Peptide P34 Research Articles

Evidence that protein corona reduces the release of antimicrobial peptides from polymeric nanocapsules in milk

The antimicrobial peptide produced by Bacillus velesensis P34 has a broad activity against Gram-positive bacteria, showing potential as natural food preservative. In this work, nanocapsules (NCs) containing the peptide P34 were produced using the polymers poly-ε-caprolactone (PCL) or Eudragit RS-100 (EUD), and their antimicrobial activities were assessed evaluating L. monocytogenes growth in synthetic media, milk and isolated milk proteins. As results, cationic and anionic nanocapsules were obtained, with zeta potential ranging from +15 to +28 mV for EUD and around −19 mV for PCL, and average diameter in the range of 104–130 nm and 224–245 nm, respectively. In the antimicrobial tests, only the P34-EUD NCs presented activity against L. monocytogenes in BHI broth, possibly due to the EUD high swelling and permeability properties, as compared with PCL. In whole and skimmed milk, the P34-EUD NCs caused no inhibition of L. monocytogenes growth, due to a possible interaction of casein proteins with the NCs surface resulting in protein corona formation, which interfered with the antimicrobial peptide release. Therefore, the application of polymeric NCs as antimicrobial delivery systems in foods could be limited by the polymer type, and the adhesion of specific matrix proteins that could form protein corona, reducing the bioactive compound release.

Inhibition of equine arteritis virus by an antimicrobial peptide produced by Bacillus sp. P34

ABSTRACT P34 is an antimicrobial peptide produced by Bacillus sp. P34, isolated from the intestinal contents of a fish from the Amazon basin. This peptide showed antibacterial properties against Gram-positive and Gram-negative bacteria and was characterized as a bacteriocin like substance. It was demonstrated that the peptide P34 exhibited antiviral activity against feline herpesvirus type 1 in vitro. The aim of this work was to evaluate P34 for its antiviral properties in vitro, using RK 13 cells, against the equine arteritis virus, since it has no specific treatment and a variable proportion of stallions may become persistently infected. The results obtained show that P34 exerts antiviral and virucidal activities against equine arteritis virus, probably in the viral envelope. The antiviral assays performed showed that P34 reduces significantly the viral titers of treated cell cultures. The mechanism of action of P34 seems to be time/temperature-dependent. This peptide tends to be a promising antiviral compound for the prevention and treatment of arteriviral infections since it has a high therapeutic index. However, more detailed studies must be performed to address the exact step of viral infection where P34 acts, in order to use this peptide as an antiviral drug in vivo in the future.

Activity of the antimicrobial peptide P34 against bovine alphaherpesvirus type 1

ABSTRACT: Previous studies have demonstrated the antimicrobial activity of the peptide P34. In this study, the antiviral potential of P34 and the in vitro mechanism of action were investigated against bovine alphaherpesvirus type 1 (BoHV1). P34 exhibited low toxicity, a high selectivity index (22.9) and a percentage of inhibition of up to 100% in MDBK cells. Results from antiviral assays indicated that P34 did not interact with cell receptors, but it was able to inhibit the viral penetration immediately after pre-adsorption. In addition, BoHV1 growth curve in MDBK cells in the presence of P34 revealed a significant reduction in virus titer only 8h post-infection, also suggesting an important role at late stages of the replicative cycle. Virucidal effect was observed only in cytotoxic concentrations of the peptide. These findings showed that the antimicrobial peptide P34 may be considered as a potential novel inhibitor of in vitro herpesviruses and must encourage further investigation of its antiherpetic activity in animal models as well as against a wide spectrum of viruses.

INFLUENCE OF PEPTIDE P34 ON GENE EXPRESSION OF LISTERIA MONOCYTOGENES AND LISTERIA SEELEGERI

<p><strong>Objective: </strong>Investigate the influence of the antimicrobial peptides P34 and nisin on the expression of genes associated with components of the cell surface of <em>Listeria monocytogenes</em> and <em>Listeria seeligeri</em>.</p><p><strong>Methods: </strong>Antimicrobial activity was determined by addition of peptide P34 and nisin (12.5 µg/ml) onto Brain Heart Infusion agar (BHI) plates previously inoculated with indicator strains (<em>L. monocytogenes</em> ATCC 7644 or <em>L. seeligeri </em>AC 82/4) after incubation for 24 h at 37 °C or 240 h at 4 °C. Ribonucleic acid (RNA) was directly extracted from bacterial colonies at the border of the inhibition zones, and the expression levels of genes D-alanine-D-alanyl carrier protein ligase<em> (dltA), </em>putative phospholipid lysinylation<em> </em>(<em>Imo 1695</em>)<em> </em>and EIIAB<sup>Man</sup> of mannose-specific PTS<em> </em>(<em>mptA</em>) were determined using real-time PCR.</p><p><strong>Results</strong><strong>: </strong>A non-significant increase in the levels of transcription of genes <em>dltA, Imo1695 </em>and<em> mptA</em> was observed for <em>L. monocytogenes </em>treated with peptide P34 or nisin. Both peptides caused a similar decrease in <em>dltA</em> gene expression in <em>L. seeligeri</em>. The expression of gene <em>Imo1695</em> significantly decreased (about 2000-fold) after treatment with the peptide P34 at 37 °C, while at 4 °C a reduction of 12-fold and 5-fold was detected for P34 and nisin, respectively. A significant decrease in <em>mptA</em> gene expression was observed by exposition to peptide P34 (31.872-fold) and nisin (16.047-fold) for 24 h at 37 °C.</p><p><strong>Conclusion: </strong>The results suggest that both peptide P34 and nisin influence the expression of genes related with the cell-surface/cell-membrane structure of <em>L. seeligeri</em> and in lesser extent <em>L. monocytogenes</em>.</p>

ANTIMICROBIAL PEPTIDE P34 INFLUENCES GENE EXPRESSION OF LISTERIA MONOCYTOGENES GROWING IN SOFT CHEESE

Objective: To evaluate whether antimicrobial substances produced by autochthonous lactic acid bacteria (LAB) from Minas Frescal cheese are able to enhance the activity of bacteriocin P34 against Listeria monocytogenes and investigate the influence of P34 in specific gene expression of this bacterium after the inoculation in Minas Frescal cheese.Methods: Bacillus sp. P34 and L. monocytogenes ATCC 7644 were used in this study. The antimicrobial peptide P34 was purified and applied (0, 800 or 6400 AU/ml) to cheese surface before inoculation with L. monocytogenes. Antimicrobial activity and synergism were detected using the agar diffusion technique. Expression levels of D-Alanine-D-alanyl carrier protein ligase (dltA), Putative phospholipid lysinylation (Imo 1695) and EIIABMan of mannose-specific PTS (mptA) mRNAs in bacteriocin-treated L. monocytogenes growing in Minas Frescal cheese were determined using real-time PCR.Results: The peptide P34 showed increased antilisterial activity when combined with culture supernatants of some selected LAB isolated from Minas Frescal cheese. The addition of peptide P34 to cheese caused a decrease of up to 3 log cycles in viable counts of artificially inoculated L. monocytogenes. The influence of peptide P34 on the expression of genes associated with components of the cell surface of L. monocytogenes was investigated by real-time PCR. A significant increase in the expression of the genes dltA, Imo 1695 and mptA was observed after 96 h in the presence of peptide P34.Conclusion: These results suggest that the peptide P34 influences the expression of genes involved in D-alanylation of teichoic acids and lipoteichoic acids and lysination of the cell membrane of phospholipids.

Liquid–liquid extraction of antimicrobial peptide P34 by aqueous two-phase and micellar systems

ABSTRACTAntimicrobial peptide P34 is a promising biopreservative for utilization in the food industry. In this work, aqueous biphasic systems (ABS) and aqueous biphasic micellar systems (ABMS) were studied as prestep for purification of peptide P34. The ABS was prepared with polyethylene glycol (PEG) and inorganic salts and the ABMS with Triton X-114 was chosen as the phase-forming surfactant. Results indicate that peptide P34 partitions preferentially to PEG-rich phase and extraction with ammonium sulfate [(NH4)2SO4], yielding a 75% recovery of the antimicrobial activity, specific activity of 1,530 antimicrobial units per mg of protein, and purification fold of 2.48. Protein partition coefficient and partition coefficient for the biological activity with (NH4)2SO4 system were 0.48 and 64, respectively. Addition of sodium chloride did not affect recovery, but decreased protein amount in the PEG-rich phase, indicating a higher partition of biomolecules. ABMS did not yield good recovery of antimicrobial activity. Purification fold using PEG–(NH4)2SO4 and 1.0 mol l−1 sodium chloride was twice higher than that obtained by conventional protocol, indicating a successful utilization of ABS as a step for purification of peptide P34.

Antiviral activity of a Bacillus sp. P34 peptide against pathogenic viruses of domestic animals.

P34 is an antimicrobial peptide produced by a Bacillus sp. strain isolated from the intestinal contents of a fish in the Brazilian Amazon basin with reported antibacterial activity. The aim of this work was to evaluate the peptide P34 for its in vitro antiviral properties against canine adenovirus type 2 (CAV-2), canine coronavirus (CCoV), canine distemper virus (CDV), canine parvovirus type 2 (CPV-2), equine arteritis virus (EAV), equine influenza virus (EIV), feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1). The results showed that the peptide P34 exhibited antiviral activity against EAV and FHV-1. The peptide P34 inhibited the replication of EAV by 99.9% and FHV-1 by 94.4%. Virucidal activity was detected only against EAV. When P34 and EAV were incubated for 6 h at 37 °C the viral titer reduced from 10(4.5) TCID50 to 10(2.75) TCID50, showing a percent of inhibition of 98.6%. In conclusion, our results demonstrated that P34 inhibited EAV and FHV-1 replication in infected cell cultures and it showed virucidal activity against EAV. Since there is documented resistance to the current drugs used against herpesviruses and there is no treatment for equine viral arteritis, it is advisable to search for new antiviral compounds to overcome these infections.

Antimicrobial activity of chitosan films containing nisin, peptide P34, and natamycin

The antimicrobial effect of edible chitosan films containing nisin, peptide P34, and natamycin was investigated. The activity of chitosan films was tested against Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella enteritidis, Clostridium perfringens, Lactobacillus acidophilus, Aspergillus phoenicis, and Penicillium stoloniferum. The addition of nisin and P34 significantly increased the activity against bacteria. Films containing natamycin showed similar inhibition than those with chitosan alone. Chitosan films were effective to control microbial growth in minimally processed pear. Fourier transform infrared spectra of the films were similar, indicating the absence of relevant interaction between the polymer and the antimicrobial agent. The films were also analyzed by scanning electron microscopy to visualize the surface topography. Chitosan films were rough and heterogeneous. The incorporation of antimicrobials agents to edible chitosan films or covering may be a feasible and attractive method for food biopreservation.

Evaluation of the immunogenicity and in vivo toxicity of the antimicrobial peptide P34

Immunogenicity and toxicity of antimicrobial peptide P34 were evaluated in vivo. BALB/c mice were inoculated intraperitoneally with peptide P34 alone and associated with Freund's adjuvant. For acute toxicity testing, different concentrations of the peptide P34 (82.5, 165.0, 247.5 and 330.0mg/kg) were orally administered. To evaluate the sub-chronic toxicity the tested dose of 0.825mg/kg/day of the peptide P34 or nisin were administered for 21 days. There were no hypersensitivity reactions or significant increase in antibody titer during the immunogenicity experiment or death of animals during the acute or sub-chronic toxicity tests. The LD50 was higher than 332.3±0.76mg/kg. No significant changes in serum biochemical parameters were observed in the animals treated with the peptide P34 unlike nisin-treated group showed a significant increase in alanine transaminase levels in comparison to controls. The group treated with 0.825mg/kg/day of nisin showed histological changes in the spleen, skin and liver. In the group treated with peptide P34 histological changes in the spleen were observed, with the presence of megakaryocytes. Few studies report the use of animal models to evaluate the in vivo toxicity of antimicrobial peptides and such investigation is an essential step to ensure it safe use in foods.

Kinetic and thermodynamic study of thermal inactivation of the antimicrobial peptide P34 in milk

The knowledge on thermal inactivation of biopreservatives in a food matrix is essential to allow their proper utilisation in food industry, enabling the reduction of heating times and optimisation of heating temperatures. In this work, thermal inactivation of the antimicrobial peptide P34 in skimmed and fat milk was kinetically investigated within the temperature range of 90–120°C. The inactivation kinetic follows a first-order reaction with k-values between 0.071 and 0.007min−1 in skimmed milk, and 0.1346 and 0.0119min−1 in fat milk. At high temperatures, peptide P34 was less resistant in fat milk, with a significant decrease in residual activity as compared with skimmed milk. At temperatures below 110°C, the fat globules seem to have protective effect to the peptide P34. Results suggest that peptide P34 is heat stable in milk with activation energy of 90kJmol−1 in skimmed milk and 136kJmol−1 in fat milk.

Antimicrobial Activity of Peptide P34 During Thermal Processing

In the present work, the influence of pH and sodium chloride concentration on thermal stability of antimicrobial peptide P34 was evaluated under different time–temperature conditions by a 22 factorial design experiment. At sterilization conditions (121 °C for 20 min), maximum retention (36%) was obtained at pH between 5.5 and 8.5 and sodium chloride concentration between 0.4 and 0.75 mol/l. For boiling conditions (100 °C for 20 min), antimicrobial activity was about 100% combining pH between 6.0 and 8.0 and salt concentration in the range of 0.65 to 1 mol/l. At low temperature pasteurization conditions (30 min at 65 °C), antimicrobial activity was not affected within the pH range from 5.0 to 8.0. For the three time–temperature conditions tested, the antimicrobial activity was minimal at more acidic or alkaline pH. Sodium chloride concentration of 0.65 mol/l increased thermostability of the peptide P34. Combination of sodium chloride and slight alkaline pH may increase the stability of peptide P34, which is essential to the proper utilization of bacteriocins in food industry.

Antilisterial activity and stability of nanovesicle-encapsulated antimicrobial peptide P34 in milk

Bacillus sp. P34, a strain isolated from aquatic environments of Brazilian Amazon basin, produces a bacteriocin-like substance (BLS) which was encapsulated in nanovesicles prepared from partially purified soy lecithin. The efficiency of free and encapsulated BLS P34 to control the development of L. monocytogenes and maintenance of antimicrobial activity was assessed over time in milk. The antimicrobial activity of free and encapsulated BLS P34 decreased approximately 50% after 4 days of storage (<4 °C) in skim and whole milk. After this period there was not significant loss of activity up to 21 days. The viable counts of Listeria monocytogenes in skim and whole milk containing 3200 AU/ml of free or encapsulated BLS P34 were always lower than those observed in controls without bacteriocin at both 30 °C and 7 °C. At 1600 AU/ml concentration, free and encapsulated BLS P34 were inhibitory to L. monocytogenes in skim milk, when compared with the control at 7 days. Nanovesicle-encapsulated and free BLS P34 shows potential use as biopreservative for application in milk-derived products.

Nanovesicle encapsulation of antimicrobial peptide P34: physicochemical characterization and mode of action on Listeria monocytogenes

Antimicrobial peptide P34, a substance showing antibacterial activity against pathogenic and food spoilage bacteria, was encapsulated in liposomes prepared from partially purified soybean phosphatidylcholine, and their physicochemical characteristics were evaluated. The antimicrobial activity was estimated by agar diffusion assay using Listeria monocytogenes ATCC 7644 as indicator strain. A concentration of 3,200 AU/mL of P34 was encapsulated in nanovesicles and stocked at 4 °C. No significant difference (p > 0.05) in the biological activity of free and encapsulated P34 was observed through 24 days. Size and PDI of liposomes, investigated by light scattering analysis, were on average 150 nm and 0.22 respectively. Zeta potential was −27.42 mV. There was no significant change (p > 0.05) in the physicochemical properties of liposomes during the time of evaluation. The liposomes presented closed spherical morphology as visualized by transmission electron microscopy (TEM). The mode of action of liposome-encapsulated P34 under L. monocytogenes cells was investigated by TEM. Liposomes appeared to adhere but not fuse with the bacterial cell wall, suggesting that the antimicrobial is released from nanovesicles to act against the microorganism. The effect of free and encapsulated P34 was tested against L. monocytogenes, showing that free bacteriocin inhibited the pathogen more quickly than the encapsulated P34. Liposomes prepared with low-cost lipid showed high encapsulation efficiency for a new antimicrobial peptide and were stable during storage. The mode of action against the pathogen L. monocytogenes was characterized.

Influence of pH and sodium chloride on kinetics of thermal inactivation of the bacteriocin-like substance P34

To investigate the kinetics of thermal inactivation of the bacteriocin-like substance P34 at different pH and sodium chloride concentration. Samples of bacteriocin were treated at different time-temperature combinations in the range of 0-300 min and 90-120°C and the kinetic parameters for bacteriocin inactivation were calculated. For all treatments, the thermal inactivation reaction fitted adequately to first-order model. D- and k-values were smaller and higher, respectively, for pH 4·5 than for 6·0 or 7·0, indicating that bacteriocin P34 was less thermostable at lower pH. At 120, 115 and 100°C, the addition of sodium chloride decreased thermal stability. For other temperatures, addition of NaCl increased stability of the peptide. The presence of greater amount of the salt (50 g l(-1) ) resulted in a higher thermal stability of bacteriocin P34, suggesting that the reduction in water activity of the solution interfered on the stability of the peptide. Based on an isothermal experiment in the temperature range of 90-120°C, and by thermal death time models, bacteriocin P34 is less heat stable at low pH and has increased thermal stability in the presence of NaCl. Addition of NaCl improved the stability of the peptide P34 at high temperatures. Studies on kinetics of thermal inactivation of bacteriocins are essential to allow their proper utilization in the food industry.

Evaluation of the in vitro cytotoxicity of the antimicrobial peptide P34

The in vitro cytotoxicity of the antimicrobial peptide P34 was evaluated in different eukaryotic cells. The food-grade bacteriocin nisin was also analysed for comparison. Vero cells were treated with different concentrations (0.02-2.5 microg x ml(-1)) of antimicrobial peptide P34 and nisin. Cell viability and plasma membrane integrity were checked by MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide], NRU (Neutral Red dye uptake) and LDH (lactate dehydrogenase) assays. The EC50 values of the peptide P34 in MTT and NRU assays were 0.60 and 1.25 microg x ml(-1) respectively, while values of nisin found were 0.50 and 1.04 microg x ml(-1). In the LDH assay, the EC50 values were 0.65 and 0.62 microg x ml(-1) for P34 and nisin, respectively. The peptide P34 revealed similar haemolytic activity on human erythrocytes (5.8%) when compared with nisin (4.9%). The effects on viability, motility and acrosomal exocytosis of human sperm were also evaluated. Nisin and P34 showed similar effects on sperm parameters. The evaluation of cytotoxicity of antimicrobial peptides is a critical step to guarantee their safe use.