Risk of exposure to aflatoxin M1 through consumption of cow's milk among children in Magadu, Morogoro.

Aflatoxin M1 (AFM1) contamination of milk affects the general population with particular attention to children who frequently consume milk as part of complementary food. This study determined AFM1 contamination of cow's milk and estimated the health risk of dietary AFM1 through consumption of cow's milk among children (6 to 36 months) in the Magadu ward of Morogoro region in Tanzania. A total of 165 mother-baby pairs were recruited and interviewed on child feeding practices with a focus on feeding of cow's milk in the past 24 h. Alongside the interview, 100 raw cows' milk samples were collected from subsampled respondent households and were analyzed for AFM1 using enzyme-linked immunosorbent assay (ELISA). The results showed that about 35% of the surveyed children consumed cow's milk in the form of plain milk, incorporated in porridge and/or tea. The amount consumed varied from 62.5 to 500 mls with a median of 125 (125, 250) mls at a frequency of 1 to 2 times a day. All raw cows' milk (100%) samples (n = 100) were found contaminated with AFM1 at concentrations ranging from 0.052 to 9.310 µg/L and median of 2.076 µg/L (1.27, 2.48). All samples were contaminated by AFM1 at levels above the limits of 0.05 µg/L of raw milk set by the Tanzania Bureau of Standard and the European Union, while 97% exceeded 0.5 µg/L set by the US Food and Drug Administration. Exposure to AFM1 due to consumption of cow's milk ranged from 0.0024 to 0.077 µg/kg bw per day with a median of 0.019 (0.0016, 0.026) µg/kg bw per day, while the margin of exposure (MOE) ranged from 5.19 to 166.76 and median 20.68 (15.33, 25.40) implying high risk of public health concern. This study recommends that advocacy on consumption of cows' milk to combat undernutrition in children should consider a holistic approach that considers the milk's safety aspect.

Effects of Turmeric Powder on Aflatoxin M1 and Aflatoxicol Excretion in Milk from Dairy Cows Exposed to Aflatoxin B1 at the EU Maximum Tolerable Levels.

Due to the climatic change, an increase in aflatoxin B1 (AFB1) maize contamination has been reported in Europe. As an alternative to mineral binders, natural phytogenic compounds are increasingly used to counteract the negative effects of AFB1 in farm animals. In cows, even low dietary AFB1 concentrations may result in the milk excretion of the genotoxic carcinogen metabolite aflatoxin M1 (AFM1). In this study, we tested the ability of dietary turmeric powder (TP), an extract from Curcuma longa (CL) rich in curcumin and curcuminoids, in reducing AFM1 mammary excretion in Holstein-Friesian cows. Both active principles are reported to inhibit AFM1 hepatic synthesis and interact with drug transporters involved in AFB1 absorption and excretion. A crossover design was applied to two groups of cows (n = 4 each) with a 4-day washout. Animals received a diet contaminated with low AFB1 levels (5 ± 1 µg/kg) for 10 days ± TP supplementation (20 g/head/day). TP treatment had no impact on milk yield, milk composition or somatic cell count. Despite a tendency toward a lower average AFM1 milk content in the last four days of the treatment (below EU limits), no statistically significant differences with the AFB1 group occurred. Since the bioavailability of TP active principles may be a major issue, further investigations with different CL preparations are warranted.

A bivalent binding aptamer-cDNA on MoS2 nanosheets based fluorescent aptasensor for detection of aflatoxin M1.

To ensure the safety of dairy products, especially milk, and consequently protect human health, accurate and simple analytical techniques are highly necessary to determine the low concentration of aflatoxin M1 (AFM1) as an important carcinogen. Herein, a novel, accurate and simple fluorescent aptasensor was designed for selective detection of AFM1 based on bivalent binding aptamer-cDNA (BBA-cDNA) structure. Moreover, MoS2 nanosheets (MoS2 NSs) were used as the fluorescent quencher and FAM-labeled complementary strand of aptamer (FAM-CS) was applied as a fluorescent probe. In this study, we achieved a new result. Unlike previous studies, in this work, the BBA-cDNA structure was not disassembled in the presence of the target. Therefore, as the AFM1 concentration increased, more targets were attached to the BBA-cDNA structure and as a result, the BBA-cDNA structure/AFM1 could not be placed on the surface of MoS2 NSs, leading to the more fluorescent intensity detection. Under optimized conditions, the developed fluorescent analytical method revealed great selectivity toward AFM1 with a limit of detection (LOD) of 0.5 nM and a linear range from 0.7 to 10 nM. This fabricated aptasensor indicated excellent analytical performance for AFM1 detection in milk samples with LOD of 0.1 nM. Overall, the proposed approach could provide an effective basis for small molecule analysis to guarantee food and human safety using appropriate aptamer sequences.

Effectiveness of Training and Use of Novasil Binder in Mitigating Aflatoxins in Cow Milk Produced in Smallholder Farms in Urban and Periurban Areas of Kenya.

Aflatoxins, which commonly contaminate animal feeds and human food, present a major public health challenge in sub-Saharan Africa. After ingestion by cows, aflatoxin B1 is metabolized to aflatoxin M1 (AFM1), some of which is excreted in milk. This study involved smallholder dairy farms in urban and periurban areas of Nairobi and Kisumu, Kenya. The objective was to determine the effectiveness of training and providing farmers with aflatoxin binder (NovaSil®) on AFM1 contamination in raw milk. A baseline survey was undertaken and 30 farmers whose milk had AFM1 levels above 20 ppt were randomly selected for inclusion in the study. Of these, 20 farmers were part of the intervention, and were given training on the usage of the NovaSil® binder, while 10 served as a control group. All farmers were visited biweekly for three months for interviews and milk samples were collected to measure the AFM1 levels. The AFM1 levels were quantified by enzyme linked immunosorbent assay. The NovaSil® binder significantly reduced AFM1 concentrations in the raw milk produced by the farmers in the intervention group over the duration of the study (p < 0.01). The control farms were more likely to have milk with AFM1 levels exceeding the regulatory limit of 50 ppt compared to the intervention farms (p < 0.001) (odds ratio = 6.5). The farmers in the intervention group perceived that there was an improvement in milk yield, and in cow health and appetite. These farmers also felt that the milk they sold, as well as the one they used at home, was safer. In conclusion, the use of binders by dairy farmers can be effective in reducing AFM1 in milk. Further research is needed to understand their effectiveness, especially when used in smallholder settings.

An optical aptasensor for aflatoxin M1 detection based on target-induced protection of gold nanoparticles against salt-induced aggregation and silica nanoparticles.

Not only intoxications of aflatoxins are significant risk for human beings, but also; the contamination with these toxins affect the economy. Therefore, developing a rapid, precise and inexpensive determination method is vitally important. Here, a colorimetric aptasensor is introduced for the detection of aflatoxin M1 (AFM1) based on the preservation of gold nanoparticles (AuNPs) against NaCl-induced aggregation by detaching of complementary strand of aptamer (CS) from the aptamer-modified streptavidin coated silica nanoparticles (SNPs) following the addition of target. So, the color of sample remains red. While, in the lack of AFM1, salt-induced aggregation of AuNPs occurs and the color of sample becomes purple as the aptamer/CS (dsDNA)-modified SNPs is stable and CS cannot bind to AuNPs. The proposed aptasensor could detect AFM1 in a linear dynamic range, 300-75,000 ng/L, with a detection limit of 30 ng/L. Also, the sensing method was effectively applied for AFM1 recognition in milk samples.

Occurrence of aflatoxin M1 in human milk samples in Vojvodina, Serbia: Estimation of average daily intake by babies.

The objectives of the study were to determine the aflatoxin M1 content in human milk samples in Vojvodina, Serbia, and to assess the risk of infants' exposure to aflatoxins food contamination. The growth of Aspergillus flavus and production of aflatoxin B1 in corn samples resulted in higher concentrations of AFM1 in milk and dairy products in 2013, indicating higher concentrations of AFM1 in human milk samples in 2013 and 2014 in Serbia. A total number of 60 samples of human milk (colostrum and breast milk collected 4-8 months after delivery) were analyzed for the presence of AFM1 using the Enzyme Linked Immunosorbent Assay method. The estimated daily intake of AFM1 through breastfeeding was calculated for the colostrum samples using an average intake of 60 mL/kg body weight (b.w.)/day on the third day of lactation. All breast milk collected 4-8 months after delivery and 36.4% of colostrum samples were contaminated with AFM1. The greatest percentage of contaminated colostrum (85%) and all samples of breast milk collected 4-8 months after delivery had AFM1 concentration above maximum allowable concentration according to the Regulation on health safety of dietetic products. The mean daily intake of AFM1 in colostrum was 2.65 ng/kg bw/day. Results of our study indicate the high risk of infants' exposure, who are at the early stage of development and vulnerable to toxic contaminants.

A qPCR aptasensor for sensitive detection of aflatoxin M1.

Aflatoxin M1 (AFM1), one of the most toxic mycotoxins, imposes serious health hazards. AFM1 had previously been classified as a group 2B carcinogen [1] and has been classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) [2]. Determination of AFM1 thus plays an important role for quality control of food safety. In this work, a sensitive and reliable aptasensor was developed for the detection of AFM1. The immobilization of aptamer through a strong interaction with biotin-streptavidin was used as a molecular recognition element, and its complementary ssDNA was employed as the template for a real-time quantitative polymerase chain reaction (RT-qPCR) amplification. Under optimized assay conditions, a linear relationship (ranging from 1.0 × 10(-4) to 1.0 µg L(-1)) was achieved with a limit of detection (LOD) down to 0.03 ng L(-1). In addition, the aptasensor developed here exhibits high selectivity for AFM1 over other mycotoxins and small effects from cross-reaction with structural analogs. The method proposed here has been successfully applied to quantitative determination of AFM1 in infant rice cereal and infant milk powder samples. Results demonstrated that the current approach is potentially useful for food safety analysis, and it could be extended to a large number of targets.

Effects of calcium montmorillonite clay and aflatoxin exposure on dry matter intake, milk production, and milk composition.

Fifteen primiparous crossbred dairy cows that were 114±14d in milk and weighed 533±56kg were used in a replicated 5×5 Latin square to test the efficacy of a calcium montmorillonite clay, NovaSil Plus (NSP; BASF Corp., Ludwigshaven, Germany), for the reduction of aflatoxin (AF) metabolite (AFM1) in milk and the effect of NSP on milk composition. Cows were housed in a freestall barn, fed once a day and milked twice a day. The experiment consisted of five 14-d periods: d 1 through 7 were considered for data collection, and d 8 through 14 were considered a wash-out phase. In each period, cows were randomly assigned to 1 of 5 dietary treatments: (1) control (CON), consisting of a basal total mixed ration (TMR); (2) high-dose NSP diet (NSP-1%), consisting of TMR plus 230 g of NSP; (3) aflatoxin diet (AFD), consisting of the TMR plus AF challenge; (4) low-dose NSP with AF (NSP-0.5%+AFD), composed of TMR plus 115 g of NSP and AF challenge; and (5) high-dose NSP with AF (NSP-1%+AFD), consisting of TMR plus 230 g of NSP and AF challenge. The AF challenge consisted of top dressing a daily dose of 100 µg/kg estimated dry matter intake (DMI); similarly, NSP was fed at 1.0 or 0.5% of estimated DMI. Milk yield and DMI were similar across treatments averaging 21.1±1.33 kg/d and 19.7±0.56 kg/d, respectively. Concentration of milk fat, protein, and lactose were similar across treatments with averages of 4.91±0.20%, 3.85±0.10%, and 4.70±0.06%, respectively. Concentration of vitamin A averaged 0.28±0.03 µg/mL and riboflavin concentration averaged 1.57±0.13 µg/mL across treatments. The concentration of minerals in milk were similar for all treatments. Cows fed CON and NSP-1% yielded the lowest concentration of AFM1 in milk with 0.03 and 0.01±0.06 µg/L. Addition of NSP reduced milk AFM1 from 1.10±0.06 µg/L with the AF diet to 0.58 and 0.32±0.06 µg/L with the NSP-0.5%+AF and NSP-1%+AF diets, respectively. Excretion of AFM1 was reduced by NSP; mean values were 24.38, 11.86, 7.38, 0.64, and 0.23, ± 1.71 µg/d, for AFD, NSP-0.5%+AFD, NSP-1%+AFD, NSP-1%, and CON, respectively. More specifically, 1.07±0.08% of the daily AF intake was transferred to the milk of cows consuming the AFD, whereas the AF transfer rates in milk from cows that consumed the NSP-0.5%+AFD and NSP-1%+AFD were 0.52 and 0.32±0.08%. Results from this research demonstrate that feeding NSP to lactating cows is an effective method to reduce the transfer and excretion of AFM1 in milk with no negative effects on dry matter intake, milk production, and composition.

An ultra-high-performance liquid chromatography-tandem mass spectrometry method for simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 in traditional Chinese medicines.

An ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 in traditional Chinese medicines (TCMs) was developed. The approach was characterized in details and a special focus was placed on the recovery rates of isolation procedure in different TCM matrices, i.e. rhizomes and roots, seeds, flowers, grasses and leaves. For this purpose, [(13)C(17)]-aflatoxinB1 was employed as the internal standard and a reliable solid phase extraction-based clean-up method was developed. The observed recovery rates of the six aflatoxins ranged from 85.6% to 117.6% in different matrices. Then, the established method was successfully applied to the determination of the six aflatoxins in various TCMs. For 30 commercial samples analyzed, 16 were contaminated with aflatoxins. The mean levels (incidence) of aflatoxins B1, B2, G1 and G2 in positive samples were 1.40 (68.8%), 1.27 (50.0%), 0.50 (43.8%) and 0.94 (43.8%) microg kg(-1), respectively. Interestingly, aflatoxin M1 was detected in two samples with the maximal content of 0.70 microg kg(-1). No sample was contaminated with aflatoxin M2. Meanwhile, a possible association between the contamination levels and the selected herbs was clarified in the present study.

Determinants of aflatoxin M1 in breast milk in a selected group of Egyptian mothers.

In Egypt, there is a paucity of biomarker data on aflatoxin (AF) exposure. The study assessed the level and frequency of breast milk AFM1 as a biomarker of maternal exposure. Breast milk samples were collected from a selected group of 388 Egyptian lactating mothers of children attending the New El-Qalyub Hospital, Qalyubiyah governorate, Egypt, during May-September 2003. Following aflatoxin extraction, AFM1 levels were assessed by high-performance liquid chromatography (HPLC) with fluorescence detection. Approximately 36% of mothers tested positive for AFM1 (median 13.5 pg ml-1, interquartile range (IQR) 10.27-21.43). Non-working status (p = 0.018, odds ratio (OR) = 2.87), obesity (p = 0.004, OR = 3.01), high corn oil consumption (p = 0.002, OR = 2.21), number of children (>1) (p = 0.025, OR = 1.99), and early lactation stage (<1 month) (p = 0.028 OR = 3.57), contributed to the occurrence of AF in breast milk. AFM1 contamination of breast milk was frequent, albeit at moderate levels. Growth and development of the infant is rapid and thus it is possible that AF exposure through breast milk has a significant health effect.

Analysis of duplicate 24-hour diet samples for aflatoxin B1, aflatoxin M1 and ochratoxin A.

In the spring and autumn of 1994, a total diet study, in which 123 participants collected duplicates of their 24-hour diets, was carried out. The goal of this study was to determine the mass fractions of a number of analytes in these duplicate diets, so as to be able to establish oral daily intake values. After measurements were carried out for pesticides, PCBs, elements, sterols, nitrate and nitrite, and fatty acids, the duplicate diet study was concluded with analyses for aflatoxin M1, aflatoxin B1 and ochratoxin A. For this purpose a method of analysis was developed, that could simultaneously determine these mycotoxins at very low levels. The method involved chloroform extraction, liquid-liquid extraction, immunoaffinity cleanup and liquid chromatography. The method was supplemented with a procedure to confirm the identity of chromatographic peaks, assumed to represent aflatoxin M1, aflatoxin B1 and ochratoxin A. The method was in-house validated. Recoveries ranged from 68-74% for aflatoxin M1 (at spiking levels from 30-120 ng/kg, c.v. 7.6%), from 95-97% for aflatoxin B1 (at spiking levels from 50-200 ng/kg, c.v. 2.8%), and from 75-84% for ochratoxin A (at spiking levels from 150-600 ng/kg, c.v. 4.3%). Limits of quantitation (defined as signal/noise = 10) were estimated to be 24, 5 and 16 ng/kg lyophilised material for aflatoxin M1, aflatoxin B1 and ochratoxin A respectively. The newly developed method was used to analyse 123 samples of 24-hour diets. Aflatoxin M1 was detectable in 48% of the samples; the toxin contents remained below the limit of quantitation in all samples. Aflatoxin B1 could be detected in 42% of the samples; in 25% of the samples the levels were above the limit of quantitation. Ochratoxin A could be quantified in all samples. The analytical results were further processed to estimate levels of intake. Intake levels for the aflatoxins were very low, and could not reliably be established. The mean ochratoxin A intake was estimated to be 1.2 ng/kg body weight per day. This is well below the tolerable daily intake established by JECFA at 14 ng/kg body weight per day. The current dietary intake of ochratoxin A in the Netherlands is concluded to pose no appreciable health risk.

Effect of ammoniation of aflatoxin B1-contaminated cottonseed feedstock on the aflatoxin M1 content of cows' milk and hepatocarcinogenicity in the trout bioassay.

The effectiveness of ammonia in inactivating aflatoxins in contaminated cottonseed was investigated. Two aflatoxin-contaminated cottonseed lots were treated separately using an atmospheric pressure, ambient temperature ammoniation procedure (APAT) or a high pressure, high temperature ammoniation procedure (HPHT), and incorporated into dairy cow rations. Isocalorific diets containing 25% defatted, dried milk from cows fed aflatoxin-contaminated cottonseed without or with APAT or HPHT treatment, or an aflatoxin-free human grade commercial milk powder, were then fed for 12 months to rainbow trout (Oncorhynchus mykiss). Aflatoxin M1 (AFM1) concentrations in milk powders without and with seed treatment were: APAT, 85 and < 0.05 microgram/kg; HPHT, 32 and < 0.05 microgram/kg. In the APAT experiment, trout consuming the diet containing milk from cows fed the aflatoxin-contaminated cottonseed had a 42% incidence of hepatic tumours; APAT cottonseed treatment reduced this to 2.5%. Positive controls were included to demonstrate trout responsiveness. AFB1 fed continuously for 12 months at 4 micrograms/kg resulted in a 34% tumour incidence, whereas positive controls fed 20 micrograms AFB1/kg, 80 micrograms AFM1/kg, or 800 micrograms AFM1/kg for 2 wk and killed 9 months later had a 37, 5.7 and 50% incidence of tumours, respectively. These data demonstrate that APAT ammonia treatment of aflatoxin-contaminated dairy cattle cottonseed feedstock abolished the detectable transfer of AFM1 or AFB1 into milk powder, and greatly reduced the carcinogenic risk posed by any carry-over of aflatoxins or their derivatives into milk. In addition, the results confirm AFM1 to be a lower level hepatocarcinogen in comparison with AFB1 in the trout carcinogenicity assay. In the separate HPHT experiment, no tumours were observed in the livers of trout fed diets containing milk from either the ammonia-treated or untreated source, or the control diet containing 8 micrograms AFM1/kg. Positive controls fed 64 micrograms AFB1/kg for 2 wk exhibited a 29% tumour incidence 12 months later. Thus in this experiment, neither AFM1 at 8 micrograms/kg nor any HPHT-derived aflatoxin derivatives that might have been carried over into milk, represented a detectably carcinogenic hazard to trout.