葡萄糖氧化酶解除黄曲霉毒素 B1应用研究

黄曲霉毒素是由真菌产生的一类剧毒物质,含黄曲霉毒素B1的饲料对小鸡的危害很大。研究中发现,用5％o25U／g（25U／mL）葡萄糖氧化酶完全可以解除饲料中500ppb浓度黄曲霉毒素B1的毒性。用含2000ppb浓度黄曲霉毒素B1和添加5‰ 25U／g葡萄糖氧化酶的饲料喂小鸡,相比对照组,实验组存活数量提高39．5％,在小鸡饮用水中加入5％e25U／mL葡萄糖氧化酶,存活数量可提高28．5％。     

Effect of probiotic fermented milk and chlorophyllin on gene expressions and genotoxicity during AFB₁-induced hepatocellular carcinoma

The aim of this study was to investigate the chemopreventive effect of probiotic fermented milk and chlorophyllin on aflatoxin B? (AFB?) induced hepatocellular carcinoma. In vivo trials were conducted on 200 Wistar rats allocated to eight groups. Rats in the positive control group were given intraperitoneal injection of aflatoxin B? at 450 μg/kg body weight twice a week for 6 weeks. The rats were sacrificed and dissected at 25th week of the experiment, and comet assay was carried out in hepatic cells to assess the genotoxicity or DNA damage. The tumour incidence was decreased by approximately one-third than AFB? control group. The expression of c-myc bax, bcl-2, cyclin D1, p53 and rasp-21 genes was also studied. A significant (P<0.05) reduction in DNA damage was observed in probiotic fermented milk with chlorophyllin group as compared to aflatoxin B? control group. The c-myc, bcl-2, cyclin D1 and rasp-21 level was found to be highest in AFB? control group as compared to the treatment group. The results advocate the enhanced protective potential of probiotic fermented milk and chlorophyllin against AFB?-induced molecular alterations in hepatic cells during carcinogenesis.     

Production and characterization of aflatoxin B2a antiserum.

The specificity and sensitivity of antiserum elicited from rabbits against aflatoxin B2a-bovine serum albumin conjugates were characterized with a radioimmunoassay (RIA) and an enzyme-linked immunosorbent assay (ELISA). Aflatoxin B1 was first converted to aflatoxin B2a and then conjugated to bovine serum albumin and horseradish peroxidase by a reductive alkylation method. The antiserum was developed in New Zealand white rabbits by multiple-site injection with the aflatoxin B2a-bovine serum albumin conjugate. Antibody titers were determined by both RIA and ELISA. Competitive RIAs with various aflatoxin analogs indicated that the antiserum was most reactive with aflatoxin B1 and slightly cross-reactive with aflatoxins B2a, B2, and M1. Competitive ELISAs showed the antiserum to be equally specific for aflatoxins B2a and B12 and less reactive with aflatoxins B2 and M1. The relative sensitivities of RIA and ELISA for aflatoxin B1 quantitation were 100 and 10 pg per assay, respectively.     

Synthesis and degradation of aflatoxins by Aspergillus parasiticus. II. Comparative toxicity and mutagenicity of aflatoxin B1 and its autolytic breakdown products

A crude mycelial protein extract from a 16-day-old culture of A. parasiticus, on purification, lost 50% of its ability to degrade aflatoxin B1. The addition of hydrogen peroxide increased this activity to 97% of that of the crude extract. Ducklings dosed orally with aflatoxin extracts from 14- and 20-day-old cultures containing 46 micrograms or more of aflatoxin B1 developed enlarged livers, haemorrhaged and died in less than 10 days, giving and LD50 of 17.5 and 17.1 micrograms aflatoxin B1 per 50 g body weight respectively for each extract. When pure aflatoxin B1 was mixed with either the crude or purified mycelial protein extract the aflatoxin B1 level was decreased by 29% as was the toxicity of the mixture. The main breakdown product of aflatoxin B1 was isolated and was shown to have an RF value of 0.34, was non-fluorescent, and was non-toxic for ducklings at oral doses as high as 400 micrograms per 50 g body weight. The mutagenic effect of aflatoxin B1 on Salmonella typhimurium was relative to its concentration. The main breakdown product of aflatoxin B1 was non-mutagenic.     

Effects of various acids and salts on growth and aflatoxin production by Aspergillus flavus NRRL 3145.

The effects of sodium chloride, sodium acetate, benzoic acid, sodium benzoate, malonic acid, and sodium malonate on growth and aflatoxin production by Aspergillus flavus were investigated in synthetic media. Sodium chloride at concentrations equivalent to or greater than 12 g/100 ml inhibited growth and aflatoxin production, while at 8 g or less/100 ml, growth and aflatoxin production were stimulated. At 2 g or less/100 ml, sodium acetate also stimulated growth and aflatoxin production, but reduction occurred with 4 g or more/100 ml. Malonic acid at 10, 20, 40, and 50 mM reduced growth and aflatoxin production (over 50%) while sodium malonate at similar concentrations but different pH values had the opposite effect. Benzoic acid (pH 3.9) and sodium benzoate (pH 5.0) at 0.4 g/100 ml completely inhibited growth and aflatoxin production. Examination of the effect of initial pH indicated that the extent of inhibitory action of malonic acid and sodium acetate was a function of initial pH. The inhibitory action of benzoic acid and sodium benzoate appeared to be a function of undissociated benzoic acid molecules. Aflatoxin reduction was usually accompanied by an unidentified orange pigment, while aflatoxin stimulation was accompanied by unidentified blue and green fluorescent spots but with lower Rf values that aflatoxins B1, G1, B2, and G2 standards.     

Effect of temperature on aflatoxin production in Mucuna pruriens seeds.

This paper describes the effect of temperature on the level of aflatoxin production in Mucuna pruriens seeds. The highest level of aflatoxin B1 (1.75 micrograms/g) was detected in the samples incubated at 25 degrees C for three weeks. At 20, 30, and 35 degrees C, aflatoxin levels were 0.30 to 0.56, 0.37 to 1.20, and 0.26 to 0.65 micrograms/g, respectively. The lowest concentration of aflatoxin B1 (0.10 to 0.29 microgram/g) was produced at 15 degrees C.     

Effect of temperature on aflatoxin production in Mucuna pruriens seeds

This paper describes the effect of temperature on the level of aflatoxin production in Mucuna pruriens seeds. The highest level of aflatoxin B1 (1.75 micrograms/g) was detected in the samples incubated at 25 degrees C for three weeks. At 20, 30, and 35 degrees C, aflatoxin levels were 0.30 to 0.56, 0.37 to 1.20, and 0.26 to 0.65 micrograms/g, respectively. The lowest concentration of aflatoxin B1 (0.10 to 0.29 microgram/g) was produced at 15 degrees C.     

Simultaneous occurrence of deoxynivalenol, zearalenone, and aflatoxin in 1982 scabby wheat from the midwestern United States.

Thirty-three samples of wheat of the 1982 crop year from Kansas and Nebraska were analyzed for deoxynivalenol, T-2 toxin, zearalenone, and aflatoxin. Deoxynivalenol was identified in 31 of 33 samples, zearalenone was identified in 3 of 33 samples, and aflatoxin B1 was identified in 23 of 31 samples. One 1982 wheat sample from Illinois and one from Texas were also contaminated with deoxynivalenol at 1,200 and 600 ng/g, respectively. None of the samples contained detectable T-2 toxin. The mean concentration of deoxynivalenol was 1,782 +/- 262 ng/g, and the concentrations of aflatoxin B1 ranged from 0.8 to 17.0 ng/g, with a mean of 3.37 +/- 0.7. Zearalenone concentrations of the three positive samples were 35, 90, and 115 ng/g. However, density segregation of two other samples which tested negative yielded light fractions, comprising less than 2% of the samples, contaminated at 230 and 254 ng of zearalenone per g; calculated zearalenone concentrations for these two samples were below the limit of detection of the method. The high frequency of aflatoxin B1 and deoxynivalenol in wheat from the 1982 crop is unprecedented, as is the simultaneous contamination of some samples with deoxynivalenol, zearalenone, and aflatoxin B1.     

Ultrastructural studies on interaction of infectious bursal disease virus (IBDV) and aflatoxin B1 on chick embryo fibroblast cell culture.

Light and electron microscopic evaluation of chick embryo fibroblast (CEF) cell culture inoculated with graded doses (0.25, 2.5 and 25 micrograms/ml medium) of aflatoxin B1 with and without infectious bursal disease virus (IBDV) was undertaken. The light microscopy revealed degeneration, detachment and necrosis of fibroblasts and multiple plaques formation in IBDV infected group without and with (0.25, 2.5 micrograms) aflatoxin B1. The cultures infected with virus, with or without 25 micrograms aflatoxin B1 showed complete detachment from glass surface. Electron microscopy of these cultures showed marked pyknotic or bizarre shaped nuclei, pronounced degenerative changes in the rough endoplasmic reticulum (RER), mitochondria and the presence of multiple vacuoles in the cytoplasm. The viruses were spherical, arrayed, complete, generally closer to nuclei and RER and indistinctly membrane bound. The viruses were either localised or scattered in the cytoplasm. Cultures containing 25 micrograms aflatoxin B1 without or infected with virus showed marked necrosis of cells. In latter group only a few viruses were seen either in infected cells or free in culture. Control cultures failed to show cytopathic changes as observed in the other three groups.     

Simultaneous occurrence of deoxynivalenol, zearalenone, and aflatoxin in 1982 scabby wheat from the midwestern United States

Thirty-three samples of wheat of the 1982 crop year from Kansas and Nebraska were analyzed for deoxynivalenol, T-2 toxin, zearalenone, and aflatoxin. Deoxynivalenol was identified in 31 of 33 samples, zearalenone was identified in 3 of 33 samples, and aflatoxin B1 was identified in 23 of 31 samples. One 1982 wheat sample from Illinois and one from Texas were also contaminated with deoxynivalenol at 1,200 and 600 ng/g, respectively. None of the samples contained detectable T-2 toxin. The mean concentration of deoxynivalenol was 1,782 +/- 262 ng/g, and the concentrations of aflatoxin B1 ranged from 0.8 to 17.0 ng/g, with a mean of 3.37 +/- 0.7. Zearalenone concentrations of the three positive samples were 35, 90, and 115 ng/g. However, density segregation of two other samples which tested negative yielded light fractions, comprising less than 2% of the samples, contaminated at 230 and 254 ng of zearalenone per g; calculated zearalenone concentrations for these two samples were below the limit of detection of the method. The high frequency of aflatoxin B1 and deoxynivalenol in wheat from the 1982 crop is unprecedented, as is the simultaneous contamination of some samples with deoxynivalenol, zearalenone, and aflatoxin B1.     

Effect of antiserum against aflatoxin B1-bovine serum albumin complex on aflatoxin B1-induced lysogenesis

Escherichia coli K12 bacteria lysogenic for the lambda phage were used to study the effect of antiserum against aflatoxin B1-induced lysogenesis. The antiserum was obtained from rabbits immunized with water in oil emulsion of aflatoxin B1-bovine serum albumin complex (AFB1-BSA). A marked reduction in the degree of lysogenesis was observed when the antiserum was added to the reaction medium prior to microsomal enzyme activation of aflatoxin B1. There was no detectable effect when the antiserum was added after aflatoxin B1 activation. The result presented suggests that the antibodies in the AFB1-BSA antiserum can interact with aflatoxin B1 prior to its activation. This implies that an immune-protective effect can only be exerted if the antibodies intervene before activation.     

Effect of Corn Steep Liquor on Mycelial Growth and Aflatoxin Production in Aspergillus parasiticus

Total aflatoxin concentrations produced by Aspergillus parasiticus, isolate 64-R8, in Czapek's broth fortified with corn steep liquor increased proportionately as the concentration of corn steep was increased from 0.5 to 8.0% (v/v) until maximal growth, as measured by dry mycelial weight, was reached. Thereafter, aflatoxin concentrations declined more rapidly than the rate of autolysis of mycelial material. Data are presented which indicate that the concentration of corn steep liquor also affects the ratio of production of aflatoxin B(1) and B(2) to that of aflatoxin G(1) and G(2). Further, this ratio also varies with time of incubation. Although both growth of the fungus and aflatoxin production are stimulated by the addition of corn steep to the basic medium, the stimulation of toxin production is much greater than fungus growth.     

Fluorescence studies of the interactions of serum albumin and rat alpha1-fetoprotein with aflatoxin B1. Specificity and binding parameters.

The interaction of bovine serum albumin and rat alpha1-fetoprotein with aflatoxin B1 has been followed by the fluorescence quenching of the protein in presence of the ligand. The binding parameters (n, number of sites and Kd, dissociation constant) have been determined for the bovine serum albumin-alflatoxin B1 system: n = 3.5 and Kd = 3.1 +/- 0.5 . 10(-5) M and for the alpha-fetoprotein-aflatoxin system: n = 4 and Kd = 3.7 +/-0.5 . 10(-5) M. The competition of anilino-naphthalene-sulfonate and aflatoxin B1 for the same hydrophobic sites on bovine serum albumin has been demonstrated. The fluorescence quenching of various proteins (lysozymes, egg-albumin, gamma-globulin) by aflatoxin B1 have shown that there is not a strict specificity of aflatoxin towards proteins.     

Survey of fungal counts and natural occurrence of aflatoxins in Malaysian starch-based foods

In a survey of starch-based foods sampled from retail outlets in Malaysia, fungal colonies were mostly detected in wheat flour (100%), followed by rice flour (74%), glutinous rice grains (72%), ordinary rice grains (60%), glutinous rice flour (48%) and corn flour (26%). All positive samples of ordinary rice and glutinous rice grains had total fungal counts below 103 cfu/g sample, while among the positive rice flour, glutinous rice flour and corn flour samples, the highest total fungal count was more than 103 but less than 104 cfu/g sample respectively. However, in wheat flour samples total fungal count ranged from 102 cfu/g sample to slightly more than 104 cfu/g sample. Aflatoxigenic colonies were mostly detected in wheat flour (20%), followed by ordinary rice grains (4%), glutinous rice grains (4%) and glutinous rice flour (2%). No aflatoxigenic colonies were isolated from rice flour and corn flour samples. Screening of aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2 using reversed-phase HPLC were carried out on 84 samples of ordinary rice grains and 83 samples of wheat flour. Two point four percent (2.4%) of ordinary rice grains were positive for aflatoxin G1 and 3.6% were positive for aflatoxin G2. All the positive samples were collected from private homes at concentrations ranging from 3.69–77.50 μg/kg. One point two percent (1.2%) of wheat flour samples were positive for aflatoxin B1 at a concentration of 25.62 μ};g/kg, 4.8% were positive for aflatoxin B2 at concentrations ranging from 11.25–252.50 μg/kg, 3.6% were positive for aflatoxin G1 at concentrations ranging from 25.00–289.38 μg/kg and 13.25% were positive for aflatoxin G2 at concentrations ranging from 16.25–436.25 μg/kg. Similarly, positive wheat flour samples were mostly collected from private homes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Some high-performance liquid-chromatographic studies of the metabolism of aflatoxins by rat liver microsomal preparations.

The metabolism of aflatoxin B1 in vitro was examined in rat liver microsomal preparations. 2. H.p.l.c. (high-performance liquid-chromatographic) systems were used. A silica column was used to separate non-polar metabolites. A system utilizing a reversed-phase column which separates both poar and non-polar metabolites was also developed. 3. The principal metabolites of aflatoxin B1 found were aflatoxin M1, aflatoxin Q1 and a compound which co-chromatographed with a degradation product of aflatoxin B1 2,3-dihydrodiol. 4. The time course of metabolism of aflatoxin B1 by microsomal preparations isolated from control and phenobarbitone-pretreated rats was examined. The rate and extent of metabolism was greater with microsomal preparations from the latter. The formation of aflatoxin Q1 was enhanced 4--5-fold by phenobarbitone pretreatment, whereas the production of aflatoxin M1 was only increased 1--2-fold. The formation of the degradation product of aflatoxin B1 2,3-dihydrodiol was increased 4--5-fold by the pretreatment with phenobarbitone. 5. The microsomal metabolism of aflatoxins M1, P1 and Q1 was examined. Aflatoxin M1 apparently underwent very limited microsomal metabolism to more polar compounds. Aflatoxin P1 was not metabolized. The situation with aflatoxin Q1 was complicated in that it was metabolized in the absence of NADPH to an unidentified metabolite. Aflatoxin B1 appeared as a metabolite of aflatoxin Q1 only when NADPH was present, and the formation of more polar metabolites was also then observed.     

Quantitation of aflatoxin B1 and aflatoxin B1 antibody by an enzyme-linked immunosorbent microassay.

A specific microtest plate enzyme immunoassay has been developed for the rapid quantitation of aflatoxin B1 at levels as low as 25 pg per assay. Multiple-site injection of rabbits with an aflatoxin B1 carboxymethyloxime-bovine serum albumin conjugate was used for the production of hyperimmune sera. Dilutions of the purified antibody were air dried onto microplates previously treated with bovine serum albumin and glutaraldehyde and then incubated with an aflatoxin B1 carboxymethyloxime-horseradish peroxidase conjugate. The amount of enzyme bound to antibody was determined by monitoring the change in absorbance at 414 nm after the addition of a substrate solution consisting of hydrogen peroxide and 2,2'-azino-di-3-ethyl-benzthiazoline-6-sulfonate. Antibody titers determined in this manner closely correlated with those determined by radioimmunoassay. Competition assays as performed by incubation of different aflatoxin analogs with the peroxidase conjugate showed that aflatoxins B1 and B2 and aflatoxicol caused the most inhibition of conjugate binding to antibody. Aflatoxins G1 and G2 inhibited the conjugate binding to a lesser degree, whereas aflatoxins M1 and B2a had no effect of the assay.     

Some mass-spectral and n.m.r. analytical studies of a glutathione conjugate of aflatoxin B1.

A system for the formation of an aflatoxin B1-reduced glutathione conjugate in vitro was developed, capable of yielding 80% conversion of aflatoxin B1 into the conjugate. A reverse-phase high-pressure-liquid-chromatography system was also devised that not only facilitates improved resolution of the compound but that, by manipulation of the pH, is also capable of an extensive purification of the compound from other aflatoxin B1 metabolites in a single step. Material produced by these techniques, after further purification, has been used in 1H-n.m.r. and mass-spectroscopic studies. Results were obtained that support the proposed linkage of the aflatoxin B1 to reduced glutathione in a 1:1 molar ratio via a thioether linkage. Amino acid analyses were also consistent with this structure. The absence of a Schiff-base linkage of aflatoxin B1 8,9-dihydrodiol to glutamate was further demonstrated by the presence of a gamma-glutamyltransferase-catalysed-transferable glutamate moiety. These data are consistent with the structure 8,9-dihydro-8-(S-glutathionyl)-9-hydroxy-aflatoxin B1.     

Production of Aflatoxin on Wheat and Oats: Measurement with a Recording Densitometer

A method has been developed for the production of aflatoxin by growing Aspergillus flavus NRRL 3145 on solid substrate wheat. Optimal yields of 900 mug of aflatoxin G(1) and 900 mug of aflatoxin B(1) per g of substrate were obtained in 4 to 5 days at 28 C. A study of aflatoxin production on hulls and groats of oats and on whole oats by A. flavus strains NRRL 2999, NRRL 3000, and NRRL 3145 revealed that aflatoxin was produced on all three substrates, although production was very slight on hulls. Strain NRRL 3145 grown on solid substrate groats produced the largest amounts of aflatoxin: 580 mug of B(1) and 450 mug of G(1) per g of substrate. A densitometric method for reading thin-layer chromatographic plates is described; this is more objective and more accurate than the visual methods previously used for the determination of all four aflatoxins.     

Aflatoxin B1 Induction of Lysogenic Bacteria

A technique for biological verification of aflatoxin B(1) was developed based on toxin-mediated induction of lysis in a lysogenic strain of Bacillus megaterium NNRL B-3695. Reduction of culture turbidity was determined at various concentrations of toxin. Incubation of 1.1 x 10(-4) g (dry weight) of cells/ml of growth medium containing 25 mug of B(1) per ml at 37 C reduced initial turbidity 0.20 absorbance units in 4 hr. If the bacterial lysate of the lysogenic strain, after a 2-hr incubation with 25 mug of B(1) per ml, was plated with a sensitive B. megaterium strain (NRRL B-3694), plaque-forming units increased approximately 150 times relative to the control. Comparable testing of the effects of aflatoxin on the nonlysogenic, sensitive strain demonstrated that 75 mug of B(1) per ml neither induced lysis nor plaque-forming units. Although induction is not an exclusive property of aflatoxin B(1), the differential response of the lysogenic and sensitive Bacillus strains to B(1) offers a unique and rapid technique for biological verification of the toxin.