Identification and characterization of extracellular enzymes secreted by Aspergillus spp. involved in lipolysis and lipid-antioxidation during katsuobushi fermentation and ripening.

A mild-flavored soup stock made from katsuobushi is an important element of traditional Japanese cuisine and is the basic seasoning responsible for the taste. Fermented and ripened katsuobushi, known as karebushi, is manufactured by simmering skipjack tuna that is then smoke-dried, fermented, and ripened in a repeated molding process by five dominant Aspergillus species. Here, our aim was to characterize and identify the lipolytic enzymes secreted by the dominant Aspergillus species, especially A. chevalieri and A. pseudoglaucus, which are involved in hydrolyzing lipids during the molding process. The crude enzyme preparations from the five Aspergillus spp. cultivated on katsuobushi solid medium hydrolyzed triglycerides in fish oil, and more saturated and unsaturated fatty acids (C16:0, C16:1, C18:0, C18:1) were produced than major polyunsaturated fatty acids (C20:5, C22:6). On the basis of ion exchange chromatograms, the composition of the lipolytic enzymes was different in the five species. There was at least one active fraction with high hydrolytic activity toward fish oil in four of the Aspergillus spp., but not A. sydowii; the lipolytic enzyme secreted by A. sydowii had quite high activity toward the artificial substrate p-nitrophenyl butyrate, but low activity toward the natural oil. The lipolytic fractions from A. chevalieri and A. pseudoglaucus were further purified by hydrophobic interaction chromatography then gel-filtration chromatography; LC-MS-MS Mascot analysis identified a variety of lipolytic enzymes, including cutinase, esterase, phospholipase, and carboxyl esterase in the lipolytic fractions from these species. The identified enzymes had 30%-70% identity to previously reported or manually annotated lipases or esterases from taxa other than Aspergillus. The different lipolytic enzymes likely acted on triglycerides in the katsuobushi fish oil. Furthermore, catalase B and Cu/Zn superoxide dismutase, which limit oxidative damage of lipids, were also identified. These antioxidant enzymes may prevent lipid oxidation and rancidity as the lipolytic enzymes hydrolyze lipids during the long fermentation and ripening process. Umami and richness tastes tended to increase in extracts from culture of protease- and peptidase-producing A. sydowii. Our results will aid in the selection and application of desirable strains of Aspergillus species as starter cultures to improve the storage and quality of fermented and ripened karebushi.