System and method for producing a silk fibroin solution and a powder containing silk fibroin

Abstract

The present disclosure relates to systems and methods for improving the production of silk solutions and powders, including silk fibroin obtained from silkworm cocoons, that can be used to improve post-harvest preservation of fresh produce and to improve the performance of packaging, including biodegradable packaging.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 191,441, filed on May 21, 2021, U.S. Provisional Application No. 63 / 212,283, filed on Jun. 18, 2021, and U.S. Provisional Application No. 63 / 231,399, filed on Aug. 10, 2021, the entire disclosures of which are incorporated herein by reference.

[0002] The present disclosure relates to systems and methods for improving the production of silk solutions containing silk fibroin from silk feedstock and the production of silk fibroin powder extracted from the silk solutions.

Background Art

[0003] One - third of the food produced in the world is discarded every year, and more than 45% of all fruits and vegetables spoil and are lost. Food waste has a significant economic, social, and environmental impact. According to the Natural Resources Defense Council (NRDC), a prominent non - profit international environmental advocacy group, the United States loses 40% of its food supply, resulting in an estimated annual economic loss of $165 billion. Embodiments of the present disclosure directly address a broad social need to reduce food waste and increase available food by extending the shelf life of fresh foods (e.g., cooked or uncooked meat, protein, carbohydrates, agricultural products, nuts, grains, seeds, dairy products, beverages, processed foods (e.g., chocolate, candy, chips, snacks, energy bars), gums, tablets, capsules, plants, roots, fungi, spores, bread, dried fruits, dried vegetables, dried foods, medical foods, flowers, plants, etc.). Embodiments of the present disclosure represent important commercial value by increasing revenues through improved distribution, waste reduction, and cost reduction associated with cold storage and transportation.

Summary of the Invention

[0004] The present disclosure relates to a system and method for improving the production of silk solutions and powders containing silk fibroin obtained from silk feedstock, which can be used to improve the post-harvest preservation of fresh food and to improve the performance of packaging including biodegradable containers.

[0005] In one embodiment, the present disclosure provides a process for producing silk fibroin in which a silk source or silk input such as silk cocoons (which may be the whole including the silkworm pupa, may be processed to remove the pupa, and / or may be cut in a specific manner), silk sheets, cocoon floss, or silk pellets, cut cocoons, shredded cocoons, silk threads and silkworm threads, silk fabrics, silk powders, silk flour, silk stuffed products, silk proteins, boiled silk, silk mats, silk webbings, silk fibers, etc. is treated with a solution or powder containing silk fibroin. For example, from Bombyx mori, the silkworm is an example of a silk source that can be used in this process.The present disclosure also applies to silk sources from silkworms other than Bombyx mori (e.g., Bombyx mandarina, Bombyx sinesis, Anaphe moloneyi, Anaphe panda, Anaphe reticulate, Anaphe ambrizia, Anaphe carteri, Anaphe venata, Anapha infracta, Antheraea assamensis, Antheraea assama, Antheraea mylitta, Antheraea pernyi, Antheraea yamamai, Antheraea polyphemus, Antheraea oculea, Anisota senatoria, Apis mellifera, Araneus diadematus, Araneus cavaticus, Automeris io, Atticus atlas, Copaxa multifenestrata, Coscinocera hercules, Callosamia promethea, Eupackardia calleta, Eurprosthenops australis, Gonometa postica, Gonometa rufobrunnea, Hyalophora cecropia, Hyalophora euryalus, Hyalophora gloveri, Miranda auretia, Nephila madagascarensis, Nephila clavipes, Pachypasa otus, Pachypasa atus, Philosamia ricini, Pinna squamosa, Rothschildia hesperis, Rothschildia lebeau, Samia cynthia and Samia ricini, and Tetragnatha versicolor) and also to silk sources from spiders or other insects. The present disclosure also applies to synthetically or recombinantly, transgenically produced silk sources, and other artificially created silk (e.g., silk from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants). Silk proteins have a unique amino acid sequence that is reproducible via synthetic means.The present disclosure relates to such types. To avoid misunderstanding, the silk cocoons described herein can be used in place of silk of the above type or any similar type of silk, regardless of whether it is natural or artificial. For example, when the present disclosure describes that silk, silk inputs, silk cocoons, or silkworm cocoons are used, it means that any of the silk sources described in this paragraph (e.g., cocoons, cocoon floss, sheets, pellets, cut cocoons, shredded cocoons, silk threads and silkworm threads, silk fabrics, silk powders, silk flour, silk stuffed products, silk proteins, boiled silk, silk mats, silk weavings, silk fibers, generated silk sources (e.g., synthetic, genetically engineered, transgenic, and other artificially produced silk), etc.) or combinations thereof can be used. In one embodiment, the silk cocoons are subjected to a degumming step, a dissolution step, a purification step, a microfiltration step, and a powdering step, resulting in a powder of a silk solution containing silk fibroin. In some embodiments, silk fibroin can be isolated from silk cocoons by the Ajisawa method or other methods using water and salts containing chaotropic agents and / or cosmotropic agents. In some embodiments, silk fibroin can be prepared according to the method described in Marelli, B., Brenckle, M., Kaplan, D. et al., Silk Fibroin as Edible Coating for Prishable Food Preservation, Sci Rep 6, 25263 (2016), https: / / doi.org / 10.1038 / srep25263, which is hereby incorporated by reference in its entirety. The microfiltration step described herein will function in any acceptable method of isolating silk fibroin from silk cocoons, including examples where silk fibroin is treated in a silk solution or as a powder. In some embodiments, silk fibroin may be as described in U.S. Patent Application Publication No. 2020-0178576A1, which is hereby incorporated by reference in its entirety.

[0006] In some embodiments, the silk fibroin present in the aqueous solution or powder can have a weight concentration (w / w) in the range of from about 0.1% (w / w) to about 1% (w / w), from 0.1% (w / w) to about 10% (w / w), from 0.1% (w / w) to about 30% (w / w), from 0.1% (w / w) to about 50% (w / w), from about 1% (w / w) to about 5% (w / w), from about 1% (w / w) to about 10% (w / w), from about 1% (w / w) to about 15% (w / w), from about 5% (w / w) to about 10% (w / w), from 5% (w / w) to about 15% (w / w), from 5% (w / w) to about 20% (w / w), from 10% (w / w) to about 30% (w / w), from 10% (w / w) to about 100% (w / w), from 50% (w / w) to about 75% (w / w), from 10% (w / w) to about 100% (w / w), from about 20% (w / w) to about 95% (w / w), from about 30% (w / w) to about 90% (w / w), from 30% (w / w) to about 100% (w / w), from about 40% (w / w) to about 85% (w / w), from about 50% (w / w) to about 80% (w / w), from about 60% (w / w) to about 99% (w / w), from about 70% (w / w) to about 99% (w / w), from about 80% (w / w) to about 99% (w / w), from about 80% (w / w) to about 100% (w / w), from about 90% (w / w) to about 99% (w / w), from about 95% (w / w) to about 99% (w / w), from about 90% (w / w) to about 100% (w / w), or from about 80% (w / w) to about 90% (w / w). In one embodiment, the percent (w / w) of silk fibroin present in the aqueous solution or powder is less than 99%. In one embodiment, the percent (w / w) of silk fibroin present in the aqueous solution or powder is less than 95%. In one embodiment, the percent (w / w) of silk fibroin present in the aqueous solution or powder is less than 60%. In one embodiment, the percent (w / w) of silk fibroin present in the aqueous solution or powder is less than 30%. In one embodiment, the percent (w / w) of silk fibroin present in the aqueous solution or powder is less than 25%. In one embodiment, the percent (w / w) of silk fibroin present in the aqueous solution or powder is less than 20%. In one embodiment, the percent (w / w) of silk fibroin present in the aqueous solution or powder is less than 19%.In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 18%. In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 17%. In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 16%. In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 15%. In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 14%. In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 13%. In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 12%. In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 11%. In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 10%. In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 9%. In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 8%. In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 7%. In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 6%. In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 5%. In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 4%. In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 3%. In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 2%. In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 1%. In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 0.9%. In one embodiment, the percentage (w / w) of silk fibroin present in the aqueous solution or powder is less than 0.8%.In one embodiment, the percent (w / w) of silk fibroin present in the aqueous solution or powder is less than 0.7%. In one embodiment, the percent (w / w) of silk fibroin present in the aqueous solution or powder is less than 0.6%. In one embodiment, the percent (w / w) of silk fibroin present in the aqueous solution or powder is less than 0.5%. In one embodiment, the percent (w / w) of silk fibroin present in the aqueous solution or powder is less than 0.4%. In one embodiment, the percent (w / w) of silk fibroin present in the aqueous solution or powder is less than 0.3%. In one embodiment, the percent (w / w) of silk fibroin present in the aqueous solution or powder is less than 0.2%. In one embodiment, the percent (w / w) of silk fibroin present in the aqueous solution or powder is less than 0.1%. Higher or lower silk fibroin contents are also possible to suit specific applications, such as the method of application, the type of product to be coated, etc.

[0007] In some embodiments, silk fibroin comprises monomers, polymers, and / or fragments of silk fibroin. As used herein, the term silk fibroin fragment also includes aggregates of silk fibroin fragments. In some embodiments, the silk film and / or silk coating is formed from silk fibroin and the silk film and / or silk coating comprises silk fibroin fragments at a particular ratio (weight / volume). In some embodiments, the silk fibroin fragments at a particular ratio have a particular molecular weight (MW). In this context, the molecular weight (MW) refers to the molecular weight of the individual silk fibroin fragments in the silk film and / or silk coating and should not be confused with the weight average molecular weight (Mw). Any method or apparatus appropriate in the art can be used to measure the various properties of silk. In one example, gel permeation chromatography (GPC) could be used to obtain the molecular weight (MW) of the silk fibroin fragments and the weight average molecular weight (Mw) of the silk.

[0008] As an example that is useful for the description, FIGS. 12 and 13 show two different exemplary graphs of the molecular weights of fibroin fragments present in silk films and / or silk coatings. The X-axis represents the molecular weight (MW), and the Y-axis represents the intensity (e.g., the number of fibroin fragments of the same molecular weight). The blue bars indicate the molecular weight (MW) range (e.g., from 50 kDa to 100 kDa) that contains a specific percentage (e.g., 10%) of fibroin fragments in the silk film and / or silk coating, as measured when the fibroin fragments are still in solution. The figures also include peaks (P), for example, FIG. 12 has one peak and FIG. 13 has two peaks. As a further example, the graph of the molecular weight (MW) of the silk film and / or silk coating will include more than two peaks. For the purposes of the present disclosure, the number of peaks is not limiting and does not affect the percentage of fibroin fragments having a specific molecular weight (MW) as described herein. The molecular weight may also be measured via other means such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or other similar techniques.

[0009] In some embodiments, none of the silk fibroin fragments have a molecular weight (MW) of less than 100 kilodaltons (kDa), less than 1% of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, about 1% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, about 5% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, about 10% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, about 15% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, about 20% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, about 25% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, about 30% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, about 35% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, about 40% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, about 45% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, about 50% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, about 55% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, about 60% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, about 65% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, about 70% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, about 75% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, about 80% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, about 85% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, about 90% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, and about 95% or more of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa.

[0010] In some embodiments, none of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, less than 1% of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, about 1% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, about 5% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, about 10% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, about 15% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, about 20% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, about 25% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, about 30% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, about 35% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, about 40% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, about 45% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, about 50% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, about 55% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, about 60% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, about 65% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, about 70% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, about 75% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, about 80% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, about 85% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, about 90% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa, and about 95% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 100 kDa.

[0011] In some embodiments, none of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, less than 1% of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, about 1% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, about 5% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, about 10% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, about 15% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, about 20% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, about 25% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, about 30% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, about 35% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, about 40% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, about 45% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, about 50% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, about 55% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, about 60% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, about 65% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, about 70% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, about 75% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, about 80% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, about 85% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, about 90% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa, and about 95% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 200 kDa.

[0012] In some embodiments, none of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, less than 1% of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, about 1% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, about 5% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, about 10% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, about 15% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, about 20% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, about 25% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, about 30% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, about 35% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, about 40% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, about 45% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, about 50% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, about 55% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, about 60% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, about 65% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, about 70% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, about 75% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, about 80% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, about 85% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, about 90% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa, and about 95% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 300 kDa.

[0013] In some embodiments, none of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, less than 1% of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, about 1% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, about 5% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, about 10% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, about 15% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, about 20% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, about 25% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, about 30% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, about 35% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, about 40% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, about 45% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, about 50% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, about 55% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, about 60% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, about 65% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, about 70% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, about 75% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, about 80% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, about 85% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, about 90% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa, and about 95% or more of the silk fibroin fragments have a molecular weight (MW) exceeding 400 kDa.

[0014] In some embodiments, between about 1% and about 10% of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, between about 1% and about 15% of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, between about 1% and about 30% of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, between about 10% and about 30% of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, between about 10% and about 50% of the silk fibroin fragments have a molecular weight (MW) of less than about 100 kDa, between about 10% and about 75% of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, between about 10% and about 95% of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, between about 15% and about 30% of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, between about 15% and about 40% of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, between about 20% and about 30% of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, between about 20% and about 35% of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, between about 30% and about 50% of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, between about 50% and about 90% of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, between about 50% and about 75% of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, between about 60% and about 75% of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, between about 75% and about 95% of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa, and between about 80% and about 95% of the silk fibroin fragments have a molecular weight (MW) of less than 100 kDa.

[0015] In some embodiments, between about 1% and about 90% of the silk fibroin fragments have a molecular weight (MW) greater than 100 kDa, between about 30% and about 90% of the silk fibroin fragments have a molecular weight (MW) greater than 100 kDa, between about 40% and about 90% of the silk fibroin fragments have a molecular weight (MW) greater than 100 kDa, between about 50% and about 90% of the silk fibroin fragments have a molecular weight (MW) greater than 100 kDa, between about 60% and about 90% of the silk fibroin fragments have a molecular weight (MW) greater than 100 kDa, between about 50% and about 85% of the silk fibroin fragments have a molecular weight (MW) greater than 100 kDa, between about 60% and about 85% of the silk fibroin fragments have a molecular weight (MW) greater than 100 kDa, between about 55% and about 80% of the silk fibroin fragments have a molecular weight (MW) greater than 100 kDa, between about 65% and about 85% of the silk fibroin fragments have a molecular weight (MW) greater than 100 kDa, between about 60% and about 80% of the silk fibroin fragments have a molecular weight (MW) greater than 100 kDa, between about 70% and about 80% of the silk fibroin fragments have a molecular weight (MW) greater than 100 kDa, between about 60% and about 99% of the silk fibroin fragments have a molecular weight (MW) greater than 100 kDa, between about 70% and about 99% of the silk fibroin fragments have a molecular weight (MW) greater than 100 kDa, between about 80% and about 99% of the silk fibroin fragments have a molecular weight (MW) greater than 100 kDa, and between about 90% and about 99% of the silk fibroin fragments have a molecular weight (MW) greater than 100 kDa.

[0016] In some embodiments, between about 0.1% and about 40% of the silk fibroin fragments have a molecular weight (MW) greater than 200 kDa, between about 0.1% and about 30% of the silk fibroin fragments have a molecular weight (MW) greater than 200 kDa, between about 0.1% and about 20% of the silk fibroin fragments have a molecular weight (MW) greater than 200 kDa, between about 0.1% and about 10% of the silk fibroin fragments have a molecular weight (MW) greater than 200 kDa, between about 0.5% and about 40% of the silk fibroin fragments have a molecular weight (MW) greater than 200 kDa, between about 0.5% and about 30% of the silk fibroin fragments have a molecular weight (MW) greater than 200 kDa, between about 0.5% and about 20% of the silk fibroin fragments have a molecular weight (MW) greater than 200 kDa, between about 0.5% and about 10% of the silk fibroin fragments have a molecular weight (MW) greater than 200 kDa, between about 1% and about 30% of the silk fibroin fragments have a molecular weight (MW) greater than 200 kDa, between about 1% and about 20% of the silk fibroin fragments have a molecular weight (MW) greater than 200 kDa, between about 1% and about 10% of the silk fibroin fragments have a molecular weight (MW) greater than 200 kDa, between about 20% and about 80% of the silk fibroin fragments have a molecular weight (MW) greater than 200 kDa, between about 40% and about 90% of the silk fibroin fragments have a molecular weight (MW) greater than 200 kDa, between about 50% and about 90% of the silk fibroin fragments have a molecular weight (MW) greater than 200 kDa, between about 60% and about 90% of the silk fibroin fragments have a molecular weight (MW) greater than 200 kDa, and between about 60% and about 80% of the silk fibroin fragments have a molecular weight (MW) greater than 200 kDa.

[0017] In some embodiments, between about 0.1% and about 3% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa, between about 0.1% and about 5% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa, between about 0.1% and about 10% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa, between about 1% and about 30% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa, between about 1% and about 10% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa, between about 1% and about 20% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa, between about 5% and about 20% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa, between about 10% and about 20% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa, between about 10% and about 30% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa, between about 10% and about 50% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa, between about 10% and about 75% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa, between about 10% and about 95% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa, between about 15% and about 30% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa, between about 20% and about 50% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa, between about 30% and about 50% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa, between about 50% and about 90% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa, between about 50% and about 75% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa, between about 60% and about 75% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa, between about 75% and about 95% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa, and between about 80% and about 95% of the silk fibroin fragments have a molecular weight (MW) greater than 300 kDa.

[0018] In some embodiments, between about 1% and about 5% of the silk fibroin fragments have a molecular weight (MW) greater than 400 kDa, between about 1% and about 10% of the silk fibroin fragments have a molecular weight (MW) greater than 400 kDa, between about 1% and about 20% of the silk fibroin fragments have a molecular weight (MW) greater than 400 kDa, between about 1% and about 30% of the silk fibroin fragments have a molecular weight (MW) greater than 400 kDa, between about 1% and about 60% of the silk fibroin fragments have a molecular weight (MW) greater than 400 kDa, between about 5% and about 10% of the silk fibroin fragments have a molecular weight (MW) greater than 400 kDa, between about 5% and about 15% of the silk fibroin fragments have a molecular weight (MW) greater than 400 kDa, between about 5% and about 20% of the silk fibroin fragments have a molecular weight (MW) greater than 400 kDa, between about 30% and about 60% of the silk fibroin fragments have a molecular weight (MW) greater than 400 kDa, between about 35% and about 55% of the silk fibroin fragments have a molecular weight (MW) greater than 400 kDa, between about 35% and about 75% of the silk fibroin fragments have a molecular weight (MW) greater than 400 kDa, between about 35% and about 85% of the silk fibroin fragments have a molecular weight (MW) greater than 400 kDa, between about 50% and about 85% of the silk fibroin fragments have a molecular weight (MW) greater than 400 kDa, between about 55% and about 80% of the silk fibroin fragments have a molecular weight (MW) greater than 400 kDa, and between about 70% and about 90% of the silk fibroin fragments have a molecular weight (MW) greater than 400 kDa.

[0019] In one aspect, the present disclosure relates to a silk manufacturing system including a plurality of processing sub-stations. Specifically, the system includes a first processing sub-station equipped with a container configured to receive silkworm cocoons and extract silk fibroin protein therefrom to generate a silk fibroin-based solution, a second processing sub-station in fluid communication with the first processing sub-station, configured to receive the silk fibroin-based solution from the first processing sub-station and purify it, a third processing sub-station in fluid communication with the second processing sub-station, configured to receive the purified silk fibroin-based solution and sterilize it, and a fourth processing sub-station in fluid communication with the third processing sub-station, configured to receive the silk fibroin-based solution and powder it. In various aspects, the systems disclosed herein may include any number and configuration of processing sub-stations as needed for a particular application.

[0020] In various embodiments of the above aspect, the system further includes a pump assembly disposed between the first processing sub-station and the second processing sub-station and configured to transfer the silk fibroin-based solution from the first processing sub-station to the second processing sub-station. The system may also include a reservoir disposed between the first processing sub-station and the second processing sub-station and configured to perform at least one of holding or conditioning the silk fibroin-based solution, for example, to adjust the temperature of the solution or the concentration of one or more components of the solution. Further, the system may further include a filtration system disposed between the first processing sub-station and the second processing sub-station and configured to filter the silk fibroin-based solution, and a heat exchange system configured to adjust the temperature of the silk fibroin-based solution either before or after any one of the processing sub-stations.

[0021] In a further embodiment, the first processing substation is configured to extract silk fibroin protein by a degumming process, a rinsing process, and a dissolving process within a single container. The second processing substation may be configured to purify the silk fibroin-based solution and / or to concentrate the silk fibroin-based solution to have a higher percentage of silk fibroin by tangential flow filtration, i.e., dialysis, or by ultrafiltration and / or diafiltration with or without using dialysis. The third processing substation may be configured to moderately wash or sterilize the silk fibroin-based solution purified by one or more of ultrafiltration, microfiltration, pasteurization, or the like. Generally, sterilization is not necessarily intended to include a solution that is completely free of bacteria or other living microorganisms, but it may be so. Another substation may be centrifugation or microfiltration for reducing turbidity. Excessive turbidity may be undesirable in a silk fibroin-based solution because it affects the adhesiveness of a coating made from the silk fibroin-based solution, hinders the barrier-forming properties of the silk solution, and / or makes the coating formed from the silk fibroin-based solution appear cloudy or milky white. For this reason, the turbidity may be maintained at less than about 1,000 optical density, including a solution concentration of silk fibroin in water of 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, or 20%, and the optical density is measured at a wavelength of 660 nm (OD660). In some embodiments, the turbidity may be maintained below a lower limit such as about 0.900, 0.800, 0.700, 0.600, 0.500, 0.400, 0.300, 0.200, 0.100, 0.050 (OD600), or any increment within that range.

[0022] Furthermore, the presence of excessive amounts of microorganisms can negatively impact the performance of the silk solution and render silk fibroin-based solutions unsuitable for human consumption or targeted uses, including pre-harvest uses, post-harvest uses, animal feed uses, or other such uses. For this reason, microorganisms need to be killed and / or substantially removed from the silk fibroin-based solution, which can range from a small level of reduction to essentially complete removal, as determined, for example, within the detection limit and / or within the range of microorganism types (e.g., for mold, yeast, enterobacteria, Staphylococcus aureus, Escherichia coli, less than 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 cfu / g, and for Salmonella and Listeria monocytogenes, a "negative / less than 25 g" reading). In one example, this may mean keeping the amount of microorganisms below 10 CFU / ml under an approved testing agency, such as, for example, the total aerobic plate count on Plate Count Agar (PCA) and / or Potato Dextrose Agar (PDA). In some cases, a third processing substation is configured to sterilize the purified silk fibroin-based solution to food-grade standards. In some cases, the third processing substation may be completely removed from the system. In those cases, or other cases, the above-mentioned substation may be able to produce a food-grade standard product and / or sterilize the silk fibroin-based solution to the levels described herein. For example, the first substation may be able to sterilize the silk fibroin-based solution by treating the solution at a certain temperature to remove microorganisms from the solution. In this example, further sterilization may not be necessary since the entire system could instead be closed. In other cases, the third processing substation may be located at different positions within the system. In some cases, the entire system may be a closed system so that there may not be a sufficient number of microorganisms present to require a third processing substation. Additionally, a fourth processing substation may be configured to powder the purified silk fibroin-based solution by spray drying, freeze drying, or similar drying and powdering methods known in the industry.

[0023] In still other embodiments, the system may further include a pretreatment system configured to condition the silkworm cocoons before or during introduction to the first processing substation, such as, for example, a shredder, a softening device, an immersion device, and / or a material handling device for shredding the silkworm cocoons. In some embodiments, the silkworm cocoons are shredded into a reduced size and shape (e.g., fragments of 0.5 to 50 cm or longer cocoon fluff, silk sheets, or 0.5 to 50 cm strands of silk bales), and / or processed or pressed. In some embodiments, the cocoons or other silk inputs are degummed, washed to remove organic and inorganic compounds, have other proteins removed, or are combined with multiple silk inputs to increase the amount of fibroin per unit mass of the silk input before being introduced into the system. This may or may not include a shredding or cutting step, or a preliminary degumming step. Further, the pretreatment apparatus may include a system for cleaning the cocoons, including separating debris from the cocoons, testing the cocoons (e.g., chemical analysis), and / or performing other quality control processes including cocoon composition evaluation.

[0024] The system may also include a post-treatment system configured to receive fibroin powder from a fourth processing sub-station. The post-treatment apparatus may include an apparatus for conditioning the fibroin powder by adding one or more additives, or silk powder from different batches having different chemical or polymer properties (i.e., molecular weight profile, turbidity, etc.). (For example, lower molecular weight fibroin may be added to higher molecular weight fibroin to enable instantiation and increased solubility, or to enable different features and properties.) The post-treatment apparatus may also add heat treatment steps or aggregation steps that can make the powder drier, wetter, denser, cleaner, and / or more instantiatable. The post-treatment apparatus may also include an apparatus for testing the fibroin powder and / or packaging the fibroin powder. The post-treatment step may be a sterile packaging method to enable fibroin powder that can be stored at room temperature.

[0025] The system may include a controller configured to communicate with various processing sub-stations (e.g., valve assemblies, sensors, switches, transmitters, drives, etc.) and control one or more of the input variables (e.g., volume, flow rate, mixing ratio, stirring speed, process timing / duration, pretreatment operations, component ratios, pH level, temperature, pressure, solution volume, solid content, etc.) of various components (e.g., cocoons, solvents, compounds, etc.), control the degumming operation (e.g., number of immersions and temperature, pressurization, stirring speed and its timing, volume control (i.e., draining and refilling of the container, recirculation)), control the rinsing operation (e.g., determination of the state of the solution, draining and refilling of the container, addition of solvent, frequency and duration of various steps, pressurization, or depressurization), control the fibroin dissolution operation (e.g., addition of a second compound and its concentration, time, temperature, pressure, stirring speed and its timing, duration, etc.), and control the output from the sub-station (e.g., flow rate, temperature, etc.).

[0026] In various embodiments of any of the aspects disclosed herein, a first processing substation includes a reaction vessel having a first inlet port configured to receive silkworm cocoons and one or more components (e.g., soda ash, chaotropic agents, catalysts, additives, or the like), a second inlet port configured to receive a solvent (e.g., water, ethanol, citric acid, etc.), and at least one outlet configured to output a silk fibroin-based solution. The reaction vessel is configured to process silkworm cocoons by at least one of degumming, scouring, and dissolving silk fibroin protein from the cocoons. The first processing substation may also include a water jacket or an oil jacket disposed around the reaction vessel and configured to provide heat exchange (e.g., heating or cooling as needed) with the vessel and its contents. The first processing substation may further include a device configured to stir the contents of the reaction vessel, such as, for example, a mixer, a diaphragm, a magnetic stirrer, an ultrasonic disruptor, a liquid pump, an air pump, a water flow, etc. Stirring may be generated through an external or internal pressure flow, which is a liquid and / or a gas. In various embodiments, the stirring device may be disposed proximate to the bottom or top surface of the reaction vessel. In various embodiments, the stirring device may be disposed at various portions of the reaction vessel (i.e., a pump at the bottom, center, and top, a stirrer at the bottom, and a pump at the top, etc.). In some embodiments, the stirring device is a mixer having an integral shaft and impeller. The impeller may be configured for axial flow, radial flow, and / or tangential flow and may be driven reversibly. Further, the impeller may be coated with a material resistant to silk fiber adhesion and / or may have a surface finish of the blades (e.g., a surface roughness below some threshold). The mixer may have a replaceable impeller, and the impeller may be configured to be suitable for a particular process and have flat blades, curved blades, inclined blades, finger blades, anchor blades, gate blades, ribbon blades, etc., having different shapes, pitches, etc.The impeller may also be configured to rise or fall within the container or within the container contents during different processing steps or between different processing steps.

[0027] In a further embodiment, the reaction vessel includes a second outlet for removing at least a portion of the solvent and any residues therein (e.g., dissolved sericin) that can be sent to waste, recycled, or reused. The reaction vessel has a glass lining and can be sized to have an aspect ratio of height to diameter defined by a working volume of 0.5 to 5.0, or more preferably 0.8 to 2, and more preferably 1.0 to 1.5. The aspect ratio can be selected to suit a particular application, such as temperature control, processing speed, desired volume, working space, etc. The volume of the vessel can range from about 0.25 liters to about 80,000 liters, preferably 0.5 liters to 5,000 liters, depending on the required batch size. Further, since the reaction vessel can have a shape other than cylindrical, the aspect ratio is the height of the vessel to its cross-sectional area (e.g., cross-sectional shapes such as rectangular, oval, etc.). The contents of the vessel can include a plurality of silkworm cocoons (with or without pretreatment), a solvent (e.g., water), and a compound. The packing density of the silkworm cocoons can vary to suit a particular application (e.g., a finished silk fibroin-based solution) and / or different silk inputs (e.g., cocoons, cocoon fluffs, etc.) and can range from about 1% to 100%, about 1% to 70%, about 1% to 50%, about 1% to 30%, about 1% to 20%, about 2% to 20%, about 2% to 15%, less than about 100%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, greater than about 1%, greater than about 5%, greater than about 10%, greater than about 15%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%. A water jacket or an oil jacket is configured to heat the contents to about 50°C to about 150°C, preferably about 75°C to about 125°C, or other temperatures suitable for a particular application.Further, the rinsing step may include performing 1 to 30 rinsing cycles, more preferably about 1 to about 10 rinsing cycles, more preferably about 3 to about 10 rinsing cycles, and more preferably about 4 to about 6 rinsing cycles, or any number of rinsing cycles that are essentially suitable for a particular application. Generally, the number of process runs, temperature, pH, and other solution characteristics may vary to suit a particular application such as the type of silk or specific output specifications.

[0028] The reaction vessel may also include a handling structure or handling device configured to control the movement and / or position of the silkworm cocoons within the vessel (e.g., to prevent the cocoons from floating). The device may include, for example, a screen or net disposed proximate to the lower portion of the vessel to separate the silkworm cocoons from the agitator and / or to prevent the silkworm cocoons from floating to the upper portion of the vessel, a chute or funnel structure in communication with the first inlet and configured to direct the silkworm cocoons to a specific location within the vessel during their introduction, a recirculation system configured to draw a portion of the solution from the lower portion of the vessel, reintroduce the solution to the upper portion of the vessel, and / or introduce fresh water to push the silkworm cocoons downward into the solution, a vertically movable sieve (e.g., a perforated plunger) disposed within the vessel and configured to "push" any solids within the solution toward the lower portion of the vessel, and one or more baffles disposed within the vessel and extending from its inner wall, the baffles being configured to direct the movement of the solution and the contents therein. In some embodiments, the movement of the silkworm cocoons may be controlled by adjusting the processing temperature between various stages of the process. For example, during the degumming operation, the contents may be heated to a temperature slightly below its boiling point to reduce the formation of air bubbles. Other methods of controlling the movement of the cocoons are described in more detail below. See, for example, FIGS. 9-11.

[0029] The first processing sub-station and system can generally include one or more valve assemblies (equipped with manual or automatic actuators) configured to control the introduction of and removal from the first processing sub-station and / or reaction vessel of any components such as, for example, silkworm cocoons, compounds, solvents, waste liquids, residues, and the final silk fibroin-based solution. The first processing sub-station and system can generally include at least one sensor configured to sense one or more of the temperature, concentration, flow rate, pH, liquid level, turbidity, particle size, molecular weight, pressurization, etc. of the solution, which can be used to control the operation of various processes (with or without human intervention).

[0030] In a further embodiment of any of the aspects disclosed herein, the second processing substation includes a filtration module that houses at least one membrane. The filtration module contains a second compound (e.g., calcium bromide, magnesium chloride, lithium acetate, lithium perchlorate, guanidinium chloride, ethanol, methanol, urea, thiourea, sodium dodecyl sulfate, lithium thiocyanate (LiSCN), sodium thiocyanate (NaSCN), calcium thiocyanate (Ca(SCN)2), magnesium thiocyanate (Mg(SCN)2), anhydrous or dihydrated calcium chloride (CaCl2), lithium chloride (LiCl), lithium bromide (LiBr), zinc chloride (ZnCl2), copper nitrate (Cu(NO2)2), copper ethylenediamine (Cu(NH2CH2CH2NH2)2(OH)2), Cu(NH3)4(OH)2, Ajisawa's reagent (CaCl2 / ethanol / water), isopropanol, 1-butanol, 2-butanol, ethyl acetate, calcium nitrate, magnesium nitrate, calcium perchlorate, calcium chlorate, calcium acetate, dibasic calcium phosphate / calcium hydrogen phosphate, calcium sulfate, calcium fluoride, ammonium fluoride, ammonium sulfate, ammonium phosphate, diammonium phosphate (dibasic ammonium phosphate), ammonium dihydrogen phosphate, ammonium acetate, ammonium chloride, ammonium bromide, ammonium nitrate, ammonium chlorate, ammonium iodide, ammonium perchlorate, ammonium thiocyanate, potassium fluoride, potassium sulfate, monopotassium phosphate, dipotassium phosphate (dibasic potassium phosphate), tripotassium phosphate, potassium acetate, potassium chloride, potassium bromide, potassium nitrate, potassium chlorate, potassium iodide, potassium perchlorate, potassium thiocyanate, sodium fluoride, sodium sulfate, monosodium phosphate (e.g., monosodium phosphate, disodium phosphate, trisodium phosphate), disodium phosphate and sodium polyphosphate (e.g., disodium monophosphate, disodium diphosphate, disodium trisphosphate, disodium tetraphosphate, trisodium phosphate), sodium acetate, sodium chloride, sodium bromide, sodium nitrate, sodium chlorate, sodium iodide, sodium perchlorate, lithium fluoride, lithium sulfate, lithium phosphate,An inlet configured to receive a silk fibroin-based solution containing a chaotropic agent such as lithium chloride, lithium bromide, lithium nitrate, lithium chlorate, lithium iodide, magnesium fluoride, magnesium sulfate, monomagnesium phosphate, mimagnesium phosphate, trimagnesium phosphate, magnesium acetate, magnesium bromide, magnesium chlorate, magnesium iodide, magnesium perchlorate, magnesium thiocyanate, monocalcium phosphate, tricalcium phosphate, octacalcium phosphate, dicalcium diphosphate, tricalcium triphosphate, calcium iodide, guanidinium nitrate, guanidinium iodide, guanidinium thiocyanate, or a combination thereof; an outlet configured to output a purified silk fibroin-based solution (i.e., the retained material) with a reduced concentration of any chaotropic agent; and a waste port configured to output a portion of a second compound (i.e., the filtration product). The filtration module is configured to remove the second compound from the silk fibroin-based solution by diafiltration or dialysis. In some cases, the flow through the module is tangential to the surface of the membrane. The silk fibroin-based solution may also experience a certain level of concentration that can be adjusted to optimize subsequent processes (e.g., sterilization or powdering). The silk fibroin-based solution can be circulated through the filtration module for a period determined by from about 1 diavolumes to at least about 12 diavolumes, preferably from about 3 diavolumes to about 10 diavolumes, and more preferably from about 5 diavolumes to about 9 diavolumes. In some cases, the concentration level of the chaotropic agent in the retained material and / or the pressure drop across the entire filtration module can also be monitored to determine the state of the process. Generally, it is desirable to obtain a level of residual chaotropic agent that is substantially undetectable by the user (e.g., odorless). However, this level varies with different agents and / or product applications and may include concentrations as low as less than 1 part per million (ppm), less than 900 ppm, less than 650 ppm, less than 400 ppm, less than 300 ppm, less than 250 ppm, and even less than 150 ppm. In some cases, to ensure that there are no contaminants or unwanted substances in the silk fibroin-based solution,Other tests are performed.

[0031] Furthermore, the filtration module may include one or more spiral membranes. However, other membrane structures such as plates and frames, hollow fibers, etc. may be used to suit specific applications (e.g., flow rate, pressure, etc.). The filtration module may include multiple stages and may include from about 1 to about 10 membranes, from about 1 to about 8 membranes, from about 3 to about 8 membranes, from about 3 to about 5 membranes. When multiple filter stages or filtration modules are used, the silk fibroin-based solution may pass through them in series, in parallel, or both to suit a specific application. The number, size, and configuration of the membranes are selected based on various system parameters (e.g., flow rate). The structure and chemistry of the membrane active layer may also vary to suit a specific application and may be structured with a molecular weight cut-off from about 1 kDa to about 300 kDa, from about 1 kDa to about 100 kDa, from about 1 kDa to about 50 kDa. Additionally, the membrane may be manufactured from polyethersulfone (PES), polyvinylidene fluoride (PVDF), polyacrylonitrile (PAN), polypropylene (PP), polyethylene terephthalate (PET), or combinations thereof.

[0032] The second processing substation may also include a heat exchange system including any valves, pumps, control devices, etc. as necessary to control the temperature of the silk fibroin-based solution during processing. For example, reducing the temperature of the silk fibroin-based solution before introducing it into the filtration module may facilitate the removal of the second compound. The second processing substation may also include one or more valve assemblies configured to direct the silk fibroin-based solution output containing the reduced second compound to at least one inlet (recirculation) or to one or more sensors (e.g., differential pressure, temperature, flow rate, salinity meter, conductivity, etc.) that communicate with the third processing substation and the controller. In some embodiments, the filtration module may include a recycle circuit for recovering the removed second compound by evaporation or the like.

[0033] In any additional embodiments of the aspects disclosed herein, the third processing substation includes a microfiltration module having an inlet configured to receive the degummed silk fibroin-based solution from the second processing substation and an outlet configured to output a sterile silk fibroin-based solution. The microfiltration module is configured to reduce turbidity and / or remove microorganisms from the degummed silk fibroin-based solution. In some embodiments, the inlet is configured to receive the silk fibroin-based solution from the first processing substation, and the outlet is configured to output the sterile silk fibroin-based solution to the second processing substation. Further, the microfiltration module may include one or more filter stages, with or without pumps, valves, and holding tanks as needed. In some embodiments, the first filter stage may be disposed upstream of the second processing substation, and the second filter stage may be disposed downstream of the second processing substation. In embodiments including one or more pumps, the pumps are configured to transfer the silk fibroin-based solution, as needed after completion of the microfiltration process, between filter stages and / or processing substations and / or to another process. Further, one or more holding tanks may be included to provide additional processing such as temperature control or concentration adjustment, as may be necessary to store the solution or to address turbidity or sterility levels.

[0034] The filter stage may include one or more spiral membranes. However, other membrane structures such as plate and frame, hollow fiber, bag filter, cartridge, etc. may be used to suit specific applications. In some embodiments, the microfiltration module may include two stages, the first stage being configured to remove large aggregates, while the second stage is configured to remove smaller aggregates and / or sterilize and reduce the turbidity of the solution. The membranes of the second stage may be configured for depth filtration or surface filtration with pore sizes in the range of 0.65 - 15 μm. The membranes of the second stage may be configured for depth filtration or surface filtration with pore sizes in the range of about 0.05 - 0.65 μm. The membranes may be made of PES, PP, or cellulose, with or without using a food-grade filter aid. The filter stage may include from about 1 to about 52 membranes.

[0035] Silk fibroin-based solutions may pass through the membranes in series, parallel, or both to suit specific applications. The membranes may have an average pore size in the range of about 0.02 μm to about 15 μm. In some embodiments, the membranes of the first filter stage may have pore sizes in the range of about 0.7 μm to about 5 μm, preferably about 0.9 μm, and about 1.4 μm, while the membranes of the second filter stage may have pore sizes in the range of about 0.05 μm to about 0.8 μm, preferably about 0.2 μm to about 0.8 μm. The silk fibroin-based solution passes through the first filter stage before passing through the second filter stage (e.g., to remove larger aggregates in the first stage). In some cases, the silk fibroin-based solution contains a minimal amount of a chaotropic agent. Additionally or alternatively, the third processing substation may include a heat exchange circuit for sterilizing the solution by pasteurization.

[0036] In yet a further embodiment of any of the aspects disclosed herein, the fourth processing substation includes a powdering device configured to receive a sterilized silk fibroin-based solution from the third processing substation and output silk fibroin protein in powder form. Further, the resulting powdered silk fibroin can have a water activity level of less than 1.0, 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1. Preferably, the water activity level is less than 0.9 to enable a powder that can be stored at room temperature from a food and microbiological perspective. The powdering device can include a spray dryer configured to receive the sterilized silk fibroin-based solution from the third processing substation and an outlet configured to output silk fibroin protein in a powdered and readily-instantiable form. In one embodiment, the spray dryer can be configured to have a high-pressure nozzle, and the spray is generated by forcing a feed, which in this case is a silk fibroin-based solution, through the nozzle orifice. Alternatively, a two-fluid nozzle spray dryer in which the spray is generated by the interaction between the feed and compressed air can be used. In a two-fluid nozzle configuration, the feed can be atomized through contact with compressed air with or without the use of a subsequent nozzle heating step. A hot drying gas can also be used to accelerate the spray engine when the spray engine contacts the feed. The hot drying gas can be configured to move at a low speed. Other spray dryer configurations can also be used. By way of non-limiting example, the spray dryer can be one of the following types, namely a high-pressure nozzle, a two-fluid nozzle, a combustion nozzle, atomization.

[0037] Instantiable can include a series of properties including, but not limited to, being fluid, being easily dispersible in a liquid, forming a stable dispersion in the liquid without stirring or shaking the powder in the liquid, but instead could be made by stirring or shaking the powder in the liquid for only a short period of time. In one embodiment, the water content of the powder needs to be about 1% to 10%, more preferably about 1.0% to 7%. The fourth processing sub-station may also include a supply container for holding a sterilized silk fibroin-based solution before processing. The supply container can be configured to process the sterilized silk fibroin-based solution before processing, for example, to facilitate powdering or to produce a more instantiable powder. The fourth processing sub-station may also include a device (e.g., inclusion of additives to make it more instantiable, or a device to assist in aggregation) or a packaging device disposed downstream of the powdering device for modifying the powdered silk fibroin protein. As an example of an aggregation device, the fourth processing sub-station may include an external fluidized bed or a fluidized bed integrated with the powdering device. The aggregation device can assist in the aggregation of the powdered silk fibroin protein, which can improve the dispersibility, instantiability, or wetting properties of the powdered silk fibroin protein. Any suitable aggregation device can be utilized. In some embodiments, the powdered silk fibroin can pass through the aggregation device after it is powdered. In other embodiments, the aggregation device may be integrated into the spray dryer such that aggregation occurs during the powdering process. In some embodiments, the aggregation device can increase the size of the powdered silk fibroin protein particles by about 5% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 150% or more, about 200% or more, about 250% or more, about 300% or more, about 350% or more, about 400% or more, about 500% or more, about 600% or more, about 700% or more, about 800% or more, about 900% or more, about 1000% or more.

[0038] In another aspect, the present disclosure relates to a method of processing silkworm cocoons to obtain food-grade silk fibroin. The method includes introducing a plurality of silkworm cocoons into a reaction vessel; introducing a solvent (e.g., water (e.g., softened water, filtered water, deionized water, tap water), ethanol, citric acid, or other suitable substance having an acidic pH) into the reaction vessel; introducing a first compound into the reaction vessel; introducing heat into the contents of the reaction vessel to facilitate degumming of the silkworm cocoons; optionally pressurizing the reaction vessel and / or optionally stirring the contents of the reaction vessel to control movement of the silkworm cocoons within the reaction vessel; removing at least a portion of the solvent and, if any, degumming residues; rinsing the degummed silk fibroin; introducing a second compound into the reaction vessel to dissolve the remaining silk fibroin protein in solution (with or without using additional solvent); filtering the contents of the reaction vessel to substantially remove the second compound and produce a purified silk fibroin-based solution (e.g., to meet a specific level or range of purity, if necessary); leading the purified silk fibroin-based solution to a sterilization process to obtain a silk fibroin-based solution of "food-grade" quality; and powdering the purified silk fibroin-based solution to obtain silk fibroin in powder form. Various parameters of the process vary to suit a particular application, such as the order, amount, and rate of introduction or removal of various components (e.g., silkworm cocoons, solvents, compounds, rinsing liquids, etc.), the operating temperature range, the processing time (e.g., the rate and timing of the stirring step(s)), the operating sequence, and the like. In various embodiments, the methods disclosed herein may incorporate any of the additional processes or steps corresponding to the systems and subsystems disclosed herein.

[0039] Embodiment 1: (A) A first processing sub-station including a container configured to receive a silk input, extract silk fibroin protein therefrom, and generate a silk fibroin-based solution such that the silk fibroin-based solution is substantially free of sericin, the first processing sub-station being configured to extract silk fibroin protein by a degumming process, a rinsing process, and a dissolution process within a single container; (B) A second processing sub-station in fluid communication with the first processing sub-station, the second processing sub-station being configured to receive and purify a silk fibroin-based solution from the first processing sub-station, the purified silk fibroin-based solution containing one or more salts or non-organic particles of less than about 650 parts per million (ppm); (C) The silk fibroin-based solution is sterilized prior to the third processing sub-station to produce a sterilized silk fibroin-based solution; (D) A third processing sub-station in fluid communication with the second processing sub-station, the third processing sub-station being a spray dryer configured to receive the sterilized silk fibroin-based solution and powder it, a silk manufacturing system.

[0040] Embodiment 2: (A) A first processing sub-station including a container configured to receive a silk input, extract silk fibroin protein therefrom, and generate a silk fibroin-based solution, the first processing sub-station being configured to extract silk fibroin protein by a degumming process, a rinsing process, and a dissolving process within a single container; (B) A second processing sub-station in fluid communication with the first processing sub-station, the second processing sub-station being configured to receive the silk fibroin-based solution from the first processing sub-station and purify it; (C) A third processing sub-station in fluid communication with the second processing sub-station, the third processing sub-station being configured to receive the purified silk fibroin-based solution and sterilize it; (D) A fourth processing sub-station in fluid communication with the third processing sub-station, the fourth processing sub-station being configured to receive the purified silk fibroin-based solution and powder it, the fourth processing sub-station being a spray dryer. A silk manufacturing system comprising the above components.

[0041] Embodiment 3: (A) A first processing sub-station configured to receive a silk input, extract silk fibroin protein therefrom, and generate a silk fibroin-based solution, wherein the first processing sub-station is configured to extract silk fibroin protein within a single container, and the first processing sub-station includes a first inlet port configured to receive a raw silk input and one or more compounds, a second inlet port configured to receive a solvent, and a reaction container including at least one outlet configured to output a silk fibroin-based solution, the reaction container being configured to process the silk input by degumming, washing, and dissolving the silk fibroin protein from the silk input; a liquid jacket disposed around the reaction container and configured to provide heat exchange with the container, the liquid jacket being configured to heat the contents to a temperature of about 50°C to about 150°C; and a stirrer mechanism configured to stir the contents of the reaction container. (B) A second processing sub-station in fluid communication with the first processing sub-station, the second processing sub-station being configured to receive the silk fibroin-based solution from the first processing sub-station and purify it, the second processing sub-station being configured to purify the silk fibroin-based solution by tangential flow filtration, the second processing sub-station including a filtration module that houses at least one membrane, the module including an inlet configured to receive a silk fibroin-based solution containing a compound, an outlet configured to output a purified silk fibroin-based solution with a reduced amount of the compound, and a waste port configured to output a portion of the compound, the filtration module being configured to remove the compound from the silk fibroin-based solution by circulating the silk fibroin-based solution through the filtration module until it reaches a volume of at least about 12 dia from about 1 dia. (C) The silk fibroin-based solution is sterilized prior to the third processing sub-station to produce a sterilized silk fibroin-based solution. (D) A third processing sub-station in fluid communication with the second processing sub-station, the third processing sub-station beingReceives a sterilized silk fibroin-based solution and is configured to powder it. The third processing sub-station is configured to powder the sterilized silk fibroin-based solution by a spray dryer. A silk manufacturing system including a third processing sub-station including one agglomeration device, and (E) a post-treatment system configured to receive silk fibroin powder from the third processing sub-station and perform at least one of adjustment of the silk fibroin powder, testing of the silk fibroin powder, or packaging of the silk fibroin powder into a container safe for use in food.

[0042] Embodiment 4: A silk manufacturing system including a first processing sub-station including a container configured to receive a silk input, extract silk fibroin protein therefrom, and generate a silk fibroin-based solution such that the silk fibroin-based solution is substantially free of sericin, the first processing sub-station being configured to extract silk fibroin protein by a degumming process, a rinsing process, and a dissolving process within a single container.

[0043] Embodiment 5: The silk manufacturing system according to any one of Embodiments 1 to 4, or any combination thereof, wherein the purified silk fibroin-based solution contains one or more salts or non-organic particles of less than about 400 ppm.

[0044] Embodiment 6: The silk manufacturing system according to any one of Embodiments 1 to 5, or any combination thereof, wherein the powdered silk fibroin-based solution contains a water activity level of less than 0.9.

[0045] Embodiment 7: The silk manufacturing system according to any one of Embodiments 1 to 6, or any combination thereof, wherein the silk input is derived from Bombyx mori silkworms.

[0046] Embodiment 8: A storage device disposed between a first processing sub-station and a second processing sub-station and configured to perform at least one of holding or adjusting a fibroin-based solution, and a pump assembly disposed between the first processing sub-station and the second processing sub-station and configured to transfer a fibroin-based solution between the first processing sub-station, the storage device, and the second processing sub-station. The silk manufacturing system according to any one of Embodiments 1 to 7, or any combination thereof.

[0047] Embodiment 9: The silk manufacturing system according to any one of Embodiments 1 to 8, or any combination thereof, wherein the second processing sub-station includes at least one spiral filtration membrane.

[0048] Embodiment 10: The silk manufacturing system according to any one of Embodiments 1 to 9, or any combination thereof, further including a heat exchange system configured to adjust the temperature of a fibroin-based solution before or after any one of the processing sub-stations.

[0049] Embodiment 11: The silk manufacturing system according to any one of Embodiments 1 to 10, or any combination thereof, wherein the second processing sub-station is configured to purify a fibroin-based solution by diafiltration.

[0050] Embodiment 12: The silk manufacturing system according to any one of Embodiments 1 to 11, or any combination thereof, wherein the second processing sub-station is configured to purify a fibroin-based solution by tangential flow filtration.

[0051] Embodiment 13: The silk manufacturing system according to any one of Embodiments 1 to 12, or any combination thereof, wherein the third processing sub-station or the fourth processing sub-station includes one flocculation device.

[0052] Embodiment 14: A silk manufacturing system according to any one of Embodiments 1 to 13, or any combination thereof, further including a post-treatment system configured to receive silk fibroin powder from a third processing sub-station and perform at least one of adjustment of the silk fibroin powder, testing of the silk fibroin powder, or packaging of the silk fibroin powder into a container safe for use in food.

[0053] Embodiment 15: A silk manufacturing system according to any one of Embodiments 1 to 14, or any combination thereof, wherein the third processing sub-station is configured to sterilize a purified silk fibroin-based solution to food-grade standards by microfiltration.

[0054] Embodiment 16: A silk manufacturing system according to any one of Embodiments 1 to 15, or any combination thereof, wherein the third processing sub-station is configured to sterilize a purified silk fibroin-based solution to food-grade standards by pasteurization.

[0055] Embodiment 17: A silk manufacturing system according to any one of Embodiments 1 to 16, or any combination thereof, further including a fourth processing sub-station in fluid communication with the second processing sub-station, the fourth processing sub-station being configured to receive a purified silk fibroin-based solution and sterilize it.

[0056] Embodiment 18: A silk manufacturing system according to any one of Embodiments 1 to 17, or any combination thereof, wherein the fourth processing sub-station includes a microfiltration module configured to receive at least one of a silk fibroin-based solution or a purified silk fibroin-based solution, remove microorganisms from at least one of the silk fibroin-based solution or the purified silk fibroin-based solution, and reduce turbidity.

[0057] Embodiment 19: The microfiltration module includes two filter stages, the first filter stage has a pore size between about 0.7 μm and about 5 μm, the second filter stage has a pore size between about 0.05 μm and about 0.8 μm, and a fibroin-based solution passes through the first filter stage before passing through the second filter stage. The silk manufacturing system according to any one of Embodiments 1 to 18, or any combination thereof.

[0058] Embodiment 20: The microfiltration module further includes one or more pumps configured to transfer a fibroin-based solution between filter stages, between processing subsystems, and, if necessary, to another process after completing a microfiltration process or any combination thereof. The silk manufacturing system according to any one of Embodiments 1 to 19, or any combination thereof.

[0059] Embodiment 21: The microfiltration module further includes one or more holding tanks, and the tanks can be configured to provide additional processing including one or more of storage of the solution, temperature control of the solution, or adjustment of the solution concentration to address turbidity or sterility levels. The silk manufacturing system according to any one of Embodiments 1 to 20, or any combination thereof.

[0060] Embodiment 22: The reaction vessel is sized to have an aspect ratio of height to diameter defined by a working volume of about 0.5 to about 5.0. The silk manufacturing system according to any one of Embodiments 1 to 21, or any combination thereof.

[0061] Embodiment 23: The reaction vessel is sized to have an aspect ratio of height to diameter defined by a working volume of about 0.8 to about 2.0. The silk manufacturing system according to any one of Embodiments 1 to 22, or any combination thereof.

[0062] Embodiment 24: The silk manufacturing system according to any one of Embodiments 1 to 23, or any combination thereof, wherein the reaction vessel further includes a handling structure for controlling at least one of the movement or position of the silk input in the vessel.

[0063] Embodiment 25: The silk manufacturing system according to any one of Embodiments 1 to 24, or any combination thereof, further including a pretreatment system configured to adjust the silk input before or during introduction into the first processing subsystem.

[0064] Embodiment 26: The silk manufacturing system according to any one of Embodiments 1 to 25, or any combination thereof, wherein the second processing subsystem further includes a heat exchange system for controlling the temperature of the silk fibroin-based solution during processing.

[0065] Embodiment 27: The silk manufacturing system according to any one of Embodiments 1 to 26, or any combination thereof, wherein the filtration module is configured to remove a second compound from the silk fibroin-based solution by circulating the silk fibroin-based solution through the filtration module until it reaches at least about 8 dia volume from about 5 dia volume.

[0066] Embodiment 28: A method for processing a silk input to obtain silk fibroin, including steps of introducing a plurality of silk inputs into a reaction vessel, introducing a solvent into the reaction vessel, introducing a first compound into the reaction vessel, introducing heat into the contents of the reaction vessel to promote degumming of the silk input, controlling the movement or positioning of the silk input in the reaction vessel, rinsing the degummed silk input, introducing a second compound into the reaction vessel to dissolve any remaining silk fibroin protein in the solution, stirring the contents of the reaction vessel, filtering the contents of the reaction vessel to substantially remove the second compound and produce a purified silk fibroin-based solution, and powdering the purified silk fibroin-based solution to obtain the purified silk fibroin in powder form.

[0067] Embodiment 29: A method for treating silk inputs to obtain silk fibroin, comprising the steps of introducing a plurality of silk inputs into a reaction vessel, introducing a solvent into the reaction vessel, introducing a first compound into the reaction vessel, introducing heat into the contents of the reaction vessel to promote degumming of silkworms, controlling the movement or positioning of the silk inputs within the reaction vessel, removing at least a portion of the solvent and any degumming residues, rinsing the degummed silk inputs, introducing a second compound into the reaction vessel to dissolve the remaining silk fibroin protein in solution, stirring the contents of the reaction vessel, filtering the contents of the reaction vessel to substantially remove the second compound and produce a purified silk fibroin-based solution, leading the purified silk fibroin-based solution to a sterilization process to obtain a sterilized silk fibroin-based solution, and powdering the sterilized silk fibroin-based solution to obtain silk fibroin in powder form.

[0068] Step 30 of Embodiment: A step of providing a reaction vessel configured to extract silk fibroin protein by a degumming process, a rinsing process, and a dissolving process in the reaction vessel, the vessel including at least one inlet port, at least one outlet port, and a liquid jacket configured to provide heat exchange between the vessel and its contents; a step of introducing a plurality of silk inputs into the reaction vessel through at least one inlet port; a step of introducing a solvent into the reaction vessel through at least one inlet port; a step of introducing a first compound into the reaction vessel through at least one inlet port; a step of heating the contents of the reaction vessel to a temperature of about 50°C to about 150°C through the liquid jacket to promote degumming of the silk inputs; a step of controlling the movement or positioning of the silk inputs within the reaction vessel; a step of removing at least a portion of the solvent and any degumming residues through at least one outlet port; a step of rinsing the degummed silk inputs; a step of introducing a second compound into the reaction vessel through at least one inlet port to dissolve the remaining silk fibroin protein to form a silk fibroin-based solution; a step of stirring the contents of the reaction vessel; a step of outputting the silk fibroin-based solution containing the second compound to a filtration module through at least one outlet port; a step of filtering the silk fibroin-based solution containing the second compound to substantially remove the second compound and produce a purified silk fibroin-based solution, the filtration module being configured to remove the second compound from the silk fibroin-based solution by circulating the silk fibroin-based solution through the filtration module until it reaches at least about 12 dia capacity from about 1 dia capacity; and a step of powdering the purified silk fibroin-based solution by a spray dryer to obtain silk fibroin in powder form such that the moisture activity level of the powder is less than 0.9. A method for treating silk inputs to obtain food-grade silk fibroin.

[0069] Embodiment 31: A step of providing a reaction vessel configured to extract silk fibroin protein by a degumming process, a rinsing process, and a dissolving process in the reaction vessel, the vessel including at least one inlet port, at least one outlet port, and a liquid jacket configured to provide heat exchange between the vessel and its contents; a step of introducing a plurality of silk inputs into the reaction vessel through at least one inlet port; a step of introducing a solvent into the reaction vessel through at least one inlet port; a step of introducing a first compound into the reaction vessel through at least one inlet port; a step of heating the contents of the reaction vessel to a temperature of about 50°C to about 150°C through the liquid jacket to promote degumming of the silk inputs; a step of controlling the movement or positioning of the silk inputs within the reaction vessel; a step of removing at least a portion of the solvent and any degumming residues through at least one outlet port; a step of rinsing the degummed silk inputs so that the silk inputs are substantially free of sericin; a step of introducing a second compound into the reaction vessel through at least one inlet port to dissolve the remaining silk fibroin protein and form a silk fibroin-based solution; a step of stirring the contents of the reaction vessel; a step of sterilizing the silk fibroin solution to obtain a sterilized silk fibroin-based solution; a step of outputting the sterilized silk fibroin-based solution containing the second compound to a filtration module through at least one outlet port; a step of filtering the silk fibroin-based solution containing the second compound to substantially remove the second compound and produce a purified silk fibroin-based solution, the filtration module being configured to remove the second compound from the silk fibroin-based solution by circulating the silk fibroin-based solution through the filtration module until it reaches at least about 12 diavolumes from about 1 diavolume, and the purified silk fibroin-based solution containing one or more salts or inorganic particles at less than about 650 parts per million (ppm); the moisture activity level of the powder is 0.A method for processing a silk input to obtain a food-grade silk fibroin, comprising the step of pulverizing a purified silk fibroin-based solution by a spray dryer to obtain silk fibroin in powder form so as to be less than 9.

[0070] Embodiment 32: A silk manufacturing system according to any one of Embodiments 28 to 31, or any combination thereof, wherein the silk input is derived from Bombyx mori silkworms.

[0071] Embodiment 33: A silk manufacturing system according to any one of Embodiments 28 to 32, or any combination thereof, wherein the packing density of the silk input in the reaction vessel is between about 1% and about 70%.

[0072] Embodiment 34: A silk manufacturing system according to any one of Embodiments 28 to 33, or any combination thereof, wherein the packing density of the silk input in the reaction vessel is greater than 5%.

[0073] Embodiment 35: A silk manufacturing system according to any one of Embodiments 28 to 34, or any combination thereof, wherein the packing density of the silk input in the reaction vessel is greater than 15%.

[0074] Embodiment 36: A silk manufacturing system according to any one of Embodiments 28 to 35, or any combination thereof, wherein the packing density of the silk input in the reaction vessel is greater than 25%.

[0075] Embodiment 37: A silk manufacturing system according to any one of Embodiments 28 to 36, or any combination thereof, wherein the filtration step includes purifying the silk fibroin-based solution by diafiltration.

[0076] Embodiment 38: A silk manufacturing system according to any one of Embodiments 28 to 37, or any combination thereof, wherein the filtration step includes purifying the silk fibroin-based solution by tangential flow filtration.

[0077] Embodiment 39: The silk manufacturing system according to any one of Embodiments 28 to 38, or any combination thereof, wherein the method further includes a step of performing a sterilization process to obtain a food-grade quality silk fibroin-based solution before the powdering step.

[0078] Embodiment 40: The silk manufacturing system according to any one of Embodiments 28 to 39, or any combination thereof, wherein the sterilization process includes a step of guiding a purified silk fibroin-based solution to a microfiltration module.

[0079] Embodiment 41: The step of guiding a purified silk fibroin-based solution to a microfiltration module includes a step of guiding the purified silk fibroin-based solution through a first microfiltration stage having a pore size between about 0.7 μm and about 5 μm, and a step of guiding the purified silk fibroin-based solution through a second microfiltration stage having a pore size between about 0.05 μm and about 0.8 μm. The silk manufacturing system according to any one of Embodiments 28 to 40, or any combination thereof.

[0080] Embodiment 42: The silk manufacturing system according to any one of Embodiments 28 to 41, or any combination thereof, further including a step of adjusting the temperature of the silk fibroin-based solution during processing.

[0081] Embodiment 43: The silk manufacturing system according to any one of Embodiments 28 to 42, or any combination thereof, further including a post-powdering step including at least one of aggregating the silk fibroin powder, conditioning the silk fibroin powder, testing the silk fibroin powder, or packaging the silk fibroin powder in a container safe for use in, for example, food.

[0082] Embodiment 44: The silk manufacturing system according to any one of Embodiments 28 to 43, or any combination thereof, wherein the filtration step includes using at least one spiral-shaped membrane.

[0083] Embodiment 45: A silk manufacturing system according to any one of Embodiments 28 to 44, or any combination thereof, including at least one inlet port including a first inlet port configured to receive a silk input, a first compound, and a second compound, and a second inlet port configured to receive a solvent, and at least one output port including a first output port configured to output a silk fibroin-based solution, and a second output port configured to output at least a portion of the solvent and any degumming residue.

[0084] Still other aspects, embodiments, and advantages of these exemplary aspects and embodiments are described in detail below. Further, both the above information and the following detailed description are merely examples useful in describing various aspects and embodiments, and are intended to provide an overview or framework for understanding the nature and characteristics of the claimed aspects and embodiments. It should be understood, therefore, that these purposes and other purposes will become apparent by reference to the following description and the accompanying drawings, together with the advantages and features of the present disclosure disclosed herein. Further, it should be understood that the features of the various embodiments described herein are not mutually exclusive and may exist in various combinations and arrangements.

[0085] In the drawings, like reference numerals generally refer to the same parts throughout different figures. Also, the drawings are not necessarily to scale; instead, emphasis is generally placed on showing the principles of the disclosure and is not intended as a definition of the limits of the disclosure. For clarity, not every component is labeled in every drawing. In the following description, various embodiments of the present disclosure are described with reference to the following drawings.

Brief Description of the Drawings

[0086]

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DETAILED DESCRIPTION OF THE INVENTION

[0087] Here, some embodiments of the present disclosure will be further described in detail below with reference to the accompanying drawings, which show some but not all embodiments of the present disclosure. In fact, the various embodiments of the present disclosure can be embodied in many different forms and should not be construed as limited to the embodiments described herein. Rather, these exemplary embodiments are provided so that the present disclosure will be thorough and complete and will fully convey the scope of the disclosure to those skilled in the art.

[0088] Unless otherwise specified or clear from the context, references to first, second, third, etc. should not be construed as meaning a particular order. (Unless otherwise specified or clear from the context) Features described as being on top of another feature may instead be below, and vice versa, and similarly, features described as being to the left of another feature may instead be to the right or vice versa. Also, although this specification may refer to measures of quantity, values, geometric relationships, etc., unless otherwise stated, all or any one or more of these may be absolute or approximate to account for possible acceptable variations, such as due to engineering tolerances.

[0089] The present disclosure relates to systems and methods for improving the production of silk fibroin-based solutions containing silk fibroin from silk cocoons.

[0090] For the present disclosure to be more readily understood, certain terms are first defined. Further definitions of the following terms and other terms are set forth throughout this specification.

[0091] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include the plural referents unless the context clearly dictates otherwise.

[0092] Unless explicitly stated or clear from the context, as used in this specification, the term "or" is inclusive and is understood to cover "and" as well as "or".

[0093] The term "and / or", as used herein, should be construed as a specific disclosure of each of two specified features or components, including or excluding the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include A and B, A or B, A alone, and B alone. Similarly, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments, namely, A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A alone, B alone, and C alone.

[0094] The terms "e.g." and "i.e." as used herein are used merely as examples and are not intended to be limiting, and should not be construed as referring only to those items explicitly listed herein.

[0095] Terms such as "at least one", "or more", "at least", "more than", etc., are understood to include, but not be limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or values greater than those described. Also included are any larger numbers or fractions in between.

[0096] Conversely, the term "no more than" includes each value less than the recited value. In one embodiment, "no more than 100" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0. Also included are any smaller numbers or fractions therebetween.

[0097] Terms such as "a plurality of", "at least two", "two or more", "at least a second", etc. are understood to include, but not be limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more, and any larger number or fraction therebetween is also included.

[0098] Throughout this specification, the word "comprising", or variations such as "comprises" or "comprising", are to be understood to mean that the recited element, integer or step, or group of elements, integers or steps, is included, but is not meant to exclude any other element, integer or step, or group of elements, integers or steps. It should be understood that whenever embodiments are described in this specification in terms of the word "comprising", similar embodiments are also provided which are described, where appropriate, in terms of the terms "consisting of" and / or "consisting essentially of". The term "consisting of" excludes any element, step, or component not specified in the claims. In one embodiment, "consisting of" is defined as closing the claim against including materials other than those recited, except for impurities ordinarily associated therewith. Claims dependent on a "consisting of" claim cannot add an element or step. The term "consisting essentially of" or "essentially consisting of" similarly has the meaning ascribed to it under United States patent law, and this term is open-ended and allows the presence of what is recited, provided the basic or novel features of what is recited are not changed by the presence of more than what is recited, but excludes prior art embodiments.

[0099] Unless otherwise specified or apparent from the context, as used herein, the term "about" refers to a value or composition within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. In one embodiment, "about" or "approximately" may mean within one or more standard deviations, according to the convention in the art. "About" or "approximately" may mean a range of up to 10% (i.e., ±10%). Thus, "about" can be understood to be within a range that is 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the recited value. In one embodiment, about 5 mg can include any amount between 4.5 mg and 5.5 mg. Further, particularly with respect to biological systems or processes, the term may mean up to one order of magnitude or up to five-fold the value. When a particular value or composition is provided in the present disclosure, unless otherwise specified, the meaning of "about" or "approximately" should be assumed to be within the acceptable error range for that particular value or composition.

[0100] Furthermore, when used hereinafter, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features without further limiting possibilities. Accordingly, the features introduced by these terms are optional features and are not intended to limit the claims in any way. As will be recognized by those skilled in the art, the present disclosure can be implemented by using alternative features. Similarly, features introduced by "in one embodiment of the present disclosure" or similar expressions are optional features without any limitation regarding further embodiments of the present disclosure, without any limitation regarding the scope of the present disclosure, and without any limitation regarding the possibility of combining such features with other optional or non-optional features of the present disclosure as such.

[0101] As described herein, any concentration range, ratio range, proportion range or integer range is to be understood to include any integer value within the recited range, and, if necessary, fractions thereof (such as 1 / 10 and 1 / 100 of an integer), unless otherwise specified.

[0102] The units, prefixes, and symbols used herein are provided using the Systeme International de Unites (SI) approved format. Numerical ranges include the numbers defining the range. Further, when multiple of the same component are described, the multiple can be referenced individually (e.g., ##a, ##b, ##c, etc.) or collectively (##).

[0103] Description Figure 1 shows a process 100 for manufacturing a silk fibroin-based solution and obtaining silk fibroin in powder form. Specifically, process 100 includes a degumming process (step 120) for extracting silk fibroin from silkworm cocoons 102, and then a dissolution process (step 140) for the degummed silk fibroin to be dissolved in a heated chaotropic agent solution. Next, process 100 includes exposing the fibroin-based solution to a purification process (step 160) in which the chaotropic agent is removed from the dissolved silk fibroin solution, and finally the silk fibroin solution is dried (step 180) to obtain silk fibroin (e.g., by powdering). The quality of the resulting silk fibroin can be improved by improving the quality of the silk fibroin-based solution.

[0104] During the manufacturing process of the silk fibroin solution, the process can be used to kill microorganisms to reduce turbidity and obtain a silk fibroin solution that meets the desired performance and safety requirements. Excessive turbidity is undesirable in a silk fibroin-based solution because it affects the adhesiveness of coatings made from the silk fibroin-based solution, hinders the barrier-forming properties of the silk fibroin-based solution, and may make the coatings formed from the silk fibroin-based solution appear cloudy or milky white. Therefore, the turbidity needs to be maintained below about 0.800 optical density measured at a wavelength of 600 nm (OD660). Thus, a method that meets these requirements is desirable, for example, it may include the integration of the sterilization steps / substations described herein (see 1110 in Figure 5A and 610a / b in Figure 7).

[0105] Generally, the various systems and subsystems described herein can be interconnected by conventional piping techniques and can include any number and combination of components such as pumps, valves, sensors, gauges, etc. to monitor and control, either manually or automatically, the various systems and processes described herein. The various components are made of materials suitable for the temperature and materials to which they are exposed and can be used with a controller as described herein.

[0106] Figure 2 shows a first subsystem 220 configured to perform a degumming operation, a dissolution operation, and a rinsing operation on a silkworm cocoon 202 to receive the silkworm cocoon 202 and obtain a silk fibroin-based solution. Ideally, the silk fibroin-based solution would be substantially sericin-free after the degumming process. As shown, the subsystem 220 includes a first inlet port 226a configured to receive the silkworm cocoon 202 and one or more components 232 (e.g., soda ash, chaotropic agent), a second inlet port 226b configured to receive a solvent (e.g., water), and a reaction vessel 222 having at least one outlet configured to output a silk fibroin-based solution. The reaction vessel 222 is configured to process the silkworm cocoon into a silk fibroin solution by at least one of degumming, rinsing, and dissolution within a single vessel lined with glass. In an alternative configuration, one or more first subsystems, and specifically one or more reaction vessels, can be provided to suit a particular application. See, for example, FIGS. 7A-7C. In some examples, multiple smaller vessels can be used to optimize the process, for example, by making the heating and cooling of the solution more efficient. In some embodiments, the subsystem 220 includes a heat exchanger 242 for conditioning the solvent prior to introduction into the vessel 222.

[0107] The first processing sub-station 220 also includes a water jacket or an oil jacket 224 disposed around a reaction vessel 222 configured to provide heat exchange (e.g., heating or cooling as needed) with the vessel 222 and its contents. The water jacket or the oil jacket 224 includes a heat exchange circuit 236 including a pump 237 for recirculating a heat transfer medium / cooling medium that is in fluid communication with one or more of steam or a coolant as needed to control the temperature of the contents of the vessel 222, e.g., in fluid communication with two heat exchangers 239a, 239b. The first processing sub-station 220 also has the ability to pressurize its contents. The first processing sub-station is configured to pressurize the contents to pounds per square inch (psi) of from about 0 psi to about 20 psi, from about 0 to about 10 psi, from about 0 to about 5 psi, from about 0.1 psi to about 20 psi, from about 0.1 psi to about 10 psi, and from about 0.1 psi to about 5 psi. The pressure can be applied during any of the steps for obtaining a silk fibroin-based solution, including degumming, rinsing, and dissolution.

[0108] The first processing sub-station 220 also includes a plurality of inputs and outputs 238, 244 for introducing and / or removing solvents, vapors, cooling water, condensates, etc., e.g., into and / or from the vessel 222 and / or the water jacket or the oil jacket 224. For example, in some embodiments, the input 238a is configured to introduce softened water 245 through an inlet 226b to a heat exchanger 242 and then into the vessel 222, the inputs 238b, 238c are configured to introduce steam into the heat exchangers 242 and 239b, respectively, and the input 238d is configured to introduce cooling water into one of the heat exchangers 239a. The outputs 244a, 244b, 244c are configured to remove condensates and cooling water from the heat exchangers 239, 242. In other embodiments, oil can be used instead of water to achieve the same cooling or heating requirements.

[0109] The first processing sub-station 220 may further include an apparatus 234 configured to stir the contents of the reaction vessel 222, such as a mixer, a diaphragm, a magnetic stirrer, an ultrasonic crusher, a liquid jet flow, an air flow, etc. In various embodiments, the stirring apparatus 234 may be disposed proximate to the bottom surface of the reaction vessel 222. In some embodiments, the stirring apparatus 234 is a mixer having an integral shaft and impeller 235. The impeller 235 may be configured for axial flow, radial flow, and / or tangential flow and may be driven in reverse. Further, the impeller 235 may be coated with a material resistant to the adhesion of silk fibers and / or may have a blade surface finish (e.g., a surface roughness below some threshold). The mixer may have an interchangeable impeller, and the impeller may be configured to be suitable for a particular process and have flat blades, curved blades, inclined blades, finger blades, anchor blades, gate blades, ribbon blades, etc. having different shapes, pitches, etc. In some embodiments, the impeller assembly includes a slidable sleeve configured to compress the cocoons and / or remove deposits on the impeller (e.g., extrude or discard the cocoons from the impeller).

[0110] In a further embodiment, the reaction vessel 222 includes a second outlet 228b for removing at least a portion of the solvent and any residue therein (e.g., dissolved sericin) that can be sent to waste, recycled, or reused. The reaction vessel 222 can be sized to have an aspect ratio of height to diameter defined by the workload. The volume of the vessel can vary to suit a particular application (e.g., final yield) and can range from about 0.2 liters to about 150,000 liters, preferably from about 0.5 liters to about 5,000 liters. The contents of the vessel can include a plurality of silkworm cocoons 202 (with or without pretreatment), a solvent 244a (e.g., water), and a compound. The water jacket or oil jacket 224 is configured to heat the contents to about 50°C to about 150°C, preferably from about 85°C to about 125°C. Generally, the number of process cycles, temperature, pH, and other solution characteristics can vary to suit a particular application such as the type of silk source.

[0111] The reaction vessel 222 may also include a handling structure or handling device 230 configured to control the movement and / or position of the silkworm cocoons 202 within the vessel 222 (e.g., to prevent the cocoons from floating). The device 230 may include, for example, a screen or net disposed adjacent to the lower portion of the vessel 222 and configured to separate the silkworm cocoons from the agitator 234, a chute or funnel structure in communication with the first inlet and configured to direct the silkworm cocoons to a specific location within the vessel 222 during their introduction, a recirculation system configured to draw a portion of the solution from the lower portion of the vessel 222, reintroduce the solution to the upper portion of the vessel 222, and / or introduce fresh water to push the silkworm cocoons downward into the solution, a vertically movable sieve (e.g., a perforated plunger or a vented floating lid) disposed within the vessel and configured to "push" any solids within the solution toward the lower portion of the vessel, one or more spray balls, and one or more baffles disposed within the vessel and extending from its inner wall and configured to direct the movement of the solution and the contents therein. In one embodiment, the device 230 includes one or more cages or nets disposed within the vessel 222 to ensure that the silkworm cocoons are spaced apart throughout the vessel 222. For example, the silkworm cocoons may be divided into a plurality of spherical or cubic cages.

[0112] The first processing sub-station 220 and the system generally include one or more valve assemblies 225, inlets 226, and / or outlets 228 (equipped with manual or automatic actuators) configured to control the introduction of and removal from the first processing sub-station and / or reaction vessel 222 of any components such as, for example, silkworm cocoons, compounds, solvents, waste liquids, residues, vapors, cooling water, and the final silk fibroin-based solution. The first processing sub-station 220 and the system generally include at least one sensor 227 configured to sense one or more of the temperature, concentration, flow rate, pH, liquid level, turbidity, particle size, molecular weight, pressure, etc. of the solution, which can be used to control the operation of various processes (with or without human intervention). Once a desired silk fibroin-based solution is obtained, which can be determined manually or via one or more sensed characteristics, the solution is directed to the next sub-station described below (e.g., via pumps, valves, etc. as needed). In various embodiments, the system described herein can include a clean-in-place (CIP) module 246 (e.g., a mobile cart) that is fluidly coupled to the sub-station and can perform maintenance on the sub-station.

[0113] Figure 3 shows a second substation 260 configured to receive a silk fibroin-based solution and filter the solution to remove the chaotropic agent from the solution. As shown, substation 260 includes a holding container 268 and a filtration module 266 that houses at least one membrane. Substation 260 is configured to receive at least one input 261 from a first substation 220 a silk fibroin-based solution containing a chaotropic agent, and at least one output 263 configured to output a purified silk fibroin-based solution with a reduced concentration of the chaotropic agent (i.e., the retained substances 270, 270'), and another outlet 274 configured to output a waste stream such as a filtration product 272 from the filtration module 266. The silk fibroin-based solution containing the chaotropic agent is introduced into the container 268 via an inlet 267a disposed thereon.

[0114] Generally, filtration module 266 is configured to remove the chaotropic agent from the silk fibroin-based solution by diafiltration. In some cases, the flow through the module is tangential to the surface of the membrane. The silk fibroin-based solution may experience levels of concentration that may be beneficial in later operations. The silk fibroin-based solution can be circulated through the filtration module for a period defined by from about 1 dia volume to at least about 12 dia volumes, preferably from about 3 dia volumes to about 10 dia volumes, and more preferably from about 5 dia volumes to about 9 dia volumes. In some cases, the concentration level of the chaotropic agent in the retained substances and / or the pressure drop across the entire filtration module can also be monitored to determine the state of the process. Filtration module 266 can include any number and type of membranes to be suitable for a particular application. In one embodiment, module 266 can include one or more spiral-shaped membranes that can be provided in multiple stages. For example, the silk fibroin-based solution can pass through the filtration module 266 and its various stages in series, in parallel, or both, to be suitable for a particular application.

[0115] The holding container 268 may include one or more inlets 267b, 267c configured to introduce a rinsing solution, such as soft water (feed 264a) or reverse osmosis permeate (feed 264b), into the silk fibroin-based solution during the filtration process. The container 268 further includes an outlet 265 for removing the solution 270 and directing the solution via a pump system 276 to the filtration module 266. The at least partially purified solution 270’ is returned (via inlet 267d) to the holding container 268, where the solution 270’ can be subjected to additional rinsing and circulation through the filtration module 266. The filtration product 272 can be output to waste (with or without further treatment) or recycled if possible. When the solutions 270, 270’ reach the desired level of purification, which is determined manually or automatically, the solutions 270, 270’ are output via a valve device 225 to another processing substation (e.g., sterilization). In some embodiments, the purified solution can be removed from the second processing system 260 via an alternative line 269 and a valve assembly 225 disposed downstream of the filtration module 266. In some embodiments, the solutions 270, 270’ are output directly via the outlet 263 to a fourth processing substation 280, 380 rather than to another substation.

[0116] The second processing substation 260 may also include a heat exchange system 262 that includes any valves, pumps, control devices, etc., as needed to control the temperature of the silk fibroin-based solution during processing. As shown in FIG. 3, the heat exchange circuit 262 is disposed in the return piping to control the temperature of at least the partially purified solution 270’ exiting the filtration module 266. However, the heat exchange circuit 262 may be disposed elsewhere to suit a particular application. The circuit 262 further includes ports 271a, 271b for introducing and / or removing a cooling (or heating) medium.

[0117] In various embodiments of the systems disclosed herein, an optional pre-filtration substation 250 may be disposed between a first processing substation 220 and a second processing substation 260. The pre-filtration substation 250 shown in FIG. 4 may include a transfer pump 252 to assist in the transfer of the silk fibroin-based solution to the second processing substation 260, one or more filtration modules 254 suitable for a particular application, and a heat exchange circuit 256. In some embodiments, the pre-filtration substation may include a valve assembly 225 configured to extract and discard a portion of the silk fibroin-based solution that may contain an excessive amount of contaminants (e.g., sericin), with or without cooling as necessary.

[0118] Figures 5A - 5E are exemplary embodiments of various third processing substations 1110, 1210, 1310, 1410, 1510 for sterilization that may be incorporated into the overall production process of a silk fibroin-based solution containing silk fibroin. These figures do not show all possible embodiments of the system and process and are generally shown in relation to the second processing substations 1160, 1260, 1360, 1460, 1560.

[0119] Generally, one of the main concerns when treating silk fibroin-based solutions is that the process does not adversely affect the silk fibroin-based solution containing silk fibroin or its performance. For example, using a filter with a pore size that is too small can damage the shear-sensitive silk fibroin in the silk fibroin-based solution, which would reduce the barrier-forming properties of the silk fibroin-based solution. In another example, the filter may remove some of the silk fibroin from the solution, modifying the molecular weight (Mw) of the silk fibroin-based solution, for example, making the Mw too high or too low by narrowing the polydispersity index (PDI). As another example, the microfiltration step will reduce the volume of the silk fibroin-based solution that should be limited. The goal of the filtration step is to provide a process that can meet all the requirements described herein without adversely affecting the performance of the silk fibroin-based solution.

[0120] One option for obtaining these results is by the sterilization process of the third processing sub-stations 1110, 1210, 1310, 1410, 1510 described herein. The third processing sub-stations shown herein use microfiltration to address these concerns and can produce a silk fibroin-based solution that is below the tolerance limit for each. Additional sterilization systems and processes are disclosed in U.S. Provisional Patent Application No. 63 / 191,441, filed May 21, 2021, which is hereby incorporated by reference in its entirety. The microfiltration process involves using multiple different types of filters (e.g., spiral, membrane, cartridge, hollow fiber, plate and frame, cartridge with O-ring), materials, membrane structures, pore sizes, etc.), different transmembrane pressure differences, and / or the number and configuration of filters (e.g., two or more filter stages arranged in series, where each filter stage can incorporate multiple filters / membranes in different configurations). Generally, the exact number and arrangement of filter stages and / or filters included in the filter stages can vary to suit a particular application, along with the pore size of the membrane, to address, for example, different flow rates, volumes, target pressure drops, target turbidity levels, sterilization levels, solvents used, etc.

[0121] Figure 5A shows an example of a sterilization process / third processing sub-station 1110 incorporated with a purification process / second processing sub-station 1160. The third sub-station 1110 is arranged downstream of the second sub-station 1160 and includes the use of two pumps 1114, two filter stages 1116, and two holding tanks 1118. As shown, the path of the silk fibroin-based solution after leaving the purification process of the second sub-station 1160 is led to the first pump 1114a, and the first pump 1114a passes the solution through the first filter stage 1116a and into the first holding tank 1118a. The second pump 1114b in fluid communication with the first holding tank 1118a transfers the silk fibroin-based solution through the second filter stage 1116b and into the second holding tank 1118b. The silk fibroin-based solution can be led to another process as required after completing the microfiltration process of the third sub-station 1110.

[0122] Figure 5B shows another example of a sterilization process / third processing sub-station 1210 where the purification process 1260 precedes the sterilization process / third processing sub-station 1210 and includes the use of one pump 1214, two filter stages 1216, and one holding tank 1218. As shown, the path of the silk fibroin-based solution after leaving the purification process of the second sub-station 1260 is led to the single pump 1214, and the pump 1214 passes the silk fibroin-based solution through the first filter stage 1216a and then through the second filter stage 1216b and into the holding tank 1218. Again, the silk fibroin-based solution can be led to another process as required after completing the sterilization process, for example, returning through the third processing sub-station 1210 for a second pass, quality testing, or including powdering.

[0123] FIG. 5C shows yet another example of the sterilization process / third processing sub-station 1310. The sterilization process is incorporated with the purification process / second processing sub-station 1360 and includes two pumps 1314, two filter stages 1316, and one holding tank 1318. As shown, the path of the silk fibroin-based solution is introduced to the first filter stage 1316a via the first pump 1314a and then to the purification process 1360. The silk fibroin-based solution exiting the purification process 1360 is directed to the second pump 1314b, which passes the purified silk fibroin-based solution through the second filter stage 1316b and into the holding tank 1318. In this embodiment, the sterilization process is performed both before and after the purification process and includes the use of two pumps, two filters, and one holding tank.

[0124] FIG. 5D shows yet another example of the sterilization process / third processing sub-station 1410. The sterilization process is performed before the purification process 1460 and includes one pump 1414, two filter stages 1416, and one holding tank 1418. As shown, the path of the silk fibroin-based solution is introduced to the first filter stage and the second filter stages 1416a, 1416b via the pump 1414 and then to the purification process 1460. The silk fibroin-based solution exiting the purification process 1460 is directed to the holding tank 1418. In this embodiment, the sterilization process 1410 occurs before the purification process 1460 and includes the use of one pump, two filters, and one holding tank. However, other quantities of pumps, filter stages, and tanks may be incorporated to suit a particular application.

[0125] Figure 5E shows another example of a sterilization process / third processing sub-station 1510 similar to some of those described above, where the purification process / second processing sub-station 1560 precedes the sterilization process 1510 and includes the use of two pumps 1514, two filter stages 1516, one holding tank 1518, and one auxiliary device 1512. As shown, the path of the silk fibroin-based solution after it leaves the purification process 1560 is directed to a first pump 1514a, which first passes the solution through and into a heat exchange module 1512 that can be used to heat and / or cool the silk fibroin-based solution prior to its introduction into the first filter stage 1516a. However, the heat exchange module 1512 would be placed after the first or second filter stage 1516a, 1516b and / or before the holding tank 1518. A second pump 1514b is in fluid communication with the first filter stage 1516a and transfers the silk fibroin-based solution through and into the holding tank 1518 through the second filter stage 1516b.

[0126] In other embodiments, different numbers and configurations (e.g., in series or parallel) of filter stages may be used. Multiple tanks or pumps may also be used to obtain the desired throughput of the filter and appropriate pressure to achieve optimal filtration. Further, the tank may include structures for further treating the silk fibroin-based solution to reduce turbidity and / or further reduce microorganisms, such as by adjusting the solution composition.

[0127] Figures 6A and 6B show alternative fourth processing substations 280, 380 for pulverizing a sterilized silk fibroin-based solution. As shown in Figure 6A, the fourth substation 280 includes a dryer feed mixer container 282 and a spray dryer 288 that are in fluid communication with a plurality of inputs and outputs 284 (e.g., soft water 284a, compressed air 284b, exhaust 284c, cooling water in 284d, and cooling water out 284e). Generally, the fourth processing substation 280 includes an input 281 configured to receive a purified and / or sterilized silk fibroin-based solution and an output 283 configured to output a silk fibroin powder that is readily instantiable. In some embodiments, the water activity level of the silk fibroin powder may be from about 0.01 to about 1.0, preferably less than 0.85. The dryer feed mixer container 282 holds the silk fibroin-based solution prior to drying and may be configured to process the silk fibroin-based solution prior to drying, for example, to enhance pulverization or to produce a more instantiable powder. The silk fibroin-based solution is transferred from the container 282 to the spray dryer 288 via a pump system 286. In some embodiments, silk fibroin-based solutions from different batches with different molecular weight profiles (e.g., lower molecular weight silk fibroin may be added to higher molecular weight silk fibroin) may be mixed together in a supply tank prior to pulverization. In some embodiments, additives will be added to the supply tank prior to pulverization. These additives may be any of the additives known to those skilled in the art, including kosmotropic components, wetting agents, anti-caking agents, anti-foaming agents, oils, sugars, desiccants, catalysts, or those shown in U.S. Patent Publication No. 2020-0178576A1, which is incorporated herein by reference.

[0128] The fourth processing sub-station 380 shown in FIG. 6B is substantially the same as the sub-station 280 of FIG. 6A as long as the sub-station 380 is in fluid communication with a plurality of inputs and outputs 384, receives a purified and / or sterilized silk fibroin-based solution via input 381, and outputs silk fibroin powder via output 383, including a dryer-feed mixer vessel 382 and a spray dryer 388 configured to do so. The fourth processing sub-station 380 includes additional equipment located downstream of or incorporated into the spray dryer 388. Specifically, the sub-station 380 includes an apparatus 391 for providing additives (if any) to the silk fibroin powder or, alternatively, for conditioning the powder, for example for performance enhancement. In one embodiment, the apparatus 391 is an agglomeration apparatus such as an external fluidized bed or a fluidized bed integrated with a powdering apparatus. The agglomeration apparatus may assist in the agglomeration of the powdered silk fibroin protein, thereby improving the dispersibility, instantability, and wettability of the powdered silk fibroin protein. Any suitable agglomeration apparatus may be utilized. The sub-station also includes a packaging apparatus 393 for appropriately packaging the silk fibroin powder.

[0129] FIGS. 7 and 8 show alternatives to the system and process for manufacturing a silk fibroin solution and obtaining silk fibroin powder therefrom.

[0130] Generally, FIGS. 7A - 7D illustrate alternative systems / processes 600, 600', 600'', 600''' for producing a silk fibroin solution. The system 600 of FIG. 7A includes an optional pretreatment sub - station 605 for pretreating silk silkworms (e.g., shredding, immersion, removal of pupae (e.g., sieving, vibrating sieve, etc.)), an optional heat - exchange sub - station 612a for adjusting the solution, and a first processing sub - station 620 downstream thereof for performing a degumming process, a rinsing process, and a dissolution process to obtain a silk - fibroin - based solution. In some embodiments, the pretreatment sub - station 605 may include a continuous immersion process in which the silk cocoons are immersed in a heated solution (e.g., water containing a first compound), and then supplied to a reaction vessel with or without dehydration. Further, during processing in the first processing sub - station 620, the reaction vessel may be drained, filled, and reheated at various stages of the process (e.g., at intermediate points) and / or of the multiple degumming processes being performed.

[0131] Downstream of and in fluid communication with the first sub - station 620 is a first sterilization module 610a of a third processing sub - station for treating the silk - fibroin - based solution before the solution is purified in a second processing sub - station 660. The second processing sub - station 660 may include an optional heat - exchange sub - station 612b. Next, the solution is directed to a second sterilization module 610b of the third processing sub - station, and then the sterilized silk - fibroin - based solution is transferred to a fourth processing sub - station 680 for powdering the silk - fibroin - based solution. The silk fibroin powder may then be directed to an optional post - treatment sub - station 615 for additional processing and / or packaging. The systems and processes described herein may include additional or different processing sub - stations as needed to be suitable for a particular application.

[0132] The systems 600', 600'' of FIGS. 7B and 7C are similar to the system 600 described above, as long as they include an optional pretreatment subsystem 605', 605'', one or more first processing subsystems 620', 620'', one or more sterilization modules 610', 610' (i.e., the third processing subsystem), a second processing subsystem 660', 660'' for purifying the solution, a fourth processing subsystem 680', 680'' for powdering the silk fibroin-based solution, and an optional post-treatment subsystem 615', 615''. Specifically, the system 600' shown in FIG. 7B incorporates a plurality of first processing subsystems (or a plurality of DRD vessels) 620a', 620b', 620c' arranged in parallel. For example, a plurality of smaller subsystems can be used in parallel to accelerate production and / or accommodate the installation area of a particular plant. The system 600'' shown in FIG. 7C also includes a plurality of first processing subsystems (or a plurality of DRD vessels) 620a'', 620''b, but they are arranged in series. In some embodiments, the system 600''' shown in FIG. 7D is used. The system 600''' includes one or more first processing subsystems 620''' for performing a degumming process, a rinsing process, and a decomposition process to obtain a silk fibroin solution, one or more second processing subsystems 660''' for purifying the solution, and one or more fourth processing subsystems 680''' for powdering the silk fibroin-based solution. Generally, the specific number and arrangement of the first processing subsystems 620, 620', 620'', 620''' can vary to suit a particular application.

[0133] FIG. 8 shows a system / process 700 for producing a silk fibroin solution, including a first processing substation 720, a second processing substation 760 disposed downstream of the first substation, a third processing substation 710 disposed downstream of the second substation, and a fourth processing substation 780 disposed downstream of the third substation. The various substations are configured such that the first substation 720 receives a plurality of cocoons, a solvent, and one or more compounds for processing to obtain a silk fibroin-based solution, the second substation is configured to filter the silk fibroin-based solution to produce a substantially purified silk fibroin-based solution by substantially removing one or more compounds, the third substation receives the purified silk fibroin-based solution and is configured to sterilize the purified silk fibroin-based solution to obtain a silk fibroin-based solution of "food grade" quality, and the fourth substation is similar to the substations described herein insofar as it is configured to powder the purified and sterilized silk fibroin-based solution to obtain silk fibroin in powder form. Many modifications and other embodiments of the present disclosure will come to mind to those of ordinary skill in the art to which this disclosure pertains, having the benefit of the teachings presented in the foregoing description and the related drawings. Accordingly, it is to be understood that the present disclosure should not be limited to the specific embodiments disclosed herein, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0134] Figures 9A - 9C show different configurations (plan and front views) of screens 930, 930', 930'' that can be integrated into the systems disclosed herein, for example, to assist in controlling the movement of cocoons, for example, generally within a first processing sub - station or specifically within a reaction vessel. The screen 930 shown in FIG. 9A includes a ring 988 having a conical basket 990 extending from the ring 988. The basket 990 includes a plurality of perforations 992 (e.g., liquid, perhaps along with other components that are generally smaller than the perforations and are usually considered desirable and / or negligible) that allow flow therethrough. Generally, the ring 988 can be configured to provide a joint surface to the device (e.g., attachment to the bottom of the container via a flange assembly), support for the basket 990, and / or means for handling the screen 930. The screen 930 shown in FIG. 9A includes an optional handle 994. The screen 930' shown in FIG. 9B also includes a ring 988' and a basket 990' extending from the ring 988'. The basket 990' generally has a tapered or frustoconical shape (also known as a pilgrim's cap) and also includes a plurality of perforations 992' formed therein. The screen 930'' shown in FIG. 9C also includes a ring 988'' and a basket 990'' extending from the ring 988''. The basket 990'' generally has a cylindrical or rectangular shape and includes a plurality of openings 992'' formed therein. In some embodiments, the basket 990'' can be constructed from a woven mesh screen with wire size, spacing, % free space, etc., selected to be suitable for a particular application disclosed below.

[0135] Generally, screens 930, 930', 930'' can be used to ensure that undesirable aspects of the fibroin solution, including silk fibroin (including fragments) and fragments contained within the silkworm cocoons (e.g., silkworms, organic matter (plant materials, soil, etc.), inorganic matter (packaging, strings, etc.)), do not flow through the outlets (928a in FIG. 10 and 1028a in FIG. 11). It should be understood that the size, shape (cylindrical, rectangular, bowl, etc.), type of perforation, opening size, free space, and distribution (e.g., whether perforations 992, 992', 992'' are arranged across the surface of the basket), and the material of the screen (e.g., stainless steel, polymer, etc.) can be varied depending on the desired location and use of the screen, as well as whether the screens 930, 930', 930'' are to be cleaned during processing. For example, as shown in FIGS. 10 and 11, screens with larger perforations can be used in parallel with screens having smaller perforations to remove different materials at different stages of the process and at different locations along the flow path.

[0136] FIG. 10 shows one embodiment of a first processing sub-station 920 (with or without a stirring device 934), where a single screen 930 is disposed at the bottom of the reaction vessel 922 immediately before the outlet 928a. FIG. 11 shows another embodiment of a first processing sub-station 1020 (with or without a stirring device 1034) in which two screens 1030', 1030'' are utilized. Specifically, the first screen 1030' is disposed at the bottom of the reaction vessel 1022 immediately before the outlet 1028a, while the second screen 1030'' is fixed within the pipe exiting the vessel 1020 and disposed downstream of the outlet 1028a, for example, via a pair of tri-clamps 1096 or a similar mechanism that allows for easy removal of the screen 930'' for cleaning or replacement. Generally, the screens 930, 930', 930'' can be fixed via flanges (preferably with gaskets), screw connections, or other suitable means. Additionally, one or more valves can be incorporated to separate the screens and / or for easy removal from the system. As described above, the screens 930', 930'' can have different perforation patterns, shapes, etc. to suit a particular application. For example, the screen 1030' disposed within the vessel 1022 can include a coarse mesh that clogs less frequently, requires less cleaning, and provides faster drainage, while the screen 1030'' disposed downstream of the vessel can include a finer mesh but is easily removable for cleaning or replacement. Generally, the screen arrangement can be selected to suit the appropriate application (e.g., ease of insertion and removal, fit within the vessel, mounting configuration, etc.).

Claims

1. A silk manufacturing system, comprising: (A) A first processing sub-station comprising a container configured to receive a silk input, extract silk fibroin protein from the silk input, and generate the silk fibroin-based solution such that the silk fibroin-based solution does not contain sericin, wherein the first processing sub-station is configured to extract the silk fibroin protein by a degumming process, a rinsing process, and a dissolving process within a single container; (B) A second processing sub-station in fluid communication with the first processing sub-station, wherein the second processing sub-station is configured to receive the silk fibroin-based solution from the first processing sub-station and purify it, and the purified silk fibroin-based solution contains one or more salts or non-organic particles less than 650 ppm; (C) The first processing sub-station and the second processing sub-station are configured to sterilize the silk fibroin-based solution so as to generate a sterilized silk fibroin-based solution before the third processing sub-station; (D) A third processing sub-station in fluid communication with the second processing sub-station, wherein the third processing sub-station is a spray dryer configured to receive the sterilized silk fibroin-based solution and powder it. The silk manufacturing system comprising the above.

2. The purified silk fibroin-based solution contains one or more salts or non-organic particles less than 400 ppm, and / or The powdered silk fibroin has a water activity level less than 0.9, and / or The silk input is derived from Bombyx mori silkworms, and / or The second processing sub-station is configured to purify the silk fibroin-based solution by diafiltration, and / or The system according to claim 1, wherein the second processing sub-station is configured to purify the silk fibroin-based solution by tangential flow filtration.

3. (i) a post-treatment system configured to perform at least one of receiving silk fibroin powder from the third processing sub-station, conditioning the silk fibroin powder, testing the silk fibroin powder, or packaging the silk fibroin powder in a food-safe container; (ii) a reservoir disposed between the first processing sub-station and the second processing sub-station and configured to perform at least one of holding or conditioning the silk fibroin-based solution; (iii) a pump assembly disposed between the first processing sub-station and the second processing sub-station and configured to transfer the silk fibroin-based solution between the first processing sub-station, the reservoir, and the second processing sub-station; comprising at least one of: (iv) the second processing sub-station includes at least one spiral filtration membrane, or (v) a heat exchange system configured to adjust the temperature of the silk fibroin-based solution before or after any one of the processing sub-stations, the system according to claim 1 or 2.

4. The system according to claim 1, wherein the third processing sub-station includes a single flocculation device.

5. A silk manufacturing system, comprising: (A) a first processing sub-station comprising a container configured to receive a silk input, extract silk fibroin protein from the silk input, and generate a silk fibroin-based solution, the first processing sub-station being configured to extract the silk fibroin protein by a degumming process, a rinsing process, and a dissolving process in a single container; (B) A second processing sub-station in fluid communication with the first processing sub-station, the second processing sub-station configured to receive and purify the silk fibroin-based solution from the first processing sub-station, (C) A third processing sub-station in fluid communication with the second processing sub-station, the third processing sub-station configured to receive and sterilize the purified silk fibroin-based solution, (D) A fourth processing sub-station in fluid communication with the third processing sub-station, the fourth processing sub-station configured to receive the purified silk fibroin-based solution and powder it, and being a spray dryer, comprising the third processing sub-station is configured to sterilize the purified silk fibroin-based solution to food grade standards by microfiltration module, and / or the third processing sub-station is configured to sterilize the purified silk fibroin-based solution to food grade standards by pasteurization, said silk manufacturing system.

6. A reaction vessel including a first inlet port configured to receive a raw silk input and one or more compounds, a second inlet port configured to receive a solvent, and at least one outlet configured to output the silk fibroin-based solution, the reaction vessel configured to process the silk input by degumming, rinsing, and dissolving the silk fibroin protein from the silk input, A liquid jacket disposed around the reaction vessel and configured to provide heat exchange with the vessel and its contents, the liquid jacket configured to heat the contents to a temperature of 50°C to 150°C, A stirrer mechanism configured to stir the contents of the reaction vessel and comprising the first processing sub-station, A filtration module for containing at least one membrane, the module comprising an inlet configured to receive the silk fibroin-based solution containing the compound, an outlet configured to output the purified silk fibroin solution with a reduced amount of the compound, and a waste port configured to output a portion of the compound, and the silk fibroin-based solution is circulated through the filtration module until it reaches at least 12 dia volumes from 1 dia volume, so as to remove the compound from the silk fibroin-based solution, the filtration module comprising the second treatment substation, The silk manufacturing system according to any one of claims 1 to 5, comprising

7. The microfiltration module includes two filter stages, the first filter stage has a pore size between 0.7 μm and 5 μm, the second filter stage has a pore size between 0.05 μm and 0.8 μm, and the silk fibroin-based solution passes through the first filter stage before passing through the second filter stage, The microfiltration module further comprises one or more pumps configured to transfer the silk fibroin-based solution between filter stages, between treatment substations, and to another process if necessary after completing a microfiltration process or any combination thereof, and / or The microfiltration module further comprises one or more holding tanks, and the tanks can be configured to provide additional treatment including one or more of storage of the solution, temperature control of the solution, or adjustment of the solution concentration to address turbidity or sterility levels. The system according to claim 5

8. A method for treating silk inputs to obtain food-grade silk fibroin, comprising introducing a plurality of silk inputs into a single reaction vessel, introducing a solvent into the single reaction vessel, introducing a first compound into the single reaction vessel, To promote the degumming of the silk input, introducing heat into the contents of the single reaction vessel; rinsing the degummed silk input; introducing a second compound into the single reaction vessel to dissolve any remaining silk fibroin protein in solution; stirring the contents of the single reaction vessel; filtering the contents of the reaction vessel to remove the second compound and produce a purified silk fibroin-based solution; powdering the purified silk fibroin-based solution to obtain the purified silk fibroin in powder form The method comprising. **Claim 9** wherein the silk input is derived from Bombyx mori silkworms and / or the packing density of the silk input in the reaction vessel is greater than 25% and / or the filtering step comprises purifying the silk fibroin-based solution by at least one diafiltration or by tangential flow filtration and / or the method further comprises performing a sterilization process to obtain a food-grade quality silk fibroin-based solution prior to the powdering step, further comprising adjusting the temperature of the silk fibroin-based solution during processing and / or the method according to claim 8, further comprising at least one post-powdering step including aggregating the silk fibroin powder, conditioning the silk fibroin powder, testing the silk fibroin powder, or packaging the silk fibroin powder in a container safe for use in food. **Claim 10** controlling the movement or positioning of the silk input within the reaction vessel and / or removing at least a portion of the solvent and any degumming residues and / or The filtration step includes using at least one spiral-shaped membrane. The sterilization process includes the step of guiding the purified silk fibroin-based solution to a microfiltration module. The step of guiding the purified silk fibroin-based solution to a microfiltration module includes: guiding the purified silk fibroin-based solution through a first microfiltration stage having a pore size between 0.7 μm and 5 μm; guiding the purified silk fibroin-based solution through a second microfiltration stage having a pore size between 0.05 μm and 0.8 μm; The method according to claim 9, comprising:

11. A method for treating a silk input to obtain food-grade silk fibroin, comprising: providing the reaction vessel configured to extract silk fibroin protein by a degumming process, a rinsing process, and a dissolving process in the reaction vessel, the vessel including at least one inlet port, at least one outlet port, and a liquid jacket configured to provide heat exchange between the vessel and its contents; introducing a plurality of silk inputs into the reaction vessel through the at least one inlet port; introducing a solvent into the reaction vessel through the at least one inlet port; introducing a first compound into the reaction vessel through the at least one inlet port; heating the contents of the reaction vessel to a temperature of 50°C to 150°C through the liquid jacket to promote degumming of the silk input; removing at least a portion of the solvent and any degumming residues through the at least one outlet port; rinsing the degummed silk input; Introducing a second compound into the reaction vessel via the at least one inlet port to dissolve the remaining silk fibroin protein and form a silk fibroin-based solution; Stirring the contents of the reaction vessel; Outputting the silk fibroin-based solution containing the second compound to a filtration module via the at least one outlet port; Filtering the silk fibroin-based solution containing the second compound to remove the second compound and produce a purified silk fibroin-based solution, wherein the filtration module is configured to remove the second compound from the silk fibroin-based solution by circulating the silk fibroin-based solution through the filtration module until it reaches at least 12 diavolumes from 1 diavolume; Powdering the purified silk fibroin-based solution using a spray dryer to obtain the silk fibroin in powder form such that the moisture activity level of the powder is less than 0.9; The method comprising the above steps.

12. The at least one inlet port comprises a first inlet port configured to receive the silk input, the first compound, and the second compound, and a second inlet port configured to receive the solvent; The at least one output port comprises a first output port configured to output the silk fibroin-based solution, and a second output port configured to output at least a portion of the solvent and any degumming residue; The method according to claim 11.

13. Controlling the movement or positioning of the silk input within the reaction vessel and / or Washing the degummed silk input such that the silk input does not contain sericin and / or Sterilizing the silk fibroin solution to obtain a sterilized silk fibroin-based solution, and / or Outputting the sterilized silk fibroin-based solution containing the second compound to a filtration module via the at least one outlet port, and / or Filtering such that the purified silk fibroin-based solution contains less than 650 ppm of one or more salts or non-organic particles, The method according to claim 11 or 12, further comprising.

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