Effects of different culture conditions on biological potential and metabolites production in three Penicillium isolates.

The genus Penicillium is well known for its importance in drug and food production. Certain species are produced on an industrial scale for the production of antibiotics (e.g. penicillin) or for insertion in food (e.g. cheese). In the present work, three Penicillium species, part of the natural mycobiota growing on various food products were selected - P. ochrochloron, P. funiculosum and P. verrucosum var. cyclopium. The objective of our study was to value these species from the point of view of production of bioactive metabolites. The species were obtained after inoculation and growth in Czapek and Malt media. Both mycelia and culture media were analyzed to monitor the production of different metabolites by each fungus and their release to the culture medium. The concentrations of sugars, organic acids, phenolic acids and tocopherols were determined. Antioxidant activity of the phenolic extracts was evaluated, as also the antimicrobial activity of phenolic acids, organic acids and tocopherols extracts. Rhamnose, xylose, fructose and trehalose were found in all the mycelia and culture media; the prevailing organic acids were oxalic and fumaric acids, and protocatechuic and p-hydroxybenzoic acids were the most common phenolic acids; γ-tocopherol was the most abundant vitamin E isoform. Generally, the phenolic extracts corresponding to the mycelia samples revealed higher antioxidant activity. Concerning the antimicrobial activity there were some fluctuations, however all the studied species revealed activity against the tested strains. Therefore, the in-vitro bioprocesses can be an alternative for the production of bioactive metabolites that can be used by pharmaceutical industry.


Introduction
Molds species especially from the genus Penicillium and similar genera (e.g. Aspergillus) are recognized nowadays as producers of several bioactive compounds and different chemicals currently used as pharmaceuticals 1 . The golden age of antibiotic discovery and their isolation from Penicillium spp. dating back to the twentieth century and, these potent drugs, indisputably saved millions of lives. Thousands of Penicillium isolates have probably been screened in bioprospecting programs since the discovery of penicillin, and new bioactive metabolites continue to be discovered from these fungi at the moment 2,3 , indicating their current importance as a source of novel bioactive molecules to be used by the pharmaceutical industry. Despite this fact, there are few studies containing a detailed chemical analysis of food contaminants, and their biotechnological potential. Since only a small number of species of the fungi kingdom is known, and most fungi produce unknown metabolites, molds constitute one of the most promising sources of new compounds with potential application in biotechnology, pharmaceutical and food industry 4 .
The Penicillium genus comprises more than 200 described species and many are common soil inhabitants, as well as food borne contaminants or food ingredients used in the preparation of cheese and sausages 5,6 . Several species of Penicillium are common contaminants on various substrates and are known as potential mycotoxin producers. Human pathogenic species are rare; however, some species, like Penicillium marneffei, are known for causing severe infections. This specific species usually affects apparently immunocompetent individuals who suffered for example from tuberculosis or HIV, affecting different organs/ tissues like the bone marrow, intestine, kidneys or lungs 7 . Opportunistic infections leading to mycotic keratitis, otomycosis and endocarditis, often following insertion of valve prosthesis, have been also reported 8 . Penicillium species also produce pigments 9 that might be used as colorants for food industry. The studied species are food borne contaminants and they are frequently used to test the molds resistance 10 . However, they have been studied in other fields. Penicillium ochrochloron is already known as a source of certain compounds such as penitrem A, B, C, D, E and F, bromopenitrem A and F and dehydro-penitrem D 4 . Filamentous fungi are often studied in the area of enzymes production. P. ochrochloron revealed bioremediation potential, since it was capable to decolorize cotton blue 11 . This species is also used for the production of (1-3) b-D-glucanases and b-glucosidase 12 . In addition, hemicellulases from Penicillium funiculosum, namely xylanases, have been already characterized 13 and cellulases from this species revealed a hydrolytic action on paper materials 14 . Penicillium verrucosum is a fungus well known as an ochratoxin producer 15 ; nevertheless, it has been also studied for lipase production 16 .
The antimicrobial activity of P. ochrochloron has been also proved through the isolation of two secondary metabolites with this potential, namely (À)2,3,4-trihydroxybutanamide and (À) erythritol 17 . Thus, this species could be used as a microorganism with antimicrobial properties. Some antioxidants have also been described in Penicillium species such as P. citrinum 18 and P. expansum 19 .
The potential already demonstrated by these species as a source of different compounds has stimulated us to study the production of other metabolites and their biological properties. The main objective of our study was to value these three Penicillium species from the point of view of the production of interesting metabolites (e.g. tocopherols or phenolic compounds) and testing their bioactivity (antioxidant and antimicrobial activities), for further application in pharmaceutical industry. For that, the studied species were grown in two different culture media -Czapek and Malt media -to further compare the produced metabolites and verify if the media exert some influence on the metabolites production and their release/or not (to predict possible extraction methods in future work). Then, the biological potential was assessed through four different assays to test the antioxidant activity and through the evaluation of the antimicrobial activity against a Gram-positive bacterium and one yeast species. Thus, we could infer that the produced compounds conferred bioactive properties to the fungi.

Growth and culture conditions
The molds were maintained on malt agar (MA) medium at 25 C for 7 d. The stock culture (7 d old) was inoculate in 150 mL of Czapek medium (NaNO 3 3 g, K 2 HPO 4 1 g, MgSO 4 .7H 2 O 0.5 g, KCl 0.5 g, FeSO 4. 7H 2 O 0.01 g, sucrose 30 g, pH 7.0 AE 2) or in 150 mL of malt medium (malt extract 50 g, Torlak, Belgrade, Serbia) in 500 mL Erlenmeyer Fasks. Media were sterilized by filtration using sterile filter papers. The inoculated Czapek (Cz) Fasks were then incubated at 28 C for 15 d on a rotary shaker (150 rpm). The inoculated Fasks containing malt medium were incubated at 28 C for 21 d stationary. The procedures were repeated four times in order to obtain enough biomass to perform all the assays. Fungal mycelia were separated from the media by Eltration using Whatman Elter paper No. 4 and washing twice with sterile distilled water. The mycelia were dried at 37 C and kept at 4 C until further use. The culture media were lyophilized and kept at 4 C until further use.

Free sugars extraction and analysis
The lyophilized samples (1 g) were spiked with the internal standard (IS) (raffinose, 5 mg/mL), and were extracted with 40 mL of 80% aqueous ethanol at 80 C for 1 h 30 min. The resulting suspension was filtered and concentrated under reduced pressure (rotary evaporator Büchi R-210) and defatted three times with 10 mL of ethyl ether, successively. After concentration, the residues were dissolved in water to a final volume of 5 mL, filtered through a 0.22 mm disposable LC filter disk, transferred into an injection vial and analyzed by HPLC consisting of an integrated system with a pump (Knauer, Smartline system 1000), degasser system (Smartline manager 5000) and auto-sampler (AS-2057 Jasco), coupled to a refraction index detector (RI detector Knauer Smartline 2300). The chromatographic separation was achieved with an Eurospher 100-5 NH 2 column (4.6 mm Â 250 mm, 5 mm, Knauer) operating at 35 C (7971 R Grace oven). The mobile phase used was acetonitrile/deionized water, 70:30 (v/v) at a flow rate of 1 mL/min, and the injection volume was 20 mL 21 . Sugars identification was made by comparing the relative retention times of sample peaks with standards. Data were analyzed using Clarity 2.4 Software (DataApex). Quantification was based on the RI signal response of each standard, using the IS method and by using calibration curves obtained from commercial standards of each compound. The results were expressed in mg per g of dry weight.

Organic acids extraction and analysis
The lyophilized samples (1.5 g) were extracted by stirring with 25 mL of meta-phosphoric acid (25 C at 150 rpm) for 45 min and subsequently filtered through Whatman No. 4 paper. Before the analysis by HPLC (Shimadzu 20A series UFLC), the sample was filtered through 0.2 mm nylon filters. Separation was achieved on a Sphere Clone (Phenomenex) reverse phase C 18 column (5 mm, 250 mm Â 4.6 mm i.d) thermostatted at 35 C. The elution was performed with sulfuric acid 3.6 mM using a flow rate of 0.8 mL/ min. Detection was carried out in a photodiode array detector (PDA), using 215 nm and 245 as preferred wavelengths 22 . The organic acids were quantified by comparison of the area of their peaks recorded at 215 nm with calibration curves obtained from commercial standards of each compound. The results were expressed in mg per g of dry weight.

Phenolic acids extraction and analysis
The lyophilized samples (1 g) were extracted with methanol:water (80:20,v/v; 30 mL) at À20 C for 1.5 h. After sonication for 15 min, the extract was filtered through Whatman No. 4 paper. The residue was then re-extracted with an additional 30 mL portion of the methanol:water mixture. Combined extracts were evaporated under reduced pressure to remove the methanol. The aqueous phase was washed with diethyl ether (2 Â 20 mL) and ethyl acetate (2 Â 20 mL). To the combined aqueous phases, anhydrous sodium sulfate was added and the extracts were filtered through Whatman No. 4 paper, evaporated to dryness and then redissolved in methanol:water (80:20, v/v). The extracts (1 mL) were filtered through a 0.22 mm disposable LC filter disk for HPLC analysis.The analysis was performed by HPLC (equipment described above). Detection was carried out in a PDA using 280 nm as the preferred wavelength 23 . The phenolic compounds were characterized according to their UV and mass spectra and retention times, and comparison with authentic standards when available. The identified phenolic acids were quantified by comparison of the area of their peaks recorded at 280 nm with calibration curves obtained from commercial standards of each compound. The results were expressed in mg per g of dry weight.

Tocopherols extraction and analysis
3,5-Di-tert-butyl-4-hydroxytoluene (BHT) solution (100 mL) and IS solution (tocol, 250 mL) were added to the sample prior to the extraction procedure. The lyophilized samples ($500 mg) were homogenized with methanol (4 mL) by vortex mixing (1 min). Subsequently, hexane (4 mL) was added and again vortex mixed for 1 min. After that, saturated NaCl aqueous solution (2 mL) was added, the mixture was homogenized (1 min), centrifuged (5 min, 4000g) and the clear upper layer was carefully transferred to a vial. The sample was re-extracted twice with hexane. The combined extracts were taken to dryness under a nitrogen stream, re-dissolved in 1 mL of hexane, dehydrated with anhydrous sodium sulfate, filtered through a 0.22 mm disposable LC filter disk, transferred into a dark injection vial and analyzed by HPLC (equipment described above for sugars analysis), and a fluorescence detector (FP-2020; Jasco) programmed for excitation at 290 nm and emission at 330 nm. Data were analyzed using Clarity 2.4 Software (DataApex). The chromatographic separation was achieved with a polyamide II (250 Â 4.6 mm) normal-phase column from YMC Waters (Japan) operating at 35 C. The mobile phase used was a mixture of hexane and ethyl acetate (70:30, v/v) at a flow rate of 1 mL/min, and the injection volume was 20 mL 24 . The compounds were identified by chromatographic comparisons with authentic standards. Quantification was based on the fluorescence signal response of each standard, using the IS method and by using calibration curves obtained from commercial standards of each compound. The results were expressed in mg per g of dry weight.

Antioxidant activity of phenolic acids extract
From the phenolic acids extract (PHA) mentioned above, successive dilutions were made from a stock solution at a concentration of 20 mg/mL for mycelia and 50 mg/mL for culture media. These methanolic solutions were then submitted to distinct in-vitro assays already described by Reis et al 20 . The results were expressed in EC 50 values (sample concentration providing 50% of antioxidant activity or 0.5 of absorbance in the Ferricyanide/ Prussian blue assay). Trolox was used as standard.
Reducing power was evaluated by Folin-Ciocalteu and Ferricyanide/Prussian blue assays. The first one measures the reduction of Folin-Ciocalteu reagent by the samples, expressed as mg of gallic acid equivalents (GAEs) per g of extract. The second one evaluates the capacity of the samples to convert Fe 3þ into Fe 2þ , measuring the absorbance at 690 nm in an ELX800 microplate reader (Bio-Tek Instruments, Inc, Winooski, VT). DPPH radical-scavenging activity was evaluated by using the same microplate reader, and calculated as a percentage of DPPH discoloration using the formula: [(A DPPH À A S )/A DPPH ] Â 100, where A S is the absorbance of the solution containing the sample at 515 nm and A DPPH is the absorbance of the DPPH solution. Inhibition of b-carotene bleaching was evaluated though the b-carotene/linoleate assay; the neutralization of linoleate free radicals avoids b-carotene bleaching, which is measured by the formula: b-carotene absorbance after 2 h of assay/initial absorbance) Â 100 20 .
Antimicrobial activity of organic acids, phenolic acids and tocopherols extracts and individual compounds The antimicrobial activity was evaluated for organic acids extract (OA), phenolic acids extract (PHA) and tocopherols extract (TOC), mentioned above, and also for the compounds identified in those extracts. The extracts (obtained using different solvents according to the polarity of the compounds to be extracted and analyzed) and pure compounds were dissolved in 1 mg/100 mL of 5% dimethyl sulfoxide (DMSO) (innocuous) before evaluation.
The Gram-positive bacterium Staphylococcus aureus (ATCC 6538) and the yeast Candida albicans (ATCC 10231) were used. The organisms were obtained from the Mycological Laboratory, Department of Plant Physiology, Institute for Biological Research ''Siniša Stanković'', Belgrade, Serbia.
Five, 10 and 20 mL of each sample (extract and pure compound) were applied on thin-layer chromatography (TLC) plates (Kieselgel 60 F254, Merck, Art. 5721) and sprayed either with freshly prepared bacterial and fungal inoculum (1.0 Â 10 5 cfu/mL) in nutrient broth (Tryptic Soy Broth; Biolife Italiana S.r.l., Milano, Italy). The plates were incubated for 24 h at 37 C and then sprayed with aqueous sol. 3% of p-iodonitrotetrazolium violet (INT) (2-(4-iodophenyl)-3-(4-nitrphenyl)-5-phenyltetrazolium chloride; Sigma), stored for another 3 h and sprayed with 70% EtOH to stop bacterial and fungal growth. White inhibition zones on a pinkish background were indicative of the antimicrobial activity of the tested extracts. The widths of these zones (mm) were the measure of efficiency. The antimicrobial activity was evaluated as the diameters of the inhibition zones with standard errors 25 . All the experiments were performed in duplicate and replicated three times. Streptomycin (Galenika, Belgrade, Serbia) and commercial fungicide fluconazole (F8929, Sigma-Aldrich, St. Louis, MO) were used as positive controls (5-20 mg/spot).

Statistical analysis
For statistical analysis, three samples were used and all the assays were carried out in triplicate. The results are expressed as mean values and standard deviation (SD), and analyzed using one-way analysis of variance (ANOVA) followed by Tukey's honestly significant difference (HSD) test with ¼ 0.05. This analysis was carried out using SPSS v. 18.0 program (SPSS Inc., Chicago, IL).

Metabolites analysis
As referred above, one way to value the studied species was the analysis of the metabolites produced by them, monitoring also the release/or not of these molecules for the culture media in order to prove the presence of such compounds and evaluate (in further works) possible methodologies of extraction. The assessed molecules were sugars (primary metabolites, however important for industry, from a nutritional point of they are energy producer and economically, sugars are probably the most efficient foodstuff), organic acids (result also from the primary metabolism, but some of them possess bioactive properties), phenolic acids (usually pointed as secondary metabolites produced when the species are under stress conditions, and reported as antioxidant and anticancer metabolites) and tocopherols (isoforms of vitamin E reported as bioactive compounds, especially as antioxidants).
The results of sugars composition of the studied mycelia and culture media are shown in Table 1. Concerning free sugars composition (of mycelia and culture media), rhamnose, xylose, fructose and trehalose were presented by all the studied samples. Noted that Cz medium has 30 g/mL of sucrose and this sugar was not detected in any of the samples corresponding to this medium. Since this is the carbon source provided to fungi, this leads us to believe that this sugar was metabolized.
Concerning organic acids, oxalic and fumaric acids were found in all the studied samples (Table 2). However, the latter was found in very low amounts (0.02-0.36 mg/g dw). Organic acids were present in higher quantities in culture media (34.94-182.29 mg/g dw), suggesting, once again, a release of the compounds from the fungus mycelium.
The phenolic acids detected in higher amounts were protocatechuic and p-hydroxybenzoic acids (Table 3). Generally, protocatechuic acid was found in all the mycelia, except in P. ochrochloron grown in malt medium. On the other hand, p-coumaric acid was only found in P. verrucosum var. cyclopium grown on malt medium (8.52 mg/g dw). The samples from MA medium were those which revealed the highest content in total phenolic acids, since P. verrucosum var. cyclopium grown on this medium revealed 85.46 mg/g dw of p-hydroxybenzoic acid.
Regarding tocopherols identification, P. ochrochloron and P. verrucosum var. cyclopium mycelia grown on Czapek medium, revealed the highest content in total tocopherols (11.53 and 40.96 mg/g dw, respectively; Table 4). P. ochrochloron was also the species with the highest content in tocopherols when inoculated in malt medium (0.84 mg/g dw).
Antioxidant activity of the phenolic acids extract As mentioned above, after the evaluation of the produced compounds, the biological properties of the species were evaluated. To test the antioxidant activity of the molds, and since there is no single method to evaluate the antioxidant activity, this property was monitored through four different assays. The results of the antioxidant properties of the phenolic extracts (PHA) obtained from the studied fungus mycelia and culture media are shown in Table 5. The species P. verrucosum var. cyclopium grown on both media revealed the highest reducing power measured by Folin-Ciocalteu assay (28.73-55.83 mg GAE/ g extract) and by Ferricyanide/Prussian blue assay (1.29-2.81 mg/ mL). This species also revealed, higher DPPH radical scavenging activity (2.31-5.45 mg/mL). Concerning b-carotene bleaching inhibition, the three fungi species, grown on Czapek medium, revealed statistically similar values (0.11-2.38 mg/mL). Analyzing data for the mentioned species grown on malt medium, the results do not differ much, being the samples with the highest b-carotene bleaching inhibition.

Antimicrobial activity of organic acids, phenolic acids and tocopherols extracts and individual compounds
Other biological activity tested to prove the bioactivity of the metabolites produced by the studied fungi was the antimicrobial potential. The OA, PHA and TOC extracts activity of the species and respective culture media was tested against the bacterium S. aureus and the yeast C. albicans, and the results were expressed as diameters of inhibition zones (Tables 6-8, Figure 1). The activity of the respective extracts was tested in three different concentrations: 5 mg/spot, 10 mg/spot and 20 mg/spot.
All the OA extracts derived both from fungal mycelia and culture media revealed antibacterial activity (it was possible to see and measure the inhibition zones; Table 6, Figure 1). Concerning the anticandidal activity, mycelia and media presented similar values, but just for the highest concentration tested (20 mg/spot). In a concentration of 5 mg/spot, only one of the culture medium revealed activity and for the concentration of 10 mg/spot there were also some samples that do not reveal any inhibition zone. All the tested individual organic acids possessed antibacterial activity against S. aureus (5.33-16.00 mm). There were some variations in Table 2. Composition in organic acids of the studied mycelia and culture media (mg/g dw; mean AE SD).   these results; however, it can be concluded that malic acid showed the best antibacterial properties for all the tested concentrations. On the other side, only oxalic and fumaric acids possessed antifungal activity against C. albicans, but lower than the commercial drug fluconazole ( Table 6, Figure 1). The results of the antimicrobial activity of PHA extracts of the three tested fungi are presented in Table 7 and Figure 1. The best antibacterial activity for the mycelia PHA extracts was achieved by P. ochrachloron mycelium, grown on Czapek media (18.00 mm). Concerning mycelia extracts from MA medium, P. verrucosum var. cyclopium revealed the highest antibacterial activity. Only the mycelia PHA extracts of P. ochrochloron grown on Cz medium and P. verrucosum var. cyclopium grown on MA medium revealed activity against C. albicans. Generally, all the tested individual phenolic acids showed similar values of antibacterial inhibition against S. aureus, and higher than the commercial drug streptomycin (Table 7, Figure 1). On the other side, only p-coumaric and cinnamic acids revealed antifungal activity against C. albicans at all the tested concentrations, the latter being the best inhibitor of this fungi species (even when compared with fluconazole).
Regarding the TOC extracts derived from fungal mycelia, the biggest inhibition zone was recorded for P. verrucosum var. cyclopium against S. aureus (13.33-16.33 mm; Table 8). However, all the tested samples revealed antibacterial activity (except P. funiculosum mycelium grown in MA medium at a concentration of 5 mg/spot). The diameter of inhibition zones for the mycelia TOC extracts of the three tested species, grown on Cz medium, towards C. albicans, varied from 5.67 to 8.67 mm ( Table 8). The TOC extracts from culture media did not reveal anticandidal activity, except P. funiculosum culture medium tested at the highest concentration (7.67 mm). TOC extracts from MA medium, generally demonstrate similar results. The studied tocopherol isoforms (a, b, g and ) on the selected Gram-positive Table 6. Antimicrobial activity of the organic acids extracts and individual compounds of the studied mycelia and culture media by TLC bioautography method (expressed as diameters of inhibition zones (mm), mean AE SD).  In each column, and for each culture medium, different letters mean significant differences between species (p50.05). Concerning the Folin-Ciocalteu assay, higher values mean higher reducing power; for the other assays, the results are presented in EC 50 values, what means that higher values correspond to lower reducing power or antioxidant potential. EC 50 : extract concentration corresponding to 50% of antioxidant activity or 0.5 of absorbance for the Ferricyanide/Prussian blue assay.

Species
bacterium and C. albicans are also presented in Table 8 and Figure 1. Only the isomers gand -tocopherol showed antibacterial activity against S. aureus with inhibition zone of 5.00-10.00 mm. All the tocopherol isomers, revealed antifungal activity against C. albicans with different diameters of inhibition (5.00-14.0 mm), depended on concentration. The commercial drug streptomycin revealed lower antibacterial activity than the tested compounds (5.00 mm), while fluconazole shown one of the best results for anticandidal activity (11.67-15.00 mm; Table 8 and Figure 1).

Discussion
One of the most abundant sugars in the studied samples was trehalose. This could be an interesting result since this sugar seems to play an important role during the oxidative stress acting as an antioxidant 26 . Thus, although it is a component of most immature sporocarps and may function as a reserve which is metabolized when the sporocarps are maturing 27 , it may also have been produced in response to stress under which the species stood. Concerning organic acids, the main detected compounds were oxalic and fumaric acid. These organic acids play an important role in human body being part of the Krebs cycle. However, oxalic acid is usually associated to species pathogenicity. For example, during a process of pathogenesis by some fungi, this acid is produced in order to make the plant more susceptible 28 . Therefore, oxalic acid may have been produced as a response to oxidative stress, since the fungi were under in-vitro conditions and, when in the presence of S. aereus or C. albicans, it may be produced as a toxic element to inhibit their growth. Fumaric acid is an unsaturated dicarboxylic acid that also possesses biological effects such as anti-inflammatory, neuroprotective and chemopreventive activity. Besides, it could be also used in food industry Table 7. Antimicrobial activity of the phenolic acids extracts and individual compounds of the studied mycelia and culture media by TLC bioautography method (expressed as diameters of inhibition zones (mm), mean AE SD).  as an antimicrobial agent for fruits and vegetables preservation 29,30 , hence the importance of its synthesis. Different aspects of organic acids working mechanism with respect to their antibacterial activity are given in the review articles of Cherrington et al. 31 and Russell 32 . The organic acids are lipid soluble in the undissociated form, in which they are able to enter the microbial cell. Once in the cell, the acid releases the proton in the more alkaline environment, resulting in a decrease of intracellular pH. This influences the microbial metabolism, inhibiting the action of important microbial enzymes and forces the bacterial cell to use energy to release protons, leading to an intracellular accumulation of acid anions. This accumulation depends on the pH gradient across the membrane. The acid anion seems to be very important regarding the antibacterial effect of organic acids and their salts. Accordingly, the production of organic acids by the fungi and their release to the media, it may be an answer to the stress conditions, once the molds were not in their habitat, and when placed in the presence of a foreign agent, they may attempt to inhibit its growth through the production of these molecules. Phenolic acids are composed of hydroxycinnamic and hydroxybenzoic acids. They have antioxidant activity as chelators and free radical scavengers with special impact over hydroxyl and peroxyl radicals, superoxide anions and peroxynitrites 33  P P P P P P P P P C C C C C C C C C P P P P P P p c 10 P P P P P P  the importance of their detection in biological sources. Phenols are also referred as molecules produced when the species are under stress conditions which may justify their production by the molds. These results could confirm that the studied species may be a source of important molecules that act as antioxidants. Comparing the antioxidant activity of the mycelium and the culture medium on which it has been developed, generally, the mycelium revealed higher antioxidant potential, as reported by other studies with different species and culture media [34][35][36] . However, there was some antioxidant activity detected in culture media, which may suggest the release of some antioxidant compounds from the mycelia like phenolic compounds ( Table 5). The antimicrobial capacity of some phenolic compounds is well-known 37,38 . They act by causing the leakage of cytoplasmic constituents such as protein, glutamate or potassium and phosphate from bacteria, which may be due to the disruption of cell peptidoglycan or damage of the cell membrane. Extracts may be more beneficial than isolated constituents, since a bioactive individual component can change its properties in the presence of other compounds present in the extract 39 . The mechanism of action involves the alteration of the permeability of the cell membrane that could result in the uncoupling of oxidative phosphorylation, inhibition of active transport and loss of pool metabolites due to the cytoplasmic membrane damage 40 . Moreover, the presence of hydroxyl groups in the phenolic compound might influence their antimicrobial effectiveness by binding to the active site of enzymes, form hydrogen bonds with enzymes and alter their metabolism; the lipid solubility and the degree of steric hindrance of the phenolic compounds might determine also their antimicrobial activity 37 . This could explain the production of these compounds by the studied fungi.

A A A A A A A A A A A A
Vitamin E is composed of eight isoforms, with four tocopherols (a-tocopherol, b-tocopherol, g-tocopherol and -tocopherol) and four tocotrienols (a-tocotrienol, b-tocotrienol, g-tocotrienoland -tocotrienol). This vitamin halts lipid peroxidation and is the only major lipid-soluble chain-breaking antioxidant found in plasma, red cells and tissues, allowing the protection of the integrity of lipid structures, mainly membranes 41 . Since malt extract contained amino acids in certain amount, the biosynthesis of tocopherols in these conditions might be enhanced. Noted that the isoform found in highest quantities was g-tocopherol, even it was not present in all the studied samples (namely in almost all culture media; Table 4). Other studies of our research group also revealed that this was the most abundant isoform present in different mycelia inoculated in different culture media 34 . Thus, it seems that various genera of fungi, obtained by in-vitro culture produce g-tocopherol in higher amounts. Often, a-tocopherol is referred as the most bioactive isoform of vitamin E. Nevertheless, other studies suggest that g-tocopherol provides different antioxidant activities in food as also in some in-vitro studies and showed higher activity in trapping lipophilic electrophiles and reactive nitrogen and oxygen species 42 . With these results we can conclude that fungi can be seen as a good source of g-tocopherol and perhaps this is a new source to explore. Studies on antimicrobial activity of tocopherol extracts from Penicillium species have not been reported earlier.
Concerning the general results for the antimicrobial activity, they were very similar between the samples. Nonetheless, there were some differences between mycelia and culture media. The latter almost not showed anticandidal activity; however, they demonstrated some activity against S. aereus, which could be an indicator (over again) of the release of the compounds with this activity for the culture media.

Conclusions
With this study we can conclude that the studied Penicillium species could be a source of numerous interesting molecules, namely sugars, organic acids, phenolic acids and tocopherols. The production of these molecules could be enhanced by changes in the culture media, once they have different chemical compositions providing different nutrients and different answers. For example, samples grown on Czapek medium were richer in sugars, organic acids and tocopherols. These results could be interesting from the standpoint of the production of some bioactive compounds, and as the analyses were extended to the culture media and it seems that some molecules were released (because they revealed the presence of some compounds and shown antioxidant and antimicrobial potential), in future works different extraction methods for certain molecules of interest could be tested. It is unquestionable that the interest in all the types of fungal species, as possible sources of new bioactive compounds, is highly increasing. Moreover, the use of experimental design can accelerate the finding of optimal growth conditions for the production of metabolites, in agreement with the previously reported related experiments 43 .
This work demonstrates that even the considered pathogenic fungi from the Penicillium genus may be valued as a source of different molecules as they may have beneficial activities. Hence, these compounds naturally produced by fungi exhibit diverse biological effects and may also possess pharmacological activities.