Mode of Action of Nisin Z against Listeria monocytogenes Scott A Grown at High and Low Temperatures

Nisin Z, a natural nisin variant, was recently isolated from Lactococcus lactis subspecies lactis NIZO 22186. The gene for this lantibiotic, designated nisZ, has been cloned, and its nucleotide sequence was found to be identical to that of the precursor nisin gene with the exception of a single mutation resulting in the substitution of Asn-27 for His-27 in the mature polypeptide (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). A K⁺ electrode was used to investigate the effect of various environmental parameters on the action of nisin Z against Listeria monocytogenes. Addition of nisin Z resulted in immediate loss of cell K⁺, depolarization of the cytoplasmic membrane, inhibition of respiratory activity, and hydrolysis and partial efflux of cellular ATP. The action of nisin Z was optimal at pH 6.0 and was significantly reduced by di- and trivalent cations. The lanthanide gadolinium (Gd³⁺) was an efficient inhibitor and prevented nisin Z activity completely at a concentration of 0.2 mM. Nisin Z-induced loss of cell K⁺ was reduced at low temperatures, presumably as a result of the increased ordering of the lipid hydrocarbon chains in the cytoplasmic membrane. In cells grown at 30°C, the action of nisin Z was prevented below 7°C, whereas in cells grown at 4°C nisin Z was able to induce K⁺ leakage at this low temperature.     

Nisin Z inhibits the growth of Candida albicans and its transition from blastospore to hyphal form

Aims: To investigate the efficacy of nisin Z, an antimicrobial peptide produced by certain strains of Lactococcus lactis against Candida albicans growth and transition. Methods and Results: Candida albicans was cultured in the presence of various concentrations of nisin Z (1000, 500, and 100 μg ml⁻¹) for different time points. Candida albicans growth was determined using the Alamar Blue assay. The yeast’s transition from blastospore to hyphal form was assessed through optical microscope observations. The effect of nisin Z on C. albicans ultrastructure was followed by scanning and transmission electron microscopy. Our results show that nisin Z inhibited C. albicans growth beginning at 500 μg ml⁻¹. This inhibition was both time- and dose-dependent. Nisin Z was also active against C. albicans transition by significantly inhibiting the transformation of C. albicans from the blastospore to hyphal form. Treatments with nisin Z lead to ultrastructural disturbances of C. albicans. Conclusion: Our findings indicate that nisin Z significantly reduced C. albicans growth and transition. These effects may have occurred through ultrastructural modifications of this yeast. Significance and Impact of the Study: For the first time, effect of nisin Z on C. albicans was investigated. These results therefore suggest that nisin Z may have antifungal properties, and could be used as an antifungal molecule.     

Potential application of the nisin Z preparation of Lactococcus lactis W8 in preservation of milk

Aims: The aim of the study is to evaluate the effectiveness of the preparation of nisin Z from Lactococcus lactis W8‐fermented milk in controlling the growth of spoilage bacteria in pasteurized milk. Methods and Results: Spoilage bacteria isolated from pasteurized milk at 8 and 15°C were identified as Enterococcus italicus, Enterococcus mundtii, Enterococcus faecalis, Bacillus thuringiensis, Bacillus cereus, Lactobacillus paracasei, Acinetobacter sp., Pseudomonas fluorescens and Enterobacter aerogenes. These bacteria were found to have the ability to survive pasteurization temperature. Except Enterobacter aerogenes, the spoilage bacteria were sensitive to the nisin Z preparation of the L. lactis W8. Addition of the nisin Z preparation to either the skim milk or fat milk inoculated with each of the spoilage bacteria reduced the initial counts (about 5 log CFU ml^?1) to an undetectable level within 8–20 h. The nisin Z preparation extended the shelf life of milk to 2 months under refrigeration. Conclusions: The nisin Z preparation from L. lactis W8‐fermented milk was found to be effective as a backup preservative to counteract postpasteurization contamination in milk. Significance and Impact of the Study: A rapid inhibition of spoilage bacteria in pasteurized skim and fat milk with the nisin Z preparation of L. lactis W8 is more significant in comparison with the commercially available nisin (nisin A). The nisin Z preparation can be used instead of commercial nisin, which is not effective in fat milk.     

Improvement of nisin production in pH feed-back controlled,fed-batch culture by <Emphasis Type="Italic">Lactococcus lactis</Emphasis>subsp<Emphasis Type="Italic">. lactis</Emphasis>

Nisin production by Lactococcus lactis subsp. lactisin fed-batch culture was doubled by using a pH feed-back controlled method. Sucrose concentration was controlled at 10 g l^–1 giving 5010 IU nisin ml^–1 compared to 2660 IU nisin ml^–1 in batch culture.     

Influence of growth conditions on the nisin production of bioengineered <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strains

Nisin production of three bioengineered strains, (LAC338, LAC339 and LAC340) with immunity (nisFEG) and/or regulation (nisRK) genes of nisin biosynthesis on plasmids in the Lactococcus lactis LL27 nisin producer, was evaluated under pH-controlled and pH-uncontrolled batch fermentations. Optimization studies showed that fructose and yeast extract yielded the highest nisin activity. The strains LAC338, LAC339, and LAC340 produced 24, 45, and 44% more nisin, respectively, than wild-type L. lactis LL27 after 12-h incubation. However, sharp decreases in the yield of nisin were observed at the late phase of fermentation with LAC339 and LL27 in contrast to LAC340 and LAC338 strains for which the high level of nisin could be maintained longer. Obviously, increasing the copy number of the regulation genes together with immunity genes in the nisin producers retarded the loss of nisin in the late phase of the fermentation.     

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°C for 6 months. In some trials, L. innocua was added to cheese milk at 10⁵ to 10⁶ 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.     

Modelling the production of nisin by Lactococcus lactis in fed-batch culture

Nisin production in batch culture and fed-batch cultures (sucrose feeding rates were 6, 7, 8, and 10 g l^–1 h^–1, respectively) by Lactococcus lactis subsp. lactis ATCC 11454 was investigated. Nisin production showed primary metabolite kinetics, and could be improved apparently by altering the feeding strategy. The nisin titer reached its maximum, 4,185 IU ml^–1, by constant addition of sucrose at a feeding rate of 7 g l^–1 h^–1; an increase in 58% over that of the batch culture (2,658 IU ml^–1). Nisin biosynthesis was affected strongly by the residual sucrose concentration during the feeding. Finally, a mathematical model was developed to simulate the cell growth, sucrose consumption, lactic acid production and nisin production. The model was able to describe the fermentation process in all cases.     

PCR detection of the structural genes of nisin Z and lacticin 481 in Lactococcus lactis subsp. lactis INIA 415, a strain isolated from raw milk Manchego cheese

A Lactococcus strain with strong antimicrobial activity was isolated from raw milk Manchego cheese during a survey on the production of bacteriocins by lactic acid bacteria present in raw milk cheeses. It was identified as Lactococcus lactis subsp. lactis, phenotypically by its morphological and physiological characteristics and genotypically by a PCR technique. When tested for tolerance to known bacteriocins produced by lactococci, it was shown to be resistant to nisin A and nisin Z. The presence of genes encoding nisin and lacticin 481 was revealed by PCR techniques with specific probes. Sequences of the respective PCR amplified fragments matched sequences reported for nisin Z and lacticin 481.     

Genetics of subtilin and nisin biosyntheses

Several peptide antibiotics have been described as potent inhibitors of bacterial growth. With respect to their biosynthesis, they can be devided into two classes: (i) those that are synthesized by a non-ribosomal mechanism and (ii) those that are ribosomally synthesized. Subtilin and nisin belong to the ribosomally synthesized peptide antibiotics. They contain the rare amino acids dehydroalanine, dehydrobutyrine, meso-lanthionine, and 3-methyl-lanthionine. They are derived from prepeptides which are post-translationally modiffied and have been termed lantibiotics because of their characteristic lanthionine bridges (Schnell et al. 1988). Nisin is the most prominent lantibiotic and is used as a food preservative due to its high potency against certain gram-positive bacteria (Mattick & Hirsch 1944, 1947; Rayman & Hurst 1984). It is produced by Lactococcus lactis strains belonging to serological group N. The potent bactericidal activities of nisin and other lantibiotics are based on depolarization of energized bacterial cytoplasmic membranes. Breakdown of the membrane potential is initiated by the formation of pores through which molecules of low molecular weight are released. A trans-negative membrane potential of 50 to 100 mV is necessary for pore formation by nisin (Ruhr & Sahl 1985; Sahl et al. 1987). Nisin occurs as a partially amphiphilic molecule (Van de Ven et al. 1991). Apart from the detergent-like effect of nisin on cytoplasmic membranes, an inhibition of murein synthesis has also been discussed as the primary effect (Reisinger et al. 1980). In several countries nisin is used to prevent the growth of clostridia in cheese and canned food. The nisin peptide structure was first described by Gross & Morall (1971), and its structural gene was isolated in 1988 (Buchman et al. 1988; Kaletta & Entian 1989). Nisin has two natural variants, nisin A and nisin Z, which differ in a single amino acid residue at position 27 (histidin in nisin A is replaced by asparagin in nisin Z (Mulders et al. 1991; De Vos et al. 1993). Subtilin is produced by Bacillus subtilis ATCC 6633. Its chemical structure was first unravelled by Gross & Kiltz (1973) and its structural gene was isolated in 1988 (Banerjee & Hansen 1988). Subtilin shares strong similarities to nisin with an identical organization of the lanthionine ring structures (Fig. 1), and both lantibiotics possess similar antibiotic activities. Due to its easy genetic analysis B. subtilis became a very suitable model organism for the identification and characterization of genes and proteins involved in lantibiotic biosynthesis. The pathway by which nisin is produced is very similar to that of subtilin, and the proteins involved share significant homologies over the entire proteins (for review see also De Vos et al. 1995b). The respective genes have been identified adjacent to the structural genes, and are organized in operon-like structures (Fig. 2). These genes are responsible for post-translational modification, transport of the modified prepeptide, proteolytic cleavage, and immunity which prevents toxic effects on the producing bacterium. In addition to this, biosynthesis of subtilin and nisin is strongly regulated by a two-component regulatory system which consists of a histidin kinase and a response regulator protein.     

An in vitro synergetic evaluation of the use of nisin and sodium fluoride or chlorhexidine against Streptococcus mutans

The objective of this study is to investigate the synergetic action between nisin and sodium fluoride or chlorhexidine against Streptococcus mutans, a primary cariogenic pathogen. In the antibacterial assay, a synergetic effect on S. mutans was found between nisin and sodium fluoride, but there was no interaction between nisin and chlorhexidine by the checkerboard, the fractional inhibitory concentration (FIC) and the fractional bactericidal concentration (FBC) tests. S. mutans survival rates showed a significant decline after treatment with a combination of nisin and sodium fluoride in a time-kill study. Scanning electron microscopy showed that the damage to S. mutans with the combined nisin and sodium fluoride treatment was the most severe among all of the different single and combined antimicrobial treatments. Furthermore, in the antibiofilm test, nisin in combination with sodium fluoride produced a stronger bactericidal effect on a S. mutans biofilm for 4 h and 16 h compared with sodium fluoride alone by confocal laser scanning microscopy. Nisin in combination with sodium fluoride exerted a high bactericidal effect on S. mutans and thereby has the potential to be used as an effective drug combination to prevent dental caries.     

Immunomodulatory efficacy of nisin—a bacterial lantibiotic peptide

Nisin is a peptide bacteriocin, grouped under the category of lantibiotics. It is naturally produced by Lactococcus lactis to eliminate other competing gram‐positive bacteria from its vicinity. Moreover under certain conditions it is reported to be effective against a broad range of gram‐negative bacteria as well. Thus, it has been widely used as a safe food preservative especially in the dairy industry. Because of its wide‐scale consumption, its effect on eukaryotic cells should be of great concern. Here we examine the immunomodulatory efficacy of nisin in vitro. MTT‐based cytotoxicity assay demonstrated nisin's cytotoxicity on human T‐cell lymphoma Jurkat cells, Molt‐4 cells and freshly cultured human lymphocytes at over 200 µM concentration (IC₅₀225 µM ). The cell death mechanism induced by nisin in all these lymphocyte types was independent of oligonucleosomal DNA fragmentation, as analyzed by agarose gel electrophoresis and comet assay. Additionally, scanning electron microscope and fluorescence microscopy demonstrated the ability of nisin to activate human PMNs in vitro. Nisin‐activated neutrophils extruded intact nuclear chromatin to form NETs, well known for neutralization of virulence factors and extermination of bacterial pathogens. Nisin's presence also elevated neutrophil intracellular superoxide levels, normally produced by activated NADPH oxidase and prerequisite to NET formation. These nisin‐induced responses in cellular representatives of two separate branches of human immune system—adaptive and innate—although leading to cell death, did not include DNA fragmentation. From these findings, we propose that nisin might trigger similar AICD mechanisms in lymphocytes and neutrophils, different from conventional apoptosis which involves DNA fragmentation. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

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     

Characterization of the Structural Gene Encoding Nisin F, a New Lantibiotic Produced by a Lactococcus lactis subsp. lactis Isolate from Freshwater Catfish (Clarias gariepinus)

Lactococcus lactis F10, isolated from freshwater catfish, produces a bacteriocin (BacF) active against Staphylococcus aureus, Staphylococcus carnosus, Lactobacillus curvatus, Lactobacillus plantarum, and Lactobacillus reuteri. The operon encoding BacF is located on a plasmid. Sequencing of the structural gene revealed no homology to other nisin genes. Nisin F is described.     

Nisin F-loaded brushite bone cement prevented the growth of Staphylococcus aureus in vivo

Aims: To determine if nisin F‐loaded self‐setting brushite cement could control the growth of Staphylococcus aureus in vivo. Methods and Results: Brushite cement was prepared by mixing equimolar concentrations of β‐tricalcium phosphate and monocalcium phosphate monohydrate. Nisin F was added at 5·0, 2·5 and 1·0% (w/w) and the cement moulded into cylinders. In vitro antibacterial activity was determined using a delayed agar diffusion assay. Release of nisin F from the cement was determined using BCA protein assays. Based on scanning electron microscopy and X‐ray diffraction analysis, nisin F did not cause significant changes in cement structure or chemistry. Cement containing 5·0% (w/w) nisin F yielded the most promising in vitro results. Nisin F‐loaded cement was implanted into a subcutaneous pocket on the back of mice and then infected with S. aureus Xen 36. Infection was monitored for 7 days, using an in vivo imaging system. Nisin F prevented S. aureus infection for 7 days and no viable cells were isolated from the implants. Conclusions: Nisin F‐loaded brushite cement successfully prevented in vivo growth of S. aureus. Significance and Impact of the Study: Nisin F incorporated into bone cement may be used to control S. aureus infection in vivo.     

Effects of pH profiles on nisin fermentation coupling with foam separation

Online foam separation was proposed to recover nisin during fermentation of Lactococcus lactis subsp. lactis ATCC 11454. Firstly, the optimal pH profile of nisin fermentation was investigated including different realkalization set values and pH drop gradients. Then the selected pH profiles of 5.75 ± 0.05 and 6.25–5.75 (±0.02) were used to perform nisin fermentation coupling with foam separation. The results showed that pH profile of 5.75 ± 0.05 was better than that of 6.25–5.75 (±0.02) for online foam separation. With the optimal pH profile, an aeration of 20 ml min⁻¹ that started at 8 h of incubation and lasted for 2 h resulted in 6.6 times higher specific productivity than that of the fermentation without aeration. Nisin synthesis was therefore prolonged with low sucrose concentration in the culture broth, which indicated that the feedback inhibition of nisin is more influential than the substrate limitation of sucrose in the late phase of nisin fermentation. Total nisin production (4,870 ± 180 IU ml⁻¹) was increased by 30.3% with online foam separation. This effective online recovery method for nisin production could be easily scaled up due to the facile operation of foaming process.     

Biosynthetic characterization and biochemical features of the third natural nisin variant,nisin Q,produced by Lactococcus lactis 61‐14

Aims: To characterize the genetic and biochemical features of nisin Q. Methods and Results: The nisin Q gene cluster was sequenced, and 11 putative orfs having 82% homology with the nisin A biosynthesis gene cluster were identified. Nisin Q production was confirmed from the nisQ‐introduced nisin Z producer. In the reporter assay, nisin Q exhibited an induction level that was threefold lower than that of nisin A. Nisin Q demonstrated an antimicrobial spectrum similar to those of the other nisins. Under oxidizing conditions, nisin Q retained a higher level of activity than nisin A. This higher oxidative tolerance could be attributed to the presence of only one methionine residue in nisin Q, in contrast to other nisins that contain two. Conclusions: The 11 orfs of the nisin producers were identical with regard to their functions. The antimicrobial spectra of the three natural nisins were similar. Nisin Q demonstrated higher oxidative tolerance than nisin A. Significance and Impact of the Study:  Genetic and biochemical features of nisin Q are similar to those of other variants. Moreover, owing to its higher oxidative tolerance, nisin Q is a potential alternative for nisin A.     

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     

Lantibiotic Immunity: Inhibition of Nisin Mediated Pore Formation by NisI

Nisin, a 3.4 kDa antimicrobial peptide produced by some Lactococcus lactis strains is the most prominent member of the lantibiotic family. Nisin can inhibit cell growth and penetrates the target Gram-positive bacterial membrane by binding to Lipid II, an essential cell wall synthesis precursor. The assembled nisin-Lipid II complex forms pores in the target membrane. To gain immunity against its own-produced nisin, Lactococcus lactis is expressing two immunity protein systems, NisI and NisFEG. Here, we show that the NisI expressing strain displays an IC₅₀ of 73±10 nM, an 8–10-fold increase when compared to the non-expressing sensitive strain. When the nisin concentration is raised above 70 nM, the cells expressing full-length NisI stop growing rather than being killed. NisI is inhibiting nisin mediated pore formation, even at nisin concentrations up to 1 µM. This effect is induced by the C-terminus of NisI that protects Lipid II. Its deletion showed pore formation again. The expression of NisI in combination with externally added nisin mediates an elongation of the chain length of the Lactococcus lactis cocci. While the sensitive strain cell-chains consist mainly of two cells, the NisI expressing cells display a length of up to 20 cells. Both results shed light on the immunity of lantibiotic producer strains, and their survival in high levels of their own lantibiotic in the habitat.     

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.     

Optimization of Non-Nutritional Factors for a Cost-Effective Enhancement of Nisin Production Using Orthogonal Array Method

In order to increase nisin production in a cost-effective manner, non-nutritional factors as well as nutritional parameters must be optimized. In this study, optimization of the most important non-nutritional factors for nisin production using orthogonal array method was performed. Optimization of temperature, agitation, age and size of inoculum, medium initial pH value and flask volume/medium volume ratio in de Man, Rogosa and Sharpe (MRS) medium in batch fermentation was accomplished. Nisin was produced by Lactococcus lactis subsp. lactis PTCC 1336 and measured by bioassay method using Micrococcus luteus PTCC 1169 as the nisin-sensitive strain. The optimum levels of non-nutritional factors for maximum nisin production and productivity were obtained as: flask volume/medium volume ratio: 5.00, medium initial pH value: 8.00, inoculum size: 1%, inoculum age: 24 h old (A = 1.7), agitation: 100 rpm and temperature: 27 °C. Under the optimized conditions, maximum nisin production and maximum nisin productivity were 599.70 IU/mL and 37.48 IU/mL/h, respectively.