Abstract: In this study, the effect of different Thymus vulgaris extract fractions on Cell Surface Hydrophobicity (CSH) and acid-base properties of three fungal strains (Penicillium commune (PDLd10), Penicillium commune (PDLd’’) and Thielavia hyalocarpa (PDLb3)) using contact angle measurement method was investigated. The main results demonstrated that all fractions tested are able to influence fungal cell physicochemical properties. Indeed, the methanolic and ethyl acetate fractions were effective in reducing CSH and made the cells more hydrophilic and highly electron donor (p<0.05) compared to the untreated ones. Whereas, the hexane-ethyl acetate treatment was found made them more hydrophobic and weakly donor electron compared to the control (p<0.05). It is also noted that the modification degree of microbial cell surface properties was fraction-dependent.
INTRODUCTION
The microbial attachment and adhesion to the surface is a crucial point of the biofilm formation process that is controlled and affected by both substrata and cell microbial surfaces physicochemical properties. These latter, which have important implications in a variety of processes, are manifested in Lifshitz-van der Waals, electrostatic (Hood and Zottola, 1995; Marques et al., 2007) and electron donor-electron acceptor properties (Oulahal et al., 2008; Seale et al., 2008).
According to literature data, cell surface hydrophobicity (Bernardes et al., 2010; El Abed et al., 2010), surface charge (Zhao et al., 2007), acid-base properties and surface topography can have an important and a significant effect on microbial adhesion. Moreover, several works have reported that the hydrophobicity cannot always explain the results of microbial adhesion to the support (Pratt-Terpstra et al., 1988; Sjollema et al., 1990) and the acid-base interactions play a very important role in this phenomenon (Hamadi and Latrache, 2008; Henriques et al., 2004). Also, Van Oss (1993) reported that the acid-base interactions are 10-100 times more important compared to other interactions.
Several methods used to characterize/assess microbial cell surface hydrophobicity include binding to aliphatic acids, hydrocarbons, microsphere assay, hydrophobic interaction chromatography and salt aggregation test (Doyle, 2000). These methods are subjected to criticism as they are indirect methods to quantify hydrophobicity of the microbial cell surfaces (Chau et al., 2009). Three methods have been used to assess the acid-base properties of cell surface, including contact angle measurement (Busscher et al., 1984; El Abed et al., 2010) combined with equation of Van Oss et al. (1988), microbial adhesion to solvents (Bellon-Fontaine et al., 1996) and acid-base titration (Fein et al., 1997).
A considerable amount of research has been focused on the effect of medicinal plants extract on the cell surface hydrophobicity of many microorganisms mainly bacteria and yeast, using different methods such us salt aggregation test (Annuk et al., 1999), cell surface hydrophobicity (Nostro et al., 2000; Polaquini et al., 2006; Rahim and Khan, 2006). To the best of our knowledge, no studies have investigated the effect of these natural products on the physicochemical characteristics of fungal surfaces. Thus, our study which is carried out for the first time was designed with the objective of evaluating the effect of three fractions of Thymus vulgaris extract on the cell surface hydrophobic/hydrophilic character of three fungi responsible of cedar wood decay and also on their electron donor-electron acceptor properties using contact angle measurement method.
MATERIALS AND METHODS
Fungal strains: Penicillium commune (PDLd10), Penicillium commune (PDLd”) and Thielavia hyalocarpa (PDLb3) were isolated from cedar wood deteriorated from an old house located in Derb Lamté in the old Medina of Fez (Morocco). These strains were identified using rDNA ITS regions 1, 5.8S and ITS regions 2, which were amplified using primers ITS 1 and ITS 4 in our laboratory (Zyani et al., 2009). The strains were grown in malt extract-agar and incubated at 25°C for 7 days.
Preparation of extract: The extract preparation was carried out as described in our previous study (Sadiki et al., 2014). Brief, 50 g of dried and ground aerial part of Thymus vulgaris L. (Labiateae) were placed in flasks, mixed with 500 mL of methanol and sonicated in an ultrasonic bath (Elma-Transsonic TI-H-15) (Frequency 35 kHz, power: 100 W, temperature: 30°C for 45 min) (Two repetitions were performed). At the end of extraction time, each mixture was filtered through Whatman paper No. 1 and then evaporated under vacuum (Temperature ≤40°C) to obtain crude extract.
Fractionation and determination of total phenolic content: The fractionation of the Thymus vulgaris methanolic extract was performed using column chromatography over silica gel (60 G). About 4 g of crude extract dissolved in methanol was mixed with silica and the solvent evaporated to leave a dry powder. Then, the elution was made using organic solvents with increasing polarity, namely hexane-ethyl acetate (50:50), ethyl acetate and methanol which allows to obtain three fractions, which are evaporated and stored at 4°C until use.
The total phenolic content of fractions obtained was determined using the Folin-Ciocalteu reagent assay, with gallic acid as standard (McDonald et al., 2001). Briefly, from each fraction, 1 mg was dissolved in methanol (1 mL). The solutions of Folin-Ciocalteu reagent 10% (v/v) and Na2CO3 5% (w/v) were prepared. Subsequently, each fraction sample (200 μL) was taken in a test tube and 1.5 mL of Folin-Ciocalteu reagent (10%) was added. Then all the test tubes were kept in a dark place for 5 min. Thereafter, 1.5 mL of Na2CO3 (5%) was added to the solution and mixed. Again, all the test tubes were kept in the dark for 2 h. The absorbance was measured for all solutions by using a UV-vis spectrophotometer at constant wavelength 750 nm.
| Table 1: | Surface energy of contact angle liquids |
Contact angle measurements, surface tension components and hydrophobicity: Initial fungal cell surface properties and the effect of different fractions tested were deduced from measured contact angles for all probe liquids including water, formamide and diiodomethane (Table 1) (Van Oss et al., 1988).
The suitable microbial lawns for angle contact measurements were prepared and deposited on membrane filter as described by El Abed et al. (2011). Briefly, the microbial cells were suspended in KNO3 (0.1 mol L–1) sterile solution, followed by centrifugation at 7000 g min–1 for 15 min at 4°C. The pellet was then washed twice with the same solution and re-suspended in 10 mL of dimethylsulfoxide 2% and the fraction tested was added to a final concentration of 0.5 mg mL–1. The tubes were mixed by vortexing and incubated under shaking conditions at 30°C for 60 min. The control was performed with DMSO 2% alone. After the contact time, the spore suspensions were deposited on a cellulose acetate membrane filter (0.45 μm) using negative pressure (Busscher et al., 1984). The filters containing the microorganisms (spores approximately 108 cell mm–2) were placed to air dry for 30 min at room temperature in order to obtain stable, so-called "Plateau" contact angles. Contact angles were measured in triplicate. Once the contact angles were measured, the Lifshitz-van der Waals (γLW) and acid-base (γAB) surface tension components were obtained by the three equations system from the application of the Young-Dupré equation to each probe liquid (Van Oss, 1994):
where, θ is the measured contact angle, γLW is the Van der Waals free energy component, γ+ is the electron acceptor component, γ¯ is the electron donor component and the subscripts S and L are solid surface and liquid phases, respectively.
The surface free energy is expressed as:
where,
The fungal surface hydrophobicity was evaluated through contact angle measurements and by the approach of Van Oss et al. (1988). In this approach, the degree of hydrophobicity of a given material (1) is expressed as the free energy of interaction between two entities of that material when immersed in water (w): ΔGiwi. If the interaction between the two entities is stronger than the interaction of each entity with water, the material is considered hydrophobic (ΔGiwi<0); conversely, for a hydrophilic material (ΔGiwi>0). The ΔGiwi is calculated through the surface tension components of the interacting entities, according to the following formula:
Statistical analysis: Within subjects analysis of variance was performed using SPSS statistics software. The p-values tested the statistical significance of each of the factors through F-tests, when p-values were lower than 0.05, these factors had a statistically significant effect at the 95% confidence level.
RESULTS
Fractionation and total phenolic contents: The hexane-ethyle acetate fraction (788 mg), ethyl actetate fraction (155 mg) and methanolic fraction (2299 mg) were recovered. The total phenolic contents of these extractives was determined (expressed by mg gallic acid g–1 of fraction) and reported in our previous work (Sadiki et al., 2015). The results showed that it varied and was solvent-dependent. Indeed, the lowest amount was given by the ethyl acetate fraction which was similar to hexane-ethyl acetate fraction with values of 142.96 and 135.46 mg gallic acid g–1 fraction, respectively. While, the highest content was observed for the methanolic fraction (170. 62 mg gallic acid g–1 fraction).
Physicochemical surface properties of fungal spores studied: The surface hydrophobicity character, the Lifshitz-Van Der Waals (γLW), acid-base, surface free energy (ΔGiwi), electron donor (γ+) and electron acceptor (γ¯) parameters of fungal spores before and after treatment with the different fractions of T. vulgaris extract were assessed by contact angle measurements and are given in Table 2 and 3.
The contact angle vis-a-vis the water (θw) formed by fungal spores lawns may be used as a qualitative indicator of cell surface hydrophobicity. According to Vogler (1998) and the approach of Van Oss et al. (1989), the degree of hydrophobicity of a given material is expressed as the free energy of interaction between two entities of that material when immersed in water (w): Thus, the surface is considered hydrophobic if θw is higher than 65° and ΔGiwi<0, whereas hydrophilic ones if θw is lower than 65° and ΔGiwi<0.
| Table 2: | Contact angles measurements and free energy (ΔGiwi) of treated and untreated fungal strains studied |
| Table 3: | Lifshitz-van der Waals (γLW) component, electron donor (γ¯) and electron acceptor (γ+) parameters of treated and untreated fungal strains studied |
Taking into account the values of water contact angle (Table 2), it can be seen that all strains studied (Penicillium commune) (PDLd10), Penicillium commune (PDLd”) and Thielavia hyalocarpa (PDLb3) are qualitatively and quantitatively hydrophilic with values of water contact angle ranging from 36.1 to 41.9° and ΔGiwi between 8.28 and 37.12 mJ m–2.
The results demonstrated also that all strains were mainly electron donor with high values of (γ¯) ranging from 36.06 mJ m–2 (P. commune PDLd10) to 51.9 mJ m–2. In addition, the finding proves that all strains were low electron acceptor comprised between 0.2 mJ m–2 (P. commune PDLd”) and 7.97 mJ m–2 (P. commune PDLd10) (Table 3).
Effect of T. vulgaris fractions on the fungal cell surface hydrophobicity and their acid-base properties: The results of the separate fractions effect on the cell surface hydrophobicity (CSH), electron donor (γ¯) and electron acceptor (γ+) characters of the studied strains are presented in Table 2 and 3.
As can be noted from these findings, all fractions tested have changed qualitively and quantitaively (p<0.05) the hydrophobicity parameter of fungal studied compared to untreated ones. Indeed, both ethyl acetate and methanolic fractions have decreased the CSH of all species (p<0.05), except in the case of P. commune PDLd10 treated with ethyl acetate fraction (p<0.05). In addition, it is observed that the methanolic fraction has more and drastically reduced the water contact angle (θw) of P. commune PDLd10, P. commune PDLd” and T. hyalocarpa PDLb3 to the values of θw = 28.03°, θw = 22.0° and θw = 20.4°, respectively compared to the untreated ones (Table 2).
The results also highlighted the effect statically significant of hexane-ethyl acetate fraction, which has highly increased the hydrophobicity of all strains studied (p<0.05). Thus, T. yalocarpa PDLb3 and P. commune PDLd10 have become more hydrophobic qualitatively and quantitatively with values of θw = 73.1°, ΔGiwi = -49.20 mJ m–2 and θw = 61.66°, ΔGiwi = -49.20 mJ m–2, respectively. While, P. commune PDLd” has became less hydrophilic than the control.
Concerning the effect of the various fractions tested on acid-base properties of fungal cells surface, the results showed that the electron acceptor and donor parameters are influenced after treatment. In fact, the methanolic fraction has slightly increased (p<0.05) the electron acceptor (γ+) parameter of all fungal strains studied. Whereas, the ethyl acetate and hexane-ethyl acetate fractions have reduced the acid property of P. commune PDLd10 and enhanced it for P. commune PDLd” and T. yalocarpa PDLb3.
In addition, it was observed that both methanolic and ethyl acetate fractions have increased the electron donor for P. commue PDLd” and T. yalocarpa PDLb3 (γ¯ = 55.4, 55.8, 58.0 and 61.0 mJ m–2, respectively) (p<0.05) and they have caued a slight increase in the case of P. commune PDLd10 (p<0.05). While, the hexane-ethyl acetate has reduced the negatively charged surface of all fungal species (p<0.05) and drastically in the case of T. yalocarpa PDLb1 and P. commune PDLd10 with values of γ¯ = 2.3 and 12.18 mJ m–2, respectively) compared to the control ones (Table 3).
Furthermore, it can be noted that the physicochemical properties (CSH and acid-base) of T. yalocarpa (PDLb3) are more influenced with treatment followed by those of P. commune (PDLd10) and P. commune (PDLd”), indicating that the reduction or enhancement effect was found to be fraction-dependent.
DISCUSSION
The study of physicochemical characteristics (hydophobicity and electron donor-electron acceptor) of microbial cell surface has an importance in several areas of research and development, such as microbial adhesion, heritage field and food control. Thus, a broad knowledge for their quantification, modification and modulation is required in order to be able to control all concerns of these fields.
Many authors reported that the microbial surface properties depend fundamentally on the chemical composition of cell surface (Djeribi et al., 2013; Latrache et al., 2002; Pelletier et al., 1997). Indeed, they showed that the hydrophobicity measured by the contact angle is directly correlated with the high ratio N/C and inversely correlated with the concentrations of O/C. Also, other authors have reported that yeasts hydrophobicity is related to the presence of proteins (Dengis and Rouxhet, 1997). Regarding acid-base properties, the electron-donor was attributed to the presence of basic groups exposed on the cell surface, such as carboxyl groups (COO¯), phosphate (PO4) phospholipids, lipopolysaccharides and lipoproteins, amines (NH2) (Briandet et al., 1999) or sulfate groups (SO3) (Pelletier et al., 1997). While, the electron acceptor is attributed to the presence of amino and acidic groups such as R or R-NH-OH and NH3 groups on the cell surface (Djeribi et al., 2013). For spores, the hydrophobic/hydrophilic character is depending to the protein/carbohydrate ratio. Thus, more hydrophobic spores tented to have greater protein/carbohydrate ratio and therefore a rough surface, contrary to the hydrophilic ones (Jeffs et al., 1999). This given proposes that fungal strains of this work have low protein/carbohydrate ratios and smooth surface. And accordingly, they seem to be essentially electron donors. Our finding corroborated those presented by other authors (El Abed et al., 2013; Van der Mei et al., 1998), showing that all microbial cell surfaces are electron-donating. However, the important electron-accepting cell surfaces can be also reported.
The evaluation of the effect of T. vulgaris extract fractions on the hydrophobicity character of three fungal strains, demonstrated that it has been significantly influenced after treatment. In fact, in one hand, both ethyl acetate and methanolic fractions have decreased the CSH of all species. In the other hand, the hexane-ethyl acetate fraction has highly increased it compared to the control. These findings could be due to the variation of protein/carbohydrate ratio which could be decreased and giving a high hydrophilic character in the case of methanolic and ethyl acetate fractions. These different modifiying effects of separate fractions noticed on fungal CSH could be due to their varied phenolic contents and especially their different bioactive compounds such as flavonoids, tannins and alkaloids (Roby et al., 2013; Sadiki et al., 2015), which are rich in hydroxyl group and were already reported on several works that exhibited a wide range of biological activities such as antimicrobial activity (Aleksic and Knezevic, 2014; Annuk et al., 1999) and antiadherence affect (Facino et al., 1990). Our results are similar in the case of methanolic and ethyl acetate fractions treatment to those of Nordin et al. (2013), who reported that Piper betle and Brucea javanica extracts have reduced the CSH of seven oral Candida. Annuk et al. (1999) has also shown that the aqueous extracts of bearberry leaves and red cranberry have reduced the hydrophobicity of ten strains of Helicobacter pylori. Moreover, our results in the case of hexane-ethyl acetate fraction are similar to those of Polaquini et al. (2006), who demonstrated the increase effect of Neem (Azadirachta indica) aqueous extract on the hydrophobicity of two strains of Candida.
According to the scientific literature, no studies have already been focused on the assessment of the herbal extracts effect on the acid-base properties of the microbial cells using the contact angle measurement. Thus, the present work seems to pioneer this effect. Based on the results, all fractions tested are found to be able to influence the acid-base properties of all studied fungal surfaces studied. Indeed, the electron acceptor was slightly and less affected. Whereas, an increase statistically significant (p<0.05) of the negative charge surface for P. commue PDLd” and T. yalocarpa (PDLb3) has been noted in the case of both ethyl acetate and methanolic fractions treatment and its decrease for all stains in the case of hexane-ethyl acetate fraction (p<0.05). It has been mentioned above that, the acid-base properties of microbial cells surface depends fundamentally on their chemical composition. Thereby, these statements could be related to the effect of phenolic components contained in these fractions on different functional groups of the cell surface, as can be attributed to the alteration of chemical compostion and thus the modification of protein/carbohydrate ratio of the fungal cells surface.
CONCLUSION
As observed, all T. vulgaris extract fractions tested were able to alter the physicochemical properties in the term of hydrophobicity and electron donor-electron acceptor of all fungal strains studied. The methanolic and ethyl acetate fractions made the cells more hydrophilic and more electron donor. While, the treatment with hexane-ethyl acetate fraction made them more hydrophobic and weakly donor electron compared to the control (p<0.05).