Effects of organic acids on mycotoxigenic fungi that produces aflatoxins and Ochratoxin A

Book of Abstracts of CEB Annual Meeting 2017[Excerpt] Mycotoxins are toxic fungal secondary metabolites that occur frequently in a great diversity of agricultural commodities and processed foods. Two of the main mycotoxins that can be found in foods are aflatoxins (AFs) and ochratoxin A (OTA). Some important producers of these mycotoxins are respectively Aspergillus flavus and Penicillium nordicum. Aspergillus flavus occurs mostly on corn, peanuts, spices, cottonseed and tree nuts; P. nordicum occurs on dry-cured meat and cheese products. [...]info:eu-repo/semantics/publishedVersio

Antifungal effect of organic acids on Aspergillus flavus and Penicillium nordicum

SFRH/BD/103245/2014 (FCT), and UMINHO/BPD/51/2015 from project UID/BIO/04469/2013 (FCT/MEC)info:eu-repo/semantics/publishedVersio

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)

Biodegradação de Ochratoxin A por Pediococcus parvulus

Algumas bactérias do ácido láctico (BAL) são capazes de destoxificar micotoxinas através de processos de adsorção às suas paredes celulares ou através de processos de biotransformação em compostos menos tóxicos. Uma das micotoxinas mais importantes encontradas em produtos agrícolas é a ocratoxina A (OTA). A OTA é conhecida principalmente pela sua nefro e carcinogenicidade, estando classificada no Grupo 2B pelo IARC. O presente trabalho descreve a destoxificação de OTA por estirpes de Pediococcus parvulus que foram isoladas de vinhos do Douro. As estirpes foram identificadas e caracterizadas utilizando uma abordagem polifásica que utilizou métodos feno e genotípicos. Para identificar e caracterizar a sua capacidade para destoxificar a OTA, as estirpes foram cultivadas em meio MRS suplementado com esta micotoxina (1 µg/mL). A concentração de OTA, a temperatura de incubação e a concentração de inóculo foram os parâmetros cujo efeito na destoxificação foi avaliado. Verificou-se que a OTA foi degradada em OT pelas estirpes de P. parvulus em todas as condições testadas e que a estirpe tipo desta espécie não apresentou essa capacidade. Ademais, a OT foi confirmada por LC-MS/MS. A conversão de OTA em OT indica que a ligação amida presente na micotoxina foi hidrolisada por uma peptidase. Verificou-se também que a taxa de biodegradação da OTA depende do tamanho do inóculo e da temperatura de incubação. Às condições ótimas (10 9 CFU/mL e 30 ºC), 50% e 90% da OTA foi degradado em 6 e 19 h, respetivamente. Por outro lado, observou-se que as células mortas de P. parvulus adsorveram apenas 1,3% da OTA, o que exclui este mecanismo na eliminação da micotoxina pelas bactérias. A biodegradação de OTA por P. parvulus UTAD 473 foi também avaliada e observada em mostos de uvas. Experiências de vinificação foram também realizadas. Uma vez que algumas estirpes de P. parvulus têm propriedades probióticas relevantes, as estirpes isoladas de vinhos do Douro podem ser de particular interesse para aplicações em alimentos e rações de forma a neutralizar os efeitos tóxicos da OTA.Agradecimentos: Este trabalho foi financiado pelo FEDER através do COMPETE e pela FCT; Ref. FCOMP-01-0124-FEDER-028029 e PTDC/AGR-TEC/3900/2012, respetivamente. Luís Abrunhosa foi apoiado através da bolsa Incentivo/EQB/LA0023/2014 do ON.2 - O Novo Norte

Mycotoxins in food and mitigation of its effects

Book of Abstracts of CEB Annual Meeting 2017[Excerpt] Mycotoxins are toxic compounds mainly produced by fungi of the genera Aspergillus, Fusarium and Penicillium. These toxins are frequently detected in many food commodities including cereals, fruit and vegetables, even after processing [1]. Since the same fungus may produce more than one toxin and the same toxin may be produced by different fungi, mycotoxins often occur as a mixture. Their ubiquitous presence represents a challenge to the health of humans, animals and the environment. Hundreds of compounds are listed as possible mycotoxins; however, only a few occur at levels that may are really challenging. The main focus of AMG is to study and develop integrated and innovative methods, supported in fundamental and applied research, for the mitigation of the occurrence of mycotoxins in food and feed, promoting complex system understanding and knowledge gaps identification. [...]info:eu-repo/semantics/publishedVersio

Anti-aflatoxigenic effect of organic acids produced by Lactobacillus plantarum

ICFC 2017 - International Conference on Food Contaminants (Book of Abstracts)Molds play an important role in food spoilage, being estimated that 5 to 10% of the world food’s production is lost due to fungal contamination. Further, certain fungal species produce highly toxic metabolites, designated as mycotoxins. Biopreservation, de fined as the control of one organism by another, has received much attention in recent years. Also, some strains of lactic acid bacteria (LAB) that demonstrated antifungal and antimycotoxin properties gained interest to be used as natural biopreservatives. In this work, it is shown that the cell free supernatant (CFS) of Lactobacillus plantarum UM55 inhibited the growth of aflatoxigenic fungi, Aspergillus flavus , by 32% and the production of aflatoxins (AFs) by 91%. These inhibitions were lost when the CFS pH was neutralized. Additionally, it was observed an increase of the inhibitions with increasing concentration of CFS. Other aflatoxigenic strains, such as A. parasiticus , A. arachidicola , A. nomius and A. minisclerotigenes were inhibited by the CFS of the bacterium in different extents. Organic acids present in CFS were quantified, with main differences between CFS and control found in the levels of lactic acid, phenyllactic acid (PLA), hydroxyphenyllactic acid (OH - PLA) and indole lactic acid (ILA). When t ested, individually against A. flavus , all the compounds were able to inhibit fungal growth and AFs production. PLA showed the stronger effects and the 90% inhibitory concentration (IC90) for fungal growth and AFs was of 11.9 and 0.87 mg/mL, respectively. AFLs IC90 for ILA, OH - PLA and lactic acid were of 1.47, 1.80, and 3.92 mg/mL, respectively. Inhibitory effects of L. plantarum UM55 seems to be related to the production of lactic acid, PLA, OH-PLA and ILASFRH/BD/103245/2014 from the Portuguese Foundation for Science and Technology (FCT). Luís Abrunhosa was supported by grant UMINHO/BPD/51/2015 from project UID/BIO/04469/2013 financed by FCT/MEC (OE). This study was supported by FCT under the s cope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684); of BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020 -Programa Operacional Regional do Norte; and under the scope of the projects RECI/BBB-EBI/0179/2012 (FCOMP - 01 - 0124 - FEDER - 027462)info:eu-repo/semantics/publishedVersio

Antifungal and antiaflatoxigenic properties of Lactobacillus species

Molds play an important role in spoilage of food products. It is estimated that 5 to 10% of the world foods production is lost due to fungal contamination. Further, certain fungal species produce highly toxic metabolites designated as mycotoxins. According to FAO, up to 25% of the worlds food crops have been estimated to be significantly contaminated with mycotoxins. Among all known mycotoxins, aflatoxins are the most relevant ones because they are carcinogenic. Biopreservation, defined as the control of one organism by another, has received much attention by researchers in recent years. In this field, lactic acid bacteria (LAB) are of great interest to be used as natural biopreservatives of food and feed since they have extensive probiotic properties and have been traditionally used in fermentation processes. The aim of this work is to demonstrate the potential of Lactobacillus species to control the occurrence of aflatoxigenic fungi and aflatoxins in agricultural commodities. For that several aflatoxigenic species such as Aspergillus flavus, A. parasiticus, A. nomius, A. tamari, A. arachidicola, and A. minisclerotigenes were inoculated in triplicate in MEA supplemented with 10% of sterile supernatants of different Lactobacillus species obtained from liquid MRS cultures. Supernatants of most active strains inactivated with heat, proteases and NaOH (for pH neutralization) were also tested and compared with untreated ones. The fungal radial growth and the aflatoxin concentration produced in each plate were recorded and compared. L. casei and L. plantarum supernatants were found to be most active. Growth of Aspergillus flavus in MEA after 7 days was reduced approx. by 31% and 25%, respectively for each bacteria. Aflatoxins B1 and B2 production were inhibited approx. by 97 and 87%, respectively. The antifungal and antiaflatoxigenic activity of those strains was reverted when supernatants were treated with proteolytic enzymes or their pH adjusted to 7

Optimization of enzymes production by Aspergillus niger in solid state fermentation of agro-industrial byproducts

The agri-food industry generates significant amounts of by-products that are often discarded or used in low-value applications. Solid by-products of lignocellulolytic structure have been proved to be suitable substrates in solid-state fermentation (SSF) to produce carbohydrases, that have many industrial applications [1]. In this work, the by-products brewer's spent grain (BSG), rice husk (RH), and vine shoot trimmings (VST) were used to carry out SSF experiments using Aspergillus niger CECT 2088 and to produce enzymes of relevance in textile industry, such as cellulases, xylanases and amylases. Factors influencing SSF performance were examined such as the substrate granulometry and the supplementation of nitrogen and phosphorous sources. In SSF experiments performed at 25ºC and 75% (w/w) moisture for 7 days, a positive effect of medium supplementation with 2% (w/w) (NH4)2SO4 and 1% (w/w) K2HPO4 was observed, leading to 2 to 10-fold increase of enzymes production using RH and BSG. Under these conditions SSF was performed with different particle sizes (1, 4 and 10 mm) and the highest enzyme activities were obtained with the highest size of BSG and RH, and with 4 mm VST. With selected granulometry of the three byproducts, a Simplex-Centroid mixtures design was performed. Optimal enzyme activities were obtained with 100% BSG for xylanase (651 U/g), -glucosidase (363 U/g), cellulase (189 U/g) and 72% (w/w) of BSG and 28% of RH for amylase (263 U/g). Besides those optimal values, the use of 50% (w/w) BSG/RH mixture resulted in high production of xylanase (553 U/g) and -glucosidase (221 U/g), and with 50% (w/w) BSG/VST a cellulase activity similar with 100% BSG was obtained. These results indicates that SSF-based bioprocessing of BSG alone or in mixtures of VST and RH, is an effective and sustainable approach to produce enzymes of textile industry interest, contributing to boost circular economy in this sector.This study was supported by the integrated project be@t – Textile Bioeconomy (TC-C12-i01, Sustainable Bioeconomy No. 02/C12-i01/2022), promoted by the Recovery and Resilience Plan (RRP), Next Generation EU, for the period 2021 – 2026; by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit; by LABBELS Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems (LA/P/0029/2020).info:eu-repo/semantics/publishedVersio

Bio-detoxification of mycotoxins by lactic acid bacteria from different food matrices

Lactic acid bacteria (LAB) play a key role in the biopreservation of a wide range of fermented food products, such as yogurt, cheese, fermented milks, meat, fish, vegetables (sauerkraut, olives and pickles), certain beer brands, wines and silage, allowing their safe consumption, which gave to these bacteria a GRAS (Generally Recognised as Safe) status. Besides that, the use of LAB in food and feed is a promising strategy to reduce the exposure to dietary mycotoxins, improving their shelf life and reducing health risks, given the unique mycotoxin decontaminating characteristic of some LAB. Mycotoxins present carcinogenic, mutagenic, teratogenic, neurotoxic and immunosuppressive effects over animals and Humans, being the most important ochratoxin A (OTA), aflatoxins (AFB1), trichothecenes, zearalenone (ZEA), fumonisin (FUM) and patulin. In a previous work of our group it was observed OTA biodegradation by some strains of Pediococcus parvulus isolated from Douro wines. So, the aim of this study was to enlarge the screening of the biodetoxification over more mycotoxins besides OTA, including AFB1, and ZEA. This ability was checked in a collection of LAB isolated from vegetable (wine, olives, fruits and silage) and animal (milk and dairy products, sausages) sources. All LAB strains were characterized phenotypically (Gram, catalase) and genotypically. Molecular characterisation of all LAB strains was performed using genomic fingerprinting by MSP- PCR with (GTG)5 and csM13 primers. The identification of the isolates was confirmed by 16S rDNA sequencing. To study the ability of LAB strains to degrade OTA, AFB1 and ZEA, a MRS broth medium was supplemented with 2.0 g/mL of each mycotoxin. For each strain, 2 mL of MRS supplemented with the mycotoxins was inoculated in triplicate with 109 CFU/mL. The culture media and bacterial cells were extracted by the addition of an equal volume of acetonitrile/methanol/acetic acid (78:20:2 v/v/v) to the culture tubes. A 2 mL sample was then collected and filtered into a clean 2 mL vial using PP filters with 0.45 m pores. The samples were preserved at 4 °C until HPLC analysis. Among LAB tested, 10 strains isolated from milk were able to eliminate AFB1, belonging to Lactobacillus casei (7), Lb. paracasei (1), Lb. plantarum (1) and 1 to Leuconostoc mesenteroides. Two strains of Enterococcus faecium and one of Ec. faecalis from sausage eliminated ZEA. Concerning to strains of vegetal origin, one Lb. plantarum isolated from elderberry fruit, one Lb. buchnerii and one Lb. parafarraginis both isolated from silage eliminated ZEA. Other 2 strains of Lb. plantarum from silage were able to degrade both ZEA and OTA, and 1 Lb. buchnerii showed activity over AFB1. These enzymatic activities were also verified genotypically through specific gene PCR and posteriorly confirmed by sequencing analysis. In conclusion, due the ability of some strains of LAB isolated from different sources to eliminate OTA, AFB1 and ZEA one can recognize their potential biotechnological application to reduce the health hazards associated with these mycotoxins. They may be suitable as silage inoculants or as feed additives or even in food industry.Acknowledgements This work was funded by FEDER funds through the COMPETE and by national funds through FCT, Ref. FCOMP-01-0124-FEDER-028029 and PTDC/AGR-T EC/3900/2012, respectively. Luís Abrunhosa received support through grant Incentivo/EQB/LA0023/2014 from ON.2 O Novo Nort

Rice husk, brewer’s spent grain, and vine shoot trimmings as raw materials for sustainable enzyme production

Solid by-products with lignocellulosic structures are considered appropriate substrates for solid-state fermentation (SSF) to produce enzymes with diverse industrial applications. In this work, brewer’s spent grain (BSG), rice husk (RH), and vine shoot trimmings (VSTs) were employed as substrates in SSF with Aspergillus niger CECT 2088 to produce cellulases, xylanases, and amylases. The addition of 2% (NH4)2SO4 and 1% K2HPO4 to by-products had a positive effect on enzyme production. Substrate particle size influenced enzyme activity and the overall highest activities were achieved at the largest particle size (10 mm) of BSG and RH and a size of 4 mm for VSTs. Optimal substrate composition was predicted using a simplex centroid mixture design. The highest activities were obtained using 100% BSG for β-glucosidase (363 U/g) and endo-1,4-β-glucanase (189 U/g), 87% BSG and 13% RH for xylanase (627 U/g), and 72% BSG and 28% RH for amylase (263 U/g). Besides the optimal values found, mixtures of BSG with RH or VSTs proved to be alternative substrates to BSG alone. These findings demonstrate that SSF bioprocessing of BSG individually or in mixtures with RH and VSTs is an efficient and sustainable strategy to produce enzymes of significant industrial interest within the circular economy guidelines.This study was funded by the Recovery and Resilience Plan (PRR), Next Generation EU, for the period 2021–2026, through the integrated project be@t—Textile Bioeconomy (TC-C12-i01, Sustainable Bioeconomy No. 02/C12-i01/2022), and by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit (DOI 10.54499/UIDB/04469/2020).info:eu-repo/semantics/publishedVersio