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LWT - Food Science and Technology 63 (2015) 714e719

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LWT - Food Science and Technology

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Sensitivity of polymerase chain reaction (PCR)-southern hybridizationand conventional PCR analysis for Halal authentication of gelatincapsules

Sahilah Abd Mutalib*, Nursheila Mustafa Muin, Aminah Abdullah, Osman Hassan,Wan Aida Wan Mustapha, Norrakiah Abdullah Sani, Mohd Yusof MaskatSchool of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor,Malaysia

a r t i c l e i n f o

Article history:Received 12 March 2014Received in revised form23 February 2015Accepted 4 March 2015Available online 13 March 2015

Keywords:Halal authenticationPCR-southern hybridizationGelatin capsulesConventional PCRPorcine DNA detection

* Corresponding author. Tel.: þ60 03 89215444; faE-mail address: sahilah@ukm.edu.my (S.A. Mutalib

http://dx.doi.org/10.1016/j.lwt.2015.03.0060023-6438/© 2015 Elsevier Ltd. All rights reserved.

a b s t r a c t

Halal authentication of gelatin capsules was conducted using polymerase chain reaction (PCR)-southernhybridization on chip and conventional PCR analysis. The primers used in PCR-southern hybridizationwere targeted on mitochondria DNA (mtDNA) of cytochrome b (cyt b) gene and its amplicon was 276 bp.In conventional PCR, three pairs of mtDNA primers targeted cyt b, cytochrome oxidase II (COII) and ATP6gene were tested, resulting of 398, 212 and 83 bp amplicons, respectively. Of 20 brands examined usingPCR-southern hybridization, 6 capsules (C1eC6) were found to be porcine DNA positive but none werepositive using conventional PCR method. The sensitivity of each primer in the detection of porcine DNAwas 0.25 ng (cyt b), 0.1 ng (COII), 0.001 ng (Olipro™ Chip) and 0.0001 ng (ATP6). Results demonstratedthat the PCR-southern hybridization on chip was useful and reliable for verifying porcine DNA in gelatincapsules compared to conventional PCR.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Halal (permissible) quality of food is required to abide in Mus-lims daily live. However, the industrialization of food processing inthe 20th and 21st centuries has exposed Muslims community tovarious ingredients such as gelatin in pharmaceutical productarising from the advancement of science and technology. As a resultin many cases, the Muslims are facing difficulties to ascertainwhichproducts are permitted or not under the Islamic law. It is also in-fluence the other communities such as Jews, allergic towardporcine ingredient and vegetarian. Thus, this study is the step ofpreventingmisconduct of pharmaceuticals products for consumer'schoice.

Gelatin is considered as a hydrocolloid which are special andunique, serving multiple functions with a wide range of applica-tions in various industries including beverages, processed foods,cosmetics and pharmaceutical products (Karim & Bhat, 2008). Theuse of gelatin in pharmaceutical products such as capsules is

x: þ60 03 89215410.).

inevitable because it helps to protect themedicines against harmfulinfluences, such as light and oxygen. There are two types of cap-sules namely soft and hard capsules. Both are different due to shellcomposition and the production process. Soft gelatin capsules havethicker shells and contain approximately 20%e30% of plasticizers inthe form of glycerol and sorbitol. High content of plasticizer willhold approximately 30% of water content. While, in hard gelatincapsules consist of pure gelatin with a sorption water content of13%e16% (Stegemann & Bornem, 2002). In term of the productionprocess, hard gelatin capsules require less stringent conditionsduring manufacture and storage than soft gelatin capsules. Detailson the production process of soft and hard gelatin capsules are wellexplained by Stegemann and Bornem (2002). The soft capsules aremainly used for liquid fillings, while hard capsules are used forpowders.

The issue of gelatin is alarming and sometimes controversial dueto commercial gelatines are limited to porcine-based since theemergences of Bovine Spongiform Encephalopathy (BSE) or madcow disease in the 1980s which restricted the use of bovine gelatin(Morrison, Clark, Chen, Talashek, & Sworn, 1999). In addition theproduction of porcine gelatin takes about 30 days, while bovinegelatin production lasted between 60 and 80 days and this affect

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the product cost. The Halal gelatin from bovine source must fulfillthe Shariah requirement such that the bovine must be slaughteredby a Muslim and processed according to Shariah Laws. The alter-native gelatin sources are gum arabic, seaweeds (carrageenan) andfishes (fish gelatin) but these could not fulfill various industriesdemand. The various use of porcine gelatin in industries isexpanding and the exposure of haram (non-Halal) gelatin is notonly towards Muslims but also other communities such as Jews,vegetarians and a number of people who are allergic toward hiddenporcine ingredients and meat sources in processed foods (Tanabe,Hase, et al., 2007).

There are a number of molecular techniques that can be used todetermine the presence of porcine DNA in food materials and toidentify disputability of the food products (Aida, Che Man, Wong,Raha, & Son, 2005;Matsunaga et al., 1999; Montiel-Sosa et al.,2000; Yoshida et al., 2009) and in feeds (Cheng, Wen, Ding, Koa, &Kuo, 2003; Corona, Lleonard, Carpio, Uffo, & Martinez, 2007; Partiset al., 2000; Tartaglia et al., 1998; Yoshida et al., 2009). Most earlytechniques were based on hybridization to specific probes which istime consuming (Chikuni, Ozutsumi, Koishikawa, & Kato, 1990;Ebbehoj & Thomsen, 1991). However, the PCR techniques usingDNA amplification of specific target gene of mitochondria DNA(mtDNA) is the method of choice due to its rapidity, specificity,sensitivity and reproducibility (Tanabe, Miyauchi, et al., 2007). Thecombination techniques of PCR and southern hybridization werereported by Sahilah et al. (2012) in Halal market surveillance ofgelatin capsules in pharmaceuticals market in Malaysia. The PCR-southern hybridization on chip was developed to detect the pres-ence of porcine DNA by hybridizing the denatured biotinylatedamplicons with specific probes immobilized onto membrane. Thebiotin-labeled amplicons bind to streptavidin-alkaline phosphataseand subsequently detected by the colorimetric substrate of nitro-blue tetrazolium/5-bromo-4-chloro-3-indoyl-phosphate (NBT/BCIP). The colored signal was captured by Scanner System whichallows the species-specific identification. Other technique forporcine DNA detection in gelatin capsules using real-time PCR wasreported by Yasemin, Pelin, and Hamide (2012). In addition to thecyt b gene, the primer targets used in conventional PCR fordetecting porcine DNA include cyt oxidase II (COII), D-loop, 12SrRNA, 16s DNA, ATP8 and ATP6 (Cheng et al., 2003; Corona et al.,2007; Partis et al., 2000; Tartaglia et al., 1998; Yoshida et al.,2009). Therefore, this study was designed to compare the sensi-tivity of PCR-southern hybridization and conventional PCR inverifying the porcine DNA present in the gelatin capsules.

2. Materials and methods

2.1. Gelatin capsules and DNA extraction

All samples (pharmaceuticals and porcine canned meats) werepurchased from Selangor during the period of August to September2011. Twenty gelatin capsules of different brands (n ¼ 20) fromlocal and international companies were examined and designatedas C1 to C20. While, the three (n ¼ 3) different brands of porcine

Table 1Oligonucleotide primers for porcine DNA detection in gelatin capsules.

Primer sequence (50 to 30) Gen

SimP-F: 50-GAC CTC CCA GCT CCA TCA AAC ATC TCA TCT TGA TGA AA-30 CytoSimP-R: 50-GCT GAT AGT AGA TTT GTG ATG ACC GTA-30

Pork 1: 50-GCC TAA ATC TCC CCT CAA TGG TA-30 CytoPork 2: 50-ATG AAA GAG GCA AAT AGA TTT TCG-30

PPA6 F: 50-CTA CCT ATT GTC ACC TTA GTT-30 ATPPPA6 R: 50-GAG ATT GTG CGG TTA TTA ATG-30

canned meats (P1eP3) were purchased from local supermarkets. Acommercial Pig Genomic DNA (Novagen®, Germany) was used aspositive control.

The capsules DNAwere extracted using QIAGEN DNeasy® Bloodand Tissue Kit (Qiagen, USA) as instructed by the manufacturer.Total of 20 capsules were minced and a total of 50e100 mg wastransferred to a 1.5 ml sterile microcentrifuge tubes. DNA wasextracted from the capsules using QIAGENDNeasy Blood and Tissuekit (Qiagen, USA) and eluted with 100 ml of AE buffer. DNA wasquantified using MaestroNano® Spectrophotometer (Maestrogen,USA) and stored at �20 �C until further analysis. All DNA of gelatincapsules and porcine cannned meats were extracted in triplicatefrom each source.

2.2. Oligonucleotide primers

The oligonucleotide primers targeting mitochondria DNA(mtDNA) regions cyt b, cytochrome oxsidase II (COII) and ATP6 wereused in conventional PCR assays (Table 1). All mtDNA primers weresynthesized and supplied from First Base Laboratories (Selangor,MY). A commercial primer targetingmtDNA cyt b gene (cyt b biotin-labeled oligonucleotide primers) for PCR-southern hybridization onchip (Olipro™ PORCINE Gene Chip Kit) was supplied by OLIPROBiotechnology Sdn. Bhd. MY.

2.3. PCR amplification using different oligonucleotide primers

Amplification of DNA using primers SimP F and SimP R targetingthe mtDNA cyt b at 398 bp (Matsunaga et al., 1999) were performedin a final volume of 50 ml containing 25 ml of DreamTaq PCR MasterMix (2X) (Fermentas, Lithuania), 1 ml of 5 mM each primer (forwardand reverse for porcine DNA), NFWand 2 ml of approximately 50 ngDNA template depending on the DNA concentration. A negative anda positive DNA control was performed by adding 2 ml of NFW andPig Genomic DNA (Novagen®, Germany) respectively. The conditionof porcine DNA amplification assay consisting of the initial dena-turation at 95 �C for 2 min, followed by 35 cycles of amplification at94 �C for 30 s (denaturation), hybridization at 55 �C for 30 s, andelongation at 72 �C for 40 s, and a final extension step at 72 �C for3 min. It was carried out in the Mastercycler® gradient thermalcycler (Eppendorf, USA). The amplification products were electro-phoresed through 2.5% (w/v) agarose gel in 1 X TAE buffer (40 mMTris-OH, 20 mM acetic acid and 1 mM of EDTA; pH 7.6) at 100 V for45 min and stained in ethidium bromide.

The PCR amplification using primers pork 1 and pork 2 for COIIat 212 bp size (Lahiff et al., 2001) were performed in a final volumeof 50 ml containing 25 ml of DreamTaq PCR Master Mix (2X) (Fer-mentas, Lithuania) 1 ml of 100 mM each primers (forward andreverse), NFW and 2 ml of approximately 50 ng DNA template. Anegative and a positive DNA control were performed as above.Mastercycler® gradient thermal cycler (Eppendorf, USA) was usedto run the PCR with a temperature program consisting of the initialdenaturation at 95 �C for 2 min, followed by 30 cycles of 94 �C for1 min, 55 �C for 1 min, 72 �C for 2 min, and a final extension step at

e target Amplicon length (bp) Reference

chrome b 398 Matsunaga et al. (1999)

chrome oxidase II 212 Lahiff et al. (2001)

6 83 Yoshida et al. (2009)

Fig. 1. Gel electrophoresis of gelatin capsules using primers supplied by Olipro™Porcine PCR kit before southern hybridization on gene chip analysis. Lane M; Marker(100 bp ladder); Lane 1e6: Capsule samples of C1eC6 (Positive of porcine DNA(276 bp) and internal control (195 bp)); Lane 7: Positive control and Lane 8: Negativecontrol.

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72 �C for 10 min. The PCR products were analyzed through 2.5% (w/v) agarose gel as mentioned above.

The porcine DNA were amplified using 83 bp target primers ofPPA6F and PPA6R (ATP6) (Tanabe, Miyauchi, et al., 2007) in a 20 mlreaction volume containing 10 ml of DreamTaq PCRMaster Mix (2X)(Fermentas, Lithuania), 1 ml of 0.4 mM each primer, 6 ml of NFW and2 ml of approximately 50 ng extracted DNA template. A negative anda positive DNA control were performed as above. PCR was alsocarried out in Mastercycler® gradient thermal cycler (Eppendorf,USA) with a temperature program consisting of the initial heatactivation at 95 �C for 9 min, followed by 45 cycles of 92 �C for 30 s,55 �C for 30 s, 72 �C for 30 s, and a final extension step at 72 �C for5min. The PCR products were separated by electrophoresis through3% (w/v) agarose gel in 1X TAE buffer (40 mM Tris-OH, 20 mMacetic acid and 1 mM of EDTA; pH 7.6) at 80 V for 1 h and stained inethidium bromide.

All agarose gels in above experiments used 100 bp DNA ladder(Fermentas, Lithuania) as the size marker and was visualized usingUV transilluminator gel documentation (AlphaImager™ EP System,India).

2.4. PCR amplification for southern-hybridization on DNA chip

The southern-hybridization on Olipro™ PORCINE Gene Chipwas started with PCR amplification by using Olipro™ Porcine PCRkit. The multiplex PCR is performed with biotin-labeled primer setsto amplify species-specific target DNA (cyt b) and internal controlsequences. Later, the corresponding pair of sequence-specificprobes are immobilized onto membrane and hybridized with thebiotinylated PCR products. PCR was conducted as manufacturer'sinstruction in a final volume of 50 ml reaction. Each reactionmixture containing 24.6 ml of Porcine Gene Chip 1 X PCRMasterMix(Olipro™, MY), 0.5 ml of Taq DNA Polymerase, 2.0 ml of approxi-mately 50 ng DNA template, and nuclease freewater (NFW) asmarkup to final volume. A negative and a positive DNA control wasperformed by adding NFW and Pig Genomic DNA (Novagen®, Ger-many), respectively. The PCR amplification was carried out inMastercycler® gradient thermal cycler (Eppendorf, USA) with atemperature program consisting of the initial denaturation at 95 �Cfor 5 min to completely denatured the DNA template, followed by45 cycles of denaturation at 95 �C for 30 s, annealing at 55 �C for30 s, polymerization at 72 �C for 30 s and finally, elongation at 72 �Cfor 5 min. Negative controls (NFW) were included in each PCRamplification, in order to verify the PCR efficiency and to detectcontamination (Sahilah, Norhayati, Norrakiah, Aminah, & WanMustapha, 2011). The amplification products were analyzed byelectrophoresis using 2.5% (w/v) agarose gel in 1 X TAE buffer(40 mM Tris-OH, 20 mM acetic acid and 1 mM of EDTA; pH 7.6) at100 V for 45 min and stained in ethidium bromide and visualizedwith UV transilluminator gel documentation (AlphaImager™ EPSystem, India). A 100 bp DNA ladder (Fermentas, Lithuania) wasused as size reference. Positive result for porcine DNAwas indicatedby a band of 276-bp, and the 195-bp internal control (IC). IC in eachPCR functioned as an indicator to ensure that all PCR assays are ingood condition or the reactions were carried without inhibitor orimpurities (personal communication).

2.5. Southern-hybridization analysis on chip

The amplicons were denatured at 95 �C for 10 min and placedinto ice block immediately. Hybridizationwas carried out followingPORCINE Gene Chip protocols (Olipro™, MY). The amplicons weremixed with reagent A on the chips and incubated in hybridizationoven (OliproOven HYB001, MY) at 70 �C for 1 h with maximumvibration. Chips were washed using reagents B, C, D, E and F to

hybridize the biotinylated amplicons with specific probes. Afterhybridization reaction, the chips were again washed for colordevelopment. Chips were rinsed using reagent G and oven-dried(OliproOven HYB001, MY) at 70 �C for 5 min. Finally, chips werescanned and positive spots identified using Scanner System (Oli-proScan, MY). Positive result for porcine DNA will be indicated byperfect matched probe-target hybrid which formed blue-purplecolor on the chips.

2.6. Interpretation of results

Positive detection of porcine DNA is showed by the grey color attwo spots in the middle, while for internal control, eleven spots atthe left and the upper part of the chip appeared, including 4 spotslocated at every corner of the chips. However, no color will beformed in the middle of the chip if the result is negative. If the colordoes not appeared at all internal control spots, results are not valid,thus the experiment should be repeated.

2.7. Detection limit of oligonucleotide primers

The detection limit of all oligonucleotides as described in Table 1was examined using Pig Genomic DNA (Novagen®, Germany). ThePCR assay condition was similar as described in the PCR amplifi-cation using different oligonucleotide primers with different con-centration of porcine DNA ranging from 0 to 150 ng. While, in PCR-southern hybridization analysis, the condition used was similar asdescribed with porcine DNA but the concentration ranged from0.00001 to 1 ng.

3. Results and discussion

In the presence study, we examined twenty gelatin capsules forthe presence of porcine DNA using PCR-southern hybridization onchip. Of twenty samples tested, six capsule samples were testedpositive for porcine DNA (C1eC6). As indicated in Fig. 1, positiveresults for porcine DNA showed by bands of 276 bp and the 195 bp(IC). Capsules C1 and C3 did not show band of 276 bp on agarose gelanalysis, but gave strong signal when it hybridized on the chip(Fig. 2). Porcine DNA positive showed two spots in themiddle of the

Fig. 2. Pattern images on porcine gene chip of capsule samples which was read and identified by scanner analysis software. Chip image results of positive porcine 2 spots at thecenter for capsule samples (C1eC6) and porcine canned meats (P1eP3) on Olipro™ Porcine Gene Chip. The other eleven spots were internal control (IC). Chip image results ofpositive control spot (PC) and negative control spot (NC) on Olipro™ Porcine Gene Chip.

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chip (Fig. 2) with eleven spots as internal control (IC). All internalcontrol for capsule samples showed clear bands on agarose gelwhich indicated the PCR assay was in a good condition. There weretwo other spots which were invisible (negative control) and it po-sition are located above the two positive spots. If the above twospots were positive to porcine DNA, this indicated the chip wascontaminated with porcine DNA prior to used. Table 2 summarizedthe porcine DNA detection on gelatin capsules and porcine cannedmeats using PCR-southern hybridization and conventional PCRanalysis. The result obtained from this study was consistent with aprevious study (Sahilah et al., 2012) which reported similarobservation on Halal market surveillance of one hundred andthirteen gelatin capsules from different pharmaceutical products

using PCR-southern hybridization on chip. In their finding, lowintensity band was observed on agarose gel may due to the most ofDNAwas degraded and loss of specific site on mtDNA target region.However, the formation of nitroblue substrate of NBT/BCIP (Fig. 2)at low amplicons concentration, hybridized with specific probes onchip membrane and give positive result. Those results wereconsistent in repeated experiments. Other studies showed thatdespite DNA being degraded and altered, it was possible to amplifysmall DNA fragments with sufficient information to allow identi-fication (Corona et al., 2007; Rodriguez et al., 2004; Teletchea,Maudet, & Hanni, 2005).

The sensitivity of PCR-southern hybridization on DNA chip wascompared to the conventional PCR techniques as described by

Table 2Summarization of porcine DNA detection on gelatin capsules and porcine canned meats using PCR-southern hybridization and conventional PCR analysis (analyzed on agarosegels).

Samples Type of samples PCR-southern hybridization on DNA chip Conventional PCR

Cytochrome b (398 bp) Cytochrome oxidase II (212 bp) ATP6 (83 bp)

C1 Hard capsule þ � � �C2 Soft capsule þ � � �C3 Hard capsule þ � � �C4 Hard capsule þ � � �C5 Soft capsule þ � � �C6 Soft capsule þ � � �P1 Porcine canned meat þ � þ þP2 Porcine canned meat þ þ þ þP3 Porcine canned meat þ þ þ þC7eC20 Hard and soft capsule All negative All negative All negative All negative

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Matsunaga et al. (1999). The amplicons of cyt b region gave a singleband of 398 bp in size. No band was observed for all capsules andporcine canned meat (P1) in repeated experiments. However, twosamples of porcine cannedmeats (P2 and P3) were showed positiveband which produced a band of 398 bp in size (Table 2). Matsunagaet al. (1999) reported these primers were useful to detect porcineDNA in a range of fresh to cook meat (100e120 �C for 30 min).However, heat treatment applied to canned food in industrialpractices could possibly damage the mtDNA resulting in loss ofspecific primer binding sites as indicated in canned meat sample ofP1 which not produced any band. This happened because there areseveral of heat treatments involved during production processes ofcanned food such as cooking, pasteurization, sterilization and so onin the condition of high pressurewhere the temperature parametermight be up to 100 �C for 10e60 min and are exposed to a pH < 4(Teletchea et al., 2005).

Fig. 3. Porcine gene chip pattern images of different porcine genomic DNA concentration. NC0.0001 ng and 7: 0.00001 ng.

The COII and ATP6 primers were subsequently used to detectporcine DNA in all gelatin capsules (C1eC20). Both primers werechosen due to its capability to detect porcine DNA in feed sampleswhich undergone heat and denaturing treatment duringmanufacturing. Similar assumption goes to gelatin capsules whichalso undergone various steps of gelatin manufacturing process.However, no specific band was observed for both primers indetecting porcine DNA in gelatin capsule. From Lahiff et al. (2001)and Yoshida et al. (2009), those primers were reported useful todetect porcine DNA in feeds (Lahiff et al., 2001; Yoshida et al., 2009)with amplicons of 212 bp and 83 bp in size, respectively. Theporcine DNA detection of PCR-southern hybridization and con-ventional PCR analysis of gelatin capsules were shown in Table 2. Asindicated in Table 2, the 6 capsules tested which were positive to-wards porcine DNA using PCR southern-hybridization analysisshowed negative results in PCR conventional analysis.

1 and NC2 were negative control; 1: 10 ng; 2: 1 ng; 3: 0.1 ng; 4: 0.01 ng; 5: 0.001 ng: 6:

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The detection limit of the commercial Olipro™ PORCINE GeneChip primer (Olipro™, MY) in the detection of porcine DNA basedon cyt b target sequence was 0.001 ng (1 pg) (Fig. 3). While, for theother different primers were 0.25 ng (cyt b), 0.1 ng (COII) and0.0001 ng (ATP6) (Lahiff et al., 2001; Matsunaga et al., 1999;Yoshida et al., 2009). Our detection limit results are consistentwith the findings reported from those other workers (Lahiff et al.,2001; Matsunaga et al., 1999; Yoshida et al., 2009) who reportedthe similar values of genomic DNA detection limit.

The detection limit of ATP6 (PPA6F and PPA6R) primers showedhigh sensitivity to detect porcine DNA which the DNA was comefrom the commercial source. However, those primers failed to showany specific band on agarose gel from gelatin capsule samples. Ourfinding was in contrast with other workers who reported theprimers were successful to detect porcine DNA in meat and bonemeal (MBM), thus, useful as an Official Methods of Feed Analysis(Matsunaga et al., 1999; Yoshida et al., 2009). They demonstratedthe ATP6 primers have high specificity and sensitivity in detectingporcine DNA in various rendering procedures in Japan. The possiblereasonwhy it failed to detect porcine DNA from gelatin capsule wasthe mtDNA not only degraded but also present in small quantitieswhich reduced the number of DNA fragments with suitable size formolecular analysis (Teletchea et al., 2005). The respective ampliconmay exist in a very low concentration and could not be seen onagarose gel analysis.

4. Conclusions

In conclusion, the PCR-southern hybridization on chip is a newinnovative approach in Halal authentication, though combinationof PCR and southern hybridization are not new techniques. Theadvantage of this technique is that, they are able to detect a verylow amount of porcine DNA amplicons after PCR process bysouthern hybridization procedures. The PCR-southern hybridiza-tion offer promising results in detecting porcine DNA in highlyprocessed products such as gelatin and other processed foods. Thisapproach demonstrated sensitive, reproducible and certainly usefulto detect porcine DNA for Halal authenticationwhich would furthervalidate the pharmaceutical products labeling for consumers.

Acknowledgments

We are gratefully thanks to Olipro Biotechnology Sdn. Bhd.,HEJIM grant (INDUSTRI-2011-052) and FRGS/1/2014/STWN10/UKM/02/4, for the assistance in this research.

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