Widespread food poisoning due to microbial contamination has been a major concern for the food industry, consumers and governing authorities. This study is designed to determine the levels of fungal contamination in edible bird nests (EBNs) using culture and molecular techniques. Raw EBNs were collected from five house farms, and commercial EBNs were purchased from five Chinese traditional medicine shops (companies A-E) in Peninsular Malaysia. The fungal contents in the raw and commercial EBNs, and boiled and unboiled EBNs were determined. Culturable fungi were isolated and identified. In this study, the use of these methods revealed that all EBNs had fungal colony-forming units (CFUs) that exceeded the limit set by Standards and Industrial Research Institute of Malaysia (SIRIM) for yeast and moulds in EBNs. There was a significant difference (p < 0.05) in the number of types of fungi isolated from raw and commercial EBNs, but no significant difference in the reduction of the number of types of fungi after boiling the EBNs (p > 0.05). The types of fungi isolated from the unboiled raw EBNs were mainly soil, plant and environmental fungi, while the types of fungi isolated from the boiled raw EBNs, unboiled and boiled commercial EBNs were mainly environmental fungi. Aspergillus sp., Candida sp., Cladosporium sp., Neurospora sp. and Penicillum sp. were the most common fungi isolated from the unboiled and boiled raw and commercial EBNs. Some of these fungi are mycotoxin producers and cause opportunistic infections in humans. Further studies to determine the mycotoxin levels and methods to prevent or remove these contaminations from EBNs for safe consumption are necessary. The establishment and implementation of stringent regulations for the standards of EBNs should be regularly updated and monitored to improve the quality of the EBNs and consumer safety.
Malayan box turtle (Cuora amboinensis) has been a wildlife-protected vulnerable turtle species in Malaysia since 2005. However, because of its purported usage in traditional medicine, tonic foods and feeds, clandestine black market trade is rampant. Several polymerase chain reaction (PCR) assays for the taxonomic detection and classification of turtle species have been proposed. These assays are based on long-length target amplicons which are assumed to break down under compromised states and, hence, might not be suitable for the forensic tracing and tracking of turtle trafficking. For the first time this paper develops a very short-amplicon-length PCR assay (120 bp) for the detection of Malayan box turtle meat in raw, processed and mixed matrices, and experimental evidence is produced that such an assay is not only more stable and reliable but also more sensitive than those previously published. We checked the assay specificity against 20 different species and no cross-species detection was observed. The possibility of any false-negative detection was eliminated by a universal endogenous control for eukaryotes. The assay detection limit was 0.0001 ng of box turtle DNA from pure meat and 0.01% turtle meat in binary and ternary admixtures and commercial meatballs. Superior target stability and sensitivity under extreme treatments of boiling, autoclaving and microwave cooking suggested that this newly developed assay would be suitable for any forensic and/or archaeological identification of Malayan box turtle species, even in severely degraded specimens. Further, in silico studies indicated that the assay has the potential to be used as a universal probe for the detection of nine Cuora species, all of which are critically endangered.
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.
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.
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.
Being the third-largest primate population has not made macaque (Macaca fascicularis sp.) monkeys less exposed to threats and dangers. Despite wildlife protection, they have been widely hunted and consumed in several countries because of their purported nutritional values. In addition to trading as pure bush meats in several places, monkey meat has been sold in meatball and soup products in Indonesia. Thus the possibility of macaque meat trafficking under the label of common meats is quite high. This paper reports the development of a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay with the shortest amplicon length for the confirmed detection of monkey meat under compromised states which are known to degrade DNA. We amplified a 120-bp region of d-loop gene using a pair of macaque-specific primers and confirmed their specificity for the target species through cross-challenging against 17 different species using a 141-bp site of an 18 S rRNA gene as an endogenous control for eukaryotes. This eliminated the possibilities of any false-negative detection with complex matrices or degraded specimens. The detection limit was 0.00001 ng DNA in a pure state and 0.1% of meat in mixed matrices and commercial meatball products. RFLP analysis further authenticated the originality of the PCR product and distinctive restriction patterns were found upon AluI and CViKI-1 digestion. A micro-fluidic lab-on-a-chip automated electrophoretic system separated the fragments with high resolution. The assay was validated for screening commercial meatball products with sufficient internal control.
Porcine gelatine is a common adulterant found in edible bird's nests (EBNs) used to increase the net weight prior to sale. This study aimed to develop indirect enzyme-linked immunosorbent assays (ELISAs) for porcine gelatine adulteration using anti-peptide polyclonal antibodies. Three indirect ELISAs were developed (PAB1, 2 and 3), which had limits of detection (LODs) of 0.12, 0.10 and 0.11 µg g(-1), respectively. When applied to standard solutions of porcine gelatine, the inter- and intra-assays showed coefficients of variation (CVs) less than 20% and were able to detect at least 0.5 ng µg(-1) (0.05%) porcine gelatine in spiked samples. The proposed ELISA offers attractions for quality control in the EBN industry.
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 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.
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.
Palm kernel cake (PKC) is a useful source of protein and energy for livestock. Recently, it has been used as an ingredient in poultry feed. Mycotoxin contamination of PKC due to inappropriate handling during production and storage has increased public concern about economic losses and health risks for poultry and humans. This concern has accentuated the need for the evaluation of mycotoxins in PKC. Furthermore, a method for quantifying mycotoxins in PKC has so far not been established. The aims of this study were therefore (1) to develop a method for the simultaneous determination of mycotoxins in PKC and (2) to validate and verify the method. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using an electrospray ionisation interface (ESI) in both positive- and negative-ion modes was developed for the simultaneous determination of aflatoxins (AFB₁, AFB₂, AFG₁ and AFG₂), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB₁ and FB₂), T-2 and HT-2 toxin in PKC. An optimum method using a 0.2 ml min⁻¹ flow rate, 0.2% formic acid in aqueous phase, 10% organic phase at the beginning and 90% organic phase at the end of the gradient was achieved. The extraction of mycotoxins was performed using a solvent mixture of acetonitrile-water-formic acid (79:20:1, v/v) without further clean-up. The mean recoveries of mycotoxins in spiked PKC samples ranged from 81% to 112%. Limits of detection (LODs) and limits of quantification (LOQs) for mycotoxin standards and PKC samples ranged from 0.02 to 17.5 μg kg⁻¹ and from 0.06 to 58.0 μg kg⁻¹, respectively. Finally, the newly developed method was successfully applied to PKC samples. The results illustrated the fact that the method is efficient and accurate for the simultaneous multi-mycotoxin determination in PKC, which can be ideal for routine analysis.
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).
A new method for the simultaneous quantification of 12 mycotoxins was developed and optimized using reverse phase high performance liquid chromatography (RP-HPLC) with a photodiode array (PDA) and fluorescence detector (FLD), a photochemical reactor for enhanced detection (PHRED) and post-column derivatization. The mycotoxins included aflatoxins (AFB(1), AFB(2), AFG(1), and AFG(2)), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB(1), FB(2), and FB(3)), T-2 and HT-2 toxins. A double sample extraction with a phosphate-buffered saline solution (PBS) and methanol was used for co-extraction of mycotoxins, and a multifunctional immunoaffinity column was used for cleanup. Optimum conditions for separation of the mycotoxins were obtained to separate 12 mycotoxins in FLD and PDA chromatograms with a high resolution. The method gave recoveries in the range 72-111% when applied to spiked corn samples. The limits of detection (LOD) were 0.025 ng/g for AFB(1) and AFG(1), 0.012 ng/g for AFB(2) and AFG(2), 0.2 ng/g for OTA, 1.5 ng/g for ZEA, 6.2 ng/g for FB(1), FB(3) and HT-2 toxin, 9.4 ng/g for FB(2) and T-2 toxin, and 18.7 ng/g for DON. In addition, the limits of quantification (LOQ) ranged from 0.04 ng/g for AFB(2) and AFG(2) to 62 ng/g for DON. The method was successfully applied to the determination of these mycotoxins in 45 cereal samples obtained from the Malaysian market. The results indicated that the method can be applied for the multi-mycotoxin determination of cereals.
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.
Migration of melamine has been determined for 41 types of retail melamine-ware products in Malaysia. This study was initiated by the Ministry of Health, Malaysia, in the midst of public anxiety on the possibility of melamine leaching into foods that come into contact with the melamine-ware. Thus, the objective of this study was to investigate the level of melamine migration in melamine utensils available on the market. Samples of melamine tableware, including cups and plates, forks and spoons, tumblers, bowls, etc., were collected from various retail outlets. Following the test guidelines for melamine migration set by the European Committee for Standardisation (CEN 2004) with some modifications, the samples were exposed to two types of food simulants (3% acetic acid and distilled water) at three test conditions (25°C (room temperature), 70 and 100°C) for 30 min. Melamine analysis was carried out using LC-MS/MS with a HILIC column and mobile phase consisting of ammonium acetate/formic acid (0.05%) in water and ammonium acetate/formic acid (0.05%) in acetonitrile (95 : 5, v/v). The limit of quantification (LOQ) was 5 ng/ml. Melamine migration was detected from all samples. For the articles tested with distilled water, melamine migration were [median (interquartile range)] 22.2 (32.6), 49.3 (50.9), 84.9 (89.9) ng/ml at room temperature (25°C), 70 and 100°C, respectively. In 3% acetic acid, melamine migration was 31.5 (35.7), 81.5 (76.2), 122.0 (126.7) ng/ml at room temperature (25°C), 70 and 100°C, respectively. This study suggests that excessive heat and acidity may directly affect melamine migration from melamine-ware products. However the results showed that melamine migration in the tested items were well below the specific migration limit (SML) of 30 mg/kg (30,000 ng/ml) set out in European Commission Directive 2002/72/EC.
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 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 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.
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.
The objective of this study was to examine the effect of washing pre-treatment on mercury concentration in fish fillet. Response surface methodology was used to investigate the influence of three variables, pH (1-6.5), NaCl (0-1% w/v) and exposure time (5-30 min) by using a three-factor central composite design. The aim was to obtain the best possible combination of these variables in order to reduce mercury in fish fillet. The experimental data were adequately fitted into a second-order polynomial model with multiple regression coefficients (R(2)) of 0.961. The results indicated that the reduction of mercury in fish flesh significantly depends on the pH of the solution used. The overall optimal condition resulting in the maximum mercury reduction in fish fillet was obtained at a combined level pH of 2.79, NaCl of 0.5% and exposure time of 13.5 min. The optimized protocol produced a solution that can reduce mercury from raw fish fillet up to 81%.