Objective: In this study, the antifungal activities, the minimum inhibitory (MIC) and fungicidal (MFC) concentrations, of carvacrol were determined vis-a-vis the growth of Penicillium expansum spore. Methodology: In addition, the surface properties of spores of the studied strain were also investigated in terms of hydrophobicity-hydrophilicity and electrons donor-acceptor properties by the contact angle method. Results: Thus, the obtained results allowed at first, to find a strong sensitivity of these spores relative to the studied essential oil component through a very low MIC and MFC. Moreover, the untreated spores of P. expansum exhibited hydrophilic characteristics both qualitatively and quantitatively with very pronounced electron donor properties. The momentaneous exposure of the spores to the carvacrol molecule has led to strengthening of the hydrophilic character of the spores surface with almost monopolar basic properties over treatment time. Conclusion: Although, the mode of action, by which the essential oil components inhibit the growth of microorganisms, is poorly defined in the scientific literature, this study contributes to elucidate the impact of these molecules and especially the carvacrol, on the surface properties of fungal spores. These physicochemical properties govern the approach and adhesion of microorganisms in biofilm formation on materials.
PDF Abstract XML References Citation
How to cite this article
The Penicillium genus, with over 300 species1, is one of the most known in the family of fungi, with Aspergillus and Paecilomyces2, in particular thanks to certain strains for their positive roles in the food industry as well as in the medical field. Indeed, the species such as P. roqueforti, P. nalgiovense or P. camemberti are widely used in the manufacturing processes of certain cheeses, when P. chrysogenum and P. notatum produce the penicillin antibiotic. However, several other species of the Penicillium genus are known for their negative roles, notably P. digitatum, P. italicum or P. expansum3-5. This latter is responsible for huge post-harvest losses of fruits6-8 and can produce, among other mycotoxins, the patulin which is one of most toxic for the human health7,9.
Many studies are regularly reported, in recent decades, on the significant antibacterial and antifungal potential of essential oils. Indeed, they are frequently used as antimicrobial, antioxidant and insecticidal agents10-12. However, few studies are carried out with the main components of these essential oils. These latter are known to be very complex volatile compounds consisting of several tens or even hundreds of different molecules. Some of them, present in high proportion, confer to these essential oils their important antimicrobial activities.
Similarly, very few studies were reported in the scientific literature on the impact of antifungal molecules on the physicochemical surface properties of pathogens fungal that may present a major risk in the food processing industries, medical field and even in the preservation of the historical monuments. These studies on the physico-chemical properties would allow to better understand the adhesion phenomenon of microorganisms and the forces that govern their approaches13-16 on the materials surfaces regularly used in these sectors. They would also help to better understand the effect of substances and molecules with antimicrobial effect, used in the treatment of materials, on the physico-chemical surface properties of these microorganisms in their vegetative and spore forms.
Thus, this study aimed to evaluate the antifungal activity of carvacrol, which is a main component of the Thymus vulgaris essential oil, on the growth of Penicillium expansum spores by determining the minimum inhibitory (MIC) and fungicidal (MFC) concentrations. In addition, the physico-chemical properties of the Penicillium expansum spores surface were also evaluated by the contact angle method before and after exposure of the spores to the MIC of carvacrol and their evolution as a function of time.
This study is the first in the recent scientific literature, which shows the effect of momentaneous exposure of fungal spores to an antifungal molecule on their surface properties in terms of hydrophobicity-hydrophilicity, electron donor-acceptor properties and interfacial free energy.
MATERIALS AND METHODS
Penicillium expansum strain, growth conditions and harvesting spores: Penicillium expansum was isolated from cedar wood decay and identified in the laboratory17. Growth was obtained at 25°C using malt extract agar. After 7 days of incubation, the spores of P. expansum were then harvested by scraping the culture surface in sterile 0.1% tween 20. The spores suspensions was concentrated by centrifugation at 10,000 g for 15 min at 4°C until a concentration of 106 spores mL1 (counted with a hemacytometer).
Antifungal activity evaluation: The antifungal activities, MIC and MFC, of the essential oil component (carvacrol) against Penicillium expansum spores were determined according to the micro-well dilution method18. Briefly, the carvacrol was diluted in sterile 1% tween 20 and was distributed in the microplate wells containing already 10 μL of the prepared spores suspension; the carvacrol was therefore diluted successively from well to well and the range of concentrations was 5-0.005%. In the control wells, the equivalent volume to that of the tested component was replaced by the solution of tween 20. Thus, the MIC, that is defined as the lowest concentration which inhibited the growth of the studied strain was determined after incubation for 48 h at 28°C. The determination of the CMF was conducted from wells that have not shown a visible growth by depositing spots (5 μL) on malt extract agar. The Petri dishes were then incubated at 28°C for 72 h and the lowest concentration that has not showed visible growth was considered as MFC.
Essential oil component: The essential oil component used in this study, for the antifungal activity and the physico-chemical surface properties of the P. expansum spores was carvacrol (99% pure) purchased from sigma-aldrich.
Momentaneous exposure treatment of the Penicillium expansum spore surface: The spore suspension concentrated to 106 spores mL1, prepared such as mentioned above was distributed in three volumes of 10 mL each. Then the tested compound was added, so as to obtain a final concentration of 0.625% for the 2 volumes. Finally, the spores were then incubated for the 2 studied time (1 and 3 h). After each incubation time, the corresponding erlenmeyer flask was removed and the spore suspension was filtered through a nitrocellulose membrane of porosity 0.45 μm. The membranes were then placed in sterile petri dishes before proceeding to the contact angles measures with the 3 liquids after 20-25 min.
Contact angle measurements and calculation of the interfacial free energy: The physico-chemical properties of the cedar wood surface were characterized by the contact angles measurements through the sessile drop technique using a goniometer apparatus19-21. The initial contact angle of each liquid was measured after drop stabilization on the solid sample surfaces. For the determination of the interfacial free energy of the solid surface (treated and untreated samples), three liquids are recommended22. They consist of two polar liquids (water and formamide) and one apolar liquid (diiodomethane) with known surface tension characteristics (Table 1). Therefore, contact angles measurments on each wood samples were made using these pure liquids. Then, all parameters of the surface physico-chemical characteristics (the Lifshitz-van der Waals component (γLW), the electron donor or Lewis base (γ‾) and the electron acceptor or Lewis acid (γ+)) allowing to determine the surface free energy of each sample (ΔGiwi) were calculated by the Youngs equation23:
where, the terms (S) and (L) denote solid surface and liquid phases, respectively.
Lewis acid-base component () is obtained by:
Moreover, the degree of hydrophobicity of each sample surface was evaluated by applying the approach23. According to this study, the degree of hydrophobicity of a given material is expressed as the free energy of interaction between two entities of that material immersed in water (w): ΔGiwi. This parameter has been calculated through the surface tension components of the interacting entities, according to the following equation:
The values of the surface tension parameters for the three pure liquids used in this study are shown in Table 1.
Statistical analysis: Data are expressed as Mean±Standard Error (SE) and analyzed using SPSS Statistics software by one-way ANOVA followed by Tukey post hoc test with significance defined at p<0.05.
Effect of carvacrol on spores growth inhibition of Penicillium expansum: The antifungal activity results of the essential oil component, after an incubation period of 72 h at 28°C, were shown in Table 2.
As it can be seen in Table 2, the carvacrol had a very important antifungal effect on the growth of P. expansum spores. Indeed, the minimum inhibitory concentration of the tested component for this strain was 0.625% of carvacrol concentration. This reflects a strong sensitivity of this fungal strain to the carvacrol molecule.
The MFC was determined by depositing spots (5 μL) from wells 9-12 in petri dishes containing sterile malt extract agar, then incubated for 72 h. The results showed that there was growth of spores only for the spots taken from the well 9. Therefore, the concentration of the well 10 is considered as the MFC. Thus, the CMF was determined at 1.25% of carvacrol concentration.
Physicochemical surface characterization of Penicillium expansum spores: To study the effect of momentaneous exposure of P. expansum spores to carvacrol on their physico-chemical surface properties, the spore suspension was treated at a concentration of 0.625% (corresponding to the MIC determined in the previous step of the antifungal essay) for 1 and 3 h.
|Table 1:||Surface tension properties of pure liquids used to measure contact angles24|
|Table 2:||Effect of carvacrol on the growth of Penicillium expansum spores|
|+: Presence of growth and -: Absence of growth|
Table 3 presents, for the three used liquids, the mean values of contact angles obtained for the untreated spore surface (control) and those exposed to carvacrol as a function of time. In Table 3, the calculations results of the intermediate surface tensions properties (the Lifshit Van der Waals (γLW) and the Lewis acid (γ+) and base (γ‾) parameters) and the interfacial free energy (ΔGiwi) obtained by using the Youngs equations23,24.
In Table 3, the water contact angle results showed an evolution of the wetting behavior of the spore surface before and after exposure to carvacrol. Indeed, the untreated P. expansum spore surface showed a very low water contact angle of the order of 26.5°. However, after 1 h of exposure to carvacrol, the spores wetting behavior evolved and given a water contact angle of 38 and 28.7° after 3 h of incubation. These obtained results, for the three samples (treated and untreated), are quite characteristics of hydrophilic surfaces.
In fact, when the value of the water contact angle exceeds 65°, the surfaces are characterized as hydrophobic and hydrophilic, when inversely the value of the water contact angle is less than 65°. Moreover, a positive value of the interfacial free energy (ΔGiwi) means that the surface is hydrophilic and a negative value indicates that it is hydrophobic24,25.
There is also an important decrease of the contact angles values with the apolar solvent (diiodomethane) after the treatment.
Electron donor-acceptor properties and the interfacial free energy of untreated and treated P. expansum spores surfaces: The surface tension components of Lifshit Van der Waals, as well as those of acid and base of Lewis are essential intermediate parameters to determine the interfacial free energy of surfaces by the Youngs equation.
The untreated surface of the P. expansum spores showed a very low electron acceptor character while the electron donor component were considerably high (Table 3).
It is known that the surfaces which have very high electron donor components are characterized as hydrophilic.
On the other hand, although already very low for the untreated spores (γ+ = 1.63 mJ m2), the exposure to carvacrol has visibly the effect of significantly (p<0.001) reducing the surface tension, corresponding to the Lewis acid, in time. Indeed, after 3 h, it was almost zero (γ+ = 0.003 mJ m2) (Table 3).
However, there was an increase of the Lifshit Van der Waals component from γLW = 33.8 mJ m2 (for control) to γLW = 40.8 mJ m2 (after 1 h) and γLW = 41.6 mJ m2 (after 3 h). The latter is inversely proportional to the diiodomethane contact angle value. In fact, the more the value of the diiodomethane contact angle is high and the more γLW is lower.
The hydrophilic or hydrophobic quantitative characterization of a material is defined by the calculated value of its surface free energy. Thus, according to the results presented in Table 3, the untreated surface of the P. expansum spores can be characterized as hydrophilic with a positive value of the interfacial free energy (ΔGiwi = 28.88 mJ m2>0).
The results also showed positive values for the interfacial free energies of the samples of both treatments after the momentaneous exposure of spores to the carvacrol molecules with ΔGiwi = 14.01 and 45.54 mJ m2, respectively for 1 and 3 h of exposure. This indicates quantitatively and in the both cases, the hydrophilic character of the treated spore surfaces, after 1 h, a decrease of the interfacial free energy. However, the latter was much more strengthened in the second time (3 h) compared to the control.
These results clearly demonstrate the impact of the P. expansum spores exposure to carvacrol on their surface tension parameters. Thus, in light of all these findings, it appears quite clearly that the untreated spores surface is both qualitatively and quantitatively hydrophilic with more electron donor than acceptor characters.
The treatment by momentaneous exposure to carvacrol allowed to strengthen the hydrophilicity of the spores surface from ΔGiwi = 28.88 mJ m2 (for control) to ΔGiwi = 45.54 mJ m2 (after 3 h).
|Table 3:||Physico-chemical surface properties of Penicillium expansum spores depending on the exposure time to carvacrol|
|Statistically different from the control, *p<0.05, **p<0.01 and ***p<0.001|
The very important antibacterial and antifungal potential of essential oils is regularly reported in the scientific literature of these last decades. At the same time, their antimicrobial activities is attributed to their major phenolic components, such as thymol, eugenol or carvacrol26. This latter is present in a very high proportion in particular in the thyme and oregon essential oils27,28.
The important activity of carvacrol against the P. expansum strain, highlighted in this present study, is in agreement with the different works related to this molecule and reported in the literature.
Indeed, several studies have reported the inhibition of the spores of Botrytis cinerea29 by carvacrol and its antifungal property against Aspergillus spp. and Penicillium spp.30. Similar results of the inhibition of Aspergillus niger (MIC = 50 and MFC = 75 μg mL1), Aspergillus flavus (MIC = 100 and MFC = 125 μg mL1), Penicillium citrinum (MIC = 150 and MFC = 175 μg mL1) and Penicillium chrysogenum (MIC = 125 and MFC = 150 μg mL1) by the cavracrol have also been reported by Abbaszadeh et al.31 in their recent study.
However, very few studies have explained the mechanism of action of these essential oils and their volatile terpene derivatives in the inhibition of the growth of microorganisms. This moreover explains why the mechanism of action by which these compounds act against microorganisms is not very well defined in the literature. But, several hypotheses have been reported by researchers and would involve the perforation of the membrane, the destruction of the cell walls and the degradation of the spores of fungi.
In the case of carvacrol, its mechanism of action would consist in disrupting the membrane of microorganisms by affecting their membrane permeability; this action is enhanced by its hydrophobic nature32,33.
In addition to its proved antifungal activity, it was also indicated by different studies that this phenolic compound presents the anti-inflammatory, antioxidant, insecticidal, antiparasitic and hepatoprotective properties34-36.
However, no study has investigated on the effect of carvacrol on the physico-chemical surface properties of microorganisms in term of hydrophobicity-hydrophilicity and electron donor-acceptor characteristics.
The importance of these surface properties, both those of microorganisms as those of materials, is now well established in the literature and their implications in the mediation of the adhesion phenomenon of microorganisms in the biofilm formation on materials.
Thus, it is matter to show, in this study, how the surface properties of P. expansum spores could be affected when they are exposed to a substance with known antimicrobial property such as carvacrol.
The initially hydrophilic character of the surface of the P. expansum spores observed in the present study is in agreement with that reported in the study of El Abed et al.37. Indeed, the authors have reported the both qualitative (θw = 45.3±1.5°) and quantitative (ΔGiwi = 15.29 mJ m2) hydrophilic characteristics of the spore surface of this fungal strain.
Similar results have also been reported for several other species of the Penicillium genus with in particular P. crustosum (θw = 11.9±1.98°, ΔGiwi = 20.2 mJ m2), P. granulatum (θw = 36.5°, ΔGiwi = 18.7 mJ m2), P. commune (θw = 17.9±0.75°, ΔGiwi = 8.5 mJ m2) and P. chrysogenum (θw = 10.4±4.54°, ΔGiwi = 6.3 mJ m2) with electron donor properties much more marked than the electron acceptor properties38.
Capizzi and Schwartzbrod39 showed, using hydrocarbons, that the hydrophobic potential of Ascaris suum eggs was 95% (for fresh eggs) and 72% (for stored eggs at -20°C) obtained with octane as a solvent.
The results have also highlighted an electron donor characteristic reinforced over time further to the treatment. In fact, when the γ‾ value is greater than 27.9 mJ m2, the material is characterized as hydrophilic and hydrophobic23 when conversely the γ‾ value is less than 27.9 mJ m2. Thus, with a value of γ‾ = 50.98±0.45>>27.9 mJ m2, the untreated P. expansum spores could be characterized as hydrophilic. This confirms the qualitative results obtained above with the water contact angle.
The physico-chemical properties of microorganisms depend on the chemical composition and the morphology of their surfaces. But they are also influenced by the different physical parameters of their environments such as temperature40 and pH41, but also by the nature of the available nutrients42,43 and even their growth phase42.
Indeed, the Lewis base component (γ‾) of the surface is often due to the presence of different chemical groups to the surface of these microorganisms and which are mostly negatively charged, such as carboxylate (COO‾), amino (NH2), phosphate or SO3 groups44,45. These chemical groups belong to the main macromolecules which constitute the external membranes of microorganisms (lipopolysaccharides, lipoproteins and phospholipids).
On the other hand, Jeffs et al.46 found in their study that spores with rugose surfaces were hydrophobic whereas those with smooth surfaces were hydrophilic. Also, these studies have shown that the chemical treatment of the aerial conidia of the genera Beauveria, Metarhizium, Paecilomyces, Tolypocladium and Verticillium with Sodium Dodecyl Sulfate (SDS) and the Formic Acid (FA) reduced the surface hydrophobicity.
Azcurra et al.47 also reported similar results on effect of the High Molecular Weight Chitosan (HMWC) and sodium alginate (NaAL) on Candida albicans hydrophobicity and adhesion to cells. Indeed, they found a decrease of 44% of the hydrophobicity in presence of HMWC and 82% in the presence of NaAL, when they used chloroform as organic medium. On the other side, they found a decrease of the hydrophobic character of the order of 30 and 19% in presence of HMWC and NaAL, respectively when they used xylene, what caused a significant decrease in the adhesion of C. albicans to epithelial cells and human fibroblasts.
However, contrary to these authors, the results showed that the hydrophilic character of the P. expansum spore surface was enhanced by their momentaneous exposure to carvacrol. This effect of carvacrol has also been reported recently on the surface of cedar wood19. Indeed, the initially hydrophobic surface of the wood has become hydrophilic after the treatment by cavacrol.
Thus, the strengthening of the hydrophilic character of the P. expansum spores surface could be due to the hydroxyl function of the carvacrol molecule. This hydroxyl group induces a stronger electron donor character of the surface through electron pairs of the oxygen atom.
Furthermore, Ultee et al.28 have suggested that the hydroxyl function of molecules such as thymol and carvacrol play an important role in the antibacterial and antifungal activity of these derivatives terpene.
The antimicrobial activities and the physico-chemical properties of carvacrol could be very effective in surface treatments in both food industry and the medical field to prevent the adhesion and biofilm formation by pathogenic microorganisms.
In conclusion, the obtained results showed the important antifungal activity of the carvacrol against P. expansum in a lower concentration. Based on the results, this molecule, which is naturally present in medicinal and aromatic plants, has the ability to inhibit the growth of P. expansum spores and to eliminate them definitively at concentrations of 0.625 and 1.25% (v/v), respectively. On the other hand, the momentaneous exposure of these spores to carvacrol has significantly (p<0.05) modified their surface properties.
These findings allowed to notice the strengthening of the hydrophilicity of the spore surface which is revealed into an increase of the wetting behavior and the interfacial free energy. If the antifungal properties of phenolic compounds of the essential oils have repeatedly been reported. This present study is the first one to enable a better understanding of the effect of these compounds on the surface properties of fungi especially in terms of hydrophobicity-hydrophilicity and electron donor-acceptor properties. Besides the bioactive potential of carvacrol against the growth of P. expansum spores and its ability to influence their physico-chemical surface properties, this essential oil component could reduce and even completely inhibit their adhesion on materials.
- Houbraken, J. and R.A. Samson, 2011. Phylogeny of Penicillium and the segregation of Trichocomaceae into three families. Stud. Mycol., 70: 1-51.
- He, S., X. Ren, Y. Lu, Y. Zhang, Y. Wang and L. Sun, 2016. Microemulsification of clove essential oil improves its in vitro and in vivo control of Penicillium digitatum. Food Control, 65: 106-111.
- Zeng, R., A. Zhang, J. Chen and Y. Fu, 2012. Postharvest quality and physiological responses of clove bud extract dip on 'Newhall' navel orange. Scientia Horticulturae, 138: 253-258.
- Kellerman, M., A. Erasmus, P.J.R. Cronje and P.H. Fourie, 2014. Thiabendazole residue loading in dip, drench and wax coating applications to control green mould and chilling injury on citrus fruit. Postharvest Biol. Technol., 96: 78-87.
- Da Rocha Neto, A.C., M. Maraschin and R.M. di Piero, 2015. Antifungal activity of salicylic acid against Penicillium expansum and its possible mechanisms of action. Int. J. Food. Microbiol., 215: 64-70.
- Da Rocha Neto, A.C., C. Luiz, M. Maraschin and R.M. di Piero, 2016. Efficacy of salicylic acid to reduce Penicillium expansum inoculum and preserve apple fruits. Int. J. Food. Microbiol., 221: 54-60.
- Sanzani, S.M., L. Schena, A. de Girolamo, A. Ippolito and L. Gonzalez-Candelas, 2010. Characterization of genes associated with induced resistance against Penicillium expansum in apple fruit treated with quercetin. Postharvest Biol. Technol., 56: 1-11.
- Wright, S.A.I., D.V. de Felice, G. Ianiri, C. Pinedo-Rivilla, F. de Curtis and R. Castoria, 2014. Two rapid assays for screening of patulin biodegradation. Int. J. Environ. Sci. Technol., 11: 1387-1398.
- Bakkali, F., S. Averbeck, D. Averbeck and M. Idaomar, 2008. Biological effects of essential oils-A review. Food Chem. Toxicol., 46: 446-475.
- Arnal-Schnebelen, B., F. Hadji-Minaglou, J.F. Peroteau, F. Ribeyre and V.G. de Billerbeck, 2004. Essential oils in infectious gynaecological disease: A statistical study of 658 cases. Int. J. Aromather., 14: 192-197.
- Burt, S., 2004. Essential oils: Their antibacterial properties and potential applications in foods-A review. Int. J. Food Microbiol., 94: 223-253.
- Barkai, H., S. El Abed, M. Sadiki, M.I. Houssaini and S.I. Koraichi, 2016. A study on the impact of the adhesion of Penicillium expansum on the physicochemical surface properties of cedar wood. J. Adhesion, 92: 341-348.
- Barkai, H., M. Sadiki, S. El Abed, M. Moustakhim, M.I. Houssaini and S.I. Koraichi, 2015. Comparison of the evolution of physico-chemical properties due to the single and combined adhesion of two species of the Penicillium genus on cedar wood. J. Mater. Environ. Sci., 6: 749-755.
- El Abed, S., M. Mohamed, B. Fatimazahra, L. Hassan, H. Abdellah, H. Fatima and I.K. Saad, 2011. Study of microbial adhesion on some wood species: Theoretical prediction. Microbiology, 80: 43-49.
- Hamadi, F., H. Latrache, M. Zekraoui, M. Ellouali and J. Bengourram, 2009. Effect of pH on surface energy of glass and Teflon and theoretical prediction of Staphylococcus aureus adhesion. Mater. Sci. Eng.: C, 29: 1302-1305.
- Zyani, M., D. Mortabit, M. Mostakim, M. Iraqui, A. Haggoud, M. Ettayebi and S.I. Koraichi, 2009. Cellulolytic potential of fungi in wood degradation from an old house at the Medina of Fez. Ann. Microbiol., 59: 699-704.
- Tian, J., Y. Chen, B. Ma, J. He, J. Tong and Y. Wang, 2014. Drosera peltata Smith var. lunata (Buch.-Ham.) C. B. Clarke as a feasible source of plumbagin: Phytochemical analysis and antifungal activity assay. World J. Microbiol. Biotechnol., 30: 737-745.
- Barkai, H., M. Sadiki, S. El Abed, E.H. EL Harchli, S. Boutahari and S.I. Koraichi, 2015. The effect of carvacrol and carvone treatments on the cedar wood surfacephysico-chemical properties. Int. J. Sci. Eng. Res., 6: 767-771.
- Chen, X., J.A. Weibel and S.V. Garimella, 2015. Exploiting microscale roughness on hierarchical superhydrophobic copper surfaces for enhanced dropwise condensation. Adv. Mater. Interfaces, Vol. 2.
- Liang, C.C., C.H. Lin, T.C. Cheng, J. Shieh and H.H. Lin, 2015. Nanoimprinting of flexible polycarbonate sheets with a flexible polymer mold and application to superhydrophobic surfaces. Adv. Mater. Interfaces, Vol. 2.
- Kaelble, D.H., P.J. Dynes and E.H. Cirlin, 1974. Interfacial bonding and environmental stability of polymer matrix composites. J. Adhesion, 6: 23-48.
- Van Oss, C.J., 1995. Hydrophobicity of biosurfaces-origin, quantitative determination and interaction energies. Colloids Surf. B: Biointerfaces, 5: 91-110.
- Van Oss, C.J., M.K. Chaudhury and R.J. Good, 1988. Interfacial lifshitz-van der waals and polar interactions in macroscopic systems. Chem. Rev., 88: 927-941.
- Vogler, E.A., 1998. Structure and reactivity of water at biomaterial surfaces. Adv. Colloid Interface Sci., 74: 69-117.
- ben Arfa, A., S. Combes, L. Preziosi-Belloy, N. Gontard and P. Chalier, 2006. Antimicrobial activity of carvacrol related to its chemical structure. Lett. Appl. Microbiol., 43: 149-154.
- De Vincenzi, M., A. Stammati, A. De Vincenzi and M. Silano, 2004. Constituents of aromatic plants: Carvacrol. Fitoterapia, 75: 801-804.
- Ultee, A., M.H.J. Bennik and R. Moezelaar, 2002. The phenolic hydroxyl group of Carvacrol is essential for action against the food-borne pathogen Bacillus cereus. Applied Environ. Microbiol., 68: 1561-1568.
- Markovic, T., P. Chatzopoulou, J. Siljegovic, M. Nikolic, J. Glamoclija, A. Ciric and M. Sokovic, 2011. Chemical analysis and antimicrobial activities of the essential oils of Satureja thymbra L. and Thymbra spicata L. and their main components. Arch. Biol. Sci., 63: 457-464.
- Martinez-Romero, D., F. Guillen, J.M. Valverde, G. Bailen and P. Zapata et al., 2007. Influence of carvacrol on survival of Botrytis cinerea inoculated in table grapes. Int. J. Food Microbiol., 115: 144-148.
- Abbaszadeh, S., A. Sharifzadeh, H. Shokri, A.R. Khosravi and A. Abbaszadeh, 2014. Antifungal efficacy of thymol, carvacrol, eugenol and menthol as alternative agents to control the growth of food-relevant fungi. J. Mycol. Med., 24: e51-e56.
- Ozogul, F., C. Kacar and I. Hamed, 2015. Inhibition effects of carvacrol on biogenic amines formation by common food-borne pathogens in histidine decarboxylase broth. LWT-Food Sci. Technol., 64: 50-55.
- Xu, J., F. Zhou, B.P. Ji, R.S. Pei and N. Xu, 2008. The antibacterial mechanism of carvacrol and thymol against Escherichia coli. Lett. Applied Microbiol., 47: 174-179.
- Kordali, S., A. Cakir, H. Ozer, R. Cakmakci, M. Kesdek and E. Mete, 2008. Antifungal, phytotoxic and insecticidal properties of essential oil isolated from Turkish Origanum acutidens and its three components, carvacrol, thymol and p-cymene. Bioresour. Technol., 99: 8788-8795.
- Manohar, V., C. Ingram, J. Gray, N.A. Talpur, B.W. Echard, D. Bagchi and H.G. Preuss, 2001. Antifungal activities of origanum oil against Candida albicans. Mol. Cell. Biochem., 228: 111-117.
- Nostro, A., A.S. Roccaro, G. Bisignano, A. Marino and M.A. Cannatelli et al., 2007. Effects of oregano, carvacrol and thymol on Staphylococcus aureus and Staphylococcus epidermidis biofilms. J. Med. Microbiol., 56: 519-523.
- El Abed, S., F. Hamadi, H. Latrache, H.M. Iraqui and S.I. Koraichi, 2010. Adhesion of Aspergillus niger and Penicillium expansums pores on Fez cedar wood substrata. Ann. Microbiol., 60: 377-382.
- El Abed, S., I.K. Saad, H. Abdellah and L. Hassan, 2013. Experimental and theoretical investigations of the adhesion time of Penicillium spores to cedar wood surface. Mater. Sci. Eng.: C, 33: 1276-1281.
- Capizzi, S. and J. Schwartzbrod, 2001. Surface properties of Ascaris suum eggs: Hydrophobic potential and lewis acid-base interactions. Colloids Surf. B: Biointerfaces, 22: 99-105.
- Kumar, G.S., M.V. Jagannadham and M.K. Ray, 2002. Low-temperature-induced changes in composition and fluidity of lipopolysaccharides in the antarctic psychrotrophic bacterium Pseudomonas syringae. J. Bacteriol., 184: 6746-6749.
- Ellwood, L.L. and D.W. Tempest, 1972. Influence of culture pH on the content and composition of teichoic acids in the walls of Bacillus subtilis. Microbiology, 73: 395-397.
- Atrih, A., G. Bacher, G. Allmaier, M.P. Williamson and S.J. Foster, 1999. Analysis of peptidoglycan structure from vegetative cells of Bacillus subtilis 168 and role of PBP 5 in peptidoglycan maturation. J. Bacteriol., 181: 3956-3966.
- Ellwood, D.C., 1970. The wall content and composition of Bacillus subtilis var. niger grown in a chemostat. Biochem. J., 118: 367-373.
- Djeribi, R., Z. Boucherit, W. Bouchloukh, W. Zouaoui, H. Latrache, F. Hamadi and B. Menaa, 2013. A study of pH effects on the bacterial surface physicochemical properties of Acinetobacter baumannii. Colloids Surf. B: Biointerfaces, 102: 540-545.
- Hamadi, F., H. Latrache, A. El Ghmari, M. El Louali, M. Mabrrouki and N. Kouider, 2004. Effect of pH and ionic strength on hydrophobicity and electron donor and acceptor characteristics of Escherichia coli and Staphylococcus aureus. Ann. Microbiol., 54: 213-225.
- Jeffs, L.B., I.J. Xavier, R.E. Matai and G.G. Khachatourians, 1999. Relationships between fungal spore morphologies and surface properties for entomopathogenic members of the general Beauveria, Metarhizium, Paecilomyces, Tolypocladium and Verticillium. Can. J. Microbiol., 45: 936-948.
- Azcurra, A.I., S.R. Barembaum, M.A. Bojanich, S.E. Calamari, J. Aguilar, L.J. Battellino and S.T. Dorronsoro, 2006. Effect of the high molecular weight chitosan and sodium alginate on Candida albicans hydrophobicity and adhesion to cells. Med. Oral. Patol. Oral. Cir. Bucal., 11: 120-125.