Halal authentication
Halal detection technologies and related analytical method approaches - PART 1: Introduction
The world of technology has blossomed, using scientific knowledge to create practical solutions essential for industries operating in real time. From developing equipment to serving various sectors, including those adhering to halal standards, technology has seen significant improvements over time. One crucial area where technology plays a pivotal role is in halal detection.
Halal detection technologies (HDTs) are all about finding evidence to ensure that products comply with halal requirements, meaning they're free from any contaminants prohibited in Islamic law. These contaminants could include substances like pork, dogs, or anything else considered nonhalal. It's not just about what goes into the product either; even substances like urine, blood, or vomit, whether from humans or animals, are taken into account when using HDTs.
Ensuring halal products isn't about religious observance; it's also about ensuring they're safe and wholesome for consumers. HDTs play a crucial role here, too, by identifying and quantifying any harmful substances that could pose health risks, such as intoxicating or poisonous chemicals. So, it's not surprising that HDTs are widely used to test products for their halal status and to ensure they're safe to consume.
But using HDTs for halal testing isn't without its challenges. Different types of samples require different methodologies, making the process more complex. With the demand for halal products on the rise, including a wide range of processed goods, there's a pressing need to establish the most effective HDTs and methods for testing. Different approaches are used depending on the nature of the sample, whether it's protein-based, fat-based, or contains alcohol. For example, protein- and fat-based samples require specialized equipment to detect nonhalal ingredients that may dissolve in water or oil.
Malaysia's Halal Management System of 2020 supports these approaches, even incorporating DNA testing for hair, skin, and meat speciation. This means that we can identify the physical properties of substances and pinpoint specific ingredients through targeted testing methods.
Analytical equipment plays a crucial role in halal testing, with techniques like real-time polymerase-chain reaction (RT-PCR) used to detect porcine DNA and high-performance liquid chromatography (HPLC) to analyze amino acid profiles. Tools like Fourier-transform infrared spectrometers (FTIR) and gas chromatography-mass spectrometers (GC/MS) are employed for substances that mix with fats and oils. These methods, when combined with multivariate data analysis (MDA), provide comprehensive guidelines for analysts worldwide, ensuring accurate and reliable halal testing results. We'll talk about the best approaches in the next post.
Further reading: https://www.sciencedirect.com/science/article/abs/pii/B9780323916622000156
Halal detection technologies and related analytical method approaches - PART 2: Approaches of halal detection technologies
As the demand for halal products grows beyond food, testing methods are expanding, too. It's not just about DNA testing anymore. Methods like HPLC and GC/MS have been successful in testing halal products. Other methods like FTIR and differential scanning calorimetry are being explored, but there's no clear guideline on which methods to use. Based on my experience handling clients' requests, I propose three approaches based on the type of sample: protein-based, fat-based, and alcohol-based. Each approach can be either targeted or profiling. Finally, a summary of these approaches is provided in the table below. We'll talk about the details of these approaches in the next post.
Further reading: https://www.sciencedirect.com/science/article/abs/pii/B9780323916622000156
Halal detection technologies and related analytical method approaches - PART 3: Protein-based approach - Identification of targeted deoxyribonucleic acid (DNA)
The protein-based approach involves identifying deoxyribonucleic acid (DNA), amino acids (AAs), polypeptides, and even proteins. Both targeted and profiling types apply to this approach.
IDENTIFICATION OF TARGETED DNA
DNA is a double-helix polynucleotide that encodes genetic information in the species-specific cell. The nucleotides comprise three components: a nitrogen base, deoxyribose sugar, and a phosphate group. Since DNA is a double-helix polynucleotide, this structure can be separated or denatured into two individual strands, each consisting of its polynucleotides. The polynucleotides are bonded by oxygen from the deoxyribose sugar of one nucleotide and phosphorus of the phosphate group from another nucleotide. This bonding creates what is known as a phosphodiester bond. The phosphodiester bond is the connector between the 3-prime (3`) carbon of one nucleotide and the 5-prime (5`) carbon of another nucleotide. In other words, the phosphodiester bond connects the nucleotides that make up each strand of the DNA molecule, forming the molecule's backbone with a 3-prime (3`) to 5-prime (5`) carbon arrangement. Each DNA strand has a reverse 3-prime (3`) to 5-prime (5`) carbon arrangement. The connection between two DNA strands is called hybridization or annealing, and the nitrogen base plays a crucial role in this process.
The nitrogen base is represented by four types: adenine, guanine, thymine, and cytosine, which attach to the 1-prime (1`) carbon of the deoxyribose sugar. Adenine and guanine belong to the purine group (2 rings), while thymine and cytosine belong to the pyrimidine group (1 ring). In the DNA molecular arrangement, purine and pyrimidine groups pair together via hydrogen bonding; for example, adenine pairs up with thymine via two hydrogen bonds, while guanine pairs with cytosine via three hydrogen bonds. Hydrogen bonds are broken by high-temperature denaturation (HDT), which separates the two DNA strands into single strands. These single strands can then identify species-specific cells for halal testing.
DNA testing has been adopted to authenticate meat and meat products and to identify non-halal gelatins in food and cosmetics. Various DNA testing methods include singleplex Polymerase Chain Reaction (PCR) or conventional PCR, duplex PCR, multiplex PCR, and RT-PCR or quantitative PCR.
In the next post, we'll delve into profiling amino acids as part of a protein-based approach. Further reading: https://www.sciencedirect.com/science/article/abs/pii/B9780323916622000156
Halal detection technologies and related analytical method approaches - PART 4: Protein-based approach - Amino Acid Profiling
Amino acids (AAs) are fundamental units of proteins, synthesised from translated DNA sequences, thus forming the building blocks of proteins. Given their significant role in protein composition, AAs are pivotal in authenticating halal sources, particularly as target analytes for profiling. The wide variety of AAs, with 17 distinct types identified in certain studies, supports a profiling approach for authentication rather than a targeted method. Researchers have utilised high-performance liquid chromatography with fluorescence detection (HPLC-FLD) to identify and quantify these AAs, following a protocol that includes freeze-drying the samples to achieve moisture levels below 10% to mitigate interference from the sample matrix. Subsequent acid hydrolysis and incubation at 110°C for 24 hours prepare the samples for analysis.
Prior to amino acid quantification, it is essential to establish calibration curves for each AA using a minimum of seven working standards spiked with an internal standard. An internal standard, such as L-aminobutyric acid (AABA), is incorporated in ultra-high-performance liquid chromatography with a diode-array detector (UHPLC-DAD) to minimise matrix effects. This protocol includes AABA spiking in the acid-hydrolysed samples. It is also advisable to avoid serial dilution to prevent systematic errors in preparing working standards. The derivatisation of AAs depends on the instrumental analysis requirements, with fluorescence derivatisation often employed for HPLC-FLD analysis. For instance, ortho-phthalaldehyde (OPA) and 9-fluorenyl-methyl chloroformate (FMOC) have been used to derivatise primary and secondary AAs, respectively. Similarly, fluorescence derivatisation agents may be applied before UHPLC-DAD injection, demonstrating the versatility of fluorescence derivatisation for both FLD and DAD.
The UHPLC or HPLC analysis of AAs typically involves eluting two mobile phases through a membrane filter to a C18 column. A common setup employs an aqueous solution of AccQ.Tag concentrate as eluent A, alongside an acetonitrile-water mixture as eluent B. This gradient setup allows optimal AA separation, achieving effective peak resolution for quantification, typically within a 10-minute elution window for satisfactory AA separation. Each AA’s peak emerges at a specific retention time, and as working standard concentrations increase, peak areas expand, creating a calibration curve with robust linearity. This calibration curve can be expressed as:
As/Ais = m Cs/Cis + c
Where As and Ais represent the peak areas of the working standard and internal standard, respectively; m denotes the calibration curve’s slope; Cs and Cis are the working and internal standard concentrations, and c is the y-axis intercept. The linearity of this calibration curve is confirmed by criteria such as a high correlation coefficient near 1, insignificant variance differences between experimental and theoretical F-values, bounded confidence intervals, and a randomly distributed residual plot.
Following AA identification and quantification, multivariate data analysis (MDA), including principal component analysis (PCA), discriminant analysis (DA), and partial least square-discriminant analysis (PLS-DA), is recommended to authenticate protein-based products. In the next post, we'll delve into targeted polypeptide analysis as part of a protein-based approach. Further reading: https://www.sciencedirect.com/science/article/abs/pii/B9780323916622000156 and https://journals.iium.edu.my/inst/index.php/hs/article/view/83/84
