Basic Requirements of Laboratory Operation for Halal Analysis

Analysis of halal food requires that the laboratories conductive the tests adhere to international guidelines and standards. Common worldwide guidelines and standards for laboratory include the ‘International Organization for Standardization 17025’ (ISO 17025), the ‘Good Manufacturing Practice’ (GMP), and ‘Good Laboratory Practice’ (GLP). In halal analysis, the laboratory shall comply with ISO 17025, GMP, and ‘Good Hygiene Practice’ (GHP) as stated in the ‘Manual Procedure for Malaysia Halal Certification’ (MPPHM). This article discusses the basic requirements for laboratory practises, specifically for halal analysis. The study compares these international guidelines  with ‘Malaysia Halal Standards’ to demonstrate that these international standards are combined with Islamic practices to produce valid test results using globally recognized best-practices. This promotes confidence in the halal laboratory’s work both nationally and internationally, and will thus improve international trade. Keywords: Halal analysis, ISO 17025, GMP, GLP, laboratory operation

Improved gel-enhanced liquid chromatography-mass spectrometry by chemometrics for halal proteomics

Numerous analytical methods for the authentication of halal meat are now well established, with gel-enhanced liquid chromatography-mass spectrometry (GeLCMS) being a popular approach. However, the selection of potential protein markers on 1-dimensional gel electrophoresis (1DE) prior to LCMS is considered problematic, because using the optical density for the selection process could introduce human error. In this study, an improved GeLCMS method assisted by multivariate principal component analysis (PCA) was developed to identify the potential protein markers for non-halal pork among halal beef and chicken. The improved GeLCMS technique allowed for the confident excising of identified protein bands prior to in-gel tryptic digestion. The inferential protein markers (myofibrillar proteins), which might be present in the samples, were determined based on the identified sequence of peptides. This chemometric-assisted GeLCMS could potentially be used as a guideline to assist chemists in analysis of any gel-based separation of biomolecules, regardless of the field of stud

Comparing the effect of heat on tropomyosin isoforms patterns from water buffalo and wild boar meat by two-dimensional gel electrophoresis

Tropomyosin is one of the most abundant proteins in meat; however, very little is known about it due to the lack of scientific literature. In this study, the spot volume of tropomyosin (TPM) isoforms, TPM2 and TPM1, in meat from water buffalo and wild boar subjected to various cook treatments were compared. We hypothesized that primary structures of the tropomyosin isoforms from both species would remain stable despite the application of heat. Proteins extracted from the treated meats were analyzed using two-dimensional gel electrophoresis and mass spectrometry. A Kruskal-Wallis test showed that there were no significant differences in protein spot volumes for all treatments; however, a significant difference was observed between species. Changes in the amino acid sequence of TPM1 were observed between the two species, indicating that the isoforms could be used as thermostable proteins or peptide markers for species identification because of their resistance to high temperatures

Discrimination of Malaysian stingless bee honey from different entomological origins based on physicochemical properties and volatile compound profiles using chemometrics and machine learning

Identification of honey origin based on specific chemical markers is important for honey authentication. This study is aimed to differentiate Malaysian stingless bee honey from different entomological origins (Heterotrigona bakeri, Geniotrigona thoracica and Tetrigona binghami) based on physicochemical properties (pH, moisture content, ash, total soluble solid and electrical conductivity) and volatile compound profiles. The discrimination pattern of 75 honey samples was observed using Principal Component Analysis (PCA), Hierarchical Clustering Analysis (HCA), Partial Least Square-Discriminant Analysis (PLS-DA), and Support Vector Machine (SVM). The profiles of H. bakeri and G. thoracica honey were close to each other, but clearly separated from T. binghami honey, consistent with their phylogenetic relationship. T. binghami honey is marked by significantly higher electrical conductivity, moisture and ash content, and high abundance of 2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde, 2,6,6-trimethyl-1-cyclohexene-1-acetaldehyde and ethyl 2-(5-methyl-5-vinyltetrahydrofuran-2-yl)propan-2-yl carbonate. Copaene was proposed as chemical marker for G. thoracica honey. The potential of different parameters that aid in honey authentication was highlighted

Hydroxyproline determination for initial detection of halal-critical food ingredients (gelatin and collagen)

Gelatin and collagen are considered halal-critical ingredients as they are typically derived from either bovine or porcine animals. Current analytical methods for determining the sources of gelatin and collagen suffer from limitations in terms of robustness and false positives in peptide matching. Thus, the aim of this study was to investigate the utility of monitoring hydroxyproline, a signature amino acid for gelatin and collagen, for identifying potentially haram foodstuffs. To determine the hydroxyproline profiles among animal- and plant-based samples, one-way univariate analysis of variance followed by pair-wise comparison was used to establish statistical significance. Multivariate chemometric analysis through principal component analysis revealed a discrete distribution pattern among 59 samples due to hydroxyproline variability. Finally, inter- and intra-laboratory comparisons demonstrated the validity and robustness of hydroxyproline determination according to ISO 17025. Thus, this preliminary identification technique will aid the identification of potentially haram foodstuffs

Shotgun proteomics approach for the establishment of peptide markers for pork

Statistically, in 2015 the largest population in the world is the Christians, nevertheless, by 2060 the population of Muslim is expected to be nearly equal to the Christians. This indicates that the halal status of any particular food as a future global concern. According to the Food and Agricultural Organisation of the United Nations 2009, the demand for meat is expected to increase drastically for the developing countries, from 26 to 37 kg of average annual per capita consumption from the year 2000 to 2030. Consequently, certain manufacturers have unethically adulterated the meat owing to the desire to generate a high-profit margin as well as to fulfil the market demand, whereby pork is added to beef. These consequences highlight a requirement for meat authentication analysis. Recently, qPCR is the most famous genomic-based method that has been employed in the routine laboratories worldwide; owing to the lower limit of detection method as compared to any other proteomic-based methods such as SDS-PAGE and ELISA. In contrast to the DNA, the peptide sequences are extremely stable, in which their intactness remain against chemical or mechanical processes. The objective of this study, therefore, is to establish the peptide markers for pork by a newly shotgun proteomics approach, which those peptide markers shall be related to the contractile proteins of meat such as myosin, actin, tropomyosin, or troponin complexes. Initially, the peptide masses of proteolytic peptides, generated from peptide mass fingerprinting of LC-MS analysis, were analysed by principal component analysis (PCA) to overview the distribution pattern of 577 peptide masses among pork, beef, and broiler. Then, the most significant peptide masses for pork were determined from a validated PCA model through a discriminant analysis of orthogonal partial least square. Consequently, only seven potential peptide markers for pork were identified, but only five peptide markers were true-positive, as confirmed by another independent tandem LC-MS/MS analysis. Subsequently, the MS/MS spectra of true-positive peptide markers were annotated for their peptide sequences and inferential proteins through de novo database search engine (MS-Taq tool of ProteinProspector 5.20.0). Furthermore, a validation study was conducted to measure the precision, detection limit, and specificity of the peptide markers in relation to their reliability and applicability. In summary, only three reliable and applicable peptide markers from contractile proteins of pork had successfully established through an advanced shotgun proteomics approach by using Agilent 1200 Series high-performance liquid chromatography hyphenated with AB Sciex 4000 QTrap mass spectrometer with a detection limit was 10% of pork. Unfortunately, the three peptide markers are not applicable to processed pork. This finding might be owing to a modification of certain amino acid in the peptide marker sequence through deamination or oxidation due to the extreme processing. Therefore, further comprehensive study is warranted for processed pork, as the established peptide markers from this study were successfully developed for raw pork not processed pork. Moreover, an advanced method validation for individual peptide marker is required, before it can be routinely implemented in the laboratory as a standard procedure for meat authentication