Mechanisms of butylated hydroxytoluene chemoprevention of aflatoxicosis--inhibition of aflatoxin B1 metabolism

Chemoprevention of toxicoses and/or cancer through the use of nutrients or pharmacologic compounds is the subject of intense study. Among the many compounds examined, food additives such as antioxidants are being considered due to their ability to reduce disease formation by either induction or inhibition of key enzyme systems. One such compound, butylated hydroxytoluene (BHT), has been found to protect against cancer formation caused by exposure to aflatoxin B₁ (AFB₁) in rodents. We have shown that dietary BHT protects against clinical signs of aflatoxicosis in turkeys, a species that is very susceptible to this mycotoxin. In this study, the effect of BHT on AFB₁ metabolism and other cytochrome P450 (CYP)-related enzyme activities in turkey liver microsomes was examined to discern possible mechanisms of BHT-mediated protection against aflatoxicosis. Ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), prototype activities for CYP1A1 and 1A2, respectively, were decreased in the BHT fed (4000 ppm) animals, while oxidation of nifedipine, a prototype activity for CYP3A4, was increased. However, BHT added to microsomal incubations inhibited these CYP activities in a concentration-related manner. Importantly, BHT inhibited conversion of AFB₁ to the reactive intermediate AFB₁-8,-9-epoxide (AFBO), exhibiting Michaelis-Menton competitive inhibition kinetics (Ki = 0.81 μM). Likewise, microsomes prepared from turkeys fed BHT were significantly less active in AFBO formation compared to those from control birds. When turkeys were fed BHT for up to 40 days, residual BHT was present in liver, breast meat, thigh meat and abdominal fat in concentrations substantially below U.S. FDA guidelines for this antioxidant, but in concentrations greater than the Ki, likely sufficient to inhibit bioactivation of AFB₁in vivo. BHT-induced hydropic degeneration in the livers of BHT fed animals was significantly greater in birds that remained on BHT treatment for up to 30 days, but this lesion diminished in animals fed for 40 days or when returned to a control diet. The data indicate that the observed chemopreventive properties of BHT in turkeys may be due, at least in part, to its ability to inhibit hepatic AFB₁ epoxidation and also that the BHT-induced hydropic degeneration is reversible and does not appear to cause long-term effects.     

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.     

Trp266 determines the binding specificity of a porcine aflatoxin B1 aldehyde reductase for aflatoxin B1-dialdehyde

Aflatoxin B₁ (AFB₁) is a severe threat to human and animal health. The aflatoxin B₁ aldehyde reductase (AFAR) family specifically catalyzes AFB₁-dialdehyde, a toxic metabolic intermediate of AFB₁, producing a nontoxic dialcohol. Although several AFARs have been found and characterized, the binding specificity of the family for AFB₁-dialdehyde remains unclear. Herein, according to the published sequence, we cloned a porcine AFAR gene. Recombinant porcine AFAR was expressed and purified from Escherichia coli as hexa-histidine tagged fusion protein. Using the cloned porcine AFAR as a model, site-directed mutagenesis combined with high performance liquid chromatography studies revealed that the substitution of Trp266 with Ala resulted in almost complete loss of catalytic activity for AFB₁-dialdehyde. Interestingly, the substitution of Met86 with Ala exhibited an obviously increased activity to the dialdehyde. Based on these results and by using molecular docking simulations, this work provides a structural explanation for why the AFAR family exhibits high specificity for AFB₁-dialdehyde. The Trp266 residue in porcine AFAR plays a critical role in stabilizing the binding of AFB₁-dialdehyde in the active pocket through the hydrophobic interaction of the side-chain indole ring of Trp266 with the fused coumarin rings of the dialdehyde molecule. The enhanced activity of M86A may be attributed to the formed π–π stacking interaction between Trp266 and the dialdehyde. In addition, other hydrophobic residues (e.g. Phe and Trp) around the dialdehyde molecule also stabilize the substrate binding. The findings may contribute to understanding the substrate specificity of the AFAR family for AFB₁-dialdehyde.     

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.     

Aflatoxin B1 and fumonisin B1 affect the oxidative status of bovine peripheral blood mononuclear cells

Mycotoxins are secondary metabolites having a high cytotoxic potential. They are produced by molds and released in food and feed. To date, the mechanisms underlying the mycotoxin-induced cytotoxicity have not been fully clarified. The induction of oxidative stress, as a possible mechanism, has been postulated. This in vitro study was focused on the effect of two widely occurring mycotoxins, aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁), on the oxidative status of bovine peripheral blood mononuclear cells (PBMC) incubated for 2 and 7 days at different levels of AFB₁ (0, 5 and 20 μg/ml) and FB₁ (0, 35 and 70 μg/ml). Reactive oxygen metabolites (ROM), intracellular thiols (SH), malondialdehyde (MDA) and gene expression of cytoplasmic superoxide dismutase (SOD) and glutathione peroxidase (GSHPX-1) were measured on PBMC after incubation. The highest concentration of AFB₁ and all concentrations of FB₁ caused an increase (p < 0.05) of intracellular ROM without any time dependent effect. Intracellular SH decreased with 20 μgAFB₁/ml (p < 0.05) and the effect was particularly marked after 7 days of exposure. Intracellular SH were not affected by FB₁ even though a lower (p < 0.05) SH level after 2 days exposure than after 7 days was observed. MDA increased (p < 0.05) in AFB₁ or FB₁ treated PBMC. The exposure to FB₁ for 7 days increased MDA (p < 0.05) only in cells treated with 70 μg/ml. Exposure of PBMC to AFB₁ reduced SOD mRNA while FB₁ decreased both SOD and GSHPX-1 mRNA abundance. These results demonstrate that, even though by different mechanisms, AFB₁ and FB₁ may induce cytotoxicity through an impairment of the oxidative status of PBMC.     

Glycine N-methyltransferase affects the metabolism of aflatoxin B1 and blocks its carcinogenic effect

Previously, we reported that glycine N-methyltransferase (GNMT) knockout mice develop chronic hepatitis and hepatocellular carcinoma (HCC) spontaneously. For this study we used a phosphoenolpyruvate carboxykinase promoter to establish a GNMT transgenic (TG) mouse model. Animals were intraperitoneally inoculated with aflatoxin B₁ (AFB₁) and monitored for 11 months, during which neither male nor female GNMT-TG mice developed HCC. In contrast, 4 of 6 (67%) male wild-type mice developed HCC. Immunofluorescent antibody test showed that GNMT was translocated into nuclei after AFB₁ treatment. Competitive enzyme immunoassays indicated that after AFB₁ treatment, the AFB₁-DNA adducts formed in stable clones expressing GNMT reduced 51.4% compared to the vector control clones. Experiments using recombinant adenoviruses carrying GNMT cDNA (Ad-GNMT) further demonstrated that the GNMT-related inhibition of AFB₁-DNA adducts formation is dose-dependent. HPLC analysis of the metabolites of AFB₁ in the cultural supernatants of cells exposed to AFB₁ showed that the AFM₁ level in the GNMT group was significantly higher than the control group, indicating the presence of GNMT can enhance the detoxification pathway of AFB₁. Cytotoxicity assay showed that the GNMT group had higher survival rate than the control group after they were treated with AFB₁. Automated docking experiments showed that AFB₁ binds to the S-adenosylmethionine binding domain of GNMT. Affinity sensor assay demonstrated that the dissociation constant for GNMT-AFB₁ interaction is 44.9 μM. Therefore, GNMT is a tumor suppressor for HCC and it exerts protective effects in hepatocytes via direct interaction with AFB₁, resulting in reduced AFB₁-DNA adducts formation and cell death.     

Aflatoxin B1 Degradation by a Pseudomonas Strain

Aflatoxin B₁ (AFB₁), one of the most potent naturally occurring mutagens and carcinogens, causes significant threats to the food industry and animal production. In this study, 25 bacteria isolates were collected from grain kernels and soils displaying AFB₁ reduction activity. Based on its degradation effectiveness, isolate N17-1 was selected for further characterization and identified as Pseudomonas aeruginosa. P. aeruginosa N17-1 could degrade AFB₁, AFB₂ and AFM₁ by 82.8%, 46.8% and 31.9% after incubation in Nutrient Broth (NB) medium at 37 °C for 72 h, respectively. The culture supernatant of isolate N17-1 degraded AFB₁ effectively, whereas the viable cells and intra cell extracts were far less effective. Factors influencing AFB₁ degradation by the culture supernatant were investigated. Maximum degradation was observed at 55 °C. Ions Mn²⁺ and Cu²⁺ were activators for AFB₁ degradation, however, ions Mg²⁺, Li⁺, Zn²⁺, Se²⁺, Fe³⁺ were strong inhibitors. Treatments with proteinase K and proteinase K plus SDS significantly reduced the degradation activity of the culture supernatant. No degradation products were observed based on preliminary LC-QTOF/MS analysis, indicating AFB₁ was metabolized to degradation products with chemical properties different from that of AFB₁. The results indicated that the degradation of AFB₁ by P. aeruginosa N17-1 was enzymatic and could have a great potential in industrial applications. This is the first report indicating that the isolate of P. aeruginosa possesses the ability to degrade aflatoxin.     

Occurrence of aflatoxins in mahua (Madhuca indica Gmel.) seeds: Synergistic effect of plant extracts on inhibition of Aspergillus flavus growth and aflatoxin production

Occurrence of aflatoxin in Madhuca indica Gmel. seeds was determined by competitive ELISA. Eighty percent of mahua seed samples were found to be contaminated with aflatoxin. Total aflatoxin content ranged from 115.35 to 400.54 ppb whereas the concentration of AFB₁ was in the range of 86.43 to 382.45 ppb. Mahua oil was extracted by cold press expeller and analysed for contamination of aflatoxin in both the oil and cake samples. Total aflatoxin and aflatoxin B₁ were 220.66 and 201.57 ppb in oil as compared to that in cake samples where it was 87.55 and 74.35 ppb, respectively. Various individual and combined plant extracts were evaluated for their efficacy against growth of Aspergillus flavus and aflatoxin production in vitro. Combination of botanicals were found to be more effective in controlling fungal growth and aflatoxin production than individual extracts. Results of the present study suggests that synergistic effect of plant extracts can be used for control of fungal growth and aflatoxin production. These natural plant products may successfully replace synthetic chemicals and provide an alternative method to protect mahua as well as other agricultural commodities of nutritional significance from toxigenic fungi such as A. flavus and aflatoxin production.     

Butylated hydroxytoluene chemoprevention of aflatoxicosis – Effects on aflatoxin B1 bioavailability, hepatic DNA adduct formation, and biliary excretion

The extreme sensitivity of turkeys to aflatoxin B₁ (AFB₁) is associated with efficient hepatic cytochrome P-450 (P450)-mediated bioactivation, and deficient glutathione S-transferase (GST) mediated detoxification. Butylated hydroxytoluene (BHT) protects against AFB₁ toxicity in turkeys through mechanisms that include competitive inhibition of P450-mediated AFB₁ bioactivation. To test whether dietary BHT alters hepatic AFB₁–DNA adduct formation, excretion, and bioavailability of AFB₁ in vivo, turkeys were given diets with BHT (4000 ppm) for 10 days, given a single oral dose of [³H]-AFB₁ (0.05 μg/g; 0.02 μCi/g), then sampled at intervals up to 24 h. Radiolabel in serum, red blood cells, liver, and breast meat was frequently lower in BHT-treated compared to control. Hepatic AFB₁–DNA adducts in BHT-treated turkeys were significantly lower at 12 and 24 h. BHT-fed birds had significant higher bile efflux, though biliary radiolabel excretion was not different from control. The amount of aflatoxin M₁ (AFM₁) excreted in the bile was lower than in control, but BHT had no effect on the biliary excretion of AFB₁, aflatoxin Q₁ or glucuronide and sulfate conjugates. Thus, the chemopreventive properties of BHT may also occur through a reduction in AFB₁ bioavailability in addition to inhibition of bioactivation.     

Screening of Lactobacillus casei strains for their ability to bind aflatoxin B1

It has been proposed that the consumption of lactic acid bacteria capable of binding or degrading foodborne carcinogens would reduce human exposure to these deleterious compounds. In the present study, the ability of eight strains of Lactobacillus casei to bind aflatoxin B₁ in aqueous solution was investigated. Additionally, the effect of addition of bile salts to the growth medium on aflatoxin B₁ binding was assessed. The eight strains tested were obtained from different ecological niches (cheese, corn silage, human feces, fermented beverage). The strains exhibited different degrees of aflatoxin binding; the strain with the highest AFB₁ binding was L. casei L30, which bound 49.2% of the available aflatoxin (4.6 μg/mL). In general, the human isolates bound the most aflatoxin B₁ and the cheese isolates the least. Stability of the bacterial–aflatoxin complex was assessed by repeated washings. Binding was to a limited degree (0.6–9.2% release) reversible; the L. casei 7R1–aflatoxin B₁ complex exhibited the greatest stability. L. casei L30, a human isolate, was the strain least sensitive to the inhibitory effects of bile salts. Exposure of the bacterial cells to bile significant increased aflatoxin B₁ binding and the differences between the strains was reduced.     

Toxic Effects of Aflatoxin B1 in Chinese Sea Bass (Lateolabrax maculatus)

This study was performed to assess the effects of dietary aflatoxin B₁ (AFB₁) on the growth, antioxidant and immune response, digestive enzyme activities, and intestinal morphology of Lateolabrax maculatus during a 56-day feeding trial. Four diets were formulated including 0, 0.1, 0.5, and 1.0 mg/kg of AFB₁. Each diet was randomly assigned to 3 fish tanks with 40 fish per tank. Results indicated that the fish’s final body weight, weight gain rate, specific growth rate, feed intake, condition factor, viscerosomatic index, hepatosomatic index, and intestinesomatic index decreased (p < 0.01) as dietary AFB₁ increased. AFB₁ levels in diets increased (p < 0.05) serum total antioxidant capacity (TAOC), superoxide (SOD), catalase, malondialdehyde (MDA), alkaline phosphatase (AKP), and lysozyme (LZM), and increased (p < 0.05) the TAOC, SOD, MDA, AKP, LZM, and immunoglobulin M in the livers of the fish. Dietary AFB₁ decreased (p < 0.05) intestinal trypsin activity and induced intestinal injury. In summary, dietary AFB₁ up to 1.0 mg/kg was toxic to L. maculatus as judged by reduced growth, enhanced antioxidant and immune response, decreased intestinal trypsin activity, and impaired intestinal morphology.     

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.     

Potential of Kale and Lettuce Residues as Natural Adsorbents of the Carcinogen Aflatoxin B1 in a Dynamic Gastrointestinal Tract-Simulated Model

Adsorption of the carcinogen aflatoxin B₁ (AFB₁) onto agro-waste-based materials is a promising alternative over conventional inorganic binders. In the current study, two unmodified adsorbents were eco-friendly prepared from kale and lettuce agro-wastes. A dynamic gastrointestinal tract-simulated model was utilized to evaluate the removal efficiency of the sorptive materials (0.5%, w/w) when added to an AFB₁-contaminated diet (100 µg AFB₁/kg). Different characterization methodologies were employed to understand the interaction mechanisms between the AFB₁ molecule and the biosorbents. Based on adsorption results, the biosorbent prepared from kale was the best; its maximum adsorption capacity was 93.6%, which was significantly higher than that of the lettuce biosorbent (83.7%). Characterization results indicate that different mechanisms may act simultaneously during adsorption. Non-electrostatic (hydrophobic interactions, dipole-dipole interactions, and hydrogen bonding) and electrostatic interactions (ionic attractions) together with the formation of AFB₁-chlorophyll complexes appear to be the major influencing factors driving AFB₁ biosorption.     

Simplified Synthesis and Stability Assessment of Aflatoxin B1-Lysine and Aflatoxin G1-Lysine

Aflatoxins B₁ (AFB₁) and G₁ (AFG₁) are carcinogenic mycotoxins that contaminate crops such as maize and groundnuts worldwide. The broadly accepted method to assess chronic human aflatoxin exposure is by quantifying the amount of aflatoxin adducted to human serum albumin. This has been reported using ELISA, HPLC, or LC-MS/MS to measure the amount of AFB₁-lysine released after proteolysis of serum albumin. LC-MS/MS is the most accurate method but requires both isotopically labelled and unlabelled AFB₁-lysine standards, which are not commercially available. In this work, we report a simplified synthetic route to produce unlabelled, deuterated and ¹³C₆ ¹⁵N₂ labelled aflatoxin B₁-lysine and for the first-time aflatoxin G₁-lysine. Additionally, we report on the stability of these compounds during storage. This simplified synthetic approach will make the production of these important standards more feasible for laboratories performing aflatoxin exposure studies.     

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.     

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.     

Chemical profile,antifungal, antiaflatoxigenic and antioxidant activity of Citrus maxima Burm. and Citrus sinensis (L.) Osbeck essential oils and their cyclic monoterpene, dl-limonene

The study deals with antifungal, antiaflatoxigenic and antioxidant activity of Citrus maxima and Citrus sinensis essential oils (EOs) and their phytochemical composition. The EOs were obtained by hydrodistillation and their chemical profile was determined through GC and GC–MS analysis. Both the EOs and their 1:1 combination showed broad fungitoxic spectrum against different food contaminating moulds. The EOs and their combination completely inhibited aflatoxin B₁ (AFB₁) production at 500 ppm, whereas, dl-limonene, the major component of EOs showed better antiaflatoxigenic efficacy even at 250 ppm. Both the oils exhibited antioxidant activity as DPPH free radical scavenger in dose dependent manner. The IC₅₀ for radical scavenging efficacy of C. maxima and C. sinensis oils were to be 8.84 and 9.45 μl ml⁻¹, respectively. The EOs were found non-mammalian toxic showing high LD₅₀ for mice (oral, acute). The oils may be recommended as safe plant based antimicrobials as well as antioxidants for enhancement of shelf life of food commodities by checking their fungal infestation, aflatoxin production as well as lipid peroxidation.     

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.     

Toxicity,biochemical effects and residue of aflatoxin B1 in marine water-reared sea bass (Dicentrarchus labrax L.)

Aflatoxicosis and resulting epizootic hepatoma have been reported among a wide range of fish where Aspergillus species-contaminated foodstuffs are incorporated into the diet. Aflatoxin B₁ (AFB₁) is among the most potent known hepatotoxins and carcinogens. Therefore, it is an important potential toxicant to the most of the popularly cultured fish species. The present study was undertaken to assess the susceptibility and toxicity of AFB₁ to sea bass (Dicentrarchus labrax L.), by behavioral and biochemical evaluations. The estimated oral acute median lethal concentration (96 h LC₅₀) of AFB₁ for sea bass was 0.18 mg/kg bwt. The abnormal behavioral responses and signs of toxicity were described. The prolonged oral administration of 0.018 mg/kg bwt AFB₁ to sea bass for 42 successive days induced a significant increase in serum transaminases and alkaline phosphatase activities, and significant decrease in plasma proteins. Residual AFB₁ was detected at high levels (≈5 ppb) in fish musculature at the end of the experimental period. We conclude that marine water sea bass is a species highly sensitive to AFB₁. In addition, consumption of sea bass reared on AFB₁-contaminated diet could have a negative health impact on human health.     

Screening a Strain of Aspergillus niger and Optimization of Fermentation Conditions for Degradation of Aflatoxin B1

Aflatoxin B₁, a type of highly toxic mycotoxin produced by some species belonging to the Aspergillus genus, such as Aspergillus flavus and Aspergillus parasiticus, is widely distributed in feed matrices. Here, coumarin was used as the sole carbon source to screen microorganism strains that were isolated from types of feed ingredients. Only one isolate (ND-1) was able to degrade aflatoxin B₁ after screening. ND-1 isolate, identified as a strain of Aspergillus niger using phylogenetic analysis on the basis of 18S rDNA, could remove 26.3% of aflatoxin B₁ after 48 h of fermentation in nutrient broth (NB). Optimization of fermentation conditions for aflatoxin B₁ degradation by selected Aspergillus niger was also performed. These results showed that 58.2% of aflatoxin B₁ was degraded after 24 h of culture under the optimal fermentation conditions. The aflatoxin B₁ degradation activity of Aspergillus niger supernatant was significantly stronger than cells and cell extracts. Furthermore, effects of temperature, heat treatment, pH, and metal ions on aflatoxin B₁ degradation by the supernatant were examined. Results indicated that aflatoxin B₁ degradation of Aspergillus niger is enzymatic and this process occurs in the extracellular environment.