Functionality of Alternative Flours as Additives Enriching Bread with Proteins

Abstract

Legume cultivation is important for a wide array of reasons, including its positive effects on the environment, the economy, and human health. Legumes have different amino acid profiles that complement those of the three globally most important staple foods (rice, corn, and wheat). Therefore, the aim of this work was to assess the functionality of legume flours (as well as hemp as an emerging hemp protein source) as enriching supplements in breadmaking. The research focused on both the nutritional and sensory evaluation of flour with the assistance of novel research techniques such as diffusing wave spectroscopy and static multiple light scattering. The nutritional value of yellow and green peas as well as chickpeas was comparable, with the most noticeable difference being total fiber content, that ranged between 6.8 and 9.7 g/100 g of flour. Hemp flour outclassed all legume flours both in terms of protein content as well as fiber, which was over quadrupled. However, it was associated with the cost of worse technological properties. Addition of all investigated flours increased the dough stability, which was proved by static multiple light scattering and a reduction in the Turbiscan Stability Index. Microrheology of the dough was improved only by the addition of yellow pea flour, which was manifested by an increase in the macroscopic viscosity index and decrease in the fluidity index. This flour had also the most beneficial properties for the bread quality, including texture and sensory perception, including appearance, taste, and overall acceptance.

1. Introduction

Bread is a principal staple food that it is made mainly from wheat flour. Its quality determines, to a large extent, the standard of nutrition of entire populations and, in consequence, of public health. Therefore, there is increasing interest in the possibility of the fortification of bread products by using alternative flours. Due to this strategy, it is possible to significantly improve the nutritional profile of bread in terms of protein, minerals, vitamin or fiber content, antioxidant potential, etc. Alternative flours can be made from various plant organs including grains, nuts, seeds, or roots. The most common sources for their production include the following: almond, amaranth, arrowroot, barley, buckwheat, maize, millet, oat, potato, quinoa, rice, rye, soybean, spelt, or tapioca, although recently flour made from insects has also been recognized [1,2,3,4,5,6].

When using alternative flours as protein sources, legumes seem to be the best solution. They are cheap, abundant, nutritious, and rich in bioactive compounds. Among them, the most commonly consumed, soybeans, beans, and chickpeas, should be mentioned. However, there is growing concern among consumers about the consumption of genetically modified plants, as well as confirmed allergies and intolerances—lower soybean value as a source of protein. As regards common beans, the presence of antinutrients negatively affects mineral bioavailability [7]. The chickpea (Cicer arietinum L.) is a widely consumed pulse around the world, as it is recognized as a valuable source of proteins, containing also minerals, vitamins, as well as phenolic acids, isoflavones, and dietary fiber. It is believed to reveal several health benefits, including positive impacts on type 2 diabetes, obesity, hypertension, colorectal cancer, and cardiovascular diseases, as well as decreases in inflammation and oxidative stress [8,9,10]. Similar nutritional and health benefits are associated with the consumption of peas (Pisum sativum L.), which are the fourth most cultivated legume crop worldwide [9,11]. In terms of the applicability of peas, there are two main types of this crop: dry or field peas and the garden pea, which is consumed (both the pods and seeds) immature [12]. Nutritional value, i.e., macro- and micronutrient contents of yellow and green peas, differs only slightly. Even their amino acid compositions are similar. The basic difference is the very different sensory values, which implies different culinary and technological suitability [13]. When looking for common, accessible, and valuable alternative sources of protein, hemp definitely cannot be left out. Cannabis sativa L. is one of the most ancient plant species, primarily used in the textile industry. Currently, due to the discovery of the possibility of medical use of cannabis oil as well as the approval of cannabis with THC content below 0.3% for food purposes in Europe, the interest in breeding this plant has increased significantly. But above all, hemp protein stands out for its high digestibility and its amino acid composition that resembles casein [14,15,16,17,18,19].

Current research on the application of legume flours in bread indicates the technological possibility of replacing up to 20% of wheat flour with legume flours. However, substitution above 10% may cause deterioration of the physicochemical properties of the dough as well as the texture and sensory quality of the bread. In order to improve the functionality of alternative flours, various pre-treatment methods are used, such as grinding to different granulations, soaking, roasting, microwave treatment, sprouting, or fermentation [20,21,22,23,24,25,26,27]. Moreover, present studies are progressing towards the development of different types of gluten-free breads as well as improving their nutritional quality beyond protein content [8,9,10,28,29]. In contrast to increasing interest in improving the quality of bread by applying biological methods, less interest in the development of understanding of physicochemical aspects of dough preparation on the quality of the final product has been observed. However, emerging analytical techniques—diffusing wave spectroscopy (DWS) and static multiple light scattering (SMLS)—could contribute to further development in this field. They are perfectly suited for the characterization of liquid dispersions, such as suspensions, emulsions, gels, etc., directly in their native state. SMLS offers the ability to investigate the dispersion state and its evolution over time. DWS employs dynamic light scattering to derive rheological properties without the application of any shear stress. This non-invasive technique, by precise measurement of the thermal motion of dispersed particles, gives information about microrheological loss G’ and storage G”” moduli [30,31,32]. Taking into consideration the above the aim, the work was designed to estimate the functionality of flours derived from defatted hemp, chickpeas, yellow peas, and green peas as protein-enriching supplements in breadmaking. The study of the flours included analyses of nutritional value, pasting characteristics, texture of obtained dispersions, and their rheological and microrheological properties. Breads were examined in terms of visual representation, color, hardness, and sensorial quality.

2. Materials and Methods

2.1. Materials

Commercial pulse and hemp flours were provided as follows:

• yellow pea (Pisum sativum)—Młyn Niedźwiady (Niedźwiady, Poland),
• green pea (Pisum sativum)—POL-ENSA (Zator, Poland), raw material sourced from the Czech Republic,
• chickpea (Cicer arietinum)—POL-ENSA (Zator, Poland), raw material sourced from Turkey,
• hemp (Cannabis sativa), residual product of hemp seed oil pressing (Sklep Ale Młyn (Gdańsk, Poland)
  were the initial working material. White wheat flours with 405 mg/100 g and 650 mg/100 g ash content, namely type 405 and 650, were used as reference material in the breadmaking process. Flours were manufactured by GoodMills Polska sp. z o.o. (Stradunia, Poland) and “Młynpol” Adam Gołębiowski, Roman Wołoszczak i wspólnicy Spólka Jawna (Gromadka, Poland), respectively. All food ingredients including fresh baking yeast and brewed table salt used for bread preparation were sourced from a local grocery store. All reagents used were of analytical grade unless otherwise stated.

2.2. Flour Characteristics

2.2.1. Proximate Composition

Nutritional value of legume and hemp flours was determined according to the PN-A-79011 standard [33], except the following macronutrients: the total nitrogen content was determined using the Kjeldahl method according to ISO 20483 [34] and was used to calculate the protein content by multiplying the result by the conversion factor of 6.25, according to Regulation (EU) No 1169/2011 of the European Parliament and of the Council [35]. Unsaturated fatty acids were determined according to the ISO 12966 standard [36]. The ash content was determined according to AACC Method 08-12.01 [37]. Total dietary fiber content was determined according to AOAC Method 991.43 [38]. Carbohydrates and energy value were calculated based on the following formulas:

Carbohydrates (g/100 g) = 100-(water + protein + fat + ash)

(1)

Energy value (kJ) = 17 [(carbohydrates-fiber) + protein] + 37∙fat + 8∙fiber

(2)

2.2.2. Pasting Characteristics

Pasting characteristics of 10% pulse (47.0 g sample weight on dry matter basis) and 25% hemp flour (117.5 g sample weight on dry matter basis) suspensions were studied with a Brabender Viscograph model Type 800446 (Duisburg, Germany). Measurement profile used: measurement system 0.07 Nm, heating rate 1.5 °C/min, cooling rate 1.5 °C/min, temperature ramp 25 (heating)—92.5 (holding)—25 °C (cooling), holding period of 20 min at 92.5 °C.

2.2.3. Rheological Properties

Rheological properties of flour pastes prepared in the Brabender Viscograph were determined using a RotoVisco1 rheometer (Haake Technik GmbH, Vreden, Germany) equipped with a Z20 DIN coaxial measurement geometry system. Measurement methodology was described in detail previously by Lewandoiwcz and coauthors [39]. Obtained flow curves were described with the Ostwald de Waele equation:

τ = K·γⁿ

(3)

where τ is shear stress Pa, K is the consistency index Pa∙sⁿ, γ is the shear rate s⁻¹, and n is the flow behavior index (a dimensionless number that indicates the closeness to Newtonian flow).

2.2.4. Water Absorption Capacity

Water absorption capacity (WAC) was determined according to the Sosulski method [40]. Briefly, 5 g of flour was suspended in an excess of water (30 mL) with vigorous stirring. The sample was allowed to settle for 10 min and resuspended by vigorous stirring. This process was repeated eight times. Afterwards, the sample was centrifuged at 1167× g for 25 min. WAC was expressed in % of water absorbed by flour sample.

2.3. Model Dough Properties

Model dough samples were prepared using identical flour proportions as for bread preparation (2.4). To this end, 23.5 g of wheat flour (type 405) and 12.5 g of wheat flour (type 650) were dispersed in 64 g of deionized water. In fortified samples, 6% of wheat flour was substituted proportionally with pulse or hemp flour. All samples were studied immediately after preparation.

2.3.1. Static Multiple Light Scattering

Static multiple light scattering (SMLS) studies were performed with assistance of Turbiscan-type TDNS (Formulaction, Toulouse, France). Samples were scanned for 2 h at 15 min interval followed by 1 h intervals for next 22 h. Samples were investigated in 20 mL vials at 30 °C.

2.3.2. Diffusing Wave Spectroscopy

Diffusing wave spectroscopy was performed with assistance of Rheolaser mtype Lab 6 Master (Formulaction, Toulouse, France). Measurements were performed in full characterization mode in 20 mL vials for 24 h. Following that, microrheological parameters were determined: elasticity index (nm⁻²), solid–liquid balance, macroscopic viscosity index (nm⁻²∙s), and fluidity index (Hz).

2.4. Bread Characteristics

Wheat bread was prepared with assistance of multi-functional food processor model MC Smart (Hoyer Handel, Hamburg, Germany). Dough samples were prepared using 550 g of water, 470 g of wheat flour (type 405), 250 g of wheat flour (type 650), 15 g of fresh yeast, and 10 g of table salt. For fortified samples, 6% of wheat flours were substituted proportionally with pulse or hemp flour. All ingredients were mixed for 4 min in the processor at moderate speed setting of 5/10 a.u. and formed bread dough was allowed rise for 90 min. Afterwards, the dough was transferred to rectangular trays and baked for 45 min at 200 °C. All measurements were performed on bread samples minimum 16 h after preparation, but not later than 20 h.

2.4.1. Color Parameters

Color measurements of bread crumb and crust were made using the Minolta Chroma Meter CR-310 (Japan) colorimeter. Measurement conditions: observer 2°, illuminant C, color space CIE L*a*b*. Furthermore, absolute color difference (ΔE) was calculated using where bread as reference. Color measurements were performed in triplicate.

2.4.2. Texture Analysis

Texture profile analysis of bread was performed with a TA-XT2 texturometer (Stable Micro Systems, Godalming, UK). Bread loaves were cut into slices of 25 mm thickness. Each sample was compressed by a cylindrical probe with a 35 mm diameter to 40% of its strain. The hardness of the bread crumb was expressed as grams of force required to compress bread crumb to target strain. Texture analysis was performed in quadruplicate.

2.4.3. Sensory Analysis

Sensory evaluation of bread was conducted by 7 previously trained experts in accredited Sensory Laboratory of Department of Food Concentrates and Starch Products at Prof. Wacław Dąbrowski Institute of Agriculture and Food Biotechnology—State Research Institute. The evaluation was conducted at 20 ± 2 °C and relative humidity 52 ± 5%.

Quantitative descriptive analysis (QDA) of bread was performed according to PN-ISO 6564 standard [41]. Following parameters were evaluated for bread crust: hardness, color, chewiness, odor intensity, foreign odor, typical odor, salty taste, typical taste, and foreign taste; while for bread crumb hardness, color, chewiness, odor intensity, foreign odor, typical odor, salty taste, typical taste, foreign taste, pore size, and moisture intensity. Each trait was evaluated by the experts one by one on a linear scale of 10 cm with boundary lines. The plotted results were converted to a numerical value in conventional units on a scale of 0–10. Moreover, the bread samples were evaluated by the sensory panel on a hedonic scale in terms of appearance, color, smell, taste, and overall acceptance.

2.5. Statistical Analysis

The data are presented as mean values of three replicates ± standard deviation (unless otherwise stated). One-way analysis of variance (ANOVA) and Tukey’s post hoc test were performed to determine statistically homogenous subsets at α = 0.05. Cluster analysis (CA) was conducted based on Ward’s method, and Euclidean distance was used as a measure of similarity. The statistical analyses were performed using Statistica 13.3 (TIBCO Software Inc., Palo Alto, CA, USA).

3. Results and Discussion

3.1. Flour Composition

The nutritional value of the legume flours investigated was similar (

Table 1. Nutritional value of investigated pulse and hemp flours.

Nutritional Value	Flour
	Yellow Pea	Green Pea	Chickpea	Hemp
Energy (kcal)	354	331	330	319
Fat (g/100 g)	2.45 b ± 0.07	2.20 ab ± 0.06	2.08 a ± 0.06	12.18 c ± 0.11
Saturates (g/100 g)	0.40 a ± 0.02	0.34 a ± 0.01	0.34 a ± 0.01	1.32 b ± 0.05
Carbohydrates (g/100 g)	61.45	61.05	59.56	45.87
Sugars (g/100 g)	6.04	6.05	5.43	2.70
Fiber (g/100 g)	7.99 a ± 1.12	9.72 a ± 1.37	6.75 a ± 0.95	43.40 b ± 6.10
Protein (g/100 g)	25.43 ab ± 1.45	21.08 a ± 1.20	21.75 a ± 1.24	28.24 b ± 1.61
Sodium (mg/kg)	14.0 bc ± 1.5	15.8 c ± 1.6	10.2 b ± 1.1	4.6 ab ± 0.5
Ash (g/100 g)	2,85 a ± 0.06	2.58 a ± 0.06	2.58 a ± 0.06	5.54 b ± 0.09
Water (g/100 g)	7.82 a ± 0.12	12.64 b ± 0.19	14.03 c ± 0.21	8.17 a ± 0.12

Values marked with the same lowercase letter do not differ significantly, p > 0.05.

), and the energy value ranged from 330 to 354 kcal per 100 g of product. These results are also comparable to white wheat flour, which has approximately 340 kcal per 100 g. The differences in fat and carbohydrate contents between investigated flours were negligible, both from a technological and dietary point of view. Significant differences were observed, however, for fiber and protein content. The richest source of fiber was green pea flour, which contained more than 20% fiber when compared to pea flour and almost half as much as chickpea flour. Nevertheless, all of the flours tested could be labeled with the nutrition claim “high fiber” in the European Union (EU). In terms of protein content, the mature form of peas had 20% more than the green one; at the same time, the chickpea flour had an intermediate value. Nevertheless, for all legume flours tested, over 20% of the energy value came from protein, which allows these products to be labeled as “high protein” in the EU. Moreover, when compared to average whole grain wheat flour, the protein content of investigated flours was at least twice as high [42]. In addition, all products were characterized by low sodium content, i.e., below the value that forces the necessity to declare it on the packaging. Hemp flour is produced in the process of pressing hemp oil, and in fact is a side stream product. Nevertheless, it is a product of high nutritional value, which was confirmed by the results shown in Table 1. Hemp flour is characterized in particular by high protein content—over 1/3 of the energy value comes from this constituent, making it a richer source of protein than all of the tested legume flours. Moreover, due to the presence of the husk, hemp flour can be a valuable source of dietary fiber (more than 40% of its content) and minerals (more than 5%). These properties, although desirable from a dietary perspective, may be a limiting factor in technological applications in which hemp flour would be the main component of a product, including bread.

The technological usefulness of flour and flour additives can be easily evaluated by pasting analysis. The investigated legume flours (Figure 1) were characterized by a typical swelling behavior for products containing starch, but their characteristics were dependent on the botanical origin of the flour. Chickpea flour gelatinized at a slightly lower temperature of 78.2 °C, both in comparison to green peas (83.3 °C) and yellow peas (82.7 °C). However considering the relatively high wheat starch gelatinization temperature [43], those differences are of minor technological importance in the breadmaking process. Nevertheless, the chickpea flour pasting process was also more efficient, which was visible in the more dynamic increase in viscosity at the initial stage of sample heating. Moreover, during the holding period the stabilization of viscosity was visible for this sample, whereas further increase in viscosity at the next stage was caused mainly by a decrease in temperature. Therefore, chickpea flour was a textbook example of a medium-type swelling characteristic pattern. The gelatinization process of both pea flour variants was identical and more restricted than in the case of chickpea flour paste, which was manifested by a continuous increase in viscosity through all phases of the study. Similarity between both pea flour variants could be expected, as the molecular structure of this polymer should be similar. The basic difference between green pea flour and pea flour concerned the final viscosity, which may be attributed to the higher starch content in pea flour (resulting from the greater maturity of this raw material). Hemp flour was characterized by much lower viscosity compared to the legume flours tested, necessitating a Brabender Viscograph test at a suspension concentration of as much as 25%. These differences arise from the very low starch content of hemp seeds, which is below 2% [44]. The course of the pasting curve of hemp flour was also different and reassembled the high-type swelling characteristics pattern. It was characterized by a low but pronounced viscosity peak, the onset of which began at 84.4 °C. Subsequently, stabilization of viscosity was observed, as well as a typical increase in the cooling phase that resulted from a decrease in the temperature of the sample. The suitability of hemp flour as a water-binding material is relatively low considering the concentration tested but giving it a hydrothermal treatment may slightly improve its technological value.

In order to further characterize the studied flour pastes from a rheological point of view, a study using a rotational rheometer was conducted. Based on the obtained experimental data (flow curves), rheological parameters of samples were determined using the Ostwald de Waele model (

Table 2. Rheological properties of investigated pulse and hemp flour pastes.

Rheological Parameter	Flour
	Yellow Pea	Green Pea	Chickpea	Hemp
Consistency index (Pa∙sn)	32.7 c ±6.3	12.1 ab ±2.7	18.7 b ±0.5	9.9 a ±1.1
Flow behavior index (-)	0.307 b ±0.015	0.373 c ±0.030	0.337 bc ±0.010	0.002 a ±0.001
Thixotropy (Pa∙s−1)	16,750 a ±6152	8599 a ±221	14,000 a ±368	10,281 a ±2234

Values marked with the same lowercase letter do not differ significantly, p > 0.05.

). The use of the aforementioned model makes it possible to determine the consistency index (K) and flow behavior index (n). The K coefficient determines the viscosity of the product at the initial stage of the test (relatively at low shear); in turn, the n value is an indicator of convergence with Newtonian flow, allowing us to determine how much a change in shear forces affects the change in viscosity of the product. In addition, the value of the area of the thixotropy hysteresis loop, which is a measure of rheological instability, was determined.

The rheological properties of the pastes prepared from legume flours were similar and corresponded to the observations made during the analysis of the pasting profile. Pea flour had the highest consistency index, followed by chickpea flour and green pea flour (analogous to the final viscosity during the Brabender Viscograph test). All of the tested samples were pseudoplastic non-Newtonian liquids, and the higher consistency index was associated with a greater tendency to thin with shear—as illustrated by a decrease in the value of the flow index. Nevertheless, all of the legume flours studied have similar technological properties, and the possible benefits of the selected raw materials may depend on the method and extent of processing of the raw material in the technological process. The rheological properties of the hemp flour paste were similar to those of legume flours, but with a reduced viscosity that was manifested by a lower consistency index and a higher flow behavior index. At the same time, this sample had a relatively high value of thixotropy, which can be associated with the presence of relatively large husk particles that were suspended in the colloidal system. This effectively led to the deterioration of viscosity during the shear of the sample. Considering the higher concentration of the tested system, the usefulness of this raw material as a water-binding substance is limited.

3.2. Model Dough Properties

Fortification of wheat bread with unconventional flour may result in a decrease in dough stability, as well as hinder its fermentation. Figure 2 presents the difference in the backscattering (BS) signal during storage of the bread dough that reflects various phenomena occurring during the ageing of the dough. At the beginning of the study to up to 18 h, one could observe sedimentation of the flour suspension (represented by the blue, green, and yellow curves). After the 18 h mark, intense fermentation took place, which resulted in an increase in volume that could be observed as an increase in BS at the top of the vial, i.e., above 40 mm. Those changes were noticeable on red curves. Simultaneously partial water separation took place, which was reflected by a decrease in the BS signal approximately in the middle of the test vial, i.e., 25 mm. Addition of all investigated flours pronounced the sedimentation, including its speed and intensity, but mostly at the beginning of the study. It should be also noted that the differences between individual flours were mostly insignificant. The addition of legume flours did not hinder the fermentation process; moreover, in the case of chickpea and hemp flour, the dough started to rise approximately 2 h earlier. Due to relatively insignificant changes between samples, further delving into the behavior of the dough requires analysis of the Turbiscan Stability Index (TSI). The TSI is the sum of all temporal and spatial variations within the sample at selected time intervals. The computed TSI value at 8 h, corresponding to a time where most of the sedimentation has occurred, but the fermentation is not yet optically visible, is presented in

Table 3. Turbiscan Stability Index, microrheological properties, and water absorption capacity of model dough suspensions.

Bread Dough	Turbiscan Stability Index (-)	Macroscopic Viscosity Index (nm−2∙s)	Solid–Liquid Balance (-)	Fluidity Index (Hz)	Water Absorption Capacity (%)
Reference	7.0	0.0053 b ± 0.0007	0.497 b ± 0.016	0.461 b ± 0.063	64 a ± 1
Yellow pea	3.5	0.0146 c ± 0.0019	0.446 a ± 0.008	0.169 a ± 0.019	107 c ± 1
Green pea	6.4	0.0052 b ± 0.0016	0.508 b ± 0.038	0.484 b ± 0.149	93 b ± 1
Chickpea	5.7	0.0033 a ± 0.0010	0.535 c ± 0.039	0.809 c ± 0.256	93 b ± 4
Hemp	3.8	0.0041 ab ± 0.0005	0.518 b ± 0.013	0.512 b ± 0.052	127 d ± 1

Values marked with the same lowercase letter do not differ significantly, p > 0.05.

. The TSI values indicate that addition of all investigated flours increased the stability of the bread dough, as the value for reference sample was the highest. Nevertheless, most notable values were computed for bread dough with the addition of yellow pea and hemp flour, indicating beneficial properties for the stabilization of wheat bread dough.

Light scattering studies of dough were followed by employing diffusing wave spectroscopy, which enables the determination of microrheological properties. Figure 3 presents the elastic index (EI) of the investigated dough specimens. The course of the changes of the EI is similar for all samples, regardless of the flour used for fortification. At the initial stage of the study (approximately 30 min), one can observe a rapid increase in EI, which can be mostly attributed to the binding of water by the protein of the flours used. Subsequently, stabilization of EI is observed, followed by a decrease in elasticity due to fermentation of the dough. Model reference dough had a similar EI to samples fortified with both pea flours. On the other hand, addition of chickpea flour led to a decrease in EI, which might be related to its weak water-binding properties (Table 3). Surprisingly, hemp flour was characterized by similar microrheological properties of the formed dough to reference and pea samples. These observations are also in line with the high water absorption of this flour. This indicates that while the pasting properties of hemp flour are poor due to a lack of starch, the rheological properties of fresh dough might be improved by its addition.

The remaining microrheological parameters, i.e., the macroscopic viscosity index, solid–liquid balance, and fluidity index, followed the same pattern as the EI. For this reason, there values that are represented as means during the stabilization period (between 5 an 10 h) and presented in Table 3. The most notable changes of the aforementioned parameters, with reference to pure wheat bread, were observed for yellow peas and chickpeas. The first substantially improved the macroscopic viscosity, thus reducing the fluidity index and partially the solid–liquid balance. Those observations were in line with the relatively high WAC of this flour. The latter caused a decrease in macroscopic viscosity of the dough, thus increasing the solid–liquid balance and fluidity index, which was reflected also by weaker WAC.

3.3. Bread Characteristics

Incorporation of pulse and hemp flour in bread dough recipes has led to noticeable differences in the color of the baked bread. The source of these changes may be attributed to the different effect of individual ingredients on the intensity of the Maillard reaction. On the other hand, the flours introduced may have a secondary coloring effect. For this reason, the results of bread color measurements have been presented separately for the crumb and the crust of the bread (

Table 4. Color parameters of bread crumb and crust in CIE L*a*b color space.

Bread Crumb	Color Parameter
	L	a	b	ΔE
Reference	72.57 b ± 1.85	−0.21 a ± 0.07	16.46 b ± 0.25	-
Yellow pea	70.91 b ± 1.03	0.52 b ± 0.06	19.64 c ± 0.13	3.66
Green pea	71.06 b ± 0.13	0.76 bc ± 0.13	16.41 b ± 0.23	1.79
Chickpea	71.00 b ± 1.88	1.63 d ± 0.23	15.36 b ± 0.64	2.65
Hemp	51.89 a ± 0.68	0.64 c ± 0.03	13.96 a ± 0.71	20.85
Bread crust
Reference	64.03 c ± 1.62	8.36 b ± 1.24	31.62 c ± 0.84	-
Yellow pea	50.84 a ± 0.51	10.02 b ± 0.57	25.72 b ± 1.09	14.55
Green pea	55.59 b ± 1.37	13.10 c ± 0.53	30.87 c ± 0.98	9.71
Chickpea	54.22 b ± 0.63	13.10 c ± 0.81	30.39 c ± 0.27	11.30
Hemp	52.30 ab ± 0.45	4.18 a ± 0.49	21.95 a ± 0.51	15.76

Values marked with the same lowercase letter do not differ significantly, p > 0.05.

). In addition, Figure 4 shows a cross-section of the tested bread samples.

The crumb of the reference pure wheat bread was characterized by a relatively high brightness (L) of 72.6% and an even balance of the green and red chromatic components (a ≈ 0), with a predominance of the yellow component (b > 0). The enrichment of bread with flours from legumes caused noticeable differences in the color of bread that an inexperienced observer is able to perceive. This is in line with previous observations for bread with other legume flours, which caused an increased intensity of color perception even at the 5% addition level [45]. Nevertheless, the change is still below the threshold that leads to an impression of two different colors (ΔE < 5). The shift in color was mostly due to a decrease in the brightness of the crumb (L) and a shift in the balance of the chromatic components from slightly green (a < 0) to slightly red (a > 0). A significant increase in the yellow component (b > 0) was also observed for the bread with pea flour. Those observations are in line with the results reported for bread with chickpea, field bean, lentil, and pea flours at the addition levels reported by Cacak-Pietrzak and coauthors [46]. The addition of the hemp flour led to more significant changes in the color of the crumb. This phenomenon was related to the dark color of the raw material. The change in the color of the bread was not only due to a decrease in the brightness, but also a decrease in the intensity of the yellow color component was observed. The aforementioned changes will have a significant and negative impact on the design of white bread. In contrast, for whole-grain products, such raw material characteristics may be desirable.

Compared to the crumb, the color changes of the bread crust were more pronounced. Overall, the crust of the tested bread was characterized by average brightness, a slight red hue, and a high saturation of yellow color. The addition of all of the raw materials used for bread fortification led to a decrease in the brightness, an increase in the proportion of the red color component, and a decrease in the proportion of the yellow color component. Only in the case of hemp flour was there a noticeable decrease in the value of the red color component, which was related to the intensity of the green color (b > 0) of the introduced raw material. This resulted in the greatest overall color difference in comparison to the reference product.

The addition of different hydrocolloids (including flours) to the bread dough will result in significant changes in the texture of the finished products. This phenomenon is related to changes in water binding, which are the result of the interaction of wheat protein and starch with proteins and possibly starch contained in the flour used for fortification. The hardness of the reference wheat bread crumb was 1108.5 g of force (

Table 5. Hardness of the bread crumb.

Bread	Hardness (g)
Reference	1108.5 a ± 67.0
Yellow pea	1278.6 a ± 187.2
Green pea	1217.4 a ± 160.2
Chickpea	1204.4 a ± 48.1
Hemp	1342.4 a ± 178.6

Values marked with the same lowercase letter do not differ significantly, p > 0.05.

). The addition of all of the tested flours caused an increase in hardness value by 95.9–233.9 g of force. The most noticeable difference was observed for hemp flour, which might be surprising considering its thickening capabilities. The increase in bread hardness by substitution of wheat flour by legumes was reported in the literature previously by various authors [20,46]. Nevertheless, the observed differences are of minor technological significance at the analyzed fortification level (6% flour substitution), but with an increase in the proportion to wheat flour—there might be a necessity to adjust the formulation of manufactured products.

The results of the sensory scoring test of investigated bread samples are presented in Figure 5. The evaluation results of hedonically perceived differentiators were quite consistent for all breads. The best score of the overall rating, as well as for all other quality differentiators, was attributed to the bread with the addition of pea flour. The ratings of the reference bread (without fortification) were also relatively high. The appearance and color of breads with the addition of legume flours were rated similar and comparable to bread without additives, while the color of bread with the addition of hemp flour was rated worse. When further analyzing the evaluation of taste and overall acceptance of breads, one should emphasize that only the bread with the addition of pea flour was rated higher than bread without additives. At the same time, bread with the addition of hemp flour was better perceived in terms of palatability than in the case of other legumes (green peas and chickpeas). Nevertheless, it should be noted that the evaluation was performed by trained experts, who theoretically are not intended for hedonic evaluation and can bias the results. On the other hand, in commercial practice, for quality control, a scoring test with trained experts is often used. Previous studies have indicated that yellow pea flour can be incorporated to wheat bread at levels up to 20% without deterioration of palatability (panel n = 60) [23]. Moreover, it was reported that for soy and a mixture of soy and barley flour, 10% substitution of wheat flour does not change the overall acceptance of wheat bread (panel n = 10) [47]. Studies on gluten-free bread indicated that 5% addition of pea protein powder increased the bread’s overall acceptance, but after that threshold, s decrease in scoring was observed [48]. On the other hand, substitution of wheat flour with grass pea, yellow lupine, or narrow-leaf lupine flours had a negative impact on the sensory acceptability of the bread [49]. Considering the above, it should be noted that at relatively low substitution levels, the sensory properties of bread fortified with legume flours may be improved, which is consistent with the literature data.

Figure 6 and Figure 7 show the results of the QDA for the crust and crumb of the bread with added legume or hemp seed flour. The most typical taste and smell (odor) was attributed to the crust of bread with the addition of pea flour, followed by bread without additives (reference). The rest of the investigated bread samples had lower and comparable typical odor intensity. On the other hand, for the bread crumb, the most typical taste and smell was attributed to bread without additives. The salty taste for the crusts and crumb of all breads was similar, which could be expected due to the low sodium content of the investigated legume and hemp flours. The crusts of breads with the addition of pea, chickpea, and hemp flour were characterized by high hardness, thus resulting in high chewiness. Relatively, the softest and most easily chewed crumb was attributed to bread with the addition of pea flour. The crust and crumb of bread with hemp flour was characterized by a darker color compared to breads with all other products (which was also confirmed by instrumental tests). In contrast, the crust of bread without additives was characterized by the lightest color. For the bread crumb, perception of moisture intensity was also assessed; however, most differences were negligible, except those for bread prepared with yellow pea flour, which had the lowest moisture evaluation by the panel.

Hierarchical cluster analysis (Figure 8) represents the similarities between samples based on the data presented in Section 3.2 and Section 3.3. The dendrogram strengthens the theory regarding the similar properties of reference and pea-fortified bread, as those formed one cluster. Nevertheless, the results for chickpeas and green peas were even more similar than the aforementioned group. Moreover, this cluster was linked closer to bread and dough with hemp flour than to the reference and yellow pea cluster. This indicated that although pure hemp pure flour had significantly different physicochemical properties, in the breadmaking process those differences were minimized.

4. Conclusions

This study assessed the usefulness of yellow pea, green pea, chickpea, and hemp flour as functional additives in the breadmaking process. The nutritional value of legume flours was comparable, with the most notable differences being the fiber and partially protein content. The hemp flour outclassed the above in that matter but revealed significantly worse pasting characteristics and rheological properties of the paste. The addition of all investigated flours improved the stability of the bread dough, with most notable results obtained for yellow pea and hemp flour. The microrheology of the dough was improved only by addition of yellow peas, whereas chickpeas had negative effect on the formed suspension, lowering its macroscopic viscosity. Differences between aforementioned flours were also highly noticeable during sensory evaluation of the bread, with the bread fortified using yellow peas being highly accepted, while the overall acceptance of the chickpea bread was the lowest. Moreover, the differences between sensory evaluations of those breads arise from numerous sensory descriptions, which was proven by quantitative descriptive analysis. The research provides an insight into the fortification of wheat bread with emerging protein sources, with an emphasis on the high nutritional, technological, and sensory value of yellow pea flour. Fortification of wheat bread with 6% of yellow pea flour not only improved its sensory acceptance, but also facilitated the breadmaking process. These observations were proved by dough stability studies by SMLS and microrheological examination of the dough by DWS. Moreover, the suitability of yellow pea flour as valuable ingredient in technological process was proven by higher viscosity in board shear rate range when compared to other legume or hemp flours.