Fibers and Polymers 2010, Vol.11, No.3, 351-356
DOI 10.1007/s12221-010-0351-y
A New Application Method of Chitosan for Improved Antimicrobial Activity on Wool Fabrics Pretreated by Different Ways Asli Demir , Buket Arik1, Esen Ozdogan1, and Necdet Seventekin1 *
. Emel Akin Vocational High School, Ege University, Bornova, .Izmir, Turkey 1 Textile Engineering Department, Ege University, Bornova, Izmir, Turkey
(Received July 6, 2009; Revised November 17, 2009; Accepted February 22, 2010)
Abstract: Antimicrobial treatments have become more important for the textile materials especially used in sportswear,
activewear, and casual wear since they can easily be contaminated by perspiration leading to bacterial growth and body odor. In this work, antimicrobial activity of chitosan in a silica matrix on pretreated wool fabrics was studied. The pretreatment processes were applied by two different ways (enzymatic and enzymatic+hydrogen peroxide). Afterwards chitosan solutions were applied to the untreated samples and to the samples that were pretreated by two different ways to give antimicrobial effects. The antimicrobial activity of wool fabrics treated in various methods was assessed before and after repeated washings (up to 10 cycles) by the application of standard test method AATCC 147-1998. The morphology of the treated fabrics was investigated by SEM and their characterizations were made by the FT-IR spectral analysis. Results revealed that pretreatment ways and chitosan application methods were quite important for adsorption and diffusion of chitosan on wool fabrics and washing stability. From the SEM images, it was clearly observed that pretreatment processes caused some degradation on the surface of the fiber; but combined processes were found to be less degradative and more effective. Keywords: Wool, Chitosan, Sol-gel coating, Antimicrobial, Washing stability
chemical properties of chitosan (Figure 1) are attributed to its polyamine character, which makes the polymer watersoluble (at acidic pH) and positively charged and gives bioadhesive properties [4,5]. Chitosan, which is a polysaccharide-based cationic biopolymer, is poly((1,4)-2-amino-2-deoxy-β-D-glucan) (CHT) usually obtained by deacetylation of chitin and chitin is a component of some fungi, exoskeleton of insects and marine invertebrates (crabs and shrimp). The chemistry of chitosan is similar to that of cellulose, but it reveals the fact that the 2hydroxyl group of the cellulose has been replaced with a primary aliphatic amino group. This polysaccharide has several useful properties, such as non-toxicity, biocompatibility, biodegradability, antimicrobial activity, chemical reactivity, and film forming ability, which makes it an important biopolymer for textile applications [6-8]. Chitosan has been found to inhibit the growth of microbes in a large body of work that has been extensively reviewed by Lim and Hudson [9]. The antimicrobial mechanism is not clear but it is generally accepted that the primary amine groups provide positive charges which interact with negatively charged residues on the surface of microbes. Such interaction causes extensive changes in the cell surface and cell permeability, leading to leakage of intracellular substances. Early works indicated that the antimicrobial effect was potent against a range of microbes, but the finishing was not durable. To improve durability and antimicrobial performance of chitosan, it must be covalently bonded to the fabric or entrapped in a matrix [9,10]. Chitosan can be applied to textile materials by different methods such as pad-dry-cure, exhaustion, or covalent bonding. Nowadays modified silica coatings, containing
Introduction
The wool fiber surface is covered by a covalently-bonded fatty layer, being responsible for the strong hydrophobicity of wool. Protease which can catalyze the degradation of different component of a wool fiber is the most common enzyme used for wool fabrics. Oxidation process applied before enzyme can prepare the fiber for enzyme digestion. For example as an oxidizing agent, hydrogen peroxide (H2O2) in an aqueous alkaline medium favors the formation of the unstable perhydroxy (-OOH) species that transfers oxygen and under these conditions the disulfide bond is attacked and the surface of wool becomes susceptible to enzymatic degradation [1-3]. The application of biopolymers such as chitosan, which is the most promising one, has attracted a great deal of scientific and industrial interest. These biopolymers can be used as possible substitutes for synthetic polymers. As a substitute for synthetic polymer in wool treatments, chitosan has been proposed, because of its unusual combination of chemical and biological properties. The most important
Figure 1. Chemical structure of chitosan. *Corresponding author:
[email protected] 351
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embedded biocides on textiles have also been used to give antimicrobial effect [11]. The gradual release of the biocides from a modified organic-inorganic silica matrix bonded onto the textile surface allows the effective antimicrobial properties of textiles to be sustained over a long time and even after several washings. Such a sol-gel finishing, however, has mostly been carried out on the laboratory level and has not yet been widely used in industrial processes [12]. Therefore, in this study antimicrobial activity of chitosan in a silica matrix were investigated with regard to parameters such as antimicrobial efficacy, wash-out and long-term behavior as well as the effect of the pre-treatment processes on the antimicrobial activity of wool samples.
Experimental Materials
100 % wool knitted fabric with a weight of 251 g/m was used in the experiments. For the pre-treatment processes, protease enzyme, non-ionic wetting agent, and hidrogen peroxide were applied. Protease enzyme (Perizym AFW) and non-ionic wetting agent were supplied from the company named Dr. Petry textile auxiliaries and H O was supplied from Merck. As an antimicrobial agent, medium molecular weight chitosan (Sigma-Aldrich) dissolved in a diluted 5 % CH COOH solution (Merck) was used in combination with the reactive organic-inorganic binder (RB) named iSys MTX (CHT). ISys MTX can be mixed with chitosan solution to any desired concentration and can form a silica matrix in condensation process. 2
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Treatment Methods
Chitosan Treatments The antimicrobial finish was applied to wool fabrics (untreated, pre-treated by process A, and pre-treated by process B) by the pad-dry-cure and by the sol-gel methods. Chitosan solutions used in these methods were freshly prepared by dissolving the biopolymer in pure water containing 5 % acetic acid. For the pad-dry-cure method, a solution consisting of 10 g/l chitosan that is equivalent to 1 % o.w.f was used. This method was applied by immersing the samples at 20 C, padding with a wet-pick-up of 90±1 %, drying at 90 for 3 min, and curing at 150 C for 1 min. For the sol-gel method, a solution consisting of 10 g/l chitosan, which is equivalent to 1 % o.w.f and 15 g/l of RB reactive binder (iSys MTX) was used. The samples (untreated, pre-treated by method A, and pre-treated by method B) were immersed in chitosan-RB sol-gel solutions at 20 C. Afterwards, the samples were padded with a wetpick-up of 90±1 %, dried at 90 for 3 min and cured at 150 C for 1 min. o
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Evaluation Methods
Antimicrobial Activity Assessment In this research, antimicrobial activity assessments were made to the washed samples as well as the non-washed samples. The washing process was made in order to check the washing stability of the samples. The washings were made repeatedly in an Atlas Launder-O-Meter Instrument. The treated fabric samples were washed repeatedly up to 10 times and the duration of the washing cycles was 30 min. In order to prevent any effect of detergent, washings were carried out in a soap solution with a concentration of 5 g/l, at 35 C, with a liquor ratio of 50:1. After washing, the samples were rinsed in cold pure water, squeezed and dried at room temperature. The antimicrobial activity was assessed after the first, fifth, and tenth washing cycles. AATCC Test Method 147-1998 was used to test the antimicrobial activity of the untreated and all treated fabrics. Two different kinds of bacteria, Staphylococcus aureus (ATCC 6538) as Gram positive bacteria and Klebsiella pneumonia (ATCC 4352) as Gram negative bacteria were studied. The medium was Trypticase Soy Agar which was prepared by heating 40 g of agar powder in 1000 ml distilled water for 25 min at 15 psi and 120 C. Test samples were cut by hand in rectangular shape (25×50 mm), they were uniformly pressed on the agar and incubated for 24 h at 37±1 C. After incubation, assessment based on the absence or presence of bacterial growth in the contact zone between the agar and the sample and on the eventual appearance of an inhibition zone which was calculated from: W = (T − D)/2 where W is the width of clear zone of inhibition in mm, T is o
Method A (Enzyme Treatments) Enzyme treatments were carried out by the exhaustion method using a Wascator machine (James H. Heal). The liquor ratio was 20:1, the temperature was 70 C and the pH was 8. As chemical agents; 1 g/l Perizym AFW and 0.5 g/l non-ionic wetting agent were used and the exhaustion process was lasted for 1 h. After the treatment, the wool samples were hand-squeezed, post-treated in a pH 4 solution at 70 C for 5 min and then rinsed in cold pure water, finally dried at room temperature. Method B (Hydrogen Peroxide Treatments of Enzyme Treated Samples) Enzyme treated wool fabrics were then treated with hydrogen peroxide by the exhaustion method using a Wascator machine (James H. Heal). The liquor ratio was 20:1, the temperature was 70 C and the pH was 9. In this bleaching treatment, 18 ml/l H O , 2 g/l non-ionic wetting agent and 2 g/l sodiumpyrophosphate were applied for 1 h. After the treatment, the wool samples were hand-squeezed, post-treated in a pH 4 solution at 70 C for 5 min and then rinsed in cold pure water, finally dried at room temperature. o
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New Method for Good Antimicrobial Activity on Wool
the total diameter of test specimen and clear zone in mm, and D is the diameter of the test specimen in mm. Following the standard method, the inhibition zone was measured in mm and the degree of bacterial growth in the nutrient medium under the specimen was assessed. The antimicrobial effect of the studied samples was described either “good”, “limited” or “insufficient”. All tests were performed in duplicate. Characterization Assesment of the Chitosan and Chitosan-RB Coating The samples were analyzed using a Fourier Transform Infrared Spectrophotometer (FT-IR), Perkin Elmer, in the region from 4000 to 800 cm and the surface morphology was evaluated by Scanning Electron Microscopy (SEM). SEM observations were made with a Phillips XL-30S FEG scanning electron microscope. -1
Results and Discussion
Effect of Pre-treatment Processes
In this study, two pre-treatment methods (Method A and Method B) were applied to the wool samples. In method A, protease enzyme was used to modify the surface of the wool fiber. To the results, protease enzyme was found to degrade the protein components of the wool fiber and deform the scale structure. On the contrary to the earlier studies on pretreatment of wool, it was observed that the amount of adsorbed protease enzyme was clearly high for wool that was subjected to a surfactant washing or posterior bleaching [4,13]. Therefore, in method B, hydrogen peroxide (H O , bleaching agent) was applied to the enzyme treated samples. This pretreatment method enabled a higher penetration of protease into wool and consequently a higher degradation level. The bleaching with H O also promoted a partial removal of the bounded fatty acid barrier of the epicuticle. So it’s concluded that a simple enzymatic treatment was not enough to remove the fatty bounded layer and promote chitosan adsorption when compared with enzyme + peroxide treatment. This fact confirmed the results of the other studies on this matter [2,4,13,14]. 2
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Antimicrobial Mechanism and Properties of Antimicrobial Coating After pre-treatment processes, chitosan (antimicrobial biopolymer) was applied to the wool samples. In this step, two methods (pad-dry-cure and sol-gel methods) were studied. From the results, sol-gel method which was able to form a modified organic-inorganic silica matrix on the sample was found to be more effective than pad-dry-cure method. The gradual release of the chitosan (Figure 1) from a modified organic-inorganic silica matrix bonded onto the textile surface allowed the effective antimicrobial properties
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The inhibition zones of the samples pretreated by method B; (a) against K. Pneumonia and (b) against S. Aureus. Figure 2.
The diameters of clear zones of inhibition of the all treated and untreated samples in mm Table 1.
S. Aureus
K. Pneumonia
0 1 2 3 0 1 2 3 Pad-dry-cure method 0 0 1 2.5 0 0 1.2 2.2 Sol-gel method 0 5 6 11.5 0 5 6.5 10 0: Untreated, 1: un-pretreated, 2: pretreated by method A, 3: pretreated by method B.
of textiles to be sustained over a long time and even after several washings. The results on the antimicrobial activity of chitosan-RB coating confirmed its direct dependence on the chitosan application method for the wool samples. On the other hand the samples treated by pad-dry-cure method indicated that an insufficient amount of chitosan was adsorbed on the fiber to achieve effective antimicrobial activity. The inhibition zone on the unwashed finished samples was lower than 2.5 mm and rapidly decreased after repeated washing. Compared to the unwashed samples there was heavy growth of bacteria in the medium under the washed samples. If the effect of pre-treatment processes was concerned, it could be concluded that the wool samples pre-treated by method B (Figure 2) showed much better antimicrobial activity and washing stability than untreated wool and pretreated by method A. In Table 1, the diameters of clear zones of inhibition of the all treated and untreated samples in mm were shown against Staphylococcus aureus and Klebsiella pneumonia.
After the washing processes the diameter of clear zones rapidly decreased and the antimicrobial activity was only observed under the samples. The antimicrobial activity of the samples finished by sol-gel method was higher than those of finished by the pad-dry-cure method. In Figure 3, the inhibition zones of the samples pretreated by method B and finished by sol-gel and pad-dry-cure method after first and tenth washing processes were shown. After the first washings, heavy growth of bacteria was observed both
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under and around the samples finished by the pad-dry-cure method. However, there was only some restricted colonies on the samples finished by sol-gel method especially on the sample pre-treated by method B. In Table 2, the antimicrobial activities of the unwashed and washed samples were given.
FT-IR and SEM Analysis
Figure 4 is the FT-IR spectra of the pure chitosan film and chitosan-RB coating film and the region from 4000 to 800 cm has been shown in detail. The main absorption bands of chitosan, 1658 cm (amide I) and 1595 cm (-NH bending) and one signal at 1582 cm were observed in the chitosan film. The absorption band at 1154 cm (anti-symmetric stretching of C-O-C bridge) was characteristic of its saccharide structure [15] and the band at 2927 cm was attributed to the symmetric stretching of -CH - of chitosan [16]. The characteristic signals of SiO appeared at 1130 and 1075 cm [12,17]. These IR peaks confirmed the formation of structural units of Si-O-Si in the chitosan-RB coating film. As could be seen in Figure 4, the intensity of absorption bands increased with the formation of chitosanRB coating film and showed a shift to higher frequencies. Figure 5 has shown the surface appearances of the treated and untreated wool samples. As seen from the images, enzymatic treatment and enzymatic+peroxide treatment -1
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The inhibition zones of the samples pretreated by method B; (a) finished by sol-gel method after first washing, (b) finished by sol-gel method after tenth washing, (c) finished by pad-dry-cure method after first washing, and (d) finished by pad-dry-cure method after tenth washing. Figure 3.
The antimicrobial activities of the unwashed and washed samples Sample Washing Size of inhibition zone (mm) Growth of bacteria in the medium under the specimen Antimicrobial effect Untreated 0 0 Heavy Insufficient 0 0 Slight, only some restricted colonies Limited 1 0 Heavy, compared to the control no growth reduction Insufficient P1 5 0 Heavy Insufficient 10 0 Heavy Insufficient 0 1 Slight, only some restricted colonies Limited 1 0 Heavy, compared to the control no growth reduction Insufficient P2 5 0 Heavy Insufficient 10 0 Heavy Insufficient 0 2,5 None Good 1 0 Heavy, compared to the control no growth reduction Insufficient P3 5 0 Heavy Insufficient 10 0 Heavy Insufficient 0 5 None Good 1 1,5 None Good S1 5 1 Slight, only some restricted colonies Limited 10 0 Heavy, compared to the control no growth reduction Insufficient 0 6 None Good 1 2 None Good S2 5 1,5 Slight, only some restricted colonies Limited 10 0 Heavy, compared to the control no growth reduction Insufficient 0 11,5 None Good 5 None Good 1 S3 Good 5 3 None Limited 10 1 Slight, only some restricted colonies P: Pad-dry-cure method, S: sol-gel method, 1: un-pretreated, 2: pretreated by method A, 3: pretreated by method B. Table 2.
New Method for Good Antimicrobial Activity on Wool
Figure 4.
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FT-IR spectra of the pure chitosan film and chitosan-RB coating film; (a) 4000~1800 cm and (b) 1800~800 cm . -1
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SEM images of the samples; (a) untreated, (b) pretreated by method A, (c) pretreated by method B, and (d) treated by chitosan-RB sol-gel solution after being pretreated by method B. Figure 5.
caused degradation on the surface of the fiber. Enzymatic treatment had a hydrolytic effect causing bond-cleavages, which made the effect more degradative. On the other hand, when the dual combination (enzymatic+peroxide) was evaluated, it could be seen that this was more effective than only enzyme application. When chitosan was taken into account, it covered the scaly surface of the fiber and gave a smoother appearance [18]. From the image of the wool treated by chitosan-RB sol-gel solution, the film coating and silica spherical particles, embedded in the fiber structure were clearly observed. As shown in the SEM images,
chitosan-silica hybrid antibacterial particles were well dispersed on the surface of wool fibers. It could be said that hydrolysis and condensation of chitosan-silica hybrid led to these well dispersed nanosized silica spherical particles [19,20]. In addition to these results, the covering of the scales of wool fibers as shown clearly in Figure 5 could be an advantage for anti-felting property of wool fabrics.
Conclusion In this study, it was found that chitosan had strong and
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long-termed antimicrobial activity on wool fabrics when applied together with a reactive organic-inorganic binder matrix (iSys MTX). Results showed that pre-treatment processes were quite effective on antimicrobial activity of wool samples. Enzyme and peroxide combination clearly promoted chitosan adsorption, leading to improved durability. The samples pre-treated by method B and finished by sol-gel coating method showed the best and washing-stable, longlasting antimicrobial activity even after 10 repeated washings. On the other hand, heavy growth of bacteria was observed both under and around the samples finished by the pad-drycure method after washing processes. So, it was concluded that, in order to get a good and durable antimicrobial effect, the wool sample must be pre-treated by method B and finished by sol-gel method that was much more suitable than the pad-dry-cure method.
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