KAJIAN METODE FISIKA-KIMIA DAN BIOLOGI MOLEKULER UNTUK ANALISIS DAGING CELENG DALAM PRODUK MAKANAN UNTUK AUTENTIKASI HALAL

ANY GUNTARTI, Prof.Dr. Abdul Rohman, MSi., Apt

2017 | Disertasi | S3 Ilmu Farmasi

ABSTRAK Indonesia adalah negara dengan komunitas muslim terbesar di dunia. Seiring dengan kesadaran untuk mengkonsumsi makanan halal, pasar makanan halal di dunia meningkat secara signifikan. Celeng merupakan jenis babi hutan dan daging celeng sering disalahgunakan dalam perdagangan. Penelitian ini bertujuan melakukan analisis daging celeng dengan menggunakan metode Gas Chromatography-Mass Spectrometry (GC-MS), Spektrofotometri Fourier Transfor Infrared (FTIR), Differential Scanning Calorimetry (DSC) dan Real-Time Polymerase Chain Reaction (real-time PCR). Sampel utama adalah daging celeng dari Kalimantan Tengah. Pada analisis secara GC-MS, sampel yang digunakan berupa lemak daging: celeng, babi, ayam, sapi, dan kambing. Lemak diambil dengan cara rendering dalam oven pada suhu 90-100 celcius selama 1-1,5 jam. Lemak yang diperoleh dilakukan proses esterifikasi dengan menggunakan NaOCH3 dan BF3 untuk mendapatkan senyawa derivat asam lemak metil ester. Pada analisis secara spektrofotometri FTIR, sampel lemak diekstraksi dari bakso referensi (daging celeng, daging sapi dan campurannya) dan produk bakso di pasaran. Lemak diambil secara Soxhlet pada suhu 69- 70 celcius, selama 5-6 jam dengan pelarut n-heksan. Pada analisis secara DSC, sampel lemak yang digunakan diperoleh dengan ekstraksi Soxhlet dari bakso referensi (daging celeng, daging sapi dan campurannya), daging ayam, babi, kambing, daging kelinci, serta bakso produk pasaran. Pada metode real-time PCR, DNA diekstraksi dari sampel bakso referensi (mengandung daging celeng, daging sapi dan campurannya), berbagai jenis daging: babi, ayam, kambing, celeng dari Timor Leste, dan bakso produk di pasaran dari Yogyakarta dan Kalimantan Tengah. Hasil analisis dengan metode GC-MS berupa kromatogram dan spektra. Kromatogram asam lemak dari sampel lemak yang diperoleh dibandingkan dengan standar fatty acid methyl ester (FAME). Parameter yang digunakan adalah waktu retensi (tR) dan % area. Sementara itu, spektra yang dihasilkan dari spektrometri massa dibandingkan dengan referensi yang ada di dalam data base instrumen. Parameter yang digunakan dalam spektra massa adalah Similarity index (SI) dan Berat molekul (BM). Pada metode spektrofotometri FTIR, data yang diperoleh berupa spektra dengan parameter serapan dan bilangan gelombang (cm-1). Pada analisis dengan metode DSC, data yang dihasilkan berupa profil kurva kristalisasi dan kurva pelelehan lemak. Parameter yang digunakan adalah suhu transisi termal (celcius), onset (celsius), offset (celsius), entalpi (J/g), dan range (celsius). Pada metode real-time PCR, data yang diambil adalah kurva amplifikasi dan kurva puncak denaturasi. Parameter yang digunakan adalah nilai Relative Fluorescent Unit (RFU) dan Threshold cycle (Ct). Hasil penelitian secara GC-MS diperoleh bahwa lemak celeng mengandung asam: (1) laurat (C12:0), miristat (C14:0), palmitoleat (C16:1), palmitat (C16:0), margarat (C17:0), linoleat (C18:2), oleat (C18:1), dan stearat (C18:0). Kandungan asam lemak tidak jenuh pada celeng yang tertinggi yaitu asam oleat 45,43%. Asam lemak jenuh tertinggi adalah asam palmitat, sebesar 19,61%. Kemometrika Principal Component Analysis (PCA) dapat mengelompokan jenis asam lemak: celeng, sapi, ayam, babi, kambing menggunakan variabel komposisi asam lemak. Metode Spektrofotometri FTIR yang dikombinasikan dengan kalibrasi partial least square (PLS) pada daerah bilangan gelombang 1250-900 cm-1 dapat digunakan untuk analisis kuantitatif lemak celeng dalam bakso sapi dengan validitas metode: nilai R2 0,9884, nilai root mean square error of calibration (RMSEC) 1,98%, nilai root mean square error prediction (RMSEP) 1,48% dan nilai root mean square error cross calibration (RMSECV) 1,39%. Spektra FTIR dengan PCA pada bilangan gelombang 1250-900 cm-1 dapat mengelompokkan lemak celeng dan lemak sapi pada produk bakso di pasaran. Lemak celeng dari Kalimantan Tengah memiliki karakteristik profil termal dan parameter fisika yang berbeda-beda, sebagaimana dianalisis dengan DSC. Analisis multivariat PCA dapat mengelompokkan karakteristik termal lemak: celeng, babi, sapi, ayam, dan kambing. Sementara itu, kalibrasi multivariat PLS dengan menggunakan variabel kristalisasi memberikan nilai R2 99,75%; nilai RMSEP= 0,22%; serta nilai RMSECV= 0,12%. Profil pelelehan memberikan nilai R2 98,90%, nilai RMSEP= 2,93% serta nilai RMSECV= 0,34%. Primer CytbAG3A, forward:5'-AGGCCGGGGCCTATATTA-3' dan CytbAG3A reverse :5'-TCTACGAGGTCTGTTCCGAT-3' telah dirancang dengan situs (website) NCBI untuk analisis DNA daging celeng. Optimasi suhu penempelan diperoleh 59 celsius dengan batas deteksi sebesar 48 pg/mikro liter. Pada uji linieritas, dengan bakso celeng 100% diperoleh nilai E = 105,7%, nilai R2 = 0,999, sementara itu uji linieritas sampel referensi bakso celeng-sapi diperoleh nilai E = 102,5%, nilai R2 = 0,945. Uji repeatability dengan 100% bakso celeng diperoleh nilai CV 10,02%, dan nilai CV sebesar 5,80% untuk sampel referensi bakso celeng 10%. Primer CytbAG3A dapat mengamplifikasi DNA celeng, sedangkan untuk DNA: sapi, babi, ayam, dan kambing tidak teramplifikasi. Primer CytbAG3A tidak mengamplifikan DNA bakso pasaran dari Yogyakarta, Kalimantan Tengah, dan Timor Leste. Sehingga, primer CytbAG3A specifik untuk celeng dari Kalimantan Tengah. Kata kunci: daging celeng, spektrofotometri FTIR, DSC, GC-MS, real time-PCR, autentikasi halal.

ABSTRACT Indonesia is the country with the biggest moslem community in the world. Along with the awarness to consume halal food, halal food market is increasing significantly in the world. One of pigs that not livestocked is wild boar meat (WBM) and is usually as adulteration in the meatballs. The purpose of this research is to use physio-chemical and biology molecular methods to identify and to quantify WBM in meatballs namely Gas Chromatography-Mass Spectrometry (GC-MS), Fourier Transfor Infrared (FTIR) spectrophotometry, Differential Scanning Calorimetry (DSC) and Real-Time Polymerase Chain Reaction (RTPCR). The main sample was wild boar meat from Central Kalimantan. For GC-MS analysis, the samples used was lipid taken from: wild boar, porcine, chicken, cow, and goat. Lipids are taken by rendering in convential oven at temperature of 90-100 celcius for 1-1.5 hours. Fat obtained was subjected to esterification process using NaOCH3 and BF3 to obtain fatty acid methyl ester (FAME) derivatives. During analysis using FTIR spectrophotometry, samples of fat from reference meatballs (wild boar meat, beef and mixtures thereof) and meatball products available in the market are obtained from Soxhlet extraction at temperature of 69- 70 celcius, for 5-6 hours. DSC analysis were used to analyze fats extracted from reference meatballs (containing of WBM, beef and mixtures thereof with known concentration of WBM), chickens, pigs, goats, rabbits, and commercially meatball products. In real-time PCR method, DNA was extracted from reference meatballs (wild boar meat, beef and mixtures thereof), and some kind of meat, namely pork, chicken, goat, wild boar from Timor Leste, and meatball products available on the market around Yogyakarta and Central Kalimantan. During analysis using GC-MS, the chromatograms of fatty acid methyl esters (FAMEs) in samples are compared with those obtained using standard FAME from Supelco. The parameters used were retention time (tR) and % area. Meanwhile mass spectra resulted from mass spectrometry were compared with those in the data base available in the instrument. The parameters used in mass spectra were similarity index (SI) and molecular weight (Mw). Using FTIR spectrophotometry method, the data obtained were FTIR spectra with main parameters of wavenumbers (cm-1) and absorbances. Data obtained using DSC method were the curves of crystallization and melting of evaluated fats. The parameters used were thermal transition temperature (celcius), onset (celcius), offset (celcius), enthalpy (J/g), and range (celcius). Data of the amplification curve and peak denaturation curve, Relative Fluorecent Unit (RFU) and Threshold cycle (Ct) were used for analysis using real-time PCR. The result of this research showed that: using GS-CM method, (1) wild boar fat contains lauric (C12:0), miristic (14:0), palmitoleic (C16:1), palmitic (C16:0), margaric (C17:0), linoleic (C18:2), oleic (C18:1), and stearic (C18:0) acids. The highest contentration of nonsaturated fatty acid was oleic (45.43%), while the palmitic acid (19.61%) was saturated fatty acid dominates. The chemometrics of principal component analysis (PCA) was successfully used for classification of meat fats based on fatty acid composition as variables. FTIR spectrophotometry method combined with multivariate calibration of Partial Least Square (PLS) in frequency regions of 1250 - 900 cm-1 could classify wild boar fat and cow fat on the commercial meatballs. DSC analysis revealed that wild boar fat from Central Kalimantan had several physio-chemical characteristics, namely fatty acid composition, volatile components, and different thermal profiles that were affected by the origin of wild boar, preparation and sampel treatment, wild boar age, and storage methods. DSC parameters combined with PLS could be used for predicting WBM in meatball with coeffisient of determination R2 value of 99.75%; root mean square error of prediction (RMSEP) of 0.22%; and root mean square error of cross validation (RMSECV) value of 0.12% for the relationship between actual value of WBM and DSC predicted value using crystallization profiles. Meanwhile, using melting profile the R2 of 98.90%; RMSEP value of 2.93%; and RMSECV value of 0.34% were obtained. Primer CytbAG3A forward: 5'-AGGCCGGGGCCTATATTA-3' and CytbAG3A reverse: 5'- TCTACGAGGTCTGTTCCGAT-3' revealed annealing temperature of 59oC with limit of detection of 48 pg. The linearity test using wild boar meatball 100% obtained efficiency value of 105.7% with R2 = 0.999. Repeatibility test with 100% wild boar meatball shows CV value of 10.02%, and using reference meatballs, CV value obtained is 5.80%. Primer CytbAG3A amplified wild boar DNA, while DNAs for cow, porcine, chicken, and goat were not amplified. Primer CytbAG3A did not amplifiy DNA extracted from commercial meatballs obtained from Yogyakarta, Central Kalimantan and Timor Leste. Therefore, the primer was specific to wild boar from Central Kalimantan. Keywords: wild boar meat, FTIR spectrophotometry, DSC, GC-MS, real-time PCR, authentication.

Kata Kunci : daging celeng, spektrofotometri FTIR, DSC, GC-MS, real time-PCR, autentikasi halal