Quinoa Sourdough Fermented with Lactobacillus plantarum ATCC 8014 Designed for Gluten-Free Muffins—A Powerful Tool to Enhance Bioactive Compounds

Lactobacillus plantarum ATCC 8014 was used to ferment quinoa flour, in order to evaluate its influence on the nutritional and rheological characteristics of both the sourdough and muffins. The quantification of carbohydrates and organic acids was carried out on a HPLC-RID system (high-performance liquid chromatography coupled with with refractive index detector), meanwhile HPLC-UV-VIS (high-performance liquid chromatography coupled with UV-VIS detector), AAS (Atomic absorption spectrophotometry), aluminum chloride colorimetric assay, Folin–Ciocalteu, and 1,1-Diphenyl-2-picrylhydrazyl radical scavenging activity (DPPH) methods were used to determine folic acid, minerals, flavonoids, total phenols, and radical scavenging activity, respectively. Two types of sourdough were used in this study: quinoa sourdough fermented with L. plantarum ATCC 8014 and quinoa sourdough spontaneous fermented. The first one influenced the chemical composition of muffins in terms of decreased content of carbohydrates, higher amounts of both organic acids and folic acid. Furthermore, higher amounts of flavonoids, total phenols and increased radical scavenging activity were recorded due to the use of Lactobacillus plantarum ATCC 8014 strain. These results indicate the positive effect of quinoa flour fermentation with the above strain and supports the use of controlled fermentation with lactic acid bacteria for the manufacturing of gluten free baked products.


Introduction
Quinoa (Chenopodium quinoa Willd) pseudo-cereal comes from the Andean region and it is considered an ancient grain [1]. Nowadays quinoa is receiving increased attention due to its RWF was hydrothermally treated in order to improve its textural characteristics, as reported by Chiș et al. [39]. Different fermentation times (0, 12, and 24 h) were used for the muffins preparation with SP sourdough or with OR sourdough. The following codes were used for the final baked muffins manufactured with SP at 0, 12, and 24 h fermentation times: SP PF 0 h, SP PF 12 h, SP PF 24 h, and OR PF 0 h, OR PF 12 h, and OR PF 24 h for muffins with OR, respectively.

Organic Acids and Glucose, Maltose, and Fructose Determination by HPLC-RID
High-performance liquid chromatography (HPLC-Agilent 1200 series, Santa Clara, CA, USA) equipped with solvent degasser, manual injector coupled with with refractive index detector (RID) (Agilent Techologies, CA, USA) was used in order to analyze the organic acids and carbohydrates amount. Briefly, 1 g of sample was mixed with 5 mL of ultrapure water, vortexed for 1 min and sonicated for 2 h at 50 °C in a heated ultrasonic bath Elmasonis E 15H (Elma Schmidbauer GmbH, Singen, Germany). After that, the samples were centrifuged at 2300 × g for 10 min, in an Eppendorf 5804 centrifuge (Hamburg, Germany), filtered through Chromafil Xtra PA-45/13 nylon filter and 20 μl were injected in the HPLC-RID system. OR-spontaneous fermented sourdough) and muffins' production.

Organic Acids and Glucose, Maltose, and Fructose Determination by HPLC-RID
High-performance liquid chromatography (HPLC-Agilent 1200 series, Santa Clara, CA, USA) equipped with solvent degasser, manual injector coupled with with refractive index detector (RID) (Agilent Techologies, Santa Clara, CA, USA) was used in order to analyze the organic acids and carbohydrates amount. Briefly, 1 g of sample was mixed with 5 mL of ultrapure water, vortexed for 1 min and sonicated for 2 h at 50 • C in a heated ultrasonic bath Elmasonis E 15H (Elma Schmidbauer GmbH, Singen, Germany). After that, the samples were centrifuged at 2300× g for 10 min, in an Eppendorf 5804 centrifuge (Hamburg, Germany), filtered through Chromafil Xtra PA-45/13 nylon filter and 20 µL were injected in the HPLC-RID system.
The compounds were separated on a Polaris Hi-Plex H, 300 × 7.7 mm column (Agilent Techologies, Santa Clara, CA, USA) using the 5 mM H 2 SO 4 mobile phase with a flow rate of 0.6 mL/min, column temperature T = 80 • C and RID temperature T = 35 • C. Elution of the compounds was made for 25 min. Data acquisition and results interpretation was performed using OpenLab software-ChemStation (Agilent Techologies, Santa Clara, CA, USA). The retention times for maltose, glucose, and fructose were 8.87 min, 10.24 min, and 10.88 min, respectively; meanwhile, the retention times for citric, lactic, and acetic acids were 9.39 min, 13.46 min, and 15.92 min, respectively.

Folic Acid Determination
Determination of folic acid was fulfilled in concordance with [40]. Briefly, an amount of 0.5 g of each sample was diluted with 5 mL of phosphate buffer (Ph = 7). After homogenization in a vortex, the mixtures were sonicated and centrifuged for 30 min at 3000 rpm (Eppendorf 5804, Hamburg, Germany). The supernatant was filtrated with nylon filter (0.45 µm) and 20 µL was injected in HPLC-UV detection (Agilent Technologies 1200 Series, Santa Clara, CA, USA).
A folic acid standard curve (y = 154.79x − 8.1463, R2 = 0.9954) having as minimum and maximum concentration 2 µg/mL and 25 µg/mL, respectively, was used in order to establish the concentration of folic acid, expressed in mg/l supernatant.

Analysis of Macro and Microelements
Atomic absorption spectrophotometry (AAS) was used in order to determine the amounts of micro and macro elements, as described by [39,40]. Briefly, 3 g of each sample was burned for 10 h in a furnace (Nabertherm B150, Lilienthal, Germany) at a temperature of 550 • C. Afterwards, the ash was recovered in HCl 20% (w/v) in a volumetric flask, in order to achieve a final volume of 20 mL. The resulted samples were analyzed by AAS (Varian 220 FAA equipment, Germany). The results were calculated considering the samples fresh weight basis and expressed as mean value (n = 3) of three independent assays.

Total Flavonoids
Total flavonoids were determined according to the aluminum chloride colorimetric assay described by [41], adapted for the 96 well microplate reader (Synergy™ HT BioTek Instruments, Winooski, VT, USA). Quercetin was used as reference standard. Briefly, 25 µL of each sample methanolic extracts was mixed for 5 min with 100 µL distilled water and 10 µL of 5% sodium nitrate (NaNO 2 ) solution. Afterwards, 15 µL of 10% aluminum chloride (AlCl 3 ), 50 µL of 1 M sodium hydroxide (NaOH) and 50 µL of distilled water were added. The detection of total flavonoids was set at λ = 510 nm. A standard curve of quercetin was used to establish the final amount of total flavonoids content (y = 0.0003x + 0.0029, R 2 = 0.9916). The results were calculated as mg of Qe (quercetin equivalent) per g of extract.

Total Phenols Assay by Folin-Ciocalteau Reagent
In order to analyze the total phenols amount, 1 g of sample was homogenized with 100 mL acidified methanol (85:15 v:v, MeOH:HCl). Atfer that, the sample was dried at 40 • C by using a vacuum rotary evaporator (Laborota 4010 digital rotary evaporator, Heidolph Instruments GmbH & Co. KG, Schwabach, Germany), according to the method described by [42,43]. Folin-Ciocalteu colorimetric method was used to evaluate the total phenols amount, as follows: 100 µL of methanolic extract was mixed with 500 µL Folin-Ciocalteu reagent, 6 mL of distilled water, and 2 mL of 15% Na 2 CO 3 , as described by [34,44]. The solution was brought up to 10 mL by adding distilled water, kept in the dark for two hours at room temperature the absorbance was read at λ = 760 nm with a UV/visible spectrophotometer Schimadzu 1700 (Shimadzu Corporation, Kyoto, Japan). A standard calibration curve of galic acid was used to establish the final amount of total phenols (y = 1.022958x + 0.08740, R 2 = 0.99614) and the results were expressed as milligrams of gallic acid equivalent (GAE) per 100 g product.

Radical Scavenging Activity by DPPH Assay
The radical scavenging activity (RSA) was analyzed by DPPH method (1,1-Diphenyl-2picrylhydrazyl) method as descriebed previously by [38]. Briefly, 0.1 mL of each methanolic extract was mixed with DPPH solution (3.9 mL), kept in the dark at room temperature for 30 min and recorded at λ = 515 nm, using an UV/visible spectrophotometer Schimadzu 1700 (Shimadzu Corporation, Kyoto, Japan). The following equation was used to calculate the radical scavening activity: where Abs DPPH = absorbance of DPPH solution; Abs Sample = absorbance of the sample.

Rheological Measurements
The SP and OR sourdoughs dynamic rheological characteristics were determined by using an Anton Paar MCR 72 rheometer (Anton Paar, Graz, Austria), supplied with a Peltier plate-plate system (P-PTD 200/Air) with temperature control and a 50 mm diameter smooth parallel plate geometry (PP-50-67300), according to the method described by [45]. The sourdough samples were analyzed before and after freezing (one week at −20 • C, and then defrosted at room temperature). Shortly, 3 g of sample 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 used in order to prevent sample moisture loss through testing. The storage modulus (G') and loss modulus (G") of each sourdough at an angular frequency of 0.628-628 rad/s −1 were tested, and the shear strain was set at a constant value of 0.1%, with 35 total measuring points, at a constant temperature of 30 • C.

Statistical Analysis
Duncan multiple comparison test by SPSS version 19 software (IBM Corp., Armonk, NY, USA) was used in order to analyze the results. All samples were analyzed in triplicates and the results were expressed as means ± standard deviations.

Results and Discussion
3.1. Carbohydrates, Organic Acids, Folic Acid, Minerals, Flavonoids, Total Phenols Content, and Radical Scavenging Activity of Quinoa Flour (QF)

Carbohydrates and Organic Acids from Quinoa Wholemeal Flour (QWF)
The values of simple carbohydrates (maltose, glucose, and fructose) and organic acids (lactic, acetic, and citric) of QWF are reported in Table 1. The main carbohydrates from QWF was glucose, followed by fructose and maltose.
The carbohydrate content in quinoa is higher than in the common cereals and might be associated with frost tolerance, as confirmed by [46] who reported that quinoa from mountain region had higher sugar content than those from the valleys region. Likewise, ref. [47] reported that also the method of extraction, the origin of quinoa seeds, cultivation, and environmental stress could also influence the sugars content.
With respect to fructose content, it is known that its consumption could induce oxidative stress, leading to different type of diseases such as obesity, hypertriglyceridemia, and cardiovascular diseases. Pasko et al. [48] showed that although quinoa seeds contain fructose, the seeds are able to reduce the oxidative stress due to its ability to increase MDA (malondialdehyde) level, indicating an intensive lipid peroxidation and protecting plasma against peroxidation. Moreover, reducing the oxidative stress could reduce the free radicals during some pathological states. In our previous study, we determined a value of 68.2% total carbohydrates [26], close to the value reported by [1] of 67.9% and [49]. The total carbohydrates content in quinoa could vary between 48.6 and 68.1% of dry matter weight and 45.16-59.78%, respectively, as reported by [50,51]. Starch, the major carbohydrate of quinoa, ranges between 32 and 69% of total carbohydrates [52] and is followed by total dietary fiber (7-9.7%) [50] and fermentable sugars (2% from the total carbohydrate amount), as reported by [53].
With respect to organic acids, lactic, and acetic acids could not be found in QWF, but citric acid had a total amount of 8.59 ± 0.30 mg/g f.w. (fresh weight). Citric acid was also identified in quinoa flour by other researchers, in the range of 210-317 mg/100 g d.w., and from 0.40 to 0.71 g/100 g f.w., respectively [47,49].

Quinoa Wholemeal Flour (QWF) Content in Folic Acid
In the present study, the amount of folic acid determined in quinoa flour was 183 ± 0.03 µg/100 g f.w. (fresh weight). The folic acid amount from QF is higher than the folic acid content of wheat flour (10.62 µg/100 g f.w.) and green lentil flour (168.36 µg/100 g f.w.), respectively, as previously reported by Păucean et al. [40]. In general, the folic acid content in pseudo-cereals such as quinoa, is higher than the amount in cereal grains which could range from 29 to 143 µg/100 g f.w. [54]. For example, the raw pearl-millet was reported to have a folic acid content of only 26.2 µg/100 g f.w. Therefore, the researchers' attention is mainly focused on using raw materials with higher contents in folic acid in food manufacturing.
Many research data show that quinoa flour is as an important nutritional natural food source due to its valuable bioactive compounds such as B vitamin group (especially folic acid, named B9 vitamin), minerals, and essential amino acids [9,50,52,55]. Total folate content of quinoa is reported to be by about ten times higher than in wheat [56].

QWF Minerals Content
Quinoa is very rich in minerals like potassium, calcium, magnesium, zinc, and iron [57,58]. Table 2 displays the mineral content of quinoa flour and its composition in the following macro and microelements: calcium (Ca), magnesium (Mg), potassium (K), iron (Fe), copper (Cu), Zinc (Zn), manganese (Mn), and chromium (Cr). The ash content of quinoa flour could range from 2.4 to 4.8% [50]. In the present study, the ash content of quinoa flour was 2.3% (results previously published [3]). From the quantitative point of view, the main mineral is K (813.92 mg/100 g f.w.), followed by Mg, Ca, Fe, Mn, Zn, and Cu. The K and Mg content amounts are close to the values reported by Silva et al. [50], 926 mg/100 g, and 249.6 mg/100 g, respectively. Variation in different quinoa flour mineral content might be due to the environmental conditions (especially soil mineral availability) [59], fertilizer soil application [50], by the plant genotype [60,61], and also by the removal of the husk during milling process [51].

QWF Flavonoids
In the present study, the flavonoids content of QWF was 997 ± 0.52 mg Qe/100 g fresh weight. Quinoa flour has high flavonoid content that could range from 36.2 to 144.3 mg/100 g dry weight basis [10] but the total amount could be influenced by the extraction temperature, the solvent type used and non-application of ultrasounds [62]. The main flavonoids from quinoa are glycosides of the flavonols, kaempferol, and quercetin [47]. De Carvalho et al. [63] proved that besides quercetin and kaempferol glycosides, protocatechuic acid and a vanillic acid glucoside were also determined in QWF. Isoflavones, particularly Daidzein and Genistein, were found in different amounts in quinoa flour from different origins [64].

OR, SP Carbohydrates, and Organic Acids Contents
The OR and SP carbohydrates and organic acids amounts are displayed in Table 3. The glucose content of SP increased in the first 8 h of fermentation due to the starch degradation and conversion into glucose but decreased afterwards. There are two possible processes that could influence the glucose decrement. Firstly, glucose consumption during Lp cellular development and secondly, glucose conversion into lactic acid through via Embden-Meyerhof-Parnas pathway (EMP). On the other side, acetic acid increased during SP fermentation, probably due to the degradation of pentose and gluconate through via the pentose phosphate (PP) pathway [30,31] With respect to SP maltose content, the amount decreased after 24 h of fermentation, as reported in Table 3, reaching a final value of 1.05 mg/g f.w. after 24 h of fermentation.
On the other side, SP fructose content decreased from 4.1 mg/g f.w. to 2.69 mg/g f.w. after 24 h of fermentation. This could be due to the use of fructose by Lp as alternative external electron acceptor and its conversion into mannitol by mannitol dehydrogenase [65].
The SP content of lactic and acetic acids after 24 h of fermentation had a total value of 8.5 mg/g f.w. and 1.40 mg/g f.w., respectively, and the Lp growth during 24 h of fermentation reached a final value of log 6.7 cfu/g [38], thus emphasizing a good adaptability of the strain in the quinoa sourdough. The SP ratio between lactic and acetic acid, named FQ (fermentation quotient) was 6.07, indicating a good ratio between the two organic acids. With respect to OR fermentation, the FQ values was 1.27. This is in line with Montemurro et al. [66] who reported a FQ fermentation of 6.1 at quinoa flour fermented with Lactobacillus plantarum 1A7 strain. In the case of spontaneous sourdough, in the first 8 h of fermentation the carbohydrates conversion was almost similar to SP. After 8 h of spontaneous fermentation, the carbohydrates conversion lags behind, probably due to the low capacity of wild microbiota to multiply and to produce organic acids. This behavior is reflected by the FQ values compared to the same values for SP and indicate low amounts of lactic and acetic acids.
With respect to the citric acid amount, after 24 h of fermentation the SP's citric acid content decreased, but there was no significant difference between OR and SP (p < 0.05), as displayed in Table 3. The decrease of citric acid amount could be explained by the ability of Lp strain to use citric acid as an energy supply [45]. However, the Lp preference for energy source is reflected mainly in the use of fructose, decreasing its amount in SP sample from 4.36 to 1.69 mg/g f.w.
Salminen et al. [67] reported that in a raw matrix that contains glucose and fructose, the heterofermentative LABs will mainly use glucose as an energy source to grow and fructose as an electron acceptor. This is in line with [29] who proved that the same strain of Lactobacillus plantarum ATCC 8014 was able to growth in different MRS media supplemented with concentrations of glucose, fructose, sucrose, and maltose, and consumed all types of carbohydrates, although glucose being the easiest fermentable sugar for this strain.
On the other hand, [30] reported an increase amount of glucose and fructose during fermentation of wheat flour with LAB strains such as Lactobacillus reuteri and Lactobacillus brevis, respectively. Furthermore, Lb. reuteri R29 was able to metabolize maltose during sourdough fermentation, resulting in a low amount in the final sample.
Overall, it can be stated that during sourdough fermentation, the utilization of carbohydrates depends on the type of LAB strain which could have preferences towards a certain type of carbohydrates and on the chemical composition of the raw matrix. The fine link between bacterial strain and its favorite substrate is defined by the relationship between LAB and the raw material [68].

SP and OR folic Acid Content
In the present study, SP folic acid content improved during 24 h of fermentation, having a final value of 648.39 µg/100 g f.w. sourdough compared with OR sourdough, where the total folic acid amount was 169.12 µg/g dough f.w., as illustrated in Figure 2. The SP folic acid content is 3.8 times higher compared with OR sourdough and this is in agreement with [69] who reported that L. plantarum CRL 2107 + L. plantarum CRL 1964 strains are able to improve folic acid content during quinoa flour fermentation.
In the present study, SP folic acid content improved during 24 h of fermentation, having a final value of 648.39 μg/100 g f.w. sourdough compared with OR sourdough, where the total folic acid amount was 169.12 μg/g dough f.w., as illustrated in Figure 2. The SP folic acid content is 3.8 times higher compared with OR sourdough and this is in agreement with [69] who reported that L. plantarum CRL 2107 + L. plantarum CRL 1964 strains are able to improve folic acid content during quinoa flour fermentation. The capacity of Lactobacillus strains to produce folic acid during fermentation is supported by a large body of literature [70][71][72][73][74]. Likewise, [55] reported that through fermentation of cereal based foods, the folate content could increase up to 700%. Furthermore, [74] showed that Lactobacillus plantarum CRL 1973 strain was able to produce folate during quinoa sourdough fermentation, due to its capability to synthesize B-group vitamins. Other strains of LAB isolated from cereals and seeds from Argen, like Lactobacillus pentosus ES124 and Lactobacillus plantarum ES137 were also reported to be able to produce high amounts of folate [75].

QP and QQ Macro and Microelements Content
During SP fermentation a significant increase of minerals was recorded ( The capacity of Lactobacillus strains to produce folic acid during fermentation is supported by a large body of literature [70][71][72][73][74]. Likewise, [55] reported that through fermentation of cereal based foods, the folate content could increase up to 700%. Furthermore, [74] showed that Lactobacillus plantarum CRL 1973 strain was able to produce folate during quinoa sourdough fermentation, due to its capability to synthesize B-group vitamins. Other strains of LAB isolated from cereals and seeds from Argen, like Lactobacillus pentosus ES124 and Lactobacillus plantarum ES137 were also reported to be able to produce high amounts of folate [75].

QP and QQ Macro and Microelements Content
During SP fermentation a significant increase of minerals was recorded (Table 4). Potassium content increased by 161.43 mg/100 g, magnesium content reached a maximum value of 294.59 mg/100 g, representing 1.5 times higher than the maximum value of the spontaneous fermented sourdough. Similar trends were recorded for Ca, Fe, and Mn contents for both SP 24 h and OR 24 h with ratios SP 24 h/OR 24 h between 1.5 and 1.6. Quinoa contains about 1% phytic acid which reduces the bioavailability of magnesium, zinc, iron, and calcium due to the strong connection between phytate and these multivalent metal ions, acting as an excellent chelator of cations. As reported by the literature, phytate chelation of mineral cations could have a negative influence on the bioavailability of essential minerals like zinc, iron, calcium, and magnesium [76].
Several studies demonstrated that fermentation with LAB led to a significant reduction of the phytic acid amount, increasing the concentration of Ca and Mg [77,78].
The increment of mineral content in quinoa sourdough is caused by the diminution of phytic acid due to the acidic pH value, which activates flour endogenous phytase and due to the phytase activity of LAB. In the conditions of our study, after 24 h of fermentation the pH value of the SP was 4.2, compared to pH value of 5.8 for OR [38]. The drop of the pH to 4.2 for SP 24 h after 24 h of fermentation, enhanced lactic acid content, and decreased the phytic acid amount leading to higher amount of minerals such as calcium, potassium, iron, zinc, magnesium, manganese, and chromium. This is in agreement with [79] who proved that during fermentation of quinoa with Lactobacillus plantarum the phytate was tremendously reduced (82-98%) and iron amount increased three to fivefold.
Highlighting this idea, [69] reported that using Lactobacillus plantarum strains for fermenting quinoa sourdough could be considered as a bio-enrichment of it, due to the ability of these strains to increase mineral bioavailability such as Ca, Fe, and Mg, through phytate degradation. This is supported also by [1] who previously demonstrated that quinoa LAB sourdough had a phytase activity 2.75 times higher that raw quinoa flour.

Total Flavonoids Content of OR and SP Sourdoughs
Total flavonoids content of OR and SP sourdoughs are illustrated in Figure 3. The SP flavonoids content increased during 24 h of fermentation, having a total value of 1551 mg Qe/100 g f.w., meanwhile OR 24 h reached a final amount of 757 mg Qe/100 g f.w. This finding is consistent with [80] who reported that during fermentation with Lactobacillus genus the total flavonoid content could improve. Through enzymatic reactions, Lactobacillus strains were able to release from glycosides flavonoids and isoflavone aglycones, respectively [81].

Rheological Measurements
The rheological alterations of OR and SP sourdoughs during fermentation and freezing are presented in Figures 4-7. G' represent the capability of materials to store the elastic deformation energy and G'' modul represent the viscous portion of the materials [45]. In general, the G'' was lower than the G' in SP and OR fresh and frozen samples, indicating that the viscous properties of the sourdoughs increased while elastic behaviour decrease with the increasing hours of fermentation. This could be justified due to the possible Lp exopolysaccharides production through sourdough fermentation, which could act as viscosifiers and texturizers, having pseudoplastic rheological behavior and being involved in the water-binding capacity of sourdoughs [27,54,82] reported that strains from Lactobacillus genus could produce exopolysaccharides through sourdough fermentation. This is in agreement with [83] who confirmed that Lactobacillus plantarum is able to produce

Rheological Measurements
The rheological alterations of OR and SP sourdoughs during fermentation and freezing are presented in Figures 4-7. G' represent the capability of materials to store the elastic deformation energy and G" modul represent the viscous portion of the materials [45]. In general, the G" was lower than the G' in SP and OR fresh and frozen samples, indicating that the viscous properties of the sourdoughs increased while elastic behaviour decrease with the increasing hours of fermentation. This could be justified due to the possible Lp exopolysaccharides production through sourdough fermentation, which could act as viscosifiers and texturizers, having pseudoplastic rheological behavior and being involved in the water-binding capacity of sourdoughs [27,54,82] reported that strains from Lactobacillus genus could produce exopolysaccharides through sourdough fermentation. This is in agreement with [83] who confirmed that Lactobacillus plantarum is able to produce exopolysaccharides in sourdough through fermentation. On the other side, [84] reported that LAB might produce 12β-glucan during the growth process and metabolism, which could positively influence the viscosity and the water holding capacity of quinoa flour.          Gelation, water-holding, and foaming capacity represent the main technological applications of quinoa flour [52]. Water absorption capacity of quinoa flour is one of the most important physicochemical properties of carbohydrates content being influenced by the intermolecular association between starchy polymers [51]. Furthermore, this characteritics could influence the water loss in pastry or bakery final baked products [52]. Quinoa main carbohydrate is represented by starch having small granules (less than 3 μm in diameter) and higher maximum viscosity and significant swelling power compared with the barley and wheat starches [51]. Compared to the wheat flour, which has a gluten network, the gluten free flours network is mainly influenced by starch properties [84].
Due to its freeze-thaw stability, quinoa starch could be successefully used as an thickener in food manufacturing where resistance to retro degradation is desired. Likewise, quinoa starch is recomended for frozen baby food manufacturing proving good freeze-thawing stability. In addition to starch, dietary fiber (7-9%), such as pectin and xyloglucans, represent another carbohydrate group with importance on the viscosity and stability of the starch paste [50,57]. Moreover, due to its high content in soluble dietary fiber (1.41-2.3 % dry weight), quinoa flour could be used to improve the texture of highly viscous food products such as dough and final baked products [85]. The GFM content in carbohydrates and organic acids are presented in Table 5. Glucose, fructose, The low elastic properties of OR and SP fresh sourdoughs could be attributed to the starch degradation which might occur during fermentation [34]. Bolívar-Monsalve et al. [82] confirmed the amylolytic activity of Lactobacillus plantarum, activity which could influence the microstructure of quinoa starch and change the pasting properties of quinoa flour. SP 24 h frozen sample had the highest storage modulus (G ) (23127.00 Pa) and loss modulus (G") (6574.7 Pa) at a a final angular frequency of 628 rad s -1 , while SP 24 h fresh sample had G value of 0.06 Pa and G" value of 1852.2 Pa, respectively, indicating that through freezing the elastic behaviour of quinoa sourdough improved due to the freeze-thaw stability of quinoa starch [50]. Another possible explanation for the G" improvement through freezing could be the reorganization of hydrogen bonds of amylose and amylopectin during the cooling period [82]. In addition, OR 24 h frozen sample registered values of 4463.80 Pa and 1161.40 Pa for G' and G", respectively, while OR 24 h fresh sample had values for G' and G" of 0.09 Pa and 1252.4 Pa supporting the idea that through freezing the viscous features of sourdough improved.
Gelation, water-holding, and foaming capacity represent the main technological applications of quinoa flour [52]. Water absorption capacity of quinoa flour is one of the most important physicochemical properties of carbohydrates content being influenced by the intermolecular association between starchy polymers [51]. Furthermore, this characteritics could influence the water loss in pastry or bakery final baked products [52]. Quinoa main carbohydrate is represented by starch having small granules (less than 3 µm in diameter) and higher maximum viscosity and significant swelling power compared with the barley and wheat starches [51]. Compared to the wheat flour, which has a gluten network, the gluten free flours network is mainly influenced by starch properties [84].
Due to its freeze-thaw stability, quinoa starch could be successefully used as an thickener in food manufacturing where resistance to retro degradation is desired. Likewise, quinoa starch is recomended for frozen baby food manufacturing proving good freeze-thawing stability. In addition to starch, dietary fiber (7-9%), such as pectin and xyloglucans, represent another carbohydrate group with importance on the viscosity and stability of the starch paste [50,57]. Moreover, due to its high content in soluble dietary fiber (1.41-2.3 % dry weight), quinoa flour could be used to improve the texture of highly viscous food products such as dough and final baked products [85]. The GFM content in carbohydrates and organic acids are presented in Table 5. Glucose, fructose, and maltose amounts decreased in the gluten free muffins manufactured with SP 24 h sourdoughs, due to its lower carbohydrates content (Table 5). This is in line with [1] who reported that the starch utilization by the heterofermentative Labs such as Lactobacillus plantarum during quinoa sourdough fermentation improved the rate of starch hydrolysis and decreased the glycemic index in bread final baked product. The results are expressed in mg/100 g fresh weight (f.w.). Small letters in common indicate no significant differences between OR and SP samples withdrawn at different moments; Big different letters indicate significant differences between SP and OR samples withdrawn at the same moment; n.d.: not detected.
The glucose and fructose content of the final baked muffins could be influenced also by maple syrup, which is considered a superior natural sweetener from the chemical point of view, being rich in minerals, flavor compounds, and antioxidant capacity [86]. Sucrose, glucose, and fructose are the main carbohydrates detected in maple syrup range between 61.2 and 65.8%, 0.13 and 0.39%, and 0.07 and 0.27%, respectively. Furthermore, the consumption of maple syrup could produce lower glucose and insulin responses, being considered a successful replacement of refined sugars in human diet [87].
The presence of the organic acids in GFM could be explained by controlled and spontaneous fermentation of SP and OR sourdoughs. The lactic content of SP PF 24 h is higher (statistically different p < 0.05) than the amount of OR PF 24 h. This was expected since the initial concentration of lactic acid was statistically different (p < 0.05) in SP 24 h sourdough than in OR 24 h sourdough (8.50 mg/100 g f.w., and 5.81 mg/100 g f.w., respectively).
The same trend was observed with respect to the acetic acid content of GFM as the initial content of SP 24 h and OR 24 h were statistically different (p < 0.05). Acetic acid is the most promising organic acid involved in the bio-preservation of bakery products [88], having an antifungal effect; meanwhile, lactic acid plays an important role in the storage and safety of the final baked goods [89]. Moreover, lactic acid could positively influence the aroma and also the texture of the final baked goods [53,90] and might also degrade the rate of starch digestion in bakery products [26]. Furthermore, the presence of lactic and acetic acids in the final baked good, formed during sourdough fermentation, has been proved to reduce insulinemic and acute glycemic responses [26].
Citric acid possesses antimicrobial activity and could be produced during fermentation of sourdough with Lactobacillus plantarum [45]. Furthermore, the addition of citric acid in the manufacture of baked leaved goods could improve their sensory characteristics, including flavor [91]. In the present study, even if Lp was able to increase the amount of citric acid, the differences were not significant (p < 0.05). The same trend was noticed in the muffins made with SP 24 h, compared with OR PF 24 h, whose values were 2.20 and 2.27 mg/g f.w., respectively (Table 5).

Folic Acid of Gluten Free Muffins (GFM)
The folic content of GFM manufactured with OR and SP sourdoughs at 0, 12, and 24 h of fermentation are illustrated in Figure 8. The folic content of GFM was influenced by the addition of the OR and SP, GFM produced with SP being statistically different (p < 0.05) from GFM produced with sourdough from spontaneous fermentation. This could be explained by the higher content in folic acid in sample SP 24 h (10.48 µg/g f.w) compared with the amount found in OR 24 h sample (3.2 µg/g f.w). This finding is consistent with [79] who proved that the use of sourdough fermented with Lactobacillus strains in bread manufacturing could counteract the thermal loss of bioactive compounds through baking process. Folate is the generic descriptor for folic acid, used to describe the folic acid and its derivatives, and it is involved in cell essential metabolism function. Folic acid is defined as a chemical form of folate and it is often used in the food fortification process [92]. The lack of folates in human body could lead to the development of different diseases, such as neural tube defects, malformations, megaloblastic anemia, cardiovascular diseases, and could play an important role in lung carcinogenesis [40,92]. Therefore, in the US, the fortification of food with folic acid is mandatory and the daily intake recommendation between 200 and 400 μg [40,92]. Moreover, recently [4] reported a lack of folate content in the diet of children diagnosed with celiac disease highlighting the necessity for mandated gluten free folate food fortification policy.

Minerals Content of Gluten Free Muffins (GFM)
As presented in Table 6, the mineral content of GFM manufactured with SP 24 h are significantly higher than OR 24 h. K, Mg, and Ca were the mainly minerals identified in the SP 24 h GFM; although, Mn, Zn, Fe, and Cu could be identified in smaller amounts.
The final results for macro/micro minerals content demonstrated that through the lactic acid fermentation of a rich source of minerals, the final content of these nutrients was enhanced in the final baked product. These results are in agreement with previous findings such as [1,20]. Folic acid ((μg/100 g) f.w.
OR PF SP PF Figure 8. Folic acid content of gluten free muffins. Small letters in common indicate no significant differences between OR and SP samples withdrawn at different moments; Big different letters indicate significant differences between SP and OR samples withdrawn at the same moment.
It is noteworthy to mention that the content of folic acid in final products like bread, noodles, and cookies was improved when QWF was used [48]. Even if, folate is a temperature sensitive vitamin and baking process could diminish its amount, [48] reported a total folate of 17-98 µg/100 g d.m. (dry matter) in noodles, 18-62 µg/100 g d.m. in cookies, and 26-41 µg/100 g d.m. in breads, respectively.
Folate is the generic descriptor for folic acid, used to describe the folic acid and its derivatives, and it is involved in cell essential metabolism function. Folic acid is defined as a chemical form of folate and it is often used in the food fortification process [92]. The lack of folates in human body could lead to the development of different diseases, such as neural tube defects, malformations, megaloblastic anemia, cardiovascular diseases, and could play an important role in lung carcinogenesis [40,92]. Therefore, in the US, the fortification of food with folic acid is mandatory and the daily intake recommendation between 200 and 400 µg [40,92]. Moreover, recently [4] reported a lack of folate content in the diet of children diagnosed with celiac disease highlighting the necessity for mandated gluten free folate food fortification policy.

Minerals Content of Gluten Free Muffins (GFM)
As presented in Table 6, the mineral content of GFM manufactured with SP 24 h are significantly higher than OR 24 h. K, Mg, and Ca were the mainly minerals identified in the SP 24 h GFM; although, Mn, Zn, Fe, and Cu could be identified in smaller amounts. The results are expressed in mg/100 g fresh weight (f.w.). Small letters in common indicate no significant differences between OR and SP samples withdrawn at different moments; Big different letters indicate significant differences between SP and OR samples withdrawn at the same moment.
The final results for macro/micro minerals content demonstrated that through the lactic acid fermentation of a rich source of minerals, the final content of these nutrients was enhanced in the final baked product. These results are in agreement with previous findings such as [1,20].
Quinoa fermentation with Lp provided in the medium a value of pH optimal for enzymatic degradation of phytic acid, leading to final baked muffins enriched in minerals such as K, Ca, Mg, Mn, Fe, Zn, and Cu ( Table 6). The successefully use of sourdough aiming to improve the minerals content of the gluten free products was prevously reported by [24,93] The elimination of gluten involves sometimes the decrease of vitamins, minerals, fibers, and folate. Gluten free products had lower minerals content compared with the conventional ones and their bioavailability could range between 10 and 70% [94]. Furthermore, people who are undergoing a gluten free diet are exposed to mineral and vitamins deficiencies mainly because of their lower content in the final products [95] and due to the presence of phytic acid, an anti-nutritional factor which decreases the bioavailability of minerals such as calcium, magnesium, iron, or zinc [74] The total flavonoids content of GFM is illustrated in Figure 9. In the final baked muffins manufactured with SP 24 h, a total content of 1561 mg Qe/100 g f.w. was determined, compared with OR PF 24 h, where flavonoids had a total amount of 1317 mg Qe/100 g f.w. The difference between the samples could be explained by the presence of SP or OR 24 h sourdoughs. Nonetheless, it is noteworthy to mention that [96] identified in buckwheat flour a total of 188 flavonoid metabolites that could positively influence the content of flavonoids in the muffins. On the other side, the thermal treatment of the final baked goods could have a negative influence on the total flavonoids content [97].
The total phenols (TF) and radical scavenging activity (RSA%) of gluten free muffins made with SP sourdough were significantly different to those made with OR sourdough (Figure 10) due to the capacity of Lp to produce higher extent of lactic acid amount in SP sample, that could influence through acidification the extractability of total phenols and the antioxidant potential. Moreover, the interaction of Folin-Ciolcateu reagent with other non-phenolic compounds like vitamins, amino acids, and proteins could also have an influence on the number of polyphenolic compounds. On the other side, the lower OR 24 h total phenols and radical scavenging activity suggested that spontaneous fermentation and quinoa endogenous enzymes were not able to decrease the pH and release antioxidant compounds [98].
OR PF 24 h, where flavonoids had a total amount of 1317 mg Qe/100 g f.w. The difference between the samples could be explained by the presence of SP or OR 24 h sourdoughs. Nonetheless, it is noteworthy to mention that [96] identified in buckwheat flour a total of 188 flavonoid metabolites that could positively influence the content of flavonoids in the muffins. On the other side, the thermal treatment of the final baked goods could have a negative influence on the total flavonoids content [97]. Figure 9. Total flavonoids of gluten free muffins. Small letters in common indicate no significant differences between OR and SP samples withdrawn at different moments; Big different letters indicate significant differences between SP and OR samples withdrawn at the same moments.
The total phenols (TF) and radical scavenging activity (RSA%) of gluten free muffins made with SP sourdough were significantly different to those made with OR sourdough (Figure 10) due to the capacity of Lp to produce higher extent of lactic acid amount in SP sample, that could influence through acidification the extractability of total phenols and the antioxidant potential. Moreover, the interaction of Folin-Ciolcateu reagent with other non-phenolic compounds like vitamins, amino acids, and proteins could also have an influence on the number of polyphenolic compounds. On the other side, the lower OR 24 h total phenols and radical scavenging activity suggested that spontaneous fermentation and quinoa endogenous enzymes were not able to decrease the pH and release antioxidant compounds [98]. OR PF SP PF Figure 9. Total flavonoids of gluten free muffins. Small letters in common indicate no significant differences between OR and SP samples withdrawn at different moments; Big different letters indicate significant differences between SP and OR samples withdrawn at the same moments.
Appl. Sci. 2020, 10, x FOR PEER REVIEW 18 of 24 Figure 10. Total phenols and radical scavenging activity of gluten free muffins. Small letters in common indicate no significant differences between OR and SP samples withdrawn at different moments; Big different letters indicate significant differences between SP and OR samples withdrawn at the same moment.
It is important to mention that the TF amount of quinoa flour and RSA were 451 ± 0.3 mg GAE/100 g f.w. and 92 ± 0.5%, respectively [38]. The total phenols amount could vary between samples as reported by [50] who proved that QF from Chile had significant higher polyphenols content compared with the flour from Mexico (319 mg GAE/100 g and 180.4 mg GAE/100 g, respectively) and could be justified by the differences in the polyphenol extraction process [47].
The TF value is close to the value reported by [99] as 464 mg GAE/100 g. With respect to RSA, other studies reported a value of it up to 71.8% [1]. The differences between the results could be due to the different extraction conditions such as the extraction solvent and due to the duration of the extraction that could influence the total phenols content [100].
Through the quinoa fermentation with Lp, the TF and RSA content improved up to 350 mg GAE/100 g f.w. and 94%, respectively [38]. This idea is supported by [98] who indicated that fermentation of quinoa flour with LAB could lead to an improvement of the antioxidant activity. Briefly, Rizello et al. indicated that Lb plantarum T6A10 strain was able to increase the radical scavenging activity from 32.7 to 84.8% during 24 h of fermentation, due to its ability to release peptides with antioxidant activity during controlled fermentation through proteolysis.
Overall, it can be stated that SP and OR 24 h sourdoughs had an important influence on the final Total phenols g GAE/ 100 g f.w Radical scavenging activity RSA (%)

Time (h)
OR PF SP PF Figure 10. Total phenols and radical scavenging activity of gluten free muffins. Small letters in common indicate no significant differences between OR and SP samples withdrawn at different moments; Big different letters indicate significant differences between SP and OR samples withdrawn at the same moment.
It is important to mention that the TF amount of quinoa flour and RSA were 451 ± 0.3 mg GAE/100 g f.w. and 92 ± 0.5%, respectively [38]. The total phenols amount could vary between samples as reported by [50] who proved that QF from Chile had significant higher polyphenols content compared with the flour from Mexico (319 mg GAE/100 g and 180.4 mg GAE/100 g, respectively) and could be justified by the differences in the polyphenol extraction process [47].
The TF value is close to the value reported by [99] as 464 mg GAE/100 g. With respect to RSA, other studies reported a value of it up to 71.8% [1]. The differences between the results could be due to the different extraction conditions such as the extraction solvent and due to the duration of the extraction that could influence the total phenols content [100].
Through the quinoa fermentation with Lp, the TF and RSA content improved up to 350 mg GAE/100 g f.w. and 94%, respectively [38]. This idea is supported by [98] who indicated that fermentation of quinoa flour with LAB could lead to an improvement of the antioxidant activity. Briefly,