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Antifungal activity of selected essential oils and biocide benzalkonium chloride against the fungi isolated from cultural heritage objects
Published Date July 2014, Vol.93:118–124,doi:10.1016/j.sajb.2014.03.016 Open Archive, Creative Commons license,Funding information Author
M. Stupar a,,
M. Lj. Grbić a
A. Džamić a
N. Unković a
M. Ristić b
A. Jelikić c
J. Vukojević a
aUniversity of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac”, Studentski trg 16, Belgrade, Serbia
bInstitute for Medicinal Plant Research “Dr Josif Pančić”, Tadeuša Košćuška 1, Belgrade, Serbia
cInstitute for Protection of Cultural Monuments in Serbia, Radoslava Grujića 11, Belgrade, Serbia
Received 20 November 2013. Revised 25 March 2014. Accepted 31 March 2014. Available online 4 May 2014.
Highlights
Essential oils can be novel biocides applicable in cultural heritage conservation.
•
Origanum vulgare essential oil displayed the strongest antifungal activity.
•
Rosmarinus officinalis and Lavandula angustifolia oils showed moderate activity.
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Antifungal efficacy of the biocide benzalkonium chloride has been confirmed.
Abstract
The antifungal activity ofOriganum vulgare,Rosmarinus officinalisandLavandula angustifolia(Lamiaceae) essential oils and biocide benzalkonium chloride was investigated against fungi isolated from stone (Bipolaris spiciferaandEpicoccum nigrum) and wooden substrata (Aspergillus niger,Aspergillus ochraceus,Penicilliumsp. andTrichoderma viride) of cultural heritage objects. Carvacrol (64.06%) was the main component ofO. vulgareessential oil, while linalool (37.61%) and linalool acetate (34.86%) dominated inL. angustifoliaessential oil. The main component ofR. officinalisessential oil was 1.8-cineole (44.28%). To determine fungistatic and fungicidal concentrations (MIC and MFC) micro-, macrodilution and microatmosphere methods were used. Mycelial growth and spore germination of fungal isolates were inhibited with different concentrations of antifungal agents. The oil ofO. vulgareand biocide benzalkonium chloride displayed the strongest antifungal activities followed byR. officinalisandL. angustifoliaessential oils. MIC and MFC values obtained in microatmosphere and microdilution method forO. vulgareessential oil ranged from 0.1 to 2.0 μL mL− 1, while forR. officinalisandL. angustifoliaranged from 10.0 to 100.0 μL mL− 1. The most susceptible fungus to essential oil treatments wasE. nigrum. MIC and MFC values for benzalkonium chloride ranged from 0.1 to 4.0 μL mL− 1. Tested isolates,A. nigerandA. ochraceus, were the most susceptible for biocide treatment.
Abbreviations
SAB, subaerial biofilm
BAC, benzalkonium chloride
EO, essential oil
QAC, quaternary ammonium compound
MIC, minimal inhibitory concentration
MFC, minimal fungicidal concentration
Keywords
Antifungal activity
Biocide
Biodeterioration
Cultural heritage
Essential oil
Lamiaceae
1 Introduction
Fungi are widely recognized as major biodeteriogens of cultural heritage. They are capable of colonizing, degrading and altering a variety of materials, including the materials which have been used through the centuries for making cultural heritage monuments and artifacts (Sterflinger, 2010). Stone monuments in moderate and humid climates are usually colonized by fungal communities dominated by dematiaceous Hyphomycetes (Sterflinger and Krumbein, 1997 and Urzì et al., 2001). Fungi on stone substrata, along with cyanobacteria and algae as phototrophic partners and heterotrophic bacteria, form specific microbial communities called subaerial biofilms (SABs) that develop on solid surfaces exposed to the atmosphere (Gorbushina and Broughton, 2009). The surfaces of stone monuments can be altered by fungal activity via hyphal penetration through the porous stone matrix (Kumar and Kumar, 1999 and Sterflinger, 2000) and by production of organic acids and pigments (Gómez-Alarcón et al., 1994 and Sharma et al., 2011). Pieces of art stored in museum depots or displayed in exhibition rooms can suffer the symptoms of fungal spoilage. Different materials used for artistic expression (such as wood, paper, textiles, leather, plastic, metal and clay) can be susceptible for fungal colonization and biodeteriorated through fungal growth and metabolic activity. Biodeterioration mechanisms of fungi are related to enzymatic hydrolysis of organic materials and the production and excretion of organic acids (Görs et al., 2007). Wooden artifacts are especially affected by fungal colonization due to cellulase production by certain filamentous fungi (Fazio et al., 2010). Prevention of mold growth on cultural heritage objects and artifacts is nowadays a significant challenge for restorers, conservators and architects (Sterflinger, 2010). Chemical treatments applied in cultural heritage conservation must be non-toxic and non-destructive (Stupar et al., 2012).
The objectives of this research were to evaluate in vitro effectiveness of the biocide benzalkonium chloride (BAC) and selected essential oils (EOs) as antifungal agents against fungi isolated from cultural heritage. BAC is widely used for the control of microbial growth in clinical and industrial environments (McBain et al., 2004). The biological activity of BAC is ascribed to its quaternary ammonium group (Mehta et al., 2007). Biocidal products containing quaternary ammonium compounds (QACs) are approved for conservation of cultural heritage monuments by the European Biocide Directive as relatively environmentally friendly (Cooke, 2002). On the other hand, EOs and their derivatives are considered to be a possible substitute for controlling different types of biological settlement (Axinte et al., 2011). Antimicrobial activity of many EOs has been reviewed by Kalemba and Kunicka (2003), and many oils have been successfully applied in different fields of microbiological control. However, reports regarding the implementation of EOs in cultural heritage conservation are very scarce (e.g. Gatenby and Townley, 2003, Chung et al., 2003 and Rakotonirainy and Lavédrine, 2005).
2 Materials and methods
2.1 Essential oils
The essential oils from selected aromatic plants from the Lamiaceae family Origanum vulgare L. (Frey + Lau, Ulzburg, Germany), Rosmarinus officinalis L. (Herba d.o.o. Belgrade, Serbia), and Lavandula angustifolia Miller (Frey + Lau, Ulzburg, Germany), were commercial samples obtained from the Institute for Medicinal Plant Research “Dr Josif Pančić”, Belgrade as a part of their collection. According to the manufacturer the quality of tested EOs corresponds to European Pharmacopeia 6 (Ph. Eur. 6.0, 2004).
2.2 Biocide
An aqueous solution of the biocide benzalkonium chloride (BAC) (50% (vol/vol)) was obtained from the Institute for Protection of Cultural Monuments in Serbia. Prior to experiment biocide was diluted in sterile distilled water to make a stock solution of final concentration 10% (vol/vol).
2.3 Tested fungal isolates
All fungi used in this study were isolated from different wooden and stone substrata of cultural heritage objects in Serbia (Table 1), identified to the species or genus level using appropriate identification keys. Isolated fungi were deposited to the Mycotheca of the Department for Algology, Mycology and Lichenology, Institute of Botany, Faculty of Biology, University of Belgrade. Isolates were maintained on malt extract agar (MEA), potato dextrose agar (PDA), stored at 4 °C and subcultured once a month.
Table 1. Fungal isolates chosen for the study of antifungal activity of selected essential oils and biocide benzalkonium chloride.
Isolates
Substrata
Reference
Aspergillus nigerTiegh Aspergillus ochraceus G. Wilh Penicillium Link sp. Trichoderma viridePers.
Wooden sculptures, Museum of Temporary Art, Serbia
2.4 Gas chromatography (GC) and gas chromatography mass spectrometry (GC/MS)
Qualitative and quantitative analyses of the EOs were performed using GC and GC–MS. GC analysis of the oil was carried out on a GC HP-5890 II apparatus, equipped with a split–splitless injector, attached to a HP-5 column (25 m × 0.32 mm, 0.52 μm film thickness) and fitted with a FID. Carrier gas flow rate (H2) was 1 mL/min, split ratio 1:30, injector temperature was 250 °C, detector temperature 300 °C, while the column temperature was linearly programmed from 40 to 240 °C (at 4 °C/min). The same analytical conditions were employed for GC–MS analysis, where a HP G 1800C Series II GCD system, equipped with a HP-5MS column (30 m × 0.25 mm, 0.25 μm film thickness) was used. The transfer line was heated to 260 °C. The mass spectra were acquired in EI mode (70 eV), in m/z range 40–400. Identification of individual EO components was accomplished by comparison of retention times with standard substances and by matching mass spectral data with those held in the Wiley 275 library of mass spectra. Confirmation was performed using AMDIS software and literature (Adams, 2007). Area percentages obtained by FID were used as the basis for quantitative analysis. The percentage composition of the oils was computed by the normalization method from the GC peak areas.
2.5 Antifungal activity assays
The antifungal activity of selected EOs, and the biocide BAC, was investigated using three methods: micro-, macrodilution and microatmosphere methods. Microdilution and microatmosphere methods were used for testing the antifungal activity of EOs, while biocide BAC was tested using micro- and macrodilution methods.
2.5.1 Microatmosphere method
The following method allows the effect of volatile fractions of the EOs to be studied. The test was performed in sterile Petri plates of (85 mm diameter) containing 20 mL of MEA (Maruzzella and Sicurella, 1960). After inoculation of the fungi, using sterile needle under the stereomicroscope (Stemi DV4, Zeiss), at the center of the MEA, the Petri plates were overturned. A sterilized filter paper disc was placed in the center of the Petri plate lid soaked with various concentrations of EOs. For O. vulgare, concentrations of EO ranged from 0.1 to 2 μL mL− 1, while for R. officinalis and L. angustifolia EO concentrations ranged from 10 to 100 μL mL− 1. Plates were incubated for 3 weeks at room temperature, during which the growth of fungal colonies was monitored weekly. After incubation period, minimal inhibitory concentrations (MICs), defined as the lowest concentration of added EO with no visible fungal growth on MEA, were determined. Minimal fungicidal concentrations (MFCs) were determined by re-inoculation of treated inoculums onto sterile MEA. The lowest concentrations of EO giving no visible growth after re-inoculation were regarded as MFCs.
2.5.2 Macrodilution method
To investigate the antifungal activity of the biocide BAC, the modified mycelia growth assay with MEA was used (Ishii, 1995). The stock solution of BAC (10%, vol/vol), was further diluted in melted MEA in Petri plates to make final concentrations of the biocide ranging from 0.1 to 5 μL mL− 1. The fungi were inoculated at the center of the MEA. Plates were incubated for 3 weeks at room temperature. MIC and MFC values were determined in the same manner as in the microatmosphere method.
2.5.3 Microdilution method
The modified microdilution technique was used to determine the antifungal activity of EOs and BAC (Hanel and Raether, 1998 and Daouk et al., 1995). Conidia were washed from the surface of the agar slants with sterile 0.85% saline containing 0.1% Tween 20 (vol/vol). The conidia suspension was adjusted with sterile 0.85% saline to a concentration of approximately 1.0 × 105 in a final volume of 100 μL per well. The inocula were stored at − 20 °C for further use. Dilutions of the inocula were cultured on solid MEA to verify the absence of contamination and to check the validity of the inocula.
Determination of the MICs was performed by a serial dilution technique using 96-well microtitre plates. Different volumes of investigated EOs and biocide BAC (10% (vol/vol)) were dissolved in malt extract broth (MEB) with fungal inoculums (10 μL) to make the same final concentrations, as those used in microatmosphere and macrodilution methods. The microplates were incubated for 72 h at 28 °C. The lowest concentrations without visible growth (under a binocular microscope) were defined as the concentrations that completely inhibited fungal growth (MICs). The minimum fungicidal concentrations (MFCs) were determined by serial subcultivation of 2 μL into microtitre plates containing 100 μL of MEB. The lowest concentration with no visible growth was defined as the MFC, indicating 99.5% killing of the original inoculum.
3 Results
3.1 Chemical composition of essential oils
The chemical compositions of Lamiaceae EOs are presented in Table 2. In the EO of O. vulgare, 20 compounds were identified (99.78% of total oil). The main component was carvacrol (64.06%). A total of 22 components were identified in the EO of R. officinalis, which represented 99.99% of the total oil. The main component was 1.8-cineole (44.28%). The other components present in significant percentage were camphor (12.54%) and α-pinene (11.62%). In L. angustifolia EO, 29 components were identified (99.53%). The main components were linalool (37.61%) and linalool acetate (34.86%).
Table 2. Composition of the essential oils of Origanum vulgare, Rosmarinus officinalis and Lavandula angustifolia.
Kovats retention index (according to Adams, 2007).
3.2 Fungal sensibility to essential oils
Among all EOs analyzed in this work, O. vulgare EO was the most effective, followed by L. angustifolia and R. officinalis. High fungistatic and fungicidal activity of O. vulgare EO was demonstrated with low MIC and MFC values, ranging from 0.1 to 2.0 μL mL− 1, obtained in both methods used. Higher MIC and MFC values, in range from 0.1 to 4.0 μL mL− 1, were obtained for biocide BAC (Fig. 1). E. nigrum was the most susceptible isolate to O. vulgare EO treatments and fungicidal effect was achieved at 0.1 μL mL− 1 (Fig. 1). On the other hand, the most resistant isolate was Trichoderma viride. However, higher MIC and MFC values for this fungus were obtained with microatmosphere method than with a microdilution method. MFC values for this fungus obtained with the microatmosphere method and microdilution method were 0.5 and 2.0 μL mL− 1, respectively (Fig. 1).
Fig. 1. Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) for Origanum vulgare essential oil (EO) and biocide benzalkonium chloride (BAC) obtained in: (A) microatmosphere and macrodilution method and (B) microdilution method. EO: MIC, MFC; BAC: MIC, ■ MFC.
R. officinalis and L. angustifolia EOs showed moderate antifungal activity and significantly lower in comparison to BAC. MIC and MFC for R. officinalis EO ranged from 10 to 100 μL mL− 1 (Fig. 2). In case of R. officinalis EO Epicoccum nigrum was also the most sensitive isolate and the growth of this fungus was completely suppressed with 10 μL mL− 1 (Fig. 2). R. officinalis EO showed strong fungistatic, but significantly lower fungicidal potential against Aspergillus ochraceus in microdilution method (Fig. 2).
Fig. 2. Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) for Rosmarinus officinalis essential oil (EO) and biocide benzalkonium chloride (BAC) obtained in: (A) microatmosphere and macrodilution method and (B) microdilution method. EO: MIC, MFC; BAC: MIC, ■ MFC.
MIC and MFC for L. angustifolia EO ranged from 20 to 100 μL mL− 1. The most sensitive isolates were E. nigrum and Penicillium sp., while Aspergillus niger and Bipolaris spicifera were the most resistant (Fig. 3).
Fig. 3. Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) for Lavandula angustifolia essential oil (EO) and biocide benzalkonium chloride (BAC) obtained in: (A) microatmosphere and macrodilution method and (B) microdilution method. EO: MIC, MFC; BAC: MIC, ■ MFC.
The biocide BAC showed strong antifungal activity against all fungal isolates. Aspergillus species were the most susceptible to BAC treatment, especially when the microdilution method was used. However, the highest MFC value with the macrodilution method (4 μL mL− 1) was for A. ochraceus (Fig. 1, Fig. 2 and Fig. 3).
4 Discussion
The aim of the present study was to evaluate the antifungal activity of selected EOs against fungi associated with the biodeterioration of cultural heritage and to compare their biocidal potential with the commonly-used biocide BAC. Antifungal agents can interfere with any stage of the fungal asexual life cycle. The potential of antifungal agents (EOs and BAC) to interfere with the first step of the fungal asexual life cycle (conidia germination) was estimated using the microdilution method, while inhibition of mycelia growth was monitored using microatmosphere and macrodilution methods. Although the microdilution method has been widely recognized as a fast and efficacious method for testing antifungal activity (Daouk et al., 1995), it is not always applicable for all fungi. In the research presented here, E. nigrum remained sterile while grown on MEA. Hence, it was excluded from this method. Due to their low solubilities in solid media, the antifungal activity of EOs was tested using the microatmosphere method. This allowed the inhibition of growth of mycelia exposed to the vapor of EO components to be estimated. Strong antifungal activity was documented for all the tested EOs. O. vulgare EO can be regarded as the strongest inhibitor, while the EOs of R. officinalis and L. angustifolia showed moderate antifungal activity. The tested fungal isolates differed in their susceptibilities to the antifungal agents. T. viride was shown to be the most resistant isolate in the mycelia growth assay, while conidia germination was inhibited on lower concentrations of antifungal agents. This could be explained by a variety of enzymes produced and secreted by T. viride mycelia that can detoxify components of EOs into inactive forms (Farooq et al., 2002). On the other hand, poroconidia of B. spicifera germinated in liquid medium enriched with these antifungal agents at concentrations which did not cause mycelia growth inhibition. Fungal melanins present in the thick cell walls of B. spicifera poroconidia have a protective role, enabling fungi to survive adverse environmental conditions (Butler and Day, 1998) and are probably responsible for the higher resistance documented for this fungus.
The antifungal activity of O. vulgare EO has been proven in several studies against a variety of fungi (Raduðienë et al., 2005 and Kocić-Tanackov et al., 2012). The antifungal properties of this oil can be ascribed to the high content of the main component, carvacrol, which was present at 64.06% in our study. Carvacrol is monoterpenoid phenol which exhibits high antimicrobial and antioxidant activities (Kalemba and Kunicka, 2003). Khosravi et al. (2011) isolated EO from wild populations of O. vulgare in Iran. That oil did not contain carvacrol and showed a low antifungal activity against Candida glabrata (H.W. Anderson) S.A. Mey & Yarrow. The antifungal activity of O. vulgare EO has been seldomly tested against fungi isolated from cultural heritage objects. However, Stupar et al. (2012) reported that O. vulgareEO can inhibit the conidia germination of Aspergillus and Penicillium species isolated from wall paintings of Gradac Monastery (Serbia) and Borego et al. (2012) showed that O. vulgare EO inhibits mycelia growth and sporulation of Aspergillus, Fusariumand Penicillium species isolated from Cuban and Argentine Documentary Heritage. According to Lis-Balchin et al. (1998), the antifungal properties of L. angustifolia EO depend on its chemical composition and percentage of the main components linalool and linalool acetate. High fungistatic and fungicidal activity of linalool and linalool acetate against fungal strains isolated from library and archive storage areas was demonstrated by Rakotonirainy and Lavédrine (2005). The antifungal activity of R. officinalis EO obtained in this research corresponds with other literature data regarding its antifungal properties (Jiang et al., 2011 and Marandi et al., 2011). However, there are no data for testing this EO against fungi isolated from cultural heritage objects. There are several reports regarding the use of natural products in the field of conservation of cultural heritage. Borego et al. (2012) reported that Pimpinella anisum L. and Allium sativum L. EOs showed the best antifungal activity against fungal strains isolated from Cuban and Argentine Documentary Heritage (A. niger, Aspergillus clavatus, Penicillium sp. and Fusarium sp.). Axinte et al. (2011)showed that cinnamaldehyde, the main component of the Cinnamon sp. EO, inhibited the growth of yeast Torula sp. and brown-rot fungus Coniophora puteana(Schumach.) P. Karst., isolated from wood and stone religious artifacts. Sakr et al. (2012) showed high antifungal properties for five EOs against yeasts isolated from the Royal Tomb Paintings at Tanis, Egypt.
The high antifungal activity of biocide BAC was demonstrated against all the fungal isolates. Conidia germination and growth of mycelia were inhibited by the low concentrations of BAC we used. In general, conidia of all fungal isolates were equally susceptible to biocide treatment and more so in comparison to mycelia growth, which had significantly lower MIC and MFC values obtained with the microdilution method. No MIC values higher than 0.25 μL mL− 1 were obtained in microdilution method, while the highest MIC values obtained in macrodilution method were 1.5 μL mL− 1 (T. viride). Our results regarding the antifungal properties of BAC correspond with other results for BAC and other QACs (e.g. Tortorano et al., 2005, Bastian et al., 2009 and Day et al., 2009). However, gram-negative bacteria and Pseudomonas species are resistant to QACs (Langsrud et al., 2003), enabling these microorganisms to colonize surfaces treated with QACs for a long period of time. Scheerer et al. (2009)suggested that use of nitrogen-containing biocides should be avoided because they enable some groups of micro- and macro-organisms to use it as a nitrogen source favoring recolonization. Also, fungi and other microorganisms may become resistant after persistent usage of the same biocide (Heinzel, 1998). To avoid increasing the biocidal resistance of some microbial strains, rotation of biocides is often recommended to inhibit the resistant microorganisms (Langsrud et al., 2003), as is involvement of potentially novel biocidal agents, such as EOs.
5 Conclusions
Our results confirmed the well-known efficacy of low concentrations of biocide BAC, and suggested the potential usage of EOs as novel biocidal agents in cultural heritage conservation. Antifungal potential of O. vulgare was higher or nearly the same as BAC, which was demonstrated in all methods used. This is probably due to high amount of phenol carvacrol in this oil. Our results suggests that, EOs, could be good alternatives for existing biocides due to their low mammalian toxicity, susceptibility to biodegradation and strong antimicrobial activity (Saxena and Mathela, 1996). Further studies are required to develop appropriate methods to apply EOs in conservation of cultural heritage objects.
Acknowledgments
This research was carried out as part of the project financially supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant No. 173032). We would like to thank Professor Dr. Steve A. Quarrie for careful reading of the manuscript and language corrections.
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MIC, 
MFC; BAC: 
MIC, ■ MFC.
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