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Extraction and application of natural silk proteinsericin from Bombyx mori as antimicrobial finish forcotton fabricsR. Rajendran a , C. Balakumar a , R. Sivakumar a , T. Amruta a & N. Devaki aa PG & Research Department of Microbiology, PSG College of Arts & Science, Coimbatore,IndiaPublished online: 20 Jul 2011.

To cite this article: R. Rajendran , C. Balakumar , R. Sivakumar , T. Amruta & N. Devaki (2012) Extraction and applicationof natural silk protein sericin from Bombyx mori as antimicrobial finish for cotton fabrics, Journal of The Textile Institute,103:4, 458-462, DOI: 10.1080/00405000.2011.586151

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Extraction and application of natural silk protein sericin from Bombyx mori asantimicrobial finish for cotton fabrics

R. Rajendran*, C. Balakumar, R. Sivakumar, T. Amruta and N. Devaki

PG & Research Department of Microbiology, PSG College of Arts & Science, Coimbatore, India

(Received 30 June 2010; final version received 28 April 2011)

In this study, we developed an effective technology for the extraction of sericin from the cocoons of Bombyx morisilk worms. Sericin was extracted with ice cold ethanol to obtain crude extract. Sericin extract was coated ontocotton fabric by a paddrycure method. FTIR characterization of the sericin-coated cotton fabric showed distinctamide peaks. The test organisms that were used in the study to assess the antimicrobial activity of sericin wereEscherichia coli and Staphylococcus aureus according to AATCC standard. The antimicrobial activity of the seri-cin thus extracted was assessed by both qualitative (agar diffusion and parallel streak method) and quantitative(percentage reduction test) methods. An inhibition zone of 28 mm and 30 mm for E. coli and S. aureus by agardiffusion method and a zone of 40 mm and 42 mm for E. coli and S. aureus by parallel streak method wereobtained. Quantitative assessment by percentage reduction test showed a reduction percentage of 89.4% and 81%for S. aureus and E. coli, respectively. Results suggested that sericin might be a valuable ingredient for the devel-opment of antimicrobial textiles.

Keywords: sericin; cotton fabric; antimicrobial textiles; Bombyx mori

Introduction

Textile goods, especially those made from naturalfibers, provide an excellent environment for microor-ganisms to grow, because of their large surface areaand ability to retain moisture. A number of chemicalshave been employed to impart antimicrobial activityto textile goods. Those chemicals include inorganicsalts, organometallics, iodophors (substances thatslowly release iodine), phenols and thiophenols,onium salts, antibiotics, heterocyclics with anionicgroups, nitro compounds, ureas and related com-pounds, formaldehyde derivatives and amines (Vigo,1983). Many of these chemicals, however, are toxic tohumans and do not easily degrade in the environment.The textile industry continues to look for eco-friendlyprocesses that substitute for toxic textile chemicals.

Silk sericin, a natural protein obtained from silk-worm cocoon has a combination of many uniqueproperties such as biodegradability, nontoxicity, oxida-tion resistance, antimicrobial activity, UV resistanceand absorbs moistures (Wu, Wang, & Xu, 2007). Nat-ural silk is composed of two kinds of proteins,namely, the crystalline fibroin and the amorphous seri-cin (Fabiani, Pizzichini, Spadoni, & Zeddita, 1996).

Sericin envelops the fibroin fiber with successivesticky layers that help in the formation of a cocoon,constitutes about 2030% of the total cocoon weight(Masahiro, Hideyuki, & Norihisa, 2000). Silk sericinis a kind of water-soluble globular protein derivedfrom Bombyx mori silkworm (Wei, Li, & Xie, 2005).Sericin is especially rich in aspartic acid (19%) as wellas serine (32%) (Kwang et al., 2003), which has ahigh content of the hydroxyl group.

Sericin has been shown to be useful as a degrad-able biomaterial, biomedical material and polymers forforming articles, functional membranes, fibers and fab-rics (Zhang, 2002). According to Yamada and Nomura(1998), sericin-coated fibers can prevent abrasive skininjuries and the development of rashes. In one study,synthetic and other fibers were coated with sericin byimmersing in a 3% aqueous solution of sericin for agiven time and drying at 100C for 3 min. The fabricswoven from the sericin-coated fibers were tested inproducts such as diapers, diaper liners and wounddressing. Kurioka, Kurioka, and Yamazaki (2004)have studied the morphology and biochemical proper-ties of sericin powder recovered from different degum-ming liquors. The functional properties of some

*Corresponding author. Email: rrajendranmicro@yahoo.co.in

The Journal of The Textile InstituteVol. 103, No. 4, April 2012, 458462

ISSN 0040-5000 print/ISSN 1754-2340 onlineCopyright 2012 The Textile Institutehttp://dx.doi.org/10.1080/00405000.2011.586151http://www.tandfonline.com

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synthetic fibers can be improved by coating with seri-cin protein (Yamada & Matsunaga, 1994). Sericin-modified polyester has been reported. Although sericinhas been known to possess a numerous beneficialproperties, its application in textile industry for anti-bacterial property enhancement has not been reportedas yet (Joshi, Wazed Ali, Purwar, & Rajendran, 2009).

The present investigation primarily aims at devel-oping an eco-friendly natural antimicrobial finishfrom sericin for textile application. Sericin wasextracted from B. mori cocoon and screened for theiractivity and the protein was applied to cotton fabrics.The fabric coated with sericin was characterized byFourier Transform Infrared Spectroscopy (FTIR). Anextensive study was conducted to assess the antimi-crobial effectiveness of protein by employing stan-dard test methods and the findings are discussed inthis paper.

Materials and methods

Materials

Silkworm cocoons of Bombyx mori ware originallyobtained from District Sericulture Center (Coimbatore,Tamil Nadu, India). The reagent-grade chemicals likesodium carbonate and sodium bicarbonate were pro-cured from Hi-Media, Mumbai, India. Fabric for treat-ment was purchased from National TextileCorporation Limited (Coimbatore, Tamil Nadu, India).Specifications of fabric are (type: 100% woven cottonfabric; bleached; warp: 20s k; weft: 20s k; ends perinch: 54; picks: 40; width: 122 cm).

Bacterial strains

Escherichia coli MTCC 25922 as a representative ofGram-negative bacteria and Staphylococcus aureusMTCC 25923 as a representative of Gram-positivebacteria were used for this study as both are referencestrains used for antimicrobial susceptibility testingaccording to AATCC standard method. The strainswere cultured on nutrient agar (Hi-Media, Mumbai,India) and incubated aerobically at 37 C overnight.

Extraction of sericin and coating onto cotton fabrics

Sericin was extracted from cocoons according tomethod proposed by Gulrajani (1993) with slightmodifications. The chopped and cleaned silk cocoonsare treated in the bath containing sodium carbonate(1.06%), sodium bicarbonate (0.84%) at 100C for 60min, keeping material to liquor (M:L) ratio 1:40. Theinsoluble fibroin was removed by filtration. The result-ing liquor was precipitated with three volumes of ice-cold ethanol and incubated for an hour at 4 C. After

incubation, the solution was centrifuged for 15 minand the precipitate was dissolved in distilled water.

Sericin was applied on the 100% cotton fabric to awet pick up of 100% using the paddrycuremethod. The cotton fabric (size: 30 cm 30 cm) wasimmersed in the solution containing sericin for 5 minand then it was passed through a laboratory paddingmangle (RGE make), which was running at a speed of20 rpm with a pressure of 1.5 kgf cm2 to removeexcess solution. After padding, the fabric was air-driedand then cured for 5 min at 70 C. The treated sam-ples were then evaluated for antimicrobial activity.

FTIR characterization

The functional group and absorbency of the sericin-coated cotton fabric was determined using FourierTransform Infrared Spectroscopy (FTIR). The FTIRspectrum of the sericin-coated cotton fabric samplewas recorded (10002500 cm1) on a ThermonicoletFTIR system with the KBr pellet technique. KBr pel-lets were prepared by grinding 1 part of the samplewith 9 parts of spectral grade KBr and pressing in anevacuated die under suitable pressure to get pellets.

Qualitative and quantitative antimicrobial activityassessment

Antibacterial activity was evaluated by both qualita-tive and quantitative test methods. The following arethe descriptions of test methods employed for thisstudy. Qualitative assessment was made by agar diffu-sion method (AATCC 147-2001) and parallel streakmethod (AATCC 147-2001). Treated and untreatedcontrol fabric samples placed in intimate contact withAATCC bacteriostasis agar, which has been previouslyinoculated (Mat culture) with an inoculum of testorganisms. After incubation, a clear area of uninter-rupted growth underneath and along the side of thetest material indicates the antibacterial effectiveness ofthe fabric.

Quantitative assessment was done by percentagereduction test (AATCC 100). Specimens of the testmaterial were shaken in a known concentration ofbacterial suspension and the reduction in bacterialactivity in standard time was measured. The efficiencyof the antimicrobial treatment is determined by com-paring the reduction in bacterial concentration of thetreated sample with that of control sample expressedas a percentage reduction in standard time.

% Reduction A BB

100 (1)

where A and B are the surviving cells (CFU/ml) forthe flasks containing the control (untreated cotton

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fabric) and test samples (sericin-treated cotton fabric),respectively, after 18 hr of contact time.

Results and discussion

Extraction of sericin and application onto cottonfabrics

In this study, cocoon shells of B. mori silkworm wasswelled with water and exploded under high tempera-ture, thus external sericin was easily removed fromfibroin. Sericin was extracted using ice-cold ethanol. Itwas observed that after the addition of ethanol to thedegummed liquor, colloidal sericin appeared in the solu-tion. This cloudy solution was subjected to centrifuga-tion and sticky, creamy state sericin was separated. Thiswas dissolved in distilled water and stored at 4 C.

The degumming process is used to eliminate theexternal sericin prior to dyeing in the silk industry. Inthis study, ice-cold ethanol was used for the extractionof sericin. Adding ethanol that is less polar than watercould reduce the solvent polarity and sericin tended tobecome less soluble, thus we could separate sericinfrom silk waste water by centrifugation. In addition,to minimize the denaturation of the protein in ethanol,the precipitation was conducted at a chilled tempera-ture below 4 C (Wu et al., 2007). In this way, wewere able to extract sericin from degumming liquorthrough an easy approach.

FTIR characterization

The IR spectrum of the fabric coated with sericin and theuntreated cotton fabric are presented in Figure 1. The ser-icin-treated cotton fabric has characteristic peaks atwavelengths of 1646 cm1 (amide-I), 1480 cm1

(amide-II), and 1234 cm1 (amide-III). These

characteristic peaks proved the coating of the sericinprotein onto the cotton fabrics as the untreated cottonfabrics does not show such characteristic peaks. Amide Iprimarily represent the C=O stretching vibration of thenine amide groups. Amide II contains contribution fromNH bending and CN stretching vibration, amide IIImainly arises from CN stretching and CO bendingvibration. Further, there is much enhanced transmittanceat 1159 cm1. The prominence of this feature may beascribed to the relatively high content of carboxylic acidand alkyl hydroxyl containing amino acid side chains insericin. The presence of peak at 1372 in both the treatedand the control fabrics was attributed to the CH bending(deformation stretch) in the cotton fabrics.

Similar results were obtained by Prasong, Yaowalak,and Wilaiwan (2009) who showed that the B. mori silkfiber with sericin showed the characteristic peaks at1649 cm1 (amide-I), 1523 cm1 (amide-II) and 1233cm1 (amide-III) whereas the silk fiber without sericindoes not have these characteristic amide peaks. Further,it was confirmed that peaks at 1460 cm1, correspond

Figure 1. FTIR graph of (a) untreated cotton fabric (control) and (b) sericin-treated cotton fabric.

Figure 2. Photograph showing zone of inhibition-agardiffusion method (a) E. coli and (b) S. aureus.

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to OH in plane bending in sericin by Arunee andNuchsirapak (2007). It was also confirmed by Zhangand Wyeth (2010) that the amide-I of sericin will haveband at 1650 cm1.

Qualitative and quantitative antimicrobial activityassessment

The results of agar diffusion method against the stan-dard test organisms S. aureus (Gram positive) andE. coli (Gram negative) are given in Figure 2. Therewas a clear zone of inhibition around the fabric treatedwith sericin against both the test organisms in contrastwith control fabric which allowed the growth oforganism. The agar diffusion showed a zone of inhibi-tion which indicated that the antimicrobial activity ofsericin-treated sample exhibited a zone of 30 mm forS. aureus and 28 mm inhibition for E. coli.

While evaluating the antimicrobial activity of seri-cin-treated cotton fabric as tested by parallel streakmethod, a clear zone of inhibition was observed forboth the test organisms. The result of parallel streakmethod is interpreted in Figure 3. In the case of theantimicrobial activity of sericin-treated cotton fabricby parallel streak method, the zone of inhibition wasobserved to be 42 mm for S. aureus and 40 mm forE. coli where as the untreated cotton fabric (control)has no antibacterial activity.

The results of the percentage reduction test areshown in Table 1. The reduction percentage for E. coliand S. aureus correspond to the bacterial numbers onthe respective control test of 9.5 106 per milliliter.The reduction percentage was found to 89.4% for S.aureus and 81% for E. coli. Brahma (2006) found thatsericin-treated PET fabric showed 51% reduction ofProteus vulgaris and 38% reduction of S. aureus.

Conclusion

In this work, we developed a simple and effectivemethod for extracting sericin from the cocoons ofB. mori silkworm using chilled ethanol precipitationmethod. We focused on studying the antimicrobialproperty of cotton fabric coated with sericin obtainedby this method. The sericin-coated fabric showed ahigh degree of bactericidal activity against test organ-isms E. coli and S. aureus used in this study. In theFTIR spectra, it showed distinct amide peaks. Infuture, we are interested in increasing the durability ofthe sericin-coated fabric using cross-linking agents, sothat the antimicrobial property of sericin is retained inthe fabric even after few washes and thus enhancingdurability. We are also interested in studying the fun-gicidal activity of sericin. By this way, we desire todevelop an eco-friendly antimicrobial textile with anovel natural antimicrobial agent.

AcknowledgementThe authors express their sincere thanks to Dr R.Vijayaraghavan, Director and Dr Beer Singh, (Sc F),Head, PD Division, DRDE Gwalior for funding the project.

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Fabric sample Test organism

Survival cells (CFU/ml)

% ReductionControl fabric Treated fabric

Cotton fabrictreated with sericin

S. aureus 9.5 106 1 106 89.4E. coli 9.5 106 1.8 106 81

Figure 3. Photograph showing zone of inhibition-parallelstreak method (a) E. coli and (b) S. aureus.

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