Culturomics and metagenomics approaches in the assessment of fungal prevalence in the context of industrial food products

With the rise of genomics, taxonomic identification practices have progressed towards the use of culture-independent methodologies, such as metagenomics. Nevertheless, it has recently been shown that the use of culture-dependent procedures not only helps discriminate and describe the microorganisms present but they are in fact invaluable as they grant information unobtainable otherwise. Furthermore, the combined use of both methodologies provides synergistic gains compared to the results obtained separately. Considering that these insights are still mostly unexploited in regard of fungal diversity, we present an evaluation of a culturomics-based approach to the study of the mycological biodiversity present in the outer layer of a semi-hard goat cheese with visible mould contamination. We have staged a two- pronged setup by metagenomics-based assessment of the Internal Transcribed Spacer region contents, and a culture-based evaluation through Sanger sequencing of the same region of a subset of the total fungal strains isolated in four culture media. We have obtained eight unique representative OTUs out of 177 isolates in the culture approach, whilst with metagenomics, out of nine ASVs obtained from bioinformatic sample treatment using Q2- ITSxpress, we have obtained five representative OTUs. Combining both results, a total 13 OTUs were identified, two being exclusive to metagenomics, three common to both methods, and five exclusive to the culture approach. Our results demonstrate that a combination of both methodologies provides better results in terms of total OTUs classified in comparison to each approach individually. Moreover, the culture-based approach provided a greater number of OTUs comparatively, as well as the number of unique OTUs phylogenetically classified.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, of LABBELS Associate Laboratory in Biotechnology, Bioengineering and Microelectromechnaical Systems (LA/P/0029/2020), and of MIRRI-PT (Pólo Norte) project (PINFRA04/84445/2020) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Pt-mBRCN/MIRRI-PT: Aims and benefits for the portuguese culture collections

Microbial Culture Collections (CCs) are biobanks that preserve and provide microorganisms to the user communities. In the Portuguese landscape, CCs differ considerably regarding size, taxonomic target group, level of organization and implemented quality management systems. Since 2017, the CCs have been working together in the Portuguese microbiological Resource Centre Network (Pt-mBRCN), which was recognised as the Portuguese MIRRI node of the ESFRI Landmark MIRRI in June 2020, to promote a common voice, share experience and foster cooperation. As part of its operation, Pt-mBRCN/MIRRI- PT aims to guarantee the quality of its microbial holdings and associated data, and to implement common procedures and quality management systems to mitigate the current resource/data fragmentation microbial resources. To accomplish these objectives, a strong investment in dedicated human resources and cutting edges technologies is required, which can be partly achieved through competitive funding, such as MIRRI-PT-Polo Norte project. An example of activities within Pt-mBRCN is the collaboration between Micoteca da Universidade do Minho (MUM) and Azorean Regional Veterinary Laboratory Micoteca (LRV-Micoteca). This is providing conditions to accelerate LRV-Micoteca strains requalification process and catalogue improvement, allowing a faster development and establishment of this emerging CC, paving the future to implement ISO 17025 accreditation system.This work was supported by the European Unions Horizon 2020 Research and Innovation programme, under the Grant Agreement no 871129. This publication reflects only the authors view and the Commission is not responsible for any use that may be made of the information it contains.This study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, and by LABBELS Associate Laboratory in Biotechnology, Bioengineering and Microelectromechnaical Systems, LA/P/0029/2020, and of MIRRI-PT (Pólo Norte) project (PINFRA04/84445/2020) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Mycobiota of São Jorge cheeses with different ripening periods

The growth of filamentous fungi in the cheese surface makes the product undesirable (and therefore disposable) and can even present a health risk due to the production of secondary metabolites, such as mycotoxins. The São Jorge cheese is a highly appreciated product from São Jorge Island, Azores, Portugal. It is made with raw cow milk and has long ripening periods, up to 36 months. This product obtained the Protected Designation of Origin (PDO) certification in 1986. Considering that the mycobiota of traditional Portuguese cheeses is understudied, the main goal of this work was to unveil the mycobiota of three São Jorge cheeses with different ripening periods (five, nine and thirty months). Direct inoculation of the cheese in three different culture media was used and the isolates were identified through molecular methods (analysis of ITS and/or partial benA). A total of 32 isolates were identified from the cheese with the lowest period of ripening, mainly Penicillium spp. ser. Camembertiorum (23 isolates), but also Aspergillus sp. (1 isolates), Scopulariopsis sp. (1 isolate), and several yeasts (7 isolates). The mycobiota of cheese with the seven months of ripening was mostly composed of Penicillium spp. ser. Camembertiorum and Saccharomyces cerevisiae, with 8 and 9 isolates, respectively. In the 30 months cheese Penicillium spp. ser. Camembertiorum were also isolated, but Scopulariopsis spp. was predominant, with 20 out of 24 isolates. Although the mycobiota was largely composed of Ascomycota, two Basidiomycota were found in the cheeses with the longest periods of ripening. Future studies will be conducted using metabarcoding techniques to disclose the uncultured mycobiota. These culture-independent techniques are less time consuming and more sensitive. They have shown to be a powerful tool to gain a better and faster understanding of the influence of the microorganisms in the cheese ripening process.Teresa Vale Dias thanks for the Ph.D. scholarship given by the Foundation for Science and Technology (FCT) - 2020.05849.BD. This study was also supported by FCT under the scope of the strategic funding of CEB (UIDB/04469/2020), LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020, CIMO (UIDB/00690/2020) and SusTEC (LA/P/0007/2020)info:eu-repo/semantics/publishedVersio

Implementation of cutting-edge technologies for the benefit of culture collections: the case of Micoteca da Universidade do Minho

Since 1890, microbial culture collections (CCs) have been providing services to the scientific community, acting as reservoirs and providers of microorganisms, including their living cells, genomes, and information, being key players for the development of new and more sustainable products, compounds, and practices. For this reason, the Organisation for Economic Cooperation and Development (OECD) recognized CCs as a key component of the scientific and technological infrastructure of the life sciences and biotechnology. Along with preservation, deposit, and transfer of microorganisms, CCs can provide additional services such as strain identification and characterization, consulting, patent deposit, and training. Through these activities, CCs play a fundamental role in different fields, including agriculture, food security and safety, genetics, industrial and medical microbiology. However, to increase knowledge and maximise the benefits of their holdings for biotechnological applications, CCs must face new challenges and embrace the cutting-edge technologies that allow them to better characterize the microbial strains in their possession. CCs must thoroughly study strain capabilities, dedicating time and resources to the research and characterization of promising strains for biotechnological applications. Furthermore, the generated information regarding function, biosafety, taxonomy, and application, among others, must be made publicly available in CCs catalogues to promote the extensive use of such promising strains. This work will present the example of Micoteca da Universidade do Minho (MUM) and how it is implementing several cutting-edge technologies for the benefit of biotechnology and to respond to client demands, following a strategy integrated in a coordinated effort inside the MIRRI-ERIC Portuguese node. By installing several technological platforms, including cryopreservation in liquid nitrogen, ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), matrix-assisted laser desorption ionizationtime-of-flight mass spectrometry (MALDI-TOF MS), and next generation sequencing (NGS), MUM will advance and improve the conservation, biochemical, physiological, and genetic characterisation of more than 1,000 strains it has available in its catalogue, while also assuming a leading role in the microbiome revolution that we are currently living.This study partially supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems (LA/P/0029/2020); and by “MIRRI PT (Pólo Norte)” project (PINFRA04/84445/2020) funded by the European Regional Development Fund under Norte2020-Programa Operacional Regional do Norte, and by IS_MIRRI21 project (grant agreement nº 871129) funded by the EU H2020 research and innovation programmeinfo:eu-repo/semantics/publishedVersio

Aflatoxins and ochratoxin A contamination during Merkén Pepper Powder production in Chile

Berry fruits of Capsicum annuum L. cv. "Cacho de Cabra are used for the manufacture of a traditional pepper powder known as Merkén, which is a Chilean spicy widely consumed at national level in Chile and also exported to different countries. Merkén contamination with Aflatoxins (AFs) and Ochratoxin A (OTA) is a main concerning for local producers and national and worldwide consumers. In the present study, AFs and OTA contamination in berry fruits of C. annuum L. cv. "Cacho de Cabra was determined at (1) harvest, (2) dried and (3) smoked stages of Merkén production, in (4) cumin and coriander seeds, used as Merkén ingredients, and in (5) the final packed Merkén produced by local farmers. In addition, Merkén obtained from local markets in the Region of La Araucanía (Chile), where obtained and evaluated. Aflatoxigenic and ochratoxigenic potential of fungal strains isolated from the abovementioned substrates were also assessed. There was no detection of AFs nor OTA on pepper pods and seeds used as Merkén ingredients. In contrast, co-occurrence of aflatoxin B1 (AFB1) and OTA were detected in c.a. 57% of final packed Merkén samples (12 out of 21 samples). Regarding AFB1, Merkén samples produced by local farmers presented contamination level from 0.19±0.26 to 1.44±0.10 g/kg; while Merkén sample purchased from local markets presented contamination level from 0.29±0.37 to 1.67±0.32 g/kg. For these samples, no AFB2, AFG1, and AFG2 were detected. Ochratoxin A contamination was detected for 100% of Merkén samples from both local producers (0.79±0.05 to 5.99±0.68 g/kg) and local markets (0.83±0.83-19.81±0.70 g/kg). There was no detection of AFs and OTA on Petri plate for Aspergillus (n=52) and Penicillium (n=129) strains isolated from pepper pods, cumin and coriander seeds and Merkén. The lack of high AFs/OTA-producer among the isolated fungal species can explain and support the absence of contamination in pepper pods. In Merkén production chain the harvest and primary processing of post-harvest (dried and smoking of pepper pods) is a key point to fungal grow but are not critical for AFs and OTA production. In contrast, the second phase of post-harvest (milling, storage) are critical points for AFs and OTA contamination. Prolonged storage under poor hygienic conditions, oscillation in the water activity, and temperature can provide suitable conditions for mycotoxins production. In addition, NaCl and capsaicinoids compounds present on pepper pods and Merkén can act as intrinsic factor up-/downregulation AFs and OTA biosynthesis in this type of substrate.info:eu-repo/semantics/publishedVersio

Additions to neotropical stereoid fungi (Polyporales, Basidiomycota): one new species of Lopharia and one new combination in Phlebiopsis

Stereoid fungi are an artificial group with mostly effused-reflexed to stipitate basidiomata, smooth hymenophore, and hyaline spores. From recent surveys in the Brazilian Atlantic Forest, Caatinga, and Cerrado, six specimens of this group had their identity tested with the nrITS and nrLSU sequences. Two of them were nested within the Lopharia s.s. clade and represent a new species L. erubescens, characterized by the dextrinoid reaction of the cystidia, and small basidia and spores. The other four were initially identified as Hjortstamia amethystea, but nested in the Phlebiopsis clade. Thus, we proposed the new combination, Phlebiopsis amethystea. We also provide keys to neotropical Lopharia s.l. and Phlebiopsis s.l. and allied species.The authors would like to thank Diogo Careli dos Santos for the donation of the collection, the Herbário Pe. Camille Torrend (URM) for the loan of the exsiccates, the Programa de Pós Graduação em Biologia de Fungos (UFPE, Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant SISBIOTA (563342/2010-2), PPBio Semi-Árido (457476/2012-5), PROTAX (562106/2010-3), Universal (472792/2011-3), PQ (307601/2015-3)), Coordenação de Aperfeiçoamento de Pesquisa e Ensino Superior (CAPES; grant Capes-SIU 008/13) and Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE; grant ATP-0021-2.03/18, APQ-0788-2.03/12) for the financial support. CAPES is also acknowledged for the master scholarship of RSC and PhD scholarship of CRSL, and FACEPE for the PhD scholarship of RSC and post-doctorate scholarship of CRSL and VXL. We also would like to thank Nordesta Reforestation & Education for the support in the field work performed at the Biological Reserve of Pedra Talhada. This study was also partially supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of the UID/BIO/04469/2019 unit and the BioTecNorte operation (NORTE-01-0145-FEDER000004) funded by the European Regional Development Fund under the scope of Norte2020-Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Capsicum spp. and the antifungal potential of capsaicinoids as safeguards for agri-food production

In recent years, cases of pathogenic fungi in agricultural crops and in animals, including humans, are growing (Paziani et al., 2020; Costa et al., 2022). Many of these pathogenic fungi are resistant to the commonly used antifungals. This scenario has created a need for new effective antifungals, particularly those based on eco-friendly approaches, such as plant secondary metabolites (e.g. capsaicinoids from Capsicum species) (Costa et al., 2022). The American Phytopathological Society recognises a biofungicide as a naturally based biochemical product that must contain naturally-occurring substances (McGrath, 2004). The use of pepper pod extracts for the production of biofungicide has been proposed in the literature (Costa et al., 2022). As a natural compound, capsaicinoids and their analogues are much less dangerous than commercial synthetic fungicides. Biofungicides present a low risk for environmental health. They could be applied in different stages of agricultural production without putting workers and the environment at health risk. In addition, since biofungicides are based on non-recalcitrant molecules, there is expected no risk of contaminating soil and groundwater. In this context, the use of natural compounds is directly related with the One Health approach, which tries to find a balance among people, animals and the environments health (World Health Organization, 2023). In fact, capsaicinoids compounds has been characterised as antifungal molecules. Capsaicinoids-based biofungicides could have the potential to improve food safety, nutritional value and overcome antimicrobial resistance, with less associated health risk. Beneficial characteristics of capsaicinoids include the demonstrated fungicidal and fungistatic activities of pure Capsicum extracts and purified capsaicinoids (Soumya and Nair, 2012; Costa et al., 2022). These molecules can be used to control the growth of pathogenic fungi in plant crops and as ecological alternatives for pest management. This work aims to review the use of pepper pod extracts, rich in capsaicinoid compounds, as a strategy for safeguarding of agrifood production. The advantages and limitations, for environmental health, of using capsaicinoids-based biofungicides will be presented and discussed in this work.info:eu-repo/semantics/publishedVersio

Hongos filamentosos y micotoxinas en ají merkén

Las micotoxinas son metabolitos secundarios producidos por hongos filamentosos. La Ocratoxina A (OTA) y las Aflatoxinas (AFs) son micotoxinas altamente tóxicas y carcinógenas que se encuentran como contaminantes en alimentos para humanos y animales. El Capsicum (ají o pimiento) es un género de plantas, dicotiledóneas nativo de las regiones tropicales y subtropicales de América, pertenecientes a la familia de las solanáceas. En Chile, el cultivo de Capsicum annuum L. cv. Cacho de Cabra se extiende desde la Región de Arica y Parinacota hasta la Región de La Araucanía, correspondiendo a 9 de las 16 Regiones del país y cubriendo aproximadamente 522 hectáreas de área plantada. Las bayas del ají Cacho de Cabra se utilizan para la producción de un polvo rojo tradicional conocido como Merkén, que es un aliño picante con sabor ahumado. El Merkén es un producto intrínsecamente asociado con el grupo étnico ancestral Mapuche. Al igual que con cualquier otro producto agroalimentario, las bayas del ají Cacho de Cabra y el Merkén procesado también son susceptibles a la infección por hongos que pueden generar contaminación con micotoxinas. Cepas de Alternaria, Aspergillus, Cladosporium, Fusarium, Penicillium y Rhizopus son contaminantes comúnmente halladas en bayas de Capsicum y en sus productos derivados. Entre éstas, cepas de los géneros Aspergillus, Fusarium y Penicillium son las más importantes en términos de capacidad de producción de micotoxinas en dicha matriz alimenticia. En 2017, el Ministerio de Salud de Chile reportó dos casos de contaminación por Merkén con micotoxinas, principalmente OTA. Conocer los puntos de infección por hongos y de contaminación con micotoxinas en la cadena de producción del ají Cacho de Cabra y Merkén, sería una contribución para la implementación de buenas prácticas agroalimentarias, conduciendo a la solución de ese problema. Sin embargo, esa información no está todavía disponible. En ese contexto, el presente trabajo presentará los resultados del potencial micotoxigénico de la micobiota perteneciente a los géneros Aspergillus y Penicillium aislada en los diferentes puntos de la cadena de producción tradicional del ají Cacho de Cabra y del Merkén y posibles puntos de contaminación con OTA y AFs. Además, se discutirán las interacciones ecológicas entre los hongos micotoxigénicos y las bayas de Capsicum annuum L. cv. Cacho de Cabra y del Merkén.info:eu-repo/semantics/publishedVersio

Evaluation of lipid extraction methods from Antarctic filamentous fungi

Purpose: The benefits of natural compounds have been studied for decades for the development of new technologies to answer the global change challenges. In order to develop these new technologies, lipids represent a great class of bioactive molecules. However, the research on lipids and their applications still present gaps about new sources as well as on the extraction methods. Filamentous fungi found in Antarctic territory could represent a new source of novel bioactive lipids. Currently Folch, Bligh & Dyer and Lewis methods are the most widely employed for extraction of lipid from different sample types. Nonetheless, choosing a single extraction method as the gold standard could represent a limitation, especially when the microorganism has not been studied yet. Taking the above into consideration, the main objective of the present study was to evaluate the best extraction method to obtain lipids from different Antarctic filamentous fungal genera. Material and methods: Three isolates of Antarctic fungi belonging to each genus: Mucor, Mortierella, Cladosporium, Penicillium and Pseudogymnoascus isolates from Fildes Bay, Antarctica, were evaluated. A total of 15 isolates were assessed. Folch, Bligh & Dyer and Lewis extraction method were performed. Extraction was monitored by recording spectra of FT-IR spectroscopy of the biomass before and after lipid extraction. Results: Folch was the best method to obtain lipids from filamentous Antarctic fungi, followed by Lewis extraction. Among the three extraction methods evaluated, Bligh & Dyer was the method that presented the lowest yield, compared to Folch and Lewis for each genus and strain. Strains of the genera Mortierella and Mucor were the ones that showed the best performance for the Folch and Lewis methods. The three Penicillium isolates were the third group with the best lipids yield for the Folch method. The strains of genera Cladosporium and Pseudogymnoascus showed better yields for the Lewis method. Conclusions: In this study it was observed that the lipids yield varies according to the extraction methods, as well as both the fungal isolate and fungal genus. Depending on the purpose and fungi taxa, to obtain lipids from Antarctic fungi Folch or Lewis extraction methods are recommended.info:eu-repo/semantics/publishedVersio

Penicillium crustosum as a potential OTA producer - new insights from whole - genome sequencing of strain MUM 16.125

Ochratoxin A (OTA) is a well-studied mycotoxin that poses severe health risks. OTA is mainly produced by Aspergillus and Penicillium species associated with food spoilage and it is present in a wide diversity of food and feed products. Recent studies have reported the presence of OTA in food matrices where known OTA producers are not present1,2. For that reason, other species such as P. crustosum are now being considered. A recent study using comparative genomic analysis3 clarified the OTA biosynthetic gene cluster composition. In order to gain insight into the secondary metabolism of P. crustosum, this study aimed to sequence and explore the complete genome of strain MUM 16.125. This strain was isolated from cheese rind sample contaminated with OTA in which no known OTA producers were present1. The genome assembly comprises 199 contigs with a total length of 30.95 Mb and contains 10975 predicted protein-coding genes. In total, 109 gene clusters potentially related with secondary metabolism were identified, including putative gene clusters for penitrem, clavaric acid or naphthopyrones biosynthesis. Nevertheless, no evidence of an OTA biosynthetic gene cluster was found. A total of 83 complete and 49 partial protein sequences from published OTA biosynthetic genes from 11 Aspergillus and 3 Penicillium species were queried against the predicted P. crustosum proteins. Only 3 strong matches were found (to a short partial P. verrucosum PKS and 2 P. thymicola chloroperoxidases) but matches to complete key genes were absent. Considering these findings, it appears that strain MUM 16.125 lacks the most common genetic pathway to produce OTA, providing important information relevant to understand the role of P. crustosum as putative OTA producer. Nevertheless, the additional secondary metabolism gene clusters found (such as penitrem, clavaric acid or naphthopyrones) highlight the potential of this strain for metabolite production, including other mycotoxins or compounds with antioxidant, anticancer or antibiotic properties.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of CEB (UID/BIO/04469/2019) and iBiMED (UIDB/04501/2020) units; and by CANCYL (POCI-01-0145-FEDER-031849) and GenomePT (POCI-01-0145-FEDER-022184) projectsinfo:eu-repo/semantics/publishedVersio