Costa, E. M.

Gens, E. J.

Malcata, F. X.

Pintado, M. E.

Tavaria, F. K.

English

Repositório Institucional da Universidade Católica Portuguesa

THE INFLUENCE OF SOME ABIOTIC FACTORS ON THE ANTIMICROBIAL POTENTIAL OF CHITOSAN F. K. Tavaria, E. M. Costa, E. J. Gens, M. E. Pintado and F.  X. MalcataEscola Superior de Biotecnologia – Universidade Católica Portuguesa, Porto, Portugal The stratum corneum of the skin of patients with atopic dermatitis is highly susceptible to colonization by Staphylococcus aureus (Arikawa et al., 2002). Biopolymers have receivedmore and more attention as alternatives to synthetic polymers in several technological processes, ranging from environmental, to food and health applications. Among them, chitosanis one of the most promising; it is produced from heterogenous alkaline de-N-acetylation of chitin. Non-chemically modified chitosan is only soluble at acid pH, but pH and acidsolvents affect its antibacterial activity. The effects of such factors upon chitosan activity have been explored by some authors (Vishu Kumar et al., 2007), but it was always found tobe strain-dependent. In addition, several external (abiotic factors) also influence its activity, thus making it quite difficult to draw clear-cut conclusions. To assess the potential use ofchitosan to prevent and control atopic dermatitis, its antibacterial activity against skin-associated bacteria was investigated, by varying such abiotic factors as pH, ionic strength,organic acids and free fatty acids.INTRODUCTIONThe Portuguese Foundation for Science and Technology (FCT, Portugal) through program PRAXIS XXI is hereby gratefully acknowledged (post-docfellowship SFRH/BPD/24222/2005). Additional funding was obtained via project BIOTEX ⎯ Bioactive Textiles using functional biopolymers(POCI/CTM/58312/2004), also funded by FCT.Effect of pHBibliographyAcklowledgementsMATERIALS & METHODSRESULTS & DISCUSSIONChitosans: Chitosan of high molecular weight (ca. 624 kDa, >75% deacetylated), medium molecular weight (ca. 591 kDa, 75-85% deacetylated) and low molecular weight (ca. 107 kDa, 75-85% deacetylated) were obtained from Sigma (St. Louis, MO, USA).Microorganisms: Three major skin-related bacteria were used: Staphylococcus aureus subsp. aureus ATCC 25923, Staphylococcus sp. ATCC 155 and Escherichia coli ATCC 25922. Antimicrobial activity: A 25 full factorial design was followed: ionic strength, pH level, organic acids, fatty acids and presence/absence of chitosan, selected according to skin conditions, were used as parameters.Ionic strength: 0.2 %(w/v) and 0.4 %(w/v); pH: 4 and 7; Organic acids: lactic and propionic acids (1 % (v/v)); Free fatty acids: oleic and palmitic acids (0.75 % (v/v)). Only high molecular weight chitosan was used at 0.5 % (v/v) as this MW was found as that which affected Gram positive skin microorganisms the most (Tavaria et al., 2008). The inoculants were added to each mixture (5% v/v)) and left to incubate for 24h at 37º C. Samples were collected at time zero, and then every 2 h up to 24h, for viable cell enumeration. The reduction rate was then determined as the variation of the colony forming units (CFU) between time zero and a given time.Statistical analysis: Repeated measures oneway ANOVA was applied, using the SPSS package (v. 16.0 for windows, SPSS, IL, USA) at a 5% level of significance.pH and the fatty acid (FFA) were the most important factors influencing anti-microbial activity.pH affected significantly theantimicrobial activity of the threeisolates; for S. aureus (Figure 1), thiseffect was less apparent than for theother microorganisms, as also reportedby Yang et al. (2005).At 0.2% NaCl (Figure 2), oleic acid (A)protected the cells from theantimicrobial action (even in thepresence of chitosan).Effect of FFAsFigure 3. Effect of organic acids (A- lactic, B- propionic) on the antimicrobial activity of chitosan upon S.epidermidis (- - pH 4 with chitosan; - - pH 4 without chitosan; - - pH 7 with chitosan; - - pH 7 withoutchitosan). Other constant conditions: 0.2% NaCl, presence of oleic acid.Effect of organic acidsPresence of NaCl and of the organic acid was only significant (p<0.05) for S. epidermidis,in the presence of chitosan.At the lower NaCl concentration (0.2%), propionic acid seemed to protect bacteria fromthe antimicrobial action of chitosan at pH 7, while lactic acid seemed to potentiate itsantimicrobial activity (Figure 3). Jo et al. (2007) described a strong inhibitory effect of lacticacid upon E. coli, while Cheng et al. (2003) demonstrated that, although some strains of E.coli exhibit an increased tolerance to lactic acid, they do not show such anadaptative/tolerant capacity towards propionic acid.ConclusionsFigure 1. Effect of pH on the antimicrobial activity ofchitosan upon S. aureus (- - pH 4 with chitosan; - -pH 4 without chitosan; - - pH 7 with chitosan; - - pH 7without chitosan). Other constant conditions: 0.2%NaCl, oleic and lactic acids present.Figure 2. Effect of free fatty acids (A- oleic, B- palmitic) on the antimicrobial activity of chitosan upon E. coliat pH 7 [- - 0.2%(v/v) NaCl with chitosan; - - 0.2%(v/v) NaCl without chitosan; - - 0.4%(v/v) NaCl with chitosan; - - 0.4%(v/v) NaCl without chitosan]. Other constant conditions: presence of lactic acid.By controlling the presence of selected abiotic factors, the action of chitosan can be targetted at inhibiting growth of unwanted microorganisms, such as S. aureus and E. coli.Chitosan was able to selectively inhibit growth of skin microorganisms, a requirement for use as effective antimicrobial compound. Chitosan applied to textiles can, in this way, change the ecology of the skin native flora, without leading to the outgrowth of pathogenic bacteria. Arikawa, J., Ishibashi, M., Kawashima, M., Takagi, Y., Ichikawa, Y. and Imokawa, G. (2002). J Invest Dermatol 119: 433-439. Cheng, H.-Y., Yu, R.-C. and Chou, C.-C. (2003. Food Res. Int. 36: 49-56.Jo, S.-C., Rim, A.-Ram, Park, H.-J., Yuk, H.-G. and Lee, S.-C. (2007). Food Control 18: 1235-1240.Tavaria, F. K., Reis, I., Paulo, M., Pintado, M. and Malcata, F.X. (2008. J Biotechnol. Submitted.Vishu Kumar, A.B., Varadaraj, M.C., Gowda, L.R. and Tharanathan, R.N. (2007). Biochim Biophys Acta 1770: 495-505.Yang, T.-C., Chou, C.-C. and Li, C.-F. (2005). Int J Food Microbiol 97: 237-245.

The influence of some abiotic factors on the antimicrobial potential of chitosan

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The influence of some abiotic factors on the antimicrobial potential of chitosan

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