Nisin-producing Lactococcus Lactis Research Articles

Combination approach of paired starter culture and lactic acid on inhibiting autochthonous lactic acid bacteria for extending kimchi shelf life

Excessive kimchi fermentation by lactic acid bacteria (LAB) during distribution and storage may negatively influence the organoleptic properties of kimchi. Thus, this study aimed to extend kimchi shelf life by inhibiting the growth of autochthonous lactic acid bacteria by applying paired starter culture, consisting of nisin-producing Lactococcus lactis WiKim0098 and nisin-resistant Leuconostoc citreum WiKim0096, and lactic acid. To maximize nisin production in the L. lactis WiKim0098 culture medium, the food grade medium composition and culture conditions were optimized using response surface methodology. Leu. citreum WiKim0096 exhibited resistance to nisin owing to high positive surface charge and was successfully used as a paired starter. In the kimchi application experiments, paired starter culture addition delayed the over-acidification (more than 1% as acidity) of kimchi for approximately 5–6 days, and when combined with lactic acid, delayed the ripening for more than 8 days compared to control kimchi fermented at 10 °C. The results of bacterial community and metabolite analysis via principal component analysis showed that paired starter culture and lactic acid inhibited the LAB growth at the early stage of kimchi fermentation. Our results suggest that combining paired starter culture with lactic acid can effectively extend the kimchi shelf life. Specifically, it is expected to be effectively applicable to kimchi products intended for export, which demand extended shelf life.

Extending shelf life of fresh noodles with nisin producer Lactococcus lactis subsp. lactis fermented sourdough and characteristic changes during storage

In this study, the effect of sourdough fermented with nisin producer Lactococcus lactis subsp. lactis CGMCC1.2030 on the shelf life and quality characteristics of fresh noodles fortified with it was investigated. The shelf life of fresh noodles with sourdough increased from 12 days to 22 days compare to the blank control during storage at 4 °C. Slight antimould activity was observed in sourdough fortified fresh noodles, and the brightened color of these noodles improved their appearance, which related to a decrease in the b* value of the noodle sheet from 12.35 to 8.47 over the storage period. Furthermore, the hardness and chewiness of sourdough-fortified fresh noodles decreased by 25.96% and 18.57%, respectively, which was lower than the 35.59% and 36.50% reduction observed in the blank control after the whole storage period. Confocal laser scanning microscopy (CLSM) analysis revealed less aggregated and more continuous gluten networks, which in combination with mitigated water migration might result in the delayed deterioration of texture quality of sourdough-fortified fresh noodles.

Nisin probiotic prevents inflammatory bone loss while promoting reparative proliferation and a healthy microbiome

Dysbiosis of the oral microbiome mediates chronic periodontal disease. Realignment of microbial dysbiosis towards health may prevent disease. Treatment with antibiotics and probiotics can modulate the microbial, immunological, and clinical landscape of periodontal disease with some success. Antibacterial peptides or bacteriocins, such as nisin, and a nisin-producing probiotic, Lactococcus lactis, have not been examined in this context, yet warrant examination because of their biomedical benefits in eradicating biofilms and pathogenic bacteria, modulating immune mechanisms, and their safety profile in humans. This study’s goal was to examine the potential for nisin and a nisin-producing probiotic to abrogate periodontal bone loss, the host inflammatory response, and changes in oral microbiome composition in a polymicrobial mouse model of periodontal disease. Nisin and a nisin-producing Lactococcus lactis probiotic significantly decreased the levels of several periodontal pathogens, alveolar bone loss, and the oral and systemic inflammatory host response. Surprisingly, nisin and/or the nisin-producing L. lactis probiotic enhanced the population of fibroblasts and osteoblasts despite the polymicrobial infection. Nisin mediated human periodontal ligament cell proliferation dose-dependently by increasing the proliferation marker, Ki-67. Nisin and probiotic treatment significantly shifted the oral microbiome towards the healthy control state; health was associated with Proteobacteria, whereas 3 retroviruses were associated with disease. Disease-associated microbial species were correlated with IL-6 levels. Nisin or nisin-producing probiotic’s ability to shift the oral microbiome towards health, mitigate periodontal destruction and the host immune response, and promote a novel proliferative phenotype in reparative connective tissue cells, addresses key aspects of the pathogenesis of periodontal disease and reveals a new biomedical application for nisin in treatment of periodontitis and reparative medicine.

Combination of High-Pressure Treatment at 500 MPa and Biopreservation with a Lactococcus lactis Strain for Lowering the Bacterial Growth during Storage of Diced Cooked Ham with Reduced Nitrite Salt.

We investigated the combined effects of biopreservation and high-pressure treatment on bacterial communities of diced cooked ham prepared with diminished nitrite salt. First, bacterial communities of four commercial brands of diced cooked ham from local supermarkets were characterized and stored frozen. Second, sterile diced cooked ham, prepared with reduced levels of nitrite, was inoculated with two different microbiota collected from the aforementioned commercial samples together with a nisin-producing Lactococcus lactis protective strain able to recover from a 500 MPa high-pressure treatment. Samples were then treated at 500 MPa for 5 min, and bacterial dynamics were monitored during storage at 8 °C. Depending on samples, the ham microbiota was dominated by different Proteobacteria (Pseudomonas, Serratia, Psychrobacter, or Vibrio) or by Firmicutes (Latilactobacillus and Leuconostoc). Applied alone, none of the treatments stabilized during the growth of the ham microbiota. Nevertheless, the combination of biopreservation and high-pressure treatment was efficient in reducing the growth of Proteobacteria spoilage species. However, this effect was dependent on the nature of the initial microbiota, showing that the use of biopreservation and high-pressure treatment, as an alternative to nitrite reduction for ensuring cooked ham microbial safety, merits attention but still requires improvement.

Impact of Nisin and Nisin-Producing Lactococcus lactis ssp. lactis on Clostridium tyrobutyricum and Bacterial Ecosystem of Cheese Matrices.

Clostridium tyrobutyricum spores survive milk pasteurization and cause late blowing of cheeses and significant economic loss. The effectiveness of nisin-producing Lactococcus lactis ssp. lactis 32 as a protective strain for control the C. tyrobutyricum growth in Cheddar cheese slurry was compared to that of encapsulated nisin-A. The encapsulated nisin was more effective, with 1.0 log10 reductions of viable spores after one week at 30 °C and 4 °C. Spores were not detected for three weeks at 4 °C in cheese slurry made with 1.3% salt, or during week 2 with 2% salt. Gas production was observed after one week at 30 °C only in the control slurry made with 1.3% salt. In slurry made with the protective strain, the reduction in C. tyrobutyricum count was 0.6 log10 in the second week at 4 °C with both salt concentration. At 4 °C, nisin production started in week 2 and reached 97 µg/g after four weeks. Metabarcoding analysis targeting the sequencing of 16S rRNA revealed that the genus Lactococcus dominated for four weeks at 4 °C. In cheese slurry made with 2% salt, the relative abundance of the genus Clostridium decreased significantly in the presence of nisin or the protective strain. The results indicated that both strategies are able to control the growth of Clostridium development in Cheddar cheese slurries.

The impact of cell-free supernatants of Lactococcus lactis subsp. lactis strains on the tyramine formation of Lactobacillus and Lactiplantibacillus strains isolated from cheese and beer

Tyramine is one of the most toxic biogenic amines and it is produced commonly by lactic acid bacteria in fermented food products. In present study, we investigated the influence of selected nisin-producing Lactococcus lactis subsp. lactis strains and their cell-free supernatants (CFSs) on tyramine production by four Lactobacillus and two Lactiplantibacillus strains isolated from cheese and beer. Firstly, we examined the antimicrobial effect of the CFSs from twelve Lactococcus strains against tested tyramine producers by agar-well diffusion assay. Six Lactococcus strains whose CFSs showed the highest antimicrobial effect on tyramine producers were further studied. Secondly, we investigated the influence of the selected six Lactococcus strains and their respective CFSs on tyramine production by tested Lactobacillus and Lactiplantibacillus strains in MRS broth supplemented with 2 g.L−1 of l-tyrosine. Tyramine production was monitored by HPLC-UV. The tyramine formation of all tested Lactobacillus and Lactiplantibacillus strains was not detected in the presence of Lc. lactis subsp. lactis CCDM 71 and CCDM 702, and their CFSs. Moreover, the remainder of the investigated Lactococcus strains (CCDM 670, CCDM 686, CCDM 689 and CCDM 731) and their CFSs decreased tyramine production significantly (P < 0.05) – even suppressing it completely in some cases – in four of the six tested tyramine producing strains.

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.

Broadening the antimicrobial spectrum of nisin-producing Lactococcus lactis subsp. Lactis to Gram-negative bacteria by means of active packaging

Cast films obtained from polyvinyl alcohol (PVOH) blended with casein hydrolysates (HCas) in a weight ratio of 1:1 were employed to carry nisin-producing L. lactis and phytic acid in order to broaden the antimicrobial spectrum of L. lactis to Gram-positive and Gram-negative spoilage and pathogen bacteria. For this purpose, the effect of the antimicrobial activity of various film formulations and combinations of films on the growth of E. coli at 37 °C for 24 h was studied. The film system that showed antimicrobial activity against Gram-negative bacteria consisted of phytic acid and L. lactis incorporated in separate films. When the active agents were in the same film the viability of L. lactis decreased considerably and it did not exert antimicrobial activity against the bacterium. Therefore, the combination of L. lactis and phytic acid in separate films was chosen as the reliable system, and the effect of its activity on the growth of Gram-negative bacteria (E. coli, Salmonella enterica, and Pseudomonas fluorescens) and Gram-positive bacteria (Listeria monocytogenes) in liquid culture medium was tested at refrigeration temperature (4 °C), and with simulated breaks in the cold chain (14 °C and 24 °C). The survival of L. lactis in coexistence with these bacteria was also studied. The film system exerted an antimicrobial effect against the Gram-negative bacteria tested, and the activity depended on the bacteria and the temperature assayed. With regard to the antimicrobial activity against L. monocytogenes, phytic acid improved the antimicrobial capacity of L.lactis. The survival of L. lactis was maintained at 7–8 log (CFU/mL) culture in liquid medium throughout the storage period.The films developed were intended to be used as coatings in the design of a double-sided active bag for a non-fermented dairy product. The bags were filled with homemade preservative-free pastry cream, and the microbiological shelf life and evolution of pH of the packaged ready-to-eat food stored at 4 °C was studied for 20 days. The results showed a reduction in the growth of spoilage bacteria and therefore an increase in the shelf life of the packaged product. The films developed could be applied in the design of packages for perishable dairy foods in order to increase their microbiological shelf life.

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.

Effect of Salmonella Enterica Serovar Enteritidison Nisin Gene Expression Induced by Lactococcuslactis on Chicken Meat

Contaminated chicken meat is measured as one of the most significant sources of Salmonella enterica serovar Enteritidis (Ses E). Using nisin-producer Lactococcuslactis (L. lactis) is a practical approach to diminish the risk of food-borne pathogens in chicken meat. The existingsurvey was done to assess the effect of SesEon nisin gene expression induced by L. lactison chicken meat. Chicken meat was divided into 4 groups of L. lactis control group (BHI media inoculated with 1.5×108 CFU/ml L. lactis), Ses Econtrol group (BHI media inoculated with 1.5×105 CFU/ml Ses E), L. lactis exposure group (BHI media inoculated with 1.5×108 CFU/ml L. lactis) and finally Ses Eexposure group (BHI media inoculated with 1.5×105 CFU/ml Ses E). Then, pH and protein contents, numbers of bacteria and nisin gene expression were examined. Growth of Ses Ein the exposure group has been decreased. L. lactis inhibits from severe changes in pH and protein contents of chicken meat. After 4 h of maintenance, amount of nisin gene expression in L. lactis subsp. lactis co-culture with SesE was 1.9 times higher than L. lactis subsp. lactis mono-culture (P =0.0008). After 48 h maintenance, amount of nisin gene expression in L. lactis subsp. lactis mono-culture was 1.1 times higher than L. lactis subsp. lactis co-culture with Ses E (P =0.042). Ses Ecan cause substantial increase in the nisin gene expression and subsequent production of nisin by L. lactis subsp. lactis.

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.

Effect of Lactococcus lactis expressing phage endolysin on the late blowing defect of cheese caused by Clostridium tyrobutyricum

Clostridium tyrobutyricum has been identified as a major species associated with the late blowing defect (LBD) of semi-hard and hard cheeses, due to undesirable butyric acid fermentation. To find new strategies to control this spoilage bacterium, we investigated the delivery of a bacteriophage endolysin by a cheese starter culture. The nisin producer Lactococcus lactis subsp. lactis INIA 415 was engineered to produce the CTP1L endolysin, encoded by the virulent bacteriophage ΦCTP1 of C. tyrobutyricum and with a demonstrated lytic activity in vitro, to the cheese matrix. The presence of the nisRK two-component regulatory system in the host strain allowed constitutive expression of the endolysin under the control of the nisA promoter (PnisA), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media. Engineered lysins with a second cell wall binding domain were also tested and shown to have improved lytic activity. Transformation of L. lactis subsp. lactis INIA 415 with endolysin delivery plasmids had a detrimental effect on its ability to produce nisin in milk, but did not affect its acidifying capacity. Transformed L. lactis subsp. lactis INIA 415 were evaluated as starters in cheeses contaminated with spores of C. tyrobutyricum. Evolution of microbiological parameters, pH and dry matter of cheeses were studied, and Clostridium metabolism and LBD in cheeses were monitored by sensory and instrumental analyses during ripening. Cheese made with the parental strain L. lactis subsp. lactis INIA 415 delayed LBD by one month, attributable to the activity of the nisin, but it was not sufficient to arrest the growth of C. tyrobutyricum during ripening completely. The use of the endolysin-producing strains in cheese manufacture as single cultures also delayed the appearance of LBD by one month, attributable to the activity of the endolysin produced in situ during ripening, because nisin activity in these cheeses was very low at day 1 and undetectable from 15 days onwards. Endolysin was more effective than nisin in inhibiting Clostridium growth, since cheeses made with the CTP1L or the chimeric derivative producers only as starters showed lower LBD symptoms, higher lactic acid levels and lower concentrations of propionic and butyric acids (associated with off-flavours) than cheese made with the parental strain. Investigation of different promoters to maximise endolysin production may help to implement CTP1L as a tool to control C. tyrobutyricum by L. lactis cheese starter and reduce LBD even further.

Multi-Functional Potential of Presumptive Lactic Acid Bacteria Isolated from Chihuahua Cheese.

The multifunctional properties of autochthonous lactic acid bacteria can be of use for enhancing the sensorial properties of food, as well as in food preservation. An initial screening for antimicrobial, proteolytic, and lipolytic capacities was done in 214 presumptive lactic acid bacteria isolates obtained from Chihuahua cheese manufacturing and during a ripening period of nine months. The antimicrobial screening was done by spot-on-the-lawn tests, using Listeria monocytogenes and Escherichia coli as indicator microorganisms; proteolysis was tested in casein-peptone agar and lipolysis in Mann–Rogosa–Sharpe (MRS)-tributyrin agar. More than 90% of the isolates hydrolyzed the casein, but only 30% hydrolyzed tributyrin; the inhibition of L. monocytogenes in the spot-on-the-lawn assay was used to select 39 isolates that had a bigger inhibition zone (>11.15 mm ± 0.3) than the control (Nisin producer Lactococcus lactis BS-10 Chr Hansen). The selected isolates were grown in MRS to obtain the neutralized cell-free supernatants and verify their antimicrobial activity by agar diffusion and the percentage of growth inhibition techniques. The selected isolates were also growth in casein peptone broth, and the cell-free supernatants were used for the determination of antioxidant activity by the radical scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) techniques. The results were analyzed to identify similarities by cluster analysis, based on their antimicrobial and antioxidant capacities. The isolates were arranged into six clusters; one cluster that included 12 isolates demonstrated L. monocytogenes (784–2811 mm2/mL AU by agar diffusion assay) and E. coli (41%–47% growth inhibition) antimicrobial activity. The isolates clustered in these groups also showed competitive inhibition of both radicals (11%–19% of DPPH and 50%–60% of ABTS). The isolates from cluster one were also identified by 16S rDNA amplification and were identified as Enterococcus faecium. Traditional products such as Chihuahua cheese can be a source or lactic acid bacteria with metabolic properties that can be used in food preparation and preservation.

The impact of l-lanthionine supplementation on the production of nisin by lactococci

The combination of cost-effective synthesis of l-lanthionine in conjunction with a fermentation process could be considered as an original approach. Innovative biosynthesis of nisin is easy to scale-up. After the selective organic synthesis of the thioether building block, cultivation medium M17 broth with glucose (0.5% w/w) was supplemented with l-lanthionine (0.78, 3.91 and 7.82 mg ml−1) to stimulate nisin biosynthesis by lactococci. The viability of nisin-producing Lactococcus lactis subsp. lactis strains (CCDM 731, NIZO R5, LTM 32, CCDM 416) and nisin production were evaluated. Nisin synthesis was significantly influenced by l-lanthionine supplementation and it increased for all the strains tested in comparison with a control. The most significant increase in nisin concentration was observed for strains CCDM 731 and LTM 32, where up to 88.6% and 90.0%, respectively, were achieved. The influence of l-lanthionine concentration was shown to be strain specific.

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.

Nisin-producing Lactococcus lactis subsp. lactis 2MT isolated from freshwater Nile tilapia in Cameroon: Bacteriocin screening, characterization, and optimization in a low-cost medium

This work was aimed to isolate bacteriocin-producing lactic acid bacteria (LAB) from freshwater fish in Cameroon, characterize the bacteriocin and investigate its production as well as growth optimization in low-cost media. From the 144 isolates obtained, two showed the ability to produce bacteriocin-like inhibitory substances. Isolate 2 MT, selected for its high bacteriocin production capacity was identified as Lactococcus lactis subsp. lactis using 16S rDNA sequencing. The bacteriocin expressed high stability to heat, pH and chemical agents. Lactococcus lactis subsp. lactis 2 MT harbored the gene for the production of nisin Z, and the bacteriocin molecular mass was estimated at 4.10 kDa. This bacteriocin also exhibited a wide range of antimicrobial activity against spoilage and pathogenic Gram-positive and Gram-negative bacteria. The fish processing byproducts-based formulated medium SP1M showed the significantly highest bacteriocin production capacity. Using Doehlert design, a sugar cane molasses concentration of 42.72 g/l, a temperature of 28.40 °C, and an incubation time of 17.05 h were recorded as the optimum conditions for bacteriocin production. The optimization led to a 4.5-fold increase in nisin, compared with the non-optimized conditions, and a 9-fold increase when compared with non-optimized MRS broth.