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International Journal of Biological Macromolecules 48 (2011) 736–741

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journa l homepage: www.e lsev ier .com/ locate / i jb iomac

ntibacterial action of acetic acid soluble material isolated from Mucor rouxii andts application onto textile

haaban Moussaa,∗, Atef Ibrahima, Adel Okbaa, Hanafy Hamzaa, Klaus Opwisb, Eckhard Schollmeyerb

Genetic Engineering and Biotechnology Research Institute, Minuofiya University, El-Sadat City, EgyptDTNW – Deutsches Textilforschungszentrum Nord-West e.V. Institut an der, Universität Duisburg – Essen, Krefeld, Germany

r t i c l e i n f o

rticle history:eceived 6 February 2011eceived in revised form 16 February 2011ccepted 24 February 2011vailable online 5 March 2011

a b s t r a c t

Acetic acid soluble material (AcSM) is a chitosan-rich fraction isolated from the fungal cell wall materials.The final step in the traditional production of fungal chitosan is the separation of chitosan from the cellwall AcSM via raising the pH to 9–10 followed by centrifugation. This step results in further undesirableeconomic and environmental effects. The goal of this paper is to avoid that by investigating the antimi-

eywords:ucor rouxii

ungal chitosanotton fabricsntibacterial activity

crobial effect of the whole AcSM from Mucor rouxii DSM-1191 cell wall and its application on cottonfabrics. The treated fabrics were characterized through monitoring the textile physical properties and forthe antibacterial activity against Escherichia coli and Micrococcus luteus. Results showed that Mucor rouxiiDSM-1191 has excellent potentials to be used for cell wall AcSM production on industrial scale with amaximum content of 40% in dry mycelia. The obtained results indicated that the physical properties ofthe treated fabrics, as well as the antibacterial activity, were improved after treatment with fungal AcSM.

. Introduction

The introduction of antimicrobial activity into medical textilesas been extensively studied to control the growing problem ofospital-related infections. The desirable biocidal materials shoulde very effective for inactivating microorganisms, not toxic toumans, and environmental friendly. Techniques such as coating,rafting, and blenching have been employed to impart antimicro-ial functions onto polymers and fabrics for protection against

nfectious disease pathogens [1]. The development of bacteria onabrics during use or storage negatively affects the fabric or inase of clothes the wearer. This dangerous effect can be limited byurable antimicrobial finishing of the fabric using broad-spectrumiocides. Biocide can be divided into two types, organic and inor-anic. Organic biocides include e.g. organic acids, tertiary alkylmines; they are often less stable at high temperatures com-ared to the inorganic biocides [2]. Chitosan, cationic copolymerf poly �-(1 → 4) linked N-acetyl-d-glucosamine is nowadays pro-

uced most often by deacetylation of chitin from exoskeleton ofhellfish wastes. One important characteristic of Mucoralean fungiZygomycetes) is that they contain chitosan as a major compo-ent in their cell walls [2–5]. There are several advantages of using

∗ Corresponding author. Tel.: +20 2 37135273; fax: +20 48 26012 66 8.E-mail address: shaus477@yahoo.de (S. Moussa).

141-8130/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.ijbiomac.2011.02.022

© 2011 Elsevier B.V. All rights reserved.

these fungi to produce chitosan. The most important is that thecell wall of zygomycetous fungi contains a large quantity of chi-tosan and the physicochemical properties of this chitosan can bemanipulated and standardized by controlling the parameters offermentation [6].

Chitosan is traditionally produced from the cell wall of fungi bya two step extraction process involving alkali and acid treatments.Proteins, lipids, and alkali-soluble carbohydrates are first dissolvedin, for example, 1 M NaOH at 121 ◦C within 15 min, and the cellwall material containing chitosan is obtained as alkali-insolublematerial (AIM). The AIM is then treated with an aqueous solutionof an acid such as 5% acetic acid at 95 ◦C for 4 h to dissolve theacetic acid soluble material (AcSM) which is generally consideredto be the “fungal chitosan” and can be precipitated by raising thepH to 9–10 followed by centrifugation [4,7–10].

The main constituents of the fungal cell wall are the polysac-charides which in general make up 80% of the dry weight.Proteins come in second with 3–20% while lipids, pigmentsand inorganic salts are present in smaller portions. The mostcommon polysaccharides are chitin, cellulose, chitosan, glu-cans and polyuronides, while the most common proteins are

glycoproteins [11].

Therefore an important task for the present research was todetermine the antibacterial activity of the AcSM from fungal cellwall on cotton fabric against gram-negative E. coli and gram positivebacteria Micrococcus luteus.

S. Moussa et al. / International Journal of Biolog

Table 1

Substrate Weight (g/m2) Threads/cm Thickness (mm)

2

2

fi

2

Efp

(4amfwC

2

mywho1wu

wmdweuai

2

swcstucso

Reduction rate (%) = × 100

Warp Weft

Cotton (100%) 250 21 18 0.57

. Materials and methods

.1. Textile material

The coating experiments were carried out using 100% cottonabric, the specific data of the material employed was summarizedn Table 1.

.2. Microorganisms and chemicals

Mucor rouxii DSM-1191 was used for the production of AcSM.. coli DSMZ-498 and Micrococcus luteus ATCC-9341 were examinedor their susceptibility and sensitivity toward the treatment withroduced AcSM.

Monochloroacetic acid and 2,3,5-triphenyltetrazolium chlorideTTC) was supplied by Merck (Darmstadt, Germany). E. coli DSMZ98 and Micrococcus luteus ATCC 9341, details of their cultivationnd inoculation are described in [12]. Standard I-nutrient brothedium (SI) and Standard I-nutrient agar (SI-agar) were purchased

rom Merck, Germany. All other chemicals used in this researchere purchased from Merck (Darmstadt, Germany) or Aldrichhemicals (Milwaukee, WI), and used without further purification.

.3. Preparation of Mucor rouxii AcSM

Mucor rouxii DSM-1191 cultures were grown in 500 ml Erlen-eyer flasks containing 200 ml of autoclaved YPD medium (0.3%

east extract, 1.0% peptone, 2.0% d-glucose and 96.7% distilledater, pH adjusted to 4.5) [13], inoculated with spore suspensionarvested from the culture on YPD plates with final concentrationf 105 spores/ml and incubated in shaking incubator set at 28 ◦C and70 rpm, for 72 h. After the desired incubation period, the myceliaere separated by vacuum filtration, washed with distilled waterntil clear filter could be obtained, and lyophilized.

3–5 g lyophilized mycelia was homogenized and deproteinizedith NaOH 1 M solution 90 ◦C, 1 h, separated of alkali-insolubleaterial (AIM) by centrifugation (4000 × g, 15 min), washed with

istilled water and centrifuged to a neutral pH (pH 7). The residuesere further extracted using (5% (v/v) acetic acid, 60 ◦C, 6 h), the

xtract slurry was centrifuged at (4000 × g, 15 min), and the insol-ble acid discarded. The pH of the supernatant fluids AcSM wasdjusted to different pH (pH 6.5) with 3 M NaOH solution as shownn Fig. 1.

.4. Preparation and treatment of cotton with AcSM

The above mentioned cotton fabrics were immersed in the AcSMolution with constant stirring for 2 h at 60 ◦C. The cotton fabricsere carried out using pad-dry-cure method. The experimental

onditions were adopted as follows: cotton fabrics were padded inolution containing AcSM with pH value (pH 6.5), and then the cot-on fabrics were squeezed between two nips and dips to a wet pick

p 100%. The cotton fabrics were dried at 80 ◦C for 3 min, and thenured at 120 ◦C for 10 min. At the end cotton fabrics were washedeveral times with water at 40 ◦C, and finally dried at ambient lab-ratory conditions [14].

ical Macromolecules 48 (2011) 736–741 737

2.5. Characterization of treated cotton fabrics

The treated and untreated cotton samples were characterizedfor:

2.5.1. Weight changesFabric weight was measured before and after treatment with

AcSM to determine the percent add-on. The percentage add-on wascalculated using the following formula:

weight add on (%) = W1 − W0

W0× 100

where W0 is the weight of sample before treatment, W1 is theweight of sample after treatment.

2.5.2. Scanning electron microscopy (SEM)Cotton fabric samples were evaluated using a Hitachi S-3200N

Variable Pressure SEM. Photos was collected at ranges from 100× to2000×. Also toward the explanation of Mucor rouxii DSM1191 AcSMantimicrobial action, micrographs of treated E. coli with fungalAcSM was captured using scanning electron microscope (Topcon-Microscope (ATB-55), Hitachi, Japan) [15], after 5 and 24 h fromthe treatment E. coli with fungal AcSM, as well as control cul-ture. Samples were prepared for SEM as described by Marrie andCosterton [16]. Briefly, samples were fixed in glutaraldehyde in0.1 M cacodylate buffer containing 0.15% ruthenium red for 3 hat 4 ◦C. The sections were then rinsed in fresh 0.1 M cacodylatebuffer for 10 min (repeated three times) and post-fixed in 1.5%osmiumtetroxide for 1 h. Samples were dehydrated in a series ofaqueous ethanol solutions (30–100%) and dried by a critical pointdryer (Tousimis Autosamdri-814, Rockville, MD) with CO2. Thespecimens were mounted on aluminium stubs with silver paste,allowed to dry for 3 h and then coated with gold/palladium using acool-sputter coater E5100 II (Polaron Instruments Inc., Hatfield, PA,USA). Sections were then examined under SEM at 20 kV. Capturedareas were selected according to the alteration in the morphologyof treated cells.

2.5.3. Antimicrobial testThe antimicrobial effect in this research was measured using

three methods: Zone of inhibition test, AATCC test method andTetrazolium/formazan test method (TTC).

2.5.3.1. Zone of inhibition test. In our study, the antimicrobial activ-ity of the treated cotton samples against Gram negative bacteriumE. coli and Gram positive bacterium Micrococcus luteus were evalu-ated by the Zone of inhibition test.

2.5.3.2. The AATCC test method. The AATCC test method wasapproved by the AATCC Committee RA31. The purpose of the AATCCtest method is to provide a quantitative procedure for the eval-uation of the degree of antibacterial activity of textile materials[17].

This standard method was used to measure the reduction ratein the number of colonies formed and provided quantitative data,which could then be converted, to the average colony forming unitsper millilitre (CFU/ml) of buffer solution in the flask. The reductionrate in the number of colonies was calculated using the followingformula:

B − A

A

where A = CFU/ml for the flask containing the treated substrate after3 h and 6 h contact time; B = CFU/ml for the flask at time zero, priorto the addition of the treated substrate.

738 S. Moussa et al. / International Journal of Biological Macromolecules 48 (2011) 736–741

Alkali insoluble material (AIM)

Freeze dried fungal mycelia

Supernatant (Proteins, lipids, alkali solluble carbohydrates) discarded

Supernatant (Chitosan, glucosamine, oligosaccharides)

Acetic acid soluble material(AcSM)

1:40 (w/v) 1M NaOH 90°Creflux 1 h

1:100 (w/v) 5% acetic acid60°C reflux 6h

Fig. 1. Extraction of AcSM material

Fr

2oiv

oacwpvv

3.3. Assays for antimicrobial activity

ig. 2. Dry weight of mycelia and acetic-acid-soluble material (AcSM) of Mucorouxii DSM1191 with incubation time.

.5.3.3. Tetrazolium/formazan test method (TTC). The redox couplef tetrazolium/formazan, acting as proton acceptor or as oxidans,s a well established indicating system for the determination of theiability of microorganisms [18].

Testing of treated textile samples were done by incubationf the textile samples to be evaluated for antimicrobial activityt 37 ◦C for 3–4 h with 10 �l of an E. coli (105 CFU/ml) cultureontaining 100 �l TTC (0.5% solution). The red formazan formedas twice separated by centrifugation, dissolved in ethanol and

hotometrical determined at 480 nm. The reduction of microbeiability can be seen from the decrease of the absorbancealue [19].

Fig. 3. SEM micrographs of: (a) blank cotton fa

from Mucor rouxii DSM-1191.

3. Results and discussion

The cell biomass dry weight and AcSM of Mucor rouxii DSM 1191increased over a period of time (Fig. 2). The fungal cell biomassincreased rapidly during the first 72 h of incubation and continuesto increase until 80 h, followed by a slow decline and the fun-gus appeared to enter the decline phase. The AcSM also increasedgradually with incubation time to 74 h, although its maximum wasreached only at about 80 h.

3.1. Fabric weight changes

Fabric weights were measured before and after treatment withAsCM solution to determine the percent add-on. Based on the pHof AcSM solution, the add-on of the treated cotton fabric was 2.53%at pH 6.5.

3.2. Surface topography of cotton fabrics

The surface of AcSM coated cotton fabrics were morphologicallyobserved by scanning electron microscope (SEM) to investigatechanges in the topography of the cotton fabrics. Fig. 3 shows theSEM micrograph of a sample of treated cotton, where the surfacewith no agglomerated particles are visible on the surface whichindicates a homogeneous distribution of the AsCM in the coatinglayer and the absence of unwanted agglomeration during formationof the resulting coatings.

Prior to the application of the AcSM to cotton fabric, itsantimicrobial activity was examined against E. coli DSMZ 498 and

bric, (b) cotton fabric treated with AcSM.

S. Moussa et al. / International Journal of Biological Macromolecules 48 (2011) 736–741 739

Fig. 4. (a) Antibacterial effect of AcSM solution against E. coli. (b) Antibacterial effect of ACSM solution against M. luteus.

coli. (

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Dti

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itM

characterization of the control E. coli samples showed typicallya normal smooth surface (Fig. 8A), whereas after treatment withAcSM for 5 h. (Fig. 8B), the cells were covered in a thick layer of thefungal AcSM swelling and severe cell wall alterations appeared.

Fig. 5. (a) Antibacterial effect of AcSM treated fabric against E.

icrococcus luteus ATCC 9341. The assays were carried out by diskiffusion method, the AATCC Test Method, and TTC test method.

The AcSM treated cotton fabric showed an excellent antimi-robial activity for both Gram-negative bacterium E. coli andram-positive bacterium Micrococcus luteus. The highest antimi-robial activity was observed against Micrococcus luteus comparingo E. coli.

Gram negative bacteria has thick layer of phospholipids slightlyhan the peptidoglycan comparing to the Gram positive which hashick layer of peptidoglycan. The negative charges of the phospho-ipids improve the adhesion power of poly cationic polymer on theell wall.

As can be seen in Fig. 4a and b different concentrations of thecSM solution showed a good antimicrobial activity against bothram-negative E. coli and Gram-positive M. luteus. Also in Fig. 5and b the different concentrations of AcSM treated cotton fabrichowed a good antimicrobial effect. The results obtained revealedhat the untreated cotton fabrics had no inhibitory effect against. coli and Micrococcus luteus. On the other hand, AcSM solutionnd fabrics treated with AcSM solution showed inhibitory effectgainst E. coli and Micrococcus luteus.

Acetic-acid-soluble material AcSM separated from Mucor rouxiiSM-1191cell wall is generally considered to be the “fungal chi-

osan”. The surrounding pH of the AcSM plays an essential role ofts application.

.4. TTC test method

TTC test method was used, which measures antimicrobial activ-ty qualitatively and quantitatively, to determine the efficiency ofhe treatment in reducing the bacterial colony counts of E. coli and. luteus. In this test, in the presence of bacteria, TTC is reduced

b) Antibacterial effect of AcSM treated fabric against M. luteus.

to red formazan. The red formazan obtained indicates the activityand viability of the bacterial cell. It was found that there is no redcolour of the treated cotton sample with AcSM and this means thatthe reduction of the bacterial count was nearly 100% (Fig. 6).

TTC test serves as indicating system for the determination ofthe viability of micro organisms, since absorbance of formazan isdirectly proportional to the amount of living bacteria as shown inFig. 7.

It was found that the antimicrobial activity of the treated cottonsample with AcSM increases with increase time of incubation.

3.5. Scanning electron microscopic analysis

Scanning electron micrographs of E. coli treated with fungalAcSM, as well as control, are illustrated in Fig. 8. Morphological

Fig. 6. Effect of incubation time of the treated cotton sample with AcSM on itsantimicrobial activity against E. coli.

740 S. Moussa et al. / International Journal of Biolog

TAfo

The antibacterial mechanisms have been proposed: (i) the ionic

Ff

Fig. 7. Absorbance of formazan for Mucor rouxii DSM-1191 AcSM.

reated cells were more swollen and enlarged than typical cells.fter 24 h from the treatment of E. coli with AcSM, cell wall was

ully disrupted (Fig. 8C); cellular debris from cell lysis of E. coli wasbserved as a mixture with cell wall residues.

ig. 8. Scanning electron micrograph of cotton fabric with E. coli treated with the Mucor rungal AcSM); (B) cotton fabric with E. coli after 5 h from the treatment with fungal AcSM

ical Macromolecules 48 (2011) 736–741

4. Conclusions

The Mucor rouxii DSM-1191 AcSM was produced and appliedto textile materials by a simply pad-cure method. The resultingtextile materials get antibacterial effect and the performance ofcoated fabrics with the fungal AcSM as an antibacterial agent wasinvestigated.

The obtained scanning electron micrographs in current studyshowed the disturbance in E. coli cell wall after treatment withfungal AcSM.

The previous studies referred that acetic acid soluble mate-rial (AcSM) as a chitosan-rich fraction from the fungal cell wallmaterials and considered to be the “fungal chitosan” [4,7–12].AcSM is a versatile material with proved antimicrobial activity.

surface interaction resulting in wall cell leakage; (ii) the inhibi-tion of the mRNA and protein synthesis via the penetration ofthe small cationic polymer into the nuclei of the microorganisms;and (iii) the formation of an external barrier, chelating metals

ouxii DSM-1191 AcSM. (A) Control (cotton fabric with E. coli without treating with; (C) cotton fabric with E. coli after 24 h from the treatment with fungal AcSM.

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S. Moussa et al. / International Journal of

nd provoking the suppression of essential nutrients to microbialrowth.

Fungal AcSM was principally interacting with the E. coli cellalls, and causing clear rough shapes in their morphogenesis. With

he extend exposure to AcSM up to 24 h, complete lyses occurred in. coli cell wall and led to the release of intercellular components.

We can conclude from the achieved data that fungal AcSM coulde successfully applied as antibacterial agent.

cknowledgements

We wish to thank the Ministry for Science and Research of theountry Northrhine-Westphalia, FRG, for financial support. Thisupport was granted within the project DTNW/Support for attain-ent of further funds.

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