RESUMEN
The ability of Listeria monocytogenes grow on ready-to-eat food is a major concern in food safety. Natural antimicrobials, such as nisin, can be used to control this pathogen, but the increasing reports of nisin tolerance and resistance make necessary novel approaches to increase its effectiveness, such as encapsulation. The goal of this study was to investigate how L. monocytogenes ATCC7644 regulates and shapes its proteome in response to sublethal doses of nisin and nisin-loaded phosphatidylcholine liposomes (lipo-nisin), compared to untreated cells growing under optimal conditions. Total proteins were extracted from L. monocytogenes cells treated for 1 h with free and lipo-nisin. As result, of 803 proteins that were initially identified, 64 and 53 proteins were differentially upregulated and downregulated respectively, in the treatments with nisin and lipo-nisin. Changes of Listeria proteome in response to treatments containing nisin were mainly related to ATP-binding cassette (ABC) transporter systems, transmembrane proteins, RNA-binding proteins and diverse stress response proteins. Some of the proteins uniquely detected in samples treated with free nisin were the membrane proteins SecD, Lmo1539 and the YfhO enzyme, which are related to translocation of L. monocytogenes virulence factors, activation of the LiaR-mediated stress defense and glycosylation of wall teichoic acid, respectively. The L. monocytogenes treated with liposome encapsulated nisin showed no expression of some stress response factors as compared with the free nisin, suggesting a reduction of stress mediated response and production of nisin-resistance factors by exposure to encapsulated nisin.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Listeria monocytogenes/efectos de los fármacos , Nisina/farmacología , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Composición de Medicamentos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Nisina/química , ProteómicaRESUMEN
The objective of this work was to investigate the effect of the addition of carboxymethyl chitosan on the structural properties and antilisterial activity of nisin-incorporated chitosan films. Chitosan and carboxymethyl chitosan solutions were prepared with different mass ratios and bacteriocin nisin was added (0, 1000 and 6000 IU/ml). Filmogenic solutions were cast, dried and their physico-chemical and antimicrobial properties were investigated. For the same chitosan/carboxymethyl chitosan mass ratio, the addition of NIS at 6000 IU/ml led to changes in the macro- and microstructure, as well as in physico-chemical properties of films. On the other hand, carboxymethyl chitosan had a plasticizing effect and enhanced the distribution of the bacteriocin within the biopolymer matrix. Moreover, nisin-incorporated blend films of chitosan and carboxymethyl chitosan were more effective against Listeria monocytogenes than their pure chitosan counterparts. This study showed that different formulations of nisin-incorporated composite films of chitosan and carboxymethyl chitosan may provide options for developing bioactive packaging to improve food safety.
Asunto(s)
Antibacterianos , Quitosano/análogos & derivados , Conservantes de Alimentos , Listeria monocytogenes/efectos de los fármacos , Nisina , Antibacterianos/química , Antibacterianos/farmacología , Quitosano/química , Quitosano/farmacología , Microbiología de Alimentos , Embalaje de Alimentos , Conservación de Alimentos , Conservantes de Alimentos/química , Conservantes de Alimentos/farmacología , Nisina/química , Nisina/farmacologíaRESUMEN
The purpose of this study was to select the promising biopreservation bacteriocin producer strain from goat milk and characterize the expressed bacteriocin, related to its physiological and biochemical properties and specificity of operon encoding production and expression of antimicrobial peptide. Brazilian goat milk was used as the source for the selection of bacteriocin-producing lactic acid bacteria. One strain (DF105Mi) stood out for its strong activity against several Listeria monocytogenes strains. Selected strain was identified based on the biochemical and physiological characteristics and 16s rRNA analysis. The bacteriocin production and inhibitory spectrum of strain DF105Mi were studied, together with the evaluation of the effect of temperature, pH, and chemicals on bacteriocin stability and production, activity, and adsorption to target cells as well as to the cell surface of bacteriocin producers. Physiological and bio-molecular analyses based on targeting of different genes, parts of nisin operon were performed in order to investigate the hypothesis that the studied strain can produce and express nisin. Based on biochemical, physiological, and 16s rRNA analysis, the strain DF105Mi was classified as Enterococcus hirae. The selected strain produces a bacteriocin which is stable in a wide range of pH (2.0-12.0), temperature (up to 120 °C), presence of selected chemicals and presents adsorption affinity to different test organisms, process influenced by environmental conditions. Higher bacteriocin production by Ent. hirae DF105Mi was recorded during stationary growth phase, but only when the strain was cultured at 37 °C. The strain's genetic analysis indicated presence of the genes coding for the production of the bacteriocin nisin. This result was confirmed by cross-checking the sensitivity of the produced strain to commercial nisin A. The strong anti-Listeria activity, bacteriocin adsorption, and stability of produced bacteriocin indicate that Ent. hirae DF105Mi presents a differentiated potential application for biopreservation of fermented dairy products.
Asunto(s)
Enterococcus hirae/aislamiento & purificación , Enterococcus hirae/metabolismo , Leche/microbiología , Nisina/metabolismo , Animales , Brasil , Enterococcus hirae/clasificación , Enterococcus hirae/genética , Cabras , Concentración de Iones de Hidrógeno , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/crecimiento & desarrollo , Nisina/química , Nisina/farmacologíaRESUMEN
Interactions between the antibiotic peptide nisin and multilamellar vesicles of phosphoglycerol lipids in different phase-states were studied using vibrational spectroscopy. The infrared amide I' band of nisin, both in solution and in the membrane-bound state, was analyzed in the temperature range comprised between 20 and 60⯰C in order to study its conformational behavior. Nisin presented mainly unordered and ß-turns conformations. Their relative populations varied according to the environment and as the temperature increased: ß turns were more favored in the membrane-bound state than in solution, but at higher temperatures the disordered conformation was dominant in both states. Spectral changes of specific infrared bands belonging to the hydrocarbon and polar moieties of lipids were also analyzed to evaluate the perturbation of the lipid membrane order. Nisin interactions with the membrane polar region induced a high restriction to water incorporation, promoting a small increase in the temperature of the lipid phase transition. Raman spectra of nisin/phosphoglycerol systems at ambient temperature were also analyzed. They revealed that the peptide incorporation to a membrane in the fluid phase caused drastic structural modifications in the hydrophobic region of the bilayer. Although nisin may be able to disrupt the hydrophobic portion of the bilayer in the gel phase, the most of the peptide molecule remained at the membrane surface interacting with the polar headgroups. This work provides evidence of a differential effect of nisin on anionic membranes, depending on the phase-state of the lipid.
Asunto(s)
Membrana Celular/química , Membrana Celular/metabolismo , Nisina/química , Nisina/metabolismo , Espectrometría Raman/métodos , Aniones , Modelos Biológicos , VibraciónRESUMEN
Nisin-loaded amaranth protein isolate:pullulan (API:PUL) nanofibers were prepared by the electrospinning method. The nisin release kinetic was evaluated at pHâ¯3.4 and 6.1 and the antimicrobial effectiveness of the electrospun mats was evaluated in apple juice and fresh cheese. The nisin API:PUL fibers with 120â¯nm average diameter reached 81.49% and 43.85% nisin release after 12â¯h at pHâ¯3.4 and 6.1, respectively. The encapsulation of nisin in electrospun fibers allowed complete bactericidal activity against Salmonella Typhimurium, L. monocytogenes and L. mesenteroides inoculated in apple juice after 48, 20 and 48â¯h, respectively. When nisin API:PUL fibers were applied to fresh cheese, microorganism inactivation was complete after 142, 120 and 170â¯h, respectively. The results demonstrated that nisin API:PUL electrospun fibers significantly reduce the bacterial population and can be used in food products for microbiological safety.
Asunto(s)
Queso , Microbiología de Alimentos/métodos , Jugos de Frutas y Vegetales , Listeria monocytogenes/efectos de los fármacos , Nisina/farmacología , Salmonella typhimurium/efectos de los fármacos , Antibacterianos/farmacología , Queso/microbiología , Queso/normas , Jugos de Frutas y Vegetales/microbiología , Jugos de Frutas y Vegetales/normas , Glucanos/química , Glucanos/farmacología , Malus/microbiología , Nanofibras/química , Nisina/químicaRESUMEN
Films and coatings based on natural polymers have gained increased interest for food packaging applications. In this work, halloysite and phosphatidylcholine liposomes encapsulating nisin were used to develop nanocomposite films of gelatin and casein. Liposomes prepared with either soybean lecithin or Phospholipon(®) showed particle size ranging from 124 to 178nm and high entrapment efficiency (94-100%). Considering their stability, Phospholipon(®) liposomes with 1.0mg/ml nisin were selected for incorporation into nanocomposite films containing 0.5g/l halloysite. The films presented antimicrobial activity against Listeria monocytogenes, Clostridium perfringens and Bacillus cereus. Scanning electron microscopy revealed that the films had a smooth surface, but showed increased roughness with addition of liposomes and halloysite. Casein films were thinner and slightly yellowish, less rigid and very elastic as compared with gelatin films. Thermogravimetric analysis showed a decrease of the degradation temperature for casein films added with liposomes. The glass transition temperature decreased with addition of liposomes and halloysite. Gelatin and casein films containing nisin-loaded liposomes and halloysite represent an interesting alternative for development of active food packaging.
Asunto(s)
Silicatos de Aluminio/química , Liposomas/química , Nanocompuestos/química , Nisina/química , Caseínas/química , Arcilla , Embalaje de Alimentos , Gelatina/química , Membranas ArtificialesRESUMEN
The aim of this work was the development and characterization of nisin-loaded nanoparticles and the evaluation of its potential antifungal activity. Candidiasis is a fungal infection caused by Candida sp. considered as one of the major public health problem currently. The discovery of antifungal agents that present a reduced or null resistance of Candida sp. and the development of more efficient drug release mechanisms are necessary for the improvement of candidiasis treatment. Nisin, a bacteriocin commercially available for more than 50 years, exhibits antibacterial action in food products with potential antifungal activity. Among several alternatives used to modulate antifungal activity of bacteriocins, polymeric nanoparticles have received great attention due to an effective drug release control and reduction of therapeutic dose, besides the minimization of adverse effects by the preferential accumulation in specific tissues. The nisin nanoparticles were prepared by double emulsification and solvent evaporation methods. Nanoparticles were characterized by dynamic light scattering, zeta potential, Fourier transform infrared, X-ray diffraction, differential scanning calorimetry, and scanning electron microscopy. Antifungal activity was accessed by pour plate method and cell counting using Candida albicans strains. The in vitro release profile and in vitro permeation studies were performed using dialysis bag method and pig vaginal mucosa in Franz diffusion cell, respectively. The results revealed nisin nanoparticles (300 nm) with spherical shape and high loading efficiency (93.88 ± 3.26%). In vitro test results suggest a promising application of these nanosystems as a prophylactic agent in recurrent vulvovaginal candidiasis and other gynecological diseases.
Asunto(s)
Antifúngicos/administración & dosificación , Antifúngicos/química , Candidiasis/tratamiento farmacológico , Nanopartículas/administración & dosificación , Nanopartículas/química , Nisina/administración & dosificación , Nisina/química , Animales , Rastreo Diferencial de Calorimetría/métodos , Candida albicans/efectos de los fármacos , Candidiasis/microbiología , Femenino , Tamaño de la Partícula , Polímeros/química , Porcinos , Vagina/microbiología , Difracción de Rayos X/métodosRESUMEN
Heat processing is the most commonly used hurdle for inactivating microorganisms in fruit juices. However, this preservation method could interfere with the organoleptic characteristics of the product. Alternative methods have been proposed and bacteriocins such as nisin are potential candidates. However, the approval of bacteriocins as food additives is limited, especially in foods from vegetal origin. We aimed to verify the stability, the effect on physico-chemical properties, and the antimicrobial activity of nisin in different fruit juices. Nisin remained stable in fruit juices (cashew, soursop, peach, mango, passion fruit, orange, guava, and cupuassu) for at least 30 days at room or refrigerated temperature and did not cause any significant alterations in the physico-chemical characteristics of the juices. Besides, nisin favored the preservation of vitamin C content in juices. The antimicrobial activity of nisin was tested against Alicyclobacillus acidoterrestris, Bacillus cereus, Staphylococcus aureus and Listeria monocytogenes in cashew, soursop, peach, and mango juices. Nisin caused a 4-log reduction in viable cells of A. acidoterrestris in soursop, peach, and mango juices after 8h of incubation, and no viable cells were detected in cashew juices. After 24h of incubation in the presence of nisin, no viable cells were detected, independently of the juices. To S. aureus, at 24h of incubation in the presence of nisin, viable cells were only detected in mango juices, representing a 4-log decrease as compared with the control treatment. The number of viable cells of B. cereus at 24h of incubation in the presence of nisin represented at least a 4-log decrease compared to the control treatment. When the antimicrobial activity of nisin was tested against L. monocytogenes in cashew and soursop juices, no reduction in the viable cell number was observed compared to the control treatment after 24h of incubation. Viable cells were four and six times less than in the control treatment, in peach and mango juices respectively. The most sensitive microorganism to nisin was A. acidoterrestris and the least sensitive was L. monocytogenes. Still, a reduction of up to 90% of viable cells was observed in peach and mango juices inoculated with L. monocytogenes. These results indicate that the use of nisin could be an alternative in fruit juice processing.
Asunto(s)
Antibacterianos/química , Conservantes de Alimentos/química , Jugos de Frutas y Vegetales/análisis , Nisina/química , Alicyclobacillus/efectos de los fármacos , Antibacterianos/farmacología , Bacillus cereus/efectos de los fármacos , Citrus sinensis/química , Conservantes de Alimentos/farmacología , Jugos de Frutas y Vegetales/microbiología , Listeria monocytogenes/efectos de los fármacos , Mangifera/química , Nisina/farmacología , Staphylococcus aureus/efectos de los fármacosRESUMEN
Three different nanoclays (bentonite, octadecylamine-modified montmorillonite and halloysite) were studied as potential carriers for the antimicrobial peptides nisin and pediocin. Adsorption occurred from peptide solutions in contact with nanoclays at room temperature. Higher adsorption of nisin and pediocin was obtained on bentonite. The antimicrobial activity of the resultant bacteriocin-nanoclay systems was analyzed using skimmed milk agar as food simulant and the largest inhibition zones were observed against Gram-positive bacteria for halloysite samples. Bacteriocins were intercalated into the interlayer space of montmorillonites as deduced from the increase of the basal spacing measured by X-ray diffraction (XRD) assay. Infrared spectroscopy suggested non-electrostatic interactions, such as hydrogen bonding between siloxane groups from clays and peptide molecules. Transmission electron microscopy did not show any alteration in morphologies after adsorption of antimicrobial peptides on bentonite and halloysite. These results indicate that nanoclays, especially halloysite, are suitable nanocarriers for nisin and pediocin adsorption.
Asunto(s)
Silicatos de Aluminio/química , Antiinfecciosos/química , Bacteriocinas/química , Nanopartículas/química , Nisina/química , Adsorción , Antiinfecciosos/farmacología , Bacteriocinas/farmacología , Arcilla , Portadores de Fármacos , Bacterias Grampositivas/efectos de los fármacos , SolucionesRESUMEN
The use of poly(lactic-co-glycolic acid) (PLGA) matrix as a biomolecule carrier has been receiving great attention due to its potential therapeutic application. In this context, we investigated the PLGA matrix capacity to incorporate nisin, an antimicrobial peptide capable of inhibiting the growth of Gram-positive bacteria and bacterial spores germination. Nisin-incorporated PLGA matrices were evaluated based on the inhibitory effect against the nisin-bioindicator Lactobacillus sakei. Additionally, the PLGA-nisin matrix stability over an 8-months period was investigated, as well as the nisin release profile. For the incorporation conditions, we observed that a 5 h incorporation time, at 30 °C, with 250 µg/mL nisin solution in PBS buffer pH 4.5, resulted in the highest inhibitory activity of 2.70 logAU/mL. The PLGA-nisin matrix was found to be relatively stable and showed sustained drug delivery, with continuous release of nisin for 2 weeks. Therefore, PLGA-nisin matrix is could be used as a novel antimicrobial delivery system and an alternative to antibiotics incorporated into PLGA matrices.
Asunto(s)
Antibacterianos/química , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/instrumentación , Ácido Láctico/química , Nisina/química , Ácido Poliglicólico/química , Antibacterianos/farmacología , Lactobacillus/efectos de los fármacos , Nanopartículas/química , Nisina/farmacología , Tamaño de la Partícula , Copolímero de Ácido Poliláctico-Ácido PoliglicólicoRESUMEN
In this paper, films based on tapioca starch and containing nisin, natamycin and glycerol were characterized in relation to their physicochemical properties, roughness and hydrophobicity. The content of glycerol affected the mechanical properties of the films studied and the roughness and it was observed an increase in WVP with the increase in glycerol content. The addition of antimicrobials affected the mechanical properties, being nisin the one that produced the greater decrease in the Young modulus. The color was highly affected by the joint presence of natamycin and nisin, which increased the yellow index. The contact angle increased with antimicrobial addition indicating a decrease in hydrophilicity. Nisin also affected the roughness of the films. Water vapor permeability was slightly reduced by the presence of natamycin. It was observed that water vapor permeability and contact angle were correlated with the roughness of the films.
Asunto(s)
Glicerol/química , Manihot/metabolismo , Natamicina/química , Nisina/química , Almidón/química , Antiinfecciosos/química , Antiinfecciosos/farmacología , Bacterias/efectos de los fármacos , Módulo de Elasticidad , Interacciones Hidrofóbicas e Hidrofílicas , Ensayo de Materiales , Permeabilidad , Agua/química , HumectabilidadRESUMEN
Antilisterial efficiency of three bacteriocins, viz, Nisin, Pediocin 34 and Enterocin FH99 was tested individually and in combination against Listeria mononcytogenes ATCC 53135. A greater antibacterial effect was observed when the bacteriocins were combined in pairs, indicating that the use of more than one LAB bacteriocin in combination have a higher antibacterial action than when used individually. Variants of Listeria monocytogenes ATCC 53135 resistant to Nisin, Pediocin 34 and Enterocin FH99 were developed. Bacteriocin cross-resistance of wild type and their corresponding resistant variants were assessed and results showed that resistance to a bacteriocin may extend to other bacteriocins within the same class. Resistance to Pediocin 34 conferred cross resistance to Enterocin FH 99 but not to Nisin. Similarly resistance to Enterocin FH99 conferred cross resistance to Pediocin 34 but not to Nisin. Also, the sensitivity of Nisin, Pediocin 34 and Enterocin FH99 resistant variants of Listeria monocytogenes to low pH, salt, sodium nitrite, and potassium sorbate was assayed in broth and compared to the parental wild-type strain. The Nisin, Pediocin 34 and Enterocin FH99 resistant variants did not have intrinsic resistance to low pH, sodium chloride, potassium sorbate, or sodium nitrite. In no case were the bacteriocin resistant Listeria monocytogenes variants examined were more resistant to inhibitors than the parental strains.(AU)
Asunto(s)
Animales , Antibacterianos/análisis , Nisina/química , Bacteriocinas/análisisRESUMEN
The effectiveness of free and nanovesicle-encapsulated nisin to control Listeria monocytogenes in Minas Frescal cheese was investigated. Commercial nisin was encapsulated into liposomes of partially purified soy lecithin. Free (0.1 mg/mL and 0.25 mg/mL) and nanovesicle-encapsulated nisin (0.25 mg/mL) were applied onto the surface of cheese samples, and L. monocytogenes was inoculated before incubation at 6-8°C for 28 days. A bactericidal effect was observed with 0.25 mg/mL free nisin; a bacteriostatic effect was observed for liposome-encapsulated nisin and 0.1 mg/mL free nisin. Free nisin was more efficient than nisin-loaded liposomes in controlling L. monocytogenes. Possible reasons for this behavior, and also the significance of nisin to soft cheeses are discussed. Nisin acted as a suitable barrier within hurdle technology, potentially extending the shelf-life and safety of fresh cheeses.(AU)
Asunto(s)
Animales , Listeria , Nisina/químicaRESUMEN
Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria, nisin being the most well-known member. Nisin inhibits peptidoglycan synthesis and forms pores at sensitive membranes upon interaction with lipid II, the essential bacterial cell wall precursor. Bovicin HC5, a bacteriocin produced by Streptococcus bovis HC5, has the putative N-terminal lipid II binding motif, and we investigated the mode of action of bovicin HC5 using both living bacteria and model membranes, with special emphasis on the role of lipid II. Bovicin HC5 showed activity against Staphylococcus cohnii and Staphylococcus warneri, but bovicin HC5 hardly interfered with the membrane potential of S. cohnii. In model membranes, bovicin HC5 was not able to cause carboxyfluorescein release or proton influx from DOPC vesicles containing lipid II. Bovicin HC5 blocked lipid II-dependent pore formation activity of nisin, and a high-affinity interaction with lipid II was observed (apparent binding constant [K(a)] = 3.1 × 10(6) M(-1)), with a 1:1 stoichiometry. In DOPC vesicles containing lipid II, bovicin HC5 was able to assemble with lipid II into a prepore-like structure. Furthermore, we observed pore formation activity of bovicin HC5, which was stimulated by the presence of lipid II, in thin membranes. Moreover, bovicin HC5 induced the segregation of lipid II into domains in giant model membrane vesicles. In conclusion, bovicin HC5 has a primary mode of action similar to that of nisin, but some differences regarding the pore-forming capacity were demonstrated.