EXTENSION

CROSS-REFERENCE TO RELATED APPLICATION

[0001 ] This application claims priority from U.S. Provisional Patent

Application No. 62/777,512, filed December 10, 2018, which is hereby incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

[0002] Aspects of the present disclosure pertain to compositions, solutions, and methods for preparing fibroin proteins and its use in extending the shelf-life of perishables and on substrates.

BACKGROUND OF THE DISCLOSURE

[0003] One third of the food produced in the world is wasted each year and over 45% of all fruits and vegetables are lost to spoilage. Food waste has massive economic, social, and environmental implications. According to the Natural

Resources Defense Council (NRDC), a prominent non-profit international

environmental advocacy group, the United States loses 40% of its food supply resulting in an estimated economic loss of $165 billion per year. Embodiments of the present disclosure directly address the broader societal need for reducing food waste and increasing food availability by extending the shelf-life of perishables.

Embodiments of the present disclosure represent significant commercial value by increasing revenue through improved distribution, reducing waste, and decreasing costs associated with cold storage and transport. [0004] Exemplary compositions of the present disclosure comprise natural and edible silk coatings that are able to extend the shelf life of perishable goods and decrease reliance on energy and cost intensive cold chains. The silk coatings of the present disclosure may be odorless, low cost, edible, compostable, and

biodegradable. When deposited onto the surface of perishable foods, these coatings are able to drastically extend the foods’ shelf life. Many of today’s state-of-the-art food preservation strategies rely on petroleum based synthetic coatings, genetic modifications, single-use packaging, and a heavy reliance on the cold-chain. While these methods have shown moderate success, they have become less sustainable to people seeking a natural way of reducing food waste with a far lower carbon footprint. Coating compositions of the present disclosure offer a unique combination of advantages to augment or replace these approaches.

[0005] Silk fibroin is a structural protein that may be produced and extracted from silkworm, spiders, or other insects. It can also be otherwise generated synthetically. Silk fibroin is naturally produced by species such as, without limitation, Antheraea mylitta; Araneus bicentenarius; Araneus ventricosus; Bombyx mori;

Bombyx mandarins; Galleria mellonella; Nephila clavipes; Nephila

madagascariensis; and Tetragnatha versicolor. Silk fibroin’s unique properties are derived from its structure, consisting of hydrophobic blocks separated by hydrophilic spacers. In its natural state, silk fibroin is organized in beta-sheets, which are formed by highly ordered crystalline regions alternated by amorphous regions. This unique structure results in high levels of strength and toughness for silk fibroin-based materials. The wide range of forms into which silk fibroin solution can be processed make it attractive for several high-tech applications, including scaffolds for tissue engineering, bone screws for fixation, and drug depots for therapeutic delivery. [0006] Among other insects, the Bombyx mori silkworm starts its life in a cocoon. A single cocoon can be made up of hundreds of meters of silk. The cocoon itself is made up largely by two proteins: fibroin and sericin. With the other component parts, the fibroin helps regulate gas diffusion in and out of the cocoon. Similarly, such regulation is achievable with silk fibroin-based materials. This is applicable to extending the shelf-life of perishables given that the main modes of spoilage relate to oxidation, degradation, and dehydration. When applied to agriculture, the tunability of silk fibroin allows for its versatility of application across different produce types. Silk fibroin forms polymeric coatings with varying degree of solubility and breathability. Fine-tuning is enabled by different production parameters such as concentration, shear force, additives, temperature, and postprocessing. By tuning these parameters, silk-fibroin structural organization may be controlled.

Structural differences ultimately affect crystallinity, elasticity, solubility and porosity which in turn alter the gas and water diffusion behavior, as well as reduce microbial growth rates. Silk coatings can be designed for different storage conditions or products of interest.

SUMMARY OF THE DISCLOSURE

[0007] In some aspects, the present disclosure relates to the following embodiments. One embodiment is directed to a food composition comprising silk fibroin, wherein the silk fibroin was previously in a powder form or in a solution in which powdered silk fibroin has been reconstituted within a solvent. Food

compositions of the present disclosure may include materials that are edible in either a cooked, prepared, processed, or raw state and that are susceptible to at least one type of damage or reduction in quality, which typically involves changes in one or more parameters, such as microbial growth, color, water content, mass loss, taste, smell, texture, viral or fungal growth, firmness, or other parameters. Non-limiting examples include cooked or uncooked meats, proteins, carbohydrates, produce, nuts, grains, seeds, dairy, beverages, processed foods (e.g., chocolates, candies, chips, snacks, energy bars), gums, tablets, capsules, plants, roots, fungi, spores, breads, dried fruits, dried vegetables, dehydrated foods, medical foods, and the like. Further examples include the peels or outer layers of foods; for example, a banana peel, avocado peel, pistachio or nut shells, citrus peel, or the like.

[0008] Another embodiment is directed to a solution containing silk fibroin. In some aspects, no more than 25% of the solution may comprise silk fibroin fragments. In some further aspects, no more than 10% of the silk fibroin fragments may have a molecular weight of over 400 kilodaltons (kDa). In some alternative further aspects, no more than 45% of the silk fibroin fragments may have a molecular weight of over 300 kDa.

[0009] Another embodiment is directed to a composition comprising a substrate. In some aspects, the substrate may be coated with at least one silk fibroin layer. In some aspects, the thickness of the at least one silk fibroin layer may range from about 12 nm to about 180 pm. Substrates of the present disclosure may include perishables, perishable items, perishable goods, or perishable products that may be edible in cooked, prepared, processed, or raw forms. These terms may be defined as something that is likely to decay, spoil, or degrade. Non-limiting examples of a perishable include food, food items, candy, meat, gum, produce, baked goods, vegetables, fruits, power bars, granola bars, cereal, chocolates, processed foods, tablets, sugars, protein powders, flavor powders, and others. Edible peels and coatings may also be included. Further examples include inedible peels and coatings, such as banana peels, citrus peels, avocado peels, pistachio or nut shells, or the like.

[0010] Another embodiment is directed to a method of preparing a food composition. In some aspects, the method may comprise spray-coating the food composition with a solution containing silk fibroin fragments. In some aspects, the silk fibroin fragments may range from 10kDa to 600kDa.

[001 1 ] Another embodiment is directed to a method of preparing a food composition. In some aspects, the food composition may comprise dip-coating the food composition with a solution containing silk fibroin fragments. In some aspects, the silk fibroin fragments may range from 10kDa to 600kDa.

[0012] Another embodiment is directed to a method of preparing a food composition. In some aspects, the food composition may comprise mixing the food composition with silk fibroin fragments. In some aspects, the silk fibroin fragments may range from 10kDa to 600kDa.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 A depicts the percentage of mass loss in avocados not treated with the silk composition (control) and avocados treated with the silk composition and further exposed to a water-annealing step (silk-water ann.) after 0, 5, and 1 1 days. Figure 1 B depicts an avocado not treated with the silk composition (control) and an avocado treated with the silk composition and further exposed to a water-annealing step (silk-water ann.) after 1 1 days. Water-annealing can refer to exposing the fibroin to water or water vapor to increase the crystallinity of the fibroin.

[0014] Figure 2A depicts the percentage of mass loss in avocados not treated with the silk composition (control) and avocados treated with the silk composition without a water-annealing step (silk-nonwater ann.) after 0, 6, and 14 days. Figure 2B depicts an avocado not treated with the silk composition (control) and an avocado treated with the silk composition without a water-annealing step (silk-nonwater ann.) after 14 days.

[0015] Figure 3 depicts the percentage of mass loss in avocados having the silk composition applied by spraying and dipping after 10 days.

[0016] Figure 4A depicts the percentage of mass loss in bananas not treated with the silk composition (control) in comparison to bananas treated with the silk composition (silk) after 9 days at room temperature. Figure 4B depicts the bananas of Figure 4A.

[0017] Figure 5A and Figure 5B depict avocados and beef not coated (uncoated) in comparison to those coated with the silk composition.

[0018] Figure 6 depicts the growth of bacteria on beef not coated (control) or coated with the silk composition and maintained for 3 days at 40°F.

[0019] Figure 7 depicts a control, untreated meat product (Control) and a meat product treated with actives such as green tea extracts or acerola and silk fibroin solution and excipients after 9 days.

[0020] Figure 8 depicts avocado firmness in control, untreated avocados and avocados coated with silk fibroin solution via spray deposition at baseline (0 days) and after 3, 5, 7, and 9 days (n = 60).

[0021 ] Figure 9A depicts control, untreated spinach and spinach coated with silk fibroin solution via spray deposition after 24 days.

[0022] Figure 9B depicts spinach mass retention in control, untreated spinach and spinach coated with silk fibroin solution via spray deposition at baseline (0 days) and after 5, 10, and 12 days.

[0023] Figure 10A depicts silk fibroin powder. [0024] Figure 10B depicts silk fibroin solution.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0025] Embodiments of the present disclosure are compositions, solutions, and methods for extending the shelf life of food compositions.

[0026] One embodiment may be directed to a food composition comprising silk fibroin. In some aspects, the silk fibroin may be a dried powder, such as in FIG.

I OA. In some further aspects, the silk fibroin may be in liquid form, such as in FIG.

I OB. In some aspects, the solution may comprise the silk fibroin powder mixed with a solvent. In some aspects, the solvent may be a liquid. In some further aspects, the solvent may be an acid with a pH under 6.0. Alternatively, the solvent may be an alcohol or water. In other aspects, the solvent may be acetic acid. In some aspects, the silk fibroin powder may be in a mixture containing an additive. In some

alternative embodiments, the liquid solvent may contain an additive. In some embodiments, both the silk fibroin powder mixture and the liquid solvent may contain additives. In some alternative aspects, the silk fibroin may be emulsified with the additive prior to being mixed into the solution. For example, liquid silk fibroin may be mixed or dry-blended with the additive prior to being mixed into the solution. In some aspects, the additive may be at least one of a sugar, a plasticizer, or a crosslinking agent. For example, the sugar additive may be a sugar-ol, a poly-ol, or a hygroscopic polymer (e.g., polyethylene glycol). In other examples, if the sugar additive is a crosslinking agent, the crosslinking agent may be photoreactive. Specifically, the crosslinking agent may be, for example, one or more of horseradish peroxidase, lysyl oxidase, disuccinimidyl suberate, disuccinimidyl glutarate, N-hydroxysuccinimide ester, or an aryl azide. In some alternative aspects, the additive may include one or more of a bacteria, a metal, an enzyme, or a biologic. For example, the metal may include one or more of an alkali metal, an alkaline earth metal, or a transition metal.

In other examples, the biologic may be an insulin glargine, infliximad, rituximab, etanercept, adalimumab, monoclonal antibodies, trastuzumab, or other biologies. In other examples, the additive may be an oligonucleotide, such as an RNA. The RNA may be tRNA, mRNA, rRNA, snRNA, srpRNA, gRNA, TERC, SL RNA, crRNA, miRNA, siRNA, or eRNA. Alternatively, in other examples, the additive may be an enzyme (i.e., an RNase or a DNase), a fatty acid, a sugar (e.g., an alcohol sugar), or a mineral. For example, the enzyme may include erepsin maltase, lactase, sucrase, disaccharidases, lingual lipase, lysozymes, salivary amylase, pepsin, gastric lipase, other lipases, hydrochloric acids, intrinsic factors, mucins, gastrins, trypsinogen, ductal cells, carboxypeptidase, elastases, and the like. In some other alternative aspects, the additive may be at least one of a coloring agent, a chelator, a ligand, an antimicrobial, a filler, a scent, or a flavor. For example, the coloring agent may be allura red, Ponceau 3R, amaranth, erythrosine, indigotine, Light Green SF, Naphthol yellow, Orange 1 , quinoline yellow, tartrazine, an E1 suit (e.g., E100, E161 b, etc.), an anthocyanin, a betacyanin, a carotenoid, or a phenolic. In other examples, the chelator may be ethylenediaminetetraacetic acid (EDTA), transferrin, or

desferrixoxamine. In other examples, the microbial may be acetic acid, benzoic acid, natamycin, nisin, nitrate, nitrite, propionic acid, sorbic acid, sulfite, or sulfur dioxide.

In other examples, the filler may be cellulose. In other alternative embodiments, the additive may be at least one of a vitamin, a nutrient, an antioxidant, and a protein. In some examples, the protein may be a peptide, an amino acid, (e.g., a post-translated amino acid), or a synthetic amino acid. A nutrient may be defined as a mineral, protein, carbohydrate, fat, Q10, glutathione, lithium, probiotic, glycine, DHA, flavonoid, or others. An antioxidant may include vitamins C and E, selenium, carotenoids, thiols, catalase, superoxide dismutase, uric acid, and ubiquinol. In some further alternative embodiments, the additive may be at least one of a green tea extract, a rosemary extract, a phenolic antioxidant, catechin, acerola, tocopherol, chamomile extract, malphigia emarginata, camellia sinensis, epicatechin, epigallocatechin, gallochatechin, epigallocatechin gallates, vitamin A, vitamin E, and/or vitamin C. In some aspects, the additive may be mixed with an accelerant or an excipient. For example, the additive may be mixed with polyethylene glycol or xylitol. In some further aspects, the additive may be emulsified with the accelerant or excipient and mixed into a silk fibroin solution. In some further aspects, the solution may be deposited onto the food composition via spray-coating. Alternatively, the solution may be deposited onto the food composition via dip-coating. In some aspects, the silk fibroin may not be annealed after or before deposition. In some further aspects, the food composition may include multiple layers of silk fibroin. For example; the food could be sprayed with silk fibroin solution, dried, and then sprayed once more. This can happen any number of times to add thickness and additional layers. In some further aspects, the food composition may comprise of multiple layers, with each layer serving a function. For example, the food may be coated with silk fibroin. Then, the silk fibroin-coated food may be itself coated by another coating that is hydrophobic or water-tight such that water may not permeate the outer layer and reach the inner silk fibroin layer. In some further aspects, a tablet-coating may be utilized, where the silk fibroin is coated while in an industrially-relevant drum. Tablet coating may additionally be utilized, as well as film-coating.

[0027] FIGs. 1 A, 1 B, 2A, 2B, 5A, 5B, 9A, and 9B, demonstrate that silk deposition on foods by spray or dip coating assists with their preservation, regardless of whether the deposition step is paired with an annealing step. Thus, these findings indicate that pairing silk deposition with an annealing step is not necessary for preserving and extending the shelf-life of food products, such as avocados. Thus, the invention unexpectedly demonstrates that an additional annealing step is not necessary for extending the shelf-life of food products. Spray or dip coating food products with silk is sufficient for optimizing their preservation. This is further corroborated by FIGs. 1 A, 2A, 3, and 4A which show that the percent mass loss of foods subjected to silk deposition is significantly reduced relative to control, irrespective of the presence of an annealing step. FIG. 8 also demonstrates that avocado firmness and is also consistently enhanced following silk treatment.

[0028] Deposition of silk on food has also been shown to slow bacterial growth, as illustrated in FIG. 6. As demonstrated in FIG. 7, deposition of silk on beef not only enhanced its preservation relative to control with regards to the look and feel of the beef, but also the bacterial growth rates (FIG. 6). This may be further enhanced with additives, such as green tea and acerola extracts, which can be seen in FIG. 7. The silk fibroin may be dissolved in acetic acid or other solvents in order to further reduce microbial growth rates, as well as keep food fresher for longer.

[0029] In some aspects of the present embodiment, all or substantially all of the external surface of the food composition may be coated with the silk fibroin. In some aspects, the silk fibroin coating may comprise silk fibroin fragments ranging from 10kDa - 600 kDa. In some aspects, the thickness of a single silk fibroin layer in the surface of the food composition may be at least 12 nm. In some further aspects, the thickness of a silk fibroin layer may be no more than 180 pm. In some further aspects, the silk fibroin coating may be comprised of multiple layers, where the silk fibroin is added to the substrate, dried, and another layer is then added. This may be done numerous times to increase the number of layers. In some aspects the thickness of at least one layer is from about 5 urn to 180 urn. In some aspects, the additional layers may be comprised of a different coating. In some other aspects of the embodiment, the silk fibroin solution may be dried via air-drying, freeze-drying, vacuum drying, or heat-drying. In some alternative aspects of the embodiment, the silk fibroin may be mixed into the food composition. In some aspects of the embodiment, the silk fibroin may slow microbial growth in the food composition. In other alternative embodiments, the silk fibroin may alter moisture transport properties within the food composition. In some further embodiments, altering moisture and gas transport properties within the food composition may extend the shelf-life of the food composition by, for example, preventing sogginess. This may be done on prepared compositions of foods, such as a traditional sandwich or granola bar.

[0030] Another embodiment may be directed to a solution containing silk fibroin. In some aspects, no more than 25% of the solution may comprise silk fibroin fragments. In some further aspects, no more than 10% of the silk fibroin fragments may have a molecular weight of over 400 kDa. In some alternative further aspects, no more than 45% of the silk fibroin fragments may have a molecular weight of over 300 kDa.

[0031 ] Another embodiment may be directed to a composition comprising a substrate. In some aspects, the substrate may comprise a perishable, a perishable item, a perishable good, or a perishable product. A perishable may comprise an item or product likely to decay, spoil, or go bad quickly. Examples of a perishable include food, food items, candy, meat, gum, produce, baked goods, vegetables, fruit, power bars, chocolates, and processed foods. In some aspects, the substrate may be coated with at least one silk fibroin layer. In some aspects, the at least one silk fibroin layer may comprise an additive. In some aspects, the additive may function to stabilize the silk fibroin layer coated on the substrate. Alternatively, the additive may stabilize other ingredients being coated on the substrate. In some aspects, the additive may be a coloring agent, an antioxidant, a microorganism, an enzyme, a protein, a sugar, an acid, or a plasticizer. In some aspects, the additive may increase the flexibility and/or elasticity of the silk fibroin layer. In some aspects, the thickness of the at least one silk fibroin layer may range from about 12 nm to about 180 pm. In some aspects, the silk fibroin layer may comprise a dried silk fibroin solution. In some further aspects, the silk fibroin solution may comprise no more than 25% silk fibroin fragments. In some aspects, the crystallinity of the silk fibroin solution may increase following its addition to the substrate. In some aspects, increasing the crystallinity of the silk fibroin solution may comprise changing the pH of the silk fibroin solution; removing water, solvents, or additives from the silk fibroin solution; water-annealing the silk fibroin solution; or adding methanol, acetic acid, and/or ethanol to the silk fibroin solution. Increasing the crystallinity of the solution may occur prior to or after deposition of the silk fibroin onto a substrate or food composition by any of the above means, or means similar.

[0032] Another embodiment may be directed to a method of preparing a food composition. In some aspects, the method may comprise spray-coating the food composition. In some alternative aspects, the food composition may comprise dip-coating the food composition. In some alternative aspects, the food composition may comprise mixing the food composition. In some aspects, the food composition may be spray-coated, dip-coated, or mixed with silk fibroin fragments. In some aspects, the silk fibroin fragments may range from 10kDa to 600kDa. [0033] Moreover, while illustrative embodiments have been described herein, the scope of any and all embodiments having equivalent elements, modifications, omissions, combinations (e.g., of aspects across various

embodiments), adaptations and/or alterations as would be appreciated by those skilled in the art based on the present disclosure. The limitations in the claims are to be interpreted broadly based on the language employed in the claims and not limited to examples described in the present specification or during the prosecution of the application. The examples are to be construed as non-exclusive. Furthermore, the steps of the disclosed methods may be modified in any manner, including by reordering steps and/or inserting or deleting steps. It is intended, therefore, that the specification and examples be considered as illustrative only, with a true scope and spirit being indicated by the following claims and their full scope of equivalents.