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Ege University Emel Akýn Vocational High School, Bornova, Izmir, Turkey. 1. Ege University Textile Engineering Department, Bornova, Izmir, Turkey. (Received ...
Fibers and Polymers 2010, Vol.11, No.7, 989-995

DOI 10.1007/s12221-010-0989-5

The Comparison of the Effect of Enzyme, Peroxide, Plasma and Chitosan Processes on Wool Fabrics and Evaluation for Antimicrobial Activity Asli Demir*, Buket Arik1, Esen Ozdogan1, and Necdet Seventekin1 Ege University Emel Akýn Vocational High School, Bornova, Izmir, Turkey 1 Ege University Textile Engineering Department, Bornova, Izmir, Turkey

(Received February 22, 2010; Revised June 29, 2010; Accepted July 2, 2010)

Abstract: Pretreated (enzymatic and enzymatic+hydrogen peroxide) knitted wool fabrics were treated with atmospheric

argon and air plasma to improve their adsorption capacity. After plasma treatments chitosan solution was applied to have antimicrobial effect on wool fabrics. The treated fabrics were evaluated in terms of washing stability as well as antimicrobial activity. The surface morphology was characterised by SEM images and FTIR analysis. From the results it was observed that atmospheric plasma treatment had an etching effect and increased the functionality of a wool surface. Atmospheric plasma treatment also enhanced the adhesion of chitosan to the surface and improved the antimicrobial activity of the wool sample. Argon was found to be more effective than air, since argon radicals played an important role in killing and removing bacteria. No significant difference in washing durability was observed in terms of plasma treatments. The samples of combined pretreatment processes had good washing durability even after 10 washing cycle. From the SEM images it was observed that combination of plasma and the other pre-treatment processes gave less damage than only one process. Keywords: Atmospheric plasma, Enzyme, Peroxide, Chitosan, Silica coating, Antimicrobial

promote the formation of new anionic groups on the fiber and to increase the surface energy of wool fibers. To achieve this, one of today’s established methods is the LTP treatment of wool. Low temperature plasma (LTP) is a totally or partially ionised gas which contains radicals, ions, photons and other excited species [6-9]. Low temperature plasma treatments modify the cuticle surface of the wool fibers, generating new hydrophilic groups as a result of hydrocarbon chain oxidation, reducing the chain length of fatty acid, improving their surface wettability, dyeability, fiber cohesion, and shrink resistance [9-12]. The oxidation process also promotes cystine oxidation in the exocuticle, converting it into cysteic acid, and thus reducing the number of crosslinkages in the fiber surface [6,13]. Chitosan can be applied to textile materials by different methods such as pad-dry-cure, exhaustion or covalent bonding. Nowadays modified silica coatings, containing embedded biocides on textiles are also used to have antimicrobial effect [14]. 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. Plasma treatment improves the antimicrobial effects too. Therefore, in this study effects of plasma treatments on antimicrobial activity of chitosan in a silica matrix were investigated and the results were compared and assessed 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.

Introduction

The wool fiber has a typical-core-shell-structure consisting of an inner protein core, cortex and surface shell, cuticle. The cuticle consists of several layers. The upper layer, epicuticle, contains lipoproteins. Lipoid part of lipoproteins is bound by the sulfoester bond with proteinous part. The lipoproteins are connected with upper layer of exocuticle. Exocuticle is cross-linked by disulfide links. This surface morphology of wool plays an important role in wool processing, since the hydrophobic nature of the cuticle and the high cross-linking density in the outermost fiber surface creates a nature diffusion barrier, which influences sorption properties [1]. So wool surface must be modified before processes to improve hydrophilicity, dyeability, antifelting and adhesion of polymers. Chitosan, which is a polysaccharide obtained by alkaline deacetylation of chitin, has excellent biological properties such as biodegradability in the human body and immunological, antibacterial, and wound-healing activities. The most important chemical properties of chitosan are attributed to its polyamine character, which makes the polymer water-soluble (at acidic pH) and positively charged and confers bio-adhesive properties [2-4]. In the case of wool treatments, the role of chitosan is to improve the dyeability, antimicrobial activity and antifelting properties. These properties enable chitosan to be used in wool finishing [5]. To enhance chitosan’s adsorption on wool fabric and increase the uniformity of its distribution, it is necessary to develop anionic groups on the wool surface. Therefore, to enhance chitosan binding, it can be useful to *Corresponding author: [email protected] 989

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Experimental

Washing Treatments

Materials

100 % wool knitted fabric with a mass of 245 g/m was used in the experiments and in the pre-treatment processes the samples were treated with atmospheric plasma, enzyme (protease), H O and non-ionic wetting agent. Protease (Perizym AFW) and non-ionic wetting agent were kindly supplied from Dr. Petry, 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 iSys MTX (CHT). ISys MTX is a reactive organic-inorganic binder (RB) that can be mixed with chitosan solution to any desired concentration and can form a silica matrix in condensation process. 2

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Enzyme Treatments

Enzyme treatments were carried out by the exhaustion method at a liquor-to-wool ratio of 20:1 using a Wascator machine (James Heal) at 70 C and pH 8 using 1 g/l Perizym AFW and 0,5 g/l non-ionic wetting agent for an hour. After the treatment, the wool samples were hand-squeezed, posttreated in a pH 4 solution at 70 C for 5 min, then rinsed in cold distilled water, and finally dried at room temperature. o

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Hydrogen Peroxide Treatments

Enzyme treated wool fabrics were used in the treatments which were carried out by the exhaustion method at a liquorto-wool ratio of 20:1 using a Wascator machine (James Heal) at 70 C and pH 9. In the treatment 18 ml/lt H O and 2 g/l non-ionic wetting agent were applied for an hour. After the treatment, the wool samples were hand-squeezed, posttreated in a pH 4 solution at 70 C for 5 min, then rinsed in cold distilled water, and finally dried at room temperature. o

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Plasma Treatments

A dielectric barrier discharge (DBD) atmospheric plasma device was used. The distance between the electrodes was 0.2 cm. The samples were placed between the electrodes and passed continuously with the speed of 0.45 m/min. In all treatments, air and argon were used under a constant power of 130 W. The samples were treated for 40 sec in the plasma.

Chitosan Treatments

Chitosan solutions were freshly prepared by dissolving the biopolymer in distilled water containing acetic acid. The antimicrobial finish was applied to wool samples by sol-gel method. In this method, a solution consisting of 10 g/l chitosan, which is equivalent to 1 % o.w.f and 15 g/l of RB was used. The samples were immersed in chitosan-RB solgel solutions at 20 C. Afterwards, the samples were wrung to a wet-pick-up of 90±1 % at 20 C, dried at 90 C and cured at 150 C for 1 min. o

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The washing durability of the samples was determined after repetitive washing in an Atlas Launder-O-Meter Instrument. The finished fabric samples were washed repetitively up to 10 times; the duration of the washing cycles was 30 min, in order to prevent any adverse effects of detergent, washings were carried out in a solution of soap with a concentration of 5 g/l, at 35 C, with a liquid ratio of 50:1. After washing, the samples were rinsed in cold distilled water, squeezed and dried at room temperature. The antimicrobial activity was assessed after the first and tenth washing cycles. o

Antimicrobial Activity Assessment

AATCC Test Method 147-1998 was used to test the antimicrobial activity of the samples. 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 (2,5×5 cm), they were uniformly pressed on the agar and incubated for 24 h at 37±1 C. After incubation, assessment was 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 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. o

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SEM and FT-IR Analysis

The structure and the appearance of the wool fibers were evaluated by Scanning Electron Microscopy (SEM). SEM observations were made with a Phillips XL-30S FEG scanning electron microscope. The samples were analyzed in Fourier Transform Infrared Spectrophotometer (FTIR), Perkin Elmer, in the region from 4000 to 650 cm to describe changes of chemical bonds and formation of new chemical groups after the plasma treatments. -1

Results and Discussion Effect of Enzyme, Peroxide, and Plasma Processes

Protease enzyme was used to remove the scale cuticles or smooth the edges. Protease enzymes penetrate and degrade the internal structure of wool during processing. Oxidation

Enzyme Peroxide Plasma and Chitosan Effect on Wool

process applied after enzyme treatment catalyzes the fibre for further applications. For example as an oxidizing agent, hydrogen peroxide (H2O2) in an aqueous alkaline medium favors the formation of the unstable per hydroxy (HO2−) species that transfers oxygen and under these conditions, the disulfide bond is attacked, but this action causes some fibre damage [15-17]. Plasma-assisted coating of surfaces is a new and very promising application for effective antimicrobial treatments. These plasma based antimicrobial treatments are in the focus

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of scientific research and development for several years [18,19]. The effect of plasma, which is an environmental process, is thought to be due to oxidation and etching reactions, which enhance the hydrophilicity of the fiber surface [20,21]. After plasma treatments carbon content of the samples reduces due to the etching effect of atmospheric air and argon plasma treatment. This fact is clearly observed by XPS analysis in our previous study [6]. On the other hand as a result of the oxidation of hydrocarbon chains located on

SEM images of the samples (a) untreated, (b) pretreated by enzyme, (c) pretreated by enzyme+peroxide, (d) pretreated by air plasma, and (e) pretreated by argon plasma. Figure 1.

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SEM images of the samples (a) pre-treated by enzyme+peroxide+air plasma and post-treated by chitosan-RB coating and (b) pretreated by enzyme+peroxide+argon plasma and post-treated by chitosan-RB coating. Figure 2.

the wool surface, an increase in carboxylic acid occurs [6,22]. In order to evaluate the changes on the surface of the untreated and treated wool samples, SEM observations were made. Figure 1 shows surface appearances of the samples. As can be seen from the images, enzyme, peroxide and atmospheric plasma treatments cause degradation on the surface of the fiber. But the dominant effects of the two processes are different. In the plasma processes, etching is dominant and causes partial degradation, such as rounding scales and microcracks [1,6,23,24]. That small amounts of substances are thought to be formed by the decomposition of lipid and protein material and are scattered over the surface of the plasma-treated wool fiber [20,24]. On the other hand, enzymatic treatment and enzymatic+peroxide treatment causes intensive degradation. When chitosan is considered, it can be observed that chitosan covers the scaly surface of the fibre and provides smoother appearance without any damage [6]. When the combinations are evaluated, it can be seen that they are more effective and less degradative than one process (Figure 2). Figure 2 shows the SEM image of the samples that chitosan-RB sol-gel solution treated after combined pretreatment processes. As seen, chitosan-silica hybrid antibacterial particles are well dispersed on the surface of wool fibres. It can be said that hydrolysis and condensation of chitosansilica hybrid leads to these well dispersed nanosized silica spherical particles [25]. Table 1.

Antimicrobial Mechanism and Properties of Antimicrobial Coating

Chitosan is known as a antimicrobial biopolymer [26]. Although the antimicrobial mechanism is not clear it is generally accepted that the primary amine groups provide positive charges which interact with negatively charged residues on the surface of bacteria. Such interaction causes extensive changes in the cell surface and cell permeability, leading to leakage of intracellular substances. In order to enhance the bonding efficiency of chitosan polymer with wool and to increase the uniformity of its distribution on surfaces, the surface energy and anionic character of a wool surface must be promoted. In this way there will be an increase in the reactivity of the wool surface and antimicrobial efficiency will be better due to the higher chitosan binding [6]. In this study different pre-treatment processes (enzyme treatment, enzyme+peroxide treatment, air plasma, argon plasma and combinations of these processes) were applied to modify the wool surface, promote chitosan binding and increase antimicrobial efficiency. In Table 1, the diameters of clear zones of inhibition of the all treated and untreated samples in mm are shown against Staphylococcus aureus and Klebsiella pneumonia. To the results enzyme+peroxide+ argon plasma pre-treatment was found to be the most efficient (Figure 3). Plasma is not only effective in killing bacteria and fungi, but also it can remove dead bacteria and viruses from the

The diameters of clear zones of inhibition of the all treated and untreated samples in mm S. Aureus

1 2 3 Chitosan-RB coating 5 6 11,5 Air plasma+chitosan-RB coating 5 6,5 12 Argon Plasma+chitosan-RB coating 5,5 7 13 1: Un-pre-treated, 2: pre-treated by enzyme, and 3: pre-treated by enzyme+peroxide.

1 5 6 7

K. Pneumonia

2 6,5 6,5 8

3 10 10,5 15,5

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Enzyme Peroxide Plasma and Chitosan Effect on Wool

The inhibition zones of the samples pretreated enzyme+ peroxide+argon plasma (a) against K. Pneumonia (b) against S. Figure 3.

Aureus.

Table 2.

samples

The antimicrobial activities of the unwashed and washed

Size of Growth of inhibition bacteria the Antimicrobial Sample Washing zone medium in under effect (mm) the specimen Untreated 0 0 Heavy Insufficient 0 5 None Good Chitosan1 1,5 Slight Limited RB coating (1) 10 0 Heavy Insufficient 0 6 None Good Chitosan1 1,5 Slight Limited RB coating (2) 10 0 Heavy Insufficient 0 11,5 None Good Chitosan1 5 None Good RB coating (3) 10 1 Slight Limited 0 5 None Good Air plasma + 1 2 None Good chitosanRB coating (1) 10 0 Heavy Insufficient 0 6,5 None Good Air plasma + chitosan1 2 None Good RB coating (2) 10 0 Heavy Insufficient 0 12 None Good Air plasma + chitosan1 6 None Good RB coating (3) 10 2 None Good 0 5,5 None Good Argon plasma+ chitosan-RB 1 2,5 None Good coating (1) 10 0 Heavy Insufficient 7 None Good Argon plasma+ 0 chitosan-RB 1 3 None Good coating (2) 10 0 Heavy Insufficient 13 None Good Argon plasma+ 0 chitosan-RB 1 7 None Good coating (3) 10 2 None Good (1) Un-pre-treated, (2) pre-treated by enzyme, and (3) pre-treated by enzyme+peroxide.

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surface of the samples [27,28]. The electrons and ions in the discharge zone of the plasma have a great etching effect on the surface of bacteria, resulting in the crack of the cell walls or membranes and the leakage of protein and nucleic acid [28]. Plasma treatment also enhances chitosan binding by promoting new anionic groups on the fiber and these new functional groups cause some changes in the surface composition. To the study, plasma treatment was found to have a sterilizing effect to some extent in the samples pre-treated by enzyme+peroxide and not post-treated with chitosan. On the other hand after the chitosan application, excellent antibacterial effects were obtained. When air and argon plasma were compared, argon plasma treatment was found to be more effective than air plasma treatment in terms of antimicrobial efficiency. So it can be said that argon radicals play an important role in killing and removing bacteria, especially against Klebsiella pneumonia, which is one of the gram negative bacteria. After the washing processes the diameter of clear zones have decreased but under the samples of combined pretreatment processes, antimicrobial effect has still been observed even after 10 washing cycle. In Table 2, the antimicrobial activities of the unwashed and washed samples are given.

FT-IR Analysis

Figures 4 and 5 show the FTIR spectra of wool samples of untreated and pre-treated by different processes before chitosan-RB treatment. The identification of chemical bonds corresponding to wool fibres are presented in Table 3.

untreated (1), enzyme+peroxide treated (2), enzyme+peroxide+air plasma treated (3), enzyme+peroxide+argon plasma treated (4)

FTIR spectra of wool samples of untreated and pretreated by different processes before chitosan-RB treatment (1800650 cm ). Figure 4.

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Conclusion

untreated (1), enzyme+peroxide treated (2), enzyme+peroxide+air plasma treated (3), enzyme+peroxide+argon plasma treated (4)

FTIR spectra of wool samples of untreated and pretreated by different processes before chitosan-RB treatment (40001800 cm ). Figure 5.

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The identification of chemical bonds of wool fibres [29] Structure Wavenumber (cm ) 3000-2800 -CH ; -CH -OH; -NH3650-3150 Amide I; amide II (-CO-NH-) 1800-1450 S-S 560-480 S-O 1200-1000 C-S (cistine) 665

Table 3.

Plasma, enzyme, peroxide and chitosan modify the wool surface in different ways. While plasma has etching effect, enzyme and peroxide have degradative effect. On the other hand chitosan covers the surface without any degradation and also gives antimicrobial effect to the samples. Plasma especially argon treatment, enzyme, peroxide, and combination of these pre-treatments increase the reactivity of the wool surface and provides more chitosan binding. Results showed that chitosan had strong and long-termed antimicrobial activity on wool fabrics when applied together with a reactive organic-inorganic binder matrix (ISys MTX). Enzyme and peroxide combination clearly promoted chitosan adsorption, leading to improved durability. Plasma treatments promoted new anionic groups on the fiber and these new functional groups increased the reactivity of the wool surface. From the SEM images it was observed that combination of plasma and the other pre-treatment processes gave less damage than only one process. Comparing the plasma treatments, argon was found to be more effective on the antimicrobial activity of the wool samples. So, it can be concluded that enzyme+peroxide+argon plasma combination provides the optimum damage and the sample pre-treated by this way and post-treated by chitosan-RB coating shows the best antimicrobial effect.

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The most important bonding (C-S) in wool fibres is associated with cistine content at 665 cm , but generally it gives a small absorption signal, so the intensity of this line is very weak in the spectra. The absorption bands at 1054, 1228, and 1231 cm are attributed to S-O bonds. The other certain absorption bands of a wool fibre, 1633 (amide I), 1575, 1577, 1582 (amide II) and 3255 cm (-NH bending) are confirmed in the spectra. The absorption bands 2972, 2926 and 2911 cm are attributed to the -CH and -CH bonds of the wool fibre. It’s observed that there is no significant difference before and after the plasma treatments. The slight modifications in some spectral ranges (4000-3500 cm ) are thought to be due to the degradation of organic components from the material structure and the spectra is consistent with the spectra that is given by Luciu [29]. -1

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