A method for extraction of reduced keratin from human hair

Abstract

The invention relates to a fast, easy, low-cost method optimized for the extraction of keratin from human hair and the keratin obtained by this method. In said method, the need for centrifugation, a second filtering process, and the use of excess chemicals is eliminated, and thus the method can be carried out at low cost. With said method, keratin reduced to small filaments is obtained, thus making the use of keratin as a biomaterial easier.

Description

[0001] A METHOD FOR EXTRACTION OF REDUCED KERATIN FROM HUMAN HAIR

[0002] Technical field

[0003] The invention relates to a fast, easy, low-cost method optimized for the extraction of keratin from human hair and the keratin obtained by this method. In said method, the need for centrifugation and a second filtering process is eliminated. With said method, keratin reduced to small filaments in the range of 0.1 -10 kDa is obtained, thus making the use of keratin as a biomaterial easier.

[0004] State of the Art

[0005] In recent years, naturally derived biomaterials have been widely used in tissue engineering and regenerative medicine due to their biological functions, structural support, excellent biocompatibility, and favourable biodegradability properties. In addition, these materials are frequently used to develop scaffolds, hydrogels, and other forms for biomedical applications, and keratin is one of these biomaterials

[0001] . Keratin is a cysteine-rich fibrous protein associated with intermediate filaments (IFs) that make up the majority of cytoskeletal and epidermal appendages such as hair, horns, feathers, wool and nails and and contains a large number of binding motifs that benefit cell attachment and proliferation in its structure, such as leucine-aspartate-valine (LDV), glutamate-aspartate-serine (EDS), and arginine-glycine-aspartate (RGD). Keratin can be extracted from wool, feather and horns, however, keratin obtained from human hair can be distinguished from its epidermal counterparts by its relatively higher cysteine content compared to keratin obtained from other species, and this feature shows that there is a disulphide bond to form a harder and more durable structure in the tissues where hair keratins are dispersed [2],

[0006] Keratins can be obtained from the cortex of human hair using reducing or oxidative chemicals. However, due to the intramolecular hydrogen bonding network of keratin in the hair and the disulphide bond of the three-dimensional network structure of keratin, it is difficult to dissolve with conventional organic solvents and the sizes of the keratin filaments obtained are not suitable for every application. Therefore, different methodologies for the extraction of keratin from human hair mainly focus on cleavage of disulphide linkages on the keratin protein backbone to increase the aqueous solubility of keratin. These methods, using reducing agents or oxidative chemicals, are typically two-step processes in which the cross-linked structure of keratins is broken down by oxidation or reduction. The most common method in the state of the art is the treatment with sodium sulphide. In this method, hair prepared by delipidisation and dried is reacted with sodium sulphide (0.125 M) at 40 °C for 4.5 hours. The reaction mixture is filtered after the reaction and centrifuged for 10 minutes (5000 xg) then dialysed against distilled water. The product obtained after dialysis is filtered using a syringe filter (0.22 urn) and freeze-dried to obtain a brown coloured flake protein fraction [3]. However, said method is both long in terms of procedure time and the yield of the product obtained after production is low. After the mentioned reaction, filtration, centrifugation, and re-filtration processes also cause a lot of material loss. Oxidizing agents such as peracetic acid, urea, or reducing agents such as thioglycolic acid are also used in the state of the art alongside sodium sulphite, but in most of the reported studies in the state of the art performed at high pH or elevated temperature, the protein is degraded [4]-

[0007] Another method used to extract keratin from human hair in the state of the art is the alkaline method. It has been known for many years that strong and hot alkaline solutions can dissolve wool. Upon processing the wool with an alkaline solution, the sulphur core begins to separate and the cystine residues decompose. [5] High concentration alkaline solution separates hydrogen from sulphate and carboxylic groups and facilitates dissolution, but damage occurs in peptide chains. The breakdown of these bonds can lead to the formation of alkali sulphide odour, which has a very unfavourable odour during the purification process. For this reason, damage and degradation of the protein backbone, high consumption of alkaline reagents and, accordingly, the need for high amounts of acid to neutralise and precipitate the protein are the main factors preventing the commercialisation and widespread use of the alkali method [6].

[0008] In the prior art patent application numbered EP2744820A2, methods for the extraction and purification of keratin protein-based biomaterials are described. In said document, thioglycolic acid solution is used for the reduction reaction and the reaction is carried out in 15 hours. After the reaction, 100 mM Tris base solution is added to the filtered hair at a ratio of 25:1 , followed by centrifugation, dialysis (duration uncertain) and lyophilisation processes. However, said document does not disclose the filament size of the extracted keratin, in addition, adding a base and centrifugation increases the cost of the method used. In addition, in said document, the extraction process takes place in more than 15 hours, which leads to serious time losses.

[0009] In the patent application numbered CN105924654A in the state of the art, a method for extracting human hair keratin using L-cysteine is described. Said method comprises adding L-cysteine solution to washed and crushed hair fibres, precipitation, centrifugation, dialysis, and freeze-drying, and the reaction temperature is stated to be in the range of 65-95 °C. However, due to the high reaction temperature, the energy consumed is also high. In addition to the centrifugation process in said method, there is no explanation regarding the keratin filaments obtained or the size of the filaments. In addition, precipitation is also included in said document and is carried out with acetic acid solution. Said method is costly due to the use of too many chemicals, high reaction temperatures and centrifugation.

[0010] In the patent application numbered TR 2014 / 02104 in the state of the art, a method for the extraction of keratin protein from hair is described. In said document, samples taken from poultry farms (with / without feathers) and slaughterhouses or hairdressers (men and women's hair) are delipidised. Then, extraction is carried out in various alkaline solutions at certain temperatures and times. To suppress colour and odour, the extracted solution is treated with H2O2 (hydrogen peroxide) and Na2SOs (sodium sulphide). Following this process, the obtained solution is centrifuged, filtration and / or ultra-filtration is applied to separate the insoluble parts. After the purification process, the suspension obtained by precipitating the crude keratin solution obtained by treating with acids is filtered and the precipitate is washed several times with distilled water and then dried by freeze drying and / or spray drying methods, thus raw keratin is obtained in dry / powder form. In said document, chemical treatment is excessive, and the method presented is costly due to the application of post-reaction filtration and centrifugation processes.

[0011] Due to reasons such as the limitations and inadequacies of the solutions in the current technique, the presence of a large number of chemical treatment processes in the methods used for keratin extraction from human hair, their cost due to the application of centrifugation and a second filtering process, the large size of the extracted keratin filaments in the current technique and the limitation of their usage areas, and also the long extraction times and the high loss of material due to the loss of many materials and chemicals, it was necessary to make an improvement in the field of keratin extraction from human hair.

[0012] Brief Description and Aims of the Invention

[0013] In the invention, a fast, easy, low-cost method optimized for the extraction of keratin from human hair and the keratin obtained by this method are explained. In said method, the need for centrifugation, a second filtering process, and the use of excess chemicals is eliminated, and thus the method can be carried out at low cost. Withsaid method, keratin reduced to small filaments is obtained, thus making the use of keratin as a biomaterial easier.

[0014] The aim of the invention is to provide fast, easy and low-cost extraction of keratin from human hair. In the method that is the subject of the invention, there is no centrifugation and a second filtration application, and since the use of chemicals is at a minimum level, keratin extraction can be performed at low cost. In addition, in said method, the hair becomes homogeneous by reacting with Na2S.9H2O (Sodium sulphide nonahydrate) solution in a short time like 4 hours.

[0015] Another aim of the invention is to obtain small keratin filaments. By means of the increased amount ofNa2S.9H20 (Sodium sulphide nonahydrate) in the method that is the subject of the invention, the keratin filaments become 0.1 -10 kDa in size. In this way, the extracted keratin filaments can be easily used as biomaterials.

[0016] Description of Drawings

[0017] Figure 1. MALDI-TOF / MS analysis of human hair keratin obtained by the method that is the subject of the invention

[0018] Figure 2. The final product FTIR spectrum for the reduced human hair keratin that is the subject of the invention Figure 3. FTIR spectrum made for the filter paper with the reaction intermediate step that is the subject of the invention

[0019] Figure 4. The final product XRD spectrum for the reduced human hair keratin that is the subject of the invention

[0020] Figure 5. FTIR analysis of undissolved hairs obtained using 0.125 M Na2S in the state of the art

[0021] Figure 6. FTIR analysis of the material obtained after centrifugation using 0.125 M Na2S in the state of the art

[0022] Detailed Description of the Invention

[0023] The invention relates to a fast, easy, low-cost method optimized for the extraction of keratin from human hair and the keratin obtained by this method. In said method, the need for centrifugation, a second filtering process, and the use of excess chemicals is eliminated, and thus the method can be carried out at low cost. Withsaid method, keratin reduced to small filaments is obtained, thus making the use of keratin as a biomaterial easier.

[0024] A method for the extraction of keratin from human hair, which is the subject of the invention, comprises the process steps of: i. washing the raw materials of human hair with water and detergent several times and treating the washed materials with ethanol for 30 minutes, ii. rinsing the delipidised hair several times with distilled water and drying it at room conditions, iii. cutting the dried hair into small pieces, iv. treating the hair cut into small pieces with a mixture of chloroform-methanol and drying it again, v. keeping the dried hair in an oil bath at 30°C-40oC in sodium sulphide nonahydrate (Na2S.9H2O) in the range of 2-5 M for 4-5 hours, vi. filtering the solution extracted from the oil bath with ordinary filter paper, vii. creating the keratin solution by applying dialysis to the filtered solution for 2- 4 days, viii. freezing the keratin solution from dialysis, and ix. obtaining keratin as a result of drying the frozen keratin solution with a lyophiliser.

[0025] In one embodiment of the invention, a method for the extraction of keratin from human hair comprises the process steps of: i. washing the raw materials of human hair with water and detergent several times and treating the washed materials with ethanol for 30 minutes, ii. rinsing the delipidised hair several times with distilled water and drying it at room conditions, iii. cutting the dried hair into small pieces of 5-6 g, iv. treating the 5-6 grams of hair with a 2:1 mixture of chloroform-methanol and drying it again, v. keeping the dried hair in 3.25 M sodium sulphide nonahydrate (Na2S.9H2O) at 30°C in an oil bath for 4 hours, vi. filtering the solution extracted from the oil bath with ordinary filter paper, vii. creating the keratin solution by applying dialysis to the filtered solution for 4 days, viii. freezing the keratin solution from dialysis at -4°C, and ix. obtaining keratin as a result of drying the frozen keratin solution with a lyophiliser.

[0026] In the process step (i) of the method that is the subject of the invention, the hair is disinfected by treating the hair with ethanol. In addition, delipidisation process is carried out by treating the hair cut in the process step (iv) with a mixture of chloroform / methyl alcohol. Keratin is obtained in the form of protein powder by lyophilisation in the last process step applied in the invention, namely (ix), thus minimizing the degradation of the protein. The filament size of the keratin obtained by said method is 0.1 -10 kDa, and thus it can be easily used as a biomaterial, in the production of scaffolds, as a bioadhesive, as a bioadditive material to increase cell growth and spread, etc.

[0027] As a result of the MALDI-TOF MS analysis performed for small weight filaments due to the keratin oligopeptides obtained by the method of the invention, which is indicated in Figure 1 , it is determined that keratin has multiple peaks below 2373 Da and the peaks around 324-1000 Da in the MS spectrum belong to CHCA (MALDI matrix). Figure 2 shows the FTIR analysis of hand-made keratin by the method of the invention, the spectra in the 700 and 1 100 cm-1 frequency range sensitive to disulphide bonds are clearly indicated. The keratin sample exhibits distinct characteristic absorption peaks at about 1030 cm1and 810 cm1. Human hair keratins produce N-H and O-H stretching vibrations observed between 3284 and 3252 cm-1. C-H stretching vibration is observed around 2930 cm1. Characteristic bands of proteins, i.e. Amid A (3284- 3252 cm’1), Amid I (1643- 1638 cm’1), Amid II (1535-1518 cm’1) and Amid III (approximately 1244 cm-1) are defined. Figure 3 shows the FTIR analysis of the material remaining on the filter paper after the reaction, and when compared to Figure 2, it is seen that there are no Amid II and Amid III peaks. In addition, no peak is observed around -C-H2, -C-H3 2900 cm1.

[0028] In the invention, X-ray diffraction (XRD) analysis was performed to examine the crystallinity of keratin. For XRD characterization, approximately 1 -2 g of each finely ground sample is randomly placed on a flat brass sample holder in an airtight environment. Operating conditions (A = 1.540 A) are determined at 40 kV and 25 mA, and data are collected in Brag-Brentano (9 / 29) horizontal geometry and scanning speed 0.50min-1. In the invention, the diffraction pattern of the reduced keratin obtained from human hair is shown in Figure 4. The peak at ~9° is significantly weaker than the apparent peak at ~20°. These peaks are associated with a-helix and [3-sheet structures, respectively.

[0029] In addition, when the FTIR analysis of the undissolved hair remaining on the filter paper after the reaction in the keratin extraction with 0.125M Na2S, which is included in the state of the art, is examined, almost all the keratin structure remains on the filter paper as seen in the FTIR result, since the post-reaction hairs are not fully dispersed in Na2S. Again, when the post-centrifugation FTIR analysis (Figure 6) of the study with Na2S prepared as 0.125M is compared with the method (Figure 2) that is the subject of the invention, it is seen that there are Amid II and Amid III peaks. In addition, a peak around -C-H2, -C-H3 2900 cm-1is observed. In this way, it is seen that most of the hair keratin remained in the filter paper with the method in the state of the art and the amount of keratin obtained is low due to the weakness of the centrifugal peaks. By means of the method that is the subject of the invention, the yield of keratin is increased, since the loss of substance is minimised. In addition, since there is no centrifugation and a second filtration process in the method used in the invention, keratin extraction can be performed in a short time.

[0030] REFERENCES

[0031] [1] Lee, H„ Noh, K„ Lee, S. C„ Kwon, l.-K., Han, D.-W., Lee, l.-S., & Hwang, Y.-S.

[0032] (2014, August 7). Human hair keratin and its-based biomaterials for biomedical applications - tissue engineering and regenerative medicine. SpringerLink. Retrieved February 21 , 2023, from https: / / link.sprinqer.eom / article / 10.1007 / s13770-014-0029-4

[0033] [2] Jialin Yu, & AbstractHuman hair keratins were among the first to be studied but it is only recently that sufficient information has been obtained to gain a basic biologic perspective of these proteins. Hair keratins are members of the intermediate filament family of p. (2017, December 2). Human hair keratins. Journal of Investigative Dermatology. Retrieved February 21 , 2023, from https: / / www.sciencedirect.com / science / article / pii / 0022202X93905Q18

[0034] [3] K;, A. V. P. A. G. I. S. C. (n.d.). Comparative study of keratin extraction from human hair. International journal of biological macromolecules. Retrieved February 21 , 2023, from https: / / pubmed.ncbi.nlm.nih.gov / 31002909 /

[0035] [4] Maximum extraction of wool proteins by thiol -urea solutions, (n.d.). Retrieved

[0036] February 22, 2023, from https: / / iournals.saqepub.eom / doi / abs / 10.1177 / 004051758705700205

[0037] [5] Gr;, B. S. (n.d.). The reaction of wool keratin with alkali. Biochimica et biophysica acta. Retrieved February 22, 2023, from https: / / pubmed.ncbi.nlm.nih.gOv / 133153111

[0038] [6] AEA;, S. A. S. T. H. B. A. A. B. (n.d.). Keratin: Dissolution, extraction and biomedical application. Biomaterials science. Retrieved February 22, 2023, from https: / / pubmed.ncbi.nlm.nih.gov / 28686242 /

Claims

CLAIMS1. A method for the extraction of keratin from human hair, comprising the process steps of: i. washing the raw materials of human hair with water and detergent several times and treating the washed materials with ethanol for 30 minutes, ii. rinsing the delipidised hair several times with distilled water and drying it at room conditions, iii. cutting the dried hair into small pieces, iv. treating the hair cut into small pieces with a mixture of chloroformmethanol and drying it again, v. keeping the dried hair in an oil bath at 30-40oC in sodium sulphide nonahydrate Na2S.9H2O) in the range of 2-5 M for 4-5 hours, vi. filtering the solution extracted from the oil bath with ordinary filter paper, vii. obtaining the keratin solution by applying dialysis to the filtered solution for 2-4 days, viii. freezing the keratin solution from dialysis, and ix. obtaining keratin as a result of drying the frozen keratin solution with a lyophiliser.

2. A method according to claim 1 , comprising the steps of: i. washing the raw materials of human hair with water and detergent several times and treating the washed materials with ethanol for 30 minutes, ii. rinsing the delipidised hair several times with distilled water and drying it at room conditions, iii. cutting the dried hair into small pieces of 5-6 g, iv. treating the 5-6 grams of hair with a 2:1 mixture of chloroform-methanol and drying it again,v. keeping the dried hair in 3.25 M sodium sulphide nonahydrate Na2S.9H2O) at 30°C in an oil bath for 4 hours, vi. filtering the solution extracted from the oil bath with ordinary filter paper, vii. obtaining the keratin solution by applying dialysis to the filtered solution for 4 days, viii. freezing the keratin solution from dialysis at -4°C, and ix. obtaining keratin as a result of drying the frozen keratin solution with a lyophiliser.

3. Keratin extracted from human hair by a method according to claim 1 or 2.

4. A keratin according to claim 3, wherein filament size is in the range of 0.1 -10 kDa.

5. A biomaterial comprising a keratin according to claim 3 or 4,

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