Mislabelling in fish products is a highly significant emerging issue in world fish trade in terms of health and economic concerns. DNA barcoding is an efficient sequencing-based tool for detecting fish species substitution but due to DNA degradation, it is in many cases difficult to amplify PCR products of the full-length barcode marker (~650 bp), especially in severely processed products. In the present study, a pair of universal primers targeting a 198 bp sequence of the mitochondrial 16s rRNA gene was designed for identification of fish species in the processed fish products commonly consumed in Malaysia. The specificity of the universal primers was tested by both in-silico studies using bioinformatics software and through cross-reaction assessment by practical PCR experiments against the DNA from 38 fish species and 22 other non-target species (animals and plants) and found to be specific for all the tested fish species. To eliminate the possibility of any false-negative detection, eukaryotic endogenous control was used during specificity evaluation. The developed primer set was validated with various heat-treated (boiled, autoclaved and microwaved) fish samples and was found to show high stability under all processing conditions. The newly developed marker successfully identified 92% of the tested commercial fish products with 96-100% sequence similarities. This study reveals a considerable degree of species mislabelling (20.8%); 5 out of 24 fish products were found to be mislabelled. The new marker developed in this work is a reliable tool to identify fish species even in highly processed products and might be useful in detecting fish species substitution thus protecting consumers' health and economic interests.
The intake of heterocyclic amines is influenced by the amount and type of meat and fish ingested, frequency of consumption, cooking methods, cooking temperature, and duration of cooking. In this study, the dietary intake of heterocyclic amines in Malaysia and their main sources were investigated. Forty-two samples of meat and fish were analysed by high-performance liquid chromatography with photodiode array detector to determine the concentration of the six predominant heterocyclic amines, namely: 2-amino-3-methylimidazo[4,5-f] quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f] quinoline(MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f] quinoxaline (4,8-DiMeIQx), 2-amino-3,7,8-trimethylimidazo[4,5-f] quinoxaline (7,8-DiMeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Dietary intake data were obtained using a food-frequency questionnaire when interviewing 600 Malaysian respondents. The level of total heterocyclic amines in food samples studies ranged from not detected to 38.7 ng g(-1). The average daily intake level of heterocyclic amine was 553.7 ng per capita day(-1). The intake of PhIP was the highest, followed by MeIQx and MeIQ. The results reveal that fried and grilled chicken were the major dietary source of heterocyclic amines in Malaysia. However, the heterocyclic amine intake by the Malaysian population was lower than those reported from other regions.
A simple method for the reduction of aflatoxins B₁ (AFB₁), B₂ (AFB₂), G₁ (AFG₁), G₂ (AFG₂) and ochratoxin A (OTA) in white pepper was studied. Response surface methodology (RSM) was applied to determine the effect of four variables, which included time (20-60 min), temperature (30-70°C), calcium hydroxide (Ca(OH)₂) (0-1%) and hydrogen peroxide (H₂O₂) (1-3%) during the washing step of white pepper. The efficacy of the method was evaluated by the determination of mycotoxins by HPLC with fluorescence detection (FD). Statistical analysis showed that the experimental data could be adequately fitted into a second-order polynomial model, with a multiple regression coefficient (R²) in the range of 0.805-0.907 for AFG₂ and AFG₁, respectively. The optimal condition was 57.8 min, 62.0°C, of 0.6% (w/v) and 2.8% (v/v) for time, temperature, Ca(OH)₂ and H₂O₂ respectively. By applying the optimum condition, the mycotoxins reduction was found to be in the range of 68.5-100% for AFB₂ and AFG₁ respectively.
The effect of 18 different chemicals, which included acidic compounds (sulfuric acid, chloridric acid, phosphoric acid, benzoic acid, citric acid, acetic acid), alkaline compounds (ammonia, sodium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide), salts (acetate ammonium, sodium bisulfite, sodium hydrosulfite, sodium chloride, sodium sulfate) and oxidising agents (hydrogen peroxide, sodium hypochlorite), on the reduction of aflatoxins B(1), B(2), G(1) and G(2) and ochratoxin A (OTA) was investigated in black and white pepper. OTA and aflatoxins were determined using HPLC after immunoaffinity column clean-up. Almost all of the applied chemicals showed a significant degree of reduction on mycotoxins (p < 0.05). The lowest and highest reduction of aflatoxin B(1), which is the most dangerous aflatoxin, was 20.5% ± 2.7% using benzoic acid and 54.5% ± 2.7% using sodium hydroxide. There was no significant difference between black and white peppers (p < 0.05).
The extent of industrial trans fatty acids (TFA) in the food supply is unknown in Malaysia, whilst TFA disclosure on food labels is not mandatory by Malaysian food standards. Supermarket foods such as dairy products, fats and oils, meat products, snack foods, soups, and confectionery are commonly cited to be major contributors of TFA in the diet. A consumer survey (n = 622) was used to develop a food listing of these 'high risk' foods. TFA content of high-risk foods were analysed by gas chromatography. Food samples (n = 158) were analysed and their total TFA content were compared with Malaysian Food Standards. A wide variation in TFA content within food categories was indicated. Of the foods containing TFA, many food labels did not cite TFA content or the use of partially hydrogenated vegetable oils (PHVO) as an ingredient. Hypothesised estimates of TFA intake from these supermarket foods in a sample day's menu providing 2000 kcal projected a minimum intake of 0.5 g and a maximum intake of 5.2 g TFA. This study found there was no voluntary disclosure of TFA content on food labels or identifying PHVO as an ingredient. It appears that health education targeting consumers to minimise TFA consumption is required supported by mandatory PHVO disclosure on the food label.
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.
Halal authentication has become essential in the food industry to ensure food is free from any prohibited ingredients according to Islamic law. Diversification of food origin and adulteration issues have raised concerns among Muslim consumers. Therefore, verification of food constituents and their quality is paramount. From conventional methods based on physical and chemical properties, various diagnostic methods have emerged relying on protein or DNA measurements. Protein-based methods that have been used in halal detection including electrophoresis, chromatographic-based methods, molecular spectroscopy and immunoassays. Polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) are DNA-based techniques that possess better accuracy and sensitivity. Biosensors are miniatured devices that operate by converting biochemical signals into a measurable quantity. CRISPR-Cas is one of the latest novel emerging nucleic acid detection tools in halal food analysis as well as quantification of stable isotopes method for identification of animal species. Within this context, this review provides an overview of the various techniques in halal detection along with their advantages and limitations. The future trend and growth of detection technologies are also discussed in this review.
Chloride reduction in crude palm oil (CPO) of greater than 80% was achieved with water washing conducted at 90°C. Inorganic chloride content in CPO was largely removed through washing, with no significant reduction in the organic chloride. Phosphorous content of CPO reduced by 20%, while trace elements such as calcium, magnesium and iron were also reduced in the washing operation. The 3-MCPDE formed in the refined, bleached and deodorised palm oil displayed (RBDPO) a linear relationship with the chloride level in washed CPO, which could be represented by the equation y = 0.91x, where y is 3-MCPDE and x represents the chloride in RBDPO refined from washed CPO. In plant scale trials using 5% water at 90°C, mild acidification of the wash water at 0.05% reduced chloride by average 76% in washed CPO. Utilising selected bleaching earths, controlled wash water temperature and wash water volume produced low chloride levels in RBDPO. Chloride content less than 1.4 mg kg-1 in plant RBDPO production was achieved, through physical refining of washed CPO containing less than 2 mg kg-1 chloride and would correspond to 3-MCPDE levels of 1.25 mg kg-1 in RBDPO. The 3-MCPDE reduced further to 1.1 mg kg-1 as the chloride level of washed CPO decreased below 1.8 mg kg-1. Chloride has been shown to facilitate the 3-MCPDE formation and its removal in lab scale washing study has yielded lower 3-MCPDE levels formed in RBDPO. In actual plant operations using washed CPO, 3-MCPDE levels below 1.25 mg kg-1 were achieved consistently in RBDPO.
A total of 126 food samples, categorised into three groups (seafood and seafood products, meat and meat products, as well as milk and dairy products) from Malaysia were analysed for polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). The concentration of PCDD/Fs that ranged from 0.16 to 0.25 pg WHO05-TEQ g(-1) fw was found in these samples. According to the food consumption data from the Global Environment Monitoring System (GEMS) of the World Health Organization (WHO), the dietary exposures to PCDD/F from seafood and seafood products, meat and meat products, as well as milk and dairy products for the general population in Malaysia were 0.064, 0.183 and 0.736 pg WHO05-TEQ kg(-1) bw day(-1), respectively. However, the exposure was higher in seafood and seafood products (0.415 pg WHO05-TEQ kg(-1) bw day(-1)) and meat and meat products (0.317 pg WHO05-TEQ kg(-1) bw day(-1)) when the data were estimated using the Malaysian food consumption statistics. The lower exposure was observed in dairy products with an estimation of 0.365 pg WHO05-TEQ kg(-1) bw day(-1). Overall, these dietary exposure estimates were much lower than the tolerable daily intake (TDI) as recommended by WHO. Thus, it is suggested that the dietary exposure to PCDD/F does not represent a risk for human health in Malaysia.
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.
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.
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.
3-Monochloropropane-1,2-diol esters (3-MCPDE) are food contaminants commonly found in refined vegetable oils and fats, which have possible carcinogenic implications in humans. To investigate this clinically, we conducted an occurrence level analysis on eight categories of retail and cooked food commonly consumed in Malaysia. This was used to estimate the daily exposure level, through a questionnaire-based case-control study involving 77 subjects with renal cancer, with 80 matching controls. Adjusted Odds Ratio (AOR) was calculated using the multiple logistic regression model adjusted for confounding factors. A pooled estimate of total 3-MCPDE intake per day was compared between both groups, to assess exposure and disease outcome. Among the food categories analysed, vegetable fats and oils recorded the highest occurrence levels (mean: 1.91 ± 1.90 mg/kg), significantly more than all other food categories (p
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.
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
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.
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.
Method validation for quantitative analysis of aflatoxins (AFs), ochratoxin A (OTA) and zearalenone (ZEA) in cereals using HPLC with fluorescence detector (FLD) is described. Mycotoxins were extracted with methanol : water (80 : 20) and purified with a multifunctional AOZ immunoaffinity column before HPLC analysis. The validation of the analytical method was performed to establish the following parameters: specificity, selectivity, linearity, limits of detection (LOD) and quantification (LOQ), accuracy, precision (within- and between-day variability), stability, robustness, measurement of performance, and measurement of uncertainty. Calibration curves were linear (r > 0.999) over the concentration range, from the LOQ to 26, 40 and 400 ng/g for AFs, OTA and ZEA, respectively. LOD and LOQ were 0.0125 and 0.05 ng/g for aflatoxin B1 (AFB1) and G1 (AFG1), 0.0037 and 0.015 ng/g for aflatoxin B2 (AFB2) and G2 (AFG2), as well as 0.05 and 0.2 ng/g for OTA and 0.5 and 2 ng/g for ZEA, respectively. The mean recovery values were 77-104% for different concentrations of AFs, OTA and ZEA in spiked cereal samples. Both intra- and inter-day accuracy and precision were within acceptable limits. This method was successfully applied for the simultaneous determination of mycotoxins for 60 cereal samples collected from Malaysian markets. Fifty per cent of the cereal samples were contaminated with at least one of these mycotoxins, at a level greater than the LOD. Only one wheat sample and two rice samples were contaminated with levels greater than the European Union regulatory limits for AFs and OTA (4 and 5 ng/g). The means and ranges of mycotoxins obtained for the cereal samples were 0.4 ng/g and 0.01-5.9 ng/g for total AFs; 0.18 ng/g and 0.03-5.3 ng/g for OTA; and 2.8 ng/g and 2.4-73.1 ng/g for ZEA, respectively. The results indicate that the method is suitable for the simultaneous determination of AFs, OTA and ZEA in cereals and is suitable for routine analysis.
The usage of porcine pepsin or other porcine derivatives in food products is a common practice in European, American and certain Asian countries although it creates issues in religious and personnel health concerns. In this study, porcine pepsin was detected using indirect ELISA that involved the anti-pep80510 polyclonal antibody raised against a specific peptide of porcine pepsin, pep80510. The sensitivity of the assay for standard porcine pepsin was 0.008 µg/g. The immunoassay did not cross-react to other animal rennet and milk proteins except for microbial coagulant from Mucor miehie. The recovery of porcine pepsin in spiked cheese curd within the range of CV < 20% while for porcine pepsin in spiked cheese whey the recovery is also within the range of CV% < 20%.
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