Introduction
Aqueous extraction processing of oilseeds is environment-friendly, and beneficial for simultaneous production of proteins and lipids (Nikiforidis, 2019; Kumar et al., 2021). Generally, aqueous extraction processing contains the following steps: 1) pretreatment (e.g., soaking, grinding, and flaking), 2) aqueous extraction (neutral and alkaline pH), 3) separating by centrifugation, and 4) subsequent processing of proteins and lipids. During aqueous extraction, lipids (mainly oil bodies, OBs) and proteins (e.g., storage proteins and various enzymes) are released into aqueous phase and interact with each other (Zhao, Chen, Chen, Kong, & Hua, 2016; Zhao, Kong, Zhang, Hua, & Chen, 2017; Chen et al., 2018). As a result, the OB-protein aggregates are formed, and floated by centrifugation as OBs cream. The proteins in the OB-removed solution are not denatured and can be used for value-added processing (such as vegetable protein beverages) (Samoto, Kanamori, & Shibata, 2011). The OBs cream, which is a kind of natural O/W emulsion rich in phospholipids, phytosterols (Gallier, Gordon, & Singh, 2012), and vitamin E (Wu et al., 2012), have been directly processed for the practical applications in foods and personal care products (Samoto et al., 2011; Nikiforidis, Matsakidou, & Kiosseoglou, 2014; Nikiforidis, 2019). Due to the oil consumption habits (e.g., stir-frying and frying), the OBs cream are also demulsified into food oil (Campbell et al., 2011; Li et al., 2016).
OBs have a neutral lipid matrix core, which is coated by a monolayer of phospholipids embedded with OB intrinsic proteins (Lin, Liao, Yang, & Tzen, 2005). Oleosins are dominant OB intrinsic proteins, whereas caleosins and steroleosins are minor ones (Lin et al., 2005). It has been clarified that phospholipids are needed to stabilize OBs, and oleosins are mandatory to avoid coalescence of OBs (Deleu et al., 2010). Many researches have proved that the oleosin-phospholipid membrane can protect OBs from several environmental stresses, such as high centrifugation speed, alkaline pH, urea, and heat (White et al., 2008; Chen, McClements, Gray, & Decker, 2012; Chen et al., 2018). Due to the high physical and chemical stability of OBs, the enzymatic method is the most efficient for demulsification of OBs in all reported methods (Campbell et al., 2011; Li et al., 2016), which uses commercial proteases to degrade the intrinsic and extrinsic proteins in OBs cream. However, the needs for commercial proteases, acid, and alkali greatly restrict its practical utilizations in industry.
Until now, several studies have confirmed that plant seed endogenous endopeptidases are contained in the isolated OBs, and they can hydrolyze the intrinsic and extrinsic proteins of OBs (Vandana & Bhatla, 2006; Zhao et al., 2016; Zhao et al., 2017; Chen et al., 2018). However, few studies have checked whether these endogenous endopeptidases can efficiently demulsify the OBs. In addition, many researches have proved that plant seeds not only contain endopeptidases but also exopeptidases (Müntz, Belozersky, Dunaevsky, Schlereth, & Tiedemann, 2001; Tan-Wilson & Wilson, 2012), but the information concerning exopeptidases in isolated OBs are limited.
In our lab, the endopeptidases and exopeptidases in isolated OBs from various oilseeds (e.g., soybean, peanut, sunflower, castor bean, rapeseed, sesame, and walnut kernel) were systematically examined, and it was found that the isolated sesame OBs contained endopeptidases and exopeptidases with high proteolytic activity. Therefore, these proteases might be directly used to demulsify the isolated sesame OBs. In this study, liquid chromatography tandem mass spectrometry (LC–MS/MS) was used to systematically identify the intrinsic proteins of sesame OBs and the proteases in isolated sesame OBs. Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Tricine–SDS–PAGE) analysis was used to explain the optimal pH and temperature of endopeptidases, and protease inhibitor assay was used to confirm the catalytic centers and pH sensitivity of these endopeptidases. Trichloroacetic acid–nitrogen soluble index (TCA–NSI) was used to quantitatively estimate the combined activity of endopeptidases and exopeptidases, and free amino acid analysis was used to quantitatively explain the activity of exopeptidases. At last, the effects of these endopeptidases and exopeptidases on the demulsification of isolated sesame OBs were examined. In all, the aims of this study were to supply a new strategy for the efficient demulsification of isolated OBs by using sesame endogenous proteases.