Demulsification of pH 7-OB into Free Oil by Endogenous Proteases
The effects of temperature and time on demulsification of pH 7-OB were examined at pH 5. pH 5 was selected due to two reasons. One was that endopeptidase activity was the highest, and the other was that less acid was needed compared to pH 4. By incubation at pH 5 and 30–80 °C for 2 h (Fig. 3a), the free oil yield peaked at 60 °C with a value of 97.29%, followed by 50 (85.99%) and 40 °C (80.58%). It was low at 30, 70, and 80 °C. The low free oil yield at 30 °C could be easily explained by the low endopeptidase activity, whereas that at 80 °C could be attributed to the thermal denaturation of endopeptidases (Fig. 1c). However, the low free oil yield at 70 °C could not be well explained at present stage. It might be due to the formation of hydrolyzed products, which had good emulsifying activity. By incubation at pH 5 and 60 °C for 1–4 h (Fig. 3b), it was found that the free oil yield sharply increased to 87.81% after 1 h, and almost achieved to the maximum value after 2 h. By incubating pH 7-OB at pH 5 and 60 °C, the oleosins were almost totally hydrolyzed by proteases, but considerable amounts of 11S and 7S globulins and 2S albumins were still remained (Fig. 1). These results clearly revealed that the efficient degradation of oleosins was the key point for the efficient demulsification of OBs, which agreed with the conclusion that oleosins were mandatory to avoid coalescence of OBs (Deleu et al., 2010).
As stated above, approximately 92% of total lipids in sesame seeds could be recovered into water extract, and approximately 87% of total lipids in water extract was distributed into floating fraction. Together with the maximum free oil yield, it was calculated that approximately 78% of total lipids in sesame seeds could be obtained as free oil by using this method. It was reported that approximately 60–70% of total lipids in sesame seeds were recovered by cold-pressing (Sarkis, Boussetta, Tessaro, Marczak, & Vorobiev, 2015; Ribeiro et al., 2016), while roasting (120–180, 20–30 min) and subsequent screw-pressing could increase it to 65–76% (Bordon et al., 2021). Immersion in water, drying, grinding, and pressing could recover 80% of total lipids (Sarkis et al., 2015), while supercritical CO2 and hexane could extract 85% (Döker, Salgin, Yildiz, Aydogmus, & Çalimli, 2010). In addition to these physical methods, enzymatic methods were also used to extract free oil from water extract of sesame seeds. Just 57.4% of total lipids were recovered by using Alcalase 2.4L (2% of sesame seeds) (Latif & Anwar, 2011), and increased to approximately 61% by using combined pectinex Ultra SPL and Alcalase 2.4L (Ribeiro et al., 2016). The highest value of 89.7% was obtained by using Pectinex (1% of water extract) and Neutrase (1% of water extract) (Tirgarian, Farmani, & Milani, 2019). Compared to these reported methods, it was found that the method in this study was better than pressing methods, but less efficient than supercritical CO2 and hexane extraction. The reported enzymatic methods needed high amount of enzymes to achieve the high oil recovery. However, commercial enzymes were not needed in our method, and the acid amount used for pH adjustment was greatly decreased due to the low content of proteins in isolated OBs. In future, further studies are needed to improve the extraction of OBs into water extract, and enhance the floating of OBs.