Determination of aflatoxin M1 levels in Iranian white and cream cheese

A screening survey on the occurrence of aflatoxin M1 (AFM1) was accomplished on 210 cheese samples composed of white cheese (116 samples) and cream cheese (94 samples) purchased from popular markets in central part of Iran (Esfahan and Yazd provinces). The quantitative analysis of AFM1 levels in the samples was performed by using the competitive enzyme-linked immunosorbent assay (ELISA) technique. Aflatoxin M1 at measurable level (50 ng/kg) was detected in 161 (76.6%) samples, consisting of 93 (80.1%) white and 68 (72.3%) cream cheese samples. The concentration of AFM1 in the samples ranged from 52.1 to 785.4 ng/kg. Comparing to legal regulation (250 ng/kg) accepted by some of the countries, 24.2% of the samples exceeded the accepted limit. Among these, the AFM1 levels in 28.4% of white and 19.1% of cream cheese samples were not in accordance with the safety limit. The results indicated that contamination of the samples with AFM1 in such a level appear to be a potential hazard for public health. This paper represents the data of the first survey on the occurrence of AFM1 in cheeses consumed in central part of Iran.     

Infants exposure to aflatoxin M1 as a novel foodborne zoonosis

Occurrence of aflatoxin M₁ (AFM₁) in infant formula milk powder (IFMP) and maternal breast milk (MBM) was investigated as a risk factor affects the health of newborns in Egypt. A total of 125 IFMP and 125 MBM samples were collected and examined for the presence of AFM₁ using competitive ELISA test. The results indicated that the relative risk (RR) of exposure to AFM₁ via consumption of MBM was higher than IFMP (RR; 1.6, 95% CI; 1.28–2.03, p = 0.0001). The mean concentrations of AFM₁ were significantly differed (p < 0.0001) between MBM (74.413 ± 7.070 ng/l) and IFMP (9.796 ± 1.036 ng/l). High frequency distributions were detected within the range of 5–25 ng/l and >50–100 ng/l in IFMP and MBM, respectively. The average daily exposure of newborns to AFM₁ via consumption of MBM and IFMP was 52.684 and 8.170 ng, respectively, with a significant difference at p < 0.0001. Consumption of raw milk by lactating mothers exhibited a significant correlation (p < 0.0001) with the presence of AFM₁ in their milk. In conclusion, this work established a pioneering concept that AFM₁ may be considered as an etiological factor for a novel foodborne zoonosis identified as Aflatoxicosis M₁.     

Assessment of aflatoxin M1 levels in pasteurised milk,raw milk,and cheese in Arak,Iran

Aflatoxins are fungal toxins, which may be found in food and due to negative health effects, they are of major concerns for human health and food industries. Aflatoxin M₁ contamination in dairy products in Arak, Iran, 2013 was evaluated using ELISA method. Sample groups were comprised of 111 samples, including pasteurized milk (n?=?56), raw milk (n?=?16) and cheese (n?=?39), and were analyzed for AfM₁ content. The results showed that 93.7% of the samples were positive with the total average concentration of 85.8?µg/L in milk and 30.39?µg/kg in cheese. Moreover, AfM₁ of 94.4% of milk samples and 92.3% of cheese samples was above the standards of Iran and EU.     

Occurrence of aflatoxin M1 in some samples of UHT,raw & pasteurized milk from Indian states of Karnataka and Tamilnadu

Aflatoxin M₁ (AFM₁) is a toxic metabolite found in the milk of lactating animals which have consumed feedstuffs contaminated with aflatoxin B₁. Ultra high temperature treated (UHT) milk is a product which is becoming popular in developing countries like India as there is a lack of proper cold storage or refrigeration facilities. In this study, 45 samples of UHT milk of popular brands prevalent in the market were analyzed for the presence of AFM₁ by reversed phase HPLC using fluorescent detector after cleanup of sample with immunoaffinity columns. All samples of plain UHT milk were positive for AFM₁ and 38% of these contained levels more than 0.5 μg/kg, the maximum permitted limit prescribed by the Codex Alimentarius Commission and by the mandatory regulations of the country, the FSSAI Regulations, 2011. In 62.5% of flavored UHT milk, AFM₁ was below detectable levels (0.02 μg L⁻¹). However, 12.5% of these samples also contained levels exceeding the maximum permitted limits. AFM₁ was present in 61.6% of the 52 raw milk samples analyzed from the two states of Karnataka and Tamilnadu with a range of 0.1–3.8 μg L⁻¹. 17.3% of these samples also exceeded the regulatory limits of the country.     

On the occurrence of aflatoxin M1 in milk and dairy products

Aflatoxins are toxic fungal metabolites found in foods and feeds. When ruminants eat AFB₁-feedstuffs, they metabolise the toxin and excrete AFM₁ in milk. To control AFM₁ in foods it is necessary to reduce AFB₁ contamination of feeds for dairy cattle by preventing fungal growth and AFB₁ formation in agricultural commodities intended for animal use. Corn and corn-based products are one of the most contaminated feedstuffs; therefore risk factor analysis of AFB₁ contamination in corn is necessary to evaluate risk of AFM₁ contamination in milk and milk products. During the corn silage production, the aflatoxins production is mostly influenced by: harvest time; fertilization; irrigation; pest control; silage moisture; and storage practices. Due to the lower moisture at harvest and to the conservation methods, the corn grain is mostly exposed to the contamination by Aspergillus species. Therefore, it is necessary to reduce the probability of this contaminant through choice of: hybrids; seeding time and density; suitable ploughing and fertirrigation; and chemical or biological control. Grains harvested with the lowest possible moisture and conservation moisture close to or less than 14% are necessary to reduce contamination risks, as is maintaining mass to homogeneous moisture. Kernel mechanical damage, grain cleaning practices and conservation temperature are also factors which need to be carefully controlled.     

The comparative metabolism and toxic potency of aflatoxin B1 and aflatoxin M1 in primary cultures of adult-rat hepatocytes

Both aflatoxin B₁ (AFB₁) and a hydroxylated metabolite, aflatoxin M₁ (AFM₁), were potent cytotoxins and genotoxins to primary cultures of rat hepatocytes. However, AFB₁ stimulated the release of lactate dehydrogenase into the culture medium and the loss of viable cells from the monolayer at lower doses than did AFM₁. The lowest toxic doses of AFB₁ and AFM₁ were 0·05–0·1 and 0·6 μg/ culture, respectively. Genotoxicity, determined by an assay for stimulation of DNA repair, was apparent at lower doses than was cytotoxicity. AFB₁ was again more potent than AFM₁, stimulating DNA repair at 0·025 μg/culture. compared to the lowest genotoxic dose of AFM₁ of 0·05 μg/culture. At higher doses (1·2–2·4 μg/culture) the responses due to both aflatoxins in the cytotoxicity and DNA-repair assays were approximately equal. The metabolism of a low dose (c. 0·17 μg/culture) of [¹⁴C]AFB₁ and [³H]AFM₁ by cultured hepatocytes differed significantly. After 1 hr, 50% of the [¹⁴C]AFB₁ remained unchanged in the culture medium, whereas about 18 hr were required for the same amount of [³H]AFM₁ metabolism to occur. [¹⁴C]AFB₁ was metabolized to AFM₁, to polar metabolites recovered in the aqueous phase after chloroform extraction, and to metabolites covalently bound to hepatocyte macromolecules. [³H]AFM₁ was also metabolized to polar metabolites and to forms bound to macromolecules. The degree of covalent binding of the aflatoxins correlated with their cytotoxicity and genotoxicity at lower doses. After a 24-hr incubation, 12·5% of the dose of [¹⁴C]AFB₁ was covalently bound to macromolecules compared to 1·5% of [³H]AFM₁. Although AFM₁ was less potent than AFB₁ in cytotoxicity, DNA-repair and covalent-binding assays using primary cultures of hepatocytes, AFM₁ was still active at relatively low doses and therefore is probably a potent hepatotoxin in vivo.     

Exposure of newborns to aflatoxin M1 and B1 from mothers’ breast milk in Ankara,Turkey

Aflatoxins (AFs) are important risks for human health due to their widespread presence in foods and environment. However, contamination risk of breast milk with different pollutants including AFs is high in today’s life conditions. Since breast milk is a major nutrient for infants, feeding of infants with safe milk is essential. Therefore, the objective of this study was to determine the levels of AF M₁ and B₁ in breast milk samples collected from 75 mothers in Ankara, Turkey. AF M₁ and B₁ levels were investigated by high performance liquid chromatography (HPLC) with a fluorescence detector following an extraction procedure. The limit of detection was found to be 5 ng/l. Both AFs were detected in diverse degrees in all breast milk samples: The level of AF M₁ were in the ranges of 60.90–299.99 ng/l, and AF B₁ were in the ranges of 94.50–4123.80 ng/l. These results pointed out the exposure of mothers and neonates to AF M₁ and B₁, and the necessity of further research on mycotoxin contamination both in foods and biological fluids as well as protection strategies.     

Activity of some respiratory enzymes and cytochrome contents in rat hepatic mitochondria following aflatoxin B1 administration

Effects of aflatoxin B₁ (AFB₁) administration (7 mg/kg body weight i.p.) on rat hepatic mitochondrial respiratory components have been examined. Succinoxidase and cytochrome oxidase activities were decreased in liver mitochondria isolated from rats 12–24 h after AFB₁ treatment. Both enzyme activities returned to normal levels after 48 h. Glutamate dehydrogenase and β-hydroxybutyrate dehydrogenase activities did not show any alterations up to 24 h and thereafter increased at 48–72 h. Succinate dehydrogenase activity was impaired by 41% at 12 h and thereafter was found to be normal. The intramitochondrial cytochrome b content declined at 24–72 h, whereas cytochrome aa₃ content was decreased maximally at 72 h after AFB₁ administration. These observations on mitochondrial enzyme activities and cytochrome contents correlate well with our earlier observations made on hepatic mitochondrial respiratory rates after AFB₁ treatment. The impairment of respiratory functions possibly results from membrane damage and selective modification of gene expression in mitochondria imparted by AFB₁.     

Study of the Ochratoxin A effect on Aspergillus parasiticus growth and aflatoxin B1 production

Aflatoxin B1 (AFB1) is a carcinogenic metabolite produced by certain Aspergillus species. Ochratoxin A (OTA) is a metabolite of Aspergillus ochraceus and Penicillium verrucosum. AFB1 and OTA are amongst the most frequent combinations of mycotoxins found in plant products. Thus, synergistic effects or interactions between the two mycotoxins could be taking place. The aim of the present study was to investigate the effect of OTA on Aspergillus parasiticus growth and AFB1 production in yeast extract sucrose (YES) medium at concentrations of 0.16, 1.6 and 16 ng OTA flask(-1). The AFB1 extracted from cultures and purified with immunoaffinity columns was then quantitated by HPLC. The recovery and detection limit of the method were 95.3% and 0.02 ng AFB1 mL(-1), respectively. Maximum AFB1 productions in cultures with OTA were observed from 9 to 12 days (76.09-82.52 ng AFB1 flask(-1)) while in control cultures (without OTA) maximum production (197.2 ng AFB1 flask(-1)) was observed on 14th day. Maximum AFB1 levels in cultures with OTA were reduced by a percentage of 58-61% compared to control cultures. Furthermore AFB1 levels in cultures with OTA were practically (92%) degraded after 18 days of incubation. Conclusively when OTA is present the production of AFB1 by A. parasiticus in YES medium is inhibited.     

Antigenotoxic effect of Saccharomyces cerevisiae on the damage produced in mice fed with aflatoxin B1 contaminated corn

The potential of Saccharomyces cerevisiae (Sc) was evaluated for reducing the micronucleated normochromatic erythrocytes (MNNE) rate in mice fed AFB₁ contaminated corn. The study included two groups fed AFB₁ contaminated corn (0.4 and 0.8 mg/kg), a control fed uncontaminated corn, another group fed uncontaminated corn and 0.3% of Sc (1 × 10⁸ live cells/g), and two groups fed AFB₁ contaminated corn (0.4 and 0.8 mg/kg) plus 0.3% Sc. Weight and MNNE were determined weekly for six weeks. Subsequently, the same determinations were made for another three-week period, but in mice receiving only a normal diet, without AFB₁ and Sc. Results in the first period revealed the following: control and Sc fed mice had similar constant weight increase, and low MNNE rate; mice fed only AFB₁ showed weight decrease and significant MNNE increase; finally, Sc improved weight gain and reduced MNNE produced by AFB₁. In the second period, results exhibited a tendency similar to that of the previous phase in the control and Sc fed mice; the weight and MNNE values improved in the other groups. We also determined the capacity of Sc for adsorbing and modifying the mycotoxin structure. The mixture was filtered to obtain two phases, and AFB₁ content was measured. Sc revealed a potent adsorbent capacity; however, chromatographic determination suggested no structural modification.     

Determination of aflatoxin B1 levels in Iranian rice by ELISA method

This study was carried out to detect the presence of aflatoxin B₁ (AFB₁) in 40 samples of Tarom rice from Iran. Enzyme-linked immunosorbent assay (ELISA) was applied to analyze AFB₁ in the samples. All the analyses were conducted twice. Aflatoxin B₁ was found in all rice samples, the concentration of AFB₁ ranged from 0.29 to 2.92?µg/kg. The AFB₁ concentration mean in the rice samples produced in 2013 was higher (P < 0.05) than the findings in rice in 2012.. However, 25 of the 40 samples exceeded the maximum prescribed limit, i.e. 2?µg/kg of European Union Regulations and also none of the samples reached the maximum prescribed limit 5?µg/kg of the Institute of Standards and Industrial Research of Iran (ISIRI) for aflatoxin B₁. Although, rice is ranked the second among cereal staples consumed food in Iran and many countries, it can make a serious health problem for people even for a small amount of aflatoxin.     

Aflatoxin B1 and aflatoxin M1 induced cytotoxicity and DNA damage in differentiated and undifferentiated Caco-2 cells

Aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) are natural mycotoxins that frequently present in food and feed and pose risks to human health. There are few data in the literature regarding the impairment of them in the intestine. Therefore, the present study investigated their cytotoxic effect on Caco-2 cells, especially the differentiated ones that resemble mature small intestinal enterocytes. Both undifferentiated (UC) and differentiated (DC) cells were treated with AFB1 and AFM1 at various concentrations for up to 72 h. Cell viability, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) production and DNA damage were determined. Data showed that AFB1 and AFM1 significantly inhibited UC and DC cell growth, increased LDH and caused genetic damage in a time- and dose-dependent manner (p < 0.05). In comparison, AFB1 was found to be more toxic than AFM1 on both UC and DC. All these cytotoxic outcomes might be associated with intracellular ROS generation, leading to membrane damage and DNA strand break. Additionally, DC was found to be more sensitive to aflatoxins, which might be due to the alteration of enzymes during cell differentiation. The present study provided the first in vitro evidence of DNA damage of DC induced by AFB1 and AFM1.     

Red ginseng extract protects against aflatoxin B1 and fumonisins-induced hepatic pre-cancerous lesions in rats

The current study was conducted to evaluate the chemoprevention effects of ginseng extract (GE) against pre-cancerous lesions in female Sprague–Dawley rats treated with aflatoxin B₁ (AFB₁) and fumonisin (FB). Six experimental groups treated for 12 weeks and included: the control group; the GE alone-treated group (150 mg/kg b.w); the group treated orally with AFB₁ (17 μg/kg b.w) during the first 2 weeks and fed FB-contaminated diet (250 mg/kg diet) during the 6th to 8th weeks; the group treated with GE during the mycotoxin protocol and continued till week 10; the group treated with GE 2 weeks before AFB₁ administration and continued till the end of FB treatment and the group treated with GE for 4 weeks after the toxin protocol stopped. The sequential mycotoxins treatment induced significant changes in serum biochemical parameters accompanied by severe histological and histochemical changes of the liver tissue. Treatment with GE during, before or after the treatment with the mycotoxins improved all biochemical parameters and histological picture of the liver. Moreover, treatment with GE after the administration of the mycotoxins was found to be more effective. It could be concluded that GE has a protective effects as pre-cancerous lesions and therapeutic effects as well.     

Aflatoxin M1 levels in UHT milk and kashar cheese consumed in Turkey

In the present study, 100 UHT milk and 132 kashar cheese samples were analyzed for aflatoxin M1 (AFM1). They were obtained from retail outlets in five big cities (Istanbul, Izmir, Konya, Tekirdag, Edirne). The occurrence and concentration range of AFM1 in the samples were investigated by competitive enzyme-linked immunosorbent assay (ELISA) method. Sixty-seven percent of the UHT milk samples and 82.6% of the kashar cheese samples contained AFM1. The positive incidence of AFM1 in the UHT milk samples and the kashar cheese samples ranged from 10 to 630 ng/kg and from 50 to 690 ng/kg, respectively. AFM1 levels in 31 (31%) UHT milk and 36 (27.3%) kashar cheese samples exceeded the maximum tolerable limit of the EC and the TFC. AFM1 levels in the samples show that there is a presence of high aflatoxin level that constitutes a human health risk in Turkey. Therefore milk and dairy products have to be controlled continuously for presence of AFM1 contamination by the Turkish public health authorities.     

Occurrence of aflatoxin B1 contamination in dairy cows feed in Iran

Aflatoxins (AFs) are secondary toxic metabolites produced by fungal species. Occurrence of aflatoxin B1 (AFB1) was determined in 210 dairy cow feed samples consisting of concentrate (n=?70), corn silage (n =?70) and alfalfa hay ( n=?70) obtained from dairy farms in central part of Iran during July–September 2014. AFB1 analysis was carried out by using the competitive enzyme-linked immunosorbent assay technique for screening and high performance liquid chromatography for confirmatory purpose. AFB1 was detected in 88 analyzed samples (41.9%), ranging from 1.87 to 19.41?μg/kg; and 70 samples (33.3%) had levels of the toxin above the Iranian national standard and European Commission limits (5?μg/kg). The frequencies of AFB1 contamination in the samples were as follows: alfalfa hay, 55.7%; concentrate, 44.3% and corn silage, 25.7%. The high occurrence of AFB1 contamination may be attributed to poor harvesting technique, environmental conditions, insect infestation or unsuitable storage conditions. Hence, the contamination with AFs could be a potential hazard for human and animal health.     

HPLC法测定三七中人参皂苷Rg1和三七皂苷R1的含量

目的：建立HPLC测定三七中人参皂苷Rg1和三七皂苷R1含量的方法。方法：色谱条件为：C18柱，流动相为乙腈-0．05％磷酸溶液(21．5：78．5)，UV检测波长为203nm。结果：此方法线性关系良好，平均加样回收率分别为人爹皂苷Rg199．54％和三七皂苷R1101．40％(n=6)。结论：本方法分离良好，结果准确可靠。     

Residues of aflatoxin B1 in broiler meat: Effect of age and dietary aflatoxin B1 levels

This study describes the effect of dietary levels of aflatoxin B1 (AFB1) and age of the birds upon the residue level in liver and muscles of broiler chicks. In three different experiments broiler chicks of 7, 14 and 28 days of age were kept for 7 days on contaminated rations having 1600, 3200 and 6400 μg/kg AFB1. AFB1 residues were detected earlier in younger birds and those fed high AFB1 dietary levels. The highest residue levels in liver and muscles of young chicks fed 6400 μg/kg AFB1 was 6.97 ± 0.08 and 3.27 ± 0.05 ng/g, respectively. Maximum residue concentration was high in birds of young age and those kept on high AFB1 ration. After withdrawal of AF contaminated rations, residues clearance was slow and AFB1 was detectable in liver and muscles of birds for longer duration in younger birds and those fed high AFB1 dietary levels. AFB1 residues in poultry tissues may buildup to high levels in areas with no regulatory limits on AFB1 levels of poultry feed and may pose a risk to consumers health.     

Occurrence of aflatoxin M1 in raw milk collected from traditional dairies in Morocco

A survey of the presence of aflatoxin M1 (AFM1) was carried out on eight traditional dairies belonging to four sectors of Fez city situated in the northern center of Morocco. Raw milk samples were collected between October 2009 and September 2010, and analyzed by LC-fluorescence detection after immunoaffinity purification. AFM1 was detected in 13 out of 48 samples (27%) at concentrations ranged between 10 and 100 ng/l. Within these positive samples, four (∼8% of the total) were above the European legislation limit of 50 ng/l. This study revealed a variation of contamination from one sector to another with a higher incidence in milk samples collected in autumn compared to those collected in other seasons suggesting a link between feeding practices, such as the use of silage and AFM1 contamination. This is the first report on AFM1 contamination in raw milk directly collected from Moroccan traditional dairies. The levels of contamination found justify more detailed and continuous monitoring to assess the public health implication and reduce consumers’ exposure to AFM1.     

HPLC法测定跌打片中人参皂苷Rg1和人参皂苷Rb1的含量

目的:建立高效液相色谱法测定跌打片中人参皂苷Rg1和人参皂苷Rb1的含量测定方法.方法:采用Luna C18色谱柱(250 mm×4.6 mm,5μm);柱温:35℃;流动相为乙腈-水,梯度洗脱;流速1.0 ml ·min-1;检测波长203 nm.结果:人参皂苷Rg1线性范围为0.340～5.094 μg(r=1.000 0),人参皂苷Rb1线性范围为0.303 ～0.454 μg(r=1.000 0);平均加样回收率:人参皂苷Rg1为97.17％,RSD=1.09％ (n =6),人参皂苷Rb1为102.63％,RSD=1.18％ (n =6).结论:该方法简便可靠,可用于跌打片的质量控制.