Limited information exists regarding adulteration of Halal-certified food by substances forbidden under Islamic law (Haram substances). This study was conducted using forensic laboratory testing to investigate the prevalence of this type of adulteration. In this large-scale survey of Halal-certified food products randomly collected from markets in Thailand, 4,829 food samples from 10 food groups were tested in the laboratory for four potentially Haram substances: porcine DNA, porcine fatty acids, ethanol, and hydroxyproline (gelatin). No samples were adulterated with porcine DNA or fatty acids. However, 62 samples (approximately 1.3%) were positive for ethanol (>0.5% for non–naturally fermented products and >1% for naturally fermented products). The hydroxyproline concentration in the samples was compared with that of a negative control. Gelatin, as indicated by the presence of hydroxyproline, was the major suspicious substance found in these products. Further investigations are required to determine whether the gelatin is of Halal origin. These results from this first large-scale postmarket surveillance of Halal-certified food products for forbidden substances reveals the important role of forensic laboratory testing for supporting Halal supervision and certification. These findings provide useful information for government agencies seeking to encourage Halal compliance by food enterprises and for Muslim consumers and Halal food importers and exporters. HIGHLIGHTS
Meat adulteration has become a serious problem in global which directly affects to food consumers and producers. Therefore, it requires a tool to authenticate meat species to ensure safety of food products. Next generation sequencing (NGS) coupled with ribosomal RNA mitochondrial DNA gene can be used to analyze mixture of meat species in multiple meat samples. Therefore, this study aims to utilize NGS coupled with rRNA gene to identify 4 meat species (cattle, chicken, fish, and pig). Three primer sets (12S-Ki, 16S-KH, and 16S-Ki) were used to amplify DNA from the four meat species. All primer sets could be successfully amplified DNA fragments which corresponded to their size expectation. 16S-KH showed better detection effect in all species comparing with others. While the 12S-Ki and 16S-Ki could not be used to amplify in fish and chicken species. This may occur due to mismatches between sequences of primers and annealed regions of these species. Library construction of all PCR amplicons were prepared and sequenced by NGS. Amplicons amplified by 12S-Ki (fish) and 16SKi (chicken and fish) could not be mapped to the database because no PCR amplicons could not be amplified. NGS coupled with 16S-KH was then evaluated for precision test. The experimental precision was directly investigated comparing the results obtained from libraries that derives from DNA of four meat species which separately amplified for 3 different runs. As expected, the number and proportion of mapped reads between three different runs were also concordant. The percentage of mapped reads ranged from 14.05% to 31.04%, 15.14% to 31.98%, and 14.21% to 33.05% (1st, 2nd, and 3rd run, respectively). This demonstrated that NGS coupled with rRNA mtDNA gene could be reliably implemented as a routine testing. This developed technique can be applied to control and monitor meat adulterations in halal meat production and industry.
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