Nisin-producing Lactococcus Lactis Strain Research Articles

Efficient Production of Nisin A from Low-Value Dairy Side Streams Using a Nonengineered Dairy Lactococcus lactis Strain with Low Lactate Dehydrogenase Activity.

Nisin is commonly used as a biopreservative in foods. For industrial production, nisin-producing Lactococcus lactis strains are usually grown to high cell densities to achieve the highest possible nisin titer. However, accumulation of lactic acid eventually halts production, even in pH-controlled fermentations. Here, we describe a nisin-producing L. lactis strain Ge001, which was obtained after transferring the nisin gene cluster from L. lactis ATCC 11454, by conjugation, into the natural mutant L. lactis RD1M5, with low lactate dehydrogenase activity. The ability of Ge001 to produce nisin was tested using dairy waste as the fermentation substrate. To accommodate redox cofactor regeneration, respiration conditions were used, and to alleviate oxidative stress and to reduce adsorption of nisin onto the producing cells, we found it to be beneficial to add 1 mM Mn2+ and 100 mM Ca2+, respectively. A high titer of 12 084 IU/mL nisin could be reached, which is comparable to the highest titers reported using expensive, rich media. Summing up, we here present a 100% natural, robust, and sustainable approach for producing food-grade nisin and acetoin from readily available dairy waste.

Unravelling the Potential of Lactococcus lactis Strains to Be Used in Cheesemaking Production as Biocontrol Agents.

This research, developed within an exchange program between Italy and Canada, represents the first step of a three-year project intended to evaluate the potential of nisin-producing Lactococcus lactis strains isolated from Italian and Canadian dairy products to select a consortium of strains to be used as biocontrol agents in Crescenza and Cheddar cheese production. In this framework, the acidification and the production of nisin in milk, and the volatile molecule profiles of the fermented milk, were recorded. The strains were further tested for their anti-Listeria monocytogenes activity in milk. The data obtained highlighted good potential for some of the tested strains, which showed production of nisin beginning within 12 h after the inoculation and reaching maximum levels between 24 and 48 h. The highest inactivation levels of L. monocytogenes in milk was reached in the presence of the strains 101877/1, LBG2, 9FS16, 11FS16, 3LC39, FBG1P, UL36, UL720, UL35. The strains generated in milk-specific volatile profiles and differences in the presence of fundamental aromatic molecules of dairy products, such as 2-butanone and diacetyl. The results highlight the interesting potential of some L. lactis strains, the producer of nisin, to be further used as biocontrol agents, although the strains need to be tested for interaction with traditional thermophilic starters and tested in real cheesemaking conditions.

Engineering Lactococcus lactis as a multi-stress tolerant biosynthetic chassis by deleting the prophage-related fragment

BackgroundIn bioengineering, growth of microorganisms is limited because of environmental and industrial stresses during fermentation. This study aimed to construct a nisin-producing chassis Lactococcus lactis strain with genome-streamlined, low metabolic burden, and multi-stress tolerance characteristics.ResultsThe Cre-loxP recombination system was applied to reduce the genome and obtain the target chassis strain. A prophage-related fragment (PRF; 19,739 bp) in the L. lactis N8 genome was deleted, and the mutant strain L. lactis N8-1 was chosen for multi-stress tolerance studies. Nisin immunity of L. lactis N8-1 was increased to 6500 IU/mL, which was 44.44% higher than that of the wild-type L. lactis N8 (4500 IU/mL). The survival rates of L. lactis N8-1 treated with lysozyme for 2 h and lactic acid for 1 h were 1000- and 10,000-fold higher than that of the wild-type strain, respectively. At 39 ℃, the L. lactis N8-1 could still maintain its growth, whereas the growth of the wild-type strain dramatically dropped. Scanning electron microscopy showed that the cell wall integrity of L. lactis N8-1 was well maintained after lysozyme treatment. Tandem mass tags labeled quantitative proteomics revealed that 33 and 9 proteins were significantly upregulated and downregulated, respectively, in L. lactis N8-1. These differential proteins were involved in carbohydrate and energy transport/metabolism, biosynthesis of cell wall and cell surface proteins.ConclusionsPRF deletion was proven to be an efficient strategy to achieve multi-stress tolerance and nisin immunity in L. lactis, thereby providing a new perspective for industrially obtaining engineered strains with multi-stress tolerance and expanding the application of lactic acid bacteria in biotechnology and synthetic biology. Besides, the importance of PRF, which can confer vital phenotypes to bacteria, was established.

The impact of commercial prebiotics on the growth, survival and nisin production by Lactococcus lactis 537 in milk

In this work we investigated the potential of a new dairy-based fermented food which contains a nisin producing Lactococcus lactis strain with biopreservative function and a prebiotic. Lc lactis 537 was incubated in ultrahigh temperature (UHT) skim milk in the presence of 8 different commercially available prebiotics (inulin, fructooligosaccharide, xylooligosaccharide, lactulose, gum arabic, maltodextrin, isomaltooligosaccharides, galactooligosaccharides) at 3% concentration. The growth of Lc. lactis 537 was found to be enhanced in the presence of inulin only, as compared to in milk without prebiotic. Acidification was minimal, with all milks with or without prebiotic remaining above pH 6. Survival during storage for 14 days at 4 °C was high with or without prebiotic, with less than a 1-log CFU/mL reduction in viability seen. Antimicrobial production by Lc. lactis 537 was evaluated and found to be significantly higher when cells were grown in the presence of inulin. A nisB mutant of Lc. lactis 537 did not have any antimicrobial activity with or without prebiotic, thus demonstrating the effect was due to nisin. The findings of this study suggest that inulin is the most promising prebiotic inclusion for the development of a milk-based symbiotic product containing Lc. lactis 537.

Control of Listeria monocytogenes on ready-to-eat ham and fresh cut iceberg lettuce using a nisin containing Lactococcus lactis fermentate

Listeria monocytogenes is a potentially deadly psychrotrophic pathogen that can cause illness through contamination of ready-to-eat (RTE) foods. In this work we evaluated the use of a fermentate consisting of cells and supernatant of a nisin producing Lactococcus lactis strain to control L. monocytogenes in foods. In addition, to determine the role of nisin production in this activity, a transposon screen was carried out to isolate a nisin-negative mutant. Several independent mutants containing transposon insertions in the nisin gene cluster were obtained which exhibited a complete loss of anti-L. monocytogenes activity in an agar-based assay. A simple media using food grade components was found to support strong growth and nisin production of Lc. lactis 537 following incubation at between 20 and 35 °C. The ability of the fermentate to control L. monocytogenes in foods was evaluated in cut iceberg lettuce and RTE sliced ham stored at 4 °C for 10 days. Initial levels of 103–104 CFU/g L. monocytogenes were reduced to below detection limit (<20 CFU/g) immediately by the fermentate and remained so for the duration of the trial. In comparison, fermentate from the nisin-negative nisB mutant was unable to reduce L. monocytogenes levels initially. It did however provide some L. monocytogenes growth inhibition in ham and faster killing in lettuce at later time points, but not to undetectable levels. These results demonstrate that nisin containing fermentate provides a simple and effective means to control L. monocytogenes on RTE foods.

No more cleaning up -Efficient lactic acid bacteria cell catalysts as a cost-efficient alternative to purified lactase enzymes.

β-galactosidases, commonly referred to as lactases, are used for producing lactose-free dairy products. Lactases are usually purified from microbial sources, which is a costly process. Here, we explored the potential that lies in using whole cells of a food-grade dairy lactic acid bacterium, Streptococcus thermophilus, as a substitute for purified lactase. We found that S. thermophilus cells, when treated with the antimicrobial peptide nisin, were able to hydrolyze lactose efficiently. The rate of hydrolysis increased with temperature; however, above 50°C, stability was compromised. Different S. thermophilus strains were tested, and the best candidate was able to hydrolyze 80% of the lactose in a 50g/L solution in 4h at 50°C, using only 0.1g/L cells (dry weight basis). We demonstrated that it was possible to grow the cell catalyst on dairy waste, and furthermore, that a cell-free supernatant of a culture of a nisin-producing Lactococcus lactis strain could be used instead of purified nisin, which reduced cost of use significantly. Finally, we tested the cell catalysts in milk, where lactose also was efficiently hydrolyzed. The method presented is natural and low-cost, and allows for production of clean-label and lactose-free dairy products without using commercial enzymes from recombinant microorganisms. KEY POINTS: • Nisin-permeabilized Streptococcus thermophilus cells can hydrolyze lactose efficiently. • A low-cost and more sustainable alternative to purified lactase enzymes. • Reduction of overall sugar content. • Clean-label production of lactose-free dairy products.

In vitro immunomodulatory effect of nisin on porcine leucocytes.

Nisin, a lantibiotic bacteriocin, has been used for years as a natural food preservative. In addition to its antimicrobial activity, nisin also shows immunomodulatory properties, and the nisin-producing Lactococcus lactis strain has been successfully tested as a probiotic in weaned piglets. However, the impact of nisin on porcine immune cells has not yet been explored. The objective of the present study was to examine the in vitro immunomodulatory effect of nisin on porcine peripheral blood leucocytes. The whole heparinized blood samples or freshly isolated peripheral blood mononuclear cells (PBMCs) were incubated with different nisin concentrations (0, 1.56, 3.125, 6.25, 12.5, 25 or 50µg/ml) for 1, 24, 48 or 72hr. Escherichia coli bacteria were used to stimulate blood phagocytes, while concanavalin A and lipopolysaccharide from E.coli were used as mitogens. Control cells remained unstimulated. MTT colorimetric assay was used to evaluate PBMCs viability and mitogenic response. Phagocyte activity and T-cell proliferation were measured by flow cytometry. Flow cytometer was also used for immunophenotyping of T cells. Cytokine levels in the culture media were determined using commercial immunoassay (ELISA) kits. The highest concentration of nisin exhibited proliferative activity (p˂0.05), stimulated interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) production (both at p˂0.001), and increased the percentage of CD4+ CD8+ T cells (p˂0.001) among unstimulated leucocytes. After cell stimulation, however, the highest nisin concentration showed antiproliferative activity (p˂0.05), decreased phagocytic functions (p˂0.05) and inhibited the synthesis of IL-6 (time- and concentration-dependent effect). As a typical bacterial product, nisin had a stronger impact on innate immune cells, and its effect on T cells was likely a consequence of the modulation of the activity of antigen-presenting cells. Nisin may be a good candidate as an immunomodulator in pig breeding.

Volatile Molecule Profiles and Anti-Listeria monocytogenes Activity of Nisin Producers Lactococcus lactis Strains in Vegetable Drinks.

This work aimed to evaluate the potential of 15 nisin producing Lactococcus lactis strains, isolated from dairy products, for the fermentation of soymilk and carrot juice. In particular, the acidification and the production of nisin in the food matrices were recorded. Moreover, three strains (LBG2, FBG1P, and 3LC39), that showed the most promising results were further scrutinized for their anti-Listeria monocytogenes activity and volatile molecules profile during fermentation of soymilk and carrot juice. Lactococcus lactis strains LBG2, FBG1P, and 3LC39 resulted the most interesting ones, showing rapid growth and acidification on both food matrices. The higher amounts of nisin were detected in soymilk samples fermented by the strain LBG2 after 24 and 48 h (26.4 mg/L). Furthermore, the rapid acidification combined with the production of nisin resulted in a strong anti-Listeria activity, reducing the pathogen loads below the detection limit, in carrot juice samples fermented by the strains LBG2 and FBG1P and in soymilk by the strain LBG2. The fermentation increased the presence of volatile molecules such as aldehydes and ketones with a positive impact on the organoleptic profile of both the fermented products. These results highlighted the interesting potential of three nisin producing L. lactis strains for the production of fermented carrot juice and soymilk. In fact, the fermentation by lactic acid bacteria, combined or not with other mild technologies, represents a good strategy for the microbiological stabilization of these products. Furthermore, the increase of molecules with a positive sensory impact, such as aldehydes and ketones, in the fermented products suggests a possible improvement of their organoleptic characteristics.

Characterization and application of newly isolated nisin producing Lactococcus lactis strains for control of Listeria monocytogenes growth in fresh cheese

This study was aimed to screen for nisin producing Lactococcus lactis strains with prominent antimicrobial and technological characteristics applicable for the dairy industry. Twelve out of 181 Lactococcus spp. strains isolated from goat and cow milk, fermented wheat and buckwheat samples were identified as L. lactis strains harbouring nisin A, Z or novel nisin variant GLc03 genes. For the first time technological characteristics of L. lactis strains harbouring nisin variant GLc03 were evaluated. All 12 isolated strains showed clear antagonistic activity against tested food spoilage and pathogenic bacteria. Moreover, strains encoding nisin Z presented favorable enzymatic activities of acid phosphatase, esterase lipase and phosphohydrolase. However, strains encoding novel nisin variant GLc03 were lacking appropriate technological characteristics being resistant to tetracycline and producing harmful β-glucosidase, therefore these strains are not applicable for the dairy food production. Two nisin A producing L. lactis strains were poor acidifiers and one strain showed resistance to tetracycline. Three strains were selected according to safety and technological criteria and were examined in fresh cheese production for control of Listeria monocytogenes growth. Listeria numbers were significantly reduced (P < 0.001) in model cheese suggesting the application of these strains for fresh cheese safety improvement.

Use of a nisin-producing Lactococcus lactis strain, combined with natural antimicrobials, to improve the safety and shelf-life of minimally processed sliced apples

The intrinsic characteristics of minimally processed fruit may favor the growth of pathogens and spoilage microorganisms. In this context, the use of natural alternatives to traditional chemical sanitizer agents may represent a useful tool to increase the shelf-life and the safety of minimally processed fruit. The aim of this study was to evaluate the application of the nisin-producing Lactococcus lactis CBM21 as a potential biocontrol agent in sliced apples combined or not with hexanal, 2-(E)-hexenal and citral through microbiological, colorimetric, textural and sensory assessments.The use of L. lactis CBM21 limited the growth of yeasts on apples below 5 log cfu/g during 28 days of storage. Moreover, this strain significantly increased the safety of the products, inhibiting the growth of Listeria monocytogenes, especially when used in combination with the proposed natural antimicrobials. No negative effects on color parameters were observed after 14 days of storage in presence of the natural antimicrobials. Furthermore, the addition of the biocontrol agent was positively perceived by panelists for product flavor and odor.Even if further studies are necessary, these results suggest that the combination of the considered “hurdles” can represent a new strategy to prolong shelf-life and ensure the safety and quality of sliced apples.

NMR resonance assignments of the lantibiotic immunity protein NisI from Lactococcus lactis.

The lantibiotic nisin is a small antimicrobial peptide which acts against a wide range of Gram-positive bacteria. Nisin-producing Lactococcus lactis strains express four genes for self-protection against their own antimicrobial compound. This immunity system consists of the lipoprotein NisI and the ABC transporter NisFEG. NisI is attached to the outside of the cytoplasmic membrane via a covalently linked diacylglycerol anchor. Both the lipoprotein and the ABC transporter are needed for full immunity but the exact immunity mechanism is still unclear. To gain insights into the highly specific immunity mechanism of nisin producing strains on a structural level we present here the backbone resonance assignment of NisI (25.8kDa) as well as the virtually complete (1)H,(15)N,(13)C chemical shift assignments for the isolated 12.7kDaN-terminal and 14.6kDa C-terminal domains of NisI.

A Food-Grade System for Production of Pediocin PA-1 in Nisin-Producing and Non–Nisin-Producing Lactococcus lactis Strains: Application To Inhibit Listeria Growth in a Cheese Model System

Food-grade heterologous production of pediocin PA-1 in nisin-producing and non–nisin-producing Lactococcus lactis strains, previously selected because of their technological properties for cheese making, was achieved. Plasmid pGA1, which contains the complete pediocin operon under the control of the strong P32 promoter and is devoid of any antibiotic marker, was introduced into L. lactis ESI 153 and L. lactis ESI 515 (Nis+). Transformation of L. lactis ESI 515 with pGA1 did not affect its ability to produce nisin. The antimicrobial activity of the pediocin-producing transformants on the survival of Listeria innocua SA1 during cheese ripening was also investigated. Cheeses were manufactured from milk inoculated with 1% of the lactic culture and with or without approximately 4 log CFU/ml of the Listeria strain. L. lactis ESI 153, L. lactis ESI 515, and their transformants (L. lactis GA1 and GA2, respectively) were used as starter cultures. At the end of the ripening period, counts of L. innocua in cheeses made with the bacteriocin-producing lactococcal strains were below 50 CFU/g in the L. lactis GA1 cheeses and below 25 CFU/g in the L. lactis GA2 ones, compared with 3.7 million CFU/g for the controls without nisin or pediocin production.

Function of Lactococcus lactis Nisin Immunity Genes nisI and nisFEG after Coordinated Expression in the Surrogate Host Bacillus subtilis

Nisin-producing Lactococcus lactis strains show a high degree of resistance to the action of nisin, which is based upon expression of the self-protection (immunity) genes nisI, nisF, nisE, and nisG. Different combinations of nisin immunity genes were integrated into the chromosome of a nisin-sensitive Bacillus subtilis host strain under the control of an inducible promoter. For the recipient strain, the highest level of acquired nisin tolerance was achieved after coordinated expression of all four nisin immunity genes. But either the lipoprotein NisI or the ABC transporter-homologous system NisFEG, respectively, were also able to protect the Bacillus host cells. The acquired immunity was specific to nisin and provided no tolerance to subtilin, a closely related lantibiotic. Quantitative in vivo peptide release assays demonstrated that NisFEG diminished the quantity of cell-associated nisin, providing evidence that one role of NisFEG is to transport nisin from the membrane into the extracellular space. NisI solubilized from B. subtilis membrane vesicles and recombinant hexahistidine-tagged NisI from Escherichia coli interacted specifically with nisin and not with subtilin. This suggests a function of NisI as a nisin-intercepting protein.

Fate of enterotoxigenic Staphylococcus aureus in the presence of nisin-producing Lactococcus lactis strain during manufacture of Jben, a Moroccan traditional fresh cheese

Abstract The inhibitory effect of nisin-producing Lactococcus lactis subsp. lactis UL730 on the growth of enterotoxigenic Staphylococcus aureus J10 during manufacture of Jben, a Moroccan traditional fresh cheese prepared from recombined milk, was investigated. With an inoculum level of 10 3 cfu mL −1 , S. aureus was absent in Jben four days after inoculation when the nisin-producing lactococcus was used as lactic starter. In contrast, it survived after that period, when the starter was non-nisin-producing. No staphylococcal thermonuclease was detected in all Jben samples made from milk inoculated with S. aureus at the level of 10 3 cfu mL −1 . With a higher inoculum of 10 5 cfu mL −1 , S. aureus was still present in Jben after manufacture and persisted during the storage of the product for 3 days in the laboratory, even when the starter used was nisin-producing. Staphylococcal thermonuclease and type C enterotoxin were detected in all Jben samples made from milk inoculated with 10 5 cfu mL −1 . Thermonuclease and enterotoxin were already produced in the coagulum, at 24 h after milk inoculation with S. aureus .

Synergistic antimicrobial effect of nisin whey permeate and lactic acid on microbes isolated from fish.

The antimicrobial activity of a combination of lactic acid and whey permeate fermented by a nisin-producing Lactococcus lactis strain was tested by the agar diffusion method using bacteria isolated from fish as test organisms. Lactic acid inhibited all bacterial strains studied, but nisin whey permeate inhibited Gram-positive bacteria only. The combination was more effective than lactic acid alone against Pseudomonas fluorescens and Staphylococcus hominis isolated from fish, and Pseudomonas aeruginosa ATCC9721 and Micrococcus luteus ATCC9341.

The Effect of Lactic Acid, Nisin Whey Permeate, Sodium Chloride and Related Combinations on Aerobic Plate Count and the Sensory Characteristics of Rainbow Trout

The inhibitory effects of 2.5 g/kg lactic acid, 30 g/kg whey permeate, 20 g/kg sodium chloride and their combinations on the total aerobic counts of minced fish were studied. Whey permeate was produced by a nisin-producingLactococcus lactisstrain. The applicability of lactic acid and whey permeate as bio-preservatives was also studied through the use of quantitative sensory profiling techniques. Bacterial growth in minced fish stored at +3 °C for 4, 7 and 10 d was inhibited effectively in samples that contained a combination of lactic acid, sodium chloride and/or whey permeate. Lactic acid enhanced fishy flavour, metallic flavour, astringency and sourness as found in minced fish but whey permeate did not affect any of the characteristics investigated.

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.

A lactococcal expression system for engineered nisins

The nisin-producing Lactococcus lactis strain FI5876 has been modified and developed for use as an expression system for engineered nisin variants. Insertional inactivation of the resident nisA gene had a polar effect on downstream genes, including those involved in nisin immunity. However, subsequent chromosomal rearrangements in this region involving a newly discovered insertion element (IS905) generated a strain that was deficient in the nisA gene product but expressed those nisin determinants necessary for prenisin maturation, secretion, and immunity. Complementation of the lesion in the nisA gene by plasmid-encoded nisA genes containing site-specific mutations resulted in the exclusive production of altered nisins containing specific amino acid substitutions.

Novel paired starter culture system for sauerkraut, consisting of a nisin-resistant Leuconostoc mesenteroides strain and a nisin-producing Lactococcus lactis strain.

Nisin-resistant Leuconostoc mesenteroides NCK293 and nisin-producing Lactococcus lactis subsp. lactis NCK401 were evaluated separately and in combination for growth and nisin production in a model sauerkraut fermentation. Strains were genetically marked and selectively enumerated by using antibiotic-containing media. The growth and survival of L. mesenteroides were similar in the presence and absence of Lactococcus lactis subsp. lactis. The growth of Lactococcus lactis subsp. lactis was not inhibited, although the maximum cell density was reduced and the population decline was more pronounced in the presence of L. mesenteroides. Nisin was detected within 24 h, and levels were relatively constant over the 12-day test period. The maximum cell populations and nisin level achieved could be altered by changing the initial cell ratios of L. mesenteroides and lactococcus lactis subsp. lactis. Isogenic nisin-producing and nisin-negative Lactococcus lactis subsp. lactis derivatives were used in combination with nisin-resistant L. mesenteroides to demonstrate that nisin levels produced in mixed culture were sufficient to retard the onset of the growth of nisin-sensitive, homofermentative Lactobacillus plantarum ATCC 14917.