Technological processes for reduction of fungal and mycotoxin contamination of nuts

Almond and hazelnut production in Portugal are of great importance for the economy of their main producing areas. However, the contamination of these nut fruits with fungi and mycotoxins poses a significant risk to food security. Mycotoxins are secondary metabolites produced by filamentous fungi that frequently contaminate these commodities. This work intended to evaluate the influence of storage conditions on the microbial and mycotoxin stability and safety of nuts throughout long-term storage. For that, almond and hazelnut samples were stored under different relative humidity (RH) conditions (60, 70 and 80%) at 25°C for up to 6 months. The storage of two almond varieties – Lauranne and Guara - under 60% and 70% RH at 25°C were further compared with storage at 4 ºC for 9 months. Microbial loads (aerobic mesophiles and yeasts and molds) were determined at post-harvest stage and after being submitted to preservation, and molds were identified morphologically and molecularly. Nuts were also subjected to multi- mycotoxin analysis by LC-MS/MS to determine their mycotoxin contamination level and profile. For hazelnuts, five fungal species were identified belonging to four fungal genera commonly associated with mycotoxin production: Alternaria, Aspergillus, Fusarium, and Penicillium. For hazelnuts, an important range of mycotoxin was detected after 6 months of storage with a remarkable cumulative amount in 80% RH at 25°C, when compared to 60% and 70% RH at 25°C. For almonds, 26 species were identified belonging to seven genera: Alternaria, Aspergillus, Cladosporium, Fusarium, Penicillium, Paecilomyces, Talaromyces. For the variety Guara, mycotoxins related to Aspergillus sect. Flavi such as Aflatoxin B1, B2, G1, G2, Averufin, Versicolorin C, Norsolorinic acid were detected only after nine months of storage at 70 and 60% RH at 25°C. Penicillium sp. mycotoxins as Quinolactacin A, Roquefortine C and Flavoglaucin were also detected. Flavoglaucin was present in all time-points and conditions of storage in important amounts. For the variety Lauranne, Penicillium sp. mycotoxins were detected such as Citrinin, Quinolactacins A and B, Roquefortines Cand D, Cyclopenin, Cyclopenol, Penitrem A, Viridicatin and Viridicatol. Mycotoxins related to Aspergillus sp. such as Aspulvinone E, Flavoglaucin, Paspalin, Asperglaucide, Asperphenamate, cyclo(L-Pro-L-Tyr) and cyclo(L-Pro-L-Val) were also detected.

A produção de amêndoa e avelã em Portugal é de grande importância para a economia das regiões produtoras. No entanto, a contaminação destes frutos com fungos e micotoxinas apresenta um elevado risco para a segurança alimentar. Micotoxinas são metabolitos secundários dos fungos frequentemente produzidos nestes frutos. O presente trabalho teve como objetivo avaliar a influência das condições de armazenamento de longo prazo na sua estabilidade e segurança microbiana. Para o efeito, amostras de amêndoas e de avelãs foram armazenadas sob diferentes condições de humidade relativa (HR) – 60, 70 e 80%, a 25 ºC durante 6 meses. O armazenamento de duas variedades de amêndoa – Lauranne and Guara – a 60% e 70% HR (25 ºC) foi também comparado com o armazenamento a 4 ºC durante 9 meses. A carga microbiana (aeróbios mesófilos) e fúngica (bolores e leveduras) foi determinada após a colheita e após preservação. Os bolores foram identificados morfológica e molecularmente. Os frutos foram também submetidos a análise multi-micotoxina por LC-MS/MS para determinação do nível e perfil de contaminação com micotoxinas. Nas avelãs foram identificados quatro géneros de fungos potencialmente produtores de toxinas: Alternaria, Aspergillus, Fusarium e Penicillium. Várias micotoxinas foram detetadas após 6 meses de armazenamento, com níveis cumulativos elevados a 80% HR. No caso das amêndoas, foram identificadas 26 espécies pertencentes a sete géneros: Alternaria, Aspergillus, Cladosporium, Fusarium, Penicillium, Paecilomyces, Talaromyces. Para a variedade Guara, foram detetadas várias toxinas relacionadas com Aspergillus sect. Flavi como Aflatoxinas B1, B2, G1, G2, averufina, versicolorina C e ácido norsolorinico após nove meses de armazenamento a 70 e 60% HR. Foram ainda detetadas várias micotoxinas de Penicillium sp. (quinolactacina A, roquefortina C e flavoglaucina). Para a variedade Lauranne, foram detetadas as micotoxinas de Penicillium sp. citrinina, quinolactacinas, roquefortinas, ciclopenina, ciclopenol, penitrem A, viridicatina e viridicatol, e as micotoxinas de Aspergillus sp. aspulvinona E, flavoglaucina, paspalina, asperglaucide, asperfenamate, ciclo(L-Pro-L-Tyr) e ciclo(L- Pro-L-Val).

Work financed by ValNuts Project (PDR2020-101-030756), as part of a community initiative promoted by PDR2020 and co-financed by FEADER, within the Portugal 2020. This work was also partially financed by CIMO (UID/AGR/00690/2019) through the FEDER under PT2020.