Quality parameters in a culture collection - Micoteca da Universidade do Minho

Tese de doutoramento em Engenharia Química e BiológicaThe biological diversity is quite important to the world, in a social, industrial, economic and scientific point of view. Nowadays it is mandatory to assure and guarantee biodiversity conservation, its success, sustainable use and the equitable share of benefits arising from the use of genetic resources through: ethical sourcing practices, and collaborations between the different Biological Resource Centres (BRCs). It is now the era of awareness of quality assurance. This work presents ways to contribute and increase the quality, knowledge, information, maintenance and preservation of biological resources, applied to filamentous fungi. The achievement of quality within the BRCs and culture collections (CC) context is a dynamic process, always evolving as well as the backlog and build-up of biological resources data that increase with each research made. An option to achieve quality is the implementation of a Quality Management System (QMS) based on the standard ISO 9001:2008, like the one here described and explained on a CC of filamentous fungi: Micoteca da Universidade do Minho (MUM). The QMS implemented at MUM in 2011 and the obtained certification, are a continuous improvement process focused on customer satisfaction. Maintenance of biological resources implies the choice of the best methods and constant search of cheaper, faster and more practical, with assured procedures and validation of preservation success. With this purpose, an assessment of preservation methods was performed, through a polyphasic approach using several techniques of fungal characterisation [macroscopic (photography), microscopic (optical, stereomicroscopy and SEM), mycotoxin screening (HPLC), enzymatic screening (spectrophotometry and specific inducer media), MALDI-TOF MS and molecular biology analysis]. It was found that long-term stored lyophilized samples were not significantly altered making this method one of the most appropriate in the case of filamentous fungi preservation. The minor changes observed were dependent on the strain to preserve. In the same context of the continuous strive for improvement, a proposal to develop new methods for the preservation of strains, specially delicate and/or recalcitrant strains of fungi was made. Perlite and alginate encapsulation were used in a specific group of selected fungal strains. For the conditions chosen, after morphological and MALDI-TOF MS analysis, it was found that perlite does not allow the viability of samples after preservation whether alginate encapsulation proved to be a better alternative to the common and well known Castellani method, i.e., preservation in water.A diversidade biológica é muito importante do ponto de vista social, industrial, económico e científico. Assegurar e garantir a conservação da diversidade biológica, o seu uso sustentável e a repartição equitativa dos benefícios resultantes da utilização dos recursos genéticos, através de: práticas de abastecimento éticas e colaborações entre os diferentes Centros de Recursos Biológicos (BRCs), é um requisito actual. Este trabalho pretende apresentar formas de contribuir e aumentar a qualidade, conhecimento, informação, manutenção e conservação de fungos filamentosos. A obtenção de qualidade dentro do contexto dos BRCs e Colecções de Cultura (CCs) é um processo dinâmico, em constante evolução, bem como o aumento crescente e acumulação de dados sobre os recursos biológicos que aumentam a cada pesquisa feita. Uma opção para alcançar a qualidade é a implementação de um Sistema de Gestão da Qualidade (SGQ) baseado na norma ISO 9001:2008, como o aqui descrito e explicado para uma CC de fungos filamentosos: Micoteca da Universidade do Minho (MUM). O SGQ implementado na MUM em 2011 e a certificação obtida, são um processo de melhoria contínua focado na satisfação do cliente. A manutenção dos recursos biológicos implica a escolha dos melhores métodos e a busca constante de outros mais baratos, rápidos, práticos, com procedimentos assegurados e validados. Com esta finalidade, foi realizada uma avaliação de métodos de preservação, através de uma abordagem polifásica com o uso de várias técnicas de caracterização fúngicas [macroscópica (fotografia), microscópica (óptica, esteromicroscópica e SEM), rastreio de micotoxinas, rastreio de produção enzimática, MALDI-TOF MS e análise de biologia molecular]. Verificou-se que as amostras liofilizadas armazenadas por longos períodos não sofrem grandes alterações o que torna este método como um dos mais apropriados na preservação de fungos filamentosos. As poucas alterações observadas são dependentes da estirpe a preservar. Foi ainda realizada uma proposta de desenvolvimento de novos métodos para a preservação de estirpes fúngicas, delicadas e/ou recalcitrantes. Foram testados: perlite e encapsulamento em alginato. Verificou-se que a perlite não permite a viabilidade das amostras após preservação, mas o encapsulamento em alginato demonstrou, após análise morfológica e por MALDI-TOF, ser uma boa alternativa ao método de preservação em água, ou seja: método Castellani

Assessment of fungi stability by MALDI-TOF ICMS following preservation on alginate encapsulated samples

Alginate-encapsulation is a commonly used, simple and cost effective, method to preserve plant samples. Since alginate has proven to protect tissues against physical and environmental damage, minimising dehydration, it presents as a good preservation technique. The application of this method for the preservation of filamentous fungi was intended to present an alternative for the commonly used preservation methods, especially to be used on recalcitrant fungal strains. Matrix-assisted laser desorption⁄ionisation time-of-flight intact cell mass spectrometry (MALDI-TOF) emerged in the late 1980s as a sound technique to investigate the mass spectrometry of molecular high-mass of organic compounds through a soft ionisation of molecules resulting in minimum fragmentation. This technique has demonstrated its high potentiality for identification of filamentous fungi species and, in some specific cases, for strain identification. One of the most interesting advantages of the technique is the possibility of analysing the intact fungal cell generating peptides and proteins profiles. A novel technique was applied on the preservation of Botrytis cinerea (MUM 10.163, 10.165, 10.167), Aspergillus ibericus (MUM 04.68) and Aspergillus brasiliensis (MUM 06.181) using the methodology of alginate encapsulation, in two different conditions: distilled water (I) and a 10% glycerol solution (II), both at 4 ºC for 1 year; the viability of these strains was studied. The assessment was made by comparison with the method of Castellani preservation in water (III), using morphologic and MALDI-TOF ICMS analyses for the analysis of the 3 preservation methods. The encapsulated samples of the strains preserved in distilled water (I), presented lower viability than those preserved in 10% glycerol (II). However, when comparing the growth from the samples preserved in water (III) with the ones encapsulated and maintained in 10% glycerol (II), we noted that the last ones presented a healthier and faster growth. From the MALDI-TOF ICMS analysis, it was observed that the strains were clustered according to species identification, and for Botrytis cinerea all presented small differences (< 5%) in the percentages of similarity, except for MUM 10.167 (>5%) when preserved in water (III). From our evaluation we were able to conclude that the success of the preservation method is strain dependent.European Community’s Seventh Framework Programme (FP7, 2007-2013), Research Infrastructures action, under the grant agreement No. FP7-228310 (EMbaRC project), FCT – Portugal for the scholarship SFRH/BD/64260/2009

The importance of structural diversity of spores in the taxonomy of Aspergillus (section Nigri)

Species of Aspergillus from section Nigri, also known as black aspergilli, are distributed worldwide and have been widely used for purposes of various types such as: biotechnological, industrial, medical, among others. They have been extensively studied for being the causing agents of biodeterioration of commodities and food. Within this section, new species have been recently described and among them Aspergillus ibericus and Aspergillus uvarum were both isolated from grapes. The polyphasic approach used in the analysis of these species, either by morphological techniques as well as by molecular methods, allowed not only their characterisation but also their consequent separation from all others in the section. Microbial taxonomy has the identification of species as one of its fundamental goals. Data such as: morphological characteristics description, physiological and biochemical properties, ecological roles, and societal risks or benefits, are key elements in any fungal identification process. But, this process is subjected to periodic changes due to frequent revisions of the taxonomic schemes, therefore becoming time consuming and more demanding and difficult, even for skilled specialists. Furthermore, each taxonomic group has specialised literature, terminology and characters. This takes place since identifications have difficulties of consensual naming, depending on the criteria used and the amount of information available when producing all data. It is increasingly becoming clear that, to better achieve a more accurate concept of species, microbial identification and authentication require a polyphasic approach to produce consistent, useful and quality data. Characterisation of morphologic and structural aspects of spores from Aspergillus strains, section Nigri, has been carried out using scanning electron microscopy (SEM) and intends to contribute to the associated data of the strains from this section. Colonies from 13 Aspergillus from section Nigri, from the Micoteca da Universidade do Minho (MUM), were grown at 25 ºC for 3 or 4 days in malt extract agar directly mounted in a sterilised SEM stub. The samples were covered with a mixture of gold and platinum (80/20%) and then examined in the SEM [NanoSEM - FEI NovaTM 200 (FEG/SEM); EDAX - Pegasus X4M (EDS/EBSD)]. A stereomicroscope (Leica MZ12.5) was used to have a clear image comparison of the size of the conidial head. Although the analysed strains presented dimensional, morphological and structural diversity, common or typical features could be inferred and related to each taxon like those represented in Fig 1. From the SEM analysis we were able to conclude that in the section Nigri of genus Aspergillus the spore wall ornamentation, and its size and shape continue to present themselves as important primary diagnostic traits for species differentiation

Aged freeze-dried ampoules of preserved biotechnological important fungi

The implementation of consistent fungal preservation techniques and appropriate quality assurance are key issues for an effective and efficient preservation. The cost and convenience of each method are important aspects to be taken into consideration such as the knowledge of all parameters capable of affecting the procedures [1]. Preservation methods currently used are highly empirical and in many instances, do not provide reliable genetic and phenotypic stability. Freeze-drying is commonly used to preserve fungal strains at room temperature, however, genetic and phenotypic alterations after long term-storage are yet unknown. Therefore, the main goal of the present experimental study is to evaluate the freeze-drying preservation method for the effective long-term preservation of strains belonging to Aspergillus section Nigri. Twenty-one strains representative of Aspergillus section Nigri were selected and preserved by freeze-drying. The strains were subjected to accelerated storage during 4 weeks at 37 ºC. These samples were morphological, physiological and genotypical analysed. In order to detect macro and micro-morphological changes, growth for seven days at 25°C on Potato Dextrose Agar, Malt Extract Agar, Czapek Yeast Extract Agar and Czapek Dox Agar was performed. The physiological changes were monitored for the detection of ochratoxin A and fumonisin B2 as described elsewhere [2,3]. In order to identify genotypic changes, DNA fingerprinting techniques using the oligonucleotides M13 and (GACA)4 were performed. All assays were evaluated at 3 points in time: before preservation (I), 2 (II) and 4 (III) weeks after preservation. For all the methodologies used to evaluate freeze-drying of fungi along time the major results are: 1) no significant changes were observed in the macro and micro-morphological analysis; 2) all strains maintained their mycotoxins production pattern, before and after ageing; 3) after ageing different DNA fingerprinting was observed. In conclusion, freeze-drying can be considered a technique of excellence to be used on the maintenance of biodiversity within the filamentous fungi, and more accurately for Aspergillus section Nigri. However, it is recommended to consider possible genetic changes after long shelf-life periods.info:eu-repo/semantics/publishedVersio

Micoteca da Universidade do Minho (MUM): a portuguese culture collection

[Excerpt] Micoteca da Universidade do Minho - MUM is a mycological culture collection that exists since 1996 and is hosted by the Biological Engineering Research Centre (www.deb.uminho.pt), a centre of excellence integrated in the Institute for Biotechnology and Bioengineering (IBB - www.ibb.pt). The mission of MUM is to provide the highest quality services to its customers, collecting, maintaining and supplying fungal strains and their associated information for teaching and research in biotechnology and life sciences, and to be a centre of knowledge, information and training in mycology, operating at a global level and under national and international regulations [1,2]. (...

Micoteca da Universidade do Minho (MUM): 15 anos de funcionamento e sua recente certificação baseada na ISO 9001:2008

A Micoteca da Universidade do Minho (MUM, www.deb.uminho.pt) é uma coleção de fungos filamentos isolados do ambiente, indústria alimentar e clínicos. Foi criada em 1996 e está sediada no Centro de Engenharia Biológica, Bmga. Esta tem a missão de fornecer serviços de elevada qualidade aos nossos clientes, coletar, manter e distribuir estirpes tiíngicas, e a informação a elas associada, para o ensino, investigação em biotecnologia e ciências da vida. A MUM é um centro de conhecimento, informação e formação em micologia funcionando à escala global de acordo com as leis e regulamentações nacionais e internacionais. Toda a atividade da MUM tem-se baseado no estado-da-arte do conhecimento científico micológico e no pioneirismo das suas orientações estmtégicas e de gestão dos recursos humanos e financeiros. Assim, desde a sua fundação, a MUM começou a participar em organizações internacionais como a ECCO, a WFCC e, mais tarde, participou ativamente na iniciativa da OCDE para a implementação de critérios de gestão de qualidade para os futuros CRBs e sua rede global. Nestes 15 anos, a MUM cumpriu mandatos nas direções da ECCO e WFCC. Esta experiência internacional permitiu estar atualmente envolvida como parceiro, entre 20 países de todos os continentes, no projeto demonstmtivo da Rede Global de Centros de Recursos Biológicos e no projeto europeu Consórcio de Centros de Recursos Microbiológicos. A introdução de parâmetros mais rigorosos de qualidade na identificação e autenticação do material biológico preservado pela MUM tem levado a um reconhecimento científico desta coleção e ao aumentando a sua competitividade ao nível mundial. Dentro de um acervo de milhares de fungos, a MUM possui cerca de 800 estirpes em catálogo cobrindo 184 espécies pertencentes a 68 géneros. A atividade da MUM reflete..., em publicações internacionais, no apoio à pós-gmduação com produção de dissertações de mestmdo e de teses de doutommento, em contmtos com a indústria e serviços, em projetos nacionais e europeus. Nas parcerias internacionais para além dos seus parceiros europeus, a MUM colabom com a Malásia, Paquistão, Chile, EUA e o Bmsil. O Bmsil é o seu parceiro mais relevante pelos projetos comuns e na formação e mobilidade de estudantes e investigadores. Com a recente certificação do sistema de gestão de qualidade da MUM na ISO 900 I :2008 esta tomou-se entre as 590 coleções de cultum de 68 países registadas na WFCC a I" coleção de cultums em Portugal e a 23" coleção de culturas a nível mundial a obter tão relevante reconhecimento. Este íàcto tomou a MUM num parceiro central no atual recente projeto da União Europeia sobre a Infraestrutura de Investigação de Recursos Microbianos (MIRRI) e que fui incluído no Road Map do European Stmtegy Forum on Research Infrastructures - ESFRI. A MUM continua como uma das coleções que seguem o modelo da excelência ao nível global e estã reforçada para estes novos e exigentes desafios que se avizinham à escala global

Implementation of a Quality Management System based on ISO9001:2008 and its importance on culture collections

Culture collections (CC), in order to contribute to a better and sounder preservation of microbial biodiversity, constantly evaluate and analyse methodologies and are faced with the increasingly need to implement and maintain quality control [2]. Micoteca da Universidade do Minho (MUM) is a filamentous fungi culture collection established in May 1996, hosted by the Biological Engineering Centre, which is a centre of excellence integrated in the Institute for Biotechnology and Bioengineering (IBB). MUM has found with the implementation of a Quality Management System (QMS), based on the standard ISO 9001:2008, a way to contribute to this continuous search of a better Quality [1]. The implementation of the QMS occurred for almost 1 year during which 3 main steps were developed. The first step was the planning of the QMS, followed by the design of the system and documental development and the third step was the implementation of the QMS with the achievement of the certification in May 2011, from the independent assessment agency (Portuguese Association for Certification - APCER with the International Certification Network - IQNet). From this date on MUM has kept on performing QMS revision and pursuing a continuous improvement. From the analysis of the data, existing since the QMS implementation, it was possible to conclude that, when comparing the first year with the second year of the System, there was an increase on the demand of services supplied, noted through the increase on the number of customers that request the service of supply and number of strains requested. MUM, is a specialised CC that keeps on gaining recognition from other Session 15: Biological resource centers: quality management systems entities and acknowledgement of its services proving that the implementation of a QMS is an asset that should set an example to other CCs

Micoteca da Universidade do Minho (MUM): implementation of a quality management system based on ISO 9001:2008

[Excerpt] Micoteca da Universidade do Minho (MUM) is a filamentous fungi culture collection established in May 1996, hosted by the Biological Engineering Centre, which is a centre of excellence integrated in the Institute for Biotechnology and Bioengineering (IBB).The mission of MUM is to provide the highest quality services to our customers, collecting, maintaining and supplying fungal strains and their associated information for teaching and research in biotechnology and life sciences. MUM intends to be a centre of knowledge, information and training in mycology, operating at a global level and under national and international regulations. In order to keep its high standards MUM has implemented a Quality Management System (QMS) based on the normative reference ISO 9001:2008.MUM has defined three processes, those being: Material Reception Process, Material Preservation Process and Material Supply Process; and has also developed a documental base for all the QMS including proceedings, standard operating procedures, forms, as well as quality objectives and goals for a continuous improvement