Aflatoxin M1 in Raw Milk,Pasteurized Milk and Cottage Cheese Collected along Value Chain Actors from Three Regions of Ethiopia

Milk is a highly nutritious and perfect natural food for humans. However, when lactating animals feed on Aflatoxin B₁ (AFB₁)-containing feed, the hydroxyl metabolite aflatoxin M₁ (AFM₁) contaminates the milk and dairy products. The objective of the current study was to assess the level of AFM₁ in raw milk, normally pasteurized milk and Ethiopian cottage cheese collected from value chain actors (producers, collectors, processors and retailers). Cross-sectional study and simple random techniques were used to collect primary samples. A total of 160 composite samples was collected; raw milk (n = 64), pasteurized milk (n = 64) and cheese (n = 32) was analyzed. Quantitative analysis of AFM₁ was conducted using enzyme-linked immunosorbent assay (ELISA). The results indicate that AFM₁ was detected in all milk products. Results along value chains show that the concentration of AFM₁ in raw milk from collectors was significantly higher than from producers, and in pasteurized milk from processors and retailers (p < 0.05). However, no significant (p > 0.05) difference was observed in cottage cheese value-chain actors in all regions. Comparison of AFM₁ mean values among all dairy products shows that raw milk had a significantly higher concentration of AFM₁ followed by pasteurized milk and cottage cheese. However, there was no significant difference between raw and pasteurized milk (p > 0.05). The mean AFM₁ contamination in milk products ranged from 0.137 to 0.319 µg/L (mean value 0.285 µg/L). The contamination percentages of AFM₁ in raw milk (62.50%), pasteurized milk (67.20%) and cottage cheese (25%) were above the regulatory limit set by the European Union (EU) (0.05 µg/L). According to USA/Ethiopian Standard (US/ES) (0.50 µg/L), 21.87%, 25% and 1% exceeded the regulatory limit for the above products, respectively. The overall prevalence (56.88%) was above the EU regulatory limit and 19.38% over US/ES regulations. Therefore, to provide accurate information about the health risk to consumers, there is a need to conduct risk assessment studies in consumers of milk and dairy products at different age groups.     

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.     

Adsorbents Reduce Aflatoxin M1 Residue in Milk of Healthy Dairy Cow Exposed to Moderate Level Aflatoxin B1 in Diet and Its Exposure Risk for Humans

This study investigated the effect of moderate risk level (8 µg/kg) AFB₁ in diet supplemented with or without adsorbents on lactation performance, serum parameters, milk AFM₁ content of healthy lactating cows and the AFM₁ residue exposure risk in different human age groups. Forty late healthy lactating Holstein cows (270 ± 22 d in milk; daily milk yield 21 ± 3.1 kg/d) were randomly assigned to four treatments: control diet without AFB₁ and adsorbents (CON), CON with 8 μg/kg AFB₁ (dry matter basis, AF), AF + 15 g/d adsorbent 1 (AD1), AF + 15 g/d adsorbent 2 (AD2). The experiment lasted for 19 days, including an AFB₁-challenge phase (day 1 to 14) and an AFB₁-withdraw phase (day 15 to 19). Results showed that both AFB₁ and adsorbents treatments had no significant effects on the DMI, milk yield, 3.5% FCM yield, milk components and serum parameters. Compared with the AF, AD1 and AD2 had significantly lower milk AFM₁ concentrations (93 ng/L vs. 46 ng/L vs. 51 ng/L) and transfer rates of dietary AFB₁ into milk AFM₁ (1.16% vs. 0.57% vs. 0.63%) (p < 0.05). Children aged 2–4 years old had the highest exposure risk to AFM₁ in milk in AF, with an EDI of 1.02 ng/kg bw/day and a HI of 5.11 (HI > 1 indicates a potential risk for liver cancer). Both AD1 and AD2 had obviously reductions in EDI and HI for all population groups, whereas, the EDI (≥0.25 ng/kg bw/day) and HI (≥1.23) of children aged 2–11 years old were still higher than the suggested tolerable daily intake (TDI) of 0.20 ng/kg bw/day and 1.00 (HI). In conclusion, moderate risk level AFB₁ in the diet of healthy lactating cows could cause a public health hazard and adding adsorbents in the dairy diet is an effective measure to remit AFM₁ residue in milk and its exposure risk for humans.     

The Presence of Aflatoxin M1 in Milk and Milk Products in Bangladesh

As milk provides both micro- and macronutrients, it is an important component in the diet. However, the presence of aflatoxin B₁ (AFB₁) in the feed of dairy cattle results in contamination of milk and dairy products with aflatoxin M₁ (AFM₁), a toxic metabolite of the carcinogenic mycotoxin. With the aim to determine AFM₁ concentrations in milk and milk products consumed in Bangladesh, in total, 145 samples were collected in four divisional regions (Sylhet, Dhaka, Chittagong, and Rajshahi). The samples comprised these categories: raw milk (n = 105), pasteurized milk (n = 15), ultra-high temperature (UHT)-treated milk (n = 15), fermented milk products such as yogurt (n = 5), and milk powder (n = 5). AFM₁ levels in these samples were determined through competitive enzyme-linked immunosorbent assay (ELISA). Overall, AFM₁ was present in 78.6% of milk and milk products in the range of 5.0 to 198.7 ng/L. AFM₁ was detected in 71.4% of raw milk (mean 41.1, range 5.0–198.7 ng/L), and in all pasteurized milk (mean 106, range 17.2–187.7 ng/L) and UHT milk (mean 73, range 12.2–146.9 ng/L) samples. Lower AFM₁ levels were found in yogurt (mean 16.9, range 8.3–41.1 ng/L) and milk powder samples (mean 6.6, range 5.9–7.0 ng/L). About one-third of the raw, pasteurized, and UHT milk samples exceeded the EU regulatory limit (50 ng/L) for AFM₁ in milk, while AFM₁ levels in yogurt and milk powder samples were well below this limit. Regarding regions, lower AFM₁ contamination was observed in Chittagong (mean 6.6, max 10.6 ng/L), compared to Sylhet (mean 53.7, max 198.7 ng/L), Dhaka (mean 37.8, max 97.2 ng/L), and Rajshahi (mean 34.8, max 131.4 ng/L). Yet, no significant difference was observed in AFM₁ levels between summer and winter season. In conclusion, the observed frequency and levels of aflatoxin contamination raise concern and must encourage further monitoring of AFM₁ in milk and milk products in Bangladesh.     

A Recent Overview of Producers and Important Dietary Sources of Aflatoxins

Aflatoxins (AFs) are some of the most agriculturally important and harmful mycotoxins. At least 20 AFs have been identified to this date. Aflatoxin B₁ (AFB₁), the most potent fungal toxin, can cause toxicity in many species, including humans. AFs are produced by 22 species of Aspergillus section Flavi, 4 species of A. section Nidulantes, and 2 species of A. section Ochraceorosei. The most important and well-known AF-producing species of section Flavi are Aspergillus flavus, A. parasiticus, and A. nomius. AFs contaminate a wide range of crops (mainly groundnuts, pistachio nuts, dried figs, hazelnuts, spices, almonds, rice, melon seeds, Brazil nuts, and maize). Foods of animal origin (milk and animal tissues) are less likely contributors to human AF exposure. Despite the efforts to mitigate the AF concentrations in foods, and thus enhance food safety, AFs continue to be present, even at high levels. AFs thus remain a current and continuously pressing problem in the world.     

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.     

Metabolism of aflatoxin B1 in turkey liver microsomes: the relative roles of cytochromes P450 1A5 and 3A37

The extreme sensitivity of turkeys to aflatoxin B₁ (AFB₁) is associated with efficient epoxidation by hepatic cytochromes P450 (P450) 1A5 and 3A37 to exo-aflatoxin B₁-8,9-epoxide (exo-AFBO). The combined presence of 1A5 and 3A37, which obey different kinetic models, both of which metabolize AFB₁ to the exo-AFBO and to detoxification products aflatoxin M₁ (AFM₁) and aflatoxin Q₁ (AFQ₁), respectively, complicates the kinetic analysis of AFB₁ in turkey liver microsomes (TLMs). Antisera directed against 1A5 and 3A37, thereby individually removing the catalytic contribution of these enzymes, were used to identify the P450 responsible for epoxidating AFB₁ in TLMs. In control TLMs, AFB₁ was converted to exo-AFBO in addition to AFM₁ and AFQ₁ confirming the presence of functional 1A5 and 3A37. Pretreatment with anti-1A5 inhibited exo-AFBO formation, especially at low, submicromolar (~ 0.1 μM), while anti-3A37, resulted in inhibition of exo-AFBO formation, but at higher (> 50 μM) AFB₁ concentrations. Metabolism in immunoinhibited TLMs resembled that of individual enzymes: 1A5 produced exo-AFBO and AFM₁, conforming to Michaelis-Menten, while 3A37 produced exo-AFBO and AFQ₁ following the kinetic Hill equation. At 0.1 μM AFB₁, close to concentrations in livers of exposed animals, 1A5 contributed to 98% of the total exo-AFBO formation. At this concentration, 1A5 accounted for a higher activation:detoxification (50:1, exo-AFBO: AFM₁) compared to 3A37 (0.15: 1, exo-AFBO: AFQ₁), suggesting that 1A5 is high, while 3A4 is the low affinity enzyme in turkey liver. The data support the conclusion that P450 1A5 is the dominant enzyme responsible for AFB₁ bioactivation and metabolism at environmentally-relevant AFB₁ concentrations in turkey liver.     

Hydrated sodium calcium aluminosilicate (HSCAS), acidic HSCAS,and activated charcoal reduce urinary excretion of aflatoxin M1 in turkey poults. Lack of effect by activated charcoal on aflatoxicosis

In one experiment, the effect of inorganic sorbents on the metabolic fate of aflatoxin B₁ (AFB₁) was studied in turkey poults. At 5 weeks of age, female poults were surgically colostomized and 9 days later orally dosed with 0.75 mg AFB₁/kg BW. Hydrated sodium calcium aluminosilicate (HSCAS), acidic HSCAS, and activated charcoal (AC) were tested, by concomitant administration with AFB₁. Urine was collected up to 48 h post-dosing and analyzed for aflatoxin M₁ (AFM₁) which was the major metabolite found in all treatment groups. Hydrated sodium calcium aluminosilicate, previously proven beneficial in alleviating aflatoxicosis in farm animals, reduced urinary AFM₁ output when orally dosed simultaneous with AFB₁. Also, acidic HSCAS and AC significantly decreased AFM₁ excretion when administered concomitantly with AFB₁. A second experiment was conducted to evaluate the ability of two types of AC to modify aflatoxicosis when added to aflatoxin (AF)-contaminated (from culture material) diets of turkey poults. Although AC was able to decrease AFM₁ excretion in the first experiment, no protective effects from AF toxicity were observed in the feeding study.     

Removal of Aflatoxin B1 by Edible Mushroom-Forming Fungi and Its Mechanism

Aflatoxins (AFs) are biologically active toxic metabolites, which are produced by certain toxigenic Aspergillus sp. on agricultural crops. In this study, five edible mushroom-forming fungi were analyzed using high-performance liquid chromatography fluorescence detector (HPLC-FLD) for their ability to remove aflatoxin B₁ (AFB₁), one of the most potent naturally occurring carcinogens known. Bjerkandera adusta and Auricularia auricular-judae showed the most significant AFB₁ removal activities (96.3% and 100%, respectively) among five strains after 14-day incubation. The cell lysate from B. adusta exhibited higher AFB₁ removal activity (35%) than the cell-free supernatant (13%) after 1-day incubation and the highest removal activity (80%) after 5-day incubation at 40 °C. In addition, AFB₁ analyses using whole cells, cell lysates, and cell debris from B. adusta showed that cell debris had the highest AFB₁ removal activity at 5th day (95%). Moreover, exopolysaccharides from B. adusta showed an increasing trend (24–48%) similar to whole cells and cell lysates after 5- day incubation. Our results strongly suggest that AFB₁ removal activity by whole cells was mainly due to AFB₁ binding onto cell debris during early incubation and partly due to binding onto cell lysates along with exopolysaccharides after saturation of AFB₁ binding process onto cell wall components.     

Aflatoxin B1 interferes with the antigen-presenting capacity of porcine dendritic cells

Aflatoxins (AFs) are harmful to animal and human health upon consumption of AF-contaminated feed or food. Among many forms of AFs, aflatoxin B₁ (AFB₁) is the most toxic and carcinogenic. In addition, AFB₁ impairs cell-mediated immunity, although the exact mechanism of this immunotoxicity is currently unknown. By far the most pivotal cells in the induction of immune responses are dendritic cells (DCs). These highly specialised cells dictate T-cell polarisation depending on the nature of the encountered antigens and environmental cues. To elucidate the effect of AFB₁ on the function of DCs, we used porcine monocyte-derived DCs (MoDCs) as a model system. A low dose of AFB₁ transiently reduced the phagocytic capacity of MoDCs. Furthermore, as compared to untreated MoDCs, AFB₁ significantly downregulated the cell surface expression of the co-stimulatory molecule CD40 at 12 h post treatment, while at 24 h the membrane expression levels of CD40 and the activation marker CD25 were significantly upregulated. Interestingly, the T-cell proliferation-inducing capacity of DCs was diminished upon AFB₁ treatment. In contrast, the cytokine secretion pattern of AFB₁-treated MoDCs was similar to mock-treated MoDCs. The results in this study indicate that a low level of AFB₁ dysregulates the antigen-presenting capacity of DCs, which could explain the observed immunotoxicity of this mycotoxin, and further stress the need to reduce AFB₁ levels in agricultural commodities.     

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.     

Modulation of aflatoxin B1 biotransformation by β-naphthoflavone in isolated rabbit lung cells

 The cytotoxic and carcinogenic mycotoxin aflatoxin (AF) B₁ (AFB₁) is biotransformed by the cytochrome P450 monooxygenases (CYP) to a number of relatively nontoxic metabolites, as well as to the ultimate toxic metabolite, AFB₁–8,9-epoxide. In a number of tissues and species, AFB₁ hydroxylation to the relatively nontoxic metabolite, AFM₁, is induced by β-naphthoflavone (BNF) treatment. Although the liver is the principal target organ for AFB₁ toxicity, the mycotoxin is also toxic and carcinogenic to respiratory tissues. To determine if BNF treatment alters the extent of pulmonary AFB₁ bioactivation by enhancing detoxification and thereby decreasing epoxidation, the effects of BNF on pulmonary AFB₁ metabolism were examined. Rabbit lung cells, isolated by protease digestion and centrifugal elutriation, were incubated with [³H]AFB₁. In nonciliated bronchiolar epithelial (Clara) cell-enriched (45–50%) fractions, [³H]AFM₁ production (pmol/mg DNA per 2 h) was increased by prior treatment of rabbits with BNF (80 mg/kg per day, 3 and 2 days before cell isolation) as follows: with 1.0 μM [³H]AFB₁; control, 10.6±2.3; BNF, 30.0±6.4; with 0.10 μM [³H]AFB₁; control, 9.4±4.7; BNF, 20.6±5.9. With 1.0 μM [³H]AFB₁, prior treatment of animals with BNF abolished formation of [³H]aflatoxicol (AFL) but not [³H]AFQ₁. The activation (epoxidation) of [³H]AFB₁ was measured indirectly as covalent binding to endogenous DNA. With 1.0 μM [³H]AFB₁, treatment of rabbits with BNF did not alter DNA binding (pmol/mg DNA per 2 h) in the Clara cell-enriched fraction: control, 103±41; BNF, 114±49. However, with 0.10 μM [³H]AFB₁, DNA binding in the same fraction was 47% lower in cells from BNF treated animals: control, 17.4±4.2; BNF, 9.3±3.9. Formation of 8,9-dihydro-8,9-dihydroxy- AFB₁, and the glutathione conjugate of the aflatoxin epoxide (AFB₁-GSH) were not detectable at the AFB₁ concentrations and time point studied, in cells from either BNF-treated or control rabbits. Incubation of isolated, unseparated lung cells from untreated rabbits with 5.0 to 50 μM BNF decreased [³H]AFB₁-DNA binding in the presence of 0.1 μM [³H]AFB₁ by 35 to 77%, while lower BNF concentrations did not alter DNA binding. In lung cells isolated from BNF treated rabbits, BNF was not detectable (i.e.<0.5 μM detection limit). Therefore, the amount of BNF present in isolated rabbit lung cells following in vivo treatment with BNF was below that required to directly inhibit AFB₁-DNA adduct formation. The decrease in AFB₁-DNA binding from rabbits treated with BNF is apparently due to the selective induction of CYP isozymes and related increases in AFM₁ formation, and not to direct inhibition of epoxidation or enhanced conjugation of AFB₁-8,9-epoxide with glutathione. Received: 5 March 1996/Accepted: 10 May 1996     

Recombinant Expression of Trametes versicolor Aflatoxin B1-Degrading Enzyme (TV-AFB1D) in Engineering Pichia pastoris GS115 and Application in AFB1 Degradation in AFB1-Contaminated Peanuts

Aflatoxins seriously threaten the health of humans and animals due to their potential carcinogenic properties. Enzymatic degradation approach is an effective and environmentally friendly alternative that involves changing the structure of aflatoxins. In this study, Trametes versicolor aflatoxin B₁-degrading enzyme gene (TV-AFB₁D) was integrated into the genome of Pichia pastoris GS115 by homologous recombination approach. The recombinant TV-AFB₁D was expressed in engineering P. pastoris with a size of approximately 77 kDa under the induction of methanol. The maximum activity of TV-AFB₁D reached 17.5 U/mL after the induction of 0.8% ethanol (v/v) for 84 h at 28 °C. The AFB₁ proportion of 75.9% was degraded using AFB₁ standard sample after catalysis for 12 h. In addition, the AFB₁ proportion was 48.5% using AFB₁-contaminated peanuts after the catalysis for 18 h at 34 °C. The recombinant TV-AFB₁D would have good practical application value in AFB₁ degradation in food crops. This study provides an alternative degrading enzyme for the degradation of AFB₁ in aflatoxin-contaminated grain and feed via enzymatic degradation approach.     

Distribution and induction of aflatoxin B1-9a-hydroxylase activity in rat liver parenchymal and non-parenchymal cells

 Chronic administration of aflatoxin B₁ (AFB₁) to rats gives rise to hepatocellular and cholangiocellular carcinomas without affecting Kupffer and endothelial cells. The enzymatic conversion of AFB₁ to AFB₁-8,9-epoxide is the critical step in the activation of the mycotoxin, while the conversion of AFB₁ to aflatoxin M₁ (AFM₁), catalyzed by the AFB₁-9a-hydroxylase, is considered to be a detoxication route for the toxin. In the present study the distribution and inducibility of AFB₁-9a-hydroxylase were analyzed in microsomes derived from freshly isolated liver parenchymal (PC) and nonparenchymal cells (i.e. Kupffer+endothelial cells, NPC). AFB₁-9a-hydroxylase activity was clearly measurable in NPC and similar to that of PC. In NPC the rate of formation of AFM₁ was higher (when incubating with 16 μM AFB₁) than or similar (with 128 μM AFB₁) to that of AFB₁-8,9-epoxide, while in PC it was significantly lower. Taken together, these results suggest that the AFB₁-9a-hydroxylase activity might be particularly important in NPC to protect these cells from AFB₁ by converting it to a significantly less mutagenic metabolite and by reducing the amount of AFB₁ available for epoxidation. Furthermore, it is shown that AFB₁-9a-hydroxylase activity is inducible by phenobarbital (only in PC), 3-methylcholanthrene, isosafrole and Aroclor 1254, thus indicating that in rat liver the conversion of AFB₁ to AFM₁ is catalyzed by members of the cytochrome 1A and 2B families. Received: 7 December 1995/Accepted: 13 February 1996     

Comparative biotransformation of aflatoxin B1 in swine,domestic fowls,and humans

Aflatoxin B₁ (AFB₁) is primarily biotransformed in the liver by cytochrome P450 (CYP) enzymes, which can yield either the genotoxic metabolite AFB₁-8,9 epoxide that causes liver carcinogenicity or less toxic compounds. The biotransformation of AFB₁ is better understood in humans, including gene expression of CYPs involved in the detoxification process. Studies on farm animals have demonstrated genes homologous to human CYPs that play similar roles in AFB₁ biotransformation. This review compares the activities of the most important CYPs related to the biotransformation of AFB₁ in humans, swine and domestic fowls (chickens, quail, turkeys and ducks), as well the main detoxification mechanisms in these species.     

Effect of the combined probiotics with aflatoxin B1-degrading enzyme on aflatoxin detoxification,broiler production performance and hepatic enzyme gene expression

In order to degrade aflatoxin B₁ (AFB₁), AFB_l-degrading microbes (probiotics) such as Lactobacillus casei, Bacillus subtilis and Pichia anomala, and the AFB_l-degrading enzyme from Aspergillus oryzae were selected and combined to make feed additive. Seventy-five 43-day-old male Arbor Acres broilers were randomly divided into 5 groups, 15 broilers for each group. The broilers were given with 5 kinds of diets such as the basal diet, 400 μg/kg AFB₁ supplement without feed additive, and 200, 400, 800 μg/kg AFB₁ supplement with 0.15% feed additive. The feeding experimental period was 30 d, which was used to determine production performance of broilers. In addition, serum, liver and chest muscle were selected for measuring AFB₁ residues, gene expressions, microscopic and antioxidant analyses. The results showed that adding 0.15% feed additive in broiler diets could significantly relieve the negative effect of AFB₁ on chicken’s production performance and nutrient metabolic rates (P < 0.05). It could also improve AFB₁ metabolism, hepatic cell structure, antioxidant activity, and many hepatic enzyme gene expressions involved in oxidoreductase, apoptosis, cell growth, immune system and metabolic process (P < 0.05). It could be concluded that the feed additive was able to degrade AFB₁ and improve animal production.     

Fungal flora and aflatoxin contamination in Pakistani wheat kernels (Triticum aestivum L.) and their attribution in seed germination

This study aimed to isolate fungal pathogens and to subsequently quantify aflatoxin (AF; B₁ + B₂ + G₁ + G₂) contamination in wheat crops grown in Pakistan. Accordingly, a total of 185 wheat samples were collected from different areas of Pakistan and numerous potent fungal pathogens were isolated. AF contamination attributed to the presence of intoxicating fungal pathogens and resulting metabolic activities were quantified using a high performance liquid chromatography-fluorescence detector coupled with postcolumn derivatization. Additionally, the effect of fungal pathogens on seed germination was also examined. The results obtained showed that 50% of tested wheat samples were found to be contaminated with a diverse range of fungal species. The rate of recurrence of fungal pathogens were Aspergillus 31%, Penicillium 9%, Fusarium 8%, Rhizopus 3%, and Alternaria 2%. The presence of Tilletia indica and Claviceps purpurea species was found to be inevident in all tested wheat samples. AFB₁ contamination was detected in 48 (26.0%) samples and AFB₂ in 13 (7.0%) samples. AFG₁ and AFG₂ were not found in any of the tested samples. The contamination range of AFB₁ and AFB₂ was 0.05–4.78 μg/kg and 0.02–0.48 μg/kg, respectively. The total amount of AFs (B₁ + B₂) found in 48 (26.0%) samples had a mean level of 0.53 ± 0.40 μg/kg and a contamination range of 0.02–5.26 μg/kg. The overall results showed that in 137 (74.0%) samples, AFs were not found within detectable limits. Furthermore, in 180 (97.2%) samples, AF levels were found to be below the maximum tolerated levels (MTL) recommended by the European Union (4 μg/kg). In five (2.7%) samples, AF contamination was higher than the MTL of the European Union. However, these samples were fit for human consumption with reference to the MTL (20 μg/kg) assigned by the USA (Food and Drug Administration and Food and Agriculture Organization) and Pakistan (Pakistan Standards and Quality Control Authority). Germination rates in healthy and contaminated wheat kernels were 84.6% and 45.2%, respectively. Based on the obtained results, it was concluded that the levels of fungal pathogen and AF contamination in Pakistani-grown wheat are not a potential threat to consumer health. However, control procedures along with a strict monitoring policy are mandatory to further minimize the prevalence of fungal carriers and the potency of AFs in crops cultivated in Pakistan.     

Aflatoxin M1 contamination and antibiotic residue in milk in Khorasan province,Iran

The aim of this study was to evaluate aflatoxin M₁ (AFM₁) contamination and antibiotic presence in milk samples in Khorasan province in Iran. During 4 months (March to June 2008), one hundred ninety-six milk samples were collected from seven dairies. The occurrence and concentration range of AFM₁ in the samples were investigated by competitive enzyme-linked immunoabsorbent assay (ELISA) method. Antibiotic presence was determined using Copan test, a broad-spectrum test capable of detecting beta-lactams, tetracyclines, sulfonamides, aminoglycosides and macrolydes. AFM₁ was found in 100% of the examined milk samples by average concentration of 77.92 ng/kg. The concentration of AFM₁ in all of the samples were lower than Iranian national standard and FDA limit (500 ng/L), but 80.6% of the samples had AFM₁ greater than the maximum tolerance limit (50 ng/L) accepted by European Union and Codex Alimentarius Commission. Statistical evaluation showed no significant difference between the mean concentrations of AFM₁ of milk samples taken from different factories (P > 0.05). Copan milk test was positive for 40.8% of the samples.