E3S Web of Conferences 141, 03006 (2020) https://doi.org/10.

1051/e3sconf/202014103006
RI²C 2019

Profiling Analysis of Fatty Acids and Collagens Obtained from

Sea Cucumbers
Pascal Budzinski1, Mananya Maimeun2, Parita Mutrakulcharoen2,3, Benjamaporn Wonganu4, and Malinee Sriariyanun2,*

1
Institute of Thermodynamics, Helmut-Schmidt-University / University of the Federal Armed Forces Hamburg, Hamburg, Germany
2Department of Mechanical and Process Engineering, The Sirindhorn International Thai-German Graduate School of Engineering
(TGGS), King Mongkut’s  University  of  Technology  North  Bangkok (KMUTNB), Bangkok, Thailand
3Department of Agro-Industrial, Food, and Environmental Technology, Faculty of Applied Science, King Mongkut’s  University  of  

Technology North Bangkok (KMUTNB), Bangkok, Thailand

4Department of Biotechnology, Faculty  of  Applied  Science,  King  Mongkut’s  University  of  Technology  North  Bangkok  (KMUTNB),

Bangkok, Thailand

Abstract. Investigations of alternative resources for production of functional foods and ingredients
containing valuable compounds with biological activities are getting more and more attention. Sea
cucumbers are aquatic functional foods with various medical and pharmaceutical effects, such as
antioxidant, antibacterial, antifungal, antiviral, anti-inflammatory and neuroprotective activities. This study
aimed to conduct profiling analysis of fatty acids and collagens extracted from four different sea cucumbers
harvested from Papua New Guinea by using Gas Chromatrography-Mass Spectrometry (GC-MS) and
Fourier Transform Infrared Spectrometer (FT-IR). Three different extraction methods in combination with
various solvents were used to find the best combination for extracting fatty acids. Enzymatic and chemical
extraction methods were applied for collagen extraction. The  highest fat recovery in this study was 85.32%
of theoretical yield with high proportions of unsaturated fatty acids up to 74.54%, and enriched with omega-
3 fatty acid. FT-IR chromatogram of sea cucumber protein extracts showed the characteristic of collagen
enriched with glycine and proline.   The nutritional analysis   of sea cucumber extracts demonstrated the
potential use as functional foods and ingredients with high benefits to human health.

Keyword. Sea cucumber, Fatty acid profiling, Collagen, Omega 3, Functional food

1 Introduction Proximate analysis revealed that wet sea cucumber

consists of 44-55% protein, 3-5% carbohydrate and 1.5%
Due to the growing needs of functional foods and fat suggesting that it is low-fat, high protein food
ingredients in the markets worldwide, finding the raw ingredients [4]. Collagen is one of the major protein found
materials with beneficially nutritional properties and in animal connective tissues. It has been described to
specific to targeted consumers gains intensive attention function in various effects, including damaged tissue
from industries. For example, the food supplements with repairing [5], antitumor [6], antioxidant [7], and
human health improvement effects obtained from animal angiotensin-converting enzyme inhibitory activity [8].
meats, i.e. nuts and herbs, may contain active chemicals, Therefore, collagen is one of major active components in
which stimulate allergenic reactions to some sensitive cosmetic recipes. Although, sea cucumber   contains low
consumers [1]. Therefore, variations of raw material quantity of fat, the fatty acid profiles are rich with high
sources are important for production and development of health-value components, such as eicosatetraenoic acid
foods and ingredients to supply the industries and (EPA) and docosahexaenoic acid (DHA) [4]. EPA and
markets. DHA have attracted much attention for the beneficial
Among marine organisms, sea cucumbers are effects on human health in reducing the risk for sudden
interesting feedstocks with pharmaceutical activities. death caused by cardiac arrhythmias and heart diseases [9,
With these characteristics, they could be used in foods as 10]. Additionally, they could be used as a wound healing
well as biomedicine industries [2]. Recently, many studies agent or an antithrombotic [10]. As the consumption of
provide scientific evidences that sea cucumbers contain DHA and EPA obtained from foods contributes only a
various biologically active compounds that provide health very small proportion to total daily need, the use of dietary
benefits, such as antioxidant, antibacterial, antifungal, antiviral, supplements containing EPA and DHA is required for
anti-inflammatory activities and neuroprotective activities total daily intakes.
[3]. However, the variations in supplied sea cucumbers in
terms of species, quantity and quality cannot secure their

*  Corresponding author Email: macintous@gmail.com

© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution
License 4.0 (http://creativecommons.org/licenses/by/4.0/).
E3S Web of Conferences 141, 03006 (2020) https://doi.org/10.1051/e3sconf/202014103006
RI²C 2019

functions as raw materials in industries. Additionally, temperature from Table 1. Then, the solvent was filtrated
other physical factors, including seasons, harvesting areas with filter paper before it was evaporated, and fat sample
and post-harvest manipulations also affect the active was collected for GC-MS analysis.
compounds in sea cucumbers [11]. Therefore, the After finding a suitable extraction method, the selected
objectives of this work targeted to the analysis of the fatty method was modified with different solvent mixtures,
acid profiles and collagen obtained from 4 sea cucumbers including acetic acid, hexane, chloroform, methanol and
harvested in Papua New Guinea. The investigation results ethanol. These solvents were selected to cover a wide
here could contribute in the selection of sea cucumbers to variety in the polarity and property of organic solvents.
supply the market needs.
2.3 Fatty acid analysis
2 Material and methods
For analysing the profiles with the GC-MS, the samples
were derivatized [15], using 2 mL of 1% H2SO4 in
2.1 sample preparation Methanol (MtOH) for each 40 µg of fat. This mixture was
boiled at 70°C for 2 h and every 30 min the sample was
Samples of dried Blackfish (Holothuria nobilis), Black vortexed. After 2 h, the sample was cooled to room
Teatfish (Actinopyga miliaris), White Teatfish temperature. 3 mL of hexane and 2 mL of deionized water
(Holothuria fuscogilva), and Elephant Trunkfish were added to separate the fatty acids from aqueous phase.
(Holothuria fuscopunctata) were delivered from Papua After centrifugation at 4,000 g for 10 min, the top layer
New Guinea to the laboratory (Courtesy provided by was collected and evaporated to weigh the recovered amounts
Wonnapob Co., Ltd., Bangkok, Thailand). The dry weight of fatty acids. For GC-MS the dry fatty acids were
and the wet weight were measured and recorded. For the dissolved in hexane to a concentration of 50 µg/µl and
wet weight, the sea cucumbers were rehydrated, by filtered with a 0.2 µm filter. The profiles of the extracted
putting them into deionized water for 4 days at 4°C. After fat were analysed with a GC-MS (QP2020, Shimadzu,
recording the weights, the sea cucumbers were sliced into Japan) using a DB-WAX capillary column (Agilent
small pieces and stored at -18°C. Technologies, Inc., CA., USA)(30 m x 0.25 mm, 0.25 µm)
[16]. The carrier gar was helium with flow rate of 1.0
2.2 Fatty acid extraction mL/min. The oven temperature programmed was initially
set at 120°C and increased to 220°C at the rate of
In order to find the best method for fat extraction from sea 10°C/min. Then, the temperature increased to 250°C with
cucumber, three different extraction methods, including the rate of 15°C/min. The 1 µl of sample was injected
Soxhlet extraction [12], Reflux and Bligh and Dyer [13], using a split mode with the split ratio of 1:30. The mass
were comparatively tested with the same solvent, a spectrometer was set to scan in range of m/Z 35-550 in
mixture of chloroform and methanol in a 1:1-ratio (v/v). electron ionization mode (EI) at 70 eV.
The ratio of sample:solvent was fixed at 10% w/v. The
setting for the different extraction methods was shown in
Table 1. Each extraction method was performed twice on 2.4 Collagen extraction
each sea cucumber. In this study, enzymatic and chemical extraction methods
Table 1. Settings for the different extraction methods. were used to recover collagen from sea cucumbers. All
these procedures were carried out at 4°C in order to reduce
Parameters Soxhlet Reflux Bligh&Dyer the enzymatic activities. In case of the enzymatic pepsin
Wet weight (g) 20 10 10 digestion [17], 200 mL of 0.5 M acetic acid were added to
Chloroform (mL) 100 50 50 20 g of sea cucumber sample, and homogenized for 20
Methanol (mL) 100 50 50 min. After homogenization, pepsin (Sigma-Aldrich, Inc.,
Temperature (°C) 84 75 30 USA) was added with a ratio of 1:100 v/w, and incubated
at 4°C for 16 h. To collect the supernatant, the mixture
For Bligh and Dyer extraction [13], the sample was was centrifuged at 4,000 g for 40 min. Collagen was
transferred to a tube. The sample and the 3 mL of solvent extracted from the supernatant by salt precipitation, 2.6 M
were homogenized for 20 min with a homogenizer (D-130, NaCl were added and stirred overnight. After
Wiggens, Straubenhardt, Germany). After homogenization, centrifugation at 4,000 g for 40 min, the precipitate was
1.8 mL water for each gram of sample was added. The tube collected and dialyzed against deionized water for 24   h.
was shaken for 2 h before the mixture was transferred to The remaining extract was called pepsin-solubilized
a centrifugation tube. After centrifugation for 10 min at collagen (PSC).
4,000 g, the top layer (aqueous phase) was discarded, the Chemical cleavage can be achieved by treatment with
lower layer (organic phase) was collected. After solvent diluted solutions of formic acid, hydrochloric acid or
evaporation, the fat remains in the tube and can be acetic acid [18].   In this work, 20 g of sample were stirred
analyzed by GC-MS. In case of Soxhlet and Reflux in 500 mL of distilled water for 30 min, the water was
extraction, the steps were conducted as described replaced again to clean up the sample. After 1 h stirring,
previously [14]. The sample was transferred into a timble the water was discarded and 500 mL of the disaggregation
in the glass apparatus. After adding the solvent, both solution (containing 4 mM ethylene-diaminetetraacetic
extractions were performed for 6 h in the waterbath at the acid (EDTA), 0.1 M Tris–HCl, pH 8.0) was added and

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stirred for 3 days. The liquid was replaced with 500 mL health problems including heart disease, obesity, diabetes
of distilled water and stirred for 2 days. Then, the and even cancer [20]. The ratios of the extracted −6 to
supernatant was collected by centrifugation at 4,000 g for −3 fatty acids from Black Teatfish and Blackfish were
10 min. Then, crude collagen fibrils were collected from 1:2 to 1:3 (Table 2), which were close to the ideal −6 to
supernatant fraction by centrifugation at 4,000 g for 40 −3 ratio.
min.
Table 2. Recovered fat yields and fatty acid profiles of Black
Teatfish and Blackfish obtained from three extraction methods.
2.5 Collagen analysis
Type of fatty
The extracted collagen was analysed by SDS-PAGE by Bligh&Dyer Reflux Soxhlet
acid
mixing with SDS-PAGE loading buffer. The mixture was Black Teatfish
denatured by heating at 95°C for 10 min. The denatured Yield (wt%
proteins were separated under electrophoresis and stained 0.49±0.05 0.38±0.14 0.18±0.06
dried  weight)
with Coomassie blue dye [19]. SFA 46.42±0.29 42.04±0.88 36.71±1.49
For FT-IR analysis, the extracted collagen fibrils were USFA 45.00±1.38 48.92±1.41 49.87±1.95
air dried at 60°C overnight and then spectrum scans were  (−3) 7.14±0.23 6.93±0.42 8.72±1.45
obtained from 4000 to 650 cm−1. FT-IR spectra were
 (−6) 19.14±0.58 18.20±1.36 19.13±0.54
carried out to compare the molecular groups of the extract
 (−9) 5.57±0.45 5.96±1 5.56±1.49
with a standard collagen FT-IR profile.
Blackfish
Yield (wt%
2.06±0.26 1.24±0.09 0.15±0.08
3 Results and discussion dried weight)
SFA 43.1±0.78 43.92±0.93 43.25±2.12
USFA 53.1±0.64 54.35±0.94 55.62±0.92
3.1 Selection of fatty acid extraction method
 (−3) 6.98±0.39 7.62±0.5 7.25±0.85
In order to select the most efficient extraction method for  (−6) 13.55±0.5 15.80±1.62 15.97±1.45
fat, the recovered yields and fatty acid profiles obtained  (−9) 3.67±1.18 3.96±2.27 5.72±3.5
from 3 different methods, including Soxhlet extraction,
Reflux and Bligh and Dyer, were compared by using the The highest amount of essential fatty acids has the
Blackfish and Black Teatfish as models. Based on the eicosapentaenoic acid (EPA, C20:5, −3) and arachidonic
recovered fat yields,  higher fat contents could be obtained
acid (ARA, C20:4, −6). The amount of EPA in the Black
from Blackfish (ranging from 1.25-2.06 wt% of dried
Teatfish was 5-6.5 wt% and the amount of ARA was 11-
weight) compared to Black Teatfish (ranging from 0.18-
14 wt% of the fat. For the Blackfish, the amount of EPA
0.49 wt% of dried weight) by using all 3 extraction
was 5-5.5 wt% and the amount of ARA was 11-14 wt%
methods (Table 2). In both sea cucumbers, Bligh and Dyer of the fat. EPA and ARA were polyunsaturated fatty acids
method provided the highest recovered fat yields
(PUFA) with important biological functions in human
compared to the other methods.
health as they are components of phospholipids of cell
In addition to fat yield, the fatty acid profiles obtained
membranes, precursors in neurotransmitter, and signaling
from 3 different extraction methods were also analysed by
molecules. They are abundantly found in brain, muscle and
using GC-MS analysis. The fatty acids were classified to
livers, and, with docosahexaenoic acid (DHA), ARA
2 main groups, saturated fatty acid (SFA) and unsaturated contributed to ~20 wt% of fatty acids in human brain [9].
fatty acid (USFA). In Black Teatfish, the SFA and USFA
The effect of different extraction methods was
contents were in similar level at 46.42% and 45.00%,
evaluated by comparing each group of beneficial fatty
respectively (Table 2). While in Blackfish, USFA was
acids. The results show that the extracted profiles for each
53.1% and SFA was 43.1%, suggesting that this sea
method were similar with small differences (Table 2).
cucumber contained high content of USFA. Using 3
Regarding to yields and profiles, Bligh and Dyer method
different extraction methods, the proportions of USFA:
was the best of the three methods. In addition, it was the
SFA in both sea cucumbers were similar, suggesting that
most time efficient method among those three methods.
the profiles of fatty acids did not much affected by
The Bligh and Dyer method had only an extraction time
extraction methods.
of 2 h, while the other methods had an extraction time of
Among USFA, there are subgroups of fatty acids
6 h. Therefore, the later part of this study, Bligh and Dyer
called omega fatty acids, i.e. omega 3 (−3). To be method was used for further fat analysis.
considered as good fatty acids with benefits to human
health, thus, the high −3 fat content should be included
in the daily meal [1, 9]. In both sea cucumbers, nearly half 3.2 Selection of fatty acid extraction solvent
of the USFA are out of −3 and −6 fatty acids (Table
In 3.1, methanol:chloroform (MtOH:Chlo) solvent was
2). Also, the ratios between −3 and −6 fatty acids in selected to be the tested solvent as suggested by other
both sea cucumbers were considered to be the studies. In this study, different solvent systems, including
recommended fats. It is estimated that the −6 to −3 hexane (Hex), toluene (Tol), methanol (MtOH), ethanol
ratio in the modern diet is 20:1 when it should ideally be (EtOH), acetic acid (Acet) and chloroform (Chlo), were
closer to 2:1 [9]. This nutrition imbalance can lead the challenged in fat extraction using the Bligh and Dyer

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method. In this experiment, White Teatfish was used as a EtOH:Chlo. While, in Elephant Trunkfish, three different
model sample for solvent type testing. mixed solvents provided the similar level of USFA. The
For the single solvent testing, including Hex, Tol and compositions of the USFAs showed clearly that the
MtOH, the best tested solvent was the protic solvent EtOH:Hex provided not only the highest yield of USFA,
MtOH with an extracted yield of 0.21 wt%, but it still it also extracted the highest amounts of −3 (11.13 wt%)
could not reach the original mixture of MtOH:Chlo with and −6 fatty acids (37.19 wt%) (Table 3). This extracted
an extracted amount of 0.49 wt% (Fig. 1). The first fat had a healthy −3 to −6 fatty acid ratio of about 1:3,
assumption for the MtOH:Chlo mixture extracting the a good ratio of healthy fat [20].
highest yield could be due to the strength of the acid-base
reaction. The reaction takes place due to the strength Table 3. Recovered fat yields and fatty acid profiles of White
difference of the acid and base given by the pKa. value. Teatfish and Elephant Trunkfish obtained from three extraction
The pKa value difference between Chlo (pKa = 25) and solvents.
MtOH (pKa = 15.5) is 9.5. With pairing the mixture of
Type of fatty acid MtOH:Chlo EtOH:Chlo EtOH:Hex
acetic acid (pKa = 4.76) and hexane (pKa =51) as solvent,
the pKa difference is 46.24, which is much higher than White Teatfish
MtOH:Chlo mixture [21]. The second assumption could Yield (wt% dried
0.49±0.02 0.50±0.01 0.78±0.11
be the amounts of available OH groups to perform a weight)
proton transfer. In this case, the MtOH is the shortest SFA 49.41±1.03 46.97±4.74 24.58±0.65
chained alcohol and offers the highest amounts of OH USFA 45.39±1.17 43.76±3.23 74.54±0.76
groups.  (−3) 4.67±0.48 4.62±0.73 11.13±0.17
However, the assumption of pKa difference did not
 (−6) 15.97±0.75 18.97±4.6 37.19±0.73
really support the results of other mixed solvents. The
extracted fat yield with Acet and Hex was even lower with  (−9) 3.3±1.19 7.82±4.1 21.29±0.96
bigger pKa difference (Fig. 1). On the other hands, Elephant Trunkfish
comparing between numbers of reactive groups available Yield (wt% dried
0.59±0.13 0.58±0.10 0.56±0.11
in MtOH and Acet in the same solvent volume, MtOH has weight)
more numbers than Acet because MtOH has less molar mass SFA 39.28±4.08 36.38±4.47 39.72±1.52
(MCH3OH = 32.04 g/mol , MCH3COOH = 60.05 g/mol). Based USFA 56.56±2.15 58.19±2.77 55.61±2.74
on the second assumption, the mixture of EtOH and Chlo
 (−3) 7.03±0.57 8.18±0.57 5.56±0.91
should extracted a lower amount than the mixture MtOH
and Chlo, because the EtOH mix provides less numbers  (−6) 24.7±4.63 24.3±4.58 24.77±1.45
of OH groups in the same solvent volume (MMtOH= 32.04  (−9) 10.6±1.81 10.34±3.36 13.9±4.33
g/mol, MEtOH = 46.07 g/mol). Interestingly, the fat yield
before fat derivatization obtained from MtOH and Acet In Elephant Trunkfish, all three mixed solvents
mix (0.96 wt%) was higher than EtOH and Acet mix (0.68 provided almost the same amounts of SFAs (36.38 to
wt%) that supported the possibility of the second 39.72 wt%) and USFA (55.56 to 58.19 wt%) (Table 3).
assumption. Against the result of the White Teatfish, the solvent
EtOH:Hex provided the lowest amount of −3 fatty acids
with 5.56 wt%. The same pattern was observed in −6
(ranging from 24.30 wt% to 24.77 wt%) (Table 3). These
results suggested that the selection of suitable solvent
could be different depending on the types of sea
cucumbers in terms of yield and fatty acid profiles.

3.3 Analysis of sea cucumber collagen

Since sea cucumbers have high collagen contents, thus it
could be potential raw materials for collagen production.
However,  before  using  sea  cucumber’s  collagens commercially,
Fig. 1. Fat extraction from White Teatfish by using different the property analysis should be conducted because they
solvent system. have wide variations and biological functions.
In this work, collagens of 4 different types of sea
Then, the pairing mix of EtOH was changed from Chlo cucumbers, including Blackfish, Elephant Trunkfish,
to Hex and the highest fat yield was up to 0.78 wt%. To Black Teatfish and White Teatfish, were extracted by
confirm the selection of extraction solvents, the enzymatic and chemical extraction methods. Using SDS-
MtOH:Chlo, EtOH:Chlo and EtOH:Hex were repeated in PAGE analysis, the molecular sizes of extracted collagens
the Elephant Trunkfish. The fat yields obtained from three could be determined by comparing with the sizes of
different mixed solvents show comparable level at 0.56- standard protein markers. The major protein bands with
0.59 wt% (Table 3). For fatty acid profiles, EtOH:Hex ~48 kDa- and ~63 kDa-sized were found and in the
extracted USFA from White Teatfish up to 74.54 wt%, chemical extraction. Whereas, ~120 kDa-sized band was
which was higher than that obtained from MtOH:Chlo and observed in the enzymatic extract. These results suggested

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that the enzymatic method was the better method to Nevertheless, the enzymatic collagen extraction
extract intact collagen due to the size of the collagen in delivered a better and consistent result of collagen
SDS-PAGE [17, 22]. The hydrolyzed or degraded forms extraction, because all the curves of the enzymatic
of collagens were observed in chemical extracts. extraction had the same peak patterns (Fig. 2). In chemical
The chemical and physical properties of collagen extraction, the height of each peak pattern varied
extracts obtained from sea cucumbers were analyzed and depending on each type of sea cucumber. The Blackfish
compared by using FT-IR (Fig. 2). All chemical and all and the Black Teatfish had a similar shape with bigger
enzymatic extracts showed the typical peaks at about 3400 peaks than the ones from Elephant Trunkfish and White
cm-1, which belonged to stretching vibration of –N-H Teatfish. In case of the White Teatfish the peaks in the
bonds, and the peak at 2900 cm-1, which represented to area between 1000 cm-1 to 1500 cm-1 were so small that
stretching vibration of –C-H bonds [17]. Many peaks they cannot be clearly seen (Fig. 2).
were found in the range starting from about 1000 cm-1 to
1600 cm-1, which belonged to the vibration of stretching
and bending of amide III –C=O bonds, stretching of amide 4 Conclusion
III –N-H bonds, bending of –C-H bonds and stretching of In this study, the fatty acids and collagens of 4 different
amide III –C-N bonds [17, 23]. These peaks indicate a types of sea cucumbers were analyzed in order to
high level of glycine and proline residues enriched in the investigate their potential use as materials for functional
collagen [17]. The last peak could be found at about 600 foods and ingredients. Comparison of fat yields, fatty acid
cm-1, which indicated the bending vibration of amide IV – profiles, simplicity and time-efficiency suggested that the
C-N bonds (Fig. 2). A comparison of these results with the suitable extraction method was Bligh and Dyer. Three
standard curve from collagen showed that they have the types of mixed solvent systems for fat extraction shown
same peak pattern and, therefore, all samples contained to be efficient ones for fat extractions were MtOH:Chlo,
collagen. EtOH:Chlo and EtOH:Hex due to high proportion of
a) essential fatty acids, especially −3 in the products. For
collagen extraction, both FT-IR and SDS-PAGE agreed
well to that enzymatic extraction method showed the
better result with intact collagen compared to chemical
extraction method. The analysis results in this work,
which identified the beneficial nutrition of sea cucumbers,
could be used as an initial investigation to evaluate the
potential of sea cucumbers in food, cosmetic and
pharmaceutical industries.
Authors would like to thank to Wonnapob Co., Ltd., Thailand
Science Research and Innovation (TSRI, Research grant number
RDG6250062) and  King  Mongkut’s  University  of  Technology  
North Bangkok (Research grant number KMUTNB-62-KNOW-
01) for financial support for this work.

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