This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues.Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. a b s t r a c tThe antimicrobial proteins lysozyme and lactoferrin were incorporated into paper containing carboxymethyl cellulose, that allowed non-covalent binding of the positively charged proteins to the paper matrix. More than 60 percent of the proteins added alone or in combination during the papermaking process were released in buffered saline. The released proteins retained their structural and functional features, indicating that the papermaking process did not affect their structure. The antimicrobial activity on common food contaminants was also retained in the released protein, and a synergism between the two proteins was evident in tests carried out with paper containing both proteins against Listeria.Tests on thin meat slices laid on paper sheets containing either or both antimicrobial proteins indicated that lysozyme was most effective in preventing growth of this particular microbiota.

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We present an easy analytical method for determination of the charge density of polyelectrolytes, including polysaccharides and other biopolymers. The basic principles of conductometric titration, which is used in the pulp and paper industry, as well as in colloid and interface science, were adapted to quantify the charge densities of a negatively charged polysaccharide (pectin) and a positively charged biopolymer (chitosan): two biomacromolecules commonly used in food and biomaterials applications. We demonstrate that this novel conductometric titration method can be easily applied in most analytical chemistry teaching laboratories, due to its ease-of-use, safety, and educational benefits. This analytical technique can be used in a wide-range of laboratory activities, and has extensive research applications in areas of chemistry involving charged biopolymers, such as food science, materials science and physical chemistry.

SUMMARYActive cellulose-based packaging materials were prepared by binding lysozyme to paper modifi ed with anionic polyelectrolytes. Polyelectrolytes improve the paper binding capacity towards the positively charged lysozyme and play a protective role towards lysozyme activity during the paper-making process. The charge density of paper was increased by incorporating carboxymethylcellulose (CMC) or polygalacturonic acid (PGA). The presence of either CMC or PGA greatly increased the amount of bound lysozyme, and the stability of its binding towards buffers or towards non-ionic chaotropes which disrupt the three dimensional structure in macromolecules (urea, 8 M). Binding of lysozyme to modifi ed papers was sensitive to anionic detergents (sodium dodecyl sulphate, 1%) and to non-chaotrope salts (NaCl, 0.5M). These data provide information about the nature of the interactions between the protein and the various types of paper, and provide insights on how to preserve the activity of lysozyme-loaded paper. The polymers used for lysozyme immobilization were found to affect only marginally the structural and functional properties of the antimicrobial protein, and to facilitate the recovery of structural features of the protein after heat treatment. Presence of a negative polyelectrolyte (and of CMC in particular) improves the thermal stability of the protein, making it resistant to thermal inactivation, even under the conditions used for drying processes without compromising the paper's mechanical properties. The activity measurements showed that paperbound lysozyme retains its lytic (and therefore, antimicrobial) activity against the cell walls of Micrococcus lysodeikticus, the target microrganism used as standard for lysozyme bioassays.