WO2019004941A1

WO2019004941A1 - A method of producing modified silk from silkworm and a feed composition for producing the same

Info

Publication number: WO2019004941A1
Application number: PCT/SG2018/050316
Authority: WO
Inventors: Leng Duei KOH; Si Yin Tee; Choon Peng TENG; Michelle Dela Cruz REGULACIO; Yin Win Khin; Xian Jun Loh; Yuan Cheng; Yong Wei Zhang; Ming-yong Han
Original assignee: Agency For Science, Technology And Research
Priority date: 2017-06-29
Filing date: 2018-06-28
Publication date: 2019-01-03
Also published as: SG11201913550QA; JP2020525040A

Abstract

According to the present disclosure, a method of producing a modified silk from a silkworm is provided. The method comprises heating a mixture comprising mulberry leaf powder and water, mixing the mixture with a protic organic compound after heating the mixture to form a feed composition comprising the protic organic compound, feeding the silkworm with the feed composition to generate the modified silk, and extracting the modified silk from the silkworm. A modified silk produced by the method as described above is also provided in the present disclosure. A feed composition for producing the modified silk from a silkworm is further provided in the present disclosure. The modified silk comprises crystallites and each of the crystallites has a length of not more than 7 nm. The protic organic compound in the feed composition is less than 1 wt% of the mulberry leaf powder in the feed composition.

Description

A METHOD OF PRODUCING MODIFIED SILK FROM SILKWORM AND A FEED COMPOSITION FOR PRODUCING THE SAME

Cross-Reference To Related Application

[0001] This application claims the benefit of priority of Singapore Patent Application No. 10201705349U, filed 29 June 2017, the content of it being hereby incorporated by reference in its entirety for all purposes.

Technical Field

[0002] The present disclosure relates to a method of producing a modified silk from a silkworm, and a modified silk produced by such a method. The present disclosure also relates to a feed composition for producing the modified silk from silkworm.

Background

[0003] Bombyx mori (B. mori) silk is a natural fibrous material produced worldwide by silkworms on an extremely large scale (120,000 tons per year). Today, B. mori silk may be the most widely used type of silk in wide-ranging and ever expanding applications that span across various industries (e.g. fashion textiles, biomedical/healthcare, consumer care electronics, sporting goods, automotive, and food industries). This is because the core filament protein of silk, which is fibroin, exhibits numerous attractive features including shine/luster, superior strength/toughness, biocompatibility, thermal stability, long-term biodegradability, etc.

[0004] The structure of fibroin may be represented by discrete β-sheet crystallites (hereinafter referred to as crystallites) embedded in a matrix of amorphous chains. Silkworm's silk contains crystallites with length of about 10 nm or more, and such crystallines are typically longer than those of spider's silk (6-6.5 nm).

[0005] Various approaches have been explored to tune or change the structures in silk, in order to alter the mechanical properties of silk. For instance, artificial spinning of silk (i.e. pulling silk from the spinneret of an insect at a controlled drawing rate) did not significantly change the length of crystallites (i.e. 11.49 nm for the artificially spun silk compared to 1 1 .65 nm for the silk used as the control). In another instance, modulated spinning of silk (i.e. allowing the insect to be spun in an electric field) also failed to change the length of crystallites (i.e. 20.2 nm for the modulated silk, 20.4 nm for the silk used as control). In addition, prior molecular modelling and simulation studies reported that crystallite length (i.e. along chain axis) in excess of 1-2 nm tends to have no effect on the mechanical properties of the silk.

[0006] There is thus a need to provide for a method of producing silk that at least ameliorates one or more of the above limitations. The silk produced from such a method should have reduced crystallite length and enhanced mechanical properties.

Summary

[0007] In a first aspect, there is provided for a method of producing a modified silk from a silkworm, comprising:

heating a mixture comprising mulberry leaf powder and water;

mixing the mixture with a protic organic compound after heating the mixture to form a feed composition comprising the protic organic compound, wherein the protic organic compound is less than 1 wt% of the mulberry leaf powder;

feeding the silkworm with the feed composition to generate the modified silk; and

extracting the modified silk from the silkworm, wherein the modified silk comprises crystallites and each of the crystallites has a length of not more than 7 nm.

[0008] In a second aspect, there is provided for a modified silk produced by the method according to the first aspect.

[0009] In a third aspect, there is provided a feed composition for producing a modified silk from a silkworm, wherein the modified silk comprises crystallites and each of the crystallites has a length of not more than 7 nm, the feed composition comprising:

mulberry leaf powder;

water; and

a protic organic compound which induces a change in each of the crystallites to have the length of not more than 7 nm for the modified silk, wherein the protic organic compound is less than 1 wt% of the mulberry leaf powder. Brief Description of the Drawings

[0010] The drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the invention. In the following description, various embodiments of the present disclosure are described with reference to the following drawings, in which:

[0011] FIG. la illustrates the mechanical properties of CA silk against control silk. CA silk represents silk obtained from silkworms fed with a feed modified to contain citric acid (CA). The modified feed in this instance contains CA at 0.05 wt% of mulberry leaf powder. Control silk represents silk obtained from silkworm fed with mulberry leaf powder without CA. Specifically, FIG. la shows the stress-strain curves of CA silk and control silk.

[0012] FIG. lb illustrates the mechanical properties of CA silk against control silk. The modified feed, for obtaining the CA silk in this instance, contains CA at 0.05 wt% of mulberry leaf powder. Specifically, FIG. lb shows the tensile mechanical properties of CA silk against control silk. Data are presented as average ± standard deviation. Statistical significance was performed using one-way analysis of variance (ANOVA), where '*' represents p<0.0l, N=5.

[0013] FIG. 2a depicts a X-ray diffraction (XRD) of control silk (left image) and the respective diffraction spectra (right image) along the meridian axis, curve-fitted into crystalline and amorphous component peaks in solid and dotted lines, respectively. The green (002) peak (represented by the shaded area in the right image) in the meridian spectra provides information on the crystallite length along the β-chain axis. The XRD curves were offset by 250 in intensity after curve fitting for easy comparison.

[0014] FIG. 2b depicts a X-ray diffraction (XRD) of CA silk (left image) and the respective diffraction spectra (right image) along the meridian axis, curve-fitted into crystalline and amorphous component peaks in solid and dotted lines, respectively. The green (002) peak (represented by the shaded area in the right image) in the meridian spectra provides information on the crystallite length along the β-chain axis. The XRD curves were offset by 250 in intensity after curve fitting for easy comparison. The CA silk in this instance is obtained using the modified feed containing CA at 0.05 wt% of mulberry leaf powder. [0015] FIG. 2c is a schematic illustration of a β-sheet crystallite, in which a is the direction along the stacking of β-sheets (i.e. crystallite thickness), b is the direction in a β-sheet perpendicular to the β-chain axis (i.e. crystallite width), and c is the direction along the β-chain axis (i.e. crystallite length).

[0016] FIG. 2d is a tabulation of crystallite dimensions of the associated component diffraction peaks in a, b and c directions, for control silk and CA silk. The CA silk in this instance is obtained using the modified feed containing CA at 0.05 wt% of mulberry leaf powder.

[0017] FIG. 2e is a tabulation of crystallite dimensions, calculated from the full-width at half-maximum of the associated component diffraction peaks in a, b and c directions, for control silk and CA silk. The CA silk in this instance is obtained using the modified feed containing CA at 0.05 wt% of mulberry leaf powder.

[0018] FIG. 3a relates to molecular dynamics simulations on the interaction of silk fibroin in the absence and/or presence of CA molecules. Specifically, FIG. 3a shows a representative silk fibroin molecule used for simulations according to embodiments disclosed herein. The unshaded regions (i.e. in white color) represent glycine while the shaded regions represent either an alanine, a serine or a tyrosine (i.e. in blue, yellow and green colors, respectively).

[0019] FIG. 3b relates to molecular dynamics simulations on the interaction of silk fibroin in the absence of CA molecules. Specifically, FIG. 3b shows a snapshot of four-chain fibroins in equilibrium at 200 ns of simulations in an explicit water shell in the absence of CA.

[0020] FIG. 3c relates to molecular dynamics simulations on the interaction of silk fibroin in the presence of CA molecules. Specifically, FIG. 3c shows a snapshot of four-chain fibroins in equilibrium at 200 ns of simulations in an explicit water shell in the presence of CA.

[0021] FIG. 3d relates to molecular dynamics simulations on the interaction of silk fibroin in the absence of CA molecules. Specifically, FIG. 3d shows the evolution of secondary structures over time for the silk fibroin simulated in FIG. 3b (the representations of different types of secondary structures in the time-evolution plots are given in the legends). [0022] FIG. 3e relates to molecular dynamics simulations on the interaction of silk fibroin in the presence of CA molecules. Specifically, FIG. 3e shows the evolution of secondary structures over time for the silk fibroin simulated in FIG. 3c (the representations of different types of secondary structures in the time-evolution plots are given in the legends).

[0023J FIG. 3f relates to molecular dynamics simulations on the interaction of silk fibroin in the presence and absence of CA molecules. Specifically, FIG. 3f is a tabulation of the percentages of crystalline and amorphous structures collected during the latter 100 ns of the molecular dynamics simulations in the presence and absence of CA molecules. "4-chains CA" represents simulations carried out with the presence of CA.

[0024] FIG. 4 depicts a summarized flow diagram of the present method.

Detailed Description

[0025] The following detailed description refers to the accompanying drawings that show, by way of illustration, specific details and embodiments in which the invention may be practised.

[0026] Features that are described in the context of an embodiment may correspondingly be applicable to the same or similar features in the other embodiments. Features that are described in the context of an embodiment may correspondingly be applicable to the other embodiments, even if not explicitly described in these other embodiments. Furthermore, additions and/or combinations and/or alternatives as described for a feature in the context of an embodiment may correspondingly be applicable to the same or similar feature in the other embodiments.

[0027] Various embodiments refer in a first aspect to a method of producing a modified silk from a silkworm. The method comprises heating a mixture comprising mulberry leaf powder and water, mixing the mixture with a protic organic compound after heating the mixture to form a feed composition comprising the protic organic compound, wherein the protic organic compound is less than 1 wt% of the mulberry leaf powder, feeding the silkworm with the feed composition to generate the modified silk, and extracting the modified silk from the silkworm, wherein the modified silk comprises crystallites and each of the crystallites has a length of not more than 7 nm.

[0028] Advantageously, the present method produces modified silk with shorter lengths of crystallites, which are more regularly and/or tightly packed, as compared to conventional silk. For example, silk produced by silkworms tend to have crystallites with lengths of about 10 nm or more. In another example, crystallites in silk produced by spiders tend to be 6-6.5 nm. Further advantageously, the modified silk has better mechanical properties over conventional silk. The improved mechanical properties include toughness, tensile strength, ductility, etc. To illustrate, modified silk produced by the present method has a toughness of 225 ± 14 MJ/m³ that is twice that of a conventional silk from a silkworm (121 ± 13 MJ/m³). To explain for the advantages, the background of silk is discussed as follows.

[0029] Silk is typically made of fibroin and sericin. Sericin is the adhesive gum coating the fibroin, thereby keeping the fibroins together. Fibroin, otherwise termed as silk fibroin, serves as the main structure of silk and may be represented by discrete β- sheet crystallites embedded in a matrix of amorphous chains, as already described above. These β-sheet crystallites, which are referred to herein as crystallites, are regions in the silk that exhibit an arrangement having perceptible organization, regularity, or orientation of its constituent elements, such that there is an order to the structure in that part of fibroin. The amorphous chains, on the other hand, are regions that exhibit no readily perceptible organization, regularity, and orientation of its constituent elements.

[0030] The crystallites are naturally formed of amino acid residues, such as alanine, glycine, serine and tyrosine, in which tyrosine is a bulky amino acid residue compared to the other three amino acids. For an amino acid residue to be incorporated into a crystallite, the functional groups of tyrosine, for example, functional groups that are capable of forming hydrogen bonds, need to be accessible for forming one or more hydrogen bonds to bind with neighboring peptide chain(s). Through the present method, however, the bulky tyrosine is prevented from being incorporated into the crystallites and this eliminates bulky tyrosine amino acids from the crystallites. The tyrosine is hindered from participating in the formation of crystallites as the protic organic compound, when present, interacts with the tyrosine to form a complex that is sterically hindered from being incorporated into the crystallites. To briefly illustrate, when citric acid (CA) as the pro tic organic compound is added to a feed composition, and the feed composition is consumed by silkworms, crystallites of the generated modified silk are substantially or completely absent of tyrosine. The citric acid interacts with tyrosine to form the complex, in this instance, a CA-tyrosine complex. The strong interaction between tyrosine and CA limits accessibility of the functional groups as mentioned above, and prevents tyrosine from forming hydrogen bond(s) with neighboring peptide chains. The bulky tyrosine residue is strongly bound to CA with a much stronger binding energy (-36.18 ± 2.61 kJ/mol) compared to the other three smaller amino acid residues (glycine: -7.06 ± 1.65 kJ/mol, alanine: -1 1.31 ± 3.55 kJ/mol, serine: -31.06 ± 3.66 kJ/mol). The stronger binding energy allows for the pro tic organic compound, e.g. citric acid, to form a complex with tyrosine over the other three amino acids. Tyrosine, accordingly, is substantially or completely excluded from being incorporated into crystallites. The complex also sterically hinders tyrosine from being incorporated into the crystallites.

[0031] In various embodiments, the feed composition of the present method may include an amount of the protic organic compound that is less than 1 wt% of the mulberry leaf powder. Including the protic organic compound in the feed composition at an amount that is 1 wt% or more, with respect to the mulberry leaf powder, may produce an adverse effect on the mechanical properties of silk. For example, when a feed composition of the present method contains citric acid that is 1 wt% or more, based on the mulberry leaf powder, the silk mechanically weakens. This is because a high amount (i.e. 1 wt% or more) of protic organic compound, such as citric acid, exerts an undesirable influence on the pH of silkworm's haemolymph (i.e. blood), which in turn affects transfer of the protic organic compound for forming the complex that prevents incorporation of tyrosine into silk. In other words, lesser protic organic compound may be available in vivo for forming the complex when the amount of protic organic compound in the feed composition, based on the mulberry leaf powder, is 1 wt% or more.

[0032] The expression "protic organic compound" as used herein refers to an organic compound that readily donates a proton (H⁺). In this regard, the expression "protic organic acid" would include Bronsted-Lowry acid, i.e. any organic acid that readily gives up a hydrogen ion. The protic organic compound may also be capable of forming one or more hydrogen bonds for forming interaction(s) with tyrosine that have a stronger binding energy over that of alanine, glycine and serine. In this manner, the protic organic compound binds to tyrosine over alanine, glycine and serine, and hinders incorporation of tyrosine into the crystallites. In various instances, the protic organic compound may have one or both of such capabilities. The protic organic compound may include weak organic acids as they tend to undergo partial dissociation, in other words, weak organic acids may have both capabilities.

[0033] With tyrosine substantially or completely eliminated from crystallites, the crystallites become even more compact, and mechanical properties of the modified silk become significantly superior over conventional silks. For instance, the modified silk produced by the present method has a toughness that even surpasses the toughness (i.e. 160-200 MJ/m³) of spider dragline silk.

[0034] Various embodiments refer in a second aspect to a modified silk produced by the method as described according to the first aspect. Various embodiments refer in a third aspect to a feed composition for producing the modified silk from a silkworm.

[0035] The definitions of certain terms are first discussed before going into details of the various embodiments.

[0036] The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.

[0037] In the context of various embodiments, the articles "a", "an" and "the" as used with regard to a feature or element include a reference to one or more of the features or elements.

[0038] In the context of various embodiments, the term "about" or "approximately" as applied to a numeric value encompasses the exact value and a reasonable variance.

[0039] As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

[0040] Unless specified otherwise, the terms "comprising" and "comprise", and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, unrecited elements.

[0041] With the above in mind, details of the various embodiments are now discussed below.

[0042] In the present disclosure, there is provided for a method of producing a modified silk from a silkworm. The method comprises heating a mixture comprising mulberry leaf powder and water, mixing the mixture with a protic organic compound after heating the mixture to form a feed composition comprising the protic organic compound, wherein the protic organic compound is less than 1 wt% of the mulberry leaf powder, feeding the silkworm with the feed composition to generate the modified silk, and extracting the modified silk from the silkworm, wherein the modified silk comprises crystallites and each of the crystallites has a length of not more than 7 nm, not more than 5 nm, not more than 4 nm, not more than 3 nm, not more than 2 nm, not more than 1 nm, etc.

[0043] According to the present method, the mulberry leaf powder and water may be first mixed before heating the mixture of mulberry leaf powder and water. The mixing of mulberry leaf powder and water may also take place with heating simultaneously. Heating of the mulberry leaf powder and water is carried out to form a leaf paste or chow that is convenient for feeding, storage and preservation.

[0044] The mulberry leaf powder and water may be mixed in a weight to volume ratio of 1 :1 to 1 :10, 1 :3 to 1 : 10, 1 :5 to 1 : 10, or 1 :8 to 1 : 10. For example, 100 g of mulberry leaf powder and 300 mL of water may be mixed. In various embodiments, the method may further comprise mixing the mulberry leaf powder and water in a weight to volume ratio of 1 : 1 to 1 :10, 1 :3 to 1 : 10, 1 :5 to 1 : 10, or 1 :8 to 1 : 10 before heating the mixture. In specific embodiments, the method as disclosed herein may further comprise mixing the mulberry leaf powder and water in a weight to volume ratio of 1 :3.

[0045] After mixing or during mixing of the mulberry leaf powder and water, the mixture may be heated. The heating may be carried out by any suitable method, for example, using a hot plate, stove heating, mixing with hot or boiling water, etc. The mulberry leaf powder may normally be dissolved just by mixing with water. Heating, advantageously, may be carried out so that the feed stays fresh throughout the duration of feeding (e.g. the feed stays fresh for a month in the refrigerator after heating). In various embodiments, the heating may be in the form of microwave heating. In various embodiments, the heating may comprise exposing the mixture to microwaves. Microwaves are a form of electromagnetic radiation with wavelengths ranging from 1 m to 1 mm.

[0046] After heating the mixture, the mixture may be mixed with the protic organic compound to form the feed composition. Mixing or addition of the protic organic compound, e.g. citric acid, after heating, avoids thermal decomposition of the protic organic compound. For example, if the mixture is heated above 175°C, citric acid may decompose into carbon dioxide and water. Hence, in various instances, the mixture may be allowed to cool down slightly, for example, to reach a temperature of 150°C before adding the citric acid. This temperature may depend on the protic organic compound used.

[0047] Mixing of the protic organic compound and the mixture may comprise adding the protic organic compound to the mixture. In various embodiments, mixing of the mixture with the protic organic compound may comprise adding less than 1 wt%, such as 0.001 to 0.9 wt%, 0.01 to 0.9 wt%, 0.001 to 0.8 wt%, 0.01 to 0.8 wt%, 0.05 to 0.9 wt%, 0.05 to 0.8 wt%, 0.5 to 0.9 wt%, 0.5 to 0.8 wt%, 0.05 to 0.5 wt%, 0.5 wt%, or 0.05 wt%, etc., of the protic organic compound to the mixture, wherein the wt% may be based on the mulberry leaf powder. In specific embodiments, mixing of the mixture with the protic organic compound may comprise adding 0.05 or 0.5 wt% of the protic organic compound to the mixture, wherein the wt% is based on the mulberry leaf powder. If too much protic organic compound is added to modify the feed, the feed may become unsuitable for silkworm consumption and the modified silk may not be produced. Accordingly, the protic organic compound mixed with the mixture may be less than 1 wt%, such as 0.001 to 0.9 wt%, 0.01 to 0.9 wt%, 0.001 to 0.8 wt%, 0.01 to 0.8 wt%, 0.05 to 0.9 wt%, 0.05 to 0.8 wt%, 0.5 to 0.9 wt%, 0.5 to 0.8 wt%, 0.05 to 0.5 wt%, 0.5 wt%, or 0.05 wt%, etc., of the mulberry leaf powder. As already mentioned above, if the amount of protic organic compound, based on the mulberry leaf powder, in the feed composition is 1 wt% or more, mechanical weakening of silk occurs as the protic organic compound, such as citric acid, exerts an undesirable influence on the pH of silkworm's blood such that transfer of the protic organic compound for forming the complex that prevents incorporation of tyrosine into silk, becomes adversely affected, where lesser protic organic compound may be available in vivo for forming the complex.

[0048] Once the feed composition comprising the protic organic compound is prepared, it may be fed to the silkworms. The silkworms may be B. mori silkworms. The silkworms then generate the modified silk. As explained above, the modified silk generated from silkworms fed with the feed composition modified to contain the protic organic compound, according to the present method, possesses improved mechanical properties that surpass conventional silks, such as spider dragline silk. As already explained above, the presence of the protic organic compound results in the formation of a complex formed from the protic organic compound and the tyrosine. The complex is formed when the protic organic compound interacts with tyrosine, for example, forming one or more hydrogen bonds between the protic organic compound and one or more of the various functional groups of tyrosine. The interaction between the protic organic compound and tyrosine to form the complex may include van der Waals interaction. The binding energy of citric acid and the respective functional groups of tyrosine, for example, can be calculated to demonstrate that the complex is formed through interactions that involve hydrogen bonding and van der Waals interaction (-OH:-12.90 ± 3.53 kJ/mol; benzene ring: -10.52 ± 3.12 kJ/mol; -COOH: - 2.08 ± 3.54 kJ/mol). The formation of the complex sterically hinders tyrosine from being incorporated into the crystallites when the modified silk generates. The complex is also sterically hindered from being incorporated into the crystallites. Without tyrosine, crystallites in the modified silk become shorter in length and more regularly and/or tightly packed, and the modified silk attains the improved mechanical properties, as compared to conventional silk. Accordingly, in various embodiments, generating the modified silk may comprise forming a complex from tyrosine and the protic organic compound which prevents the tyrosine from being incorporated into the crystallites of the modified silk. In some embodiments, generating the modified silk may comprise forming a complex from tyrosine and the protic organic compound which completely prevents all tyrosine from being incorporated into the crystallites of the modified silk. In some other embodiments, generating the modified silk may comprise forming a complex from tyrosine and the protic organic compound which substantially prevents the tyrosine from being incorporated into the crystallites of the modified silk. The term "substantially", used in the present context, means that at least 90%, at least 95%, at least 99%, or even 100%, of the tyrosine in silk is not incorporated into the crystallites.

[0049] In various embodiments, the protic organic compound may be a monoprotic or polyprotic organic compound. Monoprotic organic compound refers to a protic organic compound that donates a single proton and/or has the capability to form one hydrogen bond while polyprotic organic compounds can donate more than one proton and/or have the capability to form more than one hydrogen bond.

[0050] In various embodiments, the protic organic compound may comprise a functional group selected from the group consisting of carboxylic acid group (- COOH), sulfonic acid group (-SO2OH), hydroxyl group (-OH), thiol group (-SH), and a combination thereof. In specific embodiments, the protic organic compound may be an organic acid. The organic acid may be a weak organic acid that undergoes partial dissociation. The organic acid, including weak organic acid, may have the capability to donate one or more protons and/or form one or more hydrogen bonds. Inorganic acids are avoided as they are hazardous acids to silkworm which, if ingested, can lead to a silkworm's death. The organic acid may comprise one or more of the functional groups described above. In various embodiments, the organic acid may be selected from the group consisting of citric acid (HOC(COOH)(CH2COOH)2), acetic acid (CH3COOH), formic acid (HCOOH), benzoic acid (C₆H₅COOH), glycolic acid, oxalic acid, succinic acid, malic acid, malonic acid, nitrilotriacetic acid, and a combination thereof. In specific embodiments, the organic acid may be citric acid.

[0051] Various embodiments in a second aspect refer to a modified silk produced by the method according to the first aspect described above. The modified silk comprises crystallites and each of the crystallites has a length of not more than 7 nm. In other words, the modified silk comprises crystallites which are shorter in length compared to crystallites of conventional silk. Various features of the modified silk have already been described above. Various embodiments of the present method, and advantages associated with various embodiments of the present method, as described above may be applicable to the modified silk, and vice versa. [0052] Crystallites of the modified silk substantially or completely exclude tyrosine, as a protic organic compound (e.g. citric acid) present in the feed composition that is fed to silkworms, interacts with tyrosine to form a complex that prevents tyrosine from being incorporated into the crystallites during formation of the modified silk. In various embodiments, tyrosine is substantially or completely absent from the crystallites of the modified silk. As already described above, without tyrosine, the crystallites become more compact and mechanical properties of the modified silk are improved. The protic organic compound may be present in the feed composition in an amount, based on the mulberry leaf powder, that is already described above in various embodiments that refer to the first aspect.

[0053] Accordingly, the modified silk, in various embodiments of all aspects disclosed herein, comprises crystallites and each of the crystallites has a length of not more than 7 nm, not more than 5 nm, not more than 4 nm, not more than 3 nm, not more than 2 nm, not more than 1 nm, etc. In some embodiments of all aspects disclosed herein, each of the crystallites of the modified silk may have a length of 3 nm to 5 nm, 4 nm to 5 nm, etc. In some embodiments of all aspects disclosed herein, each of the crystallites of the modified silk may have a length of 4.69 nm. The length may be an average length in various embodiments of all aspects disclosed herein.

[0054] Various embodiments in a third aspect refer to a feed composition for producing a modified silk from a silkworm, wherein the modified silk comprises crystallites and each of the crystallites has a length of not more than 7 nm, not more than 5 nm, not more than 4 nm, not more than 3 nm, not more than 2 nm, not more than 1 nm, etc. Features of the feed composition, such as the protic organic compound, have already been described above.

[0055] The feed composition comprises mulberry leaf powder, water, and a protic organic compound. The protic organic compound may be less than 1 wt% of the mulberry leaf powder. Various embodiments of the present method and modified silk, and advantages associated with various embodiments of the present method and modified silk, as described above may be applicable to the feed composition, and vice versa.

[0056] The feed composition is advantageous for producing a modified silk from silkworm. In various embodiments, the feed composition contains a protic organic compound that induces a change in each of the crystallites to have a length of not more than 7 nm for the modified silk. That is to say, the protic organic compound induces a change in each of the crystallites such that each of the crystallites in the modified silk has a length of not more than 7 nm, not more than 5 nm, not more than 4 nm, not more than 3 nm, etc. The crystallites produced from such a feed composition, when compared to crystallites of conventional silk, are advantageously shorter in length and more regularly and/or tightly packed. Hence, the protic organic compound induces the change (decrease) in a crystallite's length and arrangement of the crystallites. The modified modified silk also has improved mechanical properties.

[0057] The protic organic compound of the feed composition forms a complex with tyrosine that is sterically hindered from being incorporated into crystallites of the modified silk generated by a silkworm. The complex also sterically hinders tyrosine from being incorporated into crystallites of the modified silk. Accordingly, the protic organic compound interacts with tyrosine to form a complex which prevents the tyrosine from being incorporated into crystallites of the modified silk in various embodiments. Without tyrosine, crystallites of the modified silk become shorter in length and more regularly and/or tightly packed, and the modified silk attains better mechanical properties. Embodiments regarding the complex have already been described above in the earlier aspects.

[0058] In various embodiments, the protic organic compound may be a monoprotic or polyprotic organic compound. The protic organic compound may comprise a functional group selected from the group consisting of carboxylic acid group (- COOH), sulfonic acid group (-SO2OH), hydroxyl group (-OH), thiol group (-SH), and a combination thereof. The protic organic compound may be an organic acid. The organic acid may comprise one or more of the functional groups described above. In various embodiments, the organic acid may be selected from the group consisting of citric acid (HOC(COOH)(CH₂COOH)₂), acetic acid (CH3COOH), formic acid (HCOOH), benzoic acid (C6H5COOH), glycolic acid, oxalic acid, succinic acid, malic acid, malonic acid, nitrilotriacetic acid, and a combination thereof. The organic acid may be a weak organic acid as already described above. The organic acid, including weak organic acid, may have the capability of donating one or more protons and/or t

15 forming one or more hydrogen bonds as already described above. In specific embodiments, the organic acid may be citric acid.

[0059] In various embodiments, the protic organic compound in the feed composition may be less than 1 wt%, such as 0.001 to 0.9 wt%, 0.01 to 0.9 wt%, 0.001 to 0.8 wt%, 0.01 to 0.8 wt%, 0.05 to 0.9 wt%, 0.05 to 0.8 wt%, 0.5 to 0.9 wt%, 0.5 to 0.8 wt%, 0.05 to 0.5 wt%, 0.5 wt%, or 0.05 wt%, etc., of the mulberry leaf powder. As already mentioned above, if the amount of protic organic compound, based on the mulberry leaf powder, in the feed composition is 1 wt% or more, mechanical weakening of silk occurs as the protic organic compound, such as citric acid, exerts an undesirable influence on the pH of silkworm's blood such that transfer of the protic organic compound for forming the complex that prevents incorporation of tyrosine into silk, becomes adversely affected, where lesser protic organic compound may be available in vivo for forming the complex.

[0060] In various embodiments, the mulberry leaf powder and water may be present in a weight to volume ratio of 1 :1 to 1 : 10, 1 :3 to 1 :10, 1 :5 to 1 : 10, or 1 :8 to 1 : 10. For example, 100 g of mulberry leaf powder may be present with 300 mL of water.

[0061] While the methods described above are illustrated and described as a series of steps or events, it will be appreciated that any ordering of such steps or events are not to be interpreted in a limiting sense. For example, some steps may occur in different orders and/or concurrently with other steps or events apart from those illustrated and/or described herein. In addition, not all illustrated steps may be required to implement one or more aspects or embodiments described herein. Also, one or more of the steps depicted herein may be carried out in one or more separate acts and/or phases.

Examples

[0062] The present disclosure relates to a method of producing a modified silk from a silkworm. The method as disclosed herein involves a feed composition modified to contain a protic organic compound, and feeding that feed composition to silkworms to generate the modified silk. The generated modified silk have crystallites that are more compact and mechanical properties of the modified silk improve significantly. The present disclosure also relates to the feed composition and a modified silk obtained from such a method.

[0063] Generally, citric acid may be used as the protic organic compound as it has stronger interactions with tyrosine to form a complex that prevents tyrosine from being incorporated into the crystallites, resulting in more compact crystallites in the modified silk. Besides citric acid, other protic organic additives including small molecular compounds can be used to greatly shorten the length of crystallites in silk. Any organic acid, including monoprotic or polyprotic organic acids, can be used as long as it can interact strongly with bulky amino acid residues to form the complex that hinders packing of the bulky residue(s) into crystallite(s) when forming the modified silk. Such organic acids can be fed to silkworms without killing them.

[0064] The organic acids may be organic compounds that contain functional groups capable of conferring acidity, and such groups may include but are not limited to carboxylic acid group (-COOH), sulfonic acid group (-S0₂OH), hydroxyl group (- OH), thiol group (-SH), etc. Typical examples of organic acids may include but are not limited to citric acid (HOC(COOH)(CH₂COOH)₂), acetic acid(CH₃COOH), formic acid (HCOOH), benzoic acid (C₆H₅COOH), glycolic acid, oxalic acid, succinic acid, malic acid, malonic acid, nitrilotriacetic acid, etc.

[0065] The concentration of the protic organic additive/compound used to form the feed composition may be less than 1 wt% of the mulberry leaf powder, such as in the range of about 0.001 to about 0.9 wt%, about 0.01 to about 0.9 wt%, about 0.001 to about 0.8 wt%, about 0.01 to about 0.8 wt%, about 0.05 to about 0.9 wt%, about 0.05 to about 0.8 wt%, about 0.5 to about 0.9 wt%, about 0.5 to about 0.8 wt%, about 0.05 to about 0.5 wt%, about 0.01 to about 0.1 wt%, about 0.5 wt%, or about 0.05 wt%, etc,

[0066] The present method, modified silk and feed composition are described in more detail, by way of non-limiting examples, as set forth below.

[0067] Example 1: Silkworm Culturing and Feed Preparation

[0068] Normal feed was prepared by mixing and microwaving mulberry leaf powder (100 g, Recorp Inc., Canada) with water (300 mL). The modified feed was prepared by mixing and microwaving mulberry leaf powder (100 g) with water (300 mL), followed by mixing with citric acid (CA) (50 mg, Fisher) at 0.05 wt% of the leaf powder. [0069] Domesticated silkworms (Bombyx mori, Carolina Biological Supply Company, USA) were cultured using the normal feed up to the second day of the fifth instar. The silkworms were then transitioned to the modified feed and continuously fed using the modified feed until the silkworms started spinning to produce the modified silk. The modified silk may be referred to as CA-modified silk or CA silk in the present disclosure.

[0070] In contrast, control silk was obtained by continuously feeding silkworms the normal feed (i.e. without CA) throughout larval stage.

[0071] The feeding concentration of CA may be varied to achieve the desired mechanical properties. By varying the concentration of CA added to the feed, the length of crystallites in silk was tunable down to 4.69 nm in length (equivalent to about 13 amino acid residues along the β-chain in the β-sheet), which was only about half the length observed in that of control silk (8.84 nm, equivalent to about 25 residues along the β-chain in the β-sheet).

[0072] Example 2a: Characterization of Modified Silk For Mechanical Properties

[0073] To determine mechanical properties of the silk, silk fibers were individually mounted onto paper frames with a 20x5 mm window for tensile tests. After fixing to the two grips on a universal tester (Instron Double Column Universal Tester 5569) equipped with a 5 N load cell (accuracy: ± 0.5%; strain rate: 2 mm min^"1), the paper frame was then cut so that the load was exerted directly on the silk fiber during testing. The tests were carried out under atmospheric air of 60% humidity at room temperature (24°C). For each sample in the tensile test, the gauge length was individually determined from the distance between the two grips at the starting position using Bluehill software on a computer connected to the tester. Slack correction was also made with the software to remove 'toe' region in the obtained stress-strain curves. The toe region of the stress-strain curves is present at the starting part of the curves (in the present instance, it is absent from FIG. 1 as the toe region has already been removed (i.e. toe compensation)). There are two ways to compensate for the toe before or after testing (i.e. preload or slack correction calculation, respectively). The latter was used for the present studies as an approach to correct all the stress-strain curves. It is a standard procedure to carry out toe compensation (i.e. remove toe region).

[0074] To determine cross-sectional area of silk fibers for tensile stress calculation, a segment of the silk was mounted on a specimen holder using carbon tape, sputter- coated with gold under vacuum, and imaged under a scanning electron microscope (SEM) (JEOL LV 6360LA). The smallest diameter along each fiber was used to derive the cross-sectional area of the silk fibroin fiber. Mechanical data were presented as average ± standard deviation. Statistical significance was performed using one-way ANOVA (*p<0.01 ; N=5).

[0075] The mechanical properties, including ultimate tensile strength (UTS), breaking strain, toughness, etc., of silk obtained from silkworms fed with CA were tested and the results are shown in FIG. la and FIG. lb. The results of CA silk for FIG. l a and FIG. lb are obtained using the modified feed having CA at 0.05 wt% of the mulberry leaf powder, and such a CA silk is denoted herein as 0.05 CA silk. In a typical feeding scenario, the feeding of CA (e.g. 0.05 wt% of the mulberry leaf powder) to silkworms resulted in the strong and tough silk, and significant strengthening and toughening effects were also observed in the 0.5 CA silk (obtained by feeding CA at 0.5 wt% of mulberry leaf powder to silkworms). The 0.05 CA silk exhibited a high UTS of 840 ± 38.7 MPa (pO.Ol) as compared to 572 ± 71.1 MPa of the control silk. The 0.05 CA silk also exhibited a high toughness of 225 ± 14 MJ/m³ as compared to 121 ± 13 MJ/m³ of the control silk (pO.01 ). As these enhancements in mechanical properties were accompanied by a significant reduction in the crystallite size (i.e. length of 4.69 nm in 0.05 CA silk vs. 8.84 run in control silk), the enhancement is attributable to the shorter crystallites, which resulted from the feeding of CA.

[0076] Example 2b: Characterization of Modified Silk For Structural Properties

[0077] To determine crystallite dimensions, a bundle of the degummed fibers were mounted on a cardboard frame and the X-ray diffraction (XRD) patterns were then collected on a Bruker AXS X8 Proteum diffractometer using a beam size of 100 μιη and a radiation wavelength of 1 .5418 A (Cu Ka). The sample-to-detector distance was 6 cm and the exposure time was 60 s. Radial integration was then carried out on the obtained XRD patterns along either the equatorial (5-40°) or meridian (5-37.5°) axis. The resultant spectra were fitted into characteristic component peaks of silk fibroin. Crystallite sizes along a, b and c directions were determined from the FWHM (full- width at half-maximum) of the (200) and (120) peaks from the equatorial spectra and the (002) peak from the meridian spectra. Calculations were performed using Scherrer equation, L = 0.9 λ /(FWHM ^χ cos Θ), where L = crystallite size along a, b or c direction, 0.9 = Scherrer's constant, λ = wavelength of incident X-ray (1.5418 A for Cu Ka radiation), Θ = peak position, and FWHM = full-width at half-maximum. Specifically, a is the direction along the stacking of β-sheets (crystallite thickness), b is the direction in a β-sheet perpendicular to the chain axis (crystallite width), and c is the direction along the β-chain axis (crystallite length).

[0078] FIG. 2a and FIG. 2b illustrate the XRD pattern of control silk and CA silk, respectively. In this instance, the CA silk is 0.05 CA silk. A β-sheet crystallite is depicted schematically (FIG. 2c) to show the intersheet (a), interchain (b), and β- chain axis (c) directions, corresponding to the crystallite thickness, width, and length, respectively. The three dimensions of the β-sheet crystallites in control silk and CA silk (tabulated in FIG. 2d) were also measured from the full-width at half-maximum (FWHM) of the associated diffraction peaks in equatorial (i.e. (200) and (120)) and meridian (i.e. (002)) directions (tabulated in FIG. 2e). The XRD results indicated that while crystallites in both silks were similar in terms of thickness and width, they were markedly different in length. The crystallites in CA silk were 4.69 nm in length (equivalent to about 13 amino acid residues along β-chain in β-sheet), which was only about half of the length observed in control silk (8.84 nm, equivalent to about 25 residues along β-chain in β-sheet). Such remarkably short crystallite length is unprecedented in naturally produced silkworm silk, and the reduced length of crystallites in CA silk was even shorter than those observed in spider dragline silks (i.e. 6-6.5 nm). As for the control silk, the crystallite dimensions were comparable to reported values for B, mori silk (e.g. 8.8 nm in length, 3.4 nm in thickness and 3.8 nm in width).

[0079] Example 3: Extraction of Silk Dope and Haemolymph, and Quantification of Citric Acid

[0080] Silkworms were dissected at the end of fifth instar in an Insect Ringer solution containing 128 mM NaCl, 1.8 mM CaCh.2H₂0, 1.3 mM KC1 and 50 mM Tris (trisaminomethane). Silk glands were then removed from the dissected silkworms, washed with distilled water, and their gland epithelial layers peeled off to obtain the silk dope. To remove the sericin in silk dope after drying, the silk dope was added to a degumming solution (5 mL) consisting of Savinase enzyme (5 mg) and Triton-X (5 mg) in 1 g L^'1 NaHC0₃ solution, and then heated at 55°C for 1 hr. The obtained fibroin from each silk dope was dried and used to quantify the content of CA. The dried silk fibroin (3 mg) was dissolved by heating in 100 of 9.3 M LiBr solution at 70°C for 3 hrs. Upon dissolution of the silk fibroin, the obtained solution was analyzed to quantify the amount of CA using spectrophotometric method. To determine the amount of CA in the silk fibroin (N=3), the solution samples (100 μί) were individually added to a mixture of 4 mL of Fe(N0₃)₃ solution and 1 mL of HN0₃, and topped up to 25 mL in a glass vial using distilled water. Upon illumination with a solar simulator at 1000 W/m² (Newport Corporation USA, model 66901) for 10 mins, the solution turned violet due to the photochromism of ferric-citrate complex. The color development was stabilized in shade for 30 mins at 4°C. The peak absorbance of the solution at 520 nm was measured using an UV-Vis (ultraviolet- visible) spectrophotometer (Shimadzu UV-3150). The actual amounts of CA were then calculated by referring to the calibration curve that was prepared using the same procedure with a series of different concentrations of CA. The data were presented as average ± standard deviation. On the other hand, the haemolymph (i.e. blood) was also extracted from silkworms prior to dissection using a needle syringe. The same method was also used to determine the amount of CA in the haemolymph (iV=3).

[0081] Example 4; Molecular Dynamics (MP) Simulation of Interaction of Citric Acid with Fibroin Molecule

[0082] MD simulations were performed to assess the influence of CA on the structure of silk. Energetic analysis confirmed the binding of CA molecules to the silk, which also prevented incorporation of tyrosine, and the simulation results showed that CA molecules were capable of inducing structural changes and altering the structural contents in silk. When the molecular folding was allowed to take place in the presence of CA, the content of β-sheets dropped (FIG. 3f). That is to say, the steric hindrance from the tyrosine-CA complex prevented the extensive formation of β-sheets. Results of the simulation were in line with experimental findings on structural contents in the silk. Detailed methods on the modeling and simulations are elaborated below. [0083] A representative fibroin molecule was first constructed using software package SYBYL 8.0 (Tripos Associates, Inc.) from a typical crystalline sequence (GX repeats, GAGAGSGAGAGAGSGAGAGSGAGAGSGAGAGSGAGSGAGAGSGAGAGSG AGAGYGAGAGSGAAS) and two identical amorphous sequences (GAGAGAGAGAGTGSSGFGPYVANGGYS GYEYAWSSESDFGTGS) at both ends of the crystalline sequence. The complete sequence is GAGAGAGAGAGTGSSGFGPYVANGGYSGYEYAWSSESDFGTGS- GAGAGSGAGAGAGSGAGAGSGAGAGSGAGAGSGAGSGAGAGSGAGAGSG AGAGYGAGAGSGAAS- GAGAGAGAGAGTGS S GFGP YV ANGGYSGYE YA WS SESDFGTGS . These sequences were adopted from the GX 6.5 and Linker 6 of B. mori silk fibroin, respectively. For MD simulations on the interaction of CA with fibroin by using Gromacs 5.0.6, four such fibroin molecules in an unfolded chain configuration were first arranged in opposite directions with an initial distance of 5 A between two neighboring molecules. Assisted Model Building with Energy Refinement (Amber03) force field was then used to parameterize the silk fibroin. An explicit water shell was further implemented to simulate the structure of silk fibroin to mimic the natural folding process of silk fibroin. The water content in the system is about 80 wt% (1 1,527 water molecules in total), similar to experimental data that works with silk having a water content of more than 70%.

[0084] Transferable intermolecular potential with 3 points (TIP3P) water model was adopted in this simulation. There were 86 CA molecules added (calculated based on the measured amount of CA in silk gland). To neutralize the fibroin in the system, 24 counter ions (Na⁺) were added. The molecular structure of CA was constructed using Materials Studio 7.0. The parameters of CA were calculated using the Antechamber module in the Amber 1 1 software based on Amber GAFF (general AMBER force field) force field. The molecular structure of CA was equilibrated through energy minimization using 1,000 steps of the steepest descent method followed by 1 ,000 steps of conjugate gradient minimization using Gromacs.

[0085] A simulation box size of 20 nm ^χ 20 nm ^χ 53 nm was adopted using the NVT ensemble at a temperature of 300 K. To simulate the interaction of CA with fibroin in this system, energy minimization was carried out first, using 1,000 steps of the steepest descent method followed by 1,000 steps of conjugate gradient minimization using Gromacs. Two equilibrations were then performed using Gromacs: an initial 1 ns simulation with a restraining spring force of 1,000 kJ-(mol-nm)^"1 followed by another 1 ns of 500 kJ-(mol-nm)^"1. The restraints allowed the amino acid side chains and water atoms to equilibrate. Subsequently, all the restraints were removed and the system was subjected to 200 ns of MD simulations for data production and analysis. Long-range electrostatic interactions were calculated by the particle mesh Ewald (PME) method. A non-bonded interaction distance cut-off of 1.2 nm was applied. A time step of 0.002 ps was adopted in all MD simulations. To analyze the structural evolution of the silk fibroin, protein secondary structures were calculated using the STRIDE algorithm, which was a built-in module in visual molecular dynamics (VMD) software. The same simulation was carried out for silk fibroin without CA as control system. The snapshots based on MD simulation results were generated using VMD software.

[0086] Energetic analysis of the interaction of CA with amino acids in representative fibroin molecule was also performed using Gromacs. Binding energy was calculated based on the trajectory of the above MD simulations during the latter 100 ns of simulation time. The amino acids used for the binding energy calculations are SER91, ALA95, GLY96, TYR97 (i.e. four representative types of amino acids on the selected crystalline domain, the numbers refer to the positions of residues in the complete sequence of fibroin). Energy data collected (during the latter 100 ns of simulation time) in each case represents the mean value from four amino acid residues for each type from the four-chain structure of silk fibroin used in the simulation.

[0087] Example 5: Discussion and Results

[0088] As demonstrated above, the method disclosed herein provides an approach to greatly shorten the crystallites in silkworm silk and produce intrinsically toughest silk by removing bulky residues in silk crystallites.

[0089] In the present method, CA is used as a non-limiting example to prepare a modified feed for silkworm by mixing CA with mulberry leaf powder. By supplementing citric acid (CA) to a silkworm's diet (e.g. mulberry leaf powder diet), and subsequently feeding the modified feed to, for example, B. mori silkworms, the size of crystallites in the silk can be significantly reduced (e.g. reducing the length to almost half of the original length) and an intrinsically toughest silkworm silk can be obtained. The amount of CA fed was less than 1 wt% of the mulberry leaf powder in the feed composition, for example, 0.05 wt% and 0.5 wt%.

[0090] The obtained intrinsically toughest silkworm silk (i.e. CA silk) is more superior to conventional silkworm silks (e.g. recombinant silk from transgenic silkworms achieved a toughness of 167.2 MJ/m³), as determined by tensile testing. What is remarkable is that the toughness of the present CA silk even surpasses that of naturally produced spider dragline silks (160-200 MJ/m³), including those from spider species Araneus diadematus and Nephila clavipes. As determined experimentally by tensile test, CA silk is able to withstand a significantly higher load compared to control silk (i.e. silk from silkworm without the modified feed), resulting in an ultimate tensile strength (UTS) of 840 ± 38.7 MPa as compared to 572 ± 71.1 MPa of control silk. CA silk also deforms plastically and extends more to giving a significantly larger breaking strain of 38.4 ± 1.89 % as compared to 31.8 ± 1.88 % of control silk. In addition to the concomitant improvements in strength and ductility, the CA silk has a toughness value of 225 ± 14 MJ/m³ that is nearly twice that of control silk (121 ± 13 MJ/m³).

[0091] The X-ray diffraction (XRD) results indicated that the significant enhancement in mechanical properties depend on the formation of much shorter and tighter-packed crystallites in the silk. The CA silk contains shorter crystallites (4.69 nm) and a reduced content of β-sheets (19.9 %, which is calculated from peak analysis i.e. based on areas under the component peaks.), both of which are about half of those for control silk (i.e. unmodified silk) (8.99 nm and 38.9%). The much shorter and tighter crystallites are achieved by removing bulky residues present for the formation of crystallites. It is known that amino acid residues of glycine (G), alanine (A), serine (S) and tyrosine (Y) are involved in formation of β-sheet crystallites of silkworm silk, in which tyrosine is the most abundant bulky residue compared to glycine, alanine and serine. An exclusion of bulky tyrosine enables a more regular packing of residues, thereby resulting in the shorter crystallites. This in turn results in better mechanical properties of the silk, including improved strength, breaking strain/elasticity, and toughness. [0092] For the purpose of demonstrating the present method, CA was selected as a model compound to illustrate its strong selective interaction with bulky tyrosine residue over small amino acid residues (glycine, alanine and serine) that are commonly present in crystallites. Experimental findings by dissection of the silkworms and analysis of gland content revealed that CA introduced to the feed was taken up into the haemolymph (i.e. blood) and transferred into silk glands. The concentration of CA in glands reached as high as about 260 μg of CA per mg of dried silk fibroin matter in the silk glands (i.e. about 26 wt% of the solid matter is CA; or 7.8 wt% in the presence of water). The length of crystallites in silk was found to have a near linear correlation to CA concentration in fibroin dope. The resulting short and compact crystallites contributed to significant enhancement of the mechanical properties of the modified silk.

[0093] Both experimental investigations and theoretical analyses have generated evidence that silk containing lengthy crystallites are of inferior strength and toughness, as they tend to incorporate bulky residues which adversely disrupt the tight structure of the crystallites. The present MD simulations demonstrated that bulky tyrosine can disrupt the tight packing of β-chains in crystallites, and removal of the bulky residues resulted in the formation of tight crystallites. The energetic analyses show that CA interacts strongly with bulky tyrosine (Y) (-36.18 ± 2.61 kJ/mol) compared to smaller amino acid residues (G, A, S) (glycine: -7.06 ± 1.65 kJ/mol, alanine: -1 1.31 ± 3.55 kJ/mol, serine: -31.06 ± 3.66 kJ/mol). With the strong CA-tyrosine interaction, a bulky CA-tyrosine complex forms to sterically hinder tyrosine from participating in the formation of β-sheets.

[0094] The present method greatly shortens the size of crystallites in silk, advantageously allowing silkworm silk to achieve enhanced strength and toughness, resulting in the toughest silkworm silk that outperforms all existing synthetic fibers in toughness. Since crystallites constitute the main components of silk, the ability to greatly shorten the size of crystallites allows better control of functional properties of silk (e.g. drug loading efficiency, release profile, etc.), as well as other material properties such as optical and thermal properties. This introduces greater versatility to the use of silk material in wide-ranging applications.

[0095] Example 6: Commercial and Potential Applications [0096] The method as disclosed herein relates to a simple, environmentally sustainable, and scalable strategy for producing intrinsically toughest silkworm silk, which is tougher than spider dragline silk. The method of intrinsic toughening silkworm silk involves forming tight crystallites within silk fibroin for enhancing the silk's structure and functionality. Specifically, the crystallites in silk constitute silk fibroin as the main component, and the crystallites can be tuned to achieve enhanced properties/functionalities for silk fibroin materials.

[0097] The present method, involving the use of CA, facilitates practical applications in textile, biomedical, and many other fields because CA is naturally occurring, commercially available, environmentally friendly and has been established safe for use in human body. With the advantages of being simple, environmentally sustainable, versatile and scalable, the present method significantly advances the silkworm silk processing technology as a sustainable platform for the production of mechanically tough and functional silk at minimal cost, and with little modification to standard industrial sericulture practices.

[0098] While the invention has been particularly shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is thus indicated by the appended claims and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced.

Claims

1. A method of producing a modified silk from a silkworm, comprising:

heating a mixture comprising mulberry leaf powder and water;

2. The method according to claim 1, further comprising mixing the mulberry leaf powder and water in a weight to volume ratio of 1 : 1 to 1 : 10 before heating the mixture.

3. The method according to claim 1 or 2, wherein the heating comprises exposing the mixture to microwaves.

4. The method according to any one of claims 1 to 3, wherein the protic organic compound mixed with the mixture is 0.001 to 0.9 wt% of the mulberry leaf powder.

5. The method according to any one of claims 1 to 4, wherein generating the modified silk comprises forming a complex from a tyrosine and the protic organic compound which prevents the tyrosine from being incorporated into the crystallites of the modified silk.

6. The method according to any one of claims 1 to 5, wherein the protic organic compound is a monoprotic or polyprotic organic compound.

7. The method according to any one of claims 1 to 6, wherein the protic organic compound comprises a functional group selected from the group consisting of carboxylic acid group (-COOH), sulfonic acid group (-SO2OH), hydroxyl group (- OH), thiol group (-SH), and a combination thereof.

8. The method according to any one of claims 1 to 7, wherein the protic organic compound is an organic acid.

9. The method according to claim 8, wherein the organic acid is selected from the group consisting of citric acid (HOC(COOH)(CH₂COOH)₂), acetic acid (CH₃COOH), formic acid (HCOOH), benzoic acid (C₆H₅COOH), glycolic acid, oxalic acid, succinic acid, malic acid, malonic acid, nitrilotriacetic acid, and a combination thereof.

10. The method according to claim 8 or 9, wherein the organic acid is citric acid.

11. A modified silk produced by the method according to any one of claims 1 to 10.

12. A feed composition for producing a modified silk from a silkworm, wherein the modified silk comprises crystallites and each of the crystallites has a length of not more than 7 nm, the feed composition comprising:

mulberry leaf powder;

water; and

a protic organic compound which induces a change in each of the crystallites to have the length of not more than 7 nm for the modified silk, wherein the protic organic compound is less than 1 wt% of the mulberry leaf powder.

13. The feed composition according to claim 12, wherein the protic organic compound is 0.001 to 0.9 wt% of the mulberry leaf powder.

14. The feed composition according to claim 12 or 13, wherein the mulberry leaf powder and water is present in a weight to volume ratio of 1 : 1 to 1 :10.

15. The food composition according to any one of claims 12 to 14, wherein the protic organic compound interacts with a tyrosine to form a complex which prevents the tyrosine from being incorporated into crystallites of the modified silk.

16. The feed composition according to any one of claims 12 to 15, wherein the protic organic compound is a monoprotic or polyprotic organic compound.

17. The feed composition according to any one of claims 12 to 16, wherein the protic organic compound comprises a functional group selected from the group consisting of carboxylic acid group (-COOH), sulfonic acid group (-S0₂OH), hydroxyl group (-OH), thiol group (-SH), and a combination thereof.

18. The feed composition according to any one of claims 12 to 17, wherein the protic organic compound is an organic acid.

19. The feed composition according to claim 18, wherein the organic acid is selected from the group consisting of citric acid (HOC(COOH)(CH₂COOH)₂), acetic acid(CH₃COOH), formic acid (HCOOH), benzoic acid (C₆H₅COOH), glycolic acid, oxalic acid, succinic acid, malic acid, malonic acid, nitrilotriacetic acid, and a combination thereof.

20. The feed composition according to claim 18 or 19, wherein the organic acid is citric acid.