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.     

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.     

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%.     

Aflatoxin Removal from Pistachio Nuts by Natural Natrolite

ABSTRACT: Natrolite was used to explore a physical and safe method to decontaminate aflatoxin-containing pistachio lots. Pistachio nuts with low, medium, and high levels of aflatoxin were subjected to a washing process with a slurry of 5% natrolite. Using thin-layer chromatography and scanning, the aflatoxin contents of the samples were measured. Aflatoxins B₁ and B₂ were found in pistachio nuts. The majority of total aflatoxins was aflatoxin B₁ Natrolite treatment resulted in a 38% to 100% reduction in aflatoxin B₁, depending on the initial aflatoxin level. Although natrolite was demonstrated to be an effective candidate to reduce aflatoxin B₁, its efficacy against aflatoxin B₂ was limited.     

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.     

Aflatoxin Inactivation Using Aqueous Extract of Ajowan (Trachyspermum ammi) Seeds

ABSTRACT: Aqueous extract of ajowan seeds was found to contain an aflatoxin inactivation factor (IF). Thin layer chromatography analysis of the toxins after treatment with IF showed relative reduction of aflatoxin G₁ > G₂ > B₁ > B₂. Quantification of toxin using a fluorotoxin meter as well as the Enzyme Linked Immuno s orb ent Assay (ELISA) confirmed these findings. An approximate 80% reduction in total aflatoxin content over the controls was observed. This observed phenomenon of reduction in total toxin was referred to as toxin inactivation. Temperature was found to influence the rate of toxin inactivation. At 45 °C, it was found to be rapid during the initial 5 h and slowed later. The IF was found to retain considerable activity even after boiling and autoclaving, indicating partial heat stability. The activity was lost below pH 4.0. Above pH 4.0, it increased gradually, reaching the maximum at pH 10.0. IF was found to be stable to gamma irradiation. Toxin decontamination in spiked corn samples could be achieved using IF. This study emphasizes the potential of ajowan IF in aflatoxin removal from contaminated food commodities. However, the biological toxicity, if any, of the IF inactivated aflatoxins needs to be confirmed, and the work in this direction is in progress.     

PREPARATION AND CHARACTERIZATION OF AFLATOXIN B2a-HEMIGLUTARATE AND ITS USE FOR THE PRODUCTION OF ANTIBODY AGAINST AFLATOXIN B1

Hemisuccinate (HS) and hemiglutarate (HG) of aflatoxin B_2a (afla B_2a) were prepared by refluxing afla B_2a with the corresponding anhydride and 4-N, N-dimethylaminopyridine in tetrahydrofuran. Two epimers of the respective HS or HG which show different chromatographic behavior and physiochemical properties were isolated and characterized. Afla B_2a-HS hydrolyzes very rapidly in aqueous solution and was not used for further study. Afla B_2a-HG hydrolyzes at a much slower rate and was selected for the coupling to protein. Using the mixed anhydride method, as much as 12 moles of afla B_2a-HG were conjugated to each mole of bovine serum albumin (BSA). The antibody obtained from rabbits immunized with afla B_2a-HG BSA is most specific to afla B₁ and shows little cross reaction with afla G₁ and aflatoxicol. The lower limit for detection of afla B₁ by radioimmunoassay using this antibody is in the range of 30–50 pg per assay.     

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.     

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.     

REDUCTION OF FUNGI AND MYCOTOXINS FORMATION IN SEEDS BY GAMMA-RADIATION

Ninety samples of maize, chick-peas and groundnut seeds collected from the Egyptian market were found to be heavily contaminated by molds. Alternaria, Aspergillus, Cladosporium, Eurotium, Fusarium, Mucor, Penicillium and Rhizopus were the most common fungal genera isolated from nondisinfected seeds . Aspergillus alutaceus, A. flavus, Fusarium verticillioides and F. oxysporum were isolated from all surface-disinfected seeds and were reported to produce ochratoxin A, aflatoxin B₁ and zearalenone, respectively. Irradiation at a dose 4.0 kGy reduced the mold growth greatly relative to unirradiated controls. There was no growth at dose 5.0 kGy. On the basis of the radiation survival data, the decimal reduction values D₁₀ for A. alutaceus, A. flavus and F. verticilliodies were 0.70. 2.10 and 0.93 kGy in maize. A dose of 5 kGy inhibited the toxigenic molds and mycotoxin formation in seeds. Aflatoxin B₁ and ochratoxin A were detected in maize and chick-peas, whereas zearalenone was detected in maize samples. Application of radiation at a dose of 6.0 kGy detoxified aflatoxin B₁ by 74.3–76.7%, ochratoxin A by 51.3–96.2% and zearalenone by about 78%.     

Aflatoxins in spices marketed in Portugal

Seventy-nine prepackaged samples of 12 different types of spice powders (five cardamom, five cayenne pepper, eight chilli, five cloves, seven cumin, five curry powder, five ginger, five mustard, 10 nutmeg, 12 paprika, five saffron and seven white pepper) were selected from supermarkets and ethnic shops in Lisbon (Portugal) for estimation of aflatoxins by immmunoaffinity column clean-up followed by HPLC. Aflatoxin B (AFB) was detected in 34 samples of prepackaged spices (43.0%). All of the cayenne pepper samples were contaminated with levels ranging from 2 to 32 µ g AFB / kg. Three nutmeg samples contained levels ranging from 1 to 5 µ g/kg, three samples had levels ranging from 6 to 20 µ g/kg, and there were two with 54 µ g/kg and 58 µ g/ kg. Paprika contained levels of aflatoxin B₁ ranging from 1 to 20 µ g/kg. Chilli, cumin, curry powder, saffron and white pepper samples had levels ranging from 1 to 5 µ g/kg. Aflatoxins were not detected in cardamom, cloves, ginger and mustard. None of the samples analysed contained aflatoxins B₂, G₁ and G₂.     

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.     

Mathematical modelling of O2 consumption and CO2 production rates of whole mushrooms accounting for the effect of temperature and gas composition

Investigation of respiration rate of fresh produce, under different gas composition and temperatures, and respective mathematical modelling is central for the modified atmosphere packaging design. This work investigates the effect of temperature (4, 8, 12, 16, 20 °C) and gas composition (O₂ between 3 to 21% and CO₂ between 0 to 15%) on respiration rate of whole mushrooms. Oxygen and carbon dioxide respiration rates increased significantly (3–4 fold) as the temperature elevated from 4 to 20 °C and were in the range of 13.23 ± 3.12 to 102.41 ± 2.132 mL kg⁻¹ h⁻¹) and 14.33 ± 1.56 to 97.02 ± 2.51 mL kg⁻¹ h⁻¹) respectively. Low O₂ and high CO₂ levels reduced O₂ consumption and CO₂ production rates of whole mushrooms on average by a circa 47–60% at all temperatures as compared to the respiration rate at ambient air. Mathematical models were developed for RO₂ and RCO₂, by combining the Arrhenius and Michaelis–Menten uncompetitive equations. These models predicted well, O₂ consumption and CO₂ production rates of whole mushrooms as a function of both temperature and gas composition.     

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.     

Extension of shelf-life of whole-wheat flour by gamma radiation

The effect of low-dose gamma irradiation (0.25–1.00 kGy) on pre-packed whole-wheat flour (atta) was assessed in terms of physico-chemical properties, nutritional quality, chapati-making quality and sensory attributes. Semi-pilot scale storage studies on irradiated pre-packed whole-wheat flour revealed that there was no adverse effect of irradiation and storage up to 6 months of whole-wheat flour treated at doses up to 1.00 kGy on total proteins, fat, carbohydrates, vitamin B₁ and B₂ content, colour index, sedimentation value, dough properties, total bacterial and mould count. Storage of wheat flour resulted in slight increase in moisture, free fatty acids, damaged starch, reducing sugars and slight decrease in gelatinization viscosity.
However, irradiation as such had no effect on any of these parameters. Irradiation at 0.25 kGy was sufficient to extend the shelf-life of atta up to 6 months without any significant change in the nutritional, functional attributes. Chapaties made from irradiated atta (0.25 kGy) were preferred even after 6 months storage, compared with the control.     

Rheological behaviour of acorn starch dispersions: effects of concentration and temperature

Rheological properties of acorn starch dispersions at different concentrations (4%, 5%, 6% and 7%) were evaluated under steady and dynamic shear conditions. The flow behaviours of the acorn starch dispersions at different temperatures (25, 40, 55 and 70 °C) were determined from the rheological parameters provided by the power law model. The acorn starch dispersions at 25 °C exhibited high shear-thinning fluid characteristics ( n  = 0.23–0.36). Consistency index (K) and apparent viscosity (η_a,100) increased with an increase in starch concentration, and were also reduced with increasing temperature. Within the temperature range of 25–70 °C, the η_a,100 obeyed the Arrhenius temperature relationship with a high determination coefficient ( R ² = 0.97–0.99), with activation energies (E_a) ranging between 16.5 and 19.0 kJ mol⁻¹. Both the power law and exponential type models were employed in order to establish the relationship between concentration and apparent viscosity (η_a,100) in the temperature range of 25–70 °C. Magnitudes of storage ( G' ) and loss ( G ") moduli increased with an increase in the starch concentration and frequency (ω). The magnitudes of G ' were higher than those of G " over most of the frequency range (0.63–62.8 rad s⁻¹). The dynamic (η*) and steady shear (η_a) viscosities of acorn starch dispersion at 7% concentration follow the Cox–Merz superposition rule.     

Aflatoxins in ginseng roots

Ginseng roots can be infected by molds during growth, harvest and storage and result in contamination with mycotoxins. In this study, an analytical method for the determination of aflatoxins B₁, B₂, G₁ and G₂, a group of structurally similar mycotoxins, in ginseng root was developed. Test samples were extracted with methanol-water (8 + 2), diluted and passed through an immunoaffinity column packed with antibodies specific for aflatoxins. The purified extract was then derivatized with a mixture of water, trifluoroacetic acid and acetic acid. Aflatoxins were then separated and quantified by reverse phase liquid chromatography (LC) with fluorescence detection. Recoveries of total aflatoxins at 2, 4, 8 and 16 ng/g added to toxin-free 4 to 5-year old dried sliced Wisconsin ginseng were 92, 77, 91 and 83% respectively; and relative standard deviations were 3.6, 8.0, 6.9 and 2.0% respectively. A total of 11 wild simulated and 12 cultivated ginseng root samples were analysed for aflatoxins. All cultivated roots were found to be free of aflatoxin contamination. Two of the wild simulated roots contained total aflatoxins B₁, B₂, G₁ and G₂ at 15.1 and 15.2 ng/g. One moldy ginseng root purchased from a grocery store was found to be contaminated with aflatoxins at 16 ng/g.     

Efficacy of Artabotrys odoratissimus oil as a plant based antimicrobial against storage fungi and aflatoxin B1 secretion

The essential oil of Artabotrys odoratissimus R.Br. was evaluated for antifungal activity against some storage fungi causing contamination of food stuffs. Minimum inhibitory concentration of the oil was found to be 750 μL L⁻¹ against Aspergillus flavus Link. It was found superior over different prevalent synthetic fungicides which inhibited the growth of A. flavus between 1000–5000 μL L⁻¹. The oil exhibited broad fungitoxic spectrum against fourteen different storage fungi. Aspergillus fumigatus was inhibited at 1000 μL L⁻¹ whereas Cladosporium cladosporioides , Curvularia lunata , Fusarium oxysporum , Helminthosporium oryzae , Macrophomina phaseolina , Microsporum gypseum , Mucor racemosus , Penicillium italicum , Pythium debaryanum , Rhizoctonia solani , Sclerotium rolfsii and Trichoderma viride at 500 μL L⁻¹. Aspergillus niger was found to be inhibited only 84.9% at 1000 μL L⁻¹. In addition, the oil showed significant efficacy in arresting aflatoxin B₁ secretion by the toxigenic strain (Navjot 4NSt) of A. flavus at 750 μL L⁻¹. The efficacy of A. odoratissimus oil as aflatoxin suppressor is being reported for the first time.     

Optimisation of the medium composition for production of protease and soybean peptides by Bacillus subtilis SHZ using response surface methodology

Responses surface methodology was employed to enhance the production of protease and soybean peptides by Bacillus subtilis SHZ. For screening of medium composition significantly influencing protease and soybean peptides yield, the two-level Plackett–Burman design was used. Among thirteen variables tested; KH₂PO₄, glucose and defatted soybean flour (DSF) were selected based on their high significant effect on both protease activity and soybean peptides yield. Then, a three-level Box–Behnken design was employed to optimise the medium composition for the production of the protease and soybean peptides in submerged fermentation. Mathematical models were then developed to show the effect of each medium composition and their interactions on the production of protease and soybean peptides. The model estimated that, the maximal protease activity (320 ± 1 U mL⁻¹) could be obtained when the concentrations of glucose, KH₂PO₄, DSF were set at 8–9 g L⁻¹, 2–3 g L⁻¹, 55–65 g L⁻¹, respectively; while a maximal yield of soybean peptides (8.5 ± 0.1 g L⁻¹) could be achieved when the concentrations of glucose, KH₂PO₄, DSF were set at 7–9 g L⁻¹, 3–4 g L⁻¹ and 55–58 g L⁻¹, respectively. These predicted values were also verified by validation experiments.