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.
A simple and rapid high performance liquid chromatographic with fluorescence detection method for the determination of the aflatoxin B1, B2, G1 and G2 in peanuts, rice and chilli was developed. The sample was extracted using acetonitrile:water (90:10, v/v%) and then purified by using ISOLUTE® multimode solid phase extraction. After the pre-column derivatisation, the analytes were separated within 3.7 min using Chromolith® performance RP-18e (100-4.6mm) monolithic column. To assess the possible effects of endogenous components in the food items, matrix-matched calibration was used for the quantification and validation. The recoveries of aflatoxins that were spiked into food samples were 86.38-104.5% and RSDs were <4.4%. The method was applied to the determination of aflatoxins in peanut (9), rice (5) and chilli (10) samples. Liquid chromatography-tandem mass spectrometry analysis using triple quadruple analyser and operated in the multiple reaction monitoring modes on the contaminated samples was performed for confirmation.
This study was conducted to characterize the growth of and aflatoxin production by Aspergillus flavus on paddy and to develop kinetic models describing the growth rate as a function of water activity (a(w)) and temperature.
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.
Twenty commercial mixed herbal drugs were examined for mycological profile. Aspergillus species were the predominant fungi found in the drugs. Other fungi harboured in the drugs with less frequency were Paecilomyces species, Eurotium species, Monascus species, Acremonium species, Penicillium species, Cladosporium species, Scopulariopsis species, Phialophora species and Fonseceae species. Fungal count was between 1.0 log(10) CFU and 2.4 log(10) CFU per gram of sample. When the drugs were incubated in 85% humidity at 25 degrees C, fungal colonies grew on only two of the drugs. The mixed herbal drugs were extracted with water and the extracts were used to grow Aspergillus parasiticus. All extracts reduced aflatoxin B(1) and aflatoxin G(1) production by 62-97%. All but two of the extracts reduced aflatoxin B(2) and aflatoxin G(2) production by 39-95%. It can be concluded that the commercial powdered mixed herbal drugs contained low number of endogenous fungi, and these drugs are inhibitory to the growth of its endogenous fungi and aflatoxins production by aflatoxigenic fungi.
The aim of this study was to model the radial growth rate and to assess aflatoxin production by Aspergillus flavus as a function of water activity (a(w) 0.82 to 0.92) and temperature (12 to 42 °C) on polished and brown rice. The growth of the fungi, expressed as colony diameter (mm) was measured daily, and the aflatoxins were analyzed using HPLC with a fluorescence detector. The growth rates were estimated using the primary model of Baranyi, which describes the change in colony radius as a function of time. Total of 2 secondary models were used to describe the combined effects of a(w) and temperature on the growth rates. The models were validated using independent experimental data. Linear Arrhenius-Davey model proved to be the best predictor of A. flavus growth rates on polished and brown rice followed by polynomial model. The estimated optimal growth temperature was around 30 °C. A. flavus growth and aflatoxins were not detected at 0.82 a(w) on polished rice while growth and aflatoxins were detected at this a(w) between 25 and 35 °C on brown rice. The highest amounts of toxins were formed at the highest a(w) values (0.90 to 0.92) at a temperature of 20 °C after 21 d of incubation on both types of rice. Nevertheless, the consistencies of toxin production within a wider range of a(w) values occurred between 25 to 30 °C. Brown rice seems to support A. flavus growth and aflatoxin production more than the polished rice.
In October 1988, 13 Chinese children died of acute hepatic encephalopathy in the northwestern state of Perak in peninsular Malaysia. The acuteness of the illness differed from previously reported outbreaks described in Kenya, India, and Thailand. Epidemiologic investigations determined that the children had eaten a Chinese noodle, loh see fun, hours before they died. The attack rates among those who had eaten the noodles were significantly higher than those who had not (P < 0.0001). The cases were geographically scattered in six towns in two districts along the route of distribution of the noodle supplied by one factory in Kampar town. Aflatoxins were confirmed in postmortem samples from patients. This outbreak has important public health implications for many developing countries.
Aspergillus flavus is the predominant species that produce aflatoxins in stored peanuts under favourable conditions. This study aimed to describe the growth and aflatoxin production by two A. flavus strains isolated from imported raw peanuts and to model the effects of temperature and aw on their colony growth rate as a function of temperature and aw in Peanut Meal Extract Agar (PMEA). A full factorial design with seven aw levels (0.85-0.98 aw) and five temperature levels (20-40 °C) was used to investigate the growth and aflatoxin production. Colony diameter was measured daily for 28 days while AFB1 and total aflatoxin were determined on day 3, 7, 14, and 21. The maximum colony growth rate, μmax (mm/day) was estimated by using the primary model of Baranyi, and the μmax was then fitted to the secondary model; second-order polynomial and linear Arrhenius-Davey to describe the colony growth rate as a function of temperature and aw. The results indicated that both strains failed to grow at temperature of 20 °C with aw <0.94 and aw of 0.85 for all temperatures except 30 °C. The highest growth rate was observed at 30 °C, with 0.98 aw for both strains. The analysis of variance showed a significant effect of strain, temperature, and aw on the fungal growth and aflatoxin production (p
In studying the ecophysiology of fungal phytopathogens, several stages are involved (in vitro, greenhouse, in planta). Most in vitro studies extensively utilise the general growth media such as Potato Dextrose Agar and Malt Extract Agar. Although the crop components in these media serve as excellent carbon sources and yield luxuriant growth, they are not naturally contaminated with Aspergillus flavus and thus might result in under- or overestimation of its actual toxigenic potentials. Empirical data on the formulation of semi-synthetic growth medium mimicking the natural crop commonly contaminated by A. flavus for the ecophysiological studies in vitro are scarce. The present work was aimed at investigating the ecophysiology of A. flavus on commercial growth media (PDA, MEA); formulating maize- and peanut-based semi-synthetic growth media using two methods of raw material preparation (milling, hot water extraction) at different concentrations (1, 3, 5, 7, 9% w/v), and comparing the ecophysiological parameters between commercial and formulated growth media. Growth rates were obtained by computing the hyphal expansion data into y = mx + c equation. AFB1 was quantified using high performance liquid chromatography with fluorescence detector. Formulated media were found to yield significantly higher growth rates when compared to commercial media. However, commercial media yielded significantly higher AFB1 when compared to all formulated media. Between the two formulations, milling yielded significantly higher growth rates and AFB1 when compared to hot water extraction. Although in vitro data cannot directly extrapolate in planta performance, results obtained in the present work can be used to gauge the actual toxigenic potential of A. flavus in maize and peanut agro-ecosystems.
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.
Pistachio nut (Pistacia vera L.) is one of the popular tree nuts in the world. Proper selection of packaging materials is necessary to prevent absorption of moisture and aflatoxin formation which will influence the overall product quality and safety. This research is undertaken to study the effect of different type of flexible packaging films on the moisture and aflatoxin contents of whole pistachio nuts during storage at ambient temperature (22-28 °C) and relative humidity of 85-100%. Five types of plastic films tested were low density polyethylene (LDPE) which serves as the control, food-grade polyvinyl chloride (PVC), nylon (LDPE/PA), polyamide/polypropylene (PA/PP) and polyethylene terephthalate (PET). The moisture content and aflatoxin content of pistachio nuts were measured using oven drying method and HPLC, respectively. Sample were analysed at 0, 2, 4, 6, 8 and 10 months during the storage period. Results showed that there was an increase in moisture content with the increase in storage time of pistachio nuts. The increase in moisture content was associated with the aflatoxin level of pistachio nuts during storage time. All the packaging materials except LDPE delayed the moisture absorption and aflatoxin formation of the product. The most suitable packaging materials for maintaining the quality and safety of pistachio nuts is PET films followed by nylon, PA/PP and PVC. The shelf-life of pistachio can be extended from 2 months (Control) to 5 months when PET is used as the packaging material.
A method for the determination of aflatoxins B1 and B2 in peanuts and corn based products is described. The samples were extracted with a mixture of acetonitrile-water (84:16), followed by multifunctional clean-up and liquid chromatography with fluorescence detection. Both calibration curves showed good correlation from 4.0 to 32.0 ppb for aflatoxin B1 (r=0.9999) and 1.2 to 9.6 ppb for aflatoxin B2 (r=0.9997). The detection limit of aflatoxins B1 and B2 were established at 0.1 and 0.03 ppb, respectively, based on signal-to-noise ratio of 3:1. Average recoveries for the determination of aflatoxins B1 and B2 at 10 and 3 ppb spiking levels, respectively ranged from 94.2 to 107.6%. A total of 20 peanut samples and corn based products were obtained from retail shop and local market around Kuala Terengganu and analyzed for aflatoxins B1 and B2 contents, using the proposed method. Aflatoxins B1 and B2 were detected in 5 out of the 9 peanuts samples and 5 out of the 11 corn based products, at levels ranging from 0.2 to 101.8 ppb.
Palm kernel cake (PKC) is the solid residue following oil extraction of palm kernels and useful to fatten animals either as a single feed with only minerals and vitamins supplementation, or mixed with other feedstuffs such as corn kernels or soy beans. The occurrence of mycotoxins (aflatoxins, ochratoxins, zearalenone, and fumonisins) in feed samples affects the animal's health and also serves as a secondary contamination to humans via consumption of eggs, milk and meats. Of these, aflatoxin B₁ (AFB₁) is the most toxically potent and a confirmed carcinogen to both humans and animals. Methods such as High Performance Liquid Chromatography (HPLC) and Liquid Chromatography-Mass Spectrometry (LC-MS/MS) are common in the determination of mycotoxins. However, these methods usually require sample pre-treatment, extensive cleanup and skilled operator. Therefore, in the present work, a rapid method of electrochemical immunosensor for the detection of AFB₁ was developed based on an indirect competitive enzyme-linked immunosorbent assay (ELISA). Multi-walled carbon nanotubes (MWCNT) and chitosan (CS) were used as the electrode modifier for signal enhancement.N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (EDC) andN-hydroxysuccinimide (NHS) activated the carboxyl groups at the surface of nanocomposite for the attachment of AFB₁-BSA antigen by covalent bonding. An indirect competitive reaction occurred between AFB₁-BSA and free AFB₁ for the binding site of a fixed amount of anti-AFB₁ antibody. A catalytic signal based on horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H₂O₂) and 3,3',5,5'-tetramethylbenzidine (TMB) mediator was observed as a result of attachment of the secondary antibody to the immunoassay system. As a result, the reduction peak of TMB(Ox)was measured by using differential pulse voltammetry (DPV) analysis. Based on the results, the electrochemical surface area was increased from 0.396 cm² to 1.298 cm² due to the electrode modification with MWCNT/CS. At the optimal conditions, the working range of the electrochemical immunosensor was from 0.0001 to 10 ng/mL with limit of detection of 0.1 pg/mL. Good recoveries were obtained for the detection of spiked feed samples (PKC, corn kernels, soy beans). The developed method could be used for the screening of AFB₁ in real samples.
The secondary metabolites produced by fungi known as mycotoxins, are capable of causing mycotoxicosis (diseases and death) in human and animals. Contamination of feedstuffs as well as food commodities by fungi occurs frequently in a natural manner and is accompanied by the presence of mycotoxins. The occurrence of mycotoxins' contamination is further stimulated by the on-going global warming as reflected in some findings. This review comprehensively discussed the role of mycotoxins (trichothecenes, zearalenone, fumonisins, ochratoxins, and aflatoxins) toward gut health and gut microbiota. Certainly, mycotoxins cause perturbation in the gut, particularly in the intestinal epithelial. Recent insights have generated an entirely new perspective where there is a bi-directional relationship exists between mycotoxins and gut microbiota, thus suggesting that our gut microbiota might be involved in the development of mycotoxicosis. The bacteria-xenobiotic interplay for the host is highlighted in this review article. It is now well established that a healthy gut microbiota is largely responsible for the overall health of the host. Findings revealed that the gut microbiota is capable of eliminating mycotoxin from the host naturally, provided that the host is healthy with a balance gut microbiota. Moreover, mycotoxins have been demonstrated for modulation of gut microbiota composition, and such alteration in gut microbiota can be observed up to species level in some of the studies. Most, if not all, of the reported effects of mycotoxins, are negative in terms of intestinal health, where beneficial bacteria are eliminated accompanied by an increase of the gut pathogen. The interactions between gut microbiota and mycotoxins have a significant role in the development of mycotoxicosis, particularly hepatocellular carcinoma. Such knowledge potentially drives the development of novel and innovative strategies for the prevention and therapy of mycotoxin contamination and mycotoxicosis.
In October 1988, a series of food poisoning cases occurred in the State of Perak in Malaysia. Most of the victims were children. Ultimately 13 children between the ages of 2.5 and 11 years died. Epidemiological investigations showed that the probable source of the poison was Loh See Fun, a noodles in the shape of a rat's tail. All the deceased ate the noodles from one supplier. Clinical and pathological findings were similar in each case. Postmortem examination was performed in 11 cases. Toxicological examination on organs in 10 cases showed a high concentration of aflatoxin in tissues of the deceased. High levels of boric acid were excreted from most of the victims. Histological examination of the liver in these cases showed necrotic changes found in aflatoxin poisoning. Combination of the epidemiological, clinical, toxicological, and pathological findings pointed to the fact that there was a common toxin or toxins responsible for the deaths. These were thought to be a combination of boric acid and aflatoxin.
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).
Human exposure to aflatoxin is through the diet, and probiotics are able to bind aflatoxin and prevent its absorption in the small intestine. This study aimed to determine the effectiveness of a fermented milk drink containing Lactobacillus casei Shirota (LcS) (probiotic drink) to prevent aflatoxin absorption and reduce serum aflatoxin B1-lysine adduct (AFB1-lys) and urinary aflatoxin M1 concentrations. The present study was a randomised, double-blind, cross-over, placebo-controlled study with two 4-week intervention phases. In all, seventy-one subjects recruited from the screening stage were divided into two groups--the Yellow group and the Blue group. In the 1st phase, one group received probiotic drinks twice a day and the other group received placebo drinks. Blood and urine samples were collected at baseline, 2nd and 4th week of the intervention. After a 2-week wash-out period, the treatments were switched between the groups, and blood and urine samples were collected at the 6th, 8th and 10th week (2nd phase) of the intervention. No significant differences in aflatoxin biomarker concentrations were observed during the intervention. A within-group analysis was further carried out. Aflatoxin biomarker concentrations were not significantly different in the Yellow group. Nevertheless, ANOVA for repeated measurements indicated that AFB1-lys concentrations were significantly different (P=0·035) with the probiotic intervention in the Blue group. The 2nd week AFB1-lys concentrations (5·14 (SD 2·15) pg/mg albumin (ALB)) were significantly reduced (P=0·048) compared with the baseline (6·24 (SD 3·42) pg/mg ALB). Besides, the 4th week AFB1-lys concentrations were significantly lower (P<0·05) with probiotic supplementation than with the placebo. Based on these findings, a longer intervention study is warranted to investigate the effects of continuous LcS consumption to prevent dietary aflatoxin exposure.
Fungi are distributed worldwide and can be found in various foods and feedstuffs from almost every part of the world. Mycotoxins are secondary metabolites produced by some fungal species and may impose food safety risks to human health. Among all mycotoxins, aflatoxins (AFs), ochratoxin A (OTA), trichothecenes, deoxynivalenol (DON and T-2 toxin), zearalenone (ZEN), and fumonisins (FMN) have received much attention due to high frequency and severe health effects in humans and animals. Malaysia has heavy rainfall throughout the year, high temperatures (28 to 31 °C), and high relative humidity (70% to 80% during wet seasons). Stored crops under such conditions can easily be contaminated by mycotoxin-producing fungi. The most important mycotoxins in Malaysian foods are AFs, OTA, DON, ZEN, and FMN that can be found in peanuts, cereal grains, cocoa beans, and spices. AFs have been reported to occur in several cereal grains, feeds, nuts, and nut products consumed in Malaysia. Spices, oilseeds, milk, eggs, and herbal medicines have been reported to be contaminated with AFs (lower than the Malaysian acceptable level of 35 ng/g for total AFs). OTA, a possible human carcinogen, was reported in cereal grains, nuts, and spices in Malaysian market. ZEN was detected in Malaysian rice, oat, barley, maize meal, and wheat at different levels. DON contamination, although at low levels, was reported in rice, maize, barley, oat, wheat, and wheat-based products in Malaysia. FMN was reported in feed and some cereal grains consumed in Malaysia. Since some food commodities are more susceptible than others to fungal growth and mycotoxin contamination, more stringent prevention and control methods are required.