The peanut supply chain in Malaysia is dominated by three main stakeholders (importers, manufacturers, retailers). The present study aimed to determine the levels and critical points of aflatoxin and fungal contamination in peanuts along the supply chain. Specifically, two types of raw peanuts and six types of peanut-based products were collected (N = 178). Samples were analysed for aflatoxins by using high-performance liquid chromatography. Results revealed that the aflatoxin contamination was significantly higher (P ≤ 0.05) in raw peanuts and peanut-based products from the retailers. However, there was no significant difference (P ≥ 0.05) in fungal contamination for both types of peanuts except for the total fungal count in raw peanuts from the retailers. Furthermore, raw peanut kernels from the retailers were the most contaminated ones ranged from
An average 50 ml breast milk samples were collected from 21 lactating primiparous mothers (range 25 to 45 years, mean 33 years), 4-8 weeks after delivery in Penang Island, Malaysia. The geometric mean concentration of the most toxic congeners, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) was 0.14 pg WHO2005-TEQ g-1 zlipid. The most abundant congeners of PCDD/Fs were octachlorodibenzo-p-dioxin (OCDD) (5.9-75.4%), followed by 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (1,2,3,4,6,7,8-HpCDD) (1.1-30.7%). The geometric mean level of total dioxins and dl-PCBs was 2.2 pg WHO2005-TEQ g-1 lipid, significantly lower than those in developed countries or highly contaminated areas. The total dioxins and dl-PCBs in pg WHO2005-TEQ levels in breast milk were significantly correlated with years of residence at potential contaminated site. The average daily intake of 11.8 pg WHO2005-TEQ kg-1 body weight was estimated for a breastfed infant at 6 months of age. This demonstrates the exposure risk to infants, especially from Penang region, to these pollutants from human milk intake are potentially high during the lactation period.
Lateral flow devices (LFDs) are straightforward scientific tools that have made substantial advances in recent years. They have been used in many fields including the meat industry to detect disease markers, determine meat freshness or meat species determination. They are, therefore, significant in the research of meat adulteration by mixed animal species, because food component authenticity is a serious concern encompassing health, economic, legal, and religious issues. Pork adulteration is one of the most crucial issues in the global meat industry. In this review, we discuss the various types of LFDs and recent research on the development of LFDs as an authenticity tool for detecting pig additives in meat-based products, and how regulatory authorities could adopt LFDs for their workflows. Despite the benefits of rapidity, simplicity, low cost, high sensitivity, and specificity, researchers face challenges when using LFD as a final confirmation test. Future directions are suggested for globalising the use of LFD as a halal authentication method.
Food contaminated with heavy metals poses a serious threat to consumers. This study aims to assess levels of lead (Pb) and cadmium (Cd) in offals of chicken, cattle, and pig as well as tin (Sn) in canned food. A total of 378 offal samples was collected from wet markets, while 218 canned food samples were purchased locally. Samples were digested using a microwave before analysis with inductively coupled plasma mass spectrometry (ICP-MS). Pb was determined, highest in cattle lung (0.11 ± 0.20 mg/kg) followed by cattle spleen (0.09 ± 0.14 mg/kg), and cattle tripe (0.09 ± 0.12 mg/kg). For Cd, the highest concentrations were in cattle liver (0.13 ± 0.12 mg/kg), pig liver (0.08 ± 0.05 mg/kg), and chicken liver (0.03 ± 0.02 mg/kg). Significant variations of Sn levels existed in different canned food categories with 3.21% samples (n = 7) exceeded the maximum level of 250 mg/kg set by Codex Alimentarius. All offal samples were below the Malaysian regulatory limits, indicating their safety for human consumption. However, Sn levels varied significantly among canned food categories, with the highest levels found in canned pineapple chunks in syrup, mixed pineapple cubes in syrup, pineapple slices and longan. Samples exceeding the maximum level set by Codex Alimentarius may pose a risk to consumers.
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%.
Natural antioxidants in spices and herbs have attracted considerable attention as potential inhibitors against the formation of mutagenic heterocyclic amines (HCAs) in heat-processed meat. In this study, the inhibitory activity of four spices/herbs and their mixtures on HCAs formation in grilled beef were examined. A simplex centroid mixture design with four components comprising turmeric, curry leaf, torch ginger and lemon grass in 19 different proportions were applied on beef samples before grilling at 240 ºC for 10 min. The HCAs were extracted from the samples using solid phase extraction (SPE) method and analysed using Liquid chromatography mass spectrometry LC-MS/MS. All spices/herbs in single or mixture forms were found to reduce total HCA concentrations in marinated grilled beef ranging from 21.2% for beef marinated with curry leaf to 94.7% for the combination of turmeric and lemon grass (50:50 w/w). At the optimum marinade formula (turmeric: lemon grass 52.4%: 47.6%), concentration of 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), Harman, Norharman and AαC were 2.2, 1.4, 0.5, 2.8 and 1.2 ng/g, respectively. The results of the mutagenic activity demonstrated that this optimised marinade formula significantly (p
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.
Camel meat production for human consumption and pet food manufacture accounts for a relatively small part of overall red meat production in Australia. Reliable statistical data for the Australian production and consumption of camel meat are not available; however, it is estimated that 300,000 feral camels roam within the desert of central Australia, with an annual usage of more than 3000 camels for human consumption, 2000 for pet food manufacture and a smaller number for live export. Despite a small Australian camel meat production level, the usage of camel meat for pet food has been restricted in recent years due to reports of serious liver disease and death in dogs consuming camel meat. This camel meat was found to contain residues of indospicine, a non-proteinogenic amino acid found in certain Indigofera spp., and associated with mild to severe liver disease in diverse animals after dietary exposure to this hepatotoxin. The extent of indospicine-contaminated Australian camel meat was previously unknown, and this study ascertains the prevalence of such residue in Australian camel meat. In this study, indospicine levels in ex situ (95 samples collected from an abattoir in Queensland) and in situ (197 samples collected from camels after field culling in central Australia) camel meat samples were quantitated using a validated ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The quantitation results showed 46.7% of the in situ- and 20.0% of the ex situ-collected camel meat samples were contaminated by indospicine (more than the limit of detection (LOD) of 0.05 mg kg(-1) fresh weight). The overall indospicine concentration was higher (p < 0.05) in the in situ-collected samples. Indospicine levels detected in the present study are considered to be low; however, a degree of caution must still be exercised, since the tolerable daily intake for indospicine is currently not available for risk estimation.
A study to quantify the free glutamic acid content of six processed foods, 44 dishes and 26 condiments available in Malaysia was performed using high-performance liquid chromatography with a fluorescence detector (HPLC-FRD). Recovery tests were carried out with spiked samples at levels from 6 to 31 mg g(-1). High recovery in different matrices was achieved ranging from 88% +/- 13% to 102% +/- 5.12%, with an average of 97% +/- 8.92%. Results from the study revealed that the average free glutamic acid content ranged from 0.34 +/- 0.20 to 4.63 +/- 0.41 mg g(-1) in processed foods, while in prepared dishes it was as low as 0.24 +/- 0.15 mg g(-1) in roti canai (puffed bread served with curry or dhal) to 8.16 +/- 1.99 mg g(-1) in dim sum (a small casing of dough, usually filled with minced meat, seafood, and vegetables, either steamed or fried). Relatively, the content of free glutamic acid was found to be higher in condiments at 0.28 +/- 0 mg g(-1) in mayonnaise to 170.90 +/- 6.40 mg g(-1) in chicken stock powder.
Detection of porcine plasma using indirect ELISA was developed using mAb B4E1 for the prevention of their usage in human food that creates religious and health conflicts. The immunoassay has a CV
Rabbit meat is receiving increasing attention because it contains a high level of proteins with relatively little fat. On the other hand, squirrel meat is served in upper-class meals in certain countries, so is sold at higher prices. The other side of the coin is rat meat, which has family ties with rabbit and squirrel but poses substantial threats to public health because it is a potential carrier of several zoonotic organisms. Recently, rat meat was mislabelled and sold as lamb after chemical modification. Thus, the chances of rabbit and squirrel meat substitution by rat meat cannot be ruled out. For the first time, a multiplex PCR assay was developed in Malaysia for the discriminatory identification of rat, rabbit and squirrel in the food chain. Rabbit (123 bp), rat (108 bp) and squirrel (243 bp) targets were amplified from ATP6 and cytb genes, along with a eukaryotic internal control (141bp). The products were sequenced and cross-tested against 22 species. A total of 81 reference samples and 72 meatball specimens were screened to validate the assay. Analyte stability was evaluated through boiling, autoclaving and micro-oven cooking. The tested lower limits of detection were 0.01 ng DNA for pure meat and 0.1% for meatballs.
The inclusion of ingredients derived from pigs in highly processed consumer products poses a significant challenge for DNA-targeted analytical enforcement, which could be overcome by using digital PCR. However, most species detection methods use digital PCR to target single-copy nuclear genes, which limits their sensitivity. In this work, we examined the performance of a nanoplate-based digital PCR method that targets multi-copy nuclear (MPRE42) and mitochondrial (Cytb) genes. Poor separation of positive and negative partitions, as well as a 'rain effect' were obtained in the porcine-specific MPRE42 assay. Among the optimization strategies examined, the inclusion of restriction enzymes slightly improved the separation of positive and negative partitions, but a more extensive 'rain effect' was observed. The high copy number of the MPRE42 amplicon is hypothesized to contribute to the saturation of the positive signal. In contrast, the porcine-specific Cytb assay achieved perfect separation of positive and negative partitions with no 'rain effect'. This assay can detect as little as 0.4 pg of pork DNA, with a sensitivity of 0.05% (w/w) in a pork-chicken mixture, proving its applicability for detecting pork in meat and meat-based products. For the MPRE42 assay, potential applications in highly degraded products such as gelatin and lard are anticipated.
The analysis of aflatoxins (B1, B2, G1 and G2) and ochratoxin A (OTA) was performed in processed spices marketed in Penang, Malaysia, using immunoaffinity columns and HPLC equipped with fluorescence detector (HPLC-FD). The processed powdered spices analysed include dried chilli, fennel, cumin, turmeric, black and white pepper, poppy seed, coriander, 'garam masala', and mixed spices for fish, meat and chicken curry. Two different studies were carried out. The limit of detection (LOD) was 0.01 ng g(-1) for each aflatoxin (AF) and 0.10 ng g(-1) for OTA (signal-to-noise ratio = 3:1). In the first study, 34 commercial processed spices analysed with a mean level, range and incidence of positive samples for total AF were 1.61 ng g(-1), 0.01-9.34 ng g(-1) and 85%, respectively, and for AFB1 were 1.38 ng g(-1), 0.01-7.68 ng g(-1) and 85%, respectively. The mean level, range and incidence of positive samples for OTA were 2.21 ng g(-1), 0.14-20.40 ng g(-1) and 79%, respectively. Natural co-occurrence of AF and OTA was found in 25 (74%) samples. In the second study of 24 commercial processed spices, the mean level, range and incidence of positive samples for total AF were 8.38 ng g(-1), 0.32-31.17 ng g(-1) and 88%, respectively, and for AFB1 were 7.31 ng g(-1), 0.32-28.43 ng g(-1) and 83%, respectively. Fifteen positive samples for total AF and two positive samples for OTA exceeded the permissible Malaysian limit of 5 ng g(-1). Contamination of both mycotoxins in spices may represent another route of exposure to consumers due to their frequent and prolonged consumption, as spices are common ingredients in popular dishes among Asian countries.
Gelatin is widely used in pharmaceuticals as a protective coating, such as soft and hard capsule shells. However, the animal source of gelatin is a sensitive issue because certain gelatins such as porcine and bovine gelatins are not welcome in Halal, Kosher and Hindus' consumer goods. Recently, we have documented DNA barcoding and multiplex PCR platforms for discriminating porcine, bovine and fish gelatins in various fish and confectionary products; but those assays were not self-authenticating and also not tested in highly refined pharmaceutical products. To address this knowledge gap, here we report a self-authenticating multiplex PCR-restriction fragment length polymorphism (RFLP) assay to identify animal sources of various gelatin in pharmaceutical capsules. Three different restriction enzymes, BsaAI, Hpy188I and BcoDI were used to yield distinctive RFLP patterns for gelatin-based bovine (26, 94 bp), fish (97, 198 bp) and porcine (17, 70 bp) DNA in control experiments. The specificity was cross-tested against 16 non-target species and the optimised assay was used to screen gelatin sources in 30 halal-branded pharmaceuticals capsule shells. Bovine and porcine DNA was found in 27 and 3 of the 30 different capsules products. The assay was suitable for detecting 0.1 to 0.01 ng total DNA extracted from pure and mixed gelatins. The study might be useful to authenticate and monitor halal, kosher, vegetarian and Hindu compliant pharmaceuticals, foods and cosmetics.
The presence of 3-monochloropropanediol esters (3-MCPDE), 2-monochloropropanediol esters (2-MCPDE) and glycidyl esters (GE) in infant formula products has raised serious concerns. They incorporate vegetable oils, particularly palm-based oils, which are well-known to contain large amounts of these process contaminants. An analysis was conducted on infant formula samples (n = 16) obtained from the Malaysian market to determine the levels of 3-MCPDE, 2-MCPDE and GE using gas chromatography-mass spectrometry (GC-MS). The method was validated, with a limit of quantification (LOQ) on instrument of 0.10 µg/g for all analytes. The median concentrations of 3-MCPDE, 2-MCPDE and GE in infant formula in this study were 0.008 µg/g, 0.003 µg/g and 0.002 µg/g respectively. The estimated dietary intakes calculated from consumption of infant formula show higher exposures to infants within the age group of 0 to 5 months, highest for GE (1.61 µg/kg bw/day), followed by 3-MCPDE (0.68 µg/kg bw/day) and 2-MCPDE (0.41 µg/kg bw/day) compared to the age group of 6 to 12 months. Only one sample, relating to GE exposure is a potential risk for both age groups with MOE value below 25,000.
Glycidyl esters (GEs) and 3-monochloropropanediol esters (3-MCPDEs) are process contaminants commonly found in refined edible oils which are often added to infant formulas. The Taiwan Food and Drug Administration (TFDA) launched regulations for GEs in infant formulas that went into effect on 1 July 2021. To investigate levels of GEs and 3-MCPDEs in infant formula powder, 45 products were sampled and analysed during 2020-2021. The contents of GEs and 3-MCPDEs in formulas of different brands significantly varied, but their concentrations in all of the formulas complied with European Union (EU) regulations. Infant formulas containing palm oil had significantly higher 3-MCPDE levels in both extracted oils and milk powder than those without palm oil. Concentrations of GEs and 3-MCPDEs in infant formula powder and extracted oils were significantly lower in products from Europe than those from Australia and New Zealand. Infants aged 0-1 years in Taiwan who consumed only infant formula showed a margin of exposure (MoE) exceeding 25,000. Mean consumer exposures to 3-MCPDEs stayed below the tolerable daily intake (TDI), while high exposures at the 95th percentile (P95) exceeded the TDI by 1.7-fold. Herein, we present the changing trends in the risk assessment results of infant formula across various countries in the decade. Implementation of regulations and mitigation strategy effectively reduced the risk of infants being exposed to GEs and 3-MCPDEs through infant formula.
This study aims to optimise the operating conditions for the supercritical fluid extraction (SFE) of toxic elements from fish oil. The SFE operating parameters of pressure, temperature, CO2 flow rate and extraction time were optimised using a central composite design (CCD) of response surface methodology (RSM). High coefficients of determination (R²) (0.897-0.988) for the predicted response surface models confirmed a satisfactory adjustment of the polynomial regression models with the operation conditions. The results showed that the linear and quadratic terms of pressure and temperature were the most significant (p < 0.05) variables affecting the overall responses. The optimum conditions for the simultaneous elimination of toxic elements comprised a pressure of 61 MPa, a temperature of 39.8ºC, a CO₂ flow rate of 3.7 ml min⁻¹ and an extraction time of 4 h. These optimised SFE conditions were able to produce fish oil with the contents of lead, cadmium, arsenic and mercury reduced by up to 98.3%, 96.1%, 94.9% and 93.7%, respectively. The fish oil extracted under the optimised SFE operating conditions was of good quality in terms of its fatty acid constituents.
A reversed-phase HPLC optimization strategy is presented for investigating the separation and retention behavior of aflatoxin B1, B2, G1, G2, ochratoxin A and zearalenone, simultaneously. A fractional factorial design (FFD) was used to screen the significance effect of seven independent variables on chromatographic responses. The independent variables used were: (X1) column oven temperature (20-40°C), (X2) flow rate (0.8-1.2 ml/min), (X3) acid concentration in aqueous phase (0-2%), (X4) organic solvent percentage at the beginning (40-50%), and (X5) at the end (50-60%) of the gradient mobile phase, as well as (X6) ratio of methanol/acetonitrile at the beginning (1-4) and (X7) at the end (0-1) of gradient mobile phase. Responses of chromatographic analysis were resolution of mycotoxin peaks and HPLC run time. A central composite design (CCD) using response surface methodology (RSM) was then carried out for optimization of the most significant factors by multiple regression models for response variables. The proposed optimal method using 40°C oven temperature, 1 ml/min flow rate, 0.1% acetic acid concentration in aqueous phase, 41% organic phase (beginning), 60% organic phase (end), 1.92 ratio of methanol to acetonitrile (beginning) and 0.2 ratio (end) for X1-X7, respectively, showed good prediction ability between the experimental data and predictive values throughout the studied parameter space. Finally, the optimized method was validated by measuring the linearity, sensitivity, accuracy and precision parameters, and has been applied successfully to the analysis of spiked cereal samples.
The presence of 3-monochloropropane-1,2 diol ester (3-MCPDE) and glycidyl ester (GE) in processed palm oils is of concern, as these oils are widely used for edible purposes. The mitigation method studied here optimizes the removal of chloride through water washing of crude palm oil (CPO), to limit the formation of 3-MCPDE. The contaminant removal obtained via washing CPO supports the quantitative findings. By utilizing 5% water in the washing step, water-soluble chlorides in CPO are removed by up to 76%, resulting in a 71% reduction of 3-MCPDE to within statutory limits. In this study, a linear correlation was developed between the chloride and the corresponding 3-MCPDE with a correlation coefficient (R2) of 0.99. Using the correlations, 1.0 mg/kg of 3-MCPDE in refined, bleached and deodorized palm oil (RBDPO) will be obtained from CPO with 1.2 mg/kg chloride with 7% wash water usage. The study also showed minor GE reduction between 7 and 11% was attained after water washing.
Response surface methodology (RSM) was used to determine the optimum experimental conditions to extract methylmercury from fish samples for GC analysis. The influence of four variables - acid concentration (3-12 M), cysteine concentration (0.5-2% w/v), solvent volume (3-9 ml) and extraction time (10-30 min) - on recovery of methylmercury was evaluated. The detection limit for methylmercury analysis using a microelectron capture detector was 7 ng g(-1) in fish samples. The mean recovery under optimum conditions was 94%. Experimental data were adequately fitted into a second-order polynomial model with multiple regression coefficients (r(2)) of 0.977. The four variables had a significant effect (p < 0.05) on the recovery of methylmercury from a reference material (BCR-463). Optimum conditions for methylmercury extraction were found using an acid concentration of 12.2 M, cysteine concentration of 2.4%, solvent volume of 1.5 ml and extraction time of 35 min. The validation of the developed method to analyze methylmercury in fish samples exhibited good agreement with mercury content in the samples.