*2.1. Chemical Composition*

The chemical composition of native and acetylated pumpkin protein concentrate (PPC) is presented in Table 1. All analysed samples differed in dry matter, fat and ash content. The dry matter content of PPC preparations ranged from 94.90% to 99.01%. This is in line with the results obtained by Rutkowski and Kozłowska [19], who reported a water content for protein preparations not exceeding 10%. In this study, the application of 1.0 and 2.0 mL/g acetic anhydride had a significant impact on the dry matter content of PPC. The protein content in the analysed commercial pumpkin preparation reached a value of 65.11%. Based on the data found in the literature [20], it can be concluded that these proteins were extracted and purified in a neutral or acidic environment. Moreover, according to Ozuna and León-Galván [5], the composition of protein preparations depends on the chemical composition and characteristics of the proteins contained in the raw material. In most cases, acetylation had no effect on the protein content. No statistically significant differences were found between the control and the most experimental samples. Along with an increase in acetic anhydride concentration from 0.0 to 2.0 mL/g, there was no observed

tendency to a decrease in the total protein share in the analysed preparations, except for the sample modified with 1.0 mL/g acetic anhydride, where a statistically significant (*p* < 0.05) decrease was noted. Under these conditions, acetylation caused a decrease in total protein content in modified samples, from 63.24 to 53.52 g/100 g. These results are in line with those of other authors who acetylated protein preparations, such as El-Adawy [21], Lawal and Adebowale [22], Lawal et al. [23] and Miedzianka et al. [24]. However, our results disagree with those of Khader et al. [25] and Zedan et al. [26] in which the total protein percentage increased after acetylation. The different research results indicate that the effect of acetylation on protein content is not unequivocal.


**Table 1.** Characteristics of native and acetylated PPC.

Values are means <sup>±</sup> standard deviation; *<sup>n</sup>* = 2; a,b,c,d—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.

> Acetylation reduced the fat content in modified PPC. As shown in Table 1, the fat content ranged from an average of 9.17 g/100 g before chemical modification to an average of 8.89 g/100 g in modified samples. However, the acetylated samples were not significantly differed in fat content. Increasing the amount of anhydride did not affect the level of fat.

> The total ash content in native PPC (8.36 g/100 g) was similar to the 9.09 g/100 g found by Zdu´nczyk et al. [27] in pumpkin oil seed cake. The results are in line with statements that plant-derived protein preparations usually have an ash content of less than 10%, because they contain relatively fewer minerals than preparations of animal origin, and their availability is additionally limited by the presence of chelating compounds. Moreover, the total ash content in protein preparations is mainly influenced by the quality of the raw material, which is largely due to the plant variety, the degree of purification and insulation conditions (pressing technology or the type of press used). The ash content in acetylated samples differed significantly (Table 1). A statistically different decreasing trend was found on the ash content for higher anhydride-to-protein ratios. It ranged from 5.49 g/100 g (in samples modified with 2.0 mL/g) to 8.68 g/100 g (in samples acetylated with 0.4 mL/g). Similar findings were observed by Lawal and Adebowale [22], who acetylated the jack bean protein. This decreasing ash content in acetylated protein preparations can be associated with more frequent removal of an excess modifying reagent (samples modified in the amount of 2.0 mL/g of protein were washed 5 times).
