Ž . International Journal of Food Microbiology 71 2001 235–244 www.elsevier.comrlocaterijfoodmicro Chitosan disrupts the barrier properties of the outer membrane of Gram-negative bacteria I.M. Helander a , E.-L. Nurmiaho-Lassila b , R. Ahvenainen a , J. Rhoades c , S. Roller c, ) a VTT Biotechnology and Food Research, FIN-02044 VTT, Espoo, Finland b DiÕision of General Microbiology, Department of Biosciences, FIN-00014 UniÕersity of Helsinki, Helsinki, Finland c School of Applied Science, South Bank UniÕersity, 103 Borough Road, London SE1 0AA, UK Received 17 January 2001; received in revised form 17 April 2001; accepted 30 June 2001 Abstract Ž . The mode of antimicrobial action of chitosan polymeric b-1,4-N-acetylglucosamine on Gram-negative bacteria was Ž . studied with special emphasis on its ability to bind to and weaken the barrier function of the outer membrane OM . Ž . Ž . Chitosan 250 ppm at pH 5.3 induced significant uptake of the hydrophobic probe 1-N-phenylnaphthylamine NPN in Ž . Escherichia coli , Pseudomonas aeruginosa and Salmonella typhimurium. The effect was reduced E. coli , salmonellae or Ž . abolished P. aeruginosa by MgCl . No NPN uptake was observed during exposure of the salmonellae to chitosan at pH 2 Ž . 7.2. Chitosan also sensitized P. aeruginosa and the salmonellae to the lytic effect of sodium dodecyl sulfate SDS ; such sensitization was not blocked by MgCl and was reversible by washing chitosan-treated cells prior to SDS exposure. 2 Chemical and electrophoretic analyses of cell-free supernatants of chitosan-treated cell suspensions showed that interaction Ž . of chitosan with E. coli and the salmonellae involved no release of lipopolysaccharide LPS or other membrane lipids. However, chitosan rendered E. coli more sensitive to the inhibitory action of dyes and bile acids used in selective media. Highly cationic mutants of S. typhimurium were more resistant to chitosan than the parent strains. Electron microscopy showed that chitosan caused extensive cell surface alterations and covered the OM with vesicular structures. Chitosan thus appeared to bind to the outer membrane, explaining the loss of the barrier function. This property makes chitosan a potentially useful indirect antimicrobial for food protection. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Chitosan; Antibacterial; Outer membrane; Gram-negative bacteria 1. Introduction Chitosan, the deacylated derivative of chitin, is a versatile biopolymer with a number of food applica- Ž . tions Shahidi et al., 1999 . Chitosan consists of polymeric 1 ™4-linked 2-amino-2-deoxy-b-D-glu- cose, but preparations and batches vary with respect ) Corresponding author. Tel.: q44-207-815-7961; fax: q44- 207-815-7999. Ž . E-mail address: rollers@sbu.ac.uk S. Roller . to their degree of deacetylation and polymerisation, and many derivatives such as N-sulfonyl chitosan or salts such as chitosan lactate with altered physico- chemical properties can be prepared. Since chitosans exhibit antibacterial and antifun- gal activity, they have received attention as potential Ž food preservatives of natural origin Chen et al., 1998; Shahidi et al., 1999; Rhoades and Roller, . 2000; Roller and Covill, 2000; Tsai et al., 2000 . Although more active against spoilage yeasts, chi- tosan has also been shown to inhibit some Gram- 0168-1605r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. Ž . PII: S0168-1605 01 00609-2