Jaqueline Garda-Buffon

Inhibition of Fusarium graminearum growth and mycotoxin production by phenolic extract from Spirulina sp.

Fusarium graminearum is a fungal species complex pathogenic occurring worldwide, mainly associated with cereal crops. The most important Fusarium mycotoxins are fumonisins, zearalenone and trichothecenes. The availability of efficient control measures that are less harmful to both the environment and the consumers is urgent. For such, phenolic acids (PAs) from natural sources are known to reduce fungal contaminations. This work aimed to identify the PAs present in a culture extract of Spirulina algae (strain LEB-18) and evaluate its effect on mycelial growth rate, glucosamine level, amylase activity and mycotoxin production by four strains of two lineages of F. graminearum. Results showed that amendment of potato dextrose media with LEB-18 extract (3% w/v), which was mainly composed by gallic acid, greatly reduced radial growth of fungal colonies compared to media containing a single PA and the control. Also, average reductions of 40% and 62% in the glucosamine levels and the amylase activity were observed. In general, the LEB-18 extract and the PAs reduced mycotoxin concentration, with an average reduction of 68% for the trichothecene mycotoxins deoxynivalenol and nivalenol.

Aflatoxins: Contamination, Analysis and Control

Aflatoxins are toxic metabolites produced by different species of toxigenic fungi, called mycotoxins. Humans can be exposed to aflatoxins by the periodic consumption of contaminated food, contributing to an increase in nutritional deficiencies, immunosuppresion and hepatocellular carcinoma (Wagacha & Muthomi, 2008). Aflatoxins (AFs) have a wide occurrence in different kind of matrices, such as spices, cereals, oils, fruits, vegetables, milk, meat, etc. Among the 18 different types of aflatoxins identified, the major members are aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2), M1 (AFM1) and M2 (AFM2) which are produced by Aspergillus flavus and/or Aspergillus parasiticus. Strains of A. flavus can vary from non-toxic to highly toxigenic and are more likely to produce AFB1 than AFG1. Strains of A. parasiticus generally have less variation in toxigenicity and produce AFB1 and varying amounts of AFB2, AFG1 and AFG2 (Coppock & Christian, 2007). Other fungi have been described in the literature as aflatoxins’ producers such as A. bombycis, A. ochraceoroseus and A. pseudotamari (Klich et al, 2000; Mishra & Das, 2003). A. flavus and A. fumigatus have also been identified as pathogens for animals and humans (Zain, 2011). The order of acute and chronic toxicity is AFB1 > AFG1 > AFB2 > AFG2, reflecting the role played by epoxidation of the 8,9-double bond and also the greater potency associated with the cyclopentenone ring of the B series, when compared with the six-membered lactone ring of the G series. AFM1 and AFM2 are hydroxylated forms of AFB1 and AFB2 (Mclean & Dutton, 1995; Wogan, 1966). In the primary fungi metabolism a lot of interrelated reactions catalyzed by enzymes occur, with the objective of promoting energy and primary metabolites (synthetic intermediates and macromolecules), ensuring the growth and reproduction of fungi. Secondary

Kinetics of deoxynivalenol degradation by Aspergillus oryzae and Rhizopus oryzae in submerged fermentation

The objective was to evaluate the capacity of Aspergillus oryzae and Rhizopus oryzae to degrade deoxynivalenol (DON) during submerged fermentation. The submerged medium utilized was sterile distilled water with 1μg mL-1 DON, added and inoculated with 4 × 106 spores mL-1 of the fungal species. Sampling was performed every 48 h to 240 h. DON analyses included residual mass, percentage and velocity of degradation. Residual mass of DON in the collected medium was extracted by liquid-liquid partition and quantified by gas chromatography through derivation with trifluoroacetic anhydride. The time required for the largest DON degradation was 96 h and 240 h by Aspergillus oryzae and Rhizopus oryzae respectively, and degradation rate were 0.62 and 0.54 μg h-1, respectively. Rhizopus oryzae caused the largest decrease in DON at around 90% in 240 h, while Aspergillus oryzae caused the most rapid degradation with a 74% reduction of DON at 96 h.

Análises quali e quantitativas de micotoxinas em águas da cadeia produtiva do arroz por CCD e CCDAE

This study validated a simple and applied method for determining mycotoxins aflatoxin B1, aflatoxin B2, ochratoxin A, zearalenone and deoxynivalenol, in water from the rice production chain. Five solvent combinations for extraction were tested, with quantification performed by TLC/HPTLC and confirmation by LC-MS/MS. Mycotoxins in water from field and rice industries were evaluated. Mycotoxin recovery levels were around 90%. Two samples from rice parboiling waste were contaminated (deoxynivalenol/aflatoxin B1, 110/9 ng mL-1; and deoxynivalenol, 100 ng mL-1). Zearalenone, deoxynivalenol and ochratoxin A (36, 30 and 28%) were carried to soaking water during parboiling.

Método QuEChERS para determinação de ocratoxina a e citrinina em arroz e farelo de arroz

The present study was carried out to establish the optimal conditions for performing ochratoxin A (OTA) and citrinin (CIT) extraction using the QuEChERS method in rice. Employing the factorial experimental design, variables that significantly influenced the extraction stages were determined. The following variables were analyzed: addition of water, acidification of acetonitrile with glacial acetic acid, as well as amounts of magnesium sulfate, sodium acetate, sodium citrate and diatomaceous earth. The best combining procedure resulted in a predictive model using more water and less diatomaceous earth. Recoveries of CIT and OTA were 78-105%.

Characterization and application of the enzyme peroxidase to the degradation of the mycotoxin DON

ABSTRACT Deoxynivalenol (DON), one of the main mycotoxins found in food matrices, has high level of toxicity. This study aimed to characterize the peroxidase enzyme extracted from rice bran to be applied to the biodegradation of DON in order to evaluate the potential peroxidase (PO) from rice bran (RB) has to degrade DON in optimal conditions. Purification and recovery factors of PO extracted from RB and purified by three-phase partitioning were 5.7% and 50%, respectively. PO had the highest level of activity in the phosphate buffer 5 mM pH 5.5 in both crude and purified forms, whose reaction temperatures were 25°C and 10°C. At the end of production, purification and characterization steps, specific activities of the bran were 115.79 U mg−1 and 4363 U g−1. Reduction in the mycotoxin DON in optimal conditions determined for PO from RB was 20.3%, a promising result when the aim is to adequate mycotoxicological levels to foods.

Evaluation of the Suitability of Analytical Methods in Trichothecene A and B Degradation

An investigation was conducted to standardize conditions for laccase and lipase activity required to maximize trichothecene degradation. The analytical methods were suitable and validated in model solutions for detection of five analytes (T-2 toxin, deoxynivalenol, nivalenol, 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol), the methods were then applied in the degradation tests of these trichothecenes. The method of salting-out assisted liquid-liquid extraction (SALLE), using three different NaCl concentration levels, obtained recoveries in the range of 47.4-103.4% at pH 5.0 and 36.6-106.8% at pH 7.0, with an intra-day relative standard deviation under 15% for the majority of the compounds. Quantification limits remained in the region of 0.07 μg mL for 15-acetyldeoxynivalenol and 0.3 μg mL for nivalenol. Finally, the suitable analytical methods were applied in a study of trichothecene degradation by enzymatic action, resulting in a reduction of 12.3% for T-2 toxin, 68.4% for deoxynivalenol, 50.2% for 3-acetyldeoxynivalenol and 45.4% for 15-acetyldeoxynivalenol.

Peroxidase from soybean meal: obtention, purification and application in reduction of deoxynivalenol levels

This study established the conditions for the extraction of the enzyme peroxidase (PO) from soybean meal (SBM). An experimental design methodology was carried out in order to evaluate the effects of stirring rate time, pH and extracting solvent volume on the enzyme extraction. By using 5 g SBM and 50 mL phosphate buffer 10 mmol L pH 4.7, 60 min stirring rate at 100 rpm, an enzyme with specific activity of 100 U mg for SBM was obtained. Two techniques of purification were tested and compared for purification of peroxidase from soybean meal: triphasic partition (TPP) and molecular exclusion chromatography. TPP showcased a greater efficiency with 50% recuperation and a purification factor of 13.6. Peroxidase in crude and pure forms was characterized for kinetics, thermodynamics and biochemistry. The parameter of thermal inactivation indicates high stability to exposure time and temperature increase, showing that enzyme activity is not altered by the presence of constituents of the reaction medium. Peroxidase in crude form represented a greater upkeep in activity, keeping 50% activity for 114 days at 0 °C. Peroxidase in pure form had greater affinity for substrate and reduced Deoxynivalenol levels by 80%, 20% more than the crude form.