Perspectives on antimicrobial properties of Paulownia tomentosa Steud. fruit products in the control of Staphylococcus aureus infections.

ETHNOPHARMACOLOGICAL RELEVANCE: Paulownia tomentosa Steud. (P. tomentosa) is a medium-sized tree traditionally used in Chinese folk medicine for the treatment of infectious diseases. It is a rich source of prenylated phenolic compounds that have been extensively studied for their promising biological activities. AIM OF THE STUDY: Due to the increasing development of antibiotic resistance, our study investigated plant-derived natural products from the fruits of P. tomentosa that could control Staphylococcus aureus infections with novel targets/modes of action and reduce antimicrobial resistance. MATERIALS AND METHODS: The ethanolic extract was fractionated and detected by liquid chromatography. The antistaphylococcal effects of the plant formulations were studied in detail in vitro by various biological methods, including microdilution methods for minimum inhibitory concentration (MIC), the checkerboard titration technique for synergy assay, fluorescence measurements for membrane disruption experiments, autoinducer-2-mediated bioassay for quorum sensing inhibition, and counting of colony-forming units for relative adhesion. Morphology was examined by transmission electron microscopy. RESULTS: Total ethanolic extract and chloroform fraction showed MICs of 128 and 32 µg/mL, respectively. Diplacol, diplacone, and 3'-O-methyl-5'-hydroxydiplacone inhibited S. aureus growth in the range of 8-16 µg/mL. Synergistic potential was shown in combination with mupirocin and fusidic acid. The ethanolic extract and the chloroform fraction destroyed the cell membranes by 91.61% and 79.46%, respectively, while the pure compounds were less active. The ethanolic extract and the pure compounds reduced the number of adhered cells to 47.33-10.26% compared to the untreated control. All tested plant formulations, except diplacone, inhibited quorum sensing of S. aureus. Transmission electron microscopy showed deformation of S. aureus cells. CONCLUSIONS: The products from the fruit of P. tomentosa showed antimicrobial properties against S. aureus alone and in combination with antibiotics. By affecting intracellular targets, geranylated flavonoids proposed novel approaches in the control of staphylococcal infections.

Significant Benefits of Environmentally Friendly Hydrosols from Tropaeolum majus L. Seeds with Multiple Biological Activities.

Tropaeolum majus L. is a traditional medicinal plant with a wide range of biological activities due to the degradation products of the glucosinolate glucotropaeolin. Therefore, the goals of this study were to identify volatiles using gas chromatography-mass spectrometry analysis (GC-MS) of the hydrosols (HYs) isolated using microwave-assisted extraction (MAE) and microwave hydrodiffusion and gravity (MHG). Cytotoxic activity was tested against a cervical cancer cell line (HeLa), human colon cancer cell line (HCT116), human osteosarcoma cell line (U2OS), and healthy cell line (RPE1). The effect on wound healing was investigated using human keratinocyte cells (HaCaT), while the antibacterial activity of the HYs was tested against growth and adhesion to a polystyrene surface of Staphylococcus aureus and Escherichia coli. Antiphytoviral activity against tobacco mosaic virus (TMV) was determined. The GC-MS analysis showed that the two main compounds in the HYs of T. majus are benzyl isothiocyanate (BITC) and benzyl cyanide (BCN) using the MAE (62.29% BITC and 15.02% BCN) and MHG (17.89% BITC and 65.33% BCN) extraction techniques. The HYs obtained using MAE showed better cytotoxic activity against the tested cancer cell lines (IC50 value of 472.61-637.07 µg/mL) compared to the HYs obtained using MHG (IC50 value of 719.01-1307.03 µg/mL). Both concentrations (5 and 20 µg/mL) of T. majus HYs using MAE showed a mild but statistically non-significant effect in promoting gap closure compared with untreated cells, whereas the T. majus HY isolated using MHG at a concentration of 15 µg/mL showed a statistically significant negative effect on wound healing. The test showed that the MIC concentration was above 0.5 mg/mL for the HY isolated using MAE, and 2 mg/mL for the HY isolated using MHG. The HY isolated using MHG reduced the adhesion of E. coli at a concentration of 2 mg/mL, while it also reduced the adhesion of S. aureus at a concentration of 1 mg/mL. Both hydrosols showed excellent antiphytoviral activity against TMV, achieving100% inhibition of local lesions on the leaves of infected plants, which is the first time such a result was obtained with a hydrosol treatment. Due to the antiphytoviral activity results, hydrosols of T. majus have a promising future for use in agricultural production.

Controlled growth of ZnO nanoparticles using ethanolic root extract of Japanese knotweed: photocatalytic and antimicrobial properties.

Synthesis of zinc oxide (ZnO) nanoparticles (NPs) was mediated by plant extracts to assist in the reduction of zinc atoms during the synthesis and act as a capping agent during annealing. The preparation used ethanolic extracts from the roots of Japanese knotweed (Fallopia japonica). Two major outcomes could be made. (i) A synergistic effect of multiple polyphenolic components in the extract is needed to achieve the capping effect of crystallite growth during thermal annealing at 450 °C characterized by an exponential growth factor (n) of 4.4 compared to n = 3 for bare ZnO. (ii) Synergism between the ZnO NPs and plant extracts resulted in superior antimicrobial activity against both Gram-positive bacteria, e.g., Staphylococcus aureus, and Gram-negative bacteria, e.g., Escherichia coli and Campylobacter jejuni. The materials were also tested for their antimicrobial activity against S. aureus under ultraviolet (UV) illumination. Also here, the photocatalyst prepared with plant extracts was found to be superior. The residues of the plant extract molecules on the surface of the catalyst were identified as the main cause of the observed differences, as proved by thermal gravimetry. Such a preparation using ethanolic extract of Fallopia japonica could serve as a more controlled synthesis of ZnO and potentially other metal oxides, with low environmental impact and high abundance in nature.

Rhodiola rosea Reduces Intercellular Signaling in Campylobacter jejuni.

Campylobacter jejuni is a major foodborne pathogen and the leading cause of bacterial gastroenteritis, i.e., campylobacteriosis. Besides searching for novel antimicrobials, identification of new targets for their action is becoming increasingly important. Rhodiola rosea has long been used in traditional medicine. Ethanolic extracts from the roots and rhizomes of the plant contain a wide range of bioactive compounds with various pharmacological activities. In this study, cultivated plant materials have been used, i.e., "Mattmark" and "Rosavine". Through optimized protocols, we obtained fractions of the initial ethanolic extracts rich in most important bioactive compounds from R. rosea, including salidroside, rosavins, proanthocyanidins (PACs), and flavonoids. The antimicrobial activity in relation to the chemical composition of the extracts and their fractions was studied with an emphasis on C. jejuni AI-2-mediated intercellular signaling. At concentration 15.625 mg/L, bioluminescence reduction rates varied from 27% to 72%, and the membrane remained intact. Fractions rich in PACs had the strongest antimicrobial effect against C. jejuni, with the lowest minimal inhibitory concentrations (MICs) (M F3 40%: 62.5 mg/L; R F3 40%: 250 mg/L) and the highest intercellular signaling reduction rates (M F3 40%: 72%; R F3 40%: 65%). On the other hand, fractions without PACs were less effective (MICs: M F5 PVP: 250 mg/L; R F5 PVP: 1000 mg/L and bioluminescence reduction rates: M F5 PVP: 27%; R F5 PVP: 43%). Additionally, fractions rich in flavonoids had strong antimicrobial activity (MICs: M F4 70%: 125 mg/L; R F4 70%: 250 mg/L and bioluminescence reduction rates: M F4 70%: 68%; R F4 70%: 50%). We conclude that PACs and flavonoids are crucial compound groups responsible for the antimicrobial activity of R. rosea roots and rhizomes in C. jejuni.

Phenolic Profile, Antioxidant Capacity and Antimicrobial Activity of Nettle Leaves Extracts Obtained by Advanced Extraction Techniques.

Nettle is a widely known plant whose high biological activity and beneficial medicinal effects are attributed to various bioactive compounds, among which polyphenols play an important role. In order to isolate polyphenols and preserve their properties, advanced extraction techniques have been applied to overcome the drawbacks of conventional ones. Therefore, microwave-assisted extraction (MAE) has been optimized for the isolation of nettle leaves polyphenols and it was compared to pressurized liquid extraction (PLE) and conventional heat-reflux extraction (CE). The obtained extracts were analyzed for their individual phenolic profile by UPLC MS2 and for their antioxidant capacity by ORAC assay. MAE proved to be the more specific technique for the isolation of individual phenolic compounds, while PLE produced extracts with higher amount of total phenols and higher antioxidant capacity. Both techniques were more effective compared to CE. PLE nettle extract showed antimicrobial activity against bacteria, especially against Gram-negative Pseudomonas fragi ATCC 4973 and Campylobacter jejuni NCTC 11168 strains. This suggests that PLE is suitable for obtaining a nettle extract with antioxidant and antimicrobial potential, which as such has great potential for use as a value-added ingredient in the food and pharmaceutical industry.

Antibiofilm Potential of Lavandula Preparations against Campylobacter jejuni.

New approaches for the control of Campylobacter jejuni biofilms in the food industry are being studied intensively. Natural products are promising alternative antimicrobial substances to control biofilm production, with particular emphasis on plant extracts. Dried flowers of Lavandula angustifolia were used to produce essential oil (LEO), an ethanol extract (LEF), and an ethanol extract of Lavandula postdistillation waste material (LEW). The chemical compositions determined for these Lavandula preparations included seven major compounds that were selected for further testing. These were tested against C. jejuni for biofilm degradation and removal. Next-generation sequencing was used to study the molecular mechanisms underlying LEO actions against C. jejuni adhesion and motility. Analysis of LEO revealed 1,8-cineol, linalool, and linalyl acetate as the main components. For LEF and LEW, the main components were phenolic acid glycosides, with flavonoids rarely present. The MICs of the Lavandula preparations and pure compounds against C. jejuni ranged from 0.2 mg/ml to 1 mg/ml. LEO showed the strongest biofilm degradation. The reduction of C. jejuni adhesion was ≥1 log10 CFU/ml, which satisfies European Food Safety Authority recommendations. Lavandula preparations reduced C. jejuni motility by almost 50%, which consequently can impact biofilm formation. These data are in line with the transcriptome analysis of C. jejuni, which indicated that LEO downregulated genes important for biofilm formation. LEW also showed good antibacterial and antibiofilm effects, particularly against adhesion and motility mechanisms. This defines an innovative approach using alternative strategies and novel targets to combat bacterial biofilm formation and, hence, the potential to develop new effective agents with biofilm-degrading activities. IMPORTANCE The Lavandula preparations used in this study are found to be effective against C. jejuni, a common foodborne pathogen. They show antibiofilm properties at subinhibitory concentrations in terms of promoting biofilm degradation and inhibiting cell adhesion and motility, which are involved in the initial steps of biofilm formation. These results are confirmed by transcriptome analysis, which highlights the effect of Lavandula essential oil on C. jejuni biofilm properties. We show that the waste material from the hydrodistillation of Lavandula has particular antibiofilm effects, suggesting that it has potential for reuse for industrial purposes. This study highlights the need for efforts directed toward such innovative approaches and alternative strategies against biofilm formation and maintenance by developing new naturally derived agents with antibiofilm activities.

Waste streams in onion production: Bioactive compounds, quercetin and use of antimicrobial and antioxidative properties.

Onion production generates abundant waste with high contents of bioactive compounds. These might have several beneficial functional properties for fortification of foods. To understand the variety and potential for further use, we examined various parts of the plants (edible/inedible waste/outer skin of onion), as well as extraction in water/ethanol and by shaking/sonication. Quercetin content and antioxidant capacity were initially determined for extracts of edible and waste parts of red, yellow and white onions, and red shallots. Ethanol extracts of the waste fraction had the highest quercetin content and antioxidant capacity. Except white onion, which contained no quercetin, the dried waste ethanol extracts contained up to 15 mg quercetin g-1 and had an antioxidant capacity of nearly 40 mg Trolox equivalents g-1. Furthermore, the dried skin ethanol extract of yellow onion, which is commercially the most available fraction, contained 8 mg quercetin g-1, with antioxidant capacity of 25 mg Trolox equivalents g-1 and high antimicrobial activity. Dried yellow onion skin showed good stability for the quercetin content under various storage conditions (4, 25, 37, 40 °C; dark/light; dry/moist air/in water). Bacteria, bacterial spores, yeast and mould counts remained unchanged for dried onion skin over 5 days under storage conditions that can promote food spoilage, indicating exceptional microbial stability. Finally, two different applications are demonstrated for dried yellow onion skin: tablets for home use (tablets as more convenient form of storage and for simple dosing in cooking), and a stabilisation additive (prolonged shelf-life of olive oil). Both represent efficient and straightforward approaches through waste prevention and food fortification.

Targeting fish spoilers Pseudomonas and Shewanella with oregano and nettle extracts.

To control Pseudomonas and Shewanella as important psychrotrophic spoilage bacteria in fish meat, we used ethanolic extracts of oregano (Origanum vulgare subsp. vulgare) and nettle (Urtica dioica), with phytochemical characterisation of the extracts and their bioactive compounds. Liquid chromatography coupled with photodiode array detection and electrospray ionisation-mass spectrometry was used for qualitative compositional determination of the extracts. Four main compounds were identified in the oregano extract, with rosmarinic acid the most abundant, followed by three glycosylated phenolics, one of which is reported for the first time in O. vulgare: 4'-O-ß-d-glucopyranosyl-3',4'-dihydroxybenzyl-4-hydroxybenzoate. Six main compounds were identified in the nettle extract, as caffeoylmalic acid and five flavonoid glycosides. These oregano and nettle ethanolic extracts showed in-vitro antimicrobial activities against selected Pseudomonas and Shewanella strains in broth and fish meat homogenate when evaluated at two inoculum concentrations. The antimicrobial activities were more pronounced for the nettle extract at the lower inoculum concentration, and for both the Shewanella strains. Growth inhibition in the fish meat homogenate was evaluated at 3.13 mg/mL and 1.56 mg/mL at 5 °C. Again, the nettle extract showed greater antimicrobial activity, which was seen as the lowest maximum growth rate, followed by the oregano extract, which was inhibitory only at 3.13 mg/mL. Finally, the extracts were applied to fish meat that was then stored at 5 °C for 9 days. Evaluation here was for the counts of the mesophilic, psychrotrophic, Pseudomonas and H2S producers. These confirmed the better antimicrobial effects of the nettle extract, especially against the H2S-producing bacteria, which included Shewanella. Both of the extracts were rich in glycosides of flavonoids and phenolic acids. The enzymatic activities of the Pseudomonas and Shewanella spoilage bacteria and their actions on the phenolic glycosides from natural sources will be further investigated.

Development of Biodegradable Whey-Based Laminate Functionalised by Chitosan-Natural Extract Formulations.

In this research, antimicrobial polysaccharide chitosan and natural extracts were used as surface coating of a plastic laminate with an integrated whey layer on the inside. The aim was to establish the biodegradable and active concept of packaging laminates. For this purpose, chitosan nanoparticles (CSNPs) with embedded rosemary or cinnamon extracts were synthesised and characterised. Additionally, a whey-based laminate was functionalised: i) chitosan macromolecular solution was applied as first layer and ii) cinnamon or rosemary extracts encapsulated in CSNPs were applied as upper layer (layer wise deposition). Such functionalised whey-based laminate was physicochemically characterized in terms of elemental surface composition, wettability, morphology and oxygen permeability. The antimicrobial activity was tested against Staphylococcus aureus, Escherichia coli, Aspergillus flavus and Penicillium verrucosum. The antioxidant properties were determined using the ABTS assay. It could be shown that after functionalization of the films with the above-mentioned strategy, the wettability was improved. Furthermore, such whey-based laminates still show excellent barrier properties, good antimicrobial activity and a remarkable antioxidative activity. In addition to the improved biodegradability, this type of lamination could also have a positive effect on the shelf-life of products packaged in such structured films.

Bioactive Characterization of Packaging Foils Coated by Chitosan and Polyphenol Colloidal Formulations.

Polypropylene (PP) and polyethylene (PE) foils, previously activated by ultraviolet (UV)/ozone, were functionalized using chitosan-extract nanoparticle dispersions. A solution of macromolecular chitosan was applied onto foils as a first layer, followed by the deposition of various extracts encapsulated into chitosan nanoparticles, which were attached as an upper layer. Functionalized foils were analyzed from a bioactive point of view, i.e., regarding antimicrobial and antioxidant activity. Desorption kinetics were also studied. Moreover, barrier properties were examined, as the most important parameter influencing antimicrobial and antioxidant activity. Finally, all these properties were correlated with different surface parameters, determined previously, in order to understand if there is any direct correlation between surface elemental composition, surface charge, contact angle, or morphology and a specific bioactive property. It was shown that great bioactive properties were introduced due to the additive effect of antimicrobial chitosan and antioxidative plant extracts. Moreover, oxygen permeability decreased significantly, and the migration of polyphenols and chitosan from the foil surface was below the OML (overall migration limit), which is very important for food industry applications. Furthermore, surface properties of foils influence to some extent the desired bioactivity.

Combination of rosemary extract and buffered vinegar inhibits Pseudomonas and Shewanella growth in common carp (Cyprinus carpio).

BACKGROUND: Aquaculture is the fastest growing food-production sector, and common carp (Cyprinus carpio) is one of the most cultivated fish species in the world. Due to its intrinsic characteristics, fish meat is highly susceptible to microbiological spoilage. Pseudomonas and Shewanella are the primary and secondary occurring microbiota during storage of fish meat, with significant contribution to spoilage with the formation of hydrolytic enzymes (lipases and proteases). RESULTS: With in vitro testing, we show that rosemary extract (Inolens4), buffered vinegar and their combination (SyneROX) exhibit antimicrobial effects against P. fragi, P. psychrophila, S. putrefaciens and S. xiaemensis at concentrations of 3.13 and 1.56 mg mL-1 . The combination was the most effective in inhibiting growth of selected bacteria in food model, and production of lipases and proteases during 9 days at 5 °C. In situ testing of antimicrobial dip treatment of carp meat determined that aerobic mesophilic, total psychrotrophic, Pseudomonas and hydrogen sulfide producer counts were reduced in all treatments, with the most prominent influence being shown by the combination and buffered vinegar. CONCLUSIONS: Our study highlights the importance of a multilevel assessment of the antimicrobial potential of biopreservatives under conditions comparable to those of the selected food. Investigation with bacteria and food model provided coherent and consistent data for the evaluation of the antimicrobial potential for carp meat. Combination of buffered vinegar (as active antimicrobial) and rosemary extract, with well-known and researched antioxidant properties but low in situ antimicrobial activity, represents a good potential for combined effect in preservation of fish meat. © 2020 Society of Chemical Industry.

Antiadhesion activity of juniper (Juniperus communis L.) preparations against Campylobacter jejuni evaluated with PCR-based methods.

The food-borne pathogen Campylobacter jejuni can cause bacterial gastrointestinal infections. Biofilm formation amplifies the risk of human infection by improving survival and persistence of C. jejuni in food processing environments and its transmission through the food chain. We aimed to control C. jejuni using an alternative strategy of low doses of Juniperus communis fruit preparations to target bacterial adhesion properties in the first step of biofilm formation. First, we defined the anti-Campylobacter activity of a juniper fruit crude extract and its fractionated biflavonoids, flavone glycosides, and purified amentoflavone, of juniper fruit essential oil and of juniper fruit postdistillation waste material extract. For accurate quantification of adherent C. jejuni, we optimised digital Polymerase Chain Reaction (PCR) and quantitative real-time PCR for construction of standard curves and quantification. We show for the first time that juniper fruit formulations can effectively inhibit adhesion of C. jejuni to polystyrene. Furthermore, ≥94% of the antiadhesion activity of juniper fruit crude extract and juniper fruit essential oil remained under food-related conditions: modified culture medium with glucose, or a stainless steel surface, or mixed co-cultures of C. jejuni and Listeria monocytogenes. This study indicates that addition of juniper fruit formulations can control growth and adhesion of C. jejuni and thus limit food chain transmission of campylobacters.

Aqueous Extracts of Wild Mushrooms Show Antimicrobial and Antiadhesion Activities against Bacteria and Fungi.

Mushrooms represent promising sources of novel bioactive compounds and can be applied as innovative strategies to control microbial contamination and infection via the food chain. We characterized aqueous extracts from 21 wild basidiomycete mushrooms and the cultivated oyster mushroom, Pleurotus ostreatus, as putative sources of antimicrobial and antiadhesive compounds. Broth microdilutions and adhesion to a polystyrene surface were evaluated on Gram-positive and Gram-negative bacteria and on fungi. The aqueous extracts tested showed antimicrobial and antiadhesive activities against these microorganisms. Biochemical analyses of the P. ostreatus extract indicated the involvement of several compounds with different molecular masses. Copyright © 2017 John Wiley & Sons, Ltd.

Attenuation of Adhesion, Biofilm Formation and Quorum Sensing of Campylobacter jejuni by Euodia ruticarpa.

Thermophilic campylobacters are a major cause of bacterial food-borne diarrhoeal disease. Adherence and biofilm formation are key elements of Campylobacter jejuni persistence in unfavourable environmental conditions. The phytochemical analysis of Euodia ruticarpa fruit ethanol solution extract (EREE) indicated that the major compounds were evodiamine (1), rutaecarpine (2) and evocarpine (9). E. ruticarpa fruit ethanol solution extract, compounds 1 and 2 as well as a mixture of quinolinone alkaloids with 41.7% of 9 were tested for antibacterial, antibiofilm and antiquorum sensing activities against C. jejuni. Minimal inhibitory concentrations varied from 64 to 1024 µg/mL. A mutant strain that lacks the functional gene coding for the CmeB efflux pump protein was the most susceptible. Interestingly, in addition to the wild-type (NCTC 11168) and cmeB mutant, also a mutant that lacks autoinducer-2 production (luxS) was able to adhere (1 h) and to produce a biofilm (24, 48 and 72 h). The subinhibitory concentrations of all preparations at least partly inhibited C. jejuni adhesion and biofilm formation with the most visible effect of the quinolinone alkaloid fraction. Using a Vibrio harveyi luminescence assay, the inhibition of autoinducer-2 production was observed in the wild-type and cmeB mutant after 48 h with the most visible effect of EREE and its fraction Q. Copyright © 2016 John Wiley & Sons, Ltd.

Anti-adhesion activity of thyme (Thymus vulgaris L.) extract, thyme post-distillation waste, and olive (Olea europea L.) leaf extract against Campylobacter jejuni on polystyrene and intestine epithelial cells.

BACKGROUND: In order to survive in food-processing environments and cause disease, Campylobacter jejuni requires specific survival mechanisms, such as biofilms, which contribute to its transmission through the food chain to the human host and present a critical form of resistance to a wide variety of antimicrobials. RESULTS: Phytochemical analysis of thyme ethanolic extract (TE), thyme post-hydrodistillation residue (TE-R), and olive leaf extract (OE) using high-performance liquid chromatography with photodiode array indicates that the major compounds in TE and TE-R are flavone glucuronides and rosmarinic acid derivatives, and in OE verbascoside, luteolin 7-O-glucoside and oleuroside. TE and TE-R reduced C. jejuni adhesion to abiotic surfaces by up to 30% at 0.2-12.5 µg mL(-1) , with TE-R showing a greater effect. OE from 3.125 to 200 µg mL(-1) reduced C. jejuni adhesion to polystyrene by 10-23%. On the other hand, C. jejuni adhesion to PSI cl1 cells was inhibited by almost 30% over a large concentration range of these extracts. CONCLUSION: Our findings suggest that TE, the agro-food waste material TE-R, and the by-product OE represent sources of bioactive phytochemicals that are effective at low concentrations and can be used as therapeutic agents to prevent bacterial adhesion. © 2015 Society of Chemical Industry.

Effects of selected essential oils on the growth and production of ochratoxin A by Penicillium verrucosum.

Essential oils from oregano (Origanum vulgare L.), mint (Mentha piperita L.), fennel (Foeniculum vulgare Mill.), and pine (Abies alba Mill.) needles and cones, and their active substances thymol, carvacrol, menthol, and anisaldehyde were tested for antifungal activity against Penicillium verrucosum. The lowest minimal inhibitory concentrations (MICs) were achieved for essential oil of oregano, followed by carvacrol, thymol, and menthol. These antifungal components were further investigated, as the main aim of our study was to elucidate the effect of natural antifungals on ochratoxin A production. During 21 days of exposure, the growth of P. verrucosum, and subsequently the production of ochratoxin A, was fully inhibited by thymol at ½ MIC (0.0625 mg mL-1), but menthol at » and ½ MIC (0.1875 and 3750 mg mL-1) showed no growth inhibition. After 21 days of incubation, the greatest inhibitory effect on ochratoxin production (inhibition was 96.9 %) was also achieved with thymol at » MIC (0.0313 mg mL-1). Essential oil of oregano (» MIC, 0.2930 µL mL-1) and carvacrol (½ MIC, 0.1953 µL mL-1) stimulate production of ochratoxin A at 13.9 % to 28.8 %, respectively. The observed antifungal effects depended on the agent, the concentration used, and the time of interaction between the agent and P. verrucosum. Our results indicate the possibility of using oregano essential oil as a substitute for artificial preservatives in certain foods, but further research is needed.