144
STUDIES ON THE EXTRACTION OF PROTEIN FROM POULTRY FEATHERS AND
THEIR ANALYSIS RESULTS
1
Tursunova Dilnozakhon Isroiljon qizi,
2
Ziyadullayev Anvar Egamberdiyevich
1
Department of Basic organic synthesis products, Faculty of Chemical technology of fuels and
organic substances, University of Tashkent Chemical-Technological Institute, Tashkent-100011,
Uzbekistan. e-mail:
dilnoza7417@mail.ru
ORCID ID:
https://orcid.org/0000-0002-4281-8383
2
Department of Basic organic synthesis products, Faculty of Chemical technology of fuels and
organic substances, University of Tashkent Chemical-Technological Institute, Tashkent-100011,
Uzbekistan. e-mail:
anvar_ziyadullayev@mail.ru
ORCID ID:
https://orcid.org/0000-0001-9864-
3132
Abstract
Poultry farming is one of the most developed industries, and it is very important to operate
the industry completely without waste. More than 20,000 chickens are slaughtered in poultry
factories per month. Taking into account that on average 100 grams of feathers are extracted from
one chicken, the main goal is to extract keratin protein by processing more than 2 tons of chicken
feathers per month. During the research, my method of extracting protein from feathers will be
discussed. In addition, determination of the amount of nitrogen in the protein isolated by a special
method, determination of the total protein and amino acids are discussed. In the results section, it
was found that 25% dry mass was extracted, it contained 17 different amino acids. It was concluded
that chicken feather protein can be used for cosmetics based on theoretical comparisons with keratin
protein. Hair is composed mainly of keratin protein and a small amount of lipid.
Keywords:
Keratin, Protein, Feathers, Amino acids, Chicken feather, Feather protein.
Introduction
We know that feather pillows are made from bird feathers. But the feather pillow is a
favorable environment for the development of fungi. Due to the fact that it is the cause of various
allergic diseases, it is not in demand among the population today.
Poultry farming is one of the main areas of agriculture. Today, poultry meat is popular
among the population. By using poultry products and their feathers in the national economy, it is
possible to reduce the amount of harmful waste emitted into the atmosphere and environment. The
main function of a bird's feather is, of course, the ability to fly. The chemical composition,
properties and research results of all birds and poultry feathers are presented in the article. By
working without waste, it is possible to bring up ecological culture, to fundamentally change the
relationship between man and the environment. On average, 100 grams of dry chicken feathers are
extracted from one chicken, and insufficient processing system is considered one of the urgent
problems. Keratin protein is isolated as a solution to the problem. The advantages of processing
poultry feathers and applying cosmetics have been proven in the world experience.
Literature review
Approximately 5 million tons of feather biomass is being generated annually from poultry
farms [1,2] which leads to serious solid waste hazards [3]. It is important to establish a feather
processing system. Considering the high protein content (90%) [4], it could provide an excellent
source of amino acids for animal feed [5], bio composites [6] and for numerous other industrial
applications [7,8]. The sodium sulfide-based extraction of keratin by dissolving chicken feather is
an efficient and economically favorable method which provide sufficient yield and also retained the
sec-ondary structure of protein [9]. The feather protein also has potent antioxidant and anticancer
properties. Recently the anti-cancerous activity of hair protein had been reported by [24].
Na
2
S + H
2
O → 2Na
+
+ HS
−
+ OH
−
Hair is composed mainly of keratin protein (90%) and a small amount of lipid (1–9%). The
diameter of hair fibers varies between 40 and 150 μm and its major structure consists of a cuticle,
cortex and medulla [10].
Keratin protein is present in the div of reptiles and birds. For example,
145
found in mammals' feathers, claws, horns and horns [11].
According to the literature analysis, it shows keratin hydrolysis peptides with molecular
mass are obtained by enzymatic method 800 to 1079 Daltons. Keratin is widely used in the
cosmetic, medical, pharmaceutical and biotechnological industry.
Considering that keratin protein obtained from feathers is an environmentally friendly
product, based on stability and biotransformation he claims that it is protein rich biomass. Keratin is
a complex biopolymer, composed of 19 amino acids linked together in ladder-like polypeptide
chains by peptide bonds [12].
Based on the sources of this article, [13] the chemically extracted particles of keratin
proteins can be used pharmacologically to formulate the medically important products like anti-
ageing cream, shampoo and wound healing creams. This study also provided a sustainable method
to remove the huge chicken feather waste biomass moderately generated by poultry industry and
production of value aided materials. The feather biomass is easily available and can be used
produce the keratin microparticles with effective bio-functional properties of pharmaceutical use.
Feather keratin has an average molecular mass of about 60500 g/mol, ranging from 59000 up to
65000 Daltons [14]. Melting point – 230 °C.
Experimental parts of articles published in international journals were studied. Fresh white
chicken feathers (about 3cm-20cm long) slaughtered adult Leghorn chickens were supplied by
Baiada Poultry Pty Ltd (Melbourne VIC, Australia). All feathers are cleaned of oils, stains, dirt, etc.
by ethanol extraction method. Cleaned feathers were conditioned using an incubator at 34 ± 1°C for
3 days and conditioned at 20 ± 2°C and 60 ± 2%. It was dried for 72 hours until the relative
humidity disappeared. Ground chicken feathers in a ratio of 1:20 to 100 ml of aqueous solutions 0.5
mol/L sodium sulfide (Na
2
S) solution (Solution A) and immersed in 8 mol/L urea (NH
2
CONH
2
) and
0.165 mol/L L-cysteine (Solution B), then adjusted to pH 10.5 using NaOH (2 mol/L). Then the
solutions are heated and stored at 40°C and required continuous stirring for 6 h at 10 g using a
magnetic stirrer, then centrifuged at 11,648 g for 20 min at 10°C. The supernatant was collected and
the particles contained were discarded. Hydrochloric acid (7 mol/L) was added to the solution until
pH 4 [38,39] and the isoelectric-keratin point was taken and left for 2 h without heating or stirring.
The aqueous phase was tested after sedimentation and the Biuret test confirmed the presence of
protein. The precipitated keratin was washed three times with deionized water, filtered and dried at
40°C. Keratin powders were taken and stored in closed light-sensitive glass containers. When the
concentration of keratin solutions was analyzed by ultraviolet radiation, the spectrophotometer was
determined to be 280 nm using the BioFotometer plus [16].
Research Methodolog
The chicken feathers were collected from a chicken process-ing plant at Jaya Gading,
Kuantan, Malaysia. Sodium sulfide, NaOH, HCl, petroleum ether and Cetrimonium bromide
(CTAB)were purchased from Sigma Aldrich (Selangore, Malaysia). Wet feathers are cleaned and
dried in a ventilated oven at 40°C for 72 hours. Chicken feathers are degreased using petroleum
ether, then washed twice with distilled water. Washed feathers are dried for 24 hours at a
temperature of 20°C and a relative humidity of 65%. After the first cleaning, the feathers are
cleaned with Cetrimonium bromide for 3 hours to remove impurities and microbial contamination.
These feathers were then cut into small pieces (2–25 cm), dried under sunlight for 48 h, and stored
at 4◦C for further use. For keratin extraction, 25 g of ground feathers were immersed in sodium
sulfide (0.5 M) at 50°C for 6 h using a mechanical stirrer. The hydrolyzate solution was filtered
twice and centrifuged to separate the supernatant. The pH of the hydrolyzate solution is neutralized
with 2N HCl to pH 3.5 and 5.5, and a thick precipitate is deposited after 24 hours. The precipitates
were collected and designated as Keratin microparticles (KM1 and KM2, respectively). To remove
salts and other impurities, KM1 and KM2 were dispersed in water and centrifuged 3 times at 12,000
rpm for 10 minutes each. Finally, the keratin precipitate was collected and lyophilized to obtain
keratin powders. The total yield of extracted keratin powder was 79.6% and 70.23% for KM1 and
KM2, respectively [13].
10 g of chicken feathers in warm soapy water and a few washed once with distilled water.
146
Washed feathers are placed in a flask and 100 ml of 10% NaOH solution is added and the process of
alkaline hydrolysis occurs. It is also hydrolyzed with reducing agents. In this way 10 g of feathers
are washed with warm soapy water, put into a distilled flask, shaken several times, and 200 ml of
0.1 N NaOH solution, 200 ml 10% solution of H
2
O
2
, Na
2
S, Na
2
SO
3
is added. The amount of
isolated protein is determined by the protein photocolorimetric method and the biuret reaction
method. As a result, up to 43 g/l of protein was extracted within 48 hours [17].
Effects of reagents
According to preliminary studies, feathers are not affected by hot and cold water when they
are carried over them in a laboratory room with 87% humidity.
When heated under the influence of sulfuric acid H
2
SO
4
, a black solution is formed and the
feather dissolves in 15 minutes.
When heated with phosphoric acid H
3
PO
4
, a purple solution was formed and the feather was
insoluble.
Liquid ammonia solution and ammonium hydroxide NH
4
OH do not cause any change in the
solution when exposed to poultry feathers.
When exposed to nitric acid HNO
3
, the feather completely dissolves and a pale-yellow
solution is formed.
When caustic soda NaOH is applied to a bird's feather for 10 minutes, the feather dissolves
and a light-yellow solution is formed. Based on preliminary studies, it was decided to hydrolyze the
feathers in an alkaline medium.
The method of extracting proteins from discarded feathers
The feathers were collected from the poultry factory located in the Fergana region of the
Republic of Uzbekistan. According to the conducted research, poultry feathers are washed, dried
and ground to a size of 0.25 cm. To separate proteins from biomaterials, their solubility property is
used. But the isolation of proteins causes more difficulties. The main difficulty in this separation is
their instability. They lose their natural "native" properties under the influence of high temperature,
strong acids and alkalis, and a lot of reagents. These processes are called denaturation. The second
difficulty encountered in the isolation of proteins is to get rid of protein molecules, other organic
compounds that mix with them and form complexes with them, lipids, carbohydrates, and nucleic
acids in complex mixtures obtained from biological material. In order to get rid of the residual oils
contained in the feather, it is cleaned using extracted gasoline. Then, feather dried with a dryer was
hydrolyzed with NaOH solution with 0.2 N pH value of 12 in a 95℃ water bath (1:20 ratio) for 2
hours. When measured on an analytical balance, it was found that 20 g of protein (25% compared to
the initial product) precipitated. When the solubility of the isolated protein was studied, it did not
dissolve under the influence of water, alcohol, acetic acid, acetonitrile, hydrochloric acid and salts
containing phosphate anion. When it was exposed to sodium alkali, it was observed that the melting
process took place. All chemicals were of analytical grade and used as received.
Figure 2 shows the original chicken feathers and the protein I extracted, which is the
minimum working version of the finished product of the Feather Keratin Start-up project. In
addition, here you can see the unpurified state of the protein in dark brown color and the purified
protein in light yellow color. The liquid in the last container reflects the state of our protein mixed
with shampoo.
3. Results and Discussion
The amino acid content of the resulting protein hydrolyzate was determined by
chromatographic method. Chromatographic analysis results are shown in Table 2. It was analyzed
according to the method of amino acid analysis using phenyl isothiocyanate derivatives. Isolation of
free amino acids. The sedimentation of proteins and peptides from the aqueous extract of the
samples was carried out in centrifuge beakers. To do this, 1 ml (exact volume) of 20% TCA was
added to 1 ml of the test sample. After 10 minutes the precipitate was separated by centrifugation at
8000 rpm for 15 minutes. After separating 0.1 ml of the supernatant, freeze-dried. The hydrolyzate
147
was evaporated, the dry residue was dissolved in a mixture of triethylamine-acetonitrile-water
(1:7:1) and dried. This operation was repeated twice to neutralize the acid. Reaction with
phenylthioisocyanate gave phenylthiocarbamyl derivatives (FTC) of amino acids according to the
method of Steven A., Cohen Daviel. Identification of amino acid derivatives was carried out by
HPLC. HPLC conditions: Agilent Technologies 1200 chromatograph with DAD detector, 75x4.6
mm Discovery HS C18 column. Solution A: 0.14 M CH
3
COONa + 0.05% TEA pH 6.4, B: CH
3
CN.
Flow rate 1.2 ml/min, absorbance 269nm. Gradient %B/min: 1-6%/0-2.5min; 6-30%/2.51-40min;
30-60%/40.1-45min; 60-60%/45.1-50min; 60-0%/50.1-55min [18].
Figure 3. Chromatogram of chicken feather protein hydrolyzate.
Table 2.
The amount of amino acids in 1 gram of substance according to the chromatogram of
protein hydrolyzate obtained from chicken feathers.
№
Name of amino acids
Protein hydrolysis isolated from chicken
feathers
Concentration mg/g
1
Asparaginic acid
56,78975
2
Glutamic acid
78,86349
3
Tserin
56,99378
4
Glycine
65,98891
5
Asparagine
0
6
Glutamine
0
7
Cysteine
46,2541
8
Threonine
37,11877
9
Arginine
44,37028
10
Alanine
37,92451
11
Proline
74,15931
12
Tyrosine
52,13104
13
Valin
79,67239
14
Methionine
13,73588
15
Histidine
55,13941
16
Isolate
43,56399
17
Leucine
97,88839
148
18
Tryptophan
0
19
Phenylalanine
26,67172
20
Lysine
22,80812
Total
890,0738
In this article, the main goal is to reduce the amount of environmental waste by promoting the
technology of processing chicken feathers, which is considered as a waste of poultry farms. The
problem exists in all poultry factories, and by reducing the amount of environmental waste, creating
an additional income by creating a zero-waste technology, and trying to solve the problem in a new
way, causes the interest of entrepreneurs to increase. If more than 25,000 broiler chickens are
slaughtered per month in one poultry factory, 625 kg of protein can be obtained by processing the
feathers separated from them with a yield of 25% in our research.
According to the account books, an entrepreneur who processed waste by selling 7.5 tons of
protein per year will have a large investment by introducing the finished product to the market. The
product mainly occupies the market of cosmetic products intended for hair, there is a significant
consumer demand, and it will stimulate the creation of additional vacancies in the labor market.
Discussion
According to our research results, 250-300 kg of protein can be extracted from 1 kg of
chicken feathers. Further research is being done to increase the yield. Unlike the method of other
scientists mentioned in the analysis of the literature, in the article, only sodium hydroxide and acetic
acid can be seen in the process of alkaline hydrolysis and precipitation of proteins. In this way, the
necessary raw materials can be reduced. We are conducting research on the introduction of feather
processing technology in the conditions of Uzbekistan.
The main goal is to use cosmetics for hair that preserves keratin protein by introducing the
technology of protein extraction from chicken feathers, and includes the following tasks:
- extraction of dry mass for the preparation of protein from chicken feather, which is considered a
local raw material;
- study of the chemical composition of dry mass obtained from chicken feathers;
- to determine the composition of the main amino acids in the dry mass obtained from chicken
feathers;
- is to develop a recipe for the preparation of special washing agents for hair based on the obtained
keratin protein.
Conclusion
One of the disadvantages of our method is the smell of acetic acid and alkaline solutions. In
addition, it is necessary to bleach the color of the obtained protein. In addition, it is necessary to
study the toxicological and allergic aspects of the isolated protein. The reason is that feathers collect
fungi in themselves, and feathers are no longer used as pillows today. Also, in the future, it was
aimed to study the content of the extracted protein more deeply, to determine the content of heavy
metals.
References
[1]. P.Manivasagan, J.Venkatesan, K.Sivakumar,S.-K. Kim, Production, characterization and
antioxidant potential of protease from Streptomyces sp. MAB18 using poultry wastes, BioMed Res.
Int. (
2013
).
[2]. P.M.Schrooyen, P.J.Dijkstra, R.C.Oberthür, A.Bantjes, J.Feijen, Partiallycarboxymethylated
feather keratins. 1. Properties in aqueous systems, J.Agric. Food Chem. 48 (9) (
2000
) 4326–4334.
[3]. A.Gupta, N.B.Kamarudin, C.Y.G. Kee, R.B.M. Yunus, Extraction of keratinprotein from
chicken feather, J. Chem. Chem. Eng. 6 (8) (
2012
) 732.
[4]. J. Csap´o, Cs. Albert, Methods and procedures for the processing of feather from poultry
slaughterhouses and the application of feather meal as antioxidant, Acta Univ. Sapientiae,
Alimentaria, 11 (
2018
) 81–96
149
[5]. J.G.Rouse, M.E. Van Dyke, A review of keratin-based biomaterials forbiomedical applications,
Materials 3 (2) (
2010
) 999–1014.
[6]. J.R.Barone, Lignocellulosic fiber-reinforced keratin polymer composites, J.Polym. Environ. 17
(2) (
2009
) 143–151.
[7]. A.Brandelli, L.Sala, S.J.Kalil, Microbial enzymes for bioconversion of poultrywaste into
added-value products, Food Res. Int. 73 (
2015
) 3–12.
[8]. P. Kumaran, A.Gupta, S.Sharma, Synthesis of wound-healing keratinhydrogels using chicken
feathers proteins and its properties, Int. J. Pharm.Pharm. Sci. 8 (
2016
).
[9]. A.J.Poole, R.E.Lyons, J.S.Church, Dissolving feather keratin using sodiumsulfide for bio-
polymer applications, J. Polym. Environ. 19 (4) (
2011
)995–1004.
10].
Kajiura Y, Watanabe S, Itou T, Nakamura K, Iida A, Inoue K, NYagi N,
Shinohara Y, Amemiya Y: Structural analysis of human hair single fibres by scanning microbeam
SAXS. J Strut Biol
2006
, 155(3):438–444.
[11]. Sionkowska A, Skopinska-Wiśniewska J, Kozłowska J, Płanecka A, Kurzawa
M:Photochemical behaviour of hydrolysed keratin. Int J Cosm Sci
2011
,33:503–508.
[12].
J. Zhang, et al., Isolation and characterization of biofunctional keratin particles extracted from
wool wastes, Powder Technol. 246 (
2013
) 356–362.
[13]. Swati Sharma, et al., Characterization of keratin microparticles from feather biomass
withpotent antioxidant and anticancer activities. International Journal of Biological
Macromolecules 104 (
2017
) 189–196
[14]. K. Murayama-Arai, R. Takahashi, Y. Yokote, K. Akahane. European Journal of Biochemistry,
132, 501–507 (
1983
).
[15]. A. Ghosh, S.R. Collie. Defence Science Journal, 64, 3, 209-221 (
2014
) .
[16]. F.Pourjavaheri, et al., Extraction of keratin from waste chicken feathers using sodium sulfide
and Lcysteine, Process Biochemistry 82 (
2019
) 205–214.
[17].
Бортников С.В., Горенкова Г.А., получение растворимой модификации кератина из
белок содержащих отходов животноводств, ХИМИЧЕСКИЕ НАУКИ (02.00.00)
[18]. Steven A., Cohen Daviel J. Amino acid analysis utilizing phenylisothiocyanata derivatives //
Jour. Analytical Biochemistry –
1988
. – V.17.-№1.-P.1-16.
[19]. Methods of control. chemical factors. Guidance on methods of quality control and safety of
biologically active food supplements. Guide R 4.1.1672-03. M.: Federal Center for State Sanitary
and Epidemiological Surveillance of the Ministry of Health of Russia,
2004
.
