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.

Acrolein as a Major Volatile in the Early Stages of Fish Oil TAG Oxidation.

Fish oil rich in docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) is known to have an unpleasant smell, even at low oxidation levels. Therefore, it is highly important to know the major volatiles formed during the early stages of fish oil oxidation. Comparative study with solid-phase microextraction (SPME) and static headspace (SHS) methods showed that 2-propenal (acrolein) was formed as the major volatile from the beginning of fish oil triacylglycerol (TAG) oxidation. The effectiveness of SPME extraction on each volatile was different from each fiber. Among the three different SPME fibers used in the present study, carboxen/polydimethylsiloxane (CAR/PDMS) was determined to be a better fiber for measuring the volatiles, including acrolein. The present study also showed that the non-selective SHS method is useful for determining the characteristic volatile formation in the early stages of fish oil TAG oxidation.

Effective Prevention of Oxidative Deterioration of Fish Oil: Focus on Flavor Deterioration.

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), both abundant in fish oil, are known to have significant biochemical and physiological effects primarily linked to the improvement of human health, especially cardiovascular and brain health. However, the incorporation of fish oil into foods and beverages is often challenging, as fish oil is very easily oxidized and can cause undesirable flavors. This review discusses this rapid formation of the fishy and metallic off-flavors, focusing especially on an early stage of fish oil oxidation. Although oxidative stability and quality of commercialized fish oil have improved over the past few years, there is a still a problem with its application: Flavor deterioration can be found even at very low oxidation levels. This review also notes the effective way to inhibit the formation of the volatile compounds responsible for the flavor deterioration.

The dietary effect of milk sphingomyelin on the lipid metabolism of obese/diabetic KK-A(y) mice and wild-type C57BL/6J mice.

Purified milk sphingomyelin (SM) was obtained from lipid concentrated butter serum (LC-BS) by successive separations involving solvent fractionation, selective saponification, and silicic acid column chromatography. The SM obtained was given to obese/diabetic KK-A(y) mice and wild-type C57BL/6J mice. SM supplementation significantly increased fecal lipids paralleled with a decrease in non-HDL cholesterol levels in the serum and neutral lipids and in cholesterol levels in the livers of KK-A(y) mice. The reduction of liver lipid levels also resulted in a decrease in the total fatty acid content of the KK-A(y) mice livers, while n-3 fatty acids derived from the conversion of α-linolenic acid (18:3n-3) increased due to SM supplementation. In contrast to the KK-A(y) mice, little change in the serum and liver lipids was observed in wild-type C57BL/6J mice. The present study suggests that SM may be effective only in subjects with metabolic disorders.

Inhibitory Effect of Dihydrosphingosine with α-Tocopherol on Volatile Formation during the Autoxidation of Polyunsaturated Triacylglycerols.

The effect of dihydrosphingosine (d18:0) on triacylglycerol (TAG) oxidation was examined with and without α-tocopherol. Three types of TAG from fish, linseed, and soybean oil were oxidized at 50°C to determine the effect of dihydrosphingosine (d18:0) with or without α-tocopherol on triacylglycerol (TAG) oxidation. The analysis of oxygen consumption and total volatile formation demonstrated a small effect of d18:0 on TAG oxidation in the absence of α-tocopherol. On the other hand, the combination of d18:0 with α-tocopherol showed strong antioxidant activity and completely inhibited volatile formation within 1400 h for soybean oil TAG, 650 h for linseed oil TAG, and 380 h for fish oil TAG.