Development and antimicrobial activity of composite edible films of chitosan and nisin incorporated with perilla essential oil-glycerol monolaurate emulsions.

Aquatic products are highly susceptible to spoilage, and preparing composite edible film with essential oil is an effective solution. In this study, composite edible films were prepared using perilla essential oil (PEO)-glycerol monolaurate emulsions incorporated with chitosan and nisin, and the film formulation was optimized by response surface methodology. These films were applied to ready-to-eat fish balls and evaluated over a period of 12 days. The films with the highest inhibition rate against Staphylococcus aureus were acquired using a polymer composition of 6 µL/mL PEO, 18.4 µg/mL glycerol monolaurate, 14.2 mg/mL chitosan, and 11.0 µg/mL nisin. The fish balls coated with the optimal edible film showed minimal changes in appearance during storage and significantly reduced total bacterial counts and total volatile basic nitrogen compared to the control groups. This work indicated that the composite edible films containing essential oils possess ideal properties as antimicrobial packaging materials for aquatic foods.

Impact of a teat disinfectant based on Lactococcus cremoris on the cow milk proteome.

BACKGROUND: Dairy cow milking practices require cleaning and disinfection of the teat skin before and after milking to ensure the safety and quality of milk and prevent intramammary infections. Antimicrobial proteins of natural origin can be valuable alternatives to traditional disinfectants. In a recent field trial, we demonstrated that a teat dip based on a nisin A-producing Lactococcus cremoris (L) had comparable efficacy to conventional iodophor dip (C) in preventing dairy cow mastitis. Here, we present the differential shotgun proteomics investigation of the milk collected during the trial. METHODS: Four groups of quarter milk samples with low (LSCC) and high somatic cell count (HSCC) collected at the beginning (T0) and end (TF) of the trial were analyzed for a total of 28 LSCC (14 LSCC T0 and 14 LSCC TF) and 12 HSCC (6 HSCC T0 and 6 HSCC TF) samples. Milk proteins were digested into peptides, separated by nanoHPLC, and analyzed by tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Tribrid mass spectrometer. The proteins were identified with MaxQuant and interaction networks of the differential proteins were investigated with STRING. The proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD045030. RESULTS: In healthy milk (LSCC), we detected 90 and 80 differential proteins at T0 and TF, respectively. At TF, the Lactococcus group showed higher levels of antimicrobial proteins. In mastitis milk (HSCC), we detected 88 and 106 differential proteins at T0 and TF, respectively. In the Lactococcus group, 14 proteins with antimicrobial and immune defense functions were enriched at TF vs. 4 proteins at T0. Cathelicidins were among the most relevant enriched proteins. Western immunoblotting validation confirmed the differential abundance. CONCLUSIONS: At T0, the proteomic differences observed in healthy milk of the two groups were most likely dependent on physiological variation. On the other hand, antimicrobial and immune defense functions were higher in the milk of cows with mammary gland inflammation of the Lactococcus-treated group. Among other factors, the immunostimulatory action of nisin A might be considered as a contributor.

Nisin A elevates adenosine to achieve anti-inflammatory activity.

Inflammation is a ubiquitous physiological status that exists during the occurrence, development and prognosis of numerous diseases. Clinical anti-inflammatory drugs mainly include antibiotics, antivirals, non-steroids and corticosteroids, and the treatments are often accompanied by side effects, including nausea, abdominal pain, allergy, nerve injury and organ dysfunction. Current studies have focused on continuously exploring efficient anti-inflammatory natural components with high biosafety, while nisin, a natural bioactive anti-microbial peptide produced by Lactococcus, has been reported to have anti-inflammatory activity via its superior anti-bacterial abilities. Several recent studies have focused on the potent direct anti-inflammation of nisin, whereas its effects and the corresponding mechanism still remain unclear. The cellular and Caenorhabditis elegans (C. elegans) models were constructed in this study to evaluate the anti-inflammatory effects of nisin A both in vitro and in vivo, while the inflammatory mechanism was further uncovered based on omics analysis. This study reveals the direct anti-inflammatory activity of nisin A and elucidates the regulatory actions of nisin A on adenosine, followed by alteration of the sphingolipid signaling pathway and purine metabolism, enhancing the deep understanding of nisin A with its anti-inflammatory capacity, providing new ideas for future nisin A-based anti-inflammatory strategies.

Nymphaea alba leaf powder effectiveness in removing nisin from fermentation broth using docking and experimental analysis.

The accumulation of nisin in the fermentation medium can reduce the process's productivity. This research studied the potential of Nymphaea alba leaf powder (NALP) as a hydrophobic biosorbent for efficient in-situ nisin adsorption from the fermentation medium by docking and experimental analysis. Molecular docking analysis showed that di-galloyl ellagic acid, a phytochemical compound found in N. alba, had the highest affinity towards nisin. Enhancements in nisin adsorption were seen following pre-treatment of NAPL with HCl and MgCl2. A logistic growth model was employed to evaluate the growth dynamics of the biosorption capacity, offering valuable insights for process scalability. Furthermore, optimization through Response Surface Methodology elucidated optimal nisin desorption conditions by Liebig's law of the minimum, which posits that the scarcest resource governs production efficiency. Fourier Transform Infrared (FTIR) spectroscopy pinpointed vital functional groups involved in biosorption. Scanning electron microscopy revealed the changing physical characteristics of the biosorbent after exposure to nisin. The findings designate NALP as a feasible adsorbent for nisin removal from the fermentation broth, thus facilitating its application in the purification of other biotechnological products based on growth and production optimization principles.

Comparative assessment of antibacterial activity of Matricaria chamomilla L. extract, nisin and of its combination against Alicyclobacillus spp.

Alicyclobacillus spp. is a potential spoiling agent of acidic products and citrus drinks, leading to sensory alterations in contaminated products and consequent economic losses. Treatments such as pasteurization eliminate vegetative cells, but also create a favorable atmosphere for spore germination. To guarantee quality and safety, the application of natural substances as bioconservatives is a considerable and promising alternative for the food industry. This study evaluated the effect of hexane extract of Matricaria chamomilla L. (HE), Nisin (N) and their combination (HE + N). These compounds are present in some studies describing their antibacterial action, but no studies were found on the association of these compounds against the species Alicyclobacillus spp. This study aimed to analyze the antioxidant activity (AA) for the DPPH• (0,23 µmol Trolox/mg) and ABTS (27.93 µmol Trolox/mg), the Checkboard test revealed synergism between HE and N with a fractional inhibitory index (FIC) of 0.068., and to study the antibacterial and sporicidal effect. The antibacterial and sporicidal activity was satisfactory against Alicyclobacillus acidoterrestris with MIC and MBC of 1.95 µg/mL and MSC of 7.81 µg/mL in analyzes using HE + N. The application in orange juice proved to be effective, with an MBC of 0.007 µg/mL. The MIC results served as a parameter for other tests carried out in this study, such as flow cytometry and Scanning Electron Microscopy (SEM), and for the evaluation of sensory characteristics with Electronic Nose (E-nose).

Synthesis and Characterization of a Novel Chitosan-Based Nanoparticle-Hydrogel Composite System Promising for Skin Wound Drug Delivery.

As a natural preservative, nisin is widely used in the food industry, while its application in biomedicine is limited due to its susceptibility to interference from external conditions. In this study, a nanoparticle-hydrogel composite system was designed to encapsulate and release nisin. Nisin nanoparticles were identified with a smooth, spherical visual morphology, particle size of 122.72 ± 4.88 nm, polydispersity coefficient of 0.473 ± 0.063, and zeta potential of 23.89 ± 0.37 mV. Based on the sample state and critical properties, three temperature-sensitive hydrogels based on chitosan were ultimately chosen with a rapid gelation time of 112 s, outstanding reticular structure, and optimal swelling ratio of 239.05 ± 7.15%. The composite system exhibited the same antibacterial properties as nisin, demonstrated by the composite system's inhibition zone diameter of 17.06 ± 0.83 mm, compared to 20.20 ± 0.58 mm for nisin, which was attributed to the prolonged release effect of the hydrogel at the appropriate temperature. The composite system also demonstrated good biocompatibility and safety, making it suitable for application as short-term wound dressings in biomedicine due to its low hemolysis rate of less than 2%. In summary, our nanoparticle-based hydrogel composite system offers a novel application form of nisin while ensuring its stability, thereby deepening and broadening the employment of nisin.

Nisin-relevant antimicrobial peptides: synthesis strategies and applications.

Small pentacyclic peptides, represented by nisin, have been successfully utilized as preservatives in the food industry and have evolved into a paradigm for understanding the genetic structure, expression, and control of genes created by lantibiotics. Due to the ever-increasing antibiotic resistance, nisin-relevant antimicrobial peptides have received much attention, which calls for a summarization of their synthesis, modification and applications. In this review, we first provided a timeline of select highlights in nisin biosynthesis and engineering. Then, we outlined the current developments in nisin synthesis. We also provided an overview of the engineering, screening, and production of nisin-relevant antimicrobial peptides based on enzyme alteration, substrate modification, and sequence mining. Furthermore, an updated summary of applications of nisin-relevant antimicrobial peptides has been developed for food applications. Finally, this study offers insights into emerging technologies, limitations and the future development of nisin-relevant antimicrobial peptides for pathogen inhibition, food preservatives, and improved health.

An Engineered Nisin Analogue with a Hydrophobic Moiety Attached at Position 17 Selectively Inhibits Enterococcus faecium Strains.

Antibiotic resistance is one of the most challenging global public health concerns. It results from the misuse and overuse of broad-spectrum antibiotics, which enhance the dissemination of resistance across diverse bacterial species. Antibiotics like nisin and teixobactin do not target an essential protein and employ a dual mode of action antibacterial mechanism, thereby being less prone to induce resistance. There is a need for the development of a potent narrow-spectrum dual-mode-acting antibiotic against human pathogens. Using nisin, a lantibiotic with potent antimicrobial activity against many pathogens, as a template, the unnatural amino acid azidohomoalanine was introduced at selected positions and subsequently modified using click chemistry with 14 alkyne-moiety containing tails. A novel nisin variant, compound 47, featuring a benzyl group-containing tail, exhibited potent activity against various (drug-resistant) E. faecium strains with an MIC value (3.8 mg/L) similar to nisin, whereas its activity toward other pathogens like Staphylococcus aureus and Bacillus cereus was significantly reduced. Like nisin, the mode of action of compound 47 results from the inhibition of cell wall synthesis by binding to lipid II and nisin-lipid II hybrid-pore formation in the outer membrane. The resistance of compound 47 against proteolytic degradation is markedly enhanced compared to nisin. Like nisin, compound 47 was hardly hemolytic even at a very high dose. Collectively, a modified nisin variant is presented with significantly enhanced target organism specificity and stability.

Multi-functional pH-sensing/antioxidant/antibacterial bioaerogels with long-term activity of loaded anthocyanin for the smart packaging of food.

Anthocyanins (ATH), which are plant pigments with potential health benefits, possess antioxidant and natural indicator properties. However, their inherent instability poses a hurdle for practical applications in the food industry. In the present study, we addressed this challenge by encapsulating ATHs in nisin/gelatin (GA)/pullulan (PUL) bioaerogels through freeze-drying. The results showed that the ATH + nisin@GA/PUL bioaerogels exhibited antibacterial activity against S. aureus and E. coli, and pH-responsiveness to the increase in biogenic amines during the spoilage of shrimp, indicating their potential as a freshness indicator. The bioaerogels also displayed sustained antioxidant effects after two months of storage at room temperature. In summary, the ATH + nisin@GA/PUL bioaerogel serves as a stable matrix for preserving the antioxidant activity of ATHs, and facilitates the indication of freshness in perishable foods. This innovative encapsulation technique represents an advancement in the utilization of ATHs in food packaging.

A nisin-inducible chromosomal gene expression system based on ICE Tn5253 of Streptococcus pneumoniae, transferable among streptococci and enterococci.

The present work reports the development and validation of a chromosomal expression system in Streptococcus pneumoniae which permits gene expression under the control of Lactococcus lactis lantibiotic nisin. The system is based on the integrative and conjugative element (ICE) Tn5253 of S. pneumoniae capable of site-specific chromosomal integration and conjugal transfer to a variety of bacterial species. We constructed an insertion vector that integrates in Tn5251, an ICE contained in Tn5253, which carries the tetracycline resistance tet(M) gene. The vector contains the nisRK regulatory system operon, the L. lactis nisin inducible promoter PnisA upstream of a multiple cloning site for target DNA insertion, and is flanked by two DNA regions of Tn5251 which drive homologous recombination in ICE Tn5253. For system evaluation, the emm6.1::ha1 fusion gene was cloned and integrated into the chromosome of the Tn5253-carrying pneumococcal strain FR24 by transformation. This gene encodes a fusion protein containing the signal peptide, the 122 N-terminal and the 140 C-terminal aa of the Streptococcus pyogenes M6 surface protein joined to the HA1 subunit of the influenza virus A hemagglutinin. Quantitative RT-PCR analysis carried out on total RNA purified from nisin treated and untreated cultures showed an increase in emm6.1::ha1 transcript copy number with growing nisin concentration. The expression of M6-HA1 protein was detected by Western blot and quantified by Dot blot, while Flow cytometry analysis confirmed the presence on the pneumococcal surface. Recombinant ICE Tn5253::[nisRK]-[emm6.1::ha1] containing the nisin-inducible expression system was successfully transferred by conjugation in different streptococcal species including Streptococcus gordonii, S. pyogenes, Streptococcus agalactiae and Enterococcus faecalis. As for S. pneumoniae, the emm6.1::ha1 transcript copy number and the amount of M6-HA1 protein produced correlated with the nisin concentration used for induction in all investigated bacterial hosts. We demonstrated that this host-vector expression system is stably integrated as a single copy within the bacterial chromosome, is transferable to both transformable and non transformable bacterial species, and allows fine tuning of protein expression modulated by nisin concentration. These characteristics make our system suitable for a wide range of applications including complementation assays, physiological studies, host-pathogen interaction studies.

Heterologous expression and purification of the phage lysin-like bacteriocin LysL from Lactococcus lactis LAC460.

The wild-type Lactococcus lactis strain LAC460 produces two bacteriocin-like phage lysins, LysL and LysP. This study aimed to produce and secrete LysL in various heterologous hosts and an in vitro cell-free expression system for further functional studies. Initially, the lysL gene from L. lactis LAC460 was cloned into Lactococcus cremoris NZ9000 and L. lactis N8 strains, with and without the usp45 signal sequence (SSusp45), under a nisin-inducible promoter. Active LysL was primarily produced intracellularly in recombinant L. lactis N8, with some secretion into the supernatant. Recombinant L. cremoris NZ9000 lysed upon nisin induction, indicating successful lysL expression. However, fusion with Usp45 signal peptide (SPUsp45-LysL) weakened LysL activity, likely due to incomplete signal peptide cleavage during secretion. Active LysL was also produced in vitro, and analysed in SDS-PAGE, giving a 42-kDa band. However, the yield of LysL protein was still low when produced from recombinant lactococci or by in vitro expression system. Therefore, His-tagged LysL was produced in Escherichia coli BL21(DE3). Western blot confirmed the intracellular production of about 44-kDa His-tagged LysL in E. coli. His-tagged active LysL was then purified by Ni-NTA affinity chromatography yielding sufficient 4.34 mg of protein to be used in future functional studies.

Effects of quaternization sites and crossing methods on the slow-release and antibacterial effects of hydroxypropyltrimethyl ammonium chloride chitosan/dialdehyde chitosan-based film.

In this study, the active food packaging film were prepared using hydroxypropyltrimethyl ammonium chloride chitosan with different substitution sites (O-HACC & N-HACC) and dialdehyde chitosan (DCS) grafted with protocatechuic acid (PA). To explore the effect of chitosan quaternization positions and crosslinking approaches on the slow-release and antibacterial properties, the double-crosslinked film were fabricated through the self-coupling reaction of PA and Schiff base reaction between amino groups on HACC and aldehyde groups on DCS. The HACC/DCS-based film exhibited stable porous three-dimensional networks with high nisin loading ratios (>90 %). With the participation of the catechol-catechol structure, the dense double-crosslinked film effectively restricted the diffusion of the water molecules, resulting in excellent slow-release properties fitting with the Korsmeyer-Peppas kinetic model. Especially, O-HACC/PA-g-DCS film, which had more reaction sites for Schiff base crosslinking than N-HACC, exhibited the equilibrium swelling ratio of 800 % at 60 h and could sustainably release nisin via non-Fickian diffusion behavior until 48 h. Moreover, the HACC/DCS-based double-crosslinked film performed good long-time antibacterial activity and preservation effects on salmon. On the 10th day of storage, the TVBN of N-HACC/PA-g-DCS and O-HACC/PA-g-DCS groups were only 28.26 ± 1.93 and 29.06 ± 1.68 mg/100 g and still lower than the thresholds.

Polyvinyl alcohol/soybean isolate protein composite pad with enhanced antioxidant and antimicrobial properties induced by novel ternary nanoparticles for fresh pork preservation.

In this study, oregano essential oil (OEO)-loaded soluble soybean polysaccharide (SSPS) -nisin nanoparticles (ONSNPs) were formulated through electrostatic attraction-driven and hydrophobic interactions utilizing SSPS, nisin, and OEO as raw materials. ONSNPs were integrated into polyvinyl alcohol (PVA) and soybean protein isolate (SPI) matrices to create composite pads (PS-ONSNPs) by physically cross-linked using a simple freeze-thaw cycling process. The effects of ONSNPs content on the structure and physicochemical properties were evaluated. The results revealed that strong intermolecular interactions between ONSNPs and the PS matrices affected the crystallinity, microstructure, and thermal stability of the pads. Upon incorporating 5 % to 15 % ONSNPs, the structure of composite pads became denser, and the mechanical properties and water resistance were enhanced. Concurrently, the PS-ONSNPs pads facilitated the protection and controlled release of OEO. Furthermore, ONSNPs significantly improved the antioxidant activity of the pads and effectively inhibited the growth of Staphylococcus aureus and Escherichia coli. The prepared PS-ONSNPs 15 % pad was applied to storage experiments of fresh pork, which could extend the shelf life of meat to 10-12 days under 4 °C storage conditions. Therefore, the composite pad devised in this research holds promise as a viable option for intelligent active packaging of fresh meat.

A water-resistant egg white/chitosan/pectin blending film with spherical-linear molecular interpenetrating network strengthened by multifunctional tannin-nisin nanoparticles.

Edible films are effective alternatives to plastic packaging, however, the hydrophilicity of edible films based on protein and polysaccharide limits the application. Therefore, we fabricated a water-stable hybrid film with a linear-spherical interpenetrating molecular topology network using egg white (EW), chitosan (CS), and pectin. Meanwhile, the nisin-tannin acid self-assembly complex nanoparticles were employed as a multifunctional cross-linker, antibacterial and antioxidant agent to improve the performance of films. The FTIR, XRD, and SEM analysis revealed that the conformation and crystalline structure rearrangement of chitosan induced by the alkaline environment provided by egg white enhanced the network structure of films, effectively avoided the addition of modifying reagents. The proposed hybrid films exhibited excellent properties, with EW/TNPCS3 showing the best overall performance. The water contact angle (WCA) increased to 105.27 ± 1.62°, and its dissolution and swelling rates were significantly lower than pure egg white and pure chitosan films. Moreover, tannin-nisin (TN) nanoparticles endowed the films with excellent antimicrobial activity against the common Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. Thus, the prepared blending films have great application potential in food preservation, especially to maintain stable performance in high humidity environment.

The synergistic effect of polyphenols and polypeptides for plant-based bioplastic film - Enhanced UV resistance, antioxidant and antibacterial performance.

The exceptional biodegradability and active biological functions of bio-based packaging materials have attracted increasing interest. In this study, a bioplastic film was developed by introducing simultaneously polyphenols (tea polyphenols, TPs) and peptides (nisin) into a soy protein isolate/sodium alginate (SPI/SA) based film-forming matrix. The research results revealed that the dynamic coordinated interaction between TPs and nisin enhanced mechanical properties, UV-resistance, and thermal stability of bioplastic films. Furthermore, the bioplastic film exhibited antibacterial activity and antioxidant properties. Significantly, biofilm growth of Staphylococcus aureus treated with TPs-5/Nisin-5 bioplastic film was inhibited by 91.12% compared to the blank group. The shelf life of beef with TPs-5/Nisin-5 bioplastic film was prolonged by 2 days because of the synergistic effect of TPs and nisin. Additionally, the bioplastic film biodegraded in the natural environment about 21 days. This environmentally friendly regeneration strategy and the integration of advantageous functions provided ideas for the development of active food packaging.

The effect of silver nanoparticles on the antimicrobial activity of cloned nisin against extensively drug-resistant Acinetobacter baumannii".

BACKGROUND: Antibiotic resistance is a global threat to human health that leads to disasters. Acinetobacter baumannii cannot be controlled by the existing antibiotics, and it became challenging. Therefore, novel antibacterial agents are required to combat such threats. The aim of this project is to find a novel antimicrobial agent to treat this multi-drug resistant bacterium. METHODS: The NisA gene was isolated from Lactococcus lactis spp. lactis and cloned into the pET-3a plasmid using Gibson cloning assembly. Purified Nisin from cloning was conjugated with silver nanoparticles. Finally, an assessment of antibacterial activity for each of the purified Nisin, Silver nanoparticles, and Nisin-Silver nanoparticles conjugate against the extensively drug-resistant A. baumannii was performed. RESULTS: Nisin was successfully purified from cloned bacteria, and the concentration was 416 µg/ml. The conjugation of nisin and silver nanoparticles was analyzed by electron microscopy. The minimum inhibitory concentration of Nisin and silver nanoparticles against A. baumannii were 104 µg/ml and 125 µg/ml, respectively. While Nisin-silver nanoparticle conjugates showed potent antimicrobial activity with MIC 125-52 µg/ml in which silver nanoparticles increased the antimicrobial activity of nisin beyond its optimum concentration (104 µg/ml). CONCLUSION: The development of new antibacterial agents is necessary to control extensively drug-resistant bacteria. Nisin-silver conjugates showed more potent antimicrobial activity than when applied separately and gave hope to combat the multi-drug resistant A. baumannii.

Glycated nisin enhances nisin's cytotoxic effects on breast cancer cells.

Antimicrobial peptides, such as nisin, are proposed as promising agents for cancer treatment. While glycation has been recognized as an effective method for enhancing various physicochemical properties of nisin, its anticancer effects remain unexplored. Therefore, we aimed to assess the anticancer potential of glycated nisin against MDA-MB-231 cells. The MDA-MB cells were treated with increasing concentrations of nisin and glycated nisin for 24, 48, and 72 h. The IC50 values for nisin were higher than those for glycated nisin. Glycated nisin at concentrations of 20 and 40 µg/mL decreased cell viability more than nisin at the same concentrations. The rate of apoptosis in the group treated with 20 µg/mL of nisin was lower compared to other treatment groups, and no significant difference in apoptosis rates was observed at different time points (p > 0.05). However, in the glycated nisin groups with concentrations of 10, 20, and 40 µg/mL, the level of apoptosis was very high after 24 h (73-81% of cells undergoing apoptosis). Overall, our study suggests that glycated nisin exhibits stronger cytotoxic effects on MDA-MB-231 cells, primarily involving the induction of apoptosis. This indicates its potential utilization as an alternative approach to address the issue of drug resistance in cancer cells.

Changes in the ability of Listeria monocytogenes to resist thermal treatment and simulated gastric condition after exposure to sequential stresses in minced meat.

In this study, Listeria monocytogenes from minced pork was evaluated for changes in resistance to thermal treatment and gastric fluid following environmental stresses during food processing. Bacteria were exposed to cold stress, followed by successive exposures to different stressors (lactic acid (LA), NaCl, or Nisin), followed by thermal treatments, and finally, their gastrointestinal tolerance was determined. Adaptation to NaCl stress reduced the tolerance of L. monocytogenes to subsequent LA and Nisin stress. Adaptation to LA stress increased bacterial survival in NaCl and Nisin-stressed environments. Bacteria adapted to Nisin stress showed no change in tolerance to subsequent stress conditions. In addition, treatment with NaCl and LA enhanced the thermal tolerance of L. monocytogenes, but treatment with Nisin decreased the thermal tolerance of the bacteria. Almost all of the sequential stresses reduced the effect of a single stress on bacterial thermal tolerance. The addition of LA and Nisin as a second step of stress reduced the tolerance of L. monocytogenes to gastric fluid, whereas the addition of NaCl enhanced its tolerance. The results of this study are expected to inform processing conditions and sequences for meat preservation and processing and reduce uncertainty in risk assessment of foodborne pathogens due to stress adaptation.

Thiolated chitosan nanoparticles encapsulated nisin and selenium: antimicrobial/antibiofilm/anti-attachment/immunomodulatory multi-functional agent.

BACKGROUND: The increase in the resistance of bacterial strains to antibiotics has led to research into the bactericidal potential of non-antibiotic compounds. This study aimed to evaluate in vitro antibacterial/ antibiofilm properties of nisin and selenium encapsulated in thiolated chitosan nanoparticles (N/Se@TCsNPs) against prevalent enteric pathogens including standard isolates of Vibrio (V.) cholerae O1 El Tor ATCC 14,035, Campylobacter (C.) jejuni ATCC 29,428, Salmonella (S.) enterica subsp. enterica ATCC 19,430, Shigella (S.) dysenteriae PTCC 1188, Escherichia (E.) coli O157:H7 ATCC 25,922, Listeria (L.) monocytogenes ATCC 19,115, and Staphylococcus (S.) aureus ATCC 29,733. METHODS: The synthesis and comprehensive analysis of N/Se@TCsNPs have been completed. Antibacterial and antibiofilm capabilities of N/Se@TCsNPs were evaluated through broth microdilution and crystal violet assays. Furthermore, the study included examining the cytotoxic effects on Caco-2 cells and exploring the immunomodulatory effects of N/Se@TCsNPs. This included assessing the levels of both pro-inflammatory (IL-6 and TNFα) and anti-inflammatory (IL-10 and TGFß) cytokines and determining the gene expression of TLR2 and TLR4. RESULTS: The N/Se@TCsNPs showed an average diameter of 136.26 ± 43.17 nm and a zeta potential of 0.27 ± 0.07 mV. FTIR spectroscopy validated the structural features of N/Se@TCsNPs. Scanning electron microscopy (SEM) images confirmed their spherical shape and uniform distribution. Thermogravimetric Analysis (TGA)/Differential Scanning Calorimetry (DSC) tests demonstrated the thermal stability of N/Se@TCsNPs, showing minimal weight loss of 0.03%±0.06 up to 80 °C. The prepared N/Se@TCsNPs showed a thiol content of 512.66 ± 7.33 µmol/g (p < 0.05), an encapsulation efficiency (EE) of 69.83%±0.04 (p ≤ 0.001), and a drug release rate of 74.32%±3.45 at pH = 7.2 (p ≤ 0.004). The synthesized nanostructure demonstrated potent antibacterial activity against various isolates, with effective concentrations ranging from 1.5 ± 0.08 to 25 ± 4.04 mg/mL. The ability of N/Se@TCsNPs to reduce bacterial adhesion and internalization in Caco-2 cells underscored their antibiofilm properties (p ≤ 0.0001). Immunological studies indicated that treatment with N/Se@TCsNPs led to decreased levels of inflammatory cytokines IL-6 (14.33 ± 2.33 pg/mL) and TNFα (25 ± 0.5 pg/mL) (p ≤ 0.0001), alongside increased levels of anti-inflammatory cytokines IL-10 (46.00 ± 0.57 pg/mL) and TGFß (42.58 ± 2.10 pg/mL) in infected Caco-2 cells (p ≤ 0.0001). Moreover, N/Se@TCsNPs significantly reduced the expression of TLR2 (0.22 ± 0.09) and TLR4 (0.16 ± 0.05) (p < 0.0001). CONCLUSION: In conclusion, N/Se@TCsNPs exhibited significant antibacterial/antibiofilm/anti-attachment/immunomodulatory effectiveness against selected Gram-positive and Gram-negative enteric pathogens. However, additional ex-vivo and in-vivo investigations are needed to fully assess the performance of nanostructured N/Se@TCsNPs.

Metal Uptake by Mitochondrial Carrier Family Proteins Using Lactococcus lactis.

Metal ion homeostasis in mitochondria is essential to maintaining proper cellular physiology. However, the ability of metals to bind off target or form complexes with multiple metabolites presents major challenges to understanding the mechanisms that govern this homeostasis. Adding further to the complexity, some of the major mitochondrial transporters have shown substrate promiscuity. In many cases, mitochondrial metals are found in the matrix compartment that is surrounded by the impermeable inner membrane. Four major classes of transporters facilitate the movement of solute across the inner membrane. These are mitochondrial carrier family, ATP-binding cassette transporters, mitochondrial pyruvate carriers, and sideroflexins. For iron, the matrix is the site of iron-sulfur clusters and heme synthesis and therefore transport must occur in a coordinated fashion with the cellular needs for these critical cofactors. Iron could be transported in numerous forms as it has been shown to form complexes with abundant metabolites such as citrate, nucleotides, or glutathione. Here, we describe assays to study iron (or any metal) transport by mitochondrial carrier family proteins expressed in Lactococcus lactis using a nisin-controlled expression system.