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Kristin Johansson | Oxygen-reducing enzymes in coatings and films for active packaging | | Oxygen-reducing enzymes in coatings and films for active packaging Kristin Johansson Oxygen-reducing enzymes in coatings and films for active packaging Oxygen-reducing enzymes This work focused on investigating the possibility to produce oxygen-scavenging packaging materials based on oxygen-reducing enzymes. The enzymes were incorporated into a dispersion coating formulation applied onto a food- in coatings and films for packaging board using conventional laboratory coating techniques. The oxygen- reducing enzymes investigated included a glucose oxidase, an oxalate oxidase active packaging and three laccases originating from different organisms. All of the enzymes were successfully incorporated into a coating layer and could be reactivated after drying. For at least two of the enzymes, re-activation after drying was possible not only Kristin Johansson by using liquid water but also by using water vapour. Re-activation of the glucose oxidase and a laccase required relative humidities of greater than 75% and greater than 92%, respectively. Catalytic reduction of oxygen gas by glucose oxidase was promoted by creating 2013:38 an open structure through addition of clay to the coating formulation at a level above the critical pigment volume concentration. For laccase-catalysed reduction of oxygen gas, it was possible to use lignin derivatives as substrates for the enzymatic reaction. At 7°C all three laccases retained more than 20% of the activity they had at room temperature (25°C), which suggests that the system is also useful for packaging of refrigerated food. ISBN 978-91-7063-516-8 Faculty of Health, Science and Technology ISSN 1403-8099 Chemical Engineering DISSERTATION | Karlstad University Studies | 2013:38 DISSERTATION | Karlstad University Studies | 2013:38 Oxygen-reducing enzymes in coatings and films for active packaging Kristin Johansson DISSERTATION | Karlstad University Studies | 2013:38 Oxygen-reducing enzymes in coatings and films for active packaging Kristin Johansson DISSERTATION Karlstad University Studies | 2013:38 ISSN 1403-8099 ISBN 978-91-7063-516-8 © The author Distribution: Karlstad University Faculty of Health, Science and Technology Department of Engineering and Chemical Sciences SE-651 88 Karlstad, Sweden +46 54 700 10 00 Print: Universitetstryckeriet, Karlstad 2013 WWW.KAU.SE Abstract This work has focused on the addition of oxygen-reducing enzymes to a dispersion coating formulation applied to a food-packaging material using conventional laboratory coating techniques. The purpose of adding the enzymes to the coating was to investigate the possibility of producing oxygen-scavenging active-packaging materials. It was shown that it is possible to incorporate various oxygen-reducing enzymes, including a glucose oxidase, an oxalate oxidase and three laccases originating from different organisms, in a coating layer and to re-activate them after drying. The laccases were also investigated with regard to their potential to function as oxygen scavengers in active packages at low temperatures. At 7°C, all three laccases retained more than 20% of the activity they had at room temperature (25°C), which suggests that the system is also useful for the packaging of refrigerated food. The surfactants present in the latex were shown to have an immediate negative effect on the oxalate oxidase, whereas the glucose oxidase was affected only after seven days of storage at 4°C. Immobilised oxalate oxidase showed a potential to function both as an oxygen scavenger and as an oxalic-acid scavenger in aqueous solutions. The addition of a platy pigment to the coating formulation had several effects on the activity of glucose oxidase. A small amount of clay was shown to hinder the pre-oxidation of glucose in the wet/semi-wet coating and a large amount of clay (more than the critical pigment volume concentration) was shown to promote the oxygen-scavenging capacity of the dry layer by introducing pores to the structure. The porous structure also had a greater water-vapour uptake, which enabled the activation of glucose oxidase by using only water vapour at a relative humidity of 75% or higher. Migration of the glucose-oxidase-containing coating was reduced by adding an extrusion-coated liner of polypropylene on top of the coating. The possibility of producing heat-sealable packaging materials with various barrier properties was investigated by extrusion coating of both sides (the enzyme-coated side and the reverse side) of the board with one of three plastics, viz. polypropylene, i polyethylene or polylactic acid. Independently of the water-vapour barrier properties of the plastic films, glucose oxidase was activated at a relative humidity of 84% and above. A comparison of the plastic materials showed that the materials that were extrusion-coated with PLA on at least one side consumed the largest amount of oxygen. To remove oxygen using laccase originating from the basidiomycete fungus Trametes versicolor, it was possible to use lignin derivatives, macromolecular by- products from the forest industry, as substrates for the enzymatic reaction. Coatings containing a lignin derivative and laccase and applied to a paper board were able to scavenge oxygen at or above a relative humidity of 92%. When the laccase and the lignin derivative lignosulphonate were added to starch-based films, the enzymatic oxidation of lignosulphonate resulted in a polymerisation that increased the stiffness and the water-resistance of the biopolymer film. ii List of papers included in the thesis I Johansson, K, Jönsson, L.J., Järnström, L. (2011). Oxygen scavenging enzymes in coatings – Effect of coating procedures on enzyme activity. Nord. Pulp Paper Res. J. 26, 197-204. II Chatterjee, R., Johansson, K., Järnström, L., Jönsson, L.J. (2011). Evaluation of the potential of fungal and plant laccases for active-packaging applications. J. Agric. Food Chem. 59, 5390-5395. III Winestrand, S., Johansson, K., Järnström, L., Jönsson, L.J. (2013). Co- immobilization of oxalate oxidase and catalase in films for scavenging of oxygen or oxalic acid. Biochem. Eng. J. 72, 96-101. IV Johansson, K., Christophliemk, H., Johansson, C., Jönsson, L., Järnström, L. (2013). The effects of coating structure and water-holding capacity on the oxygen-scavenging ability of enzymes embedded in the coating layer. Tappi J. 12, 43-52. V Johansson, K., Kotkamo, S., Rotabakk, B.T., Johansson, C., Kuusipalo, J., Jönsson, L., Järnström, L. Extruded polymer films for optimal enzymatic oxygen- scavenging performance. Submitted for publication. VI Johansson, K., Winestrand, S., Johansson, C., Järnström, L., Jönsson, L.J. (2012). Oxygen-scavenging coatings and films based on lignosulfonates and laccase. J. Biotechnol. 161, 14-18. VII Johansson, K., Gillgren, T., Winestrand, S., Järnström, L., Jönsson, L.J. Comparison of lignin derivatives as substrates for laccase-catalyzed scavenging of oxygen in coatings and films. Submitted for publication. iii Authors contribution The author of this thesis carried out 50% or more of the laboratory work and was the principal author of Papers I, IV, and VI. In Paper II, the author performed all the experiments with immobilised enzyme. The author contributed equally with S. Winestrand to the investigation and the manuscript described in Paper III. The author performed the oxygen-scavenging test and the oxygen transmission rate analysis of Paper V and was the principal author of the manuscript. The author performed about 40% of the laboratory work of paper VII and drafted the manuscript together with T. Gillgren and L. J Jönsson. The majority of the work presented in this thesis was part of the MNT-ERA.NET Nordic Innovation project 07144: ENZYCOAT II - Enzymes embedded in barrier coatings for active and intelligent packaging. The project report is available through http://www.nordicinnovation.org/sv/publikationer/enzycoat-ii/ iv List of symbols and abbreviations A Pre-exponential factor or frequency factor Abs Absorbance ABTS 2-2'-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) Ap Surface area of packaging aw Water activity b Geometry term c Concentration Cat Catalase CPVC Critical pigment volume concentration DMA Dynamic mechanical analysis E' Storage modulus Ea Activation energy EC Enzyme Commission EVOH Ethylene vinyl alcohol EPR Electron paramagnetic resonance f0 Force applied at the peak of the sine wave FAD Flavin adenine dinucleotide FDA US Food and Drug Administration GOx Glucose oxidase HRP Horseradish peroxidase IR Infrared k Chemical reaction rate constant l Path length LDPE Low density polyethylene LMS Laccase mediator system v LO Organosolv lignin LS Lignosulphonate MaL Laccase from Melanocarpus albomyces MAP Modified atmosphere packaging MtL Laccase from Myceliophthora thermophila Mn Number average molecular weight Mw Weight average molecular weight OP Oxygen permeability OTR Oxygen transmission rate OxO Oxalate oxidase P Permeability p vapour pressure over the curved surface p0 Vapour saturation pressure of a liquid PE Polyethylene PET Polyethylene terephthalate pin Partial pressure of oxygen inside the package PLA Polylactic acid pout Partial pressure of oxygen outside the package PP Polypropylene PS Polystyrene PVA Polyvinyl alcohol PVC Pigment volume concentration Q10 Temperature coefficient R Ideal gas constant rk Mean radius of curvature of the meniscus RvL Laccase from Rhus vernicifera SA-latex Styrene-acrylate latex vi SB-latex Styrene-butadiene
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