Impact of Alfalfa Sprouts (Medicago sativa L.) on the Quality of Composite Sourdough Fermented With Lactiplantibacillus plantarum ATCC 8014 and Saccharomyces cerevisiae

Abstract

Alfalfa sprouts have gained attention due to their nutritional quality and health benefits in finding new nutrient resources to fortify staple food. The sourdough fermentation is a powerful method to exploit its potential in breadmaking. This study aims to assess the performance of wheat-alfalfa sprout composite flours in sourdough fermentation. The impact of Lactiplantibacillus plantarum ATCC 8014 and Saccharomyces cerevisiae in single culture or coculture and alfalfa sprouts addition level on the quality characteristics (biochemical, nutritional, and rheological) of sourdough was tested. Alfalfa sprout flour revealed a high content of protein (38.6%), dietary fibers (33.4%), minerals, and carbohydrates that could contribute to the initiation of sourdough fermentation. At the same time, fatty acids, total polyphenols, and antioxidant activity improve the functional attributes of the product. For sourdough performance, the results show good microbial cell growth (average value of 9 log cfu/g after 24 h) for both tested microorganisms, especially as single cultures, without the negative influence of alfalfa sprout flour on cell viability. Good lactic acid yields (maximum of 5.86 mg/g) and ethanol (maximum of 6.63 mg/g) were produced, indicating good metabolic performance for both inoculants. The upward trend of total free amino acids, ranging from 1.62 to 3.5 mg/mL, was due to the addition of alfalfa sprout flour and the proteolytic activity of the strains. Elevated concentrations of potassium, magnesium, and calcium were identified in all samples, but potassium was slightly higher in yeast fermentation, while magnesium and calcium were in the lactic fermentation. Increasing total polyphenol contents (maximum of 185 mg GAE/100 g) and radical scavenging activity (maximum of 27.36%) were recorded with both alfalfa sprout addition and microbial metabolism. No negative influence of alfalfa sprout flour on sourdough viscoelastic behavior was found. The good quality of wheat-alfalfa sprout sourdough could contribute to the improvement of the final product quality.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Alfalfa (Medicago sativa L.) is a legume that is widely grown for feed and is considered to be one of the most important crops globally in terms of economic value [1, 2]. Owing to its high nutritional content, alfalfa, also known as lucerne, is acceptable for people to eat as sprouts, edible seeds, or protein concentrates [3]. In contrast to refined wheat flour (WF), dried alfalfa seeds have greater levels of protein, fat, and crude fiber and don’t contain gluten [4]. Comparing dried alfalfa seeds to dried wheat seeds has revealed higher concentrations of polyunsaturated fatty acids (PUFA), particularly essential fatty acids like linoleic acid and linolenic acid, as well as higher vitamin (riboflavin, E, and C) and mineral contents [5]. Furthermore, Bhojak et al. [6] reported that the total phenolic content of alfalfa seeds is equivalent to that of green tea, rosemary, and grape seed extracts. Although human studies are limited, adding dried alfalfa seeds to the diet regularly may help normalize blood cholesterol values in individuals with type II hyperlipoproteinemia through saponin activity [7].

Known since ancient times, sprouting seeds is a method that enhances the nutritional content and health benefits of meals. Germination is nowadays considered a biotechnological process that transforms grains into new plants, improving the nutritional content of cereals and legumes through physiological and biochemical changes [8]. Moreover, sprouted seeds are gaining popularity globally as consumers seek less processed, additive-free, natural, and nutritious foods. The increased popularity of sprouts is mostly due to their beneficial health effects. Sprouts have been linked to some biologically active compounds that may provide health advantages. Shi et al. [9] claimed that the high concentration of saponins and other beneficial bio-compounds in the sprouted seeds is the reason for the increasing intake of alfalfa sprouts. Plaza et al. [5] found that sprouting significantly increases the initial vitamin A and C content of alfalfa seeds by 1250-fold and 10-fold, respectively. Moreover, remarkable increases in some B-vitamins (B1, B2, B6) and iron and potassium during alfalfa seeds sprouting were also reported by the same study. In addition, alfalfa sprouts have been linked to a decrease in blood plasma cholesterol levels and an inhibitory impact on cholesterol absorption. The enhanced conversion of hepatic cholesterol to bile salts by alfalfa saponins has been linked to the hypocholesterolemic activity of alfalfa sprouts, resulting in their loss in the feces [10].

However, the widespread application of alfalfa seeds and sprouts in bakery products is limited for different reasons, including the pronounced taste, the dark color, and the presence of anti-nutritional factors. Food security has inevitably driven researchers to look for alternative sources of protein, with an emphasis on plants high in protein, germs, and byproducts. Less conventional food components have undoubtedly attracted attention and from a sustainable viewpoint due to their unique mix of functional and nutritional qualities. It is important to choose unconventional plants and byproducts early on. However, the majority of these matrices feature antinutritional elements that might reduce digestibility and advantageous compounds in non-bioavailable form. They also often lack fully adequate technical, rheological, and sensory attributes.

A high number of studies published in the past years have shown how sourdough fermentation is a special method for enhancing the rheological, sensorial, shelf life, and nutritional qualities of bread made with various flour blends. Moreover, the remarkable variety of flours and agricultural byproducts that have successfully undergone fermentation serves as evidence of sourdough’s incomparable potential [11]. Thereby, legume flours were among the most used during sourdough fermentation due to their valuable nutritional quality, which serves as a raw matrix for fermenting microorganisms. Curiel et al. [12] fermented 19 different types of legume flour from Italy and reported their potential in sourdough technology alone or better combined with cereal flours. Other studies showed that sourdough fermentation of legume flours boosted free amino acids, γ-amino butyric acid (GABA amino acid), polyphenols, dietary fibers, and minerals [13]. It also improved antioxidant activity and protein digestibility, and decreased the glycemic index [14, 15]. Moreover, lactic acid fermentation was found to play an important role in sprouted grain processing by reducing the impact of antinutritional factors [16]. According to their inoculum and technological setup, four types of sourdough can be distinguished: types 0, I, II, and III. Type 0 is a pre-dough or sponge dough made with baker’s yeast addition. Type 1 sourdough manufacturing procedures involve fermentations of the flour–water combination by typical daily backslopping to maintain the sourdough LAB and yeasts metabolically active. Type II sourdough production methods are one-step fermentations produced by acid-tolerant starter cultures and conducted at regulated temperatures above room temperature (30°C–37°C). Type III sourdoughs are dried sourdoughs that may be used as nonliving acidifiers and flavor carriers, whether or not incorporated in bread mixes [17].

This study aims to assess the performance of wheat-alfalfa sprout composite flours in sourdough fermentation. The impact of Lactiplantibacillus plantarum ATCC 8014 and Saccharomyces cerevisiae in single culture or coculture and alfalfa sprouts addition level on the changes of biochemical, nutritional, and rheological characteristics of sourdough was tested.

2. Materials and Methods

2.1. Materials

WF (480 type according to the ash content by Romanian classification), alfalfa sprout seeds (UE origin), and bakery yeast (Saccharomyces cerevisiae (Sc), Pakmaya (Turkey)) were purchased from local specialized stores. The alfalfa sprout flour (ASF) was obtained by milling to a fine product (< 300 μm) on a Grindomix (GM200) laboratory mill at 10.000 rot min⁻¹ for 50 s. Lactiplantibacillus plantarum ATCC 8014 (Lp) was acquired from Microbiologics (Minnesota, USA). The analytical grade reagents are from Chempur (Piekary Ślaskie, Poland).

2.2. Methods

2.2.1. Proximate Composition and Fatty Acids Profile of ASF

ASF samples were analyzed according to AACC [18]. Moisture (44-15.02), starch (76-11.01), fat (30-25.01), ash (08-01.01), dietary fiber (32-07.01), total sugar after inversion (80-68.01), ash (SR ISO 2171:2023), and proteins were measured using the Kjeldahl method (46-11.02), and the nitrogen to protein conversion factor was 5.7. For fatty acids profile, the following methods were used: ISO 12966-1:2015-01, ISO 12966-2:2017-05, ISO 12966-4:2015-07.

2.2.2. Sourdough Formulation, Lactiplantibacillus plantarum ATCC 8014 Activation, and Cell Viability

Sourdough samples (Table 1) were prepared by mixing the WF/ASF flour blend with distilled water, while the microbial inoculum was added at a level of 10⁸ cfu/g for all inoculation variants. Before the inoculation, flour blends were sterilized using the ultraviolet (UV-C) radiation method (10 min at 260 nm), while distilled water was autoclaved for 15 min at 121°C. From the 12 sourdough samples, 4 samples were fermented with Lp at 37°C for 24 h, 4 samples with bakery yeast (Sc) at 30°C for 24 h, and the last 4 samples by a co-culture of Lp and Sc at 30°C for 24 h. The flour blends were obtained by adding 5, 10, and 15 g of ASF to the corresponding amount of WF to reach 40 g of blend. The samples without ASF, containing 100% WF, were control samples. The amount of water was obtained after the determination of the WF/ASF blends’ water-holding capacity using the method of centrifugation described by Traynam et al. [19]. Consequently, DY (dough yield = dough weight  × 100/flour weight) was 187.5, 200, 212 and 225.

Table 1

Sourdough formulations and samples codifications.

Materials	LG0	LG 5	LG 10	LG 15	DG0	DG 5	DG 10	DG 15	LDG0	LDG 5	LDG 10	LDG 15
Wheat flour, WF (g)	40	35	30	25	40	35	30	25	40	35	30	25
Alfalfa sprouts flour, ASF (g)	—	5	10	15	—	5	10	15	—	5	10	15
Lactiplantibacillus plantarum ATCC 8014 suspension (mL)	8.5	9	9.5	10	—	—	—	—	4.25	4.5	4.75	5
Saccharomyces cerevisiae (g)	—	—	—	—	1	1	1	1	0.5	0.5	0.5	0.5
Water (mL)	26.5	31	35.5	40	34	39	44	49	30.25	35	39.75	44.5

Note. LG0—Lp control sample, LG 5—Lp and 5 g alfalfa sprouts, LG 10—Lp and 10 g alfalfa sprouts, LG 15—Lp and 15 g alfalfa sprouts, DG0—Sc control sample, DG 5—Sc and 5 g alfalfa sprouts, DG 10—Sc and 10 g alfalfa sprouts, DG 15—Sc and 15 g alfalfa sprouts, LDG0—Lp, Sc control sample, LDG 5—Lp, Sc and 5 g alfalfa sprouts, LDG 10—Lp, Sc, and 10 g alfalfa sprouts, LDG 15—Lp, Sc and 15 g alfalfa sprouts.

The inoculum of Lp was prepared by suspending freeze-dried cells in 10 mL of Man Rogosa Sharpe (MRS) broth and incubating them under aerobic conditions at 37°C for 48 h before subculturing them into 95 mL of MRS broth and incubating them under the same conditions. The biomass was centrifuged at 2300 × $g$ (Eppendorf R 5804 centrifuge, Hamburg, Germany) for 10 min at 4°C, rinsed three times with sterile saline solution, and inoculated in the prepared matrix to reach an initial cell count of 10⁸ cfu/g. The microbial optical density of the inoculum was assessed using the spectrophotometer Shimadzu UV-1900 (Shimadzu Scientific Instruments, Kyoto, Japan), and absorbance was measured at a wavelength of 600 nm [20].

Five grams of each sourdough sample were diluted into 45 mL of sterile saline solution (0.85% w/v; NaCl) to obtain a 10-fold dilution and homogenized at 2500 RPM using a DLAB vortex, model MX-F (DLAB Scientific Co., Ltd., Beijing). 1 mL of the serial dilutions was placed in a Petri dish with MRS agar, and it was incubated for 48 h at 37°C. For the Sc yeast growth, 0.1 mL was used in Petri dishes with YPD agar (Oxoid, CM0361 Basingstoke, UK). The yeasts were grown for 3–5 days at 30°C. To count microorganisms, the last step involved analyzing Petri plates containing colonies using a colony counter (Colony Star 8500, Funke Gerber, Berlin, Germany) [21].

2.2.3. Sourdough Analysis

2.2.3.1. pH Measurements

After being first calibrated with a reference solution, the pH was measured using a pH meter (GroLine HI1285-7, Woonsocket, RI, USA).

2.2.3.2. Determination of Total Amino Acid Content

2.2.3.2.1. Total free amino acids content determination. The method adapted after Păucean et al. [22], using ninhydrin was applied to determine the total free amino acids content. Shortly, 1 mL of extract was homogenized with 0.5 mL of phosphate buffer solution (pH 8.04) and a solution consisting of 0.5 mL of ninhydrin 2% and 0.8 mg/mL of tin (II) chloride. The mixture obtained was placed in the laboratory oven at 105°C for 10 min, and after cooling, 10 mL of distilled water was added. The last step consisted of reading the absorbance at the spectrophotometer Shimadzu 1700 (Kyoto, Japan) at a wavelength of 570 nm. Determination of total free amino acids content was performed with standard calibration procedure using alanine solutions. The calibration curve was obtained using eight calibration solutions of alanine in the range of 10–600 μg/mL.

2.2.3.3. Determination of Micro/Macroelements by Atomic Absorption Spectrophotometry (AAS)

AAS (Varian 220 FAA Atomic Absorption Spectrometer, Varian Inc., Germany) was used to identify macro- and microelements. After undergoing preliminary processing, which involved calcining the samples (3 g) for 10 h at 500 ± 100°C in a furnace (Nabertherm B150, Lilienthal, Germany), the samples were examined using the Varian 220 FAA equipment. After treating the residue with 5 mL of HCl (6 mol/L), it was dissolved in precisely 20 mL of HNO₃ 0.1 mol/L. The results of the analysis are given as mg/kg, with each number representing the average of three separate calculations [20].

2.2.3.4. Determination of Carbohydrates, Organic Acids, and Ethanol Content by HPLC-RID

The Agilent 1200 series HPLC system, outfitted with quaternary pumps, a solvent degasser, and a manual injector coupled with a refractive index detector (RID), was used to carry out the identification of carbohydrates (maltose, glucose, and fructose), organic acids (lactic, acetic, and citric), and ethanol. The 300 7.7 mm Polaris Hi-Plex H column, made by Agilent Technologies in California, USA, was used to separate the chemicals. Compounds were eluted for 25 min using the mobile phase H₂SO₄ 5 mM at a flow rate of 0.6 mL/min, column temperature T = 80°C, and RID temperature T = 35°C. The OpenLab—ChemStation system from Agilent Technologies, Santa Clara, California, USA, was utilized to analyze the results. Finally, the substances were identified by comparing the acquired retention periods to the standard values for glucose, fructose, maltose, citric acid, lactic acid, acetic acid, and ethanol (Sigma-Aldrich, Germany). In short, 2 g of the sample and 4 mL of UPW were vortexed for 1 min using a Heidolph Reax Top vortex, sonicated for 30 min using an Elmasonic E15H sonication bath, and then centrifuged at 7155 × $g$ using an Eppendorf AG 5804 (Hamburg, Germany) centrifuge for an additional 10 min. A 0.45-m nylon filter (CHROMAFIL Xtra PA-45/13) was used to filter the resultant supernatant, and 20 μL was then added to the Agilent 1200 HPLC system [20].

2.2.3.5. Analysis of Total Free Phenolic Compounds (TPC)

The method described by Chiş et al. [23] was used. Shortly, 1 g of each bread sample was homogenized with 100 mL of acidified methanol (85:15, v/v, MeOH:HCl) and dried at 40°C using a vacuum rotary evaporator (Laborota 4010 digital rotary evaporator, Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). The quantity of total phenols was determined using the Folin-Ciocâlteu colorimetric technique. The absorbance was measured at 760 nm (UV/visible spectrophotometer). The standard gallic acid calibration curve (y = 1.022958x + 0.08740, $r^2$ = 0.99614) was used for calculation, and the results were expressed as milligrams of gallic acid equivalent (GAE) per 100 g product.

2.2.3.6. Radical Scavenging Activity (RSA) by 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Assay

RSA was analyzed by the DPPH method as described previously by Chiş et al. [24]. Briefly, a Shimadzu 1700 UV/visible spectrophotometer (Shimadzu Corporation, Kyoto, Japan) was used to measure the absorbance at 515 nm after mixing 0.1 mL of each methanolic extract with 3.9 mL of DPPH solution. The RSA was calculated using the equation:$$\begin{array}{}\text{(1)}& \text{RSA\hspace{0.17em}}\%=\frac{A\text{bs}\text{DPPH}-\text{Abs}\text{Sample}}{\text{Abs}\text{DPPH}}\times 100,\end{array}$$where Abs DPPH = absorbance of DPPH solution and Abs Sample = absorbance of the sample.

2.2.3.7. Determination of Rheological Properties

The sourdough’s rheological measurements were performed using an Anton Paar MCR 302 rheometer (Anton Paar, Graz, Austria) with a parallel plate geometry (PP50) of 50 mm diameter. The technique presupposes that 3 g of each sample is placed on the device’s bottom plate, and the upper plate is lowered to a plate distance of 1 mm. The rheometer was adjusted to 25°C. The storage modulus (G′) and loss modulus (G″) were measured at an angular frequency ranging between 0.1 and 10 Hz, as well as the shear deformation was set at a value of 0.1%.

2.2.3.8. Statistical Analysis

To analyze scientific data, the Duncan multiple comparison test (SPSS version 19 software; IBM Corp., Armonk, NY, USA) was used. Lowercase letters indicate significant differences ($p$ < 0.05) between samples at the same fermentation period, whereas uppercase letters indicate significant differences between samples at various fermentation times (0–24 h). All analyses were performed in triplicate (n = 3).

3. Results and Discussion

3.1. Proximate Composition, Fatty Acids Profile, TPC, and DPPH RSA of Alfalfa Sprouts Seeds Flour

The proximate composition of ASF flour is shown in Table 2, while Table 3 shows the fatty acids profile. Generally, alfalfa seed germination is followed by a spectrum of substantial modifications in its metabolite composition, enhancing the nutritional and functional properties of alfalfa seeds [25]. The results show that ASF is an excellent source of protein (38.6%), dietary fibers (33.4%), and minerals (expressed as ash 3.43%) and supplies carbohydrates that could contribute to the initiation of sourdough fermentation.

Table 2

Proximate composition, TPC, and RSA of alfalfa sprouts seeds flour.

Compounds (g)/100 g	Alfalfa sprouts flour (ASF)
Moisture	7.90 ± 0.60
Protein	38.60 ± 3.10
Total dietary fiber	33.40 ± 6.70
Fat	12.50 ± 1.10
Starch	4.20 ± 0.30
Total sugar after inversion	4.70 ± 0.50
Ash	3.43 ± 0.21
Total polyphenols (TPC), mg GAE/100 g	230.00 ± 0.40
RSA (%)	66.51 ± 0.10

Note. Values are expressed as mean ± standard deviation.

Table 3

Fatty acids content (g/100 g) of alfalfa sprouts seeds flour.

Shorthand nomenclature	Fatty acids (g)/100 g	Alfalfa sprouts flour (ASF)
16:0	Palmitic acid	1.30 ± 0.11
18:0	Stearic acid	0.30 ± 0.15
18:1 (n − 9)	Oleic acid	1.40 ± 0.12
18:2 (n − 6)	Linoleic acid (LA)	4.70 ± 0.60
20:0	Arachidic acid	0.10 ± 0.10
18:3 (n − 3)	α-Linolenic acid (ALA)	3.50 ± 0.52
22:0	Behenic acid	0.10 ± 0.13
20:4 (n − 6)	Arachidonic acid (ARA)	0.10 ± 0.11
∑SFAs	Total saturated fatty acids	1.90 ± 0.20
∑MUFAs	Total monounsaturated fatty acids	1.50 ± 0.23
∑PUFAs	Total polyunsaturated fatty acids	8.30 ± 1.10
∑n − 3	Total Omega-3 fatty acids	3.50 ± 0.51
∑n − 6	Total Omega-6 fatty acids	4.80 ± 0.62
∑n − 9	Total Omega-9 fatty acids	1.40 ± 0.20

Note. Values are expressed as mean ± standard deviation.

The protein, fat, fatty acids, TPC, and RSA are among the remarkable properties of ASF that sustain its applicability in sourdough technology. The growth and viability of microorganisms in sourdough depend on the nutrient’s availability. Proteins, amino acids, vitamins, and minerals (especially Ca, Mg, Fe, and Zn) influence consistently the cell microbial dynamics [26]. First, the readily digested molecules that the microorganisms consume include simple sugars, vitamins, and free amino acids. After that, the proteolytic activity of the microbial cells has the main effect on their growth and viability [20]. Thus, due to the high content of proteins, dietary fibers, ash, PUFAs (especially linoleic and α-linolenic acids), and MUFAs (oleic acid being the most abundant), blends of wheat and ASFs could be a valuable raw matrix for sourdough fermentation. Moreover, ASF was reported to be a rich source of vitamins (B complex, A, C, E), phytoestrogens, and saponins [10, 27], contributing to the functional properties. In addition, during alfalfa seed germination, an intensification of the RSA is reported due to the increase in total polyphenols, vitamins, and other antioxidant compounds. In the case of the tested ASF, high contents of TPC (230 mg GAE/100 g) and RSA (66.51%) were found. Close values of TPC and RSA have been found by Francis [3]. However, some differences in the proximate composition of ASF flour might be due to the provenience, variety, and pedo-climatic conditions of the raw seeds [4]. In addition, germination time, light, and moisture could significantly influence the nutritional content of the sprouts [3, 5].

These findings indicate the use of alfalfa sprouts in sourdough fermentation to improve bread quality attributes.

3.2. Viable Cell Count and pH Variation in Sourdough Samples

Figure 1 shows significant cellular growth for Lactiplantibacillus plantarum (Lp) in all sourdough samples where lactic bacteria was the single starter culture. The initial bacterial concentration was around 7 log cfu/g in all these samples (LG0, LG5, LG10, LG15) and reached after 24 h of fermentation values between 7.95 and 9.99 log cfu/g, demonstrating a good adaptability and growth dynamic in WF/ASF blends. In the sourdough samples co-inoculated with Lp + Sc, the bacterial growth was smaller, indicating competition for substrate nutrients like fermentable carbohydrates and nitrogen sources [28]. In these samples (LDG0, LDG5, LDG10, LDG15), Saccharomyces cerevisiae (Sc) yeast growth was smaller too (Figure 1), compared to their growth in the samples inoculated with bakery yeast as a single strain (DG0, DG5, DG10, DG15).

[figure(s) omitted; refer to PDF]

Sourdough characteristics influence the microbial community, which impacts the physicochemical properties of the product, ultimately affecting its quality. Generally, the fast pH drop and accumulation of lactic or acetic acid in an undissociated, lipophilic, and membrane-diffusible state, along with the growth-inhibiting actions of ethanol, can partially limit yeast cell development in the dough containing mixed LAB cultures [29, 30]. On the contrary, the number of Sc viable cells was significant, at 9.07–10.69 log cfu/g after 24 h of fermentation at 30°C, in the samples inoculated with monoculture. Other studies have reported an increasing trend of yeast cell density vs. LAB in spontaneous fermentation of sprouted legume flours in mix with cereal flours [8, 16]. The very good cell development of Sc in the sourdough samples, especially in the case of monoculture, might be because the bakery yeast is already well adapted to dough fermentation [31]. Furthermore, in the case of co-culture, it has been shown that the anaerobic environment due to the CO₂ created by yeasts might promote the development of LAB in species like Lb. sanfranciscensis and Lb. plantarum [28].

No negative effect on the viability of Lp and Sc could be noticed due to ASF flour addition, compared to the control samples. Moreover, the co-inoculated sourdough samples with ASF flour addition (LDG 5, LDG10, LDG15) lead to the increment of both, Lp and Sc, cells count after 24 h of fermentation, having a similar behavior as the control sample.

Figure 2 illustrates the changes in pH for the sourdough samples over 24 h, starting from the initial moment (T0). The pH values of the sourdough samples ranged from 5.15 to 5.93 at T0. Addition of ASF flour slightly dropped the pH values in the unfermented (T0) samples compared to the control samples but without significant differences. As expected, due to the metabolic activity of lactic bacteria, after 24 h of fermentation, lower pH values were recorded in the sourdough samples fermented with Lp either as a single inoculum or in co-culture. In the samples with ASF, the lowest pH values ranged between 3.17 (LG5) and 3.4 (LG15), while the highest pH values were measured in samples fermented with (Sc) and ranged between 4.91 (DG5) and 5.29 (DG15). All the samples fermented with Lp (single or in co-culture) were significantly different after 24 h of fermentation compared to the initial moment. The pH value under 4.5 that was recorded in these samples indicates a proper activity of Lp that reached a final concentration of 9 log cfu/g [32]. Noteworthy, in the sourdough samples co-inoculated with Lp + Sc, lactobacilli’s metabolic activity reduces pH levels until a critical limit is reached, particularly for yeasts [30, 33], as results from cell viability showed (Figure 1(b)). In these samples, ASF addition significantly affects the pH values compared to the control sample. A similar trend was recorded in the sourdough samples inoculated with Sc monoculture.

[figure(s) omitted; refer to PDF]

3.3. Carbohydrates (Maltose, Glucose, Fructose), Organic Acids, and Ethanol Contents in Sourdough Samples

Table 4 shows the effect of fermentation time on maltose, glucose, and fructose content in sourdough samples. Significant consumption of maltose after 24 h was recorded in the samples fermented with Sc or Lp + Sc, the remaining concentrations being below 1 mg/g. After 24 h of fermentation, glucose was consumed totally in the sourdough samples with single inoculum starter (Lp or Sc), except DG0 and DG5. In the samples with co-culture (Lp + Sc), small amounts below 0.21 mg/g were recorded. As for the fructose concentrations, significant variations were found after 24 h of fermentation in all samples. These results indicate glucose and maltose consumption as the main sources of energy for both Lp and Sc, but in the case of co-inoculation, the microorganisms competed for the nutritive substrate. However, the total metabolization of glucose from LG and DG samples indicates that both the initial glucose contents from the flours and the contents resulting after maltose depletion were used by Lp and Sc. Noteworthy, to use maltose, yeast needs an active transport mechanism across the plasma membrane. In the second step, glucosidase enzymes convert maltose into two glucose molecules. However, in the sourdough containing Sc, single or co-culture, maltose depletion was significantly higher than in Lp-containing sourdough. In addition, in the case of Sc, glucose was preferred over fructose due to a greater binding specificity [31]. Concerning fructose consumption by Lp, it was probably utilized as a carbon source, with pyruvate serving as the primary metabolic connecting point of sugar metabolism [34]. Yeast could consume fructose, also contributing to acetic acid production [17].

Table 4

Effect of fermentation time on maltose, glucose, and fructose content in sourdough samples.

Sample	Maltose	Glucose	Fructose	Maltose	Glucose	Fructose
	T0 (mg/g)	T0 (mg/g)	T0 (mg/g)	T24 (mg/g)	T24 (mg/g)	T24 (mg/g)
LG0	7.68 ± 0.12abB	1.88 ± 0.02bc	0.82 ± 0.01aB	2.85 ± 0.03cA	n.d	0.33 ± 0.02aA
LG 5	14.23 ± 0.02efB	1.35 ± 0.03abc	2.40 ± 0.05cB	12.67 ± 0.07efA	n.d	0.71 ± 0.02bA
LG 10	12.01 ± 0.07dA	1.12 ± 0.01abc	1.33 ± 0.07abA	12.11 ± 0.02eA	n.d	1.82 ± 0.01dB
LG 15	11.52 ± 0.04dB	1.09 ± 0.07ab	1.35 ± 0.04abA	7.73 ± 0.04dA	n.d	2.58 ± 0.05eB
LDG0	7.18 ± 0.01aB	1.95 ± 0.03bcB	0.75 ± 0.09aB	0.12 ± 0.01cA	0.12 ± 0.01bA	0.15 ± 0.01aA
LDG 5	13.65 ± 0.03eB	2.46 ± 0.02dB	2.11 ± 0.01bcB	0.49 ± 0.04abA	0.05 ± 0.02aA	0.38 ± 0.07aA
LDG 10	11.46 ± 0.08dB	1.84 ± 0.08bcB	1.72 ± 0.03bB	0.76 ± 0.03bA	0.15 ± 0.01bA	0.40 ± 0.08aA
LDG 15	10.30 ± 0.02cB	1.66 ± 0.04bcB	2.68 ± 0.08cB	0.80 ± 0.07bA	0.21 ± 0.04cA	1.03 ± 0.04cA
DG0	6.95 ± 0.05aB	1.92 ± 0.01bcB	0.79 ± 0.02aAB	0.25 ± 0.02cA	0.21 ± 0.01A	0.36 ± 0.02aA
DG 5	13.85 ± 0.05eB	1.83 ± 0.06bcB	2.45 ± 0.05cB	0.95 ± 0.01bcA	0.25 ± 0.07cA	0.65 ± 0.01abA
DG 10	11.98 ± 0.04dB	1.05 ± 0.03ab	2.70 ± 0.06cB	0.46 ± 0.03abA	n.d	0.51 ± 0.03aA
DG 15	10.23 ± 0.01cB	0.66 ± 0.09a	2.99 ± 0.02dB	0.19 ± 0.02aA	n.d	0.48 ± 0.02aA

Note. LG0—Lp control sample, LG 5—Lp and 5 g alfalfa sprouts, LG 10—Lp and 10 g alfalfa sprouts, LG 15—Lp and 15 g alfalfa sprouts, DG 5—Sc and 5 g alfalfa sprouts, DG0—Sc control sample, DG 10—Sc and 10 g alfalfa sprouts, DG 15—Sc and 15 g alfalfa sprouts, LDG0—Lp, Sc control sample, LDG 5—Lp, Sc, and 5 g alfalfa sprouts, LDG 10—Lp, Sc, and 10 g alfalfa sprouts, LDG 15—Lp, Sc, and 15 g alfalfa sprouts. Values are expressed as mean ± standard deviation. Different lowercase letters indicate significant differences ($p$ < 0.05) between samples at the same fermentation time, and different uppercase letters indicate significant differences ($p$ < 0.05) between the same sample at different fermentation times (0, 24 h).

The high maltose concentrations in the unfermented sourdough samples could be the result of a higher amylolytic activity from the ASF addition, since during sprouting these enzymes are activated [10]. A higher amount of maltose was found by Perri et al. [16] in sprouted barley flour sourdough compared to sourdough obtained from blends of wheat and sprouted barley flour. However, the sugar profile of sprouted seeds depends on the levels of α-amylase and β-amylase released during germination and the germination time [35]. Noteworthy, different alfalfa cultivars could accumulate maltose and other soluble sugars to protect plants from low-temperature damage, preserve cell membrane integrity, relay cold signals, and regulate light-induced gene expression during acclimation [36].

Table 5 shows the effect of fermentation time on lactic acid, acetic acid, and ethanol content from sourdough samples. At the initial moment (T0), lactic acid was absent or recorded a very low content (0.11–0.19 mg/g). After 24 h of fermentation, the highest concentrations were recorded in sourdough samples fermented with Lp as the single starter, as expected. The highest contents have been found in LG0 (4.53 mg/g) and LG10 (4.1 mg/g), while in the case of co-culture (Lp + Sc), LDG15 recorded the highest value (5.86 mg/g). Lp as single or in co-culture with Sc was also found to produce lactic acid when 5% soy flour was added to wheat sourdough and the reported values were very close to those detected in this study [37]. However, it is possible that Saccharomyces cerevisiae consumed lactic acid, reducing the acidity of the sourdough environment and promoting microbial growth, as was reported by Sieuwerts et al. [38]. But, nevertheless, it is possible that the small amounts of lactic acid recorded after 24 h of fermentation in samples fermented with Sc as single inoculum were produced by LAB species which could contaminate the bakery yeast during its preparation [17].

Table 5

Effect of fermentation time on lactic acid, acetic acid, and ethanol content from sourdough samples.

Sample	Lactic acid	Acetic acid	Ethanol	Lactic acid	Acetic acid	Ethanol
	T0 (mg/g)	T0 (mg/g)	T0 (mg/g)	T24 (mg/g)	T24 (mg/g)	T24 (mg/g)
LG0	n.d	n.d	n.d	4.53 ± 0.05c	0.89 ± 0.03b	0.12 ± 0.02a
LG 5	n.d	n.d	n.d	3.15 ± 0.01b	0.22 ± 0.02a	0.05 ± 0.02a
LG 10	n.d	n.d	n.d	4.10 ± 0.04c	0.40 ± 0.07a	0.13 ± 0.02a
LG 15	n.d	n.d	n.d	3.62 ± 0.08b	0.35 ± 0.06a	0.18 ± 0.03a
LDG0	n.d	n.d	n.d	4.72 ± 0.08cd	1.21 ± 0.02c	3.09 ± 0.02b
LDG 5	0.11 ± 0.02aA	n.d	0.47 ± 0.08aA	4.63 ± 0.09cdB	0.49 ± 0.05a	3.62 ± 0.05bA
LDG 10	0.17 ± 0.08abA	n.d	0.48 ± 0.02aA	5.33 ± 0.07eB	0.53 ± 0.03a	4.72 ± 0.07cA
LDG 15	0.19 ± 0.04bA	n.d	0.55 ± 0.04bA	5.86 ± 0.05eB	0.50 ± 0.04a	5.25 ± 0.09dA
DG0	n.d	n.d	n.d	0.15 ± 0.001a	0.89 ± 0.01a	5.75 ± 0.03d
DG 5	n.d	n.d	0.87 ± 0.01eA	0.13 ± 0.002a	n.d	5.71 ± 0.06dB
DG 10	n.d	n.d	0.80 ± 0.05cdA	0.19 ± 0.001a	n.d	6.43 ± 0.03eB
DG 15	n.d	n.d	0.76 ± 0.03cA	0.2 ± 0.01a	0.50 ± 0.01c	6.63 ± 0.01eB

Note. LG0—Lp control sample, LG 5—Lp and 5 g alfalfa sprouts, LG 10—Lp and 10 g alfalfa sprouts, LG 15—Lp and 15 g alfalfa sprouts, DG0—Sc control sample, DG 5—Sc and 5 g alfalfa sprouts, DG 10—Sc and 10 g alfalfa sprouts, DG 15—Sc and 15 g alfalfa sprouts, LDG0—Lp, Sc control sample, LDG 5—Lp, Sc, and 5 g alfalfa sprouts, LDG 10—Lp, Sc, and 10 g alfalfa sprouts, LDG 15—Lp, Sc, and 15 g alfalfa sprouts. Values are expressed as mean ± standard deviation. Different lowercase letters indicate significant differences ($p$ < 0.05) between samples at the same fermentation time, and different uppercase letters indicate significant differences ($p$ < 0.05) between the same sample at different fermentation times (0, 24 h).

Acetic acid was absent in all sourdough samples at T0, but after 24 h, Lp sourdough recorded low concentrations ranging from 0.22 to 0.53 mg/g and 0.89 mg/g for the control sample. This is consistent with other findings for wheat sourdough fermented with Lp [20, 37], acetic acid being a minor metabolite of the heterofermentative pathway. However, yeast Sc. cerevisiae has been found to contribute to the acetic acid accumulation in sourdough, thus explaining the higher concentrations of acetic acid in LDG samples [30]. No significant influence of ASF flour addition was found, with only a very slight increment being detected with the raising of the ASF flour amount. On the contrary, compared to the control samples, acetic acid amounts recorded for the samples with ASF flour addition were significantly different and smaller.

Ethanol content recorded higher values in sourdough samples fermented with Sc and co-culture Lp + Sc due to the alcoholic fermentation performed by yeast. In the samples with ASF flour, the highest ethanol concentrations were determined in sourdough fermented with Sc as single inoculum and ranged between 6.63 and 5.71 mg/g, these values indicating the high ability of Sc yeast to catabolyze six-carbon sugars in the production of ethanol. The samples DG10 and DG 15 were significantly different from the control sample DG0. Results are consistent with those reported by Martău et al. [30] in sourdough fermented with Fructilactobacillus florum and S. cerevisiae. As for the sourdough samples fermented by Lp, the small ethanol amounts formed are the result of the heterofermentative pathway of lactic fermentation. Except for the samples fermented with Lp, ASF flour addition did not influence significantly the ethanol content accumulation.

3.4. Total Free Amino Acids (TFA) Content

The TFA content from sourdough samples has an upward trend after 24 h of fermentation for the samples fermented with Lp and Sc as single starters, with values close to 3.5 mg/mL for LG15 and DG15 at T24. Significant differences have been found between these samples. In the case of co-inoculation of WF/ASF blends, TFA recorded lower values, ranging between 1.62 and 2.22 mg/mL after 24 h, but significantly different ($p$ < 0.05) compared to the initial moment of fermentation (Figure 3). The lower TFA values recorded in the case of co-inoculation is probably due to the competition of yeast and bacteria for the free amino acids as nitrogen source. Generally, protein degradation by endogenous and microbial proteases boosts TFA levels. The increase in TFA is explained by the proteolytic activity of the microorganisms, the amino acids being used for their microbial dynamics [22]. Typically, sourdough fermented by Lb. plantarum showed increased concentrations of amino acids due to the intracellular peptidases secreted by the bacterial strain as well as due to the flour’s endogenous protease activated by the low pH [39]. Similarly, yeast Sc is known for high proteolysis activity and the free amino acids formed could highly influence the aroma profile [40].

[figure(s) omitted; refer to PDF]

Generally, as the results show, the free amino acids concentrations increased with increasing amounts of ASF flour addition in both unfermented and fermented samples. A large number of studies are reporting on the germinated legumes’ flour sourdough potential to enhance the peptides and free amino acids concentrations [8, 41, 42]. In the case of Medicago sativa L., recent studies are reporting that amino acids are synthesized in higher amounts due to their important role as a nitrogen source for plants and adaptation in stress conditions [36]. Thus, the combined effect of ASF flour and sourdough fermentation is a strategy to enhance sourdough and bread not only from the nutritional point of view but also by acting as volatile organic compounds precursors [43]. Aside from nutritional attributes, amino acids influence taste and flavor. However, fermentation time appears to have a stronger effect on TFA content than the ASF flour addition.

3.5. Macro/Microelements Contents

The mineral profile of sourdough showed significant ($p$ < 0.05) elevated concentrations for Ca, Mg, K, Mn, and Fe (Table 6) as the ASF flour addition increased, both at 0 and 24 h of fermentation.

Table 6

Effect of fermentation time on macro/microelements content from sourdough samples.

Minerals (mg/kg)

LG 0

LG 5

LG 10

LG 15

LDG0

LDG 5

LDG 10

LDG 15

DG0

DG 5

DG 10

DG 15

T0

Ca

98.75 ± 0.06aB

105.21 ± 0.04cB

109.37 ± 0.08eB

167.20 ± 0.06gB

101.06±bB

103.43 ± 0.05cB

105.25 ± 0.08cA

154.22 ± 0.03fB

100.24 ± 0.05bB

104.70 ± 0.08bB

107.33 ± 0.05dB

227.82 ± 0.08hB

Mg

60.56 ± 0.12aA

62.56 ± 0.01bA

73.02 ± 0.06eB

78.77 ± 0.04fA

62.86 ± 0.25bA

70.24 ± 0.02dA

78.26 ± 0.07fA

81.06 ± 0.07gA

66.41 ± 0.36cA

84.67 ± 0.2hA

83.65 ± 0.07hA

88.70 ± 0.06iA

K

100.93 ± 0.01aA

113.25 ± 0.06cA

132.38 ± 0.04dB

159.51 ± 0.08hA

102.35 ± 0.25aA

105.21 ± 0.08bA

142.45 ± 0.09eA

178.59 ± 0.07jA

101.72 ± 0.18aA

144.82 ± 0.03fA

186.28 ± 0.08kA

175.14 ± 0.03fA

Mn

0.30 ± 0.01aA

n.d.

0.19 ± 0.04a

0.36 ± 0.02abA

0.36 ± 0.04abA

0.92 ± 0.04cdA

0.68 ± 0.04bcA

1.39 ± 0.05dB

0.40 ± 0.01bA

1.27 ± 0.03dA

0.84 ± 0.05cdB

2.73 ± 0.04eB

Fe

7.05 ± 0.02dB

7.44 ± 0.04dB

4.01 ± 0.05aB

5.61 ± 0.03cA

7.52 ± 0.03dB

8.14 ± 0.03eB

4.65 ± 0.05abB

8.13 ± 0.06eB

7.18 ± 0.02dB

7.68 ± 0.03dB

5.68 ± 0.02cB

8.78 ± 0.03eB

Zn

0.04 ± 0.01aA

0.04 ± 0.01aA

0.03 ± 0.01aAB

0.04 ± 0.02aB

0.4 ± 0.01aA

0.04 ± 0.01aA

0.03 ± 0.03aA

0.65 ± 0.03bB

0.04 ± 0.01aA

0.04 ± 0.02aA

0.05 ± 0.01aB

0.11 ± 0.05abA

T24

Ca

59.28 ± 0.02dA

67.24 ± 0.04eA

81.22 ± 0.05iA

99.64 ± 0.08jA

65.04 ± 0.04dA

70.48 ± 0.05fA

75.03 ± 0.04gB

77.45 ± 0.07hA

51.05 ± 0.02bA

45.41 ± 0.04aA

50.41 ± 0.08bA

55.41 ± 0.03cA

Mg

65.33 ± 0.15aB

85.97 ± 0.08dB

65.34 ± 0.06aA

85.43 ± 0.06dB

70.04 ± 0.2bB

98.14 ± 0.03gB

94.75 ± 0.05fB

96.45 ± 0.04fB

73.05 ± 0.12cB

88.46 ± 0.07dB

86.42 ± 0.03cdB

88.75 ± 0.08deA

K

144.23 ± 0.02bB

169.64 ± 0.06dB

121.72 ± 0.05aA

178.82 ± 0.09eB

148.18 ± 0.14B

187.01 ± 0.08fB

160.15 ± 0.02cB

188.15 ± 0.07fgB

144.89 ± 0.15bB

199.15 ± 0.05hB

169.49 ± 0.07dB

189.95 ± 0.03gB

Mn

0.71 ± 0.02aB

1.15 ± 0.05d

n.d.

0.71 ± 0.03aB

0.85 ± 0.04aB

1.23 ± 0.06bB

1.11 ± 0.01bB

1.01 ± 0.03bA

0.82 ± 0.01aB

1.79 ± 0.03cB

1.16 ± 0.05bA

1.01 ± 0.04bA

Fe

3.69 ± 0.05deA

3.43 ± 0.03cdeA

1.30 ± 0.02aA

4.48 ± 0.05efB

4.08 ± 0.19eA

2.35 ± 0.02bA

3.46 ± 0.05cdeA

4.99 ± 0.05fA

3.89 ± 0.12deA

2.48 ± 0.07bcA

3.11 ± 0.03bcdA

3.79 ± 0.01deA

Zn

0.03 ± 0.02aA

0.04 ± 0.01cA

0.02 ± 0.01aA

0.05 ± 0.03dB

0.03 ± 0.02abA

0.02 ± 0.01abA

0.03 ± 0.02abA

0.03 ± 0.01bcA

0.03 ± 0.02abA

0.03 ± 0.02bcA

0.03 ± 0.01bcA

0.03 ± 0.01abB

Note. LG0—Lp control sample, LG 5—Lp and 5 g alfalfa sprouts, LG 10—Lp and 10 g alfalfa sprouts, LG 15—Lp and 15 g alfalfa sprouts, DG0—Sc control sample, DG 5—Sc and 5 g alfalfa sprouts, DG 10—Sc and 10 g alfalfa sprouts, DG 15—Sc and 15 g alfalfa sprouts, LDG0—Lp, Sc control sample, LDG 5—Lp, Sc, and 5 g alfalfa sprouts, LDG 10—Lp, Sc, and 10 g alfalfa sprouts, LDG 15—Lp, Sc, and 15 g alfalfa sprouts. Values are expressed as mean ± standard deviation. Different lowercase letters indicate significant differences ($p$ < 0.05) between samples at the same fermentation time, and different uppercase letters indicate significant differences ($p$ < 0.05) between the same sample at different fermentation times (0, 24 h).

The highest concentrations were found for K followed by Mg and Ca, but Fe contents showed high values also. In the unfermented sourdough samples, the highest K concentrations ranged between 159.51 and 178.59 mg/kg and were recorded in the samples with a 15 g ASF addition level. As for Ca and Mg, the trends were similar, with the highest values of 227.82 and 88.7 mg/kg, respectively, in the DG15 samples. The recorded values for K, Ca, and Mg were significantly different from the control samples. Compared to wheat dry seeds, Plaza [5] reported higher values of K, Fe, and Ca for alfalfa sprouts. However, the sprouting conditions (temperature, moisture, time, light) and plant breed could influence significantly the mineral content, and variations regarding their concentrations are widely reported [10, 26]. In addition, high phytase activity was reported both for Lb. plantarum and S. cerevisiae yeast, especially during prolonged fermentation, thus resulting in increased content of minerals, particularly Mg. Moreover, it was stated that phytase activity varied greatly across sourdough starters with both yeast and lactic acid bacteria. The main factors influencing microbial phytase activity are the pH values ranging between 4.8 and 5.5 and a lower content of Ca [44]. These conditions allow yeast S. cerevisiae to reduce phytic acid’s negative effects. However, acidic sourdough appears to be a superior source of readily accessible minerals, particularly magnesium, iron, and zinc [31]. In this regard, both Lp and Sc showed a high capacity to increase available minerals, mainly Mg, K, and Fe. Noteworthy, the co-inoculated sourdough samples showed the highest mineral concentrations in samples with 10 and 15 g ASF flour for K, Mg, and Fe. However, in the case of S. cerevisiae as single inoculum or co-culture, a significant decrease ($p$ < 0.05) of Ca concentrations was recorded after 24 h of fermentation. In S. cerevisiae cells, calcium ions (Ca²⁺) play an important role in regulating cell proliferation, programmed cell death, etc., and metabolic pathways are used by yeast cells to maintain calcium homeostasis [45], thus explaining the Ca drop in yeast sourdough samples.

3.6. TPC and DPPH RSA

The evolution of TPC and RSA of sourdough samples is presented in Figures 4(a) and 4(b). The results show that both sourdough fermentation time and ASF addition led to the TPC and RSA increments. After 24 h of fermentation, sourdough samples LDG15 and DG15 revealed the highest TPC (183 and 185 mg GAE/100 g) and RSA (23.84% and 27.36%). Fermentation conditions, flour matrix composition, and types of microorganisms used as starters influence soluble polyphenol content [15]. TPC and RSA significantly ($p$ < 0.05) increased with the ASF amount at 10 and 15 g addition and compared to the control samples. This is an expected result since ASF had a TPC of 230 mg GAE/100 g and 66.5% RSA (Table 2). Alfalfa sprouts contain significant amounts of phenolic chemicals (gallic and caffeic acids) and flavonoids (apigenin, kaempferol, myricetin, naringin, quercetin, rutin, daidzein, and genistein). These compounds have many biological effects, including antidiabetic, antiobesity, and antioxidant properties [10, 46]. Moreover, it was found that Medicago sativa extracts showed a strong antioxidant potential and the best activity against O₂^-, when compared to extracts of other legumes, such as Glycine max and Vigna radiata. Therefore, the phenols that are produced during seed germination may contribute to higher TPC concentrations and antioxidant activity, which would increase the nutraceutical qualities [47], of the ASF flour and thus the derived food products.

[figure(s) omitted; refer to PDF]

Concerning the inoculation variants, higher TPC was recorded for the sourdough fermented with Sc and co-culture of Lp + Sc. The results show significant differences ($p$ < 0.05) for TPC content between sourdough fermented with Lp and Sc, Lp + Sc, but only for 10 and 15 g ASF addition. Generally, microorganisms’ metabolic activity might change the amounts of bioactive compounds during fermentation thus explaining the observed results. Fermentation, through endo/exo enzymes like amylases, xylanases, and proteases can break down plant cell walls and produce beneficial compounds [48]. One important factor affecting the plant endogen enzymes release is the sourdough pH since enzymatic activity needs an optimum pH value, but no less important is that the microbial species has a great influence, also. In this context, fermentation has been shown to increase the content of extractable phenolic compounds and antioxidant activity in sourdough and fermented legume flours [15, 42]. Moreover, sourdough and yeast fermentation of rye meal contributed to increased levels of TPC [49]. Increased antioxidant activity was reported for the lactic acid fermentation of cowpea, mottled cowpea, speckled kidney bean, and tiny rice bean flours when performed by strains of Lb. plantarum. This has been related to the biotransformation between soluble phenols and the release of bound phenols. Legumes are known to have an increased content of bounded phenols. Nonetheless, there is growing agreement for the theory that lactic acid bacteria’s metabolism of phenolic substances may potentially play a part in maintaining the equilibrium of energy within cells [42].

3.7. Viscoelastic Properties

The storage modulus (G′) and loss modulus (G″) of the sourdough samples were measured at the initial moment (T0) and after T24H of fermentation at an angular frequency ranging between 0.1 and 10 Hz (Figures 5(a) and 5(b)). The materials’ ability to maintain their elastic deformation energy is shown by the storage modulus, while their viscous part is indicated by the loss modulus [20]. Generally, G′ was greater than G″, an indication that the dough behaves elastically for all sourdough samples. Moreover, the two moduli predominantly increased as the angular frequency increased, a phenomenon that can be attributed to the sourdough’s increased structure. The rheological properties of the dough are highly influenced by the gluten’s quantity and quality. It is well known that adding legume flour causes adverse consequences in the rheology of the dough. However, as the results of this study show and as was reported previously, germination appears to be a good method for improving the rheological properties of dough [50–52]. The increased quantity of protein and dietary fiber constituents in ASF flour interact with the gluten network, reducing gluten plasticity and producing a strength effect in the dough [53]. This explains why the addition of ASF flour increased the values of the two viscoelastic parameters. This rheological behavior can be seen in comparison with the control samples. Also, the increment of ASF flour addition had a similar effect on the viscoelastic parameters.

[figure(s) omitted; refer to PDF]

The improvement of dough elasticity is desirable because it contributes to better gas network development and bubble structure retention, especially when the sourdough fermentation method is applied. Sourdough fermentation could have a significant impact on dough rheology being influenced by the microbial strains and their metabolic activity. From this point of view, pH value has an important influence on the viscoelastic parameters variation. As Figures 5(a) and 5(b) shows, the two dynamic moduli decreased after 24 h of fermentation for all sourdough samples, due to the pH drop. Thus, sourdough acidification improves the interaction between water molecules and structural components like proteins and starch [20, 53]. The type of inoculum used for sourdough fermentation led to different pH values after 24 h, as was previously shown (Figure 2), and consequently, the difference between the G′ and G″ at T0 and T24 varied. When Lp was used as a single inoculum, a smaller decrease of the dynamic moduli was recorded after 24 h. The use of Sc as a fermentation starter led to higher differences between these parameters after 24 h, with the highest differences in the sourdough samples fermented with Sc as the single starter. In these sourdough samples (LDG), the pH values were the highest and probably the available water content was smaller. Hardt et al. [54] stated that G′ increased at lower water content leading to a decrease in water’s plasticizing impact. This behavior was seen also in the sourdough samples where Sc was used as fermenting agent, either single or co-culture. All control samples revealed a similar trend, thus it can be stated that the microbial strain and the fermentation process have an important impact on the rheological behavior of the sourdough, independently from the ASF addition.

4. Conclusions

The valuable chemical composition of alfalfa sprouted seeds makes it worthy of increasing attention and the combination with different fermentation types, using lactic bacteria or bakery yeast, appears a valid option to promote its use in breadmaking. No negative effect of ASF addition in wheat blends sourdough fermentation was identified.

An analysis of the differences in the microbiological and physicochemical composition of bread obtained with these sourdough will be conducted in order to fully understand the effects of these variables on the product’s quality.

Ethics Statement

This study does not involve any human or animal testing.

Author Contributions

Păuşan Delia-Elena: data curation, formal analysis, investigation, methodology, and writing – original draft. Adriana Păucean: concept, methodology, project administration, validation, visualization, and writing – review & editing. Simona Maria Man: formal analysis, investigation, resources, and validation; Maria Simona Chiş: investigation, resources, and software; Carmen-Rodica Pop: formal analysis, investigation, methodology, resources, and supervision; Anamaria Pop: formal analysis, methodology, and supervision; Floricuța Ranga: formal analysis, investigation, methodology, validation, and resources; Andreea Puşcaş: formal analysis, investigation, methodology, validation, and software; Ersilia Alexa: formal analysis, methodology, supervision, and resources; Adina Berbecea: formal analysis, methodology, and resources; Anca-Corina Fărcaş: investigation, supervision, and visualization; Vlad Mureşan: project administration, resources, and visualization.

Funding

No funding was received for this research.

Acknowledgments

The authors would like to thank the University of Agricultural Sciences and Veterinary Medicine from Cluj-Napoca, Romania, for the support in conducting the present research work.

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