A Review of Gelatin Source Authentication Methods <s> Nucleic Acid Based Method <s> Abstract Many meat products nowadays may contain several species in different proportions. To protect consumers from fraud and misdeclarations, not only a qualitative but also a quantitative monitoring of ingredients of complex food products is necessary. DNA based techniques like the polymerase chain reaction (PCR) are widely used for identification of species but no answer to the proportional amount of a certain species could be given using current techniques. In this study we report the development and evaluation of a quantitative competitive polymerase chain reaction (QC-PCR) for detection and quantification of porcine DNA using a new porcine specific PCR system based on the growth hormone gene of sus scrofa. A DNA competitor differing by 30 bp in length from the porcine target sequence was constructed and used for PCR together with the target DNA. Specificity of the new primers was evaluated with DNA from cattle, sheep, chicken and turkey. The competitor concentration was adjusted to porcine DNA contents of 2 or 20% by coamplification of mixtures containing porcine and corresponding amounts of bovine DNA in defined ratios. <s> BIB001 </s> A Review of Gelatin Source Authentication Methods <s> Nucleic Acid Based Method <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> Nucleic Acid Based Method <s> Gelatine is a collagen derivative obtained from bones and hides/skin mainly from bovine and pigs. As a consequence of the outbreak of bovine spongiform encephalopathy (BSE), the use of bovine gelatine in feed, food and pharmaceutical products has been restricted by regulatory authorities. However, no method was presently available for its specific detection. The large similarity in amino-acid sequences of collagens from different species make their immunochemical differentiation difficult when using polyclonal antibodies raised against the whole molecule [A. Venien, D. Levieux, J. Immunoassay Immunochem., in press]. To obtain bovine-specific antibodies, we immunized rabbits against putative species-specific sequences of the bovine collagen alpha 1(I) chain. Using these antibodies, an indirect ELISA was developed to allow a quick and easy differentiation between bovine and porcine gelatines. Moreover, a competitive indirect ELISA was found suitable to detect bovine gelatine in porcine gelatine purchased from laboratory chemicals suppliers down to a dilution of 2-4 parts per 1000 with CVs ranging from 5.7 to 7.7%. When testing mixtures of the largest possible range of industrial batches of bovine and porcine gelatines (skin/hides or bones origin, acid or alkaline processes, high or low Bloom) the detection limit was down to a dilution of 8 parts per 100 bovine gelatine in porcine gelatine. These ELISAs could be routinely used by pharmaceutical and food manufacturers to secure their supply chain. <s> BIB003 </s> A Review of Gelatin Source Authentication Methods <s> Nucleic Acid Based Method <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> Nucleic Acid Based Method <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> BIB005 </s> A Review of Gelatin Source Authentication Methods <s> Nucleic Acid Based Method <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> BIB006 </s> A Review of Gelatin Source Authentication Methods <s> Nucleic Acid Based Method <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> BIB007 </s> A Review of Gelatin Source Authentication Methods <s> Nucleic Acid Based Method <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> BIB008 </s> A Review of Gelatin Source Authentication Methods <s> Nucleic Acid Based Method <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> BIB009 </s> A Review of Gelatin Source Authentication Methods <s> Nucleic Acid Based Method <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> BIB010 </s> A Review of Gelatin Source Authentication Methods <s> Nucleic Acid Based Method <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> BIB011 </s> A Review of Gelatin Source Authentication Methods <s> Nucleic Acid Based Method <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> BIB012 </s> A Review of Gelatin Source Authentication Methods <s> Nucleic Acid Based Method <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> BIB013 </s> A Review of Gelatin Source Authentication Methods <s> Nucleic Acid Based Method <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> BIB014 </s> A Review of Gelatin Source Authentication Methods <s> Nucleic Acid Based Method <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> BIB015
The DNA techniques can be used for qualitative and quantitative analysis of processed food because of their stability. The technique can be used virtually in all samples because of the ubiquity of DNA. This technique is highly specific, very sensitive (up to 0.1%) and capable of detecting a small amount of certain DNA in the sample BIB001 . Other advantages of DNA-based technique are less contamination risk and larger dynamic range of detection BIB004 ). More so, the Polymerase chain reaction (PCR) techniques can be monitored by fluorescence thus eliminate detection steps BIB004 . Table 2 depicts the characteristics of bovine and porcine primers previously used in Halal authentication of gelatin source. Although universal primer can be used, a specific primer which allows shorter amplicons is better for food samples. This is because universal primer requires long PCR products with adequate DNA sequence variation for restriction analysis or single strand conformation polymorphism and thus not suitable for food samples that usually contain degraded DNA with few hundred base pairs BIB001 . In the work of BIB005 two species-specific quantitative-PCR (qPCR) assays was developed for the detection and quantitation of porcine and bovine DNA in gelatin blends and gelatin capsules. The developed assay was highly specific as it lacks cross-reactivity during amplification of each samples DNA. Also, the assay was highly sensitive to detect contamination level as low as 1.0% in gelatin blends (bovine + porcine). Analysis of gelatin capsules revealed possible discriminative detection and quantification of porcine and bovine species identities, although, less DNA was detected. BIB007 Immunochemical Bovine and porcine skin and bone gelatin that has undergone acid or alkaline BIB003 Porcine gelatin in edible bird's nests BIB009 BIB013 Gelatin sources in confectionery products  Nucleic acid based Gelatin in the capsule BIB005 ; BIB014 ; BIB010 ; BIB015 Gelatin blends BIB006 ; BIB011 Marshmallow/cake, gum-drops, desserts, jelly and Turkish delight Porcine and fish gelatins BIB012 Gelatin in capsule BIB002 Gelatin in soft candy BIB008 In another study, BIB006 reportedly used real-time PCR techniques to authenticate the source of gelatin in gum drops, marshmallows and Turkish delight. Two of 14 products from Germany were positive for porcine DNA whereas only 1 of 29 products from Turkey was positive for porcine DNA. Also,  used PCR and southern hybridisation on biochip analysis to investigate soft and hard gel capsules for Halal compliance. The result showed that 42 (without Halal logo) out of 113 capsules tested positive for porcine DNA. This evinced the suitability of PCR techniques and southern hybridisation on the biochip for monitoring haram compounds in the soft and hard capsule. Furthermore, BIB010 also used PCR-southern hybridisation on chip and convention PCR for Halal authentication of gelatin in 20 capsule brands. PCR-southern hybridisation on chip detected DNA in 6 capsules that were undetected by conventional PCR analysis. Compared to conventional PCR, PCR-southern hybridisation on the chip was more suitable and dependable for authenticating porcine DNA in gelatin capsules. BIB011 reportedly used species-specific PCR technique to validate gelatin sourced from 8 gelatin containing food products (two marshmallow, two jellies, two deserts and two cakes) and observed them to be from bovine but not porcine. BIB015 also used real-time PCR technique to validate the lack of porcine DNA in the capsule shell.