Aplikasi Metode Analisis Derivat Babi Berbasis Protein, DNA dan Metabolit untuk Autentikasi Halal

SITI RAHMATUL AINI, Prof. Dr. apt, Abdul Rohman, M.Si.; Prof. Mulyanto; Prof. Ir. Yuny Erwanto, S.Pt., MP., Ph.D., IPM.

2025 | Disertasi | S3 Ilmu Farmasi

Seiring perkembangan ilmu pengetahuan dan teknologi, serta didorong faktor ekonomi, sering ditemukan penambahan daging non halal ke dalam daging halal. Kondisi ini menyebabkan kerugian, baik material maupun spiritual, mengingat adanya larangan bagi umat Islam untuk mengonsumsi produk makanan yang mengandung daging non halal. Penelitian ini bertujuan untuk mengembangkan metode deteksi daging non halal dengan metode imunokromatografi, Real-time Polymerase Chain Reaction (Real-time PCR) dan menentukan metabolit potensial dalam identifikasi daging sapi, daging babi, dan daging celeng menggunakan teknik untargeted metabolomic LC-HRMS dan kemometrika. Pada metode imunokromatografi, dikembangkan strip uji berisi anti-Swine IgG poliklonal antibodi berlabel koloid emas yang secara visual mendeteksi daging babi dalam campuran daging halal hingga konsentrasi 1/5000 (b/b) dalam waktu 15 menit. Pengujian spesifisitas menunjukkan hasil positif pada 30 sampel daging babi, hasil negatif pada 30 sampel daging ayam, 30 sampel daging kambing, dan 24 sampel daging sapi.  Pada metode Real-time PCR, primer ND5 (forward: TCGCCTCACTCACATTAACC, reverse: GGGACTAGGCTGAGAGTGAA) mengamplifikasi DNA daging celeng pada suhu optimal 59°C dengan sensitivitas 5 pg/µL pada daging celeng (Efisiensi 109.9%, slope 3,106 dan R2 0,968) dan 781,25 pg/ µL pada bakso celeng 100% referensi (Efisiensi 96%, slope 3,421 dan R2 0,96), serta mampu mendeteksi campuran celeng pada bakso sampai dengan konsentrasi 3% (Efisiensi 83.2%, slope 3,805 dan R2 0,989). Uji keterulangan pada 100?kso celeng menunjukkan nilai koefisien variasi (CV) = 2,37%. Primer ND5 tidak mengamplifikasi DNA yang diekstraksi dari bakso pasaran di wilayah Yogyakarta. Selain DNA celeng, primer ND5 juga mengamplifikasi DNA babi. Konfirmasi hasil strip uji imunokromatografi pada ayam, kambing, sapi dan babi dilakukan dengan metode Real-time PCR yang dikembangkan dan hanya DNA babi yang teramplifikasi dengan primer ND5. Analisis metabolit secara kemometrika dengan Principal Component Analysis (PCA) dan Partial Least Square-Discriminant Analysis (PLS-DA) menunjukkan bahwa asetilkolin, DL-carnitine, C18- carnitin, dan asetil-L carnitin d tinggi memiliki kelimpahan yang tinggi pada sapi, nikotinamid, D-pantothenic acid dan L-phenylalanin yang lebih tinggi pada babi dan untuk celeng terdapat metabolit dalam intensitas intermediat seperti prolil-leucine. Sebagai kesimpulan, telah dikembangkan strip uji imunokromatografi untuk mendeteksi kandungan babi dalam waktu 15 menit pada campuran daging dengan limit deteksi 1/5000 (b/b). Primer ND5 mampu mengamplifikasi DNA daging babi dan daging celeng.  Analisis metabolomik yang dikombinasi dengan kemometrika mampu mengidentifikasi metabolit diskriminatif pada daging babi, celeng dan sapi. 

With the advancement of science and technology, and driven by economic factors, the addition of non-halal meat to halal meat is often found. This condition causes losses, both material and spiritual, considering the prohibition for Muslims to consume food products that contain non-halal meat. This research aims to develop a method for detecting non-halal meat using immunochromatography, Real-time Polymerase Chain Reaction (Real-time PCR), and to identify potential metabolites in the identification of beef, pork, and wild boar meat using untargeted metabolomic LC-HRMS techniques and chemometrics. In the immunochromatography method, a test strip containing anti-Swine IgG polyclonal antibodies labeled with colloidal gold was developed, which visually detects pork in halal meat mixtures up to a concentration of 1/5000 (w/w) within 15 minutes. Specificity testing showed positive results in 30 samples of pork, negative results in 30 samples of chicken, 30 samples of goat meat, and 24 samples of beef. In the Real-time PCR method, the ND5 primer (forward: TCGCCTCACTCACATTAACC, reverse: GGGACTAGGCTGAGAGTGAA) amplifies wild boar meat DNA at an optimal temperature of 59°C with a sensitivity of 5 pg/µL for wild boar meat (Efficiency 109.9%, slope 3.106, and R2 0.968) and 781.25 pg/µL for 100% reference wild boar meat meatballs (Efficiency 96%, slope 3.421, and R2 0.96), and is capable of detecting wild boar mixtures in meatballs up to a concentration of 3% (Efficiency 83.2%, slope 3.805, and R2 0.989). The repeatability test on 100% wild boar meatballs showed a coefficient of variation (CV) value of 2.37%. The ND5 primer does not amplify DNA extracted from market meatballs in the Yogyakarta area. Besides wild boar DNA, the ND5 primer also amplifies pig DNA..Confirmation of the immunochromatographic strip test results on chickens, goats, cattle, and pigs was performed using the developed Real-time PCR method, and only pig DNA was amplified with the ND5 primer. Metabolite analysis using chemometrics with Principal Component Analysis (PCA) and Partial Least Square-Discriminant Analysis (PLS-DA) shows that acetylcholine, DL-carnitine, C18-carnitine, and acetyl-L-carnitine have high abundance in cattle, nicotinamide, D-pantothenic acid, and L-phenylalanine are higher in pigs, and for wild boar, there are metabolites with intermediate intensity such as prolyl-leucine. In conclusion, an immunochromatographic test strip has been developed to detect pig content within 15 minutes in meat mixtures with a detection limit of 1/5000 (w/w). The ND5 primer is capable of amplifying the DNA of pork and wild boar meat. Metabolomic analysis combined with chemometrics is capable of identifying discriminative metabolites in pork, wild boar, and beef.

Kata Kunci : daging babi, daging celeng, immunochromatography, Real-time PCR, metabolomik, kemometrika, autentikasi halal