Bacterial nanocellulose loaded with bromelain and nisin as a promising bioactive material for wound debridement.

The bacterial nanocellulose (BnC) membranes were produced extracellularly by a novel aerobic acetic acid bacterium Komagataeibacter melomenusus. The BnC was modified in situ by adding carboxymethyl cellulose (CMC) into the culture media, obtaining a BnC-CMC product with denser fibril arrangement, improved rehydration ratio and elasticity in comparison to BnC. The proteolytic enzyme bromelain (Br) and antimicrobial peptide nisin (N) were immobilized to BnC matrix by ex situ covalent binding and/or adsorption. The optimal Br immobilization conditions towards the maximized specific proteolytic activity were investigated by response surface methodology as factor variables. At optimal conditions, i.e., 8.8 mg/mL CMC and 10 mg/mL Br, hyperactivation of the enzyme was achieved, leading to the specific proteolytic activity of 2.3 U/mg and immobilization efficiency of 39.1 %. The antimicrobial activity was observed against Gram-positive bacteria (S. epidermidis, S. aureus and E. faecalis) for membranes with immobilized N and was superior when in situ modified BnC membranes were used. N immobilized on the BnC or BnC-CMC membranes was cytocompatible and did not cause changes in normal human dermal fibroblast cell morphology. BnC membranes perform as an efficient carrier for Br or N immobilization, holding promise in wound debridement and providing antimicrobial action against Gram-positive bacteria, respectively.

Effect of Fermented Milk Supplemented with Nisin or Plantaricin Q7 on Inflammatory Factors and Gut Microbiota in Mice.

Lactic-acid-bacteria-derived bacteriocins are used as food biological preservatives widely. Little information is available on the impact of bacteriocin intake with food on gut microbiota in vivo. In this study, the effects of fermented milk supplemented with nisin (FM-nisin) or plantaricin Q7 (FM-Q7) from Lactiplantibacillus plantarum Q7 on inflammatory factors and the gut microbiota of mice were investigated. The results showed that FM-nisin or FM-Q7 up-regulated IFN-γ and down-regulated IL-17 and IL-12 in serum significantly. FM-nisin down-regulated TNF-α and IL-10 while FM-Q7 up-regulated them. The results of 16S rRNA gene sequence analysis suggested that the gut microbiome in mice was changed by FM-nisin or FM-Q7. The Firmicutes/Bacteroides ratio was reduced significantly in both groups. It was observed that the volume of Akkermansia_Muciniphila was significantly reduced whereas those of Lachnospiraceae and Ruminococcaceae were increased. The total number of short-chain fatty acids (SCFAs) in the mouse feces of the FM-nisin group and FM-Q7 group was increased. The content of acetic acid was increased while the butyric acid content was decreased significantly. These findings indicated that FM-nisin or FM-Q7 could stimulate the inflammation response and alter gut microbiota and metabolic components in mice. Further in-depth study is needed to determine the impact of FM-nisin or FM-Q7 on the host's health.

Effects of dietary Nisin on growth performance, immune function, and gut health of broilers challenged by Clostridium perfringens.

Nisin (Ni) is a polypeptide bacteriocin produced by lactic streptococci (probiotics) that can inhibit the majority of gram-positive bacteria, and improve the growth performance of broilers, and exert antioxidative and anti-inflammatory properties. The present study investigated the potential preventive effect of Nisin on necrotic enteritis induced by Clostridium perfringens (Cp) challenge. A total of 288 Arbor Acres broiler chickens of 1-d-olds were allocated using 2 × 2 factorial arrangement into four groups with six replicates (12 chickens per replicate), including: (1) control group (Con, basal diet), (2) Cp challenge group (Cp, basal diet + 1.0 × 108 CFU/mL Cp), (3) Ni group (Ni, basal diet + 100 mg/kg Ni), and (4) Ni + Cp group (Ni + Cp, basal diet + 100 mg/kg Ni + 1.0 × 108 CFU/mL Cp). The results showed that Cp challenge decreased the average daily gain (ADG) of days 15 to 21 (P<0.05) and increased interleukin-6 (IL-6) content in the serum (P < 0.05), as well as a significant reduction in villus height (VH) and the ratio of VH to crypt depth (VCR) (P<0.05) and a significant increase in crypt depth (CD) of jejunum (P<0.05). Furthermore, the mRNA expressions of Occludin and Claudin-1 were downregulated (P<0.05), while the mRNA expressions of Caspase3, Caspase9, Bax, and Bax/Bcl-2 were upregulated (P<0.05) in the jejunum. However, the inclusion of dietary Ni supplementation significantly improved body weight (BW) on days 21 and 28, ADG of days 15 to 21 (P<0.05), decreased CD in the jejunum, and reduced tumor necrosis factor-α (TNF-α) content in the serum (P<0.05). Ni addition upregulated the mRNA levels of Claudin-1 expression and downregulated the mRNA expression levels of Caspase9 in the jejunum (P<0.05). Moreover, Cp challenge and Ni altered the cecal microbiota composition, which manifested that Cp challenge decreased the relative abundance of phylum Fusobacteriota and increased Shannon index (P<0.05) and the trend of phylum Proteobacteria (0.05

Necrotic enteritis (NE), a severe digestive disorder in broiler chickens caused by Clostridium perfringens (Cp), a gram-positive bacterium, is a widespread issue in the global poultry industry, leading to significant economic losses. Nisin (Ni), a polypeptide bacteriocin produced by probiotic lactic streptococci, has been found to enhance daily weight gain and feed intake, while also exhibiting inhibitory effects on gram-positive bacteria and anti-inflammatory properties. In this study, a NE infection model in broilers was established to examine the potential preventive effects of Ni. These results demonstrated that Cp challenge reduced growth performance, caused inflammatory responses and intestinal apoptosis, damaged intestinal morphology and barrier function, and was accompanied by changes in the composition of the gut microbiota. Dietary supplementation with Ni improved growth performance and protected intestine against Cp challenge-induced damage in broilers. As a result, Ni may be a potential safe and effective additive for NE prevention in broiler production.

Combination antimicrobial therapy: in vitro synergistic effect of anti-staphylococcal drug oxacillin with antimicrobial peptide nisin against Staphylococcus epidermidis clinical isolates and Staphylococcus aureus biofilms.

The ability of Staphylococcus epidermidis and S. aureus to form strong biofilm on plastic devices makes them the major pathogens associated with device-related infections (DRIs). Biofilm-embedded bacteria are more resistant to antibiotics, making biofilm infections very difficult to effectively treat. Here, we evaluate the in vitro activities of anti-staphylococcal drug oxacillin and antimicrobial peptide nisin, alone and in combination, against methicillin-resistant S. epidermidis (MRSE) clinical isolates and the methicillin-resistant S. aureus ATCC 43,300. The minimum inhibitory concentrations (MIC) and minimum biofilm eradication concentrations (MBEC) of oxacillin and nisin were determined using the microbroth dilution method. The anti-biofilm activities of oxacillin and nisin, alone or in combination, were evaluated. In addition, the effects of antimicrobial agents on the expression of icaA gene were examined by quantitative real-time PCR. MIC values for oxacillin and nisin ranged 4-8 µg/mL and 64-128 µg/mL, respectively. Oxacillin and nisin reduced biofilm biomass in all bacteria in a dose-dependent manner and this inhibitory effect was enhanced with combinatorial treatment. MBEC ranges for oxacillin and nisin were 2048-8192 µg/mL and 2048-4096 µg/mL, respectively. The addition of nisin significantly decreased the oxacillin MBECs from 8- to 32-fold in all bacteria. At the 1× MIC and 1/2× MIC, both oxacillin and nisin decreased significantly the expression of icaA gene in comparison with untreated control. When two antimicrobial agents were combined at 1/2× MIC concentration, the expression of icaA were significantly lower than when were used alone. Nisin/conventional oxacillin combination showed considerable anti-biofilm effects, including inhibition of biofilm formation, eradication of mature biofilm, and down-regulation of biofilm-related genes, proposing its applications for treating or preventing staphylococcal biofilm-associated infections, including device-related infections.

Combined treatment of pulsed light and nisin-organic acid based antimicrobial wash for inactivation of Escherichia coli O157:H7 in Romaine lettuce, reduction of microbial loads, and retention of quality.

Microbial safety of fresh produce continues to be a major concern. Novel antimicrobial methods are needed to minimize the risk of contamination. This study investigated the antimicrobial efficacy of pulsed light (PL), a novel nisin-organic acid based antimicrobial wash (AW) and the synergy thereof in inactivating E. coli O157:H7 on Romaine lettuce. Treatment effects on background microbiota and produce quality during storage at 4 °C for 7 days was also investigated. A bacterial cocktail containing three outbreak strains of E. coli O157:H7 was used as inoculum. Lettuce leaves were spot inoculated on the surface before treating with PL (1-60 s), AW (2 min) or combinations of PL with AW. PL treatment for 10 s, equivalent to fluence dose of 10.5 J/cm2, was optimal and resulted in 2.3 log CFU/g reduction of E. coli O157:H7, while a 2 min AW treatment, provided a comparable pathogen reduction of 2.2 log CFU/g. Two possible treatment sequences of PL and AW combinations were investigated. For PL-AW combination, inoculated lettuce leaves were initially exposed to optimum PL dose followed by 2 min AW treatment, whereas for AW-PL combination, inoculated lettuce were subjected to 2 min AW treatment prior to 10 s PL treatment. Both combination treatments (PL-AW and AW-PL) resulted in synergistic inactivation as E. coli cells were not detectable after treatment, indicating >5 log pathogen reductions. Combination treatments significantly (P < 0.05) reduced spoilage microbial populations on Romaine lettuce and also hindered their growth in storage for 7 days. The firmness and visual quality appearance of lettuce were not significantly (P > 0.05) influenced due to combination treatments. Overall, the results reveal that PL and AW combination treatments can be implemented as a novel approach to enhance microbial safety, quality and shelf life of Romaine lettuce.

Anticancer activity of cloned Nisin as an alternative therapy for MCF-7 brest cancer cell line.

Despite advancements in treatment and detection, cancer remains one of the most common causes of death worldwide. Conventional chemotherapeutic drugs used to treat cancer have non-specific toxicity toward normal body cells, which leads to several adverse effects. Second, malignancies are known to develop resistance to chemotherapy over time. As a result, the demand for novel anticancer drugs is growing daily. The most frequent type of cancer among women is breast cancer. Utilizing cloned Nisin as an anticancer was the purpose of this study using Gibson cloning and a cell-free peptide synthesis system, then purification of the target protein. The antiproliferative effect of Nisin against a breast cancer MCF-7 cell line was also determined using an MTT assay, and viability in cell lines was measured using acridine orange and propidium iodide. Our findings demonstrate the successful isolation and cloning of the NisA, gene in addition to inducing of peptide synthesis system and then purification of a target protein. MTT assay results indicate that Nisin exhibits a high and selective cytotoxicity against the MCF-7 cell line with an IC50 value of 11.68 µg/ml. This data suggest that the NisA gene had in vitro antiproliferative effect against breast cancer cell. However, more research, including a combination of the NisA gene with other anticancer therapy in clinical use. In addition, in vivo studies are required.

Ex-vivo effects of propolis quantum dots-nisin-nanoquercetin-mediated photodynamic therapy on Streptococcus mutans biofilms and white spot lesions.

BACKGROUND: White spot lesions (WSLs) remain one of the most critical adverse sequelae of fixed orthodontic treatment, despite materials and techniques advances in orthodontics. WSLs seem to be a multi-factorial interaction including increased microbial plaque due to intrabuccal appliances that limit the oral-cleansing mechanism and change in the oral microbiome during fixed appliance wear. The aim of this study was to investigate the synergistic effect of propolis quantum dots (PQD), nisin (Nis), and quercetin nanoparticles (nQCT)-mediated photodynamic therapy (PQD-Nis-nQCT-mediated aPDT) in the eradication of Streptococcus mutans biofilms and the remineralization of WSLs ex-vivo. MATERIALS AND METHODS: The cytotoxicity of PQD-Nis-nQCT composite on human gingival fibroblasts was evaluated using neutral red. Intracellular reactive oxygen species (ROS) generation following PQD-Nis-nQCT-mediated aPDT was measured. Enamel slabs were prepared and demineralized using a demineralization solution containing S. mutans. Demineralized enamel slabs were divided into 9 groups (n = 10) and treated in the following groups: 1) Artificial saliva (negative control), 2) 2% neutral sodium fluoride gel (NSF; positive control or treatment control, 3) PQD, 4) Nis, 5) nQCT, 6) Nis-nQCT, 7) PQD-Nis-nQCT 8) Blue laser irradiation (light), 9) PQD-Nis-nQCT with irradiation (PQD-Nis-nQCT-mediated aPDT). Then, the surface changes, microhardness, and surface topography of the demineralized slabs were examined following each treatment using DIAGNOdent Pen reading, digital hardness tester, and SEM, respectively. After the determination of minimum biofilm eradication concentration (MBEC) of PQD, Nis, and nQCT by microtiter plate assay, the synergistic antimicrobial effects of PQD and Nis-nQCT were determined via evaluation of fractional biofilm eradication concentration (FBEC) index. The anti-biofilm effects of each treatment on S. mutans were assessed using a colorimetric assay. The virulence­associated gtfB gene expression was assessed following PQD-Nis-nQCT-mediated aPDT by quantitative real­time PCR. RESULTS: PQD-Nis-nQCT at 2048 µg/mL had no significant cell cytotoxicity on human gingival fibroblasts compared to the control group (P > 0.05). A significantly increased (7.6 fold) in intracellular ROS was observed following PQD-Nis-nQCT-mediated aPDT (13.9 ± 1.41) when compared to the control (1.83 ± 0.13). Following each treatment, the microhardness of the demineralized enamel surface significantly increased except for the artificial saliva (negative) and blue laser irradiation groups. The highest change in microhardness improvement was detected in the PQD-Nis-nQCT-mediated aPDT group (P < 0.05). Also, DIAGNODent Pen reading revealed the highest significant improved change in the level of mineralization degree in the PQD-Nis-nQCT-mediated aPDT group. Nis and blue light irradiation groups, like the artificial saliva-treated demineralized enamel slabs (control group), did not lead to remineralization (P > 0.05). Also, the PQD-Nis-nQCT-mediated aPDT treatment results obtained from SEM revealed that remineralization of demineralized enamel slabs in that group has significantly improved compared to the others. Light-activated nQCT, PQD, Nis-nQCT, and PQD-Nis-nQCT composite significantly reduced pre-formed biofilms of S. mutans compared with unactivated forms of test materials. The relative expression level of the virulence gtfB gene was significantly decreased (7.53-fold) in the presence of PQD-Nis-nQCT-mediated aPDT (P < 0.05). CONCLUSION: PQD-Nis-nQCT-mediated aPDT can be used for the eradication of S. mutans biofilms and remineralization of WSLs. The found in vitro efficacy should be tested further through clinical studies.

Contribution of antimicrobial photo-sonodynamic therapy in wound healing: an in vivo effect of curcumin-nisin-based poly (L-lactic acid) nanoparticle on Acinetobacter baumannii biofilms.

BACKGROUND: The biofilm-forming ability of Acinetobacter baumannii in the burn wound is clinically problematic due to the development of antibiotic-resistant characteristics, leading to new approaches for treatment being needed. In this study, antimicrobial photo-sonodynamic therapy (aPSDT) was used to assess the anti-biofilm efficacy and wound healing activity in mice with established A. baumannii infections. METHODS: Following synthesis and confirmation of Curcumin-Nisin-based poly (L-lactic acid) nanoparticle (CurNisNp), its cytotoxic and release times were evaluated. After determination of the sub-significant reduction (SSR) doses of CurNisNp, irradiation time of light, and ultrasound intensity against A. baumannii, anti-biofilm activity and the intracellular reactive oxygen species (ROS) generation were evaluated. The antibacterial and anti-virulence effects, as well as, histopathological examination of the burn wound sites of treated mice by CurNisNp-mediated aPSDTSSR were assessed and compared with silver sulfadiazine (SSD) as the standard treatment group. RESULTS: The results showed that non-cytotoxic CurNisNp has a homogeneous surface and a sphere-shaped vesicle with continuous release until the 14th day. The dose-dependent reduction in cell viability of A. baumannii was achieved by increasing the concentrations of CurNisNp, irradiation time of light, and ultrasound intensity. There was a time-dependent reduction in biofilm growth, changes in gene expression, and promotion in wound healing by the acceleration of skin re-epithelialization in mice. Not only there was no significant difference between aPSDTSSR and SSD groups in antibacterial and anti-virulence activities, but also wound healing and re-epithelialization occurred more efficiently in aPSDTSSR than in the SSD group. CONCLUSIONS: In conclusion, CurNisNp-mediated aPSDT might be a promising complementary approach to treat burn wound infections.

Antimicrobial and Preservative Effects of the Combinations of Nisin, Tea Polyphenols, Rosemary Extract, and Chitosan on Pasteurized Chicken Sausage.

ABSTRACT: This study was conducted to evaluate the antimicrobial and preservative effects of the combinations of nisin (NS), tea polyphenols (TP), rosemary extract (RE), and chitosan (CS) on pasteurized chicken sausage. An orthogonal test revealed that the most effective preservative was a mixture of 0.05% NS plus 0.05% TP plus 0.03% RE plus 0.55% CS (weight by sausage weight). This mixture had antimicrobial and antioxidant effects in pasteurized chicken sausage and extended the shelf life to >30 days at 4°C. The inhibitory effects of NS, TP, RE, and CS were also evaluated against Pseudomonas aeruginosa, lactic acid bacteria (LAB), and Staphylococcus aureus, the dominant spoilage and pathogenic bacteria in pasteurized chicken sausage. NS had the greatest inhibitory effect on LAB and S. aureus, with inhibitory zone diameters of 19.7 and 17.8 mm, respectively. TP had the largest inhibitory effect on P. aeruginosa, with a clear zone diameter of 18.2 mm. These results indicate that the combination of NS, TP, RE, and CS could be used as a natural preservative to efficiently inhibit the growth of microorganisms in pasteurized chicken sausage and improve its safety and shelf life.

Metabolic extract of the endophytic fungus Flavodon flavus isolated from Justicia brandegeana in the control of Alicyclobacillus acidoterrestris in commercial orange juice.

In this work, the antibacterial activity of a crude extract of the endophytic fungus Flavodon flavus (JB257), isolated from leaves of Justicia brandegeana, was evaluated against both the vegetative and sporulated forms of Alicyclobacillus acidoterrestris. The microdilution technique was performed in order to determine the antibacterial activity of the crude extract alone as well as in combination with the bacteriocin, nisin. The minimum inhibitory concentration (MIC) of the crude extract and nisin alone against A. acidoterrestris vegetative forms were 250 µg/mL and 31.5 µg/mL, respectively, while the minimum bactericidal concentrations (MBC) were 1000 µg/mL and 62.5 µg/mL,respectively. For A. acidoterrestris spores, treatment with the crude extract at a concentration of 500 µg/mL caused a 47% reduction in growth, while nisin at 62.5 µg/mL could reduce 100% of the growth. The in vitro evaluation of the crude extract combined with nisin against A. acidoterrestris by the Checkerboard method showed a synergistic interaction between the two compounds. In addition, greater selectivity towards bacterial cells over host cells, a human hepatocyte cell line, was achieved when the crude extract was combined with nisin, Using scanning electron microscopy, interferences in the cell membrane of A. acidoterrestris could be observed after treatment with the crude extract. The results presented in this study indicate that the crude extract of the endophyte F. flavus has biotechnological potential in the food industry, especially for the treatment of orange juices through the control of A. acidoterrestris.

Elucidating antimicrobial mechanism of nisin and grape seed extract against Listeria monocytogenes in broth and on shrimp through NMR-based metabolomics approach.

Nisin and grape seed extract (GSE) have been widely used as food preservatives; however, the mechanism against pathogens at molecular level has not been well elucidated. This work aimed to investigate their antimicrobial effect against Listeria monocytogenes and to elucidate the mechanism by NMR-based metabolomics. Nisin exhibited enhanced in vitro antilisterial effect when combined with GSE (4.49 log CFU/mL reduction). Marked change in cell membrane permeability was observed in the combination group using confocal laser scanning microscopy; this was verified by increased leakage of protein and nucleic acid. The underlying antimicrobial mechanism was revealed by NMR coupled with multivariate analysis. Significant decreases in threonine, cysteine, ATP, NADP, adenine were observed, whereas a few of metabolites such as lactic acid and γ-aminobutyric acid (GABA) increased after nisin-GSE treatment (P < 0.05). Pathway analysis further manifested that the nisin-GSE inhibited the survival of L. monocytogenes by blocking the TCA cycle, amino acid biosynthesis and energy-producing pathway. Lastly, nisin and GSE were applied to shrimp and binary combination showed remarkably antilisterial activity (1.79 log CFU/g reduction). GABA shunt and protein degradation from shrimp compensated the unbalanced glycolysis and amino acid metabolism by providing energy and carbon source for L. monocytogenes inoculated on shrimp. Thus, they were more tolerant to nisin and GSE stresses as compared to the broth-grown culture.

Nisin-Based Organic Acid Inactivation of Salmonella on Grape Tomatoes: Efficacy of Treatment with Bioluminescence ATP Assay.

The antimicrobial activity of a new nisin-based organic acid sanitizer (NOAS), developed in our laboratory, was tested against viable aerobic mesophilic bacteria and Salmonella populations inoculated on produce surfaces. The activity of NOAS was compared with 200 ppm of chlorinated wash water and a bioluminescence ATP technique to determine the efficacy of treatments compared with plate count methods. The activity of the 10% final concentration of NOAS against viable populations of 109 CFU/mL Salmonella in phosphate-buffered saline (PBS), sterile deionized distilled water, and buffered peptone water was tested in vitro and on grape tomatoes inoculated with Salmonella at 2.5 log CFU/g. A similar batch of inoculated tomatoes were treated with 200 ppm of total available chlorinated water. All treatments for inactivation of viable Salmonella in vitro was tested up to 30 min and 5 min for the attached populations on tomatoes. Inactivation of viable Salmonella at 109 log CFU/mL by 10% the NOAS solution averaged >107 log CFU/mL in PBS, sterile deionized distilled water, and buffered peptone water. Similarly, Salmonella bacteria inactivated on tomato surfaces by the NOAS solution was significantly (P < 0.05) greater than numbers on chlorinated washed tomatoes, and surviving bacterial populations on NOAS and chlorine-treated tomatoes were <1 and 4 CFU/g, respectively. A significant linear correlation coefficient (r2 = 0.99) between the bioluminescence ATP assay and aerobic plate counts of inoculated and untreated grape tomatoes were recorded but not with NOAS and chlorine-treated tomatoes, as bacterial populations were less than the minimum baseline for determination. Also, the results indicated that the NOAS solution is a better alternative antimicrobial wash solution than 200 ppm of chlorinated water.

Combination of natural antimicrobials for contamination control in ethanol production.

Presence of bacterial contaminants at levels > 107 colony forming units per milliliter (CFU/mL) during ethanol production processes reduces the alcoholic fermentation yield by 30%. Antibiotics are currently used to control contamination, but their residues may be detected in yeast extract, restricting this by-product trade to several countries. Thus, the objective of this study was to assess antimicrobial activity of the natural compounds hops extract, 4-hydroxybenzoic acid, nisin Z, and lysozyme against Lactobacillus fermentum, Leuconostoc mesenteroides, and Saccharomyces cerevisiae, aiming development of a formula. Minimum Inhibitory Concentration of each antimicrobial was determined for bacteria and subsequently, nisin (30 mg/L) and hops extract (5 mg/L) were tested together, showing inhibitory effects combining doses of each antimicrobial that were equivalent to an eightfold reduction of their original Minimum Inhibitory Concentrations (3.75 and 0.625 mg/L, respectively), resulting in a FICIndex of 0.25. Thereon, a formula containing both compounds was developed and tested in fermentation assays, promoting reductions on bacterial population and no severe interferences in yeast viability or population even at extreme doses. Therefore, these compounds have great potential to successfully substitute conventional antibiotics in the ethanol industry.

Antibacterial activity against porcine respiratory bacterial pathogens and in vitro biocompatibility of essential oils.

Bacterial respiratory infections affecting pigs such as pneumonia, pleuropneumonia, and pleurisy, are a major health concern in the swine industry and are associated with important economic losses. This study aimed to investigate the antibacterial activities of essential oils against major swine respiratory pathogens with a view to developing a potential alternative to antibiotics. Their synergistic interactions with the bacteriocin nisin was also examined. Lastly, we assessed the in vitro biocompatibility of the most efficient essential oils using a pig tracheal epithelial cell line. Of the nine essential oils tested, those from cinnamon, thyme, and winter savory were the most active against Streptococcus suis, Actinobacillus pleuropneumoniae, Actinobacillus suis, Bordetella bronchiseptica, Haemophilus parasuis, and Pasteurella multocida, with minimum inhibitory concentrations and minimum bactericidal concentrations ranging from 0.01 to 0.156% (v/v). The main component found in cinnamon, thyme, and winter savory oils were cinnamaldehyde, thymol, and carvacrol, respectively. Treating pre-formed S. suis and A. pleuropneumoniae biofilms with thyme or winter savory oils significantly decreased biofilm viability. We also observed a synergistic growth inhibition of S. suis with mixtures of nisin and essential oils from thyme and winter savory. Concentrations of nisin and cinnamon, thyme and winter savory essential oils that were effective against bacterial pathogens had no effect on the viability of pig tracheal epithelial cells. The present study brought evidence that essential oils are potential antimicrobial agents against bacteria associated with porcine respiratory infections.

The inhibitory effect of nisin on Mycobacterium avium ssp. paratuberculosis and its effect on mycobacterial cell wall.

Infection with Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) is a widespread problem in the United States and worldwide, and it constitutes a significant health problem for dairy animals with a potential effect on human health. Mycobacterium paratuberculosis is easily transmitted through consumption of contaminated milk; therefore, finding safe methods to reduce the mycobacterial load in milk and other dairy products is important to the dairy industry. The main objective of the current study was to investigate the effect of natural products, such as bacteriocins designated as "generally regarded as safe" (GRAS), on the survival of M. paratuberculosis in milk. Commercially synthesized bacteriocin (nisin) was used to examine its effect on the survival of laboratory and field isolates of M. paratuberculosis and in contaminated milk. Surprisingly, nisin had a higher minimum inhibitory concentration (MIC) against the laboratory strain (M. paratuberculosis K10), at 500 U/mL, than against field isolates (e.g., M. paratuberculosis 4B and JTC 1281), at 15 U/mL. In milk, growth of M. paratuberculosis was inhibited after treatment with levels of nisin that are permissible in human food at 4°C and 37°C. Using both fluorescent and scanning electron microscopy, we were able to identify defects in the bacterial cell walls of treated cultures. Our analysis indicated that nisin reduced membrane integrity by forming pores in the mycobacterial cell wall, thereby decreasing survival of M. paratuberculosis. Thus, nisin treatment of milk could be implemented as a control measure to reduce M. paratuberculosis secreted in milk from infected herds. Nisin could also be used to reduce M. paratuberculosis in colostrum given to calves from infected animals, improving biosecurity control in dairy herds affected by Johne's disease.

Potential of two delivery systems for nisin topical application to dental plaque biofilms in dogs.

BACKGROUND: Periodontal disease (PD) is caused by the development of a microbial biofilm (dental plaque) in the periodontium, affecting approximately 80% of dogs. Several bacterial species present in the canine oral cavity can be implicated in the development of this disease, including Enterococcus spp. To decrease antibiotic administration, a possible control strategy for dog's enterococcal PD may involve the use of the antimicrobial peptide (AMP) nisin. Nisin's inhibitory activity was evaluated against a collection of previously characterized enterococci obtained from the oral cavity of dogs with PD (n = 20), as well as the potential of a guar-gum gel and a veterinary toothpaste as topical delivery systems for this AMP. The Minimum Inhibitory (MIC) and Bactericidal Concentrations (MBC) and the Minimum Biofilm Eradication (MBEC) and Inhibitory Concentrations (MBIC) were determined for nisin and for the supplemented guar-gum gel. For the supplemented veterinary toothpaste an agar-well diffusion assay was used to evaluate its inhibitory potential. RESULTS: Nisin was effective against all isolates. Independently of being or not incorporated in the guar-gum gel, its inhibitory activity on biofilms was higher, with MBIC (12.46 ± 5.16 and 13.60 ± 4.31 µg/mL, respectively) and MBEC values (21.87 ± 11.33 and 42.34 ± 16.61 µg/mL) being lower than MIC (24.61 ± 4.64 and 14.90 ± 4.10 µg/mL) and MBC (63.09 ± 13.22 and 66.63 ± 19.55 µg/mL) values. The supplemented toothpaste was also effective, showing inhibitory activity against 95% of the isolates. CONCLUSIONS: The inhibitory ability of nisin when incorporated in the two delivery systems was maintained or increased, demonstrating the potential of these supplemented vehicles to be applied to PD control in dogs.

Cardioprotective Effects of Curcumin-Nisin Based Poly Lactic Acid Nanoparticle on Myocardial Infarction in Guinea Pigs.

Myocardial infarction (MI) is the most prevalent cause of cardiovascular death. A possible way of preventing MI maybe by dietary supplements. The present study was thus designed to ascertain the cardio-protective effect of a formulated curcumin and nisin based poly lactic acid nanoparticle (CurNisNp) on isoproterenol (ISO) induced MI in guinea pigs. Animals were pretreated for 7 days as follows; Groups A and B animals were given 0.5 mL/kg of normal saline, group C metoprolol (2 mg/kg), groups D and E CurNisNp 10 and 21 mg/kg respectively (n = 5). MI was induced on the 7th day in groups B-E animals. On the 9th day electrocardiogram (ECG) was recorded, blood samples and tissue biopsies were collected for analyses. Toxicity studies on CurNisNp were carried out. MI induction caused atrial fibrillation which was prevented by pretreatment of metoprolol or CurNisNp. MI induction was also associated with increased expressions of cardiac troponin I (CTnI) and kidney injury molecule-1 (KIM-1) which were significantly reduced in guinea pig's pretreated with metoprolol or CurNisNp (P < 0.05). The LC50 of CurNisNp was 3258.2 µg/mL. This study demonstrated that the formulated curcumin-nisin based nanoparticle confers a significant level of cardio-protection in the guinea pig and is nontoxic.

Reverse micelles as nanocarriers of nisin against foodborne pathogens.

Reverse micelles (RMs) as nanocarriers of nisin were optimized for the highest water and bacteriocin content. RMs formulated with either refined olive oil or sunflower oil, distilled monoglycerides, ethanol, and water were effectively designed. Structural characterization of the RMs was assessed using Electron Paramagnetic Resonance Spectroscopy and Small Angle X-ray Scattering in the presence and absence of nisin. No conformational changes occurred in the presence of nisin for the nanocarriers. To assess efficacy of the loaded systems, their antimicrobial activity against Staphylococcus aureus and Listeria monocytogenes was tested in lettuce leaves and minced meat, respectively. Antimicrobial activity was evident in both cases. Interestingly, a synergistic antimicrobial effect was observed in lettuce leaves and to a lesser extent in minced meat between nisin and some of the nanocarriers' constituents (probably ethanol). Our findings suggest complex interactions that take place when RMs are applied in different food matrices.

Effects of tartary buckwheat polysaccharide combined with nisin edible coating on the storage quality of tilapia (Oreochromis niloticus) fillets.

BACKGROUND: To investigate the effect of tartary buckwheat polysaccharide (TBP) combined with nisin edible coatings on the preservation of tilapia (Oreochromis niloticus) fillets, fillets were dip treated with different concentrations of TBP (5, 10 and 15 g kg-1 ) combined with nisin and stored at 4 °C for 12 days. The pH values, thiobarbituric acid contents, total volatile base nitrogen (TVB-N) content, total viable count (TVC), surface colors, textures and sensory properties of the tilapia fillets at storage were all periodically investigated. RESULTS: TBP combined with nisin-treated groups significantly improved the bacteriological, physicochemical, and sensory characteristics of the tilapia fillets to a greater extent compared to the control group and presented better quality preservation effects than nisin coating alone. Based on the limits of the TVB-N, TVC and sensory scores, the shelf life of the control tilapia fillets was 4 days, whereas that for nisin with TBP-coated fillets was 8-10 days. CONCLUSION: Edible coatings made from TBP combined with nisin are suitable for maintaining qualities and enhancing the shelf lives of tilapia fillets stored at 4 °C. © 2017 Society of Chemical Industry.

Nisin-induced expression of recombinant T cell epitopes of major Japanese cedar pollen allergens in Lactococcus lactis.

Japanese cedar pollinosis is a seasonal allergic disease caused by two major pollen allergens: Cry j 1 and Cry j 2 antigens. To develop an oral vaccine to treat pollinosis, we constructed recombinant Lactococcus lactis harboring the gene encoding fused T cell epitopes from the Cry j 1 and Cry j 2 antigens. The recombinant T cell epitope peptide was designed to contain the fused cholera toxin B subunit as an adjuvant and a FLAG tag at the C-terminus. An expression plasmid was constructed by inserting the T cell epitope peptide gene into the multiple cloning sites of plasmid pNZ8148, an Escherichia coli-L. lactis shuttle vector. The constructed plasmid was transformed into L. lactis NZ9000 for expression induced by nisin, an antibacterial peptide from L. lactis. The expression of the epitope peptide was induced with 10-40 ng/mL nisin, and the expressed T cell epitope peptide was detected by western blot analysis using an anti-FLAG antibody and an antibody against the T cell epitopes. The concentration of the epitope peptide was estimated to be ~ 22 mg/L of culture in the presence of 40 ng/mL nisin, although it varied depending on the nisin concentration, the culture time, and the bacterial concentration when nisin was added. The expression of the recombinant epitope peptide in L. lactis, an organism generally recognized as safe, as demonstrated in this study, may contribute to the development of an oral vaccine for the treatment of pollinosis.