Aflatoxin Binders I: In vitro binding assay for aflatoxin B1 by several potential sequestering agents

Nine potential proprietary sequestering agents consisting of 4 activated charcoals, 3 sodium bentonites, a calcium bentonite, and an esterified glucomannan were compared in a novel in vitro assay for aflatoxin B1 (AFB1) binding. Agents were evaluated in 10% methanol prepared as 1% stirred suspensions at pH 3, 7, 10 and pH-unadjusted, with or without AFB1 at 5 g/ml. All nine agents bound more than 95% of the 5 g of AFB1 in solution, regardless of pH. The sodium bentonites bound 98, 95, and 98% of the AFB1. The four activated charcoals bound over 99%, the calcium bentonite bound 98%, and the esterified glucomannan bound 97% of the AFB1 in solution. The results suggested that the sequestering agents tested here had sufficient potential to bind AFB1 at pH values commonly found in the gastrointestinal tracts of ruminants and other animals.This revised version was published online in October 2005 with corrections to the Cover Date.     

Aflatoxin Binders II: Reduction of aflatoxin M1 in milk by sequestering agents of cows consuming aflatoxin in feed

Sequestering agents bind dietary aflatoxin B1 (AFB1) and reduce absorption from an animal's gastrointestinal tract. As a result, they protect an animal from the toxic effects of AFB1 and reduce transfer of the metabolite, aflatoxin M1 (AFM1), into milk. Three experiments, using late-lactation Holstein cows fed AFB1-contaminated feed, were conducted to evaluate several potential sequestering agents for their abilities to prevent or reduce the transmission of AFM1 into milk. Six agents previously tested in our laboratory for AFB1 binding in vitro were evaluated in these experiments. These were: SA-20, an activated carbon (AC-A); Astra-Ben-20, a sodium bentonite (AB-20); MTB-100, an esterified glucomannan (MTB-100); Red Crown, a calcium bentonite (RC); Flow Guard, a sodium bentonite (FG); and Mycrosorb, a sodium bentonite (MS). Five of the six sequestering agents significantly (P < 0.01) reduced AFM1 contamination of milk (AB-20, 61%; FG, 65%; MS, 50%; MTB-100, 59%; and RC, 31%); whereas, AC-A, activated carbon, had no effect on AFM1 transmission at 0.25% of feed. By the first milking (1 day after cows consumed contaminated feed), AFM1 appeared in milk, then reached maximum levels after three days, and was absent from milk within four days after AFB1 was removed from the feed. Sodium bentonites at 1.2% of feed showed good potential as AFB1 binders; MTB-100, a yeast cell wall product, was equally effective at 0.05% in feed. Potential AFB1 binding agents should be evaluated experimentally to demonstrate efficacy. Our data show that sequestering agents can reduce AFM1 in milk of cows fed AFB1-contaminated feed.     

In vitro evaluation of the capacity of zeolite and bentonite to adsorb aflatoxin B1 in simulated gastrointestinal fluids

Anin vitro study using single concentration and isotherm adsorption was carried out to evaluate the capacity of Vietnamese produced zeolite and bentonite to adsorb aflatoxin B₁ (AFB₁) in simulated gastrointestinal fluids (SGFs), and a commercial sorbent hydrated sodium calcium aluminosilicate (HSCAS) was used as reference. In this study, AFB₁ solution was mixed with sorbents (0.3, 0.4 and 0.5% w/v) in SGFs at pH 3 and pH 7 and shaken for 8 h, centrifuged and the supernatant measured by Vicam fluorometer. Adsorption of AFB₁ onto zeolite and bentonite varied according to the pH of SGFs and was lower than HSCAS. Linearity between the increased amount of AFB₁ adsorbed on sorbents and the decrease of sorbent concentration was observed for bentonite and HSCAS, except for zeolite in SGFs at pH 7. The observed maximum amounts of AFB₁ adsorbed on bentonite and HSCAS were 1.54 and 1.56 mg/g, respectively. The adsorption capacities of bentonite and HSCAS for AFB₁ were 12.7 and 13.1 mg/g, respectively, from fitting the data to the Freundlich isotherm equation. Improvement in processing and purification for bentonite is needed to enhance the surface area, which would probably result in better adsorptive capacity for this sorbent.     

The effects of rumen fluid on the in vitro aflatoxin binding capacity of different sequestering agents and in vivo release of the sequestered toxin

In an in vitro experiment, aluminosilicates (Atox^® and Novasil™ Plus) and a yeast cell wall derivate (Mycosorb^®) were used as sequestering agents (SAs) to verify their capacity for binding aflatoxin B1 (AFB1) in vitro. SAs were individually mixed at three different ratios with AFB1 (1:5000, 1:50,000 and 1:500,000, w/w) in water (CTR), rumen fluid from a lactating cow with a low rumen pH (LRS) or rumen fluid from a dry cow with a high rumen pH (HRS), and then used in a 3 × 3 × 3 factorial arrangement of a completely randomized design. At the 1:500,000 AF:SA ratio Atox^® and Novasil™ Plus sequestered over 0.87 and 0.98 of the AFB1 in the CTR and rumen solutions (LRS and HRS), respectively. This efficacy decreased when the amount of clays was reduced, with higher values (P<0.001) for Atox^® compared with Novasil™ Plus (0.50 vs. 0.28 in CTR; 0.58 vs. 0.16 in LRS and 0.44 vs. 0.27 in HRS). Mycosorb^® had a lower sequestering efficacy (P<0.001) in all of tested experimental conditions, with 0.34 being the maximum value obtained in the CTR solution.     

Removal of copper, lead, and zinc from contaminated water by saltbush biomass: analysis of the optimum binding, stripping, and binding mechanism

Experiments performed on the Cu(II), Pb(II), and Zn(II) binding by saltbush biomass (Atriplex canescens) showed that the metal binding increased as pH increased from 2.0 to 5.0. The highest amounts of Cu, Pb, and Zn bound by the native biomass varied from 48-89%, 89-94%, and 65-73%, respectively. The hydrolyzed biomass bound similar amount of Pb and 50% more Cu and Zn than the native. The esterified biomass had a lower binding capacity than native; however, esterified flowers bound 45% more Cu at pH 2.0 than native flowers. The optimum binding time was 10 min or less. More than 60% of the bound Cu was recovered using 0.1 mM HCl, while more than 90% of Pb was recovered with either HCl or sodium citrate at 0.1 mM. For Zn, 0.1 mM sodium citrate allowed the recovery of 75%. Results indicated that carboxyl groups participate in the Cu, Pb, and Zn binding.     

Growth inhibition by tungsten in the sulfur-oxidizing bacterium Acidithiobacillus thiooxidans

Growth of five strains of sulfur-oxidizing bacteria Acidithiobacillus thiooxidans, including strain NB1-3, was inhibited completely by 50 microM of sodium tungstate (Na(2)WO(4)). When the cells of NB1-3 were incubated in 0.1 M beta-alanine-SO(4)(2-) buffer (pH 3.0) with 100 microM Na(2)WO(4) for 1 h, the amount of tungsten bound to the cells was 33 microg/mg protein. Approximately 10 times more tungsten was bound to the cells at pH 3.0 than at pH 7.0. The tungsten binding to NB1-3 cells was inhibited by oxyanions such as sodium molybdenum and ammonium vanadate. The activities of enzymes involved in elemental sulfur oxidation of NB1-3 cells such as sulfur oxidase, sulfur dioxygenase, and sulfite oxidase were strongly inhibited by Na(2)WO(4). These results indicate that tungsten binds to NB1-3 cells and inhibits the sulfur oxidation enzyme system of the cells, and as a result, inhibits cell growth. When portland cement bars supplemented with 0.075% metal nickel and with 0.075% metal nickel and 0.075% calcium tungstate were exposed to the atmosphere of a sewage treatment plant containing 28 ppm of H(2)S for 2 years, the weight loss of the portland cement bar with metal nickel and calcium tungstate was much lower than the cement bar containing 0.075% metal nickel.     

有机改性膨润土去除赤潮生物的初步研究

制备了有机膨润土并考察其对中肋骨条藻的去除效果,对中肋骨条藻去除效果是溴化十六烷基三甲铵改性的铁柱撑膨润土>溴化四甲基胺改性的铁柱撑膨润土>铁柱撑膨润土>溴化十六烷基三甲铵改性的钠化膨润土>溴化四甲基胺改性的钠化膨润土>钠化膨润土,有机膨润土对赤潮生物的去除能力随改性剂用量增加而提高,但改性剂用量大于1．0CEC后,增加较少。     

In vitro assessment of the effectiveness of non-nutritive sorbent materials as binding agents for boar taint compounds

Boar taint, an off-odor and an off-flavor in the meat from some uncastrated male pigs, is due to high levels of the testicular steroid hormone, androstenone, and the indole, skatole. Thus far, there are no known methods for controlling both androstenone and skatole through dietary means. We tested the adsorbent agents, cholestyramine (CH), activated carbon (AC), tween-60 (Tween), bentonite (BNT) and polyvinylpolypyrrolidone (PVPP) for binding androstenone, estrone (E(1)), estrone sulfate (E(1)S) and skatole from buffer solutions in an in vitro system. The goal was to determine the potential utility of these binding agents as feed additives to control boar taint. Michaelis-Menten analysis was utilized to determine the effectiveness of the adsorbents. At pH 7.4, E(1)S was bound to AC and CH with the highest B(max) (maximum binding), whereas Tween and AC had the greatest B(max) for E(1). The B(max) for skatole at pH 7.4 was highest for AC, CH and PVPP. AC had a higher B(max) for androstenone than CH and Tween. The B(max) values at pH 3.0 with E(1)S for AC and CH were essentially 100%, whereas the binding of Tween to E(1)S at pH 3.0 decreased by 49.5% from binding at pH 7.4 (P < 0.05). The Ad(int) values, which represent efficiency of binding, illustrated that AC bound E(1), androstenone and skatole with greater efficiency than the other binding agents at pH 7.4, whereas AC bound E(1)S as efficiently as CH. We conclude that AC was the most effective adsorbent agent for binding E(1), E(1)S, androstenone and skatole in vitro, followed by CH, Tween, PVPP and lastly BNT. These adsorbent agents may be useful for binding boar taint compounds in in vivo studies to decrease the risk of boar taint.     

Assessing the effect of chitosan on pesticide removal in grape juice during clarification by gas chromatography with tandem mass spectrometry

The main objective of this study was to assess the pesticide removal efficiency of chitosan in grape juice during clarification. The grape juice was spiked with six selected pesticides (chlorpyrifos, ethion, diazinon, fenitrothion, fenthion and phorate) and clarified with chitosan at different concentrations and at three different incubation times (1, 2 and 4 h). The effect of chitosan on the level of pesticides in grape juice during clarification was quantified using modified QuEChERS method with gas chromatography with tandem mass spectrometry (GC-MS/MS). The method for pesticide quantification has been validated. The maximum removal was obtained for chlorpyrifos (98%) and ethion (97%) at 0.5% of chitosan concentration for 1 h incubation followed by phorate (96%), fenthion (95%), fenitrothion (94%) and diazinon (86%) at 1% of chitosan concentration for 2 h incubation time. The juice was clarified with other clarifying agents such as activated carbon, gelatin, casein and bentonite and its pesticide removal efficiency was compared with chitosan. The pesticide removal level could be correlated with the properties of selected pesticides and clarifying agents. The results indicated that the chitosan can act as an effective pesticide scavenger and it leads to the potential application of chitosan in fruit juice clarification.     

Efficient solubilization, activation, and purification of recombinant Cry45Aa of Bacillus thuringiensis expressed as inclusion bodies in Escherichia coli

A cytotoxic protein Cry45Aa of Bacillus thuringiensis expressed as inclusion bodies in Escherichia coli was solubilized in 10 mM HCl. Protein concentration of saturated solution of the recombinant Cry45Aa in 10 mM HCl was about 25 times higher than that in the buffer of previous method (in 50 mM sodium carbonate buffer, pH 10.5, containing 1 mM EDTA, and 10 mM dithiothreitol). The Cry45Aa solubilized in the acidic solution was activated by pepsin as an alternative to proteinase K in the previous method. Cytotoxic activity against CACO-2 cells of the pepsin-treated Cry45Aa was almost identical to the proteinase K-treated protein. The pepsin-treated Cry45Aa was purified by cation-exchange chromatography. The concentration of the purified protein was 539 microg/ml, which was 27-fold higher than that of the activated Cry45Aa by the previously method. The cytotoxic activity of the purified protein was stable in broad pH region (pH 2.0-11.0) for 3 days, and 97% cytotoxic activity remained after incubation at 30 degrees C for 360 min.     

Potential protective effect of HSCAS and bentonite against dietary aflatoxicosis in rat: with special reference to chromosomal aberrations.

Bentonite and hydrated sodium calcium aluminsilicate (HSCAS) were added at a level of 0.5% (w/w) to the diets containing 2.5 mg aflatoxins (AF) per kg diet and fed to male mature rats for 15 successive days. Aflatoxin alone significantly decreased feed intake and altered serum biochemical parameters of liver and kidney functions. Aflatoxin caused chromosomal aberrations in bone marrow cells. Bentonite or HSCAS did not alter any of the parameters measured. The addition of bentonite or HSCAS to the AF-contaminated diet diminished most of the deleterious effects of the aflatoxin. Pathological examinations of liver and kidney proved that both bentonite and HSCAS were hepatonephroprotective agents against aflatoxicosis. The cytogenetic findings demonstrated that the addition of bentonite or HSCAS to AF-contaminated diet suppressed chromosomal aberrations. These findings indicated that bentonite and HSCAS could diminished many of the adverse effect of dietary AF in rats.     

Effects of recycled bentonite addition on soil properties, plant growth and nutrient uptake in a tropical sandy soil

The only way to increase the low CEC of sandy tropical soils over the long term is to apply high CEC materials such as 2:1 clay minerals. Acid activated bentonite is used in Thailand in the vegetable oil industry during the clarification process. The waste bentonite is discarded afterwards. The aim of the study was to compare the effects of the addition of these oil bentonites (OB) with the addition of cation beneficiated bentonite (BB) on soil properties and plant growth. Palm, rice and soybean OB, and bentonite beneficiated with calcium, magnesium, and potassium were applied at rates between 5 and 40 t ha⁻¹ to an Arenic Acrisol. Three consecutive crops of sorghum were grown in pots. Biomass and plant nutrient content were determined at each growth phase, and selective soil properties were measured at the start and the end of the study. Beneficiated bentonite was not water repellent, but the addition of OB resulted in soil water repellency. The application of bentonite at the rate of 40 t ha⁻¹ increased the cation exchange capacity (CEC) from 0.6 cmolc kg-1 in the control to 1.9 and 0.7 cmol_c kg⁻¹ in the BB and OB, respectiveley. The lower value of the CEC for OB compared to BB was probably due to the activation process and oil coating. OB applications at rates higher than 20 t ha⁻¹ did not increase biomass, and biomass decreased with increasing water repellency. The other treatments produced a higher biomass than the control. However biomass was below potential because of widespread nitrogen deficiency. Exchangeable K was exhausted in two crops, whatever the initial level, stressing the issue of K management in this soil type. Soybean OB is a promising material for soil chemical properties and biomass production, probably because of its low oil content.     

Behaviour of phenoloxidases in the presence of clays and other soil-related adsorbents

Summary The tyrosinases from Agaricus bisporus and Streptomyces eurythermus, laccases from Polyporus versicolor and Pleurotus ostreatus, and peroxidase from horseradish were strongly adsorbed on different bentonites and a clay-humus complex but less to on kaolinite and quartz. The adsorption was significantly dependent on the pH, reaching maxima in the range of the specific isoelectric points; it was less influenced by the valency and type of exchangeable cations. Most of the enzymes lost their activity when adsorbed on bentonite. The activity of desorbed enzymes was distinctly diminished when compared with free enzyme preparations. Conclusions from this behaviour were drawn as to the possible use of phenoloxidases as agents to transform phenolics in soil.     

Mycotoxins inactivation by extrusion cooking of corn flour

AIMS: To evaluate the effects of the extrusion cooking process on the inactivation of mycotoxins in corn flour. METHODS AND RESULTS: Samples of corn flour experimentally contaminated with aflatoxin B1 (AFB1) (50 ppb) and deoxynivalenol (DON) (5 ppm) were extruded. The effects of three extrusion variables (flour moisture, extrusion temperature and sodium metabisulphite addition) were analysed according to a two-level factorial design. The process was effective for the reduction of DON content (higher than 95%) under all the conditions assessed, but was only partially successful (10-25%) for the decontamination of AFB1. CONCLUSION: Extrusion cooking is effective for the inactivation of DON but is of limited value for AFB1, even if metabisulphite is added. More severe extrusion conditions are needed for the detoxification of AFB1. SIGNIFICANCE AND IMPACT OF THE STUDY: As contamination with DON occurs mainly in the field prior to harvesting and that of AFB1 is normally produced during grain storage, maize is often contaminated with DON but not with AFB1. Under these conditions, the described extrusion process can be used for the detoxification of DON. The addition of sodium metabisulphite did not significantly affect the inactivation of AFB1. Extrusion cooking is therefore an appropriate treatment for vomitoxin-contaminated maize in countries where, because of the prevailing conditions, these are the only toxins present.     

Structural of a glucomannan from Lupinus varius seed

Polysaccharides extracted from seeds of Lupinus varius with hot ethanol 85% (polysaccharide FI) and 0.1 M sodium phosphate buffer, pH 7.5 (polysaccharide FII) were fractionated and purified by ion-exchange and gel-filtration chromatography. According to methylation and hydrolysis analysis, the main chains of FI and FII consisted of (1 → 4)-linked glucomannan; only traces of branched sugar residues were detected. This is the first report on the isolation of glucomannan from L. varius seeds.     

Studies on alpha-amylase from a thermophilic bacterium. II. Thermal stability of the thermophilic alpha-amylase.

The effect of pH, mental ions, and denaturing reagents on the thermal stability of thermophilic alpha-amylase [EC 3.2.1.1] were examined. The enzyme was most stable at around pH 9.2, which is coincident with the isoelectric point of the enzyme. The stability of the enzyme was increased by the addition of calcium, strontium, and sodium ions. The addition of calcium ions markedly stabilized the enzyme. The protective effects of calcium and sodium ions were additive. At room temperature, no detectable destruction of the helical structure of the enzyme was observed after incubation for 1 hr in the presence of 1% sodium dodecylsulfate, 8 M urea or 6 M guanidine-HC1. The addition of 8 M urea or 6 M guanidine-HC1 lowered the thermal denaturation temperature of the enzyme. The enzyme contained one atom of tightly bound intrinsic calcium per molecule which could not be removed by electrodialysis unless the enzyme was denatured. The rate constants of inactivation and denaturation reactions in the absence and presence of calcium ions were measured and thermodynamic parameters were determined. The presence of calcium ions caused a remarkable decrease in the activation entropy.     

Effects of chondroitin sulphates on mineralization in vitro

1. Soluble sodium chondroitin sulphate, from bovine ribs or puppy epiphyseal plate, at a concentration 80mm in terms of glucuronate, decreased the amounts of calcium and phosphate precipitated from a solution 6.9mm in phosphate and 6.9 or 13.8mm in calcium, buffered in the pH range 6.6-8.2 with 20-25mm-collidine and 5-20mn-hydrochloric acid when incubated for 2hr. at 30 degrees , and for 0.25-24hr. when buffered at pH7.0. 2. An insoluble fraction of puppy epiphyseal plate, containing chondroitin sulphate and collagen, was found to have the same effect at lower concentrations of chondroitin sulphate and a higher calcium/chondroitin sulphate glucuronate ratio, but the formation of calcium phosphate from calcium bound to this material appeared to proceed more rapidly than from calcium in solution, when both were present in the same system. 3. The implications of these results are discussed in relation to the calcium/chondroitin sulphate glucuronate ratios found in different parts of non-calcifying and calcifying cartilage in vivo and to the calcium and phosphate gradients between blood and calcified tissue.     

The toxicity and decreased concentration of aflatoxin B in natural lactic acid fermented maize meal

AIMS: Aflatoxin B(1) (AFB(1)) is a mycotoxin which is known to frequently contaminate poorly stored food products destined for human consumption. This study was carried out to investigate the potential activity of lactic acid fermentation in reducing AFB(1) level in fermented maize meal products. METHODS AND RESULTS: Maize meal was spiked with 60 mug g(-1) AFB(1) and fermented, with or without starter culture, for 4 days at 25 degrees C. Unbound AFB(1) in solution and the pH of the media were monitored daily. A significant decrease (P < 0.05) in the level of unbound AFB(1) was observed (75% in the fourth day). Simultaneously, a progressive decrease in the pH of the media from 6.5 to 3.1 was also observed. AFB(1) was below the detection limit in commercial fermented porridge (amahewu) samples. Cytotoxicity tests on AFB(1)-spiked fermented extracts showed that those with a starter culture were comparatively less toxic (30-36%) than those with no added starter culture (24-30%). However, this difference was not significant (P > 0.05). CONCLUSIONS: These results indicate that lactic acid fermentation can significantly reduce the concentration of AFB(1) in maize to trace levels. However, the safety of fermented products has not been well studied, as the mechanism of AFB(1) removal is not well understood. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural fermentation may potentially reduce exposure to natural toxins occurring in food.     

Effects of adding calcium and sodium salts to field soils on the incidence of clubroot

The effect on clubroot of the addition to a mineral soil of calcium carbonate, calcium sulphate and sodium carbonate was studied in two field experiments during a 3-yr period. Applications of calcium carbonate (ground limestone) at 10 or 20 t/ha increased the soil pH from 6×7 to 7×9; sodium carbonate at 7×5 t/ha to pH 8×3 and calcium sulphate (gypsum) at 20 t/ha caused a slight depression in pH to 6×6. 98% of cabbage plants showed clubroot symptoms in the untreated plots after 3 yr and the percentages were 0×7, 1×6 and 66×6 for the calcium carbonate, sodium carbonate and calcium sulphate treatments respectively. Yields were significantly increased by all three materials. There were no significant differences in disease incidence or in yield when calcium carbonate was used at 10 or 20 t/ha either applied as a single application or as two half-rate applications. Soil from the calcium carbonate treated plots was used in a greenhouse experiment where the addition of inoculum of Plasmodiophora brassicae resulted in a large increase in clubroot incidence. There was no evidence that a biological suppressor was present in the high pH, low disease incidence soils.     

Chemically modified maize cobs waste with enhanced adsorption properties upon methyl orange and arsenic

The surface chemistry of maize naturasorbent was altered in this work by the modifying agents: phosphoric acid and different amines (triethanolamine, diethylenetriamine and 1,4-diaminobutane). Removal of methyl orange (25 mg l(-1)) was <50% by maize corn cobs modified by phosphorylation and higher by the quaternized samples: 68% with the 1,4-diaminobutane and 73% with the diethylenetriamine modificators. Adsorption of arsenite by the samples modified with phosphoric acid/ammonia was 11 microg g(-1), which corresponds to 98% removal from a 550 microg As l(-1) solution for an adsorbent dose of 50 mg ml(-1). The samples modified by phosphoric acid/urea removed 0.4 microg g(-1) arsenate from a 300 mug As l(-1) solution. Adsorption of methyl orange, arsenite and arsenate was superior by the chemically modified maize cobs judged against the initial naturasorbent. For comparison, removal by the commercial anion exchanger was 100% for methyl orange, 45% (5 microg g(-1)) for arsenite and 99% (5 microg g(-1)) for arsenate.