Novel Strategy for the Demulsification of Isolated Sesame Oil Bodies by Endogenous Proteases
Yeming Chen1,2,*, Huina Li2, Caimeng Zhang1,2, Xiangzhen Kong1,2, Yufei Hua1,2
aState Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China
bSchool of Food Science and Technology, Jiangnan University, Wuxi 214122, China
*Correspondence:
Yeming Chen
State Key Laboratory of Food Science and Technology
Jiangnan University
1800 Lihu Avenue, Wuxi, 214122
Jiangsu Province, China
Phone: 86-0510-85329091
Fax: 86-0510-85329091
Email: chenyeming@jiangnan.edu.cn
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.
Keywords Oil bodies · Oleosins · Aspartic endopeptidases · Subtilisin-like proteases · Metalloendopeptidase · Serine carboxypeptidases