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Food Chemistry 151 (2014) 286–292 Contents lists available at ScienceDirect Food Chemistry journal homepage:

Contents lists available at ScienceDirect

Food Chemistry

journal homepage: www.elsevi er.com/locat e/foodchem Analytical Methods Use of principal component analysis for

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

Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia article info Article history: Received 1 July 2011 Received

article info

Article history:

Received 1 July 2011 Received in revised form 6 October 2013 Accepted 12 November 2013 Available online 20 November 2013

Keywords:

Gelatine polypeptides Principal component analysis Acetone precipitation Food authenticity Adulteration

abstract

The study was aimed to differentiate between porcine and bovine gelatines in adulterated samples by utilising sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) combined with prin- cipal component analysis (PCA). The distinct polypeptide patterns of 6 porcine type A and 6 bovine type B gelatines at molecular weight ranged from 50 to 220 kDa were studied. Experimental samples of raw gel- 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- atine. There were no differences in the electrophoretic profiles of the jelly samples when the proteins were extracted with an acetone precipitation method. The simple approach employing SDS–PAGE and PCA reported in this paper may provide a useful tool for food authenticity issues concerning gelatine. 2013 Elsevier Ltd. All rights reserved.

1. Introduction

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).

0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.

chromatography/tandem mass spectrometry (HPLC–MS/MS) ( 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

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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. Materials and methods

2.1. Materials

Commercial culinary food colouring (artificial rose pink colour) and food flavouring (artificial rose flavour) (Star Brand, FFM Ber- had) and coarse grain sugar (sucrose) were purchased from a local retail store. All chemicals used were of analytical grade. The 12 studied samples of raw gelatines have been described in Table 1 .

2.2. Measurement of protein content

The protein content was measured according to the method of Bradford (1976). The measurement was performed in triplicate for 4 separate experiments (giving a total of 12 absorbance read- ings per sample). The protein content of the samples ranged from 0.18 to 0.36 mg/g.

Table 1 Characteristics of the commercial raw gelatines.

Gelatine sample

Species

Tissue

Type

Bloom

1

Porcine (PSS)

Skin

A

300

2

Porcine (PSC)

Skin

A

175

3

Porcine

Skin

A

90–110

4

Porcine

_

A

_

5

Porcine

Skin

A

_

6

Porcine

Skin

A

220

7

Bovine (BSS)

Skin

B

225

8

Bovine

Skin

B

75

9

Bovine

Skin

B

90–110

10

Bovine (CSA)

Skin

B

220

11

Bovine (CBA)

Bone

B

220

12

Bovine (CHG)

_

B

200

_ not stated.

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

Acetone precipitation was done according to Fic, Kedracka-Krok, Jankowska, Pirog & Dziezicka-Wasylewska (2010) with slight modifications. Four volumes of cold acetone ( 20 C) were added to one volume of melted jelly. The mixture was vortexed and kept overnight at 20 C. The mixture was then centrifuged at 14,000 rpm for 10 min. The supernatant was discarded and the acetone residue was air-dried from the protein pellet. The protein pellet was used for SDS–PAGE.

2.5. SDS–PAGE

SDS–PAGE was conducted as described by Laemmli (1970) on a slab gel consisting of 4% stacking gel and 6% resolving gel. The pro- tein sample ( 0.8 mg/mL) was dissolved in the sample buffer (8 M urea, 20% v/v SDS, 10 mM ETDA, 0.5 M Tris–HCI, 1.114 g/mL 2-mercaptoethanol, 0.1% v/v glycerol and 0.05% w/v bromophenol 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 sample were determined using high molecular weight standard markers for SDS electrophoresis (GE Healthcare, Buckinghamshire, UK). The standards consisted of the following proteins:

myosin (220 kDa), a 2 -Macroglobulin (170 kDa), b -Galactosidase (116 kDa), transferrin (76 kDa) and glutamic dehydrogenase (53 kDa). SDS–PAGE gels were stained with silver stain solution (Bio-Rad Laboratories, Hercules, CA), according to the manufac- turer’s instructions, in order to visualise as many polypeptide bands as possible (silver staining is more sensitive than Coomassie Brilliant Blue staining). The stained gel was scanned using a densi- tometer (GS-800 Calibrated Densitometer Bio-rad, Hercules, CA) and each band was analysed using Quantity One Software (Bio-rad, Hercules, CA). Visualisation of polypeptide bands, lane comparison and estimation of molecular weights was conducted for each sample.

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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 the variables, whereas the qualitative presence or absence of poly- peptide band molecular weight values in each variable of each sample was used as input data. The score plot was extracted from the first two principal components, PC1 (is direction of largest variance) and PC2 (is perpendicular to PC1 and against the largest variance), as they presented the maximum amount of variability in the data ( Kher, Mulholland, Green, & Reedy, 2006 ).

3. Results and discussion

3.1. Acetone precipitation

As a preliminary test, the acetone precipitation method was applied to extract the protein from homemade jelly samples that contained pure bovine gelatine as well as various extraneous ingre- dients. To visualise the results, protein precipitates were analysed using the electrophoresis conditions described above and the

using the electrophoresis conditions described above and the Fig. 2. Electrophoretic polypeptide pattern and

Fig. 2. Electrophoretic polypeptide pattern and densitometric profile of porcine (PSS) gelatine. Lane 1: molecular weight (kDa) marker; lane 2: electrophoretic 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:

61; 15: 58; 16: 53 kDa. Densitometric profile represented for lane 2. R f :

relative electrophoretic mobility.

pattern was compared to that of unadulterated bovine gelatine. The electrophoresis gel showed that the polypeptide patterns were not affected by the addition of coarse grain sugar (sucrose), artifi- cial food flavour and colour or the combination of all three ingredi- ents (data not shown). The polypeptide bands exhibited a similar pattern to that of pure bovine gelatine. In the case of adulterated jelly samples, the polypeptide bands exhibited a similar pattern to that of raw adul- terated gelatine samples. These results indicate that the acetone precipitation method was successful in extracting proteins without changing their electrophoretic mobility patterns (compared to the pattern of raw gelatine). This simple method for protein extraction

of raw gelatine). This simple method for protein extraction Fig. 1. Electrophoretic separation of polypeptides from

Fig. 1. Electrophoretic separation of polypeptides from different sources of gelatine. PSS: porcine skin gelatine type A from Sigma; PSC: porcine skin gela tine 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 t ype B from Sigma. The amount of protein loaded was 8 l g per sample, and the gel was visualised using silver staining.

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T. Nur Azira et al. / Food Chemistry 151 (2014) 286–292 289 Fig. 3. Electrophoretic polypeptide

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. R f : relative electrophoretic mobility.

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

bovine gelatine, displaying a diffuse pattern of polypeptide Fig. 4. PCA classification of bovine gelatine adulterated

Fig. 4. PCA classification of bovine gelatine adulterated with different percentage of porcine gelatine. (a) Score plot; (b) loading plot.

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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

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,

be defined as a band. The higher the sensitivity value, Fig. 5. PCA classification of porcine

Fig. 5. PCA classification of porcine gelatine adulterated with different percentage of bovine gelatine. (a) Score plot; (b) loading plot.

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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

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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