DISRUPTION OF ASPERGILLUS FLAVUS CELLS: A BEAD MILL HOMOGENIZATION METHOD

The most important mycotoxigenic fungus involved in pre- and post-contamination of crops is Aspergillus flavus which causes great health and economic loses worldwide due to production of the most potent natural hepatocarcinogen – aflatoxin B1. Contamination with this secondary metabolite is getting even worst by global climate changes and other abiotic stressors present in environment. Accordingly, researches with the aim of synthesis or identifying the antiaflatoxigenic and antifungal compounds are of interest. For such efforts realization, use and manipulation with intracellular content of A. flavus cells is necessary. The aim of this study was to apply Omni® Bead Ruptor 12 Homogenizer on disintegration of A. flavus cells, to find optimal parameters of homogenization and prepare biologically active extracts which can be used for determination of possible strategies for control of contamination with aflatoxins. Results of study showed that bead mill homogenizer Omni® Bead Ruptor 12 Homogenizer can be applied for disintegration of A. flavus mycelia and preparation of enzymatically active cell-free extracts. The homogenization mixture in 2 mL homogenization tubes should contain 100 mg of fresh wet mycelia, 1 g of precooled acid washed glass beads of 0.5 mm in diameter and 1 mL of ice-cold buffer. Such mixture should be homogenized at speed of 6 m/s during 120 s, in six cycles of 20 s with cooling of samples in ice-bath between cycles

Glucose oxidase activity and hydrogen peroxide accumulation in Croatian honeys

Ability of black locust (n = 7), chestnut (n = 3), lime (n = 5), mint (n = 3), and honeydew (n = 12) honeys to produce hydrogen peroxide upon dilution, as well as their glucose oxidase activity were investigated in the present study. The glucose oxidase (GOX) activity was determined by the standard horseradish peroxidase/o-dianisidin method, while hydrogen peroxide concentration in honey solutions of five different mass to volume ratios (1:1; 1:2; 1:4; 1:8; 1:16) by semi-quantitative method using MQuant™ peroxide test strips. The obtained results showed that chestnut, lime, honeydew and mint honeys exhibited high GOX activity (341.26 ± 128.78, 350.16 ± 124.91, 376.82 ± 69.02, 402.47 ± 60.99 µg H2O2/h g), while black locust honeys much lower GOX activity of 25.58 ± 21.87 µg H2O2/h g. The accumulation of hydrogen peroxide in serially diluted honeys has shown asymmetrical inverted U-shaped curve, where the increase in hydrogen peroxide accumulation with dilution reached a maximum point, after which its concentration rapidly declined. Hydrogen peroxide content in honey solutions of different mass to volume ratio varied from 0 to 294.1 µmol/L h. Lime and chestnut honeys generated the highest hydrogen peroxide content (264.71 ± 65.77, 245.10 ± 84.90 µmol/L h) on average, while black locust, mint and honeydew honeys at least two-fold lower amounts (113.40 ± 50.84, 127.45 ± 33.96, 112.75 ± 98.42 µmol/L h). Lack of correlation between glucose oxidase activity and hydrogen peroxide content indicates that the glucose oxidase activity does not present a reliable parameter for the prediction of hydrogen peroxide content produced in honey solutions

Does the diet style affect the creatinine excretion?

There is a growing interest in alternative diets such as vegan or lacto-ovo vegetarian diet. The choice is made mainly for ethical, health reasons or both. Creatinine is a waste product of creatine phosphate metabolism in muscles, and the excretion rate is relatively constant. Forty-nine participants were tested in total: 5 lacto-ovo vegeterians, 14 vegans, and 30 omnivores. To compare the groups, all participants had a similar diet consisting of bread, bran flakes and wheat bran. The creatinine level was measured before the initiation of the diet in the first morning void, and in the sample of 24-h urine. The results confirmed slightly higher mean creatinine levels in males (1139 ± 517 mg/L), compared to females (901 ± 539 mg/L). Regular consumption of different diets did not show statistically significant differences in datasets on either first void or 24-h samples. The highest median result after 24-h was determined in lacto-ovo vegetarians (2175 mg/L), followed by omnivores (1328 mg/L), while vegans had the lowest median (1235 mg/L). Based on these results, there is no evidence that the vegetarian or vegan lifestyle influences the creatinine excretion rate

Fullerol C60(OH)24 nanoparticles modulate aflatoxin B1 biosynthesis in Aspergillus flavus

Abstract The water soluble fullerene C60 daughter product - fullerols C60(OH)24 (FNP) possesses a great potential of modifying secondary metabolites biosynthesis. In order to clarify the extent of interaction, the impact of FNP (10, 100 and 1000 ng mL−1) on aflatoxin production and the available precursors of biosynthesis pathway from Aspergillus flavus NRRL 3251 was determined, in both the mycelia and yeast extract sucrose (YES) medium, during a 168-hour growth period at 29 °C in the dark. The FNP of 8 nm in diameter, and with a zeta potential of −33 mV affected mycelial growth at 1000 ng mL−1 while conidia production was slightly affected at 10 ng mL−1. The FNP effect on aflatoxin and it biosynthetic precursors was concentration dependent and alteration of the sterigmatocystin (ST) export from the cell was observed. Most of the monitored aflatoxin precursors, except norsolorinic acid, were detected in both mycelia and YES medium. However, observed precursor concentrations were much higher in mycelia, with exception of ST. The study shows the loss of FNP antioxidative effect after 120 hours of growth, and strong concentration dependent aflatoxigenic effect after that time. Thus, this data is relevant to guide future considerations on FNP-fungal interactions in the environments and on risk assessment

<i>Aspergillus flavus</i> NRRL 3251 Growth, Oxidative Status, and Aflatoxins Production Ability <i>In Vitro</i> under Different Illumination Regimes

Aspergillus flavus is the most important mycotoxin-producing fungus involved in the global episodes of aflatoxin B1 contamination of crops at both the pre-harvest and post-harvest stages. However, in order to effectively control aflatoxin contamination in crops using antiaflatoxigenic and/or antifungal compounds, some of which are photosensitive, a proper understanding of the photo-sensitive physiology of potential experimental strains need to be documented. The purpose of the study is therefore to evaluate the effect of visible (VIS) light illumination on growth and conidiation, aflatoxin production ability and modulation of A. flavus oxidative status during in vitro experiment. Aflatoxigenic A. flavus strain was inoculated in aflatoxin-inducing YES media and incubated under three different VIS illumination regimes during a 168 h growth period at 29 &#176;C. VIS illumination reduced A. flavus mycelia biomass yield, both during growth on plates and in liquid media, promoted conidiation and increased the aflatoxin production. Furthermore, aflatoxin production increased with increased reactive oxidative species (ROS) levels at 96 h of growth, confirming illumination-driven oxidative stress modulation activity on A. flavus cells