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This study aimed to evaluate the antibacterial efficacy of brine solution in compared to nisin and ozonated water treatments against E. coli O157:H7 in experimentally local produced soft cheese (ELPSC). A total of 25 raw cow milk samples were collected from farmers and local markets in Baghdad city and utilized to manufacturing 25 of ELPSC samples following the traditional procedure in rural Iraq without salting or heat treatment. Bacteriological analysis for potential E. coli O157:H7 contamination was performed and confirmed by cultural, biochemical, and serological tests. Antibacterial activity of brine solution (7% w/v), nisin solution (100 IU/mL), and ozonated water (0.6 ppm) was performed for positive samples after 2 h contact time at ambient temperature. Positive isolates of E. coli O157:H7 were detected and confirmed as 3 out of 25 cheese samples (12%), with initial average bacterial count of 6.146 log10 CFU/g. The initial bacterial count was significantly (P

Microbial technology offers a green alternative for the synthesis of value-added nanomaterials. In particular, fungal compounds can improve silver nanoparticle production, stabilizing colloidal nanoparticles. Based on a previous study by our group, silver nanoparticles obtained using the extracellular cell-free extracts of Phanerochaete chrysosporium (PchNPs) have shown antimicrobial and antibiofilm activity against Gram-negative bacteria. Moreover, nisin—a bacteriocin widely used as a natural food preservative—has recently gained much attention due its antimicrobial action against Gram-positive bacteria in biomedical applications. Therefore, the aim of this work was to conjugate biogenic silver nanoparticles (PchNPs) with nisin to obtain nanoconjugates (PchNPs@nis) with enhanced antimicrobial properties. Characterization assays were conducted to determine physicochemical properties of PchNPs@nis, and also their antibacterial and antibiofilm activities were studied. The formation of PchNPs@nis was confirmed by UV-Vis, TEM, and Raman spectroscopy analysis. Different PchNPs@nis nanobioconjugates showed diameter values in the range of 60–130 nm by DLS and surface charge values between −20 and −13 mV. Nisin showed an excellent affinity to PchNPs, with binding efficiencies higher than 75%. Stable synthesized PchNPs@nis nanobioconjugates were not only able to inhibit biofilm formation by S. aureus, but also showed inhibition of the planktonic cell growth of Staphyloccocus aureus and Escherichia coli, broadening the spectrum of action of the unconjugated antimicrobials against Gram-positive and Gram-negative bacteria. In conclusion, these results show the promising application of PchNPs@nis, prepared via green technology, as potential antimicrobial nanomaterials.

Biosafety has currently become an important public health issue in seafood industry of Thailand. In order to enhance quality of seafood products, three phases of study were established. In the first phase, biosafety of traditional processed squid sold in Chon Buri province, Thailand compiled from literatures between 2002 and 2019 was evaluated. The squid products represented a health hazard due to 86.5% samples, having total viable count (TVC) over the allowable limits imposed by Thailand and international food administration agencies. In the second phase, bacterial contamination during multiple steps related to dried seasoned squid preparation and storage was determined. TVC were low at approximately 102 CFU/g during a multi-step of preparation, and increased significantly (P < 0.05) to 103 CFU/g in post-prepared products during 25-day storage. Lastly, a novel preservation strategy was developed. A combination of partially purified solution from Bacillus velezensis BUU004 with nisin and the mixed herb extracts from hot pepper and lemongrass exhibited the strongest antibacterial efficacy observed by a significantly (P < 0.05) low TVC in the post-prepared squid within allowable limits recommended by the food administration authorities. This study suggests that the novel mixture has a preservative potential to improve biosafety of dried seafood products. Key words: Squid, bacteriocin, nisin, herb extract, Bacillus velezensis, synergy, biosafety.

A natural sap from mature coconut palm known as coconut neera is enriched with essential minerals and vitamins. Rapid microbial fermentation affects neera processing industries because it spoils the physicochemical properties. There are various methods in preservation that extend the shelf life of coconut neera. The addition of nisin is one of the methods which protect neera against fermentation. Therefore, the study is focused to identify the effective combination of nisin (50 ppm) with preservatives like sodium benzoate (500, and 1000 ppm), and calcium carbonate (2500, and 3000 ppm) at two different combinations in neera. At the end of 21 d, 3000 ppm calcium carbonate with 50 ppm nisin in N4 treatment had an effective reduction of 120 × 105 CFU/ml and 143 × 102 CFU/ml for total bacteria and total yeast count. The reduced microbial survival resulted in the pH of 10.45 ± 0.05, total soluble solids of 15.43 ± 0.12 °Brix, and total acidity of 1.11 ± 0.04 mg/ L, at this combination. The treatment of nisin with 3000 ppm calcium carbonate demonstrated the high red fluorescence bacterial cells than the treatment of nisin with 1000 ppm sodium benzoate. Additionally, the microorganisms in N4 treatment precipitated 65.34% Ca2+ from 79.96% in XRF intensity analysis. The synergistic effect of nisin and calcium carbonate explored their antimicrobial activity against the heterogeneous microbial population in coconut neera. The concentration of 3000 ppm calcium carbonate and nisin 50 ppm preserves the physicochemical and sensory qualities, up to 21 d at 4°C, and offer hope for the industrial-scale implementation.

Active food packaging materials that are sustainable, biodegradable, and capable of precise delivery of antimicrobial active ingredients (AIs) are in high demand. Here, we report the development of novel enzyme- and relative humidity (RH)-responsive antimicrobial fibers with an average diameter of 225 ± 50 nm, which can be deposited as a functional layer for packaging materials. Cellulose nanocrystals (CNCs), zein (protein), and starch were electrospun to form multistimuli-responsive fibers that incorporated a cocktail of both free nature-derived antimicrobials such as thyme oil, citric acid, and nisin and cyclodextrin-inclusion complexes (CD-ICs) of thyme oil, sorbic acid, and nisin. The multistimuli-responsive fibers were designed to release the free AIs and CD-ICs of AIs in response to enzyme and RH triggers, respectively. Enzyme-responsive release of free AIs is achieved due to the degradation of selected polymers, forming the backbone of the fibers. For instance, protease enzyme can degrade zein polymer, further accelerating the release of AIs from the fibers. Similarly, RH-responsive release is obtained due to the unique chemical nature of CD-ICs, enabling the release of AIs from the cavity at high RH. The successful synthesis of CD-ICs of AIs and incorporation of antimicrobials in the structure of the multistimuli-responsive fibers were confirmed by X-ray diffraction and Fourier transform infrared spectrometry. Fibers were capable of releasing free AIs when triggered by microorganism-exudated enzymes in a dose-dependent manner and releasing CD-IC form of AIs in response to high relative humidity (95% RH). With 24 h of exposure, stimuli-responsive fibers significantly reduced the populations of foodborne pathogenic bacterial surrogates Escherichia coli (by ∼5 log unit) and Listeria innocua (by ∼5 log unit), as well as fungi Aspergillus fumigatus (by >1 log unit). More importantly, the fibers released more AIs at 95% RH than at 50% RH, which resulted in a higher population reduction of E. coli at 95% RH. Such biodegradable, nontoxic, and multistimuli-responsive antimicrobial fibers have great potential for broad applications as active and smart packaging systems.

Aims: To encapsulate a purified bacteriocin into a nanovesicles and check its antibacterial effect. Background: Although the use of nano-encapsulated bacteriocins in food matrices is poorly reported, encapsulated nisin can reduce L. monocytogenes counts in whole and skimmed milk and in soft cheese. Objective: The present study deals with the extraction and purification of a bacteriocin from an isolated strain Pediococcus pentosaceus KC692718. A comparative study of the effect of free pediocin and liposome encapsulated pediocin against Listeria sp. was performed. Methods: The purification of the extracted cell free supernatant was subjected to ammonium sulphate precipitation, cation exchange chromatography followed by gel permeation chromatography. The bacteriocin activity and protein concentration were determined using Lowry’s method. The characterization of the pure pediocin was done. Liposome like nanovesicle was constructed and the stability of the liposome encapsulated pediocin was checked. Finally, the antibacterial effect was comparatively studied of the free pediocin, liposome, and liposome encapsulated pediocin simultaneously. Results: The pediocin of 3.6kDa was purified with a specific activity of 898.8. AU/mg. It remained stable from pH 2.0-8.0 was found to be moderately stable above 80°C and remain stable for one month when stored at -20°C. The encapsulated pediocin showed stability since it retained 50% of its initial activity. The encapsulated pediocin showed 89% of encapsulation efficiency. Conclusion: The encapsulated pediocin not only improved pediocin stability but also enhanced the controlled release of the antimicrobial substances, enough for inhibiting the foodborne pathogen L. monocytogenes.

Numerous beneficial microbes thrive in the oral cavity where they form biofilms on dental and mucosal surfaces to get access to nutrients, and to avoid being carried away with the saliva. However, biofilm formation is also a virulence factor as it also protects pathogenic bacteria, providing them with an environment for proliferation causing oral infections. Oral hygiene relies on mechanical removal of biofilms. Some oral care products also contain antimicrobials, but effective eradication of biofilms with antimicrobials requires both a high concentration and long exposure time. In the present communication, we investigate the potential of using miniaturized drug delivery devices, known as microcontainers (MCs), to deliver the antimicrobial peptide, nisin to an oral multi-species biofilm. MCs are loaded with nisin and X-ray micro-computed tomography reveals a full release of nisin through a chitosan lid within 15 min. Chitosan-coated MCs display substantial bioadhesion to the buccal mucosa compared to non-coated MCs (68.6 ± 14.3% vs 33.8 ± 5.2%). Confocal monitoring of multi-species biofilms reveals antibacterial effects of nisin-loaded chitosan-coated MCs with a faster onset (after 3 h) compared to solution-based delivery (after 9 h). Our study shows the potential of using MCs for treatment of multi-species oral biofilms and is encouraging for further design of drug delivery devices to treat oral diseases.

Lactococcus lactis is a gram positive bacteria that produces nisin, a polycyclic peptide, during fermentation process. In recent researches, the anticancer activity of nisin has been proven. We have studied the anticancer impact of nisin on human chronic myeloid leukemia (K562) cancer cell line, and in this study we evaluated the apoptotic mechanism of nisin in this cell line. In this research we studied the apoptotic effects of nisin using acridine orange/ethidium bromide (AO/EB) staining for assessing morphological alternations, Annexin V/PI method to assess the expression of phosphatidyl serine (PS) on plasma membrane surface and propidium-iodide (PI) staining for analysis of cell cycle. In previous research we have studied cell viability through treatment with nisin, and in this research we investigate the apoptotic mechanism by real-time PCR (RT-PCR) and western blot techniques. The diagram of cell cycle arrest indicates G1 arrest in a short time after treatment and sub-G1 arrest in a longer period of time. Annexin V/PI staining demonstrated the cells from early apoptotic stage alternate to late apoptotic stages. RT-PCR results showed that down-regulation of Bcl-2 genes and proteins that have anti-apoptotic characteristic and up-regulation of pro-apoptotic Bax genes and proteins. Nisin has anticancer impact on K562 cells through intrinsic (mitochondria) pathway by changing the expression of Bcl-2 and Bax genes. It is an effective chemotherapeutic peptide suitable for more investigation in cancer therapy.

ComX can improve bacterial competence by modulating global gene expression. Although competence induction may also be a protective mechanism under stress, this has not been investigated in detail. Here, we demonstrated that ComX improved the acid tolerance and nisin yield of Lactococcus lactis, which is an important gram-positive bacterium increasingly used in modern biotechnological applications. We found that overexpression of comX could improve the survival rate up to 36.5% at pH 4.0, compared with only 5.4% and 1.1% with the wild-type and comX knockout strains, respectively. Moreover, quantitative real-time PCR results indicated that comX overexpression stimulated the expression of late competence genes synergistically with exposure to acid stress. Finally, electrophoretic mobility shift assay demonstrated the binding of purified ComX to the cin-box in the promoters of these genes. Taken together, our results reveal a regulation mechanism by which ComX and acid stress can synergistically modulate the expression of late competence genes to enhance cells' acid tolerance and nisin yield.

Background and Objective: Strangle in the horses is the commonest and the most significant infectious disease. An antibiotic of the first choice, commonly Penicillin, is used for antimicrobial therapy. Due to side effects and resistance against used antibiotics, interest in novel antimicrobial substances from other sources, including herbal medicine as safe agents has been raised. This study evaluated the antibacterial susceptibility profile of Streptococcus equi subsp equi in the confrontation of Trans-cinnamaldehyde, 1, 8 Cineole, and Pulegone on bacterial pathogens isolated from the horse. Materials and Methods: Two hundred clinically isolates were studied by the single disk method to Ampicillin, Ciprofloxacin, Trimethoprim sulfamethoxazole, Gentamicin, Enrofloxacin, Chloramphenicol, Azithromycin, Cefotaxime, Oxytetracycline, Erythromycin, and Penicillin. Paper discs were prepared by impregnation in 10 µL essential oils main compounds (EOMC). The antibacterial activities of Trans-cinnamaldehyde, 1, 8 Cineole, and Pulegone were evaluated by microbroth dilution and disk diffusion methods against isolates of S. equi. Results & Conclusion: All the isolates were resistant to Trimethoprim-sulfamethoxazole and Cefotaxime. The maximum growth inhibition zone was related to Oxytetracycline and Ampicillin. The growth inhibition zone diameter was 30 mm, 20 mm, and 26 mm for Trans-cinnamaldehyde, Pulegone, and 1, 8 Cineole, respectively. The results of the Minimum inhibitory concentrations (MICs) and Minimum Bactericidal Concentrations (MBCs) showed that Trans-cinnamaldehyde had the highest antibacterial activity compared to other EOMC against S. equi. This study indicated that Trans-cinnamaldehyde, 1, 8 Cineole, and Pulegone revealed antibacterial properties; therefore, these main constituents of the medicinal plant could be a safe candidate for the new antibacterial products.

In this study, production of low-fat, nitrite-free sausage based on Silver carp surimi was performed. In order to replace oils, inulin fiber (IF) was used; natural preservatives such as grape pomace extract (GE) and nisin (NI) were used as nitrite replacements with modified atmosphere packaging (MAP: 70% CO2 + 30% N2). For this purpose; five treatments including, T1: control, T2: control + MAP, T3: IF 5% + MAP, T4: IF5% + GE0.5% + MAP, and T5: IF5% + GE0.5% + NI 0.5% + MAP, were prepared. The physicochemical and texture properties of sausage at the beginning of storage and chemicals (Peroxide value, pH, and color index) and microbial index (Total count and psychrotrophic bacteria) during 42 days’ storage in the refrigerator (4 ± 1°C) were evaluated. The results showed that the use of natural preservatives had no effect on the physicochemical properties of the sausage (P > 0.05), but the FI had a positive effect on the texture characteristics (the firmness increased and elasticity decreased), increased moisture and ash, and reduced the fat content of sausages (P < 0.05). All in all, the best results among the treatment containing natural preservative was T5, in all microbial and chemical tests there was no significant difference with T1 (P > 0.05). All in all, a functional product with properties such as fiber and natural antioxidants, low fat and nitrite-free can be produced.

An attempt was made, to characterize natural antibiotics or lantibiotics from unconventional sources and its antibacterial spectrum against food borne pathogens and drug resistant bacteria. Six different traditional fermented foods i.e., fermented fish, fermented soybeans, Soibum (fermented bamboo shoots), milk, idly and dosa batter were used for the isolation of bacteriocin producing Lactic acid bacteria (LAB). Among all bacterial cultures isolated from the various sources, 129 cultures have found to produce antimicrobial compounds. Nisin specific reporter bacteria was utilized as biosensor to identify the Nisin like bacteriocin, where 10 cultures found to be positive Nisin producer. Identified Nisin like bacteriocin was partially concentrated by using ammonium sulphate followed by butanol extraction. Minimum inhibitory concentration (MIC) was analyzed against food borne pathogen and drug resistant bacteria. MIC of partially purified Nisin (pp-Nisin) of all the LAB isolates against food-borne pathogens are ranged between 0.5 and 92 µg/ml respected to various Gram-positive bacteria. Similarly, the drug resistant bacteria were also inhibited by pp-Nisin (MIC ranged between 15 and 175 µg/ml). All samples of ppnisin exhibited auto induction ability. Taxonomic identification of the nisin producers was done by whole genome sequencing which reveals that cultures belongs to Lactococcus lactis ssp. lactis. Also it was found that Lactococcus lactis ssp. lactis C2d and Lactococcus lactis ssp. lactis SP2C4 harbor nisA gene and Lactococcus lactis ssp. lactis FS2 (L. lactis FS2) harbor nisQ gene. The finding of this study highlights the first case of L. lactis FS2 isolated from fermented fish harbor nisQ gene. Antibacterial activity of pp-Nisin against drug resistant LAB is also reported.

Exploration of novel material platforms to protect biological preservatives and realize intelligent regulation during fermentation is of great significance in industry. Herein, we established an intelligent responsive platform by introducing antimicrobial biomolecules (nisin) into rationally designed covalent organic frameworks (COFs), resulting in a new type of "smart formulation", which could responsively inhibit microbial contamination and ensure the orderly progression of the fermentation process. The encapsulated biomolecules retained their activity while exhibiting enhanced stability and pH-responsive releasing process (100% bacteriostatic efficiency at a pH of 3), which can ingeniously adapt to the environmental variation during the fermentation process and smartly fulfill the regulation needs. Moreover, the nisin@COF composites would not affect the fermentation strains. This study will pave a new avenue for the preparation of highly efficient and intelligent antimicrobial agents for the regulation of the fermentation process and play valuable roles in the drive toward green and sustainable biomanufacturing.

Staphylococcus aureus is one of the commonest food-borne pathogens that can cause gastroenteritis owing to having several enterotoxins. Also, biofilm formation can complicate infections caused by this microorganism. Nisin is a safe food bio preservative which is usually used as an agent to prevent pathogen growth; however, it is important to identify the exact impact of nisin on the growth of S. aureus and to determine the suitable concentration needed for elimination of this pathogen in food. In this study, after MIC determination of nisin against S. aureus ATCC 29213, this strain was treated with sub-MIC (1/2) of nisin (4 μg/ml) and transcript levels of toxin-encoding (hla, SEA, SEB, and SED) and biofilm-associated (fnb, ebpS, eno, and icaA) genes were determined using Quantitative Real-time PCR at 2, 8, and 24 h post exposure. All toxin genes were down-regulated following exposure to sub-MIC of nisin, whereas biofilm-associated genes were up-regulated. The expression levels of fnb and icaA in S. aureus were highest after 8 h (4.5-fold and 6.8-fold increase, respectively), while the expression levels of eno and ebpS genes were highest after 2 h (3.3 and 4.5-fold increase, respectively). According to these results, although transcriptional levels of toxin genes were reduced, sub-MIC concentrations of nisin could trigger the expression of biofilm-associated genes in S. aureus. This can further lead to bacteriocin tolerance such that even its higher concentrations cannot kill bacterial cells after exposure to sub-lethal doses. Therefore, it is pivotal to add appropriate concentrations of nisin to food products for preservation purposes.

High values of agitation and temperature lead to stressful conditions in the fermentations of Lactococcus lactis due to its aero-tolerant and mesophilic nature. Here, the adaptive laboratory evolution (ALE) technique was applied to increase biomass and nisin production yields by enhancing L. lactis subsp. lactis robustness at higher growth temperature and aeration rates. In two separate ALE experiments, after 162 serial transfers, optimum agitation and growth temperature of L. lactis were shifted from 40 rpm and 30 °C to 200 rpm and 37 °C, respectively. Oxidative and acid resistance were enhanced in the evolved strain. Whole-genome sequencing revealed the emergence of five single-nucleotide polymorphisms in the genome of the evolved strain in jag, DnaB, ArgR, cation transporter genes, and one putative protein. The evolved strain of L. lactis in this study has more industrial desirable features and improved nisin production capability and can act more efficiently in nisin production in stressful conditions.

Acanthamoeba spp. are free-living amoebas with worldwide distribution and play an important role as disease-causing agents in humans. Drug inability to completely eradicate these parasites along with their toxic effects suggest urgent need for new antimicrobials. Nisin is a natural antimicrobial peptide produced by Lactococcus lactis. Nisin is also the only bacteriocin approved for use in food preservation. In this work, we analyzed the effect of nisin on the growth of Acanthamoeba castellanii trophozoites. A total of 8 × 104 trophozoites were exposed to increasing concentrations of nisin to determine its activity. Changes in cell membrane and cellular cycle of trophozoites were investigated by flow cytometry, and nisin cytotoxicity in mammalian cells was evaluated in L929 cells by MTT method. After 24 h exposure to increasing nisin concentrations, an IC50 of 4493.2 IU mL−1 was obtained for A. castellanii trophozoites. However, after 72 h a recovery in amoebic growth was observed, and it was no longer possible to determine IC50. Flow cytometry analysis showed that nisin has no effect on the membrane integrity. Treatment with nisin induced cell-cycle arrest during G1 and S phases in A. castellanii trophozoites, which recovered their growth after 72 h. This is one of the first studies showing the effect of internationally approved nisin against A. castellanii trophozoites. Nisin caused cell-cycle arrest in trophozoites, momentarily interfering with the DNA replication process. The data highlight the amoebostatic activity of nisin, and suggest its use as an adjuvant for the treatment of infections caused by Acanthamoeba spp.

In the present study, we investigated the effects of thermal preservation, such as pasteurization, and nonthermal preservation, including irradiation, sodium dehydroacetate (SDHA), and nisin, on the quality of red sour soup after storage. Single‐factor experiments were used to optimize the parameters of different processing technologies, and the best irradiation dose and heating temperature were 4 kGy and 85℃, respectively. The optimal additive amounts of SDHA and nisin were 150 mg/500 g. During the shelf storage experiment, prepared red sour soup was stored at room temperature in the glass bottles, and further analyses were carried out up to 5 weeks of storage. The quality of red sour soup was evaluated by microflora and sensory analysis. The results showed that Lactobacillus, Streptomyces, Pediococcus, Pichia, Kazachstania, and Candida were the main microorganisms in all samples, and there were no harmful microorganisms. The sensorial attributes were observed, including different parameters, such as odor, organic acid content, color, taste, texture, apparent viscosity, and thixotropy. All of the data showed that the irradiated groups were more dramatically changed compared with the other groups, while these changes did not directly affect the sensory quality of the products. Consequently, irradiation could be used as an ideal quality preservation method for the red sour soup to reduce the impact of heat treatment and chemical additives on the quality of characteristic food., This article compared the effect of different preservation methods on the quality of red sour soup and provided more schemes for the storage and preservation of related fermentation products.

Background and Objective: Dairy fermented foods such as yogurt, cheese, fermented milk, butter milk, curd, butter and ghee are used as major diet ingredients in west of IRAN, such as Kermanshah province. Ghee (Kermanshahi traditional oil or roghan-e heiwâni) is traditionally produced from butter milk of yoghurt after fermentation. Review of the literature yielded no study on isolating probiotics from Kermanshahi traditional oil preparation stages. Therefore, purpose of this study was just to focus on isolating and identifying lactic acid bacteria in these products using culture and PCR-sequencing methods. Materials and Methods: Fifteen samples from each dairy products including yogurt, butter and Kermanshahi traditional oil were collected in Kermanshah province, Iran. Each sample was diluted, homogenized, and cultured on selective medium for growing of lactic acid bacteria. 16SrRNA gene sequences analysis was carried out for final confirmation of these isolates. Results: After culturing of samples on MRS and M17 under aerobic and anaerobic condition, a total of 78 strains of bacteria were isolated and identified by conventional biochemical tests. The frequency of bacteria in all samples were 48.71% for Lactobacillus, 33.33% for Streptococcus, 6.41% for Enterococcus and 6.41% for Bacillus. Lactobacillus, Streptococcus, Enterococcus, and Bacillus genus, were isolated from 84.44%, 57.78%, 11.11% and 15.56% of all three kind of samples, respectively. Conclusion: Based on our findings, Lactic acid bacteria and other potentially probiotic microorganisms are present in Kermanshahi traditional oil. Of course the potential probiotic properties of these isolates and impact of consumption of Kermanshahi traditional oil containing those on human health need to be analyzed more. By proving the presence of probiotic bacteria in Kermanshahi oil, it may be falls in the category of functional foods.

Background and Objective: Antimicrobial peptides have attracted significant attention in recent decades because of their properties, such as rapid bactericidal effects, having a wide spectrum of activity, and a rare development of drug resistance. The purpose of this study was to examine the antibacterial activity of a peptide derived from the lactoferrin isolated from camel milk against Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii. Materials and Methods: In the present study, by means of bioinformatics, an antibacterial peptide was potentially identified as candidates in lactoferrin of camel milk, and an appropriate peptide was selected based on defined criteria. The Pepsin-Camel-Lac1 peptide was synthesized. The methyl thiazolyl diphenyl-tetrazolium bromide assay was conducted to examine the toxicity of Pepsin-Camel-Lac1 against a cell line. Three pathogenic bacteria, namely S. aureus, P. aeruginosa, and A. baumannii were analyzed to assess the antibacterial activity of Pepsin-Camel-Lac1 peptide. Results: The results showed that the newly-identified peptide had no toxicity against the cell line. The minimum inhibitory concentration values of Pepsin-Camel-Lac1 against S. aureus, P. aeruginosa, and A. baumannii were 31.25 µg/mL, 31.25 µg/mL, and 62.5 µg/mL, respectively. Conclusion: It seems that the growth of S. aureus, P. aeruginosa, and A. baumannii was not affected by Pepsin-Camel-Lac1 treatment in the bacterial culture medium.

In this study, ultrasound (US) and nisin, applied individually or in combination (US + nisin), were investigated to determine their inactivation effect on Staphylococcus aureus in nutrient broth (NB) and milk. The inactivation of S. aureus by nisin at 0, 25 and 37°C followed “fast to slow” two-stage kinetics, and there was no significant difference in S. aureus reduction under these temperatures. A maximum reduction of 1.40 log10 cycles was obtained by nisin at 37°C for 15 min. The inactivation of S. aureus by US also followed “fast to slow” two-stage kinetics at 0°C, but it followed “fast–slow–fast” there-stage kinetics at 25 and 37°C. A maximum reduction of 5.87 log10 cycles was obtained by US at 968.16 W/cm2 and 37°C for 15 min. The reduction of S. aureus in milk was similar as in NB when treated at 968.16 W/cm2 and 25°C for 5–15 min. However, the reduction of S. aureus by US + nisin was similar to that obtained by either nisin or US alone, indicating that US and nisin had no added or synergistic inactivation effect on S. aureus in both NB and milk under the tested conditions.

Today, the usage of natural additives in the food matrix has increased. Natural antimicrobial compounds include peptides, enzymes, bacteriocins, bacteriophages, plant extracts, essential oils, and fermented compounds that can be used as alternatives to chemical antimicrobials. Plant extracts and essential oils contain terpenes, flavonoids, aldehydes, and phenolic compounds that cause antimicrobial and antioxidant activity. The synergistic activity of compounds synthesized from lactic acid bacteria (LAB) prevents the growth of bacteria and fungi. In addition to removing mycotoxins, LAB compounds have antioxidant and anticancer potentials and increase food safety and nutritional value. One of these antimicrobial molecules is bacteriocin, which is made by various microorganisms. Nisin is one of these bioactive peptides that are used widely in food bio-preservation. Antimicrobial peptides can be used alone or along with other compounds to enhance food security. This article reviews natural preservatives and their applications in food products.

Objective: The present study aims to examine the effects of nisin on the survival and apoptosis of the hepatoma cell line HepG2 and to investigate possible apoptosis pathways activated by nisin. Materials and methods: For this purpose, viability and apoptosis of the cells were accomplished by the nisin treatment using the MTT assay and Annexin-V-fluorescein/propidium iodide (PI) double staining, respectively. Additionally, the human apoptosis PCR array was performed to determine pathways or genes activated by nisin during possible apoptosis. Results: The results of the present study showed that nisin was able to decrease cell viability (IC50 ~ 40 µg/ml) in a dose-dependent manner and could induce apoptosis in HepG2 cells. PCR data indicated a considerable increase in the expression of genes, such as caspase and BCL2 families, involved in the induction of apoptosis. Conclusions: The data from this study showed that overexpression of genes involved in the intrinsic pathway of apoptosis, especially caspase-9 and BID, increased apoptosis in HepG2 cells treated by nisin, compared to the control group.

Lactobacillus kefiranofaciens contains two types of β-galactosidase, LacLM and LacZ, belonging to different glycoside hydrolase families. The difference in function between them has been unclear so far for practical application. In this study, LacLM and LacZ from L. kefiranofaciens ATCC51647 were cloned into constitutive lactobacillal expression vector pMG36e, respectively. Furtherly, pMG36n-lacs was constructed from pMG36e-lacs by replacing erythromycin with nisin as selective marker for food-grade expressing systems in Lactobacillus plantarum WCFS1, designated recombinant LacLM and LacZ respectively. The results from hydrolysis of o-nitrophenyl-β-galactopyranoside (ONPG) showed that the β-galactosidases activity of the recombinant LacLM and LacZ was 1460% and 670% higher than that of the original L. kefiranofaciens. Moreover, the lactose hydrolytic activity of recombinant LacLM was higher than that of LacZ in milk. Nevertheless, compare to LacZ, in 25% lactose solution the galacto-oligosaccharides (GOS) production of recombinant LacLM was lower. Therefore, two β-galactopyranosides could play different roles in carbohydrate metabolism of L. kefiranofaciens. In addition, maximal growths rate of two recombinant strains were evaluated with different temperature level and nisin concentration in fermentations assay for practical purpose. The results displayed that 37 °C and 20-40 U/ mL nisin were the optimal fermentation conditions for the growth of recombinant β-galactosidase strains. Altogether the food-grade Expression system of recombinant β-galactosidase was feasible for applications in the food and dairy industry.

Over the past decades, stem cell therapy has been investigated as a promising approach towards various diseases, including neurodegenerative disorders. Stem cells show the capability to differentiate into neuronal progenitor cells in vitro. In the present study, the differentiation potential of human-induced pluripotent stem cells (hiPSCs) into neural lineages was examined under the efficient induction media containing forskolin and 3-isobutyl-1-methyl-xanthine (IBMX) in the presence of nisin (Ni), non-essential amino acids (NEAA) and combination of those (NEAA-Ni) in vitro. The optimum concentrations of these factors were obtained by MTT assay and acridine orange (AO) staining. The effect of Ni and NEAA on the expression rate of neural-specific markers including NSE, MAP2, and ß-tubulin III was studied via immunocytochemistry (ICC) and real-time RT-PCR analyses. Our results indicated that the induction medium containing Ni or NEAA increased the gene and protein expression of NSE, MAP2, and β-tubulin III on the 14th differentiation day. On the other hand, NEAA-Ni showed a less-differentiated hiPSCs compared to Ni and NEAA alone. In conclusion, the obtained results illustrated that Ni and NEAA could be applied as effective factors for neural differentiation of hiPSCs in the future.

The aim of this work was to isolate and characterize bacteriocins produced by 2 Lactobacillus fermentum strains isolated from artisanal Mexican Cocido cheese. Fractions (F ≤3 kDa) obtained from cell-free supernatants of Lb. fermentum strains J23 and J32 were further fractionated by reversed-phase HPLC on a C18 column. Antimicrobial activities of F ≤3 kDa and bacteriocin-containing fractions (BCF), obtained from fractionation of F ≤3 kDa against 4 indicator microorganisms, were determined by the disk diffusion method and growth inhibition in milk. Subsequently, isolated BCF were analyzed by reversed-phase HPLC tandem mass spectrometry. Results showed that BCF presented antimicrobial activity against the 4 indicator microorganisms tested. For J23, one of the fractions (F3) presented the highest activity against Escherichia coli and was also inhibitory against Staphylococcus aureus, Listeria innocua, Salmonella Typhimurium, and Salmonella Choleraesuis. Similarly, fractions F3 and F4 produced by J32 presented antimicrobial activity against all indicator microorganisms. Furthermore, generation time and growth rate showed that F3 from J23 presented significantly higher antimicrobial activity against the 4 indicator microorganisms (2 gram-positive and 2 gram-negative) when inoculated in milk compared with F3 from J32. Interestingly, this fraction presented a broader antimicrobial spectrum in milk than nisin (control). Reversed-phase HPLC tandem mass spectrometry analysis revealed the presence of several peptides in BCF; however, F3 from J23 that was the most active fraction of all presented only 1 bacteriocin. The chemical characterization of this bacteriocin suggested that it was a novel peptide with 10 hydrophobic AA residues in its sequence and a molecular weight of 2,056 Da. This bacteriocin and its producing strain, J23, may find application as a biopreservative against these indicator microorganisms in dairy products.

Microbial spoilage of cheese represents a major concern from both health and economic views. The addition of food preservatives is considered the most applied strategy to ensure food quality and to control microbial contamination. It well established that natural preservatives such as Nisin and Natamycin are of great effectiveness against a wide range of microbial concerns, but the lack of wide spectrum effect induces looking for more efficient alternatives. This research suggests alternative treatments to be evaluated side by side with both Nisin and Natamycin within real cheese models to control microbial contamination during the storage period. To evaluate this, two varieties of cheese were manufactured and inoculated with a set of pathogen and cheese spoiling microorganisms. Talaga cheese batches were separately treated with extract of MRS that previously fermented by Lactobacillus helveticus CNRZ 32, Nisin and Natamycin at Free State and Chitosan Nanoparticles-loaded state to become 6 treatments (T1 – T6) other than the control (C). The same treatments were applied to Karish cheese batches, in addition to inoculation of Lactobacillus helveticus CNRZ 32 1% (v/v) in the seventh batch. Upon microbiological analyses, results show that T2; the extract loaded on Chitosan Nanoparticles completely reduced the count of all pathogens and spoiling populations after two weeks of cold storage (2 – 6oC) in Talaga cheese. In the case of Karish cheese, the 7th batch treated with Lactobacillus helveticus CNRZ 32 inoculation had no pathogenic nor spoiler growth after one week of cold storage (2 – 6oC). These results suggest that Lactic acid bacteria especially Lactobacillus helveticus CNRZ 32 can efficiently ensure the safety and quality of cheese if applied in appropriate form.

In this work, water-soluble zein/sodium caseinate (SC) nanoparticles were prepared as a delivery system of antimicrobials through anti-solvent procedure. Two model antimicrobials (nisin and thymol) were coencapsulated into zein particles by different interaction. Release properties indicate that zein colloid particles can be used not only in neutral food but also in acidic foods. It is worth mention that the release of thymol was delayed simply by adding nisin. To investigate the antimicrobial properties of zein/SC particles, Escherichia coli and Bacillus subtilis, Listeria monocytogenes were selected as gram-negative and gram-positive indicator bacteria. CO2 produced by incubated germs was detected by headspace gas chromatography. Long-term effective antimicrobial activities of zein particles in Luria broth was attained by burst release of nisin and slowly sustained release of thymol. Synergistic antimicromibial properties against L. monocytogenes of thymol and nisin loaded by zein/SC particles in milk were also observed. The antimicrobial property of zein/SC particles could enhance microbial safety of food, giving this delivery system promising applications in food industry.

Herein, a new method was demonstrated for effective immobilization of the antibacterial peptide nisin on Grifola frondosa hydrophobin (HGFI), without the need of any additional complex reaction. Hydrophobin can self-assemble as a monolayer to form continuous negative-charged surfaces with enhanced wettability and biocompatibility. Adding nisin solution to such hydrophobin surface created antibacterial surfaces. The quantification analysis revealed that more nisin could be adsorbed on the HGFI-coated than to control polystyrene surfaces at different pH values. This suggested that electronic attraction and wettability may play important roles in this process. The transmission electron microscopy, atomic force microscopy and fourier transform infrared (FTIR) analysis indicated the adsorption mode of nisin on the HGFI film, i.e., hydrophobins served as an adhesive layer for binding charged peptides to interfaces. The antibacterial activity of the treated surface was investigated via counting, a nucleic acid release test, scanning electron microscopy, and biofilm detection. These results indicated the excellent antibacterial activity of nisin adsorbed on the HGFI-coated surfaces. The activity retention of adsorbed nisin was demonstrated by immersing the modified substrates in a flowed liquid condition.

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

Nisin is a bacteriocin that is globally employed as a biopreservative in food systems to control gram-positive, and some gram-negative, bacteria. Here we tested the bioactivity of nisin A-producing Lactococcus lactis NZ9700 and producers of bioengineered variants thereof against representatives of the gram-negative genus Thermus, which has been associated with the pink discoloration defect in cheese. Starting with a total of 73 nisin variant-producing Lactococcus lactis, bioactivity against Thermus was assessed via agar diffusion assays, and 22 variants were found to have bioactivity greater than or equal to that of the nisin A-producing control. To determine to what extent this enhanced bioactivity was attributable to an increase in specific activity, minimum inhibitory concentrations were determined using the corresponding purified form of these 22 nisin A derivatives. From these experiments, nisin M17Q and M21F were identified as peptides with enhanced antimicrobial activity against the majority of Thermus target strains tested. In addition, several other peptide variants were found to exhibit enhanced specific activity against a subset of strains.

Nisin is one of the most studied lantibiotics which are antimicrobial peptides. Nowadays the knowledge about the Nisin-modification system is profound and can be explored to express and modify lantibiotics with new or specific antimicrobial features. Here we highlight recent advances that include a strategy on bypassing natural occurring resistances against antimicrobial peptides.

Stem cell therapy aims to replace damaged with healthy functioning cells in congenital defects, tissue injuries, autoimmune disorders, and neurogenic degenerative diseases. Mesenchymal stem cells (MSCs) have been applied for transplantation, but the disadvantage of MSC is that low survival rate following transplantation, so each strategy protects MSCs from cell death and increasing survival is valuable for therapy. Scaffold-based cell applications are a new approach to optimize cell delivery for wounds to enhance engraftment and paracrine activity of therapeutic stem cells. Nisin has now been shown to have antimicrobial activity against both gram-positive and gram-negative disease-associated pathogens. Preconditioning with protective factors is one strategy for protecting cells from harmful conditions and could be to enhance the survival and recovery of injured tissues. The aim of this article was to evaluate the effect of poly-L-lactic acid (PLLA) and preconditioning MSCs with Nisin in vitro cell survival. Mesenchymal stem cells were cultured and preconditioned with Nisin in different concentrations. After the evaluation of the sub-lethal dose of Nisin by MTT, in the next step, they were exposed to H2O2 or serum deprivation. Survival and anti-inflammation effects were evaluated by MTT assay and real-time PCR. Furthermore, the protein expression of TGF-β1 and FGF-2 was performed by ELISA. The 250 and 500 IU/mL of Nisin led to a significant anti-apoptotic impact on MSCs. Cell viability and proliferation in MSC-Nisin-PLLA significantly increased in comparison to the MSCs exposed in H2O2 or serum deprivation in 250–500 IU/mL Nisin. The gene expression of IL-10, TGF-β, and FGF-2 was significantly up-regulated with Nisin treatment (p

Persister cells are a subpopulation of bacteria with the ability of survival when exposed to lethal doses of antibiotics, and are responsible for antibiotic therapy failure and infection recurrences. In this study, we investigated persister cell formation and the role of nisin in combination with antibiotics in reducing persistence in Listeria monocytogenes. We also examined the expression of toxin-antitoxin (TA) systems in persister cells of L. monocytogenes to gain a better understanding of the effect of TA systems on persister cell formation. To induce persistence, L. monocytogenes were exposed to high doses of different antibiotics over a period of 24 hr, and the expression levels of TA system was genes were measured 5 hr after the addition of antibiotics by the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) method. To investigate the effect of nisin, L. monocytogenes was exposed to a combination of nisin and antibiotics. According to our results, L. monocytogenes was highly capable of persister cell formation, and the combination of nisin and antibiotics resulted in reduced persistence. qRT-PCR results showed a significant increase in GNAT/RHH expression among the studied systems. Overall, our results demonstrated the potential of the combination of nisin and antibiotics in reducing persister cell formation, and emphasized the role of the GNAT/RHH system in bacterial persistence.

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. HIGHLIGHTS

Dehydroalanine (Dha) residues are attractive noncanonical amino acids that occur naturally in ribosomally synthesised and post‐translationally modified peptides (RiPPs). Dha residues are attractive targets for selective late‐stage modification of these complex biomolecules. In this work, we show the selective photocatalytic modification of dehydroalanine residues in the antimicrobial peptide nisin and in the proteins small ubiquitin‐like modifier (SUMO) and superfolder green fluorescent protein (sfGFP). For this purpose, a new water‐soluble iridium(III) photoredox catalyst was used. The design and synthesis of this new photocatalyst, [Ir(dF(CF3)ppy)2(dNMe3bpy)]Cl3, is presented. In contrast to commonly used iridium photocatalysts, this complex is highly water soluble and allows peptides and proteins to be modified in water and aqueous solvents under physiologically relevant conditions, with short reaction times and with low reagent and catalyst loadings. This work suggests that photoredox catalysis using this newly designed catalyst is a promising strategy to modify dehydroalanine‐containing natural products and thus could have great potential for novel bioconjugation strategies., Bio‐orthogonal modification of dehydroalanine residues in peptides and proteins is achieved by photoredox catalysis with a newly designed water‐soluble IrIII complex in aqueous media and a variety of zinc benzylsulfinates as reagents.

Resistance to antibiotics is an ongoing problem in the biomedical industry. Developing active, alternative drug therapies would reduce our reliance on antibiotics that induce resistance in micro-organisms. To date, bacteriocins and antimicrobial peptides have shown a positive outcome as antibiotic substitutes and synergists apart from phage therapy, antibodies and probiotics. Bacteriocins are proteinaceous antimicrobial peptides synthesized by lactic acid bacteria extensively used as bio-preservatives and alternative to traditional antibiotics to overcome the problem of drug-resistant pathogens. Nonetheless, the use of bacteriocins has several limitations such as limited antimicrobial spectrum, requiring high dose, sensitivity to proteolytic enzymes, etc. Nanoparticles are one of the promising area of research explored to improve antimicrobial spectrum of bacteriocins. This review therefore highlights the recent developments and research pertaining to use of nanoparticles and bacteriocin conjugates to tackle the resistance crisis as well as its applications in food industry.

Nisin is a promising therapeutic candidate because of its potent activity against Gram-positive bacteria. The present study aimed to describe the in vitro and in vivo antibacterial effects of nisin against Streptococcus suis, an important zoonotic pathogen. The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of nisin against different S. suis strains ranged from 0.12 to 4.0 μg/mL and from 0.25 to 8.0 μg/mL, respectively. Time-killing curve assays illustrated that nisin killed 100% of tested virulent S. suis strains within 4 h when used at 2× MIC, which indicates the rapid bactericidal activity of nisin against the bacteria. Transmission and scanning electron microscopy revealed that nisin destroyed S. suis cell membrane integrity and affected its cellular ultrastructure, including a significantly wrinkled surface, intracellular content leakage, and cell lysis. In addition, nisin inhibited biofilm formation by S. suis in a concentration-dependent manner and exhibited strong degrading activities against preformed biofilms. More importantly, nisin displayed antimicrobial activity against S. suis infection in vivo. Upon treatment with 5.0-10 mg/kg nisin solution, the survival rates of mice challenged with a lethal dose of virulent S. suis virulent ranged 87.5-100%. Nisin significantly decreased bacterial proliferation and translocation in the mouse spleen, brain, and blood. These results indicate that nisin has potential as a novel antimicrobial agent for the clinical treatment and prevention of infection caused by S. suis in animals.

Nisin, a food-grade antimicrobial peptide produced by lactic acid bacteria has been examined for its probable interaction with the human ACE2 (hACE2) receptor, the site where spike protein of SARS-CoV-2 binds. Among the eight nisin variants examined, nisin H, nisin Z, nisin U and nisin A showed a significant binding affinity towards hACE2, higher than that of the RBD (receptor binding domain) of the SARS-CoV-2 spike protein. The molecular interaction of nisin with hACE2 was investigated by homology modeling and docking studies. Further, binding efficiency of the most potent nisin H was evaluated through the interaction of hACE2:nisin H complex with RBD (receptor-binding domain) of SARS-CoV-2 and that of hACE2:RBD complex with nisin H. Here, nisin H acted as a potential competitor of RBD to access the hACE2 receptor. The study unravels for the first time that a globally used food preservative, nisin has the potential to bind to hACE2., Highlights • A food grade natural peptide nisin can interact with the human ACE2 receptor. • For the first time, we unraveled that a commonly used food preservative nisin can block ACE2 receptor. • Binding affinity of nisin was higher than that of spike protein of SARS-CoV-2 as determined by docking studies. • It was predicted that nisin competitively binds to the receptor over the spike protein. • It could be an effective and safest therapeutic against COVID-19 infection.

Here, we report a novel, “dry”, nano-aerosol-based, antimicrobial technology using engineered water nanostructures (EWNSs) for leafy vegetable disinfection. These EWNS based nano-sanitizers are synthesized using a combined process of electrospray and ionization of aqueous solutions of generally recognized as safe active ingredients (AIs). A novel application of the EWNS to vegetable surfaces was carried out in this study. The EWNS-based nano-sanitizers were synthesized using an AI cocktail (10% hydrogen peroxide, 1% citric acid, 0.1% lysozyme and 0.0025% nisin), and their efficacy against foodborne pathogen surrogates and plant pathogens was assessed using a leafy vegetable model, namely spinach (Spinacia oleracea). The EWNSs were directed onto a coupon cut from spinach leaves, which were inoculated with microorganisms relevant to food safety, such as E. coli. It was shown that a 2 min exposure to EWNS-based nano-sanitizers significantly (P 0.05) in color or surface pH of spinach was found after 15 minutes of exposure. In summary, EWNS based nano-sanitizers are highly effective against foodborne pathogen surrogates and plant pathogens using only nanograms of nature-derived active ingredients and without an impact on produce quality.