Elmer H. Marth

Aflatoxin formation, lipid synthesis, and glucose metabolism by Aspergillus parasiticus during incubation with and without agitation

Abstract Glucose utilization, growth of mold, and synthesis of aflatoxin and total lipid by Aspergillus parasiticus were studied with cultures that were incubated statically and with agitation. With both cultural conditions, maximal toxin formation occurred at 5 days which coincided with the end of rapid mold growth and rapid uptake of glucose. The toxin concentration decreased as incubation continued. The pattern for formation and depletion of total lipid was similar to that for aflatoxin. Maximal yields of toxin and of total lipid did not coincide with maximal production of mold mycelium. Incubation with agitation enhanced mold growth, consumption of glucose, and production of aflatoxin and total lipid during the first 3 days. Generally, more growht occured in agitated cultures, but maximal yields of aflatoxin and total lipid were lower than in quiescent cultures. The need for limited, but not excessive, O2 for synthesis of aflatoxin and lipid also was demonstrated by varying the volume of medium in flasks that were incubated quiescently. Incorporation of [1-14C] glucose into aflatoxin indicated that limiting the O2 supply and thereby favoring glucose catabolism via the Embden-Meyerhof pathway enhanced toxin formation. Aflatoxin formation also was greater when oxidative respiration of the mold was restricted by a metabolic inhibitor. Results suggest that the degree of aeration of the culture is important in controlling biosynthesis of aflatoxin.

Association of aflatoxin M1 with casein

ZusammenfassungDie Gleichgewichtsdialyse wurde benutzt, um festzustellen, ob es eine Bindung zwischen Aflatoxin M1 (AFM1) and Casein gibt. Simuliertes Milchultrafiltrat (SM) mit entweder 10 oder 20 ng von AFM1/ml wurde gegen SM mit Caseinmicellen dialysiert. Nach 24 Std bei 7°C enthielt die Caseinsuspension 2,5- bzw. 2,9-mal so viel AFM1 als SM. Durchschmittlich wurden 17,9 bzw. 55,3 μg AFM1/g Casein gebunden. In einem Einzelexperiment wurde Milch, die natürlich mit AFM, kontaminiert war, mit einem eiweißabbauenden Enzym behandelt. Im Durchschnitt fand man etwa 30,7% mehr Toxin in behandelter als in unbehandelter Milch. Dieses Ergebnis weist auf eine Verbindung zwischen AFM1 and Casein hin.SummaryEquilibrium dialysis was used to determine whether aflatoxin M1 (AFM1) binds to casein. Simulated milk ultrafiltrate (SMUF) containing 10 or 20 ng/ml of AFM1 was dialyzed against SMUF containing casein micelles. After 24 h at 7°C, the casein suspension contained 2.5- or 2.9-fold, respectively, more toxin than that found in SMUF. An average of 17.9 or 55.3 μg of AFM1, respectively, per gram of casein was bound. In a separate experiment, milk naturally contaminated with AFM1 was treated with a proteolytic enzyme. An average of 30.7% more toxin was found in treated than in untreated milk. This result also suggests binding of AFM1 by milk protein.

Inhibition of growth and aflatoxin production of Aspergillus parasiticus by citrus oils

ZusammenfassungPampelmusensaft und ein Glucose-Hefeextrakt-Medium wurden beide mit 500–7000 ppm Orangenöl, Citronenöl oder d-Limonen angereichert und dann mitAspergillus parasiticus beimpft. Wachstum und Aflatoxinproduktion des Pilzes wurden stärker durch die Öle als durch d-Limonen gehemmt, obwohl dieser der Hauptbestandteil der beiden Öle ist. 2000 ppm Citronenöl bzw. 3000 ppm Orangenöl in Pampelmusensaft genügten zur starken Hemmung der Wachstums- and der Aflatoxinproduktion vonA. parasiticus während 7 Tage bei 28° C. Wenn Glucose-Hefeextrakt als Nährboden diente, dann wiesen bei 3000 ppm beide Öle gleiche Hemmung auf. Wenn beide Nahrboden nur 4 Tage bei 28° C gehalten wurden, dann waren 3500 ppm Orangendl notwendig, um Wachstum and Aflatoxinproduktion zu hemmen. Pampelmusensaft mit einem Orangenöl-Gehalt von 3500 ppm enthielt nur 14% der Aflatoxin-Menge des beimpften Saftes ohne Öl. Das Medium mit Glucose, Hefeextrakt und Orangendl hatte nur 1 % des Aflatoxin-Gehaltes der Kontrolle. Höhere Konzentrationen von Orangenöl hemmten noch stärker und verzögerten den Beginn der Konidienbildung. Wenn das Medium 7000 ppm Orangenöl enthielt, dann konnte nur geringes Pilzwachstum und Aflatoxinproduktion (0,2–0,5% der Kontrolle) beobachtet wurden; das minimale Wachstum des Pilzes geschah an der Grenzfläche Nährboden und Atmosphare.SummaryAspergillus parasiticus was inoculated into grapefruit juice and a glucose-yeast extract medium; both contained 500–7000 ppm of citrus oils that were incorporated into the media by sonication. Orange and lemon oil were more inhibitory to mold growth and aflatoxin production than was d-limonene, the main constituent of the two peel oils. After 7 days at 28° C, 2000 ppm of lemon and 3000 ppm of orange oil in grapefruit juice afforded maximum suppression of mold growth and toxin formation. When the glucose-yeast extract medium was used, 3000 ppm of either oil were needed to achieve the same result. After 4 days at 28° C, orange oil at 3500 ppm in either medium markedly inhibited mold growth (as evidenced by dry weight of mold mycelium) and aflatoxin production (only 14 and 1% of the amount normally produced in the juice and artificial medium, respectively). Higher concentrations of orange oil further reduced mold growth and aflatoxin production and also delayed the onset of sporulation, if it occurred. Although aflatoxin was detected in all samples, only 0.2 to 0.5% of the amount found in controls (without the citrus oil) was present when the medium contained 7000 ppm orange oil. The mold consistently grew, albeit very poorly, on the glass at the liquid-atmosphere interface even when the substrate contained a large amount of citrus oil.

Ultrafiltration and reverse osmosis in dairy technology : a review

Ultrafiltration and reverse osmosis processes can be useful in the dairy foods industry. When milk is processed, milk fat and casein are rejected fully (e.g., are in retentate) and thus are concentrated by ultrafiltration and reverse osmosis membranes. Lactic cultures are slow to reduce the pH of retentate because of its increased buffering capacity since concentrated calcium phosphate and proteins are present. Conditions for growth of pathogenic microorganisms and inhibition of such bacteria in ultrafiltered milk differ from those of unfiltered milk. The principal advantage of using ultrafiltered milk for conversion into such cheeses as Cheddar, cottage, Havarti, Feta, brick, Colby, and Domiati is an increase in yield of product. Additional benefits claimed for use of ultrafiltered milk in cheese making include reduction in costs of energy, equipment, and labor; improved consistency of cheese flavor; and possible production of new byproducts.

Use of sulphite or bentonite to eliminate aflatoxin M1 from naturally contaminated raw whole milk

ZusammenfassungZwei Methoden wurden für die Eliminierung des Aflatoxines M1 aus natürlich-kontaminierter, roher, voll-fetter Milch angewandt. Die erste benutzt die chemische Eliminierung durch Kaliumsulfit (K2SO3), die andere die physikalische Adsorption des Toxins an Bentonit. Die maximale Eliminierung mit K2SO3 (0.05 mol, 25 °C, 5 Std) war 45%. Es ist möglich, daß das Toxin durch das Bisulfitradikal (HSO3−) abgebaut wurde. Die maximale Eliminierung mit Bentonit (0.4 g/20 ml Milch, 25 °C, 1 Std) lag bei 89%.SummaryTwo methods for eliminating aflatoxin M1 (AFM,) from naturally contaminated raw whole milk (NCRWM) were examined. One involved chemical elimination using potassium sulphite (K2SO3), the other physical adsorption of the toxin using bentonite. Maximum elimination of AFM t (percent of total eliminated) for the chemical method was 45% using 0.05M K2SO3 (25 °C, 5 h). Possible mechanisms responsible for degradation of AFM, by K2SO3 may involve reactions between the toxin and the bisulphite radical (HSO3−. Maximum elimination of AFM1 (89%) by bentonite was achieved using 0.4 g of bentonite per 20 ml of NCRWM (25 °C, 1 h).

Aflatoxin can be degraded by the mycelium of aspergillus parasiticus.

ZusammenfassungMycel, 8 oder 16 Tage alt, und von einem Aflatoxin bildenden Stamm vonAspergillus parasiticus, konnte die Aflatoxine B1, B2, G1, und G2 abbauen. Dies ging etwas schneller vor sich, wenn 16 Tage altes anstatt 8 Tage altes Mycel anwesend war. Die Aflatoxine wurden gleichermaßen abgebaut, wenn der pH-Wert des Mediums 2.5 oder 6.0 war. 4 Tage altes Mycel konnte die Aflatoxine nicht beeinflussen.SummaryAflatoxins B1 B2, G1, and G2 were degraded by the 8- and 16-day old but not by the 4-day old mycelium of a toxigenic strain ofAspergillus parasiticus. The 16-day old mycelium degraded the toxin more rapidly than did the 8-day old mycelium. Degradation of toxin by the mycelium was similar at pH 2.5 and 6.0.

Stability of patulin at pH 6.0-8.0 and 25 °C

ZusammenfassungDie Stabilität von Patulin bei pH-Werten von 6;0, 6,5, 7,0, 7,5 und 8,0 wurde untersucht. Die Reaktionen wurden bei 25 °C und mit dem Phosphatpuffer von Sörensen durchgeführt. Die Patulin-konzentrationen wurden durch Hochdruckflüssigkeits-Chromatographie gemessen. Wurde die Restpatulin-menge gegen die Reaktionszeit aufgezeichnet, ergab sich eine scheinbare Kinetik erster Ordnung. Die Reaktionsgeschwindigkeit für das Verschwinden von Patulin erstreckte sich von 1,1 x 10−2/Std bei pH 8,0 bis auf 5,3 x 10−4/Std bei pH 6,0. Die kalkulierten Halbwert-zeiten lagen zwischen 64 Std bei pH 8,0 und 1310 Std bei pH 6,0.SummaryPatulin was tested for stability at pH values of 6.0, 6.5, 7.0, 7.5, and 8.0, using Sbrensens phosphate buffer at 25 °C. Patulin was determined by high-performance liquid chromatography. When the percentage of patulin remaining was plotted versus reaction time, apparent first-order reaction plots were obtained. Reaction rate constants for disappearance of patulin ranged from 1.1 x 10−2 h at pH 8.0-5.3 x 10−4 h at pH 6.0. Values for half-life were calculated and ranged from 64 h at pH 8.0 to 1310 h at pH 6.0.

Aflatoxin production is inhibited by selected herbal drugs

Aspergillus parasiticus was grown in the presence of selected herbal extracts: burdock, cromwell, honeysuckle, licorice and ginger. Two percent of each herb was used in an enriched medium which was inoculated with spores of the mold and incubated at 28 °C for 9 days. The herbs have been used in Chinese medicine for detoxification, reducing inflammation and treating ulcers or tumors of humans. Mycelial growth was inhibited by honeysuckle and no sporulation of the mold occurred in the presence of burdock and honeysuckle. Burdock, cromwell, ginger and licorice enhanced mycelial growth over that in the control. All the herbs inhibited accumulation of aflat oxins B1 and G1, especially extracts of the honeysuckle flower and root-stem, which inhibited both mycelial growth and aflatoxin production (B1=4% of control, G1=3.4% of control). In the presence of licorice, loss of aflatoxin from the medium during later stages of incubation was greatest.

Degradation of aflatoxin by lactoperoxidase

ZusammenfassungAflatoxin wurde in Gegenwart von 225 μm-NaCI und 50 μm-H2O2 bei 28° C durch 5, 50 bzw. 500 Einheiten von Lactoperoxidase/ml Reaktionsgemisch abgebaut. Wenn die Konzentration der Lactoperoxidase von 50 bis auf 500 Einheiten/ml Reaktionsgemisch gesteigert wurde, dann stieg die Geschwindigkeit des Aflatoxinabbaues von 3,6 bis auf 5,1%/24 Std. Lactoperoxidase baute Aflatoxin G1 1,5 mal schneller ab als Aflatoxin B1. Die Konzentration des Aflatoxins zu Beginn spielte keine Rolle beim Abbau durch, die Lactoperoxidase. Die Abbauprodukte von Aflatoxin B 1 waren chromatografisch dem Aflatoxin B2a ähnlich oder waren wasserlöslich. Gleiche Abbauprodukte, aber in größeren Anteilen, erhielt man durch das Mycel vonAspergillus parasiticus.SummaryThree concentrations of lactoperoxidase, 5, 50, and 500 units/ml of reaction mixture, degraded aflatoxin in the presence of 225 μM NaCl and 50 μM H202 at 28° C. Increasing the amount of lactoperoxidase from 50 to 500 units/ml of reaction mixture resulted in increasing the rate of degradation of aflatoxin B1 from 3.6 to 5.1%/24 h. When comparable amounts of lactoperoxidase were present, aflatoxin G1 was degraded approximately 1.5 times faster than was aflatoxin 131. At a given concentration of lactoperoxidase, aflatoxin degradation was independent of initial aflatoxin concentration. Derivatives that cochromatographed with aflatoxin B2a and derivatives that were water soluble were the major degradation products of aflatoxin B1. Similar derivatives, but in greater proportions, were noted as degradation products that resulted from activity of a blendure of mycelia ofAspergillus parasiticus.

Incorporation of [14C] acetate into aflatoxin by resting cultures of Aspergillus parasiticus in the presence of antifungal agents

SummaryResting cultures of Aspergillus parasiticus were treated with sorbic acid (200 ppm), Nα-palmitoyl-l-lysyl-l-lysine ethyl ester dihydrochloride (PLL) (300 ppm), nisin (30 ppm), nystatin (30 U/ml), dichlorvos (9 ppm), butylated hydroxyanisole (BHA) (30 ppm) and isoprothiolane (30 ppm). Incorporation of [14C]acetate into aflatoxins B1 and G1 by the mold in the presence of these antifungal agents was measured after 12 h of agitated incubation at 28°C. Nystatin and BHA effectively inhibited incorporation of the label into aflatoxin B1 (73.1 and 56.9%) and G1 (68.9 and 91.6%), respectively, whereas PLL and nisin, at the levels used, were less effective. Sorbic acid caused greater inhibition of de novo synthesis of aflatoxin B1 than of G1 while isoprothiolane exhibited the opposite effect. Because the compounds tested had dissimilar physical, chemical and antimicrobial properties, it is likely that they inhibited synthesis of aflatoxin by different mechanisms. Generally, inhibition of aflatoxin synthesis by the test chemicals under resting conditions was more pronounced than what was reported earlier when the same chemicals (at the same levels) were tested with growing cultures of the mold.