Chitosan is a linear heteropolysaccharide composed of2-amino-2-deoxy-glucose and 2-acetamido-2-deoxy-glucose units1obtained by chemically deacetylating chitin extracted from shrimp and crabshells. Low-molecular-weight chitosan oligomers have received attention becauseof their interesting biological properties, including the inhibitory effects onthe growth of fungi and bacteria2-6. Several enzymaticand chemical methods for producing chitosan oligomers have been described inliterature. The chemical methods include acid hydrolysis with either coldnitrous acid or hot hydrochloric acW, phosphoric acid8 or hydrogenfluoride9. Nordtveit et al10 used hydrogen peroxide,which, in the presence of Fe(III), generates hydroxyl radicals which cleave themolecule by nucleophilic attack.


The chemical treatment is very common and fast, but it hassome faults such as high cost, low yield and generation of acidic waste11.The enzymatic digestion is more popular because of its mild reaction conditionsand good reproducibility12. Moreover, in enzymatic process, thehydrolysis course and product distribution are subject to more facile control,in spite of the faster rate of chemical reaction6. However, the highcost of specific enzymes, such as chitosanase and chitinase, inhibits their useon an industrial scale. Recently, several hydrolytic enzymes, such as lysozyme13pectinases 14 , hemicellulase and papain 15 were found tocataylse the cleavage of the glycosidic linkage in chitosan.



Treatment with chitosan and its derivatives does not affect the dye uptake or its fastness in any significant way.


In this work, an attempt has been made to use celluloseenzymes for chitosan hydrolysis. Cellulases are already used commercially forbiopolishing in textile processing 12. Experiments have beenconducted to study the effect of enzyme concentration on the mol wt and degreeof polymerisation (DP) of chitosan. The low molecular weight (LMW) derivativeshave been characterised to study if they retain the properties characteristicof chitosan. They were then applied to cotton to study the effect on dyeuptake, dye distribution and dye retention.


Materials and Methods


Materials


Cotton fabric of 110 gsm, having 98 epi and 84 ppi was used.

Chitosan, as an initial material, was obtained from AldrichChemicals. Cellulase enzyme, Ezysoft L Soft was provided by Resil chemicals,Bangalore. The stock solution of chitosan (0.05%) was prepared in 2% aceticacid with pH being adjusted to 5-5.5 with NaOH.


Preparation of sample by enzymatic hydrolysis


To 100 ml solution of chitosan (5%) in 2% acetic acid(maintained at pH 5-5.5), 5 ml enzyme solution was added. This solution waskept in water bath at 50C and 130 rpm for 3 hr after which the contents of theflask were boiled for 10-15 min to deactivate the remaining enzyme present inthe flask. The same procedure was done with 7 ml, 9 ml, 11 ml, 13 ml and 15 mlof enzyme solution.


Solubility of samples at various pH


Chitosan is insoluble in water and soluble in aqueous diluteand organic acids. The solubility of chitosan and degraded chitosan was testedin water as well as in solutions of different pH. 2% solution of acetic acidand sodium hydroxide was prepared separately for adjusting the solution pH to3, 4, 5, 7 and 9 at room temperature.


Characterisation studies


The degree of deacetylation was determined by FTIR analysis16.FTIR spectra were recorded with KBr pellet on a Perkin Elmer Spectrum 1Bspectrophotometer. The viscosity change was investigated using an UbbelohdeViscometer. The viscosity molecular weight was calculated based on Mark Houwinkequation17 ([η] = KM v a). Here, DD isthe degree of the deacetylation of chitosan expressed as percentage.

 

Assay of antibacterial activity

Depolymerisation of chitosan was expected to affect its antimicrobial activity. Kinetics of inhibition over a 24h incubation period of chitosan and its LMW derivatives was studied against a common disease causing microbe - E coli using the optical density (O.D) method as described below:

Fresh inoculum (100 μl) which is in growth phase was inoculated to the liquid media at 37C. 100 μl of chitosan sample was added and the turbidity (optical density) of the solution was recorded after different time interval namely 0, 3, 6, 9, 12, 24 h at 610 nm using Lambda 25 UV / VIS spectrophotometer (Perkin Elmer, Inc). All the glassware, samples and culture media were sterilised in an autoclave (Yorko Scientific Industries Pvt Ltd, India).


Treatment of cotton with chitosan solutions


Pad-dry-solutions


For treatment on cotton, chitosan and degraded chitosan were dissolved in acetic acid at pH 3-5. Scoured cotton fabrics were padded using 0.5% (w/v) solution of each of chitosan (CH) and degraded samples of chitosan (DCH II, IV & VI). Samples were dried and cured in a curing chamber18.


Dyeing of treated and untreated cotton samples

Pretreatment with chitosan followed by dyeing

Cotton fabric treated with CH and DCH(VI) was dyed with two reactive dyes- Remazol Red RGB (2%) and Drimarene Orange HER (4%). 200 g/l urea, 20 g/l sodium bicarbonate and 2 g/l resist salt (meta nitro benzene sulfonic acid sodium salt) was also added to the dye bath. Cotton samples were padded 2 dips (for two minutes) and 2 nips with the dye solution, dried at 70C for 5 min and cured at 150C for 2 min. After dyeing, the samples were washed thoroughly in cold and then warm water followed by soaping in boiling water containing 1-2 g/l lissapol D for 15-20 min and thorough rinsing.


Post treatment of dyed cotton with chitosan


Same procedure was repeated with Drimarene orange HER and Remazol red dyes using exhaustion method and then treated with CH and DCH(VI) with and without crosslinker using pad-dry-cure method.


Colour strength measurement


The whiteness index and colour strength measurements were made using Gretag Macbeth Colour-Eye 7000 A using 10 observer and D 65 light source.

Antimicrobial activity of cotton samples


The antimicrobial activity of cotton samples against E coli was tested by the Agar Plate method (AATCC Test Method 100-1997). This is used to estimate the percentage reduction in number of colonies (CFU) on treated samples with reference to the untreated ones. 10 μl of bacteria from the mother culture was inoculated into a freshly prepared liquid media and fabric samples of size 1"x 1" were added to it. These were then incubated for 24 hrs at 37C. In the second step, 100 μl of the incubated solution (with the dilution to 10-6) was spread over solid culture medium in a petri dish. These agar plates were then incubated again for 18 hrs at 37C. The reduction in colony forming units (CFU), R after incubation was calculated by equation below:

R (%) = 100 (A-B) / A (i)


where A and B are the number of bacteria recovered from the blank and treated fabric samples, respectively after incubation19.


 

Results and discussion


6 LMW chitosan derivatives namely DCH (I), DCH (II), DCH (III), DCH (IV), DCH (V) and DCH (VI) were obtained by enzymatic hydrolysis of chitosan using cellulase. These derivatives were characterised in terms of the intrinsic viscosity and degree of deacetylation (DD) using FTIR spectra. The viscosity average molecular weight was determined according to Mark Houwink equation and the degree of polymerization was calculated from it.

The effect of chitosan on the colour of cotton when applied before and after dyeing has also been studied. The antimicrobial efficiency of the treated samples has also been recorded. Results are reported and discussed below.


Characterisation studies


FTIR analysis

Fig 1 shows the FTIR spectrum of CH and the 6 LMW derivatives of chitosan. The main peak assignment summarised in Table 1. It can be seen that the spectra of degraded chitosan samples- DCH (I) to DCH (VI) is not very different from that of, original chitosan (CH). DDA has been determined by FTIR using the formula [31. 918(A1320/A1420) - 12.2] where absorbance at 1320 cm-1 is due to the C-N stretching in secondary amide at 1420 cm-1 is due to C-H deformations due to removal of acyl group on deacetylation17. The results showed that chitosan samples prepared by enzymatic hydrolysis have DDA and molecular structure similar to that of original chitosan.

Other characterisation data is reported in Table 2. It can be seen that there is a remarkable reduction in intrinsic viscosity, molecular weight and degree of polymerisation of chitosan on increasing the concentration of cellulose enzyme. The mol wt goes down from 87.7 x105 Da to 0.07 x105 Da. Reduction in the DP is even more pronounced, as it goes down from ~ 50,000 to 41. Thus it can be seen that it is possible to hydrolyse chitosan using cellulases to produce low molecular weight derivatives (LMW) DCH (I), DCH (II), DCH (III), DCH (IV), DCH (V) and DCH (VI). This reduction in the mol wt and DP should improve the solubility of chitosan in an aqueous pH and facilitate more uniform application on textile substrates. High molecular weight chitosan is difficult to handle and apply uniformly because of the very high viscosity.


Solubility

Results of the solubility study are summarised in Table 3. CH is soluble only in pH below 5 as the solubility of chitosan is due to its cationic nature. In acidic medium, -NH2 group of chitosan converts into -NH3+ which helps in the solubility of chitosan but, in neutral and basic medium, it is insoluble. All LMW chitosan derivatives are water soluble up to pH 7 while DCH (IV), DCH (V) and DCH (VI) are found to be partially soluble upto pH 9. This difference is probably due to the availability of more - NH3+ ions in degraded chitosan as is evident from the increased degree of deacetylation of these samples.

Antibacterial Activity of CH and LMW chitosan


It has been reported frequently that the antimicrobial property of chitosan is directly related to the molecular weight as well as the DDA. In this case, the LMW derivatives show enormous decrease in molecular weight but a slight increase in the DDA. Therefore studies were undertaken to study their antimicrobial efficiency as compared to the original CH to study the effect of hydrolysis on this very critical property.


 

  • Kinetics of E. coli inhibition:


Rate of E coli inhibition by chitosan and its derivatives over 24 h was studied using optical measurements at 610 nm (Table 4). Higher the number of microbes, higher is the turbidity. Fig 2 shows that CH is a highly effective bactericide as nearly all microbes are destroyed within the first few hours of contact itself. Comparative values for LMW derivatives are plotted in Fig 3. It can be seen that the lower is the mol wt better is the activity. The derivative with the least mol wt continues to be active till the microbial colonies are destroyed completely. Interaction of the positively charged chitosan with the negatively charged residues at the cell wall of bacteria causes extensive cell surface alterations and alters cell permeability eventually leading to the death of microbial cells 20. On degradation of chitosan, number of -NH3+ groups (which are responsible for the antimicrobial activity) are more in comparison with non-degraded chitosan. Also the TEM pictures (Fig 4) showed that the reduction of molecular weight and hence the viscosity may also facilitate better adherence to the cell wall and perhaps even diffusion of LMW chitosan inside the microbial cell.



These results show that the antimicrobial activity of chitosan is not really dependent on the molecular weight as even when the mol wt has reduced drastically, the AM activity remains unaffected (Fig 5).

The effect of mol wt as well as the concentration of chitosan on the antimicrobial activity against E. coli was also studied. Table 5 shows that neither the molecular weight nor the concentration of chitosan and derivatives seem to have any significant effect on the antimicrobial activity, which otherwise is quite high in all the cases. Based on the results, it can be concluded that a concentration of 0.01% of CH or its LMW derivatives is quite effective in all the cases.

 


Studies on treated cotton


Having studied the properties and characteristics of chitosan and its derivatives, they were applied onto cotton fabric with a view to impart multifunctional properties to it. Cotton fabric was first treated with chitosan and its derivatives and then dyed with anionic dyes to study the effect on dye uptake. Studies were also conducted where the fabric was first dyed and then treated with chitosan. The effect of these treatments on the dye uptake, colour depth and wash fastness has been evaluated. Results are discussed below.


Pretreatment of cotton with chitosan and LMW derivatives


Chitosan and its LMW derivatives were applied to cotton fabric by pad-dry-cure method. Treated samples were then dyed with anionic reactive dyes. It was expected that the dye uptake would go up after chitosan treatment due to the availability of positively charged moieties of chitosan on cotton. For the reactive dyes, the trends observed were similar. It can be seen from the results that the dye uptake does increase on application of chitosan but the enhancement is less with the LMW derivatives, as compared to CH (Table 6). In fact lower the molecular weight, lower is the colour depth. Wash fastness of all samples also found to be low. As there is no affinity between cotton and chitosan and its derivatives, the fastness of dyeings is seen to be very poor. The possible reason for the lower shade depth and poor fastness could be increased aqueous solubility of the molecular derivatives. Due to this get washed off during dyeing itself the additional cationic sites are available for dye binding. Either a cross linking agent like citric acid or glutraldehyde or insolubilisation agents such as metal salts would have to be used to improve the washfastness.

  • Penetration of dye in yarn structure:

The cross section of yarns from treated and dyed fabric was observed under the Leica microscope, to study the penetration and distribution of chitosan and dye. It was seen that in case of untreated samples and samples treated with CH, the penetration/ distribution of dye in the yarn was not very uniform. But in case of cotton sample treated with LMW derivative DCH (VI), the distribution of dye in the yarn was very uniform. This shows that LMW derivatives of chitosan can act as a vehicle for better penetration of dye into the yarn structure. The high mol wt chitosan, because of the higher inherent viscosity does not get very uniformly distributed into the yarn. However, it was seen that, in all cases, the fibres are uniformly dyed to the core and that the dye is not restricted to the surface as may be the case if the dye was bound only to the chitosan and did not penetrate the fibre cross section.


Post treatment of dyed cotton with chitosan


Cotton fabric was first dyed and then treated with chitosan to see if the treatment would have any effect on the colour. Results for two reactive dyes are compiled in Table 7. It can be seen that treatment with chitosan or its LMW derivative does not affect the colour or the wash fastness in any way.

It can be hence be said that chitosan treatment, whether given before or after dyeing, does not affect the colour or fastness of reactive dyes in any way.

Antimicrobial activity of cotton samples


The antimicrobial activity of treated and untreated cotton samples was measured before and after dyeing by estimating the % reduction in the form of colony forming units (CFU) after 24 h incubation at 37C against E coli. Results are compiled in Table 8. The results suggest that degraded chitosan products show more antimicrobial activity in comparison to chitosan and it increases with decrease in molecular weight of chitosan. Where the CH destroys E coli up to only 24%, the most degraded chitosan DCH(VI) does it up to 56%. In case of reactive dyes, the % reduction is the maximum for CH treated dyed sample and goes down as molecular weight decreases. The reason for this decrease is the lesser no of available NH3+ groups in degraded chitosan padded cotton sample.


 

Conclusion


Several LMW derivatives could be prepared by hydrolysis of chitosan with cellulase enzyme. It was found that even though the molecular weight and DP were reduced, the essential characteristics of chitosan did not change on hydrolysis. The degree of deacetylation was found to be higher in LMW derivatives and so they showed better antimicrobial activity. The lower viscosity and better solubility of LMW derivatives facilitates easy application and better distribution of dye in the yarn structure. Treatment with chitosan and its derivatives does not affect the dye uptake or its fastness in any significant way.


Acknowledgement

The authors are thankful to the Department of Biotechnology, Government of India, for providing the financial support for carrying out this work.


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Tables


 







 







About the Authors:


The authors are associated with the Department of Textile Technology, lIT, New Delhi and with the Department of Chemistry, lIT, New Delhi, respectively.


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