Laboratory Analysis (Halal Forensic) PDF

LABORATORY ANALYSIS (HALAL FORENSIC) Assoc. Prof. ChM, Dr Mohd Sukri Hassan THE PROHIBITION OF SWINE IN ISLAM “He has only forbidden you dead meat, and blood, and the flesh of swine and that on which any other name hath been invoked besides that of Allah. But if one is fo...

LABORATORY ANALYSIS (HALAL FORENSIC) Assoc. Prof. ChM, Dr Mohd Sukri Hassan THE PROHIBITION OF SWINE IN ISLAM “He has only forbidden you dead meat, and blood, and the flesh of swine and that on which any other name hath been invoked besides that of Allah. But if one is forced by necessity, without willful disobedience, nor transgressing due limits, - then is he guiltless. For Allah is oft-forgiving, most merciful.” Al-Quran Surah 2: Verse 173 THE PROHIBITION OF SWINE IN ISLAM And eat of what Allah has provided for you [which is] lawful and good. And fear Allah , in whom you are believers. Al-Quran Surah 5: Verse 88  Processing Customers’ Genetically Confidence Modified Halal Present Issues Product Ingredients Safety & & Additives Quality Detection Method  ▪ Raw materials – animal or plant origin ▪ Slaughtering and stunning – according to Shariah or not ▪ Processing operations/equipment contamination ▪ Packaging/Storage/Transportation (containers and vessels) ▪ Food ingredients and additives Pig and its by-products (e.g., pork, lard, gelatin) ISSUES IN ▪ ▪ Enzymes (e.g.. rennet) HALAL FOOD ▪ Emulsifiers (e.g.. E471 or mono- & diglycerides) PRODUCTION ▪ Alcohol (ethanol) ▪ Biotechnology and GMOs (genetically modified organisms) ▪ Safety and quality aspects (aspect of ‘Thoyyiba’) ADULTERATION Adulteration is a legal term for a food product which fails to meet certain standards. Adulterants may be intentionally added to more expensive substances to increase visible quantities, reduce manufacturing costs and other deceptive or malicious purpose. Adulterants may also be accidentally or unknowingly introduced into the substances. Halal Food Issues from Islamic and Modern Science Perspectives. Nurrul hidayah A.Fadzlillah et al., 2011 ADULTERATION WITH PORCINE-BASED PRODUCT In some countries, food manufacturers choose to blend vegetable fats with lard to reduce production cost Lard could be effectively blended with other vegetable oils to produce shortening, margarines and other specialty food oils In other instances, adulteration with porcine products could be unintentional Gelatin SAFETY ISSUES (SOUTH KOREA) Humidifier sterilizer sold by UK firm killed or injured hundreds. Some 178 killed or injured by lung problems cause by product. South Korea says 530 people had registered claims since 2011 of lung ailments from using humidifier sterilizers marketed by Oxy Reckitt Benckiser, and similar products marketed by other firms. Reckitt Benckiser's global brands include Dettol antiseptic wash, Nurofen and Durex condoms. http://www.dailymail.co.uk/news/article-3569143/Reckitt-Benckiser-exec- slapped-S-Korea-apologising-deadly-sterilizers.html SAFETY ISSUES (SOUTH KOREA) Confrontation: Nearly 180 South Koreans have died or suffered injuries as a result of lung injuries caused by a humidifier sterilizer sold by Reckitt Benckiser http://www.dailymail.co.uk/news/article-3569143/Reckitt-Benckiser-exec-slapped-S-Korea-apologising-deadly- sterilizers.html GENETICALLY MODIFIED ORGANISM (GMO)? GMOs or Genetically Modified Organisms are living organisms whose genetic material has been artificially manipulated in a laboratory through Genetic Engineering (GE). This relatively new science creates unstable combinations of plant, animal, bacteria and viral genes that do not occur in nature or through traditional crossbreeding methods. http://www.nongmoproject.org/learn-more/what-is-gmo/ PROS AND CONS OF GMO FOODS PROS CONS Insect resistance Allergic Weather resistance Unknown long term Disease resistance health effect Prolonged shelf life Unnatural taste Increase production WHERE IS BLOOD PLASMA COME FROM? BOVINE PORCINE FISH CHICKEN WHY BLOOD PLASMA ? Binder Natural Bioactive colour compounds enhancer BLOOD Protein and PLASMA Iron Emulsifier supplement Egg replacer Fat replacer Ofori, J.A. and Hsieh, Y.P. The Use of Blood and Derived Products as Food Additives. www.intechopen.com WHAT IS BLOOD PLASMA? Liquid portion of blood remaining after the blood cells [white blood cells (WBCs), red blood cells (RBCs), and platelets] have been removed. The largest user of blood proteins in food is the meat industry By 2001, it was estimated that the food industry was utilizing 30% of the blood produced in slaughterhouses Ofori, J.A. and Hsieh, Y.P. The Use of Blood and Derived Products as Food Additives. www.intechopen.com WHERE IS STEM CELL COME FROM? WHAT IS STEM CELL? A stem cell is an immature cell that has the potential to become specialized into different types of cells throughout the body Stem cell procedures currently provide life-saving treatments for patients with leukemia, lymphoma, other blood disorders, and some solid tumor Recycled Used Cooking Oil RECENT DETECTION ADVANCEMENT The task of halal authentication cannot rely only on expertise from shariah alone, but also require other related technical fields such as food science and technology, chemistry and veterinary science. Halal autentication cannot rely solely on physical inspection and documentation anymore, but also using the latest high technology analytical instrumentation.  Various approaches to detect and quantify level of adulterants in food products: Determine the ratios between some chemical constituent and assuming that these ratios are constant in particular food products. Searching specific markers in food RECENT products, either chemical constituent or morphological components which proves DETECTION the presence of adulterant in food products. ADVANCEMENT Analytical methods derived from physical analysis by taking account the whole samples to show the adulteration effects on physic-chemical properties.  Fourier Transform Infrared (FTIR) spectroscopy Differential Scanning Calorimetry (DSC) Electronic Nose (E-nose) technology Others AUTHENTICATION TECHNIQUES FOOD AUTHENTICITY INSTRUMENTS FTIR GC-MS DIFFERENTIAL SCANNING CALORIMETRY PCR FTIR Food samples (chocolate, cake, biscuits) were analyzed to identify the differences in FTIR spectra profiles FTIR technique combined with chemometric analysis able to detect and quantify the level of lard adulterated in food samples. Offers rapid (results in 2 min), simple, accurate, reliable, and environmental friendly tool RECENT DETECTION RESEARCH FTIR FTIR FTIR  Thermoanalytical technique for monitoring DIFFERENTIAL changes in physical or chemical properties of SCANNING material by detecting the heat changes CALORIMETRY Thermogram profile show the presence of lard in food sample Relatively simple, accurate and minimum amount of sample needed DIFFERENTIAL SCANNING CALORIMETRY DIFFERENTIAL SCANNING CALORIMETRY DIFFERENTIAL SCANNING CALORIMETRY DIFFERENTIAL SCANNING CALORIMETRY DIFFERENTIAL SCANNING CALORIMETRY  GAS 37 FAMEs standard Fatty Acids identification CHROMATOGRAPHY 4 6 8 10 12 14 16 18 20 22 pA 9.387 - C4:0 9.994 - C6:0 10 11.123 - C8:0 13.084 - C10:0 14.430 - C11:0 15 16.000 - C12:0 17.740 - C13:0 19.586 - C14:0 20 21.020 - C14:1 21.476 - C15:0 22.928 - C15:1 FID1 A, Front Signal (SUZIE2014\160115- 2ND2015-01-16 11-17-25\002B0101.D) 23.362 - C16:0 24.558 - C16:1 25 25.209 - C17:0 26.388 - C17:1 27.001 - C18:0 27.672 - C18:1n9t 28.013 - C18:1n9c 28.745 - C18:2n6t 29.516 - C18:2n6c 30 30.605 - C18:3n6 30.416 - C20:0 31.266 - C18:3n3 31.412 - C20:1 32.098 - C21:0 32.943 - C20:2 33.786 - C22:0 34.076 - C20:3n6 34.739 34.947 C20:3n3 34.825 - C22:1n9 -- C20:4n6 35 35.489 - C23:0 36.440 - C22:2 36.928 - C20:5n3 37.277 - C24:0 38.466 - C24:1 40 42.295 - C22:6n3 min  0 50 pA 100 150 200 250 300 350 400 9.548 - C4:0 10 15 15.965 - C12:0 16.230 16.888 17.427 - C13:0 19.535 - C14:0 20 21.911 - C15:0 23.363 - C16:0 FID1 A, Front Signal (SUZIE2014\160115 - AISHAH 2015-01-19 09-10-13\037B3701.D) 24.325 24.506 - C16:1 Spiked lard of palm oil 25 25.143 - C17:0 26.254 - C17:1 26.959 - C18:0 27.655 - C18:1n9t 28.044 - C18:1n9c 29.104 29.248 - C18:2n6t 29.487 - C18:2n6c 30 30.319 - C20:0 30.608 - C18:3n6 31.214 C20:1 31.328 -- C18:3n3 32.870 - C20:2 33.659 - C22:0 34.883 - C22:1n9 35 37.118 - C24:0 39.786 40 min  Scores (training set)  Loadings (training set)  PCA application on collected samples at pasar malam. Projection of test lard samples (validate PCA model from training set). Projection of collected samples (unknown).  Scores plot of test lard samples  Scores plot of collected samples  PCA plot the 5 % spiked lard in palm olein has significant differs from the control and was use as database for calibration set (training set) into PCA projection technique. The collected night market samples were then projected into PCA model and found that it distributed far away from calibration samples (training set). Heated Edible Fats , are they similar? Raw FTIR of Mutton fat (M) and tallow (B) samples show high intensity between 3100 and 3340 cm-1. Raw FTIR of chicken fat (C) shows high noisy at 3359 – 3390, 2240 – 2250 and 1230 – 1900 cm-1. Observation at 2800 – 3200 cm-1 shows a similarity between chicken and lard by two identical peaks. Both mutton and tallow fats display split spectral peaks at these frequencies which are required pre-processing data before applying chemometrics FTIR spectra of all edibles fats after pre-processing using the Savitzky–Golay second derivatives The spectral ranges at 715 cm-1 and above were selected because the other regions (650 – 714 cm-1) have no spectral information because of the unvarying spectrum. Principal components analysis (PCA), k-mean cluster analysis (k-mean CA) and linear discriminant analysis (LDA) PCA of Fourier Transform Infrared Spectroscopy Linear Discriminant Analysis (LDA) of edibles fats after heat treatments Lard and chicken lying close to zero for a class are associated with the class. 80.5% of fats were correctly classified  Principal Component Regression (PCR), Orthogonal Partial Least Square Regression (OPLSR) and Linear Support Vector Machine Regression (SVMR) PCR OPLS  COMPARISON of REAL-TIME PCR and FTIR WITH CHEMOMETRICS TECHNIQUE in ANALYSING HALAL SUPPLEMENT CAPSULE Materials and Method – Comparison Real-Time and FTIR FTIR, DNA Isolation BioDrop Real – Time PCR Chemometrics The gelatins Measureme The amplification The analysis was was extracted nt was was repeated at made on the according to performed the 35 cycle, due frequency 4000- Agilent Porcine at to the analysis with 650 cm-1. The Detection Kits 260 nm – the standard DNA data were 280 nm. (17-33 cycle for analyzed with bovine and 19-34 PCA techniques cycle for porcine). using The Unscrambler X 10.3 software.  -0.100 0.000 0.100 0.200 0.400 0.500 0.600 0.700 0.800 0.300 586 679 772 864 957 1049 1142 1234 1327 1420 1512 1605 1697 1790 1883 1975 2068 2160 2253 2345 2438 2531 2623 2716 2808 2901 2994 FTIR Spectra in Gelatin Capsule of Supplement Product 3086 3179 3271 3364 3456 3549 3642 3734 3827 3919 4012 S1 S2 S4 S5 S9 S6 S11 S12 S13 S14 S17 S18  The PCA Score and Loadings Table ⚫ Normalization and smoothing of the FT-IR spectrum of gelatin capsule was shown in the figure above. ⚫ PCA manipulates the data of variables (peak height of amino acids) in the way that these variables can be displayed on an x, y coordinate system. PCA did this by calculating principal components (PC1) which are linear combination of original variables.  The figure showed the enlarged FTIR spectra at fingerprint regions. The different peaks in terms of peak intensity were used for selecting the spectral regions for the quantification and classification of gelatins capsule in supplement product. PC1 vs. PC2. PCA was pre-treatment with Savitsky-golay and peak normalization where PC1 described 89% variation of data, while PC2 and PC3 account for 7% and 3% variations, respectively. Therefore, more than 90% of variation can be described only by three PCs (The Scores Plot). The PCA loading plot described the projection of variables in the same plane as the score plot. From the figure, it was known that 694 and 3271 cm-1 were the variables giving the most contribution toward PC1, while 1558 and 1635 cm-1 were more influencing on PC2.  RESULT OF REAL-TIME PCR IN GELATIN CAPSULE OF SUPPLEMENT PRODUCT Sample Concentration Ct Value Remarks (ng/µl) Porcine Bovine Mean ± SD Mean ± SD Mean ± SD S1 4.85 ± 0.21 26.97 ± 0.38 20.54 ± 1.28 Mixture S2 3.23 ± 0.34 - 26.20 ± 0.46 Bovine S4 3.90 ± 0.02 26.41 ± 0.83 23.07 ± 0.76 Mixture S5 5.08 ± 0.06 27.20 ± 0.74 - Porcine S6 6.14 ± 0.82 26.57 ± 0.55 - Porcine S9 2.50 ± 0.17 27.69 ± 0.81 20.52 ± 1.02 Mixture S11 5.01 ± 0.14 27.73 ± 0.53 21.34 ± 0.68 Mixture S12 4.72 ± 0.16 26.89 ± 0.47 16.67 ± 0.53 Mixture S13 4.43 ± 0.01 - 24.23 ± 0.53 Bovine S14 4.67 ± 0.01 - 31.49 ± 0.75 Bovine S17 1.94 ± 0.07 - 31.38 ± 1.35 Bovine S18 3.78 ± 0.54 27.8 ± 0.28 27.81 ± 0.29 Mixture -CT 1.00 - - -  Different concentrations of gelatin capsules were detected based on the threshold cycle (Ct) of each sample which is the cycle number where the samples fluorescent curve jumps sharply upward and corresponds to the initial concentration of DNA. 12 retail products, 6 samples were found to contain porcine-bovine gelatin, 2 samples were found to contain only porcine gelatin, with the remaining 4 samples being bovine gelatin.  Real-time PCR were validated based upon repetitive elements of the porcine and bovine DNA and they allowed sensitive detection of porcine and bovine DNA at concentrations 10-5 ng/ul. The use of FTIR and PCA was investigated to compare the result of real-time PCR. From this method, wavenumber profiles can classify capsule shells made from bovine and porcine gelatin but was not successful for classification of adulteration gelatin in capsule shells. AUTHENTICATION METHODS FOR HALAL FOOD ASSESSMENT Examples of haram ingredients that can be analysed by laboratory analysis methods. (Spigel et al., 2012) Haram ingredient Identification Examples of laboratory analysis methods References Animal species Pork meat or DNA hybridization, DNA sequencing, PCR (Aida, Man, Raha, & Son, 2007; Aida, Man, pork Wong, Raha, & Son, 2005; Che Man, Aida, derivates, or Raha, & Son, 2007; Farouk et al., 2006; Farrokhi meat & Jafari Joozani, 2011; Kesmen, Sahin, & Yetim, 2007; from other Laube et al., 2007; Tanabe et al., 2007) animal species Animal fat Fat composition: FTIR, NIRS, DSC, NMR, HPLC, LC-MS(/MS), (Che Man, Gan, NorAini, Nazimah, & Tan, 2005; vegetable or GC, PTR-MS, electronic nose technology Fauzi & Mas’ud, 2009; Jee, 2002; Marikkar, Ghazali, animal fat Long, & Lai, 2003; Marikkar, Lai, Ghazali, & Che Man, 2001; Nurjuliana, Man, Mat Hashim, & Mohamed, 2011; Rohman & Che Man, 2010; Syahariza, Man, Selamat, & Bakar, 2005; Van Ruth, Rozijn, et al., 2010; Van Ruth, Villegas, et al., 2010) Proteins, peptides Protein SDS-PAGE, CE, FTIR, colorimetry, chromatography (Hashim et al., 2010; HSC, 2009; Venien & Levieux, 2005; or amino acids composition: (e.g. HPLC), immunoassays and immunoblotting (e.g. Westermeier & Naven, 2002) from pork origin collagen or ELISA), gelatine biosensors, NIRS, MS, 2D-PAGE coupled to mass from pork or spectrometric other techniques (MALDI-TOF) animals Animal origin of Natural or SNIF-NMR, IRMS & SNIF-NMR, (Ebeler, 2007; Fauzi & Mas’ud, 2009) ingredients synthetic Isotope ratio-based finger prints, chiral GC analysis, origin, chemical C/N-ratio by CHN-analyser or microbial conversion, bone or wooden origin Alcohol Alcohol GC, electronic nose technology, (Anis Najiha, Tajul, Norziah, & Wan Nadiah, 2010; Boscaini, PTR-MS Mikoviny, Wisthaler, Von Hartungen, & M€ark, 2004; Fauzi & Mas’ud, 2009) AUTHENTICATION METHODS FOR HALAL FOOD ASSESSMENT Halal authenticity issues in meat and meat products. Khadijah Nakyinsige et al., 2012 DETECTION RESEARCH RESEARCH RESEARCH Leather and Bristle Products Pig Skin Slanting shape for each hole clearly seen. FTIR Analysis on Pig Skin and Standard kulit khinzir, T-Dome %T 90 Polymers Poly(N-methyl acrylamide) Match:64.09 %T 50 Molecular Formula: Sample Prep: film/MTL/KRS-5 CAS Number: 0-00-0 Cocoamidopropyl hydroxys ultaine Match:62.82 %T 50 Trade Name: Scher Chemicals Inc. Schercotaine SCAB Poly(g-benzyl-L-glutamate) Match:62.71 %T 50 Molecular Formula: Sample Prep: film/CLF/CsI CAS Number: 0-00-0 NYLON Match:61.14 %T 50 Trade Name: Supplier: BADISCHE (BASF) Fiber Number: A0162 Poly(5-methylcaprolac tam) Match:60.48 %T 50 Molecular Formula: Sample Prep: film//CsI CAS Number: 0-00-0 3500 3000 2500 2000 1500 1000 Wavenumbers (cm-1) Pig Hair Pig hair shows two or three branches. FTIR on Pig Hair and Polymers  Adulteration and contamination of non‐halal components are major concern in food processing and production. CONCLUSION Development on methods of detection adulteration are urgently needed for halal food verification and certification.  These analytical methods have potential to be utilised by halal food authorities responsible for authentication of halal food such as JAKIM. New analytical methods of analysis developed by our research group is simple, rapid and CONCLUSION reliable for detection of pork and lard in food and other consumer products. THANK YOU