Food Bio-Based Packaging

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1		Enrico Maurizzi	--	5195	2022-10-26 12:19:10	\|
2	update references and layout	Rita Xu	Meta information modification	5195	2022-10-27 04:31:19	\|

This entry is adapted from the peer-reviewed paper 10.3390/polym14204257

Recently, academic research and industries have gained awareness about the economic, environmental, and social impacts of conventional plastic packaging and its disposal. This consciousness has oriented efforts towards more sustainable materials such as biopolymers, paving the way for the “green era” of food packaging.

biopolymers antioxidant compounds antimicrobial compounds

1. Introduction

Food technologies have played a crucial role since the beginning of human civilisation. Throughout history, the evolution of food processing and packaging has led to a constant increase of food quality and safety, improving the quality of human life ^[1]. Recently, human society has gained awareness about the impact of agri-food practices on our world, and these concerns have oriented the food sector towards the adoption of novel and sustainable technologies.

Among the main pillars of this multifaceted process, it is worth citing three lines of research that have deeply contributed to re-define the concept of “Food Technology” ^[2]:

Substitution of thermal techniques and chemical sanitisation with green alternatives in order to reduce the consumption of resources and the impact on food quality.
Extraction of added-value compounds from renewable sources (e.g., food by-products) and their application as alternatives to conventional preservatives and additives.
Development of bio-based active packaging based on renewable biopolymers, aiming to reduce the use of petroleum-derived plastics in the food packaging sector, and to prolong the shelf-life of the products, preventing the generation of food waste.

2. Bio-Based Packaging: General Considerations

Food packaging is a coordinated system aiming to preserve the safety and quality of the food products from their production to their end-use ^[3]. It plays a crucial role in human society as a fundamental component of the food supply chain ^[4].

Worldwide, it is estimated that one-third of produced food is disposed every year due to various factors including incorrect harvesting procedures, mechanical damage, and inadequate storage conditions, which result in microbial decay, oxidation, the degradation of nutrients, and loss of acceptability ^[5]. Therefore, the selection of adequate packaging solutions able to protect each targeted product and to maintain its quality is crucial to extend the food’s shelf life, thus preventing waste generation.

Conventional packaging is commonly constituted by a one-time use item, immediately discarded after reaching the intermediate or final user ^[4]. Over a broad variety of materials, fossil-based plastics have dominated the food-packaging industry since their appearance during the Second World War ^[6] thanks to their enhanced barrier and mechanical properties, chemical resistance, durability, lightweight nature, availability, and cost-effectiveness ^[7].

Currently, the global production of plastics comprises about 320 million tons/year ^[8]. Data reveal that one-third of all produced plastic is dedicated to packaging materials ^[9]. Hence, the food-packaging industry is closely involved in the production of massive amounts of plastics, generating severe economic burdens and ecological impacts.

The main concern of plastics is related to their non-sustainable nature since their source (petroleum) is not renewable (PE, PET, PP, etc.) ^[10]^[11]^[12]. Besides, single-use plastics are generally considered as not “environmentally friendly” due to their non-compostable nature and low recycling rate ^[13]. This ends up causing the accumulation of tremendous masses of waste in landfills and oceans, increasing wildlife mortality from ingestion and entanglement ^[14].

In the last few years, the awareness about the environmental impacts of plastic has grown both at personal and at community levels. On the one hand, consumers are increasingly demanding natural, high-quality foods, and food packaging that does not create pollution. On the other hand, governments are pushing towards the reduction of human impact on the environment. For example, the European Parliament focused its Sustainable Development Goals on the partial replacement of oil-based polymers with biodegradable polymers from renewable resources by 2030 (European Commission, 2015). This prompted researchers and companies to shift their efforts towards the exploration and exploitation of novel renewable resources and the development of sustainable packaging solutions, including films, coatings, and other items.

Specifically, films are thin layers of material prepared through different technologies such as solution casting or extrusion as stand-alone structures. The prepared films are used to wrap the foods or to be placed between the layers of food products. Coatings are thin layers of material which are directly applied on the food surface, and act as a barrier between the external environment and the product during transportation, processing, and storage. Coatings are applied either by dipping the product in the coating solution or by directly spraying them over the product’s surface.

These novel packaging systems are designed to perform multiple functions. Along with the “classic” packaging activity, namely the interposition of a physical barrier between food and environment, they may operate as carriers of bioactive compounds with antioxidant, antimicrobial, or nutritional properties. These “active ingredients” aim to prolong the shelf life or increase the nutritional value of the packaged product ^[15]. Moreover, the addition of bioactive compounds can result in modified physicochemical, mechanical, and barrier properties since they chemically interact with the biopolymer structure. Hence, their wide application may allow improving or even adapting the functional features of packaging solutions for a broad variety of applications ^[16].

2.1. Compostable, Biodegradable, or Renewable?

Research and industries are pushing towards the usage of biodegradable polymers for food-packaging purposes. Additionally, the extensive exploitation of renewable resources has the potential to reduce the use of oil and other fuels. However, plastics produced by renewable resources are not necessarily compostable or biodegradable, and vice versa ^[17]. For example, cellulose, starch, and gelatin also maintain their biodegradability when obtained synthetically. Equally, when castor oil monomers are polymerised to produce Nylon 9, they lose their biodegradability ^[18]. In fact, biodegradation is correlated to the chemical structure of the compound rather that its origin. In this context, it is important to clearly state the definitions of biodegradation and compostability, allowing further introduction of the concept of biopolymers.

Biodegradation broadly defines an event in which a biomass is over 90% decomposed within 6 months via the action of enzymes and/or chemical degeneration associated with living organisms such as moulds, yeasts, and bacteria [(UNI EN 13432:2002)]. This process can be conducted both in aerobic and anaerobic conditions ^[19]. Other processes such as photodegradation, hydrolysis, and oxidation may also have an impact on the structure of biomass prior to or during biodegradation ^[20]. Compostability involves a series of processes (mainly conducted in industrial conditions) that exploit biodegradation to convert organic matter into the so-called “compost”, which must completely degrade in soil within 3 months by producing water, carbon dioxide, and other inorganic compounds ^[21].

In light of these statements, it is worth noting that the large-scale synthesis of compostable bioplastic using 100% renewable resources has not been realised yet. Until now, bioplastic usually comprises more than 50% (w/w) of renewable sources ^[18]. Several bioplastics include mixtures of synthetic compounds to improve the technical properties of the final product, extending its potential applications. Despite that, the current tendency is to replace synthetic additives with natural compounds with comparable functional properties and to enhance the use of biopolymers over fossil-derived materials to produce approximately 100% renewable and biodegradable plastics.

2.2. Biopolymers

According to the European Bioplastics association, biopolymers are defined as biodegradable, compostable, and biocompatible polymers derived from renewable resources ^[22]. They are broadly regarded as the most promising sustainable alternative to petrol-based synthetic polymers for food-packaging applications due to their compostable nature and film-forming ability ^[20].

Thanks to their technical variability, biopolymers are adaptable to various packaging technologies, offering a range of package products, including cups, covers, separation layers, and food containers. In particular, they can be used to prepare composite films and multi-layered coatings to prolong the shelf-life of food products. Moreover, biopolymers are compatible with functional ingredients including nutraceuticals, antioxidants, antimicrobials, probiotics, and additives ^[23].

Biopolymers have been classified into three categories according to their sources and synthesis: (I) polymers extracted from renewable biomasses, including polysaccharides, polypeptides, and lipids; (II) polymers synthetised from chemical polymerisation of bio-monomers (e.g., polylactic acid); and (III) polymers derived from microbial fermentation (e.g., polyhydroxy alkanoates) ^[19] (Figure 1). Besides, biopolymers can be distinguished according to their hydroplastic or thermoplastic behaviour ^[3].

Figure 1. Classification of biopolymers (reproduced with copyright permission from Chen et al. ^[24]).

Most biopolymers possess remarkable technical features for packaging applications due to their chemical complexity, as shown by the studies in Table 1. A brief description of the most common biopolymers is detailed in the following sub-sections.

Table 1. Cases of study of biopolymers and their effects in food-packaging applications.

Polymers	Additives	Treatments	Solvents for the Polymers	The Effects and Advantages	References
Polybutylene succinate (PBS), Polyhydroxybutyrate (PHB), Polycaprolactone (PCL), Polylactic acid (PLA)	/	Biodegradation test of 10 months at 25, 37, and 50 °C soil and compost	/	Fast degradation of PCL in 8 weeks at 50 °C due to the activity of fungal strain of T. lanuginosus	^[21]
Poly-β-hydroxybutyrate	/	Fermentation	Oily sludge	Isolated 63 bacterial strains that can produce PHAs Presence of Bacillus coagulans in 99.96% of the cases Bacillus coagulans showed a production yield with molasses of 6.36 g/L, B. megaterium	^[25]
PLLA-15% ZIF-8 MOF	/	Extrusion	/	Polymer was not suitable for food packaging because of the high migration level of Zn²⁺ Zn²⁺ release was double in acidic simulant	^[26]
Gelatin 6% (w/v)	Glutaraldehyde (GTA) 50% (w/v of polymer)	Cross-linking	Distilled water	GTA cross-linking enhanced gelatin thermal stability and mechanical properties with pH 4.5	^[27]
Methyl cellulose (MC) 1% (w/v)	Murta berry extract (MU) 25% (w/w of the polymer)Glutaraldehyde (GA) 10–20% (w/w of polymer) Polyethylene glycol (PEG) (25% w/w of the polymer)	/	Distilled water	Cross-linking decreased the swelling index of the materials and increased mechanical properties	^[28]
Binary blend of polymers at a final concentration of 5% of gelatin (GEL) and different polysaccharides: gum arabic (GAR), methylcellulose (MC), octenyl succinic anhydride modified starch (OSA), and water-soluble soy polysaccharides (WSSP)	Glycerol 1% (w/w)	/	Distilled water	Pure OSA film had low plasticity GAR film was weak from a mechanical point of view Incompatibility between GEL and MC, especially at a 50/50 ratio GEL improved the durability and stiffness of the film	^[29]
Polylactic acid (PLA)	Nanocomposite films containing 1−5% (w/w of the polymer) of dye−clay hybrid nano pigments (DCNP)	Cationic exchange reaction between a cationic dye and C20A	Chloroform	Maximum improvement of E′ and glass transition temperature at 3% of DCNP loading level Oxygen permeability and WVP decreased in comparison to neat PLA Optimum of 3% for optical property and UV barrier	^[30]
Polylactic acid (PLA) 1% (w/v)	α-costic acid (α-CA) 7:1 (w/w of the polymer)	/	Chloroform	The plasticising effect of the sesquiterpenoid plant metabolite induced better thermal degradation Homogeneous and efficient inclusion of α-CA in PLA	^[31]
PLA latex	Nanocellulose fibrils with high lignin content (NCFHL) from 5 to 20% (w/w)	Extraction of Thuja plicata bark and fibrillation	Aqueous suspensions of NCFHL	PLA reacted with NCFHL at nearly 50% of the total area NCFHL until 10% enhanced elastic modulus and tensile strength NCFHL increased thermal stability and hydrophobicity	^[32]
Fossil-based and bio-based polycarbonate (PC)	/	Moulding	/	Bio-based PC had weak thermal resistance and low viscosity Good optical property but lower birefringence compared with the fossil PC	^[7]
Sodium alginate 1–3.5% (w/v)	200–800 mg/L of protease from Bacillus brevis 1–3.5% CaCl₂	/	Milli-Q water	∙ Best performance of immobilisation at 2.5–3% of Na-alginate and CaCl₂, with 400–600 mg/L of protease	^[33]
CMC 0.5% (w/v), gelatin (GEL) 0.05–0.25% (w/v)	Sodium benzoate 5–30% (w/v), saturate vapour of glutaraldehyde (GLA)	UV irradiation at (253.7 nm, 30 W) for 30–180 min	Aqueous solution	Best crosslinking rate with 20% SB and 180 min of UV and 0.2% gelatin, associated with exposure of GLA saturate vapour for 90 min Photo-crosslinking enhanced hydrophobicity Both crosslinking methods improved the tensile strength and contact angle of CMC film	^[34]
Chitosan 1.5% (w/v)	Glycerol 30% (w/w of the polymer) and tween 80 0.2% (w/v of essential oil)	/	Distilled water	HAE’s gave light barrier properties, higher water content and solubility EOs enhanced tensile strength	^[35]
Chitosan (CH) 2% (w/w), pea starch (S) 2% (w/w), CH:S 1:4 (w/w)	Lyophilised tannic acid (TA) 1:0.04 (w/w on polymer) or thyme extract (TE) 1:0.15 (w/w on polymer)	/	Water dispersion	TE modified the microstructural appearance of CH, due to crosslinking effect of polyphenols TA and TE gave higher resistance at the break but poor elasticity and opaque films	^[36]
Corn starch and polylactic acid (PLA) blended at a ratio of 80:20	Epoxidised cardoon oil (ECO) 3% (w/w of PLA fraction) and glycerol at 30% (w/w of starch fraction)	Melt blending process	/	Compatibility between ECO and PLA, which gave higher WVP and barrier to O₂ Poor mechanical property of the films	^[37]
Zein (Z) 15% (w/v), gelatin (G) 10% (w/v), blend ZG at different ratios (2:1, 1:1, 1:2) 15% (w/v)	Tea polyphenol 2.5%–7.5% (w/v), glycerol 0.4–0.8 mL	/	Acetic acid (AA) and water	Zein/gelatin ratio influenced mechanical property in multilayer films Multilayers were more transparent and had a higher UV barrier than neat polymers	^[38]
Microcrystalline cellulose 3% (w/w)	68% ZnCl₂ (w/w)	/	Distilled water	Transparent Zn-cellulose film crosslinked with Ca²⁺	^[39]
Polyvinyl alcohol (PVA) 5–12.5% (w/v)	Heat cross-linking with citric acid (CA) 3–12% (w/w of the polymer), Clove oil (CO) 20% (w/w of the polymer)	Electrospinning and cross-linking	Distilled water	Cross-linking process permitted to reach a swelling degree above 400% Microfibers treated with CA were highly hygroscopic Cross-link improved mechanical property and thermal stability	^[40]
Chitosan(CS) 4% (w/v)	Whey protein isolate (WPI) 4% (w/v), microcrystalline cellulose (MCC) 4% (w/v) and glycerin 10–50%	/	Distilled water	Compatibility between polymer and additives Better WVP at 1.5:1 CS/MCC ratio with 30% glycerin and 3.6 of pH	^[41]
Gelatin 6% (w/v)	Galla chinensis extract powder (GCE) 0.03–0.12 g/100 mL	/	Distilled water	GCE worked as a crosslinker for gelatin hydrogel The maximum concentration of GCE improved thermal stability and gel strength	^[42]

∙ Best performance of immobilisation at 2.5–3% of Na-alginate and CaCl₂, with 400–600 mg/L of protease

2.2.1. Polysaccharides

Polysaccharides are complex macromolecules consisting of repeated mono or disaccharide units linked via glycosidic bonds ^[43]. They are natural, easily accessible, non-toxic, and renewable.

Due to their complex structure, polysaccharides exhibit adequate mechanical resistance and high barrier to oxygen (O₂) and carbon dioxide (CO₂). The presence of hydroxyl groups lead to the formation of hydrogen bonds, responsible for inter–intra macromolecular association and thus film-forming ability. However, their hydrophilic nature entails poor moisture resistance and reduced capacity to hinder water vapour transmission ^[23]. To overcome these drawbacks, polysaccharides are modified through chemical pathways to obtain derivatives with enhanced performances or by blending them with hydrophobic materials and nanofillers.

Chitosan

Chitosan, or β-(l-4)-2-amino-2-deoxy-D-glucopyranose, is a cationic linear polysaccharide consisting of N-acetyl-glucosamine and N-glucosamine units. It derives from alkaline N-deacetylation of chitin, the second most abundant natural polysaccharide after cellulose. The primary sources of chitin are shellfish waste, insect cocoons, and fungi ^[44].

Chitosan is biodegradable, non-toxic, bio compatible, and broadly available. It is widely used for many applications in the biomedical, cosmetic, agricultural, and food sectors. The biodegradable property of chitosan results from the sensitivity of glycosidic bonds to chemical and physical breakdown, mainly due to oxidation and reactivity with enzymes (hydrolases), acids, and alkali compounds. Due to the absence of nearly positively charged amino groups, the A-A and A-D glycosidic sections are the preferred targets of hydrolysis in acidic conditions ^[45]. In general, it appears that as the acetylation levels increase, so does the degradation rate. This concept is true even for lysozyme, an enzyme present in human saliva and tears ^[46].

Chitosan is insoluble in water but soluble in acid aqueous solutions due to the protonation of the NH₂ groups. It exhibits good antimicrobial activity against Gram-positive and Gram-negative bacteria, filamentous fungi, and yeasts ^[47].

Chitosan shows excellent film-forming abilities. However, extrusion technology is inadequate to produce chitosan-based films due to the low degradation temperature of this polymer and its non-thermoplastic behaviour. As a result, the production of films is mainly conducted through the solution-casting method.

These films have good mechanical properties and effectively obstruct O₂ and CO₂ transmission ^[48]. Meanwhile, they are highly sensitive to moisture transmission, which compromises their use to preserve fresh or fatty food products. To overcome this criticism, authors investigated different strategies including chemical crosslinking and grafting with secondary components ^[49]. These methods provide an interpenetrated structural network to the resulting films, improving their hydrophobicity. Another suitable technique is blending chitosan with compatible polymers to induce a strong inter–intramolecular hydrogen bonding, which results in improved barrier and mechanical performances of the blend films ^[50].

Cellulose and Derivatives

Cellulose, or (1→4)-β-D-glucopyranosyl, is a linear chain polysaccharide in which anhydrous glucose rings ((C₆H₁₀O₅) n) are bound through β1-4 glycosidic bonds, and the number of repeat units depends on the source material ^[51]. It constitutes the most abundant biopolymer in nature and can be degraded by cellulolytic microorganisms. In nature, the synergism between cellulolytic and non-cellulolytic microorganisms leads to the complete degradation of this polymer. These microorganisms are mainly aerobic and can synthesise cellulases enzymes (cellobiohydrolases and endoglucanases), which hydrolyse the β1-4 glycosidic bonds ^[52]^[53].

Native cellulose is water-insoluble due to its structural complexity, high crystallinity, and tightly packed hydrogen bonds, and is thus unable to form stable gels. This limitation is overcome by applying an alkali treatment followed by acidification using hydrophilic agents such as chloroacetic acid, methyl chloride, or propylene oxide to produce cellulose hydroplastic and thermoplastic derivatives. Cellulose derivatives are commonly isolated from wood, hemp, cotton, and other plant components ^[39]. These derivatives have been extensively investigated to develop biodegradable composites and films due to their high abundance, non-toxicity, and stability (Figure 2).

Figure 2. Examples of possible applications of monomers of cellulose for polymer production (reproduced with copyright permission from Shaghaleh et al. ^[54]).

Hydroplastic polymers obtained from cellulose are highly hydrophilic and possess excellent gelling capacity. They include carboxy methylcellulose (CMC), methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), and others ^[55]. Films and coatings based on these polymers are transparent, odourless, resistant to oxidation, and show enhanced mechanical and gas barrier properties ^[19]. However, they are highly sensitive to water vapour transmission due to their hydrophilic nature, which limits their application to dried and low-fat foods. In this context, several strategies have been investigated to confer hydrophobicity to cellulose-based films, thus reducing their WVP value. Shahbazi et al. ^[34] applied surface modification of CMC based films via reaction with sodium benzoate and glutaraldehyde vapour, followed by photo-crosslinking or chemical-crosslinking with gelatin. Authors observed that photo-crosslinking improved hydrophobicity and water barrier property more than the chemical crosslinking. Another study tested cellulose-based films obtained via chemical crosslinking of CMC with hydroxy ethylcellulose (HEC) using citric acid ^[56].

Cellulose acetate is the most researched thermoplastic polymer derived from native cellulose. This derivative is obtained treating technical-grade cellulose with a methylene chloride-acetic acid solution to substitute hydroxyl groups with acetyl groups ^[57]. FDA tagged cellulose acetate as GRAS, which prompted the food-packaging industry to develop and test novel applications of this polymer ^[54]. Cellulose acetate is commonly used to wrap fresh products and baked goods. Cellulose acetate films and coatings are tough and resistant to puncture. Conversely, they possess relatively poor moisture barrier properties, high rigidity, and lower thermal resistance compared with conventional thermoplastics ^[58]. These criticisms can be partially solved by adding plasticisers, which impart clarity and tailored rigidity. Moreover, when employed for prolonged applications, cellulose acetate may undergo partial hydrolysis to produce acetic acid ^[59].

Starch

Starch represents the primary energy reserve biosynthesised in the plants and one of the most plentiful renewable feedstocks. Native starch consists of two types of glucose polymers: amylose, a linear polysaccharide with (1→4)-α-D-glucopyranosyl units, and amylopectin, branched amylose with (1→6)-α-D-glucopyranosyl side units. Starch has been extensively studied as a biodegradable plastic and food hydrocolloid component thanks to its renewability, biodegradability, and excellent film-forming capacity. This polymer can be easily degraded in water, since amyloglucosidase or α- and β-amylase can form complexes with starch and hydrolyse the glycosidic linkages ^[60]. This process is strongly influenced by pH, the degree of crystallinity of starch, and its retrogradation ^[61].

Starch-based films and coatings exhibit remarkable mechanical strength, elasticity, transparency, and low oxygen permeability ^[15]. The major challenges related to native starch films are brittleness and high hydrophilicity, which results in poor water vapour barrier properties. These drawbacks preclude the application of starch-based films and coatings to package foods sensitive to moisture and oxidation ^[20]. To enhance the flexibility and water resistance, food-grade plasticisers (e.g., glycerol, glycol) and hydrophobic substances can be incorporated into the film-forming solution ^[47].

Pectin

Pectin is an anionic, hydro soluble, and high-molecular-weight heteropolysaccharide. It is one of the main components of the plant cell wall, contributing to tissue rigidity and integrity.

Pectin is chemically composed by poly α-(1→4)-D-galacturonic acid chains ^[62], commonly known as homogalacturonan. Its linear structure is interrupted by rhamnose residues, on which secondary chains containing galactose, xylose, and arabinose are grafted. Consequently, pectin is composed of three different polysaccharide domains. The first domain is the homogalacturonan, which is the smooth component of the molecule. The second domain is named rhamnogalacturonan I and it is constituted by a chain of α-(1,2)-linked L-rhamnopyranose residues. The third one, rhamnogalacturonan II, is characterised by a complex and heterogeneous structure. The second and the third domains form the hairy regions of pectin ^[63] (Figure 3).

Figure 3. Comparison between (a) the traditional and (b) the modern pectin model (reproduced with copyright permission from Willats et al. ^[64]).

The carboxyl groups of galacturonic acid are partially esterified with methanol to form methoxylated groups, and can be converted to amide groups via reaction with ammonia ^[44]. According to the esterification degree (DE), pectin can be classified as low-methoxyl (<50%) and high-methoxyl (>50%) pectin. DE strongly influences the gelling properties of pectin ^[65].

The main industrial sources of pectin are orange pulp and apple pomace ^[47]. Pectin is widely applied in the food industry as a gelling, thickening, and stabilising agent for jam, drinks, and ice cream. It is recognised as safe (GRAS) by the FDA (2013) and it is well known for its biocompatibility, good gelling ability, and biodegradability. Degradation of pectin can be performed through physical (ultrasonication, radiation, photolysis, high-pressure treatment, etc.), chemical (pH differences of 3.5 allow either acid or alkali hydrolysis), and enzymatic processes (mainly pectate lyase, pectin lyase, and endo- and exo-polygalacturonase) ^[66]^[67].

The ability of pectin to form edible films and coatings has been largely investigated ^[63]. Some researchers suggested the scarce potential of pectin as a film-forming polymer due to its limited physicochemical and mechanical performances ^[68]. Despite that, several investigations have been conducted to improve pectin-based filming and coating properties. To enhance the mechanical stability of the film and the surface adhesion on the food substrate, pectin has been blended with food-grade plasticisers (e.g., glycerol, polyethylene glycol, and sucrose) and polymers (e.g., polyvinyl alcohol and cellulose derivatives). As well, pectin has been combined with hydrophobic compounds such as lipids to enhance its resistance to moisture and water vapour transmission.

2.2.2. Proteins

Proteins are complex macromolecules characterised by variable molecular structures and exertion of different functional properties ^[69]. Protein derivatives are commonly isolated from natural resources and represent promising biopolymers to produce biodegradable packaging with excellent physicochemical, optical, mechanical, and barrier performances. In particular, the enhanced capacity of protein-based packaging to control gas transmission allows hindering the loss of flavours and restricting the migration of active components ^[70]. Besides, protein-based packaging can be easily degraded in the environment, and acts as a good biofertiliser due to the high nitrogen content ^[24].

The film-forming ability of protein derivatives strongly depend on their structure (e.g., sequence of amino acids, amount of intra-protein interactions), molecular weight, solubility, and charge ^[69]. Besides, proteins can be combined with other biopolymers, resulting in composite films with improved features ^[71].

Gelatin

Gelatin is a water-soluble protein obtained through the partial hydrolysis of native collagen, a primary component of bones and connective tissues of animals. This protein consists of a triple helix structure with repeated glycine-proline-hydroxyproline units. It is composed by a mixture of α-chains (one polymer/single chain), β-chains (two crosslinked α-chains), and γ-chains (three crosslinked α-chains), with relevant variability depending on the source ^[24]. According to the synthesis method, gelatin is broadly classified as (I) Type A, derived from acid-treated collagen, and (II) Type B, obtained from alkali-treated collagen.

Among biopolymers, gelatin has the peculiar capacity to form thermo-reversible gels with a melting point close to 40 °C. This attribute, along with the abundance, prompted its widespread use in food and pharmaceutical industries as stabilising agent and for the production of biodegradable packaging ^[29].

Gelatin-based films exhibit low O₂ permeability and acceptable mechanical properties ^[72]. Additionally, gelatin can act as a carrier for natural antioxidants and antimicrobial agents. However, these films are highly sensitive to moisture and permeable to water vapour due to their hygroscopic behaviour.

Numerous studies have been conducted evaluating the incorporation of crosslinkers, strengthening nanofillers, plasticisers, vegetable oils (e.g., corn, sun flower, essential oils), and natural polyphenolic antioxidants as promising methods to improve the performances of gelatin-based films and to support their bioactivity ^[42]. In particular, the cross-linking reaction was found to affect the intermolecular forces within the triple helix structure, resulting in an interpenetrated network structure of the film matrix (IPN) ^[27]. Moreover, gelatin has been blended with other biopolymers including chitosan ^[27] and zein protein ^[38] to produce a series of unique hybrid active films. Some studies have found that crosslinking reduces the biodegradability of gelatin. Instead, blending with highly hydrophilic polymers enhances the degree of degradability with respect to pure gelatin. In general, the molecular weight of gelatin typically affects the rate of degradation ^[27].

Corn Zein

Zein is a prolamin protein mainly isolated from corn seeds. It is an alcohol-soluble and biodegradable protein, whose hydrophobic nature relies on the high density of non-polar amino acids ^[73]. Moreover, it exerts a thermoplastic behaviour and outstanding film-forming properties ^[3]. These characteristics make zein a good candidate for the development of biodegradable packaging items. This protein can be easily degraded in specific environmental conditions (neutral pH, 50–60% of humidity, temperature over 40 °C) or in presence of proteases, such as trypsin, thermolysin, and pepsin ^[74].

Zein-based films are smooth, thermally stable, and possess low WVP values ^[75]. These attributes are mainly related to the formation of hydrogen and disulfide bonds between zein chains during solvent evaporation. For this reason, zein-based films can be tailored to act as selective barriers to oxygen, carbon dioxide, and oils. Despite that, these films generally exhibit poor mechanical properties and fragility, which can compromise their wide application. Thus, many strategies have been explored to improve their structural properties, including the addition of plasticisers and combination with other polymers to produce bilayer and composite films ^[15].

2.2.3. Polylactic Acid (PLA)

Polylactic acid (PLA) is a compostable (under industrial conditions), biocompatible, and thermoplastic aliphatic polyester. This polymer can be completely degraded through a slow cleavage reaction of ester bonds. The process of biodegradation is carried out by microorganisms (Actinomycetes, other bacteria, fungi) or by degrading enzymes (proteases, cutinases, and esterases) ^[76].

PLA is obtained either through direct polycondensation of L- and/or D-lactic acid monomers or from the ring-opening polymerisation of lactide monomers. The first pathway is generally followed to produce low-molecular weight PLA, while the second method is applied to produce high-molecular weight PLA ^[20].

PLA is mainly synthetised by microbial fermentation from agricultural renewable sources such as corn, cassava, sugar beet pulp and sugarcane. Although 90% of total PLA is obtained by bacterial fermentation, the remaining 10% is synthetically produced by the hydrolysis of lactonitrile ^[77]. Currently, the annual production of PLA is estimated to be 140,000 tons, with an increasing trend due to its potential as a substitute for petroleum-based materials ^[78].

PLA properties include tensile strength, thermal stability, and gas permeability, and are comparable to those of synthetic polymers such as polypropylene, polyethylene, and polystyrene ^[30]. Moreover, PLA exhibits a better thermal processability compared with other thermoplastic biopolymers, and thus can be processed through conventional blow filming, injection moulding, fibre spinning, thermoforming, and cast filming ^[79].

PLA has been accepted as GRAS by the FDA ^[31]. As a result, this polymer has been increasingly employed in the food-packaging industry to produce disposable cutlery, plates, lids, and other items. Despite that, the high cost and the technical drawbacks, such as brittleness, low resistance to oxygen, and low degradation rate still deter the mass use of this polymer ^[3].

Considerable efforts have been made to improve PLA performances. Different blends of PLA with other natural biopolymers were tested. For example, blending with thermoplastic starch (TPS) enhanced the mechanical properties and the biodegradability rate of the biopolymer and reduced the production cost ^[37]. On the other hand, the PLA/PHB blend obtained by melt blending showed improved oxygen barrier and water resistance compared with pure PLA.

The addition of plasticisers represents another suitable strategy to improve the PLA mechanical performances. Thus, the demand for new “green” plasticisers based on natural and renewable resources such as vegetable oils is rapidly increasing ^[31].

2.2.4. Polycaprolactone (PCL)

Polycaprolactone (PCL) is a semicrystalline biodegradable but non-renewable biopolymer of synthetic origin. This polymer is synthesised through the polymerisation of ε-caprolactone at high temperature (over 120 °C) or polycondensation of hydroxycarboxylic acid, yielding PCL with different degrees of molecular weight based on the alcohols used as catalysts. The final molecular weight affects the polymer’s properties: low molecular weight results in a crystalline, brittle, and hard film; high molecular weight results in a more elastic, tough, and poorly crystalline film ^[80].

PCL is characterised by its good solubility in organic solvents (i.e., chloroform, dichloromethane, benzene, tetrahydrofuran, toluene, etc.) at ambient temperature, insolubility in water, and partial solubility in other organic solvents, such as acetone, acetonitrile, ethyl acetate, and dimethyl formamide. However, the solubility in these last solvents can be enhanced through heat thanks to the low melting point temperature (60–65 °C) ^[81]. Although the physical and mechanical qualities are low and influenced by molecular weight, the barrier properties to oxygen and water vapour are excellent. These characteristics prompt the possibility to combine this polymer with others to improve its gas barrier properties for applications in food packaging. Therefore, PCL has attracted the attention of medical research due to its non-toxicity and potential applications in drug-delivery systems ^[82].

PCL is a biodegradable polymer that can be easily degraded through chemical and enzymatic hydrolysis thanks to the presence of ester groups ^[81]. The enzymatic method is preferable due to the rapid reactions that result in a complete polymer degradation in a few days ^[83]. The composting of this polymer is particularly efficient due to the heat of the process, which can support the biodegradation process, and to the enzymes (in particular, lipase, and esterases) generated by the microorganisms involved in the process ^[80].

2.2.5. Polyhydroxy Butyrate (PHB)

Polyhydroxy butyrate (PHB) belongs to the family of the polyhydroxy-alkanoates (PHAs), a series of biodegradable, crystalline, and thermoplastic polyesters synthesised from microbial fermentation of organic biomass. It is produced by the Gram-positive bacterium Bacillus megaterium ^[25].

This polymer cannot be easily degraded by chemical treatments. Instead, it is more susceptible to thermo-mechanical degradation, oxidation, photodegradation, and enzyme and biotic degradation. The enzymes usually involved in this process are esterases, lipases, and proteases, which work through hydrolysis of ester linkage of the polymer. Biotic degradation is carried out mainly by PHB depolymerase, synthesised by Alcaligenes, Pseudomonas, Comamonas spp., and other species of bacteria, fungi, and algae ^[84].

PHB exhibits remarkable technical performances, comparable to those of polyethylene and polypropylene. Moreover, owing to its lamellar structure, it has superior water vapour barrier properties and a lower carbon footprint than conventional plastics. In fact, it is easily biodegraded by the action of PHA hydrolases depolymerases, which form (R)- and (S)-hydroxybutyrates and other non-toxic compounds under aerobic or anaerobic conditions ^[85].

These attributes make PHB a sustainable candidate for the replacement of fossil commodity polymers for short-term applications. Despite that, some criticisms, i.e., high brittleness, low thermal stability, and reduced processability still limit its widespread use ^[86]. Many attempts have been made to overcome these limitations. Arrieta et al. ^[87] blended PHB with PLA thanks to their comparable melting point temperatures, showing improved flexibility with respect to pure PHB. Additionally, extensibility can be enhanced by incorporating plasticiser or by fabricating composites through the addition of nanofillers ^[86].

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