Papel das enzimas oxidativas na deterioração fisiológica de mandioca

• Autor(a) principal: Ramos, Paula Acácia Silva
• Data de Publicação: 2011
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/1154
• topic: Mandioca; Deterioração fisiológica; Escurecimento fisiológico; Cassava; Physiological deterioration; Physiological browning; CNPQ::CIENCIAS AGRARIAS::AGRONOMIA::FITOTECNIA

The high perishability of cassava roots (Manihot esculenta Crantz) in nature make them to be consumed within a very short period after harvest. The main cause of loss is due to physiological deterioration, characterized by bluish streaks and dark pigments in the pulp, which progresses through the parenchyma. These changes are attributed to the action of peroxidase EC 1.11.1.7. (POD) and polyphenoloxidase E.C. 1.10.3.1. (PPO). This work was conducted in order to partially purify and characterize these enzymes kinetically, evaluating optimal conditions for their activities, and to determine the effect of different inhibitors on the activity of POD in order to reduce the enzymatic browning. The roots used for purification were cut to induce the activity of POD and PPO. The kinetic activity was determined under conditions of pH ranging from 2.5 to 9.0 at room temperature (25 oC) and ice (4 oC), and different temperatures. The POD and PPO extracted from cassava roots were partially purified by ammonium sulfate fractionation from 0 to 80% at intervals of 20%, followed by dialysis. We tested the following PPO substrates: caffeic acid, chlorogenic acid, p-coumaric acid, catechol, 4-methyl-catechol, DL-dopa, pyrogallol at a concentration of 15 mM. To evaluate the effect of different POD inhibitors, whole roots and free of damage were treated for one hour in solutions containing products such as ascorbic acid, sodium azide, sodium bisulfite, Na2EDTA, L-cysteine and SDS concentrations of 1, 5 and 10 mM and left at room temperature for six days. At every two days it was performed visual analysis and removed samples of 2 cm thick to evaluate the enzyme activity. Optimization of the enzymatic assay for POD and PPO of fresh cassava roots was achieved when the extracts were saturated with 60-80% and the reaction proceeded at pH 6.0 and 6.5, at temperatures of 40 ºC and 30 oC, respectively containing the higher affinity PPO substrate, chlorogenic acid. The complete inactivation ofPOD and PPO occurred at pH 2.5 at room temperature (25 oC) after 60 minutes and 30 minutes respectively. At pH 9.0 there was total inactivation of POD and PPO activities. The POD activity was not inactivated at alkaline pH, being less stable at acid pH, which proved to be effective in reducing enzyme activity, causing rapid damage to the active site. In the POD extracts, pre-incubated at 50 and 60 oC, had a reduction in activity of 30,7% at 70 minutes, and 40.7% at 60 minutes without total inactivation. At higher temperatures (70 and 80 oC) were not observed total inactivation of the enzyme. In extracts pre-incubated to PPO at 50, 60 and 70 ºC for 10 min resulted in partial inactivation of 60, 77 and 81.7%, respectively, without total inactivation in longer pre-incubation (180 minutes). There was total inactivation at 80 oC for 50 minutes. There was a relationship between peroxidase activity and browning, and all treatments were effective in increasing the shelf life of fresh cassava roots up to 4 days. On the second day there was 100% of root browning, in the control. The acidic pH was more effective in reducing the enzymatic activity of POD and PPO, showing that treatment with pre-incubation in acidic pHs are effective in reducing the activity of these enzymes. Treatment with temperatures of 80 oC for a period longer than 50 minutes, also proved to be effective in reducing the enzymatic activity of PPO. It was possible to increase the shelf life of cassava roots by treating with ascorbic acid, sodium bisulfite and L-cysteine. The browning of fresh cassava roots was reduced by applying different concentrations of the above inhibitors at concentrations of 1 mM, 5 mM or 10 mM.