Antifungal Activity of Finished Chromium Tanned Leather Containing Thyme and Tea Tree Essential Oils

Leather products, especially those often and intensively used, are an ideal environment for microbial growth. The leather protection against microbes at different stages of the tanning process does not make it completely resistant to microflora. The availability of various sources of nutrients stimulates spores to germinate, and then supports the growth of hyphae [1]. Also, a sufficiently high temperature and moisture content enhances bacterial and fungal colonisation by different microbes, such as Bacillus, Corynebacterium, Clostridium, Staphylococcus, Penicillium, Aspergillus, Paecilomyces, Candida and Cryptococcus. These microorganisms quickly colonise on the surface, as well as on clothing and footwear. Most of them are not dangerous to human health but can be responsible for the destruction of materials from which the inner parts of the products are made. The mechanism of the deterioration of leather and leather products by microbes is primarily related to enzymatic decomposition of the material. This leads to leather embrittlement, loss of elasticity, a decrease in collagen molecular weight, an unpleasant smell being felt, and spots appearing on its surface [2]. Discolorations and spotting on leather resulting from the presence of destructive microorganisms can be grey-white, violet, red, grey or even black [3, 4].

Finished leather, which is intended for the production of different leather products, should be characterised by increased microbiological resistance, consisting in a reduction in colonising microorganisms during storage and use. In order to prevent the appearance and development of various types of microorganisms and to prevent damage to leather, it is treated with certain biocides. Antimicrobial agents (biocides) used in the leather industry can be classified as quaternary ammonium compounds, isothiazoles, halogenated organic compounds, such as 2-bromo-2-nitro-propane-1,3-diol, isothiazoles and halogenated organic compounds containing heterocycles, such as derivatives of benzothiazole [1]. Unfortunately, biocides currently used in the leather industry are generally harmful to human health and the environment, and their use has been or shall be restricted or even banned. It affects consumers who are in contact with the products, penetrating through the clothes onto the consumer's skin, causing painful skin, dermatitis, including itching, irritation and redness. Therefore, the use of biocides for the protection of materials should be controlled within the scope of the dose applied and intended use of the final product [1]. Changes in consumers' preferences toward more natural products than synthetic ones stimulate also the use of natural products in the leather tanning process. In addition, numerous recent studies have reported the biological activity of essential oils: they exhibit antibacterial, fungicidal, antioxidant, and antiradical activities [5].

Essential oils are aroma products extracted from plants mainly by steam distillation. In terms of chemistry, volatile oils are complex mixtures of aliphatic and aromatic hydrocarbons, aldehydes, alcohols, esters and other constituents [6]. Depending on the type and amounts of compounds contained, essential oils show various properties, e.g. they inhibit the growth of infectious microbes, prevent fungal growth, accelerate wound healing and mitigate inflammations. Essential oils are widely used as components of drugs, biologically active additives and dietary supplements, as well as in aromatherapy and the food and cosmetics industries [5, 7]. In addition, there have been attempts worldwide to use essential oils as natural bioactive substances for protecting leathers in the tanning industry [1, 2, 5]. Širvaitytė et al. (2012) investigated the possibility of using essential oils of Thymus vulgaris as an alternative preservative for chromium tanned leather. During tests it was found that the antibacterial activity of essential oils of thyme depends on their chemical composition, mainly on the phenolic compounds. The most important phenolic compounds in thyme oil are thymol and carvacrol. The authors of this study found that if essential oils are used as a preservative for chromium leather, their amount should not be less than 3% of the wet-blue mass. Smaller amounts (0.05% and 1.0%) showed no inhibition zone, although leather samples remained resistant to the bacteria selected [5]. Also, the results of Bayramoĝlu et al.'s (2006) study showed that three essential oils of oregano had much stronger bactericidal activity than that of commercial bactericide and fennel oil. These findings suggested that oregano essential oils could be used as bactericidal agents in the leather industry [2]. Falkiewicz-Dulik observed that tea tree oil preparation showed a good antibacterial effect with regard to S. aureus and E. coli, as well as biocidal activity against yeast-like fungi C. albicans and filamentous fungi with large inhibition zones of mould growth A. brasiliensis and dermatophyte T. mentagrophytes [1].

The aim of this work was to investigate the antifungal activity of thyme and tea tree oils applied to finished chromium leather intended for leather products, especially for leather accessories. The work included the selection of essential oils, determination of doses, the application of selected preparation to different leathers, and verification of antifungal activity.

Results of the microbiological tests of the leathers selected are presented in Tables 3–6. Aspergillus niger ATCC 16404, Chaetomium globosum ŁOCK 0476 and Candida albicans ATCC 10231 growth was observed as white mycelium on the surface of the agar medium. In the case of leather I, a relationship was observed where the higher the concentration of thyme oil, the stronger the antifungal activity (Table 3). The results obtained indicate that the I 5t sample treated with thyme oil shows strong antimicrobial activity against strains A. niger, Ch. globosum and C. albicans. No visible growth was observed (magnification 50x) under the microscope, which means 0 for all strains tested. On the other hand, samples I 1h, I 2h and I 5h, treated with tea tree oil, had no inhibition effect against the strains. In this case, growth over the entire surface of all samples was observed. Therefore, the fungus growth on the all samples was rated at 5, which means heavy growth, covering the surface tested. For leather specimens finished without the use of essential oils (KON I), strong growth of all microbes tested around and on the leather was observed.

Very similar results were obtained for leather II (Table 4). In the case of thyme oil, the fungus growth was limited when a higher oil concentration was used. Microbial growth for II 5t was rated at 4, 2 and 0 for A. niger, Ch. Globosum and C. albicans, respectively. It means that no growth of Candida albicans was observed on the leather finished with thyme oil at a concentration of 5%. The use of tea tree oil again had no effect on microbial growth. The growth of the strains tested on leather II was rated at 5 in most cases, which means heavy growth, covering the surface tested. Results of the application of essential oils to leather IV are presented in Table 6. For the samples finished with thyme oil, the following relationship was obtained: the higher the concentration of thyme oil, the stronger the antifungal activity against the strain tested. In the case of A. niger, the growth was rated at 5, 4, 0 for thyme oil concentrations of 1%, 2% and 5%, respectively. The growth of Ch. globosum was rated at 2, 0, 0 for thyme oil concentrations of 1%, 2% and 5%, respectively. No C. albicans growth was observed on the test leather treated with thyme oil. Tea tree oil at concentrations of 2% and 5% showed effectiveness against C. albicans yeast only. The lower antifungal effect of tea tree oil has again been proven.

The results for leather III were slightly different (Table 5). No growth of C. albicans was observed on any of the samples tested. The application of 2% and 5% thyme oil to the leather completely inhibited the growth of all the strains tested. Exceptionally, in this case, tea tree oil showed antifungal activity. At each concentration of this essential oil, the growth of Ch. globosum was rated 2, while the growth of C. albicans was 0. Interestingly, the III KON sample also showed antifungal activity against two strains of fungi. In this case, the growth of Ch. globosum was rated 2, while that of C. albicans was 0. This proves that leather III without antimicrobial finishing most likely had antimicrobial properties. This may be due to elevated chromium values or the presence of dye that is toxic to microorganisms. According to other studies [9, 10], some dyes, e.g. natural ones, show antimicrobial activities. Also, metal complex formazan dye demonstrated a significant antimicrobial propensity in solution and on leather by exhibiting a percentage reduction in bacterial growth [11]. Moreover, the presence of chromium in tanned leather may have an effect on antimicrobial properties. Taking that into account, the composition of leather III should be carefully examined in further studies. However, at this moment it can be concluded that Aspergillus niger is a very durable and resistant fungus to various physico-chemical and biological factors. Thanks to these properties, even with the possible presence of toxic substances in leather III, the growth of A. niger was rated 5. In turn, Candida albicans is a microorganism characterised by a relatively low resistance to toxic components. Therefore, in each case described in this work, C. albicans was the most sensitive strain among the microorganisms tested, which was especially visible in the case of leather III (Table 5).

According to the results obtained in the present work, thyme essential oil has decidedly stronger antifungal activity than tea tree essential oil. Moreover, the increased antimicrobial activity of thyme essential oil was observed with an increase in the concentration of this oil applied to the leather. The leather treated with 5% thyme essential oils inhibited the growth of all strains tested: A. niger, Ch. Globosum and C. albicans. The strong properties of thyme oil were also confirmed in other research [12, 13]. The study of Chirila et al. (2017) showed that Thymus vulgaris oil exhibited high antifungal activity against Candida albicans and Aspergillus niger. Thyme oil could be the best inhibitor of fungi due to the high thymol and carvacrol content [12]. In addition, results obtained in our work showed that tea tree oil was effective only against C. albicans and only when it was applied to leather samples III and IV. The antifungal activity of tea tree oil against Candida sp. was confirmed in another study [14]. However, leather III without an antimicrobial finish also showed some antimicrobial properties, which should be analysed in further research. In conclusion, the use of thyme essential oil at a suitable concentration can effectively prevent the growth of fungi on leather materials.