A SIMPLE SOLID-PHASE RADIOIMMUNOASSAY FOR AFLATOXIN B1

A solid-phase radioimmunoassay (RIA) for aflatoxin B₁ (afla B₁) was developed. This method involved the incubation of afla B₁, both labelled and unlabelled, with immunoglobulin (IgG)-sepharose gel which was prepared by conjugation of the IgG highly specific to afla B₁ with CNBr-activated sepharose gel, followed by a filtration step. The binding capacity was determined by counting the radioactivity in the filtrate. Studies with different afla B₁ analogues revealed that the IgG-gel bound most effectively with B₁. Binding of afla B₂, G₁, G₂, and aflatoxicol to the IgG-gel was less effective in comparison with the IgG before coupling. Between 0.5–5.0 ng per assay, the displacement of radioactivity from the gel was directly proportional to the amount of afla B₁ present. Using a simple extraction procedure without clean-up step, the recovery yields for afla B₁ in the contaminated corn or wheat at levels of 5 ppb or above were above 60%.     

EFFECT OF GAMMA IRRADIATION ON AFLATOXIN B1 PRODUCTION BY ASPERGILLUS FLAVUS IN GROUND BEEF STORED AT 5C

The effect of γ-irradiation for controlling the production of aflatoxin B₁ by Aspergillus flavus in ground beef stored at 5C for 2 weeks was investigated. Aspergillus, Penicillium, Cladosporium, Mucor, Scopulariopsis, Candida and Rhodotorula were the most common fungal genera contaminating ground beef. A. flavus and A. niger were the most common Aspergillus spp. Aspergillus flavus isolates were able to produce aflatoxin B₁ in ground beef. Only 3 (20%) samples of ground beef were contaminated with aflatoxin B₁ (25–45 μg/Kg). Gamma irradiation dose levels resulted in an immediate reduction in the total numbers of A. flavus. No growth or aflatoxin B₁ production occurred at 1.50 kGy during storage.     

SYNERGISTIC EFFECT OF VITAMIN B1 ON SANITIZER AND DISINFECTANT TREATMENTS FOR REDUCTION OF BACILLUS CEREUS IN RICE

The synergistic bactericidal effects of vitamin B₁ (thiamine dilauryl sulfate) and the efficacy of commercial sanitizers for minimization of Bacillus cereus contamination in cooked rice were investigated. Sanitizer-treated rice exhibited a greater reduction than water-treated rice, while sanitizer-treated rice with Vitamin B₁ produced an even greater reduction. The treatments for B. cereus in rice included (1) 100 ppm hydrogen peroxide with 500 ppm vitamin B₁; (2) 200 ppm hydrogen peroxide with 100 ppm vitamin B₁; (3) 400 ppm hydrogen peroxide; (4) 50 ppm chlorine with 500 ppm vitamin B₁; (5) 60 ppm chlorine with 300 ppm vitamin B₁; (6) 70 ppm chlorine with 100 ppm vitamin B₁; (7) 80 ppm chlorine; and (8) 100,000 ppm ethanol with 500 vitamin B_1. All treatments completely eliminated B. cereus in rice. The sensory properties of all sanitizer-treated cooked rice did not differ significantly from the same properties for water-treated cooked rice.

PRACTICAL APPLICATIONS

The prevalence of Bacillus cereus in rice and rice products has been documented and control of the growth of B. cereus in rice is an important consideration. The results obtained from this study can be of use to rice producers in the manufacture of safe products. Although chemical disinfectants can be used to reduce the amount of B. cereus in rice, vitamin B₁ can also be used as an effective additive that reduces the amount of disinfectant use via a synergistic antimicrobial effect. The increasing use of chemical disinfectants for safety in the food industry can be reversed using our method.     

PHYTIC ACID INHIBITS the PRODUCTION of AFLATOXIN B1

Phytic acid inhibition of Aspergillus flavus aflatoxin B₁ production is well observed. Although this fungus grew well in Czapak-Dox medium, mycotoxin production was eliminated by adding a small amount of phytic acid. Possible reasons are discussed, and the importance of some metallic ions is observed. Results suggested that phytate may be an effective anti-AFB₁ agent for preventing the contamination of the fungus.     

TISSUE DISTRIBUTION AND METABOLISM OF AFLATOXIN B1-14C IN LAYER CHICKENS

The effects of administering low levels of aflatoxin B₁-¹⁴C by crop intubation daily for 14 days to layer chickens were determined. Studies on the distribution of ¹⁴C in the blood, selected organs, tissues, eggs and excreta were conducted. No toxic effects were observed in layer chickens during the course of the experiment. The layer chickens excreted 92.15% of the ¹⁴C administered. Of the ¹⁴C retained, 19.5, 16.1, 3.9, 7.2, 26.4 and 26.9% were detected in the blood, liver, heart, gizzard, breast and leg, respectively. No radioactivity was detected in egg samples collected. Chemical assay of those samples demonstrating radioactivity revealed that 81.2% of the radioactivity in these substrates was soluble in aqueous extracts while approximately 10% was ex tractable by classical extraction procedures. Treatment of the aqueous extracts for conjugated steroids by treatment with Beta-glucuronidase revealed that 31.5% of the ¹⁴C detected in the aqueous extract was a liberated glucuronide conjugate of aflatoxin M₁−¹⁴C     

REMOVAL OF AFLATOXIN B1 TOXICITY BUT NOT MUTAGENICITY BY 1 MEGARAD GAMMA RADIATION OF PEANUT MEAL

The toxic and mutagenic effects of gamma-irradiated peanut meal contaminated with aflatoxin B₁ were studied in Salmonella typhimu-rium strain TM 677, using forward mutation to 8-azaguanine resistance. After treatment with 5 to 10 M-rad gamma radiation, the contaminated peanut meal lost its toxic and mutagenic properties. Irradiation at 0.1 to 1.0 M-rad removed 75–100% of the toxicity but not mutagenicity.     

A Reasearch Note VITAMIN B6 IN PORK MUSCLE COOKED IN MICROWAVE AND CONVENTIONAL OVENS

Loin sections of pork muscle were heated to 75 or 85°C internal temperatures in microwave or conventional electric ovens. Cooking losses and time, percentage moisture and total vitamin B₆ were determined. Cooking time was longer, total cooking loss less and moisture content greater for pork cooked in an electric oven than for that cooked in a microwave oven. Differences in vitamin B₆ due to type of oven or internal temperature were small and not significant when calculated on a cooked weight basis. When calculated on a dry weight basis samples cooked in the conventional oven contained more vitamin B₆ than did those cooked by a microwave oven.     

AFLATOXIN PRODUCTION ON TWO MUSHROOM SUBSTRATES

Two fungi, Boletus edulis and Agaricus bisporus, were tested as substrates for two known aflatoxigenic fungi, Aspergillus flavus ATCC 15548 and A. parasiticus NRRL 2999. Both autoclaved substrates supported mycelial growth, sporulation, and aflatoxin production; however, the B. edulis substrate allowed more rapid mold growth and greater toxin production than did the A. bisporus substrate under laboratory conditions. Both aflatoxins B₁ and AFG₁ were produced with AFG₁ being the predominant toxin. Aflatoxins B₂ and AFG₂ were not detected. Although toxin was produced at low levels, the highest mean being 0.55 μg/g substrate for AFB₁ and AFG₁, both mushrooms apparently contained minimal nutrients for toxigenic mold growth and failed to cause antimycotic or antiaflatoxigenic responses. Routinely used aflatoxin extraction and analytical procedures appear applicable for such testing of mushrooms.     

Loss of Fuminosin B1 in Extruded and Baked Corn-Based Foods with Sugars

The objective of this work was to determine the effect of added sugars on fumonisin B₁ (FB₁) levels in baked corn muffins and extruded corn grits. Muffins containing added glucose had significantly lower FB₁ levels than muffins with sucrose, fructose, or no added sugar. Extrusion cooking of the grits resulted in significant (p< 0.05) reductions of FB₁ in all treatments relative to unextruded controls, but use of glucose resulted in greater reductions of FB₁ (45.3 to 71%) than did the use of fructose (29.5 to 53%) or sucrose (19.2 to 39%). When extrusion conditions were optimized, 92.1% loss of FB₁ was found when grits were extruded with glucose. Adding glucose to thermally processed food can result in a substantial reduction in FB₁ levels.     

INFLUENCE OF OTHER FUNGI ON AFLATOXIN PRODUCTION BY ASPERGILLUS FLAVUS IN MAIZE KERNELS

Experiments were designed to determine whether certain nontoxigenic fungi commonly isolated from maize kernels can affect aflatoxin B₁ development when inoculated with A. flavus onto individual unsterilized, and autoclaved maize kernels . Trichoderma viride and Aspergillus niger were found to be strongly antagonist inhibiting the growth of A. flavus by 87 and 66% respectively, whereas Aspergillus versicolor, Fusarium moniliforme, Paecilomyces variotii and Emericella quadrillineata inhibited the growth of A. flavus by less than 51%. Less aflatoxin B₁ was detected when A. flavus was paired with A. niger or T. viride than with the other test fungi. When A. niger or T. viride was introduced onto the kernels 72 h before inoculation with A. flavus, no aflatoxin B₁ was detected in unsterilized kernels and the levels of aflatoxin B₁ were greatly reduced from 700 ppb to 160 and 140 ppb in autoclaved kernels, respectively. When inoculation of A. flavus followed 72 h of incubation of either A. niger and T. viride, no aflatoxin B₁ was detected. However, when both A. niger and T. viride were introduced 72 h after inoculation with A. flavus, the levels of aflatoxin B₁ were reduced to 385 and 560 ppb, respectively in unsterilized and autoclaved maize kernels . Trichoderma viride and Aspergillus niger may be useful in biological control of aflatoxin contamination of maize kernels .; Accepted for Publication June 11, 1997     

Optimisation of baking parameters of chapati with respect to vitamin B1 and B2 retention and quality

Vitamin losses during processing of cereal grains and their food products play an important role in human vitamin intake. Therefore, experiments were conducted to optimise the baking parameters for chapati, a flat unleavened, hot-plate-baked product of Asian diet. In the experiments, whole-wheat flour (100% extraction rate) was used for the preparation of chapaties. Central composite rotatable design with three independent variables (thickness of dough sheet, time as well as temperature of baking) at five levels and nine dependent variables, i.e. vitamin B₁ and B₂ content, textural properties and overall acceptability scores, were used to conduct the experiments. The ranges for three independent variables were: thickness, 2.5–4.5 mm; time, 2.0–3.0 min; and temperature, 200–240 °C. Thickness of the dough sheet had a significant ( P  ≤ 0.05) positive effect on vitamin retention, while the temperature had greater negative effect as compared with the time of baking. Based on compromise optimisation, optimum conditions for baking of chapati, especially with respect to vitamin retention were: thickness of dough sheet 3.2 mm, time of baking 2.1 min and baking temperature 216.3 °C.     

Fumonisin B1 Production and Growth of Fusarium moniliforme and Fusarium proliferatum on Maize, Wheat, and Barley Grain

Two isolates each of Fusarium moniliforme and Fusarium proliferatum isolated from maize were compared for growth and fumonisin B₁ production on maize, wheat, and barley extract agars and on irradiated maize, wheat, and barley grain in relation to water availability and temperature. Growth rates of both Fusarium spp. were similar or higher on wheat and barley than on maize extract agar. Regardless of temperature, all isolates produced fumonisin B₁ only on irradiated maize grain, but not on wheat or barley. This might be a reason for the low natural occurrence of fumonisins on those grains.     

COMBINED EFFECT OF pH AND HEAT TREATMENT ON DEGRADATION OF AFLATOXINS IN DRIED FIGS

Dried figs are sensitive commodities to aflatoxin contamination. Although preventive methods are the logical solution to aflatoxin problems, once the product is contaminated, decontamination procedures are inevitable. In this study, the effectiveness of a procedure consisted of acidification/alkalization, and heat treatment in degradation of aflatoxins was evaluated. The pH of dried fig extracts was adjusted to 3.1, 3.5, 6, 8 or 10 by adding acid or base. Extracts were heated at 50, 75 or 98C for 1 or 2 h, and then the residual aflatoxin B₁, B₂, G₁ and G₂ were determined. The highest level of degradation for aflatoxin B₁ (97  ±  1%) and B₂ (87  ±  1%) were observed at pH 10 in samples heated at 98 and 50C, respectively. Some treatments resulted in 100% degradation of aflatoxin G₁ and G₂ so that they could not be detected.

PRACTICAL APPLICATIONS

Aflatoxin contamination is a serious problem for a number of processed and non-processed foods, including dried figs. This not only presents severe risks to human and animal health but also causes economic problems for countries such as Turkey, U.S.A., Greece and Spain, which produce and export dried figs. It is clear that detoxifying studies are unavoidable when the amount of crop contaminated by toxins is considered. Therefore, the food industry is in search of applications that are effective in mycotoxin detoxification and adaptable to food processes. This is the first report on degradation of aflatoxins in naturally contaminated dried figs by such a promising method.     

Efficacy of extract and essential oil of Lantana indica Roxb. against food contaminating moulds and aflatoxin B1 production

The study investigates the antifungal and antiaflatoxigenic efficacy of Lantana indica against Aspergillus flavus , a key storage fungus. The leaf essential oil of L. indica was found more active than leaf extracts. The oil absolutely inhibited the growth of A. flavus at 1.5 mg mL⁻¹ while ethanolic and chloroform extracts of leaf show MIC at 7.5 and 10.0 mg mL⁻¹ concentrations respectively. The oil also showed pronounced antiaflatoxigenic efficacy and completely inhibited the aflatoxin B₁ production at 0.75 mg mL⁻¹. The ethanolic and chloroformic extracts inhibited the aflatoxin B₁ production at 5.0 and 7.5 mg mL⁻¹, respectively while other extracts exhibited poor efficacy. The L. indica essential oil exhibited broad fungitoxic spectrum against twelve different storage moulds. The present findings may recommend the L. indica essential oil and its bioactive leaf extracts as natural preservative would of immense significance in view of the environmental and toxicological implications by indiscriminate use of synthetic pesticides .     

INFLUENCE OF MOISTURE CONTENT AND STORAGE TEMPERATURE ON THE PRODUCTION OF AFLATOXIN BY ASPERGILLUS FLAVUS EA-81 IN MAIZE AFTER EXPOSURE TO GAMMA RADIATION

The effect of gamma radiation on aflatoxin production by Aspergillus flavus EA-81 in maize with different initial moisture levels was determined over a 15-day period. The viability of A. flavus on maize decreased over time with increasing moisture contents and storage at 8C. After 45 days at 28C, levels of viable conidiospores of A. flavus increased from 4.5 × 10⁷ to about 3.0 × 10⁸ per gram of maize. Levels of aflatoxin B₁ produced by A. flavus were 10 μg kg^-1 in the maize stored at 8C after 45 days. Production of aflatoxin was highest at 40% moisture and 28C. Irradiation of 1.0 or 2.0 kGy greatly reduced the level of mold growth relative to unirradiated controls. A dose of 4.0 kGy eliminated all viable fungi. Aflatoxin B₁ production decreased with increased levels of irradiation and was negligible at 4.0 kGy. When maize was inoculated after irradiation and stored, the spore counts and aflatoxin levels were higher than in unirradiated and inoculated controls after 30 days. Apparently, the natural competitive microflora prevented growth and thus limited higher concentrations of aflatoxin in maize.     

Aflatoxin levels in dried figs, nuts and paprika for export from Turkey

Three hundred and thirteen of 2643 dried fig, two of eighty hazelnut, sixteen of twenty-eight pistachio, five of ten peanut and nineteen of twenty-three paprika samples for export from Turkey were contaminated with total aflatoxins in the range of 0.2–162.76, 5.46–6.55, 2.31–63.11, 0.75–26.36 and 1.79–6.55 μg kg⁻¹, respectively. Samples were collected from January to August 2007 and tested for aflatoxins (B₁, B₂, G₁ and G₂) by immunoaffinity column extraction using RP-HPLC. Fifty-six of the 313 dried fig, all of the contaminated hazelnut and pistachio, two of the sixteen peanut and three of the nineteen paprika samples exceeded the regulatory limits of the European Union. The ratio of the different types of aflatoxin present in each sample exhibited great variability. For example, of 313 contaminated fig samples, 159 contained only aflatoxin B₁, eighty-five contained B₁ (49.7%) + G₁ (50.3%), twenty-two contained only G₁, twenty contained B₁ (89.4%) + B₂ (10.6%), thirteen contained B₁ (73.7%) + B₂ (10.8%) + G₁ (15.5%) and fourteen contained all four types, B₁ (26%) + B₂ (2.5%) + G₁ (66.5%) + G₂ (5%).     

RESEARCH NOTE: MYCOTOXINS IN FENNEL SEED

Ten samples of fennel seed from fields in India were examined for the presence ofaflatoxins B₁, B₂, G₁ and G₂. Five of the samples were fluorescent or became fluorescent during a 12 month storage period. Only aflatoxins B₁, B₂ and G₁ were identified. One nonfluorescent sample contained detectable aflatoxin B₁ after 12 months of storage.     

INHIBITION OF ASPERGILLUS PARASITICUS GROWTH AND TOXIN PRODUCTION BY GARLIC

Mycelial growth and toxin production by Asperigillus parasiticus were inhibited by garlic concentrations of 0.3–0.4%. When the fungus was grown in broth, aflatoxin B₁ production was inhibited at a garlic concentration of 0.3% while aflatoxin G₁ production was inhibited by 0.25% garlic. When growth on rice, aflatoxin B₁ was detected at garlic concentrations of up to 2.5%. Aflatoxin G₁ was detected at 1.25% garlic concentration but not above this level.     

Comparison of Antimutagenic Activity of Phenolic Compounds in Newly Harvested and Stored Common Beans Phaseolus vulgaris against Aflatoxin B1

ABSTRACT: Phenolic compounds were extracted from recently harvested or stored black Jamapa beans ( Phaseolus vulgaris ) that were subjected or not to thermal treatment. The beans studied were cropped in the same area and were from the same lot. The highest amount of condensed tannins (CT) was found in the seed coat of recently harvested beans [222.41 ± 16mg of (+)-catechin equivalents per gram of seed coat]. After 2 yof storage, the amount of CT dropped significantly [35.8 ± 3.4 mg of (+) -catechin equivalents per gram of seed coat]. Thermal treatment significantly reduced the amount of CT in whole beans by approximately 70%. The raw seeds contained 13.76 ±1.2 mg of (+)-cat-echin equivalents per gram of seeds and a portion of CT appeared in the broth [9.4 ± 0.1 mg of (+) -catechin equivalents per gram of lyophilized broth]. The antimutagenic activity of these extracted phenolic compounds was tested against aflatoxin B₁ (AFB₁) in the Kado microsuspension assay. Newly harvested beans showed higher antimutagenic activity against AFB₁ mutagenicity than stored beans. The results suggest that to take the maximum advantage of components with biological activity present in beans, they must be used fresh.     

PURIFICATION AND PROPERTIES OF PHOSPHOLIPASE A2 ISOZYMES FROM PYLORIC CECA OF THE STARFISH (ASTERINA PECTINIFERA)

Phospholipase A₂ isozyme II (PLA₂ II), which showed different mobility on native PAGE from that of the PLA₂ isozyme I (PLA₂ 1) isolated previously, was purified from pyloric ceca of the starfish (Asterina pectinifera). The PLA₂ II mainly released oleic acid from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. N-terminal amino acid sequence of the PLA₂ II was SVYQF. Temperature and pH optima of the PLA₂ II were at around 50C and pH 9.0, respectively, and the enzyme activity was enhanced by sodium deoxycholate and 1 mM or higher concentration of Ca²⁺. The PLA₂ II did not show fatty acid specificity for hydrolysis of phosphatidylcholine (PC). Specific activity of the PLA² II was about 10 times higher than that of commercially available porcine pancreatic PLA₂. The PLA₂ II hydrolyzed PC more effectively than phosphatidylethanolamine. These characteristics of the PLA₂ II were the same as those of the PLA₂ I.