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WO2024246936A1 - Procédé de préparation de nano-peptides de kératine et son utilisation - Google Patents

Procédé de préparation de nano-peptides de kératine et son utilisation Download PDF

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Publication number
WO2024246936A1
WO2024246936A1 PCT/IN2024/050609 IN2024050609W WO2024246936A1 WO 2024246936 A1 WO2024246936 A1 WO 2024246936A1 IN 2024050609 W IN2024050609 W IN 2024050609W WO 2024246936 A1 WO2024246936 A1 WO 2024246936A1
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Prior art keywords
keratin
peptides
nano
keratin nano
peptide
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Mr Sumit BANERJEE
Dr Neha SINGH
Dr Jyoti GUPTA
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Helixion Biosciences Pvt Ltd
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Helixion Biosciences Pvt Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/465—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/10—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from hair, feathers, horn, skins, leather, bones, or the like
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/04—Animal proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/63—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from plants

Definitions

  • the present invention broadly relates to the field of biotechnology. More particularly, the present invention relates to a process for preparing water soluble and/or oil- soluble keratin nano-peptides and the water-soluble and/or oil soluble keratin nano-peptides obtained thereof. Further, the present invention also relates to the use of such keratin nano-peptides in nutraceutical, pharmaceutical and cosmetic industries. Moreover, the present invention also provides an enzyme cocktail for the hydrolysis of keratin from poultry feathers.
  • Keratin is the collective name for a family of tough proteins which are found in a number of structures. Keratin is of significant commercial value and is used in a number of industries especially the cosmetic, nutraceutical and pharmaceutical industry.
  • One source of keratin in particular — feathers — is produced in vast quantities by the poultry industry. Feathers represent from 5% to 7% of the body weight of chickens. These important by-products of the poultry industry are produced in millions of tons annually throughout the world.
  • Chicken feathers are approximately half feather fibre (barbs) and half quill (rachis) by weight. The quill is the stiff central core, to which the soft and interlocking fibres are branched. Both feather fibre and quill are made of keratin (about 90% by weight).
  • the keratin is an insoluble and highly durable protein found in hair, hoofs and horns of animals. Fibres from chicken feathers have several distinctive features such as: surface toughness, flexibility, high length to diameter ratio, hydrophobicity and a highly organized morphology characterized by its complex hierarchical structure. Further the protein fibres are effectively self-sustainable, biodegradable and continuously renewable due to their natural biopolymer origin.
  • Poultry feathers typically contain approximately 90% protein in the form of keratin.
  • Keratin a fibrous protein that is the main structural element of feather by-product is used in a wide range of industries, including food, textiles, pharmaceutical, cosmetics, leather, and poultry processing (Donato and Mija 2020; Sharma S, et., al, 2017; Lasekan A, 2013).
  • the a-keratin are mostly present in the hair, wool, horns, nails, claws, and hooves of mammals.
  • Poultry feathers typically contain approximately 90% protein in the form of P-keratin.
  • Keratin proteins have a high concentration of the sulphur-containing amino acid cysteine, which helps to create disulfide bridges between the individual molecules and contributes to the hard structure of keratin, thus, it can be challenging to digest and degrade. Therefore, the degradation of feathers can offer a low-cost source of easily absorbed protein and amino acids (McCasland and Richardson 1966). As a renewable and biodegradable biopolymer, this keratin could also be applied in a more sustainable way in value added applications by the conversion of the complex feather structure into a more appropriate form of small peptide and high-value compounds or products. Unfortunately, currently available methods to extract this nutrient are very expensive. Therefore, most of the feather waste streams are simply disposed of in landfills or through burning, which can cause negative environmental effects and reduce the sustainability of the primary commercial process.
  • “Shindai Method” is one of the most commonly used extraction protocols (Sinkiewicz 1 , 2017; Yin XC, 2013; Nakamura et al. 2002) in which chicken feather samples incubated with a specific buffer.
  • This process is time - consuming and costly due to the expensive chemicals but was predominantly used as the recovery yield of protein was high.
  • keratin can be extracted using several conventional methods such as thermal hydrolysis, chemical hydrolysis, enzymatic treatment, dissolution in ionic liquids, microwave technique, and steam explosion technique.
  • the procedures that are currently in use are complex and comparatively challenging since they use reducing agents and other chemicals for keratin extraction, followed by hydrolysis under acidic or alkaline conditions at high temperature.
  • These methods can be used effectively at the lab scale, but to scale up a process up to the pilot plant or industrial scale, factors like the costs of chemicals, elevated temperature and time required may become their limitations.
  • the existing methods mostly focus on reducing agents which cleaves disulfide bond (-S-S-) of the cystine residue to give a thiol group (-SH) in initial step.
  • fatty acid chlorides are toxic and corrosive in nature and hence, the existing methods which are mostly dependent on the use of acid chloride as acylating agents are not feasible options.
  • the reactions are carried out at high temperature of methyl esters (such as, for example, methyl myristate or methyl stearate) with various amino acids.
  • methyl esters such as, for example, methyl myristate or methyl stearate
  • these reactions are accompanied by decarboxylation and then amidation reactions, to result in the formation of fatty amides exhibiting the side chain of the amino acid, and thus in structures which are not those desired.
  • the use of a solvent comprising an alcohol functional group makes it probable that an ester will be formed as undesired byproduct.
  • a particular area of interest is the use of metal catalysts to form amides from a range of starting materials.
  • metal catalysts In addition to metal catalysts, non-metal compounds have also been found to catalyze amidation reactions successfully, with boronic acids and related compounds as the most common examples. As well as these general amidation methods there are also many protocols reported that are specific to the formation of acetamides.
  • acetamides usually involve the use of activated acylating agents, such as acetic anhydride and acetyl chloride. Although these reagents are cheap, their toxicity and hygroscopic nature make them less than ideal as acylating agents. Many procedures also use metal catalysts, both heterogeneous and homogeneous, which are often expensive and add further purification steps.
  • activated acylating agents such as acetic anhydride and acetyl chloride.
  • keratin can be extracted using several conventional methods such as thermal hydrolysis, chemical hydrolysis, enzymatic treatment, dissolution in ionic liquids, microwave technique, and steam explosion technique.
  • the procedures that are currently in use are complex and comparatively challenging since they use reducing agents and other chemicals for keratin extraction, followed by hydrolysis under acidic or alkaline conditions at high temperature.
  • These methods can be used effectively at the lab scale, but to scale up a process up to the pilot plant or industrial scale, factors like the costs of chemicals, elevated temperature and time required may become their limitations.
  • the existing methods mostly focus on reducing agents which cleaves disulfide bond (-S-S-) of the cystine residue to give a thiol group (-SH) in initial step.
  • the present inventors have devoted their significant efforts with the aim of a reliable, economical, and eco-friendly method for scalable production of keratin while retaining the functional properties of natural keratin as much as possible, from chicken feather waste.
  • the present invention resolves the problem of the conventional method, reduces the time of the completion of reaction, and improves the yield of the product.
  • the inventors of the present invention have developed a novel process that solves the aforementioned problems in the prior art by providing methods, and compositions that are economically viable for the acylation of keratin (e.g., in feathers) for the enzymatic hydrolysis of keratin (e.g., in feathers) compared to conventional methods and produce digestible and more palatable products.
  • the present invention is focused to achieve an eco-friendly, cost- effective method for the production of keratin peptides while sustaining the functional characteristics of natural keratin.
  • the present inventors have devoted their significant efforts with the aim to achieve a reliable, economical, and eco-friendly method for scalable production of keratin while retaining the functional properties of natural keratin as much as possible, from chicken feather waste.
  • the present invention resolves the problem of the conventional method, reduces the time of the completion of reaction, and improve the yield of the product.
  • Ob ject of the Invention provides a two-step process for the alkaline extraction of water-soluble keratin nano-peptides from poultry feathers.
  • An object of the present invention provides a process for the extraction of hydrolyzed keratin nano- peptides from poultry feathers.
  • An object of the present invention provides the water-soluble keratin nano-peptides obtained by the two-step process for the alkaline process.
  • An object of the present invention provides the oil-soluble keratin nano-peptides obtained by the process of the invention.
  • An object of the present invention provides the multilevel filtration process for obtaining the nano-keratin nano-peptide.
  • An object of the present invention provides a multi-level filtration system.
  • An object of the present invention provides a composition comprising the keratin nano-peptide along with pharmaceutically acceptable excipients, diluent and carriers.
  • An object of the present invention provides use of the composition as and when used in the preparation of tissue engineering scaffolds or for preparation of medicated creams or gels for management of 2nd and 3rd degree burns.
  • An object of the present invention provides the water-soluble keratin nano-peptide as and when employed in nutraceutical, pharmaceutical and cosmetics industry, or as an excipient for formulation in a drug delivery system.
  • An object of the present invention provides the oil-soluble keratin nano-peptide as and when employed in nutraceutical, pharmaceutical and cosmetics industry, or as an excipient for formulation in a drug delivery system.
  • FIG. 1 SDS-PAGE analysis of keratin hydrolysate and protein standard Marker. It shows wide range molecular weight marker (3.5 - 240 kDa) in Lane: M. Enzymatic hydrolysed peptides in Lanes 1, 2 &3. Keratin nano-peptides 1, 2 & 3 represents keratin hydrolysates prepared in 3 batches. These results show the molecular weight range of keratin hydrolysate protein between 3.5 to 30kDA.
  • FIG. 1 Thi n-layer chromatography analysis of the keratin hydrolysates.
  • FIG. 3 Mouse immunized with high dose of Keratin nano-peptides which received Evan’s Blue dye in the tail vein.
  • FIG. 4 Blood vessels in the mouse’s ear which are stained blue due to the dye injected in the tail vein (arrow pointing to the stained blood vessel)
  • Figure 5 The immunizing antigen is injected in the mouse’s ear.
  • Figure 6 The mouse’s ear did not show signs of dye leaking post injection of the immunizing agent (arrow pointing to the site of injection).
  • the antibody titre (dilution at which the response signal is indistinguishable from the background noise, i.e., when the signal is extinguished) in this case was 10’ 2 .
  • Figure 8 shows ELISA results of the mice immunized with the higher dose of 50 pg.
  • Figure 9 shows IgG and IgE responses elicited by immunization with 50 pg doses of OVA.
  • Figure 10A and 10B shows In vitro recall response to Keratin nano-peptides of (A): LN cells and (B): spleen cells from only one of four mice immunized with lower dose of Keratin nano- peptides.
  • Cells showed low-level and random proliferation in response to antigen challenge at concentrations ranging from 0.1 to 100 pg/ml.
  • the X-axis is logarithmic (logs).
  • the Y-axis shows indexed proliferation where no proliferation is indicated as 100% (dashed line; i.e., the cell population remained 100% of what was seeded in the well). The average of three replicates are plotted; standard deviation was above 50%, which is larger than the range of the means, and is not plotted as error bars.
  • Figure 12A and 12B In vitro recall response to OVA of (A): LN cells and (B): spleen cells from four mice immunized with the OVA. LN cells proliferated in response to antigen challenge at indicated concentrations. There was a statistically non- significant but dosedependent loss of viability of LN cells. The average proliferation index of four mice is plotted, error bars show standard deviation. The (+)ve control indicated that cells were live and responsive to mitogen challenge.
  • Figure 13 SEM images of effectiveness of Keratin Nano-peptides in repair of damaged hair with Fiberhance (commercially available product).
  • Figure 14 Particle size distribution keratin nano peptides sample in water.
  • the inventors of the present invention have developed a novel process wherein the first stage extraction of keratin is carried out under mild conditions to loosen tightly packed betasheet structure and efficiently break the peptide bonds of the keratin by enzymatic hydrolysis in second step.
  • the process reduces the requirements of chemicals and their cost that makes it economically and commercially viable.
  • the present invention provides a method for obtaining a good yield in a simple and easy process.
  • an aspect of the present invention provides a two-stage alkaline-enzymatic hydrolysis for the conversion of chicken feathers into keratin hydrolysate solution.
  • the inventors of the present invention have developed a significantly effective enzyme cocktail along with optimized process parameters in terms of temperature, pH and time, that helps in enhancing yield and reducing time of operations thus contributing heavily to making the process commercially workable.
  • Another aspect of the present invention offers a low-cost protease enzyme derived from fungal strain. This offers a distinct advantage over bacterial enzymes because fungi can grow on low-cost substrates and secrete large number of enzymes into culture medium which could ease downstream processing.
  • the present inventors have also developed a novel two- stage process for the production of oil-soluble keratin nano-peptides wherein the first stage comprises a two-step extraction method wherein extraction of keratin is carried out firstly under mild conditions to loosen tightly packed beta-sheet structure and secondly to efficiently break the peptide bonds of the keratin by enzymatic hydrolysis in the second step.
  • the second stage of the process comprises acylation of the enzyme hydrolyzed keratin nano-peptides obtained in the first stage of the process.
  • the process reduces the requirements of chemicals and their cost that makes it economically and commercially viable.
  • the present invention provides a method for obtaining a good yield in a simple and easy process.
  • the inventors of the present invention have developed a novel Water Phase Acylation technology by optimizing and standardizing the process where enzyme hydrolyzed keratin nano-peptides are acylated in the water phase itself making it effectively soluble in oil phase up to 25%.
  • the enzyme hydrolyzed keratin nano-peptides in water phase are the most effective and are in their natural form without much denaturation, that typically happens during conventional drying and converting to the powder format.
  • the technology developed by the present invention keeps nano-peptides in their most natural form.
  • this technology helps in completely removing the step of drying the product present in the conventional methods and brings down the operational complexity which in turn makes it commercially viable and competitive.
  • the inventors of the present invention have also developed an in house standardized and optimized Multilevel Filtration Technology that ensures that the obtained keratin peptides are standard product with a consistent size range of 0.5kDa to 30kDa. This is also critical in terms of final product functional delivery as nano peptides have a lot of function in pharmaceutical industry.
  • the multilevel filtration technology means a set of filtration system that has been designed in a way where the product after the hydrolysis process passes through this system developed by the inventors.
  • the system involves a strata of vortex filtration system in combination with multiple levels of micro and nano filtration systems placed in a way which not only helps concentrating the peptides but also helps in getting a concentrated peptides within a desired range of size in a consistent manner with a much better yield.
  • This system helps making a commercially viable and functionally deliverable product.
  • Another important aspect of the present invention relates to water soluble and oil soluble hydrolyzed keratin peptides with a wide size molecular weight range distribution from 500Da to 30kDA. This is the characteristic required in different industrial usage like cosmetics, pharmaceuticals and nutraceuticals.
  • An important embodiment of the present invention provides a process for preparing a keratin nano peptides, the process comprising the steps of: a. collecting poultry feathers, followed by hot water rinsing, sterilization and dewaxing to enhance the surface porosity of the feathers obtained in step (a); b. drying and grinding the feathers of step (a) to obtain a fine uniform powder; c. mixing the powder obtained in step (b) with an alkaline solvent and incubated at 45°C- 65°C for 1 to 3 hours in a blender with continuous stirring at 100-200 rpm to obtain the alkaline mixture; d.
  • processing step (e) comprises passing the post-enzyme hydrolysis extract obtained in step (d) through a multilevel filtration system to obtain water soluble keratin nano peptides.
  • the multilevel filtration system comprises the steps of passing the enzymatically treated alkaline mixture in a sequential manner through rotary filter, a microfiltration unit and finally through a nanofiltration unit to obtain purified keratin nano-peptides.
  • the processing step (e) comprises a) mixing the post-enzyme hydrolysis extract obtained in step (d) with a fatty acid ester in a vessel at 60-80°C overnight. b) adding a 2 nd fatty acid ester dissolved in an alcohol to the reaction mixture obtained in step (a) for 3-5 hours at temperature 60-80°C; c) raising the temperature to 80-90°C of the solution obtained in step (b) for 2- 6 hours; and d) filtering the solution obtained in step (c) to obtain the oil soluble keratin nano peptides.
  • a preferred embodiment of the present invention provides that the oil- soluble keratin nano-peptide obtained in step (d) is filtered with an inline filter membrane.
  • the enzyme cocktail of step (d) is an enzymatic concoction of at least two or more fungal or plant protease enzymes.
  • Another embodiment of the present invention provides that the two or more protease enzymes are in the concentration range of 0.2 to 3.5.
  • fungal protease enzymes are obtained from the group consisting of genera Aspergillus, Penicillium, or Rhizopus.
  • protease in enzyme cocktail is obtained from at least 2 proteases selected from aspartic, glutamic, metalloproteases, cysteine, serine, and threonine proteases or combinations thereof.
  • alkaline solvent of step (c) is selected from sodium hydroxide, potassium hydroxide and calcium hydroxide.
  • Another embodiment of the present invention provides that the alkaline solvent is in the range of 1-5%.
  • Yet another important embodiment of the present invention provides a multilevel filtration process for obtaining the nano-keratin nano- peptide, wherein the process comprises the steps of: a) passing the solution post enzyme hydrolysis extract obtained in step (e) of the process of preparing the keratin nano-peptides through a rotary filter having a sieve of at most 50p to obtain a solution free of any bigger size particles, b) passing the solution of step (a) through a microfiltration unit a ceramic filter of at most 0.2 microns and c) passing the extract from step (b) through a nanofiltration unit with a cut off of 300 kDa molecular level size to obtain the final concentrated product which is a water-soluble keratin nano peptides.
  • Yet another important embodiment of the present invention provides a multi-level filtration system comprising: a rotary filter (100) having a sieve size of 50 microns; microfiltration unit having a sieve size of .02 microns; and nanofiltration unit having a sieve size of a minimum of 300 kDa; wherein the concentrate is transported from the feed storage tank (101) through a recirculation pump (102) to the ceramic membrane system (103); the permeate is collected in the permeate tank (104) and transported by the feed pump (105) to the MCF (106); the nanofiltration high pressure pump (107) transfers the permeate to the nanofiltraton membrane system (108) and the concentrate is collected; the permeate is then stored in a permeate storage tank (109).
  • Yet another important embodiment of the present invention provides a water-soluble keratin nano- peptide obtained from the process for preparing the keratin nano peptides wherein the keratin nano- peptide has a molecular weight range of 500Da to 30kDa.
  • Yet another important embodiment of the present invention provides an oil-soluble keratin nano- peptide obtained from the process for preparing the keratin nano peptides wherein the keratin nano- peptide has a molecular weight range of 500Da to 30kDa.
  • a preferred embodiment of the present invention provides that the size of the keratin nano-peptides is in the range of l-5nm.
  • compositions comprising the keratin nano-peptide along with pharmaceutically acceptable excipients, diluent and carriers.
  • the pharmaceutically acceptable excipients or carriers are any selected from disaccharides, polysaccharides, sugar alcohol, gum, humectants, emulsifiers, emollients, preservatives, water, cellulose starch, maltodextrin, essential oils or combinations thereof.
  • composition is formulated in the form of shampoos, serums, hair and scalp care products, supplements and nutraceutical formulation.
  • Yet another important embodiment of the present invention provides the keratin nano- peptide as and when employed in nutraceutical, pharmaceutical and cosmetics industry, or as an excipient for formulation in a drug delivery system.
  • compositions as and when used in the preparation of tissue engineering scaffolds or for preparation of medicated creams or gels for management of 2nd and 3rd degree bums.
  • Another embodiment of the present invention provides that the keratin nano particle as and when used in biomaterials such as sponges, hydrogels, wound patches, films, and fibres which have been created for biomedical applications.
  • Another embodiment of the present invention provides that the keratin nano particle as and when used in the field of nanoparticle medication delivery wherein the protein polymers and protein composite materials are suitable for drug delivery systems and can improve the controller release or targeting mechanism.
  • Another embodiment of the present invention provides that the keratin nano particle is used as a protein supplement.
  • Example 1 Production of water-soluble keratin peptides from poultry feathers
  • feathers from chicken were gathered.
  • the feathers were washed and then the amino acid composition was determined by HPLC method.
  • Feathers from chicken were gathered from either poultry processing industry or from the poultry Farms. Rinsing the feathers with hot water followed by sterilization and dewaxing.
  • the feathers are dewaxed using surfactants such as sodium carbonate and sodium hypochlorite combination as a self- standardized ratio at 36.5 to 40.5°celsius. This was done at selfstandardized feather to surfactants and hypo solution ratio at self- standardized exposure time that helps in expediting the future reactions.
  • Keratin extraction from the feathers was carried out by two-stage alkaline process.
  • feathers were mixed with sodium hydroxide and incubated at 45°C- 65°C for 1 to 3 h in a blender with continuous stirring at 100-200 rpm to obtain the hydrolysate solution.
  • Sodium hydroxide was taken in the range of 1-5%.
  • This solution was then treated with a proteolytic enzyme concoction were added at 37 °C- 50°C/ 3 to 5 h in a blender with agitator to obtain the keratin hydrolysate.
  • the concentration of keratin hydrolysate protein was 80.68%, calculated through nitrogen analysis, using Kjeldahl nitrogen determination method (Table- 1).
  • amino acid profile of the obtained keratin hydrolysate was also calculated as shown in Table 2.
  • concentration of amino acids in the keratin hydrolysate the picomoles of amino acid reported by the chromatographic software was divided by the injection volume. This value was then multiplied by the volume of diluent divided by the volume derivatized.
  • Table-2 Amino acid profile of Keratin hydrolysate.
  • step (i) Sixty volumes of keratin hydrolysate obtained in step (i) was mixed with 30 volumes of a fatty acid ester i.e., 30% Isopropyl palmitate at 60-80°C and stirred overnight. Further, another fatty acid ester i.e., 10% Isopropyl myristate, was added to reaction mixture for 3-8 h at temperature 60-80°C. The solution obtained was then filtered to get the oil soluble product. The yield of the oil soluble keratin nano-peptides was 85-95% by using the method of the present invention.
  • the keratin hydrolysate of example 1 was prepared to analyse its weight and molecular mass. After the hydrolysis step, the obtained keratin protein was broken into small peptides. The size range of peptide was determined through SDS PAGE.
  • Figure 1 shows wide range molecular weight marker (3.5 - 240 kDa) in Lane: M. Enzymatic hydrolysed peptides in Lanes 1, 2 &3. Keratin nano-peptides 1, 2 & 3 represents keratin hydrolysates prepared in 3 batches. These results show the molecular weight range of keratin hydrolysate protein between 3.5 to 30kDA. Further, analysis of the enzymatic keratin hydrolysates was carried out using thin-layer chromatography. The nano-peptides with a lower molecular mass were observed as shown in Figure 2, while no streak was observed in unhydrolyzed keratin.
  • Example 4 Multi-level filtration of the keratin hydrolysate to obtain the keratin nano peptides
  • the inventors of the present invention have developed and optimized a novel multilevel filtration mechanism which post carrying out the hydrolysis as described in example 2, the separation and concentration of “Nanopeptides/Nano-actives” which were the desired final product parameters in terms of their molecular size in the nanoparticle range (500 Dalton to 30 kDA) was performed using the multi-level filtration system.
  • post enzyme hydrolysis extract was first passed through a rotary filtration system where bigger size particles were removed while the extract passed through 50-micron sieve. The filtrate was then in continuity passed through the microfiltration unit comprising a ceramic filter of 0.2 microns. Finally, the extract was passed through a nano-filtration unit with a cut off of 300 kDa molecular level size. Here, the hydrolyzed extract was concentrated to the final product which was a water soluble keratin nano-peptides.
  • This filtration chain was developed, standardized and operationally optimized to make it a commercially successful process to concentrate “Nanopeptides/nanoactives” which have high biocompatibility/ high biological activity & enhanced bio-functionality.
  • Example 5 Efficiency of keratin production by the proposed method on comparison with conventionally known methods
  • the concentration of the test item- Keratin Nano-peptides was expressed in weight-based measures with the maximum target in-well concentration set to lOOug/ml.
  • the test item was soluble in water.
  • cytotoxicity assessment For the cytotoxicity assessment, stock solutions in a concentration range were prepared by serial dilution and finally applied concentration ranged from 100 ug/ml to 5.9 ug/ml. Cells were incubated with the test item under standard conditions. After exposure, the cells were stained with pro-indium iodide and cell viability was measured by flow cytometry analysis. Following cellular stimulations, RNA was isolated and endpoint measurements were performed using GARDskin GPS.
  • test item was assayed starting from 100 ug/ml.
  • results of the cytotoxicity assessment are presented in table 4. No cytotoxicity was observed.
  • Table 4 Test item cytotoxicity assessment iii. Phenotypic Quality Control
  • Table 5 The expression of the phenotypic biomarkers in the cell cultures used for the experiments iv. Relative Viability
  • test item The relative viability of the test item, reference item and absolute viability of the unstimulated control were done and presented in table 6. All samples passed the acceptance criteria.
  • RNA Quality Control The concentration was based on maximum solubility and cytotoxicity and the observed viability was calculated based on equation in the Test method. Lastly, the absolute viability is in percentage. v. RNA Quality Control
  • RNA quality control was performed in-house by standard methods. The results are presented in table 7 below. All samples passed the acceptance criteria.
  • the test item (Keratin nano-peptide) was freely soluble at the maximum in well concentration of lOOug/ml, and no solubility issues were reported. The keratin nano-peptide did not induce cytotoxicity.
  • the positive and negative controls were correctly classified as skin sensitizer and skin non- sensitizer, respectively, in the GARDskin assay. In addition, all specified acceptance criteria were fulfilled in this study, resulting in a valid study.
  • ACA Active Cutaneous Anaphylaxis
  • IgE Immunoglobulin E
  • mice 8-week-old, 22-25g male BALB/c mice were obtained from the Laboratory Animals Facility at CSIR-CDRI after receiving permission to conduct the experiments from the Institutional Animal Ethics Committee.
  • Table 10 provides the details of the control and experimental group of mice as per the experiments performed.
  • the tissue sample was placed in 1 ml of dimethyl acetamide in 1.5 ml Eppendorff tubes and incubated at 60°C on a dry heating block for 2 hours.
  • the optical density at 608 nm was recorded against a blank which was prepared by extracting the ear pinna of another mouse sacrificed as untreated control for an unrelated experiment.
  • the popliteal and inguinal lymph nodes (LN) and spleen were harvested aseptically at necropsy.
  • Single-cell suspensions were prepared using hand-held ground glass tissue homogenizers. The cell suspensions were washed by centrifugation in sterile PBS. Erythrocytes were lysed by distilled water osmotic shock, and splenocytes/LN cells were recovered by centrifugation. They were re-suspended in RPML1640 medium purchased from Thermo Fisher Scientific, Cat. No. 11875085 containing 10% FBS. Cells were plated in 96- well culture plates at a density of 105 cells/well. Cells were exposed to keratin nano peptides and OVA at indicated concentrations overnight at 37°C in 5% CO2 incubator.
  • Concanavalin A a T-cell mitogen
  • a fluorescence-based cell proliferation assay kit (Cell Census, Sigma) was used as per the manufacturer’s instructions to estimate cell numbers in different wells after overnight incubation. The numbers did not change appreciably over negative control (no antigen challenge), except in the case of the Concanavalin A positive control.
  • Figure 4 shows a mouse immunized with the higher dose of keratin nano peptides after Evan’s Blue dye was injected in the tail vein.
  • the dye in the blood circulatory system imparted a blueish color to the blood vessels visible in the ear of the mouse, as shown in Figure 5.
  • the ear tissue itself remained pink and clear of any kind of blue stain prior to the injection of the immunizing or irrelevant antigen in the marginal ear vein.
  • mice ear was injected with lOpg of the immunizing antigen (keratin nano peptides , OVA or sterile PBS Negative Control), without adjuvant in the marginal ear vein of one ear ( Figure 6).
  • the animal was observed for 10 minutes.
  • the immunizing antigen was injected subcutaneously in the same ear and observed for another 10 minutes.
  • the irrelevant antigen OVA in mice immunized with keratin nano peptides , and vice-versa
  • the blood vessels become leaky, and the skin of the ear stains blue.
  • Alum-Adsorbed Keratin nano-peptides was a Moderate B-cell Immunogen and Allergen Enzyme-Linked Immunosorbent Assay (ELISA) was performed to study whether alum adsorbed OVA and keratin nano peptides would evoke a B cell response.
  • the ELISA was designed to capture antibodies specific to keratin nano peptides or OVA present in the blood serum of immunized mice. Accordingly, wells in the ELISA plate were coated by overnight incubation with either keratin nano peptides or OVA (lOOpg/ml) at pH 9.6. The coating solution was discarded and residual sites on the plastic surface were blocked with 5% fat- free milk.
  • Mouse serum was diluted over a range of 1:100- 1:100,000 and added to the wells for 2 hours at room temperature.
  • the wells were washed and a secondary antibody conjugated to the enzyme horseradish peroxidase (HRP) was added to the wells.
  • the secondary antibody was Ig class- specific: one antibody-enzyme conjugate would bind only mouse IgG and the other, only mouse IgE.
  • HRP horseradish peroxidase
  • the antibody titer in the mice was close to 10 6 , which may be considered as “moderate” in comparison to values as high as 10 9 -10 12 obtained by active immunization with strong immunogens. It is to be noted, however, that the apparent differences in OD produced by IgG and IgE antibodies respectively between dilutions of 10 3 -10 5 suggested that more IgE was produced by the immunization protocol.
  • keratin nano peptides is moderately immunogenic and allergenic.
  • the inventors carried out parallel experiments using chicken egg albumin or OVA as the immunizing antigen. Only the higher dose-level was tested. It is important to note that the antibody conjugates used for revealing captured antibodies to OA and keratin nano peptides were the same.
  • Figure 10 illustrates the IgG and IgE responses elicited by immunization with OVA.
  • the antibody titer in the mice was about 3xl0 5 .
  • the magnitude of the IgG and IgE response was comparable.
  • the data did not provide information about any putative differences between the magnitude of the IgG versus IgE response; and only permit the conclusion that OVA is also a “moderate” immunogen for evoking IgG and IgE responses from B cells.
  • Table 9 shows the mean comparison of the IgE responses to different doses of keratin nano peptides and OVA. It was evident that there was no statistically significant difference between animals immunized with low versus high doses of keratin nano peptides ; despite the higher dose eliciting a stronger response. However, there was a statistically significant difference between the response elicited by (the high dose of) OVA when compared with both the low and the high dose of keratin nano peptides . Further the magnitude of the difference was dose-dependent, and OVA elicited a higher (allergenic) response than keratin nano peptides at equivalent dose. Population variances, however, were also significantly different with the F value being 7.4095 and the Probability > F 0.00284.
  • lymph node (LN) cells and splenocytes recovered from the same mice as described above were exposed to different concentrations of the immunizing antigen, or to the T-cell mitogen Concanavalin A.
  • the in vitro antigen recall response was estimated by a fluorimetric assay of cell proliferation, keratin nano peptides at both low and high doses and OVA elicited a weak recall of the respective antigens.
  • Figures 11-13 depict the results graphically.
  • Both OVA and keratin nano peptides elicited moderate antibody responses; with OVA generating more antibodies of the immunoglobulin E (IgE) sub-type that is indicative of allergy. It was concluded that keratin nano peptides had lower allergenic potential than egg albumin. Both OVA and keratin nano peptides elicited weak-to-negligible T-cell responses in assays to estimate the ability of T lymphocytes recovered from immunized mice to ‘recall’ the immunizing antigen. It was concluded that keratin nano peptides is a weak T cell antigen. This conclusion was supported by observations of the antibody response, indicating that T cell help was not sufficient to enable a robust antibody response.
  • IgE immunoglobulin E
  • keratin nano peptides is hypoallergenic, with lower allergenic potential than OVA.
  • keratin nano peptides is hypoallergenic, with lower allergenic potential than OVA.
  • Example 8 Determination of the efficacy of the Keratin Nano-pep tides on hair. Due to the low molecular weight and the small size, the hydrolyzed keratin Nano Peptides go beyond the cuticle, penetrate the hair shaft and reduce damage. When the broken-down protein fills these microscopic gaps, hair gets the strength to minimize damage from chemical treatments, heat styling, mechanical manipulation and the sun. Usually, when hair undergoes chemical processes, the amino acid cysteine breaks down and results in weakened and damaged hair. Keratin Nano-Peptides is known to replace this lost cysteine. Hydrolyzed keratin Nano- Peptides increases cysteine content, which increases tensile strength and minimizes damage. The following study was done to evaluate the efficacy of active biomolecule - keratin nanopeptide on Sensory parameters and effectiveness in repair of damaged hair by analyzing hair texture through SEM, Hair breakage and reduction in split ends.
  • Test Group 1 Test Group (1) active biomolecule - keratin nano-peptide versus Control - 6 tresses.
  • Group 2 Control - 6 tresses i. Pre-treatment of Hair tresses
  • Damaging solution was prepared by adding 9% H2O2 (Quantity to be added will be double as H2O2 is 50% active); 20% Ammonia Solution (Please note: 20% of (25-28)% Active Ammonia Solution, pH around 10.5).
  • Hair tresses were dipped in this damaging solution for 1 hour. Hair tresses were removed and rinsed thoroughly with distilled/tap water of 40°C for 5 minutes to wash off any residue of Ammonia and Hydrogen Peroxide. Hair tresses were Tissue/ Towel dried and combed again gently to get rid of loose hairs. iii. Baseline Evaluations before treatment for all the groups: The hair tresses were kept at 30°C/65% RH for approximately 18 hours to condition them. There were two groups Test and Control. Baseline evaluation for Hair Sensory Attributes (Detangling, Combability, Frizz, Visual Shine, Smoothness, Softness and Bounce) was done by 6 experts.
  • the inventors have developed a medicated cream or gel for management of 2nd and 3rd degree burns that would be non-inferior to the Standard of Care (SoC).
  • the cream comprises either the water and/or oil-soluble keratin nano peptides for preparation of tissue engineering scaffolds that are readily colonized by proliferating fibroblasts, chondrocytes, cardiomyocytes and epithelial cells as prepared in examples 1 and 2.
  • Figure 14 shows the particle size distribution of keratin nano peptides sample in water. The results demonstrate that the hydrolyzed keratin nano-peptide fragments are distributed around a median diameter of approximately 120nm.
  • a method comprising steps a, b, and c encompasses a method of steps a, b, x, and c, a method of steps a, b, c, and x, as well as a method of steps x, a, b, and c.
  • a method comprising steps a, b, and c encompasses, for example, a method of performing steps in the order of steps a, c, and b, the order of steps c, b, and a, and the order of steps c, a, and b, etc.
  • the terms "about” or “approximately” are used herein to mean approximately, in the region of, roughly, or around.

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Abstract

Un procédé de préparation de nano-peptides de kératine et son utilisation. La présente invention concerne d'une manière générale le domaine de la biotechnologie. Plus particulièrement, la présente invention concerne un procédé de préparation de nano-peptides de kératine solubles dans l'eau et/ou solubles dans l'huile et les nano-peptides de kératine solubles dans l'eau et/ou solubles dans l'huile obtenus à partir de ceux-ci. En outre, la présente invention concerne également l'utilisation de tels nano-peptides de kératine dans les industries nutraceutiques, pharmaceutiques et cosmétiques. De plus, la présente invention concerne également un cocktail enzymatique pour l'hydrolyse de kératine à partir de plumes de volaille.
PCT/IN2024/050609 2023-05-26 2024-05-24 Procédé de préparation de nano-peptides de kératine et son utilisation Pending WO2024246936A1 (fr)

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CN104798981A (zh) * 2015-03-17 2015-07-29 齐鲁工业大学 一种利用碱性蛋白酶/角蛋白酶制备羽毛蛋白粉的方法
US20180263259A1 (en) * 2015-01-07 2018-09-20 Dupont Nutrition Biosciences Aps Methods and uses for keratin hydrolysis
CN109965086A (zh) * 2019-05-14 2019-07-05 华中农业大学 一种水解羽毛粉添加剂及其在动物饲料中的应用
WO2020079133A1 (fr) * 2018-10-17 2020-04-23 Bioextrax Ab Procédé de production de microfibres de kératine et d'hydrolysat de protéines à partir de plumes de volaille par hydrolyse microbienne
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US20180263259A1 (en) * 2015-01-07 2018-09-20 Dupont Nutrition Biosciences Aps Methods and uses for keratin hydrolysis
CN104798981A (zh) * 2015-03-17 2015-07-29 齐鲁工业大学 一种利用碱性蛋白酶/角蛋白酶制备羽毛蛋白粉的方法
WO2020079133A1 (fr) * 2018-10-17 2020-04-23 Bioextrax Ab Procédé de production de microfibres de kératine et d'hydrolysat de protéines à partir de plumes de volaille par hydrolyse microbienne
CN109965086A (zh) * 2019-05-14 2019-07-05 华中农业大学 一种水解羽毛粉添加剂及其在动物饲料中的应用
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KIM ET AL.: "Effect of Enzymatic and Chemical Treatments on Feather Solubility and Digestibility", POULTRY SCIENCE, vol. 81, no. 1, 2002, pages 95 - 98, XP055130594 *
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