Download extraction of keratin protein from chicken feather

EXTRACTION OF KERATIN PROTEIN FROM CHICKEN FEATHER
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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
A,Gupta. R,Perumal. R,B,M,Yunus.
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
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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
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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
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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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