2.1.1. Protein Solubility Index (PSI)

Solubility is often used as an indicator of other functional properties. It is defined as the ability of a substance to form homogeneous mixtures with other substances. The solubility of the control and modified proteins in this study was pH-dependent, as indicated in Figure 1. Over the pH range studied [2,12,16], both the unmodified and acetylated RPC were soluble almost exclusively at pH 12; however, even under this condition, the solubility was very low (below 0.5%). The acetylation of the RPC at pH 12 with a 0.4 mL/g dose of acetic anhydride caused an almost two-fold increase in PSI to 0.45%, as compared to the control sample (0.28%). This could have resulted from the covalent incorporation of the acetyl groups into the amino group of the protein, which results in a certain degree of protein unfolding (electrostatic attraction is diminished). The hydrophilic groups can be exposed, and therefore hydrophilicity increases, which improves solubility [17].

As the dose of acetic anhydride increased (from 1.0 mL/g (with a degree of acetylation of 92.24%) to 2.0 mL/g (with a degree of acetylation of 99.98%), data presented in Table 1), the protein solubility decreased, which is in line with the reports by other authors [8,22,23]. The data provided by Heredia-Leza et al. [21] show that when the degree of acetylation does not exceed 78%, the solubility of proteins increases. Thus, the solubility depends on the degree of acetylation, and, in some cases, a deep degree of acetylation is not desired. Similar results have been found for pumpkin proteins wherein a dose of 2 mL of acetic anhydride/g protein caused a decrease in the solubility from 62.44% to 49.52% [21]. Moreover, hydrogen bonds play a fundamental role in regulating protein and water interactions and rely on polarity. RPC is characterized by low polarity; thus, it cannot interact and form hydrogen bonds; therefore, the solubility of the control and acetylated samples was very low [21].



Values are means ± standard deviation; *n* = 3; a, b, c—the same letters within the same column indicate values that are not significantly different; RPC—rice protein concentrate; 0.4, 1.0, 2.0—rice protein preparations after acetylation conducted with different concentrations of acetic anhydride.

In an alkaline environment, protein molecules may unfold, increasing the exposure of the hydrophobic groups and thus changing the solubility of the protein. However, considering the low solubility of the RPC and acetylated samples, this change may have resulted from the increased degradation of the commercial protein, which resulted in fewer interactions between the protein molecules. Additionally, the high insolubility of rice protein in water could be due to extensive aggregation and crosslinking through the disulphides of one of the main fractions, glutelin [8]. Rice proteins are composed of two major hydrophobic fractions: glutelins, which are high-molecular-weight proteins composed of subunits bound by disulphide linkages and are soluble only in dilute acid or alkali, and prolamins, which are soluble in 70% ethanol [11,13]. Also, according to the results of these studies [11,13], RPC displays poor solubility in the pH range of 2–10, with a minimum at pH 5. When considering the amino acid profile (Table S2), it also can be stated that rice proteins have an acidic character; that is, the sum of the aspartic acid and glutamic acid residues (25.95 g/100 g protein) is greater than the sum of the lysine, arginine, and histidine residues (12.51 g/100 g protein). Therefore, they exhibit maximum solubility at alkaline pH. Additionally, the very low solubility of the RPC samples can be explained by the protein isolation method used and, therefore, by the enhanced hydrophobic interactions [10]. Generally, protein preparations with low solubility in water can potentially be applied to products, e.g., meat analogs, baked goods, breakfast cereals, protein bars, and pet food.
