Analysis of Lard in Meatball Broth Using Fourier Transform Infrared Spectroscopy and Chemometrics

Analysis of lard in meatball broth using Fourier transform infrared spectroscopyand chemometrics Analisis lemak babi dalam kuah bakso menggunakan spektroskopi Fourier transform infrared (FTIR) dan kemometrika Endah Kurniawati a,b, Abdul Rohman a,b,c,d , Kuwat Triyana a,d a Research Center of Halal Products, Gadjah Mada University, Yogyakarta 55281, Indonesia b Faculty of Pharmacy, Gadjah Mada University, Yogyakarta 55281, Indonesia c Centre of Research for Fiqh Science and Technology (CFiRST), Universiti Teknologi Malaysia, Malaysia d Department of Physics, Faculty of Mathematics and Natural Sciences, Gadjah Mada University, Yogyakarta 55281, Indonesia abstract Meatball is one of the favorite foods in Indonesia. For the economic reason (due to the price difference), the substitution of beef meat with pork can occur. In this study, FTIR spectroscopy in combination with chemometrics of partial least square (PLS) and principal component analysis (PCA) was used for analysis of pork fat (lard) in meatball broth. Lard in meatball broth was quantitatively determined at wavenumber region of 1018–1284 cm-1. The coefficient of determination (R2) and root mean square error of calibration (RMSEC) values obtained were 0.9975 and 1.34% (v/v), respectively. Furthermore, the classification of lard and beef fat in meatball broth as well as in commercial samples was performed at wavenumber region of 1200–1000 cm-1. The results showed that FTIR spectroscopy coupled with chemometrics can be used for quantitative analysis and classification of lard in meatball broth for Halal verification studies. The developed method is simple in operation, rapid and not involving extensive sample preparation. Bakso merupakan salah satu makanan favorit di Indonesia. Untuk alasan ekonomi (karena perbedaan harga), substitusi daging sapi dengan daging babi dapat terjadi. Dalam penelitian ini, spektroskopi FTIR dikombinasikan dengan kemometrika dari kuadrat terkecil parsial atau partial least square (PLS) dan analisis komponen utama atau principal component analysis (PCA) digunakan untuk analisis lemak babi (lard) dalam kuah bakso. Lemak babi dalam kuah bakso secara kuantitatif ditentukan pada daerah bilangan gelombang 1018-1284 cm -1. Koefisien determinasi (R2) dan nilai akar kuadrat tengah atau root mean square error kalibrasi (RMSE) yang diperoleh masing-masing adalah 0,9975 dan 1,34% (v / v). Selanjutnya, klasifikasi lemak babi dan lemak sapi dalam kuah bakso serta dalam sampel komersial dilakukan pada daerah bilangan gelombang 1200-1000 cm -1. Hasil penelitian menunjukkan bahwa spektroskopi FTIR dipasangkan dengan kemometrika dapat digunakan untuk analisis kuantitatif dan klasifikasi lemak babi dalam kuah bakso untuk studi verifikasi Halal. Metode yang dikembangkan sederhana dalam pengoperasian, cepat dan tidak memerlukan persiapan sampel yang luas. 1. Introduction Transparency in meat speciation used in food products is an ever increasing demand and is essential for the protection of consumers' right, religious credence and hard-earned fortunes (Ali, Hashim, Mustafa, Che Man, Dhadi et al., 2012; Doosti, Ghasemi Dehkordi, & Rahimi, 2011; Fajardo, González, Rojas, García, & Martín, 2010). Verification of declared components in food products is also necessary for the prevention of adulteration practice. For this purpose, some countries make regulation for assuring that food products available are safe and authentic. Therefore, detection of species fraud in meat products including meatball is important for consumer protection and food industries (Doosti et al., 2011). Saat ini. It can be prepared using beef.Transparansi dalam spesiasi daging yang digunakan dalam produk makanan adalah kebutuhan yang semakin meningkat dan sangat penting untuk melindungi hak konsumen. 2011). Erwanto. García. González. beberapa negara membuat peraturan untuk menjamin bahwa produk makanan yang tersedia aman dan otentik. atau daging ikan. terutama di Asia Timur. Currently. Chaudry. electronic nose and gas chromatography–mass spec-trometry by detection of the aroma and volatile compounds of lard present in meatball (Nurjuliana. & Che Man. et al. Verifikasi halal membutuhkan metode yang dapat diandalkan yang memastikan bahwa barang-barang non-halal seperti daging babi tidak ada dalam produk makanan (Nakyinsige. Che Man. & Sazili. Hashim. Muslim and Jew communities are not allowed to consume food products containing pig derivatives like pork. Hashim. Rohman & Che Man. babi tidak halal dan tidak kosher (Regenstein. Rohman & Che Man. Hasyim. Analysis of pork in meatball and other food products can be performed by DNA amplification present in pork using polymerase chain reaction using different targets of amplification (Ali. atau dikenal sebagai 'bakso' (Rohman. Mustafa. Che Man. Dhahi. proposed and used for analysis of pork in food products (Mursyidi. Hashim. in Indonesia. pork. Mustafa. kepercayaan agama dan keberuntungan jerih payah (Ali. Verifikasi komponen yang dinyatakan dalam produk makanan juga diperlukan untuk pencegahan praktik pemalsuan. 2011). Meatball is a processed comminuted meat which can be classified as restructured meat. 2003. Sismindary. Sismindary. Akibatnya. Bakso adalah olahan daging yang dihaluskan yang dapat diklasifikasikan sebagai daging yang direstrukturisasi. 2012. 2012). Di Indonesia. Dosti. Penggantian daging sapi dengan daging babi adalah masalah serius tidak hanya untuk alasan ekonomi tetapi juga untuk sudut pandang agama. 2012. & Regenstein. Dalam Islam dan Yahudi. Dhadi et al. Che Man. Halal verification needs a reliable method assuring that non-halal items like pork are absent in food products (Nakyinsige. 2012). unscrupulous seller replaces beef meatball with pork meatball in order to gain economical profits.. Ali. Oleh karena itu.. diusulkan dan digunakan untuk analisis daging babi dalam produk makanan (Mursyidi. Kadang-kadang. In the Middle East and other Islamic countries. & Sazili. Hal ini dapat disusun dengan menggunakan daging sapi. due to the high different prices between pork and beef.. Chaudry. 2013). di Indonesia. the adulteration practice of beef meatball with pork meatball is occurring. In Islamic and Jews. deteksi penipuan spesies dalam produk daging termasuk bakso penting bagi perlindungan konsumen dan industri makanan (Doosti et al. & Che Man. Erwanto. especially in East Asia. Di Timur Tengah dan negara-negara Islam lainnya. & Rahimi. Che . 2008). chicken. Dhahi. 2003. karena perbedaan harga yang tinggi antara daging babi dan sapi. 2010). 2013). 2008). Mustafa. Fajardo. In Indonesia. Ali. Ghasemi Dehkordi. & Che Man. Sometimes. ayam. 2011. As a consequence. 2012). salah satu makanan favorit yang dikonsumsi adalah meatball. Che Man. Untuk tujuan ini. Rojas. & Regenstein. et al. 2012). 2012. 2012). dan salah satu yang sangat populer dan banyak ditemukan di pasar Indonesia adalah bakso sapi (Purnomo & Rahardyan. praktek pemalsuan bakso daging sapi dengan bakso daging babi terjadi. The substitution of beef with pork is a serious problem not only for economic reason but also for religious point of view. babi. penjual yang tidak bermoral menggantikan bakso daging sapi dengan bakso daging babi untuk mendapatkan keuntungan ekonomis.. some analytical methods have been developed. & Martín. or known as ‘bakso’ (Rohman. sertifikasi halal telah diwajibkan untuk semua produk makanan daging dan daging berbasis impor seperti bakso. Komunitas Muslim dan Yahudi tidak diperbolehkan untuk mengkonsumsi produk makanan yang mengandung turunan babi seperti daging babi. beberapa metode analisis telah dikembangkan. and the one that is very popular and widely found in the Indonesian market is beef meatball (Purnomo & Rahardiyan. halal certification has been made mandatory for all meat and meat based imported food products like meatball. pork is not halal and not kosher (Regenstein. one of the favorite foods consumed is meatball. or fish meat. 2011). FTIR spectroscopy has been exploited for analysis of lard in the binary mixture with other animal fats with the aid of multivariate calibration in combination with discriminant analysis (Che Man & Mirghani. The adipose tissues of pig and cattle were cut into small pieces using commercial cutter in order to effectively extract lard and beef fat. Due to the different components that may be present in lard extracted from meatball broth and lard extracted from pork fat in meatball. Che Man. & Bakar. and Fourier transform infrared (FTIR) spectroscopy by analyzing pork fat or lard as a whole of matter extracted from pork (Rohman. 2005). Hasyim. Syahariza. Jinap. Selamat. Jaswir. Preparation of animal fats The pork fat (lard) and beef fat were obtained by rendering process of corresponding animal. Sismindary. FTIR spectroscopy coupled with chemometrics is promising analytical techniques to be used in the halal verification studies. Spektroskopi FTIR dipasangkan dengan kemometrika menjanjikan teknik analisis yang digunakan dalam penelitian verifikasi halal. kami mengembangkan spektroskopi FTIR yang dikombinasi dengan partial least square dan analisis komponen utama untuk kuantifikasi dan klasifikasi lemak babi dalam kuah bakso daging sapi. Hal ini cepat. 2001.Man. 2012. dan spektroskopi Fourier transform inframerah (FTIR) dengan menganalisis lemak babi atau lard secara keseluruhan dari materi yang diekstrak dari daging babi (Rohman. & Bakar. Hassan. the cut tissue was introduced into conventional oven during 3 h at 100 °C. Karena komponen yang berbeda yang mungkin ada dalam lemak babi yang diekstrak dari kuah bakso dan lemak babi yang diekstrak dari lemak babi dalam bakso. we develop FTIR spectroscopy in combination with partial least square and principal component analysis for quantification and classification of lard in broth of beef meatball. indeed. Syahariza. Selamat. 2011). 2011). Mirghani. Mustafa. Dhahi.1. Using Beaker glass. Syahariza. In this study. tidak merusak dan tidak melibatkan persiapan sampel yang melelahkan. The procedure of rendering follows with that reported in Rohman and Che Man (2009). spektroskopi FTIR telah dimanfaatkan untuk analisis lemak babi dalam campuran biner dengan lemak hewan lain dengan bantuan kalibrasi multivariat yang dikombinasi dengan analisis diskriminan (Che Man & Mirghani. Che Man. The melted fat was strained with filter . Mirghani. . Jaswir. kami mengekstrak lemak babi dari lemak babi yang terkandung dalam bakso di mana beberapa komponen lain mungkin ada dalam diekstrak lemak babi. Mustafa. In that research. 2011). It is fast. In halal authentication. Che Man. Sebelumnya. Mirghani. Mat Hashim. & Che Man. oleh karenanya model kalibrasi harus dikembangkan.. 2005. 2003). et al. Previously. Hasyim. et al. Jinap. serta untuk analisis lemak babi dalam formulasi kue dan cokelat menggunakan kalibrasi partial least square (Che Man. et al. 2012). & Mohd Said.. et al. Che Man. Hassan. Sismindary.. Dhahi. & Mohamed. Syahariza. Dalam studi ini. Mat Hashim. 2011).. 2005. 2011). we extracted lard from pork fat contained in meatball in which some other components may be present in extracted lard. & Bakar. 2003). we have analyzed pork fat in meatball products (Rohman.. 2001. Materials and methods 2. Ali. Dalam otentikasi halal. 2005). Dalam penelitian itu. & Mohamed. 2011). Ali. Sismindary. kami telah menganalisis lemak babi dalam produk bakso (Rohman. electronic nose dan spectrometry kromatografi-mass gas dengan deteksi aroma dan senyawa volatil lemak babi yang terdapat dalam bakso (Nur Juliana. 2012. the calibration model should be developed. 2. Hasyim. Mirghani. Analisis daging babi di bakso dan produk makanan lainnya dapat dilakukan oleh amplifikasi DNA yang terdapat dalam daging babi dengan menggunakan polymerase chain reaction menggunakan target amplifikasi yang berbeda (Ali. & Bakar. as well as for analysis of lard in cake and chocolate formulation using partial least square calibration (Che Man. et al. & Mohd Said. et al. not destructive and not involving laborious sample preparation. Sismindary. Selected samples. added with 100 mL hexane. USA) to identify the retention times of FAME in lard and beef fat. Sampel yang dipilih. Preparation of calibration samples A set of standards consisting of lard in beef fat was prepared by mixing of both at concentration ranges of 0–100% (v/v) of lard in beef fat. The GC condition was as reported in Rohman et al.2. 2. The fat obtained was further used for analysis of fatty acid composition and FTIR spectra analysis. beef fat along with their blends were measured using FTIR spectrophotometer. Satu set standar yang terdiri dari lemak babi dalam lemak sapi dibuat dengan mencampurkan kedunya pada rentang konsentrasi 0-100% (v / v) dari lemak babi dalam lemak sapi. Analysis of lard in meatball broth The meatball broth was taken from several markets in Yogyakarta. The mixture was shaken vigorously and the hexane phase containing fats was taken. Prosedur rendering sesuai dengan yang dilaporkan dalam Rohman dan Che Man (2009). The residue of water was removed using anhydrous sodium sulfate. Komposisi asam lemak terdiri dari lemak babi dan lemak sapi dilakukan dengan menggunakan kromatografi gas dengan detektor ionisasi nyala atau flame ionization detector (GC-FID). Lemak yang mencair diregangkan dengan kertas saring. . The spectral regions in which the variations among analytes were observed were selected for developing multivariate calibration and principal component analysis. 2. Daerah-daerah spektral di mana variasi antara analit yang diamati dipilih untuk mengembangkan kalibrasi multivariat dan analisis komponen utama. Jaringan adiposa babi dan ternak dipotong kecil-kecil menggunakan cutter komersial agar dapat mengekstrak lemak babi dan lemak sapi secara efektif. As standard of fatty acid methyl esters (FAMEs). An approximately 100 mL of meatball broth was taken. The fats obtained were subjected to FTIR measurements. Lemak babi (lard) dan lemak sapi yang diperoleh dengan proses rendering hewan yang sesuai. Residu air dihilangkan menggunakan natrium sulfat anhidrat. (2012). lemak sapi bersama dengan campuran mereka diukur dengan menggunakan spektrofotometer FTIR. kami menggunakan 37 senyawa (C4 ke C24) dari Sigma Chemicals (St. Lemak yang diperoleh digunakan untuk analisis komposisi asam lemak dan analisis spektra FTIR. MO. yang berbeda dari sampel kalibrasi. (2012). were used as validation samples.paper. The water phase was further extracted using 50 mL of hexane. jaringan potong dimasukkan ke dalam oven konvensional selama 3 jam pada 100 ° C. Sebagai standar asam lemak metil ester atau fatty acid methyl esters (FAMEs). 2. Lard. Quantification analysis of FAMEs was performed using internal normalization technique. Fatty acid composition The composition of fatty acids composed of lard and beef fat was carried out using gas chromatography with flame ionization detector (GC-FID). and the residue of water was dried with anhydrous sodium sulfate. we used 37 compounds (C4 to C24) from Sigma Chemicals (St.4. The hexane phases were collected and evaporated under vacuum rotary evaporator at 60 °C. Kondisi GC adalah seperti yang dilaporkan dalam Rohman et al. which were different from calibration samples. and subjected to liquid–liquid extraction in a separatory funnel.3. Analisis kuantifikasi FAMEs dilakukan dengan menggunakan teknik normalisasi internal. MO. digunakan sebagai sampel validasi. USA) untuk mengidentifikasi waktu retensi FAME dalam lemak babi dan lemak sapi. Menggunakan Beaker glass. Louis. Lemak babi. Louis. Lemak yang diperoleh menjadi subjek pengukuran FTIR. Canada). Sekitar 100 ml kaldu bakso diambil. 3. While. dan residu air dikeringkan dengan natrium sulfat anhidrat.13. dengan resolusi dari 8 cm-1 dan 32 pemindaian. The composition of these fatty acids in lard and beef fat was in agreement with those specified in Codex Allimentarius (2003).Kaldu bakso diambil dari beberapa pasar di Yogyakarta. Sampel ditempatkan dalam kontak yang baik dengan horisontal jumlah pantulan dilemahkan (HATR) elemen (ZnSe kristal) pada suhu kamar dikendalikan (20 ° C). Spektrum FTIR dari seluruh sampel dipindai di wilayah inframerah pertengahan 400-4000 cm-1 menggunakan ABB MB3000 FTIR spektrofotometer (Clairet Ilmiah. ditambah dengan 100 mL heksana.1 (ABB. Results and discussion 3.1. Table 1 revealed the fatty acid composition of lard and beef fat. Fase heksana dikumpulkan dan diuapkan di bawah vakum rotary evaporator pada suhu 60 ° C.6. FTIR spectral acquisition FTIR spectra of all samples was scanned in the mid infrared region of 400–4000 cm-1 using ABB MB3000 FTIR spectrophotometer (Clairet Scientific. the classification among meatball broth samples was carried out using principal component analysis with the software of Minitab (version 16.5. 2. Fatty acid analysis Fatty acid composition is one of the parameter used in the quality control of edible fats and oils. Inggris) yang dilengkapi dengan deuterated triglycine sulfat (DTGS) detektor dan KBR sebagai beam splitter. respectively.2. Beef fat contained more palmitic and oleic acids. spektrum background referensi udara baru diambil. a new reference air background spectrum was taken. Fasa air selanjutnya diekstrak dengan menggunakan 50 mL heksana. Spektrum ini dicatat sebagai nilai absorbansi pada setiap titik data dalam rangkap tiga. USA). Spectra were processed using Horizon MB FTIR software version 3. After every scan. 3. dan mengalami ekstraksi cair-cair dalam corong pemisah atau separatory funnel. Kanada).13. Northampton.0. UK) equipped with deuterated triglycine sulfate (DTGS) detector and KBR as beam splitter. Spectra diolah dengan menggunakan Horizon MB FTIR soft¬ware versi 3. Semua spektrum dijatah dengan latar belakang spektrum udara. with a resolution of 8 cm-1 and 32 scanning.1 (ABB. These spectra were recorded as absorbance values at each data point in triplicate. Statistical analysis Quantitative analysis of lard was performed using partial least square calibration with the aid of Horizon MB software (Canada) included in FTIR spectrophotometer. Northampton. All spectra were rationed against a background of air spectrum.0. 2. FTIR spectra of lard and beef fat . The fatty acid profiles can be used for differentiation of edible fats and oils due to its capability to provide the fingerprint profiles of studied fats and oils. Campuran dikocok dengan kuat dan fase heksan mengandung lemak diambil. Setelah setiap scan. while lard contained more oleic and stearic acids. The samples were placed in good contact with horizontal attenuated total reflectance (HATR) element (ZnSe crystal) at controlled ambient temperature (20 °C). & Mansor. 2010).The importance of IR spectroscopy in identification of samples originates from the much information content obtained. FTIR spectra of beef fat and lard are difficult to be differentiated. wavenumbers of 1117 and 1098 cm -1 originate from the stretching vibrations of C\O in triacylglycerols. The wavenumber of3008 cm-1orig-inates from cis-olefinic C_H. due to the different nature and composition of the both lard and beef fat. The more unsaturation degree of fats and oils. As a consequence. Table 2 described the assignment peaks and shoulders present in lard and beef fat (Guillen & Cabo. lard revealed higher peak intensity than beef fat at wavenumber of 3007 cm -1 (Che Man. however. and the possibility to assign certain absorption bands related to functional groups (Bendini et al. Herrero-Martinez. corresponding to stretching and bending vibrations of functional groups. the higher the peak intensities in that wavenumber. 2011). Lerma-Garcia. 1 is FTIR spectra of lard and beef fat. Rohman. As shown in Table 1. & Simo-Alfonso. Meanwhile.. Each peaks and shoulder indicated the functional groups responsible for infrared absorption at wavenumbers of 4000–400 cm-1. lard has more unsaturated fatty acid than that in beef fat. This wavenumber region in which lard and beef fat showed some differences was further optimized to be selected as wavenumber used for quantitative analysis of lard in meatball broth. 2007). 1117 (l) and 1098 cm -1 (m). both spectra revealed bit differences in peak/shoulder intensities and the exact wavenumbers at which the maximum absorbance was observed for each fat and oil. Ramis-Ramos. Peak assigned with (a) at 3007 cm -1 attributed from the stretching vibration of cis C_CH. Fig. . which can be used as an indicative of unsaturation degree. especially at wavenumber regions of 3007 (a). 1997. due to the capability of FTIR spectra as fingerprint tools.