Potencial de uso biotecnológico de lipase de Colletotrichum gloeosporioides e antioxidantes de Morus alba

Abstract

This work aimed to study biotechnological potential of the fungal species Colletotrichum gloeosporioides and the plant Morus alba. Production, partial purification, characterization and application tests for Colletotrichum gloeosporioides lipase as well as evaluation of the difference in antioxidant capacity in 12 varieties of Morus alba roots were screened. Maximum activity (0.742 U.mL-1) of the preparation containing lipase was obtained after fermentation of C. gloeosporioides in liquid substrate for 48 h. The lipolytic protein presented molecular mass of approximately 18 kDa, was stable at pH 5-8, holding 40% of its activity at pH 9 and about 30 and 25% at pH 10 and 11, with pH 10 being optimum pH. The enzyme was stable for five hours on optimum temperature (37 °C), stable in salts solutions (NH4+, Mg+2, Ca+2, Mn+2, Sn+2) and solvents (methanol, ethanol, isopropyl alcohol, butyl alcohol, acetonitrile, heptane e hexane) in different concentrations. The lipase presented esterification activity (70 % yield in the formation of butyl butyrate) and transesterification (yielding 5.4.10-3 U.kg-1.min-1 with the reaction between isopropyl alcohol and p-nitrophenylpalmitate). The enzyme generated free fatty acids and glycerides concentrates from all raw materials tested, with greater degrees of hydrolysis on olive oil (18.0 U), soybeans (17.8 U) and canola (15.9 U). A sequence of ultrafiltration with 10 kDa, gel filtration, and affinity chromatography with benzamidine allowed obtaining fractions partially purified. Fraction "1", obtained from affinity chromatography, reached the best combination of activity recovery (%)/purification fator (27.15/20.51) and optimum temperature around 40 °C. The physico-chemical and catalytic characteristics demonstrated applicability of this enzyme in the food industry, in the production of essential fatty acids and aromatic esters employed as flavor, as well as in detergent industry. To evaluate the biotechnological potential of M. alba, aqueous, ethanol and hexane extracts of 12 natural and hybrid varieties of M. alba roots were used. Differences between the varieties were observed, such as the phenolic content of the hexane (0.55-1.59), aqueous (2.72-6.61) and ethanol extracts (5.09 9.84 g of Gallic acid/100 g of extract). The antioxidant action of M. alba was proven by different tests, finding, for instance, 47 % of iron reduction compared to ascorbic acid. The ability of free radicals sequestration was also observed in extracts of M. alba. Ethanol was very efficient in extracting phenolic compounds and flavonoids, then ethanol extracts demonstrated the greatest antioxidant activities. The variety IZ 64 showed high phenolic content and flavonoids, as well as better ability to capture free radicals and iron reduction, showing to be the best variety for economic exploitation and use as antioxidants for food preservation or as a nutraceutical. This work has shown the potential use of lipase from C. gloeosporioides on hydrolysis of different oils for the production of fatty acids as well as in the production of butyl butyrate, flavouring the food industry with flavor of pineapple. In addition, it was observed the antioxidant potential of the roots of M. alba with IZ 64 demonstrating great potential for economic exploitation.