*3.2. Antioxidant*

Oxidative degradation is a key limiting factor in overall shelf life of many food and consumer products, as well as nutritional supplements. Many packaged products undergo quality deterioration during transport and storage due to oxidative reactions that cause lipid rancidity (e.g., mono and polyunsaturated fatty acids), color loss (e.g., carotenoids, chlorophyll, anthocyanins), and vitamin degradation (e.g., Vitamin A and its precursors, Vitamin C, Vitamin D, Vitamin E). There are several preservation strategies for packaged goods that contain oxidation-sensitive ingredients based on direct addition of antioxidants (e.g., free radical scavengers, metal chelators, singlet oxygen quenchers, oxygen scavengers) and design of appropriate packaging systems. One such preservation strategy is antioxidant active packaging, wherein an antioxidant has been incorporated into a packaging material to enhance food preservation [14]. The most widely used commercially available antioxidant active packaging technologies include oxygen scavengers, manufactured as sachets or labels [89]. However, there is emerging research in the development of migratory and non-migratory antioxidant coatings for active packaging, as surveyed in this section.

Migratory antioxidant active coatings are designed for controlled release of an antioxidant over the course of product shelf life. The majority of research for antioxidant active packaging that is designed for controlled release has been focused on blending of antioxidants with polymers followed by extrusion [4,90]. However, inclusion of antioxidants in packaging may have a negative impact on bulk material properties that may limit applications. Research in the design of active coatings for controlled release of antioxidant is promising for overcoming this challenge. Corlini *et al.* prepared antioxidant packaging materials by spray deposition of citrus oil in methanol onto the surface of PET trays and observed that plasma pretreatment enhanced the adhesion of citrus oil to the surface of PET trays, which demonstrated higher overall antioxidant activity [50]. This coating exhibited antioxidant activity with cooked turkey meat and retained activity after six months of storage [51,52]. Bolumar *et al.* coated rosemary extract directly onto LDPE plastic wrap (0.45 mg rosemary cm<sup>−</sup>2) and demonstrated significant delay in the onset of surface lipid oxidation of wrapped chicken patties. Controlled released of migratory antioxidant coatings may be enhanced by using a polymeric carrier for antioxidants rather than direct surface application [54]. For example, Lee *et al.* applied α-tocopherol to paperboard using a vinyl acetate-ethylene copolymer as a carrier for controlled release (D = 2.91 × <sup>10</sup>−<sup>11</sup> <sup>m</sup>2·s−1) and demonstrated its ability to retard oxidation in an oil-in-water emulsion [56].

Non-migratory antioxidant coatings are designed to scavenge prooxidants, such as free radicals, oxygen and transition metals, to extend the shelf life of oxidation-sensitive foods. Garces *et al.* patented an antioxidant coating composed of a polymer blend that contains plant extracts that scavenge free radicals [91]. Tovar *et al.* demonstrated that this antioxidant coating may be classified as non-migratory as it demonstrated migration that was 20 times less than the legal limits for the European Union [92]. Furthermore, this antioxidant active packaging coating derives activity from free radical scavenging in the packaging headspace, thus not requiring direct contact with the food [93–95]. Shutava *et al.* imparted free radical scavenging properties on glass slides via layer-by-layer deposition of tannic acid and poly(allylamine hydrochloride) and found that 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic

acid) (ABTS) radical scavenging activity per mole immobilized tannic acid decreased with increasing number of bilayers, most likely due to inability of ABTS to diffuse through bilayers [76]. However, increasing the number of bilayers did result in a linear increase in overall scavenging activity (mol ABTS cm<sup>−</sup>2). There has also been significant research on metal oxide coatings that have been applied to packaging materials to reduce oxygen diffusion into packaging to extend shelf life [67,96].

Non-migratory antioxidant coatings may also be applied by covalent immobilization of functional groups to the surface of packaging materials. Gallic acid was immobilized to chitosan by carbodiimide assisted reaction that resulted in an active packaging material that demonstrated significant reduction in oxidation of ground peanuts [63]. In other work, Arrua *et al.* utilized photoinitiated graft polymerization of a polymer functionalized with caffeic acid to coat polypropylene packaging materials and demonstrated its ability to prevent oxidative degradation of ascorbic acid in orange juice [79]. Photoinitiated graft polymerization has also been used to fabricate metal chelating active packaging coatings that extended the lag phase of lipid oxidation in oil-in-water emulsions by chelating transition metals, which are the most influential prooxidants in food emulsions [83,87]. Further research in this area demonstrated that active packaging coatings composed of photografted polyhydroxamate chelators retain activity in a wide range of pH values (3–5), viscosity (~1–104 CP) and competing ion (Na, Mg, Ca) conditions typically found in food and consumer products [97]. In addition to potential "clean label" regulatory benefits of non-migratory antioxidant coatings, such technologies have product quality advantages over migratory antioxidant packaging coatings, as migratory antioxidant coating technologies typically use active agents which must be used at a concentration which alters sensory perception (flavor, color, viscosity) of the packaged product. Nevertheless, technical hurdles remain in adapting non-migratory active coatings for commercial application.

#### *3.3. Biocatalytic*

The partnership of enzymes and solid support materials provides many opportunities for biocatalytic coatings for active packaging. Enzymes are proteins with enhanced specificity to a substrate, which catalyze reactions by lowering their activation energy to create products. Enzymes are employed in numerous processes used in the food, pharmaceutical, and diagnostic industries. In food processing, biocatalysts are used in production of ingredients, enhancement of product quality, and breakdown of undesirable components that may be harmful or may decrease product quality [98]. As the working conditions for biocatalytic materials can be very specific and variable with the immobilization method and material, there is much research on the immobilization of enzymes onto and into solid supports in order extend their thermostability, pH optima, and solvent stability. Integrating biocatalysts into packaging materials facilitates their use as active packaging coatings [99] which may enable so-called "in-package processing" where food constituents undergo changes to improve quality or shelf-life while in transport and storage.

The goal of many biocatalytic active packaging technologies is to control the growth of spoilage and/or pathogenic microorganisms. Lysozyme is a commonly utilized enzyme for such antimicrobial active packaging. Such antimicrobial enzymes have been incorporated into active packaging coatings via blending, non-covalent binding for controlled release, and covalent immobilization [100–102]. For additional information, refer to the comprehensive section on antimicrobial coatings of this review. Herein outlines methods for the incorporation of enzymes into biocatalytic coatings for active packaging, and a few works which have the potential for application in active packaging coatings.

Incorporation of enzymes into active packaging coatings by embedding and blending requires compatibility between the enzyme and the coating matrix. Compatibility can be achieved by surface functionalization, altering bulk material composition, and enzyme modification. Embedding and blending may simplify commercialization and scale-up as one-pot preparation methods become more available [16]; however, these techniques typically result in the enzyme becoming a part of the packaged product, reducing catalytic activity of the coating over time [103]. A targeted application for several biocatalytic active packaging coatings technologies involves the removal of oxygen for increased food shelf stability. Efforts towards incorporation of glucose oxidase in a low-density polyethylene and paper board multilaminate have included details for scaled-up production. Various compositions of LDPE, glucose oxidase, and catalase were produced in Tetra Pak's pilot plant, and showed up to 97% activity could initially be achieved even after exposure to 325 ◦C during production. Control of production parameters was key to maintaining the package's oxygen scavenging capability [104]. Johansson *et al.* worked to improve embedding the glucose oxidase and catalase oxygen scavenging pair by varying combinations of LDPE, polypropylene, and polylactic acid [55]. Variations of these embedding methods for oxygen scavenging by laccase and oxalate oxidase have shown similar results [55,57]. Recent work by Talbert *et al.* demonstrates that enzymes may be modified by hydrophobic ion pairing to be soluble and retain activity in solvents used in ink formulations, enabling the preparation of biocatalytic active packaging coatings using existing printing technology [105].

Much of the work on biocatalytic coatings for food packaging involves covalent immobilization. Covalent immobilization allows for biocatalytic coatings to interact with packaged products without being incorporated into the food matrix, enabling their regulation as contact materials rather than direct additives. Non-migratory biocatalytic coatings therefore offer a potential regulatory benefit, as there is growing demand for "clean label" products. For example, Soares and Hotchkiss developed non-migratory packaging films to de-bitter fruit juices by covalently immobilizing fungal naringinase [70,71]. Naringinase activity was maintained for 15 days storage after preparation of the biocatalytic active packaging coating, and *kM* values were lower when compared to free enzyme. Nunes *et al.*, achieved similar success by cross-linking naringinase to polyvinyl alcohol and alginate using boric acid for de-bittering [72]. In other work, β-galactosidase was bound to amine-functionalized polyethylene films by a dialdehyde tether to reduce milk lactose in package [64]. Further research demonstrated that tether length and chemistry can influence retained activity of immobilized lactase [65], but more significant enhancements in retained activity can be achieved by immobilization onto nanostructures [106].

Layer-by-layer deposition allows for more enzyme to be incorporated in a biocatalytic coating compared to traditional immobilization techniques. Indeed, increasing the number of functional layers increases total protein content. Biocatalytic coatings prepared by layer-by-layer deposition of polyethylenamine, glutaraldehyde, and lactase exhibited increased protein loading with each layer [77]. However, overall activity did not increase with layers, likely a result of reduced substrate accessibility by enzyme entrapped in sub-interfacial layers. In other work, Shutava *et al.* layered hemoglobin, PS, and catalase in order to create a physical and chemical protective barrier [68]. Although the layer-by-layer technique has been shown to increase protein loading, diffusion of substrate to enzyme can become difficult. Increasing substrate diffusion through formed layers would improve activity retention, and therefore commercial potential, of biocatalytic coatings prepared by layer-by-layer deposition.

Photografting is often used in combination with other surface modification methods for preparation of biocatalytic coatings. Garnett *et al.* published an early report on immobilization of the protease trypsin to PP, PVOH, and PS by enhancing photografting with the use of metal salts [80]. Carboiimide chemistry can be used to covalently couple enzymes (via amine groups) to carboxylic acid groups introduced to a material surface via photografted polymerization [81]. Krenkova *et al.*, functionalized poly(ethylene) glycol methacrylate to immobilize trypsin and endoproteinases for antibody analysis in enzyme reactors. In that work, 4-vinyl-2,2-dimethylazolactone was photografted to introduce a porous surface morphology with the goal of optimizing enzyme orientation to improve performance. As a result, non-specific protein binding was reduced and the protein substrate was successfully digested [82]. In other work, the hydrolase urease was immobilized to photgrafted polytetrafluoroethylene to remove urea from beverages and foods by Yamada *et al.*, After immobilization, urease exhibited lowered activity at higher protein contents due to high protein densities as a result of grafted layers [86]. Indeed, a common challenge in enzyme immobilization

is tailoring immobilization density and material chemistry to reduce adverse protein–protein and protein–surface interactions [107].

Emerging technologies in biocatalytic materials such as electrospun nanofibers and biopatterning offer new opportunities in preparing biocatalytic active packaging coatings. Electrospinning is a method by which enzymes or other active agents can be incorporated into polymer nanofibers. In one report, lactase blended into polyethylene oxide nanofibers retained up to 93% of free enzyme activity with significant retention of activity after dry storage [53]. In other work by Ge *et al.*, glucose oxidase was immobilized onto electrospun polyvinyl acetate/chitosan/tea extract fibers to reduce oxygen in packaged foods to extend shelf life [108]. The immobilized glucose oxidase in this system retained over 68% of free enzyme activity. Drug delivery with controlled release has sparked new interest in various fiber morphologies, as well as micro and nanoparticles and hydrogels. Embedding and blending is often paired with cross-linking techniques to improve retention of the support material's physical properties [109]. Biopatterning, in which specific patterns of immobilized biomolecules are defined with micron or submicron resolution, also has potential application in active packaging coatings. For example, patterning cells to the interior of microfluidic channels by photografting poly(ethylene glycol) reduced non-specific protein binding [110,111]. Creating specific patterns for enzyme immobilization can focus activity on targeted regions in a packaging matrix, thereby reducing material waste during functionalization and controlling coating costs. Because of the interdependence of the enzyme structure on biocatalytic activity, preparation of biocatalytic coatings demands a whole systems approach, with consideration given to the enzyme and the coating matrix as well as the packaged product. Consideration must be given to reducing diffusional limitations of substrate and product, as well as activity retention for increased thermostability and pH stability. The overall stability of the bound enzyme determines the success of the coating method.

#### **4. Challenges and Perspectives**

Exciting opportunities exist in the development of active packaging coatings for improving the safety, quality, and shelf life of packaged goods, which is brought about by an increasing preference and demand from consumers for additive-free foods and a continuously expanding transport chain and standards of quality driven by global commerce [112]. The global trade of active food packaging was estimated to be close to US \$9,000,000,000.00 in 2011 and it is expected to be around US \$12,000,000,000.00 in 2017 [113]. Nevertheless, the active food packaging market is currently widely dominated by oxygen scavenging and moisture absorption applications [112,114]. A number of challenges remain prior to the commercial adoption of the types of applications studied in this review. A key consideration in developing coatings for active packaging applications is considering toxicity and potential for regulatory approval of not just the active agent but any tether molecules or cross-linkers that may be employed [115]. Consideration must also be given to requirements of different regulatory agencies, whether under the jurisdiction of the Food and Drug Administration (in the United States), the European Food Safety Authority (in the European Union), or elsewhere. Non-migratory technologies produced by either covalent immobilization, cross-linked layer-by-layer deposition, or some photografted coatings offer a potential regulatory benefit in US regulated food packaging applications, as they would require approval as food contact materials rather than direct additives [116]. Migration testing using standardized simulants (water, 3% acetic acid, 15% ethanol, olive oil, iso-octane, and 95% ethanol) must be performed to quantify levels of migrants in packaged product systems [117]. It is also critical to consider the influence of the active coating on other performance characteristics of the packaging material. A benefit to coatings over bulk material modification is that bulk material properties should remain intact. However, the influence of the coating on processability, thermomechanical properties, barrier properties, and seal strength must be characterized [118]. Rigorous application tests must also be performed to ensure that neither material conversion steps nor end use result in delamination of active coatings. Many of the coatings technologies surveyed in this report have potential for scalability to roll-to-roll, high throughput

coating operations. Finally, while incorporation of active agents and specialized packaging processes will indeed increase material cost, the opportunities for new products, enhanced safety, and reduced waste of packaged goods highlight the potential for increasing product value through smart integration of active packaging coatings.

**Acknowledgments:** This material is based upon work supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under project numbers 2012-67017-30157, 2011-65210-20059, 2014-67021-21584, and 2015-67017-23119. M.J. Roman would like to acknowledge the Peter Salmon Graduate Fellowship (Department of Food Science, UMass Amherst) and Northeast Alliance for Graduate Education and the Professoriate Fellowship for their support. D.E. Wong acknowledges Agriculture and Food Research Initiative Grant No. 2015-67011-22820 for NIFA Predoctoral Fellowship support.

**Author Contributions:** Luis J. Bastarrachea wrote the overview of coating technologies and the antimicrobial applications sections and contributed to overall document formatting; Dana E. Wong wrote the biocatalytic applications part; Maxine J. Roman wrote the antioxidant applications section; Zhuangsheng Lin contributed the graphics and proofread the manuscript; and Julie M. Goddard wrote the challenges and perspectives section and edited the full text.

**Conflicts of Interest:** The authors declare no conflict of interest.


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