foods Article Development of Fortified Breads Enriched with Plant-Based Bioactive Peptides Derived from the Chia (Salvia hispanica L.) Expeller Brenda Ozón 1 , Juliana Cotabarren 1 , Florencia R. Geier 1 , M. Paula Kise 2 , Javier García-Pardo 3, * , Mónica G. Parisi 2, * and W. David Obregón 1 1 Departamento de Ciencias Biológicas, Centro de Investigación de Proteínas Vegetales (CIProVe), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 47 y 115 s/N, La Plata B1900, Buenos Aires, Argentina; brendaozon@gmail.com (B.O.); cotabarren.juliana@biol.unlp.edu.ar (J.C.); florgei.94.fg@gmail.com (F.R.G.); davidobregon@biol.unlp.edu.ar (W.D.O.) 2 Departamento de Ciencias Básicas, Instituto de Ecología y Desarrollo Sustentable (INEDES, CONICET-UNLu), Universidad Nacional de Luján, Ruta 5 y Avenida Constitución, Luján B6700, Buenos Aires, Argentina; paulakise@yahoo.com.ar 3 Institut de Biotecnologia i de Biomedicina (IBB) and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain * Correspondence: javiergarciapardo@msn.com (J.G.-P.); mparisi@unlu.edu.ar (M.G.P.); Tel.: +34-93-5868936 (J.G.-P.); +54-2323-423171 (M.G.P.) Abstract: By-products from the industrialization of oilseeds, particularly chia, can be sustainably used for the development of new functional products. In this work, wheat breads supplemented with up to 10 mg of chia expeller hydrolysate/g of flour were prepared, obtaining fortified breads with acceptability for consumption, according to a preliminary consumer research study based on an affective test employing a five-point hedonic scale of global acceptance. In this context, protein hydrolysates of the chia expeller were produced using Alcalase, reaching a degree of hydrolysis of 54.3 ± 1.6% with an antioxidant activity of 55.8 ± 0.4% after 6 h incubation at 25 ◦ C in the presence Citation: Ozón, B.; Cotabarren, J.; of the enzyme. These peptides showed appropriate techno-functional properties and chemical Geier, F.R.; Kise, M.P.; García-Pardo, compositions suitable for the further development of bakery products. Taken together, our approach J.; Parisi, M.G.; Obregón, W.D. and the development of a fortified bread with plant-based bioactive peptides provide a novel and Development of Fortified Breads eco-friendly alternative for the recovery of nutrients from agro-industrial waste. More importantly, Enriched with Plant-Based Bioactive Peptides Derived from the Chia these enriched breads could exert beneficial effects on human health by exploiting the antioxidant (Salvia hispanica L.) Expeller. Foods properties of functional peptides derived from the chia expeller. 2023, 12, 3382. https://doi.org/ 10.3390/foods12183382 Keywords: antioxidant; bioactive peptides; bread; chia expeller; chia by-product; enzymatic hy- drolysates; functional ingredient; plant-derived peptides; Salvia hispanica L. Academic Editors: Barbara Simonato and Roberta Tolve Received: 16 August 2023 Revised: 3 September 2023 1. Introduction Accepted: 6 September 2023 Wheat bread stands as a global staple food and is the main source of food and energy Published: 9 September 2023 in most developing countries [1]. However, wheat-based diets are typically poor in essential amino acid content and in micro- and macronutrients; therefore, these essential amino acids and micro- and macronutrients must be incorporated through other dietary sources. Copyright: © 2023 by the authors. Moreover, with the constant increase in the human population, food production must Licensee MDPI, Basel, Switzerland. increase substantially to meet future demands. In this context, the incorporation of plant- This article is an open access article based functional ingredients in wheat bread formulations has been shown to be an ideal distributed under the terms and approach to improve its organoleptic and nutritional properties [2–5]. conditions of the Creative Commons Chia (Salvia hispanica L.) is a traditional crop native to Central and Southern Mexico Attribution (CC BY) license (https:// and Guatemala, and nowadays, it is a cultivated plant across the landscapes of South creativecommons.org/licenses/by/ and Central America [6,7]. Notably, the oil extracted from chia seeds has emerged as 4.0/). an outstanding reservoir of α-linolenic acid and phytosterols, surpassing many other Foods 2023, 12, 3382. https://doi.org/10.3390/foods12183382 https://www.mdpi.com/journal/foods Foods 2023, 12, 3382 2 of 16 vegetable oils in their concentrations. Of remarkable significance is the abundant presence of α-linolenic acid, constituting the predominant component of the fatty acid composition within chia seeds (approximately 60% of the total) compared with the lower proportion of linoleic acid (only about 10%) [6]. Moreover, chia seeds can be considered as a “functional superfood” because they contain a high concentration of protein (15–26%) of a good nutritional quality and digestibility and a good balance of essential amino acids that are higher than most grains, especially due to the high content of cysteine and methionine amino acids [8–10]. Another important characteristic of chia seeds is the absence of gluten, which makes it more desirable to coeliac patients. It also helps to increase the satiety index and prevent cardiovascular diseases, inflammatory and nervous system disorders, and diabetes, along with contributing to human nutrition [11]. A possible approach to increase the amino acid content and the nutritional quality of wheat bread is to incorporate food processing by-products [12,13]. It is worth mentioning that between a third and a quarter of food is lost or wasted annually along the food chain. This is also the case in the industrialization of chia seeds, in which the production of cold-pressed chia oil results in large amounts of protein-rich by-products. Among them, the chia expeller can be processed to yield functionally derived peptides that can be effectively used for the development of high value-added food products [13,14]. Typically, these bioactive peptides are inactive when bound inside the parent protein, and they should be enzymatically activated to fully display its beneficial biological properties [14]. In this study, we attempted to develop a novel functional product from bread by incorporating bioactive peptides produced via the enzymatic hydrolysis of the chia expeller by-product. These peptides display antioxidant properties and are a high-nutritional-value component, improving the amino acid content in the final product. In this context, we also investigated whether the incorporation of chia expeller hydrolysates into the bread formulation may alter its functional and sensory properties. Taken together, our results demonstrate that the enrichment of bread fortified with these chia-derived peptides may be a useful strategy to improve the protein content of the bread product and simultaneously generate more economic profitability for the chia industry. 2. Materials and Methods 2.1. Materials and Reagents Protease from Bacillus licheniformis (Alcalase), sodium chloride, tris (hydroxymethyl) aminomethane, sodium dodecyl sulphate (SDS), β-mercaptoethanol (βME), Coomassie Blue G-250, N,N,N0 ,N0 -tetramethyl ethylene diamine (TEMED), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,20 -Azino-bis(3-ethylbenzothiazoline-6-sulf,onic acid) diammonium salt (ABTS), o-phthaldialdehyde (OPA), and bovine serum albumin were purchased from Sigma-Aldrich (St Louis, MO, USA). Ingredients such as wheat flour, yeast, sugar, salt, milk, butter, and oil were purchased locally. 2.2. Obtention of the Chia Expeller Protein Hydrolysate Chia expeller, provided by Greenborg S.R.L. (Lincoln, Buenos Aires, Argentina), was defatted and then grinded to produce some flour with a high protein content [4]. Chia expeller protein concentrate (PC) was obtained by mixing 40 g of defatted flour with 1000 mL of 0.01 M phosphate buffer, 0.1 M NaCl, pH 7.4, before the mixture was subjected to subsequent homogenization in a cool blender and, after 1 h at room temperature, stored at −20 ◦ C until analysis. A fraction of the PC was subjected to heat treatment at 50 ◦ C for 1 h (called “HT”) to evaluate the degree of hydrolysis (DH%) after Alcalase treatment. The enzymatic hydrolysis of PC and HT (PCH and HTH) was performed with the food-grade microbial protease Alcalase (enzyme/substrate ratio of 0.3 U g−1 ). The reaction mixture was adjusted to pH 7.0 to achieve the optimal enzymatic activity and incubated at 25 ◦ C for 2, 4, 6, 8, 10, and 24 h at 80 rpm in a shaker (PCH25 and HTH25 samples) or 50 ◦ C for 1, 2, 3, 4, 5, 6, 7, and 8 h at 80 rpm in a shaker (PCH50 and HTH50 samples) (Figure S1). All samples were withdrawn in triplicate, and the hydrolysis was stopped at the selected Foods 2023, 12, 3382 3 of 16 intervals via heating at 85 ◦ C for 15 min. The hydrolysates were then lyophilised and kept at 4 ◦ C until use. 2.3. Characterization of Hydrolysates 2.3.1. Determination of the Degree of Hydrolysis The degree of hydrolysis (DH%) was estimated by using the o-phthalaldehyde (OPA) method [15]. The OPA reagent was prepared as described by Ozón et al. [16]. A total of 10 µL of the sample and 200 µL of the OPA reagent were mixed in a 96-well flat bottom plate. The assay was performed by triplicate, including a negative control using water instead of the sample, and the absorbance at 340 nm was recorded after 2 min of incubation. A Tecan Infinite M200 PRO spectrophotometer (Männedorf, Switzerland) was used for the determinations. The DH was calculated as follows: DH% = h/htot × 100 where h is the number of hydrolysed bonds, and htot is the total number of peptide bonds per protein equivalent (which depends on the amino acid composition of the raw material). Complete hydrolysis of the sample (100%) was achieved by incubating the sample with 6 N HCl at 100 ◦ C for 24 h. 2.3.2. Evaluation of Antioxidant Activity The antioxidant activity of the chia expeller hydrolysates was evaluated by using the ABTS+ method [17] with slight modifications. The ABTS•+ solution was prepared through reacting an aqueous ABTS solution (5 mL) and potassium persulfate (K2 S2 O8 ) solution in a 2:1 ratio. After storage in the dark for 16–17 h, the blue–green radical cation solution was further diluted in phosphate buffer (pH 7.5) until its absorbance reached 1 ± 0.01 AU at 734 nm. The antioxidant compound content in PC, HT, and the hydrolysates were analysed by adapting the method to a 96-well flat bottom plate in a Tecan Infinite M200 PRO spectrophotometer (Tecan Group Ltd., Männedorf, Switzerland). A total of 190 µL of diluted ABTS•+ solution was added to a 10 µL sample or Trolox standard according to Ozón et al. [6]. The mixture was incubated for 10 min at room temperature in dark conditions, and after that, the absorbance was measured at 734 nm. ABTS+ radical scavenging activity was estimated as follows: A1 − A2 ABTS radical scavenging activity (%) = 100 − 100 × A0 The absorbance of the control without sample was expressed as A0; the absorbance in the presence of the sample and ABTS•+ and the absorbance of the sample blank without ABTS•+ was expressed as A1 and A2, respectively. To evaluate the antioxidant capacity of the breads, 1 g of ground bread crumbs were homogenised in 5 mL of a phosphate buffer (0.1 M, pH 7.0). The suspensions were homogenised in a vortex mixer for 2 min and centrifuged at 7000× g for 10 min, and the supernatant from each sample was evaluated via the ABTS method, as previously described [17]. 2.3.3. Chemical and Techno-Functional Properties of the Chia Expeller Hydrolysates PC hydrolysates produced by Alcalase hydrolysis at 25 ◦ C for 6 h (PCH25,6) were selected for subsequent analysis. The moisture (AOAC 934.01), ashes (AOAC 942.05), lipids (AOAC 920.39), dietary fibre (AOAC 985.29), and protein (micro-Kjeldahl method, AOAC 984.13) contents of the raw materials were determined according to Association of Official Analytical Chemists (2000) protocols [18], and the carbohydrate content was obtained by subtracting 100 from the sum of other components. Water retention capacity (WRC) and oil retention capacity (ORC) were determined according to Chau et al. [19] with modifications. Thus, 0.5 g of PCH25,6 were homogenised Foods 2023, 12, 3382 4 of 16 with 5 mL of water/oil via vortex for 2 min and centrifuged at 2200× g for 30 min. Water/oil on the supernatant was removed, and the remaining fibrous suspension was weighed to determine the water/oil weight gain. The WRC/ORC was expressed as grams of water/oil retained per gram of sample, considering oil density as 0.92 g/mL. final weight − initial weight WRC or ORC = initial weight Water adsorption capacity (WAC) was determined according to Chen et al. [20]. A total of 1 g of PCH25,6 was placed in a microenvironment of 98% relative humidity in equilib- rium, generated by placing 20 mL of saturated potassium sulphate saline solution in a hermetically sealed glass container at 25 ◦ C. The sample was incubated until it achieved a constant weight, reporting this capacity as the weight gain expressed in grams of water per gram of dry sample. g of adsorbed water WAC = g of CEH Emulsifying activity (EA) and emulsion stability (ES) were determined according to Chau et al. [19]. To evaluate EA, 100 mL of distilled water were added to 2 g of hydrolysates, and the mixture was homogenised at 11,000 rpm. Then, 100 mL of refined corn oil (density = 0.92 g/mL) were added and homogenised again at 11,000 rpm for 3 min in a disperser (Ultra-Turrax® , T25 Basic, IKA® , Werke, The Netherlands). The samples were placed in graduated centrifuge tubes (50 mL) and centrifuged at 170× g for 10 min, and the volume of the remaining emulsion was measured to calculate EA as follows: volume of remanent emulsion (mL) × 100 EA = initial volume of emulsion (mL) ES was determined by heating the obtained emulsions at 80 ◦ C for 30 min. The samples were cooled to room temperature and centrifuged at 170× g for 10 min. ES was expressed as the ratio of the remaining emulsion volume (mL) with respect to the original volume of the emulsion. volume of remanent emulsion (mL) × 100 ES = initial volume of emulsion (mL) 2.4. Ingredients and Breadmaking Procedure Wheat breads were prepared with the addition of different PCH:flour ratios (i.e., 1, 3, 5, and 10 mg of PCH25,6/g flour (Table S1) using the N◦ 11 program of a commercial bread oven (ATMA Easy Cook, HP4031E). Wet ingredients were first added to the recipient, and then dry ingredients were placed on the top. The mixture was homogenised for 15 min at medium speed, fermented at 30 ◦ C for 110 min with 80% relative humidity, and cooked for 50 min at 220 ◦ C. After 12 h, the breads were sliced for further analysis. 2.5. Technological and Physicochemical Parameters 2.5.1. Specific Volume Determination Specific volume (SV) was determined according to the millet seed displacement method [21]. Millet seeds were poured into a container of known volume until the bottom was covered. The test bread was then placed inside the container, followed by more millet seeds, which were levelled across the top with a spatula. The displacement of the millet seeds that were not required to fill the container were measured in a graduated cylinder and used to express the specific volume. 2.5.2. Colour Analysis The breads were cut into 2 cm wide slices, and bread colour analysis was performed using a colour spectrophotometer (HunterLab MiniScan EZ, Reston, VA, USA) according Foods 2023, 12, 3382 5 of 16 to the colour system in the L*, a*, and b* space defined by CIE-L*a*b* [22]. Calibration with black and white tiles was performed before colour measurement. The colour appearance of bread slices was quantified using three-dimensional colour spaces: lightness, colour, and hue. Lightness is described as the brightness of a surface, and colour indicates the intensity of the colour content (light green, dark green, etc.). Hue is the visual perception according to which an area appears to be similar to one of the colours: red, yellow, green, or blue. To calculate the hue, the CIE L*a*b* (CIELAB) colour space is taken into account, where the hue angle is calculated as h = arctan (b/a), between 0 and 360 degrees, corresponding to the hue circle, where (a) is the red–green component and (b) is the yellow-bluish component. In this way, h = 0 approximates the appearance of red object surfaces; h = 90 to the yellow ones, h = 180 to the green ones, and h = 270 to the blue ones. Colour difference values (∆E), which measure the changes in colour of the total colour differences, were calculated as previously described [23] by using the following equation: q ∆L2 + ∆a2 + ∆b2 2.5.3. Texture Profile Analysis The textural properties of the fortified breads were determined on three central slices (2 cm thickness) using a TA-XTplus Texture Analyser (Stable Micro Systems, Surrey, UK) with a 36 mm cylindrical probe (p/36 R) according to the 74-09 methodology proposed by the Approved Methods of the American Association of Cereal Chemists (AACC) [21]. The pre-test speed, test speed, and post-test speed values were 1, 5, and 5 mm/s, respectively, with a trigger force of 5 g. The data were measured as force (N) versus time (s) curves. The hardness (g), adhesiveness (g.s), resilience (%), cohesiveness, and springiness (%) were recorded as well. Gumminess (G) was calculated as G = hardness × cohesiveness, and chewiness (Ch) was calculated as Ch = gumminess × springiness. 2.5.4. Sensory Evaluation The sensory analysis of freshly baked bread samples (CB and B10) involved 37 un- trained panellists aged between 20 and 60 years who were regular consumers of wheat bread. This panel consisted of 18 women and 17 men, including undergraduate and gradu- ate students, as well as faculty members from the Faculty of Exact Sciences at the National University of La Plata in La Plata, Argentina. To assess the organoleptic characteristics of the breads, the panellists participated in a descriptive sensory analysis panel. The breads were baked the day before the evaluation. On the day of the evaluation, the untrained panellists were randomly divided into groups of nine people. These groups were accommodated in a room that was equipped with individual cabinets and specifically arranged for the sensory test. The panellists received basic instructions regarding the evaluation procedure, which was conducted in a single day. During the sensory evaluation, the bread samples (CB and B10) were presented as 2 cm cubes that were placed on different dishes, each labelled with random three-digit numbers. Water was provided for rinsing between tasting each sample. Following the instructions, training, and discussions related to product-oriented affective testing, each panellist received a rating form score sheet. This score sheet was based on a five-point hedonic scale for assessing the overall acceptance of the bread. The scale ranged from 5, indicating “I liked it very much”, to 1, indicating “I disliked it very much”. Additionally, panellists performed a preference test where they selected their preferred bread out of all of the options. The untrained panellists also performed a triangular test. This test is useful for evaluating overall bread differences between control samples and upon supplementation with the chia expeller-derived peptides. To perform the evaluation, three cubes were presented in random order: (i) CB-CB-B10, (ii) CB-B10-CB, (iii) B10-CB-CB. After sensory evaluation, the panellists opined on the different tastes of the bread cubes, and their answers were interpreted as “right”, “wrong”, or “do not know”. Foods 2023, 12, 3382 6 of 16 The results were interpreted according to the relationship between the number of positive judgments and the total number of judgments. A significant difference between the samples at the corresponding level of probability was considered when the number of positive judgments was greater than or equal to the value of the two-sided table [24]. 2.6. Statistical Analysis Statistical analyses (ANOVA) were performed using GraphPad Prism (v9.4.1, Graph- Pad Software Inc., San Diego, CA, USA, 2012). All experiments were conducted in triplicate of two independent experiments. Data are expressed as the mean ± standard deviation, and significant differences were determined by using Tukey’s post hoc test (p < 0.05). 3. Results and Discussion 3.1. Enzymatic Hydrolysis of Chia Expeller Using Alcalase Defatted chia expeller is a by-product with a high protein content of about 37% [14]. The optimization of the enzymatic hydrolysis of the chia expeller protein concentrate (PC) and the heat-treated sample (HT) was carried out to find the optimal digestion conditions. As observed in Figure 1, when the enzymatic hydrolysis was carried out at 50 ◦ C, a gradual increase in the degree of hydrolysis (DH) was observed as the incubation time increased in both samples (PCH and HTH), reaching a DH of 70% after 3 h of incubation (Figure 1A). No significant increases in DH were observed at longer times, reaching 90% after 8 h, indicating a certain degree of enzyme inactivation when the samples were incubated at this temperature. However, when the enzymatic hydrolysis was carried out at 25 ◦ C, the incubation time required to reach the same DH as in the test at 50 ◦ C was longer, achieving 70% DH after 10 h of incubation for both samples (Figure 1B). Furthermore, by maintaining the process for 24 h, DH only increased up to 80%. Under our experimental conditions, the faster Alcalase hydrolysis of chia proteins was achieved at 50 ◦ C incubation [16,25]. This observation aligns with the optimal temperature range for Alcalase activity, which typically falls between 50 and 65 ◦ C [26]. Additionally, we also evaluated the effect that thermal pre-treatment had on the samples. Interestingly, thermally treating the samples at 50 ◦ C for 1 h before the hydrolysis process (HTH) increased the DH by 10% in the first 4 h when the hydrolysis was carried out at 50 ◦ C, suggesting that thermal treatment can also change substrate accessibility for hydrolysis due to substrate unfolding. No significant differences were observed between the DH obtained at higher hydrolysis times. On the other hand, when the hydrolysis was carried out at 25 ◦ C, no important differences were observed in the resulting DH for both the thermally treated samples and the PC. Next, we evaluated the antioxidant activity of the chia expeller protein concentrate hydrolysates (PCHs) and the corresponding heat-treated Alcalase hydrolysates (HTHs) produced at 50 ◦ C, which was found to be the faster digestion condition. As shown in Figure 1C, the antioxidant capacity was similar for both sample groups (PCHs and HTHs) and was obtained after 2 to 8 h of hydrolysis, with values in the range 65–80%. Furthermore, the antioxidant activities produced by the chia expeller samples digested at 25 ◦ C were also comparable (Figure 1D). When we compared the antioxidant activity of the hydrolysates obtained at 25 ◦ C to those obtained at 50 ◦ C, the values obtained after 2 h of hydrolysis at 50 ◦ C were equivalent, which is in agreement with the DH values obtained. These results demonstrated the possibility of producing hydrolysates with Alcalase at room temperature, since they only require a longer hydrolysis time to obtain the same antioxidant effect as at 50 ◦ C. Furthermore, no significant differences were observed in DH and the antioxidant activity when using PC and HT as substrates. Thus, we selected the PCH sample produced after 6 h at 25 ◦ C (PCH25,6) for further characterization since this treatment showed a DH of 54.3 ± 1.6% and a high antioxidant activity (55.8 ± 0.4%). x FOR PEER REVIEW Foods 2023, 12, 3382 7 7of of 16 Figure 1. Characterization of chia expeller hydrolysates. Degree of hydrolysis (DH%) for enzymatic Figure 1. Characterization of chia expeller hydrolysates. Degree of hydrolysis (DH%) for enzymatic hydrolysis with Alcalase at 50 ◦ C (A) and 25 ◦ C (B) carried out using the OPA method. (C,D) ABTS hydrolysis with Alcalase at 50 °C (A) and 25 °C (B) carried out using the OPA method. (C,D) ABTS ◦C radical cation radical cation decolorization decolorization assay assay to to improve improve antioxidant antioxidant activity activity for for Alcalase Alcalase hydrolysis hydrolysis at at 50 50 °C (C) and and 25 ◦ 25 °CC (D). (D). In In (A–D), (A–D), the the abbreviations abbreviations are are as as follows: follows: PCH, PCH, chia chia expeller expeller protein protein concentrate concentrate (C) hydrolysate; HTH, Heat-treated chia expeller protein concentrate concentrate hydrolysate. hydrolysate. 3.2. Physicochemical and Techno-Functional Properties of the Chia Expeller Hydrolysates Next, we evaluated the antioxidant activity of the chia expeller protein concentrate The techno-functional hydrolysates (PCHs) and the properties of hydrolysates corresponding are important heat-treated Alcalase factors that need hydrolysates to be (HTHs) produced at 50 °C, which was found to be the faster digestion condition. As shownthe considered when developing novel ingredients as food additives [27]. For this reason, in proximal Figure 1C,composition and capacity the antioxidant the techno-functional was similar for properties both sample of the hydrolysates groups (PCHs and (PCH25,6) HTHs) produced and in this study was obtained after 2were to 8 hcharacterised of hydrolysis,(Tables S2 andinS3). with values As shown the range 65–80%.in Table S2, Further- a protein content of 19.5% was determined for the PCH25,6 sample. This more, the antioxidant activities produced by the chia expeller samples digested at 25 °C is slightly higher than the were alsoprotein content comparable reported (Figure 1D).in the literature When we compared for the thechia seeds (16.5%, antioxidant according activity to of the hy- the USDA National Nutrient Database for Standard Reference) [28]. drolysates obtained at 25 °C to those obtained at 50 °C, the values obtained after 2 h of As expected, hydrolysis at 50 °Cthe total were lipid content equivalent, which found is in in the PCH25,6 agreement withsample the DHderived from the values obtained. defatted expeller was dramatically reduced to 1.6%, a value that These results demonstrated the possibility of producing hydrolysates with Alcalaseis substantially lower at compared to the reference values for the chia seeds (30.7% of lipid content). Moreover, the room temperature, since they only require a longer hydrolysis time to obtain the same ash content was higher compared to the values found in chia seeds (4.8% compared to 33%, antioxidant effect as at 50 °C. Furthermore, no significant differences were observed in respectively). The amount of assimilable carbohydrates were found to be 5.9%, and the solu- DH and the antioxidant activity when using PC and HT as substrates. Thus, we selected ble fibre content stayed in the range of 30–35%, with both values being comparable to those the PCH sample produced after 6 h at 25 °C (PCH25,6) for further characterization since commonly found in the chia seeds (7.7% and 34.4%, respectively). Importantly, the protein this treatment showed a DH of 54.3 ± 1.6% and a high antioxidant activity (55.8 ± 0.4%). content observed for PCH25,6 suggests that the chia expeller hydrolysate maintains protein and soluble fibre contents comparable to those found in the chia seeds, which suggest that chia expeller hydrolysates are excellent candidates for use as dietary supplements. Extensive hydrolysates with a DH higher than 10% are commonly used for the genera- tion of functional foods, including hypoallergenic and bioactive foods. In this study, we obtained PCH25,6 derived from the chia expeller, which has the particularity of containing a high protein and fibre content. In agreement with this, the Water Retention Capacity (WRC) of this was found to be 1.92 g H2 O/g sample (Table S3). This value is in the same range to those observed in previous studies for chia seed protein isolates obtained via three Foods 2023, 12, 3382 8 of 16 distinct drying methods [29] and from flour obtained via solvent extraction or by pressing seeds [30]. In contrast, the oil retention capacity (ORC) of PCH25,6 was determined to be 2.36 g oil/g hydrolysate. This value is slightly increased compared to that reported by Capitani et al. [30], presumably due to the balance between the release of hydrophobic groups and the decrease in the molecular mass and release of ionizable groups, which probably have a negative effect on the ORC. Finally, the emulsifying activity of PCH25,6 was also determined. As shown in Table S3, we found values reported by Villanueva-Lazo et al. [31] for PCH25,6 that were slightly lower for the Alcalase hydrolysates derived from chia seeds (DH: 36.2%) than that reported for a defatted meal [30]. It is widely accepted that protein hydrolysis increases the emulsifying activity of food samples, as this has been previously reported for samples of corn, lupine, soybean, rice bran, pumpkin, oats, sesame, chickpea, peanut, rapeseed, and potato, among others [32]. 3.3. Technological Parameters of the Fortified Breads Enriched with Functional Peptides Derived from the Chia Expeller Plant-based proteins represent a good alternative to animal proteins in the production of functional peptides due to their low cost and ease of large-scale production. As described above, we have obtained chia hydrolysates produced via the enzymatic digestion of the chia expeller with Alcalase (i.e., PCH25,6). The resultant chia expeller hydrolysates displayed excellent values of emulsifying activity and water and oil retention capacities and an adequate proximal composition. Based on these results, breads enriched with different amounts of PCH25,6 (i.e., 1, 3, 5, and 10 mg of chia expeller hydrolysate/g of wheat flour) were formulated and analysed. 3.3.1. External Analysis: Crust Colour The surface browning of bread is an indicator of bread quality. As shown in Figure 2A, the crust colour of breads containing increased concentrations of the chia expeller hy- drolysates did not show significant differences compared to the bread made with wheat flour (control). In a previous study, Xing et al. [33] attributed a change in crust colour to a higher content of amino acids (specially lysine), which react with carbonyl compounds (reducing sugar in the Maillard reaction) during baking. We believe that the lack of dark- ening of the crust colour in our study is in agreement with the overall low lysine content (0.97 g/100 g) found in chia proteins [28]. 3.3.2. Alveolate and Specific Volume Analysis The specific volume and the alveolate of bread crumbs are common indices of quality as they reflect the ability of the dough to occlude air. Regarding the alveolate, bread slices were scanned, and a qualitative analysis of images was performed (see Figure 2B). The addition of increasing amounts of hydrolysates revealed a positive effect on the formation of alveoli, with larger alveoli being observed in the breads supplemented with 3, 5, and 10 mg of hydrolysate per gram of flour than those observed for the control bread (CB). In addition, a considerable increase in height was also observed in the supplemented samples, showing an increase of 1 cm compared to the CB. Interestingly, the specific volume was not significantly affected by the level of wheat flour substitution (Table 1). Only slightly higher values were recorded for the breads fortified with the chia hydrolysates (B10, B5, and B3, respectively), with mean values of 2.27 ± 0.05, 2.29 ± 0.08 and 2.25 ± 0.06 mL/g. The lowest value was observed for the control sample (CB with 2.07 ± 0.04). These results showed a minor loss of dough upon baking in the enriched formulations compared to the control bread, probably due to the high water retention capacity of the remaining mucilage in the expeller [34]. Previously, Silveira Coelho and de las Mercedes Salas-Mellado and coworkers [35] produced breads enriched with 11% of hydrated chia seeds, and they found that the enriched bread showed a significantly smaller volume than the control bread. This discrepancy was attributed to a Foods 2023, 12, 3382 9 of 16 Foods 2023, 12, x FOR PEER REVIEW decrease in the share of gluten in the bread’s dough due to a decreased ability to hold8CO of 216 during fermentation and baking. Figure2.2.Photographs Figure Photographs of of wheat wheat bread bread supplemented supplemented withwith chia chia expeller expeller functional functional peptides. peptides. (A) (A) Pho- Photographs tographs of whole of whole breadbread loaves. loaves. (B) Photographs (B) Photographs of central of central transversal transversal slices slices of eachofloaf each loaf posi- positioned tioned on on a graduated a graduated cuttingcutting board. board. Bread Bread slices produced slices produced with wheat with wheat flourflour (CB) (CB) or different or different blends blends of of wheat flour via chia hydrolysates (B1, B3, B5 and wheat flour via chia hydrolysates (B1, B3, B5 and B10). B10). Table 1. Technological and 3.2. Physicochemical characteristics of breadsProperties Techno-Functional supplemented of thewith Chiadifferent Expellerconcentrations Hydrolysates of chia expeller hydrolysates The comparedproperties techno-functional to wheat flour. of hydrolysates are important factors that need to be considered when developing novel ingredients as food additives [27]. For this reason, Bread SV (mL/g) H (g) A (g. sec) R (%) Co S (%) G Ch the proximal composition and the techno-functional properties of the hydrolysates CB 2.07 ± 0.04 2986.4 ± 936 −produced (PCH25,6) 0.90 ± 0.60in 15.31 ± 2.34 were this study ± 0.04 88.03(Tables 0.44characterised ± 2.48 S21304.2 ± 357 and S3). 1145.1 ±in As shown 306 Ta- B1 2.11 ± 0.03 ± − 0.83 ± 0.61 15.11 ± 1.24 0.44 ± 0.03 86.76 ± ble S2, a protein content of 19.5% was determined for the PCH25,6 sample. This is slightly 2696.2 546 2.95 1170.3 ± 194 1016.2 ± 175 B3 2.25 ± 0.06 b higher 2478.5 ± 687than the protein −0.71 content ± 0.61 15.71 reported ± 1.67 0.45in± the 0.03literature 87.87 ±for 2.09the1116.6 chia seeds ± 297 (16.5%, 981.2 ±accord- 261 ing to the USDA National Nutrient Database for Standard Reference) [28]. B5 2.29 ± 0.08 b 2197.4 ± 449 −0.32 ± 0.57 18.89 ± 2.45 0.50 ± 0.04 88.51 ± 1.62 1086.2 ± 180 960.4 ± 154 As expected, the total lipid content found in the PCH25,6 sample derived from the B10 2.27 ± 0.05 b 2160.9 ± 342 expeller defatted −0.59 ± 0.62 19.16 ± 1.87 reduced was dramatically 0.51 ± 0.02 91.05a±value to 1.6%, 2.63 that is±substantially 1102.1 153 1003.0 ±lower140 compared to the reference values for the chia seeds (30.7% of lipid content). Moreover,1,the Abbreviations are as follows: CB—Control bread; B1, B3, B5, and B10—Breads supplemented with 3, 5, and 10 mg of chia expeller hydrolysate/g of flour; SV—Specific volume; H—Hardness; A— Adhe- ash content was higher compared to the values found in chia seeds (4.8% compared to siveness; R—Resilience; Co—Cohesion; S—Springiness; G—Gumminess (G = H × Co); Ch— Chewiness 33%, (Ch = Grespectively). × S = H × Co × The S). amount Values showof assimilable the average ofcarbohydrates three values ± the were found standard to be 5.9%, deviation (SD). band in- the soluble dicates fibredifference a significant contentbetween stayedthe in the meansrange of 30–35%, (p < 0.05; with both values being comparable Tukey test). to those commonly found in the chia seeds (7.7% and 34.4%, respectively). Importantly, 3.3.3. Texture content the protein Profile Analysis observed for PCH25,6 suggests that the chia expeller hydrolysate A detailed maintains analysis protein and of the textural soluble properties fibre contents (Table 1) showed comparable to thosenofound significant in thedifferences chia seeds, between the hardness which suggest that chiaparameters (H) of the CB, expeller hydrolysates areB1, and B3candidates excellent bread formulations, while for use as dietary the values for B5 and B10 were considerably lower. This trend suggests that the addition supplements. of 5 and 10 mg of Extensive hydrolysates hydrolysates (with with DHhigher a DH = 54.3than± 1.6%) 10% ofarechia expeller/g commonly usedoffor flour themay gen- have eration of functional foods, including hypoallergenic and bioactive foods. In this bread, improved the textural properties of the enriched bread compared to the control study, resulting we obtained in a PCH25,6 higher specific derived volume, from the lower chia firmness, and increase expeller, which has thein the alveolate. particularity In of con- addition taining atohighchanges proteinin the andSpecific VolumeIn(SV) fibre content. and thewith agreement hardness (H),Water this, the breadRetention fortification Ca- pacity (WRC) of this was found to be 1.92 g H2O/g sample (Table S3). This value is in the same range to those observed in previous studies for chia seed protein isolates obtained via three distinct drying methods [29] and from flour obtained via solvent extraction or Foods 2023, 12, 3382 10 of 16 with 5 and 10 mg of hydrolysates also induced slight changes in the other textural properties of the bread, such as the resilience (R) and cohesiveness (Co) of the final baked product. In a previous report, Madruga et al. [5] reported a considerable reduction in the specific volume of the bread when 3 mg of chia seed hydrolysate (with DH = 30%/g of flour) was added to the bread formulation. The authors attributed these changes to a different formation of the “gluten network” due to the increase in the concentration of hydrolysate used in the formulation of the breads. In our study, the improvement in the textural parameters may be caused by the use of a chia expeller derivative with a superior degree of hydrolysis (DH = 54.3 ± 1.6%). 3.3.4. Crumb Colour Analysis Crumb colour is a key property of baked products since, together with volume and texture, it influences consumer acceptance. In our analysis, the crumb colour of the breads supplemented with hydrolysates from the chia expeller were expressed in CIE L*, a*, and b* values—corresponding to brightness, green (−)/red (+), and blue (−)/yellow (+), respectively—and compared to the standard bread (control). Table 2 summarises the colorimetric parameters obtained for the crumb of breads formulated with the chia expeller hydrolysates. In essence, no significant differences were observed in the brightness (L*) of breads B1, B3, and B5 compared to the control bread. Only breads with 10 mg of hydrolysate/g of flour showed a value of 69.39 ± 1.26, which is slightly lower compared to the control bread, which showed a value of 73.26 ± 0.74. This decrease in brightness can be attributed to the darker colour of the chia expeller hydrolysates compared to the wheat flour. However, the effect produced by baking represents only a 5% reduction compared to the control bread. A similar darkening effect was previously observed in another study after the addition of chia flour to the bread formulation [36–38]. Table 2. Colour parameters of the crumbs of breads supplemented with chia expeller hydrolysates. Bread Formulation L*2 a*2 b*2 h (◦ ) ∆E CB 73.26 ± 0.74 4.88 ± 0.39 27.33 ± 0.39 79.88 ± 0.65 0.92 B1 73.07 ± 0.50 4.13 ± 0.13 26.40 ± 0.59 81.11 ± 0.08 0.78 B3 73.54 ± 1.05 4.27 ± 0.10 26.41 ± 0.18 80.82 ± 0.15 1.07 B5 71.12 ± 1.19 4.68 ± 0.28 26.94 ± 0.33 80.15 ± 0.46 1.27 B10 69.39 ± 1.26 b 4.58 ± 0.13 26.15 ± 0.29 80.07 ± 0.17 1.30 Abbreviations are as follows: CB—Control bread; B1, B3, B5, and B10—Breads supplemented with 1, 3, 5, and 10 mg of chia expeller hydrolysate/g of flour. L*—Lightness; a*—Tendency for green–red coloration; b*— Tendency for blue–yellow coloration; h—Hue angle; ∆E—colour difference values. Values show the average of three values ± the standard deviation (SD). b indicates a significant difference between the means (p < 0.05; Tukey test). The values of the a* and b* parameters showed a slight tendency towards reddish and yellow coloration in all the formulations, including the one with the highest concentration of chia expeller hydrolysates. Despite such small changes in coloration upon baking, no substantial differences were observed between the different bread formulations. Typi- cally, the crumb colour of the bread crumbs tended to be yellow (h close to 90◦ ). In this case, the value of the h parameter did not show significant differences between the CB (79.88 ± 0.65) and the bread formulation with the highest level of chia expeller hydrolysate supplementation, B10 (80.07 ± 0.17). Regarding the colour difference values (∆E), the addition of different hydrolysate amounts slightly increased this parameter compared to the control bread. In summary, the results obtained for the chromatic parameters showed no considerable differences between the breads containing chia expeller hydrolysates and the control bread. Foods 2023, 12, 3382 11 of 16 3.4. Antioxidant Activity of Baked Breads Fortified with Chia Expeller Hydrolysates The antioxidant activity of the different bread formulations was evaluated using the ABTS method after the breads were subjected to long-term storage (up to 75 days) at −20 ◦ C. As shown in Figure 3 all fortified breads tested maintained or increased their antioxidant activity over time. Interestingly enough, the B10 bread formulation, which had 10 mg of chia expeller hydrolysate/g of flour, showed the highest antioxidant activity scores and a marked time-dependent increase in antioxidant capacity up to a value of 56 ± 3.9% after 75 days of storage at −20 ◦ C. Under the same conditions, the control bread showed a much lower antioxidant activity (44 ± 2.5%). These results are consistent with those reported by Sayed-Ahmad et al. [37] and Romankiewicz et al. [39], who demonstrated that the addition of chia seed improved the antioxidant activity of wheat bread. Similar results were reported by Zdybel et al. [38], who evaluated the antioxidant potential against DPPH free radical Foods 2023, 12, x FOR PEER REVIEW scavenging of breads added with a waste product from the pressing of chia seed 12 of oil.16In contrast, Segura-Campos et al. [40] demonstrated that the partial replacement of wheat flour by chia protein hydrolysates had no effect on the antioxidant activity of white bread, probably speculate due that to the chia effecthydrolysates expeller of high temperatures could be aduring baking, beneficial causing additive the oxidation for improving theof tryptophan antioxidant and histidine properties or thegeneration of a novel methionineof desulphuration. However, in this study, the functional breads. formulation contained only between 1 and 3 mg hydrolysates/g of flour. Figure 3. Antioxidant activity of breads supplemented with chia expeller hydrolysates. CB—Control Figure 3. Antioxidant activity of breads supplemented with chia expeller hydrolysates. CB—Con- bread; trol B1, bread; B3, B1, B3,B5, B5,and andB10—Breads B10—Breadswith with1,1,3,3,5,5,and and10 10mg mgof ofchia chiaexpeller expeller hydrolysate/g of flour. hydrolysate/g of flour. In our study, the bread formulation supplemented with 10 mg of chia expeller hy- 3.5. Sensory Test: Consumer Acceptability of Selected Bread Fortified with Chia Expeller Hydrol- drolysate/g of flour achieved increased antioxidant activity. Therefore, it is tempting to ysate (B10) speculate that chia expeller hydrolysates could be a beneficial additive for improving the To gain insights antioxidant propertiesintoofconsumer preferences a novel generation of and acceptability functional breads.criteria, consumer-ori- ented testing methods and untrained sensory panels have proven to be a valuable ap- 3.5. Sensory proach [41,42].Test: Consumer In order Acceptability to assess of Selected the consumer Bread Fortified acceptance with Chiainformation and preference Expeller for Hydrolysate the formulations (B10)treated with chia protein hydrolysates in a wheat bread, the B10 formu- lation, To whichgainhadinsights highest into consumer amount preferencesper of hydrolysate and acceptability gram of flour, wascriteria, consumer- selected for a oriented testing methods and untrained sensory panels have proven further preliminary sensory analysis [43]. This bread formulation showed a higher protein to be a valuable approach content and[41,42]. the bestIntextural order toproperties assess the compared consumer acceptance andbread. to the control preference information Moreover, this for the bread formulations formulation treated exhibited with chia a superior protein hydrolysates antioxidant capacity upon in along-term wheat bread, theAs storage. B10 formulation, summarised in which Figure had highest 4, the sensoryamount of of analysis hydrolysate the breadsper gram of flour, supplemented was with 10 selected mg of for a furtherof hydrolysate/g preliminary sensory analysis flour, 17 untrained panellists[43]. This bread awarded formulation the B10 formulation showed with thea higher best protein content and the best textural properties compared to the control bread. possible rating (“I liked it very much”), while 19 panellists gave it the second-best possible Moreover, this bread rating, formulation stating “I liked it”.exhibited Compared a superior antioxidant to the control wheatcapacity upon long-term bread, where the ratingstorage. of “I As summarised in Figure 4, the sensory analysis of the breads supplemented liked it” predominated, a positive trend was found regarding the bread fortified withwith 10 mg 10 of hydrolysate/g of flour, 17 untrained panellists awarded the B10 formulation mg of hydrolysate, which received the best score (Figure 4A). In a similar manner, when with the best carrying out the preference test (Figure 4B), 21 panellists chose bread B10 over the control bread (which was preferred by 9 panellists), while 7 panellists did not present a preference for any particular bread. Out of the 37 panellists, 24 positive responses had to have been obtained for one of the samples to ensure that there was a significant difference (p < 0.05). Foods 2023, 12, x FOR PEER REVIEW 13 of 16 Foods 2023, 12, 3382 preference. In a similar manner, Robles-Ramírez et al. [45] recently reported that the12sub-of 16 stitution of wheat flour with 60% barley flour caused a reduction in consumer preference. Interestingly, the sensory qualities of this bread were improved by adding sesame seeds to the bread formulation. possible rating (“I liked it very much”), while 19 panellists gave it the second-best possible Finally, a triangular test was also carried out to investigate whether the panellists rating, stating “I liked it”. Compared to the control wheat bread, where the rating of “I liked were able to identify fine differences between the three bread samples. As shown in Figure it” predominated, a positive trend was found regarding the bread fortified with 10 mg of 4C, only 12 panellists were able to identify the different bread samples, while 18 of them hydrolysate, which received the best score (Figure 4A). In a similar manner, when carrying pointed out an erroneous sample as different, and 7 of them found no differences. These out the preference test (Figure 4B), 21 panellists chose bread B10 over the control bread results are in agreement with the preference test since there were no significant differences (which was preferred by 9 panellists), while 7 panellists did not present a preference for any between the preference of the panellists towards a particular sample. particular bread. Out of the 37 panellists, 24 positive responses had to have been obtained Taken together, the results from the sensory evaluation suggest that both the control for one of the samples to ensure that there was a significant difference (p < 0.05). Therefore, and fortified breads present similar consumer preferences and overall sensory character- from the 21 positive responses received for the bread fortified with chia hydrolysates, it can istics since consumers could not identify which one corresponded to the formulation for- be inferred that the addition of 10 mg of hydrolysates/g of flour did not show significant tified with the chia expeller hydrolysates. differences in the preferences of the panellists with respect to the control bread. Sensoryanalysis Figure 4. Sensory analysisofofcontrol controlbreads breads (CB) (CB) and and breads breads supplemented supplemented withwith 10 mg10 of mgchia of chia ex- peller hydrolysate/g expeller hydrolysate/gof of flour (B10). flour (A) (B10). Five-point (A) Five-pointhedonic sensory hedonic sensoryevaluation. evaluation.(B) (B)Preference Preferencetest. test. NP—No preference. NP—No preference. (C) (C) Triangular Triangular test. test. In (A), the In (A), the mean mean (red (red square) square) ±±Standard StandardDeviation Deviation(SD) (SD)isis displayed over displayed over the the bars. bars. 4. Conclusions In a recent study, Madruga and coworkers [5] prepared wheat and rice breads sup- plemented with represents This study chia hydrolysates. the first In this study, attempt the authors to develop a wheatconcluded that theenriched bread product addition of chia hydrolysate flour did not affect the consumer preference. The with functional peptides derived from the chia expeller. For this purpose, we first aimedsimilarities in the observations to prepare a between our work andfrom protein hydrolysate previously defatted reported work could chia expeller be due to the via enzymatic reduced hydrolysis impact of sensory with Alcalase, attributes reaching introduced a degree by the of of hydrolysis addition of hydrolysates 54.3 ± 1.6% after 6 h of at the testedatcon- hydrolysis 25 centration. °C. This chia On the contrary, expeller a previous hydrolysate study showed (PCH25,6) by da Silva et al. [44] a notable found that antioxidant the addition activity of 55.8 of ora-pro-nobis ± 0.4%, as determined aculeata) (Pereskiavia the ABTSto salt breadMoreover, method. had a substantial impact composition the proximate on consumervalues prefer- ence. In a similar manner, Robles-Ramírez et al. [45] recently reported that of the PCH25,6 hydrolysate suggested that this additive contains a notable amount of pro- the substitution of wheat tein flour which and fibre, with 60% barley flourtocaused is comparable a reduction the values found ininraw consumer preference. chia seeds. Interest- In this context, ingly, the sensory qualities of this bread were improved by adding the techno-functional characterization of the hydrolysate showed adequate values sesame seeds to the of bread formulation. emulsifying activity, as well as water and oil retention capacities, rendering it compatible Finally, a triangular as a supplemental test was component in also breadcarried out to investigate whether the panellists were formulations. able to identify fine differences between the three bread samples. As shown in Figure 4C, only 12 panellists were able to identify the different bread samples, while 18 of them pointed out an erroneous sample as different, and 7 of them found no differences. These Foods 2023, 12, 3382 13 of 16 results are in agreement with the preference test since there were no significant differences between the preference of the panellists towards a particular sample. Taken together, the results from the sensory evaluation suggest that both the control and fortified breads present similar consumer preferences and overall sensory characteris- tics since consumers could not identify which one corresponded to the formulation fortified with the chia expeller hydrolysates. 4. Conclusions This study represents the first attempt to develop a wheat bread product enriched with functional peptides derived from the chia expeller. For this purpose, we first aimed to prepare a protein hydrolysate from defatted chia expeller via enzymatic hydrolysis with Alcalase, reaching a degree of hydrolysis of 54.3 ± 1.6% after 6 h of hydrolysis at 25 ◦ C. This chia expeller hydrolysate (PCH25,6) showed a notable antioxidant activity of 55.8 ± 0.4%, as determined via the ABTS method. Moreover, the proximate composition values of the PCH25,6 hydrolysate suggested that this additive contains a notable amount of protein and fibre, which is comparable to the values found in raw chia seeds. In this context, the techno-functional characterization of the hydrolysate showed adequate values of emulsifying activity, as well as water and oil retention capacities, rendering it compatible as a supplemental component in bread formulations. Notably, the fortification of wheat breads with different amounts of chia expeller hydrolysates (i.e., 1, 3, 5, and 10 mg of chia expeller hydrolysate/g of flour) did not affect the bread’s textural properties and provided a higher antioxidant activity even after 75 days of storage at −20 ◦ C. The most substantial enhancement in antioxidant activity was observed when 10 mg of chia expeller hydrolysate was added to the bread formulation. More importantly, the bread prepared with the highest content of hydrolysates showed acceptable sensorial attributes that were comparable to the control bread. In a preliminary consumer study, the participants enjoyed trying the bread product supplemented with the chia expeller hydrolysates, but no significant differences in consumer preference were identified between the control and fortified bread samples. This finding is in agreement with the results from a previous study by Madruga and coworkers [5], in which the authors demonstrated that the sensory characteristics of wheat and rice bread supplemented with 3 mg of chia hydrolysate/g flour were comparable to those of the control bread. Taken together, the results presented herein demonstrate that the incorporation of chia expeller hydrolysates into a novel wheat bread formulation could be a valuable way of increasing the protein content of bread. Furthermore, it positions the fortified bread as a functional wheat bread product that offers increased protein content and enhanced antioxidant activity. Ultimately, this approach facilitates the utilisation of a by-product derived from the chia oil industry, offering both significant economic and environmen- tal advantages. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/foods12183382/s1, Figure S1: Process flow diagram for obtaining and characterizing chia expeller protein hydrolysates; Table S1: Summary of the different bread formulations prepared in this study; Table S2: Proximate composition of chia expeller hydrolysates produced with Alcalase at 25 ◦ C for 6 h (PCH25,6) compared to chia seeds; Table S3: Techno-functional characterization of chia expeller hydrolysates produced with Alcalase at 25 ◦ C for 6 h (PCH25,6). Author Contributions: Conceptualization, J.C., J.G.-P., M.G.P. and W.D.O.; methodology, B.O., J.C., M.G.P. and W.D.O.; validation, B.O., F.R.G., M.P.K., J.C., M.G.P. and W.D.O.; formal analysis, B.O., J.C., M.G.P. and W.D.O.; investigation, B.O., F.R.G., M.P.K. and J.C.; resources, J.C., J.G.-P., M.G.P. and W.D.O.; writing—original draft preparation, B.O., J.C., J.G.-P., M.G.P. and W.D.O.; writing—review and editing, J.C., J.G.-P., M.G.P. and W.D.O.; visualization, B.O., J.C., F.R.G., M.P.K., J.G.-P., M.G.P. and W.D.O.; supervision, J.C., J.G.-P., M.G.P. and W.D.O.; project administration, J.C., M.G.P. and W.D.O.; funding acquisition, M.G.P. and W.D.O. All authors have read and agreed to the published version of the manuscript. Foods 2023, 12, 3382 14 of 16 Funding: This research study was funded via Departamento de Ciencias Básicas of the Universidad Nacional de Luján (UNLu) and C82 Project, Ciencia y Tecnología contra el Hambre Program— MINCYT (RESOL-2021-289-APN-MINCYT) grants to MGP and PICT-2018-03271 (2019-2021) and UNLP I+D 2020 project Code X-920, grants to WDO. Institutional Review Board Statement: Not applicable. Informed Consent Statement: All sensory panellists were informed of the procedure, and their consent for participation was obtained before the start of the sensory test. Data Availability Statement: The data used to support the findings of this study can be made available by the corresponding author upon request. Acknowledgments: This study is part of the DSc degree thesis of Brenda Ozón carried out at the UNLP. B. Ozón is a doctoral fellow from the National Agency of Scientific and Technological Promotion (ANPCyT—Argentina); J. Cotabarren and W.D. Obregón are members of the Researcher Career Program of the CONICET, and M.G. Parisi is an established researcher of UNLu. The authors acknowledge Eng. Gustavo Kreymborg from Greenborg Industries S.R.L. for his substantial contribution and valuable support (providing us the starting material—the chia expeller—required for the development of this work). Conflicts of Interest: The authors declare no conflict of interest. References 1. Kiran, A.; Wakeel, A.; Mahmood, K.; Mubaraka, R.; Hafsa; Haefele, S.M. Biofortification of Staple Crops to Alleviate Human Malnutrition: Contributions and Potential in Developing Countries. Agronomy 2022, 12, 452. [CrossRef] 2. Tolve, R.; Simonato, B.; Rainero, G.; Bianchi, F.; Rizzi, C.; Cervini, M.; Giuberti, G. Wheat Bread Fortification by Grape Pomace Powder: Nutritional, Technological, Antioxidant, and Sensory Properties. Foods 2021, 10, 75. [CrossRef] [PubMed] 3. Czarnowska-Kujawska, M.; Starowicz, M.; Barišić, V.; Kujawski, W. 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