Squalene from Fish Livers Extracted by Ultrasound-Assisted Direct In Situ Saponification: Purification and Molecular Characteristics

Abstract

An ultrasound-assisted direct in situ saponification (U-DS) process was developed to extract unsaponifiable matter (rich in squalene) from the livers of four fish species, sea bass, skipjack tuna, gray bamboo shark, and spot-tail shark. The conventional solvent extraction method was used for comparison. Box–Behnken experimental design (BBD) was adopted to optimize the independent variables including the biomass/methanol ratio (1:3 to 1:9 w/v), 50% KOH volume (1–12 mL), and sonication time (0–30 min) over the unsaponifiable yield. With the conventional process, the yield of squalene was 0.10 ± 0.02 to 5.52 ± 0.06 g (100 g)−1, whereas the U-DS process rendered the yield of 0.13 ± 0.03 to 6.86 ± 0.05 g (100 g)−1, depending on the fish species. After extraction, squalene was further concentrated (purity ≥60%) from all fish species via fractional crystallization (yield of squalene concentrate ranged from 48.35% to 74.49%) and purified using a silica gel column with a maximum recovery up to 98%. For all the fractions, components were examined using thin layer chromatography (TLC). Squalene was qualitatively and quantitatively analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC). Entire extraction processes yielded squalene with a purity of ≥94%. Fourier transform infrared (FTIR) analysis confirmed the native structure of squalene with six nonconjugated bonds, suggesting no degradation of squalene taken place during the U-DS process. Thus, the U-DS process along with fractionation and purification could be used for recovering the squalene from fish livers.