Antimicrobial Activity of Natural Agents against Saccharomyces cerevisiae

The antimicrobial (AM) activity of starch‐based films coated with linalool, carvacrol or thymol against Saccharomyces cerevisiae in vitro and/or inoculated on the surface of Cheddar cheese was investigated. In solid medium using the agar diffusion method and in experiments involving the inoculation of the microorganism on the surface of Cheddar cheese, all the films containing these AM agents in coatings demonstrated an inhibitory effect against S. cerevisiae. The results suggest that the overall inhibitory effect of linalool, carvacrol or thymol increased significantly (p ≤ 0.05) with the concentration of each of the AM agents in the film coating and that the response is linear in the concentration range of 1 to 5% (w/w) of the AM agent. Thymol had the highest AM efficacy followed by carvacrol, whereas linalool had the lowest efficacy amongst the three systems. The zones of inhibition in the agar diffusion test method at 25°C for S. cerevisiae were found to be 7.6, 7.1 and 6.1 mm for thymol, carvacrol and linalool at 1% (w/w) loading and 13.2, 12.2 and 11.2 mm at 5% (w/w) loading of the AM agents, respectively. The death rates of S. cerevisiae on Cheddar cheese wrapped in films coated with thymol, carvacrol and linalool and stored for up to 28 days at 15°C were found to be 0.044, 0.043 and 0.038 per day at 1% (w/w) loading and 0.077, 0.073 and 0.063 per day at 5% (w/w) loading of the AM agents, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Physico‐Mechanical Properties of Starch‐Based Films Containing Naturally Derived Antimicrobial Agents

Thermoplastic starch (TPS) films containing relatively low [0.75 ± 0.08% (w/w)], intermediate [1.08 ± 0.04% (w/w)] and high [3.20 ± 0.29% (w/w)] levels of the antimicrobial (AM) agents carvacrol, linalool and thymol were prepared, and their physico‐mechanical and optical properties were evaluated. Addition of these AM agents to TPS film reduced the tensile strength with increasing AM agent content, with a significant effect observed at the highest AM agent concentration. The Young's modulus and elongation at break increased with increasing AM agent concentration, especially at the highest formulation concentration of AM agent. Films having a low or intermediate formulation concentration of AM agent exhibited no significant effect on their water vapour permeability, transparency and thermal properties when compared to the control film. Although scanning electron microscope imaging suggested a significant and progressive change in the surface morphologies of the films with AM agent concentration, the overall effects on the tested properties were not significant. This suggests that the direct incorporation of AM agents into TPS films did not adversely affect the films, particularly at lower AM agent concentrations. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of antimicrobial films containing basil extracts

The properties of low‐density polyethylene (LDPE)‐based films containing either linalool or methylchavicol as antimicrobial (AM) additives were evaluated. Slight decreases in transparency, water vapour and oxygen transmission rates were found in the extruded films containing 0.34% w/w linalool or methylchavicol. The infrared (IR) spectra of the AM films were similar to that of additive‐free LDPE film. However, carbonyl peaks could also be observed in the spectra of the AM films. There was no significant difference in the degree of crystallinity and the melting temperature range of the different films. Derivative thermogravimetry mass‐loss curves showed that the thermal decomposition temperatures of the AM films were marginally lower than that of LDPE film. Electron micrographs indicated that AM LDPE‐based films exhibited no evidence of changes in microstructure to suggest that linalool and methylchavicol were not evenly distributed in the film. Copyright © 2006 John Wiley & Sons, Ltd.     

Development and evaluation of active packaging for sliced mozzarella preservation

Antimicrobial films were formed by the incorporation of nisin (NI), natamycin (NA) and a combination of both (NI + NA) into cellulose polymer. Film efficacies were evaluated in vitro against Staphylococcus aureus ATCC 6538, Listeria monocytogenes ATCC 15313, Penicillium sp. and Geotrichum sp. The films were also evaluated on sliced mozzarella cheese against moulds and yeasts, Staphylococcus sp. and psychrotrophic bacteria. Mechanical and microscopic properties of the films and the diffusion of the antimicrobial agents from the film to the cheese were also evaluated. Films containing NI showed an antimicrobial effect in vitro against S. aureus and L. monocytogenes, while films containing NA were effective in vitro against Penicillium sp. and Geotrichum sp. By the ninth day of storage at 12 ± 2°C, the count of yeasts and moulds on cheese covered with films containing NA decreased 2 log10 units compared with the count on cheese with control films. NI film did not show an effect against Staphylococcus sp., but it was effective against psychrotrophic bacteria for 6 days of storage of the cheese. The incorporation of antimicrobial compounds decreased the resistance and elongation of the films and caused changes in their molecular conformation. NI diffusion from the films to the cheese was not detected; however, time‐dependent diffusion of NA from the film containing NI + NA was measured. The incorporation of NI and NA together in the films did not show an effect. The film containing NA showed potential for application as active food packaging for sliced mozzarella cheese. Copyright © 2008 John Wiley & Sons, Ltd.     

Preparation and Characterization of Cellulose Acetate Butyrate/Organoclay Nanocomposites Produced by Extrusion

Nanocomposite films based on cellulose acetate butyrate, modified montmorillonite (Cloisite^® 30B), plasticizer (triethyl citrate) and antimicrobial compounds (carvacrol and cinnamaldehyde) were prepared by extrusion. The effects of the Cloisite^® 30B content and antimicrobial compound types on the morphology of the nanocomposite films were investigated by X‐ray diffraction and transmission electron microscopy. The thermal characteristics of films were analysed by thermogravimetry and differential scanning calorimetry; oxygen and water vapour permeability and tensile strength were determined. The film's antimicrobial behaviour against Listeria innocua, Staphylococcus aureus, Escherichia coli O157:O7 and Saccharomyces cerevisiae was investigated and determined using a viable cell count method. Nanocomposites with a Cloisite^® 30B content of 3 wt% showed better dispersion than nanocomposites with a 5 wt% content. For films with antimicrobial compounds, tensile strength and Young's modulus decreased and water vapour permeability increased (150%) because of the plasticization effect of the antimicrobial compounds (essential oils). The nanocomposites with carvacrol and cinnamaldehyde were effective against the tested Gram‐positive bacteria (reduction of at least 3.0 log CFU/ml) and yeast (reduction of at least 4.0 log CFU/ml). This study demonstrates that antimicrobial cinnamaldehyde and carvacrol can be successfully incorporated into cellulose acetate butyrate nanocomposites and that they have an inhibitory effect against microbial growth in solid medium. It shows the potential use of cellulose acetate butyrate for food packaging applications. Copyright © 2013 John Wiley & Sons, Ltd.     

Release of Thymol,Cinnamaldehyde and Vanillin from Soy Protein Isolate Films into Olive Oil

This study investigates the release of thymol, cinnamaldehyde and vanillin from soy protein isolate (SPI) films into olive oil. The SPI‐based films were casted with thymol, cinnamaldehyde and vanillin to obtain antimicrobial packaging films. The release of thymol, cinnamaldehyde and vanillin from the SPI films into olive oil at temperatures of 5°C, 20°C, 40°C and 60°C was determined by high‐performance liquid chromatography. The partition coefficients of thymol, cinnamaldehyde and vanillin between the SPI film and the olive oil were obtained. They decreased linearly with the increase of temperature. The diffusion coefficients of thymol, cinnamaldehyde and vanillin were determined by fitting the mathematical model to the experimental data. The Arrhennius equation could adequately describe the relationship between the temperature and the diffusion coefficients of thymol, cinnamaldehyde and vanillin, respectively. The activation energies for the release of thymol, cinnamaldehyde and vanillin were obtained, and they were respectively 55.92, 57.34 and 18.32 kJ mol^?1. Copyright © 2011 John Wiley & Sons, Ltd.     

Preservation of sliced ham through triclosan active film

Active packaging is an alternative to preserve perishable food. In this work, polyethylene antimicrobial active films containing different levels of triclosan (0, 2000 and 4000 mg kg^?1) were developed by extrusion. The films' efficacies were evaluated against Escherichia coli, Staphylococcus aureus, Listeria innocua, Salmonella choleraesuis and Pseudomonas aeruginosa growth using agar diffusion test and by monitoring the inhibition of E. coli and S. aureus inoculated on sliced cooked ham. The mechanical characteristics of the films were also evaluated with Universal Test Machine (Instron). The incorporation of triclosan did not affect the mechanical properties of antimicrobial films compared to the control film. The average film thickness was 82.0 µm and the tensile strength and elongation to break were 30.3 N and 46.2%, respectively. Films containing triclosan showed an antimicrobial effect in vitro against E. coli and S. aureus, with formation of an inhibition halo for both. However, this result was not observed for L. innocua, S. choleraesuis and P. aeruginosa, although, a decrease in colony density occurred around the film for both incubation temperatures (7 ± 2°C and 35 ± 2°C). Sliced ham packed with the antimicrobial films showed a reduction of 1.5 logarithmic cycles in comparison to ham in contact with a control film after 12 days of storage at 7 ± 2°C, for E. coli and S. aureus. Antimicrobial films present potential for application as active packaging materials, as they showed effective against some pathogenic microorganisms that can be transmitted by foods. Copyright © 2009 John Wiley & Sons, Ltd.     

Cellulose Acetate Butyrate Nanocomposites with Antimicrobial Properties for Food Packaging

The cellulose acetate butyrate/modified motmorillonite (Cloisite20A and Cloisite30B) nanocomposites were prepared by two different solvent casting methods. All antimicrobial nanofillers were characterized using X‐Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR) and antimicrobial components (carvacrol and cinnamaldehyde) included in the nanofillers were determined by gas and HPLC chromatography. These analyses confirmed the inclusion of antimicrobial agents in organoclay. The nanocomposite films were characterized by XRD, TGA, Differential Scanning Calorimetry (DSC), Oxygen and Water Vapor Permeability, mechanical properties and antimicrobial behavior against E coli, L.innocua, S. cerevisiae. All films presented a reduction of at least 2.0 log cfu/mL when applied film nanocomposites procedure in which antimicrobial components are included in the last stage of the process and they are not previously included in the organoclay. Copyright © 2012 John Wiley & Sons, Ltd.     

Antimicrobial activity of chitosan attached to ethylene copolymer films

Ethylene copolymer film was coated with chitosan by attachment of the polymer to the corona‐treated surface of the film, and the composite film was analysed for antimicrobial activity. The film was active against bacteria in 0.625 mM phosphate buffer; it reduced colony counts of Escherichia coli 25922 and of Listeria monocytogenes Scott A by 5 and 2–3 log₁₀, respectively after 24 h exposure. The activity of the chitosan‐coated film against bacteria in buffer was increased when silver ions were incorporated into the films as demonstrated by complete killing of Escherichia coli O157:H7 DD3795 and the chicken exudate isolate Stenotrophomonas maltophilia within 2 h in buffer. The film was active against L. monocytogenes Scott A in 0.5% buffered peptone water up to a pH of about 7.0. Tests on beef and chicken meat exudates revealed antimicrobial activity of the film against Escherichia coli O157:H7 and L. monocytogenes Scott A of about 2 and 1–2 log₁₀ reductions in colony‐forming units, respectively. The antimicrobial activity of the film against L. monocytogenes Scott A was also tested on turkey breast, and a log₁₀ reduction of about 1.7 log₁₀ units after 10 days and 1.2 log₁₀ after 15 days at 4°C was achieved. Exposure to chitosan‐coated film and 350 MPa of pressure, 55°C or 1% sodium diacetate resulted in a synergistic effect. Copyright © 2008 John Wiley & Sons, Ltd     

Immobilization of Lysozyme on Food Contact Polymers as Potential Antimicrobial Films

The objective of this work was to investigate the feasibility of incorporating an antimicrobial enzyme (lysozyme) into polymers which are suitable for food contact. Hen egg white lysozyme was immobilized on polyvinyl alcohol (PVOH) beads, nylon, 6,6 pellets and cellulose triacetate (CTA) films. Polyvinyl alcohol and nylon 6,6 yielded low activity against a suspension of dried Micrococcus lysodeikticus cells, while CTA yielded the highest activity; 1.25 cm²/ml (CTA film area to substrate volume ratio) fully hydrolyzed a 0.015% (w/v) suspension of dried cells in 30 min. The activity retention of lysozyme immobilized on CTA was 90% after one use and 60% after 20 repeated uses. The amount of enzyme added to the film during immobilization affected the activity of the immobilized lysozyme; highest activities were obtained when CTA films were formed by adding 150–250 mg lysozyme per g polymer. No significant effect of CTA film thickness on lysozyme activity was observed. Viability of M. lysodeikticus grown on tripticase soy broth (TSB) at 30°C was decreased in the presence of CTA film containing lysozyme. The film (0.01 cm²/ml TSB) was inhibitory and bactericidal against 10³ and 10⁸ c.f.u./ml M. lysodeikticus respectively. © 1997 John Wiley & Sons, Ltd.     

Development of Cellulose Eco‐nanocomposites with Antimicrobial Properties Oriented for Food Packaging

Nanocomposites based on cellulose acetate, a commercial organoclay (Cloisite30B), triethyl citrate and variable content of antimicrobial agents (thymol and cinnamaldehyde), were obtained using a solution casting technique. The properties of the nanocomposites were evaluated using X‐ray diffraction, differential scanning calorimetry, thermogravimetric analysis, mechanical (modulus of elasticity, tensile strength and elongation at break), scanning electronic microscopy, global migration and microbiological testing. A reduction of glass transition (T_g), melting temperature (T_m) and melting enthalpy (?H_m) was also observed when the content of thymol and cinnamaldehyde was increased in the cellulose acetate nanocomposites. In contrast, thermal stability, mechanical performance and morphology of material did not show important differences when the content was modified. Results of global migration were dependent of the kind of simulant used. Finally, the antimicrobial activity was dependent of the essential oil used and its content inside the nanocomposite. An important effect of organoclay on the antimicrobial activity was also observed. Copyright © 2012 John Wiley & Sons, Ltd.     

Properties of antimicrobial plastics containing traditional food preservatives

Traditional food preservatives, sodium benzoate, sodium nitrite, potassium sorbate and sodium lactate, were incorporated into synthetic plastics, low‐density polyethylene (LDPE), poly(maleic acid‐co‐olefine), polystyrene (PS) and polyethylene terephthalate (PET), aimed at producing antimicrobial packaging material for foodstuffs. The study was undertaken on plaques (thickness 2 mm) and films (thickness 70–120 µm), whose antimicrobial test results clearly differed. Plaques containing 15% sodium nitrite inhibited both Aspergillus niger and Bacillus subtilis, whereas the same concentration of sodium benzoate and potassium sorbate had activity only against B. subtilis. Sodium lactate‐containing samples did not have any antimicrobial activity and none of the samples inhibited Escherichia coli. Antimicrobial substances added into PS and PET produced the strongest activities; however, due to the brittle structure of these materials, they were not tested further. Thus, more thorough tests for antimicrobial activity, migration and oxygen and water vapour permeability were carried out using LDPE films with 2.5–15% sodium benzoate and sodium nitrite. The effects of both substances on permeability properties were negligible. Although the total migration into food simulants measured from the films in many cases exceeded the limit value of 10 mg/dm², no antimicrobial activity was observed. Copyright © 2003 John Wiley & Sons, Ltd.     

Development and evaluation of an edible antimicrobial film based on yam starch and chitosan

Edible antimicrobial films are an innovation within the biodegradable active packaging concept. They have been developed in order to reduce and/or inhibit the growth of microorganisms on the surface of foods. This study developed an edible antimicrobial film based on yam starch (Dioscorea alata) and chitosan and investigated its antimicrobial efficiency on Salmonella enteritidis. A solution of yam starch (4%) and glycerol (2%) was gelatinized in a viscoamilograph and chitosan added at concentrations of 1%, 3% and 5%. Films with and without chitosan were produced by the cast method. To evaluate the antimicrobial activity of the films, two suspensions of S. enteritidis were used in BHI medium, corresponding to counts of 2 × 10⁸ and 1.1 × 10⁶ CFU/ml. The suspensions (50 ml) were poured into flasks. The films were cut into 5 × 5 and 5 × 10 cm rectangles to be used at ratios of 1 : 1 (1 cm²/ml microorganism suspension) and 2 : 1 (2 cm²/ml). The film 30 µm thick on average. As a control, pure chitosan at an amount corresponding to that contained in the 3% and 5% films (5 × 10 cm) was added to flasks containing the microorganism suspension. Also, flasks containing only a suspension of S. enteritidis were used as control. The suspensions, in flasks, were kept at 37°C in a waterbath with agitation. Suspension aliquots were removed every hour for reading the optic density (OD₅₉₅) and plating onto PCA medium. The results showed that chitosan has a bactericidal effect upon S. enteritidis. Films treated with chitosan at different concentrations showed similar antimicrobial efficiency, in addition to being dependent on diffusion. The chitosan‐treated films caused a reduction of one to two log cycles in the number of microorganisms, whereas the pure chitosan presented a reduction of four to six log cycles compared with the control and starch film. The films showed good flexibility. Copyright © 2005 John Wiley & Sons, Ltd.     

Potassium sorbate controlled release from corn starch films

Active starch films with glycerol and potassium sorbate were obtained by casting. Native and acetylated corn starches, as well as the mixture of them in equal proportions were used and filmogenic suspensions with pH 4.5 were also prepared. Sorbate concentration decreased during film storage due to its oxidative degradation. Active films resulted more yellow and less transparent than films without sorbate. The minimum inhibitory concentration of sorbate resulted 0.3%, regardless of the starch type and the formulation pH. The use of antimicrobial package was more effective to prevent microbial growth on food surfaces than the use of conventional methods. Additive kinetic release was neither affected by the starch type nor by the formulation pH. Sorbate diffusion process was mathematically modeled satisfactorily. Active films were able to inhibit Candida spp., Penicillium spp., S. aureus and Salmonella spp. growth. Active films extended 21% the shelf life of refrigerated cheese, regardless of the formulation pH.     

Magnetron sputter deposition of (SiC)<Subscript>1−<Emphasis Type="Italic">x</Emphasis></Subscript>(AlN)<Subscript>x</Subscript> solid solution films

Magnetron sputtering of polycrystalline SiC-AlN targets was used to obtain films of (SiC)_{1− x} (AlN)_x solid solutions on silicon carbide (6H-SiC) substrates heated to a temperature in the range T = 500–1200°C. The deposits were characterized with respect to structure, composition, and optical absorption. It is demonstrated that the films obtained on 6H-SiC substrates at T ≥ 1000°C possess a single crystal structure. The compositions of (SiC)_1−x (AlN)_x films are close to those of the corresponding SiC-AlN targets.     

Extruded Blends of Chitosan and Ethylene Copolymers for Antimicrobial Packaging

Chitosan was melt‐processed with an ethylene methyl acrylate copolymer ionomer and with an ethylene vinyl acetate copolymer to create antimicrobial extruded films. The key to obtaining a successful antimicrobial blend was the use of solid chitosonium acetate that remained after evaporation of water from the chitosan solution. When solid free‐flowing powders of chitosonium acetate were formed by spray drying chitosan solutions, blended in an extruder (at 2.5% and 4% chitosan) with Elvax® 3175 ethylene vinyl acetate copolymer, and extruded through a film die onto a chill roll to form films, the films exhibited antimicrobial activity against Escherichia coli 25922, Salmonella enterica serovar Enteritidis Nal^R, and Listeria monocytogenes Scott A. The log₁₀ reductions in CFU/ml after 24 h in a shake‐flask test were near 2 for films containing 4% chitosan. This melt‐blending/extrusion approach is expected to open applications for chitosan‐based antimicrobial packages and articles that were impossible or impractical with chitosan coatings. Copyright © 2011 John Wiley & Sons, Ltd.     

Effect of organic vapour interaction on permeation rate through polymer films

The permeability coefficients (P) of linalool, citral ethyl butyrate, d-limonene and octanal permeating through LDPE and ionomer films were measured alone as individual and as a component of a mixture at a temperature of 23 °C and in saturated water vapour. Values of P for ionomer film were approximately two orders of magnitude below those for LDPE for the compounds tested. Both PET and PETG films were found to be much better barriers against the compounds tested than LDPE and ionomer films. In general, the permeability, diffusion and solubility coefficients (P. D, and S) values measured in mixture were substantially less than the values obtained individually. Permeability behaviour of these compounds at low concentrations in the mixture was similar to that of permanent gases. Reduction of solubility coefficients in mixture permeation was greater with the higher solubility coefficients. These results provide a better understanding of the mechanism of vapour diffusion through polymers.     

Incorporation of organic acids turns classically brittle zein films into flexible antimicrobial packaging materials

This study aimed to turn classically brittle zein films into flexible antimicrobial ones by the use of lactic (LA), malic (MA) and tartaric acids (TA). The most effective plasticizer was LA (400% elongation at break [EB] at 4%), while MA (189% EB at 4.5%) and TA (68% EB at 5%) showed moderate and limited plasticizing effects, respectively. The LA- and MA-loaded films maintained their flexibility during 30-day storage at 4°C or 25°C. Fourier transform infrared (FTIR) analysis suggested that the plasticization of LA and MA could be related to secondary structural changes in zein such as increased α-helix and random coils (mainly by MA) and spaced/modified intermolecular (only by LA) and intramolecular (mainly by MA) β-sheets. Atomic force and scanning electron microscopy showed that LA and MA gave more homogenous and smoother films than TA. Films with LA showed the highest water vapour permeability followed by those of control, MA- and TA-loaded films. Films with 3%–4% LA or MA formed clear zones on Listeria innocua and Klebsiella pneumonia, but only films with LA formed clear zones on Escherichia coli. All OA-loaded films gave unclear zones on Staphylococcus aureus in disc-diffusion tests, but this bacterium was inactivated rapidly in antimicrobial tests based on surface inoculation tests. LA is the best OA to develop flexible antimicrobial films from zein, an industrial by-product that films could not have been utilized as a widespread packaging material due to their brittleness.     

Evaluation of Different Conditions of Contact for Caprolactam Migration from Multilayer Polyamide Films into Food Simulants

Caprolactam, the polyamide 6 (PA‐6) monomer, can migrate during food processing. At cooking temperatures, migration is accelerated and plastic components could degrade, giving off low molecular mass compounds, which can migrate into food. In this work, caprolactam migration from multilayer films containing PA‐6 for meat foodstuffs and cheese packaging was performed at contact conditions of 40°C/10 days and 100°C/30 min. The migration into water ranged from 0.89 to 1.22 mg/dm² and 0.92 to 1.21 mg/dm², into 3% acetic acid from 1.29 to 1.74 mg/dm² and 1.13 to 1.62 mg/dm² and into olive oil from 1.18 to 1.98 mg/dm² and 0.50 to 0.80 mg/dm² for films intended for meat foodstuffs for 10 days at 40°C and 30 min at 100°C, respectively. Among PA‐6 films used for cheese, caprolactam migration into water ranged from 0.17 to 0.91 mg/dm² and 0.74 to 1.04 mg/dm², into 3% acetic acid from 1.15 to 1.26 mg/dm² and 1.11 to 1.37 mg/dm² and into olive oil from 0.23 to 0.83 mg/dm² and 0.37 to 0.56 mg/dm² for 10 days at 40°C and 30 min at 100°C, respectively. Caprolactam migration evaluation into water and 3% acetic acid at 100°C/30 min could replace the need to apply the test at 40°C/10 days, since similar results were obtained under both conditions. In the case of PA‐6 for meat foodstuffs, caprolactam migration into olive oil was highly affected by different conditions of contact, showing values two to three times higher at 40°C/10 days than at 100°C/30 min. For cheese films, caprolactam migration into olive oil was higher at 40°C for 10 days. Copyright © 2013 John Wiley & Sons, Ltd.     

Evaluation of alginate and zein films as a carrier of natamycin to increase the shelf life of kashar cheese

The aim of this study was to develop alginate and zein films containing natamycin, a natural antifungal agent, in order to limit/prevent the mould growth on the surface of kashar cheeses. The films were prepared by casting, and characterized in terms of antimicrobial and mechanical properties (tensile strength, elongation-at-break, and elastic modulus), and their morphology was examined by scanning electron microscopy (SEM). Mechanical properties of the zein films were found to be weaker than the alginate films. SEM analysis indicated that alginate films have a more regular structure than zein films, and a more homogenous distribution was observed at lower concentrations of natamycin. The antifungal activities of both films increased as the natamycin concentration (100, 200, 500, 1000, 2000, and 4000 ppm) increased; however, alginate films exhibited relatively high antifungal activity. The effects of films on the shelf life of kashar cheeses inoculated with Aspergillus niger and Penicillium camemberti were investigated during their storage under refrigerator conditions for 45 days. At high-natamycin concentrations, zein films showed higher antifungal activity against both fungi at the end of the storage period.