Multidimensional Evaluation of Local Rye Bread Fortified with Whey as a Model for Food Waste Valorization: From Recipe Development to Consumer Acceptance

Abstract

The growing demand for functional and sustainable foods has driven food innovation, enhancing its nutritional value. This study aimed to develop a nutritious bread using local rye from the Trás-os-Montes region of Portugal and incorporating whey, a by-product of the dairy industry, as a replacement for water. Three bread formulations were tested: a traditional recipe with 37.5% rye flour and water (Control—CTR); the same recipe using whey instead of water (Rye Whey—RW); and a formulation with 100% local rye and whey replacing water (Full Rye Whey—FRW). Nutritional composition was assessed, including moisture, ash, protein, dietary fiber, sodium, potassium, lipids, and carbohydrates. Sensory analysis included both quantitative descriptive analysis and consumer acceptance testing. Microbiological quality was also evaluated. Whey-containing samples showed lower moisture and increased levels of ash, lipids, carbohydrates, and potassium. RW had the highest protein content (6.54 ± 0.28 g/100 g, p < 0.05), while FRW exhibited the highest dietary fiber (6.96 ± 0.15 g/100 g, p < 0.05). RW demonstrated a balanced nutritional and sensory profile, with high consumer acceptance. Overall, the combination of local rye and whey presents a promising strategy for producing nutritious bread while valorizing local agricultural resources and dairy by-products. These findings support sustainable food production practices and contribute to circular economy approaches.

1. Introduction

The food industry is one of the largest global markets, continuously shaped by innovation and evolving consumer demands. According to Innova Market Insights (2025), 10 major food and beverage trends are shaping product development, highlighting a shift towards personalized nutrition, gut health, the reinvention of traditional foods, and the valorization of local and sustainable ingredients [1]. Simultaneously, circular economy principles are fostering the reintroduction of underutilized regional crops and by-products, contributing to the sustainability and resilience of agri-food systems [2].

Rye (Secale cereale) is an underexploited cereal historically cultivated in the Trás-os-Montes region of Portugal. This cereal is well adapted to poor soils and extreme climatic conditions, thriving in marginal agricultural areas [3,4]. Despite its resilience and historical presence, rye production in Portugal has declined sharply, particularly between 1999 and 2019 [4,5]. In contrast, rye remains central to diets in Northern Europe, particularly in countries such as Finland, Germany, and Poland, where its nutritional benefits and role in traditional food culture are widely recognized [6]. From a nutritional perspective, rye is rich in dietary fiber, particularly arabinoxylans (6–12%), β-glucans (1.5–2.6%), fructans (4–8%), and lignans, many of which have prebiotic activity and support the growth of beneficial gut bacteria such as Lactobacillus spp. and Bifidobacterium spp. [7]. It also contains complex carbohydrates (starch, 55–70%), plant-based proteins (approximately 9–12%), and micronutrients such as iron, zinc, and B vitamins [8]. Moreover, rye contains phenolic compounds, such as ferulic acid and caffeic acid, which have antioxidant, anti-inflammatory, and anticancer potential [9]. Its fiber matrix helps regulate glycemic response, prolong satiety, and modulate gut microbiota, particularly enhancing the production of short-chain fatty acids (SCFAs), such as butyrate, which support intestinal barrier function and immune regulation [6,10]. Despite its favorable nutritional profile and functionality, rye remains underused in the Portuguese baking industry, where its use is mostly traditional, and innovation, though emerging, is still limited.

Simultaneously, the dairy industry, particularly in northern and inland regions of Portugal, produces substantial volumes of whey as a by-product of cheese manufacturing. The valorization of whey aligns with current trends in the functional food sector, notably the increasing demand for ingredients with added nutritional value [11,12,13]. In this context, the European whey protein market reached USD 1.98 billion in 2022 and is expected to attain USD 4.41 billion by 2030 [14]. These projections highlight a sustained increase in demand for high-protein, functional ingredients, representing an untapped opportunity for developing potentially functional and sustainable foods. Whey represents approximately 85–90% of the original milk volume and retains around 55% of its original nutrients [15]. Once considered a waste by-product with substantial environmental impact, primarily due to its high biochemical (BOD) and chemical oxygen demand (COD), attributed principally to its lactose content (which accounts for 70–75% of total solids), cheese whey is now increasingly recognized as a valuable resource within circular economy strategies [16]. Whey contains high-quality proteins, including β-lactoglobulin, α-lactalbumin, and immunoglobulins, which offer high digestibility, a balanced amino acid profile, and functional properties such as emulsification, gelling, and foaming [13]. It is also a source of calcium, phosphorus, and B-complex vitamins, and exhibits antimicrobial, antihypertensive, and immunomodulatory properties [17].

Whey valorization is increasingly emphasized in sustainable food innovation, with applications in beverages, dairy alternatives, and baked goods. In baking, the incorporation of whey protein has been shown to improve textural properties, such as hardness, chewiness, and fracturability, which may contribute to product stability and enhance protein content [18,19]. However, according to the best of our knowledge, no studies have yet been published on the use of whey proteins in solution for the preparation of rye bread, highlighting a clear gap in both scientific research and commercial innovation.

To address this gap, the present study aimed to develop and characterize innovative rye-based bread formulations using whey protein in solution as a replacement for water, thereby exploring the combined potential of two underused regional resources from the Trás-os-Montes region within a circular economy framework. Specifically, three formulations were developed: (i) a control bread composed of wheat, rye, and water; (ii) a rye-wheat bread in which water was replaced with whey in solution; and (iii) a formulation made exclusively with local rye and whey. These breads were subjected to detailed physicochemical, nutritional, microbiological, and sensory analyses to assess the impact of whey incorporation on bread composition, structure, and consumer perception. This study addresses a combination of foods—whey in solution and rye flour—that has been rarely explored in the literature, particularly in non-wheat matrices with reduced gluten content. By tackling this technological challenge, the work contributes to expanding the use of dairy by-products and promotes the diversification of cereal-based products. Moreover, it supports the valorization of local agricultural and dairy resources, offering new opportunities for sustainable food innovation and regional economic development.

2. Materials and Methods

This is an experimental study in which rye-based bread samples were prepared and evaluated at the Kitchen Lab and the Sensory Analysis Laboratory of the University of Trás-os-Montes and Alto Douro (UTAD). All procedures were conducted in sanitized facilities, following good manufacturing practices to ensure hygiene and food safety.

2.1. Whey Preparation from Fresh Cheese Production

Whey was extracted from fresh cheese production conducted in the Kitchen Lab (Vila Real, Portugal). The procedure was adapted from artisanal techniques to ensure standardization and reproducibility for food application. In each batch, 4 L of pasteurized full-fat cow’s milk (Leite Vigor, Porto, Portugal) was used and heated to 36 °C. After reaching the target temperature, 1.5 mL of calcium chloride (diluted in chlorine-free water) was added under gentle stirring. The addition of calcium chloride aimed to enhance casein coagulation by promoting micellar aggregation and slightly lowering the pH. Subsequently, 1.5 mL of rennet diluted in water was incorporated, and the mixture was gently stirred to ensure homogeneity. Coagulation proceeded at ambient temperature (20–22 °C), and curd development was monitored by visual inspection and physical assessment. Once a firm curd was achieved, it was cut into small pieces to facilitate syneresis and promote efficient whey separation. The mixture was allowed to decant for a sufficient period to ensure proper separation of whey from the curd. The resulting whey was then clarified by sieving through a standard kitchen sieve to remove residual curd particles. The clarified whey was collected and stored at 4 °C until further use in bread formulations.

2.2. Formulation and Preparation of Rye-Based Breads

Three experimental bread formulations (Figure 1) were prepared using type 85 rye flour sourced from a regional producer in Bragança, Portugal (Moagem do Loreto). Fresh-pressed baker’s yeast (Saccharomyces cerevisiae) was used as the leavening agent in all formulations. The control formulation (CTR) was prepared with 2.5 kg of rye flour, 1.5 kg of wheat flour, 60 g of salt, 40 g of bread improver, and 200 g of fresh-pressed baker’s yeast (Saccharomyces cerevisiae). A total of 2.7 L of water was used as the liquid component. The second formulation, Rye Whey (RW), used the exact quantities of rye and wheat flour as the control, along with identical amounts of salt, improver, and yeast. In this case, 2.3 L of cow’s milk whey replaced water as the liquid component. The third formulation, Full Rye Whey (FRW), was composed exclusively of rye flour (4 kg), with 60 g of salt, 40 g of bread improver, and 150 g of yeast. This formulation also used 2.3 L of cow’s milk whey as the liquid input. The whey used in the RW and FRW formulations was freshly produced from cow’s milk cheese-making, stored at 4 °C until use, and brought to room temperature prior to incorporation into the dough. Whey production was performed either on the same day or the day before the bread preparation to ensure freshness and consistency.

In all cases, the liquid ingredients (whey or water) were adjusted to a temperature of 25 °C before incorporation. Dough mixing was performed in a planetary mixer (Ferneto, Vagos, Portugal). All dry ingredients were first combined, and the initial volume of liquid was gradually introduced. The mixing time was set at 20 min for the 100% rye formulation and 13 min for the rye-wheat blends. Once the desired dough texture was achieved, the dough was lightly dusted with flour and transferred to a proofing chamber (Ramalhos, Agueda, Portugal) set to 30 °C and 90% relative humidity, where it remained for approximately 50 min. After the first fermentation, the dough was divided into portions of approximately 700 g and shaped into loaves. A second proofing was carried out at 35 °C under the same relative humidity conditions for about 20 min.

Immediately before baking, the loaves were inverted, dusted with flour, and scored with a blade. Baking was conducted in a steam-injected deck oven (Ramalhos, Portugal) that had been preheated for at least 2 h to ensure the steam system’s functionality. The oven was set to 240 °C for the top heating element and 230 °C for the bottom. Upon loading the loaves into the oven, steam was injected for 2 s to promote crust development. Baking lasted approximately 1 h and 15 min, with an additional 30 min required for the denser 100% rye loaves. Baking progress was monitored throughout, and doneness was assessed by tapping the base of each loaf to check for a hollow sound, indicating that it was fully baked.

2.3. Nutritional Analysis

The nutritional composition of the bread samples was determined through standardized and validated analytical methods. Moisture content was assessed by drying approximately 5 g of sample at 103 ± 2 °C in a drying oven (Binder FD 115, BINDER GmbH, Tuttlingen, Germany) until a constant weight was achieved. Ash content was determined by incinerating 2.5 g of sample in a muffle furnace (Nabertherm L 40/11, Nabertherm GmbH, Lilienthal, Germany) at 550 °C until a constant mass was obtained. Crude protein content was quantified using the Dumas combustion method and a nitrogen analyzer (LECO FP 628, LECO Corporation, St. Joseph, MI, USA). Approximately 0.1 g of the sample was combusted to determine its nitrogen content, which was then converted to protein using a nitrogen-to-protein conversion factor of 6.25, as per EU Regulation 1169/2011 [20]. Dietary fiber was determined using the enzymatic-gravimetric AOAC 985.29 method [21] on a 3 g sample, involving enzymatic digestion followed by gravimetric quantification. Fat content was measured by pulsed Nuclear Magnetic Resonance (NMR) spectroscopy (Oxford Instruments MQC 23, Oxford Instruments plc, Abingdon, UK). A 2.5 g aliquot of the sample was dried and stabilized at 50 °C before analysis. Fat content was automatically calculated by comparing the NMR signal of the sample with a calibration curve generated from a certified reference standard. Carbohydrate content was calculated by difference, based on the measured contents of moisture, ash, protein, fat, and dietary fiber, following the formula [20,22]

Carbohydrates (%) = 100 − [Moisture (%) + (%) + Protein (%) + Fat (%) + Dietary Fiber (%)]

Sodium concentration was determined by flame atomic absorption spectrometry (FAAS) (Analytik Jena, Jena, Germany) after acid digestion of the sample, which was then incinerated at 550 °C. Potassium was quantified by inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7900, Agilent Technologies, Inc., Santa Clara, CA, USA) after nitric acid digestion using a DigiPrep MS digestion system (SCP Science, Baie-D’Urfé, QC, Canada) on the sample. All analyses were performed in triplicate, and the results are reported as mean values ± standard deviation.

2.4. Microbiological Analysis

A total of 5 g of leavened dough was mixed with 45 mL of Tryptone Salt (TS) solution (Himedia, Mumbai, India), homogenized using a stomacher, and subjected to serial 10-fold dilutions. Total aerobic mesophilic bacteria were enumerated on Plate Count Agar (PCA, Liofilchem, Teramo, Italy) after incubation at 30 °C for 72 h and at 7 °C for 10 days, per the ISO 4833-1:2013 standard [23]. Lactic acid bacteria were counted on de Man, Rogosa, and Sharpe (MRS) agar (Scharlau, Sentmenat, Spain), with incubation at 30 °C for 72 h, as per ISO 15214:1998 [24]. Molds and yeasts were enumerated according to the Portuguese Standard number NP 3277-1 [25], using Chloramphenicol Glucose Agar (CGA) (VWR Chemicals, Avantor, Inc., Radnor, PA, USA). The plates were incubated at 25 °C for 5 days. The microbiological results were expressed as log₁₀ colony-forming units per gram of sample (log CFU/g).

2.5. Sensorial Analysis

2.5.1. Sensory Evaluation of Rye Bread by Quantitative Descriptive Analysis (QDA)

The sensory evaluation of rye bread samples was conducted by a trained panel comprising 11 non-smoking individuals with an average age of 20 years. All panelists were faculty members or students from the University of Trás-os-Montes e Alto Douro (UTAD). The sessions took place in the sensory analysis laboratory, which was equipped according to ISO 8589:2007 standards [26]. All participants provided written informed consent before participating in the study, which was conducted in accordance with the principles outlined in the Declaration of Helsinki (study approved by the UTAD Ethical Committee—Doc 138-CE-UTAD-2024).

Samples were served at room temperature on a blank plate, and still mineral water and paper napkins were provided for palate cleansing between samples. Before the Quantitative Descriptive Analysis (QDA), a free profile was conducted with the trained panel to elicit and establish a consensual vocabulary of relevant sensory attributes. This preliminary session enabled the identification and selection of descriptors that best characterized the sensory differences among the bread samples. Based on this consensus vocabulary, the final sensory evaluation form was structured into five sections: manual texture and appearance (crust), manual texture and appearance (crumb), aroma (orthonasal), flavor and in-mouth texture, and aftertaste. QDA was then used to quantify the intensity of each descriptor across the different formulations. Panelists rated the intensity of each descriptor using a five-point ordinal scale. The scale ranged from 1 to 5, where 1 corresponded to “very weak intensity”, 2 to “weak intensity”, 3 to “moderate intensity”, 4 to “strong intensity”, and 5 to “very strong intensity”.

2.5.2. Consumer Evaluation

A consumer sensory evaluation was conducted to assess the acceptability of a sample with better commercial viability, utilizing whey protein in solution to replace water (Rye Whey—RW). A total of 109 participants were included in the study, comprising 75 women and 34 men. Participants ranged in age from 18 to 66 years, with an average age of 29 years. Consumers originated from various regions of Portugal, including Vila Real, Porto, Braga, Guimarães, Lisbon, Madeira, and the Azores, as well as international locations such as Spain, France, Brazil, Chile, and Poland.

The evaluation took place in a sensory analysis laboratory at the UTAD, conducted under controlled conditions. Each participant received the anonymized bread sample, served in uniform portions at room temperature, and was asked to evaluate the following four sensory attributes: appearance, aroma, flavor, and texture. For each attribute, participants used a five-point hedonic scale to express their degree of liking. The scale ranged from 1 to 5, where one corresponded to “disliked very much”, 2 to “disliked”, 3 to “neither liked nor disliked”, 4 to “liked”, and 5 to “liked very much”. Consumers were instructed to indicate the attributes they perceived and to rate their intensity accordingly. The questionnaire was administered individually, and palate cleansers (still water and unsalted crackers) were provided between samples to avoid carryover effects. Before participation, all individuals provided written informed consent. The study was conducted in accordance with the ethical standards for sensory and consumer research, ensuring the voluntary and anonymous nature of participation, with no personal identifying data collected.

2.6. Statistical Analysis

The results were presented as means ± standard deviation (SD). Statistical analysis for nutritional composition and microbiology was performed using one-way analysis of variance (ANOVA). Comparison of means was achieved using a Tukey HSD test (“Honestly Significantly Different”) at a significance level of 5% (p ≤ 0.05).

For the data obtained from the trained sensory panel, descriptive statistics and the non-parametric Kruskall–Wallis test were applied to evaluate differences among the bread samples based on the rated intensity of each sensory descriptor. When significant differences were identified (p ≤ 0.05), Tukey’s HSD post-hoc test was used for multiple comparisons to determine which samples differed significantly.

Regarding the consumer evaluation, frequency and intensity data for each sensory attribute were compiled and analyzed. Sensory profiles of the sample were illustrated using radar (spider) plots. The most frequently cited and intensely rated descriptors were identified to characterize consumer perception.

All statistical analyses were performed using IBM SPSS Statistics (Version 31, IBM Corp., Armonk, NY, USA).

3. Results

3.1. Nutritional Composition

The data presented in

Table 1. Nutritional composition of different bread formulations.

Composition	CTR	RW	FRW	p
Moisture (g/100 g)	40.23 ± 1.19 a	28.27 ± 4.29 b	31.80 ± 1.93 b	0.005
Ashes (g/100 g)	1.55 ± 0.05 b	2.10 ± 0.13 a	2.06 ± 0.05 a	<0.001
Lipids (g/100 g)	0.70 ± 0.04 b	0.96 ± 0.05 a	0.89 ± 0.07 a	0.003
Fiber (g/100 g)	4.77 ± 0.12 b	5.33 ± 0.40 b	6.96 ± 0.15 a	<0.001
Protein (g/100 g)	5.66 ± 0.15 b	6.54 ± 0.28 a	5.05 ± 0.16 c	<0.001
Carbohydrates (g/100 g)	47.13 ± 1.21 b	53.17 ± 1.76 a	53.27 ± 1.65 a	0.005
Sodium (g/100 g)	0.30 ± 0.02	0.36 ± 0.02	0.36 ± 0.04	0.070
Potassium (g/100 g)	0.20 ± 0.01 c	0.32 ± 0.01 b	0.36 ± 0.01 a	<0.001

CTR—Control; RW—Rye Whey; FRW—Full Rye Whey. Means with different superscript letters (lowercase) differ significantly, p < 0.05.

show the impact of whey incorporation on the nutritional profile of rye bread formulations. Whey incorporation significantly influenced the nutritional properties of the bread formulations, reducing moisture and increasing carbohydrates, ash, lipids, and potassium, especially in RW bread, while sodium levels remained unchanged. FRW showed the highest fiber content, suggesting an additive effect of rye and whey, while RW had the highest protein content and FRW the lowest.

3.2. Microbiological Analysis

Table 2. Counts (log UFC/g) for mesophilic microorganisms, molds, yeasts, and lactic acid bacteria (LAB) in whey and the different formulations before baking.

Microorganisms	CTR	RW	FRW	p	Whey
Mesophilic	7.74 ± 0.27	7.64 ± 0.00	7.23 ± 0.08	0.086	3.15 ± 0.07
Molds and Yeasts	8.66 ± 0.08	8.59 ± 0.03	8.03 ± 0.10	0.650	-----
LAB	7.74 ± 0.15	7.57 ± 0.13	7.29 ± 0.05	0.075	-----

CTR—Control, RW—Rye Whey, FRW—Full Rye Whey.

presents the microbial counts of the different bread formulations immediately before baking (leavened) and of the whey in solution. Microbiological analysis did not reveal significant differences among the bread formulations. Mesophilic bacterial counts were consistently high, with values ranging from 7.2 to 7.7 log CFU/g. Similarly, molds and yeasts ranged from 8.03 to 8.66 log CFU/g, and lactic acid bacteria (LAB) counts ranged from 7.29 to 7.74 log CFU/g, with no statistically significant differences. These results indicate that whey supplementation, as well as the use of rye flour, did not exert a discernible impact on the microbial populations present in the bread at the time of analysis. This observation is relevant because it demonstrates that the incorporation of whey did not compromise microbial stability during the leavening process.

3.3. Sensory Profile Characterization

A trained sensory panel evaluated the bread samples (CTR, RW, and FRW) based on 26 descriptors encompassing visual, textural, aromatic, and flavor-related attributes (

Table 3. Average score and standard deviation for each descriptor considering the three bread samples sensory evaluations (Control—CTR, Rye Whey—RW, and Full Rye Whey—FRW).

Domains	Descriptors	CTR	RW	FRW	p
Crust	Color	3.2 ± 1.3 a	3.6 ± 0.7 a	4.7 ± 0.5 b	0.001
	Hardness	4.7 ± 0.5 a	2.2 ± 0.9 a	4.7± 0.6 b	<0.001
	Roughness	4.0 ± 0.9 a	2.9 ± 1.1 a,b	3.3 ± 0.9 b	0.044
Crumb	Color	2.9 ± 1.0 a,b	2.0 ± 0.8 a	3.6 ± 1.3 b	0.006
	Hardness	2.7 ± 1.2 a	2.5 ± 0.9 a	3.8 ± 0.6 b	0.007
	Friability	3.5 ± 0.8	2.7 ± 1.2	3.1 ± 1.5	0.420
	Roughness	2.5 ± 0.8	2.2 ± 0.8	1.9 ± 1.1	0.367
	Density	2.7 ± 1.0	2.6 ± 1.0	3.5 ± 1.3	0.142
	Porosity	4.0 ± 0.8 a	2.6 ± 0.7 b	1.9 ± 0.8 b	<0.001
Aroma	Yeast	3.1 ± 1.0	2.7 ± 0.8	2.3 ± 1.0	0.073
	Toasted	2.1 ± 1.1	2.3 ± 1.2	3.0 ± 1.1	0.089
	Flour	2.2 ± 1.2	2.5 ± 1.0	2.6 ± 1.0	0.715
	Cereal	2.7 ± 0.9	3.4 ± 0.9	3.2 ± 0.8	0.169
	Lactic	1.7 ± 0.6	2.0 ± 0.8	1.5 ± 0.7	0.329
Flavor	Moisture	2.5 ± 1.0	3.1 ± 1.2	2.9 ± 1.1	0.543
	Hardness	4.0 ± 1.0 a	2.5 ± 0.7 b	4.0 ± 1.3 a	0.002
	Sweet	2.0 ± 1.1	2.4 ± 1.1	2.6 ± 1.4	0.497
	Salty	2.1 ± 0.7	2.5 ± 0.8	2.0 ± 1.1	0.378
	Acidic	1.7 ± 0.8	1.9 ± 1.0	1.9 ± 1.1	0.959
	Astringent	2.1 ± 1.0	1.5 ± 0.7	1.7 ± 1.0	0.313
	Cereal	3.0 ± 1.1	2.7 ± 1.1	3.4 ± 1.0	0.362
	Toasted	3.2 ± 1.3 a,b	2.6 ± 1.2 a	4.0 ± 0.9 b	0.028
Aftertaste	Sweet	2.1 ± 1.2	2.5 ± 0.9	2.6 ± 1.3	0.534
	Acidic	1.9 ± 0.9	1.6 ± 0.7	1.8 ± 1.1	0.829
	Cereal	2.7 ± 1.1	2.8 ± 1.2	3.1 ± 1.1	0.743
	Toasted	3.4 ± 1.2	2.4 ± 1.4	3.6 ± 1.0	0.060

Means with different letters (lowercase) differ significantly, p < 0.05.

). To visualize the overall sensory profiles, a radar chart (Figure 2) was constructed based on the average scores for each descriptor.

To further explore the relationships among sensory descriptors and nutritional parameters among the samples, a Principal Component Analysis (PCA) was conducted using the average values of the two replicates per formulation (Figure 3). The total percentage of total variability explained by the PCA is 41.1%, dividing the samples into three distinct groups. On the left is the CNT sample, and on the right quadrants are the FRW and RW samples, mainly characterized by whey imprinted in the sensory and nutritional attributes.

Following the trained panel sensory characterization, a consumer study was conducted to assess overall perception and product acceptance of the RW sample. The evaluation was based on a five-point hedonic scale, covering five attributes: Appearance (4.3 ± 0.6), Aroma (4.1 ± 0.8), Flavor (4.2 ± 0.8), Texture (4.1 ± 0.9), and Overall Liking (4.2 ± 0.6) (

Table 4. Results of consumer sensory analysis of Rye Whey bread (n = 109).

Scale	Appearance	Aroma	Flavor	Texture	Overall Liking
1	0%	0%	0%	0%	0%
2	0%	5%	5%	8%	0%
3	9%	15%	11%	13%	7%
4	56%	50%	48%	44%	63%
5	35%	31%	37%	35%	29%

).

4. Discussion

This study demonstrated the feasibility of incorporating whey in solution into rye bread formulations as a strategy to enhance nutritional value and promote ingredient circularity. The key contributions were the following: (1) the development of innovative recipes that reduce water usage—an essential resource—by substituting it with whey proteins in solution, in alignment with circular economy principles; and (2) the use of locally sourced rye, supporting biodiversity and fostering environmental sustainability.

The incorporation of in-solution whey into rye bread formulations led to a significant reduction in moisture content compared to the control bread (p < 0.05), likely due to the lower protein concentration and water-binding capacity of liquid whey when compared to whey protein powders [27], which are more effective at retaining moisture during baking [27,28,29]. This suggests that replacing water with fresh whey alters water-binding dynamics, resulting in greater water loss. In contrast, a significant increase in ash content was observed in the whey-containing breads (p < 0.05), consistent with previous studies [27] and attributed to the natural mineral content of whey—such as calcium, potassium, phosphorus, magnesium, and sodium—which remains after baking [28,30,31,32,33]. Furthermore, a statistically significant rise in potassium levels was also confirmed, reinforcing that liquid whey not only enhances the overall mineral profile but also contributes explicitly to higher levels of bioavailable macrominerals in the final product.

Potassium helps counteract the harmful effects of excessive sodium intake, is widely recognized as a significant risk factor for high blood pressure, making the sodium-to-potassium ratio a key indicator of the nutritional quality of food products. In Portugal, national legislation limits the sodium content in bread to a maximum of 0.55 g per 100 g. Other authors have also tested the impact of salt reduction on bread properties [34]. All samples analyzed in the present study comply with Portuguese salt regulations.

Regarding macronutrients, protein content was the most relevant result. Supplementation with in-solution whey resulted in a statistically significant increase in protein content, from 5.66% in the control bread to 6.54% in the whey-supplemented rye bread (p < 0.05), confirming whey’s potential as a protein enhancer in bakery products. While the observed increase is more modest compared to studies using whey protein concentrates or isolates—such as those by Komeroski, et al. [35] and Camargo, Silva, Komeroski, Kist, Rodrigues, Rios, Silva, Doneda, Schmidt, and Oliveira [28], which reported increases of up to 50%—this study highlights the value of using minimally processed, locally sourced fresh whey to replace the use of fresh water. Unlike industrial protein powders, whey in solution aligns with circular economy principles by promoting waste valorization and sustainable resource use, supporting environmentally responsible food production.

A statistically significant increase in lipid content was observed in the samples formulated with whey (p < 0.05). This slight elevation in lipid content may be attributed to residual lipids naturally present in whey. Although present in low concentrations, these residual lipids contribute to the overall fat content of the final product. This effect is considerably less pronounced than when whey protein concentrates (WPCs) are used [28,29,35].

Finally, the incorporation of in-solution whey into rye bread formulations led to a statistically significant increase in carbohydrate content—approximately 6% higher than in the control bread (p < 0.05)—primarily due to the natural lactose present in whey [30,31]. Unlike studies using whey protein powders, which often report reductions in carbohydrate content due to flour displacement (e.g., Camargo et al. [28] and Wani et al. [29]), the present study maintained the flour content while replacing only water, thus preserving the carbohydrate matrix. This approach supports a stable nutritional profile, with the added benefit of increased protein and minerals, without compromising the functional and sensory roles of carbohydrates. Additionally, a significant increase in fiber content was observed in the FRW formulation, reaching 6.96%, which was 2.19% and 1.63% higher than in the CTR and RW breads, respectively (p < 0.05). This enhancement is attributed to the naturally higher fiber content of rye flour compared to wheat flour [36,37], which further contributes to the nutritional value of the final product.

To provide further context to these findings, we compared the nutritional profile of our formulations with that of commercially available rye breads. According to the Portuguese Food Composition Table [38], standard rye breads typically contain 5.9 g/100 g of protein, 4.1 g/100 g of dietary fiber, and approximately 0.25 g/100 g of potassium. In contrast, our RW formulation exhibited a higher protein content (6.5 g/100 g) and higher fiber levels (5.33 g/100 g). Both RW and FRW formulations also presented higher potassium contents, reinforcing the added mineral value imparted by whey incorporation. This comparative perspective illustrates that our breads exceed industry nutritional standards, offering a superior alternative in terms of protein, fiber, and mineral content.

The incorporation of whey in rye bread not only enhances key nutritional components such as protein, minerals, and fiber but may also provide additional health benefits linked to these nutrients, including improved cardiovascular health through better sodium–potassium balance and positive effects on gut health due to increased dietary fiber [36,37]. Moreover, the use of whey as a liquid ingredient promotes sustainability by valorizing a dairy industry by-product that might otherwise contribute to environmental waste [39,40]. This circular economy approach supports more responsible resource utilization, reducing food waste and encouraging the use of local ingredients, thereby reinforcing regional food system resilience and contributing to broader environmental benefits.

Microbiological analysis of the bread formulations, conducted prior to baking, revealed no significant differences between CTR, RW, and FRW samples across the tested microbial groups. These levels are within the expected microbial load for leavened doughs under standard fermentation conditions. The absence of significant differences suggests that the incorporation of whey in solution did not increase microbial proliferation in the dough. This is noteworthy since whey, despite containing nutrients like lactose and proteins, did not compromise microbial stability during dough leavening. The microbiological profile observed aligns with safety standards, ensuring that the dough remained microbiologically safe prior to baking. Moreover, the baking process (cooking dough at 240 °C) effectively eliminates vegetative microbial cells, ensuring the safety of the final product.

According to the sensory analysis by the trained panel, the FRW sample stood out with a more intense and structured profile, showing significantly higher scores (p < 0.05) for crust color and toasted flavor. It also achieved moderate to high scores in descriptors such as crumb color, crust roughness, and aroma and flavor of cereals, while receiving lower scores for acidity, yeast and lactic aromas, saltiness, and astringency. The CTR formulation was characterized by high crumb porosity and crust hardness, resulting in a firmer texture with milder aroma and flavor notes. In contrast, the RW sample showed lower intensity across most descriptors, though it had the smoothest flavor profile, indicated by its lowest score in flavor astringency. Principal Component Analysis (PCA) confirmed these distinctions, with FRW associated with structure and toasted notes, CTR with texture and yeast aroma, and RW displaying a milder yet distinctive sensory identity, suggesting potential for targeted refinement.

Consumer testing revealed a generally positive response to the RW sample, with most attributes receiving average scores of around 4. Notably, up to 63% of consumers rated Overall Liking as 4, and 37% gave the highest score (5) for Flavor. Low scores (below 3) were infrequent. This contrasts with the trained panel’s more detailed discrimination, suggesting that consumers favored simpler, well-integrated sensory profiles. Despite its subtler characteristics in expert evaluation, RW effectively met consumer expectations, highlighting its potential as a consumer-preferred option. These findings suggest that the milder yet more balanced profile of RW offers a solid foundation for further optimization, combining innovation with broad appeal.

The intensified crust color and toasted flavor of FRW are consistent with previous studies, indicating that whey protein incorporation can enhance crust coloration and flavor intensity in bakery products [27]. Together with increased crumb color and crust roughness, these attributes suggest that the addition of whey in FRW contributed to a more structured and intense sensory identity without negatively affecting texture. In contrast, the RW sample exhibited a milder sensory profile, with generally lower intensity across descriptors, but a notably smoother flavor, as indicated by low astringency scores. This profile aligns with findings by Zandona et al. [39], who demonstrated that substituting water with liquid whey up to 50% improved textural parameters and nutritional aspects while maintaining consumer sensory acceptance. Indeed, the consumer test showed a generally positive response to the RW sample, confirming that a balanced, less intense profile can better meet consumer expectations.

The CTR formulation was marked by a firmer texture and higher crumb porosity, as reported in [41]. The firmer texture, milder aroma, and flavor notes differentiated CTR from whey-enriched samples, confirming the influence of formulation differences on bread quality. These results reinforce that the level and type of whey incorporation play a decisive role in shaping both the sensory characteristics and consumer perception of bread. The distinct profiles of RW, FRW, and CTR demonstrate that whey can be strategically used to modulate flavor intensity, crust development, and textural balance, offering a versatile approach to enhancing bakery formulations without compromising acceptance or product quality.

While the present study offers valuable insights into the sensory and nutritional impact of incorporating whey into rye bread formulations, certain limitations must be acknowledged. The absence of instrumental texture analysis and specific rheological measurements restricts a deeper understanding of the mechanical properties underlying the observed sensory differences. Nonetheless, the work stands out for its innovative approach—applying whey as a liquid ingredient in traditional rye-based formulations—and for its high potential for industrial scalability, given the simplicity of the substitutions and the positive consumer response. Future research should focus on thorough texture characterization, shelf-life evaluation, product scale-up, and strategies to promote industry adoption and consumer awareness, thereby further refining product quality and maximizing market impact.

5. Conclusions

This study demonstrated the feasibility of incorporating whey into rye bread formulations as a strategy to enhance nutritional value and promote ingredient circularity. The use of whey resulted in significant increases in ash, potassium, protein, fiber, and carbohydrate contents, while reducing moisture levels. Notably, the RW formulation offered a balanced profile with the highest protein content and favorable sensory attributes, receiving positive consumer evaluations. Meanwhile, the FRW sample stood out for its structured texture and complex sensory character, particularly appealing in trained panel analysis. These findings highlight the potential of whey-enriched rye breads, particularly RW, as innovative, nutritious, and sensorially accepted bakery products that support sustainable food systems through the valorization of local and underutilized resources.

Looking forward, these results open promising avenues for small-scale and artisanal bread production, where local sourcing of rye and whey can add value and differentiate products in niche markets focused on sustainability and nutrition. Future research should explore the scalability of these formulations, shelf-life stability, and the integration of whey sourced directly from local dairies, promoting circular economy practices and reinforcing regional food chains. Additionally, integrating instrumental texture analysis and rheological studies will strengthen product optimization and industrial application. By advancing these aspects, whey-enriched rye breads can contribute to sustainable alternatives with positive impacts on local communities and promote environmental resilience.