Characterization of Collagen from Eggshell Membrane
Collagen was extracted by acid-pepsin digestion and isolated by salt precipitation from eggshell membrane. The characteristics of eggshell membrane collagen were investigated with amino acid analysis, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Fourier transforms infrared spectroscopy and differential scanning calorimetry. The amino acid composition of the eggshell membrane collagen is rich in glycine, proline and hydroxyproline. Electrophoresis revealed two different α (α1 and α2) chains. FTIR showed regions of amides A, B, I, II and III were 3325, 2926, 1653, 1550 and 1240 cm-1, respectively. Analysis of differential scanning calorimetry revealed that thermal denaturation temperature of eggshell membrane collagen was 55.10°C and collagen of eggshell membrane retains intermolecular crosslinks after extraction process. Collagen of eggshell membrane was typical type I collagen and may be applicable to variety of usage including functional food, cosmetic, biomedical and pharmaceutical industries.
Collagen isolated from the skins and bones of land-based animals is capable
of utilization, mainly it is from bovine and swine. Non denatured collagens
from these sources are widely and diversely used in food, cosmetics, biomedical
and pharmaceutical industries (Ogawa et al., 2004).
After the outbreaks of bovine spongiform encephalopathy and foot-and-mouth disease
crisis, the restrictions on collagen trade have been established and we need
alternative safe sources of collagen (Devore et al.,
2004). Another problem of bovine collagen application is the risk of autoimmune
and allergic reactions, about 2-3% of the population is allergic in this regard.
According to the facts mentioned above, many researchers have done a lot of
work to investigate the possibility of new source of collagen from aquatic life.
Their researches involve optimal isolation methods from different kinds of fish
and the functional properties of marine source collagens (Takeshi
and Nobutaka, 2002; Sadowska et al., 2003;
Jongjareonrak et al., 2005; Wang
et al., 2008; Woo et al., 2008).
As a kind of common industrial waste, eggshell can be readilycollected anywhere
in plenty. Eggshell membranes are composed of protein fibers between egg white
and inner surface of eggshell. Eggshell membrane protein has high percentages
of certain amino acids such as arginine, glutamic acid, methionine, valine,
cystine and proline. The presence of hydroxyproline in hydrolysates of membrane
layers suggest that membrane layers fiber structure consists of collagen as
component (Wong et al., 1984). Biochemical and
immunological tests have verified the deduction. Eggshell membrane primarily
contains type I collagen, type V collagen and type X collagen (Wong
et al., 1984; Arias et al., 1992),
all the three kinds of collagen can be used in various fields. It has been determined
that about 10% of total proteinaceous constituent in eggshell membrane structure
Eggshell membrane will be an alternative potential important source of collagen
for application in foods, cosmetics and biomedical materials, if we can verify
the similar character of collagen from eggshell membrane and land-based mammalians
tissue. But few are known about the chemical properties of eggshell membrane
collagen, although eggshell membrane has been shown to contain collagenous protein
(Wong et al., 1984). In China, eggshell is consistent
available as by-product from food industry, that is about 4 million tons per
year. Eggshell membrane collagen has been proved to be of very low autoimmune
and allergic reaction (Long et al., 2004). According
to the result of research on characteristic of eggshell membrane and biosafety,
the possibility of application in functional foods, cosmetic and other industries
The aim of the research is to investigate the characteristics of collagen extracted
from eggshell membrane with amino acid analysis, Sodium Dodecyl Sulphate-Polyacrylamide
Gel Electrophoresis(SDS-PAGE), Fourier Transforms Infrared (FTIR) spectroscopy
and Differential Scanning Calorimetry (DSC) and compare its characteristic with
collagen from land-based mammalians.
MATERIALS AND METHODS
Materials: Raw eggshell membrane: obtained from commercial eggs, the membrane is peeling off manually and consisted of inner and outer membranes. Pepsin (1:10000): purchased from Sigma Inc. All reagents used in this study were analytical grade.
Extraction of collagen from eggshell membrane: The collagen was extracted
with the method of Wong et al. (1984) with a slight
modification. The manipulations were performed at 4°C. The eggshell membranes
were homogenized with a homogenizer. To remove non-collagenous proteins, fresh
eggshell membranes were soaked in 0.1 N NaOH at a sample/alkaline solution ratio
of 1:10 (w/v) for 24 h. The alkaline solution was changed every 6 h. Then, alkali-treated
eggshell membrane was washed with cold deionized water until neutral or faintly
basic pH of wash water was obtained. Then the precipitate was subjected to pepsin
digestion at 0.5% for 24 h in 0.5 M acetic acid. The digest was centrifuged
at 10,000 g for 45 min and the precipitate was washed with three 50 mL portions
of 0.5 M acetic acid and discarded. The supernatant and washes were pooled together
and filtered through a fritted disk funnel. Solid NaCl was added to the solution
to achieve a final concentration of 0.9 M and stirred for 12 h. The precipitate
was collected by centrifugation (10,000 g, 45 min), redissolved in 0.5 M acetic
acid and dialyzed exhaustively against the 0.5 M acetic acid, 0.1 M acetic acid
and deionized water. The dialysates were then lyophilized.
Amino acid analysis: A 5 mg aliquot of collagen was dissolved in 3 mL of 6 N HCl and hydrolyzed in vacuum-sealed glass tubes at 110°C for 24 h using a dry bath incubator. Hydrolyzed samples were filtered through glass filters and the filtrates dissolved in citric acid buffer (pH 2.2) and injected into an amino acid auto analyzer (Amino acid analyzer 835, Hitachi Co., Japan).
SDS-polyacrylamide gel electrophoresis (SDS-PAGE):Electrophoresis patterns
were measured with the method of Guo (2005), using Mini-Protein
3 (Bio-Rad Laboratories, Hercules, California). Polyacrylamide gel was prepared
with 5% stacking gel and 12% resolving gel.
The collagen samples were dissolved in the sample buffer (0.5 M TrisHCl,
pH 6.8, containing 4% SDS, 20% glycerol) in the presence of 10% β-ME. Samples
were loaded onto the gel. After electrophoresis, gel was stained with Coomassie
brilliant blue R-250 in 15% v/v) methanol and 5% (v/v) acetic acid). Molecular
weight markers were used to estimate the molecular weight of protein bands.
Type I collagen of pig bone was used as control.
Fourier transforms infrared spectroscopy (FTIR): FTIR spectra analysis was performed with 1.5 mg collagen in approximately 150 mg potassium bromide (KBr). All spectra were obtained from 4000 to 400 cm-1 at a data acquisition rate of 4 cm-1 by using a FTIR spectrophotometer (FTIR-8400S, Shimadzu, Japan).
Differential scanning calorimetry: The denaturation temperature (Td)
of collagen was determined with DSC using a Perkin-Elmer DSC-6. The samples
(collagens of eggshell membrane) were prepared with slightly modified method
of Rochdi et al. (2000) and Komsa-Penkova
et al. (1999).
Temperature calibration was carried out with Indium thermogram. The samples (5-10 mg) were weighed accurately and sealed into aluminium pans. The samples scan speed is at 5°C min-1 over the range of 40-100°C. An empty pan was used as the reference. Total denaturation enthalpy (ΔH) was estimated by measuring the area in the DSC thermogram. The maximum transition temperature (Td) was estimated from the thermogram.
RESULTS AND DISCUSSION
Amino acid composition: The results of amino acid analyses of eggshell
membrane collagen were listed in Table 1. Compared with the
analysis result of bovine-skin type I collagen (Jiang, 2006),
the amino acid composition of eggshell membrane collagen is of similar ratio
to the former, i.e., the amino acid profile is similar. Glycine is the most
prevalent amino acid in eggshell membrane collagen at 30.27%. The stabilization
of conformation requires the occurrence of glycine residues at one of every
three position in specific amino acid sequence of peptide chain. The contents
of proline and hydroxyproline may be important for structural integrity of collagen.
The proportions of hydroxyproline and proline residues are 9.28 and 11.94 residues
per 100 amino acids residues, respectively. There are small proportion of cysteine,
methionine, histidine, phenylalanine and tyrosine residues with 0.34, 0.76,
0.80, 0.81 and 0.92 residues per 100 amino acids residues, respectively. The
amino acid composition of collagen consists of numerous repeating Gly-Y-X residues
in a triple helical conformation (Woo et al., 2008).
Consequently, we can deduce that the structure of eggshell membrane collagen
is a (Gly-Pro-Hyp)n pattern with the consideration of these facts.
||Amino acid compositions of eggshell membrane collagen and
|Amino acid composition of bovine-skin type I collagen, cited
from Jiang (2006)
||SDS-PAGE pattern of collagen from eggshell membrane. Lane
A: Type I collagen of eggshell membrane; Lane B: The pig-bone type I collagen;
Lane marker: Protein molecular weight standards
And eggshell membrane collagen possesses the most common triplet helical configuration
in 3 dimension space.
SDS-PAGE profile of eggshell membrane collagen: Electrophoretic profile
of eggshell membrane collagen was laid out by SDS-PAGE with pig-bone collagen
||Fourier transform infrared spectrum of eggshell membrane collagen
Type I collagen extracted from mammalian is mostly consisted of two α
chains (2:1 ratio of α1 and α2) and β-component
(Lee et al., 2002). Similar to type I collagen
of pig-bone, the eggshell membrane collagen comprised at least two different
α-chains (α1 and α2) with similar mobilities
to pig-bone collagen. The result indicates that eggshell membrane collagen might
be type I collagen primarily. Figure 1 shows the SDS-PAGE
profile of eggshell membrane collagen (type I) and eggshell membrane type I
collagen consists of α1 chain (MW is about 130 kDa) and α2
chain (MW is about 116 kDa). The ratio of α1 (I) and α2
(I) chains is approximate 1:1.
Fourier transform infrared spectroscopy: FTIR spectroscopy has been
used to study changes in the secondary structure of collagen (Friess
and Lee, 1996).
Amide I band is associated with stretching vibrations of carbonyl groups (C=O
bond), within 1600-1700 cm-1, along the polypeptide backbone (Payne
and Veis, 1988) and it is the most useful for infrared spectroscopic analysis
of the secondary structure of proteins (Surewicz and Mantsch,
1988). The FTIR spectrum of eggshell membrane collagen is shown in Fig.
2. The main absorption bands are amide A (3325 cm-1), amide B
(2926 cm-1), amide I (1653 cm-1), amide II (1550 cm-1)
and amide III (1240 cm-1). Amide A band is related to NH stretch
coupled with hydrogen bond, amide B is related to CH2 asymmetrical
stretch. Amide II is associated with NH bending and CN stretching. Amide III
is related to CN stretching and NH and is involved with the triple helical structure
of collagen. All of these are similar to those results of research about collagen
from deep-sea redfish, yellowfin tuna dorsal skin and Nile perch (Muyonga
et al., 2004; Wang et al., 2008; Woo
et al., 2008).
||The denaturation temperature (Td) and total denaturation
enthalpy (ΔH) of eggshell membrane collagen
Denaturation temperature (Td): The Td of eggshell
membrane collagen can be used as an effective index of assessing the stability
of eggshell membrane type I collagen. With increase in temperature (thermal
depolymerization process), the hydrogen bonds within collagen are broken progressively
and finally, the triple helix structure of collagen maintained by hydrogen bonds
is converted into the random coil conformation of gelatin (Wang
et al., 2008). The collagen Td of different animal species
seems to be correlated with the content of amino acids (proline and hydroxyproline).
The higher the amino acid content, the higher the stability of helices (Wong,
DSC thermogram of collagen from eggshell membrane is shown in Fig.
3. The Td is determined to be about 55.10°C for eggshell
membrane type I collagen. The value of ΔH of eggshell membrane collagen
is 20.3304 J g-1. The enthalpy changes associated with collagen denaturation
processes depend on positional preferences of ionized residues of Gly-X-Y group
and formation of hydrogen bonds of inner coil-coiled α-chains (Usha
and Ramasami, 2004; Cao and Xu, 2008). The Td
of eggshell membrane collagen is lower than pig skin collagen (60°C) and
bovine skin (63-65°C), higher than aquatic life collagen. Body collagen
stability is correlated with environmental and body temperature.
This study investigated the characteristics of collagen extracted from eggshell
membrane with the methods of amino acid analysis, SDS-PAGE, FTIR and DSC. The
collagen can be classified as type I collagen according to the results and analysis
of amino acid composition and SDS-PAGE tests. The results of FTIR spectra and
denaturation temperature show that eggshell membrane collagen maintain their
triple helical structures well, be of higher thermal stability. Eggshell membrane
collagen should be thought to be of similar characteristics as other sources.
Therefore, the results suggest that collagen of eggshell membrane should be
a potential resource alternative to mammalian collagen for commercial applications.
Eggshell membrane collagen may be applicable to variety of application, such
as functional food, cosmetic, biomedical and pharmaceutical industries.
This research was supported by the National Natural Science Foundation of China (Grant No. 30871954).
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