**1. Introduction**

Bread is the most consumed food product in the world, with its contribution to the human diet having the greatest importance [1]. However, because most people prefer white bread, the nutritional benefits are limited because it is obtained from refined wheat flour [2,3]. Moreover, due to the large quality variation of wheat flour used in bread-making, bakery producers usually add different types of additives to wheat flour, especially chemicals, to improve product quality from a technological point of view [4]. Nowadays, one of the trends in bread making is to improve the nutritional quality of bread by substituting wheat flour with other flour types without affecting the quality of the final products. Specialist attempts are made to balance the content of vitamins, minerals, and fibers lost through wheat refining without adding any chemical compound to the bakery products [5]. Moreover, an attempt is made to improve the quality of proteins in baked

**Citation:** Ungureanu-Iuga, M.; Atudorei, D.; Codin ˘a, G.G.; Mironeasa, S. Rheological Approaches of Wheat Flour Dough Enriched with Germinated Soybean and Lentil. *Appl. Sci.* **2021**, *11*, 11706. https://doi.org/10.3390/app 112411706

Academic Editors: Anabela Raymundo and Stephen Grebby

Received: 30 October 2021 Accepted: 7 December 2021 Published: 9 December 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

goods by adding various ingredients which contain essential amino acids that are deficient in wheat flour [6,7]. To correct these nutritional deficiencies, different grain flours can be used in bread making, such as legumes, oilseeds, pseudocereals, etc. [5,7,8].

The substitution of wheat flour with different legumes types improves the nutritional value of bread by decreasing the glycemic index and by increasing the fiber, mineral content, and protein quality [9]. However, the addition of legume flours to wheat flour has the disadvantage that they contain some antinutritive factors, such as tannins, phytates, or trypsin inhibitors which may affect the nutritional value of bread [8]. Furthermore, the addition level in wheat flour is limited due to their gluten dilution effect which affects dough viscoelastic structure and its ability to retain gases during fermentation which can lead to bakery products of poor quality [7]. Thus, it is more convenient to use legume flours in a germinated form, a process that has a positive effect on the nutritional profile of legumes, but also their sensory profile. During legume germination, many positive changes occur, as follows: a decrease in antinutritive factors contained in legumes, an increase in protein content, an improvement in the availability of sodium, magnesium, iron, zinc, a decrease in lipids and carbohydrates content [10–12]. Additionally, the amount of volatile organic compounds, such as 2-methylbutanal and dimethyltrisulfite increases which leads to the intensification of legume grains' flavor. At the same time, their sweetness intensifies [13], improving the sensory characteristics. In addition, during germination the enzymatic activity of legume grains increases which may improve wheat flour quality if it is enzymatically deficient without adding other chemical additives during bread making [14].

Two legumes with a superior nutritional profile that may be used in bread making are lentil and soybean. Lentils boast a significant content of vitamins, minerals, carbohydrates, dietary fiber, and a low glycemic index [15]. Lentils are also rich in proteins (21–31%) which contain all essential amino acids (39.3 g of essential amino acids per 100 g of protein); they are a source of glutamic acid, lysine, arginine, leucine, acid aspartic [16]. Lentils also contain many micronutrients, of which vitamin B9, zinc, and iron have the highest weight. Lentils contain the highest amount of polyphenols, compared to all other vegetables [17]. Regarding health aspects, medical studies have shown that the consumption of lentils has benefits on cardiovascular diseases and cancer prevention [18], but also has implications in promoting slow and moderate postprandial blood glucose increase [19,20]. Soybean is boasted as having a significant amount of high-quality protein (38–55%), essential amino acids, lipids (20%), and carbohydrates (27%) [21–23]. The phytochemicals present in soybean are of great interest for health because studies have shown that they lower the amount of cholesterol, have an anticarcinogenic capacity, and contribute to bone health [24].

Soybean and lentil flour often appear in specialized studies due to the possibility of being used as an ingredient in various bakery products [1,7]. The use of lentil and soybean flours as partial substitutes in wheat flour is justified by both nutritional and sensory aspects. Their use, particularly in the germinated form, may improve the quality of bakery products even from a technological point of view, especially if the wheat flour used has enzymatic deficiencies. According to the results obtained by Zhang et al. [25], native red lentil flour incorporation in wheat dough led to higher water absorption and mechanical weakening, while dough development time, stability and minimum torque, and cooking stability were lower compared to the control and increased with the addition level, due to the influence of the chemical compounds of the ingredient added. In the study of Marchini et al. [26] on the effects of lentil flour in the wheat dough, it is stated that the addition level increase caused water absorption rises, dough stability reduction, delayed protein weakening, and worsening of dough pasting consistency which could be related to the lower swelling power of pulses compared to wheat. The incorporation of germinated lentil flour in Sangak bread determined water absorption, dough development time, and softness degree increases compared to the control, while the stability of dough did not differ significantly. Wheat bread fortification with defatted soybean led to higher water absorption and dough extensibility and lower dough stability compared to the control according to results presented by Mashayekh et al. [27]. The addition of native

and germinated soybean flours in wheat dough increased water absorption, maximum consistency time, and dough stability, while dough maximum resistance to extension and extensibility was not significantly affected [28]. The rise of the 7S protein fraction extracted from native and germinated soybean in wheat dough was related to the increment of water absorption and extensographic maximum resistance to extension, especially in the case of protein extracted from native soybeans [29].

This study aimed to optimize the formulation of germinated soybean and lentil flours that can be added to refined wheat flour of low alpha-amylase activity to improve dough rheological properties. For the optimal combination between the soybean and lentil germinated flour, the dough microstructure was analyzed by using epifluorescence light microscopy (EFLM). To our knowledge, no other studies have examined soybean and lentil germinated flour addition to wheat flour in a combined form. The importance of their use in bread making derives from the valuable nutritional composition of these legumes, but also the technological advantages of their use in a germinated form.

#### **2. Materials and Methods**

#### *2.1. Materials*

Refined wheat flour of the 650 type (harvest 2020) provided by the S.C. Dizing S.R.L. company (Brusturi, Neamt, Romania) was used. Germinated legume flours were obtained from lentil (*Lens culinaris Merr*) and soybean (*Glycine max* L.) which were germinated for 4 days, lyophilized and milled before they were used in the wheat flour according to the method reported in our previous studies [12,14].

The flours were analyzed according to the international ICC standard methods: ash content (ICC 104/1), moisture content (ICC 110/1), fat content (ICC 136), protein content (ICC 105/2) [30]. To be certain that the germinated soybean (SGF) and lentil (LGF) flour may be used in bread making, they were analyzed also from a microbiological point of view according to the following methods: molds and yeast according to SR ISO 7954:2001 [31], mycotoxins by using an ELISA kit (Prognosis Biotech, Larissa, Greece) and *Bacillus cereus* according to the SR EN ISO 7932:2005 [32]. The wheat flour has been also analyzed for its wet gluten content and gluten deformation index according to SR 90:2007 method [33].

Soybean germinated flour (SGF) at ratios of 5, 10, 15, and 20% and lentil germinated flour (LGF) at ratios of 2.5, 5, 7.5, and 15% were mixed with wheat flour and coded as SGF 5, SGF 10, SGF 15, SGF 20 and LGF 2.5, LGF 5, LGF 7.5 and LGF 10, respectively. Wheat flour without SGF or LGF was used as a control (C).

#### *2.2. Dough Rheological Properties*

#### 2.2.1. Empirical Dough Rheological Properties during Mixing and Extension

Dough rheological properties during mixing and extension were analyzed by using an Alveo Consistograph (Chopin Technologies, Cedex, France) according to ICC 171 and ICC 121 standards [30] respectively. The Consistograph test was made to determine dough rheological properties during mixing: water absorption capacity (WA), tolerance to kneading (Tol), consistency of the dough after 250 s (D250), and 450 s (D450). The Alveograph test was performed to determine dough rheological properties during extension: maximum pressure (P), dough extensibility (L), baking strength (W), and configuration ratio of the Alveograph curve (P/L).

#### 2.2.2. Empirical Dough Rheological Properties during Fermentation and Falling Number

Empirical dough rheological properties during fermentation were analyzed using the Rheofermentometer device (Chopin Rheo, type F3, Villeneuve-La-GarenneCedex, France) according to the standard method AACC89–01.01 [34]. The Rheofermentometer parameters analyzed for the dough samples obtained by kneading of 250 g mixed flours, 7 g compressed yeast of the *Saccharomyces cerevisiae* type, and 5 g salt according to the Consistograph water absorption value were: the total CO2 volume production (VT, mL), the maximum height of gaseous production (H'm, mm), volume of the gas retained in the dough at the end of

the test (VR, mL) and retention coefficient (CR, %). The falling number values expressed in s were determined by using a Falling number device (FN 1305, Perten Instruments AB, Stockholm, Sweden) according to ICC 107/1 method [30].

#### 2.2.3. Dynamic Dough Rheological Properties

The dynamic dough rheological properties were obtained with a HAAKE MARS 40 device (Termo-HAAKE, Karlsruhe, Germany) with a 2 mm gap and parallel plate geometry of 40 mm diameter, according to previous works [14,35,36]. The dough samples were placed between rheometer plates and analyzed for the storage modulus (G ), loss modulus (G"), and loss tangent (tan δ) at a frequency of 1 Hz. Additionally, the maximum gelatinization temperatures were analyzed for the dough samples during heating from 25 to 100 ◦C at a rate of 4 ◦C per min at a fixed strain of 0.001 and a frequency of 1 Hz.

#### *2.3. Dough Microstructure*

The epifluorescence light microscopy (EFLM) images of dough with and without the best combination between the soybean and lentil germinated flour addition in wheat flour were analyzed with a Motic AE 31 (Motic, Optic Industrial Group, Xiamen, China) equipped with catadioptric objectives LWD PH 203 (N.A. 0.4). The images and dough samples preparation were obtained according to methods reported in our previous studies [14,37,38]. The dough sample was immersed in a fixing solution made of 1% rhodamine B for protein coloring and 0.5% fluorescein for starch coloring for at least 1 h.

### *2.4. Statistical Analysis*

All the measurements were done in duplicate. Analysis of variance (ANOVA) was applied to compare mean values of the samples with SGF and LGF respectively, at different addition levels. Statistically significant differences were considered at *p* < 0.05 by the Tukey test. For this purpose, XLSTAT for Excel 2021 version (Addinsoft, New York, NY, USA) software was used.

Then, an experimental design was performed to identify single and combined effects of factors on the responses. The study of SGF and LGF addition levels effects on wheat dough characteristics and the optimization were performed on a trial version of Design Expert software (Stat-Ease, Inc., Minneapolis, MN, USA). A full factorial design with two factors varied at five levels, SGF addition at 0, 5, 10, 15, and 20% and LGF addition at 0, 2.5, 5, 7.5, and 10%, and Response Surface Methodology (RSM) with a two-factor interaction (2FI) model were used. The responses considered were the following: FN—falling number, WA—water absorption, Tol—tolerance to kneading, D250—dough consistency after 250 s, D450—dough consistency after 450 s, P—dough tenacity, L—dough extensibility, W—baking strength, P/L—curve configuration ratio, H'm—maximum height of gaseous production, VT—total CO2 volume production, VR—the volume of the gas retained in the dough at the end of the test, CR—retention coefficient, G —elastic modulus, G"—viscous modulus, tan δ—loss tangent, Ti—initial gelatinization temperature, Tmax—maximum gelatinization temperature.

The effects of SGF and LGF addition levels on dough properties were evaluated through mathematical modeling. The most suitable model to predict data variation for each response was selected according to *F*-test results, coefficient of determination (*R*2), and adjusted coefficients of determination (*Adj.-R*2). The effects of factors and their interactions were underlined using Analysis of Variance (ANOVA), considering a significance level of 95%.

SGF and LGF addition levels optimization was done by applying the desirability function. The coded and real values of factors are listed in Table 1.


**Table 1.** Coded vs. real values of factors.

A: SGF—soybean germinated flour (%), B: LGF—lentil germinated flour (%).

The goals established for the factors and responses considered, along with their lower and upper limits are presented in Table 2. The differences among the optimal and control sample were tested using the Student-*t*-test, at a significance level of 95%, by using XLSTAT for Excel 2021 version (Addinsoft, New York, NY, USA) software.



**Table 2.** *Cont.*


A: SGF—soybean germinated flour (%), B: LGF—lentil germinated flour (%), FN—falling number, WA—water absorption, Tol—tolerance to kneading, D250—dough consistency after 250 s, D450—dough consistency after 450 s, P—dough tenacity, L—dough extensibility, W—baking strength, P/L—curve configuration ratio, H'm maximum height of gaseous production, VT—total CO2 volume production, VR—the volume of the gas retained in the dough at the end of the test, CR—retention coefficient, G —elastic modulus, G—viscous modulus, tan δ—loss tangent, Ti—initial gelatinization temperature, Tmax—maximum gelatinization temperature.

#### **3. Results**
