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EXTRACTION OF KERATIN PROTEIN FROM CHICKEN FEATHER 1

Arun Gupta , Ramanan Perumal, Rosli Bin Mohd Yunus and Nuruldiyanah Binti Kamarudin

Faculty of Chemical and Natural Resources Engineering Universiti Malaysia Pahang, Gambang Campus, 25100, Kuantan, Pahang, Malaysia Email: [email protected]

ABSTRACT The present research was conducted to extract keratin protein from chicken feathers. Protein is an important nutrient needed by our body to maintain body structures and is an important ingredient for cosmetic products. Chicken feathers have high level of keratin protein content and can become a suitable protein source. The main processes involved are first dissolving chicken feathers using different reducing agents and later on separating the protein from chemicals. Reducing agents used are potassium cyanide, thioglycolic acid and sodium sulphide. Once the feathers are dissolved using reducing agents, ammonium sulfate solution is added to the solution for the precipitation of protein. The precipitated protein is washed with water several times and sodium hydroxide solution is used to obtain protein back in the solution form. Out of three different reducing agents used, sodium sulfide gives the highest efficiency in dissolving chicken feathers since the feathers are dissolved in a very short period of time. After the methods of precipitation, washing and dissolving the protein solution, the percentage of keratin protein is evaluated by means of biuret test and FTIR analysis. The analysis by FTIR confirmed the presence of carboxyl acid and amino groups in the protein solution. The biuret test helps in determining the concentration of protein obtained from different methods. Thus these two tests confirm the presence of protein in the solution. From this research, it can be concluded that protein can be extracted from chicken feathers. The keratin protein solution can be used for several purposes such as anti-aging cream, shampoo, and conditioner and for medical purposes such as bone replacement and bone graft. Keywords: chicken feather, reducing agents, protein precipitation, analysis INTRODUCTION The present research work is regarding extracting natural keratin protein from chicken feathers by using different reducing agents. The reducing agents help in decreasing the stability of keratin fibers in the solid form found in feathers. These reagents will break down disulphide bonds, hydrogen bonds and salt linkages of the keratin fibers in order to dissolve it into protein solution. Currently there is an increasing interest in the development of materials that are environment friendly, obtained from renewable resources. The main

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renewable materials are obtained from polysaccharides, lipid and proteins. Proteins are polymers formed by various amino acids capable of promoting intra- and inter-molecular bonds, allowing the resultant materials to have a large variation in their functional properties. Feathers are bio-resource with high protein content more than 750 g per kg crude protein. Poultry slaughterhouses produce large amounts of feathers. Further, burning feathers in special installations is economically ineffective. Uncontrolled disposal of feathers is environmentally unacceptable. Five percent of the body weight of poultry is feathers; The slaughterhouse with a capacity of 50,000 birds, can easily produce 2-3 tones of dry feathers per day. The keratin from feather shows an elevated content of the amino acids glycine, alanine, serine, cysteine and valine, but lower amounts of lysine, methionine and tryptophan. Reducing agents used in this research for reduction of disulfide bonds are thioglycolic acid, potassium cyanide, and sodium sulfide. The reductants act very quickly and without bringing about any chemical alteration or damage to the protein yield. Products prepared from the solutions behave as true proteins, and not as products of hydrolysis. Their solutions are precipitated by ordinary protein precipitants such as sulfosalicylic acid, and ammonium sulfate. The ability to flex in multiple directions without tearing is an important quality of keratin and it also provides a tough and fibrous matrix in tissues (Sheen and Judy P, 2002). According to David et.al (1934), chicken feather keratins can be converted to natural protein soluble in alkali or acid and digestible by trypsin and pepsin. This was accomplished by breaking the disulfide bonds of the keratin. The β-keratins from feather have β-pleated sheets twisted together, and then stabilized and hardened by disulfide bonds. So by breaking these disulfide bonds in feathers, the strength of the keratin in the chicken feathers can be reduced thus become soluble and converted to natural protein. As per David.et.al (1935) oxidizing agents such as bromine, permanganate and hydrogen oxide act very slowly in breaking the disulfide bonds thus slowing down the protein extraction process. But in contrast, the reducing agents act very quickly and dissolve keratin only at alkaline reaction (pH 10 to 13), but the action is not due to alkali alone. Products prepared from these reactions behave as true proteins and not as products of hydrolysis. Few other previous researchers have worked on the extraction process of keratin. Schrooyen.et.al.(2001) have studied the effect of the addition of various quantities of SDS on the rate of aggregation of the polypeptide chains and the rate of oxidation of cysteine residues during dialysis. Yamauchi et al.(1996) investigated the extraction of wool keratins with an aqueous solution of urea, 2-mercaptoethanol, and sodium dodecyl sulfate (SDS). They found that the surfactant, SDS, accelerated the extraction and increased the extraction yield. It also stabilized the aqueous protein solution after removal of urea by dialysis against water 2

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containing 2-mercaptoethanol (0.08 wt%). The surfactant forms a complex with the keratin and is removed by dialysis much slower than other low molecular mass compounds. Arai.et.al (1982), determined the amino acid sequence of a single polypeptide chain, B-4, from fowl feather barbs. The B-4 chain was found to consist of 96 amino acid residues and to have a molecular weight of 10206 in the S-carboxymethylated form. Blackburn.et.al (1951) has studied the effect of different organic acids on the structure of wool protein. There are two types of keratin, alpha-keratin and beta-keratin. Alpha-keratins are found in the soft tissues protein fibers of sheep wool, hair and skin. It is twisted together and formed like a rope strand. Alpha-keratins amino acids sequences rich in cysteine and poor in hydroxyproline and proline. Beta-keratins are found in the hard tissues protein fibers such as bird feathers, nails, fish scales and others. The beta-keratins amino acids sequences is rich in small uncharged glycine and alanine and poor in cysteine, proline and hydroxyproline. Table 2.1: The Amino Acid Composition of Chicken Feathers Amino Acid Aspartic Acid Threonine Serine Proline Glutamic Acid Glycine Alanine Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalnine Lysine Histidine Arginine

uM/mg Protein*1 0.358 0.345 1.292 0.875 0.624 1.008 0.411 0.618 0.088 0.017 0.376 0.570 0.102 0.267 0.039 0.001 0.377

% Amino Acid in Protein 4.76 4.11 13.57 1.01 9.18 7.57 3.66 7.24 2.11 0.025 4.93 7.48 1.85 4.11 0.57 0.016 6.57

*Based on sample as 100% protein 1Micro mole per milligram of protein Keratin is insoluble in water and organic compound. The chemical properties of keratin are weak acids and bases. It is characterized by cystine content in the sequence of keratin amino acids and it can be hydrolysed, reduced and oxidized. High strength of keratin is

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influenced by the two cysteine molecules bonded by disulphide bonds Krystyna et.al ( 2007). Keratin protein fraction is used in the formulation of anti-wrinkle treatment cream, sulfite hair straightener, conditioning shampoo and other personal care Kelly.et.al( 2003).

MATERIAL AND METHODS Pre-treatment of the feathers

Chicken feathers are collected from chicken processing plants and soaked in ether for 24 hr. The main purpose is to clean the feathers from stains, oil and grease before processing it. The feathers are then washed with soap water and dried under sunlight. The dried feathers are then blended and kept carefully in sealed plastic bag Dissolving of chicken feathers

2L of 0.5M sodium sulfide solution is prepared in a 2L conical flask. 50g of the blended chicken feathers are weighed and added to the sodium sulfide solution. The solution is heated to the temperature of 30 o C, pH is maintained between 10-13 and the solution is continuously stirred for 6 hours. The solution is then filtered and centrifuged at 10,000 rpm for 5 minutes. The supernatant liquid carefully collected then filtered using filter paper to make it particle free. Preparation of ammonium sulfate solution

700g of ammonium sulfate is dissolved in 1L deionized water. The solution is stirred until all the ammonium sulfate particles are dissolved. The solution is then filtered to make it particle free. Protein precipitation

The feather filtrate solution collected earlier placed in a beaker and stirred. Ammonium sulfate solution is added slowly drop wise. The ratio of feather filtrate solution and ammonium sulfate solution added is 1:1. The solution is then centrifuged at 10,000 rpm for 5 minutes and the solids particles are carefully collected. The supernatant liquids are collected separately and step 2 and 3 are repeated with it. Protein purification

The solid particles collected are added into 100ml deionized water and stirred (washing). The solution is then centrifuged at 10,000 rpm for 5 minutes and the solids are gathered carefully. The collected solid particles are then dissolved in 100ml of 2M sodium hydroxide solution. The solution is then centrifuged again at 10,000 rpm for 5 minutes and all the liquids are collected carefully and stored while the solids are discarded. The precipitating, washing and dissolving steps are repeated 3 times.

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Biuret test

1% copper sulphate solution and 1% potassium hydroxide solution are prepared. The 5ml of the solution collected is mixed with potassium hydroxide solution with 1:1 ratio. Three drops of copper sulphate solution is added to the mixture solution. Changes in the solution observed and recorded. The solution is analyzed under uv-vis to obtain its absorbance. Analysis of the sample

The solution collected after purification is analyzed in FTIR and its wavelength graph obtained and compared with the standard graph. The solution fully precipitated using ammonium sulfate, the solids are separated weighed and its weight recorded. The whole process repeated with 0.5M thioglycolate solution and 0.5M potassium cyanide solution replacing sodium sulfide solution. Both thioglycolate and potassium cyanide solutions are prepared with 0.1N sodium hydroxide.

RESULTS AND DISCUSSION Observation

Chicken feathers dissolved completely in sodium sulfide solution, where as only partially dissolved in potassium cyanide and thiglycolate. Around 45% feathers in potassium cyanide solution and 70% feathers in thioglycolate solution can be filtered out after 6 hours of reaction. This concludes that sodium sulfide reduces chicken feather efficiently than the other two reducing agents. Standard ASTM (1997) testing was followed to test the presence of protein. The presence of protein is confirmed by biuret test. The solution turned purple after reagent is added and this is only possible if peptide bonds are present in it. The more peptide bonds in it, the higher the intensity of the purple color as seen in figure 1. Also the difference between the purple colors of the different solutions is visible seen in figure 1. This is in accordance with absorbance of biuret solution and amount of protein obtained in the end of the research. The higher the amount of chicken feather dissolved the higher the protein obtained.

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Absorbance of Biuret Test Solutions

Table 1: Absorbance of biuret test solutions and amount of protein obtained PROTEIN SAMPLES

ABSORBANCE

AMOUNT OF PROTEIN

A (Thioglycolate solution)

0.326

4.42 g

B (Potassium cyanide solution)

0.742

14.86 g

C (Sodium sulfide solution)

1.435

26.50 g

Sample A

Sample B

Sample C

Figure 1: Visible differences of biuret solutions of all three samples.

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Figure 2: Percentage of protein by using different reducing agents.

Figure 3: FTIR result obtained for the final product.

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According to Beer lambert law: A= εІc A= absorbance ɛ = molar absorption coefficient ( mol-1 dm3 cm-1). I = length of the solution light passes through (cm) c= concentration of the solution (mol dm-1) Absorbance is proportional to the concentration of a solution. So increasing the absorbance also shows higher protein concentration. It was observed that the highest absorbance is in sodium sulfide reacted solution and lowest is in the thiglycolic acid reacted solution. The dissolving rate of feathers in thioglycolate solution and potassium cyanide solution is low because the reaction will be highest only if the solution is highly alkaline with pH of the solution is in the range 10 to 13. This is because in the alkaline state the proton will be removed from the amino group and the ionic bond formed by electrostatic attraction of the NHs+ group of the diamino acids and the COO- group of the dicarboxylic acids can be broken. Due to some reasons these ionic bond must be broken first to reduce the disulfide bonds of the keratin and dissolve the feathers. The sodium sulfide solution is readily alkaline not like thiglycolate solution or potassium cyanide solution in which sodium hydroxide has to be added to make it alkaline with the pH between 10 to 13. This explains why even though both solutions are reducing agents they cannot reduce the disulfide bonds. Because without alkaline state proton cannot be removed thus cannot break the ionic bond. So sodium hydroxide plays an important role in dissolving feathers. So in order to maximize the dissolving ability of potassium cyanide and thioglycolate solution the exact amount of sodium hydroxide need to be calculated. The total mass of the protein obtained by using different reducing agents are as follows sodium sulfide (53%), potassium cyanide (29.6 %) and thioglycolic acid (8.8%) .This is in accordance with the result of the UV-Vis analysis. The protein sample also analyzed using Fourier transform infrared spectroscopy. The graph obtained matched the standard graph of protein solution. The wavelength obtained from the infrared spectroscopy confirmed C-N bond, N-H bond, and C=O bond are in the sample thus the presence of carboxyl group and amino groups the two groups that will only present in amino acids are undeniable. Therefore the sample confirmed true protein.

CONCLUSION The present research is done to find an alternative source of keratin protein. Since chicken feathers consist of 90% crude protein and pose an environmental problem due to its time consuming decomposition, it is an ideal material to obtain keratin protein. The chicken feathers were first dissolved using reducing agents and protein precipitated out from the 8

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solution using ammonium sulfate. In this research three different reducing agents, thiglycolic acid, potassium cyanide and sodium sulfide were used to dissolve the chicken feathers. The dissolving ability of all three agents were compared and found that sodium sulfide has the highest efficiency since it does dissolve all the feathers completely in a short period of time. Whereas, thioglycolic acid is least efficient in dissolving the feathers. It is also observed that the dissolving ability of thiglycolic acid and potassium cyanide mainly depends on the pH of the solution, which is controlled by the amount of sodium hydroxide added to the solution. Ammonium sulfate as expected precipitated protein and at the same time used to purify the protein. The presence of the protein was confirmed first by the biuret test where the reagent changed to purple color in the presence of peptide bonds. The Fourier transform infrared spectroscopy (FTIR) also confirmed the presence of amino and a carboxyl group in the sample, the two groups confirms the presence of amino acids. Thus the product obtained at the end of the research confirmed true keratin protein without the presence of any foreign materials.

REFERENCES Alexander, P. and Hudson, R. F.(1954). Wool: Its Chemistry and Physics, Chapter 5. London: Chapman and Hall Ltd. Arai, K. M., R.Takahashi, Y.Yokote and K. Akahane,(1983). Aminoacid sequence of feather keratin from fowl. European Journal Biochemistry, 32, 501–510. Astbury, W. T., Tr. Faraday Sot., 29, 193 (1933); Fundamentals of fiber structure, London (1933). ASTM. (1997). Standard test methods for wool content of raw wool. In Annual book of ASTM standards (pp. 1–5). Designation (D584-96), Philadelphia. Blackburn.S and A. G. Lowther (1951) The Action of Organic Acids on some Fibrous Proteins: the Oxidation of Wool Keratin. Biochem J. 1951 49(4):554-559 David R. Goddard, Leonor Michaelis. (1934). A Study of keratin. Journal of biological chemistry. The rockweller institute of medical research. David R. Goddard, Leonor Michaelis. (1935). Derivatives of keratin. Journal of biological chemistry. The rockweller institute of medical research. Harrap,B.S and E. F. Woods. (1963). Soluble derivatives of feather keratin. Journal of Protein chemistry divison of protein chemistry, wool research laboratories Australia.

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R.J. Kelly and A.D. Roddick-Lanzilotta (2006). Personal care formulations containing keratin. US Patent, US 20060165635. Retrieved February 26, 2011 from http://appft1.uspto.gov/ Schrooyen, P. M. M., P.J.Dijkstra, R.C.Oberthur, A.Bantjes, and J. Feijen (2001). Stabilization of solutions of feather keratins by sodium dodecyl sulfate. Journal of Colloid and Interface Science, 240, 30–39. Yamauchi, K., Yamauchi, A., Kusunoki, T., Khoda, A., and Konishi, Y.(1996), J. Biomed. Mater. Res. 31, 439 . Yamauchi, A., & Yamauchi, K. (2002). Formation and properties of wool keratin films and coatings. In A. Gennadios (Ed.), Protein based films and coatings (pp. 253–274). Boca Raton, FL, USA: CRC Press. Bibliography: Dr Arun Gupta is presently working as a Senior Lecturer in Faculty of Chemical & Natural Resources Engineering, University Malaysia Pahang. His area of expertise is in process modeling, simulation, wood composites and extraction and separation of protein. He have done his PhD on “Modeling and Optimization of MDF Hot pressing”, from University of Canterbury, New Zealand. Earlier he worked for two years in wood composite industry as a development officer in India and for another two years in Keratec.Pvt.Ltd, Biotechnology Company in New Zealand. At present he is supervising two PhD and two master’s students. He received two Gold Medals for his work on, “Nano-Wood Composite”,(INPEX-2011 exhibition, USA, and BioMalaysia 2010) and Silver medal in MTE-2011 exhibition, for his research on, “Developing new method to extract keratin protein from chicken feather”.

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