The Impact of Fermented Quinoa Sourdough with Enterococcus Strains on the Nutritional, Textural, and Sensorial Features of Gluten-Free Muffins

Abstract

Gluten-free baked goods exhibit reduced texture and taste characteristics compared to their gluten-containing counterparts. As a result, there is a renewed interest in the fermentation of gluten-free cereals with lactic acid bacteria, which is associated with an improvement in the final baked goods. Quinoa is garnering growing attention due to its different nutrients and bioactive substances, and it is notably employed to build gluten-free goods. In the present study, quinoa flour was fermented with Enterococcus strains (E. gallinarum SL2 and E. mundtii SL1), and further used in the manufacturing of gluten-free muffins. Several analyses were performed on the obtained sourdoughs and muffins, including a viscosity study, a textural and sensory analysis, and a polyphenol, organic acid, and carbohydrate content analysis. The results showed that the fermented quinoa flour exhibited enhanced nutritional value, with increased levels of organic acids such as lactic and acetic acid, as well as improved polyphenol content. The sensory and textural analyses revealed that both Enterococcus strains positively impacted the sensory characteristics and texture of the muffins. Notably, muffins prepared with E. mundtii SL1 demonstrated superior elasticity and overall taste. These results suggest that fermentation with these strains can significantly improve the nutritional profile and sensory quality of gluten-free baked goods, offering a promising approach for the development of healthier and more appealing gluten-free products.

1. Introduction

Gluten, which is a mixture of seed storage proteins called prolamins, can be found in cereal grains like wheat, barley, rye, oats, and their derivatives [1,2]. Gluten creates a viscoelastic network that helps create a light and airy structure, enhancing the baking quality of these cereals [3]. Gluten proteins are rich in proline and glutamine, and as a result, they are broken down into peptides by digestive proteases in the gastrointestinal tract [4].

Currently, the prevalence of gluten-related disorders (GRDs), such as celiac disease, wheat allergy, and gluten intolerance, is steadily rising to reach 5% of the global population [5,6]. These illnesses are distinguished by intestinal and/or extraintestinal signs, an increase in particular antibodies such as anti-gliadin and anti-tissue transglutaminase, and the presence of HLA-DQ2/DQ8 haplotypes [7].

So far, the only effective and widely available treatment for GRDs is a gluten-free (GF) diet. Adhering to this diet, however, may be challenging for many celiac disease patients due to a variety of causes [8,9]. These include a lack of nutrition awareness, a lack of gluten-free products, social pressure, temptation, and an aversion to the taste of dishes manufactured from alternative grains. These variables are linked to noncompliance with the gluten-free dietary process [5].

Generally, gluten-free baked goods are characterized by reduced textural and sensory properties compared to their gluten-containing counterparts. As a result, there is a renewed interest in the fermentation of gluten-free cereals with lactic acid bacteria, which is correlated with a greater quantity of bioactive compounds and with advantages on texture, shelf life, the preservation of foods, and the sensory characteristics of the final baked goods, opening new frontiers in food manufacturing research. In this sense, quinoa is receiving increased attention due to its various nutrients and bioactive compounds and is mainly used to design gluten-free products [10,11].

These rationales suggest that sourdough could enhance the quality of gluten-free baked goods (BGs). Sourdough is a centuries-old method of preparing BGs that contributes to its improved quality. The qualities of the dough and BGs are influenced by sourdough fermentation, which also delays staling and enhances flavor. Additionally, the dough’s rheology, the flavor of the BG, and the microbial and grain enzymes are all impacted by the acidification of the dough. Lactic acid bacteria (LAB) during sourdough fermentation can produce an interesting combination of carboxylic acids, depending on the varied flour substrates [12].

In addition to having fungicidal or fungistatic action against some fungi and yeast that cause bread to deteriorate, organic acids and other metabolites generated by LAB can also prevent the formation of harmful bacteria [10,12].

For the manufacture of gluten-free products, cereals like sorghum, teff, millet, and wild rice as well as pseudocereals like quinoa, canihua, chia, and amaranth are used [11]. Quinoa is a grain crop that has gained popularity in recent years in the conception of gluten-free products due to its high nutrient content, phytochemical qualities, and health advantages. Quinoa grain has a high concentration of amino acids, fiber, minerals, vitamins, saponins, and phenolics that can help alleviate various biological diseases in the human body [10,11].

Therefore, the aim of the present research was to study the adaptability of two different strains, Enterococcus mundtii SL1 and Enterococcus gallinarum SL2, on quinoa flour. The obtained sourdoughs were characterized regarding their nutritional and rheological characteristics. Furthermore, the sourdoughs were used for the preparation of gluten-free muffins, which in turn were evaluated for their nutritional, sensory, and textural properties.

2. Material and Methods

2.1. Strains and Culture Conditions

The microorganisms used throughout this study were obtained from the Physiopathology, Molecular Genetics and Biotechnology laboratory at the Faculty of Science, Casablanca. The strains Enterococcus mundtii SL1 and Enterococcus gallinarum SL2 were cultivated in MRS broth and incubated at 37 °C for 24 h.

2.2. Screening for Amylolytic Activity

The amylolytic activity was evaluated according to [13]. Briefly, the strains were grown in modified MRS broth with 1% starch added. The broth was inoculated with 2% overnight culture and incubated at 37 °C for 24 h. After incubation, the culture was centrifuged at 8000× g for 10 min and the supernatant was kept to determine the starch content using iodine. The absorbance was measured at 580 nm using a Synergy HT microplate reader (BioTeK^®, Winooski, VT, USA). The amylolytic activity was measured by the determination of the starch hydrolysis rate following the equation

$$Starchhydrolysisrate={\displaystyle \raisebox{1ex}{$A_0-A_1$}\!\left/ \!\raisebox{-1ex}{$A_0$}\right.}\times 100$$

(1)

where A₀ represents the absorbance of the initial culture containing starch, and A₁ the absorbance after 24 h of incubation.

2.3. Fermentation of Quinoa

2.3.1. Sourdough Preparation

The sourdough was prepared by mixing the quinoa flour previously sterilized under UV with sterile distilled water (1:1, w:v) according to the protocol described by [6], with some modifications. The bacteria were cultivated in the MRS media and incubated at 37 °C for 24 h. The cells were collected by centrifugation at 12,000 rpm for 15 min, washed, suspended in sterile distilled water, and added to the sourdough to reach an initial number of cells of 10⁷ UFC/g.

The fermentation was carried out for 24 h at 37 °C on an orbital agitator (Heidolph Unimax 1010 Inkubator 1000, Schwabach, Germany) at 170 rpm. During fermentation, samples were taken at different time intervals (0, 12, and 24 h).

2.3.2. Cell Growth and pH Determination of Sourdoughs

Cell viability was determined by suspending 1 g of each sourdough in 9 mL of sterile NaCl solution at 0.9% and homogenizing using a vortex. Then, 1 mL was diluted in another tube with 9 mL of saline solution, and this procedure was repeated for both microorganisms four and six times until the viability was measurable on plates. The plates were incubated at 37 °C for 48 h.

The colonies were counted using a colonial counter (Colony Star 8500, Funke Gerber, Berlin, Germany).

The pH value of each sourdough was determined using a WTW pH meter (Weilheim, Germany).

2.3.3. Organic Acid and Sugar Determination Using HPLC-RID (Refractive Index Detector)

The organic acids and sugars were determined following the protocol previously described by Plessas et al., 2023, with some modifications [14]. Briefly, a 1 g sample with 4 mL distilled water was vortexed for 1 min, followed by 30 min ultrasonic treatment at room temperature, then centrifugation at 10,000 rpm for 10 min. The supernatant, containing the extracted sugars and organic acids, was filtered through a nylon filter (pore size 0.45 μm), and 20 μL was injected into the HPLC system. The quinoa flour was treated in the same way and served as a control. In addition to the quinoa flour, the supernatant of the strains was injected directly.

An analysis was carried out using an HP-1200 liquid chromatograph equipped with a degasser for the solvents, a quaternary pump, a manual injector, and an RID detector (Agilent Technologies, Santa Clara, CA, USA). The separation of the sugars and organic acids was carried out on a column Polaris Hi-Plex H, 300 × 7.7 mm (Agilent Technologies, USA). The mobile phase was 5 mM of a H₂ SO₄ solution with a flow rate of 0.6 mL/min, the column temperature was T = 80 °C and detector RID temperature T = 35 °C, and the elution time was 20 min. Data acquisition was performed with OpenLab—ChemStation (Agilent Technologies, USA).

2.3.4. Phenolic Compound Determination Using HPLC-DAD-MS-ESI+

The phenolic compounds were determined following the protocol previously described by Gabriele et al., 2023, with some modifications [15].

A 1 g sample with 4 mL methanol was vortexed for 1 min, followed by 30 min ultrasonic treatment at room temperature, then centrifugation at 10,000 rpm for 10 min. The supernatant, containing the extracted polyphenols, was filtered through a nylon filter (pore size 0.45 μm), and 20 μL was injected into the HPLC system. In addition, the quinoa flour was treated similarly and served as a control.

An analysis was carried out using an HP-1200 liquid chromatograph equipped with a quaternary pump, autosampler, DAD detector, and MS-6110 single quadrupole API-electrospray detector (Agilent Technologies, USA). The positive ionization mode was applied to detect the phenolic compounds; then, a different fragmentor, in the range 50–100 V, was applied. The column was a Kinetex XB-C18 (5 μm; 4.5 × 150 mm i.d.) from Phenomenex, Torrance, CA, USA.

The mobile phase was (A) water acidified by formic acid at 0.1% and (B) acetonitrile acidified by formic acid at 0.1%. The following multistep linear gradient was applied: start with 5% B for 2 min; increase B from 5% to 90% in 20 min; hold for 4 min at 90% B; then in 6 min, arrive at 5% B. The total analysis time was 30 min, the flow rate was 0.5 mL/min, and the oven temperature was 25 ± 0.5 °C.

A mass spectrometric detection of positively charged ions was performed using the Scan mode. The applied experimental conditions were the following: gas temperature 350 °C, nitrogen flow 7 L/min, nebulizer pressure 35 psi, capillary voltage 3000 V, fragmentor 100 V, and m/z 120–1500.

Chromatograms were recorded at wavelengths λ = 280 nm and λ = 350 nm, and data acquisition was conducted with the Agilent ChemStation software 1.0.

2.3.5. Rheological Parameters

The rheological parameters of the sourdoughs were determined by the method described in [16]. The measurements were carried out with an Anton Paar MCR 72 rheometer (Anton Paar, Graz, Austria) equipped with a Peltier plate–plate system (P-PTD 200/Air) supplied with a temperature controller (T  =  30 °C) along with a 50 mm diameter smooth parallel plate geometry (PP-50-67300). A total of 3 g of each sourdough was applied on the lower plate, and the upper one was lowered to a plate distance set at a gap of 1 mm. Silicone oil was applied after the excess dough was removed to prevent sample drying during testing. The storage modulus (G′) and loss modulus (G′) of each leaven at an angular frequency of 0.628–628 rad/s⁻¹ was tested, and the shear strain was set at a constant value of 0.1%, with 35 measuring points in total.

2.4. Preparation and Analysis of Gluten-Free Muffins

2.4.1. Muffin Preparation

Quinoa flour fermented for 0, 12, and 24 h was used in making the muffins. First, rice flour was subjected to hydrothermal treatment, considered a bakery improver [17]. After that, rice-treated flour, corn starch, baking powder, inulin, buckwheat flour, an egg yolk, and a sourdough starter were mixed for two minutes in a blender (KitchenAid^® Precise Heat Mixing Bowl, Ohio, OH, USA) at medium speed. The egg white was whisked and homogenized with maple syrup and slowly added into the first mixture. Thirty grams were used to fill the specific baking trays. An electric oven (Zanolli, Verona, Italy) was used to bake the muffins at 200 °C for 10 min and then at 180 °C for 5 min. After baking, the muffins were cooled at the bakery of the pilot station (Faculty of Food Technology) and used for further studies.

2.4.2. Organic Acid Determination Using HPLC-RID (Refractive Index Detector)

The muffins’ organic acid and sugar profile was determined as described above (Section 2.3).

2.4.3. Phenolic Compound Determination Using HPLC-DAD-MS-ESI+

The organic acid and sugar profiles of the muffins were determined as described above (Section 2.4).

2.4.4. Textural Analysis

An analysis of the textural properties of the muffins was carried out using the CT3 texture analyzer (Brookfield Engineering Labs, Middleboro, MA, USA) and according to the method described by Goswami et al. (2015) with slight modifications [18]. The CT3 texture analyzer was equipped with a 10 kg load cell and a TA25/1000 probe (50.8 mm diameter, AOAC Standard Clear Acrylic 23 g, length 20 mm). The muffin samples were subjected to a double compression test under the following conditions: 50% target strain, 1 mm s⁻¹ test and post-test speed, 5 g trigger load, and time of recovery of 5 s. Hardness, resilience, elasticity, cohesion, gum, and chewiness were used as indicators for the texture profile analysis.

Each muffin sample was cut manually with a stainless-steel knife, resulting in 25 × 25 × 25 mm (l × w × h) breadcrumb samples. The samples were tested immediately after preparation to avoid drying out the crumbs. Specific texture parameters were determined using Texture Pro CT V1.6 software (Brookfield Engineering Labs, Middleboro, MA, USA).

2.4.5. Sensory Analysis

The sensory analysis was carried out at the Laboratory for Sensory Analysis (Faculty of Food Technology, USAMV, Cluj Napoca, Romania). We established tasting sheets to facilitate the study and processing of the results obtained subsequently. The sensory characteristics of the muffins, such as appearance, color, texture, taste, flavor, and overall acceptability, were analyzed by 15 panelists (60% female and 40% male, aged 19 to 45 years). A nine-point hedonic scale was used to assess these sensory characteristics.

It should be mentioned that since the sensory analysis was limited to muffins and did not present any special dangers to the subjects, an ethical assessment was not required. To take into consideration any potential allergies, all participants were given complete details about the components of the muffins they evaluated. Prior to conducting the sensory evaluation, written consent was obtained from all participants involved in this study. Participants were informed about the purpose of the study, the nature of their involvement, and the voluntary nature of their participation. They were assured of the confidentiality of their personal information (names and signatures).

2.5. Statistical Analysis

All samples were analyzed in triplicates and the results were expressed as means ± standard deviations, n = 3. The statistical evaluation was carried out using Graph Prism Version 8.0.1. (GraphPad Software) through a one-way ANOVA (Tukey multiple comparisons tests).

3. Results and Discussion

3.1. Screening for Amylolytic Activity

LAB are used in food manufacturing due to the many beneficial compounds they produce, such as some enzymes that improve the structure and the organoleptic properties of food [13,19]. In our study, only two showed amylolytic activity, E. mundtii SRBG1 and E. gallinarum SRBG2. While amylolytic activity is important in fermentation and improving starch-based food quality [20], most LAB do not possess this property [13]. Yao Hou et al., 2022, reported the lack of amylolytic activity in Enterococcus faecium NMCC-M6 and Enterococcus lactis NMCC-M7 [21].

3.2. Cell Growth and pH Determination of Sourdoughs

LAB are one of the most frequently used microorganisms in sourdough fermentation, and the use of appropriate starter cultures is of great importance.

During fermentation, bacterial growth gradually increased and reached a final concentration of 9.37 and 9.24 CFU/mL in quinoa fermented by Enterococcus mundtii SRBG1 (SL1) and Enterococcus gallinarum SRBG3 (SL2), respectively (Figure 1).

At the same time, the pH gradually decreased from 6.11 to 4.3 for SL1 and from 6.08 to 4.65 for SL2. This decrease in pH is caused by the production of organic acids, particularly lactic acid. With the continued metabolic activities of LAB, the pH reaches values lower than <4.3 at the end of fermentation, which can also be seen in several studies on different substrates [22,23,24].

3.3. Organic Acid and Sugar Determination Using HPLC-RID (Refractive Index Detector)

Table 1. (A) The quantity of maltose, glucose, citric acid, and lactic acid in sourdough fermented by Enterococcus mundtii SRBG1 (SL1) and Enterococcus gallinarum SRBG3 (SL2) after 0, 12, and 24 h of fermentation. (B) The organic acid content of the quinoa flour and the supernatant (after 24 h of cultivation).

(A)
Samples		Glucose	Maltose	Citric Acid	Lactic Acid	Acetic Acid
SL1	0 h	36.560 ± 0.12 a	1.203 ± 0.32 a	2.446 ± 0.01 a	n.d.	n.d.
	12 h	43.266 ± 0.11 a,b	1.333 ± 0.21 a,b	3.949 ± 0.01 b	1.564 ± 0.02 a	1.103 ± 0.01 a
	24 h	54.415 ± 0.35 b	2.201 ± 0.01 b	4.426 ± 0.02 c	4.642 ± 0.01 b	2.345 ± 0.03 b
SL2	0 h	44.185 ± 0.10 a	1.474 ± 0.05 a	2.396 ± 0.01 a	n.d.	n.d.
	12 h	47.186 ± 0.11 a,b	2.065 ± 0.10 a,b	3.849 ± 0.05 b	0.663 ± 0.02 a	0.564 ± 0.01 a
	24 h	60.383 ± 0.23 b	2.133 ± 0.11 b	4.105 ± 0.01 c	3.866 ± 0.02 b	1.901 ± 0.02 b
(B)
		Quinoa Flour (mg/g)		Cell-Free Supernatant (mg/mL)
				SL1		SL2
Citric acid		2.59 ± 0.1 a		1.941 ± 0.2 b		1.901 ± 0.2 b
Lactic acid		n.d.		4.596 ± 0.3 a		3.383 ± 0.2 a
Acetic acid		n.d.		2.070 ± 0.2 a		1.907 ± 0.1 a

n.d.: Not detected. ^a,b,c: Different letters in the same column indicate statistically significant differences (p < 0.05) between time points and strains.

show the values of the carbohydrates and organic acids of the sourdoughs, quinoa flour and cell-free supernatants. The results emphasized that the glucose and maltose content increased during fermentation; this could be explained by the hydrolysis of starch by the action of amylase. This is consistent with the results of the amylolytic activity obtained in our study. Our results are in agreement with Chiş et al.’s (2020) study [5].

Regarding organic acids, their content in the sourdough produced by SL1 after 24 h of fermentation was 1.15 times higher than the sourdough produced by SL2. Lactic, citric, and acetic acids were abundant in both sourdoughs and the cell-free supernatants compared to the unfermented flour, which contained only citric acid. This can confirm that organic acids come from the fermentation of sugars by our bacteria.

The capacity of LAB to produce bioactive molecules has been confirmed by several studies [25,26,27]. Organic acids are produced from carbohydrates through fermentation and have a great influence on the final flavor of baked goods, being considered a flavoring product.

3.4. Phenolic Compound Determination Using HPLC-DAD-MS-ESI+

Table 2. The quantity of phenolic components in the sourdoughs fermented by Enterococcus mundtii SRBG1 (SL1) and Enterococcus gallinarum SRBG3 (SL2) in µg/g.

Rt (min)	Phenolic Compound	SL1			SL2
		0 h	12 h	24 h	0 h	12 h	24 h
3.01	2-Hydroxybenzoic acid	1664.444	2006.462	2542.151	1584.264	1865.134	2441.033
3.89	2,3-Dihydroxybenzoic acid	359.662	477.316	662.898	343.602	438.296	603.060
4.57	Gallic acid	237.250	324.568	433.776	218.097	273.295	383.931
9.47	Protocatechuic acid	106.272	111.387	230.469	117.692	124.116	250.573
13.42	Vanillic acid	88.784	72.010	68.917	128.280	126.257	150.407
11.89	Chlorogenic acid	20.899	23.869	40.353	30.552	33.819	37.532
14.21	Quercetin-rhamnosyl-rhamnosyl-glucoside	189.164	183.669	176.987	275.446	260.447	225.994
14.54	Quercetin-xylosyl-rutinoside	110.159	90.259	75.854	165.255	169.561	142.236
14.83	Kaempferol-rhamnosyl-rhamnosyl-glucoside	551.963	461.672	417.863	445.336	404.349	353.411
15.24	Quercetin-xylosyl-glucoside	109.862	116.545	122.931	101.546	105.110	107.189
15.54	Quercetin-rutinoside (Rutin)	111.644	100.209	97.833	130.950	101.546	100.655
16.23	Kaempferol-rutinoside	142.088	72.290	54.172	123.822	67.092	54.024
16.72	Quercetin-glucuronide	60.113	56.252	54.234	98.427	97.685	94.566
21.39	Quercetin	5.017	7.690	15.116	7.542	11.848	32.788
23.27	Kaempferol	11.106	14.224	27.590	11.403	14.373	29.818
	Total phenolics	3768.427	4118.422	5021.144	3788.087	4092.928	5007.217

shows the quantity of phenolic compounds in the sourdoughs. The results revealed that fermentation did not affect the profile of the phenolic compounds but rather affected the content of these compounds.

Phenolic acids such as hydroxybenzoic, gallic, protocatechuic, vanillic, and chlorogenic acids were detected in the sourdoughs and unfermented quinoa flour. Regarding the flavonoids, nine different conjugated and free flavonoids were identified in the sourdoughs and unfermented quinoa flour. Quercetin-rhamnosyl-rhamnosyl-glucoside, quercetin-xylosyl-rutinoside, kaempferol-rhamnosyl-rhamnosyl-glucoside, rutin, kaempferol-rutinoside, and quercetin-glucuronide experienced a slight decrease after 24 h of fermentation, while quercetin and kaempferol increased.

This could be explained by the bioconversion of their bound or conjugated forms to their free forms due to (i) the breaking of bonds within the components of the cell wall and (ii) the activities of enzymes, such as β-glucosidase, decarboxylases, esterases, hydrolases, and reductases. In their free form, phenolic compounds have greater bioaccessibility and the potential to increase antioxidant activity [28]. Bhanja Dey et al. (2016) also reported that although abundant in quinoa, these phenolic compounds are classified as bound because they are covalently linked to other cell wall constituents such as hemicelluloses, which can limit their bioavailability [29]. Therefore, fermentation can help convert free phenolic compounds, thereby increasing their bioavailability.

Our results followed the study carried out by [30], which showed that polyphenol content increased significantly with fermentation time. It is suggested that this may be due to proteolytic activities that contribute to the release of free polyphenol bonds.

3.5. Rheological Parameters

Dough rheology has become an area of great interest in the cereal industry given its implication in the quality of baked products. During bread making, the dough experiences a variety of deformations and stresses of varying degrees, which are essential for evaluating the viscoelastic behavior of the dough. Particularly during fermentation, extensional deformation is an important element which has a major impact on the rheological characteristics and consistency of the final product. Figure 2 shows the sourdoughs’ viscoelastic properties after 0, 12, and 24 h of fermentation. The results showed that with an increase in the angular frequency (ω), there was an increase in the storage modulus (G′), which represents the elastic characteristics and the loss modulus (G″), which represents the viscous properties.

The dough mixtures had different water absorption capacities and moisture contents, which affected the dynamic moduli. As a result, the water content was the same in each experiment. On the other hand, for both sourdoughs, G′ was higher than G″, and also increased with increasing ω. Therefore, each sourdough sample exhibited weak gel-like (stable elastic) behavior. The strongest gel-like behavior was observed in the two sourdoughs produced after 24 h of fermentation, with final values of G′ 4936.2 ± 12.7 and G″ 2338.4 ± 9.1 for the sourdough produced by SL1 and G′ 4225.2 ± 11.9 and G″ 1726.7 ± 10.1 for the sourdough produced by SL2.

This same behavior has been reported in several studies where different types of flours were analyzed in dough production, such as aleurone, durum wheat, psyllium, amaranth flour, corn starch, and isolate peas. This could be justified by the possibility of strains to produce exopolysaccharides during fermentation, which could act as viscosifiers and texturizers, having a pseudoplastic rheological behavior and being involved in the water retention capacity of sourdough [31]. Several studies have reported that lactic acid bacteria can produce exopolysaccharides through sourdough fermentation [32,33,34]. On the other hand, Wolter et al. (2014) reported that LAB could produce β-glucan during their growth process and metabolism, which could positively influence quinoa flour’s viscosity and water retention [35].

3.6. Organic Acid and Carbohydrate Analysis in Muffins Using HPLC-RID

The content of carbohydrates and organic acids in the muffins is presented in

Table 3. The quantity of maltose, glucose, citric acid, and lactic acid in muffins prepared by sourdoughs (SL1) and (SL2) in mg/g.

Sample		Glucose	Maltose	Citric Acid	Lactic Acid	Acetic Acid
SL1	0 h	36.374 ± 0.02 a	10.317 ± 0.43 a	1.434 ± 0.01 a	n.d.	n.d.
	12 h	33.585 ± 0.21 b	8.465 ± 0.14 b	1.290 ± 0.01 b	0.515 ± 0.05 b	0.332 ± 0.01 b
	24 h	30.398 ± 0.15 c	7.317 ± 0.10 c	0.634 ± 0.02 c	0.669 ± 0.01 a	1.543 ± 0.03 a
SL2	0 h	31.647 ± 0.30 a	10.154 ± 0.39 a	1.303 ± 0.01 a	n.d.	n.d.
	12 h	17.192 ± 0.13 b	6.812 ± 0.22 b	0.444 ± 0.05 b	0.399 ± 0.02 b	0.846 ± 0.01 b
	24 h	19.357 ± 0.10 b	6.271 ± 0.12 c	0.205 ± 0.01 c	0.406 ± 0.02 b	0.541 ± 0.02 c

n.d.: Not detected. ^a,b,c: Different letters within a column indicate statistically significant differences (p < 0.05) between time points and strains.

. The amounts of glucose and maltose were reduced in the gluten-free muffins prepared with the two different sourdoughs.

The glucose content of the baked muffins could also be influenced by maple syrup, a chemically superior natural sweetener, rich in minerals and aromatic compounds and having an antioxidant capacity. Sucrose and glucose were the main carbohydrates detected in the maple syrup at levels between 61.2 and 65.8% and 0.07 and 0.27%, respectively.

The controlled fermentation of the sourdoughs could explain the presence of organic acids in the muffins. The lactic acid content of the muffin prepared by SL1 in 24 h was higher (statistically different p < 0.05) than that in the muffin prepared by SL2 in 24 h. This was expected since the initial lactic acid concentration was statistically different (p < 0.05) in the 24 h SL1 sourdough compared to the 24 h SL2 sourdough.

The same trend was observed regarding the acetic acid content, as the initial contents of SL1 in 24 h and SL2 in 24 h were statistically different (p < 0.05). Acetic acid is the most promising organic acid involved in the bio-preservation of bakery products, having an antifungal effect. As for lactic acid, it plays an important role in the storage and safety of final baked goods. Moreover, it could positively influence the aroma and texture, as well as degrade the starch digestion rate, in the final baked goods. Additionally, the presence of lactic and acetic acids in final baked products formed during sourdough fermentation has been proven to reduce acute insulinemic and glycemic responses.

Citric acid has been reported to have antimicrobial activity and could be produced during the fermentation of sourdough with LABs. Moreover, its addition in bakery products could improve their sensory characteristics, including flavor [5].

3.7. Phenolic Compound Determination Using HPLC-DAD-MS-ESI+

Table 4. The quantity of phenolic components in muffins prepared by sourdoughs (SL1) and (SL2) in µg/g.

Rt (min)	Phenolic Compound	SL1			SL2
		0 h	12 h	24 h	0 h	12 h	24 h
3.01	2-Hydroxybenzoic acid	213.933	405.106	578.434	293.281	344.911	526.805
3.89	2,3-Dihydroxybenzoic acid	27.756	60.352	98.777	46.671	55.475	84.739
4.57	Gallic acid	42.508	62.137	101.275	45.839	53.571	70.702
9.47	Protocatechuic acid	18.953	26.924	67.609	28.470	27.756	32.872
13.42	Vanilic acid	7.771	12.053	29.660	11.696	10.031	13.481
11.89	Chlorogenic acid	0.000	0.000	0.000	0.000	0.000	0.000
14.21	Quercetin-rhamnosyl-rhamnosyl-glucoside	20.907	20.462	18.828	28.630	21.501	23.580
14.54	Quercetin-xylosyl-rutinoside	14.076	11.403	12.591	13.630	9.472	11.106
14.83	Kaempferol-rhamnosyl-rhamnosyl-glucoside	40.658	42.738	37.391	44.074	31.303	37.243
15.24	Quercetin-xylosyl-glucoside	8.136	10.957	12.888	15.710	8.136	13.630
15.54	Quercetin-rutinoside (Rutin)	22.838	30.263	30.709	37.540	25.956	40.807
16.23	Kaempferol-rutinoside	6.799	13.482	20.313	14.373	9.769	21.353
16.72	Quercetin-glucuronide	9.621	9.769	12.888	16.155	13.036	17.343
21.39	Quercetin	7.987	9.918	11.403	11.551	12.294	14.967
23.27	Kaempferol	14.076	17.195	17.789	13.036	13.779	15.413
	Total phenolics	456.019	732.758	1050.555	620.657	636.991	924.040

shows the phenolic component content of the muffins prepared by the SL1 and SL2 sourdoughs. The results showed a persistence of all polyphenols and flavonoids in the cooked muffins except for chlorogenic acid, which completely disappeared.

A study carried out on the evaluation of the polyphenol composition and antioxidant activity of amaranth, quinoa, buckwheat, and wheat demonstrated that there were no polyphenols detected in their gluten-free control bread and, therefore, the introduction of pseudocereals could be considered as a tool to increase the polyphenol content of these products [36]. Despite the negative impact of cooking on the polyphenol content of pseudocereals, bread made from quinoa and buckwheat flour still contains flavonoids in significant quantities, particularly 100% quinoa and sprouted buckwheat breads. These characteristics are highly desirable in gluten-free products because their nutritional quality has been reported to be of concern [37]. Thus, besides people with gluten intolerance, these products can also represent healthy alternatives for the general population.

3.8. Textural Analysis

Texture plays an essential role in the appreciation of a product. Various parameters such as hardness, resilience, elasticity, cohesion, gumminess, and chewiness were evaluated for all samples and are shown in

Table 5. Textural analysis of muffins.

		Hardness (N)	Resilience (mJ)	Elasticity (mm)	Cohesion	Chewiness (N)	Mastication (kg·mm)
SL2	0 h	26.28 ± 1.3 a	0.08 ± 0.0 a	5.01 ± 0.1 a	0.33 ± 0.2 a	8.67 ± 0.2 a	4.41 ± 0.01 a
	12 h	13.97 ± 1.9 b	0.10 ± 0.3 a	5.50 ± 0.2 a	0.41 ± 0.1 a	5.72 ± 0.6 b	3.20 ± 0.02 b
	24 h	11.38 ± 0.6 c	0.17 ± 0.2 b	7.36 ± 0.1 b	0.65 ± 0.1 b	7.39 ± 0.4 a	5.54 ± 0.01 c
SL1	0 h	27.74 ± 0.0 a	0.07 ± 0.2 a	4.76 ± 0.2 a	0.31 ± 0.1 a	8.59 ± 0.2 a	4.16 ± 0.07 a
	12 h	14.19 ± 0.2 b	0.11 ± 0.2 a	5.01 ± 0.0 a	0.42 ± 0.2 a	5.95 ± 0.5 b	3.03 ± 0.02 b
	24 h	07.40 ± 1.4 c	0.12 ± 0.4 a	5.30 ± 0.1 a	0.56 ± 0.1 b	4.14 ± 0.6 c	2.22 ± 0.01 c

^a,b,c: Different letters within a column indicate statistically significant differences (p < 0.05) between time points and strains.

. A decrease in hardness was observed for the analyzed muffin samples made by the addition of sourdoughs SL1 and SL2, giving the final products total values of 7.4 and 11.38 N, respectively. The hardness values obtained in the final products prepared by the addition of the SL1 and SL2 sourdoughs were statistically different.

The chewiness values for the muffins prepared by sourdough SL1 decreased from 8.59 N to 4.14 N compared to the muffins prepared by sourdough SL2, which reached a value of 7.39 N and showed proportional patterns with hardness.

Mastication describes the difficulty level required to chew food and form the bolus before swallowing. Statistical analysis confirmed significant differences between the samples, showing improved chewing when 24 h SL1 sourdough was added.

Elasticity is the height that the food recovers during the time between the end of the first compression and the start of the second compression. This is an important mechanical characteristic that has been associated with resilience. The latter is the ratio of recoverable energy when the first squeeze is relieved, with a lower value indicating that the muffins need more time to return to their initial shape and chew. In the present study, elasticity values increased when the muffins were made with the SL1 and SL2 sourdoughs, reaching values of 5.3 and 7.36 mm, and a similar pattern was observed with their resilience values.

The texture profile of the sample showed that sourdough could influence the characteristics of the final bakery products, improving their texture. This result is also confirmed by Campo et al. (2016), who found that sourdough using Lb. helveticus influenced the textural properties of bread, improving its elasticity [38]. This is also in accordance with Rizzello et al. (2016), who reported that leavening with LABs could enhance the hardness and elasticity of final bakery products [39]. Also, Novotni et al. (2013) confirmed that Lactobaciluus plantarum could be used for the production of sourdough, influencing the hardness and firmness of the crumb and giving the final product superior textural characteristics [40].

3.9. Sensory Analysis

This analysis allowed panelists to taste and appreciate the qualities, scents, and aromas of the muffins. This tasting is traditionally made up of three stages that require the use of our five senses (sight, touch, hearing, smell, and taste). The sensory scores of our muffins are shown in Figure 3. Panelists rated the 24 h SL1 as having a good taste, flavor, smell, and texture. The overall acceptability was 8.4, which placed the sample on the positive side of the hedonic scale, as shown in Figure 3. This result highlighted the fact that sourdough is mainly used as a flavor enhancer in the baking industry, but is also a texture improver. The idea that the fermentation of gluten-free flours by BLs could lead to the production of aromatic compounds and generate specific aroma profiles and odor compositions influencing the final results of the aroma and quality of bakery products has also been supported by several studies [5].

4. Conclusions

In the present study, quinoa flour fermented with Enterococcus mundtii SL1 and Enterococcus gallinarum SL2 improved the resulting sourdoughs’ rheological characteristics and nutritional content.

The findings indicated that the amounts of glucose and maltose increased during fermentation. The amylase-induced hydrolysis of starch may explain this. This is in line with the findings of our study’s amylolytic activity. In addition to carbohydrates, lactic, citric, and acetic acids were abundant in both sourdoughs. Regarding rheological parameters, the sourdoughs’ viscous qualities were enhanced, most likely because of exopolysaccharides.

The addition of Enterococcus to the sourdough enhanced the final baked muffins’ nutritional properties. Conversely, the texture profile of the samples showed that sourdough could influence the characteristics of the final bakery products, improving their texture. Finally, regarding sensory analysis, panelists rated the SL1 after 24 h as having a good taste, flavor, smell, and texture. The overall acceptability score was 8.4, which placed the sample on the positive side of the hedonic scale.

Further research is required to determine the ideal combination of quinoa flour and Enterococcus strains to produce gluten-free goods.