*2.3. Degree of Acetylation and In Vitro Digestibility*

As shown in Table 3, along with an increase in acetic anhydride concentration from 0.0 to 2.0 mL/g, there was a higher degree of N-acetylation in analysed preparations. This phenomenon was due to the number of ε-amino groups involved in the acetylation reaction being reduced severely with the increase in the anhydride level, leading to an increase in the degree of N-acetylation. Moreover, this increase might be correlated with process conditions and the source of protein subjected to chemical modification [32]. Additionally, Achouri and Zhang [33] stated that the extent of amino group acetylation in a polypeptide mixture depends markedly on the amount of acetic anhydride used. However, 78.67% of the ε-amino groups was acetylated with 2.0 mL/g. This means that a complete blockage of the amino acid residues is possible only in the presence of a high dose of acetic anhydride.

**Table 3.** Degree of N-acetylation and in vitro digestibility of native and acetylated PPC.


Values are means <sup>±</sup> standard deviation; a,b,c—the same letters within the same row were not significantly different; PPC—pumpkin protein concentrate; 0.4, 1.0, 2.0—pumpkin protein preparations after acetylation conducted with different concentrations of acetic anhydride.

After digestion of the analysed preparations, protein nitrogen extraction ranged from 21.4% (for samples acetylated with dose 0.4 mL/g) only up to 25.31% (for samples

acetylated with dose 1.0 mL/g) (Table 3). The amount of protein nitrogen determined in the supernatant obtained after the two-stage digestion contained nitrogen compounds and not those extracted from the sample proteins. Thus, the determination should reflect the presence of short peptides, possible to the absorption by the intestine enterocytes. The digestion step did not include digestion by the enzymes excreted by enterocytes. The cleavage specificity of porcine pepsin includes peptides with an aromatic amino acid, preferentially at carboxylic groups of the amino acid, especially if the other residue is also an aromatic or a dicarboxylic amino acid and does not destroy bonds containing valine, alanine or glycine linkages [34], while trypsin cleaves the peptide chain on the C-terminal side of lysine and arginine amino acid residues. Similar findings were reported by Shukla [35]. However, the applied method is definitely more precise than the method proposed by Salgó et al. [36] because that determines only peptides, not protein, extracted from the studied material. Thus, the results obtained should not be compared with those obtained for pH changes after placement of the sample in the intestine solutions. We showed that the use of 0.4 mL/g acetic anhydride significantly reduces the digestibility of modified PPC in comparison to native PPC, while acetylation with 1.0 mL/g acetic anhydride significantly improves the digestibility of PPC. Therefore, the effect of acetylation on the digestibility of PPC preparations cannot be clearly determined. However, according to Bergner et al. [37], acetylation of proteins does not reduce either apparent or true N digestibility. Additionally, this modification of food proteins does not influence the nutritive value.
