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Arunmozhivarman et al Int. J. Pure App. Biosci. 5 (5): 1085-1091 (2017) ISSN: 2320 – 7051
DOI: http://dx.doi.org/10.18782/2320-7051.5337 ISSN: 2320 – 7051
Int. J. Pure App. Biosci. 5 (5): 1085-1091 (2017)
Research Article

Extraction and Molecular Characterization of Collagen from Poultry Meat

Processing by-Product (Chicken Skin)

K. Arunmozhivarman*, Robinson J. J. Abraham, V. Appa rao and M. Parthiban

Department of Livestock Products Technology (Meat Science), Madras Veterinary College, Tamil Nadu
Veterinary and Animal Sciences University, Chennai – 600 007,
*Corresponding Author E-mail: arunmozhivet@gmail.com,
Received: 27.07.2017 | Revised: 30.08.2017 | Accepted: 6.09.2017

ABSTRACT
A study was conducted to utilize less value chicken skin, a by-product obtained during poultry
meat processing to extract collagen and to determine type and level of collagen available in it.
Collagen was extracted with 0.5M acetic acid containing 1% pepsin after pretreatment of
samples to remove non collagenous protein and fat, using 0.1N NaOH and 20% ethanol. The
extracted collagen was lyophilized and characterized. SDS-PAGE study revealed that collagen
had molecular pattern with two α chain (α 1 and α 2) and one ß chain which is indicative of
Type-I collagen. UV spectrum analysis of collagen was done and found that no contamination
with non collagen protein. The amino acid analysis showed that glycine was the major
component and less amount of lysine, isoleucine. FTIR study showed the characteristic amide
band at 3305.19 cm-1, 2922.52 cm-1, 1633.98 cm-1, 1549.08 cm-1, 1238.07 cm-1 for amide A, B, I,
II and III of collagen respectively. The micro architecture studies of collagen using Scanning
Electron Microscopy confirmed that the collagen is fibrillar in structure. The result of our study
clearly indicated that chicken skin has significant amount of Type-I collagen which can be
extracted using acetic acid with pepsin method.
Key words: Chicken skin, by-product utilization, collagen extraction, Type I collagen, value
addition.

INTRODUCTION which type one collagen was the predominant

Collagen is the major structural protein present which contributes 90% of the total collagen.
in vertebrates and constitutes about 30% of the The chicken skin is the one of the major by
total animal protein. They were widely products that is generated during poultry meat
distributed in the skin, bones, tendons, processing process. Commercially the chicken
vascular system and intra muscular connective skin are used to prepare animal meal for
tissues where they contribute to the stability animal feed formulation where as a small
and structural integrity of the tissues and proportion is incorporated in to meat emulsion
organs7. The structure and function of 28 or used as a source of fat mainly for soup
distinct vertebrate collagen types were well preparation5.
understood and have been identified20. Among
Cite this article: Arunmozhivarman, K., Abraham, R.J.J., Appa rao, V. and Parthiban, M., Extraction and
molecular characterization of collagen from poultry meat processing by-product (chicken skin), Int. J. Pure
App. Biosci. 5(5): 1085-1091 (2017). doi: http://dx.doi.org/10.18782/2320-7051.5337

Copyright © Sept.-Oct., 2017; IJPAB 1085

Arunmozhivarman et al Int. J. Pure App. Biosci. 5 (5): 1085-1091 (2017) ISSN: 2320 – 7051
Chicken skin contains 3% collagen3 in which lyophilization and stored at refrigerator for
approximately 75% is type one collagen and further analysis.
15% type III collagen1. Collagen products are Analytical Procedure:
mainly manufactured from skin and bones of SDS–PAGE was done using 8% stacking gel
animals. The most common raw material used and a 5% resolving gel12. The amino acid
in collagen products are porcine skin and bone. profile of the extracted collagen was analyzed
Due to objection against to use pork in by HPLC. A 100 mg sample of extracted
collagen products, alternative resources gained collagen was hydrolysed in 8 ml of 6M HCl at
tremendous attention from the researcher and 110 °C for 22 h. The ultraviolet absorption
poultry processing by-product might be used spectra of the chicken skin and feet collagens
as alternative resource4. Avian collagen is were recorded by an Epoch ™ Microplate
considered as one of the alternative source for spectrophotometer. The collagen was
various skin care and medical application dissolved in 0.5M acetic acid to obtain a
instead of conventional bovine and porcine concentration of 1 mg/ml. Prior to
collagen13. Hence, this study was conducted measurement, a base line was set with 0.5M
for utilization of chicken skin for collagen acetic acid.
extraction and characterizes collagen to see the Samples were prepared for FTIR
possibilities to use in product preparation. according to the method previously described9.
10 mg of collagen was mixed with
MATERIALS AND METHODS approximately 100 mg of potassium bromide
Preparation samples: (KBr). All spectra were obtained from 4000 to
Chicken skin was collected from in and around 400 cm-1 at a data acquisition rate of 4 cm-1 per
vepery, chennai area. They were taken to the point. The SEM observations were made at 15
laboratory in the icebox and thoroughly kV accelerating voltage with a high vacuum
washed with distilled water. The superficial (HV) mode.
adhesive fat from the chicken skin was
manually removed, cut into small pieces and RESULTS AND DISCUSSION
minced by using meat mincer. Collagen yield, most probably depends on the
Collagen Extraction: proportion of fractions of different protein in
The collagen from the pre weighed minced the samples used for collagen extraction10. The
skin samples were extracted 18 with proper yield of collagen from chicken skin in this
modification at 4°C. Fat and non collagenous study was 10-12 percent. The amino acid
protein were removed by soaking the minced composition plays a major role in physical
sample for 24 hours in 20% ethanol and 0.1N properties of collagen. The chicken skin
NaOH by changing the solution at every 8 collagen had approximately 31.25% of glycine
hours interval. The ratio of sample solution as the major amino acid followed by histidine
was maintained at 1:20 (w/v). Then the (20.24%), threonine (9.32%), arginine (8.18
collagen from the samples was solubilized by %), serine (6.54%), alanine (4.98%), valine
soaking in 0.5M acetic acid containing 1% (4.39%), leucine (4.35%) glutamic acid
pepsin (1:3000) at 1:30 w/v ratio for 48 hours. (3.18%), methionine (2.82%), tyrosine
Then the collagen was precipitated by addition (1.38%), phenylalanine (1.03%), aspartic acid
of NaCl and collected by centrifugation at 4°C (1.18%), lysine (0.82%) and isoleucine
in refrigerator centrifuge. The extracted (0.36%). The analysis of amino acid
collagen was purified by dialysis followed by composition of collagen from extracted
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Arunmozhivarman et al Int. J. Pure App. Biosci. 5 (5): 1085-1091 (2017) ISSN: 2320 – 7051
chicken skin revealed that, glycine is abundant that collagen complex with hydrogen bonding
in collagen, constitutes around 30 percent of between free N-H stretches attaches with
the total amino acid content. However, the hydrogen in polypeptide chain.
value may vary according to species and body The peaks of amides „I‟ and „II‟ of
parts8. chicken skin collagen was 1633.98 cm-1,
The protein patterns of collagen on SDS- 1549.08 cm-1 respectively. It was found that
PAGE is shown figure 1. The collagen from the amide „I‟ band, with characteristic
chicken skin comprised of two different α- frequency in the range from 1600 to 1700 cm-1
chains (α1 and α2) with slightly different was mainly associated with the stretching
mobility and one ß chain which indicated that vibrations of the carbonyl groups (C=O bond)
the collagen from the skin, might be type I along the polypeptide backbone19 and was the
collagen11. Acetic acid was used as solvent for sensitive marker of the peptide secondary
collagen extraction in which no fragments less structure24.
than 116 kDa were observed in the In addition, the absorption peaks
electrophorogram and the molecular structures around 1451–1450 cm-1 were also found. This
of collagen were well maintained during considerably corresponded to pyrrolidine ring
extraction process21. vibration of proline and hydroxyproline17. The
The ultra-violet spectra of the collagen band in the spectrum between 1200 and 1300
(figure 2) in this study had high intensity cm-1 are unique “fingerprint” of collagen
absorbance ranging from 230 to 240 nm and molecular conformation attributed to particular
no or less absorption peak at 280 nm. The tripeptide (Gly-Pro-Hyp)n 9. Moreover, the N-
result indicated that high efficiency of non- H stretching band (3300 cm-1) of denatured
collagenous protein removal2.Collagen collagen (gelatin) was not detected. These
commonly has a low amount of tyrosine, results implied the collagen still preserved as
which could absorb UV-light at 280 nm6,25. native conformation during purification
Fourier transform infrared process. The absorption of amide „III‟ for
spectroscopy of collagen of chicken skin collagen at 1238.07 cm-1 wavelength indicates
(figure 3) showed characteristic peaks of the existence of helical structure 15.
amide A, B, I, II, III at 3305.19 cm-1, 2922.52 In the present study, the SEM images
cm-1, 1633.98 cm-1, 1549.08 cm-1 and 1238.07 (figure 4) of chicken skin collagen at low
cm-1 respectively. The absorption magnification showed fibrillar like structure
characteristics of amide „A‟ is commonly and further at high magnification, the collagen
associated with N-H stretching vibration this to be regular and uniform with net working of
occurs in the wave range of 3400-3440 cm-1. porous and honey-comb like structures on the
The absorption peak of Amide „A‟ band for surface20,23. The pore size, porosity and surface
collagen from chicken skin found at 3305.19 areas are widely recognized as important
cm-1. This decrease in absorbance may be due parameters for a collagen based biomaterial22.
to the N-H group which is involved with H- Generally, uniform and regular network
bond in peptide chain, the position starts to structure of collagen as drug carrier is
shift to lower frequencies14,16. propitious, for well proportioned distribution
The amide „B‟ peak of collagen from of drugs26.Based on the finding of this study
chicken skin found to be 2922.52 cm-1 which is collagen which could be extracted efficiently,
related to asymmetrical stretch of CH27. chicken skin collagen can be suited to
Similar absorption between collagen suggested preparation of collagen based products.
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Arunmozhivarman et al Int. J. Pure App. Biosci. 5 (5): 1085-1091 (2017) ISSN: 2320 – 7051

Electrophoresis (SDS-PAGE)

Fig. 1: Molecular pattern of chicken skin collagen on SDS- Polyacrylamide Gel

4
3.5
collagen
3
skin
absorbance

2.5
2
1.5
1
0.5
0
200 250 300 350 400 450 500 550 600
nm

Fig. 2: UV spectrum analysis of collagen

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Arunmozhivarman et al Int. J. Pure App. Biosci. 5 (5): 1085-1091 (2017) ISSN: 2320 – 7051

Fig. 3: FTIR spectra of collagen from chicken skin

2500 X (High magnification) 500 X (Low magnification)

Fig. 4: Scanning Electron Microscopy image of collagen from skin

CONCLUSION Acknowledgement
The present study concluded that chicken skin I am thankful and grateful to the Director,
can be effectively utilized to extract Type I Project co-ordinators of All India Co-ordinated
collagen and the process can be optimized by Research Project on Post Harvest Engineering
Technology, Central Institute of Post Harvest
inclusion of pepsin during extraction. Proper
Engineering and Technology, Ludhiana-
collection and utilization of these wastes also 141001 and to the Indian Council of
prevent dumping of wastes in to environment Agricultural Research, New Delhi-110001 for
and thereby reduce environmental hazards. providing necessary funds for the research
The extracted collagen can be used to develop work. The authors are thankful to the Dean of
biomaterial like collagen sheet and it can be Madras Veterinary College, Tamil Nadu
used for various clinical conditions (e.g wound Veterinary and Animal Sciences University,
Chennai-600 051, India for providing
healing), which require further investigation.
necessary facilities to carry out research work.
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