In Vitro Digestibility (IVPD)
The vitro digestibility of samples by thermal treatment conditions was determined using the method of Hao et al [22] and Di et al. [21]. The vitro gastric and intestinal digestion were simulated by the pepsin and trypsin, respectively. The digestive base fluid (simulated intestinal fluid (SIF) and simulated gastric fluid (SGF)) was prepared according to Hao et al. before the experiment [22]. The experimental steps were as follows: the sample solution (1g 100mL-1) and an equal amount of SGF solution and pepsin (2000 U mL-1) were mixed, then the pH of mixture solution was adjusted to 2.0 with 1 M HCl. Thereafter, the mixture was incubated in Shaking Water Bath with 150 rpm at 37 ºC. The digestive solution was collected at different time including 0, 1, 5, 10, 30, 60 and 120 min. The gastric digestion was terminated by boiling water bath immediately for 10 min. The digestion solution was centrifugated at 4700 × g for 15 min at 8 °C. Then, the bicinchoninic acid (BCA) method was used to determine the protein content in supernatant [21]. The vitro digestibility rate of walnut meal flour was expressed according to the following equation.
\(\text{IVPD}\left(\%\right)=\frac{PC_{0}-PC_{t}}{PC_{0}}\times 100\)(6)
Where PC0 was the initial protein content; PCt was the protein content after simulated in vitro digestion.
SIF solution and sample solution digested by pepsin was mixed in a ratio of 1:1 (v/v) and the trypsin solution with the activity of 1000 U mL-1 was added at the same time. The pH of mixture solution was adjusted to 7.0 with 1 M NaOH. Then, the mixture was incubated and oscillated in a water bath at 37 ºC for 0, 1, 5, 10, 30, 60 and 120 min. Finally, the intestinal digestion was ended in ice bath for 30 min. After centrifugation, the protein content of supernatant was measured as above described. All data was done in triplicate.