Polyphasic identification of Aspergillus section Nigri preserved under mineral oil at URM culture collection

[Excerpt] The species of Aspergillus section Nigri are isolated from different environments. However, úe main habitat of úese species is the soil. According Samson et al. (2007) there are 19 species of Aspergil/zs section Nigri accepted. The species must be delineated based on a polyphasic approach, including morphology, physiology, profile of secondary metabolites and molecular biology (Samson and Varga,2009). Additionally, according to Santos et al. (2010a,2010b) it is clearer that spectral analyses add value to the polyphasic approach. It generates quahty data which are accurate and useful when some of the methods described above presented limitations. Matrix-Assisted Laser Desorptiorúonisation Time-Of-Flight Intact Cell Mass Spectrometry (MALDI-TOF ICMS) is a spectral technique that analyses the chemical cellular composition of microorganisms providing rapid and discriminatory fingerprints for identification

Production of polygalacturonases by aspergillus section Nigri strains in a fixed bed reactor

Polygalacturonases (PG) are pectinolytic enzymes that have technological, functional and biological applications in food processing, fruit ripening and plant-fungus interactions, respectively. In the present, study a microtitre plate methodology was used for rapid screening of 61 isolates of fungi from Aspergillus section Nigri to assess production of endo- and exo-PG. Studies of scale-up were carried out in a fixed bed reactor operated under different parameters using the best producer strain immobilised in orange peels. Four experiments were conducted under the following conditions: the immobilised cells without aeration; immobilised cells with aeration; immobilised cells with aeration and added pectin; and free cells with aeration. The fermentation was performed for 168 h with removal of sample every 24 h. Aspergillus niger strain URM 5162 showed the highest PG production. The results obtained indicated that the maximum endo- and exo-PG activities (1.18 U·mL-1 and 4.11 U·mL-1, respectively) were obtained when the reactor was operating without aeration. The microtitre plate method is a simple way to screen fungal isolates for PG activity detection. The fixed bed reactor with orange peel support and using A. niger URM 5162 is a promising process for PG production at the industrial level.M.H.C. Maciel thanks to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) and Erasmus Mundus External Cooperation Window Lote 17

Characterization of Tannase from Penicillium montanense URM 6286 under SSF Using Agroindustrial Wastes, and Application in the Clarification of Grape Juice (Vitis vinifera L.)

Tannase is an enzyme that hydrolyzes esters and lateral bonds of tannins, such as tannic acid, releasing glucose and gallic acid and stands out in the clarification of wines and juices. Fungi of the genera Aspergillus and Penicillium are excellent producers of this enzyme. The search for fungi that produce high levels of tannase as well as new substrates for the enzyme production by the SSF is required. The objectives of this study were to evaluate the production of tannase by Aspergillus and Penicillium species through SSF using leaves and agroindustrial waste barbados cherry and mangaba fruit as substrate, select the best producer, optimize production, characterize the crude enzyme extract, and apply it the clarification of grape juice. Selecting the best producer was performed by planning Placket-Burman and RSM. P. montanense showed highest activity with 41.64 U/mL after 72 h of fermentation residue using barbados cherry, with 3.5% tannic acid and 70% moisture. The enzyme showed the highest activity at pH 9.0 and 50 ∘ C. The tannase of P. montanense was stable over a wide pH range and temperature and, when applied to grape juice, showed higher efficiency by reducing 46% of the tannin content after incubation 120 m

Produção e caracterização parcial de pectinases de Aspergillus Niger por fermentação em Estado sólido da palma forrageira e da casca do maracujá

Microrganismos isolados e identificados, quando preservados por processos adequados podem ser armazenados por longos períodos. Existem métodos de preservação que asseguram a viabilidade, morfologia, fisiologia e genética, com o objetivo de manter a cultura por períodos, conservando as características genéticas e propriedades industriais. Aspergillus niger é a espécie mais comumente utilizada para a produção industrial de enzimas pectinolíticas. As enzimas pectinolíticas são um grupo heterogêneo de enzimas que hidrolisam as substâncias pécticas presentes nos vegetais. Este trabalho teve como objetivo avaliar a viabilidade, autenticar taxonomicamente 25 culturas de A. niger estocadas sob óleo mineral na Micoteca URM e produzir pectinases por fermentação em estado sólido (FES) da palma forrageira e da casca do maracujá utilizando a cultura selecionada. Todas as culturas reativadas foram viáveis e autenticadas taxonomicamente, apesar do longo período de estocagem, que variou de 1 a 41 anos. Quanto à capacidade pectinolítica, 23 culturas apresentaram halo de degradação ao redor da colônia, evidenciando a capacidade de degradar pectina. A cultura que apresentou maior halo de degradação foi URM4645 (18 mm), sendo selecionada para as FES, onde foram avaliadas as atividades de endopoligalacturonase (endo-PG), exopoligalacturonase (exo-PG), pectina liase (PL) e pectinesterase (PE). A atividade máxima obtida foi de 66,19 U/g de endo-PG, 3,59 U/g de exo-PG e 40.615,62 U/g de PL, nos tempos de 96, 24 e 72 horas de FES da palma forrageira, respectivamente. A melhor condição para a produção das enzimas foi obtida com 10,0 g do substrato, 107 esporos/g a 28ºC com 96 horas. A atividade ótima de endo-PG e PL foi em pH 5,0 a 50ºC e exo-PG em pH 7,0 a 40ºC. Endo-PG e exo-PG foram estáveis em uma faixa de pH 3,5-11,0 e a 50ºC e a 80ºC, respectivamente, e PL em pH 5,0 e a 50ºC. Utilizando a casca do maracujá a atividade máxima obtida foi de 31,35 U/g de endo-PG, 7,98 U/g de exo-PG, 551.299,39 U/g de PL e 447,93 U/g de PE, nos tempos de 96 horas de FES para a endo-PG e PL e 72 horas para a exo-PG e PE. A melhor condição para a produção das quatro enzimas foi obtida com 5,0 g do substrato, 30% de umidade a 34ºC com 96 horas. Endo-PG apresentou atividade ótima em pH 7,5 a 40ºC, exo-PG e PL em pH 7,0 a 80°C e PE em pH 3,5 a 30°C. Endo-PG foi estável na faixa de pH 7,0-8,0 e a 40ºC, enquanto exo-PG e PL foram estáveis na faixa de pH 6,0-8,0 e 6,0-7,5 e a 60-70ºC, respectivamente. PE foi estável na faixa de pH 3,5-5,0 e a 30-60º

Production, Characterization of Tannase from Penicillium montanense URM 6286 under SSF Using Agroindustrial Wastes, and Application in the Clarification of Grape Juice (Vitis vinifera L.)

Tannase is an enzyme that hydrolyzes esters and lateral bonds of tannins, such as tannic acid, releasing glucose and gallic acid and stands out in the clarification of wines and juices. Fungi of the genera Aspergillus and Penicillium are excellent producers of this enzyme. The search for fungi that produce high levels of tannase as well as new substrates for the enzyme production by the SSF is required. The objectives of this study were to evaluate the production of tannase by Aspergillus and Penicillium species through SSF using leaves and agroindustrial waste barbados cherry and mangaba fruit as substrate, select the best producer, optimize production, characterize the crude enzyme extract, and apply it the clarification of grape juice. Selecting the best producer was performed by planning Placket-Burman and RSM. P. montanense showed highest activity with 41.64 U/mL after 72 h of fermentation residue using barbados cherry, with 3.5% tannic acid and 70% moisture. The enzyme showed the highest activity at pH 9.0 and 50°C. The tannase of P. montanense was stable over a wide pH range and temperature and, when applied to grape juice, showed higher efficiency by reducing 46% of the tannin content after incubation 120 m

Purification of polygalacturonases produced by Aspergillus niger using an aqueous two-phase system

The partitioning and purification of polygalacturonases (PG) produced by Aspergillus niger URM 5162 were investigated in aqueous two-phase systems (ATPS), formed by polyethylene glycol and phosphate salts (PEG/phosphate). To evaluate the effect of the 4 independent variables – molar mass of polyethylene glycol (PEG) (400–8000 g/mol – MPEG), PEG concentration (12.5–17.5%, w/w – CPEG), phosphate concentration (15–25%, w/w, CPHOS) and pH (6.0–8.0) – on the 4 response variables: partition coefficient (K), activity yield (Y), purification factor (PF) and selectivity (S), a factorial design (24) was used. The endo-polygalacturonases (endo-PG) and exo-polygalacturonase (exo-PG) were preferentially partitioned in the top phase. For endo- and exo-PG, the highest values for the response variables K (1.23 and 2.40), Y (74.04% and 17.97%), PF (8.18 and 1.98) and S (24.68 and 48.07), respectively, were obtained for a CPEG of 12.5% (w/w), MPEG of 8000 g/mol, and CPHOS of 25% (w/w) at pH 6.0. These conditions were considered the most suitable for the purification of PG produced by A. niger URM 5162. Furthermore, the most important independent variables for endo- and exo-PG were CPHOS and MPEG, respectively. All independent variables studied and their interactions significantly influenced the response variables. According to these results, the PEG/phosphate system is a useful cost-effective alternative for purification of PG produced by A. niger URM 5162.M.H.C. Maciel thanks to Conselho Nacional de Desenvolvimento Cientlfico e Tecnologico (CNPq, Brazil), Fundacao de Amparo a Ciencia e Tecnologia do Estado de Pernambuco (FACEPE/APQ-0290-2.12/10), Financiadora de Estudos e Projetos (FINEP/ref. 2083/07) and Erasmus Mundus External Cooperation Window Lote 17. The authors thank the Laboratorio de Tecnologia de Bioativos (LABTECBIO), Federal Rural University of Pernambuco (Recife, Brazil). M.H.C. Maciel, C.A. Ottoni, C. Santos and N. Lima also thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the Project "BioEnv - Biotechnology and Bioengineering for a sustainable world", REF. NORTE-07-0124-FEDER-000048." Co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER