Influence of acetylation degree and molecular weight of homogeneous chitosans on antibacterial and antifungal activities Islem Younes a, ⁎, Sabrine Sellimi a , Marguerite Rinaudo b , Kemel Jellouli a , Moncef Nasri a a Laboratory of Enzyme Engineering and Microbiology, University of Sfax, National School of Engineering, P.O. Box 1173, 3038 Sfax, Tunisia b Biomaterials Applications, 6, rue Lesdiguières, 38000 Grenoble, France abstract article info Article history: Received 6 February 2014 Received in revised form 21 April 2014 Accepted 27 April 2014 Available online 6 May 2014 Keywords: Homogeneous chitosan Acetylation degree Molecular weight Antibacterial activity Antifungal activity The results given in the literature are conflicting when considering the relationship between antimicrobial activ- ity and chitosan characteristics. To be able to clarify, we prepared fifteen homogeneous chitosans with different acetylation degrees (DA) and molecular weights (MW) by reacetylation of a fully deacetylated chitin under ho- mogeneous conditions. They were tested at different pH values for their antimicrobial activities against four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella typhi), four Gram-positive bacteria (Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis and Micrococcus luteus) and three fungi (Aspergillus niger, Fusarium oxysporum and Alternaria solani). Chitosans markedly inhibited growth of most bacteria and fungi tested, although the inhibitory effect depends on the type of microorganism and on the chitosan characteristics (DA and MW) with minimum inhibitory concentrations in the range of 0.001 to 0.1 w%. Considering chitosan efficiency on bacteria, our series of data clearly show that the lower DA and the lower pH give the larger efficiency. Antibacterial activity was further enhanced for Gram-negative bacteria with decreasing MW, whereas, opposite effect was observed with the Gram-positive. Concerning the antifungal activity, the influ- ence of chitosan characteristics was dependent on the particular type of fungus. Fungal growth decreased with increasing MW for F. oxysporum and decreasing DA for A. solani, but no MW or DA dependences were observed with A. niger. © 2014 Elsevier B.V. All rights reserved. 1. Introduction The growing consumer demand for foods without chemical preser- vatives has focused efforts in the discovery of new natural antimicro- bials (Wang, 1992). In this context, the unusual antimicrobial activity of chitosan and their derivatives against different groups of microorgan- isms, such as bacteria, yeast and fungi has received considerable atten- tion in recent years. Their antibacterial and antifungal properties are specially recognized in the field of food packaging to avoid the use of chemical preservatives and to produce edible antimicrobial films due to the good film forming properties of chitosan. Chitosan is a copolymer of D-glucosamine and N-acetyl-D- glucosamine obtained from chitin. The term chitosan usually refers to a family of chitin derivatives obtained after partial deacetylation to not only varying degrees but also different molecular weights. When the average degree of acetylation (DA expressed as molar percentage) becomes lower than 50 mol%, the product becomes soluble in acidic conditions due to the protonation of the \NH 2 group at the C-2 position of glucosamine units (Rinaudo, 2006). It is the only existing natural positively charged polysaccharide. There are two types of chitosans: (1) chitosans derived from chitin by deacetylation under heterogeneous conditions and (2) chitosans de- rived from a highly deacetylated chitosan (or poly-D-glucosamine) by partial reacetylation under homogeneous conditions (Hirano et al., 1981; Maghami and Roberts, 1988). Deacetylation reaction performed under heterogeneous conditions gives an irregular distribution of N- acetyl-D-glucosamine and D-glucosamine residues and some blockwise acetyl group distribution along polymeric chains. So, their solubility and degree of aggregation could have variable behavior towards biolog- ical activities (even if two samples have the same average DA). The physicochemical properties of such chitosans appear different from those of randomly acetylated chitosans obtained under homogeneous conditions (Aiba, 1991). Until now, in biological activity studies, such characteristics for chitosan have not been considered seriously even if chitosan has attracted considerable interest in many fields. Moreover, chitosan has several advantages over other disinfectants, as it possesses a higher antimicrobial activity, a broader spectrum of activity, a higher kill rate, and lower toxicity towards mammalian cells (Franklin and Snow, 1981; Takemono et al., 1989). International Journal of Food Microbiology 185 (2014) 57–63 ⁎ Corresponding author. Tel.:+216 74 274 088. E-mail address: islem.younes@gmail.com (I. Younes). http://dx.doi.org/10.1016/j.ijfoodmicro.2014.04.029 0168-1605/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro