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    • 专利摘要:
    Method of obtaining keratins and bioplastics from keratinous residues of animals. A procedure for obtaining keratin is described, from keratinous agro-industrial waste. A keratin obtainable by means of said procedure is also described, characterized by being soluble in an alkaline medium and with average molecular weight fractions greater than 10 kDa. Finally, a method for obtaining bioplastics from keratin obtained from keratinous agro-industrial waste by the described obtaining procedure is also described. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2801025A1
    申请号:ES201930591
    申请日:2019-06-26
    公开日:2021-01-07
    发明作者:D'arlas Bidegain Borja Fernández
    申请人:Universidad Publica de Navarra；
    IPC主号:
    专利说明:

    [0004] TECHNICAL FIELD OF THE INVENTION
    [0006] The present invention refers to a process for obtaining keratin, from keratinous residues, preferably agroindustrial, from animals. The present invention also refers to the keratin obtainable by said process, characterized by being soluble in an alkaline medium and with average molecular weight fractions greater than 10 kDa. Finally, the present invention refers to a method for obtaining bioplastics from keratin obtainable from keratinous agro-industrial waste by the process of the invention.
    [0008] STATE OF THE ART
    [0010] Wool, plumage and other materials rich in keratin are currently considered agro-industrial waste and are made up of 80-90% keratin. Keratins are used as additives in cosmetics and can be used as materials for the development of biomaterials (bioabsorbable patches, artificial skin, pharmacological excipients, etc.) and are being investigated as possible precursors of bioplastics, fibers and tissues (Moore et al. 2006; Ma et al. 2016).
    [0012] Current keratin extraction methodologies are either very aggressive, obtaining hydrolysates or peptides of low molecular mass used as additives in cosmetics, or they involve the use of high concentrations of sulfur compounds such as thiols (thioglycolic acid, mercaptoethanol, etc.) or salts sulfur (sodium sulfide) combined with other salts such as ureas and thioureas. These methods also involve tedious and expensive keratin purification processes, in order to separate them from the rest of the sulfur reagents (Goddart et al. 1934). These procedures often include treatments such as dialysis that involve contamination of large volumes of water, long periods of time, and materials such as dialysis membranes (Goddart et al. 1934; Tonin et al. 2007; Ma et al. 2016;) .
    [0014] On the other hand, several documents of the state of the art describe obtaining keratins, or peptides derived from it, in an acid medium. Thus, European patent application EP1265570A1 refers to a method for obtaining water-soluble peptides from keratin materials such as hair, hooves or feathers, which involves oxidation with peracetic acid or hydrogen peroxide in an acid medium, so that the disulfide bridges transform into sulfonic groups; This step is followed by filtration, neutralization of the aqueous solution with a base such as ammonium hydroxide, and the addition of a water-miscible solvent such as methanol, ethanol, acetone, or tetrahydrofuran.
    [0016] On the other hand, the American patent application US4495173A describes the treatment of previously ground wool fibers, by means of formic acid and hydrogen peroxide, at room temperature for one day. Other oxidative methodologies of wool have been described for the extraction of keratins with paracetic acid and percarbonate (Brown et al.
    [0017] 2016).
    [0019] Other documents describe the treatment of keratinous substrates in an alkaline medium. Thus, document US5276138A describes a process for obtaining a keratin solution from animal hair, in an alkaline liquid medium containing more than 20% hydrogen peroxide, at pH 8-9, followed by acidification to form a gel and washing with a polar solvent to form keratin powder.
    [0021] Furthermore, patent application WO9001023A compares various conditions for hydrolyzing a material rich in keratin, such as feathers or wool, among others, specifically in: water; 1% sodium hydroxide; 1% sodium hydroxide together with 1% hydrogen peroxide; 1% sodium hydroxide (without hydrogen peroxide) in autoclave at 120 ° C; or plain water in an autoclave at 120 ° C. But in no case does it test or describe the use of hydrogen peroxide in a pressurized container. He concludes that the optimal ratio of substrate to peroxide is 5: 1, but requires reaction times of several days to achieve acceptable yields.
    [0023] No references have been found to the use of hydrogen peroxide in small concentrations, that is, between 0.1-0.5 M (0.25-1.25%) at basic pH and in a pressurized container.
    [0025] On the contrary, in the patent application US4495173A (Matsunaga et al.), It is highlighted (p.
    [0026] 2, paragraph 45) that "the oxidation reaction occurs in a liquid medium using oxidizing agents in excess of the disulfide bonds in the keratin-rich material, generally in amounts of two equivalents or more, preferably 4-10 equivalents" US4495173A also states that the reaction is feasible under acidic or basic conditions, "preferably weakly acidic conditions", as well as that "the conditions of temperature and pressure are not critical", and that "atmospheric pressure is sufficient, but that the reaction can be carried out also carried out under reduced pressure ”.
    [0027] Other patents also mention the use of hydrogen peroxide in an alkaline medium, but without considering the effect of pressure and using very high concentrations of peroxide (US5276138A, Yamada et al. 1994).
    [0029] As shown here and, contrary to what is expected according to the prior art, pressurization is a fundamental axis in the efficiency of the method object of the present invention and, therefore, it is of special interest.
    [0031] In addition, the procedures for the extraction or solubilization of keratins in oxidative media known in the state of the art, such as those mentioned above, lead to the obtaining of degraded or hydrolyzed keratins in the form of peptides of molecular sizes of the order of 1,000 daltons (Da) or frequently even lower, often with an appreciable proportion of free oligopeptides and amino acids. Small molecular masses are not optimal to achieve good mechanical properties, so it is desirable to minimize hydrolysis and degradation of proteins.
    [0033] On the other hand, other types of keratin extraction procedure are based on reducing media, and lead to keratins that are little modified with respect to the native forms, but that present very little solubility in aqueous medium, so they are not good candidates for preparation. of bioplastics.
    [0035] Regarding the use of protein derivatives in the formation of plastic materials, the state of the art describes the application of proteins in the biomedical area to form films by reducing their bisulfide bonds and subsequent evaporation of the solvent used (US6110487A). There are also patents that protect the formation of hydrogels, for biomedical purposes, from keratin hydrolysates (not oxidized derivatives, nor chemically functionalized) cross-linked with different cross-linking agents (WO2003086491B1). On the other hand, the use of specific proteins cross-linked with silicones in order to develop biocompatible films has also been described (US6989437B2). On the other hand, patent US2814851 protects the thermoplastic processing of reduced keratins and in the presence of different reducing agents and water. Similarly another patent (US2436156A) protects the use of reduced keratins to form films and fibers by coagulation in baths. Finally, patent US922692A protects keratinous materials with thermoplastic properties, although their mechanical properties are not defined as plastic. However, the use of some toxic crosslinkers such as reducing agents, silicones or acrylics results in materials that do not always have the characteristics of a hydrogel, mechanical properties and biocompatibility necessary for many applications.
    [0036] The object of the present invention is, therefore, to develop an improved keratin production procedure that allows the treatment of a greater quantity of treated substrate per unit mass of reagent in less time, avoiding the production of contaminating by-products and with yields. elevated.
    [0038] It is also the object of the present invention to obtain keratins with a molecular size greater than 10 KDa, which can be used to obtain bioplastics that can be processed by various routes such as coagulation or solvent evaporation, as well as thermally through techniques such as extrusion. , injection, compression molding, blown or electrospun.
    [0040] BRIEF DESCRIPTION OF THE FIGURES
    [0042] Figure 1 . Crosslinking of protein chains by alcoholysis and crosslinking of glycerol.
    [0044] Figure 2 . Comparison of the extraction methodology and solubility of the resulting keratins. Extraction by reduction and obtaining of keratins with a ) sulfhydryls (-SH) and reoxidation and reformation of b ) bisulfides ( RSSR) and the insolubility of the resulting keratins. Extraction by oxidation with H 2 O 2 and formation of soluble c ) sulphonates and d ) sulphinates.
    [0046] DETAILED DESCRIPTION
    [0048] A first aspect of the present invention describes the obtaining of keratins useful for the manufacture of protein bioplastics.
    [0050] For the purposes of the present invention, a protein bioplastic is a polymeric material with plastic properties that is obtained from natural protein derivatives.
    [0052] Said obtaining procedure is carried out from waste, preferably agro-industrial waste, rich in keratinous materials derived from the horn proteins of animals (hair, wool, feathers, horns and hooves) that undergo an oxidation process under pressure conditions - autogenous or forced.
    [0054] Thus, a first aspect of the present invention refers to a process for obtaining keratin comprising:
    [0056] i. introduce the residue in a container with an aqueous solution of H 2 O 2 with a concentration of 0.05 M to 0.50 M at pH between 8 and 11, so that the residue and H 2 O 2 are in a range of mass ratio of at least 5: 1;
    [0057] ii. increase the pressure of the container to more than 1 atmosphere and heat homogeneously between 40 ° C and 70 ° C for 0.5 to 6 hours;
    [0058] iii. Separate the remaining solid to obtain a keratin rich solution comprising a mixture of keratin polypeptides, oligopeptides and peptides.
    [0060] The process for obtaining keratins of the invention is characterized, therefore, by using an aqueous medium at a basic pH, using low concentrations of hydrogen peroxide as oxidant in a pressurized reactor.
    [0062] In the process for obtaining keratins of the invention, hydrogen peroxide is preferably used in concentrations in the range 0.1-0.3 M (0.3-0.9%) in a basic medium (pH between 8 and 11) in Pressurized vessel conditions for efficient dissolution of keratins from wool, feathers, animal hair and other keratinous debris.
    [0063] The pressure can be autogenous, by sealing the container, so that the temperature and the release of O 2 and other gases and vapors increase the pressure, or controlled by any external means including valves and other mechanisms.
    [0065] In a preferred embodiment, in step (ii) the pressure is increased by sealing the container. In this way, the oxygen created during the reaction, by the decomposition of the peroxide, and the vapors generated, increase and maintain the pressure inside. This results in yields equivalent to those obtained without pressurization, but with times of greatly reduced reaction.
    [0067] In another embodiment, in step (ii) the pressure is increased by means of an external action, by means of an external compressor and a pressure regulating valve
    [0069] In a preferred embodiment, in step (ii) the container is heated homogeneously by constant mechanical stirring.
    [0071] The reaction temperature can range from 40-70 ° C, although temperatures around 50 ° C are normally used. Under pressurized conditions, the decomposition of peroxide into oxygen is minimized, and its reaction with keratin is favored, minimizing around 10 times the amount of peroxide required for the dissolution and extraction of keratin, as can be deduced from of the examples.
    [0073] The solid residue can be separated, according to step (iii) of the process of the invention, by a simple operation such as filtration or decantation.
    [0075] The process for obtaining keratin of the present invention allows the use of various residues, preferably of agro-industrial origin. Specifically waste agroindustrial products from animals such as hair, wool, horns, feathers, eggshells, skin, nails, claws, or a mixture of these.
    [0077] In a preferred embodiment, the agro-industrial waste comprising at least 75% by weight of keratin is washed and ground, prior to its introduction into the container in step (i).
    [0079] In a preferred embodiment, washing and grinding the residue comprises
    [0081] - wash the residue with soap and water;
    [0082] - rinse with water,
    [0083] - dry at a temperature between 35 ° C and 60 ° C, and
    [0084] - grind by mechanical means.
    [0086] In a preferred embodiment, the process for obtaining keratin according to the invention further comprises:
    [0088] iv. subjecting the keratin-rich solution, obtained in step (iii), to acidification to a pH of between 2.5 and 4.5 causing the keratin to precipitate; v. separate the precipitated keratin by decantation.
    [0090] In a more preferred embodiment, the process for obtaining keratin according to the invention further comprises:
    [0092] saw. concentrating the precipitated keratin obtained in step (v) by washing with an aqueous solution comprising a water-miscible organic solvent;
    [0093] vii. precipitate in an acid medium with a pH between 2.5 and 4.5;
    [0094] viii. optionally repeat steps (vi) and (vii).
    [0096] The water miscible organic solvent is a polar protic or aprotic solvent. Preferably said water miscible organic solvent is a short chain alcohol, a ketone or an ether. Most preferably, the water-miscible organic solvent is acetone, tetrahydrofuran, or a C1-C6 alcohol. Even more preferably, the water-miscible organic solvent is acetone, tetrahydrofuran, ethanol, isopropanol, or a mixture of these.
    [0098] In particular, in a more preferred embodiment, the water-miscible organic solvent used is isopropanol or ethanol, or a mixture of both, in a percentage comprised between 25% and 50% (v / v) in the aqueous solution.
    [0100] Hydrogen peroxide can maintain a certain oxidizing power in a basic medium, especially under pressurized conditions, before decomposing, for a sufficient reaction time (between 0.5 and 6 hours, and preferably around 2 hours) to oxidize the bisulfides and promote the dissolution of keratin which, as detailed below, can be obtained in high yields.
    [0102] In the process for obtaining keratins, which are presented here at high proportions of solubilized but minimally degraded keratin, presenting fractions with molecular masses between 10 and 30 KDa, according to polyacrylamide gel electrophoresis tests. This minimal degradation of keratin, manifested by the high molecular weight of its dissolved forms, is another difference from commonly used keratin solubilization techniques. In this way, the main starting reagent is hydrogen peroxide (hydrogen peroxide, H 2 O 2 ) which is used in low concentration.
    [0104] H 2 O 2 is considered one of the attractive reagents by the industry due to its clean nature, as it does not release reaction by-products. The pressurization of the container where the keratin extraction is carried out is a key element in the efficiency of the process with the proposed reagents and conditions, an efficiency much higher than those previously described. Another key point of this procedure for obtaining keratins is that the keratin purification processes are minimized as there are no toxic by-products such as sulfur derivatives. With this procedure, therefore, lengthy dialysis processes can be avoided.
    [0106] The process for obtaining keratins of the present invention provides an oxidation of the bisulfide bonds of keratinous materials (KSSK, where K is a keratin peptide radical) with hydrogen peroxide (H 2 O 2 ). The keratins thus obtained tend to solubilize in an alkaline medium, due to the breaking of the salt bonds and a partial hydrolysis of peptide bonds (Goddart et al. 1934).
    [0108] Specifically, in a basic medium, H 2 O 2 tends to decompose according to the following reactions (Gutiérrez-Ríos, 1994a):
    [0110] H; 0; 4 O H "- * H; 0 4 HO;" (1)
    [0112] 2HO: ‘- + 20H" 4 0; (2)
    [0114] Y
    [0116] 3HO: "4 H; 0 4 2e ~ - * 3ÚH '
    [0118] On the other hand, sulfides (S2-) tend to oxidize in a basic medium, giving sulfites: (Gutiérrez-Ríos, 1994b):
    [0119] Thus, we can describe the oxidation process as follows:
    [0121] As can be seen, the advantage of this extraction route is that the main by-products of the reaction are water, oxygen and small amounts of hydroxyls. During the course of the reaction, the constant bubbling of O 2 is observed, which indicates that a significant part of the decomposition of H 2 O 2 occurs through reaction (2). Furthermore, experimentally an increase of 0.2-1.0 units in the pH scale is observed during the reaction, which reaffirms this hypothesis. When carrying out the extraction reaction in a pressurized medium, route (2) is avoided and the presence of oxidative radicals that attack the keratin is favored. This increases efficiency, allowing the treatment of a much larger mass of waste with a smaller peroxide can, reaching mass ratios of waste to be treated and peroxide of up to 12: 1, as demonstrated in the examples.
    [0123] In addition to the advantages indicated regarding the extraction of keratin since it is a very clean method, another advantage of the process of the invention is the solubility of the keratins obtained. On the contrary, keratins extracted by reductive methods tend to re-oxidize, again forming disulfide bonds, crosslinking the macromolecules and preventing the solubility of the keratins.
    [0125] On the contrary, the keratins obtainable by oxidation with H 2 O 2 in an alkaline medium according to the process of the invention, tend to maintain their solubility in an alkaline medium due to the incorporation of sulfonate and sulphinate groups.
    [0127] Thus, with the process of the invention, a greater quantity of treated substrate rich in keratin is achieved per unit mass of reagent (peroxide), with mass proportions of residue and reagent of at least 5: 1, in less time, avoiding the production of contaminating by-products and maintaining, however, high yields of at least 65% by weight of keratin obtained in relation to the total mass of waste treated.
    [0128] In addition, thanks to the reduction in reaction time, the keratins obtained have a molecular size greater than 10 KDa,
    [0130] Another aspect of the invention therefore relates to a keratin obtainable in the production process according to the invention.
    [0131] Preferably, the keratin obtainable in the production process according to the invention is characterized by being highly soluble and having a solubility greater than 40 mg / ml in alkaline aqueous medium with a pH between 9 and 11 and because the average molecular weight of the keratin in solution is between 10 and 30 kilodaltons. The keratins obtainable in the production process according to the invention have an isoelectric point similar to that of other keratins (at pH 4-5. They have a colloidal behavior at that pH, instantly obtaining, when the pH is lowered, their coagulation in the form of aggregates. flagelliform or powdery, suggesting a high molecular mass
    [0133] In the process for obtaining keratin according to the invention, unlike the techniques generally applied in the state of the art, the reaction product is found in slightly alkaline water (pH = 8-11) and comprises high proportions of keratin in dissolution. In addition, this procedure uses hydrogen peroxide (hydrogen peroxide, H 2 O 2 ) in low concentrations. H 2 O 2 is considered one of the reagents most used by the industry due to its clean nature, as it does not release reaction by-products.
    [0135] Another difference and advantage of the process for obtaining keratin according to the invention is that it minimizes the purification processes of the keratin obtained as there are no toxic by-products, such as sulfur derivatives.
    [0137] Furthermore, the keratins obtained in this procedure are very poorly degraded, presenting large molecular sizes (10-30 kDa), which makes them especially suitable and advantageous for use in the manufacture of bioplastic materials.
    [0139] The keratins obtained by the process of the invention, due to their very high solubility in aqueous medium and high molecular weights (between 10 and 30 kDa), are particularly advantageous for forming bioplastics by means of crosslinking techniques and the addition of agents. plasticizers described herein.
    [0141] Thus, another aspect of the invention refers to a method of manufacturing bioplastics obtained by means of chemical modifications of the keratins obtained, which facilitate their thermal processing, by means of chemical (macromolecular modification of keratin) and physical (plasticization) functionalizations.
    [0143] In this sense, some of the possible modifications that the keratins obtained in the procedure for obtaining the invention may undergo to produce bioplastic materials are:
    [0146] - additions of amino groups, and sulfur groups of keratins, to unsaturated carbonyl groups of crosslinking agents (such as aldehydes and ketones), or
    [0147] - addition of amino groups, and sulfur groups of keratins to unsaturated carbon-carbon groups, or
    [0148] - alcoholysis and ammonolysis reactions,
    [0149] - formation of ionic bonds through electrostatic interactions (ionic surfactant-protein type).
    [0151] More specifically, the bioplastics manufacturing methods of the invention include the reaction of terminal amino groups (K-NH 2 ) with unsaturations of oils, fats or fatty acids (R 1 R 2 = R 3 R 4 ), through the reactions type addition:
    [0153] K-NH 2 + RlR2 = R3R4 ^ K-NH- (R2) Rl-R3 (R4) H (6)
    [0155] Other modifications include partial crosslinking by Mannich addition, in which the reaction between amino groups of keratin (K 1 K 2 NH) with alpha carbons (K 3 K 4 -CH-CO-K 5 of other macromolecules) keratin, native or modified, through the mediating action of an aldehyde or ketone (R 1 -CO-R 2 ) through:
    [0157] K 1 K 2 NH R 1 -CO-R 2 + K 3 K 4 -CH-CO-K 5 ^ Ki K2N-C (Ri) (R2) -C (K3) (K4) -CO-K5. (7)
    [0159] This procedure is analogous to the one used in the past in the preparation of plastics derived from casein from milk using formaldehyde.
    [0161] One of those reactions that can give rise to protein bioplastics, such as those that are intended to be protected here, are the macromolecular extensions from oxidized keratins that can give rise to the reaction between amino groups of keratin (HNR 3 R 4 ) , sulfinitate residues (K-SO 2 Na), and ketones or aldehydes to give the crosslinks based on aminomethyl sulfones:
    [0163] K-SO 2 Na (R 1 -CO-R 2 + HNR 3 R 4 ) ^ K-SO 2 -CR 1 R 2 -NR 3 R 4 ) (8)
    [0165] Also oxidized keratins can react by condensation of sulfinic acids with aldehydes, in the presence of keratin peptide groups to give sulphonyl amides as described by Olijsma et al. (1967):
    [0167] K-SO 2 H (R 1 -CO-H R 2 HNCOR 3 ) ^ K-SO 2 -CHR 2 -NR 3 COR 4 ) (9)
    [0169] where K indicates the different parts of the keratin skeleton, and R the different chemical structures that give rise to these reactions.
    [0171] Other modifications of the keratins obtained in the process of the invention also include the formation of ionic bonds (for example, between quaternary ammonium groups of basic amino acids such as arginine and lysines with carboxylate groups from fatty acids; and / or between carboxylate groups of proteins and alkylammonium groups from surfactant agents).
    [0173] Finally, the keratins obtained in the process of the invention can also be modified by transesterification, ammonolysis or alcoholysis reactions, after a heat treatment, by means of which the peptide groups are attacked by hydroxyl from polyols, amines or other high-mass macromolecules. molecular. Figure 2 presents a diagram of the proposed chemical mechanism of action, as well as evidence of the impact of crosslinking on mechanical properties and solubility in water.
    [0175] Also a reason for protection in the present application is the bioplastics derived from the keratins and derivatives described above in combination with plasticizers, such as other biopolymers, such as natural polyesters or polyhydroxyalkanoates. Biopolymers that aim to improve their thermal processability, as well as their final properties.
    [0177] Therefore, the keratins obtained in the method for obtaining the invention can be chemically modified, to produce pellets and fibers of bioplastics that can be processed by means of common techniques in the plastics industry.
    [0179] Thus, another aspect of the invention relates to the use of keratins obtainable in the process for obtaining the invention for the manufacture of bioplastic.
    [0181] Another aspect of the invention, therefore, refers to a method of manufacturing a bioplastic comprising:
    [0183] to. dissolving the keratin obtained in the method of obtaining the invention in an aqueous solution with a pH between 9 and 11 that comprises at least one plasticizing agent and, optionally, at least one keratin crosslinking agent;
    [0184] b. coagulate the keratin to obtain said bioplastic.
    [0186] A plasticizing agent for the purposes of the present invention is defined as a substance or compound that causes the reaction of other substances or compounds causing the formation of a plastic.
    [0188] Crosslinking agent for the purposes of the present invention is defined as compounds that are capable of reacting with the keratin chains obtainable in the process of the present invention, forming a network of keratin chains.
    [0190] Cross-linking techniques include the use of reagents such as glycols or aldehydes (such as formaldehyde and glutaraldehyde). Convenient plasticizers include alcohols or polyalcohols, such as glycerin, as well as oils (such as sunflower oil, olive oil, etc.), fatty acids (such as oleic acid, succinic acid, etc.) and surfactants or surfactants (such as sodium dodecyl sulfate).
    [0192] In one embodiment the plasticizing agent is selected from the group consisting of a compound or substance that comprises hydroxyl groups, a compound or substance that comprises amino groups, a compound or substance that comprises carbon-carbon double bonds, a hydrophilic polymer, and a surfactant.
    [0194] In a preferred embodiment, the plasticizing agent is selected from the group consisting of a polyol, a polyether, a polyethylene or polypropylene polyoxide, a water-soluble polyurethane, an anionic surfactant, an oil, and a fatty acid.
    [0196] Preferably, in the method of manufacturing bioplastics according to the present invention, the plasticizing agent is a compound or substance comprising unsaturated carbon-carbon groups. Preferably, said compound or substance comprising unsaturated carbon-carbon groups is an oil or a fatty acid. Preferred plasticizers are sunflower oil, olive oil, oleic acid, succinic acid, among others.
    [0198] In another preferred embodiment, said plasticizing agent is a compound or substance that comprises hydroxyl groups. Preferably, said hydroxyl-comprising compound or substance is a polyol.
    [0200] Examples of glycols useful in the method of the invention are, among others: glycerol, sorbitol, ethylene glycol, 1,4-butanediol, 1,3-propanediol, poly (ethylene) glycol, poly (propylene) glycol (and their copolymers).
    [0202] In another preferred embodiment, said plasticizing agent is a compound or substance that comprises amino groups.
    [0204] In another preferred embodiment, said plasticizing agent is a surfactant. A preferred surfactant is sodium dodecyl sulfate
    [0206] In one embodiment of the invention, the plasticizer is a hydrophilic organic polymer. Examples of such polymers useful as plasticizing agent in the invention are, among others, polyethers, polyurethanes, polyacrylates, etc.
    [0208] In a preferred embodiment said crosslinking agent is a compound or substance comprising carbonyl groups or a sugar. Preferably, said crosslinking agent is an aldehyde, ketone, carboxylic acid or sugar.
    [0210] Examples of sugars useful in the method of the invention are, among others: glucose, fructose, mannose, sucrose and lactose.
    [0213] Examples of useful aldehydes in the method of the invention are, among others: formaldehyde, acetaldehyde and glutaraldehyde.
    [0215] Examples of ketone useful in the method of the invention are, among others: acetone, propanone, butanone, etc.
    [0217] Examples of carboxylic acids useful in the method of the invention are, among others: gallic acid, saturated fatty acids, etc.
    [0219] In a preferred embodiment the aqueous solution of step (a) comprises a hydroxide, more preferably sodium hydroxide.
    [0221] In a preferred embodiment of the method of manufacturing a bioplastic according to the invention, the coagulation of step (b) comprises spreading the solution obtained in step (a) in the form of a liquid sheet and subjecting it to evaporation of the solvent, to obtain said bioplastic in film form.
    [0223] The thickness of the liquid sheet will be adapted to the quantity to be produced and the final thickness of the bioplastic sought.
    [0225] In another preferred embodiment of the method of manufacturing a bioplastic according to the invention, the coagulation in step (b) comprises spinning the keratin from the solution obtained in step (a), by electrospinning, or by coagulation spinning in a coagulating medium, to obtain said bioplastic in the form of threads or pellets.
    [0227] The coagulating agent or medium can act in a different way, according to the method of the invention.
    [0229] In one embodiment the coagulating agent or medium is an aqueous solution comprising an agent with a very high cationic charge and a low molecular weight, in particular up to 1000 KDa. In this way the negative charges of the solubilized keratins are neutralized and their low molecular weight allows a rapid diffusion in the medium and around the solubilized keratin fractions.
    [0231] Examples of such a coagulation medium are aqueous solutions with quaternary polyamines, diallylmethylammonium chlorides and dicyandiamide resins.
    [0233] Also the coagulating medium can be comprised of a weak acid and viscosity modifying agents. Said viscosity modifiers are, in general, polymers that change viscosity through relaxation or contraction of polymer chains with changes in temperature.
    [0235] Thus, in another preferred embodiment of the method of manufacturing a bioplastic according to the invention, the coagulation of step (b) comprises co-precipitating the keratin, the agent plasticizer and, optionally, the crosslinking agent, from the solution obtained in step (a), by acidifying the medium to a pH between 2.5 and 4.5, to obtain said bioplastic in the form of a lump or mass.
    [0237] Preferably, the method of manufacturing a bioplastic according to the present invention further comprises subjecting the obtained bioplastic (whether in a laminar, thread or mass form) to a temperature above its melting point and then extruding it and cool it to obtain the desired bioplastic shape.
    [0239] In another preferred embodiment of the method of manufacturing a bioplastic according to the present invention, the bioplastic is subjected to a heat treatment at a temperature between 70 ° C and 100 ° C. In a preferred embodiment, said heat treatment lasts between 2 and 24 hours. This heat treatment favors the macromolecular extension and the modification of its hydrophobicity, degradability and mechanical properties.
    [0241] Another embodiment of the invention relates to a bioplastic obtainable by the manufacturing method of the invention described herein.
    [0243] Examples 3 and 4 show the obtaining of keratins by the process of the invention
    [0244] On the other hand, example 5 shows the manufacture of bioplastics with said keratins, by crosslinking with formaldehyde and acetone. Keratins treated with formaldehyde show the formation of amino-methylene bonds between terminal amino groups, as well as the formation of sulfo-amino-methylenic bonds, as described by Lujan-Montelongo et al. (2015) and Olijnsma et al. (1967) for low molecular mass compounds. The presence of these bonds would explain the decrease in solubility after treatment with formaldehyde. These materials are of interest in many fields such as the field of hydrogels for ophthalmology or as drying agents for biodegradable diapers.
    [0246] On the other hand, the reaction of the keratins obtained in the process of the invention with materials that comprise double bonds, such as fatty acids, makes it possible to modulate their aqueous solubility, intermolecular interactions and thermal processing.
    [0248] Finally, after treating the keratins obtainable according to the process of the invention with glycerol, water-insoluble bioplastics are obtained (Figure 1) that, in addition, have exceptional mechanical properties with tenacities of up to 19 MJ / m3.
    [0250] Thus, the procedure for obtaining keratins and the method for manufacturing bioplastics from them, described in the present invention, is summarized in: 1) Washing and separation of fibroin from lanolin; 2) Grinding the fibroin; 3) Oxidation and dissolution of fibroin; in reactors with autogenous pressure (either forced or controlled pressure, more than 1
    [0253] atmosphere, with suitable devices on the container) 4) Filtration of the protein solution from the remaining solid fibroin and 5) Precipitation of the protein. The process between stages 3 and 5 takes place in only two reactors (extractive separator and decanter), which simplifies the process. During the process (usually after step 4) or later, these proteins are chemically modified with crosslinking agents and / or extenders (aldehydes, ketones, etc.), or with reactive plasticizers (oils, unsaturated fatty acids, surfactants, etc. ) and physically mixed with other plasticizers (glycerol, sorbitol, gallic acid, water, mannoses, glucose, saturated fatty acids, polyethers, sucrose, polyacrylates, polyurethanes, etc.), in order to obtain materials that can be processed in bioplastics. The liquid and solid mixtures thus formed can be subjected to heat treatments in order to develop intermolecular interactions and extensions to improve the mechanical properties and modify their solubility.
    [0255] EXAMPLES
    [0257] Reference Example 1: This example is not representative of the invention, as it does not use a pressurized container. 100.2 g of chicken feather is washed with 20.5 g of liquid soap (Buga) immersed in 15 liters of water. After 16 h, rinse with a continuous stream of water. After decanting, it is dried in an oven at 50 ° C. The feather is ground in a mill (<1mm). 0.64 g of washed and ground pen are taken and placed in an 18 ml vial. 15 ml of 1M H 2 O 2 with pH 11 are added to obtain a 1.2: 1 weight ratio between the mass of wool treated and the mass of peroxide used. The vial is closed, fitted with a small hole, and brought to 50 ° C for 2 h. Homogenization is carried out with magnetic stirring and sporadic stirring. After this time, the vial is cooled, water is added, and filtered through filter paper, obtaining a solid residue of 22% of the initial plume, that is, a solubilization of 78%.
    [0259] Reference Example 2: This example is not representative of the invention, as it does not use a pressurized container. 102 g of manhair sheep's wool is washed with 21 g of liquid soap (Buga) immersed in 15 liters of water. After 16 h, rinse with a continuous stream of water. After decanting, it is dried in an oven at 50 ° C. The wool is ground in a mill (<1mm). 0.62 g of washed and shredded wool are taken and introduced into an 18 ml vial. 15 ml of 1M H 2 O 2 with pH 11 are added to obtain a 1.2: 1 weight ratio between the mass of wool treated and the mass of peroxide used. The vial is closed, fitted with a small hole, and brought to 50 ° C for 2 h. Homogenization is carried out with magnetic stirring and sporadic stirring. After this time the vial is cooled, water is added, and
    [0262] It filters through filter paper obtaining a solid residue of 28% of the initial wool, that is to say a solubilization of 72%.
    [0264] Example 3. Reaction under autogenous pressure. 0.85 g of washed and ground wool are taken and introduced into a 20 ml vial. 17 ml of a 0.29 M aqueous H 2 O 2 solution are introduced to obtain a 5: 1 weight ratio between the mass of wool treated and the mass of peroxide used, and a few drops of NaOH (0 , 1-0.2 ml, approx.) Concentrated until reaching pH 10. The vial is immediately closed and the mouth is sealed with hot-melt adhesive thus guaranteeing the pressurization of the container. The vial is placed in an oil bath at 50 ° C and kept immersed with sporadic shaking for 1h. The contents of the vial are diluted in 200 ml of water to stop the reaction and filtered. The content of the solid wool residue is weighed, resulting in 0.137 g, that is, a solubilization yield of 84%. This experiment shows that, compared to Example 2, very high yields can be achieved in a pressurized system by treating a higher mass of wool per unit mass of peroxide used.
    [0266] Example 4. Reaction under autogenous pressure. 0.85 g of washed and ground wool are taken and introduced into a 20 ml vial. 17 ml of an aqueous solution of H 2 O 2 at 0.12 M are introduced to obtain a 12.2: 1 weight ratio between the mass of wool treated and the mass of peroxide used, and a few drops of NaOH are added (0.1-0.2 ml, approx.) Concentrated until reaching pH 11. The vial is immediately closed and the mouth is sealed with hot-melt adhesive, thus guaranteeing the pressurization of the container. The vial is placed in an oil bath at 50 ° C and kept immersed with sporadic shaking for 1h. The contents of the vial are diluted in 200 ml of water to stop the reaction and filtered. The content of the solid wool residue is weighed, resulting in 0.293 g, ie a solubilization yield of 65%. This experiment shows that, compared to Example 2, very high yields can be achieved in a pressurized system by treating a higher mass of wool per unit mass of peroxide used.
    [0268] Example 5: A pure oxidized keratin film (125 mg) obtained according to example 4 is immersed in 40 ml of 37% formaldehyde and a drop of HCl (3N), to give a solution of pH = 2-3. The film begins to swell until it practically dissolves. After 24 hours, the solution is poured into a silicone mold and allowed to evaporate in a hood under a constant flow of air. A whitish precipitate is obtained corresponding to keratin partially crosslinked with formaldehyde. This keratin is less water soluble than the original keratin due to chain extensions and crosslinks mediated by formaldehyde to give sulfonamines, as well as those due to the classic reactions between amine groups and aldehydes.
    [0270] References
    [0272] Brown, EM; Pandya, K .; Taylor, MM; Liu, CK 2016 . Comparison of methods for extraction of keratin from waste wool . Agricult. Sci. 7, 670-679.
    [0274] Gacén-Guillen, J., 1964 . Chemical aspects of wool bleaching with hydrogen peroxide: Chemical modification of keratin . Bulletin of the Institute for Textile Research and Industrial Cooperation 39, 43-70.
    [0276] Goddard DR, Michaelis, L., 1934 . A study on Keratin . J. Biol. Chem. 106, 605-614.
    [0278] Gutiérrez-Ríos, E., 1994 a. Inorganic chemistry. chap. 33, Ed. Reverté, Barcelona (Spain), p.
    [0279] 809.
    [0281] Gutiérrez-Ríos, E., 1994 b. Inorganic Chemistry . chap. 21, Ed. Reverté, Barcelona (Spain), p.
    [0282] 472.
    [0284] Ma, B .; Hou, X .; Yang, Y. 2016 . Pure keratin membrane and fibers from chicken feather .
    [0285] Intern. J. biol. Macromol. 89, 614-621.
    [0287] Tonin, C .; Aluigi, A .; Vineis, C .; Varesano, A .; Montarsolo, A .; Ferrero, F. 2007 . Thermal and structural characterization of poly ( ethylene-oxide) / keratin blend films . J. Therm. Anal. Lime.
    [0288] 89, 601-608.
    [0290] Moore, GRP; Martelli, SM; Gandolfo, CA; Pires, ATN; Laurindo, JB 2006 . Keratin of frango penalties: extragao, characterize and obtain from films. Science. Tecnol. Aliment., Campinas, 26 (2), 421-427.
    [0292] Lujan-Montelongo, JA; Estevez, AO; Fleming, FF 2015 . Alkyl Sulfinates: Formal Nucleophiles for Synthesizing TosMIC Analogs . European J Org Chem. 7, 1602-1605
    [0293] Olijnsma, T .; Engberts, JBFN; Strating, J. 1967 . The Mannich condensation of sulfinic acid with aldehydes and carboxyamides, solfonamides, or lactams . Part IV. Rec. Trav. Chim. 86, 463-473.
    [0296] PATENTS
    [0298] - US922692A (1907). Byron B Goldsmith. Thermoplastic keratin composition.
    [0300] - US2436156A (1943). Upson Robert William (E I du Pont de Nemours and Co.).
    [0301] Preparation of shaped objects, filaments, and the like.
    [0303] - US2814851A (1953). Laurence R B Hervey (Rubberset Co). Keratin treating process and products thereof.
    [0305] - US4495173A (1985). Kinjiro Matsunaga, Takeo Okumura, Rikio Tsushima. Preshampoo type hair treatment composition.
    [0307] - US6110487A (1997). Scott F. Timmons, Cheryl R. Blanchard, Robert A. Smith.
    [0308] (Keraplast Technologies Ltd). Method of making porous keratin scaffolds and products of same.
    [0310] - WO2003086491B1 (2002). Dyke Mark E Van (Southwest Res Inst). Tissue defect dressings comprising a keratin network.
    [0312] - US6989437B2 (2002). Dyke Mark E Van (t Technologies Ltd). Methods for producing, films comprising, and methods for using heterogeneous crosslinked protein networks.
    [0313] - WO2003068204A1 (2002). Helmut Buschmann, Claudia Pütz (Grünenthal GmbH). Synthesis of substituted sulfonyl amines.
    one
    权利要求:
    Claims (21)
    [1]
    1. Procedure for obtaining keratin that includes:
    i. introducing a residue comprising at least 75% by weight of keratin in a container with an aqueous solution of H 2 O 2 at a concentration of 0.05M to 0.50M, at pH between 8 and 11, so that the washed residue and ground and e1H 2 O 2 are in a mass ratio range of at least 5: 1;
    ii. increase the pressure of the container to more than 1 atmosphere and heat evenly to between 40 ° C and 70 ° C, for 0.5 to 6 hours;
    iii. filter the remaining solid to obtain a keratin rich solution comprising a mixture of keratin polypeptides, keratin oligopeptides and keratin peptides.
    [2]
    2. Process for obtaining keratin according to claim 1, wherein in step (ii) the pressure is increased with the hermetic closure of the container.
    [3]
    3. Method for obtaining keratin according to claim 1 or 2, in which the agro-industrial waste is hair, wool, horns, feathers, eggshells, skin, nails, claws, or a mixture of these.
    [4]
    4. Process for obtaining keratin according to any one of claims 1 to 3, wherein in step (ii) the container is heated homogeneously by constant mechanical stirring.
    [5]
    5. Procedure for obtaining keratin according to any of claims 1 to 4, wherein prior to step (i) the residue is subjected to a process comprising:
    - wash the residue with soap and water;
    - rinse with water;
    - dry at a temperature between 35 ° C and 60 ° C; Y
    - grind by mechanical means.
    [6]
    6. Process for obtaining keratin according to any of claims 1 to 5, further comprising:
    iv. subjecting the solution rich in keratin, obtained in step (iii), to an acidification to a pH between 2.5 and 4.5 causing the precipitation of keratin;
    v. separate the precipitated keratin by decantation or cold filtration.
    2
    [7]
    7. Process for obtaining keratin according to claim 6, further comprising:
    saw. concentrating the precipitated keratin obtained in step (v) by washing with an aqueous solution comprising a water-miscible organic solvent; and vii. reprecipitate the keratin in an acid medium with a pH between 2.5 and 4.5;
    viii. optionally repeat steps (vi) and (vii).
    [8]
    8. The process for obtaining keratin according to claim 7, wherein the water-miscible organic solvent used is selected from the group consisting of acetone, tetrahydrofuran, and a C 1 -C 6 alcohol, or a mixture of these.
    [9]
    9. Keratin obtainable according to the method of any of claims 1 to 8.
    [10]
    10. Keratin according to claim 9, characterized by having a solubility greater than 40 mg / ml in alkaline aqueous medium with a pH between 9 and 11 and in that the average molecular weight of keratin in solution is between 10 and 30 kilodaltons.
    [11]
    11. Use of keratin according to any of claims 9 or 10 for the manufacture of bioplastics
    [12]
    12. Method of manufacturing a bioplastic comprising:
    to. adding a plasticizing agent and, optionally, at least one keratin crosslinking agent in an aqueous solution with a pH of between 9 and 11 comprising keratin obtainable according to the process of any of claims 1 to 8;
    b. coagulate the bioplastic obtained.
    [13]
    13. Method of manufacturing a bioplastic according to claim 12, wherein the aqueous solution comprises NaOH.
    [14]
    14. Method of manufacturing a bioplastic according to any of claims 12 or 13, wherein the plasticizing agent is selected from the group consisting of: a compound or substance comprising hydroxyl groups, a compound or substance comprising amino groups , a compound or substance comprising carbon-carbon double bonds, a hydrophilic polymer, and a surfactant.
    [15]
    15. Method of manufacturing a bioplastic according to any of claims 12 to 14, wherein the crosslinking agent is selected from the group consisting of: a compound or substance comprising carbonyl groups or a sugar.
    [16]
    16. Method of manufacturing a bioplastic according to any of claims 12 to 15, wherein the coagulation of step (b) comprises spreading the solution obtained in step (a) in the form of a liquid sheet and subjecting it to evaporation of the solvent, to obtain said bioplastic in the form of a film.
    [17]
    17. Method of manufacturing a bioplastic according to any one of claims 12 to 15, in which the coagulation of step (b) comprises spinning the keratin from the solution obtained in step (a), by electrospinning, or by spinning by coagulation in a coagulating liquid medium, to obtain said bioplastic in the form of threads or pellets.
    [18]
    18. Method of manufacturing a bioplastic according to any of claims 12 to 15, wherein the coagulation of step (b) comprises co-precipitating the keratin, the plasticizing agent and, optionally, the crosslinking agent, from the solution obtained in step (a), by acidification of the medium to a pH between 2.5 and 4.5, to obtain said bioplastic in bulk form.
    [19]
    19. Method of manufacturing a bioplastic according to any of claims 12 to 18, further comprising subjecting the bioplastic obtained to a temperature above its melting point and then extruding and cooling it to obtain the desired bioplastic shape.
    [20]
    20. A method of manufacturing a bioplastic according to any one of claims 12 to 19, wherein the bioplastic is subjected to a heat treatment at a temperature between 70 ° C and 100 ° C.
    [21]
    21. Bioplastic obtainable according to any one of claims 12 to 20.
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    同族专利:
    公开号 | 公开日
    ES2801025B2|2021-06-09|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO1990001023A1|1988-07-19|1990-02-08|American Biogenetics Corporation|Method for solubilizing keratinaceous materials using alkaline hydrogen peroxide solution|
    US5276138A|1990-09-17|1994-01-04|Kurashiki Boseki Kabushiki Kaisha|Process for solubilizing animal hair|
    RU2012131067A|2012-07-20|2014-01-27|Федеральное государственное бюджетное учреждение науки Институт биохимии им. А.Н. Баха Российской академии наук|METHOD FOR PRODUCING BIOPOLYMERS FROM HYDROLYSES OF KERATIN CONTAINING RAW MATERIALS AND BIOPOLYMERS OBTAINED BY THIS METHOD|
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