Food Chemistry 151 (2014) 286–292

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Use of principal component analysis for differentiation of gelatine

sources based on polypeptide molecular weights
T. Nur Azira a, Y.B. Che Man a, R.N. Raja Mohd Hafidz a, M.A. Aina a, I. Amin a,b,⇑
a
Laboratory of Halal Science Research, Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
b
Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: The study was aimed to differentiate between porcine and bovine gelatines in adulterated samples by
Received 1 July 2011 utilising sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) combined with prin-
Received in revised form 6 October 2013 cipal component analysis (PCA). The distinct polypeptide patterns of 6 porcine type A and 6 bovine type B
Accepted 12 November 2013
gelatines at molecular weight ranged from 50 to 220 kDa were studied. Experimental samples of raw gel-
Available online 20 November 2013
atine were prepared by adding porcine gelatine in a proportion ranging from 5% to 50% (v/v) to bovine
gelatine and vice versa. The method used was able to detect 5% porcine gelatine added to the bovine gel-
Keywords:
atine. There were no differences in the electrophoretic profiles of the jelly samples when the proteins
Gelatine polypeptides
Principal component analysis
were extracted with an acetone precipitation method. The simple approach employing SDS–PAGE and
Acetone precipitation PCA reported in this paper may provide a useful tool for food authenticity issues concerning gelatine.
Food authenticity Ó 2013 Elsevier Ltd. All rights reserved.
Adulteration

1. Introduction chromatography/tandem mass spectrometry (HPLC–MS/MS)

Gelatine is a high molecular weight protein obtained by the
partial hydrolysis of collagen, which is the main protein found in
animal skin and bones. The gelling properties of gelatine (e.g., gel
strength, gelling time, setting and melting temperature and viscos-
ity) and its surface behaviour (e.g., formation and stabilisation of
foams and emulsions, adhesive properties and dissolution behav-
iour) have justified its use in food products (Schrieber & Gareis,
2007). However, high-quality gelatine is frequently derived from
bovine and porcine processing by-products (Gomez-Estaca,
Montero, Fernandez-Martin, & Gomez-Guillen, 2009). This can be
a problem for certain consumers, such as those prohibited to
consume any porcine-based products e.g. Muslims and Jews, those
concerned about the occurrence of bovine spongiform encephalop-
athy (BSE) disease or swine influenza and sensitised individuals
who are prone to allergic reactions.
Thus, the establishment of methods for gelatine sources deter-
mination may be useful. There are several analytical methods that
have been established in order to distinguish the source of gelatine,
either in a pure form or in food products such as NanoUPLC-ESI-Q-
TOF-MS (Yilmaz et al., 2013), Real-time PCR (Cai, Gu, Scanlan,
Ramatlapeng, & Lively, 2012; Demirhan, Ulca, & Senyuva, 2012;
Tasara, Schumacher, & Stephan, 2005), Fourier transform infrared
(FTIR) spectroscopy (Hashim et al., 2010), high performance liquid
⇑ Corresponding author. Tel.: +60 3 89472435; fax: +60 3 89426769.
E-mail address: aminis@upm.edu.my (I. Amin).
(Zhang et al., 2009), enzyme-linked immunosorbent assay (ELISA)
(Doi, Watenabe, Shibata, & Tanabe, 2009; Venien & Levieux,
2005a,b), Reversed-phase (RP) HPLC (Nemati, Oveisi, Abdollahi, &
Sabzevari, 2004) and calcium phosphate precipitation test (Hidaka
& Liu, 2003). However, the above-mentioned methods are expen-
sive, time consuming, need high sample purity and some of the re-
sults obtained are not reproducible. In addition, the complexity of
the processed products (i.e. confectionery) making the test incom-
patible with the most abovementioned methods. Consequently, it
is imperative to discover alternative methods that are able to elim-
inate the limitation. In the last decade, polyacrylamide gel electro-
phoresis (PAGE) has been established as an easy, fast and simple
method of species identification in products such as milk in cheese
(Mayer, 2005), raw fish (Chen et al., 2010) and smoked, gravid
(Mackie et al., 2000) and cooked fish (Etienne et al., 2000). Savage,
Richardson, Jolley, Hargin, and Steward (1995) used this technique
to differentiate between mechanically recovered and hand
deboned meat for beef, lamb, chicken and turkey, for labelling
and authenticity issues. It seems that the PAGE technique offers
an approach to the development of a simple and reliable method
for determining the authenticity and sources of gelatine. In addi-
tion, this technique is also suitable for routine measurements, as
it does not require highly sophisticated equipment as well as able
to offer simplicity in sample preparation which does not need high
sample purity. This circumstance is important in case whereby dif-
ficult to extract pure protein from certain samples especially in
processed foods. Furthermore, this method is preferable to the

0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.11.066
 T. Nur Azira et al. / Food Chemistry 151 (2014) 286–292 287

determination of gelatine sources based on the detection of spe-
cies-specific DNA with the polymerase chain reaction method, as
DNA could be degraded during the gelatine manufacturing process
(Doi et al., 2009).
In this study, we used sodium dodecyl sulphate (SDS)–PAGE to
separate gelatine polypeptides on a slab gel, based on their molec-
ular weights; the gel was then used to identify the prominent
bands, which provide a signature for specific gelatine sources.
Samples that displayed similar patterns had a similar number of
bands, located in similar molecular weight regions; those that dis-
played dissimilar patterns had different band numbers and the
bands were located at different molecular weights. This technique
was also applied for the analysis of samples that contained differ-
ent percentages of bovine and porcine gelatine.
Differentiation based on molecular weight and band numbers
can be enhanced by employing chemometric analysis to present
a clear classification. Moreover, the application of chemometric
analysis for analysing food is common (Cordella, Moussa, Martel,
Sbirrazzouli, & Lizzani-Cuvelier, 2002). Furthermore, SDS–PAGE
has been used in many studies for species-specific detection
(Montowska & Pospiech, 2007), but to the best of our knowledge,
there is no report in which PCA was utilised to analyse the molec-
ular weights of gelatine polypeptides measured using electropho-
resis. Therefore, the aim of this study was to utilise SDS–PAGE, in
combination with PCA, to detect the signs of adulteration in
samples of bovine and porcine gelatine. The usefulness of acetone
precipitation in extracting the proteins from the experimental
samples of jelly was also examined.
2.3. Preparation of experimental samples
2.3.1. Control samples
One hundred percent of porcine and bovine gelatine solution
which contain same amount of protein concentration was
prepared. Two set of experimental samples of raw gelatine were
prepared by adding porcine gelatine, in a proportion ranging from
5% to 50% (v/v), to bovine gelatine and vice versa. These sample
mixtures were then subjected to SDS–PAGE.
2.3.2. Jellies
Homemade jellies were prepared as experimental samples to
evaluate the efficiency of the protein extraction method and also
to mimic the commercial method that being used for production
of this product. In addition, homemade jellies were used to inves-
tigate the influence of main ingredients (sucrose, colour and fla-
vour additives) on the gelatine polypeptide pattern. Gelatine
powder that contains required protein content was weighed and
mixed with 50 g of table sugar in a beaker. The mixture was soaked
in 250 mL deionised water for 10 min to cause the gelatine to
swell. The mixture was then heated in a water bath until all of
the ingredients were completely dissolved. Two hundred and
twenty five micro litres of artificial pink food colour and artificial
rose flavour, respectively were added and the solution was mixed
well. The duplicate of mixtures of bovine and porcine gelatine
(10 mL) were prepared at a proportion ranging from 5% to 50%
(v/v). The jellies were then placed in storage at 4 °C to solidify.

2.4. Protein extraction

2. Materials and methods

Acetone precipitation was done according to Fic, Kedracka-Krok,
Jankowska, Pirog & Dziezicka-Wasylewska (2010) with slight
2.1. Materials
modifications. Four volumes of cold acetone ( 20 °C) were added
to one volume of melted jelly. The mixture was vortexed and kept
Commercial culinary food colouring (artificial rose pink colour)
overnight at 20 °C. The mixture was then centrifuged at
and food flavouring (artificial rose flavour) (Star Brand, FFM Ber-
14,000 rpm for 10 min. The supernatant was discarded and the
had) and coarse grain sugar (sucrose) were purchased from a local
acetone residue was air-dried from the protein pellet. The protein
retail store. All chemicals used were of analytical grade. The 12
pellet was used for SDS–PAGE.
studied samples of raw gelatines have been described in Table 1.

2.5. SDS–PAGE
2.2. Measurement of protein content
SDS–PAGE was conducted as described by Laemmli (1970) on a
The protein content was measured according to the method of slab gel consisting of 4% stacking gel and 6% resolving gel. The pro-
Bradford (1976). The measurement was performed in triplicate tein sample (0.8 mg/mL) was dissolved in the sample buffer (8 M
for 4 separate experiments (giving a total of 12 absorbance read- urea, 20% v/v SDS, 10 mM ETDA, 0.5 M Tris–HCI, 1.114 g/mL
ings per sample). The protein content of the samples ranged from 2-mercaptoethanol, 0.1% v/v glycerol and 0.05% w/v bromophenol
0.18 to 0.36 mg/g. blue). The mixture was then vortexed until the protein pellet was
completely dissolved. Electrophoresis was performed in a Mini
Protean II tetra cell (Bio-Rad Laboratories, Hercules, CA, USA) at
80 V for 2 h. The molecular weights of the bands in the protein
Table 1 sample were determined using high molecular weight standard
Characteristics of the commercial raw gelatines. markers for SDS electrophoresis (GE Healthcare, Buckinghamshire,
Gelatine sample Species Tissue Type Bloom UK). The standards consisted of the following proteins:
myosin (220 kDa), a2-Macroglobulin (170 kDa), b-Galactosidase
1 Porcine (PSS) Skin A 300
2 Porcine (PSC) Skin A 175 (116 kDa), transferrin (76 kDa) and glutamic dehydrogenase
3 Porcine Skin A 90–110 (53 kDa). SDS–PAGE gels were stained with silver stain solution
4 Porcine _⁄ A _⁄ (Bio-Rad Laboratories, Hercules, CA), according to the manufac-
5 Porcine Skin A _⁄
turer’s instructions, in order to visualise as many polypeptide
6 Porcine Skin A 220
7 Bovine (BSS) Skin B 225
bands as possible (silver staining is more sensitive than Coomassie
8 Bovine Skin B 75 Brilliant Blue staining). The stained gel was scanned using a densi-
9 Bovine Skin B 90–110 tometer (GS-800 Calibrated Densitometer Bio-rad, Hercules, CA)
10 Bovine (CSA) Skin B 220 and each band was analysed using Quantity One Software (Bio-rad,
11 Bovine (CBA) Bone B 220
Hercules, CA). Visualisation of polypeptide bands, lane comparison
12 Bovine (CHG) _⁄ B 200
and estimation of molecular weights was conducted for each
_⁄not stated. sample.
288 T. Nur Azira et al. / Food Chemistry 151 (2014) 286–292

2.6. Statistical analysis

To classify the experimental samples of adulterated gelatine,

PCA was run using Unscrambler 9.7 (Camo, USA) software. Multi-
variate PCA is a data reduction technique that compresses the
number of correlated variables into a smaller number of uncorre-
lated variables called principal components (PC) (Peng, Wang,
Zhu, & Chen, 2011). PCA is a very useful tool for application in
the food industry (Lee, Noh, Bae, & Kim, 1998) that can be used
for the detection of adulterants and for quality control (Chambery,
Monaco, Maro, & Parente, 2009; Marina, Che Man, & Amin, 2010).
There are usually two outputs of PCA: (i) the score plot shows the
locations of the samples in the PC that are able to detect the sample
patterns, grouping similarities and differences, and (ii) the loading
plot interprets the relationships between the variables (Kara,
2009). A PC plot is also used to reveal important features of the
data set, such as abnormal patterns (e.g., contamination) and the
presence of outliers (Lee et al., 1998). For example, several studies
(Hashim et al., 2010; and Nemati et al. 2004) have utilised PCA to
analyse data derived from FTIR and HPLC for the differentiation of
gelatine sources.
Sixteen molecular weight regions of 160, 145, 135, 125, 120,
114, 106, 96, 87, 83, 76, 70, 64, 61, 58 and 53 kDa were set as Fig. 2. Electrophoretic polypeptide pattern and densitometric profile of porcine
the variables, whereas the qualitative presence or absence of poly- (PSS) gelatine. Lane 1: molecular weight (kDa) marker; lane 2: electrophoretic
peptide band molecular weight values in each variable of each polypeptide pattern of PSS; band and peak 1: 160; 2: 145; 3: 135; 4: 125: 5:
120; 6: 114; 7: 106: 8: 96: 9: 87; 10: 83; 11: 76; 12: 70; 13: 64; 14:
sample was used as input data. The score plot was extracted from
61; 15: 58; 16: 53 kDa. Densitometric profile represented for lane 2. Rf:
the first two principal components, PC1 (is direction of largest relative electrophoretic mobility.
variance) and PC2 (is perpendicular to PC1 and against the largest
variance), as they presented the maximum amount of variability in pattern was compared to that of unadulterated bovine gelatine.
the data (Kher, Mulholland, Green, & Reedy, 2006). The electrophoresis gel showed that the polypeptide patterns were
not affected by the addition of coarse grain sugar (sucrose), artifi-
3. Results and discussion cial food flavour and colour or the combination of all three ingredi-
ents (data not shown).
3.1. Acetone precipitation The polypeptide bands exhibited a similar pattern to that of
pure bovine gelatine. In the case of adulterated jelly samples, the
As a preliminary test, the acetone precipitation method was polypeptide bands exhibited a similar pattern to that of raw adul-
applied to extract the protein from homemade jelly samples that terated gelatine samples. These results indicate that the acetone
contained pure bovine gelatine as well as various extraneous ingre- precipitation method was successful in extracting proteins without
dients. To visualise the results, protein precipitates were analysed changing their electrophoretic mobility patterns (compared to the
using the electrophoresis conditions described above and the pattern of raw gelatine). This simple method for protein extraction

Fig. 1. Electrophoretic separation of polypeptides from different sources of gelatine. PSS: porcine skin gelatine type A from Sigma; PSC: porcine skin gelatine type A from
China; CHG: cow gelatine from Malaysia; CBA: cow bone gelatine type B from China; CSA: cow skin gelatine type B from China and BSS: bovine skin gelatine type B from
Sigma. The amount of protein loaded was 8 lg per sample, and the gel was visualised using silver staining.
 T. Nur Azira et al. / Food Chemistry 151 (2014) 286–292
Fig. 3. Electrophoretic polypeptide pattern and densitometric profile of bovine
(BSS) gelatine. Lane 1: molecular weight (kDa) marker; lane 2: electrophoretic
polypeptide pattern of BSS; band and peak 1: 135; and 2: 110 kDa. Densito-
metric profile represented for lane 2. Rf: relative electrophoretic mobility.
Fig. 4. PCA classification of bovine gelatine adulterated with different percentage of porcine gelatine. (a) Score plot; (b) loading plot.
289
from food products, such as jelly, will offer a useful tool for analysis
of food authenticity in the future. However, a poor electrophoretic
profile of polypeptides could be obtained if this method applies on
complex commercial gelatine processed product such as gummy
and marshmallow, as the interaction of gelatine with other compo-
nents within the food matrices in those products will interfere the
gelatine polypeptides (Montowska & Pospiech, 2013).
3.2. Electrophoretic profile of gelatine polypeptides
The electrophoretic separation of polypeptides on the gel was
represented by bands present in different molecular weight
regions, with differing intensities. The electrophoretic profile of
gelatine polypeptides from bovine and porcine samples is shown
in Fig. 1.
Generally, commercial available gelatines are the heteroge-
neous compounds of a (100 kDa), b (200 kDa) and c-chains
(300 k Da) with molecular weight lest than 300 kDa (Zhang, Li, &
Shi, 2005). The distributions of these chains are determined by
the conditionings process and the intensity of hydrolysis used.
Commonly, there are two processes used in commercial gelatine
production; (i) acidic treatment which is used for less covalently
cross linked in porcine skin and (ii) alkali treatment is used for
more complex collagens found in bovine hides (Karim & Bhat,
2009). Two types of treatment aforementioned obtain type A and
type B gelatine, respectively.
In the porcine gelatine, polypeptides ranged in molecular
weight from 53 to 220 kDa and were more heterogeneous than
those of bovine gelatine, displaying a diffuse pattern of polypeptide
290 T. Nur Azira et al. / Food Chemistry 151 (2014) 286–292
bands in the lane. This finding was consistent with that of
Schrieber & Gareis (2007), who found that polypeptides in type A
gelatine have a wider distribution of molecular weights than those
in type B gelatine. The samples of porcine gelatine contained about
16 prominent bands, with molecular weights of approximately
160, 145, 135, 125, 120, 114, 106, 96, 87, 83, 76, 70, 64, 61, 58
and 53 kDa (Fig. 2) by silver staining. Three of these bands (120,
114 and 64 kDa) were consistent with the results of Waber,
Steinhart, and Paschke (2010), who found 3 distinct bands with
molecular weights of 120, 117 and 60 kDa by Coomassie Brilliant
Blue (CBB) staining. The presence of other bands in our result
probably related to the high sensitivity of silver stain that able to
detect low abundance of polypeptides. Bovine gelatine had 2
prominent bands, which were visible at approximately 135 and
110 kDa (Fig. 3).
The observed electrophoretic mobility was compatible with
Eysturskaro, Haug, Ulset, and Draget (2009) observations that
made by using size exclusion chromatography–multi angle laser
light scattering (SEC–MALLS) analysis. They reported that bovine
type B gelatine exhibited well-defined peak compared to porcine
type A gelatine. Cole and Roberts (1996) also reported that acid
process calf skin gelatine exhibited similar molecular weight pro-
file with pig skin gelatine by containing discrete bands with molec-
ular weight less than a-chain. While the major molecular weight
fractions for alkali process bovine bone gelatine is in the a-chain
region (Muyonga, Cole, & Duodu, 2004). Hence, it can be suggested
that the acid process produced gelatine containing peptides with
large random distribution of molecular weight compared to alkali
process. The distinct pattern of polypeptide bands seen in porcine
Fig. 5. PCA classification of porcine gelatine adulterated with different percentage of bovine gelatine. (a) Score plot; (b) loading plot.
gelatine could be used as an indicator to distinguish it from either
type B gelatine or other sources of gelatine.
3.3. Electrophoretic patterns of experimental samples
Despite careful observations of each lane, pure versus adulter-
ated samples were neither easily nor clearly differentiated, espe-
cially when the percentage of porcine gelatine was high. No
significant differences were observed between the numbers of
bands in the samples with a high percentage of adulteration; how-
ever, there were differences in the intensities of the bands between
samples with different levels of adulteration. The electrophoretic
profile of each adulterated gelatine sample was compared with
unadulterated gelatine for identification. To overcome the difficul-
ties in differentiating between pure and adulterated samples,
SDS–PAGE analysis in combination with PCA was employed for
classification purposes; this analysis was performed based on the
percentage of adulteration, rather than the level at which discrim-
ination could be achieved.
3.4. Principal component analysis
3.4.1. Adulteration of bovine gelatine with porcine gelatine
The presence or absence of each polypeptide band (molecular
weight) in each experimental sample was calculated using PCA.
This was done by defining the band detection parameter of sensi-
tivity in the Quantity One Software (Bio-Rad, Hercules, CA). The
sensitivity determines the minimum signal intensity in the image
that will be defined as a band. The higher the sensitivity value,
 T. Nur Azira et al. / Food Chemistry 151 (2014) 286–292
the more bands will be detected. In this study the sensitivity set-
ting is 40.00. We did not set it over 40.00 as the background noise
was erroneously detected as bands. The detected band was deter-
mined as presence and undetected band as absence.
Based on 16 variables, the adulterated samples were classified
into 3 groups (Fig. 4(a)). Unadulterated bovine gelatine samples
were assigned to group I, at the end of negative side. Bovine gela-
tine samples that were adulterated with 5–30% porcine gelatine
were grouped together in between groups I and III, although they
were still in the negative side, indicating that the polypeptide pat-
terns are similar to those of unadulterated bovine gelatine. The bo-
vine gelatine adulterated with 40–50% porcine gelatine was
grouped together with pure porcine gelatine, in group III at the
end of the positive side. PC 1 and PC 2 accounted for 49% and
22% of the variation, respectively; thus, 71% of the variance was ac-
counted for by the first two PCs. The numbers on the score plot
represent the percentage of adulteration, with 0% representing
the unadulterated samples. The grouping pattern in the PC showed
that the adulterated samples were moved from the end of negative
side (pure bovine gelatine) to the end of positive side (pure porcine
gelatine). As the proportion of porcine gelatine increased, the sam-
ples were plotted increasingly toward the right side of the PC. The
results of this study show that it is possible to detect 5% porcine
gelatine in bovine gelatine.
The PCA loading plot is the projection of the variables onto the
reflection plane of the score plot. The value of the loading plot (for
the molecular weight regions) is that it highlights the importance
of the contribution of each variable to the sample classification
in the PCA. The farther from the origin a variable is placed, the
higher the contribution of that variable to the PCA model (Marina
et al., 2010). As can be seen in the loading plot in Fig. 4(b), the
molecular weight regions that most strongly contributed to the
separation of the samples were 160, 96 and 87 kDa based on PC
1 and 145, 106, 83 and 53 kDa based on PC 2. All of the regions de-
scribed above were similar with regard to the location of the poly-
peptide bands in the porcine gelatine, which indicates that the
porcine gelatine bands had a strong influence on the discrimina-
tion between pure and adulterated samples.
3.4.2. Adulteration of porcine gelatine with bovine gelatine
Based on the results shown in Fig. 5(a), PCA could not differen-
tiate between the experimental samples when the porcine gelatine
was adulterated with bovine gelatine. The samples were classified
into two groups, where group I represents the pure bovine gelatine
and group II represents the bovine gelatine samples with 5–50%
adulteration with porcine gelatine as well as pure porcine gelatine.
In the PCA, 70% and 10% of the variation were accounted for by PC 1
and PC 2, respectively; thus, 80% of the variance was accounted for
by the first two PCs. The classification of the samples into the 2
groups described above showed that the SDS–PAGE method used
in this study was not capable of determining the percentage of
adulteration. The prominent bands of porcine gelatine polypep-
tides overlapped with the prominent bands of bovine gelatine
polypeptides, which made it impossible for the SDS–PAGE tech-
nique to differentiate between them. Porcine polypeptide bands
were the main variables that contributed to the differentiation of
samples seen in Fig. 5(b). The location of scatter point 30b⁄ showed
an unusual pattern, which led us to declare it as an outlier. The re-
sults of this study suggest that PCA is a useful tool for the analysis
of SDS–PAGE data to present better classification of samples in
food adulteration cases.
4. Conclusions
The SDS–PAGE technique was used to differentiate between
porcine and bovine gelatine. Sixteen prominent polypeptides
291
derived from porcine gelatine were found to indicate the adultera-
tion of bovine gelatine with porcine gelatine. The new approach
was employed based on analysing SDS–PAGE data with PCA to
clearly classify the percentage of adulteration in the samples.
According to the best of our knowledge, this is the first report in
which PCA was utilised to analyse the molecular weights of gela-
tine polypeptides measured using electrophoresis. This study
showed that the presence of 5% porcine gelatine in bovine gelatine
can be detected. The PCA provide good differentiation of samples
with 71% of the variation accounted by the first two PCs. Extraction
of proteins with cold acetone of our homemade jelly had no effect
on the electrophoretic profile of the gelatine polypeptides. How-
ever, a risk of poor electrophoretic profile of the gelatine may be
observed in commercial processed product as the heterogonous
mixture of ingredients. A simple method of protein extraction with
cold acetone, followed by SDS–PAGE and analysis with PCA would
provide a meaningful tool for food authenticity analysis of food
products such as gelatine. Further studies are needed to employ
the approach reported in this paper to additional food matrices
for the purpose of determining the purity of gelatine in the
product.
Acknowledgements
The authors are grateful for financial support under the
Research University Grant Scheme (RUGS) (Projects No: 02-01-
07-0031RU & 05-02-10-0935RU) from Universiti Putra Malaysia,
Serdang, Selangor, Malaysia.
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