Proceeding The 1st IBSC: Towards The Extended Use Of Basic Science For Enhancing

Health, Environment, Energy And Biotechnology 111

ISBN: 978-602-60569-5-5

Collagen from Sea Cucumber (Stichopus variegatus) as an Alternative Source of Halal Collagen

MH Khirzin1, Sukarno1, ND Yuliana1, LY Susanti2*, E Chasanah3, YN Fawziya3

Food Science and Technology Department, Bogor Agricultural University, Indonesia
1
2
Science Education Department, IAIN Jember, Indonesia
3
Research and Development Center for Marine and Fisheries Product Processing and Biotechnology, Ministry of Marine Affairs and
Fisheries Jakarta, Indonesia
*Correspondence : laily.yunita@ymail.com

Abstract—Collagen is a kind of proteins which becomes the main component of teeth, muscles, flesh, bone, and skin
layer. It is generally used in various fields such as food, cosmetics, pharmaceutical and medical. Collagen which was
commercialized in the market is derived from skin and bones of land-based mammalian such as calf, pig, and sheep. While it
was being reported that there were many diseases spread by land-based mammalian in the last few years such as mad cow
disease and avian flu. Besides that, pigs is forbidden to be consumed by moslem since Islam teaches them to consume halal
food. Marine organism has a potential to be used as an alternative source of halal collagen. One of them is sea cucumber.
Sea cucumbers, especially Stichopus sp, has a potential to be used as an alternative source of halal collagen instead of
mammals. Nevertheless, the extraction of sea cucumber collagen still relies on pepsin enzyme from pig derivatives. The
exploration of source and extraction process is urgently needed to gain halal collagen. Therefore, the aim of this study was to
explore sea cucumber Stichopus variegatus as a source of halal collagen with acid extraction method. Research and
Development Center for Marine and Fisheries Product Processing and Biotechnology, Ministry of Marine Affairs and
Fisheries Jakarta (sponsors). This study was divided into two steps: extraction and characterization of collagen. Extraction of
collagen produced yield of 16.40%. Characterization of collagen showed that the major components of amino acid were
glycine (169 residues/1000residues), glutamic acid (111residues/1000 residues), proline (67 residues/1000 residues),
alanine (64 residues/1000 residues) and molecular weight of collagen was 130.33 kDa. Collagen from sea cucumber
Stichopus variegatus had similar characteristics to commercial collagen even though the yield of extraction was lower. This
kind of collagen can be used as an alternative source of halal collagen either based on the source or the extraction process.
Keywords—collagen, halal, sea cucumber.

INTRODUCTION vastus, Stichopus cloronotus, Stichopus quadrifascinatus,

and Stichopus variegatus [8].
Collagen is a kind of connective tissue protein in the Recent researches reported that sea cucumber has a
form of fiber which constructs one third parts of the great potential to be used as a source of collagen,
vertebrate body and becomes the main component of including Stichopus japonicus [9], Stichopus vastus [10],
teeth, muscle, bone, and skin layer. Collagen has an Stichopus monotuberculatos [11], and Bohadschia
unique structure for triple helix-shaped primary structure Bivitatta [12]. Collagen from sea cucumber has
which means 3 polypeptide chains spiral Gly-X-Y where physicochemical characteristics that resemble to
X and Y are usually composed of proline or alanine [1]. commercial collagen. In general, collagen which is
Collagen also has an unusual amino acid, namely extracted from sea cucumber uses PSC (pepsin
hydroxyproline and hydroxylysine, both formed by the solubilized collagen) method. This method needs pepsin
hydroxylation process. Collagen divided into 28 types, enzyme which is obtained from porcine (pig). The using
but 90% of them are I, II, III, and IV types [2]. of pepsin enzyme on collagen extraction makes the
Collagen is widely used in various fields such as gained collagen not halal although it has been derived
food, cosmetics, pharmaceutical and medical. The nature from halal sources. There are 3 kinds of collagen
characteristics of collagen are biodegradable, extraction methods, namely acidic, basic, and enzymatic.
biocompatible and high antigenicity so that collagen is The exact method of collagen extraction from sea
often chosen as raw material [3]. Collagen that had been cucumber needs to be done so that the product can be
commercializing in the market is collagen derived from stated completely halal, either based on the source or the
skin and bones of land-based mammalian such as pig extraction method. Therefore, the purpose of this study
skin (46%), beef bones (23%), and chicken. While it was was to explore the potential of sea cucumber Stichopus
being reported that there were many diseases spread by variegatus collagen by using ASC (acid solubilized
land-based mammalian in the last few years such as mad collagen) method as a source of halal collagen.
cow disease and avian flu [4].
Islam teaches moslem to consume halal food which is MATERIALS and METHODS
good for man health as Allah said in QS. Al-Baqarah Sea cucumbers Stichopus variegatus (average body
168-and 173. Awareness of the importance of halal and weight 300 g) were purchased from local fisherman in
healthy food in society is increasing in the last few Lampung Province, Indonesia. The fresh body wall of S.
decade. It is proved by the attempts of people to find the variegatus were dissected free of adherent tissues with
alternative sources of halal collagen. Accordingly, it is tweezer, cut into small pieces and stored in polybag at -
necessary to explore source of collagen which more 20 0C until used. The collagen were extracted from body
safety instead of collagen from pis, chicken, and cow. wall of S. variegatus according to the procedures
Marine organism has a great potential to be developed as described by Park et al [9] with some modifications. All
a source of halal collagen, such as fish, shellfish, and sea procedures were performed at 4 0C. A hundred gram of
cucumbers. sea cucumbers were homogenized with 1000 ml of
Sea cucumber is one of echinoderm (thorn-skined distilled water and stirred slowly for 30 minutes. The
animals), but not all of it has thorns. The thorn is actually water was replaced and the extraction in water was
a skeleton which is composed of lime embedded in the repeated once for 1 hour. Then the water was replaced
skin [5]. Sea cucumber has spherical and cylindrical with 200 ml of 50% ethanol and stirred slowly for 30
shape with body length about 10-30 cm. Sea cucumber minutes. Samples were replaced with distilled water and
has vary color from dark, brown, gray, red, dark green then incubated with 1000 ml of a disaggregating solution
and orange. It movement was very slow. It is found in the containing 0.1 M Tris-HCl pH 8.0, and 4 mM
bottom of the sea with a depth of 1-40 m [6]. There are ethylenediaminetetraacetic acid (EDTA) overnight. The
about 23 species that have been identified in Indonesia liquid was decanted and replaced with 1000 ml of
[7]. Sea cucumbers wich has highest economic value are distilled water, in which the samples were stirred slowly
from genus Holothuria, Muelleria, and Stichopus. for 30 minutes.
Species from genus of Stichopus such as Stichopus

Food Technology Collagen from Sea Cucumber (Stichopus variegatus) as an Alternative Source of Halal Collagen
 Proceeding The 1st IBSC: Towards The Extended Use Of Basic Science For Enhancing
112 Health, Environment, Energy And Biotechnology
ISBN: 978-602-60569-5-5

The disaggregated collagen fibrils was collected and

treated with 1000 ml of 0.1 M NaOH for 48 hour in order
to remove non-collagenous materials effectively and to
minimize the effect of endogenous proteinases on
collagen fibrils. The residue after alkali extraction was
thoroughly rinsed with distilled water and then soaked in
1000 ml of 0.5 M acetic acid for 48 hour. The suspension
was filtered through cheesecloth and then filtrate was
precipitated using 1 M of NaCl overnight. The filtrate
was centrifuged at 10.000g for 60 minutes and then pellet
was dissolved in 0.5 M acetic acid. It was then dialyzed
in 0.1 M acetate buffer overnight. Buffers should be
periodically replaced. The pellet was finally lyophilized
using a freeze dryer (Christ alpha 1-2 LD, USA). Amino Figure 2: SDS-PAGE of collagen from 1. sea cucumber
acid of collagen which was extracted from S. variegatus Stichopus variegatus (this research); 2. sea cucumber
was characterized by using HPLC instrument (High Bohadschia bivitatta (Siddiqui et al. 2013); 3. calf
Performance Liquid Chromatography), while its skin (Zhong et al. 2015); 4. Chicken feet (Liu et al.
2001); M = marker.
molecular weight was characterized by using SDS-PAGE
instruments (Sodium Deodecyl Sulphate Poly-Acrilamide
Gel Electrophoresis). DISCUSSION
Table 1. Amino Acid Composition of Collagen from Sea Collagen and its derivatives are currently the
Cucumber Stichopus variegatus Compared With Sea intermediate materials which are widely used in various
Cucumber Stichopus japonicus, Calf, and Pig Skin industries both medium and large scale. Collagen
Collagen (Residues/1000Residues) demand of the world continues to grow year by year.
Amino
Sea Sea
Calf skin Pig skin Collagen is most widely used in cosmetic and medical
acid
cucumber cucumber
collagen3 collagen4 fields in Indonesia. However, the supply of collagen
collagen1 collagen2
Indonesia still depends on import nowadays. Domestic
Asp 58 59 45 44 production can not compete with the global market since
Thr 37 33 18 16 the amount of production is not sufficient to fulfill the
demand collagen. Besides, quality of collagen which was
Ser 26 44 33 33
extracted in our country is still lower than international
Glu 111 109 75 72 standards.
Pro 67 95 121 123 The main source of collagen which is usually used are
from land-based mammalian such as calf, pig, and sheep.
Gly 169 325 330 341
During the past 10 years, many emerging issue of animal
Ala 64 112 119 115 diseases happened in Asian sountry, such as madcow
Cys 0 0 0 0
disease and avian flu which attacks cattle and poultry.
Even, collagen from cow and chicken is plentifully
Val 18 23 21 22 produced in Indonesia. The number of outstanding
Met 8 9 6 6 collagen from pig is also increase gradually. That is why
the issue of halal source of collagen becomes urgent in
Ile 15 21 11 10
society.
Leu 23 18 23 22 Islam teaches Moslem to be careful in choosing food
Tyr 12 5 3 1 and beverages. Islam recommends to consume food
which is lawful and good for health. It is stated in QS.
Phe 13 3 3 12 Al-Baqarah 168 and 173. It is also re-emphasized in QS.
Lys 10 7 26 27 An-Nahl 115. Halal means the souce and manufacturing
process in making food and beverages are appropriate
His 5 4 5 5
with what Islam was taught about. Food which was
Arg 66 55 50 48 derived from unhealthy source is harmful for man health.
Source: (This research), (Saito et al. 2002), (Giraud et al. 2000), (Ikoma et al. 2003).
1 2 3 4 A lot of reports about disease in land-based mammalian
the past decade encourages moslem to find halal sources
of collagen.
Fig. 2 shows SDS-PAGE pattern of collagen from the Indonesia has vast areas of oceans beyond the
body wall of sea cucumber S. variegatus that has mainland. Based on that fact, the potential of marine
electrophoretic pattern of type I collagen consisting of resources should be able to meet the needs of collagen
major component α1 of approximately 130.33 kDa. Based over the country. The exploration of marine resources as
on its molecular weight, collagen from S. variegatus a source of collagen has been actually researched and
showed a similar molecular weight to α1 chain of developed as from squid, skin, bones, and swim bladder
collagen from S. monotuberculatos (137 kDa [11]), S. of fish. Beside those sources, sea cucumber also has great
japonicus (135 kDa [13]), P. californicus (138 kDa [17]), potential to be developed since the production of sea
and Bohadschia bivitatta (138 kDa [12]). Previous result cucumber in Indonesia per year reach about 184 thousand
suggested that the collagen from sea cucumber is type I, tons. Sea cucumber is commonly exported in fresh and
consisting of an (α)2β heterotrimer or an (α) homotrimer dried form. The resale value of sea cucumber will may be
[16]. The addition of mercaptoethanol did not affect the increase only if sea cucumber is processed into
SDS-PAGE pattern, suggesting that there was no intermediate materials such as collagen.
molecular species with disulphide bonds. Type I collagen Stichopus variegatus is potentially to be developed as
usually consists of heterologous α1 and α2 chains forming a source of collagen based on this research result.
triple helix as (α1)2α2, where β and γ component of higher Research in Europe and Japan has been widely reported
molecular weight are dimer and trimer of α chain, that Stichopus sp has a high content of collagen. It is also
respectively [13]. Liu et al [20] pointed that collagen confirmed by this research. Mass production and ongoing
from chicken feet consisted of two type collagen, one is exploration will be able to meet the needs of collagen in
type I and another is type II, but the major component is Indonesia so that collagen produced in this country can
type I. All collagen types consisted of three polypeptide compete with imported collagen. Yet, it takes great effort
chain (α-chains) in close association. and cooperation of all parties to reach it.
Food Technology Collagen from Sea Cucumber (Stichopus variegatus) as an Alternative Source of Halal Collagen
 Proceeding The 1st IBSC: Towards The Extended Use Of Basic Science For Enhancing
Health, Environment, Energy And Biotechnology 113
ISBN: 978-602-60569-5-5

CONCLUSION Ahmed, N. Absar, M.D.Z.I. Sarker,

“Physicochemical and biochemical properties of
Collagen of Stichopus variegatus had a yield of pepsin solubilised collagen isolated from the
16.40 % dry basis with the primary amino acid integrument of sea cucumber Stichopus vastus,” J.
composition in the form of glycine, proline, glutamic Food. Process. Pres. Vol. 2, pp. 1-10, 2013.
acid, and alanine with a molecular weight of 130.33 kDa.
It had similar chemical characteristics to cow, pig, and [11] M. Zhong, T. Chen, C. Hu, C. Ren, “Isolation and
chicken collagen. It can be used as an alternative source characterization of collagen from body wall of sea
of halal collagen. Researchers suggest that physical cucumber Stichopus monotuberculatos,” J. Food.
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tested and compared with commercial collagen. In [12] Y.D. Siddiqui, E.M. Arief, A. Yusoff, A.H. Suzina,
addition, the extraction methods of collagen need to be S.Y. Abdullah, “Isolation of pepsin solubilised
reviewed in order to gain high yield and good quality collagen from crude collagen extracted from body
wall of sea cucumber Bohadschia spp,” Int. J.
ACKNOWLEDGMENT Pharm. Sci. vol. 5(2), pp. 555-559, 2013
This research was supported by Research and [13] F.X. Cui, H.X. Chang, J.L. Zhao, Q.Z. Yong, D.
Development Center for Marine and Fisheries Product Ping, Y.F. Xue, G. Xin, “Characterization and
Processing and Biotechnology, Ministry of Marine subunit composition of collagen from the body wall
Affairs and Fisheries Jakarta. of sea cucumber Stichopus japonicus,” Food. Chem.
Vol. 100, pp. 1120-1125, 2007.
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Food Technology Collagen from Sea Cucumber (Stichopus variegatus) as an Alternative Source of Halal Collagen