Lactic Acid Fermentation Improved Textural Behaviour , Phenolic Compounds and Antioxidant Activity of Chia ( Salvia hispanica L . ) Dough

Ana Yanina Bustos*, Carla Luciana Gerez, Lina Goumana Mohtar Mohtar, Verónica Irene Paz Zanini, Mónica Azucena Nazareno, María Pía Taranto and Laura Beatriz Iturriaga Research and Transference Centre of Santiago del Estero (CITSE – CONICET – UNSE), RN9, km 1125, AR-4206 Santiago del Estero, Argentina San Pablo T University, Av. Solano Vera and Villa Nougués, AR-4129 Tucumán, Argentina Reference Centre for Lactobacilli (CERELA-CONICET), Chacabuco 145, AR-4000 San Miguel de Tucumán, Argentina 4 Institute of Bionanotechnology (INBIONATEC-CONICET), RN9, km 1125, AR-4206 Santiago del Estero, Argentina


Introduction
Chia (Salvia hispanica L.) is an annual plant belonging to the Lamiaceae family and native to southern Mexico and northern Guatemala (1).Recently, due to its functional and nutritional att ributes, scientists have revived their interest in chia.In fact, chia contains an important amount of vitamins, minerals, antioxidant compounds and polyunsaturated fatt y acids, particularly the omega-3 and omega-6, which are found in the tissues of the central nervous system (2).Thus, the incorporation of chia in different foods such as pound cakes (3), wheat bread (4) and gluten-free bread (5) has recently been reported.However, despite its valuable nutritional and functional characteristics, the incorporation of chia in bakery products could adversely aff ect the gluten network and gas retention of dough, leading to low quality products (4,6).Due to this undesired consequences, it is necessary to propose strategies that allow the enrichment of bakery products with this ingredient without detrimental technological effects.
Sourdough is a mixture of fl our (principally rye and wheat fl our) and water that is fermented with selected lactic acid bacteria (LAB).Numerous studies have reported positive eff ects of wheat and rye sourdough addition on bread quality (fl avour, crumb structure, dough volume) and shelf life (7)(8)(9)(10).Additionally, LAB fermentation can improve macromolecule hydrolysis, thus enhancing the digestibility and nutritional quality of bread (11,12).Nevertheless, specifi c factors have to be controlled to reach an optimal fermentation process, with the type of fl our and the lactic culture being the most relevant (12,13).The fl our aff ects the technological and nutritional quality of the baked products and the development of the microbial fermentation (13).Thus, the selection of a suitable starter strain allows a bett er utilization of particular fl our (13).In this sense, to our knowledge, there are no data on sourdough prepared from chia fl our and on the selection of adequate starter strains for preparation of sourdough bread.The aim of this work is to select autochthonous LAB strains to be used for chia sourdough fermentation.Then, rheological behaviour, organic acid production, phenolic compound content and antioxidant activity of chia sourdough made with the selected starter culture will be evaluated and used to supplement wheat bread.A technological and functional profi le analysis of bread will be carried out to compare the advantages of the sourdough fermentation with respect to the use of unfermented chia fl our.

Proximate analysis of fl our
Moisture content was quantifi ed according to AOAC method (14) from the diff erence between the initial and fi nal mass of the samples subjected to a temperature of 130 °C.Proteins were determined by the Kjeldahl method (15).Briefl y, the samples were digested in concentrated sulfuric acid with a catalyst, the nitrogen was converted to ammonium sulphate, and sodium hydroxide was added to release ammonia, which was distilled in standardized hydrochloric acid for quantifi cation by titration.The protein content was calculated from total nitrogen using a factor of 5.7 for wheat and chia fl our.For ash determination, the calcination in muffl e furnace (KLS 30/11; Thermconcept, Bremen, Germany) at 500 °C was employed and the ash mass was calculated from the diff erence between the initial and fi nal mass of the samples (16).For fat quantifi cation, the Soxhlet method ( 16) was employed using anhydrous petroleum ether as solvent.

Microbiological analysis and isolation of LAB
LAB were isolated from chia fl our naturally fermented at 30 or 37 °C for 24 h with a dough yield (Y) of 300, calculated as follows: Y =(m(dough)/m(fl our))•100 /1/ Chia dough was made and propagated following a protocol commonly used for wheat fl our sourdough, without the addition of starter cultures or baker's yeast (12).Aft er this fi rst fermentation, fi ve refreshment steps were carried out by mixing 25 % (by mass) of the previously fermented dough with fl our and water (Y=300), and incubating for 24 h at 30 or 37 °C.Samples for pH measurement were taken aft er fermentation and processed as follows: 10 g of each sample was blended with 90 mL of distilled water and mixed on a digital hot plate magnetic stirrer (model MS-H-Pro; Scilogex, LLC, Rocky Hill, CT, USA) and then the mixture was centrifuged at 9000×g (model Z 326 K; Hermle Labortechnik, Wehingen, Germany) for 15 min.The pH was measured in the supernatant using a pH meter (pH 209; Hanna Instruments, Póvoa de Varzim, Portugal).Aft er fi ve refreshments, the acidifi cation rate was stable, and fermented chia dough was used for presumptive LAB isolation as follows: 10 g of chia dough was homogenized with 90 mL of sterile saline solution and cultivated in MRS agar (Sigma-Aldrich) supplemented with cycloheximide (0.1 g/L).Plates were incubated at 30 or 37 °C for 48 h.Autochthonous bacteria were randomly selected from the plates containing between 30 and 300 colonies.Individual colonies were purifi ed by scratching on MRS agar plates.Pure cultures were stored at -70 °C in MRS broth with 15 % (by mass per volume) glycerol for further examination.Fift y presumptive LAB selected (Gram-positive, catalase-negative, non-motile rods and cocci, and able to acidify the culture medium) were cultivated in MRS broth at 30 or 37 °C (depend-ing on the isolation temperature) for 24 h.Cells were harvested by centrifugation (4000×g for 10 min; model Z 326 K; Hermle Labortechnik) and washed twice with saline solution.Chia fl our (100 g) and 200 mL of tap water, containing the above cellular suspension (cell density in the dough of 7 CFU/g) were used to give an Y=300.Dough samples were fermented at 30 or 37 °C for 24 h, according to the optimal growth temperature of the lactic strains.LAB growth was determined by the plate dilution method using MRS agar, the plates were incubated at 30 or 37 °C for 48 h, and the results were expressed as log CFU/g.The pH of the dough was determined as described above, and the concentrations of total free amino acids in the dough samples were determined using the o-phthalaldehyde (OPA) method (17).Briefl y, 0.5 g of dough were blended with 2 mL of 0.75 M of trichloroacetic acid, homogenized and kept at 4 °C for 30 min.Aft erwards, the samples were centrifuged (9000×g, 1 min, model Z 326 K; Hermle Labortechnik) and 10 μL of supernatant were blended with 200 μL of OPA reagent (10.75 mL of distilled water, 1.25 mL of 20 % (by mass per volume) SDS, 12.5 mL of sodium tetraborate, 50 mL of β-mercaptoethanol and 20 mg of OPA dissolved in 500 μL of methanol).The reaction mix was kept at room temperature for 5 min and absorbance was determined at 340 nm using the UV-Vis spectrophotometer Jasco V-630 (Medson, Paczkowo, Poland).Calibration curve was plott ed using the glutamic acid standard in the range of 0.2 to 5.0 mM.

Genotypic identifi cation of selected LAB
The genotypic identifi cation of the selected strain was determined on the basis of sequencing of the variable (V1) region of the 16S rDNA, as previously described (18).The oligonucleotides for PCR reactions were: PLB16, 5′ -A G A-GTTTGATCCTGGCTCAG-3′, and MLB16, 5′-GGCTGCT-GGCACGTAGTTAG-3′.PCR amplifi cation consisted of 30 cycles for 30 s at 94 °C, 30 s at 50 °C and 1 min at 72 °C.Final PCR products were purifi ed using a commercial kit (AccuPrep ® PCR Purifi cation Kit; Bioneer Corporation, Genbiotech, Buenos Aires, Argentina) and subjected to sequencing (Sequencing Service, Science and Technology Center, CONICET, Tucumán, Argentina).The resulting sequences were analysed on-line using the NCBI BLAST algorithms (19) and the Ribosomal Database Project (20).

Preparation of chia sourdough with the selected starter
Chia sourdough was prepared using the selected strain, L. plantarum C8.Sourdough was fermented at 37 °C for 24 h.LAB growth, sourdough preparation and pH determination were carried out identically as described above, except for fermentation, which was performed at 37 °C (optimal growth temperature of the selected strain).Lactic acid quantifi cation was performed by amperometry using an electrochemical system of three glassy carbon electrodes (3 mm; CH Instruments, Inc., Austin, TX, USA) optimized for the detection of lactic acid, as previously reported (21).Acetic and phenyllactic (PLA) acids were measured as previously described (10) by HPLC (model 2350; ISCO, Lincoln, NE, USA) using an Aminex HPX-87H ion-exclusion column (300 mm×7.8 mm; Bio--Rad, Hercules, CA, USA).Organic acid mass fractions were expressed in g or μg per kg of dough.

Fundamental rheological measurements of chia sourdough
A controlled stress rheometer (AR 2000; TA Instruments, New Castle, DE, USA) was used for the dynamic rheological assays.The control soft ware Rheology Advantage Data Analysis Program (22) was used to obtain directly the data of storage modulus (G'), loss modulus (G''), complex modulus (G*), and the tangent of loss angle (tan δ).The dough samples (0 and 24 h of fermentation) were fi xed between the plates (d=40 mm with a gap of 1.5 mm), the overlaying dough was removed, and the measurements started aft er 10 min.A solvent trap device (TA Instruments) was used to avoid the sample evaporation during the measurement.Frequency sweeps were performed using a strain value in the linear viscoelastic region and a frequency range of 0.1-10 Hz at 25 °C.A Peltier plate system (TA Instruments) att ached was used to control temperature.

Extract preparation
Ten g of chia dough (at 0 and 24 h of fermentation) and bread were dried at 50 °C for 12 h (model TDC70; Tecnodalvo S.R.L., Santa Fe, Argentina), then manually crumbled and ground in a traditional stone mortar to obtain dust samples.For extraction, 1 g of powder sample of dough (2.4 % moisture) and bread (5.3 % moisture) was blended with 10 mL of 80 % (by volume) aqueous methanol solution for 2 h at room temperature.Samples were aft erwards centrifuged (9000×g for 15 min, model Z 326 K; Hermle Labortechnik).The supernatants (extracts) were stored at -20 °C until use.

Total phenolic compounds and their identifi cation by capillary zone electrophoresis
Total phenolic contents were determined colorimetrically using Folin-Ciocalteu reagent, as previously described by Ezekiel et al. (23).Absorbance of the mixture was determined at 725 nm using the spectrophotometer UV-Vis Jasco V-630 (Medson).Calibration method was used to determine the phenolic compound concentration by means of the calibration plot performed with GA in the concentration range of 0.09 to 12 mmol/L.Results were expressed in mg of gallic acid equivalents (GAE) per g.
The identifi cation of phenolic compounds present in the dough (at 0 and 24 h of fermentation) was performed by capillary zone electrophoresis on Agilent Capillary Electrophoresis System G1601A (Agilent Technologies Deutschland GmbH & Co. KG, Waldbronn, Germany) equipped with a UV detector set at 206 nm.Separation and analysis were carried out on an uncoated fused silica capillary tubing (50 μm i.d. and 56 cm total length; Agilent Technologies) at 25 °C.The operating buff er was composed of sodium tetraborate, 25 mM, pH=9.0.Kaempferol, galangin, quercetin, and coumaric, benzoic, caff eic, ferulic, chlorogenic and dihydroxybenzoic acids were used as standards.

Antioxidant activity
Antioxidant activity (AA) of the dough and bread extracts was determined based on the DPPH method (24).
Diff erent aliquots of each extract (prepared as described above) were added to methanolic solution of DPPH (initial absorbance A 515 nm =1.20).The decrease in the absorbance was monitored during 30 min using a UV-Vis spectrophotometer Jasco V-630; Medson).AA (%) was calculated according to the following equation: where A S is the absorbance of the solution in a steady state and A 0 is the absorbance of the DPPH solution prior to the addition of the sample.The absorbance of the system at the steady state was estimated by mathematical fi tting of kinetic curves using GraphPad Prism version 5.00 for Windows (25).The IC 50 values indicate the concentration of the sample required to decrease the absorbance at 515 nm by 50 %.

Ferric reducing ability of plasma assay
The ferric reducing ability of plasma (FRAP) assay was carried out according to Benzie and Strain (26).To each reaction, 0.05 mL of the extract, prepared as described above, was added to 0.95 mL of the reagent mixture, shaken vigorously and allowed to stand at room temperature in the dark for 20 min.Aft erwards, absorbance was measured, and the fi nal results were expressed in μg of Trolox equivalents (TE) per g.

Wheat bread preparation using chia sourdough
Chia sourdough fermented by L. plantarum C8 was used for the production of wheat bread.The diff erent formulations are listed in Table 1.Traditional bread from wheat fl our and wheat bread containing chia fl our were manufactured and used as the controls.Commercial dry yeast S. cerevisiae (Calsa, Yeast Company S.A., Buenos Aires, Argentina) was used as leavening agent for all dough types (7 log CFU per g of dough).The following baking schedule was adopted: mixing for 10 min (fi rst 2 min at slow speed and 8 min at increased speed).The dough samples were individually placed in aluminium pans and fermented at (30±1) °C and 85 % of relative humidity for 60 min.Aft er fermentation, the dough was baked in a batch oven (180 °C for 20 min, model OD-38D; BLU Tec, Buenos Aires, Argentina), and the bread loaves were cooled at room temperature for 120 min.The bread loaves were packed in polyethylene bags and stored at 30 °C.Specifi c loaf volume measurement and the texture profi le analysis were carried out 2 h aft er baking.Loaf volumes were measured by the rapeseed displacement method (3).Specifi c loaf volumes were calculated by dividing the loaf volume by the loaf mass, and the results were expressed in mL/g.The texture profi le was determined using the universal testing machine TA.XTplus Texture Analyzer (Stable Micro Systems, Godalming, UK) and following a procedure reported previously (6) with some modifi cations.A double compression test was applied, and a 20-mm diameter cylinder probe (TA-11) was employed.Samples were prepared by cutt ing 2-cm high slices, allowing only crumb texture measurements.Bread slices were compressed to 50 % of their original height.The textural parameters reported were: fi rmness (g), chewiness (g) and cohesiveness.

Data analysis
All measurements were conducted in at least three independent assays, and the obtained results were reported as mean values with their respective standard deviations.The analysis of variance (ANOVA) and Fisher's least signifi cant diff erence (LSD) post-hoc tests were performed using InfoStat (27).All statistical analyses were conducted at a signifi cance level of p<0.05.

Proximate composition of fl our
First, in order to characterize the raw material used in this work, the proximate composition of chia and wheat fl our was determined.Protein ((25.1±2.1)% by mass), ash ((6.7±0.2) % by mass) and fat ((19.4±1.5)% by mass) contents in chia fl our were higher than in wheat fl our ((14.6±1.1),(0.7±0.3) and (1.6±0.1)% by mass, respectively).These results are consistent with previous reports: 9-23 % of proteins and 25-35 % of lipids (3).It was reported that chia seeds from Argentina are mainly (60-70 %) composed of linolenic acid (28), whose consumption was related to benefi cial eff ects on human health (2).These data indicate that the addition of chia fl our to wheat bread could dramatically improve its protein and fat content.Furthermore, the high ash level of chia fl our makes it a good supplement to wheat fl our.

Isolation and selection of LAB from chia sourdough
In order to select a starter to be used for chia dough fermentation, autochthonous LAB strains were isolated from chia sourdough.Fift y strains (33 cocci and 17 lactobacilli) able to acidify the MRS broth, Gram-positive, catalase-negative and non-motile, were presumptively classifi ed as LAB.Lactobacilli are usually selected as sourdough starters mainly due to their competitiveness and adaptation to this particular environment; thus 17 lactobacilli were further selected to be inoculated in chia sourdough.The acidifi cation capacity (ΔpH) and total free amino acid values of the fermented chia sourdough were taken into account for the fi nal selection.Before fermentation, the pH of the dough was (6.3±0.1).The 17 strains decreased the pH of the dough with ΔpH values (diff erence between

Selected chia sourdough starter
Chia fl our was fermented by L. plantarum C8 to obtain sourdough.Aft er 24 h of fermentation, cell counts of 9.2 CFU/g and fi nal pH=4.3 were reached.The pH decrease was associated with the production of organic acids.In fact, a gradual increase in lactic acid was observed during the fermentation, reaching a value of 12.3 g per kg dough aft er 24 h (Fig. 1).Additionally, acetic acid (1.0 g per kg of dough) and PLA (23.8 μg per kg of dough) were also detected in chia sourdough (at 24 h) (data not shown).PLA is an antimicrobial agent produced by some strains of LAB able to prevent fungal spoilage (10).At the end of fermentation, the value of fermentation quotient, given by the lactic/acetic acid ratio was 12.This value is in the range of those previously reported for sourdough made with quinoa and rye (12,29).

Os cillatory testing of chia sourdoughs
Fig. 2 shows the frequency sweeps corresponding to the chia sourdough at the beginning and the end of fermentation.The oscillatory frequency sweeps carried out in the linear viscoelastic domain indicate that storage, loss and complex moduli had an increasing trend in all the frequency range applied, and a solid-like behaviour was also predominant (G'>G'') in both studied samples.G* showed diff erences between the samples; unfermented sourdough showed the highest values at the frequency of 1 Hz ((9149±819) Pa), while aft er 24 h of fermentation, it reached a value of (6902±420) Pa (data not shown).The spectrum analysis suggests that the fermentation of chia caused a loss of consistency and an increase in the viscous character with respect to that of the unfermented dough (at 0 h).A similar eff ect on the rheological properties of dough made with chestnut and chia fl our following hydrocolloid addition was reported (5).In addition, a decrease in the complex shear modulus and elasticity of amaranth sourdough made with L. plantarum or L. paralimentarius strains and buckwheat sourdough prepared with L. plantarum, L. brevis or L. paralimentarius strains was reported (30,31).These rheological changes could be partially att ributed to the increase in the proteolytic activity observed in chia dough aft er fermentation by C8 strain, as was previously reported for wheat and rye sourdough (7,9).However, chemically acidifi ed dough (wheat or amaranth, among others) showed the opposite behaviour, resulting in more elastic dough, probably because the acidifi cation is instantaneous and not progressive as in the sourdough process, and in this way, microbial and fl our enzymes are not activated (30,32).

Phenolic compounds and total antioxidant activities of chia sourdough
The presence of phenolic compounds in the diet is benefi cial to health mainly due to their antioxidant activities (2,33).As shown in Table 2, the total phenolic content signifi cantly increased (from 14.90 to 20.80 mg GAE per g) in chia sourdough aft er 24 h of fermentation.In order to identify the phenolic compounds present in the fermented and unfermented chia dough, capillary electrophoresis was performed (Fig. 3).The phenolic content and  profi le of chia sourdough (at 0 and 24 h of fermentation) were strongly modifi ed by lactic acid fermentation, as was observed in the electropherograms.In fact, 14 compounds were modifi ed, out of which nine had higher concentrations and fi ve showed the opposite behaviour aft er fermentation.Although we investigated the presence of coumaric, benzoic, caff eic, ferulic, chlorogenic and dihydroxybenzoic acids as well as of kaempferol, galangin and quercetin, compounds previously reported in chia products (2,34), only two (ferulic and chlorogenic acids) compounds coincided with the tested standards (Fig. 3).Chlorogenic acid was only found aft er lactic acid fermenta-  tion (2.5 mg/g), while ferulic acid increased from 3.9 mg/g in unfermented dough to 5.6 mg/g in 24-hour fermented sourdough.
The increase of phenolic compounds by lactic acid fermentation was also observed in cereals such as rye, barley and oat, and in a variety of vegetables (e.g.cucumbers, cabbages, olives, carrots, French beans, marrows, artichokes, capers and eggplants) (33,35).In this work, 1.33-fold increase of total phenolic acids during lactic fermentation and 1.44-fold increase of ferulic acid were observed.Konopka et al. (36) reported that in whole meal and white wheat and rye sourdough (prepared with L. casei and L. brevis) 1.0 to 13.5 % of bound ferulic acid was released after fermentation.Hole et al. (33) showed that the bioavailability of the dietary phenolic acids in the fl our from whole grain barley and oat groat signifi cantly improved aft er fermentation with LAB.In fact, the highest increments of 27.4-and 25.3-fold were observed in whole grain barley and oat groat, respectively, fermented with three probiotic strains (L.johnsonii LA1, L.reuteri SD2112 and L. acidophilus LA-5).Remarkably, L. plantarum is the LAB strain most frequently isolated from niches where phenolic compounds are abundant, and its ability to metabolize them has been reported (37,38).During sourdough fermentation, esterase activities of LAB and fl our endogenous enzymes are able to hydrolyse the complex phenolic compounds and their glycosylated forms into free phenolic acids (37,39).
The antioxidant activity of chia sourdough was also evaluated (Table 2).The activity of the extract of chia sourdough on DPPH followed fi rst-order kinetics, the fermented dough showing the highest antioxidant eff ectiveness (Fig. 4).By both complementary methods employed, the antioxidant activity aft er 24 h of fermentation increased from 33.3 to 39.6 % with respect to the initial values (Table 2).In fact, the activity of the sourdough, determined by the DPPH radical scavenging (AA and IC 50 ), reached 85.9 % ( IC 50 =2.0mg/mL), while at the beginning of the fermentation it was 52 % (IC 50 =3.1 mg/mL).The IC 50 found in our study is in line with that reported by Scapin et al. (24) in chia seed extract.Similar trend was observed with the FRAP method; the antioxidant activity improved from 14.1 to 21.1 mg of TE per g aft er fermentation.Other authors (13,29) also reported an increase in the antioxidant activity in sourdough prepared from diff erent types of fl our such as rye, whole wheat and spelt, among others, with respect to that of the unfermented dough and additionally demonstrated the capacity of LAB to release antioxidant peptides through the proteolysis of native cereal proteins.

Bread evaluation tests
Chia sourdough prepared with L. plantarum C8 was used for the production of wheat bread, while the wheat bread loaves with and without chia fl our were used as controls.In accordance with the above results, the highest phenolic content (1.13 mg/g) was found in the wheat bread with chia sourdough (Table 2).Its antioxidant activity was 1.3 times higher than of the bread with chia fl our and 7 times greater than in wheat bread.Therefore, sourdough addition eff ectively improved the mass fraction of phenols and antioxidant activity of traditional bread.Specifi c volumes of the diff erent types of bread are listed in Table 3. Chia fl our or sourdough addition slightly reduced the specifi c volume of the loaves compared to wheat bread.Similar behaviour was observed in the bread containing 5 to 11 % (by mass) of chia fl our (4,6).As reported previously, the addition of chia decreased the amount of gluten present in the formulation and consequently reduced the retention of air bubbles in the dough (6).On the other hand, the utilization of wheat sourdough has been reported both to decrease and increase the bread volume (32).It has been suggested that the infl uence of the sourdough on bread volume is due mainly to the enzymatic reactions that develop during fermentation, since the pH gradually drops, allowing the activation of amylolytic and proteolytic enzymes, among others, to modify the dough components.
The addition of chia sourdough reduced the fi rmness and chewiness of the crumb with respect to that of wheat bread.The decrease in fi rmness could be explained by the rheological behaviour previously described (Fig. 1), where the lower values of storage module (G') of sourdough than of unfermented dough could decrease bread fi rmness.In this sense, a decrease in chewiness and fi rmness had previously been reported when smaller amount of buckwheat sourdough was added to the bread (30).On the contrary, other authors observed the opposite behaviour in chia bread (4).
Cohesiveness is a textural parameter that is linked to the integration of components of a sample when it is sub- WB=wheat bread, WB-CF=wheat bread with unfermented chia fl our, WB-CS=wheat bread with chia sourdough; variables with the same lett er in superscript in the same column are not signifi cantly diff erent (p<0.05)jected to deformation.In both the wheat bread with chia sourdough and the wheat bread with chia fl our, an important increase of cohesiveness with respect to that of wheat bread was observed, without signifi cant diff erences between them.

Conclusions
Recently, chia fl our has been studied due to its nutritional and functional characteristics, and its incorporation in diff erent foods is frequently reported.Chia sourdough fermented by a selected Lactobacillus plantarum strain was characterized.Aft er 24 h of lactic acid fermentation, a loss of consistency and an increase in the viscous character were observed.Additionally, phenolic compound concentration and antioxidant activity were enhanced by lactic acid fermentation.Finally, the use of this chia sourdough improved the overall characteristics of traditional wheat bread, including its physical and antioxidant properties, which were even bett er than those observed in the bread with unfermented chia fl our.

Fig. 3 .
Fig. 3. Electropherograms of: a) fermented chia sourdough, and b) unfermented dough.Each arrow corresponds to a peak whose area was modifi ed and the percentage of change is indicated above.*ferulic acid, × chlorogenic acid

Fig. 4 .
Fig. 4. Kinetic curves of DPPH scavenging of extracts of sourdough at 0 h (dots) and aft er 24 h of fermentation (triangles)

Table 1 .
Mass fractions of ingredients in diff erent bread loaves before and aft er fermentation) ranging from (1.50±0.05) to (2.0±0.1).The initial concentration of total free amino acids was (9.2±0.5)μM per g of dough, while the fi nal concentration ranged from (19.5±0.9) to (28.3±1.1)μM per g of dough.The highest ΔpH value (2.0) and total free amino acid concentration ((28.3±1.1)μM per g of dough) were observed in the dough fermented by C8 strain, which was further genotypically identifi ed as Lactobacillus plantarum C8 and used as single starter for sourdough preparation.
WB=wheat bread, WB-CF=wheat bread with unfermented chia fl our, WB-CS=wheat bread with chia sourdough.The wheat/chia fl our ratio was 4:1 in both formulations the pH

Table 2 .
Total phenolic content and antioxidant activities of the sourdough and bread loaves GAE=gallic acid equivalent, AA=antioxidant activity against DPPH, TE=Trolox equivalent, WB=wheat bread, WB-CF=wheat bread with unfermented chia fl our, WB-CS=wheat bread with chia sourdough, n.d.=not detected; variables with the same lett er in superscript in the same column are not signifi cantly diff erent (p<0.05)