**3. Results and Discussion**

#### *3.1. Preparation and Characterization of WPI Hydrogels Containing TAs*

WPI is a promising cross-linking component for the preparation of hydrogels containing various biologically active compounds. Previously, hydrogels based on various WPI concentrations were synthesized and their properties were studied. [6]. Two types of TAs (polygalloyl glucoses—ALSOK 02, polygalloyl quinic acids—ALSOK 04) were used for the fabrication of the WPI hydrogels. The main differences in these preparations are varying amounts of hydroxyl groups and chemical structure. Based on the literature data, TA concentrations in WPI hydrogels were selected and hydrogels with differing TAs contents were synthesized 1.5; 3.0; 6.0 and 12.0 mg per mL, which corresponds to the TA/WPI ratios were 0.0375/0.075/0.15/0.30 in the hydrogels. [27,28]. Hydrogels were obtained by heating the solution to 90 ◦C for 30 min. Such a short exposure to high temperatures does not lead to pathological changes in the TA structure [29].

The gelification process of WPI-TAs solutions was carried out at pH 7 in deionized water. It is assumed that the incorporation of an additional small TAs amount into the WPI hydrogel structure (maximum TA/WPI ratio of 0.30) does not affect the hydrogel pI, since WPI is the prevailing constituent of hydrogels. According to previously published studies [30] the pI of hydrogels obtained at a pH above the native protein pI (pI 5.2) shifts to a more acidic range (pI 4.1) due to the electrostatic repulsion of negatively charged groups of glutamic and aspartic acids and corresponding deprotonation of lysine amino acid residues.

To understand the functional properties of WPI-TA hydrogels, it is necessary to determine their structure and identify the binding nature of the protein and polyphenols. FTIR measurements are a sensitive tool for detecting conformational changes in the secondary structure of a protein [31]. In the present study FTIR-spectra of WPI-TAs hydrogels were measured from a solid dried condition to exclude pronounced stretching vibrations of

water molecules in the 3673–2942 cm−<sup>1</sup> range and a deformation band of water in the 1644 cm−<sup>1</sup> region. Figure 1 shows the FTIR spectra of unmodified WPI hydrogel and hydrogels with various TA concentrations. In the spectrum of the unmodified WPI hydrogel (burgundy line), we observed three strong bands at 3208, 1673, and 1545 cm−1, which correspond to vibrations for amide A, amide I, and amide II, respectively [32]. In the vibrational spectrum region of amide I, stretching vibrations of the COO- of the Asn and Gln side residues and NH3 <sup>+</sup> deformation vibrations of amino acids containing additional NH2-groups in the side chain (Asp, Glu, Lys, and Arg) are manifested. This overlap of the amino acid residues absorption bands with the Amide I absorption band makes it very sensitive to the intermolecular H-bonds manifestation. A signal change of the amide I absorption band makes it possible to determine the conformational protein change.

**Figure 1.** FTIR spectra of whey protein isolate (WPI) hydrogels with ALSOK 02 (**a**) and ALSOK 04 (**b**). Tannic acid (TA)/WPI ratio: 0.0 (burgundy line); 0.0375 (red line); 0.075 (green line); 0.15 (purple line); 0.30 (yellow line), TA (black line). The main diagnostic bands are magnified for FTIR spectra of WPI-ALSOK 02 (**c**) and WPI-ALSOK 04 (**d**) hydrogels. The dotted lines (**c**,**d**) indicate the main vibration signals (amide A, amide I, amide II) of the control WPI hydrogel without TAs (burgundy lines).

FTIR spectra of hydrogels with different TA contents showed similar bands to that of the WPI hydrogel control spectrum. It indicates that new covalent bonds were not created. A similar result was reported by Ferraro, et al. (2015), who studied the nature of the interaction between rosmarinic acid (natural polyphenol) and milk whey proteins through non-covalent bonds in detail [33].

The spectral lines of hydrogels with TAs revealed broadening of the vibrational signal at 3208 cm−1, which indicates the formation of intermolecular H-bonds (Figure 1c,d). For hydrogels containing polygalloyl glucose (ALSOK 02) the broadening of the symmetric vibration signal of -NH and -OH groups into Amide A is more pronounced than for hydrogels with the same content of the polygalloyl quinine acid (ALSOK 04). H-bonds are the main binding force of WPI and hydrophilic substances [34]. Vibrational signals of Amide I and Amide II are considered the basis of the WPI signal and confirm the presence of whey proteins. A change in the secondary structure of the protein is usually explained by broadening of Amide I and a shift of Amide II. When more ALSOK 02 is added into hydrogels, the peaks of Amide I bending vibrations are shifted by 7 cm−<sup>1</sup> (from 1545 cm−<sup>1</sup> to 1538 cm−1) towards a lower wavenumber (Figure 1c). This indicates a change in the nature of the side amino group vibrations of Asp, Glu, Lys, and Arg due to the formation of intermolecular H-bonds with the polyphenols. The same phenomenon occurred for Amide II; the maximum shift was observed from 1673 cm−<sup>1</sup> to 1657 cm−<sup>1</sup> for a hydrogel with ALSOK 02/WPI ratio 0.30 (Figure 1b,d). For hydrogels containing ALSOK 04, the shifts of stretching vibrations of Amide I and Amide II groups were more significant than for hydrogels with ALSOK 02, perhaps due to the contribution of closely spaced signals of stretching vibrations of carboxyl groups and stretching of the C=C aromatic bonds of uncrosslinked ALSOK 04. The maximum shift was up to 25 cm−<sup>1</sup> and was observed also for hydrogels with ALSOK 04/WPI ratio 0.30 (Figure 1d). The shift of Amide I and Amide II indicates the presence of an electrostatic interaction between WPI and TA, and not chemical reactions [31]. For the WPI-ALSOK 02 complex, the formation of intermolecular H-bonds is more characteristic than for the WPI-ALSOK 04, which directly depends on the chemical structure of TAs and their ability to ionize in water. Thus, a hydrolysable polygalloyl glucose (ALSOK 02) with a large number of hydroxyl groups interacts better with protein than polygalloyl quinic acid (ALSOK 04). Thus, in all cases, non-specific binding between polyphenols and WPI is confirmed, without additional covalent bond formation during the hydrogels' preparation.
