Produção, recuperação e avaliação de pectinases de Aspergillus niger LB-02-SF obtidas em biorreator de tambor rotativo

Abstract

In the present work, the production of pectinases by Aspergillus niger LB-02-SF in solid-state cultivation was studied using a rotating drum bench bioreactor. Furthermore, extraction and concentration of enzymes by ultrafiltration were evaluated. Formulations with the concentrated extract, KCl and glycerol were assayed with respect to the maintenance of activity level and the efficiency in the treatment of fruit juices. With respect to the production of enzymes in bioreactor, the operational parameters evaluated were agitation, cultivation temperature, occupation volume (30, 50 and 70%) and specific air flow (0.18, 0.36, 0.54, 0.72 e 0.90 LkgM – liter of air per kilogram of medium per minute). Tests without and with agitation of the bioreactor (1 rpm per 5 minutes each 120 minutes) indicated that, with occupation volume of 30%, agitation leads to increasing fungal growth and decreasing enzyme production, whereas with 50% of occupation an opposite behavior was noticed. With 70% of occupation volume, no significant difference was observed among the pectinolytic activities achieved with and without agitation, the production being hindered in both cases by the insufficient aeration of medium. The interval between agitation events was evaluated for an occupation volume of 70% and no significant difference in enzyme production was observed for the intervals of 90 and 120 minutes (standard condition). For the interval of 60 minutes, the activity was reduced in approximately 58%. The cultivations with temperature control at 34oC and without control indicated that enzyme production was favored with the natural increase of medium temperature, an aspect that could be related to a stress condition that would lead to a higher enzyme production. Specific air flows between 0.18 and 0.90 LkgM were evaluated for 30 and 50% of bioreactor occupation volume. Larger fungal growth was observed when the bioreactor was loaded with the smallest occupation volume. With the intermediate flow of 0.54 LkgM, the highest enzyme activities were achieved for both occupation volumes; however, the occupation volume of 50% resulted the higher activity of 178.2 U/g, with an activity of 133.4 U/g being measured for 30% of occupation. The results achivied indicated that for a higher enzyme production, the bioreactor should be conducted with 50% of occupation volume, specific airflow of 0.54 LkgM and interval between the agitations of 120 minutes. In the study on the extraction of pectinases from the solid medium with water pH 4, under agitation, it was observed that extraction times from 15 to 120 minutes presented similar results. The extraction temperatures of 20, 30 and 40oC did not influence protein extraction, but pectinase activity was reduced in 40% at 40oC, possibly due to the low enzyme thermostability. Among the extraction ratios of solid/liquid assessed, the value 1/10 resulted in the highest enzyme activity solubilized, with recovery of 81% of the pectinolytic activity that was present in the medium. In the concentration tests, ultrafiltration of concentrated extract with no previous treatment resulted in 59% of enzyme activity recovery and in a final permeate flux of about 11 L/m²/h. On the other hand, the operation protocol that included the use of activated charcoal, microfiltration and ultrafiltration presented enzyme recovery of 74% and final permeate flux of 24 L/m2/h, results that demonstrate the importance of pre-treatments in this process. Previously to the preparation of enzyme formulations, a volume of 30 L of enzyme extract was 103-fold concentrated by ultrafiltration, with 69% of enzyme recovery, and a final 663-U/mL extract was obtained. This concentrated extract was formulated with 20, 30, 40 and 50% (w/w) of glycerol and 2% (w/w) of KCl and evaluated for 59 weeks with respect to the enzyme activity conservation at 5ºC. At the end of the test period, the blank sample (with no additives) presented an activity decrease of 25% and also microbial contamination, whereas the formulations preserved 100% of the initial activity along the experiment. The 50%-glycerol formulation was used for Bordô grape maceration and for depectinization of apple, Rosé grape and blackberry juices, showing statistically similar results to those obtained with the commercial preparations Novozym 33095 and Pectinex Ultra SP-L used as reference. The results attained in this work pointed out to the possibility of scaling-up this process, from the enzyme production in solid-state cultivation to the achievement of an enzyme formulation that has shown potential to be used in the industry of fruit juice.