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).