ofListeria monocytogenes: bioassay usingPediococcus pentosaceous FBB 61-2 as the sensitive indicator strain; visualization and enumeration of cells by microscopic image analysis; and viability of adhered cells as determined by lodonitrotetrazolium violet uptake and crystallization. The activity of adsorbed nisin was highly dependent upon conditions of adsorption. The highest antimicrobial activity of adsorbed nisin occurred with high concentrations of nisin (1.0 mg ml^–1) and brief contact times (1 h) on surfaces of low hydrophobicity. Sequential adsorption of a second protein (-lactoglobulin or bovine serum albumin) onto surfaces consistently resulted in decreased nisin activity. These data provide direction for the development of applications to limit microbial attachment on food contact surfaces through the use of adsorbed antimicrobial peptides.     

Microplate bioassay for nisin in foods,based on nisin-induced green fluorescent protein fluorescence

A plasmid coding for the nisin two-component regulatory proteins, NisK and NisR, was constructed; in this plasmid a gfp gene (encoding the green fluorescent protein) was placed under control of the nisin-inducible nisF promoter. The plasmid was transformed into non-nisin-producing Lactococcus lactis strain MG1614. The new strain could sense extracellular nisin and transduce it to green fluorescent protein fluorescence. The amount of fluorescence was dependent on the nisin concentration, and it could be measured easily. By using this strain, an assay for quantification of nisin was developed. With this method it was possible to measure as little as 2.5 ng of pure nisin per ml in culture supernatant, 45 ng of nisin per ml in milk, 0.9 microg of nisin in cheese, and 1 microg of nisin per ml in salad dressings.     

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 (10⁵ to 10⁶ 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 10⁴ 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.     

Microplate Bioassay for Nisin in Foods, Based on Nisin-Induced Green Fluorescent Protein Fluorescence

A plasmid coding for the nisin two-component regulatory proteins, NisK and NisR, was constructed; in this plasmid a gfp gene (encoding the green fluorescent protein) was placed under control of the nisin-inducible nisF promoter. The plasmid was transformed into non-nisin-producing Lactococcus lactis strain MG1614. The new strain could sense extracellular nisin and transduce it to green fluorescent protein fluorescence. The amount of fluorescence was dependent on the nisin concentration, and it could be measured easily. By using this strain, an assay for quantification of nisin was developed. With this method it was possible to measure as little as 2.5 ng of pure nisin per ml in culture supernatant, 45 ng of nisin per ml in milk, 0.9 μg of nisin in cheese, and 1 μg of nisin per ml in salad dressings.     

Oxidation of Lanthionines Renders the Lantibiotic Nisin Inactive

The peptide antibiotic nisin A belongs to the group of antibiotics called lantibiotics. They are classified as lantibiotics because they contain the structural group lanthionine. Lanthionines are composed of a single sulfur atom that is linked to the β-carbons of two alanine moieties. These sulfur atoms are vulnerable to environmental oxidation. A mild oxidation reaction was performed on nisin A to determine the relative effects it would have on bioactivity. High-mass-accuracy Fourier transform ion cyclotron resonance mass spectrometry data revealed the addition of seven, eight, and nine oxygens. These additions correspond to the five lanthionines, two methionines, and two histidines that would be susceptible to oxidation. Subsequent bioassays revealed that the oxidized form of nisin A had a complete loss of bactericidal activity. In a competition study, the oxidized nisin did not appear to have an antagonistic affect on the bioactivity of nisin A, since the addition of an equal molar concentration of the oxidized variant did not have an influence on the bactericidal activity of the native antibiotic. Electron microscopy data revealed that the oxidized forms were still capable of assembling into large circular complexes, demonstrating that oxidation does not disrupt the lateral assembly mechanism of the antibiotic. Affinity thin-layer chromatography and fluorescence microscopy experiments suggested that the loss of activity is due to the inability of the oxidized form of nisin to bind to the cell wall precursor lipid II. Given the loss of bioactivity following oxidation, oxidation should be an important factor to consider in future production, purification, pharmacokinetic, and pharmacodynamic studies.Lantibiotics are ribosomally synthesized peptide bacteriocins that undergo extensive posttranslational modifications to yield unusual amino acids, like lanthionine, methyllanthionine, 2,3-didehydroalanine, 2,3-didehydrobutyrine, and S-[aminovinyl] cysteine (8). The name lantibiotic is derived from the presence of the posttranslationally modified lanthionine residues. Nisin A (3,351.5 Da), produced by Lactococcus lactis, belongs to this class of antibiotics and is further subclassified as a type A(I) lantibiotic. Type A(I) lantibiotics are cationic and have a rigid ring conformation separated by areas of flexibility. Another well-studied lantibiotic, gallidermin, also belongs to this class of lantibiotics and has significant homology to nisin A in the first two lanthionine rings, A and B (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Schematic of the covalent structures of nisin A and gallidermin. The N-terminal rings A and B are believed to be responsible for binding to lipid II.The antibiotics in this class have drawn considerable attention for their bactericidal potential as preservatives and for their potential for treating Staphylococcus and Streptococcus infections. Nisin A has been used for over 40 years in Europe as a preservative in the food industry and was approved for use in the United States by the FDA in 1988. Its uses include controlling the growth of various bacteria in pasteurized cheese and liquid egg ingredients, as well as preserving salad dressings (12), canned foods (10, 32), and, most recently, ground beef (24). Other lantibiotics, including gallidermin and epidermin, have been shown to be useful as treatments for acne and in the maintenance of oral health (19, 21). Recent literature shows that both nisin A and gallidermin can be used to treat and/or prevent mastitis in bovines (3, 25a), and they are currently marketed as wipes.Lantibiotics have multiple modes of bactericidal activity (2, 16). In the case of nisin A, the sensitivity of the host bacterium has been shown to be dependent on the charge states of its cell wall and membrane (1, 6, 25). More importantly, the bactericidal activity is attributed to lipid II abduction (4, 5, 7). A novel mechanism of antimicrobial activity for nisin A has been described, in which it binds to lipid II and sequesters it into large complexes. These complexes aid in the abduction of lipid II from the growth zones of bacteria, where lipid II is required for new cell wall formation (16). A novel lipid II binding motif for nisin A has been characterized by nuclear magnetic resonance (NMR) (18) in which the N-terminal portion of nisin A, lanthionine rings A and B, interacts with the pyrophosphate, the peptidoglycan MurNAc, and the first isoprene of lipid II.An oxidized form of nisin A was characterized using bactericidal assays, high-mass-accuracy Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), chromatography methods, and electron and fluorescence microscopy techniques. The objectives of this study were to determine changes in the biophysical properties of oxidized nisin A as they relate to its bactericidal activity, its ability to interact with bacterial membranes, its capacity for lateral assembly, and its ability to bind to lipid II.     

Mode of action of leucocin K7 produced by <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> K7 against <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and its potential in milk preservation

Objectives

To investigate the mode of action of leucocin K7 against Listeria monocytogenes and to assess its inhibitory effect on Lis. monocytogenes in refrigerated milk.

Results

A bacteriocin-producing strain, Leuconostoc mesenteroides K7, was isolated from a fermented pickle. The bacteriocin, leucocin K7, exhibited antagonistic activity against Lis. monocytogenes with an MIC of 28 µg/ml. It was sensitive to proteaseS and displayed good thermal stability and broad active pH range. Leucocin K7 had no effect on the efflux of ATP from Lis. monocytogenes but triggered the efflux of K⁺ and the intracellular hydrolysis of ATP. It also dissipated the transmembrane electrical potential completely and transmembrane pH gradient partially. It 80 AU/ml inhibited the growth of Lis. monocytogenes by 2.3–3.9 log units in milk; when combined with glycine (5 mg/ml), it completely eliminated viable Lis. monocytogenes over 7 days

Conclusion

Leucocin K7 shows different mode of action from nisin and may have potential application in milk preservation.