Novel strategy for the demulsification of isolated sesame oil bodies by
endogenous proteases
Abstract
Oleosins are mandatory to avoid coalescence of oil bodies (OBs), so
commercial proteases are used to efficiently demulsify OBs into food
oil. However, the commercial proteases and pH regulators (acid and
alkali) greatly restrict this method in industry. In this study,
aspartic endopeptidases, subtilisin-like proteases,
metalloendopeptidase, and serine carboxypeptidases were identified in
isolated sesame OBs by liquid chromatography tandem mass spectrometry
(LC–MS/MS). Tricine–sodium dodecyl sulfate–polyacrylamide gel
electrophoresis and protease inhibitor assay revealed that aspartic
endopeptidases exerted high activity against oleosins in a pH range of
3−6 and a temperature range of 40−70 °C, while subtilisin-like proteases
exhibited sharp optimum at pH 5. Metalloendopeptidase contributed to the
low activity against oleosins at pH 7−9. Trichloroacetic acid–nitrogen
soluble index and free amino acid analyses quantitatively revealed that
the activity of serine carboxypeptidases was high at pH 3−5, and optimal
at pH 4; the combined activity of aspartic endopeptidases and
subtilisin-like proteases was optimal at pH 5. By incubating the
isolated sesame OBs at pH 5 and 60 °C for 2 h, approximately 97% of
total lipids were recovered as free oil. At last, LC−MS/MS analysis gave
deep insight into the intrinsic proteins of sesame OBs: three kinds of
oleosins with molecular weights around 17 kDa, and four kinds around 15
kDa; besides 27 kDa caleosin, four kinds of oil body-associated proteins
and one kind of peroxygenase-like protein also around 27 kDa; in
addition to 39 kDa steroleosin, 11-beta-hydroxysteroid
dehydrogenase-like 6 also around 39 kDa.