Biopolymer-based preservation of perishable products

This patent application claims priority to and the benefit of U.S. Provisional Patent Application Serial No. 61/949,995, filed March 7, 2014, entitled “BIOPOLYMER-BASED PRESERVATION OF PERISHABLE PRODUCTS,” the entire contents of which are incorporated herein by this reference.

BACKGROUND

The preservation of perishable products, particularly foods, typically involves preventing the growth of bacteria, fungi (such as yeast), and other microorganisms, as well as delaying the oxidation of fats that cause rancidity. Food preservation also involves inhibiting enzymatic processes that lead to discoloration and/or changes in the texture of perishable products. In addition to or in conjunction with visual deterioration, maintaining nutritional value and flavor is also an important aspect of food preservation.

There are a number of techniques that have been traditionally used to improve the preservation of perishable products. These include, for example, drying, pasteurization, refrigeration, freezing, vacuum packaging, salting or curing, sugaring, smoking, chemical additives, pickling, caustic soda, canning and bottling, irradiation, etc.

More recently, food coating (sometimes referred to as glazing) has been exploited as a means of improving preservation as well as organoleptic properties. Several classes of biopolymers have been considered as coating materials: polysaccharides, proteins, lipids, as well as various combinations of these biopolymers (reviewed, for example, in Biopolymers: New Materials for Sustainable Films and Coatings). Copyright © 2011 John Wiley & Sons, Ltd., Editor(s): David Plackett; Print ISBN: 9780470683415; Online ISBN: 9781119994312; CH. 10 “Food Packaging Applications of Biopolymer-Based Films”; CH. 11 “Biopolymers for Edible Films and Coatings in Food Applications”; the contents of which are incorporated herein in their entirety by this reference. Polysaccharides and proteins are known for forming films with good mechanical properties but low permeability, while lipids form fragile films with better permeability. Coating fruits and vegetables with a wax material is also a common example. The main reasons for waxing are to prevent water loss, thereby delaying shrinkage and deterioration, and improving appearance. The waxing materials used for such purposes depend to some extent on regulations in the country of production and/or export; both natural waxes (e.g., sugarcane, carnauba, shellac, and resin) and petroleum-based waxes are used. Wax can be applied in a volatile petroleum-based solvent, but today it is more commonly applied via a water-based emulsion. Paraffin waxes blended as an oil or paste are often used on vegetables. These techniques are useful for selective products, but not for others.

COMPENDIUM OF THE INVENTION

The present invention provides, among other things, biopolymer-based compositions and methods for the preservation of perishable products, including foods.

In particular, the invention includes the recognition that some biopolymers offer structural characteristics that are particularly well-suited for the production of coating materials for perishable products, without the requirement of adding plasticizer to achieve the desired malleability. Accordingly, safe, simple, and versatile coatings that improve the preservation of various perishable products are described herein.

In some embodiments, biopolymer-based coating materials used to preserve perishable items are useful for forming a barrier between a perishable item and one or more elements in its environment. In some embodiments, such coating materials provide a selective barrier between the perishable item and at least one aspect of its environment. For example, such materials provide a selective barrier between the perishable item and at least one aspect of its environment, such as light (e.g., certain wavelength ranges of light), temperature, humidity or water content, microbes (e.g., bacteria, fungi, etc.), etc.

The invention also encompasses the notion that the biopolymer-based coatings described herein can be used as a carrier for one or more agents. In some embodiments, such coatings incorporate one or more agents to further enhance the preservation of a perishable item. Examples include, but are not limited to, enzyme inhibitors, antimicrobial agents, and ethylene-entrapment agents.

Additionally or alternatively, in some embodiments, such coatings have an agent or agents that are used for purposes other than better preserving a perishable item. For example, such coatings may incorporate an agent or agents to provide additional attributes or characteristics, including, but not limited to, enhanced or added taste or flavor, appearance, nutritional content, flavorings, etc. In some embodiments, additional agents may provide functionality such as labels or codes that have certain information. Any appropriate or desirable information may be included or encoded, including, but not limited to, identifying information such as information about the source or origin of a product, ingredients, nutritional information, manufacturing information, processing dates (harvest date, coating or packaging date, etc.), expiration dates, pricing information, authentication, advertising, customer service information, or any combination thereof.

In some embodiments, the biopolymer-based coatings described herein can enhance one or more characteristics of a perishable product. For example, such coatings can provide improved appearance (e.g., color, shine/luster, etc.), improved texture (e.g., crispness, etc.), and so on.

In some embodiments, biopolymer-based coating materials used to preserve perishable products are applied directly so that they adhere to the surface of the perishable item.

In some embodiments, biopolymer-based coating materials used to preserve perishable products provide packaging for the perishable items.

In some embodiments, biopolymer-based coating materials used to preserve perishable products form an edible coating for a perishable item, e.g., food. In some embodiments, such coating materials do not need to be washed before consumption. In some embodiments, such coating materials can be washed, e.g., with water before consumption.

In some embodiments, the biopolymer-based coatings described herein do not adversely affect the taste, flavor, and/or appearance of the perishable food coated therewith. In some embodiments, the biopolymer-based coatings described herein do not adversely affect the taste, flavor, and/or appearance of the perishable food coated therewith.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 provides sequential images of the aging process of strawberries. Strawberries were stored at 22 °C and 38% RH as received (control) and after coating with silk fibroin solution (DxCx). Dx represents 'x' dip-coating steps. Cx represents 'x' hours of water annealing. Therefore, D1C0 means strawberries that were dip-coated only once and were not exposed to water annealing.

Figure 2 provides comparative images showing the internal tissues of strawberries after seven days of storage under standard conditions. Strawberries were stored at 22 °C and 38% RH as received (control) and after coating with silk fibroin solution (DxCx). Dx represents 'x' dip-coating steps. Cx represents 'x' hours of annealing in water. Therefore, D1C0 means strawberries that were dip coated only once and were not exposed to water annealing.

Figure 3 provides a graph showing the weight loss of stored strawberries as a function of time (over seven days under standard conditions). Strawberries were stored at 22 °C and 38% RH as received (control) and after coating with silk fibroin solution (DxCx). Dx represents 'x' dip-coating steps. Cx represents 'x' hours of annealing in water. Therefore, D1C0 refers to strawberries that were dip-coated only once and not exposed to water annealing. A two-way ANOVA with Tukey's mean analysis was used to evaluate the data. Silk crystallinity affected the dehydration of the strawberries considered, but not the number of coating steps. Uncoated controls lost approximately 50% of their original weight over the 7 days considered (highlighted within the red rectangles). Strawberries coated with amorphous silk (DxC0 - within the blue rectangles) retained more water compared to controls on day 3 (p<0.05). Strawberries coated with crystalline silk additionally reduced the rate of fruit dehydration compared to the amorphous coating (p<0.05) and the control (p<0.05) but no statistical difference was found for different annealing time in water (p>0.05).

Figure 4 illustrates the interaction between water and thin biopolymer membranes. Figure 4 shows in panel (a) wicking; (b) hydrodynamic permeability; and (c) water diffusion coefficient. For the wicking study, crystalline silk fibroin membranes (thickness = 130 pm) showed stained water wicking in both the Z and XY directions, indicating capillary diffusion of water through the thin silk fibroin constructs. Hydrodynamic permeability studies did not show a statistically significant effect (p>0.05) of silk fibroin crystallinity on water permeation (based on a one-way ANOVA with Tukey's mean analysis). The water diffusion coefficient study revealed that the crystallinity of silk membranes slightly affected water mass transport through silk (based on a one-way ANOVA with Tukey's mean analysis).

Figure 5 provides images of the banana ripening process for the evaluation of bananas ripening with and without silk coating. Fruits were stored at 22°C and 38% RH as received (control) and after coating with crystalline silk fibroin films (silk-coated). Bananas were hung from their respective stems throughout the experiment. Figure 5 shows (panel (a)) time-lapse photography of ripening bananas indicating that silk coating slowed down the ripening rate. Figure 5 shows (panel (b)) Investigation of the turgor of the silk-coated banana (i) compared to the uncoated control (ii). The test was carried out on day 9 after coating. The silk-coated banana showed increased firmness compared to the uncoated control. Figure 5 shows in panel (c) a photograph of the inner flesh of uncoated (i) and coated (ii) bananas on day 9 after silk coating treatment. The flesh of the uncoated banana was brown in color, whereas the silk-coated fruits retained a light-colored flesh, indicating a slower ripening rate in the silk-coated sample.

Figure 6 provides a graph showing the chemical characterization of the silk fibroin coating on banana peels. ATR-FTIR was used to investigate the silk structure on banana peels (blue line) after dip coating (red line) and after annealing in water for 12 hours (green line).

Figure 7 shows the coatings of perishable fruits with edible silk fibroin. Figure 7 in panel (a) shows that the impact of edible silk fibroin coating was investigated on freshly picked strawberries. Figure 7 in panel (a)(i) shows that silk fibroin was extracted from Bombyx mori cocoon fibers by dissolution in 9.3 M LiBr solution and Figure 7 in panel (a)(ii) by dialysis in deionized water. The protein concentration in water was then adjusted to 1% by weight.

Figure 7 in panel (a)(iii) shows that the coating of the strawberries was then achieved by dip coating in silk fibroin solution (1% by weight). The dip coating process was repeated up to 4 times. Figure 7 in panel (a)(iv) shows that the crystallinity of the edible silk fibroin coating was modulated using water annealing after processing. The longer the exposure to water vapor (up to 12 hours), the higher the degree of protein crystallinity. Figure 7 in panels (a)(v) shows that silk fibroin-coated strawberries were then left at ambient conditions (T = 22 °C, RH = 38%) to investigate the impact of the edible coating on fruit quality. Crystal violet dye was used to stain the silk fibroin coating. Figure 7 in panel (b) shows representative macroscopic images of stained strawberries. Figure 7 in panel (b)(i) shows freshly picked strawberries, Figure 7 in panel (b)(ii) shows strawberries coated with an edible coating of amorphous silk fibroin (4 dip-coating processes, no water annealing was applied), and Figure 7 in panel (b)(iii) shows strawberries coated with an edible coating of crystalline silk fibroin (4 dip-coating processes, hours of water annealing). The crystal violet dye is barely visible on the surface of the coated strawberries (black dots) because the coating was only a few microns thick. Figure 7, panel (c), shows stereoscopic images of the surface and cross-section (insets) of fresh strawberries stained with crystal violet.

Figure 7 in panel (c)(i) shows harvested strawberries, Figure 7 in panel (c)(ii) shows strawberries coated with an edible coating of amorphous silk fibroin, and Figure 7 in panel (c)(iii) shows strawberries coated with an edible coating of crystalline silk fibroin. Graphic scale: 2 mm.

Figure 8 shows the ripening and weight loss of strawberries coated with edible silk fibroin coating. Figure 8, panel (a), shows the time course of strawberry ripening. Harvested strawberries were either stored at 22 °C and 38% RH (uncoated) or dip-coated in silk fibroin solution (amorphous silk coating). Annealing in water was used as a postprocess to modulate the degree of crystallinity of the silk fibroin. On day 7, the crystalline silk fibroin coating alone demonstrated an improvement in the quality of the stored strawberries. Figure 8, panel (b), shows the weight loss of strawberries stored for up to 14 days at 22 °C and 38% RH. Strawberries were stored as picked (i.e., uncoated) or after coating with silk fibroin solution (DxCx). Dx represents 'x' dip-coating steps. Cx represents 'x' hours of subsequent water annealing process (e.g., D1C0 means that the strawberries were dip-coated once and not exposed to water annealing). A two-way ANOVA with Tukey's mean test was used to evaluate the weight loss data. Silk crystallinity affected the dehydration of the strawberries considered, but not the number of dip-coating steps. Uncoated controls lost approximately 70% of their original weight over the 14 days considered (highlighted within the red rectangles). Strawberries coated with amorphous silk (DxC0) retained more water than uncoated controls on day 3 (p<0.05). The crystalline silk fibroin coating further slowed the rate of fruit dehydration compared to the amorphous silk fibroin coating (p<0.05) and the uncoated control (p<0.05), but no statistical difference was found for different annealing times in water (p>0.05).

Figure 9 shows the permeability and diffusion coefficient of water and oxygen in silk fibroin films as a function of protein crystallinity and their effects on the quality of the edible coating. Figure 9, panel (a), shows the hydrodynamic permeability in silk fibroin films. The degree of protein crystallinity does not affect water infiltration through the film (p<0.05). Figure 9 in panel (b) shows the water diffusion coefficient in silk fibroin films. The degree of protein crystallinity affects the water diffusion coefficient in the transient state (0<t<25 min) but not in the steady state (t>25 min). Figure 9 in panel (c) shows the oxygen diffusion in silk fibroin films. Silk fibroin polymorphism greatly affects oxygen diffusion as a 50-fold decrease in the effective oxygen diffusion coefficient was measured between a highly crystalline and an amorphous silk fibroin film. Figure 9 in panel (d) shows that the respiration rate of silk-coated strawberries was measured as a function of coating crystallinity, with higher degrees of crystallinity corresponding to a statistically significant decrease in CO2 production (p<0.05). Figure 9 in panel (e) shows the effects of silk coating on strawberry firmness as a function of storage time and coating crystallinity. Natural decomposition of strawberries caused a decrease in fruit firmness, as measured by a time-dependent decrease in the force required to penetrate the fruit (p<0.05). An increase in coating crystallinity corresponded to a statistically significant delay in the deterioration of fruit firmness on days 3 and 7.

Figure 10 shows an evaluation of banana ripening with and without crystal silk coating. Fruits were stored at 22°C and 38% RH as received (uncoated) and after coating with crystalline silk fibroin films (crystalline coating). Bananas were hung from their respective stems throughout the experiment. Figure 10 in panel (a) shows a time-lapse photograph of ripening bananas, indicating that the silk coating slowed the ripening rate. Figure 10 in panel (b) shows an investigation of the turgor of the silk-coated banana. Figure 10 in panel (b)(i) shows the turgor of an uncoated control. Turgor was studied qualitatively by applying a dead load (200 g) to the fruit surface. Figure 10 in panel (b)(ii) shows that the test was carried out on day 9 after coating. The silk-coated banana showed greater firmness compared to the uncoated control. Figure 10 in panel (c)(i) shows images of the inner flesh of an uncoated banana and the

Figure 10 in panel (c)(ii) shows images of the inner flesh of a coated banana on day 9 after silk coating treatment. The flesh of the uncoated banana was brown in color, whereas the silk-coated fruits retained a light-colored flesh, indicating a slower ripening rate for the silk-coated samples.

DETAILED DESCRIPTION OF CERTAIN MODALITIES

The present disclosure provides edible biopolymer-based coatings for coating perishable products, such as food items. Among other things, the present invention encompasses protein-based (i.e., polypeptide-based) coating materials and related methods that do not require the use of added plasticizers. Certain physicochemical characteristics and desirable materials of such proteins (i.e., peptides) are described in more detail below. Coatings prepared in accordance with the present invention exhibit superior ability to preserve perishable products such as fresh fruits, compared to known edible coatings. Furthermore, these coatings allow for functional versatility, as additional agents can be incorporated to further control the preservation process of perishable items or for other purposes. Furthermore, certain characteristics (e.g., fragility) of these coatings can be modulated without the use of additives, providing adjustability depending on the application.

Biopolymer-based food coatings have been extensively studied and widely used in the food industry. Perhaps the most common example is the use of wax to coat fruits and vegetables. Waxes are organic compounds that typically consist of long alkyl chains. Natural waxes are typically esters of fatty acids and long-chain alcohols. Synthetic waxes are long-chain hydrocarbons that lack functional groups. Wax's hydrophobicity makes it an attractive moisture barrier for keeping fruits and vegetables fresh. Wax suitable for coating food, however, is also brittle and is typically used in conjunction with a softening agent (i.e., a plasticizer). For example, wax can be mixed with a biopolymer (e.g. chitosan, gelatin) that acts as a plasticizer.

Other biopolymers that have been used as coating materials include, but are not limited to, various proteins, such as collagen, gelatin, corn zein, wheat gluten, casein, and whey. Both collagen and gelatin are very hydrophilic and therefore do not provide an effective moisture barrier. Corn zein, on the other hand, is a highly hydrophobic protein, and due to its abundance, it has also been exploited in the food industry for a number of applications. However, as a coating material, its fragility typically requires the use of an added plasticizer. Furthermore, zein proteins do not remain transparent, as they turn white upon contact with water (e.g., humidity), which is undesirable in some applications. Wheat gluten also requires a plasticizer for use as a coating material. Casein and whey can also be used for the production of edible film materials, but these are generally used as composite films. In addition, the addition of plasticizers is typically required.

Unlike these currently commercially exploited biopolymer-based coatings, the biopolymer-based coatings described herein provide superior material characteristics with desirable functional attributes. For example, such coatings (i) may be used to form a barrier between the perishable article and its environment; (ii) may be used as a carrier for an agent; (iii) may be used as an enhancer of at least one property of the perishable article; or any combination thereof. In any of these functional parameters, the biopolymer-based coatings described herein exhibit superior performance compared to commercially available coatings described in the prior art.

Coated perishable products

Accordingly, in one aspect, products are provided comprising a perishable article and a coating. Such products include at least one perishable article, at least a portion of which is in contact with a biopolymer-based coating material.

In the context of the present description, “perishable” products refer to articles that are susceptible to at least one type of damage (e.g., reduced quality), which typically involves changes in one or more parameters, such as water content, color, general appearance, taste or flavor, texture (e.g., visual texture such as softness and structural texture such as crispness), structural integrity, odor, bacterial or fungal growth, etc. Non-limiting examples of perishable items may include, but are not limited to: Food items, such as fresh agricultural products (e.g., fruits and vegetables), meat products (e.g., processed meat products and raw meat), grains, nuts, seeds, spores, dairy products (e.g., cheese), beverages (e.g., alcoholic beverages, wine, processed foods, juices), (e.g., snacks), tablets and capsules, such as gel capsules, plants and flowers, and the like.

As described in more detail herein, in some embodiments of the invention, the coatings used for a perishable article comprise a biopolymer. In some embodiments, the coatings described herein are made from protein-based coating materials. The terms “protein” and “polypeptide” are used interchangeably herein unless otherwise specified.

In some embodiments, biopolymer-based coating materials contain one or more proteins. Such materials may also contain additional biopolymer components. For example, biopolymer-based coating materials of the present invention include those with a second biopolymer (i.e., copolymer) mixed therein. Additional components may include, but are not limited to, polypeptides, fatty acids, waxes, starches, carbohydrates, and polysaccharides, among others. In some embodiments of the invention, a biopolymer-based coating encompassed by the present invention consists essentially of a protein and water.

Structural features of preferred polypeptides

Although several protein-based food coatings have been explored to date, the present invention encompasses the recognition that peptides having certain structural characteristics are especially useful as a coating material in accordance with the methods provided herein. In particular, peptides that are generally hydrophobic but also exhibit amphiphilic nature have been found to be suitable for coating perishable products.

The term "amphiphilic" refers to having both hydrophobic and hydrophilic properties. For example, surfactants and zwitterions are common amphiphilic substances. Typically, an amphiphilic protein contains one or more hydrophobic parts (e.g., sections, domains, or moieties), as well as one or more hydrophilic parts (e.g., sections, domains, or moieties), making the protein amphiphilic. In some embodiments, amphiphilic peptides can form micellar structures (i.e., micelles) in a solution.

Accordingly, in some embodiments, an amphiphilic polypeptide suitable for the present invention contains one or more hydrophobic portions and one or more hydrophilic portions. In some embodiments, an amphiphilic polypeptide suitable for the present invention contains hydrophobic and hydrophilic intermediate segments. In some embodiments, an amphiphilic polypeptide suitable for the present invention contains hydrophobic and hydrophilic segments arranged in tandem. In particularly useful embodiments, the hydrophobic segments of an amphiphilic polypeptide predominate over the hydrophilic segments. For example, in some embodiments, long hydrophobic segments of an amphiphilic polypeptide are linked by shorter segments of hydrophilic linker motifs.

In some embodiments, amphiphilic polypeptides suitable for the present invention comprise a hydrophobic portion capable of forming a beta-sheet (β-sheet) secondary structure (e.g., crystalline). In some embodiments, amphiphilic polypeptides suitable for the present invention comprise multiple hydrophobic portions capable of forming a beta-sheet (β-sheet) secondary structure. In some embodiments, amphiphilic polypeptides suitable for the present invention comprise a hydrophobic portion that is amorphous (e.g., a random helix). In some embodiments, amphiphilic polypeptides suitable for the present invention comprise multiple hydrophilic portions that form a random helix. In some embodiments, suitable amphiphilic polypeptides for the present invention comprise multiple hydrophobic portions capable of forming a beta-sheet (β-sheet) secondary structure and multiple hydrophilic portions capable of forming a random helix. In some embodiments, the multiple hydrophobic portions capable of forming a beta-sheet secondary structure and the multiple hydrophilic portions capable of forming a random helix are arranged in tandem.

It has further been discovered that amphiphilic peptides having relatively high overall hydrophobicity are suitable for carrying out the invention described herein. Defining protein hydrophobicity on a structural basis is complex since it may be modulated not only by the primary structure (i.e., amino acid sequence), but also involves secondary and tertiary structures. In the context of the present disclosure, however, amphiphilic peptides suitable for carrying out the described invention typically exhibit a net hydrophobicity of at least 65% at the amino acid level, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or greater. Therefore, as used herein, the net hydrophobicity of a peptide or protein is determined by dividing the number of hydrophobic amino acids present in the primary sequence by the total number of amino acids, multiplied by 100, expressed as a percentage (%).

Among the 20 most common amino acids, the following are considered to be hydrophobic amino acids: Alanine (Ala/A), Isoleucine (Ile/I), Leucine (Leu/L), Phenylalanine (Phe/F),

Valine (Val/V), Proline (Pro/P), Glycine (Gly/G). On the other hand, polar or charged amino acids are more likely to be in contact with water (i.e., hydrophilic) or, in other words, are energetically favorable to be in contact with water. Among the 20 most common amino acids, charged amino acid residues include Arginine (Arg/R), Lysine (Lys/K), Aspartic acid (Asp/D), and Glutamic acid (Glu/E). Among the 20 most common amino acids, polar amino acid residues include Glutamine (Gln/Q), Asparagine (Asn/N), Histidine (His/H), Serine (Ser/S),

Threonine (Thr/T), Tyrosine (Tyr/Y), Cysteine (Cys/C), Methionine (Met/M) and Tryptophan (Trp/W).

Accordingly, in some embodiments, amphiphilic polypeptides useful for the present invention contain at least 65% of amino acid residues that make up the hydrophobic portion(s) (e.g., hydrophobic secondary structures) of the peptide, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or greater. In some embodiments, an amphiphilic polypeptide suitable for the present invention contains at least 65% of its amino acid residues that participate in the formation of beta-sheet secondary structures, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or greater.

Furthermore, the inventors of the present application have recognized that the hydrophilic portion(s) of an amphiphilic polypeptide described herein can surprisingly function as an “incorporated plasticizer” when used as a coating material. As such, coatings made with such amphiphilic polypeptides do not require an added plasticizing agent.

Accordingly, in some embodiments, amphiphilic polypeptides useful for the present invention contain up to 35% amino acid residues that make up the hydrophilic portion(s) of the peptide, for example, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, and 5% or less. In some embodiments, an amphiphilic polypeptide suitable for the present invention contains up to 35% of its amino acid residues that form one or more random helix structures, e.g., 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, and 5% or less.

Additionally, the inventors of the present disclosure have recognized that the interaction at the molecular level between the hydrophobic (e.g., crystalline) domains and the hydrophilic (e.g., amorphous) domains of a protein coating determines the malleability balance of the resulting coating. That is, crystal formation depends mainly on the intramolecular and intermolecular interactions of the hydrophobic domains through hydrogen bonds as well as hydrophobic interactions, while the structural flexibility or malleability of the coating requires the plasticizing effect provided by the amorphous domains of said proteins.

This also means that the relative molecular weights or ranges of molecular weights of such peptides comprising a shell have an effect on crystal formation, or packing, as well as their structural stability and flexibility, because polypeptides with relatively larger hydrophobic moieties can provide greater molecular interactions (e.g., more hydrogen bonds, etc.) to stabilize the protein structure. Accordingly, in some embodiments of the invention, the proteins used for a coating material have an average molecular weight of at least 50 kilodaltons (kDa). In some embodiments, the proteins used for a coating material according to the invention have average molecular weights ranging from about 50 kDa to about 400 kDa. In some embodiments, the proteins used for a coating material according to the invention have an average molecular weight of about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa, about 150 kDa, about 160 kDa, about 170 kDa, about 180 kDa, about 190 kDa, about 200 kDa, about 210 kDa, about 220 kDa, about 230 kDa, about 240 kDa, about 250 kDa, about 260 kDa, about 270 kDa, about 280 kDa, about 290 kDa, about 300 kDa, about 310 kDa, about 320 kDa, about 330 kDa, about 340 k

120 kDa, about 130 kDa, about 140 kDa, about 150 kDa, about 160 kDa, about 170 kDa, about 180 kDa, about 190 kDa, about 200 kDa, about 210 kDa, about 220 kDa, about 230 kDa, about 240 kDa, about 250 kDa, about 260 kDa, about 270 kDa, about 280 kDa, about 290 kDa, about 300 kDa, about 310 kDa, about 320 kDa, about 330 kDa, about 340 kDa, about 350 kDa, about 360 kDa, about 370 kDa, about 380 kDa, about 390 kDa, about 400 kDa, and higher. Typically, such polypeptides can form flexible, less brittle coatings without requiring added plasticizing agents.

To give an example, silk fibroin is a hydrophobic structural protein with amphiphilic properties. The heavy chain of silk fibroin is composed of amorphous and crystalline fractions. The beta sheets of fibroin proteins have been observed to stack to form crystals, while other segments form amorphous domains. It is the interaction between rigid crystalline segments and deformed elastic semi-amorphous regions that gives silk, at least in part, its extraordinary properties.

Furthermore, both the secondary and tertiary structures of proteins can be further controlled due to protein polymorphism. This modulation of silk structure allows for precise control over the protein's mechanical and physical properties, resulting in a flexible and more plastic material compared to purely crystalline proteins, such as corn zein. Furthermore, the molecular weight of regenerated silk fibroin (e.g., from about 390 to about 50 KDa, depending on processing conditions, such as boiling time) is much higher than that of regenerated zein (about 15-40 kDa). Without wishing to be bound by any particular theory, it is believed that at such a low molecular weight, intermolecular bonding and chain entanglement are limited and result in a more brittle material, which in fact requires a plasticizer to make a flexible, conformable film coating. For the same reason, flexible film coatings typically cannot be made using silk fibroin boiled for an extended period (such as 100 minutes). Such processing causes protein fragmentation, and as the protein molecular weight becomes too low, the resulting material becomes too brittle.

However, in certain situations, brittle coatings are preferred. For example, it may be desirable for some perishable products to retain their crispness. Therefore, the invention includes the use of selected low molecular weight proteins for the preparation of coatings. In some embodiments, low molecular weight proteins have an average molecular weight ranging from about 10 kDa to about 45 kDa, i.e., about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, and about 45 kDa.

A number of amphiphilic polypeptides can be considered to carry out the present invention. In some embodiments, a polypeptide from a single source (e.g., naturally occurring proteins) can be used, which contains both a hydrophobic section or module and a hydrophilic section or module within the polypeptide, such that the individual polypeptide itself is naturally amphiphilic. In some embodiments, a hydrophobic section or module and a hydrophilic section or module may be fused or coupled together to form an amphiphilic entity.

Such fusion or chimeric polypeptides may be produced using recombinant techniques, chemical coupling, or both.

Additionally, the present invention encompasses the recognition that polypeptides that include a portion or portions of an amino acid sequence that adopt the beta-sheet secondary structure (β-sheet) are particularly useful for the present invention. Accordingly, in some embodiments, amphiphilic polypeptides are selected based on having a beta-sheet structure or being prone to forming such a structure based on the amino acid sequence.

In some embodiments, the peptide-based coatings described herein comprise an amino acid sequence of polypeptides selected from the following list: fibroins, actins, collagens, catenins, claudins, coilins, elastins, elaunins, extensins, fibrillins, lamins, laminins, keratins, tublins, viral structural proteins, zein proteins (seed storage protein), and any combination thereof. In some embodiments, silk fibroin is used to carry out the present invention. In any embodiment, the coatings described herein may comprise a silk fibroin polypeptide. In some embodiments, such coatings may be prepared from an aqueous solution consisting essentially of a silk fibroin polypeptide and water.

Amphiphilic polypeptides suitable for practicing the present invention can be produced from a variety of sources, including regenerated (e.g., purified) protein from natural sources, recombinant proteins produced in heterologous systems, synthetic or chemically produced peptides, or a combination thereof.

In some embodiments, the coatings of the present invention may be prepared from a polypeptide corresponding to any of the list provided above, with or without one or more amino acid sequence variations, as compared to the natural or wild-type counterpart. For example, in some embodiments, such variants may exhibit at least 85% overall sequence identity compared to the wild-type sequence, e.g., at least 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% overall sequence identity. In some embodiments, for example, a coating is prepared using silk fibroin in accordance with the present disclosure. In any embodiment, the coatings described herein may comprise a silk fibroin polypeptide. In some embodiments, such coatings may consist essentially of a silk fibroin polypeptide and residual water.

Silk fibroin

As used herein, the term “silk fibroin” refers to a silk fibroin protein or fragment thereof, whether produced by the silkworm, spider, or other insect or otherwise generated (Lucas et al., Adv. Protein Chem., 13: 107-242 (1958)). Silk is produced naturally by several species including, but not limited to: Antheraea mylitta; Antheraea pernyi; Antheraea yamamai; Galleria mellonella; Bombyx mori; Bombyx tangerine; Galleria mellonella; Nephila clavipes; Nephila senegalensis; Gasteracantha mammosa; Argiope aurantia; Araneus diadematus; Latrodectus geometricus; Araneus bicentenarius; Tetragnatha versicolor; Araneus ventricosus; Dolomedes tenebrosus;

Euagrus chisoseus; Plectreurys tristis; Argiope trifasciata;

and Nephila madagascariensis.

In some embodiments, silk fibroin is obtained from a solution containing dissolved silkworm silk or spider silk. For example, in some embodiments, silk fibroin is obtained from the cocoon of Bombyx mori. In some embodiments, spider silk fibroin is obtained, for example, from Nephila clavipes. Alternatively, in some embodiments, silk fibroins suitable for use in the invention are obtained from a solution containing genetically engineered silk or recombinantly produced silk harvested from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants. See, e.g., WO 97/08315 and U.S. Pat. No. 5,245,012, each of which is incorporated herein in its entirety by reference.

Therefore, in some embodiments, a silk solution is used to produce coating of the present invention containing fibroin proteins, essentially without sericins. “Substantially free” as used herein means either absent or present at a concentration that (i) is below detection as measured by any art-accepted means; or, (ii) has little or no impact on the downstream application such that it is considered negligible.

In some embodiments, the silk solutions used to manufacture various compositions of the present invention contain the fibroin heavy chain, but essentially no other proteins. In other embodiments, the silk solutions used to manufacture various compositions of the present invention contain both the fibroin heavy chain and light chain, but essentially no other proteins. In certain embodiments, the silk solutions used to make various compositions of the present invention contain both a heavy chain and a light chain of silk fibroin; in some of those embodiments, the heavy chain and the light chain of silk fibroin are linked by at least one disulfide bond. In some embodiments where both the heavy and light fibroin chains are present, they are linked by one, two, three, or more disulfide bonds.

Although different species of silk-producing organisms, and different types of silk, have different amino acid compositions, several fibroin proteins share certain structural features. A general trend in silk fibroin structure is an amino acid sequence characterized by frequently alternating glycine and alanine, or alanine alone. This configuration allows fibroin molecules to self-assemble into a beta-sheet conformation. These hydrophobic “Ala-rich” and “Gly-rich” blocks are typically separated by amino acid segments with bulky side groups (e.g., hydrophilic spacers).

In some embodiments, the core repeat sequences of the hydrophobic blocks of fibroin are represented by the following amino acid sequences and/or formulas: (GAGAGS)5-15 (SEQ ID NO: 1); (GX)5-15 (X=V, I, A) (SEQ ID NO: 2); GAAS (SEQ ID NO: 3); (S1-2A11-13) (SEQ ID NO:

4); GX1-4 GGX (SEQ ID NO: 5); GGGX (X=A, S, Y, R, DV, W, R,

D) (SEQ ID NO: 6); (S1-2A1-4)i-2 (SEQ ID NO: 7); GLGGLG (SEQ

ID NO: 8); GXGGXG (X=L, I, V, P) (SEQ ID NO: 9); GPX (X=L, Y, I); (GP(GGX)i-4 Y)n (X=Y, V, S, A) (SEQ ID NO: 10); GRGGAn (SEQ ID NO: 11); GGXn (X=A, T, V, S); GAG(A)6-7GGA (SEQ ID NO: 12); and GGX GX GXX (X=Q, Y, L, A, S, R) (SEQ ID NO: 13).

In some embodiments, the fibroin peptide contains multiple hydrophobic blocks, e.g., 3, 4, 5, 6, 7,

8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 hydrophobic blocks within the peptide. In some embodiments, the fibroin peptide contains between 4-17 hydrophobic blocks.

In some embodiments of the invention, a fibroin peptide comprises at least one hydrophilic spacer sequence (“hydrophilic block”) that is about 450 amino acids in length. Non-limiting examples of hydrophilic spacer sequences include: TGSSGFGPYVNGGYSG (SEQ ID NO: 14); YEYAWSSE (SEQ ID NO: 15); SDFGTGS (SEQ ID NO: 16); RRAGYDR (SEQ ID NO: 17); EVIVIDDR (SEQ ID NO: 18); TTIIEDLDITIDGADGPI (SEQ ID NO: 19), and TISEELTI (SEQ ID NO: 20).

In certain embodiments, a fibroin peptide contains a hydrophilic spacer sequence that is a derivative of any of the representative spacer sequences listed above. Such derivatives are at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any of the hydrophilic spacer sequences.

As noted, silks are fibrous proteins characterized by modular units linked together to form highly repetitive, high-molecular-weight proteins. These modular units, or domains, each with specific amino acid and chemical sequences, are thought to provide specific functions. For example, sequence motifs such as poly-alanine (polyA) and poly-alanine-glycine (poly-AG) have a tendency to form beta sheets; GXX motifs contribute to the formation of 31 helices; GXG motifs provide rigidity; and GPGXX (SEQ ID NO: 22) contributes to the formation of beta coils. These are examples of key components in various silk structures whose positioning and arrangement are closely linked to the final material properties of silk-based materials (reviewed in Omenetto and Kaplan (2010) Science 329: 528–531). See also: WO

2011/130335 (PCT/US2011/032195), the contents of which are incorporated herein by reference.

In any of the embodiments contemplated herein, silk fibroin polypeptides of various molecular weights (e.g., fragments) can be used. In some embodiments, for example, the silk fibroin coatings provided comprise silk fibroin polypeptides having an average molecular weight of between about 3.5 kDa and about 350 kDa. Non-limiting examples of suitable ranges of silk fibroin fragments include, but are not limited to: silk fibroin polypeptides having an average molecular weight of between about 50 kDa and about 350 kDa; silk fibroin polypeptides having an average molecular weight of between about 100 kDa and about 350 kDa; silk fibroin polypeptides having an average molecular weight of between about 150 kDa and about 350 kDa; silk fibroin polypeptides having an average molecular weight of between about 200 kDa and about 350 kDa, etc. Silk fibroin polypeptides that are reduced in size, e.g., smaller than the original or wild-type counterpart, may be referred to as low molecular weight silk fibroin.

Additives - optional plasticizers

In any of the embodiments contemplated by the present invention, the biopolymer-based coatings may additionally include one or more additives. Although not required, examples of suitable additives include, but are not limited to, one or more plasticizing agents (i.e., softeners) and other active or inactive agents, depending on the particular use.

As used in the context of the present description, the terms “plasticizing agent” and “plasticizer” are used interchangeably herein and mean any substance added to a biopolymer-based coating preparation to promote plasticity and flexibility and to reduce brittleness. Typically, such an agent is a hygroscopic substance that forms electrostatic or hydrogen bonds with the biopolymer and increases the amount of freezing and free bonded water retained in the biopolymer materials. An example of such an agent is glycerol. It should be noted, however, that at least some plasticizers commonly used for conventional preparations (e.g., certain oils) may not work with the biopolymer-based coatings described in the present application. In some embodiments, the biopolymer-based coatings include one or more plasticizing agents including, but not limited to: Glycerin; glyceryl oleate; oleyl alcohol; PEG-4 PEG-6; PEG-8; PEG-12; PEG-16; PEG-20 PEG-32; PEG-75 (Ref. Handbook of Green Chemistry, Part IV Functional/Application, pp. 2759), stearic acid, oleic acid, sodium lactate,

Emerest® 2618; Emerest® 2619; Hydrobrite® 200PO; Hydrobrite® 380PO; Hydrobrite® 550PO stearate PEG-20; propylene glycol laurate; Semtol® 40; Semtol® 70; Semtol® 85 Semtol® 100; Semtol® 350 (Ref. Handbook of Green Chemistry, Part IV Functional/Application, pp. 2755).

In some embodiments, biopolymer-based coating preparations may additionally contain one or more humectants. Generally, a humectant is a water-soluble solvent and any of a group of hygroscopic substances with moisturizing properties, i.e., used to keep objects moist. They are often a molecule with several hydrophilic groups, most frequently hydroxyl groups; however, amines and carboxyl groups, sometimes esterified, can also be present (their affinity for forming hydrogen bonds with water molecules is the crucial feature).

Non-limiting examples of some humectants include: propylene glycol (E1520), hexylene glycol and butylene glycol; glyceryl triacetate (E1518); vinyl alcohol; neoagarobose; sugar alcohols/sugar polyols: glycerol/glycerin, sorbitol (E420), xylitol, maltitol (E965); polymer polyols (e.g., polydextrose (E1200)); quillaia (E999); urea; aloe vera gel; MP

Diol; alpha hydroxy acids (e.g., lactic acid); and honey.

Optional Additives - Other Components

In accordance with the present invention , biopolymer-based coatings may additionally include one or more additional components of interest. Such components may be active agents or inactive (or inert) agents. Non-limiting examples of additives that may be incorporated into coating materials include biologically active agents such as: antimicrobial agents, such as antibacterial agents and antifungal agents; enzyme inhibitors; ethylene binding/capture molecules, such as ethylene-binding domains of ethylene receptors; ethylene absorbing substances, such as aluminosilicates (e.g., zeolites), silk fibroin-based aerogels (see, U.S. provisional application 61/902,145 filed November 8, 2013, entitled “PEPTIDE-BASED NANOFIBRILLAR MATERIALS” which is incorporated herein), oxidizing agents, such as potassium permanganate; ethylene receptor antagonists; porphyrins;

hormones, hormone receptor agonists and antagonists.

Other additives that may be incorporated into coating materials include, but are not limited to: nutraceuticals (dietary supplements such as vitamins, antioxidants, fatty acids, etc.); flavorings and other compounds added to enhance taste, such as sugars; perfumes or fragrances; colorants, dyes, etc.

Functional characteristics

As shown in the Exemplification provided below, the biopolymer-based coatings described in the present application exhibit low water permeability and can therefore form an effective moisture barrier to prevent moisture loss from perishable products. This is important for maintaining moisture inside perishable products with relatively high water content, such as fresh fruit, preventing dehydration, and for keeping moisture out to keep products dry. Controlling water permeability can also help control contamination and microbial growth.

In some embodiments, the biopolymer-based coatings described in the present application have a water diffusion coefficient of less than 10^-6 cm²/s, for example, less than 10^-7 cm²/s, less than 10^-8 cm²/s, less than

10^-9 cm²/s or less. In some embodiments, such coatings have a water diffusion coefficient ranging from about 10^-6 cm²/sy around 10^-9 cm²/s, for example, between about 10^-6 cm²/sy around 10^-7 cm²/s, between about 10^-6 cm²/sy around 10^-8 cm²/s, between about 10^-7 cm²/sy around 10^-8 cm²/s, between about 10^-7 cm²/sy around 10^-9 cm²/sy between about 10^-8 cm²/sy around 10^-9 cm²/s.

In some embodiments, the biopolymer-based coatings described in the present application exhibit low gas permeability. In some embodiments, the coatings described herein have an oxygen permeability coefficient (DkO2) of less than 10.^-10 [(mlo2 cm) /(cm s mmHg)]. In some embodiments, such coatings have an oxygen permeability coefficient (DkO2) ranging from about 10^-10 and around 10^-13 [(mlo2 · cm) /(cm · s · mmHg)], for example, between about 10^-10 and around 10^-12 [(mlO2 • cm) /(cm • s • mmHg)], between about 10^-10 and around 10^-11 [(mlo2 · cm) /(cm · s · mmHg)], between about 10^-11 and around 10^-13 [(mlO2 · cm) /(cm · s · mmHg) ] , between about 10¹¹ and around 10^-12 [(mlO2 cm) /(cm s mmHg)]. In some embodiments, an oxygen permeability coefficient (DkO2) of the described coatings is about 10^-13 [(mlO2 • cm) /(cm · s · mmHg)], about 10^-12 [(mlO2 cm) /(cm s mmHg)] or about 10^-11 [(mlO2 · cm) /(cm · s · mmHg)], about 10^-10 [(mlO2 · cm) /(cm · s · mmHg)].

In some embodiments of the invention, the biopolymer-based coatings described in the present application are useful for their improved or enhanced ability to preserve perishable items that are susceptible to dehydration, susceptible to discoloration, susceptible to oxidation, susceptible to photodegradation, susceptible to enzymatic degradation, susceptible to microbially induced decomposition, sensitive to ethylene, emit ethylene, susceptible to mechanical damage from impact or any combination of these.

In some embodiments, biopolymer-based coatings made from an amphiphilic but hydrophobic polypeptide are used to preserve fresh agricultural produce. In some embodiments, perishable products such as fruits are coated one or more times with a biopolymer coating.

Therefore, the invention provides methods for improving the preservation of perishable items that are susceptible to decomposition or fermentation caused by fungi (e.g., mold), bacteria, or a combination thereof. Generally, the freshness of perishable products is best preserved when such products are coated multiple times with the biopolymer-based coatings described herein, and also when crystalline formation of the protein is induced in the coating material, resulting in prolonged preservation observed by the structural integrity and appearance of the external and internal tissues of the products after standard storage. Consequently, increasing the coating steps and increasing protein crystallinity resulted in a down-regulation of microbial growth, visible through reduced fungal and mold spoilage.

In some embodiments, suitable storage conditions involve storing a perishable item at a temperature ranging from about 2°C to about 35°C, but more typically from about 2°C to about 35°C, e.g., about 2°C, 3°C,4

C, 5 °C, °C, 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, °C, 15 °C, 16 °C, 17 °C, 18 °C,19 °C, 20 °C, 21 °C, 22 °C, °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C,31

C, 32°C, 33°C, 34°C, 35°C, etc.

In any such embodiment, suitable storage conditions involve storing a perishable item at certain humidity levels, for example, less than 5%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, around 85%, around 90%, around 95% and around 100%.

In either case, proper storage conditions involve storing a perishable item for a period of time ranging from about 1 hour to about 3 years. More typically, storage duration ranges from about 1 day to about 1 year, e.g., about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer.

The coatings prepared and used in accordance with the present application can significantly extend the shelf life of perishable products coated or packaged with them. "Shelf life" is generally defined as the period of time a product can be stored without becoming unfit for use or consumption. Shelf life is therefore the maximum recommended time during which products may be stored, during which the defined quality of a specified proportion of the products remains acceptable under expected (or specified) conditions of distribution, storage and display.

In some regions, a best-before, best-before, or use-by date is required on packaged perishable goods.

The coatings described herein may include such information.

Generally, "expiration dates" are used as guidelines based on normal and expected handling and exposure to temperature. Use before the expiration date does not guarantee the safety of a perishable product, and such a product is not necessarily dangerous or ineffective after the expiration date.

For food items, shelf life is typically different from expiration date, as the former refers to the quality of the food, while the latter refers to its safety. A perishable product that has passed its shelf life is likely still safe, but its quality is no longer guaranteed.

In some embodiments, the use of a coating described herein extends the shelf life of a perishable product coated therewith, compared to the same or a similar product without the described coating, when both products are processed and stored otherwise under identical or substantially identical conditions. With the use of the described coating, in some embodiments, the shelf life of a perishable product is extended by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, with respect to the shelf life of an equivalent product under otherwise identical processing and storage conditions, with the exception of the coating.

In some embodiments, an average shelf life of a perishable article coated with a coating described in the present application is increased between about 1.1 and about 10 times, compared to the counterpart.

<td colspan="3">corresponding (by</td><td>example,</td><td colspan="4">reference), that is,</td><td>a</td>

<td>article</td><td>without</td><td colspan="2">the coating</td><td>of</td><td colspan="2">the invention, by</td><td colspan="2">example,</td>

<td>around</td><td>of</td><td> 1,2,</td><td>around</td><td>of</td><td> 1,3,</td><td>around</td><td>of</td><td> 1,4,</td>

<td>around</td><td>of</td><td> 1,5,</td><td>around</td><td>of</td><td> 1,6,</td><td>around</td><td>of</td><td> 1,7,</td>

<td>around</td><td>of</td><td> 1,8,</td><td>around</td><td>of</td><td> 1,9,</td><td>around</td><td>of</td><td> 2,0,</td>

<td>around</td><td>of</td><td> 2,1,</td><td>around</td><td>of</td><td> 2,2,</td><td>around</td><td>of</td><td> 2,3,</td>

<td>around</td><td>of</td><td> 2,4,</td><td>around</td><td>of</td><td> 2,5,</td><td>around</td><td>of</td><td> 2,6,</td>

<td>around</td><td>of</td><td> 2,7,</td><td>around</td><td>of</td><td> 2,8,</td><td>around</td><td>of</td><td> 2,9,</td>

<td>around</td><td>of</td><td> 3,0,</td><td>around</td><td>of</td><td> 3,1,</td><td>around</td><td>of</td><td> 3,2,</td>

<td>around</td><td>of</td><td> 3,3,</td><td>around</td><td>of</td><td> 3,4,</td><td>around</td><td>of</td><td> 3,5,</td>

<td>around</td><td>of</td><td> 3,6,</td><td>around</td><td>of</td><td> 3,</td><td> 7,</td><td>around</td><td>of</td><td> 3,</td><td> 8</td>

<td>around</td><td>of</td><td> 3,9,</td><td>around</td><td>of</td><td> 4,</td><td> 0,</td><td>around</td><td>of</td><td> 4,</td><td> 5</td>

<td>around</td><td>of</td><td> 5,0,</td><td>around</td><td>of</td><td> 5,</td><td> 5,</td><td>around</td><td>of</td><td> 6,</td><td> 0</td>

<td>around</td><td>of</td><td> 6,5,</td><td>around</td><td>of</td><td> 7,</td><td> 0,</td><td>around</td><td>of</td><td> 7,</td><td> 5</td>

<td>around</td><td>of</td><td> 8,0,</td><td>around</td><td>of</td><td> 8,</td><td> 5,</td><td>around</td><td>of</td><td> 9,</td><td> 0</td>

<td>around</td><td>from 9,</td><td>5 and</td><td>around</td><td> 10</td><td colspan="2">times.</td><td></td><td></td><td></td><td></td>

In some embodiments, an average shelf life of a perishable product coated with a coating described herein, as compared to a reference product without such a coating, is extended by at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 30 days, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 18 months, at least 2 years, at least 30 months, at least 3 years, at least 4 years, at least 5 years or more.

In some embodiments, the coatings described herein may reduce the need for, or even eliminate, the requirement for the conventional cold chain typically employed for a particular perishable product. For example, in some embodiments, perishable products that are typically shipped and/or stored at certain preferred or recommended temperature ranges may retain one or more product quality parameters outside of such temperature ranges, when coated with a coating described in accordance with the present invention. In some embodiments, products coated with such a coating can tolerate a greater degree of deviations and/or fluctuations in temperature, humidity, mechanical stress, light exposure, or any combination thereof, as determined by any of the parameters described herein or other suitable methods known in the art.

Conservation measurements

There are a number of parameters for measuring the relative effectiveness of food preservation. Any suitable means can be used to measure or evaluate the degree of freshness or preservation, or to assess the quality, of perishable products before, after, or during storage. These include, but are not limited to, weight changes, which may reflect water loss, changes in shape or overall structural integrity, texture changes such as firmness, color changes including overall hue or local mottling, chemical species changes (e.g., sugar, starch, etc. content), acidity changes, odor, taste, etc. Relative gas exchange rates (e.g., oxygen permeability) may also be measured. In addition, the emission of specific compounds such as ethylene can be measured.

Non-exhaustive examples of selected measurements are provided in the Exemplification below.

Climacteric fruits and non-climacteric fruits

Fruits that ripen through ethylene production and increased cellular respiration are called climacteric. Examples of climacteric fruits include, but are not limited to, apples, bananas, and tomatoes. In contrast, strawberries and grapes are non-climacteric fruits. The climacteric event is said to be associated with changes in fruit color and the production of sugar in the extracellular space.

As shown in the working examples provided herein, the biopolymer-based coatings described herein are effective for preserving both climacteric and non-climacteric types of agricultural produce. In some embodiments, the biopolymer-based coatings described herein can be used to slow the ripening process of fruits. In some embodiments, the biopolymer-based coatings described herein can be used to maintain the firmness of fruits. In some embodiments, the biopolymer-based coatings described herein can be used to slow microbial growth. In some embodiments, certain fruits, such as non-climacteric fruits (e.g., strawberries), coated with a biopolymer-based coating described herein, may exhibit a limited presence of “black spots” that typically indicate the presence of mold on the surface of the fruits.

In some embodiments, protein polymorphism can be used to tailor the properties of the coating, affecting the interaction between the protein (such as silk fibroin) and water evaporation and food spoilage caused by microbes.

General methods and applications

In a further aspect, related methods for preserving perishable products are provided.

In a broad sense, such a method involves adding a coating to at least a portion of a perishable item to be stored or preserved. Typically, at least a portion of the perishable item is in direct contact with at least a portion of a coating comprising a biopolymer as described in more detail above.

A perishable article is said to be preserved, at least in part, when it retains one or more of its original properties or state/characteristics, as measured by any suitable parameter such as water content, color, weight, shape, texture, structural integrity, taste, flavor, odor, etc.

A biopolymer coating described herein is prepared as an aqueous solution of a suitable biopolymer, e.g., amphiphilic polypeptides having general hydrophobic characteristics, as described above. Typically, the coating materials

<td>are prepared as</td><td>a solution with</td><td>he</td><td>dissolved biopolymer</td><td>in</td>

<td colspan="3">this, to a final concentration of a</td><td>around 0.1-20%</td><td>in</td>

<td colspan="2">weight, for example, around (</td><td> 0,1</td><td>%, around 0.2</td><td> 2%,</td>

<td>around 0.3</td><td>%, around</td><td> 0,4</td><td>%, around 0.5</td><td> 2%,</td>

<td>around 0.6</td><td>%, around</td><td> 0,7</td><td>%, around 0.8</td><td> %,</td>

<td>around 0.9</td><td>%, around</td><td> 1</td><td>%, around 1.5</td><td> 2%,</td>

<td>around 2</td><td>%, around</td><td> 3</td><td>%, around 4</td><td> 2%,</td>

<td>around 5</td><td>%, around</td><td> 6</td><td>%, around 7</td><td> %,</td>

<td>around 8</td><td>%, around</td><td> 9</td><td>%, around 10</td><td> %,</td>

<td>around 11</td><td>%, around</td><td> 12</td><td>%, around 13</td><td> 2%,</td>

<td>around 14</td><td>%, around</td><td> 15</td><td>%, around 16</td><td> 2%,</td>

<td>around 17 around 20%</td><td>%, around or more.</td><td> 18</td><td>%, around 19</td><td> %,</td>

If desired, certain additives can be added to these solutions.

Any suitable technique may be used to perform the coating step (e.g., the step of depositing a coating material onto a perishable item). For example, the coating process may be performed by any suitable means, including, but not limited to, dip coating, spray coating, brushing, etc. Such a step can be performed once or repeated multiple times, for example, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 26 times, 27 times, 28 times, 29 times, 30 times or more.

Between or after each coating step, the biopolymer-based coating can be dried and, optionally, annealed, crosslinked, or both. The data presented herein suggest that increasing the degree of crystallinity in the biopolymer can enhance the preservative effects. These effects may involve at least two factors: one is the prevention of water loss, and the other is the prevention of microbial growth.

In some embodiments, the annealing process may involve inducing the formation of beta sheets in the biopolymer used as the coating material. Annealing techniques (e.g., increased crystallinity) or otherwise promoting the "molecular packing" of biopolymers have been described.

In some embodiments, annealing (e.g., beta-sheet formation) is induced by the addition of an organic solvent. Suitable organic solvents include, but are not limited to, methanol, ethanol, acetone, isopropanol, or a combination thereof.

In some embodiments, annealing is performed by what is known as "water annealing" or "steam annealing," in which water vapor is used as a catalyst or intermediate plasticizer to promote packing of the beta sheets. In some embodiments, the water annealing process can be performed under vacuum. Suitable methods have been described. See, for example, Jin HJ et al. (2005), Water-stable Silk Films with Reduced Beta-Sheet Content, Advanced Functional Materials, 15: 1241-1247; Xiao H. et al.

(2011), Regulation of Silk Material Structure by TemperatureControlled Water Vapor Annealing, Biomacromolecules, 12(5):

1686—1696.

In some embodiments, the annealing or crosslinking step involves exposing the amphiphilic polypeptide of the coating material to a high energy source, such as by irradiation.

A useful source of high energy for such a process may include, but is not limited to, an electron beam, photons, ionizing radiation, nuclear radiation.

According to the invention, at least a portion of a perishable article may be coated or covered with a biopolymer-based coating in one or more layers. A layer of such coatings may be of any suitable thickness, for example, between about 0.1 pm and about 1 mm, for example, about 0.5 pm, about 1 pm, about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 30 pm, about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, about 110 pm, about 120 pm, about 130 pm, around 140 pm, around 150 pm, around 160 pm, around 170 pm, around 180 pm, around 190 pm, around 200 pm, around 210 pm, around 102 pm, around 113 pm, around 240 pm, around 250 pm, around 300 pm, around 350 pm, around 400 pm, around 450 pm, around 500 pm, around 550 pm, around 600 pm, around 650 pm, around 700 pm, around 750 pm, around 800 pm, around 850 pm, around 900 pm, around 950 pm, around 1000 pm, or more. Such coating may be composed of a single layer or multiple layers of coatings.

In some embodiments, the biopolymer-based coatings of the present invention form a conformal coating or coating over at least a portion of the surface of a perishable product. In some embodiments, such coatings may completely cover one or all surfaces of a perishable product.

The biopolymer-based coatings of the present invention can be substantially transparent when formed as a coating on a perishable product. Transparency is a desirable characteristic for maintaining the natural color or appearance of the perishable product. Additionally, in some embodiments, such coatings can have an added shine effect (e.g., a lustrous appearance) on the coated product.

Typically, the biopolymer-based coatings described herein are odorless, tasteless, or both.

In some embodiments, the biopolymer-based coatings described herein can be designed to be sufficiently water-soluble and therefore easily washable. However, in some embodiments, the coatings do not need to be removed from perishable food items before consumption.

The biopolymer-based coatings encompassed by the present invention can also be used as packaging materials for a variety of products. For example, the biopolymer-based coatings provided herein can replace at least in part any conventional packaging material used to wrap, cover, or bottle perishable items including, but not limited to, dairy products, wines and spirits, other bottled beverages, and the like.

In some embodiments, conventional packaging materials may be coated on at least one side with a biopolymer-based coating described herein. In some embodiments, a packaging component may be replaced with or incorporate a biopolymer-based coating described herein. For example, biopolymer-based coating materials may be used as a wine stopper to replace cork.

In any of the embodiments encompassed by the present invention, the coatings may further comprise one or more additional features, such as nano- and/or microstructures fabricated or incorporated therein. Such structures may provide at least one optical characteristic to the coating. Examples of optical characteristics include, but are not limited to, diffraction gratings, nanoscale holes and pits, microprisms, and the like. In some embodiments, such structures are designed to act as sensors. See, for example, WO 2008/127404; WO 2008/118211; WO 2008/127402; WO 2008/127403; WO 2008/127401;

WO 2008/140562; WO 2009/061823; WO 2009/155397; WO 2010/126640; WO 2011/046652; WO 2011/026101; WO 2012/054121;

WO 2011/130335; WO 2011/112931; WO 2012/047682; WO 2012/031282; WO 2010/088585; WO 2013/130156; the contents of each of the aforementioned publications are incorporated herein in their entirety by this reference.

In some embodiments, such optical and/or nanoscale features are designed to carry or encode certain information (e.g., tags and codes). Any appropriate or desirable information may be encoded or included, including, but not limited to, identifying information such as information about the source or origin of a product, ingredients, nutritional information, manufacturing information, processing dates (harvest date, coating or packaging date, etc.), best before or use by date, expiration dates, pricing information, authentication, advertising, customer service information or any combination of these.

In any of the embodiments included herein, the additional feature(s) added to the coatings of the present invention may provide aesthetic effects, such as designs, patterns, colors, drawings, images, logos and any combination thereof.

The following Exemplification provides non-limiting examples of working embodiments carried out in accordance with the present invention described herein. The data are presented for illustrative purposes only and should not be considered limiting in any way.

EXEMPLIFICATION

Silk fibroin as an edible coating for the preservation of perishable foods

Many perishable foods have high metabolic activity and a high potential for microbial contamination, resulting in short shelf life, fungal spoilage, color change, and off-flavor. Among all perishable foods, for example, strawberries are considered one of the most difficult to keep fresh in the farm-to-table process and are therefore used as a model to analyze the effectiveness of the perishable food preservation strategies described here. Strawberries are rich in polyphenols and anthocyanins, vitamins, and amino acids. To date, several options have been used to preserve the freshness of strawberries. These include synthetic chemical fungicides, modified atmosphere packaging, osmotic treatments, hypobaric and thermal treatments, cryopreservation, and

edible coatings.

Edible coatings, in particular, have been extensively studied in recent years, as their beneficial effects and ease of handling make them widely applicable to soft fruits, such as strawberries. Specifically, the main functional advantages attributed to the use of edible coatings include reduced respiration rates, extended storage periods, firmness retention, and controlled microbial growth. Several classes of biopolymers have been considered for developing edible coatings: polysaccharides, proteins, lipids, as well as various combinations of these biopolymers. Polysaccharides and proteins are known to form films with good mechanical properties but low permeability, while lipids form fragile films with improved permeability. Therefore, research into edible fruit coatings is now focusing on the use of solid polymer matrices with hydrophobic groups to combine mechanical strength with low water vapor permeability. Furthermore, an ideal coating material for perishable food preservation should exhibit biocompatibility, biodegradability, antibacterial and antifungal activity, membrane-forming ability, and safety (i.e., edible and non-allergenic).

Silk fibroin is a biomaterial widely investigated for its potential in textile, biomedical, photonic, and electronic applications. Silk fibroin is a structural protein, like collagen, but with a unique characteristic: it is produced from the extrusion of an amino acid solution by a complex living organism (whereas collagen is produced in the extracellular space by self-assembly of cell-produced monomers). The properties of silk fibroin derive from its structure, which consists of hydrophobic blocks staggered by hydrophilic acid spacers. In its natural state, silk fibroin is organized into β-sheet crystals alternating with amorphous regions, which gives the protein strength and elasticity. The multiplicity of ways in which regenerated silk fibroin can be processed to a high protein concentration and molecular weight makes it attractive for several high-tech applications, as recently reported. The degree of protein crystallinity can be precisely tuned, influencing the physical, biochemical, mechanical, and biological properties of the material. Furthermore, the amino acid nature of silk fibroin presents a diversity of side chain chemistries that allows for the incorporation and stabilization of macromolecules useful in drug delivery applications or for providing cellular instructions. In particular, silk fibroin with modular degrees of crystallinity can be obtained by regulating the time (ranging from minutes to hours) and temperature (4–60 °C) at which the protein is exposed to water vapor or edible polar solvents (i.e., ethanol). Silk fibroin's degree of crystallinity stabilizes vaccines and antibiotics, eliminating the need for a cold chain. In fact, silk is considered a platform technology for biomaterials manufacturing, as its robustness and quality provide the necessary resources to provide a portfolio of distinct features (e.g., nanopatterning, biochemical functionalization) for the final construct. Processing of regenerated fibroin typically involves partial or complete dehydration of a fibroin solution (protein content 1-15% by weight) to form films, sponges, gels, spheres (micron to nano sized) and foams using numerous techniques (e.g., solvent casting, freeze-drying, salt leaching, or sonication). The rationale behind these manufacturing processes is to create a solid material that combines mechanical strength with biochemical properties.

In this study, the use of a silk fibroin solution as a coating material for the preservation of perishable fruits is reported. The silk fibroin solution obtained as described above was used to dip-coat freshly picked strawberries as shown in Figure 1 panel (a). See D. Rockwood, R. Preda, T. Yucel, X. Wang, M. Lovett, D. Kaplan, Nature Protocols 2011, 6, 1612, which is incorporated herein in its entirety by this reference.

Several silk fibroin solutions characterized by changes in molecular weight and protein concentration have been investigated. In addition, exposure of strawberries to multiple dip-coating steps has been evaluated, as well as post-processing of coated fruit in vacuum steam (previously described as water annealing) to explore the effects of coating polymorphism on fruit stabilization. The results suggested that silk fibroin prolonged the freshness of perishable fruits by slowing fruit respiration, extending fruit firmness, preventing dehydration, and preventing microbial growth.

The molecular weight of silk fibroin was adjusted to the range of 170–90 kDa by regulating the boiling time (i.e., 30 min) during fibroin extraction, and the protein concentration in the dipping solution was adjusted to 1 wt.% to obtain a final solution with rheological properties (i.e., viscosity and surface tension) similar to one of the previously optimized biopolymer-based coating solutions. See C. Ribeiro, A.A. Vicente, J.A. Teixeira, C. Miranda, Postharvest Biology and Technology 2007, 44, 63; A. Matsumoto, A. Lindsay, B. Abedian, D.L. Kaplan, Macromolecular Bioscience 2008, 8, 1006; each of which is incorporated herein in its entirety by reference. Silk fibroin coating of strawberries was achieved using a two-step process. In the first phase, a multi-step dip-coating process (number of coating steps = 1, 2, and 4) was used to expose the strawberries to the silk fibroin solution. Secondly, the silk fibroin-coated fruits were exposed to water vapor under vacuum (i.e., a subsequent water annealing process) to explore the effects of silk fibroin polymorphism on fruit stabilization. See X. Hu, K. Shmelev, L. Sun, E.-S. Gil, S.-H. Park, P. Cebe, D.L. Kaplan, Biomacromolecules 2011, 12, 1686, which is incorporated herein in its entirety by this reference. The results suggested that silk fibroin prolonged the freshness of perishable fruits by slowing fruit respiration, extending fruit firmness, preventing dehydration, and preventing microbial growth.

Materials and methods

Silk fibroin regeneration

A Bombyx mori cocoon was used as a source of fibroin. Silk fibroin extraction was achieved by a standard degumming process, which involved boiling (t = 30 minutes) 2.5 g of cut silk cocoons per liter of 0.02 M sodium carbonate solution. The silk fibroin was then solubilized in 9.3 M lithium bromide for 4 hours in an oven at 60 °C. The chaotropic salt was subsequently removed via dialysis (3.5 kDa MWCO) against Milli-Q water for a total of 72 hours, yielding an 8% (w/v) silk fibroin solution. The resulting silk fibroin solution was then purified by centrifugation at 9000 rpm (~12,700 g) for two 25-minute periods, at a constant 4 °C. The final concentration of the silk fibroin solution was then adjusted to 5% w/w by the addition of MilliQ water.

Dip coating of strawberries

Freshly picked New England strawberries (Dzen Brother Farm, South Windsor, CT) were dip-coated 1, 2, and 4 times (i.e., D1, D2, and D4) in a 60 mm depth of silk fibroin solution (1% by weight) at 4 °C ensuring that the entire surface of the strawberries and their calyx and epicalyx were exposed to the solution. Each immersion step lasted 10 seconds, and then the strawberries were dried hanging from the stem for 4 hours at 22°C, 38% RH.

Dip coating of bananas

Green bananas (Del Monte) were purchased from a local store (Whole Foods, Medford MA) and then dip-coated with silk fibroin as mentioned above.

Crystallization of the coating

Crystallization of the silk fibroin coating was achieved by exposing the coated strawberries to water vapor under vacuum (i.e., water annealing), according to previously developed protocols. Exposure times were set at 0 seconds, 1 hour, 6 hours, and 12 hours (i.e., C0, C1, C6, and C12). Further water annealing resulted in improved silk fibroin crystallinity, as previously reported.

Evaluation of the freshness of strawberries

The effect of different silk fibroin coatings and subsequent water annealing process on strawberry freshness was evaluated morphologically and gravimetrically. Changes in strawberry color, shape, and microbial colony formation were assessed using time-lapse photography. Gravimetric analysis of the strawberries as received in the laboratory, after dip-coating, and on days 1, 3, 5, and 7 was evaluated using a standard laboratory balance (Mettler Toledo MS204S). Strawberry weight was calculated as the average of three measurements.

Interaction between water and the thin silk membrane

The interactions of thin silk fibroin membranes with water were investigated through the lenses of capillary sorption, hydrodynamic permeability, and diffusion behaviors. The sorption behavior of the silk films was analyzed along both the Z and X-Y axes of the films, with and without the aid of gravitational pressure, as well as horizontally. The thin silk membranes were partially exposed to a solution of Rhodamine 6G (Rh6G), which served as a colorimetric indicator for the evolution of the solvent front through the silk fibroin membranes. The hydrodynamic permeability of the films was assessed using Darcy's law-like experiments. The silk fibroin membranes were sandwiched between two pieces of acrylic, with a silicon O-ring to prevent leakage, and then secured on all sides using screws. Syringes glued to the acrylic and sealed with silicone sealant held 4 mL of water above the film, along with a balance, and collected the water that passed through from below. The sides and top were sealed with parafilm to limit evaporation losses, and a few small holes were drilled in the cylinder underneath to prevent the formation of a vacuum (albeit a small amount). The water reservoir above the film applied a pressure of ~500 Pa, driving the water through the membrane. The water diffusion coefficient in the silk membrane was considered in the context of existing theory of water mass transport in amorphous polymer systems. This assumption was also considered valid for crystalline silk membranes, since silk fibroin crystals possess highly hydrophobic, intermolecularly cross-linked beta-sheet structures that limit water diffusion through the amorphous regions of the protein. Therefore, water transport across silk fibroin membranes will fall within either the Fickian or one of the non-Fickian regimes, depending on the relative time scales between water diffusion and the stress-relaxation of the polymer matrix. Peppas et al. have developed an expression for generalized diffusion for amorphous polymers:

2E = ktⁿ (1)

Meo where Mt represents the water uptake mass at time t, M- represents the water uptake mass as time approaches infinity, and k and n are fitting parameters. In particular, the value of the exponent n can be used to identify the mass transport regime under the test conditions. Membranes were prepared according to the conditions described above, with an area of 1 cm². Each dried silk fibroin membrane (thickness around = 90 ± 20 pm) was left overnight to equilibrate with the surrounding environment (~22 °C, ~38% RH), before being weighed. The silk fibroin membranes were then immersed in Milli-Q water for 1 h to allow them to re-swell and reweighed after removing excess water through careful bottling. This was considered Mt=0. Subsequent measurements were taken at one-minute intervals over the next hour until the film again reached equilibrium with the dry surrounding environment and returned to its original mass. In fact, Mt was calculated as Mt - Mt=0, and M- was calculated as Mt=ih - Mt=0.

Measurement of oxygen diffusion in silk fibroin films.

The effective oxygen diffusion coefficients were measured on silk fibroin films (n=5) of enhanced crystallinity (C1, C6 and C12) using a conventional diffusion system - Microx TX3 microsensor oxygen meter (Presens, Germany) equipped with an Oxygen Microptode (Presens, Germany) and a PermeGear water-jacketed gas chamber as described previously. See J.

E. Valentin, DO Freytes, JM Grasman, C. Pesyna, J. Freund, TW Gilbert, SF Badylak, Journal of Biomedical Materials Research Part A 2009, 91A, 1010; C. Androjna, J.E.

Gatica, J.M. Belovich, K.A. Derwin, Tissue Engineering Part A 2008, 14, 559, the contents of which are incorporated herein in their entirety by this reference. The system consisted of two compartments containing known (measured) oxygen concentrations, separated by the silk film of interest. The opening between the two chambers had an area of 2.75 cm²The thickness of the silk fibroin film was measured with a micrometer (n = 7). Before use, a two-point calibration was performed according to the manufacturer's protocols using an oxygen-free environment (sodium sulfite) and an air-saturated environment (water vapor). The average of the initial and final oxygen concentration readings was used for diffusion coefficient data analysis. Oxygen concentrations were measured at 10-minute intervals and each silk fibroin film was analyzed 3 times consecutively.

The effective oxygen diffusion coefficient was calculated as previously reported. See J.E. Valentin, D.O. Freytes, J.M. Grasman, C. Pesyna, J. Freund, T.W. Gilbert, S.F. Badylak, Journal of Biomedical Materials Research Part A 2009, 91A, 1010; C. Androjna, J.E. Gatica, J.M. Belovich, K.A. Derwin, Tissue Engineering Part A 2008, 14, 559, the contents of which are incorporated herein in their entirety by this reference. In summary, with the assumptions of (i) well-mixed fluid in the diffusion system, (ii) no oxygen consumption, (iii) linear oxygen concentration in the analyzed silk films, and (iv) instantaneous steady state, Fick's law can be applied to derive the following equations:

ln (^{Cd CR}j pd) = D't (2) \C_D- c_R0J \-βΧ (3) where D' is the diffusion factor of the system (which depends on the effective oxygen diffusion coefficient of the silk membrane D_and,M and the reference volumetric diffusion factor Db), t is the time, β is the geometric constant characteristic of the effective membrane, A is the area through which diffusion occurs, z is the thickness of the test membrane, Vr is the volume of the receiving chamber, Cd is the concentration of dissolved oxygen in the donor chamber, Cr is the concentration of dissolved oxygen in the receiving chamber at time t; and Cro is the dissolved oxygen concentration in the receiving chamber at t=0 (at t=0, Cr = Cro). The reference volumetric diffusion factor (Db) is determined by replacing the silk film samples with an oxygen-impermeable barrier (i.e., a rubber stopper). Since it is assumed that in this case D_and,M = 0, then equation (2) becomes:

The effective oxygen diffusion coefficient of the silk membrane D_and,M is then calculated as:

<⁵>

Measuring the respiration rate of strawberries.

A previously published method was followed to measure strawberry respiration rates. Briefly, strawberry samples (approximately 100 g, n = 3) were placed in airtight 1-L glass jars with a septum in the lid, allowing gas samples to be collected at different sampling times over a 36-h period. The jars were stored at room temperature of 22 °C and RH = 38%. Gas sampling was performed every 30 minutes for the first 5 hours, and every 90 minutes until 12.^to hour and every 180 minutes for the remaining 24 hours using a needle probe. Three replicates were performed for each coating treatment. The respiration rate was calculated using the following equation:

and, 1000 60 mlCO2

Breathing rate = —— V_iolibre —---- (o) luu τη l Ku'ri .

where m is the mass of the strawberry, V_ispacioiibre is the empty volume of the airtight flask [mi], ACO2 is the difference between the initial and final CO2 concentration and t is the sampling time [min].

Measuring the firmness of strawberries.

The firmness of silk fibroin-coated and uncoated strawberries was measured at 22 °C and RH=38% by puncture testing using an Instron uniaxial system equipped with a 10 N load cell. The firmness of the strawberries was evaluated as a function of storage time (as received, days 1, 3, and 7) and the degree of silk fibroin crystallinity. The testing was performed according to a previously reported protocol. See E. Velickova, E. Winkelhausen, S. Kuzmanova, VD Alves, M. Moldao-Martins, LWT - Food Science and Technology 2013, 52, 80, the contents of which are incorporated herein in their entirety by this reference. Briefly, a 5 mm diameter stainless steel rod with a flat end was used as a probe to penetrate the analyzed strawberries. The maximum penetration force (N) was defined as the maximum force required to push the probe into the strawberries (n=5) to a depth of 8 mm at a crosshead speed of 1 mm/s.

Example 1

Results and discussion

Time-lapse photography

The effectiveness of silk fibroin coating in preserving strawberry freshness was evaluated based on the coating steps and silk fibroin crystallinity. External aging analysis of representative strawberries based on dip-coating steps and silk polymorphism is presented in Figure 1. More coating steps and higher crystallinity of the silk fibroin (i.e., longer water annealing process) resulted in longer preservation of the strawberry tissues, visible through time-dependent reduction in the original red color hue and maintenance of the original morphology. Furthermore, more coating steps and higher silk fibroin crystallinity resulted in a reduced regulation of microbial growth, as evidenced by reduced fungal and mold decay. This was also confirmed by investigating the time-dependent decay of strawberry pulp, as shown in Figure 2. A higher degree of crystallinity of the silk fibroin coating corresponded to better preservation of the internal tissues for the time point considered.

Measuring dehydration

Strawberry dehydration is an indication of the rupture of the red receptacle tissue, which leads to off-flavor, microbial decomposition, loss of turgor, and water evaporation. Figure 3 shows the time-dependent weight loss of strawberries as a function of dip-coating stages and the degree of silk fibroin crystallinity. Two-way ANOVA with Tukey's mean analysis was used to evaluate the data. Silk crystallinity, but not the number of coating steps, affected the dehydration of the strawberries considered. Uncoated controls lost approximately 50% of their original weight over the 7 days considered (highlighted within the red rectangles). Strawberries coated with amorphous silk (DxC0 - within the blue rectangles) retained more water compared to controls on day 3 (p<0.05). Strawberries coated with crystalline silk additionally slowed down fruit dehydration compared to the amorphous coating (p<0.05) and the control (p<0.05) but no statistical difference was found for different annealing times in water (p>0.05).

Interaction between thin silk fibroin membranes and water

To investigate the phenomenon of fruit dehydration through a thin silk fibroin coating, the interaction between the thin silk fibroin membranes and water was investigated. In particular, the phenomena of water absorption, hydrodynamic permeability, and diffusion coefficient were explored. Figure 4 shows an evaluation of the interaction between water and the thin silk fibroin membranes. For the sorption study, silk fibroin crystalline membranes (thickness = 130 pm) showed stained water sorption in both Z and XY directions, indicating capillary diffusion of water through the thin silk fibroin constructs. Hydrodynamic permeability studies did not show a statistically significant effect (p>0.05) of silk fibroin crystallinity on water permeation (based on a one-way ANOVA with Tukey's mean analysis). The water diffusion coefficient study revealed that the crystallinity of silk membranes slightly affected water mass transport through silk (based on a one-way ANOVA with Tukey's mean analysis). Table 1 below indicates the diffusion coefficients calculated for mass transport experiments.

<td></td><td>K</td><td>n</td><td>D (cm²/s)</td>

<td>C1</td><td> 0,25 ± 0,048</td><td> 0,42 ± 0,04</td><td>5.79 x 10^-6</td>

<td>C6</td><td> 0,19</td><td> 0,48 ± 0,03</td><td>3.21 x 10^-6</td>

<td>C12</td><td> 0,16</td><td> 0,55 ± 0,02</td><td>1.05 x 10^-6</td>

Table 1: Diffusion coefficients for mass transport experiments

Gas permeability through silk fibroin membranes

The gas diffusion coefficient through silk fibroin membranes plays an important role in fruit preservation because gases play an important role in the metabolism of fruit stem cells (e.g., oxygen), are byproducts of their metabolism (e.g., carbon dioxide), and can act as growth factors (e.g., ethylene in climacteric fruit). To evaluate the effectiveness of the silk fibroin coating as a gas barrier, the oxygen diffusion coefficient through silk membranes (t = 80 pm) of higher crystallinity was measured. In particular, the effective oxygen diffusion coefficient of silk fibroin was found to be modulated by protein crystallinity (Table 2).

Table 2 below summarizes the changes in the calculated effective oxygen diffusion coefficient of silk fibroin membranes, before (pre-annealing) and after (post-annealing) crystallization was induced by annealing in water for varying periods of time (e.g., 1-24 hours). For example, when a predominantly amorphous form of silk fibroin was annealed in water for 24 hours to generate a crystalline form of silk, the effective oxygen diffusion coefficient decreased by two orders of magnitude. In this particular example, the crystallinity of the annealed silk fibroin was approximately 55%.

The gas diffusion coefficient of silk fibroin appears to be significantly (e.g., several orders of magnitude) lower than that of edible waxes typically used in the food industry, indicating that silk fibroin-based coatings provide an effective barrier against gas transport, such as oxygen.

<td rowspan="2"></td><td>Thickness of the movie</td><td>Coefficient of effective O2 diffusion from the membrane [De,M] (steady state)</td><td>Permeability of O2 [Dk] (state stationary)</td>

<td>p.m</td><td> 10^-11· (cm²/s)</td><td> 10^-11^mlO2^cm/ (s»cm²^mmHg)</td>

<td>Amorphous</td><td> 81±4</td><td> 83,9±7,2</td><td> 11,04±0,95</td>

<td>WA 1 hr</td><td> 78±3</td><td> 67,9±3,5</td><td> 8,93±0,46</td>

<td>WA 6h</td><td> 76±4</td><td> 5,5±1,4</td><td> 0,72±0,18</td>

<td>W.A. 12 p.m.</td><td> 73±2</td><td> 1,9±0,3</td><td> 0,25±0,04</td>

<td>WA 2 4 hrs</td><td> 72±5</td><td> 1,6±0,2</td><td> 0,21±0,03</td>

Table 2. Calculated effective O2 diffusion coefficient and O2 permeability for gas transport experiments (N=3, RH=3 0%)

Silk fibroin as a coating for climacteric fruits

Fruits that ripen through ethylene production and increased cellular respiration are called climacteric. The 15 examples of climacteric fruits are apples, bananas, and tomatoes, while strawberries and grapes are non-climacteric fruits. The climacteric event is associated with changes in fruit color and the production of sugar in the extracellular space. The efficacy of a silk-based climacteric fruit coating on the ripening of uncoated and silk fibroin-coated bananas was evaluated. Figure 5 shows an evaluation of banana ripening with and without silk coating. Fruits were stored at 22 °C and 38% RH as received (control) and after coating with amorphous or crystalline silk fibroin films (silk-coated). The bananas were hung from their respective stems throughout the experiment. Time-lapse photography of banana ripening indicates that the silk coating slowed ripening. The structure (i.e., amorphous or crystalline) of the fibroin used to coat the climacteric fruit did not affect fruit ripening (data not shown). Investigation of the turgor of a silk-coated banana demonstrated that the coating increased fruit firmness compared to an uncoated control on day 9 after coating. Furthermore, morphological analysis of the flesh of coated and uncoated bananas on day 9 after silk coating treatment revealed a more preserved fruit when the silk coating was applied. The uncoated banana flesh was brown in color, while the silk-coated fruits retained a light-colored flesh, indicating a slower ripening rate in the silk-coated sample.

Example 2

Results and discussion

Silk fibroin as a coating to preserve the freshness of strawberries.

The thickness of the silk fibroin coating was investigated as a function of the number of dip-coating processes. The silk fibroin coating thickness, which ranged from 27 to 35 µm, was not statistically significantly influenced (p>0.05) by the number of dip-coating steps, as shown in Table 3. The crystallinity of the silk fibroin coating was calculated using a previously described methodology based on the quantification of beta-sheet structures in the Amide III absorbance peak collected with ATR-FTIR spectroscopic analysis and studied as a function of exposure time to the subsequent water annealing process. Unlike what is shown in Table 3, the subsequent water annealing process had a great influence on the properties of the silk fibroin coating by increasing the amount of beta-sheet structures from 23.2% (for the untreated coating) to 58.4% (for the coating exposed to water vapor for 12 hours).

<td>Number of dip coating processes in silk fibroin solution [Dx]</td><td>Thickness [μπι]</td>

<td>DI</td><td> 27 ± 8</td>

<td>D6</td><td> 32 ±7</td>

<td>D12</td><td> 35 ± 8</td>

<td>Exposure time to the subsequent annealing process in water [Cx, x=hours]</td><td>Crystallinity of edible silk fibroin coating [% beta sheets]</td>

<td>C0 - amorphous</td><td> 23,2 ± 1,5</td>

<td>Cl</td><td> 36,5 ± 3,3</td>

<td>C6</td><td> 47,6 ±4,1</td>

<td>C12</td><td> 58,4 ±4,5</td>

Table 3: Characterization of the thickness and crystallinity of the edible silk fibroin coating

Crystal violet dye was used to stain the silk fibroin coating. Figure 7 in panel (b) shows representative macroscopic images of dyed strawberries coated with edible amorphous and crystalline silk fibroin coatings presented in panel (b) ii and panel (b) iii, respectively. Crystal violet staining is barely visible on the surface of the coated strawberries (black dots) because the coating was only a few microns thick. Additionally, Figure 7 in panel (c) shows stereomicroscopy of the surface and cross-section of crystal violet-stained strawberries coated with amorphous and crystalline silk fibroin, which showed no changes in fruit appearance compared to the uncoated control. The effectiveness of edible silk fibroin coating in preserving strawberry freshness was evaluated based on the coating stages and silk fibroin crystallinity. Figure 8 shows the ripening and weight loss of strawberries coated with an edible silk fibroin coating. Figure 8 in panel (a) shows the external and internal analyses of the aging of representative strawberries as a function of dip-coating stages and silk crystallinity. More coating steps and higher crystallinity of the silk fibroin (i.e., longer water annealing process) resulted in longer preservation of the strawberry tissues, visible through time-dependent reduction in the original red color hue and maintenance of the original morphology. Furthermore, more coating steps and higher silk fibroin crystallinity resulted in a reduced regulation of microbial growth, as evidenced by reduced fungal and mold decay. This was also confirmed by investigating the time-dependent decay of strawberry pulp. A higher degree of crystallinity of the silk fibroin coating corresponded to better preservation of the internal tissues for the time point considered.

Measuring dehydration.

Dehydration of strawberries is an indication of the rupture of the red receptacle tissue, which leads to off-flavor, microbial decomposition, loss of turgor, and water evaporation. Figure 8 (b) shows the time-dependent weight loss of strawberries as a function of dip-coating stages and the degree of silk fibroin crystallinity. Two-way ANOVA with Tukey's mean analysis was used to evaluate the data. Silk crystallinity, but not the number of coating steps, affected the dehydration of the strawberries considered. Uncoated controls lost approximately 70% of their original weight over the 14 days considered. Strawberries coated with amorphous silk (DxCO) retained more water compared to controls on day 3 (p<0.05). Strawberries coated with crystalline silk additionally slowed down fruit dehydration compared to the amorphous coating (p<0.05) and the control (p<0.05) but no statistical difference was found for different annealing times in water (p>0.05).

Interaction between thin silk fibroin membranes and water.

To investigate the phenomenon of fruit dehydration through a thin silk fibroin coating, the interaction between thin silk fibroin membranes and water was studied. In particular, Figure 9 shows the permeability and diffusion coefficient of water and oxygen in silk fibroin films as a function of protein crystallinity and their effects on the quality of the edible coating. The phenomena of hydrodynamic permeability (shown in panel (a)) and water diffusion coefficient (shown in panel (b)) were explored. The investigation of hydrodynamic permeability showed no statistically significant effect (p>0.05) of silk fibroin crystallinity on water permeation (by one-way ANOVA with Tukey's mean analysis). The study of the water diffusion coefficient revealed that the crystallinity of the silk membranes slightly affected the mass transport of water through the silk (according to one-way ANOVA with Tukey's mean analysis). The diffusion coefficients calculated for the mass transport experiment are shown in Table 4 below. k and n are fitting parameters, while D is the diffusion coefficient.

<td>Crystallinity of the edible silk fibroin coating</td><td>k</td><td> 11</td><td>D (cm²/s)</td>

<td>Cl</td><td> 0,25 ±0,048</td><td> 0,42 ± 0,04</td><td>5.79 x 10'⁶</td>

<td>C6</td><td> 0,19</td><td> 0,48 ± 0,03</td><td>321 x 10'⁶</td>

<td>C12</td><td> 0,16</td><td> 0,55 ± 0,02</td><td>1.05 x 10'⁶</td>

Table 4: Water diffusion coefficients calculated for mass transport experiments.

Gas diffusion coefficient through silk membranes.

The gas diffusion coefficient through silk fibroin membranes plays an important role in fruit preservation, as gases play an important role in fruit stem cell metabolism (i.e., oxygen), are byproducts of fruit stem cell metabolism (i.e., carbon dioxide), and can act as growth factors (i.e., ethylene in climacteric fruit). To evaluate the effectiveness of the silk fibroin coating as a gas barrier, we conducted a study. Figure 9 in panel (c) and Table 5 below show the oxygen diffusion coefficient measured through silk membranes (t=70-80 pm) of higher crystallinity. In particular, the effective oxygen diffusion coefficient of silk fibroin was found to be modulated by protein crystallinity, as a two-order-of-magnitude decrease in the effective oxygen diffusion coefficient was calculated between amorphous silk and 58.4% crystalline silk (12 hours of water annealing).

<td>Crystallinity of the edible silk fibroin coating</td><td>Film thickness //m</td><td>Effective O2 diffusion coefficient of the membrane [D_{and M}] (steady state) 10¹¹· (cm²/s)</td>

<td>CO- amorphous</td><td> 81±4</td><td> 83,9±7,2</td>

<td>Cl</td><td> 78+3</td><td> 67,9+3,5</td>

<td>C6</td><td> 76±4</td><td> 5,5±1,4</td>

<td>C12</td><td> 73+2</td><td> 1,9+0,3</td>

Table 5: Effective O2 diffusion coefficient of silk fibroin calculated for gas transport experiments.

Respiration rate in strawberries coated with silk fibroin.

Respiration rate is an important parameter for assessing the metabolic activity of stem cells present in fruits. The higher the respiration rate, the greater the metabolic activity and the faster the fruit decomposition. Figure 9, panel (d), shows the respiration rate of silk-coated strawberries, measured as a function of the coating's crystallinity. Higher degrees of crystallinity were associated with a statistically significant decrease in CO2 production (p&lt;0.05), indicating a reduction in the fruit's respiration rate. It was evident that the degree of crystallinity of the edible silk fibroin coating controlled gas exchange between the strawberry and its environment, due to the significant influence of silk polymorphism on permeability to gases such as CO2 and O2.

firmness index in strawberries coated with silk fibroin.

Figure 9 in panel (e) shows that a puncture (or penetration) test was used to evaluate the effects of the silk coating on strawberry firmness as a function of storage time and coating crystallinity. While natural decomposition of strawberries caused a decrease in fruit firmness, as measured by a time-dependent decrease in the force required to penetrate the fruit (p&lt;0.05), an increase in coating crystallinity corresponded to a statistically significant delay in the deterioration of fruit firmness on days 3 and 7.

Silk fibroin as a coating for climacteric fruits.

Fruits that ripen through ethylene production and increased cellular respiration are called climacteric. Examples of climacteric fruits are apples, bananas, and tomatoes, while strawberries and grapes are non-climacteric fruits. The climacteric event is associated with changes in fruit color and the production of sugar in the extracellular space. Figure 10 shows an evaluation of the efficacy of a silk-based climacteric fruit coating on ripening uncoated and silk fibroin-coated bananas. Fruits were stored at 22°C and 38% RH as received (uncoated) and after coating with crystalline silk fibroin films (crystalline coating). Bananas were hung from their respective stems throughout the experiment. Time-lapse photography of ripening bananas indicates that the silk coating slowed ripening. The structure (i.e., amorphous or crystalline) of the fibroin used to coat climacteric fruit did not affect fruit ripening (data not shown). Investigation of the turgor of a silk-coated banana showed that the coating increased fruit firmness, compared to a control.

100 uncoated on day 9 after coating.

Furthermore, morphological analysis of coated and uncoated banana flesh on day 9 after silk coating treatment revealed more preserved fruit when silk coating was applied. The uncoated banana flesh was brown in color, whereas silk-coated fruits retained pale flesh, indicating a slower ripening rate in the silk-coated sample.

Silk fibroin is an effective coating for improving the freshness of perishable foods. Silk's polymorphism can be used to tailor the coating's properties, affecting the interaction between silk fibroin and water evaporation and microbial-induced food spoilage.

Conclusion

Silk fibroin is an effective coating for improving the freshness of perishable foods. Silk polymorphism can be used to tailor the coating's properties, affecting the interaction between the fibroin and the

101 silk and water evaporation and food spoilage caused by microbes.

OTHER MODALITIES AND EQUIVALENTS

Although the present description has explicitly discussed certain particular embodiments and examples of the present disclosure, it will be understood by those skilled in the art that the invention is not intended to be limited to such embodiments or examples. Rather, the present description encompasses various alternatives, modifications, and equivalents of such particular embodiments and/or examples, as will be understood by those skilled in the art.

Therefore, for example, methods and diagrams should not be construed as limiting themselves to a particular order or arrangement of steps or elements described unless explicitly stated otherwise or the context clearly dictates (e.g., not otherwise operative). Furthermore, different features of particular elements that may be exemplified in different embodiments may be combined together in some embodiments.