SUPPLEMENTARY MATERIAL
Biochemical and radical scavenging properties of sea cucumber (Stichopus vastus) collagen
hydrolysates
Md. Zainul Abedina, Alias A. Karima*, Aishah A. Latiffb, Chee-Yuen Ganc, Farid Che Ghazalid,
Sahena Ferdosha, Md. Jahurul Haque Akandaa, Wahidu Zzamana, Md. Rezaul Karime, Md. Zaidul
Islam Sarkerf
a
Food Biopolymer Research Group, Food Technology Division, School of Industrial Technology,
Universiti Sains Malaysia, 11800 Penang, Malaysia.
b
Doping Control Centre, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia.
c
Centre for Advanced Analytical Toxicology Services, Universiti Sains Malaysia, 11800 USM,
Penang, Malaysia.
d
School of Health Sciences, Universiti Sains Malaysia Health Campus, 16150 Kubang Kerian,
Kelantan Darul Naim, Malaysia.
e
Faculty of Industrial Sciences and Technology, Universiti Malaysia Pahang, Gambang, Kuantan,
Pahang, Malaysia.
f
Department of Pharmaceutical Technology, Faculty of Pharmacy, International Islamic University
Malaysia, Kuantan Campus, 25200 Kuantan, Pahang, Malaysia.
*Corresponding Author: Email: akarim@usm.my (A.A. Karim)
Tel: +604 653 2268; Fax: +604 657 3678
Abstract
The molecular mass distribution, amino acid composition, and radical scavenging activity of collagen
hydrolysates prepared from collagen isolated from the sea cucumber Stichopus vastus were
investigated. β and α1 chains of the collagen were successfully hydrolyzed by trypsin. The molecular
mass distribution of the hydrolysates ranged from 5 to 25 kDa, and they were rich in glycine, alanine,
glutamate, proline, and hydroxyproline residues. The hydrolysates exhibited excellent radical
scavenging activity. These results indicate that collagen hydrolysates from S. vastus can be used as a
functional ingredient in food and nutraceutical products.
KEYWORDS: sea cucumber; integument collagen; trypsin hydrolysis; collagen hydrolysates;
biochemical and radical scavenging properties
1
Experimental
Materials and chemicals
Live freshly harvested sea cucumbers (Stichopus vastus) weighing 830 g (average body weight) were
purchased from the Fishery Department of Malaysia, Terengganu, Malaysia. The voucher specimen
(PPSK/USM/6139058-01-2009-SVT) samples were placed in oxygenated ice cold container for
transportation to our laboratory in Universiti Sains Malaysia, Pulau Pinang, Malaysia. The sea
cucumbers were dissected immediately after arrival to collect the integuments which were then stored
at -80 °C until extraction. All chemicals and reagents used were of analytical grade.
Pepsin-solubilised collagen (PSC) isolation procedure
Collagen from the integument of sea cucumber (Stichopus vastus) was isolated as previously
described by Liu et al. (2010) with a slight modification as previously described in our preceding
publication (Abedin et al., 2013).
Collagen hydrolysates production procedure
The freeze-dried collagen (3%, w/v) was dissolved in 50 mM phosphate buffer, pH 8.0 and the
dissolved collagen was digested with trypsin at 50 °C for 3 h (trypsin/collagen ratio of 1:20, w:w) as
described by Liu et al. (2011). The pH of the reaction mixture was maintained at 8.0 by continuous
addition of 1 M NaOH solution suitable for the enzyme action. Trypsin mediated digestion of
collagen was stopped by heating the reaction mixture at 100°C for 5 min and then cooled down with
running tap water. Following centrifugation at 5000g (30 min, 4°C), the supernatants containing
collagen hydrolysates were collected to freeze drying (FreeZone12, Labconco Corporation, Kansas
City, MO, USA) and the freeze-dried hydrolysates were stored in a refrigerator until analysis.
SDS-PAGE
To investigate the molecular weight distribution of collagen hydrolysates, SDS-PAGE was conducted
through 20% separating gel and 4% stacking gel (Laemmli, 1970). Briefly, Laemmli sample buffer
was used to dissolve the collagen hydrolysates and boiled the sample solutions for 3 min. The Mini
PROTEAN(R) tetra system (Bio-Rad Laboratories, Hercules, CA, USA) was used to conduct
electrophoresis at 120 V. After electrophoresis, staining and destaining were performed. Pre-stained
narrow range protein markers were used to estimate the molecular weight of the hydrolysates. The
image of the gel was captured using FUJIFILM luminescent image analyzer LAS-3000 (Fujifilm,
Tokyo, Japan). Multi Gauge v3.0 software (Fujifilm, Tokyo, Japan) was used to analyze the
electrophoresis pattern of hydrolysates.
Procedure for determination of amino acid composition
2
The collagen hydrolysates samples were hydrolyzed under vacuum with 6 M HCl at 110 °C for 24 h
and amino acid composition of the major hydrolysates were determined using a Waters1525 Binary
HPLC system amino acid analyzer (Waters, Milford, Massachusetts, USA).
Radical scavenging activity assay
Radical scavenging activity of the collagen hydrolysates was assessed using 2,2’-azinobis-(3ethylbenzothiazoline-6-sulfonic acid, ABTS) modified version of the method of Re et al. (1999).
Freshly prepared ABTS solution (3.9 mL containing potassium persulphate, 80% ethanol and distilled
water) and 0.1 mL collagen hydrolysates (1 mg/mL) were transferred into test tubes and incubated at
23 °C for 6 min in the dark. Methanol and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid), an antioxidant like vitamin E were used as control and reference, respectively. A
spectrophotometer (Molecular Devices SpectraMax M5, Sunnyvale, CA) was engaged to measure the
absorbance of control and sample at 734 nm.
The following equation was used to estimate the percentage of RSc activity:
% RSc  100 ×
Abs ctrl  Abs smpl
(S1)
Abs ctrl
Where, %RSc = Percentage radical scavenging of collagen hydrolysates, Abs ctrl =
Absorbance of methanol with ABTS, Abs smpl = Absorbance of sample.
Statistical analysis
All experiments were conducted in triplicate. The electrophoresis patterns of the hydrolysates were
analysed using Multi Gauge v3.0 software (Fujifilm, Tokyo, Japan).
References
Abedin, M.Z., Karim, A.A., Ahmed, F., Latiff, A.A., Gan, C.Y., Ghazali, F.C. & Zaidul, I.S.M.
(2013). Isolation and characterization of pepsin solubilised collagen from the integument of sea
cucumber (Stichopus vastus). Journal of the Science of Food and Agriculture, 93, 1083-1088.
Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature (London), 227, 680–685.
Liu, Z., Oliveira, A.C. & Su, Y.C. (2010). Purification and characterization of pepsin solubilised
collagen from skin and connective tissue of giant red sea cucumber (Parastichopus californicus).
Journal of Agricultural and Food Chemistry, 58, 1270-1274.
3
Liu, Z., Su, Y.C. & Zeng, M. (2011). Amino acid composition and functional properties of giant red
sea cucumber (Parastichopus californicus) collagen hydrolysates. Journal of Ocean University of.
China, 10, 80-84.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. & Rice-Evans, C. (1999). Antioxidant
activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology &
Medicine, 26, 1231-1237.
4
Table S1. Amino acid composition of collagen hydrolysates from sea cucumber (Stichopus vastus)
(Residues/1000 Residues) compared to sea cucumber (Parastichopus californicus) collagen
hydrolysates, salmon and trout collagen peptides.
Amino acids
Asp
Ser
Glu
Gly
His
Arg
Thr
Ala
Cys
Tyr
Val
Met
Lys
Ile
Leu
Phe
Pro
Hyp
Sea cucumber
(S. vastus) collagen
hydrolysate
59
40
97
322
6
63
45
108
0
7
25
9
4
18
25
8
91
73
Sea cucumber
(P. californicus)
collagen
hydrolysatea
61
57
102
313
8
44
41
92
9
11
25
12
6
25
24
13
103
54
Source:
a
Liu et al. (2011); bSaito et al. (2009)
5
Salmon collagen
peptidesb
Trout collagen
peptidesb
53
51
79
369
8
65
20
100
0
2
14
12
22
10
16
12
104
55
54
50
81
369
9
70
23
89
0
4
19
15
19
11
21
10
95
54
Table S2. ABTS radical scavenging activity of collagen hydrolysates from sea cucumber (Stichopus
vastus).
Sample
Reference (Trolox)
Collagen hydrolysates
ABTS(%)
radical
scavenging activity
2.6 ± 0.2
71.3 ± 1.8
*Values were means ± standard deviation of triplicate determinations.
6
Figure S1. SDS-PAGE pattern of collagen (a) and its hydrolysates (b) from sea cucumber (Stichopus
vastus). Lane 1 and 4: Broad and narrow range protein marker; Lane 2: integument collagen; Lane 3:
hydrolysates of integument collagen hydrolysed by trypsin (0.05 M phosphate buffer pH 8.0, 50 °C, 3
h and E/S ratio of 1:20 (w:w)).
7