Processing of protein using ultrasonic vibration energy

Abstract

A composition of and method of making a proteinaceous article formed from a first proteinaceous substrate and second proteinaceous substrate bonded together such that the properties of the first and second proteinaceous substrates is not degraded.

Description

PROCESSING OF PROTEIN USING ULTRASONIC VIBRATION ENERGY FIELD

[0001] The present disclosure relates to the field of proteinaceous substances that may be melted, combined, and molded to form a molded article. BACKGROUND

[0002] Functional proteins such as silk fibroin and keratin are some of the strongest natural materials and are widely used in the textile and food industries. Proteins are responsible for many functions in nature and have tunable properties such as strength, flexibility, degradability, optical transparency, acoustic transmission, enzymatic activity, and more. Engineering applications for proteins have been explored extensively in recent history such as spinning, extrusion, and molding. One key problem with thermally processing proteins is that they typically degrade or denature prior to melting limiting their processability and the ability to form end use articles. Even if the proteinaceous material is thermally moldable into an article, the resulting article is characterized by undesirable properties due to the thermal degradation of the starting material. The degradation and denaturation causes them to lose their secondary and tertiary structure such as beta sheet and alpha helix conformations which is key to their performance and function. As such, there remains a need to mold and fuse proteinaceous substances into articles with properties that are not adversely affected by thermal processing. SUMMARY

[0003] The present disclosure provides a method to fuse discreet proteinaceous substrates into an article including providing a first proteinaceous substrate and a second proteinaceous substrate; bonding said first and second proteinaceous substrates into a first article using an effective amount of ultrasonic energy; and optionally shaping the first article into the second article. The first and second proteinaceous substrate are independently in the form of one or more particles, fibers, films, non-woven meshes, woven meshes, or membranes.

[0004] In a different embodiment, the present disclosure provides an ultrasonically bonded proteinaceous article including a first starting material and a second starting material. The first starting material includes a first proteinaceous substrate, and the second starting material includes a second proteinaceous substrate.The first and second starting materials are bonded together with ultrasonic energy to form the bonded proteinaceous article. Further, the first and second starting materials are independently in the form of one or more particles, fibers, films, woven- and non- woven meshes or membranes prior to being melted and bonded together to form the proteinaceous article.

[0005] According to one embodiment (“Embodiment 1”), the disclosure relates to a method to fuse discreet proteinaceous substrates into an article comprising: providing a first proteinaceous substrate; and a second proteinaceous substrate; wherein said first and second proteinaceous substrates are independently in the form of one or more particles; pellets, filaments, a fiber, a film, a non-woven mesh, a woven mesh or a membrane; bonding said first and said second proteinaceous substrates into a first article using an effective amount of ultrasonic energy; and optionally shaping the first article into a second article.

[0006] Embodiment 2 is a method according to Embodiment 1 wherein said bonding step is conducted substantially free of any solvent.

[0007] Embodiment 3 is a method according to Embodiment 1 or 2 wherein the first article is in a flowable state.

[0008] Embodiment 4 is a method according to any one of Embodiments 1 to 3 wherein said bonding and / or shaping is by ultrasonic compression molding, ultrasonic welding, ultrasonic injection molding, ultrasonic extrusion, 3D additive manufacturing and any combination thereof.

[0009] Embodiment 5 is a method according to any one of Embodiments 1 to 4 wherein said first proteinaceous substrate and said second proteinaceous substrate are in the form of a plurality of dry powder particles.

[0010] Embodiment 6 is a method according to any one of Embodiments 1 to 5 wherein the average particle size of the dry powder particles is less than 400 µm.

[0011] Embodiment 7 is a method according to any one of Embodiments 1 to 6 wherein said first proteinaceous substrate, said second proteinaceous substrate or both first and second proteinaceous substrates are naturally-isolated or recombinantly- produced spheroproteins (globular), enzymes, scleroproteins (fiber-forming), membrane proteins, and any combination thereof.

[0012] Embodiment 8 is a method according to Embodiment 7 wherein the scleroproteins are keratins (α-, β- and γ-keratin), collagens, fibrins, silk fibroins, silk spidroins, elastins, and any combination thereof.

[0013] Embodiment 9 is a method according to Embodiment 8 wherein the first proteinaceous substrate, the second proteinaceous substrate or both said first and second proteinaceous substrates are silk fibroin, silk spidroin and combinations thereof.

[0014] Embodiment 10 is a method according to any one of Embodiments 1 to 7 wherein the first proteinaceous substrate, the second proteinaceous substrate or both said first and second proteinaceous substrates are plant proteins, plant protein isolates, plant protein hydrolysates, wheat proteins (gliadin), casein, whey, gelatin, egg white proteins, zein, resilin (dragonfly), and any combination thereof.

[0015] Embodiment 11 is a method according to any one of Embodiments 1 to 10 wherein the average molecular weight of said first or said second proteinaceous substrate is between 1 kDa and 500 kDa.

[0016] Embodiment 12 is a method according to any one of Embodiments 1 to 11 wherein said bonding is conducted at a temperature of 120 °C or less.

[0017] Embodiment 13 is a method according to any one of Embodiments 1 to 12 wherein said bonding step is conducted at a pressure of from 5 psi (34.5 kPa) to 30,000 psi (207 MPa).

[0018] Embodiment 14 is a method according to any one of Embodiments 1 to 13 wherein said bonding time ranges from 0.1 second to 60 seconds.

[0019] Embodiment 15 is a method according to any one of Embodiments 1 to 14 wherein said first proteinaceous substrate is a powder, a fiber, a filament, a pellet, a film, a non-woven mesh or a woven mesh and said second proteinaceous substrate is a powder, a pellet, a filament, a fiber, a film, a non-woven mesh or a woven mesh.

[0020] Embodiment 16 is a method according to any one of Embodiments 1 to 15 wherein said first article or said second article is in the form of a fiber, a pellet, a film, a tube, a membrane, a non-woven article, a woven article, a solid three-dimensional object, or a porous 3-dimensional object.

[0021] Embodiment 17 is a method according to any one of Embodiments 1 to 16 further comprising step (d) – repeating steps (a) to (c) a plurality of times; wherein said first proteinaceous substrate, said second proteinaceous substrate or both said first and said second proteinaceous substrates are optionally said first article or said second article.

[0022] Embodiment 18 is an ultrasonically bonded proteinaceous article comprising: a first starting material comprising a first proteinaceous substrate; and a second starting material comprising a second proteinaceous substrate; wherein the first and second starting materials are bonded together with ultrasonic energy to form thebonded proteinaceous article; wherein the first and second starting materials were independently in the form of one or more particles, a fiber, a film, a non-woven mesh, a woven mesh, or a membrane prior to being melted and bonded together to form the proteinaceous article.

[0023] Embodiment 19 is the article of Embodiment 18 wherein each of the first starting material, the second starting material, and the bonded proteinaceous article comprise a mean activation energy as measured according to ASTM E1641-18; wherein a change in the mean activation energy from at least one of the first and second starting material to the bonded proteinaceous article is an increase of less than 15% compared to the at least one of the first and second starting materials.

[0024] Embodiment 20 is the article of Embodiment 18 or 19 wherein each of the first starting material, the second starting material, and the bonded proteinaceous article comprise an activation energy of degradation at 10% conversion measured according to ASTM 1631-18; wherein a change in an activation energy of degradation at 10% conversion of at least one of the first and second starting materials to the activation energy of degradation at 10% conversion of the bonded proteinaceous article is a decrease of less than 15% as compared to the activation energy of degradation at 10% conversion of the at least one of the first and second starting materials.

[0025] Embodiment 21 is the article of any one of Embodiments 18 to 20 wherein the bonded proteinaceous article comprises an activation energy of degradation at 10% conversion of less than 185 kJ / mol as measured according to ASTM 1631-18.

[0026] Embodiment 22 is the article of any one of Embodiments 18 to 21 wherein each of the first starting material, the second starting material, and the bonded proteinaceous article comprise an α-helix and random coil content; wherein a change in the α-helix and random coil content from at least one of the first and second starting materials to the bonded proteinaceous article is a decrease of less than 15% as compared to the α-helix and random coil content of the at least one of the first and second starting materials.

[0027] Embodiment 23 is the article of any one of Embodiments 18 to 22 wherein each of the first starting material, the second starting material, and the bonded proteinaceous article comprise a β-sheet content as measured using Fourier-transform Infrared Spectroscopy (FTIR); wherein a change in the β-sheet content from at least one of the first and second starting materials to the bonded proteinaceous article is an increase of less than 15% as compared to the β-sheet content of the at least one of the first and second starting materials.

[0028] Embodiment 24 is the article of any one of Embodiments 18 to 23 wherein the bonded proteinaceous article has a mean bond strength greater than 600 kPa as determined according to ASTM D3436.

[0029] Embodiment 25 is the article of any one of Embodiments 18 to 24 wherein the bonded proteinaceous article has a compressive strength of at least 75 MPa.

[0030] Embodiment 26 is the article of any one of Embodiments 18 to 25 wherein the bonded proteinaceous article has a flexural strength at maximum strain of at least 65 MPa as measured at 25oC at ambient relative humidity with a loading rate of 2 mm / min.

[0031] Embodiment 27 is the article of any one of Embodiments 18 to 26 wherein each of the first starting material, the second starting material, and the bonded proteinaceous article comprise a flexural strength; wherein a change in the flexural strength from at least one of the first and second starting materials to the bonded proteinaceous article is a decrease of less than 30% as compared to the flexural modulus of the at least one of the first and second starting materials.

[0032] Embodiment 28 is the article of any one of Embodiments 18 to 27 wherein the bonded proteinaceous article has a flexural modulus of at least 6 GPa.

[0033] Embodiment 29 is the article of any one of Embodiments 18 to 28 wherein each of the first starting material, the second starting material, and the bonded proteinaceous article comprise a yellowness index as determined by ASTM E313-20; wherein a change in the yellowness index from at least one of the first and second starting materials to the bonded proteinaceous article is an increase of less than 60% as compared to the yellowness index of the at least one of the first and second starting materials.

[0034] Embodiment 30 is a bonded proteinaceous article comprising at least one of the following: a. an activation energy of degradation at 10% conversion of less than 165 kJ / mol as measured according to ASTM 1631-18; and b. a moisture content of less than 6 wt% based on the total weight of the bonded proteinaceous article.

[0035] Embodiment 31 is the bonded proteinaceous article of Embodiment 30 further comprising at least one of the following: a.) a mean bond strength of at least 600 kPa; b.) a flexural strength at maximum strain of at least 65 MPa; c.) a flexural modulus of at least 6.0 GPa; d.) a compressive strength of at least 75 GPa; or e.) any combination of a, b, c, or d.

[0036] Embodiment 32 is the bonded proteinaceous article of Embodiment 30 or Embodiment 31 wherein the bonded proteinaceous article comprises 0 wt% to 20 wt% β-sheet content based on the total weight of the bonded proteinaceous article.

[0037] Embodiment 33 is the bonded proteinaceous article of any one of Embodiments 30 or 31 further comprising a yellowness index of 65 or less as measured by ASTM E313-20.

[0038] Embodiment 34 is the bonded proteinaceous article of any one of Embodiments 30 to 32 further comprising 0.01 wt. % to 90 wt. % of one or more additives; wherein wt. % is based on the total weight of the bonded proteinaceous article.

[0039] Embodiment 35 is the bonded proteinaceous article of Embodiment 34 wherein the one or more additives are polymers, mixtures of proteins, composites, drugs, active ingredients, pigments, dyes, flavoring agents, fillers, radiopaque fillers, conductive fillers, porogens, desiccants, bioactive agents such as thromboresistant agents, antibiotic agents, anti-tumor agents, anti-viral agents, anti-angiogenic agents, angiogenic agents, anti-inflammatory agents, cell cycle regulating agents, and any combination thereof.

[0040] Embodiment 36 is the bonded proteinaceous article of any one of Embodiments 31 to 35 wherein said bonded proteinaceous article comprises or more proteins including, but not limited to plant proteins, plant protein isolates, plant protein hydrolysates, wheat proteins (gliadin), casein, whey, gelatin, egg white proteins, zein, resilin (dragonfly), scleroproteins, keratins (α-, β- and γ-keratin), collagens, insulin, fibrins, silk fibroins, silk spidroins, elastins, and any combination thereof.

[0041] Embodiment 37 is the bonded proteinaceous article of Embodiment 36 wherein said one or more proteins are naturally-isolated or recombinantly-produced silk fibroin, silk spidroin, and any combination thereof.

[0042] Embodiment 38 is the bonded proteinaceous article of any one of Embodiments 31 to 37 wherein said one or more proteins have an average molecular weight ranging from about 1 kDa to about 500 kDa.

[0043] Embodiment 39 is the bonded proteinaceous article of any one of Embodiments 31 to 38 wherein the bonded proteinaceous article is in the form of bone screws, artificial corneas, stents, tips and hubs for delivery systems, contact lenses, screws, pins, frames, grids used for products in the sustainability industry, drug delivery devices, time release capsules, floss picks, encapsulation of a substance for release inthe environment, stents, films, needles, glaucoma drainage devices, or other suitable articles.

[0044] The foregoing Examples are just that and should not be read to limit or otherwise narrow the scope of any of the inventive concepts otherwise provided by the instant disclosure. While multiple examples are disclosed, still other embodiments will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative examples. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature rather than restrictive in nature. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The accompanying drawings are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments, and together with the description serve to explain the principles of the disclosure.

[0046] FIG.1 is an illustration of an ultrasonic injection molding process.

[0047] FIG.2 is a graphical image comparing mean yellowness index vs. treatment method.

[0048] FIG.3 is a graphical image comparing treatment methods and the impact on β-sheet content, α-helix content, and random coil content.

[0049] FIG.4 is a graphical image comparing treatment methods vs. % change (relative to the starting material at 10% conversion) in activation energy between thermally molded and ultrasonically molded articles.

[0050] FIG.5 is a graphical image showing flexural strength (MPa) at maximum strain between the starting materials, the present ultrasonically molded articles, and a thermally molded article. DETAILED DESCRIPTION Definitions and Terminology

[0051] This disclosure is not meant to be read in a restrictive manner. For example, the terminology used in the application should be read broadly in the context of the meaning those in the field would attribute such terminology.

[0052] With respect to terminology of inexactitude, the terms “about” and “approximately” may be used, interchangeably, to refer to a measurement that includes the stated measurement and that also includes any measurements that are reasonably close to the stated measurement. Measurements that are reasonably close to thestated measurement deviate from the stated measurement by a reasonably small amount as understood and readily ascertained by individuals having ordinary skill in the relevant arts. Such deviations may be attributable to measurement error, differences in measurement and / or manufacturing equipment calibration, human error in reading and / or setting measurements, minor adjustments made to optimize performance and / or structural parameters in view of differences in measurements associated with other components, particular implementation scenarios, imprecise adjustment and / or manipulation of objects by a person or machine, and / or the like, for example. In the event it is determined that individuals having ordinary skill in the relevant arts would not readily ascertain values for such reasonably small differences, the terms “about” and “approximately” can be understood to mean plus or minus 10% of the stated value. Description of Various Embodiments

[0053] Persons skilled in the art will readily appreciate that various embodiments of the present disclosure can be realized by any number of methods and apparatus configured to perform the intended functions. It should also be noted that the accompanying drawing figures referred to herein are not necessarily drawn to scale but may be exaggerated to illustrate various embodiments of the present disclosure, and in that regard, the drawing figures should not be construed as limiting.

[0054] The present disclosure provides a method of bonding discrete proteinaceous substances together to form an article. To form an article from two or more discrete proteinaceous substances, the substances must be bonded or otherwise melted together to form a cohesive molded component. To bond the substances, thermal methods of melting and molding may be used. Thermal methods include thermal welding, thermal molding, and 3D additive manufacturing. These thermal methods may include using heat from an exothermic reaction or other convective heat source to melt each proteinaceous substance into a flowable state. However, heating the substances may degrade the structure of any proteins within the substances such that the molded article formed has lost desirable properties.

[0055] The method of the present disclosure may include using ultrasonic energy via ultrasonic bonding, ultrasonic compression molding, ultrasonic welding, ultrasonic injection molding, or any other suitable method using ultrasonic energy to fuse or melt the proteinaceous substances into a flowable state. Ultrasonic energy may melt the substances such that any proteins within the substances are minimally degraded duringmelting. By avoiding degradation, the molded product may maintain the strength and functionality of high molecular weight chains of the proteins.

[0056] Ultrasonic methods of melting may use high-frequency vibrations to melt substances. An ultrasonic tool may be used to subject a substance to vibrations at ultrasonic frequencies. The ultrasonic vibration may cause concentrated molecular vibration between layers of the substance. Friction between the molecules may heat and melt the substance without degrading proteins. To melt the substances of the present disclosure, an ultrasonic tool may have a nominal output frequency of 30 kHz at 1000 Watts power may be used, such as a model SUREWELD™ 1000, available from SONOBOND Inc., West Chester, PA.

[0057] The bonding may be conducted at a pressure equal to or less than 30,000 psi (~207 MPa), 20,000 psi, (~138 MPa), 10,000 psi , 5000(~69.0 MPa), 5,000 psi, 4000 (~34.5 MPa), 4,000 psi, 3000 (~27.6 MPa), 3,000 psi, 2000 (~20.7 MPa), 2,000 psi, (~13.8 MPa), 1000 psi (~6.90 MPa), 500 psi (~3.45 MPa, 400 psi (~2.76 MPa), 300 psi (~2.07 MPa), 200 psi (~1.38 MPa), 100 psi (~690 kPa), 50 psi (~345 kPa), 20 psi (~138 kPa), 10 psi (~69.0 kPa) or 5 psi (~34.5 kPa), or within any range encompassed by the forgoing endpoints. It should be noted that bonding via injection molding is conducted at higher pressures (for example, up to about 30,000 psi (~207 MPa)) while bonding by welding is conducted at lower pressures (for example, about 100 psi (~690 kPa) or less).

[0058] In one embodiment, the bonding may be conducted at a temperature of about 120°C, 110°C, 100°C, 90°C, 80°C, 70°C, 75°C, 60°C, 50°C, 40°C, 30°C, 20°C, 10°C, 5°C, 2°C or within any range encompassed by the forgoing endpoints.

[0059] In another embodiment, the ultrasonic bonding may be conducted at a vibration frequency of from about 15 kHz to about 40 kHz.

[0060] In another embodiment, the ultrasonic bonding time may be equal to or less than about 60 seconds, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, 5 seconds, 1 second, or 0.1 second, or within any range encompassed by the forgoing endpoints. Proteinaceous Substrates

[0061] The term “protein” as used herein is meant to refer to complex substances that consist of amino acid residues joined by peptide bonds. As used herein, the term “proteinaceous” refers to a material that includes one or more proteins, polypeptides, and the like, whereby bonding between the material is formed through the use of asufficient application of ultrasonic energy. In one embodiment, the proteinaceous material comprises one or more proteins, polypeptides, and the like whereby bonding between the material is formed through the use of a sufficient application of ultrasonic energy. In another embodiment, the proteinaceous material consists essentially of one or more proteins, polypeptides, and the like whereby bonding between the material is formed through the use of a sufficient application of ultrasonic energy. The proteinaceous substance may be in the form of a solid-state composition, such as a foam or powder. The present methods and compositions can use any number of proteinaceous substrates such as proteins, polypeptides, enzymes, and the like with the proviso that ultrasonic energy is used for weld / mold together at least one first proteinaceous substrate to a second substrate. In another embodiment, both the first and second substrates are both proteinaceous. In another embodiment, the proteinaceous substrates may be naturally-isolated or recombinantly-produced spheroproteins (globular), enzymes, scleroproteins (fiber-forming), membrane proteins, and any combination thereof. As used herein, a “scleroprotein” is a fibrous protein having polypeptide chain(s) which form filamentous and / or sheet-like structures with a low solubility in water. Scleroproteins include several superfamilies of fibrous proteins such as keratin, collagen, elastin, and fibrin.

[0062] As used herein, a “fiber-forming polypeptide” is a polypeptide or protein that includes one or more domains having amino acid sequences associated with the formation of fibrous protein having superior mechanical properties as compared to the unordered protein structure. Examples of fibrous proteins in nature having superior mechanical properties include silkworm fibroin (for example, produced from the larvae of Bombyx mori) and dragline spider silks (for example, produced from Latrodectus hesperus) as well as recombinantly produced silk-like polypeptides. In one embodiment, the fiber-forming polypeptide is a silk fibroin, a silk spidroin, and / or recombinantly produced polypeptides derived from silk fibroins / spidroins.

[0063] The amino acid sequences of the main polypeptides associated with the mechanical strength of various silk fibroins / spidroins are known. For example, the heavy fibroin protein from Bombyx mori (Zhou et al., Nucleic Acids Res (2000) 28(12):2413-9; GENBANK® Accession No. AAF76983.1) consists of layers of β-sheets. GENBANK ® is the National Institute of Health (NIH, Bethesda, MD) genetic sequence database, an annotated collection of all publicly available DNA sequences (Nucleic Acids Research, 2013 Jan;41(D1):D36-42). GENBANK® is part of the International Nucleotide Sequence Database Collaboration, which comprises the DNA DataBank of Japan (DDBJ), theEuropean Nucleotide Archive (ENA), and GENBANK® at NCBI (National Center of Biotechnology Information). These three organizations exchange data on a daily basis.

[0064] The amino acid sequences of the spider dragline silk polypeptides have also been reported. They are produced by major ampullate glands of Latrodectus hesperus and include both MaSp1 (Ayoub et al., PLoS One (2007) 2(6):e514; GENBANK® Accession No. ABR68856.1) and MaSp2 (GENBANK® Accession No. ABR68855.1). These silk spidroins are extremely large proteins (200–370 kDa; made up almost entirely of repeated blocks of amino acids (aa) flanked by short (<150 aa) non-repetitive amino (N)- and carboxy (C)-terminal domains (Ayoub et al., supra). Dragline silk polypeptides MaSp1 and MaSp2 are characterized by short glycine-rich regions (Gly-Gly- Xaa, where Xaa represents a subset of amino acids typically Ala, Gln, or Tyr) followed by a stretch of multiple alanines (poly-A; typically 4-10 residues in length) that is characteristic of both MaSp1 and MaSp2 proteins. The ubiquitous poly-A stretches are associated with forming hydrophobic crystalline domains (β-sheets) that are responsible for the high tensile strength of the fiber (Ayoub et al., supra).

[0065] In one embodiment, the proteinaceous substrate is a fiber-forming polypeptide such as silk fibroin (e.g., obtained from the processing of silkworm cocoons or recombinantly expressed) or silk spidroin (e.g., spider dragline silk) polypeptides (farmed or recombinantly expressed). Processing from natural silk sources may include one or more purification steps to remove other proteins / materials that may be undesirable for further processing. For example, silk fibroin isolated from silkworm cocoons will often be degummed to remove most (if not all) of the sericin protein. As used herein, “degummed silk fibroin” may have an amount of residual sericin between 0.5 wt. % to 25 wt. %, 1 wt. % to 20 wt. %, 2 wt. % to 15 wt. %, 3 wt. % to 10 wt. %, or 4 wt. % to 5 wt. %, or within any range encompassed by the foregoing, wherein wt. % is based on the total weight of the silk fibroin sample.

[0066] In another embodiment, the proteinaceous substrate particles (made of silk fibroin / spidroin) may be amorphous (e.g., very low or minimal crystallinity) or particles having a defined amount of crystallinity. In one embodiment, the proteinaceous substrate particles are silk fibroin / spidroin particles that are substantially amorphous (very low or no measurable β-sheet content). In another embodiment, the fiber-forming polypeptide particles are substantially amorphous and an amount of β-sheet crystallinity of 20 wt. % or less, 15 wt% or less, 10 wt% or less, 5 wt% or less, 2 wt% or less, 1 wt% or less or 0.5 wt% or less, wherein wt. % is relative to the total weight of the fiber- forming polypeptide composition. In another embodiment, the fiber-forming polypeptideparticles are substantially amorphous and an amount of β-sheet crystallinity from 0.5 wt. % to 20 wt. %, 2 wt. % to 15 wt. %, or 5 wt. % to 10 wt. %, or within any range encompassed by the foregoing, wherein wt. % is relative to the total weight of the fiber- forming polypeptide composition. The amount of β-sheet content (wt%) may be measured according to the methods described in U.S. Patent Appl. Publ. No. US 2021 / 0381129 A1 to Kaplan et al., hereby incorporated by reference in its entirety.

[0067] In one embodiment, the particle size of fiber-forming polypeptide particles (e.g., silk fibroin particles) may have an average particle size ranging from about 20 nm to about 1000 µm (i.e., 1 mm), about 50 nm to about 350 µm, about 100 nm to about 250 µm, about 250 nm to about 150 µm, about 500 nm to 100 µm, about 750 nm to about 50 µm, or about 1 µm to 10 µm, or within any range encompassed by the foregoing.

[0068] In another embodiment, the proteinaceous substrates are plant proteins, plant protein isolates, plant protein hydrolysates, wheat proteins (gliadin), casein, whey, gelatin, egg white proteins, zein, resilin (dragonfly), and any combination thereof. In another embodiment, the scleroproteins are keratins (α-, β- and γ-keratin), collagens, fibrins, silk fibroins, silk spidroins, elastins, and any combination thereof. In a further embodiment, the first proteinaceous substrate, the second proteinaceous substrate or both said first and second proteinaceous substrates are naturally-isolated and / or recombinantly-produced silk fibroin, silk spidroin and combinations thereof.

[0069] The average molecular weight of the proteinaceous substrate is preferably at least 1 kDa, 5 kDa, 10 kDa, 30 kDa, 50 kDa, 75 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa or 500 kDa. In one embodiment, the average molecular weight of the proteinaceous substrate may from range from 1 kDa to 500 kDa, 5 kDa to 500 kDa, 5 kDa to 400 kDa, 10 kDa to 400 kDa, 20 kDa to 400 kDa, 30 kDa to 400 kDa or 30 kDa to 350 kDa, or within any range encompassed by the foregoing.

[0070] The proteinaceous substrate may be amorphous, semi-crystalline or crystalline. In one embodiment, the proteinaceous substrate is a mixture of amorphous, semi-crystalline and / or crystalline proteins. In another embodiment, the proteinaceous substrate is amorphous and includes no more than 20%, 15% or 10% β-sheet content. In another embodiment, the proteinaceous substrate is semi-crystalline or crystalline, having a β-sheet content greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or 55%, or within any range encompassed by the foregoing, such as 20% to 55%, 25% to 50%, 30% to 45%, or 35% to 40%.Additives

[0071] The composition of the molded / welded / bonded protein article may further include additives, such as other polymers, mixtures of proteins, composites, fillers, drugs, active ingredients, pigments / dyes, flavoring, other additives, and combinations thereof.

[0072] Suitable additives may include radiopaque filler, conductive filler, porogens, dyes, desiccant, flavoring, other proteins, bioactive agents, fertilizer, pH, and responsive polymer.

[0073] In one embodiment, suitable bioactive agents include thromboresistant agents, antibiotic agents, anti-tumor agents, anti-viral agents, anti-angiogenic agents, angiogenic agents, anti-inflammatory agents, cell cycle regulating agents, chemically modified equivalents, and combinations thereof.

[0074] The molded / welded protein article may include an amount of additives from 0.01 wt. %, 0.1 wt. %, 1 wt. %, 15 wt. %, 30 wt. %, 50 wt. %, 70 wt. %, or 90 wt. %, or within any range encompassed by the foregoing, such as 0.01 wt. % to 90 wt. %, 0.1 wt. % to 70 wt. %, 1 wt. % to 50 wt. %, or 15 wt. % to 30 wt. %, wherein wt. % is based on the total weight of the molded protein article. Molding / Welding the Proteinaceous Substrates

[0075] To form a proteinaceous article, at least one proteinaceous substrate may be melted into a fluid state, optionally combined with additives, and then molded to form a first proteinaceous article. In one embodiment, the proteinaceous article may include two proteinaceous substrates.

[0076] In yet another embodiment, the first article may be further shaped into a second article. The first article may be post treated with anneal agents, further crystalized, machined or drilled, laser imprinted, or coated with sealant / coatings to form the second article.

[0077] The proteinaceous substrate may be molded / welded in under wet or dry conditions. In one embodiment, the proteinaceous substrate is molded / welded into an article under substantially dry conditions. In a preferred embodiment, the proteinaceous substrate is molded / welded into an article wherein the water content present during ultrasonic molded / welding is no more than 10 wt%, 9 wt%, 8 wt%, 7 wt%, 6 wt%, 5 wt%, 4 wt%, 3 wt%, 2 wt%, 1 wt% or 0.5 wt%, or within any range encompassed by the foregoing based on the total weight of the proteinaceous substrate.

[0078] The proteinaceous substrate may have a variety of form factors prior to ultrasonic molding / welding including, but not limited to powders, films, sheets, membranes, pre-formed 3D objects, and any combination thereof. In a preferred embodiment, the proteinaceous substrate is in the form of a powder or film.

[0079] International Patent Appl. Pub. No. WO20117368 A2 to Kaplan et al. discloses thermal fusion and molding of amorphous silk fibroin protein particles. Previous work has demonstrated thermal compression methods that produce a range of valuable articles with properties comparable or even superior to those of the materials fabricated by solution based-processes. However, these methods are very time and energy intensive and require very specific conformation and composition of the silk protein. The silk powder must be of an amorphous conformation with a very uniform particle size and a moisture content greater than 3 wt%. The present ultrasonic process is fast and low energy and can be used to mold protein particles of high β-sheet content and low moisture content. In some embodiments, the ultrasonic process may be solvent free. The ultrasonic process may also be substantially solvent free, for example, less than 3 wt% solvent content. In addition, ultrasonic fusion can be extended to other protein-based materials including, but not limited to keratin, zein, and casein.

[0080] Ultrasonic fusion and molding of conventional polymers has been well described in the art (Janer et al., Ultrasonics 102 (2020): 106038). However, the present disclosure of uses ultrasonic fusion and molding to fuse, flow, and / or mold proteins. The general principle of the technology uses high-power ultrasound to melt, fuse, and mold raw materials into three dimensional formed components. There are three general technologies that use this principle. Ultrasonic welding, ultrasonic compression molding, and ultrasonic microinjection molding. In this application the ultrasonic welding is more suitable for flat sheet forms such as films and fibers but can also apply to powders or composites of powders, films, woven fabrics, non-woven fabrics, and / or fibers. The welding process can involve welding of two similar substrates such as two protein films but can also involve welding, encapsulation, or over-molding of dissimilar substrates such as protein films welded to polymer or metal sheets, wires, or fibers encapsulated in a protein sheet or any combination of aforementioned forms. The ultrasonic compression molding is more suitable for pellets or powders but can also apply to rods or ground fibers. The ultrasonic injection molding is more suitable for pellets or powders but can also apply to ground fibers and can include over molding of micro molded parts onto flat sheets or fibers.

[0081] Turning to FIG.1, a representative ultrasonic injection molding system / process is illustrated (1500). A proteinaceous substrate (1540) is placed in contact with a sonotrode providing a suitable amount of ultrasonic energy in the direction indicated by the arrows (1510). The system includes a mold (1530) having a molding chamber (1520), and a gate (1525). A plunger (1550) compresses the ultrasonically treated sample which is now in a flowable state. The plunger (1570, compressed position) pushes the sample through the gate (1525) into the molding chamber in a flowable state. The ultrasonically molded final article (1580) can be of any shape, such as a dogbone (1590) for tensile testing. In another embodiment, the flowable material can also be incorporated into an additive manufacturing process, such as 3D printing, layer-by-layer manufacturing, and the like. In another embodiment, proteinaceous articles formed from said additive manufacturing process can be further processed into a desired shape using any number of technics, such as subtractive manufacturing by removing portions of the 3D shaped article.

[0082] While example proteins are of the fiber forming fibroin class of materials this technology is not limited to these proteins. In some embodiments the protein composition includes zein or casein or a mixture of fibroin / zein, fibrion / casein, zein / casein proteins. In some embodiments the composites are a mixture of protein and non-proteinaceous substrates such as salts, antioxidants, vitamins, pharmaceuticals, vaccines, dyes, molecular tracers, or enzymes. Due to the nature of the application of the ultrasonic energy there are a few desirable properties that affect fusibility or proteins such as protein structure, melt temperature, denaturation temperature, degradation temperature, elastic modulus, and chemical makeup. Desired characteristics of the final article such as strength, flexibility, transparency, or acoustic transmission would dictate protein incumbent properties such as molecular weight, density, chemistry, and conformation. In general, starting materials must be compacted to a sufficient starting density to allow transmission of the ultrasonic vibrational energy, the degradation temperature must be above process exposure temperature to avoid burning, the moisture content must be low to avoid foaming defects from water evaporation, and the starting material must be stiff enough to transmit the ultrasonic energy efficiently and avoid plowing and or flashing of the melted material.

[0083] To be molded using ultrasonic energy, powder starting materials may have a preform density from 0.6 to 0.9 g / cc, 0.5 to 0.8 g / cc, or 0.6 to 0.7 g / cc, or within any range encompassed by the foregoing. For film starting materials, the preform density may be 1 to 1.4 g / cc, 1.1 to 1.25 g / cc, or 1.15 to 1.2 g / cc, or within any rangeencompassed by the foregoing. Suitable starting materials may have a moisture content up to 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt% or 10 wt%, or within any range encompassed by the foregoing. In addition to suitable preform density and moisture content, starting materials molded using ultrasonic energy may have a molecular weight of at least 1 kDa, 5 kDa, 10 kDa, 20 kDa, 40 kDa, 60 kDa, 80 kDa, 100 kDa, 120 kDa, 140 kDa, 160 kDa, 200 kDa, 300 kDa, 450 kDa or 500 kDa. In addition to suitable preform density and moisture content, starting materials molded using ultrasonic energy may have a molecular weight from 1 kDa to 500 kDa, 20 kDa to 450 kDa, 30 kDa to 350 kDa, 40 kDa to 180 kDa, 60 kDa to 160 kDa, 80 kDa to 140 kDa, or 100 kDa to 120 kDa, or within any range encompassed by the foregoing endpoints. Additionally, exemplary starting materials may have a compressive modulus from 0.2 GPa to 0.5 GPa, 0.25 GPa to 0.45 GPa, or 0.30 GPa to 0.40 GPa, or within any range encompassed by the foregoing. Properties of Ultrasonically Bonded / Molded Proteinaceous Articles

[0084] Ultrasonic fusion and molding has the potential to generate equivalent and possibly superior properties to conventional thermal welding or compression molding in a much faster and more energy efficient way. The ultrasonic molding / bonding method disclosed herein produces articles that have not been substantially degraded due to excessive heat treatment. These differences can be determined by measuring the changes in activation energy between the starting material and the resulting molded / bonding, the relative yellowness in the final articles, changes in the crystallinity (such as decreases in α-helix and random coil content with increases in β-sheet content), the bond strength of the molded / bonded article, the flexural strength of the molded article, and the flexural modulus of the molded article.

[0085] Properties of interest in a final article but generally are mechanical strength, minimal brittleness, optical transparency, minimal yellowing. All of these are demonstrations of minimal degradation of the starting material. Additionally, the ultrasonic welding applies localized fusion with a sharp weld transition zone with no change in mechanical properties, color, transparency, or molecular conformation outside of the weld zone. FIG.2 demonstrates the ultrasonic welding method minimizes yellowing of the untreated film and with an increase in Yellowness Index (per ASTM E313) less than 50 where the thermal fusion showed higher browning and a Yellowness Index change greater than 70. FIG.3 is an illustration of the how different treatment methods impact β-sheet content, α-helix content, and random coil content of fused silkfibroin. The β-sheet, α-helix and random coil content of an un-fused silk fibroin is measured and graphed as a base line for comparison of the fusion techniques. Based on the graph of FIG.3, there is minimal change in β-sheet content for the ultrasonically fused silk fibroin compared to the non-fused silk fibroin. However, the thermally fused silk fibroin spectra indicates a significant decrease in random coil and an increase in beta sheet content from the un-fused silk fibroin, indicating a structural change due to the process method.

[0086] In one embodiment, the present ultrasonically formed molded / bonded articles exhibit less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% decrease in α-helix and random coil content compared to the respective starting substrate (for example, see FIG.3; FTIR peak at ~1640 cm-1).

[0087] In another embodiment, the present ultrasonically molded / bonded articles exhibit less than a 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% increase in β-sheet content (i.e., as measured at a wavenumber of 1618 cm-1) compared to the respective starting substrate (for example, see FIG.3; FTIR peak at ~1618 cm-1).

[0088] Another measure of how fusion methods affect the properties of a proteinaceous material is by tracking any change in activation energy, or the amount of energy a material requires to undergo a reaction. An increase in activation energy may indicate a structural or chemical change to the material. FIG.4 shows a graph of the activation energy of a fused film based on the treatment method. Further, FIG.5 depicts the percent change in activation energy of thermally molded and ultrasonically molded articles relative to the starting material at 10% conversion. Based on the results of FIGs. 4 and 5, there is minimal change (< 5 kJ / mol) in the activation energy at 10% conversion of the ultrasonically fused film from the untreated film. Conversely, the thermal welded sample showed >20 kJ / mol increase in activation energy indicating a structural or chemical change of the material. Thermally molded / bonding articles typically exhibit a change (i.e., increase) in mean activation energy between the starting material and the molded / bonding final article in excess of a 20% (for example, see FIGs.4 and 5).

[0089] In one embodiment, the present ultrasonically formed proteinaceous articles may have an increase in mean activation energy (relative to the starting material) of less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%. In another embodiment, the present ultrasonically formed proteinaceous articles have the activation energy of degradation at10% conversion (measured according to ASTM E1631-18) less than 185 kJ / mol, 180 kJ / mol, or 175 kJ / mol (for example, see FIG.4).

[0090] In another embodiment, the present ultrasonically formed proteinaceous articles have a change (relative change measured between the activation of the molded / bonded article and the starting material) in activation energy of degradation at 10% conversion (measured according to ASTM E1631-18) of less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% (for example, see FIGs.4 and 5).

[0091] The compressive strength of a material is a measure of the resistance a material can withstand before breaking. To measure the compressive strength of a material, the amount of stress versus the amount of strain on the material may be measured. In one embodiment, the bonded article has a compressive strength of at least 75 MPa, 80 MPa, 85 MPa, 90 MPa or 100 MPa.

[0092] Further, by taking the slope of the stress v. strain curve, a compressive modulus may be calculated. Compressive modulus may indicate the ability of a material to resist physical changes when exposed to stress. In another embodiment, the present ultrasonically molded / bonded article has a compressive modulus of at least 0.4 GPa, 0.5 GPa, 0.6 GPa, 0.7 GPa, 0.8 GPa, 0.9 GPa, 1.0 GPa, 2 GPa, 3 GPa, 4 GPa or 5 GPa.

[0093] In one embodiment, the ultrasonically molded / bonded articles have a mean bond strength greater than 300 kPa, 350 kPa, 400 kPa, 450 kPa, 500 kPa, 550 kPa, 600 kPa, 650 kPa, 700 kPa, 750 kPa, 800 kPa, 850 kPa, 900 kPa, 950 kPa or 1 MPa.

[0094] The present ultrasonically bonded / molded proteaceous material may have a similar or greater flexural strength, or the stress at failure in bending, as compared to the starting proteaceous material (un-fused). As illustrated in Figure 5, the un-fused starting material has a flexural strength of about 90 MPa. After ultrasonic fusion, the starting materials used in Examples 2 and 3, discussed below, have a flexural strength greater than 90 MPa. Conversely, the starting material that underwent thermal fusion has a flexural strength of about 60 MPa, indicating that thermal fusion degraded the flexural strength of the starting material.

[0095] In one embodiment, the present bonded / molded articles have a flexural strength at maximum strain of at least 65 MPa, 70 MPa, 75 MPa or 80 MPa.

[0096] In one embodiment, the present ultrasonically bonded / molded articles have a decrease in flexural modulus, or the slope of the slope of the line of the linearportion of the flexural stress strain curve, compared to the starting substrate of no more than 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5%.

[0097] In another embodiment, the present ultrasonically bonded / molded articles have a flexural modulus of at least 6.0 GPa, 6.5 GPa, 7.0 GPa, 7.5 GPa or 8.0 GPa.

[0098] In another embodiment, the present ultrasonically molded / bonded articles have an percent increase in the yellowness index (relative to the starting substrate as measured by ASTM E313-20) of less than 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5%.

[0099] In another embodiment, a bonded proteinaceous article is provided comprising at least one of the following: a. an activation energy of degradation at 10% conversion of less than 165 kJ / mol as measured according to ASTM 1631-18; and b. a moisture content of less than 6 wt% based on the total weight of the bonded proteinaceous article.

[0100] In another embodiment, the bonded proteinaceous article further comprises at least one of the following: a. a mean bond strength of at least 600 kPa; b. a flexural strength at maximum strain of at least 65 MPa; c. a flexural modulus of at least 6.0 GPa; d. a compressive strength of at least 75 GPa; or e. any combination of a, b, c, or d. In another embodiment, the bonded proteinaceous article further comprises 0 wt% to 20 wt% β-sheet content based on the total weight of the bonded proteinaceous article.

[0101] In another embodiment, the bonded proteinaceous article further comprising a yellowness index of 65 or less as measured by ASTM E313-20.

[0102] In another embodiment, the bonded proteinaceous article further comprising 0.01 wt. % to 90 wt. % of one or more additives; wherein wt. % is based on the total weight of the bonded proteinaceous article. In a further embodiment, the one or more additives are polymers, mixtures of proteins, composites, drugs, active ingredients, pigments, dyes, flavoring agents, fillers, radiopaque fillers, conductive fillers, porogens, desiccants, bioactive agents such as thromboresistant agents, antibiotic agents, anti-tumor agents, anti-viral agents, anti-angiogenic agents, angiogenic agents, anti-inflammatory agents, cell cycle regulating agents, and any combination thereof.

[0103] In another embodiment, said bonded proteinaceous article comprises or more proteins including, but not limited to plant proteins, plant protein isolates, plant protein hydrolysates, wheat proteins (gliadin), casein, whey, gelatin, egg white proteins, zein, resilin (dragonfly), scleroproteins, keratins (α-, β- and γ-keratin), collagens, insulin, fibrins, silk fibroins, silk spidroins, elastins, and any combination thereof. In a further embodiment, said one or more proteins are naturally-isolated or recombinantly- produced silk fibroin, silk spidroin, and any combination thereof.

[0104] In another embodiment, said one or more proteins have an average molecular weight ranging from about 1 kDa to about 500 kDa. End Use Applications

[0105] The present molded / bonded proteinaceous materials may be used to mold a variety of different articles and applications. In one embodiment, a non-limiting list of articles may include, but is not limited to bone screws, artificial corneas, stents, tips and hubs for delivery systems, contact lenses, screws, pins, frames, grids used for products in the sustainability industry, drug delivery devices, time release capsules, floss picks, encapsulation of a substance for release in the environment, stents, films, needles, glaucoma drainage devices, or other suitable articles. TEST METHODS

[0106] It should be understood that although certain methods and equipment are described below, other methods or equipment determined suitable by one of ordinary skill in the art may be alternatively utilized.

[0107] Bulk Density

[0108] Bulk Density was calculated by dividing the mass of a sample by the sample volume. Mass was measured using an analytical balance (Ohaus Pioneer, Parsippany, NJ). Thickness and Diameter was measured using Calipers (Mutioyo, Aurora, IL).

[0109] Compressive Strength

[0110] Compressive Strength was measured by applying a compressive force perpendicular to the top surface of the molded component using a tensile testing machine (Instron 5500R, Norwood, MA).

[0111] Compressive Modulus

[0112] Compressive Modulus was calculated as the slope of the linear portion of the compressive stress strain curve as described in the compressive strength method.

[0113] Flexural Strength

[0114] The three-point bending tests were carried out on an RSA-G2 Dynamic Mechanical Analyzer(TA Instruments, New Castle DE) in flexural test mode at 25C and ambient RH in a simple axial deformation with a loading rate of 2mm / min. The specimen has a length of 12 mm, a width of 6.5 mm, and a support span of 10 mm.

[0115] Flexural Modulus

[0116] Flexural Modulus was calculated as the slope of the line of the linear portion of the flexural stress strain curve as described previously.

[0117] Yellowness Index

[0118] Yellowness index (“Y.I.”) was assessed per ASTM E313-20 using X-Rite Model Ci6x Spectrophotometer (Grand Rapids, MI).

[0119] Moisture Content

[0120] Moisture Content was measured per ASTM D7191-18 using the Brookfield VaporPro XC (Arizona Instrument, 1912 West 4th Street, Tempe, AZ 85281) set to 120 °C for 60 minutes.

[0121] Activation Energy

[0122] Activation Energy was measured per ASTM E1641-18 Standard Test Method for Decomposition Kinetics by Thermogravimetry Using the Ozawa / Flynn / Wall Method, using TA Instruments (New Castle, DE) TGA Q5000IR model with an initial sample mass of 3 mg, ramp rates of 5, 10, 20 and 40°C / min to 600°C in Nitrogen.

[0123] Bond Strength

[0124] Bond Strength measurement follows ASTM D3136 with modified geometry, two sheets of film approximately 100 µm thick were overlapped and welded with 2000x Branson ultrasonic welder with a weld area of approximately 40 mm2. The bond area was measured using a Leica Microsystems M205 C Stereomicroscope. A 2 mil Kapton sheeting was added as a backer and the samples were die cut to a dimension of 6.35 mm x 50 mm and tested in a Instron (5500R) with a gage length of 20 mm and a cross head speed of 0.2 mm / second. The bond strength was calculated as the maximum load divided by the bond area. Sample failure mode was noted as either bond failure, weld edge failure, or leg failure.

[0125] Fourier-transform Infrared Spectroscopy (FTIR)

[0126] FTIR spectroscopy (FTIR) was carried out using a Bruker Alpha II spectrometer equipped with a ZnSe ECO Attenuated Total Reflection (ATR) cell. For each measurement, 32 scans were averaged across a spectra from 600 cm-1to 4000 cm-1, at a resolution of 2 cm-1.

[0127] Quantitation of secondary structure was performed via deconvolution of amide I peak from FTIR spectra using method of Guo et. al., Nat Mater. Jan;19(1):102- 10 (2020)) or U.S. Patent Appl. Publ. No.2021 / 0381129 A1 to Kaplan et al.). Briefly, for FTIR spectra, the spectral corrections and decomposition were performed using commercial software, the spectra were first smoothed with a 5-point triangle smoothing method and then baseline corrected using a cubic spline for the amide I band from 1720cm-1to 1580cm-1. The deconvolution was carried out by fitting gaussian peaks with floating area and full width at half maximum to four primary peaks (if present) assigned to a secondary structures respectively: 1620 cm-1(β-sheet), e.g., 1642 or 1652 cm-1(random coil / helix), 1685 cm-1(β-turn), and 1698 cm-1(β-sheet). Peak areas were then summed to create a total and the percentage of a given secondary structure determined by calculating the respective ratio of the peak or peak(s) to the sum and multiplying by 100. EXAMPLES

[0128] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions. Fusion of two substrates by welding with ultrasonic energy

[0129] Ultrasonic welding was performed using a model 2000X Branson ultrasonic welder with an output of 20kHz, a 3x12 mm horn and a flat 2C metal anvil. The apparatus is comprised of an ultrasound generator, a transducer, and acoustic unit, a horn, a horn actuator, and an anvil. The transducer transforms high-frequency electrical signals into kinetic energy (wave motion). The acoustic unit includes a booster and a horn. The booster modifies the incoming wave from the transducer amplifying or reducing the wave amplitude, whereas the horn transfers vibrational energy to the raw polymer, causing a temperature rise. The horn actuator provides the pressure to fuse two substrates together. The first substrate (“substrate 1”) was placed on the metal anviland a second substrate (“substrate 2”) was placed on top of substrate 1 and the layers were secured in place. The horn was lowered, and the layers were welded together with a burst of ultrasonic energy (e.g., 0.15 s, amplitude 100% fixed, 20 psi pressure, 2500W). Formation of molded articles with ultrasonic energy

[0130] An ultrasound molding machine (SONORUS 1G®, fabricated by Ultrasion S.L., Barcelona, Spain) was used to produce molded examples. This apparatus is based on an ultrasound generator, a transducer, an acoustic unit, and a plunger. The generator (or ultrasonic power supply) converts electrical energy into ultrasonic frequency (30 kHz). The transducer transforms high-frequency electrical signals into kinetic energy (wave motion). The acoustic unit includes a booster and a sonotrode. The booster modifies the incoming wave from the transducer amplifying or reducing the wave amplitude, whereas the sonotrode transfers vibrational energy to the raw polymer, causing a temperature rise. The plunger provides the pressure to move the melt material (flowable) to the mold, thus obtaining the desired shape. To facilitate easy loading of the ultrasonic molder, the powder was formed into a friable unbonded powder tablet with a density of approximately 0.8 g / cm3by light compaction in a cylindrical mold 7.14 mm in diameter. The plasticizer chamber is fed with the pressed powder, and ultrasonic vibration is transmitted from the sonotrode to the material. The plunger presses the material, and it flows through the feeding channel filling the mold that has been heated to a specific temperature. The temperature may be from room temperature to 120°C (i.e., 120°C or less, where about 120°C, a preferred temperature may be 80°C. is typically used). The settings used for ultrasonic molding are provided in Table 3. Fusion of two substrates by welding with thermal energy

[0131] A 3 ton hydraulic heated platen press (Wabash 3 Ton Press Model P3H- 12-X, fabricated by Wabash MPI, Wabash, Indiana) was used to produce thermal welded film samples. The press was equipped with 6”x5” heated steel platens machined flat and parallel within 0.001 inch / ft and equipped with a programmable controller with HMI to carefully control compression conditions. The film samples were cut to desired dimensions and overlapped in a modified lap-shear geometry described previously. The samples were placed between two sheets of 2 mil Kapton sheeting to protect the steel platens and places between the two heated steel platens andsubjected to the desired temperature and pressure for the desired time. The pressure was normalized for the actual sample dimensions placed within the platens. Formation of molded articles with thermal energy

[0132] A 50 ton hydraulic heated platen press (OEM Model 0128 / 035700 / HELC, fabricated by OEM Press Systems, Fullerton CA) was used to produce thermal compression molded samples. The press was equipped with 14”x14” heated steel platens machined flat and parallel within 0.001 inch / ft and equipped with a programmable controller with HMI to carefully control compression conditions. Platens were protected using ¼” thick aluminum sheet and 2 mil Kapton sheet on both upper and lower platens. A cylindrical stainless steel mold consisting of a solid base with a 28.75 mm diameter by 100 mm tall cavity machined into it with a precision fit stainless steel moveable plunger was used to produce a 28.75 mm diameter x 1.14 mm thick cylinder. The stainless steel mold was heated in a laboratory oven at 145°C for 8 hours to ensure uniform temperature distribution through the mold. 1 g of powder was preformed into tablets 28.75 mm diameter at room temperature under 70 MPa pressure for 1 minute and then conditioned as specified in each example. The preformed tablets were placed in the compression mold within the heated press and subjected to the desired temperature, pressure, and time as specified in each example. Example 1 – Fusion of Crystalline Fibroin Fibers by Ultrasonic Welding

[0133] Eight layers of a silk fibroin woven fabric (100% Chinese Habotai Silk, 0.2 mm thick, Jacquard Products; Rupert, Gibbon & Spider Inc., Healdsburg, CA) were fused by ultrasonic welding. FTIR confirmed the woven fibroin fabric was predominantly β-sheet crystalline form as has been reported elsewhere (for example Hu et.al., supra or Guo et al., supra). The substrates were fused using the protocol described above for fusion of two substrates by welding with ultrasonic energy using the following settings: 0.15 s, amplitude 100% fixed, 20 psi pressure, 2500W. The result was a bonded article with a bond area of 40 mm2, a thickness of 0.1 mm, and a bond strength of 763 kPa (111 psi). Comparative Example 1 – Attempted Fusion of Crystalline Fibers by Thermal Heat and Compression

[0134] Thermal fusion was attempted using of two layers of the silk fibroin woven fabric (Jacquard Products) described in Example 1. Using bonding conditions ofUS20160089854A1 to Omenetto et al., the fabric layers were overlapped and compressed between hot plates at 120°C and 135 psi (0.93 MPa) for 30 s. The resulting fabric did not bond and exhibited effective bond strength= 0. No further characterization was undertaken as the films did not physically fuse to form an article.

[0135] Additionally, the fabric layers were compressed between hot platens in a hydraulic press at 175°C and 10k psi (~69 MPa) pressure for 30 minutes. The elevated heat (175°C) was observed to yellow the fibers. No further characterization was undertaken as the films did not physically fuse to form an article.

[0136] Together these examples show that the ultrasonic fusion is different than thermal fusion in that crystalline fibroin fibers and woven fabrics can be fused using ultrasonic welding in a fast, low temperature, low pressure manner and cannot be fused by thermal welding as demonstrated in the above examples. Example 2- Fusion of Crystalline Fibroin Film by Ultrasonic Welding

[0137] Fibroin films were made per methods described in Rockwood et al. "Materials fabrication from Bombyx mori silk fibroin." Nature protocols 6.10 (2011): 1612-1631. Briefly, 17.5 mL of 7% aqueous silk solution was poured into a 100 mm Petrie dish and allowed to dry overnight at ambient conditions protected from any airflow. Films were approximately 100 µm thick and able to be removed from the Petrie dish easily using tweezers. Crystallized films were made by soaking the cast films in a 80% methanol / 20% DI water solution for thirty minutes. The initial films were characterized using techniques described in the test methods section. FTIR confirmed the MeOH treated cast film was predominantly β-sheet crystalline form as has been reported elsewhere (for example Hu et al., supra or Guo et al., supra). The respective moisture content of the film was 5.5 wt%, the density of the film was 1.22 g / cm3, the color by Yellowness index was 31.

[0138] Two of these film substrates were overlapped and fused using the protocol described above (“Fusion of two substrates by welding with ultrasonic energy”) for fusion of two substrates by welding with ultrasonic energy with the specific settings: 0.15 seconds, amplitude 100% fixed, 20 psi pressure, 2500W. The result was a bonded article with a bond area of 40 mm2, a thickness of 0.115 mm, and a bond strength of 1044 kPa and flexural strength of 95 MPa. The final fused respective moisture content of the film was 5.5 wt%, the density of the film was 0.96 g / cm3, the color by Yellowness index was 56.8.Comparative Example 2 – Attempted Fusion of Crystalline Film by Thermal Heat and Compression

[0139] Fibroin films were made per methods described in Rockwood et al., supra. Briefly, 17.5 mL of 7% aqueous silk solution was poured into a 100 mm Petrie dish and allowed to dry overnight at ambient conditions protected from any airflow. Films were approximately 100 µm thick and able to be removed from the Petrie dish easily using tweezers. Crystallized films were made by soaking the cast films in a 80% methanol / 20% DI water solution for thirty minutes. The initial films were characterized using techniques described in the test methods section. FTIR confirmed the MeOH treated cast film was predominantly β-sheet crystalline form as has been reported elsewhere for example Hu et al., supra. The respective moisture content of the film was 5.5 wt%, the density of the film was 1.22 g / cm3, the color by Yellowness Index was 31.

[0140] Using bonding conditions of US20160089854A1 to Omenetto et al., the films were overlapped and compressed between hot plates at 120°C and 135 psi (0.93 MPa) for 30 seconds (s). The resulting films did not bond and exhibited effective bond strength = 0. No further characterization was undertaken as the films did not physically fuse to form an article.

[0141] Together these examples show that the ultrasonic fusion is different than thermal fusion in that crystalline fibroin films can be fused using ultrasonic welding in a fast, low temperature, and low-pressure manner, and cannot be fused by thermal welding as demonstrated in the above examples. Example 3- Fusion Amorphous Fibroin Films by Ultrasonic Welding

[0142] Fibroin films were made per methods described in Rockwood et al., supra. Briefly, 17.5 mL of 7% aqueous silk solution was poured into a 100 mm Petrie dish and allowed to dry overnight at ambient conditions protected from any airflow. Films were approximately 100 µm thick and able to be removed from the Petrie dish easily using tweezers. The initial films were characterized using techniques described in the test methods section. FTIR confirmed the cast film was predominantly amorphous form as has been reported elsewhere (for example Hu et.al., supra or Guo et. al., supra). The respective moisture content of the film was 5.90 wt%, the density of the film was 1.39 g / cm3, the color by Yellowness Index was 11, the activation energy of degradation at 10% conversion was 160 kJ / mol, and the flexural strength was 90 MPa.

[0143] Two of these film substrates were overlapped and fused using the protocol described in Example 1 for fusion of two substrates by welding with ultrasonic energywith the specific settings: 0.15 s, amplitude 100% fixed, 20 psi pressure, 2500W. The result was a bonded article with a bond area of 40 mm2, a thickness of 0.380mm, and a bond strength 1114 kPa, flexural strength of 100 MPa, flexural modulus of 7.5 GPa. FTIR confirmed the ultrasonically fused cast film was predominantly amorphous form as has been reported elsewhere (for example, Hu et.al., supra or Guo et. al., supra). The final fused respective moisture content of the film was 5.9 wt%, the density of the film was 0.76 g / cm3, the color by Yellowness index was 21, the activation energy of degradation at 10% conversion was 163 kJ / mol. The relative change in characterized moisture content, Yellowness Index, activation energy, and flexural strength was 0%, 48%, 2%, and 10%; respectively. FTIR Spectra of the initial films, fused films, and % change of each conformation are shown in Table 1. Comparative Example 3 – Fusion of Amorphous Films by Thermal Heat and Compression

[0144] Fibroin films were made per methods described in Rockwood et al., supra. In summary 17.5 mL of 7% aqueous silk solution was poured into a 100-mm Petrie dish and allowed to dry overnight at ambient conditions protected from any airflow. Films were approximately 100 µm thick and able to be removed from the Petrie dish easily using tweezers. The initial films were characterized using techniques described in the test methods section. FTIR confirmed the cast film was predominantly amorphous form as has been reported elsewhere (for example Hu et.al., supra or Guo et. al., supra). The respective moisture content of the film was 5.90 wt%, the density of the film was 1.39 g / cm3, the color by Yellowness index was 11, the activation energy of degradation at 10% conversion was 160 kJ / mol, and the flexural strength was 90 MPa.

[0145] Using bonding conditions of US20160089854A1 to Omenetto et al., the films were overlapped and compressed between hot plates at 120°C and 135 psi (0.93 MPa) for 30 s. The resulting films did not bond and exhibited effective bond strength=0. No further characterization was undertaken as the films did not physically fuse to forma an article.

[0146] The films were then conditioned at a relative humidity of 75% at 24°C for 30 minutes to increase the moisture content to 10%. The films were then rapidly removed from the humidity chamber to avoid dehydration. Using bonding conditions of US20160089854A1to Omenetto et al., the films were overlapped and compressed between hot plates at 120°C C and 135 psi (0.93 MPa) for 30 s. The resulting films didnot bond and exhibited effective bond strength = 0. No further characterization was undertaken as the films did not physically fuse to form an article.

[0147] The films were then conditioned at a relative humidity of 75% at 24°C for 30 minutes to increase the moisture content. The films were then rapidly removed from the humidity chamber to avoid dehydration and using bonding conditions of US20160089854A1 to Omenetto et al., the films were overlapped and compressed between hot plates at 175°C and 10k psi (69 MPa) for 30 s. The result was a bonded article with a bond area of 330 mm2, a thickness of 0.35 mm.

[0148] FTIR confirmed the thermally fused cast film was predominantly crystalline β-sheet form as has been reported elsewhere (for example, Hu et.al., supra or Guo et. al., supra). The final fused respective moisture content of the film was 4.50 wt.%, the density of the film was 1.4 g / cm3, the color by Yellowness Index was 72, and the activation energy of degradation at 10% conversion was 201.8 kJ / mol. Furthermore, the final bonded article a flexural strength of 60 kPa, the sample was too brittle to perform lap shear testing. The relative change in characterized moisture content, Yellowness Index, activation energy, and flexural strength was -31%, 85%, 21%, and - 33%; respectively. FTIR Spectra of the initial films, fused films, and % change of each conformation are shown in Table 1. Table 1. Change in conformation as a function of process method

[0149] Together these examples show that ultrasonic fusion is different than thermal fusion in that dry amorphous fibroin films can be fused using ultrasonic welding in a fast, low temperature, low pressure manner and cannot be fused by thermal welding. The examples further show that amorphous fibroin films can only be thermally welded after being humidified and welded at an elevated temperature and high pressure for an extended period of time such that there is yellowing of the film as shown in Figure 2, the material becomes brittle and loses flexural strength as shown in Figure 3, the protein conformation changes from predominantly amorphous to predominately beta sheet crystalline as shown in Figure 4 and Table 1, and activation energy of degradation at 10% conversion increases as shown in Figure 5. The measured changes as a result of thermal processing (increase in yellowness, increase in activation energy, increase in crystallinity, loss in mechanical strength) demonstrate that there must be some degree of protein degradation in order to get sufficient mechanical fusion even with the increased plasticization and molecular mobility provided by the humidification of the amorphous silk films. The lack of change in yellowness, activation energy, crystallinity, and mechanical strength of the ultrasonic fusion demonstrates the ability to fuse the films without degrading the protein demonstrating the difference between thermal and ultrasonic fusion. Example 4- Fusion and Molding of Amorphous Fibroin Powder into an Article by Ultrasonic Compression

[0150] Amorphous Silk Fibroin: Lyophilized cakes, with an average molecular weight of 173 kDa made from amorphous silk solution and lyophilized per general methods described Rockwood et al., supra. The lyophilized samples were then cryomilled using SPEX™ SamplePrep 6870 FREEZER / MILL™ (SPEX Sample Prep Inc., Metuchen, NJ) at −196 °C with 5 min pre-cool, a rate of 15 cps, 1 cycle, and a 2- minute cool down. The cryomilled particles were sieved with a 0.5 mm sifting screen using a Geotech Sand Shaker with a 20OPN Screen (0.5080 mm)(Geotech Environmental Equipment, Inc., Denver, CO). The average particle size was 100 µm and the tap density of the powders was 0.3 g / cm3. FTIR confirmed the powder was predominantly amorphous form as has been reported elsewhere (for example Hu et.al., supra or Guo et. al., supra).

[0151] The initial powders were compacted into preformed tablets using the protocol described above for preforming of powders and characterized using techniques described in the test methods section. The respective moisture content of the preformwas 2.90 wt%, the density of the preform was 0.80 g / cm3, the color by Yellowness Index was 15.9, and the activation energy of degradation at 10% conversion was 143.2 kJ / mol.

[0152] The tablet was fused using the protocol described above for ultrasonic injection molding into a molded cylinder 8 mm in diameter and 4 mm thick using the settings provided in Table 3. The final fused respective moisture content of the cylinder was 2.90 wt%, the density of the cylinder was 1.32 g / cm3, the color by Yellowness Index was 62, and the activation energy of degradation at 10% conversion was 146 kJ / mol. Furthermore, the final fused article produced had a compressive strength greater than 100 MPa. The relative change in characterized moisture content, Yellowness Index, and activation energy of degradation at 10% conversion was 0%, 20.4%, 289%, and 2%; respectively. Comparative Example 4– Fusion and Molding of Amorphous Fibroin Powder into an Article by Thermal Compression Molding

[0153] Cryomilled amorphous silk fibroin powders prepared as described in Example 4 were used. The initial cryomilled powders were compacted into preformed tablets using the protocol described above (Formation of molded articles with thermal energy) for preforming of powders and characterized using techniques described in the test methods section. The preform was preheated at 145°C for 30 minutes. The respective moisture content of the preform was 2.90 wt%, the density of the preform was 0.80 g / cm3, the color by Yellowness Index was 15.9, and the compressive strength was 1 MPa.

[0154] Using molding conditions of Guo et al. (supra), the preform was placed in a preheated stainless steel mold at 145°C for 30 minutes and then compressed into a cylinder at 145°C at a pressure of 91600 psi (~632 MPa) for 30 minutes. The article failed to fuse and form a cohesive tablet with an effective compressive strength similar to the starting powder. The article was not characterized further.

[0155] A second sample of cryomilled powder (prepared as described above) was conditioned at a relative humidity of 75% at 24°C for 30 minutes to increase the moisture content. The humidified powder was then compacted into preformed tablets using the protocol described above (Formation of molded articles with thermal energy) for preforming of powders and characterized using techniques described in the test methods section. The respective moisture content of the preform was 7.5 wt%, thedensity of the preform was 0.80 g / cm3, the color by Yellowness index was 15.9, and the compressive strength was 1 MPa.

[0156] Using molding conditions of Guo et al. (supra), the moisture conditioned preform was placed in the previously described stainless steel mold preheated at 145°C and compressed into a cylinder at a pressure of 91600 psi (~632 MPa) and a temperature of 145°C for 30 minutes. The final fused respective moisture content of the cylinder was 7.35 wt%, the density of the cylinder was 1.35 g / cm3, the color by Yellowness Index was 67.43, and the activation energy of degradation at 10% conversion was 167.80 kJ / mol. Furthermore, the final fused article produced had a compressive strength of 160 MPa. The relative change in characterized moisture content, Yellowness Index, and activation energy was -2%, 323%, 289%, and 15.78%; respectively. Consistent with previous work this example shows that removal of bound water from amorphous silk nanoparticles (ASN) by preheating results in failure of thermal molding, which however can be reversed by moisture treatment to regain bound water.

[0157] Together these examples show that ultrasonic fusion is different than thermal fusion in that dry amorphous fibroin powders can be fused using ultrasonic energy and cannot be fused by thermal energy. Amorphous fibroin powders can only be thermally molded after being humidified and molded at an elevated temperature and pressure for an extended period of time as demonstrated in the above examples. Example 5 – Fusion of a Casein Globular Protein Substrate to Itself

[0158] Casein: Powder with a molecular weight (Mw) in the range of 19-25 kDa, density of 0.79 g / cm3(23 °C), and an average particle size of approximately 300 µm was purchased from Sigma-Aldrich (cat# C3400-500G; St. Louis, MO). Casein powder was successfully fused and formed into articles with a solid density of 1.24 g / cm3in the form of a compression molded cylinder 8 mm in diameter and 4 mm thick and an injection molded rod 3mm in diameter and 6mm long using the ultrasonic molding procedures previously described above (Formation of molded articles with ultrasonic energy; settings provided in Table 3). This example demonstrates that ultrasonic fusion applies to a wide variety of proteins including the globular structure. Example 6 – Fusion of a Zein Globular Protein Substrate to Itself

[0159] Zein: Powder with a molecular weight (Mw) in the range of 14-24 kDa, density of 0.4 g / cm3(23 °C), an average particle size of approximately 30 µm waspurchased from Sigma-Aldrich (cat.# Z3625-500g, St. Louis, MO). Zein powder was successfully fused and formed into an article with a solid density of 1.07 g / cm3in the form of a compression molded cylinder 8 mm in diameter and 4 mm thick, an injection molded rod 3mm in diameter and 6mm long, an injection molded threaded rod 1.8 mm in diameter and 6 mm long, a hollow rod 1.8 mm in diameter, 6 mm long, with a wall thickness of 0.90 mm, and guitar pick 1.14 mm thick rounded triangle with side length approx.25 mm using the ultrasonic molding procedures previously described (Formation of molded articles with ultrasonic energy; settings provided in Table 3). This example demonstrates that ultrasonic fusion applies to a wide variety of proteins including the globular structure. Example 7 – Fusion of Protein Mixtures

[0160] Fibroin, Casein, and Zein powders as previously described in Example 1 to 6 were mixed by combining two powders in equal ratio and mixing by hand until uniformly distributed. Powders were successfully fused and formed into articles with densities in Table 2 in the form of a cylinder 8 mm in diameter and 4 mm tall, and rod 3 mm in diameter and 6 mm long (molding settings provided in Table 3). These examples demonstrate that you can not only fuse proteins to themselves, but you can also fuse different proteins together. Fibroin fused with zein or casein demonstrates a scleroprotein and a globular protein can be fused and molded into an article. Zein fused with casein demonstrates you can fuse two globular proteins together. Table 2. Mixed Proteinaceous Substrates – Powder Densities and Solid Article DensitiesExample 8 – Ultrasonic Injection Molding of Zein with Vitamin E Additive

[0161] Zein powder as previously described was mixed with 1 wt% vitamin E (Sigma Aldrich, cat.# T3251, St. Louis, MO) by dissolving 20 mg / mL Vitamin E in isopropyl alcohol, mixing this solution into the zein powder at a ratio of 0.5 mL / gram ofpowder. The vitamin solvent was mixed into the powder initially using a Vortex Genie laboratory mixer (Scientific Industries Inc., Bohemia, NY) for 1 minute and then capped in a falcon tube overnight to allow the solvent to adequately diffuse into the powder. The powder mixture was then poured into an aluminum weigh boat and covered with a wipe (KIMWIPES®, Kimberly Clark Technical, Roswell, GA) and allowed to dry overnight in a laboratory hood at ambient conditions prior to processing. The Zein powder with 1 wt% vitamin E was then ultrasonically injection molded into a guitar pick as previously described (molding settings provided in Table 3). This article demonstrates the ability to mold additives and delicate cargo such as vitamins along with the protein for use of stabilization and delivery of a wide variety of compounds. Example 9 - Over Molding of Protein onto a Porous Substrate

[0162] A layer of polyethylene film 15 mm wide, 40 mm long, and 25 µm thick was placed across a microinjection tensile bar mold (per ASTM D1708-18: Standard Test Method for Tensile Properties of Plastics by Use of Microtensile Specimens) and the zein powder was ultrasonically injection molded per previously described methods to produce a solid protein tensile bar over molded on a porous polymeric sheet (molding settings provided in Table 3). This example demonstrates that one can mold a solid protein article onto another article to provide desirable features such as but not limited to rigid mechanical support. Example 10 – Encapsulation of a Suture within a Molded Protein Article

[0163] An expanded polytetrafluoroethylene (ePTFE) suture (GORE-TEX® Suture; W.L. Gore & Associates Inc., Newark, DE) was placed between two layers of the amorphous fibroin film and the two fibroin layers were fused together as previously described in Example 3 producing a suture encapsulated in two layers of protein film. This example demonstrates that one can mold a solid protein article around another article such as but not limited to a suture or wire to integrates features such as but not limited to anchoring of a suture, lead wire, or fiber optic wire. Example 11 – Powder Bed Fusion of Amorphous Fibroin Powder on a Crystalline Woven Fibroin Fabric

[0164] Amorphous fibroin powder as previously described in Example 4 was placed between two layers of the crystalline fibroin woven fabric as described inExample 1 and fused via ultrasonic welding using the procedure described above (Fusion of two substrates by welding with ultrasonic energy).

[0165] A solid fused article the size of the ultrasonic horn, 3 mm x 12 mm, was produced. This example demonstrates one can fuse a combination of forms together for example but not limited to a powder and a woven fabric.Table 3 - Ultrasonic Injection Mold Settings Used For Molded Samples

[0167] In summary, these examples demonstrate that ultrasonic fusion of proteins enables fast, low temperature fusion of materials that would typically degrade prior to melting into a variety of form factors. This is different than previous disclosures that specifically state that that one cannot fuse high β-sheet crystalline substrates oramorphous substrates with a moisture content <3% because thermal reflow potential does not exist (US20160089854 A1 to Omenetto et al.; Akiyama and Hirabayashi, Polymer 35.11 (1994): 2355-2358.). Further the long time, high temperature, and higher pressure required to fuse amorphous materials with a moisture content >3% are such that they cause degradation in the form of yellowing, loss in mechanical properties, change in protein conformation, and increase in activation energy of degradation. The examples demonstrate that a variety of different proteins (e.g., scleroproteins and globular), a variety of forms (fibers, woven meshes, films, powders), at a range of conformation (e.g. amorphous or crystalline), and a range of moisture content (e.g. <3% and >3%), can be formed to make a variety of structures (e.g., multilayer fused films, molded cylinder, rod, threaded rod, guitar pick), with a fast, low energy process with minimal degradation.

Claims

CLAIMS What is Claimed Is:

1. A method to fuse discreet proteinaceous substrates into an article comprising a. providing i. a first proteinaceous substrate; and ii. a second proteinaceous substrate; wherein said first and second proteinaceous substrates are independently in the form of one or more particles; pellets, filaments, a fiber, a film, a non-woven mesh, a woven mesh or a membrane; b. bonding said first and said second proteinaceous substrates into a first article using an effective amount of ultrasonic energy; and c. optionally shaping the first article into a second article.

2. The method of claim 1 wherein said bonding step is conducted substantially free of any solvent.

3. The method of any one of the preceding claims wherein the first article is in a flowable state.

4. The method of any one of the preceding claims wherein said bonding and / or shaping is by ultrasonic compression molding, ultrasonic welding, ultrasonic injection molding, ultrasonic extrusion, 3D additive manufacturing and any combination thereof.

5. The method of any one of the preceding claims wherein said first proteinaceous substrate and said second proteinaceous substrate are in the form of a plurality of dry powder particles.

6. The method of claim 5 wherein the average particle size of the dry powder particles is less than 400 µm.

7. The method of any one of the preceding claims where said first proteinaceous substrate, said second proteinaceous substrate or both first and second proteinaceous substrates are naturally-isolated or recombinantly-produced spheroproteins (globular),enzymes, scleroproteins (fiber-forming), membrane proteins, and any combination thereof.

8. The method of claim 7 wherein the scleroproteins are keratins (α-, β- and γ- keratin), collagens, fibrins, silk fibroins, silk spidroins, elastins, and any combination thereof.

9. The method of claim 8 wherein the first proteinaceous substrate, the second proteinaceous substrate or both said first and second proteinaceous substrates are silk fibroin, silk spidroin and combinations thereof.

10. The method of any one of claims 1 to 7 wherein the first proteinaceous substrate, the second proteinaceous substrate or both said first and second proteinaceous substrates are plant proteins, plant protein isolates, plant protein hydrolysates, wheat proteins (gliadin), casein, whey, gelatin, egg white proteins, zein, resilin (dragonfly), and any combination thereof.

11. The method of any one of the preceding claims wherein the average molecular weight of said first or said second proteinaceous substrate is between 1 kDa and 500 kDa.

12. The method of any one of the preceding claims wherein said bonding is conducted at a temperature of 120 °C or less.

13. The method of any one of the preceding claims wherein said bonding step is conducted at a pressure of from 5 psi (34.5 kPa) to 30,000 psi (207 MPa).

14. The method of any one of the preceding claims wherein said bonding time ranges from 0.1 second to 60 seconds.

15. The method of any one of the preceding claims wherein said first proteinaceous substrate is a powder, a fiber, a filament, a pellet, a film, a non-woven mesh or a woven mesh and said second proteinaceous substrate is a powder, a pellet, a filament, a fiber, a film, a non-woven mesh or a woven mesh.

16. The method of any one of the preceding claims wherein said first article or said second article is in the form of a fiber, a pellet, a film, a tube, a membrane, a non-woven article, a woven article, a solid three-dimensional object, or a porous 3-dimensional object.

17. The method of any one of the preceding claims further comprising step (d) – repeating steps (a) to (c) a plurality of times; wherein said first proteinaceous substrate, said second proteinaceous substrate or both said first and said second proteinaceous substrates are optionally said first article or said second article.

18. An ultrasonically bonded proteinaceous article comprising: a. a first starting material comprising a first proteinaceous substrate; b. a second starting material comprising a second proteinaceous substrate; wherein the first and second starting materials are bonded together with ultrasonic energy to form the bonded proteinaceous article; wherein the first and second starting materials were independently in the form of one or more particles, a fiber, a film, a non-woven mesh, a woven mesh, or a membrane prior to being melted and bonded together to form the proteinaceous article.

19. The article of claim 18, wherein each of the first starting material, the second starting material, and the bonded proteinaceous article comprise a mean activation energy as measured according to ASTM E1641-18; wherein a change in the mean activation energy from at least one of the first and second starting material to the bonded proteinaceous article is an increase of less than 15% compared to the at least one of the first and second starting materials.

20. The article of any one of claim 18 or 19, wherein each of the first starting material, the second starting material, and the bonded proteinaceous article comprise an activation energy of degradation at 10% conversion measured according to ASTM 1631-18; wherein a change in an activation energy of degradation at 10% conversion of at least one of the first and second starting materials to the activation energy of degradation at 10% conversion of the bonded proteinaceous article is a decrease ofless than 15% as compared to the activation energy of degradation at 10% conversion of the at least one of the first and second starting materials.

21. The article of any one of claims 18 to 20, wherein the bonded proteinaceous article comprises an activation energy of degradation at 10% conversion of less than 185 kJ / mol as measured according to ASTM 1631-18.

22. The article of any one of claims 18 to 21, wherein each of the first starting material, the second starting material, and the bonded proteinaceous article comprise an α-helix and random coil content; wherein a change in the α-helix and random coil content from at least one of the first and second starting materials to the bonded proteinaceous article is a decrease of less than 15% as compared to the α-helix and random coil content of the at least one of the first and second starting materials.

23. The article of any one of claims 18 to 22, wherein each of the first starting material, the second starting material, and the bonded proteinaceous article comprise a β-sheet content as measured using Fourier-transform Infrared Spectroscopy (FTIR); wherein a change in the β-sheet content from at least one of the first and second starting materials to the bonded proteinaceous article is an increase of less than 15% as compared to the β-sheet content of the at least one of the first and second starting materials.

24. The article of any one of claims 18 to 23, wherein the bonded proteinaceous article has a mean bond strength greater than 600 kPa as determined according to ASTM D3436.

25. The article of any one of claims 18 to 24, wherein the bonded proteinaceous article has a compressive strength of at least 75 MPa.

26. The article of any one of claims 18 to 25, wherein the bonded proteinaceous article has a flexural strength at maximum strain of at least 65 MPa as measured at 25oC at ambient relative humidity with a loading rate of 2 mm / min.

27. The article of any one of claims 18 to 26, wherein each of the first starting material, the second starting material, and the bonded proteinaceous article comprise a flexural strength; wherein a change in the flexural strength from at least one of the first and second starting materials to the bonded proteinaceous article is a decrease of less than 30% as compared to the flexural modulus of the at least one of the first and second starting materials.

28. The article of any one of claims 18 to 27, wherein the bonded proteinaceous article has a flexural modulus of at least 6 GPa.

29. The article of any one of claims 18 to 28, wherein each of the first starting material, the second starting material, and the bonded proteinaceous article comprise a yellowness index as determined by ASTM E313-20; wherein a change in the yellowness index from at least one of the first and second starting materials to the bonded proteinaceous article is an increase of less than 60% as compared to the yellowness index of the at least one of the first and second starting materials.

30. A bonded proteinaceous article comprising at least one of the following: a. an activation energy of degradation at 10% conversion of less than 165 kJ / mol as measured according to ASTM 1631-18; and b. a moisture content of less than 6 wt% based on the total weight of the bonded proteinaceous article.

31. The bonded proteinaceous article of claim 30 further comprising at least one of the following: a. a mean bond strength of at least 600 kPa; b. a flexural strength at maximum strain of at least 65 MPa; c. a flexural modulus of at least 6.0 GPa; d. a compressive strength of at least 75 GPa; or e. any combination of a, b, c, or d.

32. The bonded proteinaceous article of claim 30 or claim 31 wherein the bonded proteinaceous article comprises 0 wt% to 20 wt% β-sheet content based on the total weight of the bonded proteinaceous article.

33. The bonded proteinaceous article of any preceding claim further comprising a yellowness index of 65 or less as measured by ASTM E313-20.

34. The bonded proteinaceous article of any one of the preceding claims further comprising 0.01 wt. % to 90 wt. % of one or more additives; wherein wt. % is based on the total weight of the bonded proteinaceous article.

35. The bonded proteinaceous article of claim 34 wherein the one or more additives are polymers, mixtures of proteins, composites, drugs, active ingredients, pigments, dyes, flavoring agents, fillers, radiopaque fillers, conductive fillers, porogens, desiccants, bioactive agents such as thromboresistant agents, antibiotic agents, anti- tumor agents, anti-viral agents, anti-angiogenic agents, angiogenic agents, anti- inflammatory agents, cell cycle regulating agents, and any combination thereof.

36. The bonded proteinaceous article of any one of the preceding claims wherein said bonded proteinaceous article comprises or more proteins including, but not limited to plant proteins, plant protein isolates, plant protein hydrolysates, wheat proteins (gliadin), casein, whey, gelatin, egg white proteins, zein, resilin (dragonfly), scleroproteins, keratins (α-, β- and γ-keratin), collagens, insulin, fibrins, silk fibroins, silk spidroins, elastins, and any combination thereof.

37. The bonded proteinaceous article of claim 33 wherein the one or more proteins are naturally-isolated or recombinantly-produced silk fibroin, silk spidroin, and any combination thereof.

38. The bonded proteinaceous article of any one of the preceding claims wherein said one or more proteins have an average molecular weight ranging from about 1 kDa to about 500 kDa.

39. The bonded proteinaceous article of any one of the preceding claims wherein the bonded proteinaceous article is in the form of bone screws, artificial corneas, stents, tips and hubs for delivery systems, contact lenses, screws, pins, frames, grids used for products in the sustainability industry, drug delivery devices, time release capsules, floss picks, encapsulation of a substance for release in the environment, stents, films, needles, glaucoma drainage devices, or other suitable articles.

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