2.5 In vitro protein digestibility (IVPD)
The IVPD of control and fermented meals was determined using the pH-drop method according to Tinus et al. (2012). A three-enzyme assay containing 31 mg chymotrypsin (bovine pancreas ≥ 40 units/mg protein), 16 mg trypsin (porcine pancreas 13,000–20,000 BAEE units/mg protein), and 13 mg protease (Streptomyces griseus ≥ 15 units/mg solid) in 10 mL of water was prepared (fresh daily), kept in a 37℃ water bath, and adjusted to pH 8.0 ± 0.05 with 0.1 M NaOH and HCl. The sample suspensions were prepared by dispersing 62.5 ± 0.5 mg protein in 10 mL water. The samples were stirred at 37℃ for 1 h before pH adjustment to pH 8.0 ± 0.05 with 0.1 M NaOH and HCl. The protein digestibility measurements were done by adding 1 mL of the enzyme cocktail into the sample suspensions and recording changes in pH every 30 s for a total of 10 min. The equation below was followed to calculate the IVPD of each sample:
\(\text{IVPD\ }\left(\%\right)=\ 65.66+18.10\ \times\ \text{pH}_{10\ min}\)(3)
Where \(\text{pH}_{10\ min}\) is the changes in pH since the addition of the enzyme solution to the end of the 10 min measuring period.
2.6 Statistical analysis
The fermentation was made in triplicate using separate plates and spore suspensions (n=3). A three-way analysis of variance (ANOVA) was used to study the statistical differences between samples as a function of meal type, fungal strains, and fermentation time with a significance level ofp =0.05. A post-hoc Tukey’s test was used to detect statistical differences in fermentation time. All statistics were performed using the SPSS Version 28.0 software (IBM Corp. NY, IL, USA).