Influences on the antimicrobial activity of surface-adsorbed nisin

The efficacy of the antimicrobial peptide nisin was examined after adsorption to silica surfaces. Three protocols were used to evaluate nisin's activity against adhered cells ofListeria monocytogenes: bioassay usingPediococcus pentosaceous FBB 61-2 as the sensitive indicator strain; visualization and enumeration of cells by microscopic image analysis; and viability of adhered cells as determined by lodonitrotetrazolium violet uptake and crystallization. The activity of adsorbed nisin was highly dependent upon conditions of adsorption. The highest antimicrobial activity of adsorbed nisin occurred with high concentrations of nisin (1.0 mg ml^–1) and brief contact times (1 h) on surfaces of low hydrophobicity. Sequential adsorption of a second protein (-lactoglobulin or bovine serum albumin) onto surfaces consistently resulted in decreased nisin activity. These data provide direction for the development of applications to limit microbial attachment on food contact surfaces through the use of adsorbed antimicrobial peptides.     

In vitro inhibition of microbial flora of fish by nisin and lactoperoxidase system

AIMS: To test the antimicrobial effects of nisin and lactoperoxidase system (LP system) against sardines flora. This study is part of a programme designed to investigate the preservability of fish using these inhibitors as potential biopreservatives. METHODS AND RESULTS: Antimicrobial effects of nisin and LP system alone or in combination were tested by the agar diffusion method against bacterial strains isolated from sardines (Sardina pilchardus). Nisin inhibited only Gram-positive bacteria, whereas LP system inhibited all strains studied. The combination of nisin (100 IU ml-1) and LP system (10 level) was significantly more effective than LP system or nisin alone against all strains, excepting Aeromonas salmonicida subsp. salmonicida and Vibrio alginolyticus. CONCLUSION: These results clearly demonstrated the efficiency of LP System-nisin combination for inhibiting spoilage flora of fish. SIGNIFICANCE AND IMPACT OF THE STUDY: Because LP system has a broad activity spectrum, it may be an interesting additional hurdle to improve the safety of food preservation by nisin. Combination of nisin and LP system could be of great interest as biopreservatives for fish and fish products.     

Improved method for quantification of the bacteriocin nisin

Nisin, a bacteriocin produced by Lactococcus lactis subsp. lactis , is used in some types of food preservation due to its inhibitory action on Grampositive bacteria and their spores. A commonly used agar diffusion bioassay technique for quantification of nisin in food samples was modified to increase its sensitivity, accuracy and precision. Several variables were evaluated. Results showed Micrococcus luteus as the most sensitive organism tested, a lower agar concentration (0·75% compared 1·5%) increased the sensitivity of the assay (21% improvement over standard method), and incorporation of 1% Na₂HPO₄ buffer into the bioassay agar made it possible to prevent false inhibitory zones from developing due to the low pH of the test solutions. This resulted in a 57% improvement in accuracy and a 12% improvement in precision compared to the standard method     

Suppression of Listeria monocytogenes colonization following adsorption of nisin onto silica surfaces.

Nisin is an antimicrobial peptide proven to be an effective inhibitor of gram-positive bacteria. It is known that nisin can adsorb to various surfaces and still retain much of its original activity (M. A. Daeschel, J. McGuire, and H. Al-Makhlafi, J. Food Prot. 55:731-735, 1992). In this study, nisin films were allowed to form on silanized silica surfaces and then exposed to medium containing Listeria monocytogenes. Representative areas were selected from each surface, and images of resident listeriae were obtained at 4-h intervals for 12 h. During this time, cells on surfaces that had been in contact with a high concentration of nisin (1.0 mg/ml) exhibited no signs of growth and many displayed evidence of cellular deterioration. Surfaces treated with a lower concentration of nisin (0.1 mg/ml) had a smaller degree of inhibition. In contrast, both protein-free surfaces and those with films of heat-inactivated nisin allowed attached L. monocytogenes cells to grow and reproduce. These studies, when repeated with a nisin-resistant strain of L. monocytogenes, resulted in no inhibition of growth on surfaces with adsorbed nisin. The bactericidal effect of adsorbed nisin was also studied with iodonitrotetrazolium violet, a tetrazolium salt, which is reduced to a red formazan crystal by viable bacteria. Crystals were visible in 95% of the cells adhered to control surfaces but were present in less than 20% of the cells on surfaces with adsorbed nisin. These data indicate that adsorbed nisin may have potential for use as a food grade antimicrobial agent on food contact surfaces.     

Use of a sensitive EnVision +-based detection system for Western blotting: avoidance of streptavidin binding to endogenous biotin and biotin-containing proteins in kidney and other tissues

Western blotting remains a central technique in confirming identities of proteins, their quantitation and analysis of various isoforms. The biotin-avidin/streptavidin system is often used as an amplification step to increase sensitivity but in some tissues such as kidney, "nonspecific" interactions may be a problem due to high levels of endogenous biotin-containing proteins. The EnVision system, developed for immunohistochemical applications, relies on binding of a polymeric conjugate consisting of up to 100 peroxidase molecules and 20 secondary antibody molecules linked directly to an activated dextran backbone, to the primary antibody. This study demonstrates that it is also a viable and sensitive alternative detection system in Western blotting applications.     

Development of a bioactive packaging cellophane using Nisaplin as biopreservative agent

AIMS: Production of a nisin-containing cellophane-based coating to be used in the packaging of chopped meat. METHODS AND RESULTS: The adsorption of nisin to cellophane 'P' type surface was studied at 8, 25, 40 and 60 degrees C using different concentrations of nisin. Then, the antimicrobial activity of adsorbed nisin to cellophane surface was determined in fresh veal meat for effectiveness in reducing the total aerobic bacteria. The adsorption of nisin to cellophane was higher at 8 degrees C. The developed bioactive cellophane reduced significantly the growth of the total aerobic bacteria (by ca 1.5 log units) through 12 days of storage at 4 degrees C. CONCLUSIONS: Bioactive cellophane packaging could be used for controlling the microbial growth in chopped meat. SIGNIFICANCE AND IMPACT OF THE STUDY: Nisin-adsorbed bioactive cellophane would result in an extension of the shelf life of chopped meat under refrigeration temperatures.     

Effects of prebiotic oligosaccharides and trehalose on growth and production of bacteriocins by lactic acid bacteria

AIMS: To investigate the effects of two prebiotics and trehalose on the production of bacteriocins. METHODS AND RESULTS: Four carbohydrates [dextrose, fructo-oligosaccharides (FOS), raffinose, and trehalose] were used as the sole carbon source in a simple broth. Five bacteriocin-producing strains of bacteria, including those producing nisin, enteriocin, and other bacteriocins, were used, and their inhibitory activities when grown on each carbohydrate were determined. The inhibitory activity assay was performed using the agar well diffusion method, and Lactobacillus sakei JCM 1,157(T) was used as the indicator strain. Effective enhancement of bacteriocin production was observed with FOS and trehalose incubation. CONCLUSIONS: The results suggest that FOS and trehalose can effectively enhance the production of the five kinds of bacteriocins evaluated in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: This study offers useful information for not only a new application of FOS and trehalose, but also the potential improvement of food preservation.     

Production and utilization of polyclonal antibodies against nisin in an ELISA and for immuno-location of nisin in producing and sensitive bacterial strains

Specific nisin polyclonal antibodies (PAb) were produced in rabbits using nisin Z produced by Lactococcus lactis subsp. lactis biovar diacetylactis UL 719. Antisera were obtained from white female New Zealand rabbits that were first immunized with a nisin Z-keyhole limpet haemocyanin conjugate and boosted with free nisin Z. Nisin-specific PAb were purified by affinity chromatography with a yield of 15 mg specific antinisin 100 ml-1 serum. The detection limit of the ELISA test for nisin Z was 0.75 ng ml-1 in buffer but was 1.7 and 3.5 ng ml-1 in milk and complex media broth spiked (5, 10, 20 microg ml-1) with nisin Z, respectively. In nisin Z-spiked samples, the average concentration was between 90 and 107% of actual added amount. In contrast, when the bioassay (microtitration method) was used, only 50-63% of nisin Z biological activity could be detected. In addition, the affinity-purified nisin PAb, antirabbit IgG gold conjugate and transmission electron microscopy were successfully used to locate nisin Z on producing cells and to observe its bactericidal effects against sensitive cells.     

Immunodot detection of nisin Z in milk and whey using enhanced chemiluminescence

A highly specific antisera was produced in New Zealand white rabbits against nisin Z, a 3400 Da bacteriocin produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719. A dot immunoblot assay was then developed to detect nisin Z in milk and whey. As few as 1·5 10⁻¹ international units per ml (IU ml⁻¹), corresponding to 0·003 μg ml⁻¹ of pure nisin Z, were detected in carbonate-bicarbonate buffer within 6 h using chemiluminescence. When milk and whey samples were tested, approximately 0·155 μg ml⁻¹ (7·9 IU ml⁻¹) of nisin Z was detected. The detection limit obtained was lower than that of traditional methods including microtitration and agar diffusion.     

Properties of nisin Z and distribution of its gene, nisZ, in Lactococcus lactis.

Two natural variants of the lantibiotic nisin that are produced by Lactococcus lactis are known. They have a similar structure but differ in a single amino acid residue at position 27; histidine in nisin A and asparagine in nisin Z (J.W.M. Mulders, I.J. Boerrigter, H.S. Rollema, R.J. Siezen, and W.M. de Vos, Eur. J. Biochem, 201:581-584, 1991). The nisin variants were purified to apparent homogeneity, and their biological activities were compared. Identical MICs of nisin A and nisin Z were found with all tested indicator strains of six different species of gram-positive bacteria. However, at concentrations above the MICs, with nisin Z the inhibition zones obtained in agar diffusion assays were invariably larger than those obtained with nisin A. This was observed with all tested indicator strains. These results suggest that nisin Z has better diffusion properties than nisin A in agar. The distribution of the nisin variants in various lactococcal strains was determined by amplification of the nisin structural gene by polymerase chain reaction followed by direct sequencing of the amplification product. In this way, it was established that the nisZ gene for nisin Z production is widely distributed, having been found in 14 of the 26 L. lactis strains analyzed.     

Evaluation of agar diffusion bioassay for nisin quantification

The agar diffusion bioassay is the most widely used method for the quantification of nisin, due to its high sensitivity, simplicity, and cost-effectiveness. This method is based on the measurement of the inhibition zone produced in nisin-sensitive microorganisms. The size of the zone is affected by many factors, such as nisin-sensitive strain, amount of added agar and surfactant, and pre-diffusion step. This research aims to evaluate the effects of nisin-sensitive strains and pre-diffusion on the accuracy and precision of nisin quantification. Three strains of nisin-sensitive microorganisms (Micrococcus luteus, Lactobacillus sakei, Brochothrix thermosphacta) were tested along with three different incubation processes. The best combination was the method using L. sakei as an indicator strain with pre-diffusion at 4 °C for 24 h. Compared with M. luteus and B. thermosphacta, L. sakei gave more accurate and reproducible results. Moreover, the pre-diffusion step resulted in larger inhibition zones and more precise results. Finally, the best combination was validated and compared with the method that is usually used and the result showed that the method using L. sakei with pre-diffusion gave more accurate and precise results.     

Novel method to extract large amounts of bacteriocins from lactic acid bacteria.

Antimicrobial peptides, bacteriocins, produced by lactic acid bacteria were adsorbed on the cells of producing strains and other gram-positive bacteria. pH was a crucial factor in determining the degree of adsorption of these peptides onto cell surfaces. In general, between 93 and 100% of the bacteriocin molecules were adsorbed at pHs near 6.0, and the lowest (< or = 5%) adsorption took place at pH 1.5 to 2.0. On the basis of this property, a novel isolation method was developed for bacteriocins from four genera of lactic acid bacteria. By using this method we made preparations of pediocin AcH, nisin, sakacin A, and leuconocin Lcm1 that were potent and concentrated. This method produced a higher yield than isolation procedures, which rely on precipitation of the bacteriocins from the cell-free culture liquor. It is simple and can be used to produce large quantities of bacteriocins from lactic acid bacteria to be used as food biopreservatives.     

Novel method to extract large amounts of bacteriocins from lactic acid bacteria.

Antimicrobial peptides, bacteriocins, produced by lactic acid bacteria were adsorbed on the cells of producing strains and other gram-positive bacteria. pH was a crucial factor in determining the degree of adsorption of these peptides onto cell surfaces. In general, between 93 and 100% of the bacteriocin molecules were adsorbed at pHs near 6.0, and the lowest (< or = 5%) adsorption took place at pH 1.5 to 2.0. On the basis of this property, a novel isolation method was developed for bacteriocins from four genera of lactic acid bacteria. By using this method we made preparations of pediocin AcH, nisin, sakacin A, and leuconocin Lcm1 that were potent and concentrated. This method produced a higher yield than isolation procedures, which rely on precipitation of the bacteriocins from the cell-free culture liquor. It is simple and can be used to produce large quantities of bacteriocins from lactic acid bacteria to be used as food biopreservatives.     

Nisin adsorption on hydrophilic and hydrophobic surfaces: evidence of its interactions and antibacterial activity

Study of peptides adsorption on surfaces remains a current challenge in literature. A complementary approach, combining X‐ray photoelectron spectroscopy (XPS) and time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) was used to investigate the antimicrobial peptide nisin adsorption on hydrophilic and hydrophobic surfaces. The native low density polyethylene was used as hydrophobic support and it was grafted with acrylic acid to render it hydrophilic. XPS permitted to confirm nisin adsorption and to determine its amount on the surfaces. ToF‐SIMS permitted to identify the adsorbed bacteriocin type and to observe its distribution and orientation behavior on both types of surfaces. Nisin was more oriented by its hydrophobic side to the hydrophobic substrate and by its hydrophilic side to the outer layers of the adsorbed peptide, in contrast to what was observed on the hydrophilic substrate. A correlation was found between XPS and ToF‐SIMS results, the types of interactions on both surfaces and the observed antibacterial activity. Such interfacial studies are crucial for better understanding the peptides interactions and adsorption on surfaces and must be considered when setting up antimicrobial surfaces. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Improved antimicrobial activity of nisin-incorporated polymer films by formulation change and addition of food grade chelator

AIMS: The following polymers were developed: polyethylene (PE), a PE and polyethylene oxide (70% PE and 30% PEO; PE + PEO) blend, PE and nisin (PE + nisin), PE, nisin, and EDTA (PE + nisin + EDTA), and PE + PEO with nisin (PE + PEO + nisin). METHODS AND RESULTS: Of the polymers tested, PE and PE + PEO did not exhibit any antimicrobial activity against Brochothrix thermosphacta (BT); however, PE + nisin, PE + nisin + EDTA, and PE + PEO + nisin did. Beef surfaces were experimentally inoculated with 3.50 log10 cfu/cm2 of BT, vacuum packaged with each of the five polymers, and held at 4 degrees C for 21 d. After 3 d at 4 degrees C, BT was reduced > 1.70 log(10) by PE + nisin and > 3.50 log(10) with PE + nisin + EDTA or PE + PEO + nisin. By 21 d at 4 degrees C, BT was reduced to 0.30 log(10) cfu/cm(2) when treated with PE + PEO + nisin. CONCLUSION: It appears that PE + PEO + nisin or PE + nisin + EDTA were more effective for reducing BT, as compared to polymers composed of PE + nisin. SIGNIFICANCE AND IMPACT OF THE STUDY: Nisin-incorporated polymers may control the growth of undesirable bacteria, thereby extending the shelf life and possibly enhancing the microbial safety of meats.     

Bioassay of Mildiomycin and a Rapid, Cost-Effective Agar Plug Method for Screening High-yielding Mutants of Mildiomycin

Summary A simple, reliable and low-cost agar diffusion bioassay for quantitative determination of mildiomycin was developed using a strain of Rhodotorula rubra AS 2.166 as the indicator organism and potato dextrose agar at pH 7.0 as the test medium. With equivalent precision and accuracy to HPLC analysis, this method was applied to analyse mildiomycin in complex culture broth during the fermentation process. A modified agar plug method based on the bioassay was constructed for rapid and efficient screening of high-yielding mutants of mildiomycin. Within four weeks, a high production strain, the mildiomycin productivity of which was 75.5% higher than the parent strain, was obtained from 15,000 mutants.     

Bioassay for nisin in milk, processed cheese, salad dressings, canned tomatoes, and liquid egg products

A sensitive nisin quantification bioassay was constructed, based on Lactococcus lactis chromosomally encoding the nisin regulatory proteins NisK and NisR and a plasmid with a green fluorescent protein (GFP) variant gfp(uv) gene under the control of the nisin-inducible nisA promoter. This strain, LAC275, was capable of transducing the signal from extracellular nisin into measurable GFPuv fluorescence through the NisRK signal transduction system. The LAC275 cells detected nisin concentrations of 10 pg/ml in culture supernatant, 0.2 ng/ml in milk, 3.6 ng/g in processed cheese, 1 ng/g in salad dressings and crushed, canned tomatoes, and 2 ng/g in liquid egg. This method was up to 1,000 times more sensitive than a previously described GFP-based nisin bioassay. This new assay made it possible to detect significantly smaller amounts of nisin than the presently most sensitive published nisin bioassay based on nisin-induced bioluminescence. The major advantage of this sensitivity was that foods could be extensively diluted prior to the assay, avoiding potential inhibitory and interfering substances present in most food products.     

Bioassay for Nisin in Milk, Processed Cheese, Salad Dressings, Canned Tomatoes, and Liquid Egg Products

A sensitive nisin quantification bioassay was constructed, based on Lactococcus lactis chromosomally encoding the nisin regulatory proteins NisK and NisR and a plasmid with a green fluorescent protein (GFP) variant gfp_uv gene under the control of the nisin-inducible nisA promoter. This strain, LAC275, was capable of transducing the signal from extracellular nisin into measurable GFPuv fluorescence through the NisRK signal transduction system. The LAC275 cells detected nisin concentrations of 10 pg/ml in culture supernatant, 0.2 ng/ml in milk, 3.6 ng/g in processed cheese, 1 ng/g in salad dressings and crushed, canned tomatoes, and 2 ng/g in liquid egg. This method was up to 1,000 times more sensitive than a previously described GFP-based nisin bioassay. This new assay made it possible to detect significantly smaller amounts of nisin than the presently most sensitive published nisin bioassay based on nisin-induced bioluminescence. The major advantage of this sensitivity was that foods could be extensively diluted prior to the assay, avoiding potential inhibitory and interfering substances present in most food products.     

Impact of pectin esterification on the antimicrobial activity of nisin‐loaded pectin particles

The relationship between pectin structure and the antimicrobial activity of nisin‐loaded pectin particles was examined. The antimicrobial activity of five different nisin‐loaded pectin particles, i.e., nisin‐loaded high methoxyl pectin, low methoxyl pectin, pectic acid, dodecyl pectin with 5.4 and 25% degree of substitution were tested in the pH range of 4.0–7.0 by agar‐diffusion assay and agar plate count methods. It was found that the degree of esterification of carboxyl group of galacturonic acid in pectin molecule is important for the antimicrobial activity of nisin‐loaded pectin particles. Nisin‐loaded particles prepared using pectic acid or the pectin with low degree of esterification exhibit higher antimicrobial activity than nisin‐loaded high methoxyl pectin particles. Pectins with free carboxyl groups or of low degree of esterification are the most suitable for particles preparation. Moreover, nisin‐loaded pectin particles were active at close to neutral or neutral pH values. Therefore, they could be effectively applied for food preservation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:245–251, 2017     

Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy.

The theoretical basis of a new technique for measuring equilibrium adsorption/desorption kinetics and surface diffusion of fluorescent-labeled solute molecules at solid surfaces has been developed. The technique combines total internal reflection fluorescence (TIR) with either fluorescence photobleaching recovery (FPR) or fluorescence correlation spectroscopy (FCS). A laser beam totally internally reflects at a solid/liquid interface; the shallow evanescent field in the liquid excites the fluorescence of surface adsorbed molecules. In TIR/FPR, adsorbed molecules are bleaching by a flash of the focused laser beam; subsequent fluorescence recovery is monitored as bleached molecules exchange with unbleached ones from the solution or surrounding nonilluminated regions of the surface. In TIR/FCS, spontaneous fluorescence fluctuations due to individual molecules entering and leaving a well-defined portion of the evanescent field are autocorrelated. Under appropriate experimental conditions, the rate constants and surface diffusion coefficient can be readily obtained from the TIR/FPR and TIR/FCS curves. In general, the shape of the theoretical TIR/FPR and TIR/FCS curves depends in a complex manner upon the bulk and surface diffusion coefficients, the size of the iluminated or observed region, and the adsorption/desorption/kinetic rate constants. The theory can be applied both to specific binding between immobilized receptors and soluble ligands, and to nonspecific adsorption processes. A discussion of experimental considerations and the application of this technique to the adsorption of serum proteins on quartz may be found in the accompanying paper (Burghardt and Axelrod. 1981. Biophys. J. 33:455).