Enzyme Stabilization in Hierarchical Biocatalytic Food Packaging and Processing Materials

The partnership of biocatalysts and solid support materials provides many opportunities for bioactive packaging and bioprocessing aids beneficial to the agricultural and food industries. Biocatalysis, or reactions modulated by enzymes, allows bioactive materials to assist in bringing a substrate to product. Enzymes are proteins which catalyze reactions by lowering the activation energy required to drive the production of a desired product. Enzymes are commonly utilized in food processing as catalysts with specificity in order to enhance product quality through the production of beneficial food components, and to break down undesirable components that may be harmful or may decrease product quality. Enzymes are proteins with specificity for a substrate, that under the ideal conditions will speed bioprocessing by lowering the activation energy required to create a product. As the working conditions for biocatalytic materials can be very specific, enzymes are often immobilized and incorporated onto and into solid supports in order extend their thermostability and pH optima as well. Integrating biocatalysts into food packaging also allows for extended use and clean-labeling when non-migratory, which may enable “in-package processing” where food constituents undergo changes to improve quality or shelf-life while in transport and storage. Immobilized enzymes are more readily recovered, regenerated, and reused – decreasing overall energy, material, and environment costs. Introducing biocatalytic materials to solid polymeric supports requires varied techniques in order to maintain activity. However, disadvantages to immobilizing techniques lead to activity loss and are attributed to denaturation, incorrect orientation, low protein loading, and material incompatibility. Denaturation and incorrect orientation are characteristic of proteins on hydrophobic surfaces. Covalent immobilization allows for food products to interact with non-migratory biocatalytic coatings without incorporating them into the food matrix. Cross-linker compatibility is an essential part of covalent attachment too. Cross-linkers utilize various functional groups to attach active ingredients to solid surfaces and stabilize polymers. This work progresses through the advantages of covalent and non-covalent enzyme immobilization. First, lactase was immobilized for a bioactive packaging application by Layer by Layer conjugation to low-density polyethylene. Increasing layer deposition increased total protein loading, but did not increase activity per layer. Next, lactase was blended and embedded into polyethylene oxide for an electrospun nanofiber storage and dosing system maintaining up to 92% of free enzyme activity. Embedding and blending is often paired with cross-linking techniques to aid the support material maintain its physical properties. Immobilization of chymotrypsin onto nylon 6,6 demonstrated the benefit of nanoscale materials on retained activity, where the bulk material is water-insoluble. And finally, chymotrypsin was encapsulated by emulsion electrospinning into polycaprolactone with poly(vinyl alcohol) to increase biocompatibility with the solid support. These studies further demonstrate the robustness of enzymes incorporated into packaging materials is dependent on the processing technique and solid support and tether material. Food and agriculture have recently turned to nanomaterials in processing and packaging due to the increased surface area to volume ratio, ease of manufacturing scale-up, and maintained or even improved mechanical stability of nanomaterials. Increased surface area provides for more functional surfaces. A combination of the nanoscale and curvature provided by nanofibers allows enzymes to behave like their free enzyme counterparts. Often nanomaterials may be made uniformly, which also benefits increasing processing efficiency for all industries. The immobilization method must reduce diffusion limitations as well as aid activity retention for increased thermostability and pH stability by taking into account environmental interactions. Herein outlines methods for the incorporation of biocatalytic materials in active packaging by combining the benefits of enzyme immobilization at the nanoscale with complementary material interactions