A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> Our study on the effects of different gelatins on calcium phosphate precipitation demonstrated that bovine bone gelatin and porcine skin gelatin could be distinguished using a pH drop method. The induction time was more remarkably increased by the addition of bovine bone gelatin than in the case of porcine skin gelatin. The difference between these two gelatins was significant at the concentrations of 0.5 and 2.0 mg/ml. The effects on hydroxyapatite transformation were biphasic. Gelatins increased the value at a lower concentration, but they decreased it after reaching a peak concentration. The peak concentration was 0.5 mg/ml for bovine bone gelatin, but was 4.0 mg/ml for porcine skin gelatin. Two commercial gelatin products were studied. One had properties close to bovine gelatin, and the other had properties close to porcine gelatin. This simple method may be useful in analyzing the origin of gelatin products. <s> BIB001 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> Gelatin is a collagen derivative, which has a large application in the pharmaceutical, food and adhesive industries as well as photography. The large similarity in structure and properties of gelatins from different origins makes their differentiation difficult. Certain chemometric methods, such as principal component analysis (PCA), can help to classify and characterize gelatin components. In this study 14 bovine and 5 porcine gelatins were examined. The analysis procedure involved complete hydrolysis of samples by classic acid hydrolysis in order to release their amino acid residues. Separation and determination of amino acids was achieved by reversed-phase (RP) HPLC following pre-column derivatisation. Orthophtaldialdehyde (OPA) and 4-chloro-7-nitro benzofurazane (NBD-Cl) were used as derivatisation reagents. From the 20 peaks detected by HPLC analysis, one was very typical in bovine gelatin. Peak height, area, area percentage and width were used to make matrixes. Principal component analysis with the MATLAB program was used to differentiate these gelatins. PCA on matrix of height, width and total matrix were resulted in good differentiation between bovine and porcine gelatins. <s> BIB002 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> Sequence analysis of bovine and porcine collagen type I indicates that some partial sequences are specific for bovine or porcine collagen type I, which can be used as marker peptides for gelatin differentiation. Bovine and porcine gelatins were digested with trypsin. The digest mixtures were analyzed with high performance liquid chromatography/mass spectrometry ( HPLC/MS ). Marker peptides were identified by the method of peptide sequence tag. The results indicated that the bovine and porcine. gelatins could be differentiated according to the sequences of marker peptides. Further analysis indicated that some prolines in the marker peptides were hydroxylated, which should be taken into account to increase the confidence in peptide identification. The relative abundance of marker peptides in gelatin of different molecular weight range was also investigated. The results indicate that identification of the marker peptides in the digested gelatins by HPLC/MS is an alternative method for differentiation of bovine and porcine gelatins. <s> BIB003 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> In this study, a convenient, sensitive and specific real-time PCR assay was described for the species identification and their quantification in raw and cooked meat products. Specific primers and TaqMan probes were designed on the mitochondrial ND2, ND5 and ATP 6-8 genes for donkey, pork and horse, respectively, and the performance of the method was tested. In the results, no cross-reaction was observed between the donkey and pork species specific primer-probe systems and non-target species (bovine, ovine, chicken and turkey). Only one cross reaction was observed between the horse species specific primer-probe set and 100ng pork DNA at the ct 33.01 level (corresponding to 0.01ng horse DNA). The real-time quantitative assay used in this study allowed the detection of as little as 0.0001ng template DNA from pure meat for each species investigated and experimental meat mixtures. In conclusion, it can be suggested that the TaqMan probe assay used in this research might be a rapid and sensitive method for the routine meat species identifications studies in raw or cooked meat products. <s> BIB004 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> Species identification of fragmentary bone, such as in rendered meat and bone meal or from archaeological sites, is often difficult in the absence of clear morphological markers. Here we present a robust method of analysing genus-specific collagen peptides by mass spectrometry simply by using solid-phase extraction (a C18 ZipTip®) for peptide purification, rather than liquid chromatography/mass spectrometry (LC/MS). Analysis of the collagen from 32 different mammal species identified a total of 92 peptide markers that could be used for species identification, for example, in processed food and animal feed. A set of ancient (>100 ka@10°C) bone samples was also analysed to show that the proposed method has applications to archaeological bone identification. Copyright © 2009 John Wiley & Sons, Ltd. <s> BIB005 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> Gelatin is a mixture of polypeptides obtained by hydrolysis of collagen primarily from bovine and porcine skin and bones. The similarity between different gelatins makes it difficult to trace their species origin. In this work, a new method for differentiation between bovine and porcine gelatin was developed based on detection and identification of marker peptides in digested gelatins. Sequence alignment analysis indicates that bovine and porcine Type I collagen contain differential sequences. The gelatins were digested by trypsin, and the resulting peptides were analyzed by high performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). The marker peptides specific for bovine and porcine were successfully detected in the digested bovine and porcine gelatin, respectively. Comparative analysis indicated that more marker peptides could be detected in gelatin with a higher mean molecular weight. It was found that proline hydroxylation was a key factor affecting the peptide identification. For peptides such as GPPGSAGSPGK and GPPGSAGAPGK detected in digested bovine and porcine gelatin, respectively, the sequence should be verified manually since the mass shift caused by proline hydroxylation can be confused with the mass difference between Set and Ala residues. The results indicate that detection of marker peptides in the digested gelatin sample using HPLC-MS/MS is an effective method to differentiate between bovine and porcine gelatin. (C) 2009 Elsevier Ltd. All rights reserved. <s> BIB006 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> In order to classify unknown gelatin into their species of origin, a simple and rapid method for the qualitative determination was developed using Fourier transform infrared (FTIR) in combination with attenuated total reflectance (ATR) and discriminant analysis. The spectra were analysed using a chemometric method, principal component analysis (PCA), to classify and characterise gelatin compounds using regions of the FTIR spectra in the range of 3290–3280 cm−1 and 1660–1200 cm−1 as calibration models. Results from PCA, which were subsequently represented by the Cooman’s plot showed a clear distinction between gelatin samples of bovine and porcine origins. This qualitative approach, besides providing a rapid determination of the source of gelatin, may also be established based on a second derivative study of the FTIR spectrum to alleviate any doubt of the gelatin source for applications in the food and pharmaceutical industries. <s> BIB007 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> Gelatin used for capsule shells can be derived from porcine origin. Some religion like Islam prohibited their followers to consume any products containing porcine derivatives including porcine gelatin in the products. Consequently, some rapid and reliable techniques are continuously developed for detection of porcine gelatin in some pharmaceutical products like capsule shells. The aim of this study was to evaluate the possibility to use amino acid profile in combination with chemometrics of principal component analysis for detection and classification between gelatin in capsule derived from porcine and bovine. Based on score plot of first principal components (PC1) and second principal components (PC2), porcine and bovine gelatins in capsule could be apparently distinguished. Key words: bovine gelatin; porcine gelatin; amino acid profile; principal component analysis <s> BIB008 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> Abstract Detection and quantitation of material from porcine and bovine species in gelatin and gelatin capsules is required for health safety concerns and for some religious practices. In this study, two species-specific qPCR assays were developed based upon repetitive elements. They allowed sensitive detection of porcine and bovine DNA at as low as 1 pg/mL. The lack of cross-reactivity when the sets were used to amplify DNA from the other species indicates high specificity of the assay. When binary gelatin blends containing various amounts of porcine and bovine gelatin were prepared and analyzed by the qPCR assays, the determined ratios of porcine material to bovine material were very close to their theoretical values, and a contamination level as low as 1% of the other species in the gelatin blends could be determined. When evaluated in gelatin capsules, although significantly less DNA was detected, determination of porcine and bovine species identities and estimation of the relative abundance of each species was possible. Therefore, the porcine and bovine species-specific qPCR assays described here represent simple, reliable and sensitive DNA-based tests for determination and quantitation of the species of origin from highly processed products. <s> BIB009 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> A commercially available real-time PCR, based on a multi-copy target cytochrome b (cyt b) using porcine specific primers, has been validated for the Halal/Kosher authentication of gelatine. Extraction and purification of DNA from gelatine were successfully achieved using the SureFood® PREP Animal system, and real-time PCR was carried out using SureFood® Animal ID Pork Sens kit. The minimum level of adulteration that could be detected was 1.0% w/w for marshmallows and gum drops. A small survey was undertaken of processed food products such as gum drops, marshmallows and Turkish delight, believed to contain gelatine. Of fourteen food products from Germany, two samples were found to contain porcine gelatine, whereas of twenty-nine samples from Turkey twenty-eight were negative. However, one product from Turkey contained porcine DNA and thus was not Halal, and neither was the use of porcine gelatine indicated on the product label. <s> BIB010 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> Gelatin is widely used in some food products, including soft candy (one of food products preferred by children) . Most of the gelatin available in the market derived from pigs . Some religions like Islam and Jews prohibited their followers to consume any food products containing pig derivatives, including porcine gelatin. Therefore, it is necessary to develop some rapid and reliable methods for detection of porcine gelatin in soft candy . The purpose of this study was to differentiate and classification the gelatin sources (porcine or bovine) in soft candy based on amino acid profiles combined with chemometrics of p rincipal component analysis (PCA) . Separation and determination of amino acid was conducted by reversed-phase HPLC using a fluorescent detector, after being derivatized with o rtho -phtalaldehyde in 2-mercaptoethanol ( OPA/2-MCE). Parameters of peak height percentage of each amino acids from each sample were analyzed by PCA . Based on PC1 and PC2, porcine and bovine gelatins in soft candy could be apparently distinguished. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ ::: table.MsoNormalTable ::: {mso-style-name:"Table Normal"; ::: mso-tstyle-rowband-size:0; ::: mso-tstyle-colband-size:0; ::: mso-style-noshow:yes; ::: mso-style-priority:99; ::: mso-style-qformat:yes; ::: mso-style-parent:""; ::: mso-padding-alt:0in 5.4pt 0in 5.4pt; ::: mso-para-margin-top:0in; ::: mso-para-margin-right:0in; ::: mso-para-margin-bottom:10.0pt; ::: mso-para-margin-left:0in; ::: line-height:115%; ::: mso-pagination:widow-orphan; ::: font-size:11.0pt; ::: font-family:"Calibri","sans-serif"; ::: mso-ascii-font-family:Calibri; ::: mso-ascii-theme-font:minor-latin; ::: mso-fareast-font-family:"Times New Roman"; ::: mso-fareast-theme-font:minor-fareast; ::: mso-hansi-font-family:Calibri; ::: mso-hansi-theme-font:minor-latin; ::: mso-bidi-font-family:"Times New Roman"; ::: mso-bidi-theme-font:minor-bidi;} <s> BIB011 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> This study was conducted to explore the differentiation of bovine and porcine gelatins before and after pepsin hydrolysis based on peptide pattern from spectroscopic and electrophoretic analysis due to development of the halal food products analysis. In this study, pepsin (EC 3.4.23.1) was used to hydrolyze the two sources of gelatin with consideration to its ability to digest up to 20% of ingested amide bonds by cleaving preferentially after the N-terminal of aromatic amino acids such as phenylalanine , tryptophan , and tyrosine . In this study, we expect to produce the fragment of gelatins with differentiation in relative molecular weights. Gelatins fragments then analyzed by UV-Vis and FTIR spectroscopy to characterize the functional groups on each source of gelatins, followed by SDS-PAGE ( Sodium Duodecylsulphate Polyacrylamide Gel Electrophoresis ) to identify the molecular weight of the resulting fragments. In UV-Vis spectroscopy, both gelatin source before and after hydrolysis had different absorbance at 229 nm and 240 nm showing the proportion of C=O amida and differences in two -dimensional conformation of the peptide . In terms of FTIR spectra, both gelatin have wavenumber at 3300-3400 cm -1 (NH stretching), 1600 cm -1 (C=O stretching, amida), 1500 cm -1 (C-N stretching), and at 620-767 cm -1 (OCN bending). This indicates that the relative amino acid compositions from two sources of gelatins were relatively different. In contrast, SDS-PAGE analysis does not give a real differentiation, except for porcine gelatin, that fragments which on 2 hour incubation show two peptide fragments with molecular sizes below 36,2 kDa and 28.6 kDa. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ ::: table.MsoNormalTable ::: {mso-style-name:"Table Normal"; ::: mso-tstyle-rowband-size:0; ::: mso-tstyle-colband-size:0; ::: mso-style-noshow:yes; ::: mso-style-priority:99; ::: mso-style-qformat:yes; ::: mso-style-parent:""; ::: mso-padding-alt:0in 5.4pt 0in 5.4pt; ::: mso-para-margin-top:0in; ::: mso-para-margin-right:0in; ::: mso-para-margin-bottom:10.0pt; ::: mso-para-margin-left:0in; ::: line-height:115%; ::: mso-pagination:widow-orphan; ::: font-size:11.0pt; ::: font-family:"Calibri","sans-serif"; ::: mso-ascii-font-family:Calibri; ::: mso-ascii-theme-font:minor-latin; ::: mso-fareast-font-family:"Times New Roman"; ::: mso-fareast-theme-font:minor-fareast; ::: mso-hansi-font-family:Calibri; ::: mso-hansi-theme-font:minor-latin; ::: mso-bidi-font-family:"Times New Roman"; ::: mso-bidi-theme-font:minor-bidi;} <s> BIB012 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> We presented a novel nanoUPLC-MS(E) workflow method that has potential to identify origin of gelatin in some dairy products; yoghurt, cheese and ice cream. In this study, the method was performed in two steps. In the first step, gelatin was extracted from these products before the MS-sample preparation. In the second step, tryptic gelatin peptides were separated and analyzed with ultra-performance liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry (nanoUPLC-ESI-q-TOF-MS(E)). The novelty of this setup was that it functioned in a data independent acquisition mode and that alternate low and elevated collision energy was applied to acquire precursor and product ion information. This enabled accurate mass acquisition on the peptide level to identify the gelatin peptides. The marker peptides specific for porcine and bovine could be successfully detected in the gelatin added to the dairy products analyzed, revealing that the detection of marker peptides in the digested gelatin samples using nanoUPLC-ESI-q-TOF-MS(E) could be an effective method to differentiate porcine and bovine gelatin in the dairy products. <s> BIB013 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> 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 principal 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 gelatine 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 gelatine. 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. <s> BIB014 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> Consumption of food products derived from porcine sources is strictly prohibited in Islam. Gelatin, mostly derived from bovine and porcine sources, has many applications in the food and pharmaceutical industries. To ensure that food products comply with halal regulations, development of valid and reliable analytical methods is very much required. In this study, a species-specific polymerase chain reaction (PCR) assay using conserved regions of mitochondrial DNA (cytochrome b gene) was performed to evaluate the halal authenticity of gelatin. After isolation of DNA from gelatin powders with known origin, conventional PCR using species-specific primers was carried out on the extracted DNA. The amplified expected PCR products of 212 and 271 bp were observed for porcine and bovine gelatin, respectively. The sensitivity of the method was tested on binary gelatin mixtures containing 0.1%, 1%, 10%, and 100% (w/w) of porcine gelatin within bovine gelatin and vice versa. Although most of the DNA is degraded due to the severe processing steps of gelatin production, the minimum level of 0.1% w/w of both porcine and bovine gelatin was detected. Moreover, eight food products labeled as containing bovine gelatin and eight capsule shells were subjected to PCR examination. The results showed that all samples contained bovine gelatin, and the absence of porcine gelatin was verified. This method of species authenticity is very useful to verify whether gelatin and gelatin-containing food products are derived from halal ingredients. <s> BIB015 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> Porcine gelatine is a common adulterant found in edible bird’s nests (EBNs) used to increase the net weight prior to sale. This study aimed to develop indirect enzyme-linked immunosorbent assays (ELISAs) for porcine gelatine adulteration using anti-peptide polyclonal antibodies. Three indirect ELISAs were developed (PAB1, 2 and 3), which had limits of detection (LODs) of 0.12, 0.10 and 0.11 µg g–1, respectively. When applied to standard solutions of porcine gelatine, the inter- and intra-assays showed coefficients of variation (CVs) less than 20% and were able to detect at least 0.5 ng µg–1 (0.05%) porcine gelatine in spiked samples. The proposed ELISA offers attractions for quality control in the EBN industry. <s> BIB016 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> Halal authentication of gelatin capsules was conducted using polymerase chain reaction (PCR)-southern hybridization on chip and conventional PCR analysis. The primers used in PCR-southern hybridization were 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 ATP6 gene were tested, resulting of 398, 212 and 83 bp amplicons, respectively. Of 20 brands examined using PCR-southern hybridization, 6 capsules (C1–C6) were found to be porcine DNA positive but none were positive using conventional PCR method. The sensitivity of each primer in the detection of porcine DNA was 0.25 ng (cyt b), 0.1 ng (COII), 0.001 ng (Olipro™ Chip) and 0.0001 ng (ATP6). Results demonstrated that the PCR-southern hybridization on chip was useful and reliable for verifying porcine DNA in gelatin capsules compared to conventional PCR. <s> BIB017 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> The amino acid compositions of bovine, porcine and fish gelatin were determined by amino acid analysis using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate as derivatization reagent. Sixteen amino acids were identified with similar spectral chromatograms. Data pre-treatment via centering and transformation of data by normalization were performed to provide data that are more suitable for analysis and easier to be interpreted. Principal component analysis (PCA) transformed the original data matrix into a number of principal components (PCs). Three principal components (PCs) described 96.5% of the total variance, and 2 PCs (91%) explained the highest variances. The PCA model demonstrated the relationships among amino acids in the correlation loadings plot to the group of gelatins in the scores plot. Fish gelatin was correlated to threonine, serine and methionine on the positive side of PC1; bovine gelatin was correlated to the non-polar side chains amino acids that were proline, hydroxyproline, leucine, isoleucine and valine on the negative side of PC1 and porcine gelatin was correlated to the polar side chains amino acids that were aspartate, glutamic acid, lysine and tyrosine on the negative side of PC2. Verification on the database using 12 samples from commercial products gelatin-based had confirmed the grouping patterns and the variables correlations. Therefore, this quantitative method is very useful as a screening method to determine gelatin from various sources. <s> BIB018 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> Abstract Background Halal products are the only permissible products that can be consumed by Muslim population all over the world. Halal certification process is becoming critical due to processing advancements and conflict of opinions among various Islamic schools of thought. Multiple Halal certification standards are framed by regulatory bodies in different countries, but there is inconsistency about few issues among these standards. One such conflict is Istihalah, which deals with the alteration in physicochemical nature of food and render a non-permissible food (Haram) to an acceptable form (Halal) for Muslims. There is dire need to understand the grey areas, such as the concept of Istihalah, to develop unanimous International Halal Standards complying with the rulings of all Islamic schools of thought. Scope and approach To understand the Istihalah, scientific and Islamic literature from various schools of thought, has been reviewed. The primary objective of this study is to explain the concept of Istihalah with respect to the physicochemical alteration of food matrix and rulings of various Islamic schools, leading to generate a consensus for international Halal certification standards. Key findings and conclusions Istihalah is an agreed concept among Islamic schools of thought but the definition for the extent of such alteration (to be considered as Istihalah) is not agreed. This concept should not be used for Halal Certification as it may create ambiguous situation for the global acceptability and credibility of a certificate. <s> BIB019 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> Pharmaceutical dosage forms containing gelatin are recently of high attention in regard to its porcine content. In Muslim countries, halal products assurance is strictly regulated, not only for food but also for pharmaceutical products. This study aimed to develop gelatin identification and verification method in capsule shells by performing simple simultaneous protein- and nucleic acid based-identification methods. Identification and verification performing polyacrylamide gel electrophoresis (PAGE)—coupled with principle component analysis, and polymerase-chain-reaction (PCR)-restriction fragment length polymorphism (RFLP) were carried out. Gelatin was obtained by extracting the capsules using cold acetone; the precipitate was used directly for PAGE, and was used as well for DNA extraction. The results showed that the porcine gelatin reference showed 12 major bands of peptide profile, with predominant bands at ±200 and ±100–135 kDa on 8 % Tris-glycine gel, while bovine gelatin only showed four major bands. Further, PCR-RFLP result showed two specific bands of the porcine gelatin DNA at 228 and 131 bp, whereas the bovine gelatin DNA bands occurred at 316 and 44 bp. The results demonstrated that the capsule shell samples used in this study contained the following: sample 1 was verified contain porcine gelatin, whereas the three other samples identified contain bovine gelatin. Although the simultaneous approaches seemed need to be improved, it is crucial to maintain their low cost and simple protocol. <s> BIB020 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> Competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for rapid detection of porcine gelatin in edible bird's nest (EBN). Three ELISAs were developed by using polyclonal rabbit antibodies against porcine species-specific amino acid sequences of collagen α2 (I) chain (pAb1 and pAb2) and α1 (I) chain (pAb3). The limit of detection (IC15) of the three ELISAs was 0.033, 0.082 and 0.052 μg/mL respectively. The median inhibitory concentration (IC50) of pAb1, pAb2 and pAb3 was 0.265, 0.394 and 0.228 μg/mL respectively, as well as able to recognise porcine and bovine gelatins. pAb1 showed slight cross-reactivity with cave nest and egg white, while pAb2 exhibited slight cross-reactivity with blood cave nest and egg white. No cross-reactivity was observed with EBNs and egg white for pAb3. The recoveries of porcine gelatin spiked EBNs were in the range of 62.8–125.4% with intra- and inter-day coefficient of variants (CVs) of 2.9–5.4% and 4.7–9.6% respectively when using pAb3. Taking into account all abovementioned factors, pAb3 appeared sufficient for EBN authentication. <s> BIB021 </s> A Review of Gelatin Source Authentication Methods <s> INTRODUCTION <s> The presence of pig derivatives, such as porcine gelatin, in any products is prohibited to be consumed by Muslim community. This study is intended to develop a specific primer from mytochondrial D-loop capable of amplifying DNA from porcine gelatin in commercial capsule shells. Two pairs of primers designed from mitochondrial D-loop region were tested in order to confirm the primer specificity in gelatin sources (pork, beef, and catfish) and fresh tissue (pig, cows, goat, chickens, and rat). Primers were then used to perform sensitivity test of six dilution series (1000, 200, 100, 10, 5, and 1 pg/µL) of porcine gelatin and porcine capsule shell. The amplification was also performed on capsule shell from porcine-bovine mixture gelatin at 0, 10, 20, 30, 40, 50, and 100% concentration. The repeatability test was performed by measuring amplification capsule shells from porcine–bovine gelatin mixture. Real time polymerase chain reaction method using primers designed was further applied to analyze capsule shells purchased from markets. From two primers have been designed specifically, only primer D-Loop 108 (forward: 5’-CGT ATG CAA AAA ACC ACG CCA-3’; reverse: 5’-CTT ACT ATA GGG AGC TGC ATG-3’) had the capability to identify the presence of porcine DNA in fresh tissue and gelatin sources at optimum annealing temperature of 58.4ºC. Sensitivity of the developed method expressed as limit of detection of DNA in gelatin and capsule shells is 5 pg. <s> BIB022
As at 2013, the global Halal market size was about 580 to 660 billion US Dollars and has great potential to increase in the future year BIB019 . Apart from increasing Muslim population and Halal awareness, Halal market is driven by development of Halal ingredients authentication methods that strengthening consumers' confident BIB020 . Gelatin is among the most studied Halal ingredient because of its vast usage in pharmaceutical and food products. Gelatin is a hydrocolloid with unique properties and can function as gelling, thickener, foaming agent, plasticizer, texture and binding agent  . The sources of most commercial gelatin are from mammalian (bovine and mostly porcine) bone and hide BIB015 . Consumers' concern about gelatin source is due to cultural and religious beliefs. For examples, Muslims and Jews reject porcine based food derivatives, Chinese traditional medicine use only gelatin from donkey skin for treatment of some disease BIB002 ) and vegetarians avoid animal based products. Increase in Halal awareness among Muslims has called for great need of food-source authentications. Gelatin and gelatin based products are currently classified as doubtful because haram (porcine) gelatin is the most abundant. Traceability of gelatin source has been a great task in Halal field. Other reasons for gelatin source authentication include: (i) protection of consumer from food frauds due to false labeling and avoid unfair market competition BIB004 ) and (ii) health issues. The use of bovine carrying prion proteins has been linked to bovine spongiform encephalopathy (mad cow disease) causing immune response that led to fatal neurodegenerative disease (spongy degeneration of the brain and spinal cord) in cattle BIB008 . Also, bovine and porcine gelatins is risky to gelatin-allergic patients BIB011 . Gelatin contains protein, peptides or nucleic acids BIB020 . Proteins, peptide derivatives and nucleic acids are favorable means for tissue identification in samples even after it has undergone heating, for instance, in processed food BIB005 ). However, large similarities between structures of gelatin from different sources make their differentiation difficult BIB002 . Many gelatin source authentication methods have been developed including: spectroscopic method BIB007 BIB012 , immunochemical method BIB016 BIB021 , nucleic acid based method BIB009 BIB010 BIB020 BIB017 BIB015 BIB022 , mass spectrometric method HPLC/MS BIB013 BIB003 BIB006 ), chromatographic-chemometric method BIB018 , electrophoretic analysis BIB014 ) and chemisorption BIB001 . We have reviewed gelatin source authentication methods and identified their achievements and challenges. Current gelatin source authentication methods are time consuming and expensive, thus required development of cheap and quick analytical methods. Likewise, gelatin transformation during processing can potentially hinder the efficiency of the currently developed gelatin authentication methods. This work will serve as a quick reference guide for future researchers.