*4.2. General Experimental Procedure*

Figure 6 shows the experimental procedure of the hydrogels fortified with CBJ, the characterization of hydrogels samples, storage stability, determination of total bioactive compounds (TBC) and antioxidant activity (AA), color measurement, and half-life time analysis. *Gels* **2022**, *8*, x FOR PEER REVIEW 14 of 20

**Figure 6.** General experimental procedure. **Figure 6.** General experimental procedure.

### *4.3. CBJ Preparation*

of a solid gel.

*4.3. CBJ Preparation* Blueberries were washed with tap water to remove dirt, and later, the juice was ex‐ tracted in a tabletop juicer (JE2001, Nex, Barcelona, Spain). Immediately, the juice was filtered through a nylon cloth (0.8 mm mesh) to remove skin and seeds. Then, the juice was concentrated through three cryoconcentration cycles using the centrifugal method described previously by Casas‐Forero et al. [16]. Hence, CBJ corresponds to the cryocon‐ centrated sample acquired in the final cycle. After the third cycle, the CBJ reached values of 45 °Brix and a 4.1 pH. Thus, the cryoconcentrated juice was stored until use as an in‐ Blueberries were washed with tap water to remove dirt, and later, the juice was extracted in a tabletop juicer (JE2001, Nex, Barcelona, Spain). Immediately, the juice was filtered through a nylon cloth (0.8 mm mesh) to remove skin and seeds. Then, the juice was concentrated through three cryoconcentration cycles using the centrifugal method described previously by Casas-Forero et al. [16]. Hence, CBJ corresponds to the cryoconcentrated sample acquired in the final cycle. After the third cycle, the CBJ reached values of 45 ◦Brix and a 4.1 pH. Thus, the cryoconcentrated juice was stored until use as an ingredient in the elaboration of hydrogel samples.

### *4.4. Preparation of Hydrogel Products*

gredient in the elaboration of hydrogel samples.

*4.4. Preparation of Hydrogel Products* For the commercial hydrogels products based on confectionery foods, four types of samples were prepared, gelatin gel (GG), aerated gelatin gel (AGG), gummy (GM), and aerated gummy (AGM). For GG and AGG samples, the hydrogels with CBJ were per‐ formed according to Casas‐Forero et al. [15], with slight modifications. Separately, the For the commercial hydrogels products based on confectionery foods, four types of samples were prepared, gelatin gel (GG), aerated gelatin gel (AGG), gummy (GM), and aerated gummy (AGM). For GG and AGG samples, the hydrogels with CBJ were performed according to Casas-Forero et al. [15], with slight modifications. Separately, the gelatin powder (3 g for GG and 8 g for AGG) was hydrated in 70 mL distilled water at

gelatin powder (3 g for GG and 8 g for AGG) was hydrated in 70 mL distilled water at room temperature for 10 min. Then, the solutions were mechanically stirred (300 rpm) for

rpm) for 5 min at 40 °C. Later, the gel solution with CBJ was poured into plastic vessels (approximately 34 mm inner diameter and 15 mm height). For AGG samples, air bubbles were incorporated using an Oster® mixer (Oster 2532, 250‐watt, 6‐speed, Rianxo, Spain) for 9 min, and then, the aerated gel solution with CBJ was deposited into plastic vessels. Finally, the GG and AGG samples were maintained overnight at 4 °C until the formation

For GM and AGM samples, separately, 6 g of gelatin powder was hydrated in 25 mL distilled water for 10 min at room temperature. Then, the solution was heated at 60 °C and mechanically stirred at 300 rpm until complete dissolution (≈10 min). The gelatin solution was cooled and maintained until at 40 °C. In parallel, a syrup solution was prepared with 350 mL of distilled water, 350 mL of sucrose, and 300 mL of glucose syrup. Thus, once the solution was homogenized with constant agitation, it was heated for 5 min at 110 °C, and then, it was cooled to 50 °C. After, 30 g of CBJ and 45 g of syrup were mixed with the gelatin solution, and the mixture was stirred (300 rpm) for 5 min at 40 °C. Thus, for GM, the gel solution with CBJ and syrup was placed into plastic vessels. For AGM, the aeration process was performed for 4 min using an Oster® mixer (Oster 2532, 250‐watt, 6‐speed,

room temperature for 10 min. Then, the solutions were mechanically stirred (300 rpm) for 10 min at 60 ◦C. After, the solutions were cooled and maintained for 5 min at 40 ◦C, and later, 30 g of CBJ was added to each solution, and the solution (with CBJ) was stirred (300 rpm) for 5 min at 40 ◦C. Later, the gel solution with CBJ was poured into plastic vessels (approximately 34 mm inner diameter and 15 mm height). For AGG samples, air bubbles were incorporated using an Oster® mixer (Oster 2532, 250-watt, 6-speed, Rianxo, Spain) for 9 min, and then, the aerated gel solution with CBJ was deposited into plastic vessels. Finally, the GG and AGG samples were maintained overnight at 4 ◦C until the formation of a solid gel.

For GM and AGM samples, separately, 6 g of gelatin powder was hydrated in 25 mL distilled water for 10 min at room temperature. Then, the solution was heated at 60 ◦C and mechanically stirred at 300 rpm until complete dissolution (≈10 min). The gelatin solution was cooled and maintained until at 40 ◦C. In parallel, a syrup solution was prepared with 350 mL of distilled water, 350 mL of sucrose, and 300 mL of glucose syrup. Thus, once the solution was homogenized with constant agitation, it was heated for 5 min at 110 ◦C, and then, it was cooled to 50 ◦C. After, 30 g of CBJ and 45 g of syrup were mixed with the gelatin solution, and the mixture was stirred (300 rpm) for 5 min at 40 ◦C. Thus, for GM, the gel solution with CBJ and syrup was placed into plastic vessels. For AGM, the aeration process was performed for 4 min using an Oster® mixer (Oster 2532, 250-watt, 6-speed, Rianxo, Spain). Finally, the aerated gel solution with CBJ and syrup was placed into plastic vessels. Thus, the GM and AGM were solidified overnight at room temperature.

### *4.5. Characterization of Hydrogels Samples*

### 4.5.1. Physicochemical Parameters

Total soluble solids (TSS) expressed as ◦Brix were analyzed using a digital PAL-1 refractometer (PAL-3, ≈1 mL, range: 0–93 ◦Brix, precision: ±0.1 ◦Brix, Atago Inc., Tokyo, Japan). The pH was measured using a digital pH meter (HI 2210, Hanna Instruments, Woonsocket, RI, USA). The moisture content was obtained gravimetrically by vacuum drying at 60 ◦C in a vacuum oven (3618–1CE, Lab Line Instruments Inc., Melrose Park, IL, USA) for 24 h (AOAC, 20.013, 2000). The water activity (*aw*) was determined using a dew-point hygrometer (AquaLab Model 4TE, Decagon Devices Inc., Pullman, WA, USA). The density was measured by the flotation method according to Zúñiga and Aguilera [31], and the results were expressed as kg/m<sup>3</sup> . Gas hold-up (*ε*) of AGG and AGM was obtained by comparing the density of the aerated sample (*ρAGG* or *ρAGM*) with the respective gas-free sample (*ρGG* or *ρGM*) density. The *ε* was calculated according to Equation (1).

$$
\varepsilon(\%) = \left(1 - \frac{\rho\_{AGG} \text{ or } \rho\_{AGM}}{\rho\_{GG} \text{ or } \rho\_{GM}}\right) \ast 100\tag{1}
$$

where *ρAGG*, *ρAGM*, *ρGG,* and *ρGM* are the density of aerated gelatin gel (AGG), aerated gummy (AGM), gelatin gel (GG), and gummy (GM), respectively.

### 4.5.2. Rheological Properties

The rheological properties were evaluated by a rotational-type rheometer (Physica MCR300, Anton Paar GmbH, Stuttgart, Germany) using a parallel plate geometry (50 mm diameter) with a 1 mm gap. The apparent viscosity of the samples was determined at 25 ◦C with a shear rate of 0.1 to 100 s−<sup>1</sup> [27]. The frequency sweep measurement was performed from 0.1 to 100 Hz at a constant stress of 3.0 Pa within the linear viscoelastic region at 25 ◦C [52].

### 4.5.3. Mechanical Properties

The uniaxial compression test was carried out using a texture analyzer (TAXT plus100, Stable Micro Systems Ltd., Surrey, UK) with a load cell of 5.0 kg. The samples were compressed with a 50 mm diameter cylindrical aluminum probe (P50) at a constant speed

of 1 mm/s up to a compression strain of 70% [53]. The true stress (*σH*) and true strain (*εT*) were calculated from force-time curves by Equations (2) and (3), respectively.

$$
\sigma\_H = F \left[ \frac{h\_o - h}{h\_o \ A} \right] \tag{2}
$$

$$\varepsilon\_T = -\ln\left[\frac{h\_o}{h\_o - h}\right] \tag{3}$$

where *F* is the compression force (N), *A* is the cross-sectional area of the sample (m<sup>2</sup> ), and *h<sup>o</sup>* and *h* are the initial and final height after compression (m), respectively.

### 4.5.4. Optical Microscopy

The samples were cut into thick slices of 3 mm with a razor blade and the slices were placed on a slide. Photomicrographs were acquired using an Olympus Trinocular Microscope (Olympus Co., Tokyo, Japan) coupled to a digital camera, Olympus LC 20 (Olympus Co., Munster, Germany) with an objective lens Nikon 10× [12].

### *4.6. Storage Stability Study (SSS)*

Specifically, GG and AGG were maintained at 4 ◦C in the dark in a refrigerated incubator (FOC 215E, Velp Scientific Inc., Milano, Italy), and GM and AGM were stored at 25 ◦C in the dark in a thermostatic chamber (Memmert UF110, Memmert, Schwabach, Germany). For each sample and the control (CBJ), total bioactive compounds (TBC), antioxidant activity (AA), and color were analyzed at 0, 7, 14, 21, 28, and 35 days of storage.

### *4.7. Determination of Total Bioactive Compounds (TBC)*

Total polyphenol content (TPC) was measured according to the Folin–Ciocalteu method described by Waterhouse [54], with minor modifications. Gallic acid (GA) was used as standard. An amount of 100 µL of the sample was mixed with 500 µL of 10-fold diluted Folin–Ciocalteu reagent. Then, the solution was vigorously mixed with 1500 µL of Na2CO<sup>3</sup> (20% *w*/*v*). After 90 min in the dark at room temperature (incubation), the absorbance was recorded at 760 nm. TPC was calculated as milligrams of GA equivalents (GAE) per 100 g of sample (mg GAE/100 g).

Total monomeric anthocyanin content (TAC) was quantified by the differential pH method according to Lee et al. [55], with some modifications. Cyanidin-3-O-glucoside (C3G) was used as standard. An amount of 200 µL of the sample was mixed with 800 µL of KCl (pH 1.0, 0.025 M) and 800 µL of CH3COONa (pH 4.5, 0.4 M) buffers. After 30 min in the dark at room temperature (incubation), the absorbance was measured at 510 and 700 nm. TAC was calculated as milligrams of C3G equivalents per 100 g of sample (mg C3G/100 g).

Total flavonoid content (TFC) was determined using the aluminum chloride colorimetric method described by Dewanto et al. [56], with modifications. Catequin (C) was used as standard. An amount of 250 µL of the sample was mixed with 1000 µL of distilled water and 75 µL of NaNO<sup>2</sup> (5% *w*/*v*). After 6 min in the dark at room temperature (incubation), 75 µL of AlCl<sup>3</sup> (10% *w*/*v*), 500 µL of NaOH (1 M), and 600 µL of distilled water were added to the solution, and later, the absorbance was measured at 510 nm. TFC was calculated as milligrams of C equivalents (CEQ) per 100 g of sample (mg CEQ/100 g).

### *4.8. Determination of Antioxidant Activity (AA)*

The DPPH assay was assessed using the method reported by Brand-Williams et al. [57], with minor modifications. An amount of 150 µL of the sample was mixed with 2850 µL of DPPH methanolic solution. The mixture was kept in the dark at room temperature for 30 min (incubation), and the absorbance was measured at 515 nm.

The ferric reducing antioxidant power (FRAP) assay was performed according to Benzie and Strain [58], with some modifications. Briefly, FRAP reagent was prepared with 50 mL of CH3COONa buffer (pH 3.6, 300 mM), 5.0 mL of TPTZ (10 mM in HCl (40 mM)), and 5.0 mL of FeCl3·6H2O (20 mM) (10:1:1 ratio), and then the solution was incubated at 37 ◦C. An amount of 150 µL of the sample was mixed with 2850 µL of FRAP reagent. The solution was kept in the dark at 37 ◦C for 30 min (incubation), and the absorbance was measured at 593 nm.

For DPPH and FRAP assays, Trolox (T) was used as the standard curve, and the results were expressed as µmol Trolox equivalents (TE) per 100 g of sample (µM TE/100 g).

TBC (TPC and TAC) and AA (DPPH and FRAP) values were measured by spectrophotometric analysis (T70 UV/Vis spectrophotometer, Oasis Scientific Inc., Greenville, SC, USA).

### *4.9. Color Measurement*

Color analysis was performed through CIELab coordinates (L\*: darkness–lightness, a\*: green-red axis, b\*: blue-yellow axis) using a colorimeter (CM-5, Konica Minolta, Osaka, Japan), with illuminant D65 and an observer angle of 10◦ , where the samples were filled in a glass cuvette, and thus, the CIELab values were measured [59].

### *4.10. Kinetics and Half-Life Time Analysis*

First-order kinetics was used to describe the degradation TBC and AA content [22]. The reaction rate constant (*k*) and half-life time (*t*1/2) were calculated according to Equations (4) and (5), respectively.

$$\frac{\mathbf{C}\_{\mathbf{f}}}{\mathbf{C}\_{o}} = e^{-k \cdot \mathbf{f}} \tag{4}$$

$$t\_{1/2} = \frac{\ln 2}{k} \tag{5}$$

where *C<sup>o</sup>* is the initial TBC and AA content, and *C<sup>t</sup>* is the TBC and AA content at time *t* (days).

## *4.11. Statistical Analysis*

All experiments were replicated three times and measurements were carried out in triplicate, and the data were expressed as mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) was performed for each variable to identify differences between samples, and least significant difference (LSD) tests were performed for the comparison of means at a significance level of 5% (*p* ≤ 0.05) using Statgraphics Centurion XVI software (v. 16.2.04, StatPoint Technologies Inc., Warrenton, VA, USA).

**Author Contributions:** Conceptualization, N.C.-F., R.N.Z. and G.P.; methodology, N.C.-F. and I.T.-M.; software, N.C.-F. and I.T.-M.; validation, N.C.-F., R.N.Z., G.P. and P.O.-P.; formal analysis, N.C.-F. and I.T.-M.; investigation, N.C.-F., R.N.Z., G.P. and P.O.-P.; resources, G.P. and P.O.-P.; data curation, N.C.-F. and I.T.-M.; writing—original draft preparation, N.C.-F. and I.T.-M.; writing—review and editing, N.C.-F., R.N.Z., G.P. and P.O.-P.; visualization, N.C.-F., I.T.-M., G.P. and P.O.-P.; supervision, G.P. and P.O.-P.; project administration, G.P. and P.O.-P.; funding acquisition, N.C-F., G.P. and P.O.-P. All authors have read and agreed to the published version of the manuscript.

**Funding:** Patricio Orellana-Palma acknowledges the financial support of ANID-Chile (Agencia Nacional de Investigación y Desarrollo de Chile) through the FONDECYT Postdoctoral Grant 2019 (Folio 3190420).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data is contained within the article.

**Acknowledgments:** Nidia Casas-Forero would like to thank the Vice-Chancellery for Research and Graduate at Universidad del Bío-Bío for the scholarship granted to complete her doctoral studies.

**Conflicts of Interest:** The authors declare no conflict of interest.
