1. Introduction

Byproducts of various industries have long been used as raw materials. Demand for such natural resources is continuously increasing (1). Raw materials such as skins and hides are byproducts of the meat industry and are converted into valuable leather products such as bags, shoes, wallets, briefcases or backpacks in tanneries (2). The leather industry is of global economic importance. To obtain high-value leather products, the raw materials (hides and skins) must be properly preserved. Since raw hides or skins have high water and protein content, these raw materials become vulnerable to bacterial activity. To overcome bacterial deterioration on hides and skins, raw hides and skins are traditionally cured with salt or brine after the animal is slaughtered (3). The undesirable bacterial population may consist of halophilic or non-halophilic bacteria which may come from the animal itself or from environmental contamination. Additionally, salt which is used in the salt curing process may contain halotolerant microorganisms, slightly halophilic bacteria, moderately halophilic bacteria, extremely halophilic archaea and fungi which can contaminate raw skins and hides (4–11).

Previous studies demonstrated that halophilic microorganisms on skins and hides are responsible for red heat, red discoloration, holes, problems on the grain surface and deterioration of hides and skins (12–15).

Among these bacterial groups, moderately halophilic bacteria include a wide variety of bacteria (16). These bacteria may be Gram-negative or Gram-positive, aerobic or facultative anaerobic. It has been reported that these bacteria may abundantly grow in saline systems such as saltern crystalliser ponds, saline soils, the Dead Sea and evaporated ponds. Moderately halophilic microorganisms may secrete different enzymes such as proteases, lipases, cellulases and chitinases (17–19). These microorganisms may grow under conditions including 3–15% sodium chloride concentration, 0–45°C and pH 5–10 (13, 20). In recent years, although the potential enzyme production profiles of halophilic bacteria have led to a focus on their industrial use, protease and lipase producing moderately halophilic bacteria are undesirable in the leather industry due to their potential for causing defects on the final product and possible economic losses.

There are some studies examining which species of moderately halophilic bacteria can grow on hides and skins (5–8, 15, 21–23). Molecular techniques and phenotypic characterisation methods allow new species belonging to the moderately halophilic bacteria to be identified more easily. For example, Thalassobacillus pellis sp. nov. and Salimicrobium salexigens sp. nov. were reported as newly identified moderately halophilic species from salted skin samples over the past decade (21, 22).

There are also studies focused on bacterial numbers of moderately halophilic microorganisms and their potential to cause defects on hides. The results of studies investigating the abundance of moderately halophilic bacteria on hide samples showed high numbers which could possibly cause hide and skin damage (6–8, 13). In a recent study, the correlation between bacterial population and their possible defects was examined. Salted skins with red and yellow areas, mucoid appearance, bad smell and hair slip were demonstrated to have 10⁵–10⁸ colony forming units (CFU) g^–1 moderately halophilic bacteria (15). Moreover, in other studies the presence of Gram-positive moderately halophilic isolates was found to be higher than Gram-negative moderately halophilic isolates on salted sheep and goat skins (6, 7). It was reported that 41 isolates were Gram-positive and 36 isolates were Gram-negative out of 77 moderately halophilic bacteria isolated from salted sheep skins (6). In another study, 32 Gram-positive and 7 Gram-negative moderately halophilic bacteria were isolated from salted goat skin samples (7). These studies showed that Gram-positive moderately halophilic bacteria are abundant on salted skins.

It is of great importance for the leather industry to control moderately halophilic bacteria in order to gather maximum high-quality yield. A variety of methods against halophilic bacteria such as direct electric current, antimicrobial agents or bacterial toxins (7, 24–26) have been examined in the literature. The antibacterial efficacy of lichens is known, but there is a lack of literature on their activities against halophilic bacteria. Lichens are symbiotic organisms consisting of algae and fungus. These organisms produce some secondary metabolites with various biological activities. Recently, several lichen extracts have been examined against Bacillus species and Enterococcus durans isolated from soak liquor samples. A mixed culture of soak liquor and tank surface samples were tested with lichen extracts. These studies indicated that lichen extracts are successful for controlling bacterial growth (27–31).

Taking into consideration the potential harmful effects of proteolytic and lipolytic Gram-positive moderately halophilic bacteria, two species, Staphylococcus saprophyticus subsp. saprophyticus (TR5) and Salinicoccus roseus (KV3), which were isolated in a previous study (6), were selected as test bacteria. The main goal of this study is to examine the potential antibacterial efficacy of selected lichen species (Usnea sp., Platismatia glauca, Ramalina farinacea, Evernia divaricata, Bryoria capillaris, Hypogymnia tubulosa, Pseudevernia furfuracea and Lobaria pulmonaria) against these moderately halophilic bacteria.

2. Materials and Methods

3. Results

In this study, acetone extracts of lichen samples identified as P. glauca, R. farinacea, E. divaricata, B. capillaris, H. tubulosa, Usnea sp., P. furfuracea and L. pulmonaria based on morphological and anatomical features were evaluated for their antibacterial activities against two moderately halophilic bacteria from salted sheepskin samples. The test concentrations applied in the experiments were 240 μg ml^–1, 120 μg ml^–1, 60 μg ml^–1, 30 μg ml^–1 and 15 μg ml^–1 (five dilutions) or 240 μg ml^–1, 120 μg ml^–1, 60 μg ml^–1, 30 μg ml^–1, 15 μg ml^–1, 7.5 μg ml^–1, 3.75 μg ml^–1, 1.875 μg ml^–1, 0.9375 μg ml^–1 and 0.46875 μg ml^–1 (10 dilutions).

Before the experimental study design, some preliminary experiments were carried out to examine the presence of antibacterial efficacy. Then, samples having potential antibacterial efficacy were screened up to five or ten dilutions. Acetone was selected as the solvent in order to extract the most active compounds having potential antibacterial efficacy. Acetone is a preferred solvent due to its capability to dissolve both polar and nonpolar compounds.

No antibacterial effect for the acetone extracts of L. pulmonaria, B. capillaris and P. furfuracea was recorded for TR5 and KV3 during preliminary screening studies. For this reason, the figures belonging to the acetone extracts of L. pulmonaria, B. capillaris, P. glauca and P. furfuracea are not included in this paper. On the other hand, the extracts of R. farinacea, Usnea sp., E. divaricata and H. tubulosa had high antibacterial efficacies at certain concentrations against the test bacteria.

According to our results, acetone extracts of H. tubulosa, R. farinacea, E. divaricata and Usnea sp. were found to successfully suppress the bacterial growth of TR5. Extracts of L. pulmonaria, B. capillaris, P. furfuracea and P. glauca had no efficacy against TR5. Four tested concentrations (240 μg ml^–1, 120 μg ml^–1, 60 μg ml^–1 and 30 μg ml^–1) of the acetone extracts of H. tubulosa had high antibacterial efficacies against TR5 with inhibition percentages of 93.71 ± 2.68%, 94.05 ± 0.68%, 94.59 ± 0.27% and 94.72 ± 0.75%, respectively. The 15 μg ml^–1 treatment group had no notable antibacterial activity against TR5 with an inhibition ratio of only 32.50 ± 6.84% (Figure 2). No statistically significant difference was detected among the 240 μg ml^–1, 120 μg ml^–1, 60 μg ml^–1 and 30 μg ml^–1 groups. In addition, statistically significant differences were found when the control group and all treatment groups were compared (p <0.05) (Figure 2).

Antibacterial efficacy was lower for the extracts of R. farinacea against TR5. The inhibition percentages were 70.95 ± 9.75%, 76.10 ± 1.85%, 48.20 ± 4.39%, 23.86 ± 5.48% and 18.36 ± 0.64% for all tested concentrations, respectively. From these results, test concentrations of 240 μg ml^–1 and 120 μg ml^–1 may be evaluated as slightly effective for controlling growth of TR5. According to statistical analyses, there was no difference between 240 μg ml^–1 and 120 μg ml^–1 and also 30 μg ml^–1 and 15 μg ml^–1 treatment groups. All groups were statistically different in comparison to control group (p <0.05) (Figure 3).

The acetone extracts of E. divaricata at concentrations of 240 μg ml^–1, 120 μg ml^–1 and 60 μg ml^–1 inhibited bacterial growth of TR5 with inhibition ratios of 82.02 ± 0.14%, 89.49 ± 2.26% and 79.42 ± 1.14%. At lower concentrations, little suppression was detected on the growth (31.38% and 30.53%, respectively). Statistical analyses revealed that there was no significant difference among 240 μg ml^–1, 120 μg ml^–1 and 60 μg ml^–1 and also between 30 μg ml^–1 and 15 μg ml^–1 treatment groups. All groups were significantly different when compared to controls (p <0.05) (Figure 4).

The extracts belonging to Usnea sp. from 240 μg ml^–1 to 7.5 μg ml^–1 showed great inhibitory effect against TR5. The inhibition percentages for the tested concentrations of 240 μg ml^–1 to 7.5 μg ml^–1 were respectively: 99.36 ± 0.04%, 85.16 ± 2.75%, 97.81 ± 0.78%, 98.25 ± 0.26%, 98.12 ± 0.23% and 97.19 ± 0.54%. Inhibition ratios were observed to be below 50% for 3.75 μg ml^–1, 1.875 μg ml^–1, 0.9375 μg ml^–1 and 0.46875 μg ml^–1 concentrations (37.56 ± 0.49%, 28.08 ± 1.86%, 8.07 ± 2.82% and 3.15 ± 2.70%, respectively). When compared to the control group, all treatment groups were found to be significantly different except 0.46875 μg ml^–1 (p <0.05). In group comparisons, there was no statistically significant difference among 240 μg ml^–1, 60 μg ml^–1, 30 μg ml^–1, 15 μg ml^–1 and 7.5 μg ml^–1 (Figure 5).

Similar to the results obtained in TR5, there was considerable inhibition by the extracts of H. tubulosa, R. farinacea, E. divaricata and Usnea sp. against KV3, especially by the acetone extracts of H. tubulosa. Significant inhibition was achieved even at very low concentrations including the seventh dilution (3.75 μg ml^–1). The inhibition ratios were recorded as: 90.59 ± 2.68%, 90.99 ± 0.41%, 91.56 ± 0.18%, 93.57 ± 0.27%, 96.86 ± 0.32%, 97.44 ± 0.14% and 86.26 ± 18.25%, respectively. The lower concentrations had no inhibition against KV3. In statistical analyses, there were significant differences in 240 μg ml^–1, 120 μg ml^–1, 60 μg ml^–1, 30 μg ml^–1, 15 μg ml^–1, 7.5 μg ml^–1 and 3.75 μg ml^–1 treatment groups when compared to control groups (p <0.05). There was no significant difference between treatment groups of 240 μg ml^–1, 120 μg ml^–1, 60 μg ml^–1, 30 μg ml^–1, 15 μg ml^–1, 7.5 μg ml^–1 and 3.75 μg ml^–1. Likewise, no statistically significant difference was detected among 1.875 μg ml^–1, 0.9375 μg ml^–1, 0.46875 μg ml^–1 and control groups (Figure 6).

Acetone extracts of R. farinacea showed noticeable inhibition for the first four of the concentrations (240 μg ml^–1, 120 μg ml^–1, 60 μg ml^–1 and 30 μg ml^–1). The inhibition percentages for these concentrations were respectively recorded as: 92.73 ± 3.86%, 92.17 ± 1.26%, 95.92 ± 0.29% and 93.79 ± 1.10%. The inhibition rates for the concentrations 240 μg ml^–1, 120 μg ml^–1, 60 μg ml^–1 and 30 μg ml^–1 were not statistically different, indicating dose-independent inhibition. However, these treatment groups and the 15 μg ml^–1 group were statistically different when compared to the control group (p <0.05). Lower concentrations below 30 μg ml^–1 had no appreciable efficacy against KV3 (inhibition ranging from 19.86 ± 5.28% to 1.91 ± 2.57%). No significant difference was found for the sixth dilution and below (7.5 μg ml^–1, 3.75 μg ml^–1, 1.875 μg ml^–1, 0.9375 μg ml^–1 and 0.46875 μg ml^–1) in comparison to the control group (Figure 7).

The most remarkable results of the present study were obtained with Usnea sp. against KV3. As seen in Figure 8, all tested concentrations were highly effective to suppress the growth of KV3. Even the tenth dilution (0.46875 μg ml^–1) of the tested lichen extract had noticeable inhibition rates. The inhibition percentages were: 94.25 ± 6.98%, 94.02 ± 3.65%, 95.75 ± 0.30%, 96.88 ± 0.38%, 97.88 ± 0.22%, 97.97 ± 0.39%, 97.85 ± 0.29%, 97.57 ± 0.06%, 95.57 ± 0.49% and 83.55 ± 2.71% for all tested concentrations, respectively. There was no statistically significant difference between the first nine concentrations (240 μg ml^–1, 120 μg ml^–1, 60 μg ml^–1, 30 μg ml^–1, 15 μg ml^–1, 7.5 μg ml^–1, 3.75 μg ml^–1, 1.875 μg ml^–1 and 0.9375 μg ml^–1), but they were found to be significantly different compared to controls (p <0.05). The 0.46875 μg ml^–1 treatment group also had statistically significant difference when compared to controls (p <0.05) (Figure 8).

The acetone extracts of E. divaricata also gave remarkable results, although not as much as Usnea sp. The bacterial growth of KV3 was suppressed by these extracts up to the sixth dilution. At the first six concentrations tested (240 μg ml^–1, 120 μg ml^–1, 60 μg ml^–1, 30 μg ml^–1, 15 μg ml^–1 and 7.5 μg ml^–1), the inhibition percentages were recorded between 97.58 ± 0.53% and 72.72 ± 6.01%. The tested concentrations below 3.75 μg ml^–1 had no considerable inhibition ratios for KV3. In statistical analyses, there were significant differences among 240 μg ml^–1 vs. control, 120 μg ml^–1 vs. control, 60 μg ml^–1, 30 μg ml^–1, 15 μg ml^–1, 7.5 μg ml^–1 treatment groups vs. control, and 3.75 μg ml^–1, 1.875 μg ml^–1, 0.9375 μg ml^–1, 0.46875 μg ml^–1 vs. control (p <0.05). On the other hand, no statistical differences were observed among the 60 μg ml^–1, 30 μg ml^–1, 15 μg ml^–1 and 7.5 μg ml^–1 treatment groups. The same situation was recorded among the treatment groups of 3.75 μg ml^–1, 1.875 μg ml^–1, 0.9375 μg ml^–1 and 0.46875 μg ml^–1 (Figure 9).

4. Discussion

It is known that moderately halophilic bacteria, mostly from salt, can be found in high numbers on hides or skins. Due to their ability to produce enzymes such as protease and lipase that have devastating effects on hides and skins, more attention should be paid to these bacterial populations in leather production processes and methods should be applied to prevent their excessive growth. Given the high population and possibly high degradative effects of halophilic bacteria, salt curing does not seem to be the only solution to control them. Alternative strategies have to be investigated in order to obtain high-quality leather for economic benefit.

For this purpose, many researchers have tested various possible antimicrobial substances against halophilic bacteria isolated from the leather industry. Halocins, which are antimicrobial peptides produced by halophilic archaea, have been evaluated to control halophilic archaea with possibly degradative properties on leather and the potential efficacy of halocins against extremely halophilic bacteria has been reported (33, 34). Vreeland et al. indicated the antimicrobial effect of bile salt solution (0.025 g 100 ml^–1) against Haloarcula hispanica, Haloferax gibbonsii and Haloferax mediterranei and showed the potential protective effect of bile salt solutions on cured hides up to 45 days (35). Gehring et al. evaluated porcine bile in brine-curing solution against halophilic archaeal strains with positive results (36).

The potential suppressive effect of electric current application has been reported against extremely halophilic archaea and moderately halophilic bacteria (5, 25). Caglayan et al. reported the antibacterial efficiency of different electric current applications on moderately halophilic Staphylococcus saprophyticus, Bacillus pumilus, Bacillus licheniformis, Gracilibacillus dipsosauri and Idiomarina loihiensis isolated and identified from salted skins (37). In another study, different levels of direct and alternating electric current treatments were examined for preventive effects against skin deterioration and the inactivation of the growth of a mixed moderately halophilic bacterial culture including Chromohalobacter israelensis, Chromohalobacter canadensis, Halomonas halodenitrificans, Staphylococcus nepalensis and Halomonas halmophila isolated from salted sheep and goat skin samples (38).

In the literature, there are also studies investigating plant materials as eco-benign materials for their possible antibacterial effects. Sivakumar et al. reported the antimicrobial activity of myrobalan (Terminalia chebula Retz.) application along with salt utilisation on the short-term preservation of raw hides and skins (39). It is well known that lichen substances have potential antibacterial activities. To the best of our knowledge, the antibacterial efficacy of the acetone extracts of lichen species against moderately halophilic bacteria has not been studied in the literature.

In this study, acetone extracts of L. pulmonaria, B. capillaris and P. furfuracea had no efficacy against our test bacteria. However, H. tubulosa, R. farinacea, E. divaricata and Usnea sp. extracts have noteworthy suppressive effects. Usnea sp. acetone extracts were observed to have the highest antibacterial activity against KV3 among the selected lichen species even at the lowest concentration tested. In the literature there are many studies of the antibacterial efficacy of H. tubulosa, R. farinacea, E. divaricata and Usnea sp. against various bacteria, especially Gram-positive ones. However, some Gram-negative bacteria are reported to be resistant to Usnea sp. Here, we determined the antibacterial effect of lichens on Gram-positive test bacteria.

More detailed studies report many chemical compounds for various lichen species. For example, Usnea sp. has usnic acid, thamnolic acid, atranorin and barbatic acid; R. farinacea has evernic acid, atranorin, usnic acid and chloroatranorin (40, 41); P. furfuracea was reported to have evernic acid, atranorin, usnic acid, physodalic acid and chloroatranorin. Some compounds seem to be common to many lichens, such as evernic acid, atranorin and chloroatranorin (40, 42, 43). Atranorin is generally found in most lichen species including our tested lichen species. However, based on our results, atranorin does not appear to have selectivity for antibacterial activity against the tested bacterial strains since our screening experiments showed that not all of the tested lichen species were effective against KV3 and TR5. The presence of usnic acid has been indicated in the literature in some lichen species such as Usnea sp., P. furfuracea, E. divaricata and R. farinacea. Nevertheless, in our study P. furfuracea had no efficacy against our test bacteria which points to a lack of antibacterial efficacy of usnic acid.

The most successful lichen in our study was Usnea sp. In the literature, Usnea sp. has been reported to have other lichen metabolites such as thamnolic acid, barbatic acid, diffractic acid, evernic acid and squamatic acid. Its antibacterial efficacy may be due to one or more of the aforementioned metabolites, or the metabolites may have synergistic effect when applied together. Another possible scenario could be the difference in the amounts of lichen metabolites in different lichen species.

As seen in this study, extracts gathered from lichen species may exhibit varying efficacies against bacteria depending on the concentrations. Future studies to answer the question of which metabolite is effective on which bacteria may open up horizons. It can be difficult to predict what the effects on a mixed culture microorganism population might be. For example, in the mixed culture study performed by Berber et al. (27) when Usnea sp. acetone extracts were tested on the total bacterial population obtained from soaking liquors, successful results were obtained in some samples, but not in others.

5. Conclusions

The need for potent new antibacterial agents is emphasised in the literature. The presence of high populations of moderately halophilic bacteria on hides and skins inevitably leads to defects on finished products despite salt- or brine-curing methods applied to raw hides and skins. Many lichen species have been used for years for various purposes including their antibacterial potential. This study demonstrated that acetone extracts of H. tubulosa, R. farinacea, E. divaricata and Usnea sp. have antibacterial effects against certain moderately halophilic bacteria. Noteworthy antibacterial activity was detected in Usnea sp. acetone extracts against KV3 even at the lowest concentration tested. These results suggest that proteolytic and lipolytic moderately halophilic bacteria may be controlled by these ecological materials. Chemical analyses will be needed to determine which compound(s) are responsible for the antibacterial efficacy of the lichen species. These compounds could be applied onto hides and skins in microencapsulated or sprayed forms along with salt or brine during the storage period of raw stock.