GREEN PRODUCTION METHOD OF KERATIN PEPTIDES FROM WASTE CHICKEN FEATHERS

Abstract

The invention relates to the production of water-soluble keratin peptides by subjecting waste chicken feathers to ultrasound-assisted enzymatic hydrolysis.

Description

1 TARIFF GREEN PRODUCTION OF KERATIN PEPTIDES FROM WASTE CHICKEN FEATHERS METHOD Technical Field to Which the Invention Relates 5 The invention relates to food technology, biotechnology, and the transformation of waste into value-added products. It relates to areas of conversion, particularly ultrasonic-assisted conversion of waste chicken feathers. Obtaining water-soluble keratin peptides by enzymatic hydrolysis It is related to the invention. The keratin peptides produced within the scope of this invention are used in food supplements and nutraceuticals. The product is used in the production of functional foods and cosmetics. 10 State of the Art Keratin is a fibrous substance found in animal tissues that has high mechanical strength and Keratin is a structural protein with very low water solubility. Keratin is found in hair, fur, wool, nails, The basic protein in biological materials such as hooves, horns, and poultry feathers is 15 It constitutes one of its components. Keratin contains a high amount of cysteine. Due to the presence of amino acids, there are numerous disulfide bonds between protein chains. occurring; in addition, hydrogen bonds, hydrophobic interactions and Interchain cross-links cause keratin to exhibit a stable and resistant structure. This strong molecular structure increases the mechanical strength of keratin. together with keratin, dissolves in water by conventional proteolytic enzymes. This makes it difficult to break down and become biologically available. Therefore, keratin is used in food, dietary supplements, nutraceuticals, cosmetics, and pharmaceuticals. In order to be used in biomaterial and packaging applications, it must first be natural. its structure is broken down, made soluble or reduced to a lower molecular weight 25 It needs to be converted into peptides with a certain weight. Poultry farming and chicken meat production result in a significant amount of chicken. Feather waste occurs. Although the majority of chicken feathers are made of keratin. However, the direct use of these wastes results in keratin being insoluble and indigestible. Its resistance to wear is limited. Chicken feathers are used in some applications. It is burned, stored, or used in animal feed production as a low-value protein. considered as a source or from various physical and chemical processes They are disposed of by passing them through [processing]. However, uncontrolled storage and disposal 2 These processes lead to environmental problems, bad odor formation, microbial contamination, and This can lead to an increase in the organic waste load. Therefore, chicken feathers... high value-added protein, keratin, keratin hydrolysate or keratin peptide from waste materials Various methods have been developed to transform them into products. In the current state of the technique, keratin is extracted from feathers, wool, hair, and similar sources. The main methods used for separation are chemical, thermal, pressure-assisted, microbial, and Enzymatic methods are used. Keratin in chemical extraction methods. breaking of disulfide bonds in its structure and making protein chains soluble For the purpose of bringing about this, strong acids, bases, reducing agents, oxidizing agents, 10 Surfactants and chaotropic agents are used. Alkali hydrolysis These methods typically use strong solvents such as sodium hydroxide or potassium hydroxide. bases are used and keratin-containing material is maintained at high pH values ​​for a specific period. The material is subjected to heat treatment for a period of time. In these methods, the keratin structure can be broken down. and water-soluble protein fractions can be obtained, however, strong alkaline 15 These conditions lead to the degradation of some amino acids, resulting in undesirable changes in amino acid structures. changes occur and the nutritional properties of the product decrease This can be the reason. Additionally, pH adjustment and neutralization are necessary after the procedure. because it needs to be done, additional acid consumption and wastewater with high salt content This formation occurs. In acidic hydrolysis methods, hydrochloric acid and 20 using similar strong acids, keratin protein is broken down at high temperatures. This ensures their breakdown. Acidic hydrolysis effectively breaks down keratin chains. Although it can break down particles, it has long processing times, high temperature requirements, and corrosion resistance. such as the need for robust equipment and the necessity of neutralization at the end of the process. It has disadvantages. Also, as a result of intensive hydrolysis, large parts of the peptide structures are damaged. 25 It is possible for them to be broken down into free amino acids. This situation, a keratin hydrolysate rich in low molecular weight peptides is obtained This can produce an undesirable result in applications where it is intended to be achieved. The degradation of some amino acids during acid treatment can affect the color and of the product. changes in odor and 30 in products intended for oral use Negative impact on sensory perception is also among the known problems. In reduction-based keratin extraction methods, mercaptoethanol and sodium are used. bisulfite, sodium metabisulfite, cysteine, or similar compounds containing thiol groups 3 Reduction of disulfide bonds between keratin molecules using These processes can increase the solubility of keratin, however dialysis, filtration, precipitation or to remove the reducing agents used Multi-stage purification processes are required, especially for food or... 5% of products intended for oral consumption remain in the reaction medium. Removing chemical residues is important. In addition, reducing agents... in terms of cost of materials, environmental burden, unpleasant odor and process safety The challenges it creates can limit the industrial use of the method. In oxidative extraction methods, peracetic acid, hydrogen peroxide, or different 10 Oxidation of disulfide bonds in keratin using oxidizing agents and The aim is to make the protein soluble. Oxidative processes affect the protein. by enabling the unfolding of its structure, cysteine ​​and other sulfur-containing amino acids This can cause changes in its chemical structure. Therefore, the obtained The product has exactly the amino acid profile of the starting material (15). It may not be able to protect. The use of strong oxidizing agents can affect the reaction. control, equipment safety, chemical waste generation and product purification It also requires additional processing in terms of maintenance. In some applications, oxidative and reducing processes are used. The methods are applied sequentially or together, thus achieving higher keratin levels. The aim is to dissolve it to a certain extent. However, the chemical used in this approach is 20 The variety and number of process steps are increasing. In the current state of the art, ionic liquids, urea solutions, detergents, and chaotropic liquids are used. Methods for dissolving keratin using certain substances It is found there. Urea breaks the hydrogen bonds between protein chains and hydrophobic 25 It weakens the interactions, causing the keratin structure to open up. Sodium Surfactants such as dodecyl sulfate and ionic liquids also target keratin chains. It can increase its solubility. However, the solvents and auxiliary materials used may affect its solubility. Chemicals need to be removed from the final product. Especially in food. Control of solvent residues in nutraceutical and cosmetic applications is of great importance. 30 Because of this, these methods require extensive purification processes. This is heard in dialysis, membrane filtration, sedimentation and repeated washing, etc. These processes increase process time and production cost. 4 Hydrolysis of keratin-containing materials under high temperature and pressure. Methods involving feathers, wool, hair, hooves, or similar materials are also known. Keratinized materials are placed in a pressurized reactor in the presence of water and at a specific temperature. It is hydrolyzed under pressure and time conditions. Pressurized thermal treatment, keratin while being able to break down its structure and increase digestibility, 5 high energy consumption, the need for a specialized reactor, and precise process conditions. It is necessary to control the situation. Excessive temperature and processing time affect amino acids. acid degradation, darkening of product color, unwanted tastes and odors This can lead to the formation of... Also, the material obtained by these methods... It may not become completely water-soluble, and the product may contain partially insoluble keratin 10. It may include fractions. Keratin or keratin hydrolysate can be obtained from poultry feathers using subcritical water. This is also among the known methods. In subcritical water processes, the water is treated like normal water. 15 at the pressure conditions where it is kept above its boiling point and in liquid state. It is used. Under high temperatures, the ionization properties of water change and Water forms a reaction medium that supports the hydrolysis of protein structures. This method is environmentally friendly because it reduces the use of chemical solvents. Although considered advantageous, it is resistant to high temperatures and pressures. Equipment is required. Additionally, 20 is obtained as a result of the high-temperature application. The wide molecular weight distribution of the resulting hydrolysate indicates a specific peptide This necessitates additional fractionation in applications where a certain size is targeted. It is possible. Microwave-assisted treatments allow the keratin structure to open up in a shorter time and then 25 This is another technique used to facilitate hydrolysis processes. Microwave energy causes rapid and volumetric heating within the material. It contributes to the degradation of the keratin structure. Microwave pretreatment. Subsequently, hydrolysis can be carried out with proteolytic enzymes such as alkalase. However, microwave power, cooking time, and temperature should be appropriately adjusted to 30 If not controlled, amino acids like cysteine, which are sensitive to heat and processing conditions, can become infected. Losses can occur in the acids. Also, the microwave process is not homogeneous. industrial implementation and transfer to large-scale production systems This can create technical challenges on a large scale. In microbial methods, keratinase-producing species such as Bacillus and Streptomyces are used. biodegradation of chicken feathers using microorganisms This is provided by keratinase enzymes secreted by microorganisms. By breaking down the keratin structure, it releases soluble proteins, peptides, and amino acids. This enables microbial hydrolysis to eliminate the need for strong chemicals. Although it is possible to reduce this, most applications require long fermentation times. It is heard that controlling the growth conditions of the microorganism ensures sterility. the requirement is for cells and metabolic byproducts from the fermentation medium removal, product standardization, and potential microbial contamination risks This is among the disadvantages of the method. Also, 10 in different production batches Degree of hydrolysis and product composition depending on microorganism activity. It can vary. In direct enzymatic hydrolysis methods, keratinase, alkalase, from subtilisin, papain, bromelain or similar proteolytic enzymes Enzymatic methods are used. They are milder compared to chemical hydrolysis. Applicability under various temperature and pH conditions and selectivity towards specific peptide bonds 15 It is advantageous because it can be displayed. However, disulfide in the natural keratin structure bonds, hydrogen bonds and hydrophobic interactions of proteolytic enzymes in proteins This restricts access to the chains. Therefore, only the enzyme can be used. In this case, the hydrolysis yield may remain low or the process may take a long time to complete. This may require a long time and the use of high amounts of enzymes. The keratin structure... 20 alkali, acid, reducing agent, oxidizing agent, heat or before enzymatic hydrolysis Pre-processing using mechanical energy is therefore a common practice. Ultrasound therapy involves the physical processing of keratin-containing materials and proteins. 25 used to make its structure more suitable for enzymatic hydrolysis It is one of the methods. Cavitation is the process by which ultrasonic energy is produced in a liquid medium. High shear forces, microcurrents, and local pressure changes resulting from this effect. This occurs. These effects can increase the surface area of ​​the feather material, and the protein... It can support the disruption of weak interactions between the chains and the enzyme It can facilitate access to the keratin structure. In the literature, ultrasound treatment of urea 30 and when used in combination with solutions containing cysteine, the keratin in chicken feathers It has been reported to increase resolution. However, in the applications in question... Product purification due to the use of ultrasound in combination with chemical solvents. and chemical residues need to be removed. Ultrasound and enzymatic 6 Methods in which hydrolysis is used sequentially are also known. One of these methods is... In this section, chicken feathers are primarily processed under a high alkaline pH, Then protease enzyme and ultrasound are applied, and after the process, a strong acid is used. pH is neutralized using this method. In such methods, chicken feathers... While it is possible to break it down and increase its digestibility, initially 5 Using strong alkalis followed by neutralization involves chemical consumption. This leads to the formation of waste salts. Furthermore, in some reported applications... the degree of hydrolysis is limited and the molecular weight of the resulting peptides distribution of low molecular weight peptide profile targeted for oral use It does not appear to be entirely suitable. 10 Keratin hydrolysate from chicken feathers for oral or cosmetic use. In a known method of production, chicken feathers are treated with hydrochloric acid for a long time and hydrolyzed at high temperature, the process then continues at even higher temperature The process continues, the pH of the product is neutralized with sodium hydroxide, and 15 is obtained. The liquid obtained is converted into powder through spray drying. This is how it is obtained. A significant portion of the resulting product consists of free amino acids. High free amino acid ratio, preservation of keratin-derived biologically active peptides It can be limiting in the applications where it is targeted. Also, acidic hydrolysis The intense decomposition that occurs as a result affects the taste and aroma characteristics of the product. can have negative effects and lead to sensory acceptance problems when used orally. It can open. In the current state of the art, keratin hydrolysate is obtained from chicken feathers. Although there are numerous methods for this purpose, a significant portion of these methods are 25 strong acid, strong base, reducing agent, oxidizing agent, ionic liquid, urea or It appears that surfactants are used. This involves no chemical processing or... Methods that reduce chemical use involve high temperature, high pressure, and long duration. fermentation time, special equipment requirements, or low hydrolysis efficiency, etc. Problems are encountered. Furthermore, a 30% loss of products obtained from existing methods... in this section, the ratio of free amino acids is high, the ratio of peptides is low, or there are fewer peptides. It is observed that the molecular weight distribution is broad. This indicates that it is soluble in water. in terms of highly digestible, low molecular weight keratin peptides This makes it difficult to obtain a rich and suitable product for oral use. 7 Therefore, it enables the utilization of chicken feather waste; strong acid, strong a base that reduces the use of reducing or oxidizing chemicals; moderate temperature and neutral pH. This can be achieved under certain conditions; making the keratin structure accessible to the enzyme. enabled by its high peptide ratio and low free amino acid content, controlled molecular weight distribution, high water solubility and high 5 Improved technology that enables the production of digestible keratin peptides. The need for new production methods continues. Brief Description and Objectives of the Invention The invention describes the production of 10 liters of water from waste chicken feathers using an ultrasound-assisted enzymatic hydrolysis method. A method for obtaining soluble keratin peptides is described. The aim of the invention is to transform chicken feathers, which are released as waste, into a value-added protein. The aim is to ensure its transformation into its source. This purpose is to use the starting raw material. By using keratin-rich waste chicken feathers, 15 is being carried out. One aim of the invention is to remove harmful and potent chemicals from chicken feathers. The goal is to obtain keratin peptides without using other methods. The resulting technique is to produce keratin. its structure was created by applying ultrasound and endoproteases within a phosphate buffer at 20°C. It is obtained through its decomposition. One aim of the invention is to make the resistant structure of the keratin molecule suitable for enzymatic hydrolysis. The goal is to achieve this. Hydrogen bonds between keratin chains are formed at a frequency of 35 kHz and 215 kHz. The enzyme is weakened by ultrasonic energy applied at a power of W, and the protein structure of the enzyme is affected by 25 W. Access is being facilitated. One aim of the invention is to produce keratin hydrolysate with a high degree of hydrolysis. This is achieved by combining ultrasound treatment with conditions of pH 7.0 and 50 °C. The application of endoprotease, preferably alkalase enzyme, is crucial. 30 One aim of the invention is to create a product with a high peptide content and a low free amino acid content. The goal is to obtain a hydrolyzed keratin product. This feature is achieved through controlled enzymatic hydrolysis and 8 As a result of the necessary purification processes, the product consists mainly of peptides. This is achieved through its formation. Another aim of the invention is to produce keratin peptides with low molecular weight. With an average molecular weight in the range of 500-600 Da, the vast majority of which are 1000 5 The product, consisting of peptides under Da, was determined by ultrasound and enzymatic hydrolysis. It is obtained under these conditions. Another aim of the invention is to create a water-soluble solution that can be used in different product formulations. The goal is to provide keratin peptides that can participate in the process. Separation of solid and liquid phases, centrifugation, 10 Dialysis and drying steps lead to the purification of the final product and its powder form. It makes it possible to make it usable. One aim of the invention is to create a product suitable for oral use and exhibiting high digestibility. The goal is to develop a keratin peptide product. Within this scope, a low molecular weight peptide structure of 15 And with approximately 94% in vitro protein digestibility, the product's biological use It increases its potential. One aim of the invention is to study beta-keratin-derived peptides found in chicken feathers. The goal is to obtain a product rich in nutrients. Using chicken feathers as raw material instead of wool. 20 The selection of this product, which has a peptide profile different from existing products that are predominantly alpha-keratin-based, It makes its creation possible. Another aim of the invention is to utilize the resulting keratin peptides in dietary supplements, nutraceuticals, The aim is to enable its use in functional food and cosmetic products. In powder form, 25 Water-soluble and low molecular weight product structure, suitable for different compositions and This facilitates their inclusion in application methods. Explanation of the Figures Figure 1. 30 peptides identified in the keratin peptide product according to their molecular weights. A graph showing the relative abundance distribution. 9 Detailed Description of the Invention The invention is for transforming waste chicken feathers into a value-added protein source. water-soluble keratin peptides by ultrasound-assisted enzymatic hydrolysis This method involves applying ultrasound to chicken feathers to obtain keratin. The hydrogen bonds in its structure are weakened, and the endoprotease, preferably alkalase 5, is activated. Access of the enzyme to the protein structure is facilitated. Enzymatic hydrolysis This is followed by solid-liquid phase separation, centrifugation, dialysis, and drying. processed to produce a powder with high peptide content and low free amino acid content. A hydrolyzed keratin product is obtained. Here, the dialysis process is one aspect of the invention. This is carried out in the application. The resulting product has an average molecular weight of 10. Beta-keratin is in the 500-600 Da range, with the vast majority being below 1000 Da. It contains peptides of the same origin, is water-soluble, and has 94% in vitro protein content. It demonstrates digestibility. Thanks to these properties, the product is suitable for oral use. as appropriate in food supplements, nutraceuticals and functional food products, as well It is becoming possible to evaluate it in cosmetic formulations. 15 The method described in the invention; i. Adding phosphate buffer to chicken feathers and the resulting mixture mixing, ii. Applying ultrasound to a mixture containing chicken feathers and phosphate buffer, 20 iii. Endoprotease, preferably alkalase enzyme, is added to the ultrasound-treated mixture. being done, iv. Keratin is extracted by applying ultrasound-assisted enzymatic hydrolysis to the mixture. obtaining the hydrolysate, v. After the hydrolysis process, the mixture is subjected to heat treatment to remove enzyme 25. termination of activity, vi. Separation of the liquid and solid phases of the mixture by decantation, vii. Centrifugation of the separated liquid phase, viii. Drying the liquid phase obtained as a result of centrifugation and processing it into powder form. Obtaining keratin peptides 30 It includes the steps involved in the process. In an application of the invention, the method that is the subject of the invention; i. 0.05 M phosphate at a ratio of 1:50 (w / v) with a pH of 6.8-7.0 applied to chicken feathers. Adding the buffer and mixing the resulting mixture, ii. The chicken feather-phosphate buffer mixture is mixed at a temperature of 50-55 °C for 35 minutes. Ultrasound 5 at kHz frequency and 215 W power for 60-70 minutes. implementation, iii. Following ultrasound application, endoprotease, preferably alkalase, is added to the mixture. the enzyme is added and the mixture is heated at a temperature of 50-55 °C and a frequency of 35 kHz. and ultrasound-assisted enzymatic hydrolysis at 215 W power for 3-3.5 hours implementation, 10 iv. Degree of hydrolysis resulting from ultrasound-assisted enzymatic hydrolysis: OPA According to the method, keratin hydrolysate is obtained at a concentration of 55-60%. v. After the enzymatic hydrolysis is complete, the mixture is heated to 90 °C. enzyme activity is reduced by applying heat treatment for a period of minutes. termination, 15 vi. Decantation of liquid and solid phases in heat-treated mixture separation from each other vii. The separated liquid phase is heated at +4 °C and centrifuged at 12000 × g for 10-15 minutes. centrifuging for a period of minutes, viii. The liquid phase obtained as a result of centrifugation is placed in the lyophilizer at 55-60 °C for 20 minutes. drying in a drying cabinet at a certain temperature or in a spray dryer and obtaining keratin peptides in powder form It includes the steps involved in the process. In one application of the invention, the method described in the invention; 25 i. 0.05 M phosphate at a pH of 7.0 in a 1:50 (w / v) ratio applied to chicken feathers. Adding the buffer and mixing the resulting mixture, ii. The mixed chicken feather-phosphate buffer mixture was heated at 50 °C, 35 kHz Ultrasound application at a specific frequency and 215 W power for 60 minutes, iii. After ultrasound application, endoprotease (0.02 g enzyme / 1 30) was added to the mixture. (g chicken feathers), preferably with the addition of alkalase enzyme and the mixture at 50 °C Ultrasound at a temperature of 35 kHz frequency and 215 W power for 3 hours. Application of assisted enzymatic hydrolysis, 11 iv. Degree of hydrolysis resulting from ultrasound-assisted enzymatic hydrolysis: OPA According to the method, keratin hydrolysate is obtained at a concentration of 55-60%. v. After the enzymatic hydrolysis is complete, the mixture is heated to 90 °C. enzyme activity is reduced by applying heat treatment for a period of minutes. termination, 5 vi. Decantation of liquid and solid phases in heat-treated mixture separation from each other vii. The separated liquid phase was 10 at +4 °C and centrifugal force of 12000 × g. centrifuging for a period of minutes, viii. The liquid phase obtained as a result of centrifugation is placed in the lyophilizer at 60 °C for 10 minutes. drying in a drying cabinet at a certain temperature or in a spray dryer and obtaining keratin peptides in powder form It includes the steps involved in the process. The chicken feathers used in the invention were heat-treated and had their feather roots ruptured, resulting in 15... It is in powder form. In the method described in the invention, 60 minutes of ultrasound before hydrolysis... The application involves changes in protein structure (unfolding, hydrogenation). the enzyme to be used will be affected (break down) by the disulfide bonds. It is a preliminary step to ensure it takes effect effectively. At this stage, furthermore... The reaction medium becomes homogenous and the system reaches its operating temperature of 50 °C in 20 minutes. It is stabilized by being heated. The enzyme is released when the reaction medium reaches 50 °C. After the equilibrium is achieved, it is added and the hydrolysis process takes 3 hours at this temperature. This is performed under ultrasound guidance for a specified period of time. The hydrolyzed keratin product obtained within the scope of this invention has a content of 94.5-99% by weight. peptides in proportion and 1-5.5% free amino acids and amino compounds, It also contains phosphate salts. The free amino acids and amino acids in question... Taurine constitutes 17.29% of the compounds, and the peptides present in the product... The average molecular weight of the resulting hydrolyzed keratin product is 542.60 Da. protein fraction consisting of 94.5-99% peptide fraction by weight and 1-1.5% free peptides. It consists of amino acids, and its in vitro protein digestibility is 94.36%. It has been determined. INFOGEST static in vitro digestion process (Brodkorb et al., 2019) Subsequently, the hydrolysis level of the product was determined to be in the range of 35-40%. Hydrolyzed keratin peptide obtained as a result of the process applied within the scope of the invention. 12 The yield of the product is approximately 32%. For example, if 30 g of chicken feathers are used. Approximately 9.6 g of hydrolyzed keratin peptides are obtained. The invention concerns the total viable count and the presence of mold and yeast on a hydrolyzed keratin product. Counting experiments were carried out, and the analysis revealed a total of 55 living organisms. The CFU / g concentration was defined as 10 CFU / g, while the number of molds and yeasts was <10 CFU / g. In silico studies were conducted on the peptides revealed within the scope of the invention. In the analyses, the peptides matched the specific allergen sequences examined with 90% accuracy. It was determined that it did not show any signs. This indicates that 90% of the peptide sequence was of known allergen 10. This refers to a 90% overlap in sequences. In other words, it indicates a match similarity. When it is 90%, 90% of the peptide sequences correspond to known allergen sequences. Similar means only 10% are not similar. Known It is considered not to be an allergen because it does not contain sequences similar to those of allergens. In addition, these peptides are angiotensin-converting enzyme (ACE) inhibitors and 15 It has the potential to exert a dipeptidyl peptidase-IV (DPP-IV) inhibitor effect. It has been foreseen that hydrolyzed keratin will be obtained from waste chicken feathers and used in production. The absence of human keratin means the product is made from naturally derived materials that do not contain human keratin. It ensures that it has the necessary structure. Table 1. Bioactivities of peptides in hydrolyzed keratin (activity value ≥ 0.5%). those) Peptide Sequence Status* Bioactivity** WAK KE ACE inhibitor (0.5) RF KE ACE inhibitor (0.5), dipeptidyl peptidase IV inhibitor (0.8) SEC ID NO:1 RB <%1; C ACE inhibitor (0.5), dipeptidyl peptidase IV inhibitor (0.8) SEC ID NO:2 RB <%1; C ACE inhibitor (0.875), dipeptidyl peptidase IV inhibitor (0.75) 13 SEC ID NO:3 RB <%1; C ACE inhibitor (0.75), dipeptidyl peptidase IV inhibitor (0.9375) SEC ID NO:4 RB <%1; C ACE inhibitor 0.5263) dipeptidyl peptidase IV inhibitor 0.89467) SEC ID NO: 5 RB <%1; C Dipeptidyl peptidase IV inhibitor (0.5) SEC ID NO:6 RB <%1; C ACE inhibitor (1.25), dipeptidyl peptidase IV inhibitor (1.0) SEC ID NO:7 RB <%1; C Dipeptidyl peptidase IV inhibitor (0.8125) SEC ID NO: 8 RB <%1; C Dipeptidyl peptidase IV inhibitor (0.6667) SEK ID NO:9 RB <1%; C ACE inhibitor (0.8333) SEC ID NO:10 RB <%1; C ACE inhibitor (0.8889), dipeptidyl peptidase IV inhibitor (0.6667) SEC ID NO: 11 RB <%1; C ACE inhibitor (0.6364), dipeptidyl peptidase IV inhibitor (0.6667) SEC ID NO: 12 RB <%1; C ACE inhibitor (0.75), dipeptidyl peptidase IV inhibitor (0.6667) SEK ID NO:13 RB <1%; C ACE inhibitor (0.7143) SEC ID NO: 14 RB <%1; C Dipeptidyl peptidase IV inhibitor (0.5455) SEC ID NO: 15 RB <%1; C ACE inhibitor (1.25), dipeptidyl peptidase IV inhibitor (1.0833) SEC ID NO: 16 RB <%1; C ACE inhibitor (1.1111), dipeptidyl peptidase IV 14 inhibitor (0.5556) SEC ID NO: 17 RB <%1; C ACE inhibitor (0.8889), dipeptidyl peptidase IV inhibitor (0.8889) SEC ID NO: 18 RB <%1; C ACE inhibitor (0.5), dipeptidyl peptidase IV inhibitor (0.9286) SEC ID NO: 19 RB <%1; C Dipeptidyl peptidase IV inhibitor (0.8462) SEC ID NO: 20 RB <%1; C ACE inhibitor (0.875), dipeptidyl peptidase IV inhibitor (1.00) SEC ID NO: 21 RB <%1; C ACE inhibitor (0.6154), dipeptidyl peptidase IV inhibitor (0.5385) SEK ID NO:22 RB <1%; C ACE inhibitor (0.5556) SEC ID NO: 23 RB <%1; C Dipeptidyl peptidase IV inhibitor (0.5556) SEC ID NO: 24 RB <%1; C ACE inhibitor (0.8), dipeptidyl peptidase IV inhibitor (1.0) SEK ID NO:25 RB <1%; C ACE inhibitor (0.5) SEC ID NO: 26 RB <%1; C ACE inhibitor (0.5625), dipeptidyl peptidase IV inhibitor (0.9375) SEC ID NO: 27 RB <%1; C ACE inhibitor (0.5), dipeptidyl peptidase IV inhibitor (0.5833) SEC ID NO: 28 RB <%1; C ACE inhibitor (0.5556), dipeptidyl peptidase IV inhibitor (0.9444) SEC ID NO: 29 RB <%1; C ACE inhibitor (0.625), dipeptidyl peptidase IV inhibitor (0.5625) SEC ID NO: 30 RB <%1; C ACE inhibitor (1.1111), dipeptidyl peptidase IV inhibitor (1.0), neprilisin inhibitor (0.5556) SEC ID NO: 31 RB <%1; C Dipeptidyl peptidase IV inhibitor (0.9091) SEC ID NO: 32 RB <%1; C ACE inhibitor (0.7), dipeptidyl peptidase IV inhibitor (0.7) SEC ID NO: 33 RB <%1; C Dipeptidyl peptidase IV inhibitor (0.7273) SEC ID NO: 34 RB <%1; C ACE inhibitor (0.5333), dipeptidyl peptidase IV inhibitor (0.5333) SEC ID NO: 35 RB <%1; C ACE inhibitor (0.5714), dipeptidyl peptidase IV inhibitor (0.6429) SEC ID NO: 36 RB <%1; C Dipeptidyl peptidase IV inhibitor (0.8333) SEC ID NO: 37 RB <%1; C ACE inhibitor (0.8667), dipeptidyl peptidase IV inhibitor (0.8667) SEC ID NO: 38 RB <%1; C ACE inhibitor (1.3636), dipeptidyl peptidase IV inhibitor (0.8182) SEC ID NO: 39 RB <%1; C ACE inhibitor (0.75), dipeptidyl peptidase IV inhibitor (0.625) SEC ID NO: 40 RB <%1; C ACE inhibitor (0.75), dipeptidyl peptidase IV inhibitor (0.875) SEK ID NO:41 RB <1%; C ACE inhibitor (0.5833), 16 dipeptidyl peptidase IV inhibitor (0.8333) SEC ID NO: 42 RB <%1; C ACE inhibitor (0.5833), dipeptidyl peptidase IV inhibitor (0.5833) SEK ID NO:43 RB <1%; C ACE inhibitor (0.5333) SEC ID NO: 44 RB <%1; C ACE inhibitor (0.5714), dipeptidyl peptidase IV inhibitor (0.6429) SEC ID NO: 45 RB <%1; C ACE inhibitor (0.75), dipeptidyl peptidase IV inhibitor (0.9375) SEC ID NO: 46 RB <%1; C ACE inhibitor (0.6667), dipeptidyl peptidase IV inhibitor (0.6667) SEC ID NO: 47 RB <%1; C ACE inhibitor (0.8333), dipeptidyl peptidase IV inhibitor (0.5) SEC ID NO: 48 RB <%1; C ACE inhibitor (1.1333), dipeptidyl peptidase IV inhibitor (0.8667) *KE: Those that match the library RB<%1: Sequences of peptides with a relative abundance of <1% within the peptide fraction. **The numbers in parentheses represent the value of “a”. The value of “a” is the peptide value.** It is a value that indicates the density of bioactive sequences within it. In order to determine the water solubility of the keratin peptide product, a specific amount was used. The keratin peptide product is dissolved in pure water to obtain a homogeneous starting solution. The samples were prepared. Analyses were performed at room temperature, approximately 25 °C. Pure water was used as the solvent in solubility determination, and the natural pH value of the solution was calculated. This was taken as the basis and no additional pH adjustment was made (Andersen et al., 10 (1969; Andersen et al., 1970). For the purpose of separating the insoluble fraction. 17 The prepared sample was centrifuged, and the resulting product from centrifugation was obtained. The supernatant was analyzed. The total amount of peptides present in the starting solution was determined. The amount of dissolved peptides in the supernatant was determined by Dunn (1995). The water solubility of the keratin peptide product was determined according to the described method. The percentage of peptides determined in the supernatant compared to the amount of peptides present in the initial solution is 5%. the ratio of the total peptide amount to the resulting value and multiplying the value by 100 calculated in this way. As a result of the analyses performed, keratin peptide The water solubility value of the product was determined to be 99.39%. This result indicates that... Within the scope of this invention, the keratin peptide product obtained exhibits high levels of absorption in pure water. It is soluble to a certain extent, and the insoluble fraction is approximately 0.61%. It shows that it remains. The amount of free amino acids present in the hydrolyzed keratin sample in powder form, using liquid chromatography-sequential mass spectrometry (LC-MS / MS) method The samples were analyzed. The analyses were performed using an Agilent 1260 Infinity liquid chromatography system with Jet 15. Agilent 6460 Triple Quadrupole with Stream electrospray ionization source. An MS / MS system was used. As part of the free amino acid analysis, each 200 mg of the sample was weighed and 5 mL of ultrapure water containing 1% formic acid was added to it. Extraction solution was added. The resulting mixture was left for 5 seconds. It was vortexed and then centrifuged at 9000 rpm for 5 minutes. 20 50 µL of the supernatant obtained after centrifugation is taken and placed in an Eppendorf tube. transferred to a test tube, 50 µL of internal standard solution (ISTD) and 700 µL of amino acids were added. Solvent was added. The prepared sample was centrifuged at 1350 rpm for 5 minutes. The resulting clear filtrate was processed and injected into an LC-MS / MS system. The samples were transferred to glass vials. The types of free amino acids present in the sample and 25 The quantities were determined by LC-MS / MS analysis. Within the keratin peptide product... Free taurine level is the amount of taurine detected in the sample as a result of the analysis. the ratio of the total amount of free amino acids detected to the resulting value It was calculated by multiplying by 100. Accordingly, the free taurine level is as follows: Determined using the formula: 30 Free taurine percentage (%) = (Amount of taurine detected / Total free amino acids) quantity) × 100 18 Table 2. Free amino acids determined in keratin hydrolysate by LC-ESI-MS / MS method. concentrations and molar distributions of acid and amino compounds (Concentrations were determined in nmol / g by LC-ESI-MS / MS analysis. Molar ratio) (%) the concentration of each compound in terms of determined free amino acids and amino acids. (Calculated by dividing by the total concentration of the compounds.) 5 Proteinogenic free amino acid / other free amino compounds Concentration (nmol / g) Total determined free amino acids and amino compounds Molar ratio (%) A. Proteinogenic free amino acids L-Tryptophan 258.2929 0.70 L-Phenylalanine 752.2202 2.03 L-Tyrosine 1218.9232 3.30 L-Leucine 1831.4911 4.95 L-Isoleucine 686.1471 1.86 L-Methionine 430.9051 1.17 L-Valine 1085.8536 2.94 L-Glutamic acid 3547.8480 9.59 L-Aspartic acid 1795.7893 4.86 L-Threonine 2230.3745 6.03 L-Cool 3247.0535 8.78 L-Alanine 3977.5911 10.76 L-Glycine 3220.7602 8.71 Trans-4-Hydroxy-L- Proline 111.7006 0.30 L-Asparagine 886.9716 2.40 L-Proline 1136.4353 3.07 L-Glutamine 311.5491 0.84 L-Histidine 102.1270 0.28 L-Arginine 1013.6090 2.74 B. Other free amino compounds Taurine 6395.7441 17.29 19 GABA 131.2207 0.35 3-Aminoisobutyric acid 11.7786 0.03 L-2-Aminoadipic acid 25.8578 0.07 Ethanolamine 743.4810 2.01 β-Alanine 734.3092 1.99 L-2-Aminobutyric acid 37.1091 0.10 Sarkozy 218.7013 0.59 L-Homocitrulline 14.5911 0.04 L-Citrulline 168.2696 0.46 O-Phosphoethanolamine 170.4288 0.46 DL-Homocysteine ​​14.1465 0.04 L-Cystatinine 470.8646 1.27 TOTAL 36982.1448 100.00 When the molecular weight distribution of the keratin peptide product, which is the subject of the invention, is examined, total peptide content of peptides with molecular weight below 500 Da It has been determined that it constitutes 46.46%. Molecular weight between 500 Da and 1000 Da. The percentage of peptides present was determined to be 53.54%. Accordingly, the product contains 5 peptides. all of the peptides have a molecular weight below 1000 Da It has been determined. Table 3. Molecular weight distribution of peptides in hydrolyzed keratin. Molecular weight (In) Relative abundance (%) 477 13.36 796 12.2 332 8.87 287 8.21 558 6.55 847 5.25 403 4.32 257 4.16 536 4.07 580 4.04 594 3.22 922 3.19 830 3.11 903 3.08 547 3.06 259 2.93 575 2.73 374 2.64 553 2.58 321 2.44 Table 4. Crude protein, fat, and keratin peptides obtained by the method described in the invention. Heavy metal analysis results. Industrial Applicability of the Invention The invention involves the production of hydrolysate material from waste chicken feathers using ultrasound-assisted enzymatic hydrolysis. It relates to a method for obtaining soluble keratin peptides and its application to industry. It is applicable. 10 The invention is not limited to the above descriptions, and a person skilled in the field can easily make further discoveries. It can demonstrate different applications of the invention. These are the claims and demands of the invention. It should be evaluated within the scope of the protection granted. 21 REFERENCES -Anderson, RA, Conway, HF, Pfeifer, VF, & Griffin, EL Jr. (1969). Gelatinization of Corn Grits by Roll- and Extrusion-Cooking. cereal science Today, 14, 4–7, 11–12. - Anderson, R.A., Conway, H.F., & Peplinski, A.J. (1970). Gelatinization of 5 Corn Grits by Roll Cooking, Extrusion Cooking and Steaming. Starch / Stärke, 22, 130–135. https: / / doi.org / 10.1002 / star.19700220408 - Brodkorb, A., Egger, L., Alminger, M., Alvito, P., Assunção, R., Ballance, S., Bohn, T., Bourlieu-Lacanal, C., Boutrou, R., Carrière, F., Clemente, A., Corredig, M., Dupont, D., Dufour, C., Edwards, C., Golding, M., Karakaya, 10 S., Kirkhus, B., Le Feunteun, S., Lesmes, U., Macierzanka, A., Mackie, A. R., Martins, C., Marze, S., McClements, D. J., Ménard, O., Minekus, M., Portmann, R., Santos, C. N., Souchon, I., Singh, R. P., Vegarud, G. E., Wickham, M. S. J., Weitschies, W., & Recio, I. (2019).INFOGEST static in vitro simulation of gastrointestinal food digestion. Nature Protocols, 14(4), 15 991–1014.https: / / doi.org / 10,1038 / s41596-018-0119-1 - Dunn, M. J. 1995. Determination of total protein concentration in Protein Purification Handbook a Practical Approach, Harris, E. L. V. ve Angal, S. (Eds.), Oxford University Press, England. 25

Claims

22 REQUESTS 1. Obtaining water-soluble keratin peptides from chicken feathers. It is a method aimed at; its characteristic is; i. Adding phosphate buffer to chicken feathers and the resulting mixture mixing, 5 ii. Applying ultrasound to a mixture containing chicken feathers and phosphate buffer, iii. Adding endoprotease enzyme to the ultrasound-treated mixture, iv. Keratin is extracted by applying ultrasound-assisted enzymatic hydrolysis to the mixture. obtaining the hydrolysate, v. After the hydrolysis process, the mixture is subjected to heat treatment to remove enzyme 10. termination of activity, vi. separating the liquid and solid phases of the mixture by decantation separation, vii. Centrifugation of the separated liquid phase, viii. Drying of the liquid phase obtained as a result of centrifugation and powdering 15 obtaining keratin peptides in form It includes the steps of the process.

2. It is a method according to claim 1, and its characteristic is; i. 0.05 M of a solution with a pH of 6.8-7.0 at a ratio of 1:50 (w / v) to chicken feathers. Adding phosphate buffer and mixing the resulting mixture, 20 ii. The chicken feather-phosphate buffer mixture is mixed at a temperature of 50-55 °C, Ultrasound at 35 kHz frequency and 215 W power for 60-70 minutes. implementation, iii. Following ultrasound application, endoprotease is added to the mixture, preferably Alkalase enzyme is added and the mixture is heated at 50-55 °C, 35 kHz. ultrasound-assisted at a certain frequency and 215 W power for 3-3.5 hours. application of enzymatic hydrolysis, iv. Degree of hydrolysis resulting from ultrasound-assisted enzymatic hydrolysis: OPA According to the method, keratin hydrolysate is obtained at a concentration of 55-60%. v. After the completion of enzymatic hydrolysis, the mixture is heated to 90 °C 30 Enzyme activity is reduced by applying heat treatment at a temperature for 5 minutes. termination, vi. Decantation of liquid and solid phases in heat-treated mixture separation by means of 23 vii. The separated liquid phase is 10- at +4 °C and centrifugal force of 12000 × g. centrifuging for a period of minutes, viii. The liquid phase obtained as a result of centrifugation is placed in the lyophilizer at 55-60 °C in a drying cabinet at a certain temperature or in a spray dryer drying and obtaining keratin peptides in powder form 5 It includes the steps of the process.

3. It is a method according to claim 2, and its characteristic is; i. 0.05 M phosphate at a pH of 7.0 in a 1:50 (w / v) ratio applied to chicken feathers. Adding the buffer and mixing the resulting mixture, ii. The chicken feather-phosphate buffer mixture is mixed at a temperature of 50 °C, 35 10 Ultrasound at a frequency of kHz and a power of 215 W for 60 minutes. implementation, iii. After ultrasound application, endoprotease (0.02 g) was added to the mixture. (enzyme / 1 g chicken feather) enzyme is added to the mixture and heated to 50 °C. Ultrasound 15 at temperature, 35 kHz frequency and 215 W power for 3 hours Application of assisted enzymatic hydrolysis, iv. Degree of hydrolysis resulting from ultrasound-assisted enzymatic hydrolysis: OPA According to the method, keratin hydrolysate is obtained at a concentration of 55-60%. v. After the enzymatic hydrolysis is complete, the mixture is heated to 90 °C Enzyme activity was reduced by heat treatment at a temperature of 20 for 5 minutes. termination, vi. Decantation of liquid and solid phases in heat-treated mixture separation by means of vii. The separated liquid phase was 10 at +4 °C and centrifugal force of 12000 × g. centrifuging for 25 minutes viii. The liquid phase obtained as a result of centrifugation is placed in the lyophilizer at 60 °C in a drying cabinet at a certain temperature or in a spray dryer drying and obtaining keratin peptides in powder form It includes the steps of the process.

4. A method according to any of claims 1-3, the characteristic of which is; the aforementioned 30 The endoprotease is an alkalase.

5. A method according to any of claims 1-3, the characteristic of which is; the chicken in question. their feathers, heat-treated and with their hair follicles destroyed, in powder form. It is the fact that. 24 6. Keratin peptide obtained by a method according to any of claims 1-3.

7. A keratin peptide according to claim 6, characterized by its composition of 94.5-99% by weight. peptides and 1-5.5% free amino acids and amino compounds, It also contains phosphate salts.

8. It is a keratin peptide according to claim 7, and its characteristic is that the free amino acid in question is 5 The amino acid taurine constitutes 17.29% of the acids and amino compounds.

9. According to claim 8, it is a keratin peptide and its characteristic is that the average molecular weight of the peptides is... Its weight is 542.60 da.

10. According to claim 6, a keratin peptide reacts with angiotensin-converting enzyme (ACE). Its use as an inhibitor and dipeptidyl peptidase-IV (DPP-IV) inhibitor. 10 20 30

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