This paper describes a short-amplicon-based TaqMan probe quantitative real-time PCR (qPCR) assay for the quantitative detection of canine meat in chicken nuggets, which are very popular across the world, including Malaysia. The assay targeted a 100-bp fragment of canine cytb gene using a canine-specific primer and TaqMan probe. Specificity against 10 different animals and plants species demonstrated threshold cycles (Ct) of 16.13 ± 0.12 to 16.25 ± 0.23 for canine DNA and negative results for the others in a 40-cycle reaction. The assay was tested for the quantification of up to 0.01% canine meat in deliberately spiked chicken nuggets with 99.7% PCR efficiency and 0.995 correlation coefficient. The analysis of the actual and qPCR predicted values showed a high recovery rate (from 87% ± 28% to 112% ± 19%) with a linear regression close to unity (R(2) = 0.999). Finally, samples of three halal-branded commercial chicken nuggets collected from different Malaysian outlets were screened for canine meat, but no contamination was demonstrated.
Food forgery has posed considerable risk to public health, religious rituals, personal budget and wildlife. Pig, dog, cat, rat and monkey meat are restricted in most religions, but their sporadic adulteration are rampant. Market controllers need a low-cost but reliable technique to track and trace suspected species in the food chain. Considering the need, here we documented a lab-on-a-chip-based multiplex polymerase chain reaction (PCR) assay for the authentication of five non-halal meat species in foods. Using species-specific primers, 172, 163, 141, 129 and 108-bp sites of mitochondrial ND5, ATPase 6 and cytochrome b genes were amplified to detect cat, dog, pig, monkey and rat species under complex matrices. Species-specificity was authenticated against 20 different species with the potential to be used in food. The targets were stable under extreme sterilisation (121°C at 45 psi for 2.5 h) which severely degrades DNA. The assay was optimised under the backgrounds of various commercial meat products and validated for the analysis of meatballs, burgers and frankfurters, which are popular fast food items across the globe. The assay was tested to detect 0.1% suspected meats under commercial backgrounds of marketed foods. Instead of simplex PCR which detects only one species at a time, such a multiplex platform can reduce cost by at least fivefolds by detecting five different species in a single assay platform.
A sensitive and selective gas chromatography with mass spectrometry method was developed for the simultaneous determination of three organophosphorus pesticides, namely, chlorpyrifos, malathion, and diazinon in three different food commodities (milk, apples, and drinking water) employing solid-phase extraction for sample pretreatment. Pesticide extraction from different sample matrices was carried out on Chromabond C18 cartridges using 3.0 mL of methanol and 3.0 mL of a mixture of dichloromethane/acetonitrile (1:1 v/v) as the eluting solvent. Analysis was carried out by gas chromatography coupled with mass spectrometry using selected-ion monitoring mode. Good linear relationships were obtained in the range of 0.1-50 μg/L for chlorpyrifos, and 0.05-50 μg/L for both malathion and diazinon pesticides. Good repeatability and recoveries were obtained in the range of 78.54-86.73% for three pesticides under the optimized experimental conditions. The limit of detection ranged from 0.02 to 0.03 μg/L, and the limit of quantification ranged from 0.05 to 0.1 μg/L for all three pesticides. Finally, the developed method was successfully applied for the determination of three targeted pesticides in milk, apples, and drinking water samples each in triplicate. No pesticide was found in apple and milk samples, but chlorpyrifos was found in one drinking water sample below the quantification level.
Wider availability but lack of legal market trades has given feline meat a high potential for use as an adulterant in common meat and meat products. However, mixing of feline meat or its derivatives in food is a sensitive issue, since it is a taboo in most countries and prohibited in certain religions such as Islam and Judaism. Cat meat also has potential for contamination with of severe acute respiratory syndrome, anthrax and hepatitis, and its consumption might lead to an allergic reaction. We developed a very short-amplicon-length (69 bp) PCR assay, authenticated the amplified PCR products by AluI-restriction digestion followed by its separation and detection on a lab-on-a-chip-based automated electrophoretic system, and proved its superiority over the existing long-amplicon-based assays. Although it has been assumed that longer DNA targets are susceptible to breakdown under compromised states, scientific evidence for this hypothesis has been rarely documented. Strong evidence showed that shorter targets are more stable than the longer ones. We confirmed feline-specificity by cross-challenging the primers against 10 different species of terrestrial, aquatic and plant origins in the presence of a 141-bp site of an 18S rRNA gene as a universal eukaryotic control. RFLP analysis separated 43- and 26-bp fragments of AluI-digest in both the gel-image and electropherograms, confirming the original products. The tested detection limit was 0.01% (w/w) feline meat in binary and ternary admixed as well as meatball matrices. Shorter target, better stability and higher sensitivity mean such an assay would be valid for feline identification even in degraded specimens.
This paper examines the processing steps of extracting palm oil from fresh fruit bunches in a way that may impact on the formation of chloropropandiol fatty esters (3-MCPD esters), particularly during refining. Diacylglycerols (DAGs) do not appear to be a critical factor when crude palm oils are extracted from various qualities of fruit bunches. Highly hydrolysed oils, in spite of the high free fatty acid (FFA) contents, did not show exceptionally high DAGs, and the oils did not display a higher formation of 3-MCPD esters upon heat treatment. However, acidity measured in terms of pH appears to have a strong impact on 3-MCPD ester formation in the crude oil when heated at high temperatures. The differences in the extraction process of crude palm oil from current commercial processes and that from a modified experimental process showed clearly the effect of acidity of the oil on the formation of 3-MCPD esters. This paper concludes that the washing or dilution step in palm oil mills removes the acidity of the vegetative materials and that a well-optimised dilution/washing step in the extraction process will play an important role in reducing formation of 3-MCPD esters in crude palm oil upon further heat processing.
This study was conducted to investigate on the effect of different sampling regions of palm-refined oils and fats on the 2- and 3-monochloropropanediol fatty acid esters (MCPDE) and glycidol fatty acid esters (GE) levels. The American Oil Chemists' Society (AOCS) Official Method Cd 29a-13 on the determination of MCPDE and GE in edible oils and fats by acid transesterification was successfully verified and optimised, with slight modification using 7890A Agilent GC system equipped with 5975C quadrupole detector. The determined limits of detection (LOD) for MCPDE were 0.02 mg kg-1 and 0.05 mg kg-1 for GE. The method performance has showed good recovery between 80% and 120% for all pertinent compounds with seven replicates assayed in three separate days. Round robin test with two European laboratories, i.e. Eurofins and SGS, has shown compliance results with those of the present study. Among the sampling regions, only one refinery located in the central region of Malaysia showed a significant increment of the MCPDE and GE levels after refining process. The GE level averaging at 2.5 mg kg-1 was slightly higher than that of 3-MCPDE averaging at 1.3 mg kg-1. Both esters were preferentially partitioned into the liquid phase rather than the solid phase after fractionation. However, the overall results exhibited no direct correlation between the esters content and the different sampling locations of the palm oil products in Malaysia. Analysis of total chlorine content also displayed significant variations between sampling locations which clearly show its effect on the chlorine content in the CPO samples.
As a widely consumed beverage, coffee tends to be a target for intentional adulteration. This study describes the application of modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) coupled to liquid chromatography-high-resolution mass spectrometry (LC-HRMS) for simultaneous screening, identification, and quantification of undeclared phosphodiesterase 5 (PDE5) inhibitors in instant coffee premixes (ICPs). The mass spectrometer was operated in auto MS/MS acquisition for simultaneous MS and MS/MS experiments. Qualitative establishments from the suspected-target screening and targeted identification processes led to an unambiguous analyte assignment from the protonated molecule ([M+H]+) precursor ion which is subsequently used for quantification of 23 targeted PDE5 inhibitors. The analytical method validation covered specificity, linearity, range, accuracy, limit of detection (LOD), limit of quantification (LOQ), precisions, matrix effect (ME), and extraction recovery (RE). The specificity was established using the optimised chromatographic separation as well as the distinguishable [M+H]+ precursor ion. The linearity of each target analyte was demonstrated with a coefficient of determination (r2) of >0.9960 over the expected range of sample concentrations. The accuracy ranged from 88.1%-119.3% with LOD and LOQ of <70 ng/mL and 80 ng/mL, respectively. Excellent precisions were established within 0.4%-9.1% of the relative standard deviation. An insignificant ME within -5.2% to +8.7% was achieved using three different strategies of chromatography, sample extraction, and sample dilution. The RE was good for all target analytes within 84.7%-123.5% except for N-desethylacetildenafil at low (53.8%) and medium (65.1%) quality control levels. The method was successfully applied to 25 samples of ICPs where 17 of them were found to be adulterated with PDE5 inhibitors and their analogues. Further quantification revealed the total amount of these adulterants ranged from 2.77 to 121.64 mg per sachet.
A polymerase chain reaction (PCR) assay for the assessment of dog meat adulteration in meatballs was developed. The assay selectively amplified a 100-bp region of canine mitochondrial cytochrome b gene from pure, raw, processed and mixed backgrounds. The specificity of the assay was tested against 11 animals and 3 plants species, commonly available for meatball formulation. The stability of the assay was proven under extensively autoclaving conditions that breakdown target DNA. A blind test from ready to eat chicken and beef meatballs showed that the assay can repeatedly detect 0.2% canine meat tissues under complex matrices using 0.04 ng of dog DNA extracted from differentially treated meatballs. The simplicity, stability and sensitivity of the assay suggested that it could be used in halal food industry for the authentication of canine derivatives in processed foods.
The presence and distribution of endocrine-disrupting chemicals (EDCs) in the mariculture fish from Pulau Kukup, Johor of Malaysia have been studied along with the impact on human health. Six different species of mariculture fish were collected, due to their high consumption in the Asian region-especially Malaysia, to assess their levels of EDCs. The highest concentration of EDCs detected in the muscle was dexamethasone (2.37-15.84 ng/g) and (0.77-13.41 ng/g), in the liver was dexamethasone (<2.54-43.56 ng/g) and progesterone (2.23-9.78 ng/g), and in the reproductive organ are dexamethasone (<2.54-37.23 ng/g) and caffeine (0.21-18.92 ng/g). The human health risk assessment in the current study suggested that there is no potential risk to the consumer because the hazard index was below 1 (HI
5-Hydroxymethylfurfural (HMF) content is an indicator of the purity of honey. High concentrations of HMF in honey indicate overheating, poor storage conditions and old honey. This study investigated the HMF content of nine Malaysian honey samples, as well as the correlation of HMF formation with physicochemical properties of honey. Based on the recommendation by the International Honey Commission, three methods for the determination of HMF were used: (1) high performance liquid chromatography (HPLC), (2) White spectrophotometry and (3) Winkler spectrophotometry methods. HPLC and White spectrophotometric results yielded almost similar values, whereas the Winkler method showed higher readings. The physicochemical properties of honey (pH, free acids, lactones and total acids) showed significant correlation with HMF content and may provide parameters that could be used to make quick assessments of honey quality. The HMF content of fresh Malaysian honey samples stored for 3-6 months (at 2.80-24.87 mg/kg) was within the internationally recommended value (80 mg/kg for tropical honeys), while honey samples stored for longer periods (12-24 months) contained much higher HMF concentrations (128.19-1131.76 mg/kg). Therefore, it is recommended that honey should generally be consumed within one year, regardless of the type.
This study determined the heavy metals (HMs) accumulation in different vegetables in different seasons and attributed a serious health hazard to human adults due to the consumption of such vegetables in Jhansi. The total amounts of zinc (Zn), lead (Pb), nickel (Ni), manganese (Mn), copper (Cu), cobalt (Co), and cadmium (Cd) were analysed in 28 composite samples of soil and vegetables (Fenugreek, spinach, eggplant, and chilli) collected from seven agricultural fields. The transfer factor (TF) of HMs from soil to analysed vegetables was calculated, and significant non-carcinogenic health risks due to exposure to analysed heavy metals via consumption of these vegetables were computed. The statistical analysis involving Principal Component Analysis (PCA) and Pearson's correlation matrix suggested that anthropogenic activities were a major source of HMs in the study areas. The target hazard quotient of Cd, Mn, and Pb for fenugreek (2.156, 2.143, and 2.228, respectively) and spinach (3.697, 3.509, 5.539, respectively) exceeded the unity, indicating the high possibilities of non-carcinogenic health risks if regularly consumed by human beings. This study strongly suggests the continuous monitoring of soil, irrigation water, and vegetables to prohibit excessive accumulation in the food chain.
Metal contaminations in commercial fish have become a great public health concern worldwide including Bangladesh. The current study was conducted to provide preliminary evidence of nine metals in three commercially significant fish namely Pampus argenteus, Sardinella longiceps and Tenualosa ilisha collected from four coastal stations- Kuakata, Pathorghata, Cox's Bazar, and Pirojpur, and eight stations of five rivers- Padma, Meghna, Jamuna, Katcha, and Nobogonga in Bangladesh. High magnitudes of Pb (0.74-4.59 mg/kg ww), Cd (0.07-0.24 mg/kg ww), and Mn (0.45-2.03 mg/kg ww) were recorded in the sampling stations that exceeded the maximum permissible limits (MPL) proposed by different recognized organizations. Significant mean differences of metal concentrations were observed (p
The study aimed to establish the detection method for bound 3-, 2-MCPD, and glycidol using accelerated solvent extraction (ASE) and gas chromatography mass spectrometry (GC-MS). The ASE was modified for reduced solvent volume and process time to extract lipid from the chocolate spread, infant formula, potato chips, and sweetened creamer. The solvent selected for ASE was a mixture of iso-hexane and acetone at 100°C with the lipid and analyte recovery ranging from 96.9% to 98.6% and 84.1% to 107.5%, respectively. The derivatisation of analytes was adopted from the AOCS method Cd29a-13 for GC-MS analysis. The results showed that the coefficient of determination (R2) of all analytes was >0.99. The limit of detection (LOD) was 0.1 mg kg-1 expressed in lipid basis for both bound 3- and 2-MCPD and 0.2 mg kg-1 expressed in lipid basis for bound glycidol. The limit of quantitation (LOQ) was 0.3 mg kg-1 expressed in lipid basis for both bound 3- and 2-MCPD and 0.6 mg kg-1 expressed in lipid basis for bound glycidol. A blank spiked with 3-monochloropropanediols fatty acid esters (MCPDE) and 2-MCPDE (0.3, 2.1, and 7.2 mg kg-1) and glycidol esters (0.6, 4.7, and 16.6 mg kg-1) were chosen for accuracy and precision tests. The recoveries were 91.7% to 105.9%. Both repeatability and within-laboratory reproducibility of the analysis were within the acceptable level of precision ranging from 1.7% to 16%. This is the first time that a full validation procedure extending to both accuracy and precision tests has been carried out for sweetened creamer and chocolate spread. Overall, the combined protocol of ASE and AOCS Cd29a-13 was successfully validated for both solid and liquid food samples with lipid content from 10% to 30%.
3-Monochloropropane-1,2-diol (3-MCPD) esters and glycidyl esters (GE) are heat-induced contaminants which form during oil refining process, particularly at the high temperature deodorization stage. It is worth to investigate the content of 3-MCPD and GE in fries which also involved high temperature. The content of 3-MCPD esters and GE were monitored in fries. The factors that been chosen were temperature and duration of frying, and different concentration of salt (NaCl). The results in our study showed that the effect was in the order of concentration of sodium chloride food during frying should be monitored.
Short wave near infrared spectroscopy (NIR) method was used to detect the presence of lard adulteration in palm oil. MicroNIR was set up in two different scan modes to study the effect of path length to the performance of spectral measurement. Pure and adulterated palm oil sample were classified using soft independent modeling class analogy (SIMCA) algorithm with model accuracy more than 0.95 reported for both transflectance and transmission modes. Additionally, by employing partial least square (PLS) regression, the coefficient of determination (R2) of transflectance and transmission were 0.9987 and 0.9994 with root mean square error of calibration (RMSEC) of 0.5931 and 0.6703 respectively. In order to remove the uninformative variables, variable selection using cumulative adaptive reweighted sampling (CARS) has been performed. The result of R2 and RMSEC after variable selection for transflectance and transmission were improved significantly. Based on the result of classification and quantification analysis, the transmission mode has yield better prediction model compared to the transflectance mode to distinguish the pure and adulterated palm oil.
Cattle, buffalo, and porcine materials are widely adulterated, and their quantification might safeguard health, religious, economic, and social sanctity. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays have been documented but they are just suitable for identification, cannot quantify adulterations. We described here a quantitative tetraplex real-time PCR assay with TaqMan Probes to quantify contributions from cattle, buffalo, and porcine materials simultaneously. Amplicon-sizes were very short (106-, 90-, and 146-bp for cattle, buffalo, and porcine) because longer targets could be broken down, bringing serious ambiguity in molecular diagnostics. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 27 frankfurters and 27 meatballs reflected 84-115% target recovery at 0.1-10% adulterations. Finally, a test of 36 commercial products revealed 71% beef frankfurters, 100% meatballs, and 85% burgers contained buffalo adulteration, but no porcine was found in beef products.
A contaminated aquatic environment may end up in the food chain and pose risks to tourist health in a tourist destination. To assess the health risk for tourists that visit St. Martine Island, which is a popular domestic and foreign tourist destination in Bangladesh, a study is undertaken to analyse the level of heavy metal contamination from chromium (Cr), manganese (Mn), copper (Cu), zinc (Zn), arsenic (As), cadmium (Cd), lead (Pb), mercury (Hg) and iron (Fe) in six of the most consumed fish (L. fasciatus, R. kanagurta, H. nigrescens, P. cuneatus, P. annularis and S. rubrum) and five crustacean species, which consist of a shrimp (P. sculptilis), a lobster (P. versicolor) and three crabs (P. sanguinolentus, T. crenata and M. victor) captured. The samples were analysed for trace metals using atomic absorption spectrometer, and the concentrations of the metals were interpreted using the United State Environmental Protection Agency (USEPA) health risk model. The muscle and carapace/exoskeleton of shrimp, lobster and crabs were analysed and contained various concentrations of Pb, Hg, As, Cr, Cd, Fe, Cu, Zn and Mn. The hierarchy of the heavy metal in marine fish is Fe > Cd > Zn > Pb > Cu > Cr > Mn > Hg. The concentrations of Pb in the species R. kanagurta, H. nigresceus and S. rubrum were above the food safety guideline by Australia, New Zealand and other legislations in most marine fish and crustaceans. Crabs showed higher mean heavy metal concentrations than shrimp and lobster. Acceptable carcinogen ranges were observed in three fish species (R. kanagurata, H. nigresceus and S. rubrum) and one crustacean species (P. sculptilis) samples.
The present study, aimed at observing the total concentration of mercury (Hg) in edible finfish species with an implication to human health risk, was carried out from the Setiu mangrove wetlands on the east coast of Peninsular Malaysia. Out of 20 species observed, the highest Hg concentrations were found among carnivores-fish/invertebrate-feeders, followed by omnivores and carnivores-invertebrate-feeders, while the lowest concentrations in herbivores. The Hg concentrations varied widely with fish species and body size, from 0.12 to 2.10 mg/kg dry weight. A positive relationship between body weight and Hg concentration was observed in particular for Toxotes jaculatrix and Tetraodon nigroviridis. Besides the permissible range of Hg concentration up to 0.3 mg/kg (cf. United States Environmental Protection Agency (USEPA)) in majority of species, the carnivore feeders such as Acanthopagrus pacificus, Gerres filamentosus, and Caranx ignobilis have shown excess amounts (> 0.40 mg/kg flesh weight) that raising concerns over the consumption by local people. However, the weekly intake of mercury-estimated through the fish consumption in all three trophic levels-suggests that the present Hg concentrations are still within the range of Provisional Tolerable Weekly Intake (PTWI) reported by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Perhaps, a multi-species design for Hg monitoring at Setiu wetlands would be able to provide further insights into the level of toxicity transfer among other aquatic organisms and thereby a strong health risk assessment for the local communities.
Peanuts are widely consumed as the main ingredient in many local dishes in Malaysia. However, the tropical climate in Malaysia (high temperature and humidity) favours the growth of fungi from Aspergillus section Flavi, especially during storage. Most of the species from this section, such as A. flavus, A. parasiticus and A. nomius, are natural producers of aflatoxins. Precise identification of local isolates and information regarding their ability to produce aflatoxins are very important to evaluate the safety of food marketed in Malaysia. Therefore, this study aimed to identify and characterize the aflatoxigenic and non-aflatoxigenic strains of Aspergillus section Flavi in peanuts and peanut-based products. A polyphasic approach, consisting of morphological and chemical characterizations was applied to 128 isolates originating from raw peanuts and peanut-based products. On the basis of morphological characters, 127 positively identified as Aspergillus flavus, and the other as A. nomius. Chemical characterization revealed six chemotype profiles which indicates diversity of toxigenic potential. About 58.6%, 68.5%, and 100% of the isolates are positive for aflatoxins, cyclopiazonic acid and aspergillic acid productions respectively. The majority of the isolates originating from raw peanut samples (64.8%) were aflatoxigenic, while those from peanut-based products were less toxigenic (39.1%). The precise identification of these species may help in developing control strategies for aflatoxigenic fungi and aflatoxin contamination in peanuts, especially during storage. These findings also highlight the possibility of the co-occurrence of other toxins, which could increase the potential toxic effects of peanuts.
The detection of 3- and 2-MCPD ester and glycidyl ester was transformed from selected ion monitoring (SIM) mode to multiple reaction monitoring (MRM) mode by gas chromatography triple quadrupole spectrometry. The derivatization process was adapted from AOCS method Cd 29a-13. The results showed that the coefficient of determination (R2) of all detected compounds obtained from both detection mode was comparable, which falls between 0.997 and 0.999. The limit of detection and quantification (LOD and LOQ) were improved in MRM mode as compared to SIM mode. In MRM mode, the LOD of 3- and 2-MCPD ester was achieved 0.01 mg/kg while the LOQ was 0.05 mg/kg. Besides, LOD and LOQ of glycidyl ester were 0.024 and 0.06 mg/kg respectively. A blank spiked with MCPD esters (0.03, 0.10 and 0.50 mg/kg) and GE (0.06, 0.24 and 1.20 mg/kg) were chosen for repeatability and recovery tests. MRM mode showed better repeatability in area ratio and recovery with relative standard deviation (RSD %) food samples from different category were performed by repeated injections in both detection modes. Briefly, the contaminants from crude palm oil, mustard and olive oil were present in minute amount which below the LOD or LOQ in both detection modes. Sample from chocolate and infant formula products showed certain level of MCPD esters and GE, and their detection was more precisely quantitated based on MRM mode. Besides, margarine products showed a higher level of contaminations due to the high fat content in these products. MRM mode detection was proven to provide precise data with low RSD % in different food matrices. MRM mode detection was robust and selective for MCPD esters and GE analyses, it should be applied to determine the concentration of MCPD esters and GE contaminations in food.