Title of Invention	A BIOACTIVE KERATIN-SILICA MATRIX AND A PROCESS FOR THE PREPARATION THEREOF
Abstract	The invention provides a keratin-silica matrix by hydrolyzing keratin polypeptides with known silica salt, thereby ensuring not only the antimicrobial property of the product, but also to obtain the product in pH range of 7.5 - 9.0 without neutralization with acid. The product exhibits a molecular weight in the range of 2 KDa and 6 KDa, particle size 2000 and 5000 nm and pH 7.5 - 9.0. The product finds enormous potential as an important bio-alternative to replace the conventional fillers and binders for leather finishing. It may also be used as retanning agent in leather processing that will influence the uptake of subsequently applied auxiliaries such as dyestuffs and fatliquors. Besides, this matrix may also be used for pickle free .tannage. This may also be used as chromium free tanning agent and as exhaustive aid in conventional chrome tanning process.

Title of Invention

A BIOACTIVE KERATIN-SILICA MATRIX AND A PROCESS FOR THE PREPARATION THEREOF

Abstract

The invention provides a keratin-silica matrix by hydrolyzing keratin polypeptides with known silica salt, thereby ensuring not only the antimicrobial property of the product, but also to obtain the product in pH range of 7.5 - 9.0 without neutralization with acid. The product exhibits a molecular weight in the range of 2 KDa and 6 KDa, particle size 2000 and 5000 nm and pH 7.5 - 9.0. The product finds enormous potential as an important bio-alternative to replace the conventional fillers and binders for leather finishing. It may also be used as retanning agent in leather processing that will influence the uptake of subsequently applied auxiliaries such as dyestuffs and fatliquors. Besides, this matrix may also be used for pickle free .tannage. This may also be used as chromium free tanning agent and as exhaustive aid in conventional chrome tanning process.

Full Text	Field of the Invention The present invention relates to a bio-active keratin-silica matrix for industrial applications. More particularly, the present invention relates to silica embedded keratin matrix exhibiting bioactivity. The invention further provides a process for the preparation of the said novel keratin-silica matrix. The product of the present invention is envisaged to have enormous potential application as filler-cum-binder in leather finishing industry to improve the aesthetics and protection characteristics of the finished leathers. Thus, the matrix provides an important bio-alternative to replace the conventional fillers and binders for leather finishing. The keratin-silica matrix could also be used as retanning agent in leather processing that will influence the uptake of subsequently applied auxiliaries such as dyestuffs and fatliquors. Besides, this matrix may also be used as exhaustive aid in chrome tanning process. In addition, this novel product could also be used in chromium free and pickle free leather tanning process. Background of the Invention Keratins belong to the family of fibrous structural proteins. It is essentially characterized by disulfide linkage that confers additional strength and rigidity by permanent, thermally-stable crosslinking. While this protein exhibits toughness and insolubility, several attempts have been made to hydrolyze it to ensure appropriate utilization of the protein, which is abundantly available in animal sources, for different industrial purposes. Blanchard, et al. (U.S. Pat. Nos. 6,159,496, 6,274,163) have provided a method of oxidative cleavage of disulfide linkage for using the product in biomedical application. Cowsar (U.S. Pat. Application No. 20030228353) has provided a method of isolation of biologically active keratin peptides by breaking the disulfide linkage with oxidants followed by formulating composition for pharmaceutical or topical administration. All these disclosures relate to partial or complete cleavage of the disulfide linkage of keratin to utilize the resulting peptides for biomedical or pharmaceutical applications. The biological activity of these peptides may relate to cell growth activation or inhibition, healing activity of the damaged skin, to stimulate growth of fibroblasts useful for the treatment of damaged, aged or diseased epithelial tissue and skin. The major limitation of these products is that they are essentially insoluble keratin degradative substances, which do not find application in leather processing industry and other water based processing industries because of their insolubility in aqueous medium. This has prompted researchers to provide soluble products by way of hydrolyzing keratinous substances, whereby the resulting keratin hydrolysate finds wider industrial applications as reported by Karthikeyan et al (Journal of Scientific and Industrial Research, 66, 710, 2007). Several attempts have been made to prepare keratin hydrolysate by alkali hydrolysis method. Our earlier work (Indian Patent No. 180394) provided a process for hydrolyzing keratinous wastes by controlled alkali hydrolysis to get a soluble hydrolysate suitable for leather processing particularly in filling cum retanning and as an exhaustive aid in chrome tanning process in leather industry. Fleischner (U.S. Pat. No. 4,818,520) provided a method of alkaline hydrolysis of keratinous substances for cosmetic applications. However, the hydrolysate produced by alkali hydrolysis requires further treatment of neutralization to ensure that it is suitable for industrial applications, such as leather processing. Flork and Michel (US4874893), Kutsakova et.al (RU2289945) and Masakazu et.al (JP2134348) prepared keratin hydrolysate by acid hydrolysis method. But the product exhibits a pH in the range of 1.0-2.0. Hence, this product also requires neutralization to ensure that it can be used effectively. Savolainen (U.S. Pat. No. 5,262,307) provided a method of enzymatic hydrolysis of keratinous substances to produce keratin hydrolysate that requires further treatment for application in animal feed and cosmetic industries. Thus the major limitation associated with all these hitherto known hydrolysis methods is that the product requires further treatment for industrial application. Moreover, the products do not exhibit any antimicrobial property, thereby limiting the shelf life of the products to not more than 3-6 months. This limitation of these products eventually affects the economy of application adversely. Conventionally, silicates have been used for the preparation of various industrial products exhibiting antimicrobial property. Sugizaki et al (US 6544641) has provided a method for the preparation of silicate-containing sheet having antimicrobial and antifouling properties. Hidenobu et al (US 6592858) has provided a fibre structure with antifungal and antibacterial properties suitable for textiles, where the fibre structure comprising a zeolite layer and an alkyl silicate layer. Morin (GB 100163) obtained the first patent of silica tanning. Fernald and Her (US 2395472) provided a process for the production of acid soluble double silicates of an alkali metal and a metal for leather tanning. The use of silicates for the preservation hides and skins is reported by Munz (Journal of the American Leather Chemists Association, 102, 16, 2007). Alkali silicates also have been used to improve the exhaustion of added auxiliaries, especially of chrome tannins in leather processing as reported by Munz (Journal of the American Leather Chemists Association, 98, 159,2003). No prior art is available on any product comprising keratin and silica, embedded in the form of a matrix. The product obtained with all the hitherto known hydrolysis methods require further treatment for industrial applications especially in leather processing industry. Moreover, the products do not exhibit any antimicrobial property, thereby limiting the shelf-life of the product to not more than 3 to 6 months. Whereas, in the present invention, the keratin polypeptides are hydrolyzed with silica salt, thereby ensuring not only the antimicrobial property of the product, but also obtaining the product in the pH range of 7.5 to 9.0 without further neutralization with acid and enabling to store the product for longer periods of time upto more than 9 months. Objects of the Invention The main objective of the present invention is thus to provide a novel bioactive keratin-silica matrix, which obviates the limitations of the hitherto known prior art as detailed above. Another objective of the invention is to provide a hydrolysis process to obtain keratin-silica matrix at pH 7.5-9.0 without treatment with acid. Still another objective of the present invention is to provide a bioactive keratinous product exhibiting antimicrobial property. Yet another objective of the present invention is to provide a product comprising keratin embedded with silica. Still another objective of the present invention is to provide a process to hydrolyze keratinous substance with silica to provide keratin-silica matrix. Yet another objective of the present invention is to provide a bioactive keratin-silica matrix exhibiting a neutral to slightly alkaline pH range. Still another objective of the present invention is to provide a bioactive keratin-silica matrix, exhibiting a molecular weight not more than 6KDa. Summary of the invention Accordingly, the present invention provides a bioactive keratin-silica matrix, characterized in that it comprises bio- molecules such as peptides and amino acids embedded with silicate species, the said matrix having a molecular weight in the range of 2KDa - 6KDa and particle size in the range of 2000-5000nm and exhibiting pH in the range of 7.5-9.0. The present invention further provides a process for the preparation of the said bioactive keratin-silica matrix, wherein the steps comprise: [a] treating keratinous material with 75-150% w/v of a solvent of the kind such as herein described for a period of 30 to 120 min followed by drying at a temperature in the range of 35 to 70 degree C for a period of 60 to 300 min to obtain defatted keratinous material; [b] digesting the defatted keratinous material as obtained in step [a] with 20 to 35% w/w of a silica salt in aqueous medium, at a pH in the range of 11.0 to 14.0, at a temperature of 100 to 140 degree C and 15 to 35 psi pressure and continuing the reaction till the pH of the solution is in the range of 7.5 to 9.0, to obtain keratin-silica matrix in liquid form; [c] optionally, drying the keratin-silica liquid as obtained in step [b] at a temperature in the range of 110 to 250 degree C, to obtain keratin-silica matrix in powder form. Detailed description of the invention Keratinous material is treated with 75-150% w/v of known solvent for a period of 30-120 min and then dried at a temperature in the range of 35-70°C for a period of 60-300 min. The defatted keratinous material, thus obtained, is digested with 20-35% w/w of silica salt in the presence of 200-400%w/v of water at a pH in the range of 11.0-14.0. The temperature and pressure for the digestion are maintained in the range of 100-140°C and 15-35psi pressure respectively. The reaction is continued till the pH of the solution is in the range of 7.5-9.0 to obtain keratin-silica matrix in liquid form. The liquid is optionally subjected to drying at a temperature in the range of 110 -250 °C to obtain keratin-silica matrix in the powder form. The characteristic features of the keratin-silica matrix are presented below. The keratin-silica matrix consists of biomolecules (peptides and amino acid composition) being embedded with silicate species through electrostatic interaction between positively charged peptides and negatively charged silicate anions induce the formation of silica leading to obtain a product at pH 7.5-9.0 in a manner to inhibit microbial growth. The particle size of the matrix determined by ZetaSizer ranges from 2000 and 5000nm and the molecular weight determined by Sodium Dodecyl Sulphate - Poly Acryl amide Gel Electrophoresis (SDS-PAGE) is in the range of 2KDa and 6KDa. The antimicrobial property of the keratin-silica matrix was tested against the test strains using disc diffusion method. In an embodiment of the present invention, the keratinous material used may be selected from horn meal, hoof meal, feather meal, animal hair, wool, human hair. In another embodiment of the present invention, the solvent used may be selected from acetone, hexane, xylene. In yet another embodiment of the present invention, the amount of water used may be in the range of 200-400% by volume on the weight of the keratinous material. In still another embodiment of the present invention, the silica salt used may be selected from sodium silicate, potassium silicate, lithium silicate, sodium aluminum silicate, potassium aluminum silicate, sodium iron silicate, potassium iron silicate, sodium zirconium silicate, potassium zirconium silicate, sodium zinc silicate, potassium zinc silicate, aluminum silicate either alone or in any combination thereof. In still another embodiment of the present invention, the known method of drying may be such as spray drying, drum drying. In yet another embodiment of the present invention, the matrix exhibits antimicrobial property. The inventiveness of the present invention lies in providing a keratin-silica matrix by hydrolyzing keratin polypeptides with known silica salt, thereby ensuring not only the antimicrobial property of the product, but also to obtain the product in pH range of 7.5 and 9.0 without further neutralization with acid, caused by the electrostatic interaction between positively charged peptides and negatively charged silicate anions. The following examples are given by way of illustration only and therefore should not be construed to limit the scope of the present invention. EXAMPLE -1 Horn meal weighing I kg, was treated with 1 lit of acetone for 45 min and dried at 45°C for 120 min. The dried and defatted horn meal was charged into an autoclave along with 2.5 lit of water, 150gms of sodium silicate and 100 gms of potassium aluminium silicate. pH of the solution was found to be 12.0. The autoclave was switched on to reach the pressure at 15 psi. As soon as the temperature was recorded to be 110°C, the autoclave was switched off and the reactants were allowed to cool down without releasing the pressure. The pH of the slurry was monitored continuously till it was found to be 8.0. The resulting keratin-silica matrix liquid was collected. The liquid was finally drum dried at a temperature of 130°C to obtain keratin-silica matrix powder, which was stored in a plastic container. The product was subjected to characterization and it exhibited the following characteristics. Molecular weight determined by SDS-PAGE Analysis - 4.5 KDa Particle size determined by ZetaSizer - 3800 nm Antimicrobial property determined by disc diffusion method- presence of zone of inhibition 30 gm of the keratin-silica matrix powder was used as filler to prepare a base coat mixture for leather finishing and applied on cow leather. The leather exhibited excellent covering and fastness properties. EXAMPLE - 2 Feather meal weighing 1.5 kg, was treated with 2.0 lit of hexane for 60 min and dried at 60°C for 90 min. The dried and defatted feather meal was charged into an autoclave along with 3.0 lit of water and 150gms of potassium silicate and 100 gms of sodium iron silicate, 50 gms sodium zinc silicate. pH of the solution was found to be 13.0. The autoclave was switched on to reach the pressure at 20 psi. As soon as the temperature was recorded to be 120°C, the autoclave was switched off and the reactants were allowed to cool down without releasing the pressure. The pH of the slurry was monitored continuously till it was found to be 8.5. The resulting keratin-silica matrix liquid was collected. The product was subjected to characterization and it exhibited the following characteristics. Molecular weight determined by SDS-PAGE Analysis - 4 KDa Particle size determined by ZetaSizer - 3200 nm Antimicrobial property determined by disc diffusion method- presence of zone of inhibition 150 gms of keratin-silica matrix liquid was treated with delimed goat skins in chromium free tanning process. The leather showed a shrinkage temperature of 68°C. EXAMPLE - 3 Horn and hoof meal weighing 2 kg, was treated with 1.75 lit of acetone for 60 min and dried at 35°C for 300 min. The dried and defatted horn and hoof meal was charged into an autoclave along with 4.5 lit of water and 200gms of potassium silicate, 50 gms sodium aluminium silicate and 100 gms of lithium silicate. The pH of the solution was found to be 14.0. The autoclave was switched on to reach the pressure at 25 psi. As soon as the temperature was recorded to be 130°C, the autoclave was switched off and the reactants were allowed to cool down without releasing the pressure. The pH of the slurry was monitored continuously till it was found to be 9.0. The resulting keratin-silica matrix liquid was collected. The liquid was finally spray dried at a temperature of 180°C to obtain keratin-silica matrix powder, which was stored in a plastic container. The product was subjected to characterization and it exhibited the following characteristics. Molecular weight determined by SDS-PAGE Analysis - 3 KDa Particle size determined by ZetaSizer - 2700 nm Antimicrobial property determined by disc diffusion method - presence of zone of inhibition 40 gms of keratin-silica matrix powder was used as exhaustive aid in chrome tanning process. The exhaustion of chromium in the spent tan liquor was found to be around 90%. EXAMPLE - 4 Goat hair weighing 1.5 kg, was treated with 2.0 lit of hexane for 60 min and dried at 40°C for 200 min. The dried and defatted goat hair was charged into an autoclave along with 3.0 lit of water and 150gms of sodium silicate, 100 gms of aluminum silicate, 50 gms potassium zirconium silicate and 50 gms sodium zirconium silicate. The pH of the solution was found to be 13.0. The autoclave was switched on to reach the pressure at 35 psi. As soon as the temperature was recorded to be 140°C, the autoclave was switched off and the reactants were allowed to cool down without releasing the pressure. The pH of the slurry was monitored continuously till it was found to be 7.5. The resulting keratin-silica matrix liquid was collected. The liquid was finally spray dried at a temperature of 250°C to obtain keratin-silica matrix powder, which was stored in a plastic container. The product was subjected to characterization and it exhibited the following characteristics. Molecular weight determined by SDS-PAGE Analysis - 2KDa Particle size determined by Zeta Sizer - 2000 nm Antimicrobial property determined by disc diffusion method- presence of zone of inhibition. 100 gms of keratin-silica matrix powder was used as retanning agent on wet blue cow crust leather. The retanned leather exhibited excellent fullness and improved physical characteristics. EXAMPLE - 5 Human hair weighing 1.0 kg, was treated with 1.0 lit of xylene for 60 min and dried at 40°C for 200 min. The dried and defatted horn meal was charged into an autoclave along with 3.0 lit of water and 200gms of sodium silicate. The pH of the solution was found to be 11.0. The autoclave was switched on to reach the pressure at 15 psi. As soon as the temperature was recorded to be 100°C, the autoclave was switched off and the reactants were allowed to cool down without releasing the pressure. The pH of the slurry was monitored continuously till it was found to be 7.5. The resulting keratin-silica matrix liquid was collected. The liquid was finally spray dried at a temperature of 110°C to obtain keratin-silica matrix powder, which was stored in a plastic container. The product was subjected to characterization and it exhibited the following characteristics. Molecular weight determined by SDS-PAGE Analysis - 6 KDa Particle size determined by ZetaSizer - 4950 nm Antimicrobial property determined by disc diffusion method- presence of zone of inhibition. 200 gms of keratin-silica matrix powder was used as retanning agent on wet blue cow crust leather. The retanned leather exhibited excellent fullness and improved physical characteristics. Advantages of the Invention: 1. This process makes use of keratin waste to produce valuable product. 2. This process does not involve use of any toxic chemicals for the preparation of keratin-silica matrix. 3. The bioactive keratin peptides can be stored at long periods without microbial contamination. 4. The product contains protein and silica as the major part, therefore these bioactive peptides can be used as filler cum binding agent in leather finishing. 5. Since the matrix has pH in the range of 7.5 to 9.0 it can also be used as retanning agent and exhaustive aid in chrome tanning process. 6. The keratin-silica matrix may also be used in pickle free tanning process. 7. This bioactive keratin-silica matrix treated leathers are stable for longer period of time upto more than 9 months. We claim: 1. A bioactive keratin-silica matrix, characterized in that it comprises keratinous materials selected from bio- molecules and/or peptides and/or amino acids embedded with silicate species, wherein the ratio of the keratinous material to silicate species is in the range of 65 to 80% w/w, the said matrix having a molecular weight in the range of 2KDa - 6KDa and particle size in the range of 2000-5000nm and exhibiting pH in the range of 7.5-9.0. 2. A keratin-silica matrix as claimed in claim 1, wherein the matrix exhibits antimicrobial property. 3. A keratin-silica matrix as claimed in claim 1, wherein the keratinous material used is selected from horn meal, hoof meal, feather meal, animal hair, wool, human hair, nails. 4. A keratin-silica matrix as claimed in claim 1, wherein the bio-molecules used are selected from the hydrolysates obtained from the said keratinous materials. 5. A keratin-silica matrix as claimed in claim 1, wherein the amino acids used are selected from Arginine, Lysine, Leucine, Isoleucine, Glycine, Serine, Methionine, Phenylalanine, Threonine, Valine, Alanine, Aspartate, Glutamate, Cystine, Proline, Tyrosine and Histidine. 6. A keratin-silica matrix as claimed in claim 1, wherein the silica salt used is selected from sodium silicate, potassium silicate, lithium silicate, sodium aluminum silicate, potassium aluminum silicate, sodium iron silicate, potassium iron silicate, sodium zirconium silicate, potassium zirconium silicate, sodium zinc silicate, potassium zinc silicate, aluminum silicate either alone or in any combination thereof. 7. A process for the preparation of bioactive keratin-silica matrix as claimed in claim 1, wherein the steps comprise: [a] treating keratinous material with 75-150% w/v of a solvent of the kind such as herein described for a period of 30 to 120 min followed by drying at a temperature in the range of 35 to 70 degree C for a period of 60 to 300 min to obtain defatted keratinous material; [b] digesting the defatted keratinous material as obtained in step [a] with 20 to 35% w/w of a silica salt in aqueous medium, at a pH in the range of 11.0 to 14.0, at a temperature of 100 to 140 degree C and 15 to 35 psi pressure and continuing the reaction till the pH of the solution is in the range of 7.5 to 9.0, to obtain keratin-silica matrix in liquid form; [c] optionally, drying the keratin-silica liquid as obtained in step [b] at a temperature in the range of 110 to 250 degree C, to obtain keratin-silica matrix in powder form. 8. A process as claimed in claim 5, wherein the solvent used is selected from the group consisting of acetone, hexane and xylene. 9. A process as claimed in claim 5, wherein the amount of water used is in the range of 200 to 400% by volume on the weight of the keratinous material. 10. A process as claimed in claim 5, wherein the known method of drying is such as spray drying, drum drying. 11. A bioactive keratin-silica matrix and a process for the preparation thereof substantially as herein described with reference to the foregoing examples.

Full Text

Field of the Invention
The present invention relates to a bio-active keratin-silica matrix for industrial applications. More particularly, the present invention relates to silica embedded keratin matrix exhibiting bioactivity. The invention further provides a process for the preparation of the said novel keratin-silica matrix. The product of the present invention is envisaged to have enormous potential application as filler-cum-binder in leather finishing industry to improve the aesthetics and protection characteristics of the finished leathers. Thus, the matrix provides an important bio-alternative to replace the conventional fillers and binders for leather finishing. The keratin-silica matrix could also be used as retanning agent in leather processing that will influence the uptake of subsequently applied auxiliaries such as dyestuffs and fatliquors. Besides, this matrix may also be used as exhaustive aid in chrome tanning process. In addition, this novel product could also be used in chromium free and pickle free leather tanning process.
Background of the Invention
Keratins belong to the family of fibrous structural proteins. It is essentially characterized by disulfide linkage that confers additional strength and rigidity by permanent, thermally-stable crosslinking. While this protein exhibits toughness and insolubility, several attempts have been made to hydrolyze it to ensure appropriate utilization of the protein, which is abundantly available in animal sources, for different industrial purposes. Blanchard, et al. (U.S. Pat. Nos. 6,159,496, 6,274,163) have provided a method of oxidative cleavage of disulfide linkage for using the product in biomedical application. Cowsar (U.S. Pat. Application No. 20030228353) has provided a method of isolation of biologically active keratin peptides by breaking the
disulfide linkage with oxidants followed by formulating composition for pharmaceutical or topical administration. All these disclosures relate to partial or complete cleavage of the disulfide linkage of keratin to utilize the resulting peptides for biomedical or pharmaceutical applications. The biological activity of these peptides may relate to cell growth activation or inhibition, healing activity of the damaged skin, to stimulate growth of fibroblasts useful for the treatment of damaged, aged or diseased epithelial tissue and skin. The major limitation of these products is that they are essentially insoluble keratin degradative substances, which do not find application in leather processing industry and other water based processing industries because of their insolubility in aqueous medium.
This has prompted researchers to provide soluble products by way of hydrolyzing keratinous substances, whereby the resulting keratin hydrolysate finds wider industrial applications as reported by Karthikeyan et al (Journal of Scientific and Industrial Research, 66, 710, 2007).
Several attempts have been made to prepare keratin hydrolysate by alkali hydrolysis method.
Our earlier work (Indian Patent No. 180394) provided a process for hydrolyzing keratinous wastes by controlled alkali hydrolysis to get a soluble hydrolysate suitable for leather processing particularly in filling cum retanning and as an exhaustive aid in chrome tanning process in leather industry. Fleischner (U.S. Pat. No. 4,818,520) provided a method of alkaline hydrolysis of keratinous substances for cosmetic applications. However, the hydrolysate produced by alkali hydrolysis requires further
treatment of neutralization to ensure that it is suitable for industrial applications, such as leather processing.
Flork and Michel (US4874893), Kutsakova et.al (RU2289945) and Masakazu et.al (JP2134348) prepared keratin hydrolysate by acid hydrolysis method. But the product exhibits a pH in the range of 1.0-2.0. Hence, this product also requires neutralization to ensure that it can be used effectively.
Savolainen (U.S. Pat. No. 5,262,307) provided a method of enzymatic hydrolysis of keratinous substances to produce keratin hydrolysate that requires further treatment for application in animal feed and cosmetic industries.
Thus the major limitation associated with all these hitherto known hydrolysis methods is that the product requires further treatment for industrial application. Moreover, the products do not exhibit any antimicrobial property, thereby limiting the shelf life of the products to not more than 3-6 months. This limitation of these products eventually affects the economy of application adversely.
Conventionally, silicates have been used for the preparation of various industrial products exhibiting antimicrobial property. Sugizaki et al (US 6544641) has provided a method for the preparation of silicate-containing sheet having antimicrobial and antifouling properties. Hidenobu et al (US 6592858) has provided a fibre structure with antifungal and antibacterial properties suitable for textiles, where the fibre structure comprising a zeolite layer and an alkyl silicate layer.
Morin (GB 100163) obtained the first patent of silica tanning. Fernald and Her (US 2395472) provided a process for the production of acid soluble double silicates of an alkali metal and a metal for leather tanning. The use of silicates for the preservation hides and skins is reported by Munz (Journal of the American Leather Chemists Association, 102, 16, 2007). Alkali silicates also have been used to improve the exhaustion of added auxiliaries, especially of chrome tannins in leather processing as reported by Munz (Journal of the American Leather Chemists Association, 98, 159,2003).
No prior art is available on any product comprising keratin and silica, embedded in the form of a matrix. The product obtained with all the hitherto known hydrolysis methods require further treatment for industrial applications especially in leather processing industry. Moreover, the products do not exhibit any antimicrobial property, thereby limiting the shelf-life of the product to not more than 3 to 6 months. Whereas, in the present invention, the keratin polypeptides are hydrolyzed with silica salt, thereby ensuring not only the antimicrobial property of the product, but also obtaining the product in the pH range of 7.5 to 9.0 without further neutralization with acid and enabling to store the product for longer periods of time upto more than 9 months.
Objects of the Invention
The main objective of the present invention is thus to provide a novel bioactive keratin-silica matrix, which obviates the limitations of the hitherto known prior art as detailed above.
Another objective of the invention is to provide a hydrolysis process to obtain keratin-silica matrix at pH 7.5-9.0 without treatment with acid.
Still another objective of the present invention is to provide a bioactive keratinous product exhibiting antimicrobial property.
Yet another objective of the present invention is to provide a product comprising keratin embedded with silica.
Still another objective of the present invention is to provide a process to hydrolyze keratinous substance with silica to provide keratin-silica matrix. Yet another objective of the present invention is to provide a bioactive keratin-silica matrix exhibiting a neutral to slightly alkaline pH range.
Still another objective of the present invention is to provide a bioactive keratin-silica matrix, exhibiting a molecular weight not more than 6KDa.
Summary of the invention
Accordingly, the present invention provides a bioactive keratin-silica matrix, characterized in that it comprises bio- molecules such as peptides and amino acids embedded with silicate species, the said matrix having a molecular weight in the range of 2KDa - 6KDa and particle size in the range of 2000-5000nm and exhibiting pH in the range of 7.5-9.0.
The present invention further provides a process for the preparation of the said bioactive keratin-silica matrix, wherein the steps comprise:
[a] treating keratinous material with 75-150% w/v of a solvent of the kind such as herein described for a period of 30 to 120 min followed by
drying at a temperature in the range of 35 to 70 degree C for a period of 60 to 300 min to obtain defatted keratinous material;
[b] digesting the defatted keratinous material as obtained in step [a] with 20 to 35% w/w of a silica salt in aqueous medium, at a pH in the range of 11.0 to 14.0, at a temperature of 100 to 140 degree C and 15 to 35 psi pressure and continuing the reaction till the pH of the solution is in the range of 7.5 to 9.0, to obtain keratin-silica matrix in liquid form;
[c] optionally, drying the keratin-silica liquid as obtained in step [b] at a temperature in the range of 110 to 250 degree C, to obtain keratin-silica matrix in powder form.
Detailed description of the invention
Keratinous material is treated with 75-150% w/v of known solvent for a period of 30-120 min and then dried at a temperature in the range of 35-70°C for a period of 60-300 min. The defatted keratinous material, thus obtained, is digested with 20-35% w/w of silica salt in the presence of 200-400%w/v of water at a pH in the range of 11.0-14.0. The temperature and pressure for the digestion are maintained in the range of 100-140°C and 15-35psi pressure respectively. The reaction is continued till the pH of the solution is in the range of 7.5-9.0 to obtain keratin-silica matrix in liquid form. The liquid is optionally subjected to drying at a temperature in the range of 110 -250 °C to obtain keratin-silica matrix in the powder form.
The characteristic features of the keratin-silica matrix are presented below.
The keratin-silica matrix consists of biomolecules (peptides and amino acid
composition) being embedded with silicate species through electrostatic interaction
between positively charged peptides and negatively charged silicate anions induce the formation of silica leading to obtain a product at pH 7.5-9.0 in a manner to inhibit microbial growth. The particle size of the matrix determined by ZetaSizer ranges from 2000 and 5000nm and the molecular weight determined by Sodium Dodecyl Sulphate - Poly Acryl amide Gel Electrophoresis (SDS-PAGE) is in the range of 2KDa and 6KDa. The antimicrobial property of the keratin-silica matrix was tested against the test strains using disc diffusion method.
In an embodiment of the present invention, the keratinous material used may be selected from horn meal, hoof meal, feather meal, animal hair, wool, human hair.
In another embodiment of the present invention, the solvent used may be selected from acetone, hexane, xylene.
In yet another embodiment of the present invention, the amount of water used may be in the range of 200-400% by volume on the weight of the keratinous material.
In still another embodiment of the present invention, the silica salt used may be selected from sodium silicate, potassium silicate, lithium silicate, sodium aluminum silicate, potassium aluminum silicate, sodium iron silicate, potassium iron silicate, sodium zirconium silicate, potassium zirconium silicate, sodium zinc silicate, potassium zinc silicate, aluminum silicate either alone or in any combination thereof.
In still another embodiment of the present invention, the known method of drying may be such as spray drying, drum drying.
In yet another embodiment of the present invention, the matrix exhibits antimicrobial property.
The inventiveness of the present invention lies in providing a keratin-silica matrix by hydrolyzing keratin polypeptides with known silica salt, thereby ensuring not only the antimicrobial property of the product, but also to obtain the product in pH range of 7.5 and 9.0 without further neutralization with acid, caused by the electrostatic interaction between positively charged peptides and negatively charged silicate anions.
The following examples are given by way of illustration only and therefore should not be construed to limit the scope of the present invention.
EXAMPLE -1
Horn meal weighing I kg, was treated with 1 lit of acetone for 45 min and dried at 45°C for 120 min. The dried and defatted horn meal was charged into an autoclave along with 2.5 lit of water, 150gms of sodium silicate and 100 gms of potassium aluminium silicate. pH of the solution was found to be 12.0. The autoclave was switched on to reach the pressure at 15 psi. As soon as the temperature was recorded to be 110°C, the autoclave was switched off and the reactants were allowed to cool down without releasing the pressure. The pH of the slurry was monitored continuously till it was found to be 8.0. The resulting keratin-silica matrix liquid was collected.
The liquid was finally drum dried at a temperature of 130°C to obtain keratin-silica matrix powder, which was stored in a plastic container.
The product was subjected to characterization and it exhibited the following
characteristics.
Molecular weight determined by SDS-PAGE Analysis - 4.5 KDa
Particle size determined by ZetaSizer - 3800 nm
Antimicrobial property determined by disc diffusion method- presence of zone of
inhibition
30 gm of the keratin-silica matrix powder was used as filler to prepare a base coat
mixture for leather finishing and applied on cow leather. The leather exhibited
excellent covering and fastness properties.
EXAMPLE - 2
Feather meal weighing 1.5 kg, was treated with 2.0 lit of hexane for 60 min and dried at 60°C for 90 min. The dried and defatted feather meal was charged into an autoclave along with 3.0 lit of water and 150gms of potassium silicate and 100 gms of sodium iron silicate, 50 gms sodium zinc silicate. pH of the solution was found to be 13.0. The autoclave was switched on to reach the pressure at 20 psi. As soon as the temperature was recorded to be 120°C, the autoclave was switched off and the reactants were allowed to cool down without releasing the pressure. The pH of the slurry was monitored continuously till it was found to be 8.5. The resulting keratin-silica matrix liquid was collected.
The product was subjected to characterization and it exhibited the following characteristics.
Molecular weight determined by SDS-PAGE Analysis - 4 KDa Particle size determined by ZetaSizer - 3200 nm
Antimicrobial property determined by disc diffusion method- presence of zone of
inhibition
150 gms of keratin-silica matrix liquid was treated with delimed goat skins in
chromium free tanning process. The leather showed a shrinkage temperature of
68°C.
EXAMPLE - 3
Horn and hoof meal weighing 2 kg, was treated with 1.75 lit of acetone for 60 min
and dried at 35°C for 300 min. The dried and defatted horn and hoof meal was
charged into an autoclave along with 4.5 lit of water and 200gms of potassium
silicate, 50 gms sodium aluminium silicate and 100 gms of lithium silicate. The pH of
the solution was found to be 14.0. The autoclave was switched on to reach the
pressure at 25 psi. As soon as the temperature was recorded to be 130°C, the
autoclave was switched off and the reactants were allowed to cool down without
releasing the pressure. The pH of the slurry was monitored continuously till it was
found to be 9.0. The resulting keratin-silica matrix liquid was collected. The liquid
was finally spray dried at a temperature of 180°C to obtain keratin-silica matrix
powder, which was stored in a plastic container.
The product was subjected to characterization and it exhibited the following
characteristics.
Molecular weight determined by SDS-PAGE Analysis - 3 KDa
Particle size determined by ZetaSizer - 2700 nm
Antimicrobial property determined by disc diffusion method - presence of zone of
inhibition
40 gms of keratin-silica matrix powder was used as exhaustive aid in chrome tanning process. The exhaustion of chromium in the spent tan liquor was found to be around 90%.
EXAMPLE - 4
Goat hair weighing 1.5 kg, was treated with 2.0 lit of hexane for 60 min and dried at
40°C for 200 min. The dried and defatted goat hair was charged into an autoclave
along with 3.0 lit of water and 150gms of sodium silicate, 100 gms of aluminum
silicate, 50 gms potassium zirconium silicate and 50 gms sodium zirconium silicate.
The pH of the solution was found to be 13.0. The autoclave was switched on to
reach the pressure at 35 psi. As soon as the temperature was recorded to be 140°C,
the autoclave was switched off and the reactants were allowed to cool down without
releasing the pressure. The pH of the slurry was monitored continuously till it was
found to be 7.5. The resulting keratin-silica matrix liquid was collected. The liquid
was finally spray dried at a temperature of 250°C to obtain keratin-silica matrix
powder, which was stored in a plastic container.
The product was subjected to characterization and it exhibited the following
characteristics.
Molecular weight determined by SDS-PAGE Analysis - 2KDa
Particle size determined by Zeta Sizer - 2000 nm
Antimicrobial property determined by disc diffusion method- presence of zone of
inhibition.
100 gms of keratin-silica matrix powder was used as retanning agent on wet blue
cow crust leather. The retanned leather exhibited excellent fullness and improved
physical characteristics.
EXAMPLE - 5
Human hair weighing 1.0 kg, was treated with 1.0 lit of xylene for 60 min and dried at 40°C for 200 min. The dried and defatted horn meal was charged into an autoclave along with 3.0 lit of water and 200gms of sodium silicate. The pH of the solution was found to be 11.0. The autoclave was switched on to reach the pressure at 15 psi. As soon as the temperature was recorded to be 100°C, the autoclave was switched off and the reactants were allowed to cool down without releasing the pressure. The pH of the slurry was monitored continuously till it was found to be 7.5. The resulting keratin-silica matrix liquid was collected. The liquid was finally spray dried at a temperature of 110°C to obtain keratin-silica matrix powder, which was stored in a plastic container.
The product was subjected to characterization and it exhibited the following characteristics.
Molecular weight determined by SDS-PAGE Analysis - 6 KDa
Particle size determined by ZetaSizer - 4950 nm
Antimicrobial property determined by disc diffusion method- presence of zone of
inhibition.
200 gms of keratin-silica matrix powder was used as retanning agent on wet blue cow crust leather. The retanned leather exhibited excellent fullness and improved physical characteristics.
Advantages of the Invention:
1. This process makes use of keratin waste to produce valuable product.
2. This process does not involve use of any toxic chemicals for the preparation of keratin-silica matrix.
3. The bioactive keratin peptides can be stored at long periods without microbial contamination.
4. The product contains protein and silica as the major part, therefore these bioactive peptides can be used as filler cum binding agent in leather finishing.
5. Since the matrix has pH in the range of 7.5 to 9.0 it can also be used as retanning agent and exhaustive aid in chrome tanning process.
6. The keratin-silica matrix may also be used in pickle free tanning process.
7. This bioactive keratin-silica matrix treated leathers are stable for longer period of time upto more than 9 months.

We claim:
1. A bioactive keratin-silica matrix, characterized in that it comprises keratinous materials selected from bio- molecules and/or peptides and/or amino acids embedded with silicate species, wherein the ratio of the keratinous material to silicate species is in the range of 65 to 80% w/w, the said matrix having a molecular weight in the range of 2KDa - 6KDa and particle size in the range of 2000-5000nm and exhibiting pH in the range of 7.5-9.0.
2. A keratin-silica matrix as claimed in claim 1, wherein the matrix exhibits antimicrobial property.
3. A keratin-silica matrix as claimed in claim 1, wherein the keratinous material used is selected from horn meal, hoof meal, feather meal, animal hair, wool, human hair, nails.
4. A keratin-silica matrix as claimed in claim 1, wherein the bio-molecules used are selected from the hydrolysates obtained from the said keratinous materials.
5. A keratin-silica matrix as claimed in claim 1, wherein the amino acids used are selected from Arginine, Lysine, Leucine, Isoleucine, Glycine, Serine, Methionine, Phenylalanine, Threonine, Valine, Alanine, Aspartate, Glutamate, Cystine, Proline, Tyrosine and Histidine.
6. A keratin-silica matrix as claimed in claim 1, wherein the silica salt used is selected from sodium silicate, potassium silicate, lithium silicate, sodium
aluminum silicate, potassium aluminum silicate, sodium iron silicate, potassium iron silicate, sodium zirconium silicate, potassium zirconium silicate, sodium zinc silicate, potassium zinc silicate, aluminum silicate either alone or in any combination thereof.
7. A process for the preparation of bioactive keratin-silica matrix as claimed in
claim 1, wherein the steps comprise:
[a] treating keratinous material with 75-150% w/v of a solvent of the kind such as herein described for a period of 30 to 120 min followed by drying at a temperature in the range of 35 to 70 degree C for a period of 60 to 300 min to obtain defatted keratinous material;
[b] digesting the defatted keratinous material as obtained in step [a] with 20 to 35% w/w of a silica salt in aqueous medium, at a pH in the range of 11.0 to 14.0, at a temperature of 100 to 140 degree C and 15 to 35 psi pressure and continuing the reaction till the pH of the solution is in the range of 7.5 to 9.0, to obtain keratin-silica matrix in liquid form;
[c] optionally, drying the keratin-silica liquid as obtained in step [b] at a temperature in the range of 110 to 250 degree C, to obtain keratin-silica matrix in powder form.

8. A process as claimed in claim 5, wherein the solvent used is selected from the group consisting of acetone, hexane and xylene.
9. A process as claimed in claim 5, wherein the amount of water used is in the range of 200 to 400% by volume on the weight of the keratinous material.
10. A process as claimed in claim 5, wherein the known method of drying is such as spray drying, drum drying.
11. A bioactive keratin-silica matrix and a process for the preparation thereof substantially as herein described with reference to the foregoing examples.

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=rQL1NYgbVFH1a9QHOYFWrA==&loc=+mN2fYxnTC4l0fUd8W4CAA==

« Previous Patent

Next Patent »

Patent Number

271039

Indian Patent Application Number

1241/DEL/2008

PG Journal Number

06/2016

Publication Date

05-Feb-2016

Grant Date

29-Jan-2016

Date of Filing

19-May-2008

Name of Patentee

COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH

Applicant Address

ANUSANDHAN BHAVAN, RAFI MARG, NEW DELHI-110 001,INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	SEHGAL PRAVEEN KUMAR	CENTRAL LEATHER RESEARCH INSTITUTE, ADYAR, CHENNAI-600 020 INDIA
2	RAMESH RAMAKRISHNAN	CENTRAL LEATHER RESEARCH INSTITUTE, ADYAR, CHENNAI-600 020 INDIA
3	KARTHIKEYAN RAJAN	CENTRAL LEATHER RESEARCH INSTITUTE, ADYAR, CHENNAI-600 020 INDIA.
4	BALAJI SRINIVASAN	CENTRAL LEATHER RESEARCH INSTITUTE, ADYAR, CHENNAI-600 020 INDIA
5	CHANDRABABU NARASIMHAN KANNAN	CENTRAL LEATHER RESEARCH INSTITUTE, ADYAR, CHENNAI-600 020 INDIA

PCT International Classification Number

C14C 3/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA