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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
Universitas Gadjah Mada, 2017 | Diunduh dari http://etd.repository.ugm.ac.id/
KAJIAN METODE FISIKA-KIMIA DAN BIOLOGI MOLEKULER

UNTUK ANALISIS DAGING CELENG DALAM PRODUK MAKANAN
DISERTASI
Untuk memenuhi sebagian persyaratan

mencapai derajat Doktor (Dr.)
Diajukan Oleh:
ANY GUNTARTI
14/373785/SFA/101
Kepada
PROGRAM PASCASARJANA
PROGRAM STUDI ILMU FARMASI
FAKULTAS FARMASI
UNIVERSITAS GADJAH MADA
YOGYAKARTA
2017
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°C 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°C, 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 (t R) 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 (°C), onset (°C),
offset (°C), entalpi (J/g), dan range (ᵒC). 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°C dengan batas
deteksi sebesar 48 pg/µL. 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°C 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°C, 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 (t R) 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 (°C), onset (°C), offset (°C), enthalpy (J/g), and range (ᵒC). 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 non-
saturated 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.
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DAFTAR ISI
HALAMAN JUDUL i
HALAMAN PENGESAHAN ii
HALAMAN PERNYATAAN iii
KATA PENGANTAR iv
MOTTO dan PERSEMBAHAN v
DAFTAR ISI vi
DAFTAR TABEL X
DAFTAR GAMBAR xii
DAFTAR LAMPIRAN xv
DAFTAR SINGKATAN xvii
ABSTRAK xviii
ABSTRACT xx
BAB I PENDAHULUAN
A. Latar Belakang masalah 1
1. Perumusan masalah 5
2. Keaslian dan Keterbaruan Penelitian 6
3. Manfaat Penelitian 6
4. Urgensi Penelitian 8
B. Tujuan Penelitian 9
BAB II TINJAUAN PUSTAKA 10
A. Kajian Pustaka 10
1. Halal dan Analisis Komponen non-Halal dalam produk 10
2. Babi Domestika (Babi Ternak) dan Babi Hutan (Celeng) 12
3. Hewan Sapi 14
4. Lemak dan minyak 17
5. Gas Chromatography-Mass Spectrometry (GC-MS) 21
6. Spektrofotometri Fourier transform infrared (FTIR) 24
7. Differential Scanning Calorimetry (DSC) 29
8. Polymerase chain reaction (PCR) 32
9. Bakso 37
10. Kemometrika 38
B. Landasan Teori 40
C. Hipotesis 45
BAB III METODE PENELITIAN 46
A. Alat dan Bahan 46
Bahan 46
Alat 47
B. Jalannya Penelitian
1. Analisis daging celeng dengan metode GC-MS 48
a. Ekstraksi lemak 48
b. Esterifikasi 48
c. Analisis dengan GC-MS 49
d. Analisis data 49
2. Analisis daging celeng dalam bakso sapi dengan 50
spektrofotometri FTIR
a. Penyiapan sampel 49
b. Ekstraksi lemak 51
c. Analisis instrumentasi 51
d. Analisis data 52
3. Analisis dengan Differential Scanning Calorimetry (DSC) 53
a. Penyiapan sampel bakso referensi 53
b. Analisis instrumen dengan DSC 53
c. Analisis Data 54
4. Analisis daging celeng dalam bakso sapi dengan real-time 55
PCR
a. Isolasi DNA 54
b. Desain primer dengan BLAST 56
c. Penyiapan sampel 57
d. Optimasi suhu penempelan primer dengan real-time 58
PCR
e. Identifikasi DNA celeng pada bakso sapi referensi 59
f. Uji specifisitas primer terhadap DNA celeng 59
g. Uji batas deteksi 59
h. Uji linieritas metode 59
i. Uji keterulangan (repeatability) 60
j. Uji daging celeng dalam bakso pasaran 60
k. Analisis data real-time PCR 60
BAB IV. HASIL DAN PEMBAHASAN 62
A. Analisis profil asam lemak celeng dengan Gas 62
Chromatography-Mass spectrometry (GC-MS)
1. Ekstraksi Lemak 62
2. Komposisi Asam lemak standar dan asam lemak celeng 64
3. Perbandingan komponen lemak celeng, babi, ayam, sapi dan 70
kambing.
4. Principal Component Analysis (PCA) asam lemak metil 75
ester .
B. Analisis lemak celeng dalam bakso sapi secara 80
spektrofotometri FTIR
1. Ekstraksi lemak dengan Soxhlet 80 80
2. Analisis spektra FTIR 83
3. Optimasi bilangan gelombang untuk model kalibrasi PLS 88
4. Analisis kuantitatif dengan kalibrasi Partial Least Square 90
(PLS)
5. Pengelompokkan dengan Principal Component Analysis 94
(PCA)
C. Analisis pemalsuan daging celeng dengan Differential 98
Scanning Calorimetry (DCS)
1. Karakterisasi Termal Lemak Celeng dan Lemak Sapi 99
2. Karakterisasi Termal Lemak Celeng dan lemak hewani lain. 104
3. Profil lemak celeng dan lemak sapi beserta campurannya 108
4. Analisis kuantitatif lemak celeng dan lemak sapi beserta 117
campurannya dengan Partial Least Square (PLS).
5. Pengelompokkan lemak celeng dan lemak hewani lainnya 121
dengan Principle Component Analysis (PCA).
D. Analisis DNA celeng dengan Real-Time Polymerase Chain 129
Reaction (real-time PCR)
1. Isolasi DNA dalam daging segar dan dalam bakso referensi 130
2. Desain Primer 132
3. Optimasi suhu penempelan primer 134
4. Identifikasi DNA celeng pada bakso sapi referensi campuran 138
celeng:sapi
5. Uji batas deteksi DNA bakso celeng referensi 100%. 138
6. Uji linieritas 140
7. Uji keterulangan (repeatability) 143
8. Uji spesifisitas primer celeng dari Kalimantan Selatan 144
9. Uji spesifisitas primer terhadap DNA celeng dari Timor 147
Leste
10. Identifikasi DNA Celeng Pada Bakso Pasaran 149
BAB V. KESIMPULAN DAN SARAN 153
A. Kesimpulan 153
B. Saran 155
DAFTAR PUSTAKA 156

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KAJIAN METODE FISIKA-KIMIA DAN BIOLOGI MOLEKULER UNTUK ANALISIS DAGING CELENG

DALAM PRODUK MAKANAN

KAJIAN METODE FISIKA-KIMIA DAN BIOLOGI MOLEKULER

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