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Assessment of antibacterial activity of lining leather treated with silver doped hydroxyapatite
International Biodeterioration & Biodegradation 105 (2015) 262e267
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International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Assessment of antibacterial activity of lining leather treated with silver doped hydroxyapatite Meruyert Koizhaiganova a, *, Ihsan Yas¸a b, Gürbüz Gülümser c a
Denizli Vocational School of Technical Sciences, Pamukkale University, Kınıklı, Denizli 20013, Turkey Basic and Industrial Microbiology Department, Faculty of Science, Ege University, Bornova, Izmir 35100, Turkey c Leather Engineering Department, Engineering Faculty, Ege University, Bornova, Izmir 35100, Turkey b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 June 2015 Received in revised form 25 September 2015 Accepted 25 September 2015 Available online xxx
In this study, silver doped hydroxyapatite (Ag-HA) prepared by the microwave method was applied as the finishing agent to leather, and its antibacterial effect was investigated. The structural properties of the Ag-HA treated leather were determined using Fourier Transform Infrared Spectroscopy (FTIR) and Raman Spectroscopy. In the Raman and FTIR results, the presence of the PO3 4 band of HA clearly indicated the application of 0%e5% Ag-HA to the surface of the finished leathers, and xMeO (Ag2O) peaks showed that silver was present in the film on the leather surface. The antibacterial activity of the leathers were assessed by means of the parallel streak method (qualitative) and the test of percentage of microbial reduction (quantitative). According to the results of these tests, leather treated with Ag-HA containing 2% or more of silver (Ag) showed good antibacterial activity, and it was decided that Ag doped hydroxyapatite could be used as an antibacterial agent on lining leathers. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Silver doped hydroxyapatite Antibacterial activity Lining leather Coating
1. Introduction It is known that if certain functional additives are applied to the surface of leather during the finishing process, the final leather products will have the desired surface properties; consequently, adding suitable antimicrobial agents to leather surfaces can provide powerful antimicrobial functions (Lkhagvajav et al., 2015). Synthetic antimicrobial agents such as quaternary ammonium salts, chlorinated phenols, silylquaternary compounds, tolysulfone compounds, polyvinyl pyridines, diammonium rings, etc., have been used industrially. However some of them may in some degree be harmful and toxic to the environment and to human health (Türkan et al., 2013). Therefore, there is much interest in finding ways to formulate new types of safe and ecological materials. Hydroxyapatite (HA) has been widely used as a biomaterial in dentistry, orthopedics and bioengineering, because of its good biocompatibility and bioactivity (Tanaka et al., 2003). It possesses a similar structure to the major mineral constituent of human hard bone and can also serve as a carrier of antibiotics to reduce bacterial infections in the bone-implant interface. By taking advantage of its ion-exchange capabilities, HA can be effective in controlling
* Corresponding author. E-mail address: [email protected] (M. Koizhaiganova). http://dx.doi.org/10.1016/j.ibiod.2015.09.017 0964-8305/© 2015 Elsevier Ltd. All rights reserved.
microorganisms with the introduction of a transient metal ion such as silver (Oh et al., 2003). Silver has been known as a disinfectant for many years and has a broad spectrum of antibacterial activity while exhibiting low toxicity towards mammalian cells. Various studies reported that the silver ions in the HA coatings play an important role in preventing or minimizing bacterial adhesion (Stanic et al., 2011; Chen et al., 2007). Recently, the antibacterial effects of silver-substituted HA have been investigated with different materials, including textiles (Lv et al., 2006). Ag doped HA is a new antibacterial agent for enhancing antibacterial performance and can be used to inhibit bacteria in leather as well. To the best of our knowledge it has not so far been used for the leather industry. In this study, the silver doped hydroxyapatite was prepared by the microwave method and its performance as the antibacterial agent on lining leather was investigated. The 0%e5% Ag-HA treated leathers were characterized by using Fourier Transform Infrared Spectroscopy (FTIR) and Raman spectroscopy. Antibacterial activity of the leather treated with the Ag-HA was revealed by the parallel streak method and by the percentage of bacterial reduction tests.
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263
D e is the diameter of the test specimen in mm.
2. Materials and method 2.1. Preparation and application of Ag-HA to lining leather Analytical grade calcium hydroxide (Ca(OH)2, Merck), diammonium hydrogen phosphate ((NH4)2HPO4, Merck), and silver nitrate (Ag(NO)3, Fluka) were used for the preparation of the Ag-HA in accordance with the literature (Rameshbabu et al., 2006). Samples of goat lining leather processed by traditional methods were treated by spraying with solutions of hydroxyapatite with 0.5%, 1%, 1.5%, 2%, 3% and 5% added silver and with pure hydroxyapatite solution, in a finishing mixture of 1:1:1 HA:lacquer:water with 5% by total volume of penetrator, allowing the mixture to penetrate the leather. Later the leather samples were dried at 105 C for 15 min in a drying oven and ironed in an ironing press at 100 C in order to form a thin film. The processed leather samples were placed in polyethylene bags and stored in a refrigerator in preparation for microbiological studies. 2.2. Characterization of Ag-HA treated lining leather The functional groups present in the 0%e5% Ag-HA treated leather were ascertained by Fourier Transform Infrared Spectroscopy (FTIR, Perkin Elmer, Spectrum 100, USA). For this purpose, the IR spectrum of the leather samples were taken in the range of 400 cm1e4000 cm1, 4 cm1 resolution, at room temperature. Raman analysis was performed by Raman spectrometer (Perkin Elmer, Raman Station 400, USA) in the range of 200 cm1e4000 cm1, 4 shift/cm1 interval time, at room temperature. The Raman spectra of silver were checked from the Raman spectral database (Rasmin, 2015).
The qualitative test Percentage of Bacterial Reduction was performed according to the general guides of the AATCC 100 test method (AATCC 100, 2004). 2 2 cm2 leather samples which had been treated with HA solutions with different amounts of Ag added were placed in empty sterile Petri dishes in accordance with the test method. The test microorganisms E. coli and S. aureus were activated in MHB at 37 C for 18e20 h. Fresh bacterial cultures were prepared from the activated cultures by dilution, 8.1 105 CFU/ml for E. coli, and 7.3 105 CFU/ml for S. aureus. 200 mL of the test bacteria mixture was inoculated on to the leather samples treated with pure HA or 0.5%e5% Ag-HA. One hour later, the samples were placed in 250 ml flasks containing 50 ml of physiological saline (0.85% NaCl), 0.4% sodium thioglycoliate and 1% Tween 20, and stirred for 1e2 min at 37 C so that the microorganisms on the samples passed into the solution (Gallant-Behm et al., 2005; Wright et al., 1999). Serial dilutions were made by taking 1 ml quantities of the mixture from the flasks in order to determine the effects. The 1 ml quantities of serial dilution as (101, 102) were taken on to sterile Petri dishes and the microorganisms in the sample were counted by the plate-colony count method (Kim S. and Kim H., 2006; Kasuga et al., 2011). Müller Hinton Agar (MHA) was used as a medium, and a 2 ml/100 ml solution of TTC (triphenyl tetrazolium chloride) was added to the medium in order to see the microorganism colonies more easily. Equation (2) was used to calculate the reduction in bacteria on the untreated leather samples and those treated with HA with various concentrations (0%e5%) of added silver.
% Reduction ¼ ½ðA BÞ=A 100
(2)
2.3. Antibacterial assessment The antibacterial activity was evaluated using the parallel streak method (AATCC 147, 2004) as the qualitative test. The microorganisms utilized for the test were Staphylococcus aureus ATCC 6538-P, Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 29212 as Gram positive bacteria, Escherichia coli ATCC 12228, Salmonella typhimurium CCM 5445, Klebsiella pneumoniae CCM 2318 and Pseudomonas aeruginosa ATCC 27853 as Gram negative bacteria and the yeast Candida albicans ATCC 10239. Test microorganisms used in this study were activated in Muller Hinton Broth (MHB) in a shaking water bath at 37 C for 24 h. The bacterial solution was then transferred to the surface of a Muller Hinton Agar (MHA) plate by making four consecutive parallel streaks (60 mm length, 10 mm distance from each other). The leather sample treated with Ag-HA (25 50 mm) was then placed transversely across the four streaks and the plate was incubated at 37 C for 24 h (AATCC 147, 2004; Würtz, 2004; Barros et al., 2011; Fernandes et al., 2013). Other assays were performed using samples of leather without treatment and treated with pure HA or 0% Ag-HA, which were used as control. Incubated plates were examined for interruption of growth along the streaks of inoculum beneath the specimen and for a clean zone of inhibition beyond its edge (AATCC 147, 2004; Fernandes et al., 2013). The average width of a zone of inhibition along a streak on either side of the test specimen was calculated using Equation (1):
W ¼ ðT DÞ=2
(1)
where W e is the width of the clear zone of inhibition in mm T e is the total diameter of the test specimen and the clear zone in mm
A ¼ the number of microorganisms on the control samples of leather after 1 h B ¼ the number of microorganisms on the treated samples of leather after 1 h The procedures of both tests were slightly modified due to their application to leather (Fernandes et al., 2013; Lkhagvajav et al., 2015). 3. Results 3.1. FTIR analysis of the leathers FTIR spectra were taken of the leather samples in order to determine the chemical differences between the control sample and the treated samples. The aim of this scanning was to determine changes in the functional groups in the leather resulting from the finishing operation in which HA with 0%e5% added Ag had been applied. The FTIR spectra and the functional groups obtained from the leather as a result of the FTIR scanning are presented in Fig. 1 and Table 1. As can be seen in Table 1, all the functional groups relating to chrome crust leather are visible in the FTIR spectra of the leathers. Vibrations in the ranges 835 cm1e837 cm1, 1447 cm1e1449 cm1 and 3000 cm1 e 2500 cm1 are attributed to hydrogen bonds of associated functional groups, OeH, CeH (Valeika et al., 2010). Peaks in the ranges 1655 cm1 e 1658 cm1, 1547 cm1 e 1549 cm1 and 1236 cm1e 1237 cm1 are attributed to I, II and III amide bands respectively (Aslan, 2014). The area of peaks in the range 1632 cm1 corresponds to the deformation of amide II (eNH2). Peaks of the CeO band were observed at intervals
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the functional groups in these spectra obtained as a result of Raman scanning of the leather samples. The functional groups of the chrome crust leather can be seen in the Raman spectra obtained. In addition, xMeO (Ag2O) peaks at wavelength intervals of 515 cm1, 671 cm1 and 723 cm1 were observed in the leathers treated with 1%, 3% and 5% Ag-HA respectively. This verified the presence of silver in the film on the surface of the leather (Ciobanu et al., 2012). However, the characteristic xMeO (Ag2O) band was not observed in the Raman spectra of the leathers treated with 0.5% Ag-HA (data not shown). This is thought to be because the amount of silver per cm2 of the leather was very small. Moreover, the biocide in this study was applied by spraying. This may have led to a non uniform distribution of the biocide in the leather.
5 % A g -H A
3 % A g -H A
2 % A g -H A
Adsorbance
1 .5 % A g -H A
1 % A g -H A
3.3. Antibacterial properties of leather treated with Ag-HA 0%e5% Ag-HA was applied to the leather during the finishing process in this study and antibacterial activity was revealed by qualitative and quantitative tests. The parallel streak method was performed as the qualitative test of antibacterial activity for all samples. Table 3 shows the antibacterial activity of the control leather samples and the leather samples treated with pure HA and HA with 1%e3% added Ag against S. aureus, B. subtilis, E. faecalis as Gram positive bacteria, E. coli, S. typhimurium, K. pneumoniae and P. aeruginosa as Gram negative bacteria and the yeast C. albicans. After appropriate incubation periods, no inhibition zone was seen around and beneath the control leather sample. On the contrary, although no inhibition zones were seen around the leather samples treated with pure HA, there were no bacterial colonies directly under the sample in the contact area. By visually inspecting the samples coated using 1% Ag-HA, no significant differences from the leathers treated with pure HA were noticed. The treatment of the leather samples with 2% of Ag-HA was effective against E. coli, P. aeruginosa and S. aureus, as inhibition zones were seen around these samples. All microorganisms tested, except C. albicans, were found to be sensitive at the concentrations of Ag-HA of 3%, because the leather specimens treated with 3% of Ag-HA displayed antibacterial effects and zones of inhibition were in the range of 1.0e2.0 mm. The fact that no growth of any tested bacteria was observed around and beneath the leather samples containing concentrations 3% of Ag-HA was evaluated as a result of the antibacterial effect of Ag-HA. It is apparent that greater concentrations of Ag-HA used resulted in an increase in antibacterial activity in the treated leather samples. In the results of the tests, leathers treated with hydroxyapatite containing silver in a proportion of 3% showed antibacterial activity
0 .5 % A g -H A
p u re H A
co n tro l
1000
2 000
3 000
40 00
Wave number, cm-1 Fig. 1. FTIR spectra of control and 0%e5% Ag-HA treated leathers.
of 1031 cm1e1032 cm1 and 1080 cm1e1081 cm1 in all samples, while in the spectra of the leathers treated with 3% and 5% Ag-HA the CeO band was observed at 1161 cm1 (Aslan, 2013). At the same 1 1 time, the PO3 was observed 4 band of HA at 1200 cm e1202 cm in all samples except for the control sample (Rameshbabu et al., 2006). Thus, the FTIR spectra clearly distinguished the leather samples with the 0%e5% Ag-HA finishing coating applied. 3.2. Raman analysis of the leathers The control leather sample and those treated with 0%e5% AgHA were examined by Raman spectroscopy in order to determine chemical changes. The purpose was to use Raman spectroscopy to detect changes in functional groups in the leather which had occurred as a result of treatment with Ag-HA and which were not apparent from FTIR. Fig. 2 and Table 2 show the Raman spectra and
Table 1 Functional groups observed in FTIR spectra of control and 0%e5% Ag-HA treated leathers. Functional groups
Control
Pure HA
0.5% Ag-HA
1% Ag-HA
1.5% Ag-HA
2% Ag-HA
3% Ag-HA
5% Ag-HA
1031 1081 1161 1202 1237 1336 1449 1548 1632 2853 2923 3308
1031 1081 1161 1202 1236 1336 1448 1547 1632 2853 2924 3302
Wave number (cm1) ¼CeH CeO CeO CeO PO4 CeN CeN eCeH NeO eNH2 CeH CeH eOH
837 1032 1080
835 1031 1080
1031 1080
1032 1080
1031 1080
1031 1080
1236 1335 1447 1547 1632 2854 2924 3301
1202 1237 1337 1448 1549 1633 2853 2923 3311
1201 1236 1335 1448 1547 1632 2853 2923 3303
1201 1236 1335 1448 1547 1632 2853 2923 3309
1202 1237 1336 1448 1548 1632 2853 2923 3309
1200 1236 1335 1448 1548 1632 2853 2923 3311
M. Koizhaiganova et al. / International Biodeterioration & Biodegradation 105 (2015) 262e267
against all of the bacteria after a period of 24 h. The parallel streak method is accepted as a quick and simple method in the qualitative assessment of antibacterial effectiveness. This test has been used in many studies, and after qualitative determination of the antibacterial activity of the material, either the ASTM E 2149-01 or the AATCC 100 test is used to determine the level at which the antibacterial activity is effective. In our study, the AATCC 100 test was used as a quantitative test method, and the test results are given below. The leather samples coated with 1%e5% of Ag-HA were chosen according to the qualitative test results to perform the percentage of bacterial reduction test. This quantitative test was performed at 60 min contact time between the sample and the test bacteria inoculate. A non-coated leather sample was used as a control and was subjected to a parallel test using an identical inoculate of the test bacteria. The results obtained showed that the leather coated with 1%e5% Ag-HA exhibited considerable antibacterial activity, as a significant reduction of CFU/ml was observed after 60 min. The leather samples treated with 2% of Ag-HA demonstrated the highest antibacterial activity in 60 min against S. aureus with 100% bacterium removal. The same concentration of Ag-HA applied to leather was enough to exhibit the excellent percentage reduction of E. coli of 100%. Table 4 presents a survey of the results obtained, confirming that an increase in the silver concentration in Ag-HA led to an increase in the antibacterial activity of the leather coated with Ag-HA. In our study, a 100% reduction was seen after 1 h in resistance to the two test microorganisms of the 2% Ag-HA which we applied to the leather. As the main goal of this study was to gain antibacterial characteristics for leather with a simple application, this result was considered to be encouraging for the leather industry.
5% Ag-HA
Intensity
3% Ag-HA
1% Ag-HA
pure HA
control
600
265
900
Wave number, cm-1 Fig. 2. Raman spectra of control and 0%e5% Ag-HA treated leathers.
4. Discussion Table 2 Functional groups observed in Raman spectra of control and 0%e5% Ag-HA treated leathers. Functional groups
Control
Pure HA
1% Ag-HA
3% Ag-HA
5% Ag-HA
515
671
723
Wave number (cm1) yXmetal-O
e
e
The chemical changes occurring in the functional groups in the control leather and in the leathers finished by applying 0%e5% AgHA were determined by means of FTIR and Raman spectroscopy. Rameshbabu et al. (2006) observed the characteristic bands for HA as phosphate bending and stretching at 900 cm1e1200 cm1 in FTIR spectra of Ag-HA dried in an oven. Nath et al. (2010) stated that
Table 3 Antibacterial properties of the leather samples according to AATCC 147-2004. Specimens
E. coli
K. pneumoniae
P. aeruginosa
S. typhimurium
S. aureus
B. subtilis
E. faecalis
C. albicans
xa ob ob 1.0 2.0
xa ob ob ob 1.0
xa ob ob 1.0 2.0
xa ob ob ob 1.0
xa ob ob ob 1.0
xa ob ob ob ob
Mean zones of inhibition (mm) Control Pure HA 1.0% Ag-HA 2.0% Ag-HA 3.0% Ag-HA a b
xa ob ob 0.5 2.0
xa ob ob ob 1.0
x e no inhibition zone, microorganism growth beneath the specimen. o e no inhibition zone, but no microorganism growth beneath the specimen.
Table 4 Bacterial reduction on leather samples treated with different concentrations of Ag-HA in a 60 min test time. Initial number of bacteria
Escherichia coli 35.2 103
Reduction rate (%)
Staphylococcus aureus 27.2 103
Reduction rate (%)
5.7 103 2.6 103 0.1 103 0 0 0
54.39% 98.24% 100% 100% 100%
1.14 103 0.28 103 0.18 103 0 0 0
75.44% 84.21% 100% 100% 100%
Specimens Control 1.0% Ag-HA 1.5% Ag-HA 2.0% Ag-HA 3.0% Ag-HA 5.0% Ag-HA
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the AgeO band of the Ag2O and AgO phases could not be determined with the FTIR in the range of 400 cm1e4000 cm1, and for this reason, apart from the change in the vibration peak of the OeH group at 3750 cm1, the FTIR result did not show that silver had been added. By taking into account the characteristic peaks in the FTIR and Raman spectra obtained in our study, it is clearly shown that a finishing coating containing Ag-HA has been applied to the surface of the leathers, and that the film on the surface of the leather contains silver. No study was found in the literature relating to the application of HA with added Ag to leather, and therefore comparisons were made with studies on the antibacterial effects of this substance on textile. In a study by Lv et al. (2006), HA with added Ag and Ti was applied to textile and its antibacterial activity against E. coli, S. aureus and C. albicans was determined by means of the disk diffusion test. It was found that a 3 mm inhibition zone was formed around samples treated with HA with 0.5% added Ag, and a 4 mm inhibition zone formed around samples treated with HA with 0.5% Ag and 5% Ti. Kasuga et al. (2011) coated cotton and polypropylene fabrics with a hydroxyapatite nanocomposite with added Ag and Ti, and determined its antibacterial activity against E. coli, S. aureus and P. aeruginosa. It was determined that P. aeruginosa was inhibited after 3e6 h, and S. aureus and E. coli were inhibited after 18 h. When an antibacterial powder based on calcium phosphate with 5% Agþ and 14% Zn2þ ions added was applied to textile fabric and its antibacterial quality against the bacterium E. coli was examined, test results showed that the fabric samples maintained their antibacterial effectiveness even after 20 washings (Çavdar, 2009). Zhang et al. (2010) stated that the antibacterial mechanism of Ag-HA could be explained by the interaction of positively charged silver approaching negatively charged bacteria, resulting in its entry to the bacterial cell by breaking down the cell wall. Here, silver reacts with lysine, arginine and cysteine, breaking down the carboxylic (eCOOH) and thiole (eSH) groups. In this way, active proteins coagulate and the activity of basic metabolic enzymes in the bacterium falls. At the same time, silver and the eSH group of the enzyme form an SeAg bound which prevents respiration in the bacterium. In addition, Ag causes nucleic acid to coagulate and connects with DNA molecules or catalyzes to form free radicals by breaking down the chemical structure of the DNA molecules. It was found in this research that Ag-HA provides effective antibacterial properties to leather. Its performance was increased by increasing the concentration of Ag. The concentrations of Ag-HA which were effective against all tested bacteria in both tests were 3% and higher of Ag in Ag-HA. 5. Conclusion The antibacterial characteristics of the control leathers and those treated with 0%e5% Ag-HA were compared by means of the parallel streak method (qualitative) and the test of percentage reduction in the number of bacteria (quantitative). It was found in this research that Ag-HA provides effective antibacterial properties to leather and its performance was increased by increasing the concentration of Ag. According to the results of the parallel streak method the minimum level for obtaining antibacterial leather was established at 3% of Ag in Ag-HA. Moreover, quantitative test results showed that concentrations of Ag-HA which were effective against all tested bacteria were 2% and higher of Ag in Ag-HA with 100% of reduction of both tested bacteria. The results are promising for AgHA use in applications as antibacterial coating on leather. The use of leather materials with these characteristics in the manufacture of shoes is of great importance for foot health and hygiene. There have been no studies on the application of Ag-HA to shoe lining leather, so that our study is the first, and important
results have been obtained. The application of this material to leather is quick and easy, and provides it with antibacterial performance. In addition, we foresee that it will be possible to apply this material to leathers other than shoe lining leathers. Acknowledgment We would like to thank the Research Foundation of Ege University for their financial support (Project N 09MÜH080). References AATCC Test Method 147-2004, 2004. Antibacterial Activity Assessment of Textile Materials: Parallel Streak Method. AATCC Technical Manual. American Association of Textile Chemists and Colorists, Research Triangle Park, NC. AATCC Test Method 100-2004, 2004. Antibacterial Finishes on Textile Materials: Assessment of. AATCC Technical Manual. American Association of Textile Chemists and Colorists, Research Triangle Park, NC. Aslan, A., 2013. Improving the dyeing properties of vegetable tanned leathers using chitosan formate. Ekoloji 22 (86), 26e35. Aslan, A., 2014. Improved dyeing properties of chrome-tanned leathers and parameters of the waste water using chitosan formate. Ekoloji 23 (91), 75e82. Barros, M.C., Fernandez, I.P., Pinto, V., Ferreira, M.J., Barreiro, M.F., Amaral, J.S., 2011. In: Jimnez, A., Zairov, G.E. (Eds.), Chitosan as Antimicrobial Agent for Footwear Leather Components, Biodegradable Polymers and Sustainable Polymers. Nova Science, pp. 151e162. Chen, W., Oh, S., Ong, A.P., Oh, N., Liu, Y., Courtney, H.S., Appleford, M., Ong, J.L., 2007. Antibacterial and osteogenic properties of silver-containing hydroxyapatite coatings produced using a sol gel process. J. Biomed. Mater. Res. Part A 82 (4), 899e906. Ciobanu, C.S., Iconaru, S.L., Coustumer, P.L., Constantin, L.V., Predoi, D., 2012. Antibacterial activity of silver-doped hydroxyapatite nanoparticles against grampositive and gram-negative bacteria. Nanoscale Res. Lett. 7, 324. Çavdar, A., 2009. Development of Silver Doped Nano Scaled Bio Antimicrobial Materials for Textile Industry, Master of Science Thesis. Anadolu University Graduate School of Sciences, Advanced Technologies Program Nanotechnology, Eskisehir, Turkey. Fernandes, I.P., Amaral, J.S., Pintoc, V., Ferreirac, M.J., Barreiro, M.F., 2013. Development of chitosan-based antimicrobial leather coatings. Carbohydr. Polym. 98, 1229e1235. Gallant-Behm, C.L., Yin, H.Q., Liu, S., Heggers, J.P., Langford, R.E., Olson, M.E., Hart, D.A., Burrell, R.E., 2005. Comparison of in vitro disc diffusion and time killkinetic assays for the evaluation of antimicrobial wound dressing efficacy. Wound Repair Regen. 13 (4), 413e421. Kasuga, E., Kawakami, Y., Matsumoto, T., Hidaka, E., Oana, K., Ogiwara, N., Yamaki, D., Sakurada, T., Honda, T., 2011. Bactericidal activities of woven cotton and nonwoven polypropylene fabrics coated with hydroxyapatite-binding silver/titanium dioxide ceramic nanocomposite “Earth-plus”. J. Int. J. Nanomed. 6, 1937e1943. Kim, S., Kim, H., 2006. Antibacterial performance of colloidal silver-treated laminate wood flooring. Int. Biodeterior. Biodegrad. 57, 155e162. € 2015. Characterization Lkhagvajav, N., Koizhaiganova, M., Yasa, I., Çelik, E., Sari, O., and antimicrobial performance of nano silver coatings on leather materials. Braz. J. Microbiol. 46 (1), 41e48. Lv, G., Li, Y., Yang, A., Zhang, X., Yang, W., Li, J., 2006. Preparation and antibacterial activity of silver ions-substituted hydroxyapatite/titania. Mater. Sci. Forum 510e511, 78e81. Nath, S., Kalmodia, S., Basu, B., 2010. Densification, phase stability and in vitro biocompatibility property of hydroxyapatite-10 wt% silver composites. J. Mater. Sci. Mater. Med. 21, 1273e1287. Oh, K.S., Kim, K.J., Jeong, Y.K., Choa, Y.H., 2003. Effect of fabrication processes on the antimicrobial properties of silver doped nano-sized Hap. Key Eng. Mater. 240e242, 583e586. Rameshbabu, N., Sampath Kumar, T.S., Prabhakar, T.G., Sastry, V.S., Murty, K.V.G.K., Prasad Rao, K., 2006. Antibacterial nanosized silver substituted hydroxyapatite: synthesis and characterization. J. Biomed. Mater. Res. Part A 80 (3), 581e591. Rasmin, Raman Spectra Database of Minerals and Inorganic Materials, Online access 20.09.2015, http://riodb.ibase.aist.go.jp/rasmin/E_index.htm Stanic, V., Janackovic, D., Dimitrijevic, S., Tanaskovic, S.B., Mitrica, M., Pavlovic, M.S., Krstic, A., Jovanovic, D., Raicevic, S., 2011. Synthesis of antimicrobial monophase silver-doped hydroxyapatite nanopowders for bone tissue engineering. Appl. Surf. Sci. 257, 4510e4518. Tanaka, Y., Hirata, Y., Yoshinaka, R., 2003. Synthesis and characteristics of ultrafine hydroxyapatite particles. J. Ceram. Process. Res. 4 (4), 197e201. Türkan, M.F., Aslan, A., Ali Yapıcı, N., Meriçli Yapıcı, B., Bilgi, S.T., 2013. Assessment of antimicrobial activity of natural leathers treated with Pseudevernia Furfuracea (L.) Zopf extracts. Tekstil ve Konfeksiyon 23 (2), 176e180. _ J., Beleska, K., 2010. Estimation of chrome-free tanning Valeika, V., Sirvaityt e, method suitability in conformity with physical and chemical properties of leather. Mater. Sci. (Med ziagotyra) 16 (4), 330e336. Wright, J.B., Lam, K., Hansen, D., Burrel, R.E., 1999. Efficacy of topical silver against
M. Koizhaiganova et al. / International Biodeterioration & Biodegradation 105 (2015) 262e267 fungal burn wound pathogens. Am. J. Infect. Control 27 (4), 344e350. Würtz, M., 2004. Microbiological tests on shoes and materials. World Footwear 18 (2). March/April 2004.
267
Zhang, Y., Yin, Q.S., Zhang, Y., Xia, H., Ai, F.Z., Jiao, Y.P., Chen, X.Q., 2010. Determination of antibacterial properties and cytocompatibility of silver-loaded coral hydroxyapatite. J. Mater. Sci. Mater. Med. 21, 2453e2462.
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Assessment of antibacterial activity of lining leather treated with silver doped hydroxyapatite Meruyert Koizhaiganova a, *, Ihsan Yas¸a b, Gürbüz Gülümser c a
Denizli Vocational School of Technical Sciences, Pamukkale University, Kınıklı, Denizli 20013, Turkey Basic and Industrial Microbiology Department, Faculty of Science, Ege University, Bornova, Izmir 35100, Turkey c Leather Engineering Department, Engineering Faculty, Ege University, Bornova, Izmir 35100, Turkey b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 June 2015 Received in revised form 25 September 2015 Accepted 25 September 2015 Available online xxx
In this study, silver doped hydroxyapatite (Ag-HA) prepared by the microwave method was applied as the finishing agent to leather, and its antibacterial effect was investigated. The structural properties of the Ag-HA treated leather were determined using Fourier Transform Infrared Spectroscopy (FTIR) and Raman Spectroscopy. In the Raman and FTIR results, the presence of the PO3 4 band of HA clearly indicated the application of 0%e5% Ag-HA to the surface of the finished leathers, and xMeO (Ag2O) peaks showed that silver was present in the film on the leather surface. The antibacterial activity of the leathers were assessed by means of the parallel streak method (qualitative) and the test of percentage of microbial reduction (quantitative). According to the results of these tests, leather treated with Ag-HA containing 2% or more of silver (Ag) showed good antibacterial activity, and it was decided that Ag doped hydroxyapatite could be used as an antibacterial agent on lining leathers. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Silver doped hydroxyapatite Antibacterial activity Lining leather Coating
1. Introduction It is known that if certain functional additives are applied to the surface of leather during the finishing process, the final leather products will have the desired surface properties; consequently, adding suitable antimicrobial agents to leather surfaces can provide powerful antimicrobial functions (Lkhagvajav et al., 2015). Synthetic antimicrobial agents such as quaternary ammonium salts, chlorinated phenols, silylquaternary compounds, tolysulfone compounds, polyvinyl pyridines, diammonium rings, etc., have been used industrially. However some of them may in some degree be harmful and toxic to the environment and to human health (Türkan et al., 2013). Therefore, there is much interest in finding ways to formulate new types of safe and ecological materials. Hydroxyapatite (HA) has been widely used as a biomaterial in dentistry, orthopedics and bioengineering, because of its good biocompatibility and bioactivity (Tanaka et al., 2003). It possesses a similar structure to the major mineral constituent of human hard bone and can also serve as a carrier of antibiotics to reduce bacterial infections in the bone-implant interface. By taking advantage of its ion-exchange capabilities, HA can be effective in controlling
* Corresponding author. E-mail address: [email protected] (M. Koizhaiganova). http://dx.doi.org/10.1016/j.ibiod.2015.09.017 0964-8305/© 2015 Elsevier Ltd. All rights reserved.
microorganisms with the introduction of a transient metal ion such as silver (Oh et al., 2003). Silver has been known as a disinfectant for many years and has a broad spectrum of antibacterial activity while exhibiting low toxicity towards mammalian cells. Various studies reported that the silver ions in the HA coatings play an important role in preventing or minimizing bacterial adhesion (Stanic et al., 2011; Chen et al., 2007). Recently, the antibacterial effects of silver-substituted HA have been investigated with different materials, including textiles (Lv et al., 2006). Ag doped HA is a new antibacterial agent for enhancing antibacterial performance and can be used to inhibit bacteria in leather as well. To the best of our knowledge it has not so far been used for the leather industry. In this study, the silver doped hydroxyapatite was prepared by the microwave method and its performance as the antibacterial agent on lining leather was investigated. The 0%e5% Ag-HA treated leathers were characterized by using Fourier Transform Infrared Spectroscopy (FTIR) and Raman spectroscopy. Antibacterial activity of the leather treated with the Ag-HA was revealed by the parallel streak method and by the percentage of bacterial reduction tests.
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D e is the diameter of the test specimen in mm.
2. Materials and method 2.1. Preparation and application of Ag-HA to lining leather Analytical grade calcium hydroxide (Ca(OH)2, Merck), diammonium hydrogen phosphate ((NH4)2HPO4, Merck), and silver nitrate (Ag(NO)3, Fluka) were used for the preparation of the Ag-HA in accordance with the literature (Rameshbabu et al., 2006). Samples of goat lining leather processed by traditional methods were treated by spraying with solutions of hydroxyapatite with 0.5%, 1%, 1.5%, 2%, 3% and 5% added silver and with pure hydroxyapatite solution, in a finishing mixture of 1:1:1 HA:lacquer:water with 5% by total volume of penetrator, allowing the mixture to penetrate the leather. Later the leather samples were dried at 105 C for 15 min in a drying oven and ironed in an ironing press at 100 C in order to form a thin film. The processed leather samples were placed in polyethylene bags and stored in a refrigerator in preparation for microbiological studies. 2.2. Characterization of Ag-HA treated lining leather The functional groups present in the 0%e5% Ag-HA treated leather were ascertained by Fourier Transform Infrared Spectroscopy (FTIR, Perkin Elmer, Spectrum 100, USA). For this purpose, the IR spectrum of the leather samples were taken in the range of 400 cm1e4000 cm1, 4 cm1 resolution, at room temperature. Raman analysis was performed by Raman spectrometer (Perkin Elmer, Raman Station 400, USA) in the range of 200 cm1e4000 cm1, 4 shift/cm1 interval time, at room temperature. The Raman spectra of silver were checked from the Raman spectral database (Rasmin, 2015).
The qualitative test Percentage of Bacterial Reduction was performed according to the general guides of the AATCC 100 test method (AATCC 100, 2004). 2 2 cm2 leather samples which had been treated with HA solutions with different amounts of Ag added were placed in empty sterile Petri dishes in accordance with the test method. The test microorganisms E. coli and S. aureus were activated in MHB at 37 C for 18e20 h. Fresh bacterial cultures were prepared from the activated cultures by dilution, 8.1 105 CFU/ml for E. coli, and 7.3 105 CFU/ml for S. aureus. 200 mL of the test bacteria mixture was inoculated on to the leather samples treated with pure HA or 0.5%e5% Ag-HA. One hour later, the samples were placed in 250 ml flasks containing 50 ml of physiological saline (0.85% NaCl), 0.4% sodium thioglycoliate and 1% Tween 20, and stirred for 1e2 min at 37 C so that the microorganisms on the samples passed into the solution (Gallant-Behm et al., 2005; Wright et al., 1999). Serial dilutions were made by taking 1 ml quantities of the mixture from the flasks in order to determine the effects. The 1 ml quantities of serial dilution as (101, 102) were taken on to sterile Petri dishes and the microorganisms in the sample were counted by the plate-colony count method (Kim S. and Kim H., 2006; Kasuga et al., 2011). Müller Hinton Agar (MHA) was used as a medium, and a 2 ml/100 ml solution of TTC (triphenyl tetrazolium chloride) was added to the medium in order to see the microorganism colonies more easily. Equation (2) was used to calculate the reduction in bacteria on the untreated leather samples and those treated with HA with various concentrations (0%e5%) of added silver.
% Reduction ¼ ½ðA BÞ=A 100
(2)
2.3. Antibacterial assessment The antibacterial activity was evaluated using the parallel streak method (AATCC 147, 2004) as the qualitative test. The microorganisms utilized for the test were Staphylococcus aureus ATCC 6538-P, Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 29212 as Gram positive bacteria, Escherichia coli ATCC 12228, Salmonella typhimurium CCM 5445, Klebsiella pneumoniae CCM 2318 and Pseudomonas aeruginosa ATCC 27853 as Gram negative bacteria and the yeast Candida albicans ATCC 10239. Test microorganisms used in this study were activated in Muller Hinton Broth (MHB) in a shaking water bath at 37 C for 24 h. The bacterial solution was then transferred to the surface of a Muller Hinton Agar (MHA) plate by making four consecutive parallel streaks (60 mm length, 10 mm distance from each other). The leather sample treated with Ag-HA (25 50 mm) was then placed transversely across the four streaks and the plate was incubated at 37 C for 24 h (AATCC 147, 2004; Würtz, 2004; Barros et al., 2011; Fernandes et al., 2013). Other assays were performed using samples of leather without treatment and treated with pure HA or 0% Ag-HA, which were used as control. Incubated plates were examined for interruption of growth along the streaks of inoculum beneath the specimen and for a clean zone of inhibition beyond its edge (AATCC 147, 2004; Fernandes et al., 2013). The average width of a zone of inhibition along a streak on either side of the test specimen was calculated using Equation (1):
W ¼ ðT DÞ=2
(1)
where W e is the width of the clear zone of inhibition in mm T e is the total diameter of the test specimen and the clear zone in mm
A ¼ the number of microorganisms on the control samples of leather after 1 h B ¼ the number of microorganisms on the treated samples of leather after 1 h The procedures of both tests were slightly modified due to their application to leather (Fernandes et al., 2013; Lkhagvajav et al., 2015). 3. Results 3.1. FTIR analysis of the leathers FTIR spectra were taken of the leather samples in order to determine the chemical differences between the control sample and the treated samples. The aim of this scanning was to determine changes in the functional groups in the leather resulting from the finishing operation in which HA with 0%e5% added Ag had been applied. The FTIR spectra and the functional groups obtained from the leather as a result of the FTIR scanning are presented in Fig. 1 and Table 1. As can be seen in Table 1, all the functional groups relating to chrome crust leather are visible in the FTIR spectra of the leathers. Vibrations in the ranges 835 cm1e837 cm1, 1447 cm1e1449 cm1 and 3000 cm1 e 2500 cm1 are attributed to hydrogen bonds of associated functional groups, OeH, CeH (Valeika et al., 2010). Peaks in the ranges 1655 cm1 e 1658 cm1, 1547 cm1 e 1549 cm1 and 1236 cm1e 1237 cm1 are attributed to I, II and III amide bands respectively (Aslan, 2014). The area of peaks in the range 1632 cm1 corresponds to the deformation of amide II (eNH2). Peaks of the CeO band were observed at intervals
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the functional groups in these spectra obtained as a result of Raman scanning of the leather samples. The functional groups of the chrome crust leather can be seen in the Raman spectra obtained. In addition, xMeO (Ag2O) peaks at wavelength intervals of 515 cm1, 671 cm1 and 723 cm1 were observed in the leathers treated with 1%, 3% and 5% Ag-HA respectively. This verified the presence of silver in the film on the surface of the leather (Ciobanu et al., 2012). However, the characteristic xMeO (Ag2O) band was not observed in the Raman spectra of the leathers treated with 0.5% Ag-HA (data not shown). This is thought to be because the amount of silver per cm2 of the leather was very small. Moreover, the biocide in this study was applied by spraying. This may have led to a non uniform distribution of the biocide in the leather.
5 % A g -H A
3 % A g -H A
2 % A g -H A
Adsorbance
1 .5 % A g -H A
1 % A g -H A
3.3. Antibacterial properties of leather treated with Ag-HA 0%e5% Ag-HA was applied to the leather during the finishing process in this study and antibacterial activity was revealed by qualitative and quantitative tests. The parallel streak method was performed as the qualitative test of antibacterial activity for all samples. Table 3 shows the antibacterial activity of the control leather samples and the leather samples treated with pure HA and HA with 1%e3% added Ag against S. aureus, B. subtilis, E. faecalis as Gram positive bacteria, E. coli, S. typhimurium, K. pneumoniae and P. aeruginosa as Gram negative bacteria and the yeast C. albicans. After appropriate incubation periods, no inhibition zone was seen around and beneath the control leather sample. On the contrary, although no inhibition zones were seen around the leather samples treated with pure HA, there were no bacterial colonies directly under the sample in the contact area. By visually inspecting the samples coated using 1% Ag-HA, no significant differences from the leathers treated with pure HA were noticed. The treatment of the leather samples with 2% of Ag-HA was effective against E. coli, P. aeruginosa and S. aureus, as inhibition zones were seen around these samples. All microorganisms tested, except C. albicans, were found to be sensitive at the concentrations of Ag-HA of 3%, because the leather specimens treated with 3% of Ag-HA displayed antibacterial effects and zones of inhibition were in the range of 1.0e2.0 mm. The fact that no growth of any tested bacteria was observed around and beneath the leather samples containing concentrations 3% of Ag-HA was evaluated as a result of the antibacterial effect of Ag-HA. It is apparent that greater concentrations of Ag-HA used resulted in an increase in antibacterial activity in the treated leather samples. In the results of the tests, leathers treated with hydroxyapatite containing silver in a proportion of 3% showed antibacterial activity
0 .5 % A g -H A
p u re H A
co n tro l
1000
2 000
3 000
40 00
Wave number, cm-1 Fig. 1. FTIR spectra of control and 0%e5% Ag-HA treated leathers.
of 1031 cm1e1032 cm1 and 1080 cm1e1081 cm1 in all samples, while in the spectra of the leathers treated with 3% and 5% Ag-HA the CeO band was observed at 1161 cm1 (Aslan, 2013). At the same 1 1 time, the PO3 was observed 4 band of HA at 1200 cm e1202 cm in all samples except for the control sample (Rameshbabu et al., 2006). Thus, the FTIR spectra clearly distinguished the leather samples with the 0%e5% Ag-HA finishing coating applied. 3.2. Raman analysis of the leathers The control leather sample and those treated with 0%e5% AgHA were examined by Raman spectroscopy in order to determine chemical changes. The purpose was to use Raman spectroscopy to detect changes in functional groups in the leather which had occurred as a result of treatment with Ag-HA and which were not apparent from FTIR. Fig. 2 and Table 2 show the Raman spectra and
Table 1 Functional groups observed in FTIR spectra of control and 0%e5% Ag-HA treated leathers. Functional groups
Control
Pure HA
0.5% Ag-HA
1% Ag-HA
1.5% Ag-HA
2% Ag-HA
3% Ag-HA
5% Ag-HA
1031 1081 1161 1202 1237 1336 1449 1548 1632 2853 2923 3308
1031 1081 1161 1202 1236 1336 1448 1547 1632 2853 2924 3302
Wave number (cm1) ¼CeH CeO CeO CeO PO4 CeN CeN eCeH NeO eNH2 CeH CeH eOH
837 1032 1080
835 1031 1080
1031 1080
1032 1080
1031 1080
1031 1080
1236 1335 1447 1547 1632 2854 2924 3301
1202 1237 1337 1448 1549 1633 2853 2923 3311
1201 1236 1335 1448 1547 1632 2853 2923 3303
1201 1236 1335 1448 1547 1632 2853 2923 3309
1202 1237 1336 1448 1548 1632 2853 2923 3309
1200 1236 1335 1448 1548 1632 2853 2923 3311
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against all of the bacteria after a period of 24 h. The parallel streak method is accepted as a quick and simple method in the qualitative assessment of antibacterial effectiveness. This test has been used in many studies, and after qualitative determination of the antibacterial activity of the material, either the ASTM E 2149-01 or the AATCC 100 test is used to determine the level at which the antibacterial activity is effective. In our study, the AATCC 100 test was used as a quantitative test method, and the test results are given below. The leather samples coated with 1%e5% of Ag-HA were chosen according to the qualitative test results to perform the percentage of bacterial reduction test. This quantitative test was performed at 60 min contact time between the sample and the test bacteria inoculate. A non-coated leather sample was used as a control and was subjected to a parallel test using an identical inoculate of the test bacteria. The results obtained showed that the leather coated with 1%e5% Ag-HA exhibited considerable antibacterial activity, as a significant reduction of CFU/ml was observed after 60 min. The leather samples treated with 2% of Ag-HA demonstrated the highest antibacterial activity in 60 min against S. aureus with 100% bacterium removal. The same concentration of Ag-HA applied to leather was enough to exhibit the excellent percentage reduction of E. coli of 100%. Table 4 presents a survey of the results obtained, confirming that an increase in the silver concentration in Ag-HA led to an increase in the antibacterial activity of the leather coated with Ag-HA. In our study, a 100% reduction was seen after 1 h in resistance to the two test microorganisms of the 2% Ag-HA which we applied to the leather. As the main goal of this study was to gain antibacterial characteristics for leather with a simple application, this result was considered to be encouraging for the leather industry.
5% Ag-HA
Intensity
3% Ag-HA
1% Ag-HA
pure HA
control
600
265
900
Wave number, cm-1 Fig. 2. Raman spectra of control and 0%e5% Ag-HA treated leathers.
4. Discussion Table 2 Functional groups observed in Raman spectra of control and 0%e5% Ag-HA treated leathers. Functional groups
Control
Pure HA
1% Ag-HA
3% Ag-HA
5% Ag-HA
515
671
723
Wave number (cm1) yXmetal-O
e
e
The chemical changes occurring in the functional groups in the control leather and in the leathers finished by applying 0%e5% AgHA were determined by means of FTIR and Raman spectroscopy. Rameshbabu et al. (2006) observed the characteristic bands for HA as phosphate bending and stretching at 900 cm1e1200 cm1 in FTIR spectra of Ag-HA dried in an oven. Nath et al. (2010) stated that
Table 3 Antibacterial properties of the leather samples according to AATCC 147-2004. Specimens
E. coli
K. pneumoniae
P. aeruginosa
S. typhimurium
S. aureus
B. subtilis
E. faecalis
C. albicans
xa ob ob 1.0 2.0
xa ob ob ob 1.0
xa ob ob 1.0 2.0
xa ob ob ob 1.0
xa ob ob ob 1.0
xa ob ob ob ob
Mean zones of inhibition (mm) Control Pure HA 1.0% Ag-HA 2.0% Ag-HA 3.0% Ag-HA a b
xa ob ob 0.5 2.0
xa ob ob ob 1.0
x e no inhibition zone, microorganism growth beneath the specimen. o e no inhibition zone, but no microorganism growth beneath the specimen.
Table 4 Bacterial reduction on leather samples treated with different concentrations of Ag-HA in a 60 min test time. Initial number of bacteria
Escherichia coli 35.2 103
Reduction rate (%)
Staphylococcus aureus 27.2 103
Reduction rate (%)
5.7 103 2.6 103 0.1 103 0 0 0
54.39% 98.24% 100% 100% 100%
1.14 103 0.28 103 0.18 103 0 0 0
75.44% 84.21% 100% 100% 100%
Specimens Control 1.0% Ag-HA 1.5% Ag-HA 2.0% Ag-HA 3.0% Ag-HA 5.0% Ag-HA
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the AgeO band of the Ag2O and AgO phases could not be determined with the FTIR in the range of 400 cm1e4000 cm1, and for this reason, apart from the change in the vibration peak of the OeH group at 3750 cm1, the FTIR result did not show that silver had been added. By taking into account the characteristic peaks in the FTIR and Raman spectra obtained in our study, it is clearly shown that a finishing coating containing Ag-HA has been applied to the surface of the leathers, and that the film on the surface of the leather contains silver. No study was found in the literature relating to the application of HA with added Ag to leather, and therefore comparisons were made with studies on the antibacterial effects of this substance on textile. In a study by Lv et al. (2006), HA with added Ag and Ti was applied to textile and its antibacterial activity against E. coli, S. aureus and C. albicans was determined by means of the disk diffusion test. It was found that a 3 mm inhibition zone was formed around samples treated with HA with 0.5% added Ag, and a 4 mm inhibition zone formed around samples treated with HA with 0.5% Ag and 5% Ti. Kasuga et al. (2011) coated cotton and polypropylene fabrics with a hydroxyapatite nanocomposite with added Ag and Ti, and determined its antibacterial activity against E. coli, S. aureus and P. aeruginosa. It was determined that P. aeruginosa was inhibited after 3e6 h, and S. aureus and E. coli were inhibited after 18 h. When an antibacterial powder based on calcium phosphate with 5% Agþ and 14% Zn2þ ions added was applied to textile fabric and its antibacterial quality against the bacterium E. coli was examined, test results showed that the fabric samples maintained their antibacterial effectiveness even after 20 washings (Çavdar, 2009). Zhang et al. (2010) stated that the antibacterial mechanism of Ag-HA could be explained by the interaction of positively charged silver approaching negatively charged bacteria, resulting in its entry to the bacterial cell by breaking down the cell wall. Here, silver reacts with lysine, arginine and cysteine, breaking down the carboxylic (eCOOH) and thiole (eSH) groups. In this way, active proteins coagulate and the activity of basic metabolic enzymes in the bacterium falls. At the same time, silver and the eSH group of the enzyme form an SeAg bound which prevents respiration in the bacterium. In addition, Ag causes nucleic acid to coagulate and connects with DNA molecules or catalyzes to form free radicals by breaking down the chemical structure of the DNA molecules. It was found in this research that Ag-HA provides effective antibacterial properties to leather. Its performance was increased by increasing the concentration of Ag. The concentrations of Ag-HA which were effective against all tested bacteria in both tests were 3% and higher of Ag in Ag-HA. 5. Conclusion The antibacterial characteristics of the control leathers and those treated with 0%e5% Ag-HA were compared by means of the parallel streak method (qualitative) and the test of percentage reduction in the number of bacteria (quantitative). It was found in this research that Ag-HA provides effective antibacterial properties to leather and its performance was increased by increasing the concentration of Ag. According to the results of the parallel streak method the minimum level for obtaining antibacterial leather was established at 3% of Ag in Ag-HA. Moreover, quantitative test results showed that concentrations of Ag-HA which were effective against all tested bacteria were 2% and higher of Ag in Ag-HA with 100% of reduction of both tested bacteria. The results are promising for AgHA use in applications as antibacterial coating on leather. The use of leather materials with these characteristics in the manufacture of shoes is of great importance for foot health and hygiene. There have been no studies on the application of Ag-HA to shoe lining leather, so that our study is the first, and important
results have been obtained. The application of this material to leather is quick and easy, and provides it with antibacterial performance. In addition, we foresee that it will be possible to apply this material to leathers other than shoe lining leathers. Acknowledgment We would like to thank the Research Foundation of Ege University for their financial support (Project N 09MÜH080). References AATCC Test Method 147-2004, 2004. Antibacterial Activity Assessment of Textile Materials: Parallel Streak Method. AATCC Technical Manual. American Association of Textile Chemists and Colorists, Research Triangle Park, NC. AATCC Test Method 100-2004, 2004. Antibacterial Finishes on Textile Materials: Assessment of. AATCC Technical Manual. American Association of Textile Chemists and Colorists, Research Triangle Park, NC. Aslan, A., 2013. Improving the dyeing properties of vegetable tanned leathers using chitosan formate. Ekoloji 22 (86), 26e35. Aslan, A., 2014. Improved dyeing properties of chrome-tanned leathers and parameters of the waste water using chitosan formate. Ekoloji 23 (91), 75e82. Barros, M.C., Fernandez, I.P., Pinto, V., Ferreira, M.J., Barreiro, M.F., Amaral, J.S., 2011. In: Jimnez, A., Zairov, G.E. (Eds.), Chitosan as Antimicrobial Agent for Footwear Leather Components, Biodegradable Polymers and Sustainable Polymers. Nova Science, pp. 151e162. Chen, W., Oh, S., Ong, A.P., Oh, N., Liu, Y., Courtney, H.S., Appleford, M., Ong, J.L., 2007. Antibacterial and osteogenic properties of silver-containing hydroxyapatite coatings produced using a sol gel process. J. Biomed. Mater. Res. Part A 82 (4), 899e906. Ciobanu, C.S., Iconaru, S.L., Coustumer, P.L., Constantin, L.V., Predoi, D., 2012. Antibacterial activity of silver-doped hydroxyapatite nanoparticles against grampositive and gram-negative bacteria. Nanoscale Res. Lett. 7, 324. Çavdar, A., 2009. Development of Silver Doped Nano Scaled Bio Antimicrobial Materials for Textile Industry, Master of Science Thesis. Anadolu University Graduate School of Sciences, Advanced Technologies Program Nanotechnology, Eskisehir, Turkey. Fernandes, I.P., Amaral, J.S., Pintoc, V., Ferreirac, M.J., Barreiro, M.F., 2013. Development of chitosan-based antimicrobial leather coatings. Carbohydr. Polym. 98, 1229e1235. Gallant-Behm, C.L., Yin, H.Q., Liu, S., Heggers, J.P., Langford, R.E., Olson, M.E., Hart, D.A., Burrell, R.E., 2005. Comparison of in vitro disc diffusion and time killkinetic assays for the evaluation of antimicrobial wound dressing efficacy. Wound Repair Regen. 13 (4), 413e421. Kasuga, E., Kawakami, Y., Matsumoto, T., Hidaka, E., Oana, K., Ogiwara, N., Yamaki, D., Sakurada, T., Honda, T., 2011. Bactericidal activities of woven cotton and nonwoven polypropylene fabrics coated with hydroxyapatite-binding silver/titanium dioxide ceramic nanocomposite “Earth-plus”. J. Int. J. Nanomed. 6, 1937e1943. Kim, S., Kim, H., 2006. Antibacterial performance of colloidal silver-treated laminate wood flooring. Int. Biodeterior. Biodegrad. 57, 155e162. € 2015. Characterization Lkhagvajav, N., Koizhaiganova, M., Yasa, I., Çelik, E., Sari, O., and antimicrobial performance of nano silver coatings on leather materials. Braz. J. Microbiol. 46 (1), 41e48. Lv, G., Li, Y., Yang, A., Zhang, X., Yang, W., Li, J., 2006. Preparation and antibacterial activity of silver ions-substituted hydroxyapatite/titania. Mater. Sci. Forum 510e511, 78e81. Nath, S., Kalmodia, S., Basu, B., 2010. Densification, phase stability and in vitro biocompatibility property of hydroxyapatite-10 wt% silver composites. J. Mater. Sci. Mater. Med. 21, 1273e1287. Oh, K.S., Kim, K.J., Jeong, Y.K., Choa, Y.H., 2003. Effect of fabrication processes on the antimicrobial properties of silver doped nano-sized Hap. Key Eng. Mater. 240e242, 583e586. Rameshbabu, N., Sampath Kumar, T.S., Prabhakar, T.G., Sastry, V.S., Murty, K.V.G.K., Prasad Rao, K., 2006. Antibacterial nanosized silver substituted hydroxyapatite: synthesis and characterization. J. Biomed. Mater. Res. Part A 80 (3), 581e591. Rasmin, Raman Spectra Database of Minerals and Inorganic Materials, Online access 20.09.2015, http://riodb.ibase.aist.go.jp/rasmin/E_index.htm Stanic, V., Janackovic, D., Dimitrijevic, S., Tanaskovic, S.B., Mitrica, M., Pavlovic, M.S., Krstic, A., Jovanovic, D., Raicevic, S., 2011. Synthesis of antimicrobial monophase silver-doped hydroxyapatite nanopowders for bone tissue engineering. Appl. Surf. Sci. 257, 4510e4518. Tanaka, Y., Hirata, Y., Yoshinaka, R., 2003. Synthesis and characteristics of ultrafine hydroxyapatite particles. J. Ceram. Process. Res. 4 (4), 197e201. Türkan, M.F., Aslan, A., Ali Yapıcı, N., Meriçli Yapıcı, B., Bilgi, S.T., 2013. Assessment of antimicrobial activity of natural leathers treated with Pseudevernia Furfuracea (L.) Zopf extracts. Tekstil ve Konfeksiyon 23 (2), 176e180. _ J., Beleska, K., 2010. Estimation of chrome-free tanning Valeika, V., Sirvaityt e, method suitability in conformity with physical and chemical properties of leather. Mater. Sci. (Med ziagotyra) 16 (4), 330e336. Wright, J.B., Lam, K., Hansen, D., Burrel, R.E., 1999. Efficacy of topical silver against
M. Koizhaiganova et al. / International Biodeterioration & Biodegradation 105 (2015) 262e267 fungal burn wound pathogens. Am. J. Infect. Control 27 (4), 344e350. Würtz, M., 2004. Microbiological tests on shoes and materials. World Footwear 18 (2). March/April 2004.
267
Zhang, Y., Yin, Q.S., Zhang, Y., Xia, H., Ai, F.Z., Jiao, Y.P., Chen, X.Q., 2010. Determination of antibacterial properties and cytocompatibility of silver-loaded coral hydroxyapatite. J. Mater. Sci. Mater. Med. 21, 2453e2462.