Sustainable Integration of Ora-Pro-Nobis (Pereskia aculeata Miller) in Gluten-Free and Lactose-Free Sweet Bread: Impacts on Quality and Functional Properties

Abstract

Ora-pro-nobis (OPN) enriches gluten- and lactose-free bread, while improving nutritional quality and sustainability due to its high nutritional value, adaptability to diverse climates, and low resource requirements for cultivation. This study evaluated the impact of incorporating different concentrations of OPN (0–24%) on the physicochemical (e.g., centesimal composition, specific volume, and color analysis), functional (e.g., total phenolic compounds and antioxidant capacity), and sensory quality (e.g., acceptance test and purchase intent) of gluten-free and lactose-free sweet bread. The results revealed that the addition of OPN led to a 63% increase in protein content and a 65% increase in ash content (p < 0.05). Higher OPN concentrations also enhanced the specific volume by up to 35% (p < 0.05), yielding softer and more voluminous loaves. Texture analysis showed reductions in crumb hardness and chewiness by up to 74.8% and 59.4%, respectively (p < 0.05), attributed to OPN’s water retention and gas-trapping abilities during fermentation. Furthermore, OPN addition resulted in a darker crust and a dark green crumb, with a remarkable increase in total phenolic compounds (up to 464%) and antioxidant capacity (up to 503%) (p < 0.05). Sensory evaluations indicated that OPN did not affect the overall impression compared to the control bread (p > 0.05), with all samples achieving purchase intention scores >3.0 points. Thus, incorporating OPN in gluten-free and lactose-free bread not only enhances nutritional and functional properties but also supports sustainable food production, presenting an innovative solution for consumers with dietary restrictions seeking health-oriented, eco-friendly products.

1. Introduction

Bread is one of the most widely consumed foods across the Americas [1], traditionally made from wheat flour or other types of flour, mixed with liquid, and produced through fermentation and/or baking processes. Additional ingredients may be included, provided they do not alter the bread’s essential characteristics. Bread can come in various forms, with different toppings, fillings, shapes, and textures [2]. In recent years, there has been a growing demand for gluten-free and lactose-free bread, driven by an increasing prevalence of dietary restrictions, food intolerances, and a consumer preference for more inclusive food options [3].

Some individuals may experience an adverse reaction to the proteins present in wheat flour, such as those with celiac disease who exhibit an immune response to gluten [3]. Obtaining gluten-free bread is a significant challenge, as no flour provides the same technological properties of structure, volume, and texture as wheat flour in bread production. This is due to the properties imparted by gluten, such as elasticity, which contributes to gas retention during fermentation, resulting in proper dough expansion and a soft, airy crumb [4]. Among potential substitutes, rice flour and cassava starch can be alternatives to wheat flour; however, challenges exist in terms of the quality, stability, and acceptability of the resulting products [5]. For example, gluten-free bread often suffers from lower specific volume, denser crumbs, and reduced shelf life, which limits its appeal to consumers. Furthermore, the lack of gluten impacts the nutritional profile of these products, creating a need for innovative formulations to address these gaps.

In addition to gluten allergy, there are individuals who are lactose intolerant due to the absence of the lactase enzyme. Consequently, a portion of the population has restrictions on consuming foods that contain milk, such as sweet bread [6]. One of the main strategies is lactose hydrolysis, which involves pre-hydrolyzing lactose before consuming milk and its derivatives. However, the use of lactose-free milk presents a challenge related to production costs, which may hinder the affordability of sweet bread for consumers.

As consumer demand shifts toward healthier, more nutritious, and environmentally sustainable foods, incorporating functional and sustainable ingredients into bread formulations offers a promising solution. Unconventional food plants (UFPs), such as Pereskia aculeata Miller, popularly known as Ora-pro-nobis (OPN), are gaining attention for their potential to enhance both the nutritional value and sustainability of food products [7]. Despite its high nutritional value, OPN has been underutilized due to challenges in processing, potential flavor and texture alterations, cultural factors, and limitations in large-scale cultivation. However, its incorporation into gluten-free and lactose-free bread can address specific gaps in the literature by simultaneously improving nutritional content, functional properties, and consumer acceptance, while offering an eco-friendly alternative [8]. For example, OPN has been successfully integrated into pasta formulations, enriching the product with protein, dietary fiber, calcium, and iron, while reducing cooking loss compared to conventional pasta [7]. OPN has also been used in a variety of other food products, including salads, soups, omelets, pies, and as a flour for enriching cakes [8]. These successful applications demonstrate the potential of OPN to enhance the nutritional profile of various foods, making it an ideal candidate for enriching gluten-free and lactose-free products.

Furthermore, the use of OPN supports more sustainable production practices. As a hardy plant, OPN requires minimal agricultural inputs, such as water and pesticides, which reduces its environmental footprint compared to conventional ingredients. By utilizing locally sourced, underexploited plants like OPN, the reliance on imported ingredients and highly processed alternatives is reduced, further contributing to sustainability [9].

Considering the growing pressures on global food systems—including population growth, climate change, and resource depletion—there is an urgent need for innovative ingredients and sustainable practices to enhance environmental resilience and resource efficiency. Thus, this study aimed to develop gluten-free and lactose-free sweet bread enriched with different concentrations of OPN, evaluating its physicochemical, functional, and sensory qualities. The incorporation of OPN not only improves the bread’s nutritional profile but also promotes more sustainable production practices, increasing the availability of eco-friendly food options for consumers with dietary restrictions.

2. Materials and Methods

2.1. Experimental Design: Formulation of Gluten-Free and Lactose-Free Sweet Bread Enriched with OPN

Breads from each treatment were produced in three independent replications to ensure reliable and reproducible results. This implies that the complete bread production process was carried out three times for each treatment. For each replicate, analyses were performed in triplicate, as described in Section 2.4. To produce the bread, the following ingredients were used: water, commercial rice flour (Urbano Agroindustrial Ltd.a, Guarulhos, SP, Brazil), commercial potato starch (VITAO Alimentos, Curitiba, PR, Brazil), commercial cassava starch (AMAFIL Alimentos, Cianorte, PR, Brazil), fresh OPN leaves (with a proximate composition of 86.80 ± 1.98 g of water/100 g; 3.60 ± 0.71 g of protein/100 g; 1.88 ± 0.34 g of ash/100 g, and 0.32 ± 0.12 g of lipids/100 g, supplied by producers from the city of Barbacena, Minas Gerais, Brazil), egg, sugar, soybean oil, salt, and biological yeast. Five formulations were prepared, varying the concentration of fresh OPN leaves (0, 6, 12, 18, and 24% in relation to the total amount of flours), as presented in

Table 1. Formulation of gluten-free and lactose-free sweet bread enriched with different concentrations of Ora-pro-nobis.

Ingredients	F1 (0% OPN)	F2 (6% OPN)	F3 (12% OPN)	F4 (18% OPN)	F5 (24% OPN)
Rice flour (g)	190	190	190	190	190
Potato starch (g)	150	150	150	150	150
Cassava starch (g)	80	80	80	80	80
Water (g)	400	400	400	400	400
Eggs (g)	100	100	100	100	100
Sugar (g)	50	50	50	50	50
Oil (g)	40	40	40	40	40
Ora-pro-nobis (g)	0	25	50	75	100
Yeast (g)	10	10	10	10	10
Salt (g)	5	5	5	5	5

. It is worth noting that the percentage of each ingredient in the formulation was calculated in relation to the total amount of flours (rice flour, potato starch, and cassava starch), following the baking protocol.

Manufacturing Process of Gluten-Free and Lactose-Free Sweet Bread Enriched with OPN

The bread manufacturing process was carried out following the procedures described by Sandri et al. [10]. Initially, the dry ingredients (mixture of rice flour, potato starch, cassava starch, sugar, salt, and yeast) were added to a mixer, and the liquid ingredients were carefully added (water, egg, and soybean oil), along with fresh OPN leaves, which were used in their natural form (not dried or ground). These leaves were sanitized following a standard protocol with chlorinated water (200 mg/L for 10 min) before being incorporated into the mixture. The mixture was stirred for 4 min at a speed of 110 rpm. Subsequently, the dough was shaped and placed in an incubator at 37 °C and 85% relative humidity for approximately 40 min for the fermentation process. After this period, the samples were baked in an electric oven at a temperature of 170 °C for 25 min. Then, the bread was cooled to room temperature (23 °C) and stored in appropriate packaging for further analysis. All analyses were performed within a period of up to 12 h after bread production to ensure the evaluation of the bread’s freshness and its original quality attributes.

2.2. Determination of the Physicochemical and Functional Properties of Gluten-Free and Lactose-Free Sweet Bread Enriched with OPN

2.2.1. Determination of the Centesimal Composition

The moisture content was determined according to method 44-15A of the AACC [11], and the protein content was evaluated using method 991.22 according to AOAC [12], using a conversion factor of 6.25. The total lipid content was determined by the Am 5-04 method [13], the ash content according to method 986.25 [12], and the carbohydrate content was obtained by the difference, considering the fiber content.

2.2.2. Specific Volume Evaluation

To determine the specific volume of the bread, the apparent volume of the bread was initially determined using the rapeseed displacement method (AACC method 10-05.01) [11]. Then, the specific volume was calculated by dividing the apparent volume of the bread by its respective mass, according to Equation (1) (Equation (1)).

$$\mathrm{S}\mathrm{p}\mathrm{e}\mathrm{c}\mathrm{i}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{ }\mathrm{v}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e}({\mathrm{c}\mathrm{m}}^3/\mathrm{g})={\displaystyle \frac{\mathrm{a}\mathrm{p}\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{ }\mathrm{v}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e}\mathrm{ }\mathrm{o}\mathrm{f}\mathrm{ }\mathrm{b}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{d}}{\mathrm{b}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{d}\mathrm{ }\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s}}}$$

(1)

2.2.3. Evaluation of Crust and Crumb Color

The analysis of the crust and crumb color of the bread was performed using a Color i5 colorimeter (XRite, Grand Rapids, MI, USA) with D65 illuminant and a 10° angle. The samples were carefully cut, and measurements were taken in triplicate at 3 different points, resulting in a total of nine readings per sample. The surface and crumb color of the bread were described using the CIELab scale: L* (from black to white), a* (from green to red), and b* (from blue to yellow). Additionally, the total color difference (ΔE) of the crust and crumb of the bread was determined, as shown in Equation (2), using the sample without OPN as a reference.

$$∆E=\sqrt{{(∆L\mathrm{*})}^{²}+{(∆a\mathrm{*})}^{²}+{(∆b\mathrm{*})}^{²}}$$

(2)

2.2.4. Texture Profile Analysis (TPA)

The texture profile analysis (TPA) was performed using a TA.XT plus texture analyzer equipped with Exponent Software v.6.1.4, following the procedure described by Chikpah et al. [14]. The textural parameters determined from the force–time graph of TPA were hardness (g, given by the maximum force of the first compression cycle), cohesiveness (given by the ratio between the positive force area of the second compression cycle and the positive force area of the first compression cycle), elasticity (calculated by dividing the detected height distance of the second compression cycle by the original compression distance), resilience (calculated by dividing the ascending energy of the first compression by the descending energy of the first compression), and chewiness (given by the product of hardness, cohesiveness, and elasticity).

2.2.5. Determination of Total Phenolic Compounds and Antioxidant Capacity

Extraction for analysis of total phenolics and antioxidant capacity was performed according to the methodology described by Gonçalves et al. [15]. The quantification of total phenolic compounds was performed according to the method described by Singleton and Rossi [16], and the total phenolic content was expressed in mg of gallic acid equivalents (GAE) per 100 g of sample—(mg GAE/100 g). For antioxidant capacity, the assays with the 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical were performed according to the method described by Re et al. [17], and the results were expressed in µmol of Trolox equivalent (TE) per 100 g of sample—(µmol TE/100 g).

2.3. Sensory Evaluation

Sensory analysis was carried out at the Department of Food Technology at the Federal University of Viçosa. Initially, the project was approved by the research ethics committee with CAAE 33433620.0.0000.5153 code. For the evaluation, three separate batches of each bread type were produced. These batches were randomly served to 90 untrained participants (39 men and 51 women, aged 19 to 57 years) who were selected from among UFV students and employees. These participants did not have gluten allergenicity and lactose intolerance, and this was verified through a questionnaire applied prior to the sensory analysis. In addition, they were informed that they would be evaluating gluten-free and lactose-free bread enriched with Ora-pro-nobis.

Sensory analysis was performed using an affective test with a nine-point hedonic scale (1 = extremely disliked to 9 = extremely liked) for the attributes of the appearance, color, aroma, taste, texture, and overall impression of the breads. According to sensory characteristics, the parameters were evaluated based on flavor intensity for taste, visual appeal for color, overall aspect for appearance, aroma intensity for aroma, consistency for texture, and the combination of all attributes for the overall impression. Initially, samples measuring 2.5 cm × 2.5 cm × 2.5 cm were placed on white plates and identified with random three-digit numbers. During testing, samples were presented separately in balanced order, and participants were instructed to rinse their mouths with water between samples to minimize any residual effects. In addition, purchase intent was also assessed using a five-point scale, ranging from certainly would not buy (1) to certainly buy (5).

2.4. Statistical Analysis

The procedures and experiments were conducted in three repetitions, and the results obtained from all the analyses were expressed as mean ± standard deviation. The analysis of variance (ANOVA) was performed to compare the effects among different treatments, and Tukey’s test was used to assess significant differences between them at a 5% level of significance (p-value < 0.05). The statistical analyses were conducted using the Statistica software, version 7.0 (StatiSoft Inc., Tulsa OK, USA).

3. Results and Discussion

3.1. Centesimal Composition

Figure 1 presents the composition of the five samples of gluten-free and lactose-free sweet breads, enriched with different concentrations of Ora-pro-nobis (OPN), including the control bread without OPN (0 to 24%). It was observed that, as the concentration of OPN increased, the protein content (2.4 to 3.9%, p < 0.05) and ash content (0.89 to 1.47%, p < 0.05) gradually increased, while the carbohydrate content, calculated by difference and considering the fiber content, decreased (44.5 to 39.3%, p < 0.05). Additionally, no difference in moisture content was found for the samples with added OPN (p > 0.05). However, compared to the control sample (0% OPN), the F5 sample (24% OPN) had a higher water content (increased from 51.1% to 54.3%). Lastly, a low lipid concentration was found in the samples ranging from 0.91% to 1.06%, with no significant differences among them.

Overall, these results suggest that the addition of OPN, known for its remarkable nutritional profile [18], specifically in this study, 27.3% of protein and 14.2% of minerals (on a dry weight basis), can be an effective strategy to increase the protein and mineral content in gluten-free and lactose-free sweet breads. These findings are supported by the literature, which highlights that, on a dry weight basis, OPN leaves are rich in proteins (25–28%), minerals (14.2–20.1%), and total dietary fiber (39.1%) and contain notable levels of calcium (3420 mg/100 g), magnesium (1900 mg/100 g), manganese (46.4 mg/100 g), zinc (26.71 mg/100 g), and iron (14.18 mg/100 g) [19]. Additionally, these leaves are a source of vitamins A, C, and folic acid, contributing to their high nutritional value [8,19]. These characteristics highlight OPN as a highly nutritious plant, making it an excellent ingredient for enhancing the nutritional quality of food products [20]. In comparison, similar studies highlight the potential of OPN, such as improving protein, dietary fiber, calcium, and iron content in pasta, along with its cooking characteristics [7]. OPN mucilage has also been effective as an emulsifier and fat replacer in meat products, reducing fat content, while maintaining good sensory acceptance and a good nutritional profile [21]. These findings reinforce the value of OPN as a functional and nutritious ingredient in food applications.

3.2. Specific Volume and Color of Crust and Crumb

The specific volume (Figure 2) is directly correlated with the amount of gas retained by the bread mass during the fermentation and baking process. Thus, the higher the specific volume, the softer and lighter the bread. It is observed that, with increasing OPN concentration, there was a significant increase in the specific volume of the breads (p < 0.05), with values ranging from 1.30 cm³/g for the control formulation (F1) to 1.75 cm³/g for the formulation with the highest OPN concentration (F5—24% OPN) (Figure 2). This result indicates that the addition of OPN to gluten-free bread can be an interesting strategy to increase the amount of gas retained in the dough, likely due to OPN’s water retention and ability to trap gas, making the bread softer and more voluminous. Thus, enriching with OPN can be a promising alternative to produce gluten-free and lactose-free sweet breads, as these formulations usually have low volume [22]. In this case, the addition of OPN can improve the appearance characteristics of these products, making them more appealing to consumers seeking healthier and more nutritious options.

Figure 3 presents the results of the color parameters (L*, a*, and b*) for the crust and crumb of the breads with added OPN. The increase in OPN concentration significantly impacted the color of the crust and crumb of the bread, decreasing the values of L*, a*, and b* (p < 0.05), indicating lower color saturation compared to the control bread (without OPN). This led to the development of a dark greenish color in both the crust and crumb of the breads, due to the concentration of green compounds present in OPN leaves.

The color of the crust plays a crucial role in the product’s attractiveness for consumers. A darker color is often preferred by consumers, as it suggests a more intense Maillard reaction, which contributes to the flavor and aroma of the bread [23]. However, in gluten-free bread, achieving this darker crust is particularly challenging, as lighter colors are more common. In this regard, the results indicate that the addition of OPN can help improve the crust color, making it darker and more appealing to consumers. On the other hand, the greenish color of the crumb could be perceived negatively, as it deviates from the typical white-yellowish color expected for sweet bread [24]. This unusual color, caused by the chlorophyll in OPN, may reduce consumer interest, particularly in markets where traditional expectations for sweet bread appearance are strong. Studies have shown that deviations from expected visual attributes can directly impact product acceptance, emphasizing the importance of considering consumer preferences during product development. Moreover, addressing these limitations through targeted marketing strategies is essential to position the product as a functional and nutritious option that aligns with health-conscious consumer values. From a comparative perspective, the results align with previous findings that protein addition in bread formulations significantly influences crust coloration. For instance, Pico et al. [24] demonstrated that higher protein levels enhance Maillard reactions, leading to darker crusts. This effect was observed across gluten-free breads enriched with both animal and vegetable proteins, where differences in amino acid composition influenced crust coloration [24,25]. However, while the dark crust color may enhance consumer acceptance, the greenish crumb represents a critical limitation. Unlike the findings for animal proteins in gluten-free breads, which produced more favorable crust tones [24], the green hue in OPN-enriched bread could alienate consumers unfamiliar with such attributes. This highlights the need to emphasize the nutritional and functional benefits of OPN during product marketing to mitigate potential negative perceptions associated with color deviations.

For summarizing the results,

Table 2. Total color difference (ΔE) of the crust and crumb of different formulations of gluten-free and lactose-free sweet breads enriched with different concentrations of Ora-pro-nobis.

Sample	Total Color Difference (ΔE)
	Bread Crust	Bread Crumb
F1 (0% OPN)	Reference *	Reference *
F2 (6% OPN)	7.3 ± 2.1 d	12.6 ± 2.5 c
F3 (12% OPN)	14.3 ± 2.2 c	18.9 ± 3.1 b
F4 (18% OPN)	20.9 ± 1.1 b	23.2 ± 1.5 ab
F5 (24% OPN)	24.7 ± 2.3 a	26.9 ± 2.7 a

* Reference sample: sample used as a reference for comparing the ΔE of the crust and crumb of the breads. Different letters indicate significant differences between samples according to Tukey’s test at a 5% level of significance (p < 0.05).

presents the total color difference (ΔE) of the crust and crumb of gluten-free and lactose-free sweet bread enriched with different concentrations of Ora-pro-nobis. The control sample (F1, 0% OPN) was used as a reference for the comparative evaluation of the impact of OPN addition on ΔE. The results showed that the addition of OPN significantly affected the color of both the crust and crumb of the bread. According to the interpretation by Choi et al. [26], ΔE values greater than 2 confirm a visible color difference between the samples. Therefore, based on the obtained values (Table 2), it can be observed that, from 6% OPN onwards, consumers would likely be able to perceive a color difference in this sample compared to the control sample. Furthermore, the higher the concentration of OPN, the greater the total color difference compared to the control sample (maximum differences of up to 24.7 and 26.9 for the crust and crumb, respectively).

Figure 4 shows the images of the crust and crumb of the bread, illustrating the impact of OPN addition on the color characteristics of the bread. As the concentration of OPN increased, the intensity of the dark green color observed (Figure 4) also increased. As mentioned earlier, the dark green color is not characteristic of sweet bread. However, consumers have associated this color with products that have high nutritional value, due to the use of less processed flour with higher mineral and fiber content [27]. Therefore, to ensure the success of the product, it is crucial for companies responsible for producing these foods to market them appropriately, emphasizing the concept of functional, nutritious, healthy, and clean label foods.

3.3. Texture Profile Analysis (TPA)

The texture of bread is a critical factor that directly impacts product acceptability. In traditional bread, it is mainly influenced by the formation of the gluten network, which traps carbon dioxide gas during fermentation, resulting in increased volume and reduced crumb hardness [28].

Table 3. Texture profile analysis (TPA) of gluten-free and lactose-free sweet breads enriched with different concentrations of Ora-pro-nobis.

Sample	Hardness (g)	Cohesiveness	Elasticity	Resilience	Chewability
F1 (0% OPN)	230 ± 22 a	0.48 ± 0.04 c	0.93 ± 0.01 c	0.12 ± 0.01 b	102.6 ± 13.2 a
F2 (6% OPN)	179 ± 14 b	0.56 ± 0.04 bc	0.95 ± 0.01 bc	0.14 ± 0.01 b	94.3 ± 12.2 a
F3 (12% OPN)	132 ± 15 c	0.63 ± 0.04 b	0.95 ± 0.02 abc	0.18 ± 0.02 a	79.0 ± 14.2 ab
F4 (18% OPN)	102 ± 14 c	0.71 ± 0.03 a	0.97 ± 0.01 ab	0.20 ± 0.01 a	70.6 ± 10.7 b
F5 (24% OPN)	58 ± 11 d	0.74 ± 0.04 a	0.98 ± 0.02 a	0.22 ± 0.02 a	41.6 ± 9.7 c

Different letters indicate significant differences between samples according to Tukey’s test at the 5% level (p < 0.05).

presents the results of the TPA of gluten-free and lactose-free sweet breads enriched with different concentrations of OPN.

In comparison to the control sample (F1), it was observed that hardness and chewiness decreased as the concentration of OPN increased (reductions of up to 74.8% and 59.4%, respectively) (p < 0.05). These results can be attributed to the ability of OPN to retain water and trap gases produced during fermentation, minimizing water and air loss during baking. This happens because the mucilage in OPN contains various hydrophilic groups, such as hydroxyl groups, which are capable of forming hydrogen bonds with water molecules, contributing to its high water and air retention capacity [21,29]. Consequently, these effects contributed to the softening of the bread crumb texture. Additionally, the cohesiveness and elasticity of the breads enriched with OPN increased with the increasing concentration of OPN (maximum increases of 54.2% and 5.4%, respectively) (p < 0.05), suggesting that these breads exhibited a more elastic and cohesive crumb structure, which may be correlated with the formation of an additional protein network (enriched by OPN). Regarding the resilience of the breads enriched with OPN, an increase (up to 83.3%) was also observed with the increasing addition of OPN (p < 0.05), suggesting that OPN can help maintain the original height of the bread and prevent it from becoming dense or compact.

Comparatively, these results help explain the increase in the specific volume of the breads enriched with OPN. Similarly, other studies also report that the increase in specific volume of bread has a direct effect on reducing hardness [30]. Therefore, the OPN-enriched breads showed a positive impact on texture with higher volumes, resulting in a softer and more porous crumb, which can positively influence the acceptance of these products.

3.4. Total Phenolic Compounds

The results show a progressive increase in the concentration of total phenolic compounds with the increase in OPN concentration in the breads (Figure 5). Formulation F1, which did not contain OPN, had the lowest content of phenolic compounds (p < 0.05), with an average of 25.3 mg GAE/100 g. In contrast, formulation F5 showed average values of up to 142.8 mg GAE/100 g, representing a 464% increase (p < 0.05).

Previous studies have highlighted the presence of phenolic compounds in OPN leaves, particularly caffeic acid, which accounts for approximately 49% of the total phenolic content [8], thus justifying the results found in this study. Therefore, the use of OPN as a source of functional enrichment in food products can be a promising alternative for the food industry, which is increasingly seeking to meet consumer demand for healthy products. Additionally, it should be emphasized that the addition of OPN to gluten-free and lactose-free breads can contribute to diversifying the offering of nutritious foods for individuals with dietary restrictions, such as gluten allergy or lactose intolerance. In line with this, previous studies have shown that the inclusion of unconventional ingredients in food formulations can result in improved nutritional profiles and higher antioxidant activity compared to conventional formulations [31].

3.5. Antioxidant Activity (ABTS and DPPH)

Figure 6 presents the results of the antioxidant capacity evaluated by the ABTS and DPPH methods for gluten-free and lactose-free sweet breads enriched with different concentrations of Ora-pro-nobis. Based on the results obtained, a significant increase in antioxidant capacity was observed for both ABTS and DPPH radicals with increasing OPN concentration (p < 0.05). Sample F5, with the highest concentration of OPN (24%), showed the highest antioxidant capacity (p < 0.05) by both the ABTS and DPPH methods (not significantly different from sample F4), with values of 1188 µmol TE/100 g (an increase of 503% compared to F1) and 481.21 µmol TE/100 g (an increase of 303% compared to F1), respectively. In contrast, sample F1, which did not contain OPN, exhibited the lowest values (p < 0.05) (Figure 6).

Thus, the addition of OPN to bread was considered satisfactory, as it increased the content of phenolic compounds as well as the antioxidant activity of the final products. This strategy has also been highlighted in other foods [32], and the antioxidant activity has been correlated with the presence of flavonoids, proteins, and caffeic acid found in OPN [8]. Therefore, the addition of OPN in bread may be promising for the food industry aiming to enhance the antioxidant activity of these products, consequently offering health benefits to consumers, as antioxidant action is important in preventing diseases associated with oxidative stress [33]. However, further studies are needed to determine the optimal dose of OPN to achieve these benefits, as well as in vivo analyses to confirm the impact of this addition on the functionality of the product.

3.6. Sensory Evaluation

Table 4. Sensory evaluation of gluten-free and lactose-free sweet breads enriched with different concentrations of Ora-pro-nobis.

Sample	Sensory Attributes						Purchase Intent
	Appearance	Color	Aroma	Taste	Texture	Overall Impression
F1 (0% OPN)	7.7 ± 1.0 a	8.1 ± 0.7 a	7.1 ± 1.1 a	6.3 ± 1.1 a	6.1 ± 1.1 a	6.7 ± 1.0 a	3.4 ± 0.8 a
F2 (6% OPN)	7.0 ± 1.2 a	7.6 ± 0.8 ab	6.4 ± 0.8 a	6.1 ± 1.1 a	6.4 ± 1.4 a	6.1 ± 1.1 a	3.3 ± 0.8 a
F3 (12% OPN)	7.0 ± 1.0 a	7.4 ± 0.8 ab	6.7 ± 1.0 a	6.4 ± 1.3 a	6.1 ± 1.1 a	6.4 ± 0.8 a	3.4 ± 0.8 a
F4 (18% OPN)	7.1 ± 1.2 a	7.6 ± 0.8 ab	6.7 ± 0.8 a	6.4 ± 1.4 a	6.4 ± 1.4 a	6.7 ± 1.1 a	3.4 ± 0.8 a
F5 (24% OPN)	7.3 ± 1.1 a	7.3 ± 0.5 b	6.3 ± 1.4 a	6.0 ± 1.6 a	6.6 ± 1.5 a	6.0 ± 1.4 a	3.0 ± 0.9 a

Different letters indicate significant differences between samples according to Tukey’s test at a 5% level of significance (p < 0.05).

presents the results of the sensory evaluation of the breads, for the attributes of appearance, color, aroma, taste, texture, and overall impression, as well as purchase intention. Overall, no differences were observed among the formulations for the attributes of appearance, aroma, taste, texture, and overall impression (p > 0.05). This result is important, as it indicates that the addition of OPN did not interfere with the overall acceptance of the breads. Furthermore, average scores above 6.0 were observed for the overall impression, indicating that the products were accepted by the participants. In the study by Silva et al. [34], which evaluated the addition of OPN flour to bread, similar results were obtained, indicating that the addition of OPN can be well accepted by consumers as long as the concentration of OPN added is appropriate.

However, regarding the attribute of color, despite formulation F5 (24% OPN) obtaining a high average score (7.3), this value was lower compared to the control breads (F1, without OPN, average 8.1). Suggesting that the dark green color (more evident in sample F5) negatively affected the color of these breads. Therefore, it is important for the food industry to work on the visual presentation of the bread, using marketing strategies, such as incorporating a more attractive visual on the product packaging, highlighting the nutritional benefits and the presence of OPN. Additionally, other strategies can be implemented, such as the use of natural colorants that can improve the bread’s color without compromising its nutritional and sensory quality. Regarding purchase intention, all formulations had average scores (≥ 3.0) without significant differences among them (p > 0.05), indicating that consumers would have an intention to purchase these products. Therefore, it is important for bakeries to invest in the visual presentation of the bread, using marketing strategies, such as attractive packaging that highlights the nutritional benefits and the presence of OPN in the products. In addition, the use of advanced techniques, such as electronic nose or electronic tongue, to analyze the characteristic components introduced by OPN could be explored in future studies.

4. Conclusions

The incorporation of OPN into gluten-free and lactose-free sweet bread effectively enhanced the nutritional and functional profile of the product, increasing protein and mineral content, specific volume, and antioxidant capacity, while also reducing crumb hardness and chewiness. These improvements contribute to a softer and more voluminous product—an advantageous feature for the baking industry. Although the addition of OPN affected the bread’s crumb color, which may pose a challenge for consumer acceptance in traditional markets, this limitation can be mitigated through strategic marketing or the incorporation of natural colorants. Importantly, sensory evaluations indicated that the overall acceptance and purchase intent were not negatively impacted.

These findings highlight the potential of OPN as a functional ingredient to diversify product offerings for individuals with dietary restrictions, such as gluten allergy or lactose intolerance, while promoting sustainability by utilizing plant-based, eco-friendly raw materials. For the baking industry, OPN-enriched bread offers a promising avenue to meet the growing consumer demand for healthier and more sustainable food options.

Future research should explore the industrial scalability of OPN-enriched bread, with a particular focus on shelf-life analysis, stability tests, and the exploration of other unconventional plant-based flours (UFPs) to further diversify gluten-free formulations. Additionally, in vivo studies are necessary to evaluate the nutritional and functional impacts of OPN consumption on consumer health, providing robust evidence for its potential as a health-promoting ingredient.