Effect of Lactic Fermentation on Antioxidant Capacity of Rye Sourdough and Bread

Transcription

1 Food Sci. Technol. Res., 16 (6), , 2010 Effect of Lactic Fermentation on Antioxidant Capacity of Rye Sourdough and Bread Iuliana Banu, Ina Vasilean and Iuliana Aprodu Dunarea de Jos Galati University, Faculty of Food Science and Engineering, 111, Domneasca St., , Galati, Romania Received May 16, 2010; Accepted July 12, 2010 Effect of sourdough fermentation, by different starter culture (lactic acid bacteria Lactococcus lactis ssp. Lactis, Weissella confusa, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus helveticus, and yeast Kluyveromyces marxianus subsp. Marxianus), on antioxidant capacity of rye dough and bread was determined and compared with spontaneous fermentation. The total phenolic content and antioxidant activities assessed using Trolox equivalent antioxidant capacity (TEAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity expressed in terms of Trolox equivalents and of percentage of discoloration, were measured. The results indicate that antioxidant activity of the rye sourdough highly depends on the type of inoculum used for fermentation and the levels of bioactive compounds change during the bread making process. Fermentation process and type of starter culture are tools to increase the bioactive compounds with antioxidant activities of the rye bread. Keywords: whole rye flour, sourdough, bread, antioxidant capacity To whom correspondence should be addressed. Introduction Cereal processing can decrease or increase the levels of the bioactive compounds, and can change their bioavailability (Hansen et al., 2002; Liukkonen et al., 2003; Katina et al., 2005; Katina et al., 2007; Andersson et al., 2009; Villauenga et al., 2009; Sands et al., 2009; Coda et al., 2010; Dordevic et al., 2010). The most important processes in grain processing are milling and bread making, but flaking, malting, extrusion and puffing germination are also important when considering the extranutritional constituents of the processed grain, with effects on human health (Liukkonen et al., 2003; Dewettinck et al., 2008). Process variables such as: heat, shear, water content and distribution, enzymes activity, microorganisms, presence of secondary metabolites, etc. influence the product bioactivity (Liukkonen et al., 2003). Recently many studies have focused on the influence of different processing on the nutritional quality of the cereal products. Iyer and Tomar (2009) and Sands et al. (2009) suggested that the fortification induced by fermentation is a novel concept with high potential of enriching food products in bioactive compounds through bacterial strain selection, delivery engineering, and metabolic engineering. Rye is an important sources of dietary fibres (arabinoxylans, fructans, β-glucans) and bioactive compounds, such as cinnamic acids, alkylresorcinols, lignans, sterols, vitamins, and minerals (Andersson et al., 2009; Villauenga et al., 2009). Many studies focused lately on determining the total antioxidant capacity of the cereals (Beta et al., 2005; Pathirana and Shahidi, 2007; Heinio et al., 2008; Serpen et al., 2008; Hung et al., 2009; Villauenga et al., 2009; Dordevic et al., 2010). Cereal grains contain a wide range of chemical classes with antioxidant activity. Rye and wheat extracts have shown potential antioxidant properties as rye and wheat phenolics appear to serve as powerful antioxidants through radical scavenging (Pathirana and Shahidi, 2007; Dordevic et al., 2010). In case of rye bread-making process, the use of the sourdough is essential due to the multiple advantages, such as: leavening of dough, improving dough properties by inhibition of α-amylase, increasing bread flavor and taste, improving nutritional value of the final product, extending the shelf life of sourdough bread by longer mold-free period, preventing the rope in bread and antistaling (Hansen, 2006). The sourdough fermentation can be used to modify levels of bioactive compounds (folat, tocopherol and tocotrienol, sterols, alk(en)yresorcinols, lignans, phenolic acids, thiamin), the increase or decrease levels of some compounds depending on the nature of the compound and the type of the sourdough obtaining process (Liukkonen et al., 2003; Katina

2 572 et al., 2005; Katina et al., 2007). Katina et al. (2007) investigated the synergistic effects on bioactive compounds when the sourdough and germination were combined. They showed that the levels of folates, free phenolic acids, total phenolic compounds, lignans and alkylresorcinols were increased through fermentation and rye germination when compared to rye. The increase of folates and methanol-soluble phenolic compounds contents was also reported by Liukkonen et al. (2003) in case of studying rye germination. Moreover, Dordevic et al. (2010) studied the influence of fermentation by Lactobacillus rhamnosus and Saccharomyces cerevisiae on antioxidant activities and total phenolic compounds of rye, buckwheat, barley and wheat germ. Coda et al. (2010) used Lactobacillus plantarum and Lactococcus lactis subsp. lactis for sourdough fermentation of cereals (rye, wheat, oat, rice and millet), pseudo-cereals (buckwheat, amaranth and quinoa) and leguminous (chickpea and soy). They reported that the type of fermentation clearly had effect on potentially bioactive constituents of cereals. Even if the antioxidant activity of the cereals was intensively studied, the information about the influence of the fermentation with lactic acid bacteria on the content of bioactive compounds is still scarce. The objective of the present study was to assay the evolution of the antioxidant activity during the whole process of making the sourdough rye bread. The rye sourdough, dough, proofed dough and bread were characterized in terms of antioxidant activity and total phenolic content. Since the use of more than one method is recommended to give a comprehensive prediction, the antioxidant activity of 80% methanol dough and bread extracts was evaluated as free radical scavenging activities against ABTS + radical cations (Trolox equivalent antioxidant capacity) and against 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH RSA) expressed in terms of Trolox equivalents and in terms of percentage inhibition of free radical formation. The phenolic content was determined using the Folin Ciocalteu method. Materials and Methods Materials Commercial whole rye flour (falling number of 250 s) retailed on the local market (Galati, Romania) was used in this study. The inoculum used for preparing the rye sourdough consisted of four lactic acid bacteria (LAB) strains from MIUG collection, Faculty of Food Science and Engineering (Galati, Romania): Lactococcus lactis ssp. Lactis (UGAL2), Weissella confusa (UGAL1), Lactobacillus plantarum (15GAL), Lactobacillus brevis (16GAL), and the commercial strains Lactobacillus plantarum and Lactobacillus brevis (DI-PROX MTTX), Lactobacillus helveticus (LH-B02) and K. marxia- I. Banu et al. nus subsp. marxianus (LAF-4). The leavener used for dough preparation was the compressed yeast Saccharomyces cerevisiae. Sourdough fermentation Sourdough was prepared by well mixing in a large beaker the whole rye flour with tap water and inoculum to get a dough yield (DY, mass of dough/ mass of flour 100) of 300. After covering with aluminum foil, the beakers were placed in an incubator at 40 for 16 h, in case of the samples prepared with Lactobacillus helveticus and K. marxianus subsp. marxianus, and at 30 for 20 h, in all other cases. The control dough prepared without inoculum, was incubated at 30 for 20 h. The inoculum size was ufc/100 g dough in case of LAB from MIUG collection, while for the commercial strains, the producer (EDR Ingredients Romania and Chr Hansen) recommendations were followed. Bread-making Dough formulations are detailed in Table 1. The dough was prepared at 28 by mixing all ingredients in a laboratory mixing device. After fermentation at 28 for 30 min in a laboratory proofer, the dough was divided in two pieces which were molded and placed in baking trays. The proofed dough was obtained after a final leavening of 45 min; the trays were afterwards introduced into the oven. The samples were baked at 260 for 40 min (the steam tap was turned on s before placing the samples into the oven). ph and acidity ph measurements were made according to Romanian standard methods 90/2007 by means of a Hanna digital ph-meter. The total titratable acidity (TTA) value was determined as the amount of 0.1 N NaOH solution (ml) used for neutralization of 10 g sample (Romanian standard methods 90/2007). Extraction and sample preparation Samples of the whole rye flour, sourdough and dough after sourdough incorporation, proofed dough and bread were collected and analyzed. Dough and bread samples were dried, ground, and sieved through a 60-mesh screen. For determination of the total phenol contents, the samples were extracted with acidified methanol (HCl:methanol:water, 1:80:10, v:v:v) at room temperature for 2 h on a magnetic stirrer. The mixture was centrifuged at 3,000 rpm for 10 min (Beta et al., 2005) and Table 1. Formulations used to prepare the dough I (with 20% sourdough) and dough II (with 40% sourdough). Dough I Dough II Rye flour, g Sourdough, g Added water, ml Salt, g Yeast, g Total water, ml

3 Effect of Lactic Fermentation on Antioxidant Capacity of Rye Sourdough and Bread 573 the supernatant was used for determination of total phenolics. In case of antioxidant activity the extraction was made using a 80% aqueous methanol solution, for 2 h at 37. Samples were afterwards centrifuged at 10,000 rpm for 15 min (Villauenga et al., 2009). The supernatant was used for determination of antioxidant capacity. Total phenolic content The total phenolic content (TFC) was determined using the Folin-Ciocalteu method (Beta et al., 2005). The extract (0.2 ml) was added to 1.5 ml of freshly diluted 10-fold Folin Ciocalteu reagent. The mixture was allowed to equilibrate for 5 min and was then mixed with 1.5 ml of sodium carbonate solution (60 g/l). After incubation at room temperature for 90 min, the absorbance of the mixture was measured at 725 nm. Acidified methanol was used as the blank. Ferulic acid was used as the standard. Trolox equivalent antioxidant capacity (TEAC) assay TEAC was determined following the procedure of Re et al. (1999) and modified by Villauenga et al. (2009). The extract (20 μ/l) was added to 1.48 ml ABTS + (with an absorbance of ± at 734 nm). The absorbance of the mixture was measured immediately and after 6 min at 734 nm. All measurements were made at 30 Appropriate solvent blanks were run in each assay. The TEAC of extracts was expressed as percentage of inhibition of absorbance at 734 nm using Trolox standard curve. DPPH radical scavenging activity (DPPH RSA) assay expressed in terms of Trolox equivalents DPPH RSA was determined using the method described by Villauenga et al. (2009). The extract (0.1 ml) was added to 0.25 ml of the methanolic DPPH solution (10 mg DPPH in 25 ml 80% methanol) and 2 ml 80% methanol. The mixture was shaken vigorously and allowed to stand at room temperature in the dark for 20 min. The decrease in absorbance of the resulting solution was monitored at 515 nm after 20 min. The absorbance of a mixture made up of 0.25 ml DPPH solution and 2.1 ml 80% methanol was used as blank. The Trolox standard solution (concentrations ranging from 0.1 to 2.0 mm) was prepared in 80% methanol and assayed in the same conditions. DPPH scavenging activity was expressed in terms of Trolox equivalents, on the basis of percentage inhibition of absorbance at 515 nm of standards and samples. DPPH radical scavenging activity assay expressed in terms of percentage of discoloration Antioxidant activity was measured using the method proposed by Hung et al. (2009). The extract (0.1 ml) was mixed with 3.9 ml DPPH solution ( mol/l), kept in the dark at room temperature and the absorbance was recorded at 515 nm after exactly 30 min (A sample/t=30 ). Blank was prepared using 3.9 ml DPPH and 0.1 ml methanol and its absorbance was measured at t = 0 (Acontrol/t=0). Antioxidant activity was calculated according to the following formula: %DPPH scavenging = (1 A sample/t=30 /Ac ontrol/t=0 ) 100 (1) Statistical analysis was carried out using the software Statistica 6.0 for Windows. All experiments were carried out in triplicate and the average values are reported together with standard deviations. Analysis of variance (ANOVA) was used to evaluate the significant difference within each individual experiment performed with different starter cultures. Results and Discussion ph and TTA The intensity of lactic acid and alcoholic fermentation occurring in the sourdough fermentation highly depends on the rye spontaneous microflora and on the starter culture composition. The ph of sourdough obtained through spontaneous fermentation was much higher (ph 5.44), compared to the fermentation with starter culture when the ph varied between 3.84 and 4.11 (Table 2). Accordingly, the TTA values of sourdoughs prepared by adding inoculum were significantly higher compared to the control sample, but lower to the ones reported by Katina et al. (2007) for the rye sourdough fermented with Lb. brevis, Lb. plantarum, and combined starter culture of S. cerevisiae, Lb. plantarum and Lb. brevis (TTA values ranged between 15.6 and 17.5). The most important compounds responsible for ph decrease are lactic and acetic acids. As is produced through homo- and heterolactic fermentation by LAB, the lactic acid content was always reported to be higher compared to the acetic acid (Vuyst and Neysens, 2005). The typical content of lactic acid is mg/100 g sourdough and for acetic acid Table 2. ph and TTA values of sourdough. Fermentation type ph TTA Spontaneous fermentation (Control sample) LH-B02 (Lb. helveticus) + LAF-4 (K. marxianus subsp. marxianus) DI-PROX MTTX (Lb. plantarum + Lb. brevis) GAL (Lb. plantarum) + 16GAL (Lb. brevis) UGAL1 (W. confusa) UGAL2 (Lc. lactis ssp. Lactis)

4 574 mg/100 g sourdough (Hansen, 2006). Katina et al. (2005) showed that the final acidity of the sourdough is influenced by the characteristics of the rye, so that the whole rye flour with low falling number are better fermented and allow getting more acidic sourdough. Total phenolic content The total phenol compound content of the whole rye flour was mg ferulic acid/100 g d.m. The experiments run by Dordevic et al. (2010) on rye indicated a TFC of 13.2 mg gallic acid/g d.m. extract when the extraction was made with 70% (v/v) ethanol. The difference between the two results occurs mainly because of the extraction conditions. Generally, it is difficult to compare data in terms of total phenolic content with the literature because of the different method employed for extraction. The extraction solvent (acetone, ethanol or methanol) and extraction duration affect the TFC. As shown in Table 3, the highest concentration of TFC (590.8 mg ferulic acid/100 g d.m.) was found for the sourdough started with DI-PROX MTTX which was 2.2 fold higher than the control sample, while the lowest concentration found for the sourdough started with inoculum was mg ferulic acid/100 g d.m. in case of UGAL2. As shown previously by Rodriguez et al. (2009), the levels of bioactive compounds can be modified during fermentation by the metabolic activity of LAB. Katina et al. (2007) suggested that the fermentation induced structural breakdown of cereal cell walls, leading to the liberation and/or synthesis of various bioactive compounds. During fermentation, enzymes I. Banu et al. derived from rye (amylases, xylanases and proteases) and LAB contributes to the modification of grain composition, and bound phenolics may released by enzymatic treatment of samples prior to extraction. Moreover, Dordevic et al. (2010) indicate that the type of fermentation influence the levels of most bioactive compounds. The TFC of the dough decreases to and mg ferulic acid/100 g d.m. after adding 20 and 40% sourdough prepared with DI-PROX MTTX. Afterwards a slight increase of TFC was registered for the proofed dough, due to the metabolic activity of LAB and yeast. Analyzing the results in Table 3, one can see that the TFC values of the proofed dough highly depend on the type of inoculum. Finally, the bread with 20 and 40% sourdough prepared with DI-PROX MTTX had and mg ferulic acid/100 g d.m. Concerning the bread obtained with 20 and 40% sourdough UGAL2, the TFC was and mg ferulic acid/100 g d.m., respectively. ABTS scavenging activity (TEAC value) TEAC value of the whole rye flour was 3.7 μmol Trolox/g d.m, increased to 5.6 μmol Trolox/g d.m after 20 h of spontaneous fermentation (dough control sample), and decreased to 4.2 μmol Trolox/g d.m. after proofing (Table 4). Higher TEAC values are reported by Villauenga et al. (2009) for rye dough made from dark flour (extraction rate of 100%) and brown flour (extraction rate 92%), after 48 h of fermentation and 3 h of proofing, 8.81 μmol Trolox/g d.m. and 7.39 μmol Trolox/g d.m., respectively. TEAC values of rye bread depended on Table 3. Total phenol content of the sourdough, dough, proofed dough (dough fermented after molding) and bread samples. Sourdough ± ± ± ± ± ± 4.1 Dough I ± ± ± ± ± ± 2.9 Dough II ± ,2 ± ± ± ± ± 2.4 Proofed dough I ± ± ± ± ± ± 3.2 Proofed dough II ± ± ± ± ± ± 4.3 Bread I ± ± ± ± ± ± 2.1 Bread II ± ± ± ± ± ± 3.1 Table 4. TEAC values of the sourdough, dough, proofed dough (dough fermented after molding) and bread samples. Sourdough 5.6 ± ± ± ± ± ± 0.5 Dough I 4.3 ± ± ± ± ± ± 0.2 Dough II 5.0 ± ± ± ± ± ± 0.3 Proofed dough I 4.2 ± ± ± ± ± ± 0.3 Proofed dough II 6.1 ± ± ± ± ± ± 0.4 Bread I 3.3 ± ± ± ± ± ± 0.2 Bread II 5.5 ± ± ± ± ± ± 0.4

5 Effect of Lactic Fermentation on Antioxidant Capacity of Rye Sourdough and Bread 575 flour extraction rate (Villauenga et al., 2009) since the milling process caused a reduction in the amount of antioxidants and bioactive compounds in flours by the removal of outer layer of the kernel. TEAC values of the sourdough obtained with starter cultures were higher compared to the control sample, i.e. the antioxidant activity of the sourdoughs fermented with 15GAL + 16GAL and LH-B02 + LAF-4 were about 2 and 1.3 fold higher. When comparing the wheat flour bread, non-conventional flour bread, and non-conventional flour sourdough bread, Coda et al. (2010) obtained the following TEAC values: 4.02 μmol Trolox/g, 6.74 μmol Trolox/g, and 8.49 μmol Trolox/g, respectively. In case of all analyzed bread making stages, the highest TEAC values were obtained for the samples with 15GAL + 16GAL and DI-PROX MTTX. In particular for the sourdough prepared with 15GAL + 16GAL and DI-PROX MTTX, the TEAC values were 11.5 μmol Trolox/g d.m., and 10.1 μmol Trolox/g d.m., respectively (Table 4). DPPH radical scavenging activity (DPPH RSA) assay expressed in terms of Trolox equivalents The DPPH RSA of dough obtained through spontaneous fermentation (6.1 μmol Trolox/g d.m.) was almost 2-fold higher than the whole rye flour (3.2 μmol Trolox/g d.m.). Villauenga et al. (2009) reported for the rye dough made from dark flour, after 48 h of fermentation and after 3 h of proofing, about 3 μmol Trolox/g d.m. and 4 μmol Trolox/g d.m., respectively. The DPPH RSA values of the sourdoughs obtained with starter cultures were, in all cases, higher than the dough obtained through spontaneous fermentation (control sample). The highest and the lowest DPPH RSA values were obtained for the samples fermented with 15GAL + 16GAL (14.3 μmol Trolox/g d.m.), and with UGAL2 (7.8 μmol Trolox/g d.m.). In case of all studied samples, the DPPH RSA values of the rye breads I and II were slightly decreased compared to the proofed doughs I and II, respectively (Table 5). These results suggest that the DPPH RSA is influenced by the thermal processing. Antioxidant activity measured by DPPH showed the same trends as did ABTS + method, but TEAC values were slightly lower. DPPH radical scavenging activity assay expressed in terms of percentage of discoloration Our results showed significant DPPH radical inhibition ability of the whole rye flour; the antioxidant activity expressed in term of percentage of discoloration was 8.4 %. A discoloration of the mixture occurs when DPPH is reduced to non-radical DPPH in the presence of an antioxidant or a hydrogen donor. The TFC of the rye explains the DPPH radical inhibition (Table 2). The fermentation increased the DPPH radical scavenging activity, probably due to the increased levels of easily extractable phenolic compounds (Liukkonen et al., 2003; Katina et al., 2005). Higher DPPH radical scavenging activity was obtained in case of sourdough fermented with DI- PROX MTTX (19.1%) and 15GAL + 16GAL (16.8%) (Table 6), in direct correlation with the TFC (Table 3). Dordevic et Table 5. DPPH RSA values of the sourdough, dough, proofed dough (dough fermented after molding) and bread samples. Sourdough 6.1 ± ± ± ± ± ± 0.4 Dough I 3.7 ± ± ± ± ± ± 0.2 Dough II 4.1 ± ± ± ± ± ± 0.1 Proofed dough I 3.5 ± ± ± ± ± ± 0.2 Proofed dough II 5.2 ± ± ± ± ± ± 0.2 Bread I 3.1 ± ± ± ± ± ± 0.2 Bread II 4.9 ± ± ± ± ± ± 0.2 Table 6. DPPH RSA values (% inhibition) of the sourdough, dough, proofed dough (dough fermented after molding) and bread samples. Sourdough 13.3 ± ± ± ± ± ± 0.7 Dough I 9.0 ± ± ± ± ± ± 0.2 Dough II 9.3 ± ± ± ± ± ± 0.7 Proofed dough I 8.8 ± ± ± ± ± ± 0.4 Proofed dough II 9.1 ± ± ± ± ± ± 0.6 Bread I 7.5 ± ± ± ± ± ± 0.5 Bread II 8.1 ± ± ± ± ± ± 0.5

6 576 al. (2010) reported DPPH radical scavenging activities of 10.6% for rye and of 15.1% for the samples fermented with Lb. rhamnosus. Conclusions The phenolic content and antioxidant capacity of the rye can be modulated during the bread making process. The sourdough fermentation increased the antioxidant capacity, probably due the increased levels of easily extractable phenolic compounds. The type of fermentation and the metabolic activity of LAB influence the levels of bioactive compounds that enable the increase of the phenolic content and antioxidant capacity of the sourdough, dough and rye bread. Acknowledgements This work was supported by CNCSIS UE- FISCSU, project number PNII IDEI ID_500/2008. References Andersson, R., Fransson, G., Tietjen, M. and Aman, P. (2009). Content and molecular-weight distribution of dietary fiber components in whole-grain rye flour and bread. J. Agric. Food Chem., 57, Asro. (2008). Metode de analiza a cerealelor si produselor de macinis. SR 90:2007, Bucuresti. Beta, T., Nam, S., Dexter, J. and Sapirstein, H. (2005). Phenolic content and antioxidant activity of pearled wheat and roller milled fractions. Cereal Chem., 82, Coda, R., Rizzello and G., Gobbetti, M. (2010). Use of sourdough fermentation and pseuso-cereals and leguminous flours for the making of a functional bread enriched of γ-aminobutyric acid. Int. J. Food Microbiol., 137, Dewettinck, K., van Bockstaele, F., Kuhne, B., van de Walle, D., Courtens, T.M. and Gellynck, X. (2008). Nutritional value of bread: influence of processing, Food Interaction and Consumer Perception. J. Cereal Sci., 48, Dordevic, T.M., Marincovic, S.S. and Brankovic, S.D. (2010). Effect of fermentation on antioxidant properties of some cereals and pseudo cereals. Food Chem., 119, Hansen, A. (2006). Sourdough bread, Taylor and Francis Group, LLC, pp. 183(1-21). Hansen, H.B., Andreasen, M.F., Nielsen, M.M., Larsen, L.M., Knudsen, K.E.B., Meyer, A.S., Christensen, L.P. and Hansen, A. (2002). Changes in dietary fibre, phenolic cids and activity of endogenous enzymes during rye bread-making. Eur. Food Res. Technol., 214, I. Banu et al. Heinio, R.L., Liukkonen, K.H., Myllymaki, O., Pihlava, J.M., Adlercreutz, H., Neinonen, S.M. and Poutanen, K. (2007). Quantities of phenolic compounds and their impacts on the perceived flavour attributes of rye grain. J. Cereal Sci., 47, Hung, P.V., Maeda, T., Miyatake, K. and Morita, N. (2009). Total phenolic compounds and antioxidant capacity of wheat graded flours by polishing method. Food Res. Int., 42, Iyer, R. and Tomar, S.K. (2009). Folate: a functional food constituent. J. Food Sci., 74, Katina, K., Arendt, E., Liukkonen, K.H., Autio, K., Flander, L. and Poutanen, K. (2005). Potential of sourdough for healthier cereal products. Trends Food Sci. Technol., 16, Katina, K., Liukkonen, K.H., Norja, A.K., Adlercreutz, H., Heinonen, S.M., Lampi, A.M., Pihlava, J.M. and Poutanen, K. (2007). Fermentation-induced changes in the nutritional value of native or germinated rye. J. Cereal Sci., 46, Liukkonen, K.H., Katina, K., Wilhelmsson, A., Myllymaki, O., Lampi, A.M., Kariluoto, S., Piironen, V., Heinonen, S.M., Nurmi, T., Adlercreutz, H., Peltoketo, A., Pihlava, J.M., Heitaniemi, V. and Poutanen, K. (2003). Process-induced changes on bioactive compounds in whole grain rye. Proc. Nutr. Soc., 62, Pathirana, C.L. and Shahidi, F. (2007). The antioxidant potential of milling fractions from breadwheat and durum. J. Cereal Sci., 45, Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. and Evans, C.R. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med., 26(9/10), Rodriguez, H., Curiel, J.A., Landete, J.M., Rivas, B., Felipe, F.L., Cordoves, C.G., Mancheno, J.M. and Munoz, R. (2009). Food phenolic and lactic acid bacteria. Int. J. Food Microbiol., 132, Sands, D., Morris, C., Dratz, E., Pilgeram, A. (2009). Elevating optional human nutrition to a central of plant breeding and production of plant-based foods. Plant Science, 177, Serpen, A., Gokmen, V., Pellegrini, N. and Fogliano, V. (2008). Direct measurement of the total antioxidant capacity of cereal products. J. Cereal Sci., 48, Villauenga, C.M., Michalska, A., Frias, J., Piskula, M.K., Vidal- Valverde, C. and Zielinski, H. (2009). Effect of flour extraction rate and baking on thiamine and riboflavin content and antioxidant capacity of traditional rye bread. J. Food Sci., 74, Vuyst, L., Neysens, P The sourdough microflora: biodiversity and metabolic interactions, Trends Food Sci. Technol., 16,