Sensitivitas Real-Time Polymerase Chain Reaction dengan Primer Tanabe dalam Mendeteksi Gelatin Babi pada Confectionery
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Accepted
22 June 2023
Published
27 June 2023
Keywords:
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confectionery, LOD, porcine gelatin, real-time PCR
Abstract
Gelatin is commonly used as a gelling and thickening agent in confectionery products and is considered a critical material in terms of its halal status. Real-time Polymerase Chain Reaction (real-time PCR) is frequently employed as an analytical tool to detect traces of pork in food items. Although the real-time PCR method using the Tanabe primer and Internal Positive Control (IPC) has been validated effectively, its sensitivity or Limit of Detection (LOD) for confectionery products has yet to be determined. This study aims to determine the sensitivity of real-time PCR with Tanabe primers and IPC towards the confectionery products (gummy candy, marshmallow, and lozenges). Confirmation of this method on commercial marshmallow products known to contain porcine gelatin was also carried out. In this study, the LOD (% w/w) of porcine gelatin in bovine gelatin was initially determined. The findings revealed that the LOD (% w/w) was 0.01% with a cycle threshold (Ct) value of 39.20±1.72. The next step involved determining the LOD (% w/w) of porcine gelatin in various confectionery products such as lozenges, gummy candy, and marshmallows. The LOD (% w/w) for lozenges and gummy candy was found to be 0.1% with Ct values of 40.93±0.15 and 38.72±0.18, respectively. Marshmallows exhibited an LOD of 0.01% with a Ct value of 41.14±2.96. Finally, this method was applied to commercial confectionery products containing porcine gelatin, and the real-time PCR effectively detected porcine gelatin with high sensitivity.
References
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Sultana S, Hossain MAM, Zaidul ISM, Ali ME. 2018. Multiplex PCR to discriminate bovine, porcine, and fish DNA in gelatin and confectionery products. LWT-Food Sci Technol 92: 169-176. https://doi.org/10.1016/j.lwt.2018.02.019
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Yang CT, Ghosh D, Beaudry F. 2018. Detection of gelatin adulteration using bioinformatics, proteomics and high-resolution mass spectrometry. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 35: 599-608. https://doi.org/10.1080/19440049.2017.1416680
Yayla MEA, Doğan CE. 2021. Development of a new and sensitive method for the detection of pork adulteration in gelatin and other highly processed food products. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 38: 881-891. https://doi.org/10.1080/19440049.2021.1902574
Yustinadewi PD, Yustiantara PS, Narayani I. 2018. Teknik perancangan primer untuk sekuen gen MDR-1 varian 1199 pada sampel buffy coat pasien anak dengan LLA. Metamorfosa: J Biol Sci 5: 105-111. https://doi.org/10.24843/metamorfosa.2018.v05.i01.p16
Aryani D, Saifullah S, Bayu Murti Y. 2015. Pembuatan chewable lozenges ekstrak daun legundi (Vitex trifolia L.) dengan variasi proporsi basis gliserin-gelatin. Trad Med J 20: 98-104.
Arizona K, Laswati DT, Rukmi KSA. 2021. Studi pembuatan marshmallow dengan variasi konsentrasi gelatin dan sukrosa. Agrotech 3: 11-17. https://doi.org/10.37631/agrotech.v3i2.279
Azira TN, Man YBC, Hafidz RNRM, Aina MA, Amin I. 2014. Use of principal component analysis for differentiation of gelatine sources based on polypeptide molecular weights. Food Chem 151: 286-292. https://doi.org/10.1016/j.foodchem.2013.11.066
Bitskinashvili K, Gabriadze I, Kutateladze T, Vishnepolsky B, Mikeladze D, Datukishvili N. 2019. Influence of heat processing on DNA degradation and PCR-based detection of wild-type and transgenic maize. J Food Qual 2019: 5657640. https://doi.org/10.1155/2019/5657640
[BPS] Badan Pusat Statistik. 2020. Statistik Perdagangan Luar Negeri Impor 2020 Jilid I. Badan Pusat Statistik, Jakarta.
[BSN] Badan Standardisasi Nasional. 2021. Analisa biomarker molekuler − Deteksi bahan turunan hewan pada pangan dan bahan pakan menggunakan real-time PCR − Bagian 3: Metode deteksi DNA babi. Badan Standardisasi Nasional, Jakarta.
Brezna B, Piknova L. 2013. Real-time PCR methods for identification of animal or plant species. Caister Academic Press, Burgos. 253-271. https://doi.org/10.21775/9781910190159
Demirhan Y, Ulca P, Senyuva HZ. 2012. Detection of porcine DNA in gelatine and gelatine-containing processed food products−Halal/Kosher authentication. Meat Sci 90: 686-689. https://doi.org/10.1016/j.meatsci.2011.10.014
Global Islamic Economic Report. 2022. State of the global islamic economy report unlocking opportunity. New York: Dinarstandard. https://www. dinarstandard.com/post/state-of-the-global-islamic-economy-report-2022 [20 April 2022].
Guo S, Xu X, Zhou X, Huang Y. 2018. A rapid and simple UPLC-MS/MS method using collagen marker peptides for identification of porcine gelatin. RSC Adv 8: 3768-3773. https://doi.org/10.1039/C7RA12539A
Graboski AM, Galvagni E, Manzoli A, Shimizu FM, Zakrzevski CA, Weschenfelder TA, Steffens J, Steffens C. 2018. Lab-made electronic-nose with polyaniline sensor array used in classification of different aromas in gummy candies. Food Res Int 113: 309-315. https://doi.org/10.1016/j.foodres.2018.07.011
Grand View Research. 2020. Gelatin market size, share & trends analysis report by raw material (pig skin, bovine hide, cattle bones), by function (thickener, stabilizer, gelling agent), by application, by region, and segment forecasts, 2020-2027. California: Grand View Research. https:// www.grandviewresearch.com/press-release/global-gelatin-market [20 April 2021].
Hassan N, Ahmad T, Zain NM. 2018. Chemical and chemometric methods for halal authentication of gelatin: An overview. J Food Sci 83: 2903-2911. https://doi.org/10.1111/1750-3841.14370
Heryani. 2020. Validasi metode analisis DNA babi pada produk pangan dengan primer terseleksi dan exogenous internal positive control pada real-time PCR [Tesis]. Bogor: Institut Pertanian Bogor.
Kamandi N, Dana MG, Ghavami M. 2022. Molecular identification of gelatin origin in pastilles and jelly products collected from tehran markets. J Food Biosci Technol 12: 11-18.
Kang SSN, Lee HG, Kim H. 2018. Development and comparison of a porcine gelatin detection system targeting mitochondrial markers for Halal authentication. LWT-Food Sci Techol 97: 697-702. https://doi.org/10.1016/j.lwt.2018.07.062
Karni M, Zidon D, Polak P, Zelevsky Z, Shefi O. 2013. Thermal degradation of DNA. DNA Cell Biol 32: 298-301. https://doi.org/10.1089/dna.2013.2056
Mano J, Nishitsuji Y, Kikuchi Y, Fukudome S, Hayashida T, Kawakami H, Kurimoto Y, Noguchi A, Kondo K, Teshima R, Takabatake R, Kitta K. 2017. Quantification of DNA fragmentation in processed foods using real-time PCR. Food Chem 226: 149-155. https://doi.org/10.1016/j.foodchem.2017.01.064
Masiri J, Benoit L, Barrios-Lopez B, Thienes C, Meshgi M, Agapov A, Dobritsa A, Nadala C, Samadpour M. 2016. Development and validation of a rapid test system for detection of pork meat and collagen residues. Meat Sci 121: 397-402. https://doi.org/10.1016/j.meatsci.2016.07.006
Mohamad NA, Sheikha AFE, Mustafa S, Mokhtar NFK. 2013. Comparison of gene nature used in real-time PCR for porcine identification and quantification: A review. Food Res Int 50: 330-338. https://doi.org/10.1016/j.foodres.2012.10.047
Nikzad J, Shahhosseini S, Tabarzad M, Nafissi-Varcheh N, Torshabi M. 2017. Simultaneous detection of bovine and porcine DNA in pharmaceutical gelatin capsules by duplex PCR assay for Halal authentication. DARU J Pharm Sci 25: 3. https://doi.org/10.1186/s40199-017-0171-3
Omar S, Hasan M, Abu-Romman S, Ramadan H, Qatatsheh AA, Al-Dmoor H. 2018. Design and validation of short-amplicon length PCR assay for the detection of porcine gelatin in commercial candy and marshmallow products. Curr Res Nutr Food Sci J 6: 742-747. https://doi.org/10.12944/CRNFSJ.6.3.16
Posik DM, Bustamante AV, Lyall V, Peral GP, Padola NL, Giovambattista G. 2016. Species identification of a suspected bone found in blood sausage. Forensic Sci Criminol 1: 1-2. https://doi.org/10.15761/FSC.1000103
Salamah N, Erwanto Y, Martono S, Rohman A. 2021. The employment of real-time polymerase chain reaction using species-specific primer targeting on D-loop mitochondria for identification of porcine gelatin in soft candy. Indones J Chem 21: 852-859. https://doi.org/10.22146/ijc.60413
Stahl-Zeng J, Sage A, Taylor P, Netto JD, Zhang T. 2019. Advances in LC−MS/MS methods for allergen testing, meat speciation, and gelatin speciation. J AOAC Int 102: 1309-1315. https://doi.org/10.5740/jaoacint.19-0059
Sultana S, Hossain MAM, Azlan A, Johan MR, Chowdhury ZZ, Ali ME. 2020. TaqMan probe based multiplex quantitative PCR assay for determination of bovine, porcine and fish DNA in gelatin admixture, food products and dietary supplements. Food Chem 325: 126756. https://doi.org/10.1016/j.foodchem.2020.126756
Sultana S, Hossain MAM, Zaidul ISM, Ali ME. 2018. Multiplex PCR to discriminate bovine, porcine, and fish DNA in gelatin and confectionery products. LWT-Food Sci Technol 92: 169-176. https://doi.org/10.1016/j.lwt.2018.02.019
Supiah K. 2018. Preparation and processing of religious and cultural foods. Woodhead Publishing Series in Food Science, Technology and Nutrition, Cambridge. 310-321. https://doi.org/10. 1016/B978-0-08-101892-7.00016-X
Wardani DP, Arifin M, Abraha K. 2019. The revised method of quantitative detection of animal-origin bovine and porcine gelatin difference using surface plasmon resonance-based biosensor. Mater Sci Forum 948: 146-152. https://doi.org/10.4028/www.scientific.net/MSF.948.146
Yang CT, Ghosh D, Beaudry F. 2018. Detection of gelatin adulteration using bioinformatics, proteomics and high-resolution mass spectrometry. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 35: 599-608. https://doi.org/10.1080/19440049.2017.1416680
Yayla MEA, Doğan CE. 2021. Development of a new and sensitive method for the detection of pork adulteration in gelatin and other highly processed food products. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 38: 881-891. https://doi.org/10.1080/19440049.2021.1902574
Yustinadewi PD, Yustiantara PS, Narayani I. 2018. Teknik perancangan primer untuk sekuen gen MDR-1 varian 1199 pada sampel buffy coat pasien anak dengan LLA. Metamorfosa: J Biol Sci 5: 105-111. https://doi.org/10.24843/metamorfosa.2018.v05.i01.p16
Authors
HibaturrahmanS. N., KusnandarF., YulianaN. D., & HeryaniH. (2023). Sensitivitas Real-Time Polymerase Chain Reaction dengan Primer Tanabe dalam Mendeteksi Gelatin Babi pada Confectionery. Jurnal Teknologi Dan Industri Pangan, 34(1), 119-126. https://doi.org/10.6066/jtip.2023.34.1.119
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