Isolation and purification of an enzyme hydrolyzing ochratoxin A from aspergillus niger

Ochratoxin A is a mycotoxin produced by several Aspergillus and some Penicillium species which may be present in food and feed products. It can be enzymatically hydrolyzed into ochratoxin α and l-β-phenylalanine, thereby decreasing its toxicity. The ochratoxin A degradation capacity of Aspergillus niger is well known and here we report the isolation and purification of a novel enzyme from A. niger that hydrolyzes this mycotoxin. A wheat germ medium supplemented with ochratoxin A was used to produce the enzyme, which was purified from culture filtrate by acetone precipitation and anion exchange chromatography. An overall purification of 2.5-fold with a recovery of 68% and a final specific activity of 36 U/mg was obtained. The enzyme is a metalloenzyme as it was inhibited at 10 mM EDTA, whereas PMSF had no effect. The ochratoxin A hydrolytic enzyme presented a V max of 0.44 μM/min and a K m of 0.5 mM when the reaction was carried out at pH 7.5 and 37°C.Fundação para a Ciência e a Tecnologia (FCT

Use of Pediococcus parvulus to remove ochratoxin A from wine

A ocratoxina A (OTA) é uma micotoxina que pode ser encontrada no café, vinho tinto e frutos secos mas a sua principal fonte são os cereais e os seus derivados. É produzida principalmente por Penicillium verrucosum, Aspergillus westerdijkiae, Aspergillus carbonarius e Aspergillus niger. Estas duas últimas espécies são muito comuns em uvas e são responsáveis pela presença de OTA nos vinhos. Na Europa, o vinho é a segunda principal fonte dietética desta micotoxina, estando por isso estabelecido um limite máximo para a sua presença em vinho de 2 g/kg [1]. Para que os vinhos possam cumprir com esse limite, é necessário implementar medidas preventivas ou correctivas. O presente trabalho estudou a aplicação em mostos e em vinhos de uma bactéria láctica (Pediococcus parvulus) que tem a capacidade de hidrolisar a OTA em compostos não tóxicos. Desta forma, pretendeu-se avaliar se a bactéria seria capaz de eliminar esta micotoxina nas referidas matrizes. Para tal, suplementou-se mosto e vinho sintético com OTA comercial. De seguida, inocularam-se, em triplicado, matrazes com 100 mL de mosto ou de vinho com 109 CFU/mL da bactéria. Ensaios em que se inoculou a bactéria juntamente com uma levedura enológica, ensaios sem a bactéria e só com a levedura foram também realizados. Os matrazes foram incubados a 25 ºC no escuro durante 30 dias e amostras foram recolhidas ao longo do tempo para analisar a presença de OTA. Os açúcares totais, ácido málico, ácido láctico e etanol foram também determinados. No vinho sintético, verificou-se que o Pediococcus parvulus não foi capaz de eliminar a OTA embora tenha convertido o ácido málico em ácido láctico. Nos mostos, verificou-se que o Pediococcus parvulus foi capaz de eliminar esta micotoxina. Ao fim de 3 dias a bactéria tinha degradado 50% da OTA presente no mosto, ao fim de 6 dias essa percentagem atingiu os 80%. Nos ensaios realizados com a bactéria e S. cerevisiae em simultâneo, verificou-se uma tendência similar - 66% da OTA foi eliminada nos primeiros 3 dias e ao fim de 6 dias essa percentagem atingiu os 78%. Num dos ensaios conseguiu-se eliminar a totalidade da OTA do mosto ao fim de um período de 3 dias. No mosto, verificou-se ainda que os níveis de açúcares consumidos pela bactéria foram muito baixos, indicando que estes ficam disponíveis para a levedura possa levar a bom termo a fermentação alcoólica. Em estudos futuros pretende-se avaliar o impacto de Pediococcus parvulus na qualidade do vinho produzido.FEDER através do Programa Operacional Factores de Competitividade ‐ COMPETE e por fundos nacionais através da Fundação para a Ciência e a Tecnologia ‐FCT, ref. FCOMP‐01‐0124‐FEDER‐028029 e PTDC/AGR‐TEC/3900/2012, respectivamente. Este trabalho também foi financiado pelo IBB/CGB‐UTAD e Centro de Química de Vila Real (CQ‐VR)

Active whey protein edible films and coatings incorporating lactic acid bacteria for fungi control in cheese

info:eu-repo/semantics/publishedVersio

Avaliação da eficiência de diferentes produtos enológicos na remoção de ocratoxina A de vinho

As micotoxinas são metabolitos secundários tóxicos produzidos por certos fungos, sendo a ocratoxina A (OTA) das mais importantes. A presença de OTA nos vinhos pode constituir um risco para a saúde dos consumidores, sendo por isso aconselhado que se tomem medidas para atingir níveis seguros para o consumo humano [1]. De acordo com o Regulamento n.º 1881/2006 da Comissão Europeia, o limite máximo para a OTA em vinho é de 2 µg/kg [2]. Sendo assim, foi objetivo deste trabalho conhecer a eficiência de diferentes produtos enológicos na remoção de OTA de vinhos, bem como o seu impacto nas suas características organoléticas. Foram testados onze produtos enológicos diferentes, com origem mineral, sintética, microbiana, vegetal e animal, de forma a avaliar a sua eficiência na remoção de OTA de vinhos. Os ensaios foram realizados em vinhos artificialmente suplementados com OTA numa concentração final de 10 µg/L. O produto enológico mais eficiente na remoção de OTA do vinho branco (80%) é composto por gelatina, bentonite e carvão ativado. Reduções entre 10-30% foram também obtidas com o caseinato de potássio, paredes de células de levedura e proteína de ervilha. Com a aplicação de bentonite, carboximetilcelulose, polivinilpolipirrolidona e quitosana não se verificou nenhuma remoção considerável de OTA dos vinhos brancos. Estes resultados podem fornecer informações úteis para os produtores de vinho, ajudando-os na seleção do produto enológico mais adequado para a remoção de OTA de vinhos brancos, reduzindo a toxicidade do vinho e melhorando simultaneamente a segurança alimentar e qualidade do produto final.Agradecimentos: Este trabalho foi financiado por fundos FEDER através do Programa Operacional Factores de Competitividade - COMPETE e por fundos nacionais através da Fundação para a Ciência e a Tecnologia -FCT, ref. FCOMP-01-0124-FEDER-028029 e PTDC/AGR-TEC/3900/2012, respetivamente. Luís Abrunhosa recebeu apoio através da bolsa Incentivo/EQB/LA0023/2014 do ON.2 O Novo Norte

Effect of Gamma radiation on mycotoxins solutions

Due to the high toxicity of mycotoxins, many methods have been used to reduce or eliminate them from food and feed. Gamma radiation is one technique that has been investigated with some promising results in degradation of some mycotoxins from food. The aim of this study was to clarify the effect of gamma irradiation on aflatoxin B1 (AFB1) aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2), ochatoxin A (OTA) and zearelone (ZEA). The effect of the presence of moisture during the irradiation process was evaluated. Solutions with the same initial mycotoxin concentration were submitted to gamma radiation doses ranging from 0 to 10.0 kGy, at distinct moisture level – dehydrated, in water, and in methanol:water solution. Mycotoxins levels were determined by high-performance liquid chromatography with fluorescence detection (HPLC-FL), and photochemical post-column derivatization (for aflatoxins). The results showed degradation of mycotoxins with doses above 3.0 kGy, but only when irradiated in aqueous environment. With dehydrated samples, no significant reduction was observed. The results showed that gamma radiation was effective in reducing the mycotoxins concentration, but the presence of water (mainly due to the formation of hydroxyl radicals) had a very significant effect.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/79364/201, SFRH/BPD/43922/2008Programa Operacional do Norte (Adl. Portugal) - ChestNutsRad (contract number 13198

Radiosynthesis and in vivo evaluation of a 18F-labelled styryl-benzoxazole derivative for β-amyloid targeting

The formation of β-amyloid deposits is considered a histopathological feature of Alzheimer′s disease (AD). In vivo molecular imaging by means of amyloid-avid radiotracers will allow for an early and conclusive diagnostic of AD. Herein, we describe the radiosynthesis of the radiofluorinated styryl benzoxazole derivative [18F]-[2-[N-methyl-N-(2′-fluoroethyl)-4′-aminostyryl]benzoxazole] ([18F]-1) and its pre-clinical evaluation, including metabolic and biodistribution studies in male Wistar rats. The in vivo biological evaluation of [18F]-1 showed that this new radiotracer has a moderate brain uptake with a slow brain washout and a poor in vivo stability

Evaluation of zearalenone in vitro removal by Saccharomyces cerevisiae strains isolated from bovine forage

Zearalenone (ZEN) is a mycotoxin that has relatively low acute toxicity. However, it is a potent oestrogen, interfering with the reproductive tract of animals. Among other effects, ZEN decreases animals fertility, and induces fibrosis in the uterus, breast cancer and endometrial carcinoma (Zinedine et al., 2007). Anti-mycotoxin additives (AMA) are defined as a group of products that, when added to animal feed, are capable of adsorbing, inactivating, or neutralizing mycotoxins in the gastrointestinal tract of animals. One example of these products are adsorbents based on yeast cell walls, a safe and beneficial animal feed additive (Abreu et al., 2008). When based on active cells, yeast based products also act as a probiotic, contributing to improve the general animal health because it stimulates their immune system and promotes the integrity of intestinal mucosa (Albino et al., 2006). Strains of Saccharomyces cerevisiae isolated from silage were tested for their ZEN removal capability. Their effect on - and b-zearalenol (-ZOL and b-ZOL) was also tested. Strains were grown on YPD separately supplemented with ZEN, -ZOL and b-ZOL, and their elimination from culture media was quantified over time by HPLC-FL.This study was carried out with grants from CYTED (Acción 109ac0371), CNPq, CAPES-DS and FAPUR/UFRRJ (Brazil). Luís Abrunhosa was supported by grant SFRH/BPD/43922/2008 from Fundação para a Ciência e Tecnologia – FCT, Portugal.. Authors also acknowledge the support of the Society for Applied Microbiology – SfAM

Rhamnolipids inhibit aflatoxins production in Aspergillus flavus by causing structural damages in the fungal hyphae and down-regulating the expression of their biosynthetic genes

Aflatoxins are hepatotoxic and carcinogenic fungal secondary metabolites that usually contaminate crops and represent a serious health hazard for humans and animals worldwide. In this work, the effect of rhamnolipids (RLs) produced by Pseudomonas aeruginosa #112 on the growth and aflatoxins production by Aspergillus flavus MUM 17.14 was studied in vitro. At concentrations between 45 and 1500 mg/L, RLs reduced the mycelial growth of A. flavus by 2340% and the production of aflatoxins by 93.999.5%. Purified mono-RLs and di-RLs exhibited a similar inhibitory activity on fungal growth. However, the RL mixture had a stronger inhibitory effect on aflatoxins production at concentrations up to 190 mg/L, probably due to a synergistic effect resulting from the combination of both congeners. Using transmission electron microscopy, it was demonstrated that RLs damaged the cell wall and the cytoplasmic membrane of the fungus, leading to the loss of intracellular content. This disruptive phenomenon explains the growth inhibition observed. Furthermore, RLs down-regulated the expression of genes aflC, aflE, aflP and aflQ involved in the aflatoxins biosynthetic pathway (6.4, 44.3, 38.1 and 2.0-fold, respectively), which is in agreement with the almost complete inhibition of aflatoxins production. Overall, the results herein gathered demonstrate for the first time that RLs could be used against aflatoxigenic fungi to attenuate the production of aflatoxins, and unraveled some of their mechanisms of action.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER 000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. The authors also acknowledge the support of the i3S Scientific Platform Histology and Electron Microscopy (HEMS), member of the PPBI (PPBI POCI-01-0145-FEDER-022122). A.I. Rodrigues and L. Abrunhosa acknowledge FCT for the grant SFRH/BD/111600/2015 and the Assistant Research contract CEECIND/00728/2017, respectively.info:eu-repo/semantics/publishedVersio

Biodegradation of ochratoxin A by Pediococcus parvulus isolated from Douro wines

Lactic acid bacteria (LAB) are a promising solution to reduce exposure to dietary mycotoxins because of the unique mycotoxin decontaminating characteristic of some LAB. Ochratoxin A (OTA) is one of the most prominent mycotoxins found in agricultural commodities. The present work reports on the ability of Pediococcus parvulus strains that were isolated from Douro wines that spontaneously underwent malolactic fermentation to detoxify OTA. These strains were identified and characterised using a polyphasic approach that employed both phenotypic and genotypic methods. When cultivated on OTA-supplemented MRS media, OTA was biodegraded into OTα by certain P. parvulus strains. The presence of OTα was confirmed using LC-MS/MS. The conversion of OTA into OTα indicates that the OTA amide bond was hydrolysed by a putative peptidase. The rate of OTA biodegradation was found to be dependent on the inoculum size and on the incubation temperature. Adsorption assays with dead P. parvulus cells showed that approximately 1.3% ± 1.0 of the OTA was adsorbed onto cells wall, which excludes this mechanism in the elimination of OTA by strains that degrades OTA. Under optimum conditions, 50% and 90% of OTA was degraded in 6 and 19 h, respectively. Other LAB strains that belonged to different species were tested but did not degrade OTA. OTA biodegradation by P. parvulus UTAD 473 was observed in grape must. Because some P. parvulus strains have relevant probiotic properties, the strains that were identified could be particularly relevant to food and feed applications to counteract the toxic effects of OTA.This work was funded by FEDER funds through the "Programa Operacional Factores de Competitividade - COMPETE" and by national funds through "Fundacao para a Ciencia e a Tecnologia - FCT", Ref. FCOMP-01-0124-FEDER-028029 and PTDC/AGR-TEC/3900/2012, respectively. The authors also thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the Project "BioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes, REF. NORTE-07-0124-FEDER-000028" co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER. Luis Abrunhosa was supported by the grant SFRH/BPD/43922/2008 from FCT