CHARACTERIZATION OF ACID-SOLUBLE COLLAGEN FROM THE SKIN OF HAMMERHEAD SHARK (SPHYRNA LEWINI)
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
bs_bs_banner
Journal of Food Biochemistry ISSN 1745-4514
CHARACTERIZATION OF ACID-SOLUBLE COLLAGEN FROM THE
SKIN OF HAMMERHEAD SHARK (SPHYRNA LEWINI)
CHANG-FENG CHI1, BIN WANG2,3, ZHONG-RUI LI2, HONG-YU LUO2, GUO-FANG DING2 and
CHANG-WEN WU1
1
National Engineering Research Center of Marine Facilities Aquaculture, School of Marine Science, Zhejiang Ocean University, Zhoushan, Zhejiang
316004, China
2
School of Food and Pharmacy, Zhejiang Ocean University, Qixiangtai Road 51, Zhoushan, Zhejiang 316004, China
3
Corresponding author. ABSTRACT
TEL: +86-580-2554536;
FAX: +86-580-2554781; The characteristics of the acid-soluble collagen (ASC) from the skin of hammer-
EMAIL: wangbin4159@hotmail.com head shark (Sphyrna lewini) (ASC-H) were investigated and compared with those
of calf skin collagen (CSC). ASC-H with a yield of 4.23 ± 0.54% (based on the wet
Received for Publication September 16, 2012 weight of skins) contained Gly (227 residues/1,000 residues) as the major amino
Accepted for Publication June 18, 2013
acid and had imino acids of 205 residues/1,000 residues. Amino acid composition,
sodium dodecyl sulfate polyacrylamide gel electrophoresis pattern and Fourier
doi:10.1111/jfbc.12042
transform spectroscopy (FTIR), confirmed that ASC-H was mainly composed of
type I collagen. The peptide map of ASC-H was different from that of CSC, sug-
gesting the differences in amino acid sequences and conformation. Td of ASC-H
was 16.89C, which was similar to those of cold-water fishes but significantly lower
than those of tropical fish species and mammals. ASC-H exhibited high solubility
in pH (1-4) and low NaCl concentrations (≤3%). In addition, the lyophilized col-
lagen displayed loose, fibrous and porous ultrastructure.
PRACTICAL APPLICATIONS
At present, large quantities of by-products, accounting for 50–70% of the original
raw material, are generated during the process of aquatic products processing
industry. Therefore, optimal use of these by-products is a promising way to
protect the environment and produce value-added products to increase the
revenue of fish processors. Recently, many scientists have focused their interests on
isolation and characterization of collagens extracted from skins of aquatic organ-
isms. However, no information regarding acid-soluble collagen (ASC) from skins
of Hammerhead shark (Sphyrna lewini) has been reported. The aim of this study
was to isolate and characterize ASC from skin of Hammerhead shark in compari-
son with type I collagen from calf skin (CSC). Therefore, the collagen extracted
from the skin of hammerhead shark could bring considerably economic benefits
as a substitute for mammalian collagen.
gen was traditionally isolated from by-products (skins and
INTRODUCTION
bones) of land-based animals such as cows, pigs and poultry
Collagen is the major component of extracellular matrix (Liu et al. 2012), and has been widely utilized as material for
and is vital for mechanical protection of tissues, organs and food additives, cosmetics, biomedical materials, pharmaceu-
physiological regulation of cellular environment. Till now, ticals and experimental reagents because of its excellent bio-
over 29 types of collagens have been identified (named compatibility, biodegradability and weak antigenicity
types I-XXIX) from various animal tissues, and each type (Zhang et al. 2006; Shoulders and Raines 2009; Liu et al.
has a distinctive amino acid sequence and molecular struc- 2010). However, the outbreaks of bovine spongiform
ture to play a unique role in the tissue (Shoulders and encephalopathy, transmissible spongiform encephalopathy
Raines 2009; Liu et al. 2010; Matmaroh et al. 2011). Colla- and foot-and-mouth disease had resulted in anxiety among
236 Journal of Food Biochemistry 38 (2014) 236–247 © 2013 Wiley Periodicals, Inc.
C-F. CHI ET AL. COLLAGEN FROM THE SKIN OF HAMMERHEAD SHARK
users of collagen and collagen-derived products from land China and authenticated by Prof. Sheng-long Zhao (Zheji-
animal origins (Zeng et al. 2009). Additionally, collagen ang Ocean University, Zhoushan, China). The voucher
obtained from pig cannot be used as ingredients of some specimen (No. DC022) has been deposited in School of
foods for religious reasons, such as Jews and Muslims Food and Pharmacy, Zhejiang Ocean University.
(Regenstein and Zhou 2007). Therefore, the global demand
for collagen from alternative sources such as aquatic
Pretreatment of Skin
animals has been increasing over the years because of their
availability, lack of dietary restriction or risk of disease The skins of hammerhead shark were manually removed
transmission and possibility of high collagen yields. with a filleting knife and washed with cold distilled water.
At present, large quantities of by-products including The clean skins were cut into small pieces (2 mm × 8 mm)
skins, bones, scales, fins, head, guts and frame, accounting using a scissor. The prepared shark skins were mixed with
for 50–70% of the original raw material (Kittiphattanabawon 0.1 M NaOH at a skin/alkali solution ratio of 1:15 (w/v) to
et al. 2005), are generated in fish shops and processing fac- remove noncollagenous proteins. The mixture was continu-
tories. Therefore, optimal use of these by-products is a ously stirred for 24 h at 4C, and the alkali solution was
promising way to protect the environment, to produce changed every 6 h. Then the treated skins were washed with
value-added products to increase the revenue of fish proces- cold distilled water to achieve the neutral pH. Residual fat
sors and to create new job/business opportunities. Recently, was removed by 15% (v/v) butyl alcohol with a sample/
many scientists have focused their interests on isolation and solution ratio of 1:20 for 48 h with a change of solution
characterization of collagens extracted from skins of aquatic every 12 h. Defatted skins were thoroughly washed with dis-
organisms, such as ocellate puffer fish (Nagai et al. 2002), tilled water.
black drum and sheepshead seabream (Ogawa et al. 2003),
brown backed toadfish (Senaratne et al. 2006), Baltic cod
ASC-H
(Sadowska et al. 2003), Nile perch (Muyonga et al. 2004),
bigeye snapper (Jongjareonrak et al. 2005a), skate (Hwang ASC-H was extracted according to the method of Nagai and
et al. 2007), channel catfish (Liu et al. 2007), grass carp Suzuki (2000) with some modifications. Defatted skins were
(Zhang et al. 2007), deep-sea redfish (Wang et al. 2007), soaked in 0.5 M acetic acid with a solid to solvent ratio of
dusky spinefoot, sea chub, eagle ray and stingray (Bae et al. 1:15 (w/v) for 24 h with continuous stirring. The mixtures
2008), largefin longbarbel catfish (Zhang et al. 2009), were filtered with two layers of cheesecloth. In the presence
and flatfish (Heu et al. 2010). Collagen from shark of 0.05 M Tris-HCl buffer (pH 7.5), the collagen in the fil-
skins and cartilages, such as Mustelus griseus (Chen et al. trate was precipitated by adding NaCl to a final concentra-
2006), Chiloscyllium punctatum and Carcharhinus limbatus tion of 2.6 M. The resultant precipitate was collected using
(Kittiphattanabawon et al. 2010a,b) also was reported. CR21G refrigerated centrifuge (Hitachi, Ltd., Tokyo, Japan)
These collagens from fish skins were mainly type I collagen by centrifugation at 15,000 × g for 60 min at 4C. The pre-
([α1(I)]2α2) with low denaturation temperatures and cipitate was dissolved in a minimum volume of 0.5 M acetic
imino acid contents. acid and dialyzed against 0.1 M acetic acid for 24 h followed
Hammerhead shark (Sphyrna lewini), belonging to the by distilled water for 36 h, and the dialysate was freeze-
Family Triakidae, is a kind of commercially valuable fishery dried. The extraction yield of ASC-H was calculated, based
resource. However, large quantities of skins from hammer- on the weight of freeze-dried collagen, in comparison with
head shark after cutting off the fins are discarded as waste the wet weight of the skins of S. lewini used for extraction.
due to lower economic value. These by-products represent
an environmental problem to the fishing industry. However,
Proximate Analysis
no information regarding collagen from skins of hammer-
head shark has been reported. The aim of this study was to Moisture, ash, fat and protein contents of collagen powder
isolate and characterize acid solubilized collagen (ASC) from skins of hammerhead shark were determined accord-
from skins of hammerhead shark (ASC-H), and to compare ing to the methods of AOAC (2003) with the method
the characters of ASC-H with those of the type I collagen numbers of 950.46B, 920.153, 960.39 (a) and 928.08, respec-
from calf skin (CSC). tively. The converting factor of 6.25 was used for calculation
of protein content.
MATERIALS AND METHODS
Materials Determination of Amino Acid Composition
Hammerhead shark (S. lewini) was purchased from In order to determine the amino acid composition, freeze-
Nanzhen market in Zhoushan City, Zhejiang province of dried collagen was dissolved in distilled water to obtain a
Journal of Food Biochemistry 38 (2014) 236–247 © 2013 Wiley Periodicals, Inc. 237
COLLAGEN FROM THE SKIN OF HAMMERHEAD SHARK C-F. CHI ET AL.
concentration of 1 mg/mL, and an aliquot of 50 mL was
Ultraviolet-Vis Spectra
dried and hydrolyzed in vacuum-sealed glass tube at 110C
for 24 h in the presence of constant boiling 6 mol/L HCl, Ultraviolet (UV) absorption spectrum of ASC-H was mea-
which contained 0.1% phenol and used norleucine (Sigma- sured using a spectrophotometer (UV-1800, Mapada
Aldrich, Inc., St. Louis, MO) as the internal standard. After Instruments co., Ltd, Shanghai, China) from 190 to 400 nm.
hydrolysis, samples were again vacuum-dried, dissolved in The sample was prepared by dissolving in 0.5 M acetic acid
application buffer and injected into an automated amino solution with a collagen/solution ratio of 1:1,000 (w/v).
acid analyzer (Model 835-50 Amino Acid Analyzer, Hitachi,
Ltd.). Determinations were performed in triplicate and data
corresponded to mean values. Standard deviations were in FTIR
all cases lower than 2%.
The infrared (IR) spectra (450–4,000/cm) of ASC-H were
recorded in potassium bromide (KBr) disks with a Fourier
Electrophoretic Pattern and Peptide transform IR spectrophotometer (Nicolet 6700, Thermo
Mapping of ASC-H Fisher Scientific, Madison, WI). One milligram of dry
sample was mixed with 100 mg of dry KBr, and the mixture
SDS-PAGE. Electrophoretic patterns were measured was pressed into a disk for spectrum recording.
according to the method of Laemmli (1970) with a slight
modification using 7.5% separating gel and 4% stacking gel.
The samples (about 20 μL) were mixed with the sample Viscosity of Collagen Solution
loading buffer (60 mM Tris-HCl, pH 8.0, containing 25%
The viscosity of collagen solution was determined according
glycerol, 2% sodium dodecyl sulfate (SDS), 0.1% bromo-
to the method of Kittiphattanabawon et al. (2005) with
phenol blue) at 4:1 (v/v) ratio in the presence of β-ME then
some modifications. ASC-H was dissolved in deionized
applied to sample wells and electrophoresed along with type
water to obtain a concentration of 0.6% (w/v), and 500 mL
I CSC (Sigma-Aldrich, Inc.) and high molecular weight
solution was subjected to viscosity measurement using
marker (Shanghai Institute of Biochemistry, the Chinese
NDJ-8S viscometer (Jingtian Instruments co., Ltd, Shang-
Academy of Sciences, Shanghai, China) in an electrophore-
hai, China). ASC-H solution was heated from 4 to 40C with
sis instrument (AE-6200, ATTO Corporation, Tokyo,
a heating rate of 4C/min. At the designated temperature, the
Japan). The electrophoresis was carried out for about 4 h at
solution was held on for 30 min prior to viscosity determi-
a constant voltage of 100 V. After electrophoresis, gel was
nation. The relative viscosity was calculated in comparison
stained with 0.1% (w/v) Coomassie Blue R-250 in 45%
with that obtained at 4C. Td was defined as the temperature
(v/v) methanol and 10% (v/v) acetic acid.
which caused the 50% decrease in the relative viscosity of
collagen solution.
Peptide Mapping of Collagen. Peptide mapping of
collagen was examined by the method of Zhang et al. (2007)
with some modifications. The collagen samples were dis- Solubility
solved in 0.5 M acetic acid, pH 2.5, at a concentration of
The solubility of ASC-H was determined by the method of
3.5 M. The reaction mixtures were incubated at 37C for 3 h
Montero et al. (1991) with a slight modification. ASC-H
after the addition of trypsin (Sigma-Aldrich Co. LLC) with
was dissolved in 0.5 M acetic acid to obtain a final concen-
an enzyme/substrate ratio of 1/2 (w/w) to collagen solu-
tration of 3 mg/mL and the mixture was stirred at 4C for
tions. The sodium dodecyl sulfate polyacrylamide gel elec-
24 h. Thereafter, the mixture was centrifuged at 5,000 × g
trophoresis (SDS-PAGE) sample buffer was added to the
for 15 min at 4C, and the supernatant was used for solubil-
digestion sample, and the mixture was boiled for 5 min to
ity study.
terminate the reaction. Using 7.5% and 12% separating gels,
SDS-PAGE was performed to separate peptides generated by
the protease digestion. Effect of pH on Solubility. ASC-H solution (3 M,
Another collagen sample was dissolved in 0.2 M sodium 8 mL) was transferred to a 50 mL centrifuge tube, and the
phosphate buffer, pH 7.8, at a concentration of 3.5 M. Also, pH value was adjusted with 6 M NaOH/HCl to obtain the
the digested collagen solution was obtained by the same final pH ranging from 1 to 10. The volume of solution was
method as mentioned above except that the incubation time made up to 10 mL by deionized water previously and
was 3 min instead of 3 h. Peptide mapping of CSC was con- adjusted to the same pH as the ASC-H solution. The solu-
ducted in the same manner, and the peptide patterns were tion was centrifuged at 15,000 × g for 60 min at 4C. Protein
compared. content in the supernatant was measured, and the relative
238 Journal of Food Biochemistry 38 (2014) 236–247 © 2013 Wiley Periodicals, Inc.C-F. CHI ET AL. COLLAGEN FROM THE SKIN OF HAMMERHEAD SHARK
solubility was calculated in comparison with that obtained TABLE 1. PROXIMATE COMPOSITION OF FRESH SKIN OF SPHYRNA
at the pH giving the highest solubility. LEWINI AND ASC-H (G/100 G)
Fresh skin of S. lewini (%) ASC-H (%)
Effect of NaCl Concentration on Solubility. ASC-H Moisture 40.85 ± 2.39 4.56 ± 0.45
Ash 25.68 ± 1.98 8.13 ± 1.09
solution (6 M, 5 mL) was mixed with 5 mL of NaCl in
Lipids 14.59 ± 0.79 1.68 ± 0.03
0.5 M acetic acid at various concentrations to give the final
Proteins 18.88 ± 1.80 85.65 ± 1.23
concentrations of 0, 1, 2, 3, 4, 5 and 6%. The mixture was
stirred continuously at 4C for 30 min, followed by centri- ASC-H, acid-soluble collagen from the skin of hammerhead shark.
fuging at 15,000 × g for 60 min at 4C. Protein content in the
supernatant was measured, and the relative solubility was
calculated as previously described. Proximate Analysis
The proximate compositions of the fresh skin of hammer-
head shark and its collagen were shown in Table 1. The skin
Collagen Ultrastructure was rich in protein and ash with the contents of
18.88 ± 1.80 g/100 g and 25.68 ± 1.98 g/100 g, respectively.
Collagen was redissolved in 0.5 M acetic acid at a concen-
Compared with the raw material, the collagen was consider-
tration of 5% (w/v), followed by dialyzing against distilled
ably higher in protein (85.65 ± 1.23%) but lower in lipid
water (the molecular weight cutoff of dialysis bag was
(1.68 ± 0.03%), ash (8.13 ± 1.09%) and moisture (4.56 ±
1,000 Da). The collagen was lyophilized, and the sample was
0.45%). By the process of extraction, about 88.48% lipid
sputter coated for 90 s with gold using a JFC-1200 fine
and 68.34% ash were removed from the raw material, sug-
coater (JEOL Ltd., Tokyo, Japan). The morphologies of the
gesting that the methods used to extract collagen from the
electrospun fibers and membrane were observed under a
fresh skin of S. lewini were effective.
scanning electron microscope TM-1000 (Hitachi, Ltd.,
Tokyo, Japan).
Amino Acid Composition of ASC-H
The amino acid composition of ASC-H expressed as resi-
Statistical Analysis dues per 1,000 total amino acid residues was shown in
All experiments were performed in triplicate (n = 3), and an Table 2. Gly (227 residues/1,000 residues) was confirmed to
ANOVA test (using SPSS 13.0 software, SPSS Inc., Chicago, be the major amino acid of ASC-H, and the data were
IL) was used to compare the mean values of each treatment. accord with the Gly content of type I collagen (Arnesen and
Significant differences between the means of parameters Gildberg 2002); other amino acids of higher content were
were determined by using Duncan’s multiple range tests followed by Pro (129 residues/1,000 residues), Glu (102
(P < 0.05). residues/1,000 residues), Ala (97 residues/1,000 residues)
and Hyp (76 residues/1,000 residues). As described by
Muyonga et al. (2004), Gly is the most dominant amino
acid in collagen, and all members of the collagen family are
RESULTS AND DISCUSSION
characterized by domains with repetitions of the proline-
rich tripeptides, Gly-X-Y, involved in the formation of the
Extraction of ASC-H
triple helix, except for the first 14 amino acid residues from
The ASC-H was isolated with a yield of 4.23 ± 0.54% (based the N-terminus and the first 10 amino acid residues from
on the wet weight), and the ASC-H extraction condi- the C-terminus of the collagen molecules (Foegeding et al.
tions were determined to be 0.5 M acetic acid at 4C for 1996), where X is generally proline and Y is mainly hydroxy-
24 h. The yield of ASC-H was lower than those of ASC proline. Moreover, ASC-H had glycine (227 residues/1,000
from the skins of brownbanded bamboo shark (9.38%) residues) as the major amino acid, but its Gly content was
(Kittiphattanabawon et al. 2010a), bigeye snapper (6.4%) significantly lower than those of ASCs from skins of striped
(Jongjareonrak et al. 2005b) and brownstripe red snapper catfish (309 residues/1,000 residues) (Singh et al. 2011),
(9.0%) (Jongjareonrak et al. 2005a), respectively, on the balloon fish (353 residues/1,000 residues) (Huang et al.
basis of wet weight. The yield differences might be attrib- 2011), unicorn leatherjacket (321 residues/1,000 residues)
uted to the differences in fish species, tissue structure and (Ahmad and Benjakul 2010), deep-sea redfish (309 residues/
composition, biological conditions, and preparative 1,000 residues) (Wang et al. 2008) and largefin longbarbel
methods (Muyonga et al. 2004; Jongjareonrak et al. 2005b; catfish (317 residues/1,000 residues) (Zhang et al. 2009).
Singh et al. 2011). Furthermore, the amino acid composition of ASC-H
Journal of Food Biochemistry 38 (2014) 236–247 © 2013 Wiley Periodicals, Inc. 239COLLAGEN FROM THE SKIN OF HAMMERHEAD SHARK C-F. CHI ET AL.
TABLE 2. AMINO ACID COMPOSITION OF ASC-H AND CSC through its hydroxyl group (Mizuno et al. 2003). Further-
(RESIDUES/1000 RESIDUES) more, the imino acid content contributes to the thermal sta-
Amino acid ASC-H CSC bility of the helix structure of collagen because of the fact
Hyp 76 94 that the Pro and Hyp rich zones of the molecules are most
Asp 52 45 likely to be involved in the formation of junction zones sta-
Thr 27 18 bilized by hydrogen binding (Jongjareonrak et al. 2005a,b;
Ser 29 33 Huang et al. 2011). Therefore, the collagen helices of ASC-H
Glu 102 75 might be less stable than those of collagen from mammalian
Pro 129 121
skin (CSC: 215 residues/1,000 residues) (Table 2) because of
Gly 227 330
Ala 97 119
the lower imino acid content. In addition, the difference in
Cys 0 0 the imino acid content amongst the animals was associated
Val 29 21 with the different living environments (Nalinanon et al.
Met 16 6 2010; Kittiphattanabawon et al. 2010b).
Ile 22 11
Leu 30 23
Tyr 4 3 Electrophoretic Pattern and Peptide
Phe 20 3 Mapping of ASC-H
Hyl 9 7
Lys 37 26
His 9 5
SDS-PAGE. Apart from amino acid composition, the
Arg 72 50 properties of collagen are also influenced by the distribution
Total 1000 1000 of the molecular weights, structures and composition of its
Imino acid 205 215 subunits. As shown in Fig. 1, ASC-H and typical type I col-
ASC-H, acid-soluble collagen from the skin of hammerhead shark; lagen (CSC) possessed similar protein pattern, comprising
CSC, calf skin collagen. α1- and β-chains (dimmers of α1-chains) as their major
proteins. Nevertheless, the faint band of α2-chain was
noticeable in ASC-H. Furthermore, γ-chain (trimers of
showed relatively low contents of Met, Tyr, Hyl, His and Cys α1-chains) was also found in both collagens in a small
that were characteristics of all collagens. amount. The β- and γ-chains were also observed in collagen
The amounts of imino acid (Pro and Hyp) are important of bigeye snapper (Kittiphattanabawon et al. 2005), ocellate
for the structural integrity of collagens. The imino acid (Pro pufferfish (Nagai et al. 2002), back drum, sea bream and
and Hyp) content of ASC-H was 205 residues/1,000 residues,
which was relatively higher than those of ASCs from balloon
fish (179 residues/1,000 residues) (Huang et al. 2011), tiger
puffer (177.1 residues/1,000 residues), dusky spinefoot (134.5
residues/1,000 residues), eagle ray (193.2 residues/1,000 resi-
dues), Yantai stingray (197 residues/1,000 residues) (Bae et al.
2008), cod (130.3 residues/1,000 residues) and black drum
(199.8 residues/1,000 residues) (Ogawa et al. 2003), but lower
than those of ASCs from sheepshead seabream (205.1
residues/1,000 residues) (Ogawa et al. 2003), red stingray
(217 residues/1,000 residues) (Bae et al. 2008), striped catfish
(206 residues/1,000 residues) (Singh et al. 2011), brownstripe
red snapper (212 residues/1,000 residues) (Jongjareonrak
et al. 2005a), brownbanded bamboo shark (203 residues/
1,000 residues), blacktip shark (196 residues/1,000 residues)
(Kittiphattanabawon et al. 2010a) and Nile tilapia (210
residues/1,000 residues) (Zeng et al. 2009).
It was reported that pyrrolidine rings of Pro and Hyp
imposed restrictions on the conformation of the polypep-
tide chain and helped to strengthen the triple helix (Bae
et al. 2008; Huang et al. 2011). In particular, Hyp is believed
to play a key role in the stabilization of the triple-stranded FIG. 1. SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL
collagen helix because of its hydrogen bonding ability ELETROPHORESIS PATTERN OF COLLAGENS
240 Journal of Food Biochemistry 38 (2014) 236–247 © 2013 Wiley Periodicals, Inc.C-F. CHI ET AL. COLLAGEN FROM THE SKIN OF HAMMERHEAD SHARK
FIG. 2. PEPTIDE MAPPING OF TRYPSIN DIGESTED FROM ACID-SOLUBLE COLLAGEN FROM THE SKIN OF HAMMERHEAD SHARK (ASC-H) (A) AND
CALF SKIN COLLAGEN (CSC) (B) USING 12% (LANE: 1–4) AND 7.5% SEPARATING GELS (LANE: 5–8). LANES 1 AND 5: PROTEIN MARKER. LANES 2
AND 6: COLLAGENS WITHOUT TRYPSIN DIGESTION. LANES 3 AND 7: COLLAGENS WITH TRYPSIN DIGESTION AT pH 2.5. LANES 4 AND 8:
COLLAGENS WITH TRYPSIN DIGESTION AT pH 7.8
sheephead sea bream (Ogawa et al. 2003), and Nile perch component might dimerise into the β-component and
(Muyonga et al. 2004). Based on the SDS-PAGE pat- form β12-dimer.
tern (Fig. 1) and previous reports (Chen et al. 2006;
Kittiphattanabawon et al. 2010a), ASC-H might be type I
Peptide Mapping of ASC-H
collagen although the band intensity of α1-chain was far
higher than twofold of that of α2-chain, and the same result The denatured ASC-H digested by trypsin at pH 2.5 and 7.8
was found on the SDS-PAGE patterns of type I collagen was examined by SDS-PAGE using 7.5 and 12% separating
from other elasmobranches (Hwang et al. 2007; Bae gels to directly compare the pattern of peptide fragments
et al. 2008). Hwang et al. (2007) speculated that the with CSC. As shown in Fig. 2A, the bands of β-chains of
lower band intensity of α2-chain was because that α2 ASC-H as well as other high molecular weight components
Journal of Food Biochemistry 38 (2014) 236–247 © 2013 Wiley Periodicals, Inc. 241COLLAGEN FROM THE SKIN OF HAMMERHEAD SHARK C-F. CHI ET AL.
UV-Vis Spectra Analysis
Generally, the maximum absorption wavelength of protein
in the near UV region is 280 nm. Fig. 3 showed the UV
absorption spectrum of ASC-H at the wavelength 190–
400 nm. ASC-H exhibited a maximum absorbance at
220 nm as shown in Fig. 3, similar to those of collagens
from the shins of bullfrog (236 nm) (Li et al. 2004), channel
catfish (232 nm) (Liu et al. 2007), largefin longbarbel catfish
FIG. 3. ULTRAVIOLET ABSORPTION SPECTRUM OF ACID-SOLUBLE (233 nm) (Zhang et al. 2009) and ornate threadfin bream
COLLAGEN FROM THE SKIN OF HAMMERHEAD SHARK (ASC-H) (230 nm) (Nalinanon et al. 2011). The result was consistent
with the amino acid composition of ASC-H containing
almost entirely disappeared after the digestion by the low amount of Tyr (four residues/1,000 residues) with
trypsin at pH of 2.5, 37C for 3 h. In addition, the band maximum absorbance at 280 nm. The maximum absor-
intensity decreases of the α-chains (α1 and α2) of ASC-H bance at 220 nm might also be related to the groups C = O,
were observed with concomitant generations of lower MW -COOH, and CONH2 in polypeptide chains of collagen
peptide fragments ranging broadly from 97.4 to 14.4 kDa (Edwards et al. 1997).
(Lanes 3 and 7). When comparing peptide hydrolysis pat-
terns of ASC-H with that of CSC, it could be found that
FTIR
CSC was more tolerant to digestion by trypsin at pH 2.5, as
high MW components (β- and α-components) of ASC-H The infrared spectrum of ASC-H was shown in Fig. 4 and
were almost entirely digested, while those of CSC still similar to those of collagens from skins of other fish species,
remained to some extent. Peptide maps of collagens were such as Nile perch (Muyonga et al. 2004), channel catfish
reported to differ among sources and species (Mizuta et al. (Liu et al. 2007), deep-sea redfish (Wang et al. 2008),
1999). Thus, ASC-H and CSC might be fairly different in brownbanded bamboo shark (Kittiphattanabawon et al.
primary structure in terms of sequence and composition of 2010a), striped catfish (Singh et al. 2011) and swim bladders
amino acids. (Liu et al. 2012).
Moreover, both ASC-H and CSC were hydrolyzed to a The amide A band is associated with the N-H stretching
greater extent when the pH increased from 2.5 to 7.8. This frequency. A free N-H stretching vibration occurs in the
result was attributed to the activity of the trypsin that was range 3,400–3,440/cm, and when the NH group of a peptide
greater for the collagen in neutral or faintly basic medium is involved in a hydrogen bond, the position is shifted
than in acid medium. to lower frequency (Doyle et al. 1975). In the Fourier
FIG. 4. FOURIER TRANSFORM INFRARED
SPECTRA OF ACID-SOLUBLE COLLAGEN
FROM THE SKIN OF HAMMERHEAD SHARK
(ASC-H)
242 Journal of Food Biochemistry 38 (2014) 236–247 © 2013 Wiley Periodicals, Inc.C-F. CHI ET AL. COLLAGEN FROM THE SKIN OF HAMMERHEAD SHARK
transform spectroscopy (FTIR) spectrum of the ASC-H
(Fig. 4), the amide A band was found at 3,429.9/cm, which
indicated that fewer NH groups of ASC-H were involved in
hydrogen bonding. The result was in accordance with the
low amount of imino acids, especially Hyp (Table 2), which
was believed to play a key role in the formation of hydrogen
bond through its hydroxyl group. The amide B band was
found at 2,927.1/cm, showing the asymmetrical stretch of
CH2 (Abe and Krimm 1972).
The wavenumbers of the amide I, amide II and amide III
FIG. 5. THERMAL BEHAVIORS OF ACID-SOLUBLE COLLAGEN FROM
bands are directly associated with the configuration of col- THE SKIN OF HAMMERHEAD SHARK (ASC-H) MEASURED BY
lagen. The amide I band with the characteristic strong VISCOSITY CHANGE IN DEIONIZED WATER. TD WAS THE
absorbance in the range of 1,600–1,700/cm was mainly DENATURATION TEMPERATURE
related to the C=O stretching vibration along the polypep-
tide backbone, and it could be a sensitive marker of the pep-
tide’s secondary structure (Muyonga et al. 2004). The amide (19.4C), and was much lower than those of tropical fish
I band of ASC-H was found at the wavenumber of 1,649.1/ species, such as common mackerel (26.1C), eel (29.3C), Japa-
cm. Furthermore, the amide II band, which is caused by the nese seabass (26.5C), skipjack tuna (29.7C), ayu (29.7C) and
N–H bending vibration coupled with a C-N stretching Nile perch (36.5C) (Nagai et al. 2002; Muyonga et al. 2004;
vibration, normally occurs at 1,550–1,600/cm, and the shift Jongjareonrak et al. 2005a). Td of ASC-H was lower than those
to lower wavenumbers suggests the existence of hydrogen of collagens from calf and pig skins (37C) and chick sternal
bonds (Duan et al. 2009; Ahmad and Benjakul 2010). The cartilage (43.8C) (Huang et al. 2011). The result further
amide II band of ASC-H was found at the wavenumber of proved that the helices of ASC-H were less stable than those of
1,540.2/cm, which further confirmed that part of hydrogen mammalian collagens. Moreover, this finding was in agree-
bonding existed in ASC-H. In addition, the amide III band ment with Rigby’s report that the thermal stability of collagen
(1,220–1,320/cm) is associated with N-H deformation and was correlated with environmental and body temperatures
C-N stretching vibrations. The amide III band of ASC-H (Rigby 1968).
was found at the wavenumber of 1,242.4/cm. The triple
helical structure of ASC-H was confirmed from the absorp- Solubility of ASC-H
tion ratio between amide III and 1,452/cm bands, which
was approximately equal to 1.0 (Pleis et al. 1996; Heu et al. The effects of the pH and NaCl concentration on the solu-
2010). bility of ASC-H were shown in Fig. 6.
Viscosity of Collagen Solution Effect of pH on Collagen Solubility
With increasing temperature, the hydrogen bonds of colla- The effect of pH on the solubility of ASC-H in 0.5 M acetic
gen were gradually broken. Consequently, the triple helix acid was depicted in Fig. 6A. The highest solubility of
structure of collagen organized by hydrogen bonds was con- collagen was observed at pH 2. Generally, ASC-H was solu-
verted into the random coil configuration of collagen by the bilized to a greater extent in acidic pH ranging from 1
process of thermal depolymerisation, which was accompa- (82.35 ± 2.23%) to 4 (85.64 ± 3.72%), and significant
nied by changes in physical properties, such as viscosity, decrease in solubility was observed when pH ranged from 5
sedimentation, diffusion, light scattering and optical activity (72.42 ± 3.33%) to 7 (31.29 ± 3.78%) and the solubility of
(Usha and Ramasami 2004). ASC-H reached the minimum at pH 7.0. However, the
ASC-H exhibited a rapid loss of viscosity with heating from change of solubility became obscurely at pH ranging from 7
4 (1,480.80 ± 43.35 mPa·s) to 32C (389.40 ± 101.85 mPa·s) to 11. It is known that the net charge residues of protein
and remained low viscosity above 32C (Fig. 5). The denatur- molecules are greater, and the solubility is increased by
ation temperature (Td), which was defined as the temperature the repulsion forces between chains as the pH is lower or
at which the change in viscosity was half completed, was higher than pI. In contrast, total net charges of protein mol-
determined by viscosity measurement. Td of ASC-H was ecules are zero and hydrophobic–hydrophobic interaction
16.89C. Td of collagen from the skin of hammerhead shark, increases, thereby leading to the precipitation and aggrega-
which inhabits in an ocean temperature of 3–8C, was similar tion at pI. The pI of ASC-H was obtained at pH around 7
to those of cold-water fish, such as Argentine hake (10.0C), which was consistent with the report that collagen has
Baltic cod (15.0C), Alaska Pollack (16.8C) and chum salmon isoelectric points at pH 6–9 (Foegeding et al. 1996). In
Journal of Food Biochemistry 38 (2014) 236–247 © 2013 Wiley Periodicals, Inc. 243COLLAGEN FROM THE SKIN OF HAMMERHEAD SHARK C-F. CHI ET AL.
balloon fish (Huang et al. 2011), Nile tilapia (Zeng et al.
2009) and bigeye snapper (Kittiphattanabawon et al. 2005).
Effect of NaCl Concentration on
Collagen Solubility
The effect of NaCl concentration on the ASC-H solubility
was shown in Fig. 6B. The high solubility of ASC-H in
0.5 M acetic acid was maintained in the presence of NaCl
up to 3% (89.76 ± 1.98%). Solubility of this collagen
decreased gradually when the NaCl concentration exceeded
3%, and it was reduced to 45.67 ± 3.72% when the NaCl
concentration was 6%. The result was in accordance with
the reports that the solubility of collagens from the skins of
yellowfin tuna, dusky spinefoot, sea chub, eagle ray, red
stingray, yantai stingray, brownstripe red snapper, bigeye
snapper and striped catfish in acetic acid solution generally
decreased with an increase in NaCl concentration
(Kittiphattanabawon et al. 2005; Jongjareonrak et al. 2005a;
FIG. 6. RELATIVE SOLUBILITY (%) OF ACID-SOLUBLE COLLAGEN
FROM THE SKIN OF HAMMERHEAD SHARK (ASC-H) AS AFFECTED BY Bae et al. 2008; Singh et al. 2011). The decrease in solubility
DIFFERENT pH (A) AND NaCl CONCENTRATION (B). BARS of ASC-H could be described as being due to a “salting out”
REPRESENTED THE STANDARD DEVIATION (N = 3) effect, which occurred at relatively high NaCl concentra-
tions (Asghar and Henrickson 1982). An increase in ionic
strength causes a reduction in protein solubility by enhanc-
addition, the result that ASC-H had lowest solubility in the ing hydrophobic–hydrophobic interactions between protein
neutral pH range which was in accordance with the solubil- chains and increasing the competition for water with the
ity of collagens from skins of trout (Montero et al. 1991), ionic salts, thereby leading to the induced protein precipita-
brownstripe red snapper (Jongjareonrak et al. 2005a), tion (Jongjareonrak et al. 2005a; Bae et al. 2008).
A B
FIG. 7. ULTRASTRUCTURE ANALYSIS OF
COLLAGEN FROM THE SKIN OF SPHYRNA
LEWINI (A: ×100; B: ×150; C: ×300; D:
C D ×1500)
244 Journal of Food Biochemistry 38 (2014) 236–247 © 2013 Wiley Periodicals, Inc.C-F. CHI ET AL. COLLAGEN FROM THE SKIN OF HAMMERHEAD SHARK
AOAC. 2003. Official Methods of Analysis of AOAC International,
Collagen Ultrastructure
17th Ed., Association of the Official Analytical Chemists
Freeze-drying has been proved to be the most advantageous (AOAC) International, Gaithersburg, MD.
process to manufacture homogeneous porous collagen ARNESEN, J.A. and GILDBERG, A. 2002. Preparation and
devices. As shown in Fig. 7, the lyophilized ASC-H was characterisation of gelatin from the skin of harp seal (Phoca
loose, fibrous and porous structure because of the evapora- groendlandica). Bioresour. Technol. 92, 191–194.
tion of fluid, just like collagens from the skin of grass carp ASGHAR, A. and HENRICKSON, R.L. 1982. Chemical,
(Zhang et al. 2007), the coelomic wall of Sipunculida (Su biochemical, functional, and nutritional characteristics of
collagen in food systems. Adv. Food Res. 28, 232–372.
et al. 2009) and the scales of Lates calcarifer (Sankar et al.
BAE, I., OSATOMI, K., YOSHIDA, A., OSAKO, K.,
2008). Uniform and regular network structures of sponges
YAMAGUCHI, A. and HARA, K. 2008. Biochemical
as drug carriers are propitious, not only for well-
properties of acid-soluble collagens extracted from the skins
proportioned distribution for other drugs, but also for
of underutilised fishes. Food Chem. 108, 49–54.
evaporation of fluid. Taking the network structure into
CHEN, S.R., CAI, Y.P., ZHOU, Q., ZHANG, Q.B. and CAO, M.J.
account, the collagen sponge of ASC-H might also have 2006. Study on collagen from shark skin and bone. Food Sci.
good properties for distribution of the drugs as carriers. Technol. Int. 6, 173–178.
According to the applications, the pore size of collagen DOYLE, B.B., BENDIT, E.G. and BLOUT, E.R. 1975. Infrared
could be adjusted on water content during preparation spectroscopy of collagen and collagen-like polypeptides.
(Yaylaoglu et al. 1999). Biopolymers 14, 937–957.
DUAN, R., ZHANG, J., DU, X., YAO, X. and KONNO, K. 2009.
Properties of collagen from skin, scale and bone of carp
CONCLUSION (Cyprinus carpio). Food Chem. 112, 702–706.
EDWARDS, H.G.M., FARWELL, D.W., HOLDER, J.M. and
Acid solubilized collagen from the skin of S. lewini (ASC-H) LAWSON, E.E. 1997. Fouriertransform Raman spectroscopy
was isolated and characterized. Amino acid composition, of ivory: II. Spectroscopic analysis and assignments. J. Mol.
SDS-PAGE pattern and FTIR confirmed that ASC-H was Struct. 435, 49–58.
mainly composed of type I collagen. ASC-H exhibited high FOEGEDING, E.A., LANIER, T.C. and HULTIN, H.O. 1996.
solubility in acidic pH (1–4) and had higher solubility when Collagen. In Food Chemistry, 3rd Ed. (O.R. Fennema, ed.) pp.
the NaCl concentration was lower than 3%. Therefore, the 902–906, Marcel Dekker, New York.
collagen extracted from the skin of hammerhead shark HEU, M.S., LEE, J.H., KIM, H.J., JEE, S.J., LEE, J.S., JEON, Y.J.,
could bring a considerably economic benefits as a substitute SHAHIDI, F. and KIM, J.S. 2010. Characterization of acid-
for mammalian collagen (such as those from pig skin and and pepsin-soluble collagens from flatfish skin. Food Sci.
calf skin) (Zhang et al. 2007; Su et al. 2009), and further Biotechnol. 10, 27–33.
studies should be carried out to demonstrate the functional HUANG, Y.R., SHIAU, C.Y., CHEN, H.H. and HUANG, B.C.
properties of the collagen. 2011. Isolation and characterization of acid and
pepsin-solubilized collagens from the skin of balloon fish
(Diodon holocanthus). Food Hydrocolloid. 25, 1507–1513.
HWANG, J.H., MIZUTA, S., YOKOYAMA, Y. and YOSHINAKA,
ACKNOWLEDGMENTS R. 2007. Purification and characterization of molecular
This work was financed by National Natural Science Foun- species of collagen in the skin of skate (Raja kenojei). Food
dation of China (No. 31001109), the State-level Spark Chem. 100, 921–925.
Program (2010GA700088) and the Special Program for the JONGJAREONRAK, A., BENJAKUL, S., VISESSANGUAN, W.,
NAGAI, T. and TANAKA, M. 2005a. Isolation and
Science and Technology Plan of Zhejiang Province
characterisation of acid and pepsin-solubilised collagens from
(2010C13SAA00054).
the skin of Brownstripe red snapper (Lutjanus vitta). Food
Chem. 93, 475–484.
JONGJAREONRAK, A., BENJAKUL, S., VISESSANGUAN, W.
and TANAKA, M. 2005b. Isolation and characterisation of
REFERENCES
collagen from bigeye snapper (Priacanthus marcracanthus)
ABE, Y. and KRIMM, S. 1972. Normal vibrations of crystalline skin. J. Sci. Food Agric. 85, 1203–1210.
polyglycine I. Biopolymers 11, 1817–1839. KITTIPHATTANABAWON, P., BENJAKUL, S.,
AHMAD, M. and BENJAKUL, S. 2010. Extraction and VISESSANGUAN, W., NAGAI, T. and TANAKA, M. 2005.
characterisation of pepsin-solubilised collagen from the skin Characterisation of acid-soluble collagen from skin and bone
of unicorn leatherjacket (Aluterus monocerous). Food Chem. of bigeye snapper (Priacanthus tayenus). Food Chem. 89,
120, 817–824. 363–372.
Journal of Food Biochemistry 38 (2014) 236–247 © 2013 Wiley Periodicals, Inc. 245COLLAGEN FROM THE SKIN OF HAMMERHEAD SHARK C-F. CHI ET AL. KITTIPHATTANABAWON, P., BENJAKUL, S., NALINANON, S., BENJAKUL, S. and KISHIMURA, H. 2010. VISESSANGUAN, W., KISHIMURA, H. and SHAHIDI, F. Collagens from the skin of arabesque greenling 2010a. Isolation and characterisation of collagen from the (Pleurogrammus azonus) solubilised with the aid of acetic acid skin of brownbanded bamboo shark (Chiloscyllium and pepsin from albacore tuna (Thunnus alalunga) stomach. punctatum). Food Chem. 119, 1519–1526. J. Sci. Food Agric. 90, 1492–1500. KITTIPHATTANABAWON, P., BENJAKUL, S., NALINANON, S., BENJAKUL, S., KISHIMURA, H. and VISESSANGUAN, W. and SHAHIDI, F. 2010b. Isolation OSAKO, K. 2011. Type I collagen from the skin of ornate and characterization of collagen from the cartilages of threadfin bream (Nemipterus hexodon): Characteristics and brownbanded bamboo shark (Chiloscyllium punctatum) and effect of pepsin hydrolysis. Food Chem. 125, 500–507. blacktip shark (Carcharhinus limbatus). LWT-Food Sci. OGAWA, M., MOODY, M.M., PORTIER, R.J., BELL, J., Technol. 43, 792–800. SCHEXNAYDER, M.A. and LOSSO, J.N. 2003. Biochemical LAEMMLI, U.K. 1970. Cleavage of structural proteins during properties of blackdrum and sheephead seabream skin the assembly of the head of bacteriophage T4. Nature 227, collagen. J. Agric. Food Chem. 51, 8088–8092. 680–685. PLEIS, A.M.G., GOISSIS, G. and DAS-GUPTA, D.K. 1996. LI, H., LIU, B.L., GAO, L.Z. and CHEN, H.L. 2004. Studies on Dielectric and pyroelectric characterization of anionic and bullfrog skin collagen. Food Chem. 84, 65–69. native collagen. Polym. Eng. Sci. 36, 2932–2938. LIU, D., LIANG, L., REGENSTEIN, J.M. and ZHOU, P. 2012. REGENSTEIN, J.M. and ZHOU, P. 2007. Collagen and gelatin Extraction and characterisation of pepsin-solubilised collagen from marine by-products. In Maximising the Value of Marine from fins, scales, skins, bones and swim bladders of bighead by-Products (F. Shahidi, ed.) pp. 279–303, Woodhead carp (Hypophthalmichthys nobilis). Food Chem. 133, Publishing Limited, Cambridge, UK. 1441–1448. RIGBY, B.J. 1968. Amino-acid composition and thermal stability LIU, H.Y., LI, D. and GUO, S.D. 2007. Studies on collagen from of the skin collagen of the Antarctic ice-fish. Nature 219, the skin of channel catfish (Ictalurus punctaus). Food Chem. 166–167. 101, 621–625. SADOWSKA, M., KOLODZIEJSKA, I. and NIECIKOWSKA, C. LIU, Z., OLIEIRA, A.C.M. and SU, Y.C. 2010. Purification and 2003. Isolation of collagen from the skins of Baltic cod (Gadus characterization of pepsin-solubilised collagen from skin and morhua). Food Chem. 81, 257–262. connective tissue of giant red sea cucumber (Parastichopus SANKAR, S., SEKAR, S., MOHAN, R., RANI, S., californicus). J. Agric. Food Chem. 58, 1270–1274. SUNDARASEELAN, J. and SASTRY, T.P. 2008. Preparation MATMAROH, K., BENJAKUL, S., PRODPRAN, T., and partial characterization of collagen sheet from fish (Lates ENCARNACION, A.B. and KISHIMURA, H. 2011. calcarifer) scales. Int. J. Biol. Macromol. 42, 6–9. Characteristics of acid soluble collagen and pepsin soluble SENARATNE, L.S., PARK, P.J. and KIM, S.K. 2006. Isolation and collagen from scale of spotted golden goatfish (Parupeneus characterization of collagen from brown backed toadfish heptacanthus). Food Chem. 129, 1179–1186. (Lagocephalus gloveri) skin. Bioresour. Technol. 97, MIZUNO, K., HAYASHI, T. and BACHINGER, H.P. 2003. 191–197. Hydroxylation-induced stabilization of the collagen triple SHOULDERS, M.D. and RAINES, R.T. 2009. Collagen structure helix. Further characterization of peptides with 4 and stability. Annu. Rev. Biochem. 78(1), 929–958. (R)-hydroxyproline in the Xaa position. J. Biol. Chem. 278, SINGH, P., BENJAKUL, S., MAQSOOD, S. and KISHIMURA, 32373–32379. H. 2011. Isolation and characterisation of collagen extracted MIZUTA, S., YAMASA, Y., MIYAGI, T. and YOSHINAKA, R. from the skin of striped catfish (Pangasianodon 1999. Histological changes in collagen related to textural hypophthalmus). Food Chem. 124, 97–105. development of prawn meat during heat processing. J. Food SU, X.R., SUN, B., LI, Y.Y. and HU, Q.H. 2009. Characterization Sci. 64, 991–995. of acid-soluble collagen from the coelomic wall of MONTERO, P., JIMENNEZ-COLMENERO, F. and Sipunculida. Food Hydrocolloid. 23, 2190–2194. BORDERIAS, J. 1991. Effect of pH and the presence of NaCl USHA, R. and RAMASAMI, T. 2004. The effects of urea on some hydration properties of collagenous material from and n-propanol on collagen denaturation: Using DSC, trout (Salmo irideus Gibb) muscle and skin. J. Sci. Food Agric. circular dicroism and viscosity. Thermochim. Acta 409, 54, 137–146. 201–206. MUYONGA, J.H., COLE, C.G.B. and DUODU, K.G. 2004. WANG, L., AN, X., XIN, Z., ZHAO, L. and HU, Q. 2007. Characterisation of acid soluble collagen from skins of young Isolation and characterization of collagen from the skin of and adult Nile perch (Lates niloticus). Food Chem. 85, 81–89. deep-sea redfish (Sebastes mentella). J. Food Sci. 72, NAGAI, T. and SUZUKI, N. 2000. Isolation of collagen from fish E450–E455. waste material-skin, bone and fins. Food Chem. 68, 277–281. WANG, L., AN, X.X., YANG, F.M., XIN, Z.H., ZHAO, L.Y. and NAGAI, T., ARAKI, Y. and SUZUKI, N. 2002. Collagen of the HU, Q.H. 2008. Isolation and characterisation of collagens skin of ocellate puffer fish (Takifugu rubripes). Food Chem. from the skin, scale and bone of deep-sea redfish (Sebastes 78, 173–177. mentella). Food Chem. 108, 616–623. 246 Journal of Food Biochemistry 38 (2014) 236–247 © 2013 Wiley Periodicals, Inc.
C-F. CHI ET AL. COLLAGEN FROM THE SKIN OF HAMMERHEAD SHARK YAYLAOGLU, M.B., YILDIZ, C., KORKUSUZ, F. and HASIRCI, longbarbel catfish (Mystus macropterus). Food Chem. 115, V. 1999. A novel osteochondral implant. Biomaterials 20, 826–831. 1513–1520. ZHANG, Y., LIU, W.T., LI, G.Y., SHI, B., MIAO, Y.Q. and WU, ZENG, S.K., ZHANG, C.H., LIN, H., YANG, P., HONG, P.Z. X.H. 2007. Isolation and partial characterization of and JIANG, Z.H. 2009. Isolation and characterisation pepsin-soluble collagen from the skin of grass carp of acid-solubilised collagen from the skin of Nile (Ctenopharyngodon idella). Food Chem. 103, 906–912. tilapia (Oreochromis niloticus). Food Chem. 116, ZHANG, Z.K., LI, G.Y. and SHI, B. 2006. Physicochemical 879–883. properties of collagen, gelatin and collagen hydrolysate ZHANG, M., LIU, W. and LI, G. 2009. Isolation and derived from bovine limed split wastes. J. Soc. Leather. characterisation of collagens from the skin of largefin Technol. Chem. 90, 23–28. Journal of Food Biochemistry 38 (2014) 236–247 © 2013 Wiley Periodicals, Inc. 247
You can also read
