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International Journal of Biological Macromolecules 47 (2010) 454–457

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

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

nticandidal action of fungal chitosan against Candida albicans

hmed A. Tayela,∗, Shaaban Moussaa, Wael F. El-Trasb, Dierk Knittel c,laus Opwisc, Eckhard Schollmeyerc

Genetic Engineering and Biotechnology Research Institute, Minufiya University, El-Sadat City, EgyptDepartment of Hygiene & Preventive Medicine (Zoonoses), Faculty of Veterinary Medicine, Kafrelsheikh University, 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 23 May 2010eceived in revised form 15 June 2010

a b s t r a c t

The anticandidal activity of four fungal chitosan types, produced from Mucor rouxii DSM-1191, againstthree Candida albicans strains was determined. The most bioactive chitosan type, to inhibit C. albicansgrowth, had the lowest molecular weight (32 kDa) and the highest deacetylation degree (94%). Water

ccepted 29 June 2010vailable online 13 July 2010

eywords:ntifungalandidicidal action

soluble types had stronger anticandidal activity than soluble types in 1% acetic acid solution. Scanningelectron micrographs of treated C. albicans with fungal chitosan proved that chitosan principally interactwith yeast cell wall, causing severe swelling and asymmetric rough shapes, and subsequent cell wall lyseswith the prolonging of exposure time. Fungal chitosan could be recommended for C. albicans control asa powerful and safe alternative to synthetic and chemical fungicides.

icrobial chitosanatural biocides

. Introduction

Chitosan [poly �-(1,4)-2-amino-2-deoxy-d-glucose] is theeacetylated derivative of chitin, which proved to act as a naturalntimicrobial compound. Traditionally, chitosan can be obtainedrom crustacean shells (crabs, shrimp and crayfishes) either byhemical or microbiological processes, and alternatively, it can beroduced from some fungi (Aspergillus niger, Mucor rouxii, Peni-illium notatum) [1]. Commercial production of chitosan fromeacetylated crustacean chitin using strong alkali appears to have

imited potential for industrial acceptance because of seasonal andimited supply; processing difficulties resulted in a large amountf waste of concentrated alkaline solution causing environmen-al pollution and inconsistent physico-chemical properties. Fungalhitosan production is avoiding most of the previous restrictionsnd disadvantages recorded during crustacean chitosan produc-ion.

Due to its biodegradable, biocompatible and nontoxic char-cteristics, chitosan has recently gained more interest for the

pplications in food and pharmaceutics. Among other properties,he antimicrobial activity of chitosan has been pointed out as onef its most promising properties [2]. Among the earliest applica-ions of chitosan was to remove harmful metal ions from industrial

∗ Corresponding author at: Department of Industrial Biotechnology, Geneticngineering and Biotechnology Research Institute, Minufiya University, P.O. Box9/22857, El-Sadat City, Egypt. Tel.: +20 10 196 9909; fax: +20 48 260 1266–8.

E-mail address: tayel ahmad@yahoo.com (Ahmed A. Tayel).

141-8130/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.ijbiomac.2010.06.011

© 2010 Elsevier B.V. All rights reserved.

waste waters and suspended solids from food processing wastes[3].

It can also be used as a thickening agent in beverages and semi-solid foods, clarifying agent in wine and juice processing [4], amineral and lipid binder, as a flavor and color carrier, and for theproduction of coatings and edible antimicrobial films [5].

Candida albicans is a fungus that could be located on the skin andin mucous membranes such as the vagina, mouth, or rectum [6,7].This fungus can also travel through the blood stream and affect thethroat, intestines, and heart valves. Among the various human fun-gal pathogens, attacking immunocompromised patients, C. albicansaccounts for the majority of systemic infections with mortality ratesranging from 50% to 100% [8]. The systemic mycoses and especiallythose fungi that cause opportunistic infections, such as C. albicans,pose imperative problems for the clinician who must select from ashort list of antifungal agents to achieve cure [9]. The encounteredlimitations for the usage of a specific antifungal include: First, asboth the fungus and host are eukaryotic, the number of compoundsexclusively toxic for the fungus is small. Second, amphotericin-B,the “gold standard” of antifungals for the treatment of the systemicmycoses [10], regularly causes some degree of toxicity because italso binds to similar targets of host cells. Third, successful treat isfrequently influenced by the low sensitivity of available detectionmethods, especially in the case of invasive aspergillosis where labo-

ratory diagnosis by blood culture most often fails. Fourth, resistanceto fluconazole, the azole which is commonly used to treat severalmycoses, is reported with increasing frequency [11].

Most of the time, Candida infections of the mouth, skin, orvagina occur for no exactly apparent reason. A common cause of

A.A. Tayel et al. / International Journal of Biological Macromolecules 47 (2010) 454–457 455

Table 1Physico-chemical characteristics of fungal chitosan types used in the study.

Chitosan types Physico-chemical characteristics

Color Viscosity (cP) Solubility Degree of deacetylation % Molecular weight (kDa)

CTS 1 White 2.9 Water 94 32CTS 2 Creamy 5.4 Acetic acid 1% 86 184

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TS: chitosan type.

nfection may be the use of antibiotics that destroy beneficial, asell as harmful, microorganisms in the body, permitting Candida

o multiply in their place.The purpose of this current work was to evaluate the antican-

idal action of fungal chitosan against C. albicans species and toescribe the probable mechanisms of its action.

. Materials and methods

All chemicals and reagents used in current study, unless otherource indicated, were purchased from Sigma Chemical Co. (St.ouis, MO).

.1. Characteristics of fungal chitosan

Mucor rouxii DSM-1191 was used for the production of chi-osan. Subsequent to various treatments of alkaline extractionrom mycelial growth, different chitosan types were obtainednd characterized according to Synowiecki and Al-Khateeb [12].he physio-chemical characteristics of produced chitosans wereetermined according to AOAC [13] (color, viscosity and solu-ility), whereas the molecular weights were determined by gelermeation chromatography (GPC) using refractive index detectorPN-1000, Postnova Analytics, Eresing, Germany). The deacetyla-ion degree was determined by titration according to the methodf Donald and Hayes [14].

Four produced chitosan types were examined to evaluate theirnticandidal activity against C. albicans strains (Table 1).

.2. Yeast strains

Three C. albicans strains, i.e. C. albicans-A (ATCC-10231), C.lbicans-H (isolated from human tongue) and C. albicans-C (iso-ated from chicken comb) were obtained from the Department ofygiene and Preventive Medicine (Zoonoses), Kafrelsheikh Uni-ersity, Egypt The identity of examined strains was confirmedccording to the method of Barnett et al. [15]. Yeast strains wererown and maintained on Sabouraud Dextrose Agar (SDA, Merck,armstadt, Germany). Inoculums for the assays were prepared byiluting scraped cell mass in 0.85% NaCl solution and counting usingaemocytometer, cell number was then adjusted to 105 cell/mlsing above-mentioned saline solution.

.3. Anticandidal activity evaluation

The method of Ellof [16] was applied to determine the minimalnhibitory concentrations (MIC) of different chitosan types toward. albicans strains. Using a tissue culture test plates (96 wells), serialilutions of fungal chitosans, all of them were dissolved in 1% aceticcid, in Sabouraud Dextrose broth were prepared and transferred

nto the plate wells to obtain final concentrations in the range of.1–5.0 mg/ml including the inoculums of yeasts. Yeast inoculums100 �l) were added to each well and the plates were then incu-ated at 37 ◦C for 24 h. SDA plates were then inoculated with a

oopful from each well at the end of incubation period. Antimicro-

88 38id 1% 84 138

bial activity was confirmed by the addition of 20 �l of 0.5% triphenyltetrazolium chloride (TTC, Merck, Darmstadt, Germany) aqueoussolution. MIC was defined as the lowest concentration of chitosanthat inhibited visible growth on SDA, as confirmed by the TTC stain-ing (dead C. albicans cells are not stained by TTC). All tests werecarried out in triplicates to verify the results.

2.4. Micrograph capture

Toward the explanation of chitosan antimicrobial action, micro-graphs of treated C. albicans with fungal chitosan was capturedusing scanning electron microscope (SEM; S-500, Hitachi, Japan),magnification: 5000×, after 6 and 24 h from the treatment withchitosan MIC, as well as control culture. Samples were preparedfor SEM as described by Marrie and Costerton [17]. Briefly, theywere fixed in glutaraldehyde in 0.1 M cacodylate buffer containing0.15% ruthenium red for 3 h at 4 ◦C. The sections were then rinsed infresh 0.1 M cacodylate buffer for 10 min (repeated three times) andpost-fixed in 1.5% osmiumtetroxide for 1 h. They were dehydratedin a series of aqueous ethanol solutions (30–100%) and dried by acritical point dryer (Tousimis Autosamdri-814, Rockville, MD) withCO2. The specimens were mounted on aluminium stubs with silverpaste, allowed to dry for 3 h and then coated with gold/palladiumusing a cool-sputter coater E5100 II (Polaron Instruments Inc., Hat-field, PA, USA). Sections were then examined under SEM at 20 kV.Captured areas were selected according to the alteration in themorphology of treated cells.

3. Results

3.1. Fungal chitosans characteristics

The physico-chemical characteristics of chitosan types pro-duced from Mucor rouxii DSM-1191 after different extractionprocesses are illustrated in Table 1. Observable variations betweenchitosan types were recorded; two of examined types were char-acterized with water solubility, i.e. CTS 1 and CTS 3, whereas theother types (CTS 2 and CTS 4) could be solubilized in 1% acetic acidsolution (pH 4.3). The water solubility was allied to the decreasein chitosan molecular weight and the increase of deacetylationdegrees (Table 1).

On the other hand, acetic acid soluble chitosans were character-ized with high molecular weights, 184 and 138 kDa for CTS 2 andCTS 4, and comparatively low deacetylation degrees, 86 and 84%,respectively.

3.2. Anticandidal activity of fungal chitosans

All fungal chitosan types could effectively inhibit the growthof C. albicans examined strains. The anticandidal potency varied

among chitosan types (Fig. 1). CTS 1 was the most effective typeto inhibit the growth of all C. albicans strains. The recorded mini-mal inhibitory concentrations (MIC) from CTS 1 toward C. albicansstrains were 2.0, 1.75 and 1.25 mg/ml against C. albicans-A, C.albicans-H and C. albicans-C, respectively. Control solution of 1%

456 A.A. Tayel et al. / International Journal of Biolog

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ig. 1. Minimal inhibitory concentrations of fungal chitosan types against C. albicanstrains. A: C. albicans (ATCC-10231); H: C. albicans from human tongue; C: C. albicansrom chicken comb. MIC: minimal inhibitory concentrations (mg/ml).

cetic acid did not exhibit an anticandidal activity against C. albicanstrains.

CTS 3 was the second best anticandidal type compared to theemaining chitosan types. Both CTS 1 and CTS 3 were water soluble.n the other hand, the acetic-solution soluble types (CTS 2 andTS 4) showed less effectual anticandidal activity, although both ofhem could completely inhibit C. albicans growth, but with higher

IC values than those of water soluble types.Regarding the sensitivity of C. albicans strains toward fungal

hitosan types, C. albicans-C was the most sensitive, whereas C.lbicans-A was the most resistant strain to all examined chitosanypes. All of the obtained results regarding chitosan MIC were val-dated and confirmed using TTC staining of treated cells.

.3. Scanning electron microscopic analysis

Scanning electron micrographs of C. albicans-A treated with CTS, as well as control, are illustrated in Fig. 2. The micrographsronounced that typical (untreated) cells had a normal smoothurface (Fig. 2A), whereas after treatment with chitosan for 6 h.Fig. 2B), swelling and severe cell wall alterations appeared. Treatedells were more puffy and enlarged than typical cells with obviousntense distortion in their surfaces.

After 24 h from the treatment of C. albicans with chitosan (CTS), cell wall was fully disrupted (Fig. 2C); cellular debris from cell

ysis of C. albicans was observed as a mixture with cell wall residues.

ig. 2. Scanning electron micrograph of C. albicans-A treated with minimalnhibitory concentration (MIC) of fungal chitosan CTS 1. (A) Control (untreated) cells;B) after 6 h from the treatment with chitosan; (C) after 24 h from the treatment withhitosan. Treated CTS 1 concentration was 2 mg/ml.

ical Macromolecules 47 (2010) 454–457

4. Discussion

Chitosan is a safe biopolymer, approved by the Food and DrugAdministration (FDA) for use in dietary supplements, biomedical,pharmaceutical, agricultural and nutritional fields. It has also beenreported that the degradation of products of pure chitosan are non-toxic, nonimmunogenic, and noncarcinogenic [18].

The potential advantages of chitosan production from fun-gal sources, over the traditional production from marine-livingresiduals, include the independence of seasonal factor, wide scal-ing production, simplicity of extraction process, cost and timesaving, simultaneous extraction of chitin and chitosan, and theabsence of proteins contamination which could cause antipathyreaction in individuals with shellfish allergies [19,20]. Chitosan isproved to be an eco-friendly antimicrobial agent toward numerousspecies of yeast, molds, Gram positive and Gram negative bacteria[21,22].

Regarding the physico-chemical characteristics of examinedfungal chitosan types, in the current study, and their influence ontheir antifungal activity, both of the molecular weight and deacety-lation degree had a clear impact on the anticandidal potentiality.Types having lower molecular weights and higher deacetylationdegrees, i.e. CTS 1 and CTS 3, were more effective than other types.Also, solubility of chitosan in water fortified its candidicidal activitycompared to soluble chitosan in acetic acid solution. The obtainedresults are in accordance with the findings of No et al. [23].

In addition to the preference of having a water soluble chitosanto avoid any toxic, corrosive or mutagenic effect of the probableused solvents [24], the forceful antifungal action of water-solubletypes may be attributed to their high ability, which correlated tolow molecular weights, to attach to yeast cell wall and performtheir disruptive effects.

Many explanations were proposed about the antimicrobialactivity of chitosan [25]; it was suggested that the interferencebetween chitosan and microbial cells could be on the cell surface,which leads to increased permeability and irregularity of cell wallswhich lead to subsequent leakage of intracellular components, orinside the cell, which may inhibit DNA and RNA synthesis and directcell death.

The obtained scanning electron micrographs in current study(Fig. 2) clearly show the disturbance in C. albicans cell wall aftertreatment with chitosan at its MIC value. Chitosan was princi-pally interacting with the yeast cell walls, and causing observableirregular rough shapes in their morphogenesis. With the pro-long exposure to chitosan up to 24 h, mostly comprehensive lysesoccurred in C. albicans cell wall and led to the release of intercellularcomponents.

The growth of most fungal strains, except those containingchitosan as cell-wall constituent, was reported to be affected bychitosan at 1.0 mg/ml [26]. Treatment with chitosan resulted incell wall softening and, subsequently, an overall alteration of yeastmorphology [27].

The SEM observations provided morphological evidence of thepotent permeabilizing activity of the chitosan [28]. Numerous stud-ies have shown that chitosan is not only effective in halting thegrowth of the pathogen, but also induces marked morphologi-cal changes, structural alterations and molecular disorganizationof the fungal cells [24,29,30]. Although it was reported [31,32]that the exact mechanisms for the antimicrobial effect of chitosan,chitin, and any other derivatives are unknown, several mechanismshave been proposed involving some kind of damage or interac-

tion with the cell membrane. One such proposed action is thepositively charged chitosan interacting with the negative cell mem-brane which in turn alters its permeability, allowing leakage ofintracellular material to the media [21,33]. The polycationic featureof chitosan and chitosan derivatives is fundamental for antifungal

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A.A. Tayel et al. / International Journal of

ctivity against Candida spp.; the added functional groups mask theationic amino groups [34].

The high susceptibility of C. albicans to interact with chitosanould be attributed to its inclusion of sialic acid in cell wall con-tituent, presumably as terminal residues of glycoprotein glycans35]. Sialic acid-rich glycoprotein binds selectins (a family of celldhesion molecules that bind sugar polymers) in humans and otherrganisms. Michael [36] reported that the application of chitosano yeast cell suspensions resulted in changes in yeast morphologynd cell flocculation rates. These changes are suggestive that phys-ological changes also occurred, thereby resulting in a change inurface charge.

We can conclude from the achieved data that fungal chitosan,specially water soluble types with low molecular weights and higheacetylation degrees, could be effectively applied for the control of. albicans invasion as a safe and ecofriendly alternative to syntheticnd chemical antifungal agents.

cknowledgements

Always and foremost, thanks are indebted to ALLAH forever. Weish to thank the Ministry for Science and Research of the coun-

ry Northrhine-Westphalia, FRG, for financial support. This supportas granted within the project DTNW/Support for attainment of

urther funds. Also, we are grateful to Prof. Hanafy A. Hamza, GEBRI-U, for his generous scientific help and support during this work

n Egypt.

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