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Rutin and quercetin transformation during preparation of buckwheat sourdough bread
Journal of Cereal Science 69 (2016) 71e76
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Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Rutin and quercetin transformation during preparation of buckwheat sourdough bread Lea Luksi c a, Giovanni Bonafaccia b, Maria Timoracka c, Alena Vollmannova c, Janja Tr cek d, Tina Ko zelj Nyambe d, Valentina Melini b, Rita Acquistucci b, Mateja Germ a, Ivan Kreft e, * a
Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia Food and Nutrition Research Centre (CRA-NUT), Via Ardeatina 546, I-00178 Roma, Italy Department of Chemistry, Slovak Agricultural University of Nitra, Tr. A. Hlinku 2, SK-94901 Nitra, Slovak Republic d Department of Biology, Faculty of Natural Sciences and Mathematics, University of Maribor, Koroska cesta 160, SI-2000 Maribor, Slovenia e Slovenian Forestry Institute, Vecna pot 2, SI-1000 Ljubljana, Slovenia b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 October 2015 Received in revised form 20 February 2016 Accepted 23 February 2016 Available online 27 February 2016
Sourdough bread was prepared from flour of the common buckwheat (Fagopyrum esculentum) and of Tartary buckwheat (Fagopyrum tataricum) to follow the transformation of rutin and quercetin in these sourdoughs. During Tartary buckwheat sourdough fermentation, there was conversion of rutin to quercetin. The Tartary buckwheat flour contained 14.6 mg/g rutin and 1.9 mg/g quercetin as dry matter. The sourdough starter contained 1.5 mg/g rutin and an unexpectedly high 12.5 mg/g quercetin. The sourdough contained 3.2 mg/g rutin and 8.1 mg/g quercetin. In the Tartary buckwheat sourdough bread there was no rutin, whereas there was 5.0 mg/g quercetin. Thus, during the sourdough fermentation, the rutin was completely degraded. However, despite the long fermentation time (sourdough, 10 h; bread dough, 5 h), most of the quercetin remained in the dough and appeared in the baked bread. In contrast to Tartary buckwheat bread, neither rutin nor quercetin was present in common buckwheat bread. Information on the persistence of quercetin in sourdough bread is important for designing breads with high concentrations of flavonoids and good functional value. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Tartary buckwheat (Fagopyrum tataricum) Common buckwheat (Fagopyrum esculentum) Rutin Quercetin
1. Introduction Common buckwheat (CB) (Fagopyrum esculentum Moench) and Tartary buckwheat (TB) (Fagopyrum tataricum (L.) Gaertn.) are used in different parts of the world to make various food products. The grain of both of these cultivated buckwheat species contains up to 27% fibre (Bonafaccia et al., 2003). Buckwheat groats are a prebiotic food because they can, for example, increase the lactic acid bacteria in the intestine because of their content of resistant starch (Skrabanja et al., 1998, 2001). Buckwheat is also an important source of anti-oxidant activity in functional foods (Holasova et al.,
Abbreviations: a*, range of colour between red and green; ANOVA, analysis of variance; b*, range of colour between yellow and blue; CB, common buckwheat; DM, dry mass; DPPH, 2,2-diphenyl-1-picrylhydrazyl; HPLC, high performance liquid chromatography; L*, brightness of colour; ND, not detected; ORACFL, oxygen radical absorbing capacity; PCL, photochemiluminescence; PCR, polymerase chain reaction; TB, Tartary buckwheat. * Corresponding author. E-mail address: [email protected] (I. Kreft). http://dx.doi.org/10.1016/j.jcs.2016.02.011 0733-5210/© 2016 Elsevier Ltd. All rights reserved.
2002). This arises from the presence of the flavonoids rutin and quercetin in the buckwheat grain and products, because of their anti-oxidant and anti-inflammatory effects. It has been reported that TB contains about 100-fold more rutin than does CB (Fabjan et al., 2003). Rutin and quercetin are also present in baked biscuits made from CB and TB (Wieslander et al., 2011). Buckwheat products decrease cholesterol levels and improve lung capacity in humans (Wieslander et al., 2011; Sikder et al., 2014; Yang et al., 2014). Extracts from CB and TB can also protect DNA from damage caused by hydroxyl radicals (Cao et al., 2008; Vogrin ci c et al., 2010). One of the ways to prepare bread involves sourdough fermentation, which can be accompanied by the formation of flavour compounds such as lactic acid and acetic acid that have an impact on dough processing and the preparation of sourdough bread (Michalska et al., 2008). However, the combined effects of sourdough fermentation and the baking process on the flavonoid concentrations and anti-oxidant properties of CB and TB sourdough starter, bread dough and sourdough bread have not yet been
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studied. Therefore, the objective of the present study was to investigate the effects of sourdough fermentation and the baking process on rutin and quercetin content and on the anti-oxidant activities of CB and TB. More precisely, the aim was to determine whether sourdough fermentation and thermal processing affect the conversion of rutin to quercetin. We were also interested in determining how much rutin and quercetin remain in CB and TB products after sourdough fermentation and the baking process. These findings will contribute to a better understanding of the value of buckwheat products for human consumption. 2. Materials and methods
of each type of bread (CB or TB) were included in the study, made independently from the beginning (including making the sourdough out of four different starter samples). All four were used for the measurement of anti-oxidant activity, and three randomly selected loaves were used for other measurements. 2.4. Measurement of loaf volumes For the determination of loaf volumes, the rapeseed displacement method was used according to AACC method number 1005.01 (AACC International, 2008). Specific loaf volumes were obtained by dividing the volume by the loaf weight (expressed as g/ cm3).
2.1. Materials and sample preparation 2.5. Colour measurements The CB and TB flours were prepared in a Rangus mill (Vrhpolje near Sentjernej, Slovenia) using the CB grain cv. ‘Pyra’ and TB grain from a domestic strain that originated in Luxembourg (Bonafaccia et al., 2003). The CB and TB were harvested in 2012. The initial sourdough starter was obtained from Sweden (Bageriet Andreas € Ostlund, Uppsala, Sweden), and was stored in a glass jar in a refrigerator (5 C). Before use in this study, this sourdough starter was refreshed twice weekly for 6 months by addition of an equal volume of TB flour suspension (1:1; v/v; TB flour: preboiled and cooled tap-water). This was done to adapt the starter to the flavonoid-rich material. The sourdough starter was kept in a refrigerator (5e8 C). 2.2. Determination of sourdough starter microorganisms To identify the bacteria present, diluted sourdough starter was plated onto deMan, Rogosa and Sharpe (MRS) agar medium (SigmaeAldrich, St. Louis, Missouri, US) and incubated for 3 days at 30 C under anaerobic conditions. Each type of morphologically different colony was further purified to isolate the pure strains. The isolates were grown in liquid MRS medium, and the biomass was harvested and used for DNA isolation using NucleoSpin Tissue kits (MachereyeNagel, Düren, Germany). The 16S rRNA bacterial genes were specifically amplified by polymerase chain reaction (PCR) using the primers 16S_27f (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 16S_rH1542 (50 -AAGGAGGTGATCCAGCCGCA-30 ). The PCR products were cleaned up using NucleoSpin gel and PCR Clean-up kits (MachereyeNagel, Düren, Germany) and their partial sequences were determined by Eurofins Genomics. The sequences were analysed using the Blastn programme available from the National Center for Biotechnology Information (NCBI) database. The isolates were assigned to the species to which the 16S rRNA gene sequences showed the highest identity. In this way, two morphologically different species were isolated and identified as Lactobacillus heilongjiangensis and Pediococcus parvulus. The sequences were deposited in the EMBL/GenBank/DDBJ database under accession numbers LN650641 (L. heilongjiangensis) and LN650642 (P. parvulus). Fresh baker's yeast was purchased from Bonopan (Logatec, Slovenia). 2.3. Sourdough bread production The sourdough bread was prepared in two steps. On the first day, a mixture of 50 g sourdough starter, 90 g flour and 150 g tapwater at room temperature (25 C) was prepared. The mixture was stored in a refrigerator (5 C) and allowed to ferment for 10 h. On the second day, 25 g tap-water, 8 g sucrose, 5 g salt, 4 g fresh baker's yeast and 150 g flour were added to the mixture. The dough was placed in baking forms on baking paper and left to rise for 5 h. The bread was then baked in an oven at 200 C for 1 h. Four bread loaves
Colour measurements were performed on the bread crumb and crust. The lightness (L*) and red (þa*) and yellow (þb*) colorimetric indices were established according to the CIELAB colour system (CIE, 1986) using a Chroma Meter CR 300 Minolta (Konica Minolta, Inc., Tokyo, Japan) equipped with a pulsed xenon lamp and illuminant D65. A white plate (X ¼ 91.98, Y ¼ 93.97, Z ¼ 110.41) was used to standardize the instrument. Three independently prepared loaves of each type of bread were measured. 2.6. Preparation of methanol extracts The concentrations of rutin and quercetin and the anti-oxidant activity were determined for the CB flour, TB flour, sourdough starter, sourdough before and after the 10-h incubation in a refrigerator (5 C), bread dough before and after the 5-h rising at room temperature, and in the two different types of sourdough bread (i.e., CB, TB). Bread samples were prepared at room temperature (25 C). After preparing the dough and baking the bread, the loaves (four each of the two types of bread) were cooled and cut into pieces, which were stored for analysis in a freezer at 20 C. All of the samples prepared were freeze-dried and milled for analysis. To prepare the methanol extracts, 50 mL 80% aqueous methanol (HPLC grade, SigmaeAldrich Corporation, St. Louis, MO, USA) was added to 1 g of each milled sample. The mixtures were shaken at room temperature for 8 h (250 rpm). The samples were then filtered through filter paper (130 g/m2; Filtrak, Thermalbad Wiesenbad, Germany) and kept at 5 C for further analysis. The methanol extracts of each type of bread were made using three independently prepared loaves. 2.7. Anti-oxidant activity assays The anti-oxidant activities were determined on the basis of photochemiluminescence (PCL) and fluorescence (ORACFL) assays. The PCL assay was used to measure the anti-oxidant activity of extracts against superoxide anion radicals generated from luminol (a photosensitizer) when exposed to UV light, assessed with a Photochem instrument (Analytik Jena, USA Inc., Delaware, OH, USA). The anti-oxidant activities of the sample extracts were measured using integral anti-oxidant capacity of water-soluble substances kits from Analytik Jena (USA Inc., Delaware, OH, USA). Chemiluminescence evolution was monitored using the PCL soft control and analysis software. The lag time (expressed in seconds) was used as a measure of radical-scavenging activity. The antioxidant capacities were estimated by comparison with a Trolox standard (0.5e3 nmol of Trolox) and are expressed as mg/mg Trolox equivalents. The anti-oxidant index was obtained by dividing the anti-oxidant capacity by the lag time and multiplying by 1000 (anti-oxidant activity/lag time 1000).
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2.8. Determination of rutin and quercetin concentrations The filtrates were injected into a high performance liquid chromatography (HPLC) system that consisted of an HPLC chromatograph (Alliance 2695, Waters, Milford, USA), a LiChroCART Purospher RP C18 column (5 mm, 250 4.6 mm; Merck, Darmstadt, Germany) and a DAD 2996 UV detector (Waters, Milford, USA). The column temperature was 30 C. The gradient elution was carried out at a flow rate of 1 mL/min. Solvent A was acetonitrile, and solvent B was 0.1% phosphoric acid. The gradient was as follows (% A): 0e3 min, 40%; 3e8 min, 40%e5%; 8e10 min, 5%. Rutin and quercetin were detected at 365 nm and their concentrations were calculated on the basis of the calibration curves made using rutin and quercetin standards (Acros Organics, Waltham, MA, USA) prepared in methanol (gradient elution grade; SigmaeAldrich Corporation, St. Louis, USA). The limit of detection (LOD) for rutin was 1.12 mg/mL and for quercetin 1.01 mg/mL. The limit of quantification (LOQ) for rutin was 3.69 mg/mL and for quercetin 3.33 mg/ mL. The results are expressed as mg/g dry weight. Rutin can also be expressed as quercetin equivalents. For this purpose, the rutin concentration was divided by a factor of 2.02. 2.9. Statistical analysis The data are expressed as means ± standard deviation of the results for independently prepared bread loaves. ANOVA was used for the statistical analysis, and the data were considered to be significantly different when P < 0.05. 3. Results and discussion 3.1. Bread loaf volumes The specific volume is the ratio of the volume to the weight, and this has been adopted as a reliable measure of bread quality (Houben et al., 2012). The data here showed that the specific volume of the loaves from the CB sourdough bread (0.90 ± 0.04 g/cm3) was lower than that from the TB sourdough bread (1.10 ± 0.02 g/ cm3). The absence of gluten in the buckwheat flours had a strong impact on the bread's rheological properties. Because of the low carbon dioxide binding activity during rising, the volumes of gluten-free breads are generally lower in comparison with bread containing gluten-containing cereals (Houben et al., 2012). In this respect, the properties of the CB bread are also different from those of the TB bread. This is not completely unexpected, because CB and TB are different botanical species. The chemical compositions of the CB and TB flours were not very different apart from the large difference in rutin concentrations in the grain (Bonafaccia et al., 2003; Fabjan et al., 2003). The molecular basis for the differences in the loaf volumes has not yet been examined. Investigation of the possible impact of the flavonoids on the loaf volume was outside the scope of this study. 3.2. Colour measurements The colour measurements for the sourdough bread preparation are shown in Table 1. Comparisons of the colour values (L*, a*, b*) of the CB and TB crust and crumbs of the sourdough bread were performed. There were no significant differences in the L* values of CB and TB crumb or crust. The L* gives a measure of the brightness of the sample, and this parameter ranges from 0 (black) to 100 (white). Comparing the colour of the bread crumb, there were significant differences in the a* and b* values between the CB and TB bread crumb. Comparing the colours of the bread crust, there was a significant difference in the a* value but not the b* value
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between the CB and TB bread crust (Table 1). The lower a* value for the crust and crumb of the TB sourdough bread indicated that it had a stronger green tinge compared with the CB bread. The higher b* value for the crumb of the TB sourdough bread indicated that its colour was more yellowish than that of the CB sourdough bread. To our knowledge, the colour of TB bread in comparison to CB bread or other bread products has not been measured previously. Our measurements confirm that TB bread is more greenish-yellow than CB bread, confirming visual observations of a green-yellow tinge to TB food products. This reported colour appears to be connected with the high concentrations of rutin and quercetin in the TB products (Vombergar et al., 2014). 3.3. Effect of the bread-making process on anti-oxidant activity Comparisons of the anti-oxidant activities in these samples during the sourdough bread-making process were performed for each buckwheat type (Tables 2 and 3). During the CB bread-making process, there were significant differences between the antioxidant activity (expressed as mg/mg of trolox equivalents) of the sourdough starter, bread dough and bread. There were also differences in the anti-oxidant activity of the sourdough starter, bread dough and bread in the TB sourdough bread-making process. Comparing the CB and TB samples, there were significant differences in anti-oxidant activity between the two types of flour, in the sourdough starter before fermentation, in the bread dough before rising and in the bread dough after rising (Tables 2 and 3). Among the CB samples, the highest anti-oxidant activity was observed in the bread dough and bread and the lowest in the CB flour. Here, the anti-oxidant activity increased from flour to sourdough starter, and further to bread dough and sourdough bread. This might be because of the synthesis of substances with antioxidant properties, including certain Maillard reaction products that occur in bread during thermal treatment (Zhang et al., 2010). In contrast, for the TB samples, the highest anti-oxidant activity was measured in the flour and sourdough, and the lowest in the sourdough bread (Tables 2 and 3). Flavonoids in TB could interfere with the Maillard or other reactions in the bread, but identification of the mechanism behind these differences requires further investigation. A similar drop in anti-oxidant activity was established for yeasted TB bread (Vogrin ci c et al., 2010). However, Vogrin ci c et al. (2010) reported different values for yeasted wheat bread, where the DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging capacity rose from 0% in the dough after mixing to 0.54% in the dough just before baking, and to 2.88% in the bread loaf crumb. A significant decrease in anti-oxidant activity in TB flour as a result of various thermal treatments such as roasting, steam-pressure heating, and microwaving, has been reported (Zhang et al., 2010). A small decrease in the anti-oxidant activity in CB flour roasted for 10 min at 200 C has also been noted (Yasuda and Nakagawa, 1994). The CB and TB samples differed in their anti-oxidant properties; however, the molecular basis for this remains unknown. The changes in anti-oxidant activity in CB were similar to those reported for wheat (Vogrin ci c et al., 2010), although in the case of the CB used in this study, they were at a much higher level. 3.4. Impact of sourdough bread production procedure on rutin and quercetin concentrations Comparisons of the samples of each buckwheat species during the sourdough bread-making process (Table 4) were performed. In CB flour, the rutin and quercetin concentrations were below the detection limit of the method applied, although quercetin appeared in the CB sourdough at the beginning of the fermentation from the
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Table 1 Crumb and crust colour values of the CB and TB sourdough bread. Buckwheat flour
Bread sample
Colour values L*
Crust
Common Tartary Common Tartary
Crumb
a*
43.31 41.15 48.01 46.54
± ± ± ±
2.23 1.64 0.52 0.77
A A A A
12.04 9.50 4.12 1.09
b* ± ± ± ±
0.59 0.71 0.04 0.20
B A B A
21.94 21.45 9.39 20.13
± ± ± ±
0.15 A 0.979 A 0.05 A 0.53 B
Data are means ± standard deviation (n ¼ 3). Samples were prepared from three independent loaves. Means with different letters within the same loaf part (crust or crumb) are significantly different (P < 0.05). L*, brightness of colour; a*, range of colour between red and green; b*, range of colour between yellow and blue.
Table 2 Rutin, quercetin and antioxidant activity (expressed in mg/mg of trolox equivalents) in the CB and TB flours and the sourdough starter. Buckwheat sample
Rutin (mg/g)
Quercetin (mg/g)
Antioxidant activity
Common, flour Tartary, flour Sourdough starter
ND 14.69 ± 0.84 1.54 ± 0.00
ND 1.94 ± 0.79 12.53 ± 0.68
57.54 ± 5.87 A 88.45 ± 6.70 B 75.56 ± 3.28
Data are means ± standard deviation (n ¼ 3, antioxidant activity n ¼ 4, independent loaves). Means with different letters in the same column are significantly different (P < 0.05). ND: not detected.
present in the TB seeds (Yasuda and Nakagawa, 1994). The expected value for the mixture of the sourdough starter and flour was a total of 4.65 mg quercetin (including the quercetin in the rutin molecules), as calculated from the concentrations in the respective ingredients (35% sourdough starter, 65% CB flour), which represent the dry weights of the sourdough before fermentation. The levels of rutin during the CB sourdough bread-making process and in CB sourdough bread itself were below the detection limit (Tables 2 and 4). The TB flour contained more rutin than quercetin, but in other
Table 3 Antioxidant activities of CB and TB sourdough before and after 10-h fermentation, bread dough before and after 5-h rising, and sourdough bread. Buckwheat flour
Antioxidant activity (mg/mg of trolox equivalents) Sourdough
Common Tartary
Bread dough
Bread
Before fermentation
After fermentation
Before rising
After rising
63.42 ± 2.43 aA 76.40 ± 6.55 bB
68.58 ± 5.61 aA 73.78 ± 2.36 bA
79.66 ± 3.48 bB 66.64 ± 2.03 abA
79.53 ± 4.63 bB 65.64 ± 4.23 abA
77.15 ± 3.45 bA 50.25 ± 21.99 A
Data are means ± standard deviation (n ¼ 4, independent loaves). Means with different small letters in a row or with different capital letters in the column are significantly different (P < 0.05).
Table 4 Rutin and quercetin concentrations in CB and TB sourdough before and after 10-h fermentation, bread dough before and after 5-h rising, and sourdough bread. Buckwheat flour
Flavonoid
Flavonoid concentration (mg/g) Sourdough
Common Tartary
Rutin Quercetin Rutin Quercetin
Bread dough
Bread
Before fermentation
After fermentation
Before rising
After rising
ND 1.49 ± 0.03c 3.29 ± 0.09c 8.15 ± 0.10b
ND 1.26 ± 0.08b 2.72 ± 0.11b 8.45 ± 0.75b
ND 0.72 ± 0.09a 2.62 ± 0.09a 8.70 ± 0.40b
ND 0.75 ± 0.02a 2.40 ± 0.12a 8.85 ± 0.27b
ND ND ND 5.08 ± 0.40a
Data are means ± standard deviation (n ¼ 3). Extracts were prepared from independent loaves. Means with different letters in the row are significantly different (P < 0.05). ND, not detectable.
TB sourdough starter (Tables 2 and 4). The high concentrations of quercetin in the sourdough before fermentation might be because of the contribution of quercetin from the sourdough starter, where high concentrations of rutin and quercetin were measured. The sourdough starter had a total of 1.54 mg/g rutin and 12.53 mg/g quercetin. The rutin molecule is composed of a quercetin part and a sugar part; taking into account the mass of each part, the concentration of rutin is divided by 2.02 to obtain the concentration of the quercetin component of rutin. By calculating the quercetin equivalents of rutin, the total amount of quercetin (including the quercetin in the rutin molecules) was 13.29 mg/g. In the CB sourdough before fermentation (i.e., in the mixture of sourdough starter and flour), there was 1.49 mg/g quercetin. The transformation of rutin to quercetin is expected because of the rutin-degrading enzymes
TB samples taken during the sourdough bread-making process, the levels of these two flavonoids were reversed; i.e., there was more quercetin than rutin. This might be because of the transformation of rutin into quercetin during the bread-making process by the activity of the rutin-degrading enzymes, which are active in the buckwheat sample after addition of water and yeast to the flour (Yasuda and Nakagawa, 1994; Suzuki et al., 2002; Vogrin ci c et al., 2010). At the beginning of the fermentation (i.e., in the fresh mixture of sourdough starter and flour), the TB sourdough had a total of 9.78 mg/g quercetin equivalents of rutin and quercetin. In comparison, the expected value for the mixture of the sourdough starter and flour was 10.64 mg quercetin equivalents, as calculated from the concentrations in the respective ingredients (35%
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sourdough starter, 65% TB flour), which make up the dry weight of sourdough before fermentation. No rutin remained in the bread and the quercetin levels were also decreased. The concentration of quercetin in the dough depends on its content in TB flour, the transformation of rutin to quercetin, and the degradation of quercetin itself. The lower concentrations measured in the breads of both of these buckwheat species might also be related to the integration of phenolic compounds into starch (amylose) structures, which transform their structure during the baking process and can incorporate some smaller molecules during this transformational process (Skrabanja et al., 2000; Goderis et al., 2014; Ryno et al., 2014). It is a possibility that the pH of the dough may also have some impact on the release of quercetin. It is possible that under the conditions of high flavonoid concentrations in TB, the molecules can protect each other from degradation. A similar tendency was reported by Vogrin ci c et al. (2010), where in yeasted dough and bread samples with low initial concentrations of rutin and quercetin, the rutin was degraded completely; in contrast, when the initial concentrations of rutin and quercetin were high, about half of the rutin from the dough remained in the bread. The high concentration of quercetin in the sourdough is possibly because of the contribution of quercetin from the sourdough starter. The data in the present study are in agreement with those of Vogrin ci c et al. (2010), and showed that in sourdough bread, the transformation of rutin and quercetin proceeds similarly to that in yeasted bread. A similar concentration of quercetin (5.08 mg/g) remained in the sourdough bread as in their yeasted bread (5.00 mg/g). During sourdough fermentation and the baking process, the rutin disappeared completely and none was found in the bread. This was different from yeasted bread, where about 0.44 mg/ g rutin remained in the bread (Vogrin ci c et al., 2010). Surprisingly, the sourdough starter showed high concentrations of rutin (1.54 mg/g DM) and quercetin (12.53 mg/g DM) (Table 2). There was much more quercetin in the TB sourdough starter than in the TB flour. Moreover, the total concentration of quercetin contained in the TB sourdough starter (quercetin plus the quercetin part of the rutin molecule) was higher than the total quercetin in the dry matter of the TB flour. This indicates that microorganisms mainly used substances other than these flavonoids for their metabolic processes. Through degradation of other substances as energy sources for bacterial metabolism, the concentrations of quercetin (bound and free) increased (Table 2). However, it is also possible that the bacteria used the sugars released after the degradation of the rutin molecules. Rutin molecules were degraded during the sourdough fermentation (Table 4). It is not known if this is an effect of the rutinase enzyme in the TB flour (Yasuda and Nakagawa, 1994; Suzuki et al., 2002), or if there are enzymes with the same effects that are produced by the sourdough microorganisms. If the degradation of the rutin molecules during the sourdough fermentation was a result of the TB rutinase, this means that the TB rutinase molecules persisted and remained active for at least 10 h under the conditions of sourdough fermentation. Because all of the rutin molecules were degraded during the sourdough and bread dough fermentation and rising, we cannot establish what occurs to the rutin molecules during the baking. This is different to TB yeasted bread, where some rutin persists in the dough during rising and appears in the bread even after baking (Vogrin ci c et al., 2010). The type of sugar moiety is an important determinant for the intestinal uptake of dietary quercetin glycosides (Arts et al., 2004). It has also been reported that quercetin from the diet is widely distributed in animal tissues (De Boer et al., 2005). As far as is known, quercetin is an anti-oxidant with similar bioavailability and similar health-maintaining effects to rutin (Sikder et al., 2014).
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From this standpoint, the transition of all of the rutin and the appearance of this as quercetin in sourdough bread are acceptable. Indeed, the improved content of quercetin is a welcome addition to the other substances in sourdough bread that have healthpromoting effects. 4. Conclusions We have shown that the combined effects of sourdough fermentation and the baking process preserve quercetin, but not rutin, in CB and TB bread doughs and the sourdough bread. During sourdough fermentation, there was conversion of rutin to quercetin. In the CB samples, the anti-oxidant activity increased from flour to sourdough, and then further to bread dough and sourdough bread. In contrast, in the TB samples, the highest anti-oxidant activity was for the flour and sourdough, and the lowest was for the sourdough bread. Despite its quercetin content, the TB sourdough bread had a lower anti-oxidant activity than the CB bread. The finding that quercetin remains in the TB sourdough bread is important for consideration of the potential importance of TB sourdough bread products in health-preserving nutrition. The molecular and structural bases of the differences in the specific loaf volumes of the CB and TB sourdough breads, and the possible impact of the quercetin content on loaf volume, and antioxidant activity, are suggested lines for further research. Acknowledgements This study was financed by the Slovenian Research Agency, through programmes P1-0143 “Biology of Plants” (P1-0212) and P3-0395 “Nutrition and Public Health”, and projects L4-7552 and J4-5524, supported by EUFORINNO 7th FP EU Infrastructure Programme (RegPot No. 315982). The research leading to these results has received funding from the European Community under project ITEM 26220220180: Building Research Centre “AgroBioTech”. The authors are grateful to Dragan Abram for skilful technical assistance. References AACC International, 2008. Approved Methods of the American Association of Cereal Chemistry, eleventh ed. American Association of Cereal Chemists, St Paul. Arts, I.C.W., Sesink, A.L.A., Faassen-Petersa, M., Hollman, P.C.H., 2004. The type of sugar moiety is a major determinant of the small intestinal uptake and subsequent biliary excretion of dietary quercetin glycosides. Brit. J. Nutr. 91 (6), 841e847. Bonafaccia, G., Marocchini, M., Kreft, I., 2003. Composition and technological properties of the flour and bran from common and tartary buckwheat. Food Chem. 80, 9e15. Cao, W., Chen, W.J., Suo, Z.R., Yao, Y.P., 2008. Protective effect of ethanolic extracts of buckwheat groats on DNA damage caused by hydroxyl radicals. Food Res. Int. 41, 924e929. CIE International Commission on Illumination, 1986. Publication CIE, n.15.2 Wien, Austria. De Boer, V.C.J., Dihal, A.A., Van der Woude, H., Arts, I.C.W., Wolffram, S., Alink, G.M., Rietjens, I.M., Keijer, J., Hollman, P.C., 2005. Tissue distribution of quercetin in rats and pigs. J. Nutr. 135 (7), 1718e1725. Fabjan, N., Rode, J., Kosir, I.J., Wang, Z., Zhang, Z., Kreft, I., 2003. Tartary buckwheat (Fagopyrum tataricum Gaertn.) as a source of dietary rutin and quercitrin. J. Agric. Food Chem. 51 (22), 6452e6455. Goderis, B., Putseys, A., Gommes, C.J., Bosmans, G.M., Delcour, J.A., 2014. The structure and thermal stability of amylose-lipid complexes: a case study on amylose-glycerol monostearate. Crys. Grow. Des. 14 (7), 3221e3233. Holasova, M., Fiedlerova, V., Smrcinova, H., Orsak, M., Lachman, J., Vavreinova, S., 2002. Buckwheat e the source of antioxidant activity in functional foods. Food Res. Int. 35, 207e211. Houben, A., Hochstotter, A., Becker, T., 2012. Possibilities to increase the quality in gluten-free bread production: an overview. Eur. Food Res. Tech. 235 (2), 195e208. Michalska, A., Amigo Benavent, M., Zielinski, H., Dolores del Castillo, M., 2008. Effect of bread making on formation of Maillard reaction products contributing to the overall antioxidant activity of rye bread. J. Cereal Sci. 48 (1), 123e132.
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Ryno, L.M., Levine, Y., Iovine, P.M., 2014. Synthesis, characterization, and comparative analysis of amylose-guest complexes prepared by microwave irradiation. Carbohydr. Res. 383, 82e88. Sikder, K., Kesh, S.B., Das, N., Manna, K., Dey, S., 2014. The high antioxidative power of quercetin (aglycone flavonoid) and its glycone (rutin) avert high cholesterol diet induced hepatotoxicity and inflammation in Swiss albino mice. Food Funct. 5 (6), 1294e1303. Skrabanja, V., Laerke, H.N., Kreft, I., 1998. Effect of hydrothermal processing of buckwheat (Fagopyrum esculentum Moench) groats on starch enzymatic availability in vitro and in vivo in rats. J. Cereal Sci. 28, 209e221. Skrabanja, V., Laerke, H.N., Kreft, I., 2000. Protein-polyphenol interactions and in vivo digestibility of buckwheat groat proteins. Pflügers Arch. Eur. J. Physiol. 440 (5), 129e131. € rck, I.M.E., 2001. Nutritional properties Skrabanja, V., Liljeberg, E.H.G.M., Kreft, I., Bjo of starch in buckwheat products: studies in vitro and in vivo. J. Agric. Food Chem. 49 (1), 490e496. Suzuki, T., Honda, Y., Funatsuki, W., Nakatsuka, K., 2002. Purification and characterization of flavonol 3-glucosidase, and its activity during ripening in tartary buckwheat seeds. Plant Sci. 163 (3), 417e423.
Vogrin ci c, M., Timoracka, M., Melichacova, S., Vollmannova, A., Kreft, I., 2010. Degradation of rutin and polyphenols during the preparation of Tartary buckwheat bread. J. Agric. Food Chem. 58 (8), 4883e4887. Vombergar, B., Kreft, I., Horvat, M., Vorih, S., 2014. Ajda e Buckwheat. Zalo zba kme cki glas, Ljubljana. Wieslander, G., Fabjan, N., Vogrin ci c, M., Kreft, I., Janson, C., Spetz-Nystrom, U., €ck, D., 2011. Eating cookies is Vombergar, B., Tagesson, C., Leanderson, P., Norba associated with the reduction in serum levels of myeloperoxidase and cholesterol: a double blind crossover study in day-care centre staffs. Tohoku J. Exp. Med. 225, 123e130. Yang, N., Li, Y.M., Zhang, K.S., Jiao, R., Ma, K.Y., Zhang, R., Guixing, R., Zhen-Yu, C., 2014. Hypocholesterolemic activity of buckwheat flour is mediated by increasing sterol excretion and down-regulation of intestinal NPC1L1 and ACAT2. J. Funct. Foods 6, 311e318. Yasuda, T., Nakagawa, H., 1994. Purification and characterization of rutin-degrading enzymes in tartary buckwheat seeds. Phytochemistry 37, 133e136. Zhang, M., Chen, H., Li, J., Pei, Y., Liang, Y., 2010. Antioxidant properties of Tartary buckwheat extracts as affected by different thermal processing methods. Food Sci. Tech. 43, 181e185.
Contents lists available at ScienceDirect
Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Rutin and quercetin transformation during preparation of buckwheat sourdough bread Lea Luksi c a, Giovanni Bonafaccia b, Maria Timoracka c, Alena Vollmannova c, Janja Tr cek d, Tina Ko zelj Nyambe d, Valentina Melini b, Rita Acquistucci b, Mateja Germ a, Ivan Kreft e, * a
Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia Food and Nutrition Research Centre (CRA-NUT), Via Ardeatina 546, I-00178 Roma, Italy Department of Chemistry, Slovak Agricultural University of Nitra, Tr. A. Hlinku 2, SK-94901 Nitra, Slovak Republic d Department of Biology, Faculty of Natural Sciences and Mathematics, University of Maribor, Koroska cesta 160, SI-2000 Maribor, Slovenia e Slovenian Forestry Institute, Vecna pot 2, SI-1000 Ljubljana, Slovenia b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 October 2015 Received in revised form 20 February 2016 Accepted 23 February 2016 Available online 27 February 2016
Sourdough bread was prepared from flour of the common buckwheat (Fagopyrum esculentum) and of Tartary buckwheat (Fagopyrum tataricum) to follow the transformation of rutin and quercetin in these sourdoughs. During Tartary buckwheat sourdough fermentation, there was conversion of rutin to quercetin. The Tartary buckwheat flour contained 14.6 mg/g rutin and 1.9 mg/g quercetin as dry matter. The sourdough starter contained 1.5 mg/g rutin and an unexpectedly high 12.5 mg/g quercetin. The sourdough contained 3.2 mg/g rutin and 8.1 mg/g quercetin. In the Tartary buckwheat sourdough bread there was no rutin, whereas there was 5.0 mg/g quercetin. Thus, during the sourdough fermentation, the rutin was completely degraded. However, despite the long fermentation time (sourdough, 10 h; bread dough, 5 h), most of the quercetin remained in the dough and appeared in the baked bread. In contrast to Tartary buckwheat bread, neither rutin nor quercetin was present in common buckwheat bread. Information on the persistence of quercetin in sourdough bread is important for designing breads with high concentrations of flavonoids and good functional value. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Tartary buckwheat (Fagopyrum tataricum) Common buckwheat (Fagopyrum esculentum) Rutin Quercetin
1. Introduction Common buckwheat (CB) (Fagopyrum esculentum Moench) and Tartary buckwheat (TB) (Fagopyrum tataricum (L.) Gaertn.) are used in different parts of the world to make various food products. The grain of both of these cultivated buckwheat species contains up to 27% fibre (Bonafaccia et al., 2003). Buckwheat groats are a prebiotic food because they can, for example, increase the lactic acid bacteria in the intestine because of their content of resistant starch (Skrabanja et al., 1998, 2001). Buckwheat is also an important source of anti-oxidant activity in functional foods (Holasova et al.,
Abbreviations: a*, range of colour between red and green; ANOVA, analysis of variance; b*, range of colour between yellow and blue; CB, common buckwheat; DM, dry mass; DPPH, 2,2-diphenyl-1-picrylhydrazyl; HPLC, high performance liquid chromatography; L*, brightness of colour; ND, not detected; ORACFL, oxygen radical absorbing capacity; PCL, photochemiluminescence; PCR, polymerase chain reaction; TB, Tartary buckwheat. * Corresponding author. E-mail address: [email protected] (I. Kreft). http://dx.doi.org/10.1016/j.jcs.2016.02.011 0733-5210/© 2016 Elsevier Ltd. All rights reserved.
2002). This arises from the presence of the flavonoids rutin and quercetin in the buckwheat grain and products, because of their anti-oxidant and anti-inflammatory effects. It has been reported that TB contains about 100-fold more rutin than does CB (Fabjan et al., 2003). Rutin and quercetin are also present in baked biscuits made from CB and TB (Wieslander et al., 2011). Buckwheat products decrease cholesterol levels and improve lung capacity in humans (Wieslander et al., 2011; Sikder et al., 2014; Yang et al., 2014). Extracts from CB and TB can also protect DNA from damage caused by hydroxyl radicals (Cao et al., 2008; Vogrin ci c et al., 2010). One of the ways to prepare bread involves sourdough fermentation, which can be accompanied by the formation of flavour compounds such as lactic acid and acetic acid that have an impact on dough processing and the preparation of sourdough bread (Michalska et al., 2008). However, the combined effects of sourdough fermentation and the baking process on the flavonoid concentrations and anti-oxidant properties of CB and TB sourdough starter, bread dough and sourdough bread have not yet been
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studied. Therefore, the objective of the present study was to investigate the effects of sourdough fermentation and the baking process on rutin and quercetin content and on the anti-oxidant activities of CB and TB. More precisely, the aim was to determine whether sourdough fermentation and thermal processing affect the conversion of rutin to quercetin. We were also interested in determining how much rutin and quercetin remain in CB and TB products after sourdough fermentation and the baking process. These findings will contribute to a better understanding of the value of buckwheat products for human consumption. 2. Materials and methods
of each type of bread (CB or TB) were included in the study, made independently from the beginning (including making the sourdough out of four different starter samples). All four were used for the measurement of anti-oxidant activity, and three randomly selected loaves were used for other measurements. 2.4. Measurement of loaf volumes For the determination of loaf volumes, the rapeseed displacement method was used according to AACC method number 1005.01 (AACC International, 2008). Specific loaf volumes were obtained by dividing the volume by the loaf weight (expressed as g/ cm3).
2.1. Materials and sample preparation 2.5. Colour measurements The CB and TB flours were prepared in a Rangus mill (Vrhpolje near Sentjernej, Slovenia) using the CB grain cv. ‘Pyra’ and TB grain from a domestic strain that originated in Luxembourg (Bonafaccia et al., 2003). The CB and TB were harvested in 2012. The initial sourdough starter was obtained from Sweden (Bageriet Andreas € Ostlund, Uppsala, Sweden), and was stored in a glass jar in a refrigerator (5 C). Before use in this study, this sourdough starter was refreshed twice weekly for 6 months by addition of an equal volume of TB flour suspension (1:1; v/v; TB flour: preboiled and cooled tap-water). This was done to adapt the starter to the flavonoid-rich material. The sourdough starter was kept in a refrigerator (5e8 C). 2.2. Determination of sourdough starter microorganisms To identify the bacteria present, diluted sourdough starter was plated onto deMan, Rogosa and Sharpe (MRS) agar medium (SigmaeAldrich, St. Louis, Missouri, US) and incubated for 3 days at 30 C under anaerobic conditions. Each type of morphologically different colony was further purified to isolate the pure strains. The isolates were grown in liquid MRS medium, and the biomass was harvested and used for DNA isolation using NucleoSpin Tissue kits (MachereyeNagel, Düren, Germany). The 16S rRNA bacterial genes were specifically amplified by polymerase chain reaction (PCR) using the primers 16S_27f (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 16S_rH1542 (50 -AAGGAGGTGATCCAGCCGCA-30 ). The PCR products were cleaned up using NucleoSpin gel and PCR Clean-up kits (MachereyeNagel, Düren, Germany) and their partial sequences were determined by Eurofins Genomics. The sequences were analysed using the Blastn programme available from the National Center for Biotechnology Information (NCBI) database. The isolates were assigned to the species to which the 16S rRNA gene sequences showed the highest identity. In this way, two morphologically different species were isolated and identified as Lactobacillus heilongjiangensis and Pediococcus parvulus. The sequences were deposited in the EMBL/GenBank/DDBJ database under accession numbers LN650641 (L. heilongjiangensis) and LN650642 (P. parvulus). Fresh baker's yeast was purchased from Bonopan (Logatec, Slovenia). 2.3. Sourdough bread production The sourdough bread was prepared in two steps. On the first day, a mixture of 50 g sourdough starter, 90 g flour and 150 g tapwater at room temperature (25 C) was prepared. The mixture was stored in a refrigerator (5 C) and allowed to ferment for 10 h. On the second day, 25 g tap-water, 8 g sucrose, 5 g salt, 4 g fresh baker's yeast and 150 g flour were added to the mixture. The dough was placed in baking forms on baking paper and left to rise for 5 h. The bread was then baked in an oven at 200 C for 1 h. Four bread loaves
Colour measurements were performed on the bread crumb and crust. The lightness (L*) and red (þa*) and yellow (þb*) colorimetric indices were established according to the CIELAB colour system (CIE, 1986) using a Chroma Meter CR 300 Minolta (Konica Minolta, Inc., Tokyo, Japan) equipped with a pulsed xenon lamp and illuminant D65. A white plate (X ¼ 91.98, Y ¼ 93.97, Z ¼ 110.41) was used to standardize the instrument. Three independently prepared loaves of each type of bread were measured. 2.6. Preparation of methanol extracts The concentrations of rutin and quercetin and the anti-oxidant activity were determined for the CB flour, TB flour, sourdough starter, sourdough before and after the 10-h incubation in a refrigerator (5 C), bread dough before and after the 5-h rising at room temperature, and in the two different types of sourdough bread (i.e., CB, TB). Bread samples were prepared at room temperature (25 C). After preparing the dough and baking the bread, the loaves (four each of the two types of bread) were cooled and cut into pieces, which were stored for analysis in a freezer at 20 C. All of the samples prepared were freeze-dried and milled for analysis. To prepare the methanol extracts, 50 mL 80% aqueous methanol (HPLC grade, SigmaeAldrich Corporation, St. Louis, MO, USA) was added to 1 g of each milled sample. The mixtures were shaken at room temperature for 8 h (250 rpm). The samples were then filtered through filter paper (130 g/m2; Filtrak, Thermalbad Wiesenbad, Germany) and kept at 5 C for further analysis. The methanol extracts of each type of bread were made using three independently prepared loaves. 2.7. Anti-oxidant activity assays The anti-oxidant activities were determined on the basis of photochemiluminescence (PCL) and fluorescence (ORACFL) assays. The PCL assay was used to measure the anti-oxidant activity of extracts against superoxide anion radicals generated from luminol (a photosensitizer) when exposed to UV light, assessed with a Photochem instrument (Analytik Jena, USA Inc., Delaware, OH, USA). The anti-oxidant activities of the sample extracts were measured using integral anti-oxidant capacity of water-soluble substances kits from Analytik Jena (USA Inc., Delaware, OH, USA). Chemiluminescence evolution was monitored using the PCL soft control and analysis software. The lag time (expressed in seconds) was used as a measure of radical-scavenging activity. The antioxidant capacities were estimated by comparison with a Trolox standard (0.5e3 nmol of Trolox) and are expressed as mg/mg Trolox equivalents. The anti-oxidant index was obtained by dividing the anti-oxidant capacity by the lag time and multiplying by 1000 (anti-oxidant activity/lag time 1000).
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2.8. Determination of rutin and quercetin concentrations The filtrates were injected into a high performance liquid chromatography (HPLC) system that consisted of an HPLC chromatograph (Alliance 2695, Waters, Milford, USA), a LiChroCART Purospher RP C18 column (5 mm, 250 4.6 mm; Merck, Darmstadt, Germany) and a DAD 2996 UV detector (Waters, Milford, USA). The column temperature was 30 C. The gradient elution was carried out at a flow rate of 1 mL/min. Solvent A was acetonitrile, and solvent B was 0.1% phosphoric acid. The gradient was as follows (% A): 0e3 min, 40%; 3e8 min, 40%e5%; 8e10 min, 5%. Rutin and quercetin were detected at 365 nm and their concentrations were calculated on the basis of the calibration curves made using rutin and quercetin standards (Acros Organics, Waltham, MA, USA) prepared in methanol (gradient elution grade; SigmaeAldrich Corporation, St. Louis, USA). The limit of detection (LOD) for rutin was 1.12 mg/mL and for quercetin 1.01 mg/mL. The limit of quantification (LOQ) for rutin was 3.69 mg/mL and for quercetin 3.33 mg/ mL. The results are expressed as mg/g dry weight. Rutin can also be expressed as quercetin equivalents. For this purpose, the rutin concentration was divided by a factor of 2.02. 2.9. Statistical analysis The data are expressed as means ± standard deviation of the results for independently prepared bread loaves. ANOVA was used for the statistical analysis, and the data were considered to be significantly different when P < 0.05. 3. Results and discussion 3.1. Bread loaf volumes The specific volume is the ratio of the volume to the weight, and this has been adopted as a reliable measure of bread quality (Houben et al., 2012). The data here showed that the specific volume of the loaves from the CB sourdough bread (0.90 ± 0.04 g/cm3) was lower than that from the TB sourdough bread (1.10 ± 0.02 g/ cm3). The absence of gluten in the buckwheat flours had a strong impact on the bread's rheological properties. Because of the low carbon dioxide binding activity during rising, the volumes of gluten-free breads are generally lower in comparison with bread containing gluten-containing cereals (Houben et al., 2012). In this respect, the properties of the CB bread are also different from those of the TB bread. This is not completely unexpected, because CB and TB are different botanical species. The chemical compositions of the CB and TB flours were not very different apart from the large difference in rutin concentrations in the grain (Bonafaccia et al., 2003; Fabjan et al., 2003). The molecular basis for the differences in the loaf volumes has not yet been examined. Investigation of the possible impact of the flavonoids on the loaf volume was outside the scope of this study. 3.2. Colour measurements The colour measurements for the sourdough bread preparation are shown in Table 1. Comparisons of the colour values (L*, a*, b*) of the CB and TB crust and crumbs of the sourdough bread were performed. There were no significant differences in the L* values of CB and TB crumb or crust. The L* gives a measure of the brightness of the sample, and this parameter ranges from 0 (black) to 100 (white). Comparing the colour of the bread crumb, there were significant differences in the a* and b* values between the CB and TB bread crumb. Comparing the colours of the bread crust, there was a significant difference in the a* value but not the b* value
73
between the CB and TB bread crust (Table 1). The lower a* value for the crust and crumb of the TB sourdough bread indicated that it had a stronger green tinge compared with the CB bread. The higher b* value for the crumb of the TB sourdough bread indicated that its colour was more yellowish than that of the CB sourdough bread. To our knowledge, the colour of TB bread in comparison to CB bread or other bread products has not been measured previously. Our measurements confirm that TB bread is more greenish-yellow than CB bread, confirming visual observations of a green-yellow tinge to TB food products. This reported colour appears to be connected with the high concentrations of rutin and quercetin in the TB products (Vombergar et al., 2014). 3.3. Effect of the bread-making process on anti-oxidant activity Comparisons of the anti-oxidant activities in these samples during the sourdough bread-making process were performed for each buckwheat type (Tables 2 and 3). During the CB bread-making process, there were significant differences between the antioxidant activity (expressed as mg/mg of trolox equivalents) of the sourdough starter, bread dough and bread. There were also differences in the anti-oxidant activity of the sourdough starter, bread dough and bread in the TB sourdough bread-making process. Comparing the CB and TB samples, there were significant differences in anti-oxidant activity between the two types of flour, in the sourdough starter before fermentation, in the bread dough before rising and in the bread dough after rising (Tables 2 and 3). Among the CB samples, the highest anti-oxidant activity was observed in the bread dough and bread and the lowest in the CB flour. Here, the anti-oxidant activity increased from flour to sourdough starter, and further to bread dough and sourdough bread. This might be because of the synthesis of substances with antioxidant properties, including certain Maillard reaction products that occur in bread during thermal treatment (Zhang et al., 2010). In contrast, for the TB samples, the highest anti-oxidant activity was measured in the flour and sourdough, and the lowest in the sourdough bread (Tables 2 and 3). Flavonoids in TB could interfere with the Maillard or other reactions in the bread, but identification of the mechanism behind these differences requires further investigation. A similar drop in anti-oxidant activity was established for yeasted TB bread (Vogrin ci c et al., 2010). However, Vogrin ci c et al. (2010) reported different values for yeasted wheat bread, where the DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging capacity rose from 0% in the dough after mixing to 0.54% in the dough just before baking, and to 2.88% in the bread loaf crumb. A significant decrease in anti-oxidant activity in TB flour as a result of various thermal treatments such as roasting, steam-pressure heating, and microwaving, has been reported (Zhang et al., 2010). A small decrease in the anti-oxidant activity in CB flour roasted for 10 min at 200 C has also been noted (Yasuda and Nakagawa, 1994). The CB and TB samples differed in their anti-oxidant properties; however, the molecular basis for this remains unknown. The changes in anti-oxidant activity in CB were similar to those reported for wheat (Vogrin ci c et al., 2010), although in the case of the CB used in this study, they were at a much higher level. 3.4. Impact of sourdough bread production procedure on rutin and quercetin concentrations Comparisons of the samples of each buckwheat species during the sourdough bread-making process (Table 4) were performed. In CB flour, the rutin and quercetin concentrations were below the detection limit of the method applied, although quercetin appeared in the CB sourdough at the beginning of the fermentation from the
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Table 1 Crumb and crust colour values of the CB and TB sourdough bread. Buckwheat flour
Bread sample
Colour values L*
Crust
Common Tartary Common Tartary
Crumb
a*
43.31 41.15 48.01 46.54
± ± ± ±
2.23 1.64 0.52 0.77
A A A A
12.04 9.50 4.12 1.09
b* ± ± ± ±
0.59 0.71 0.04 0.20
B A B A
21.94 21.45 9.39 20.13
± ± ± ±
0.15 A 0.979 A 0.05 A 0.53 B
Data are means ± standard deviation (n ¼ 3). Samples were prepared from three independent loaves. Means with different letters within the same loaf part (crust or crumb) are significantly different (P < 0.05). L*, brightness of colour; a*, range of colour between red and green; b*, range of colour between yellow and blue.
Table 2 Rutin, quercetin and antioxidant activity (expressed in mg/mg of trolox equivalents) in the CB and TB flours and the sourdough starter. Buckwheat sample
Rutin (mg/g)
Quercetin (mg/g)
Antioxidant activity
Common, flour Tartary, flour Sourdough starter
ND 14.69 ± 0.84 1.54 ± 0.00
ND 1.94 ± 0.79 12.53 ± 0.68
57.54 ± 5.87 A 88.45 ± 6.70 B 75.56 ± 3.28
Data are means ± standard deviation (n ¼ 3, antioxidant activity n ¼ 4, independent loaves). Means with different letters in the same column are significantly different (P < 0.05). ND: not detected.
present in the TB seeds (Yasuda and Nakagawa, 1994). The expected value for the mixture of the sourdough starter and flour was a total of 4.65 mg quercetin (including the quercetin in the rutin molecules), as calculated from the concentrations in the respective ingredients (35% sourdough starter, 65% CB flour), which represent the dry weights of the sourdough before fermentation. The levels of rutin during the CB sourdough bread-making process and in CB sourdough bread itself were below the detection limit (Tables 2 and 4). The TB flour contained more rutin than quercetin, but in other
Table 3 Antioxidant activities of CB and TB sourdough before and after 10-h fermentation, bread dough before and after 5-h rising, and sourdough bread. Buckwheat flour
Antioxidant activity (mg/mg of trolox equivalents) Sourdough
Common Tartary
Bread dough
Bread
Before fermentation
After fermentation
Before rising
After rising
63.42 ± 2.43 aA 76.40 ± 6.55 bB
68.58 ± 5.61 aA 73.78 ± 2.36 bA
79.66 ± 3.48 bB 66.64 ± 2.03 abA
79.53 ± 4.63 bB 65.64 ± 4.23 abA
77.15 ± 3.45 bA 50.25 ± 21.99 A
Data are means ± standard deviation (n ¼ 4, independent loaves). Means with different small letters in a row or with different capital letters in the column are significantly different (P < 0.05).
Table 4 Rutin and quercetin concentrations in CB and TB sourdough before and after 10-h fermentation, bread dough before and after 5-h rising, and sourdough bread. Buckwheat flour
Flavonoid
Flavonoid concentration (mg/g) Sourdough
Common Tartary
Rutin Quercetin Rutin Quercetin
Bread dough
Bread
Before fermentation
After fermentation
Before rising
After rising
ND 1.49 ± 0.03c 3.29 ± 0.09c 8.15 ± 0.10b
ND 1.26 ± 0.08b 2.72 ± 0.11b 8.45 ± 0.75b
ND 0.72 ± 0.09a 2.62 ± 0.09a 8.70 ± 0.40b
ND 0.75 ± 0.02a 2.40 ± 0.12a 8.85 ± 0.27b
ND ND ND 5.08 ± 0.40a
Data are means ± standard deviation (n ¼ 3). Extracts were prepared from independent loaves. Means with different letters in the row are significantly different (P < 0.05). ND, not detectable.
TB sourdough starter (Tables 2 and 4). The high concentrations of quercetin in the sourdough before fermentation might be because of the contribution of quercetin from the sourdough starter, where high concentrations of rutin and quercetin were measured. The sourdough starter had a total of 1.54 mg/g rutin and 12.53 mg/g quercetin. The rutin molecule is composed of a quercetin part and a sugar part; taking into account the mass of each part, the concentration of rutin is divided by 2.02 to obtain the concentration of the quercetin component of rutin. By calculating the quercetin equivalents of rutin, the total amount of quercetin (including the quercetin in the rutin molecules) was 13.29 mg/g. In the CB sourdough before fermentation (i.e., in the mixture of sourdough starter and flour), there was 1.49 mg/g quercetin. The transformation of rutin to quercetin is expected because of the rutin-degrading enzymes
TB samples taken during the sourdough bread-making process, the levels of these two flavonoids were reversed; i.e., there was more quercetin than rutin. This might be because of the transformation of rutin into quercetin during the bread-making process by the activity of the rutin-degrading enzymes, which are active in the buckwheat sample after addition of water and yeast to the flour (Yasuda and Nakagawa, 1994; Suzuki et al., 2002; Vogrin ci c et al., 2010). At the beginning of the fermentation (i.e., in the fresh mixture of sourdough starter and flour), the TB sourdough had a total of 9.78 mg/g quercetin equivalents of rutin and quercetin. In comparison, the expected value for the mixture of the sourdough starter and flour was 10.64 mg quercetin equivalents, as calculated from the concentrations in the respective ingredients (35%
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sourdough starter, 65% TB flour), which make up the dry weight of sourdough before fermentation. No rutin remained in the bread and the quercetin levels were also decreased. The concentration of quercetin in the dough depends on its content in TB flour, the transformation of rutin to quercetin, and the degradation of quercetin itself. The lower concentrations measured in the breads of both of these buckwheat species might also be related to the integration of phenolic compounds into starch (amylose) structures, which transform their structure during the baking process and can incorporate some smaller molecules during this transformational process (Skrabanja et al., 2000; Goderis et al., 2014; Ryno et al., 2014). It is a possibility that the pH of the dough may also have some impact on the release of quercetin. It is possible that under the conditions of high flavonoid concentrations in TB, the molecules can protect each other from degradation. A similar tendency was reported by Vogrin ci c et al. (2010), where in yeasted dough and bread samples with low initial concentrations of rutin and quercetin, the rutin was degraded completely; in contrast, when the initial concentrations of rutin and quercetin were high, about half of the rutin from the dough remained in the bread. The high concentration of quercetin in the sourdough is possibly because of the contribution of quercetin from the sourdough starter. The data in the present study are in agreement with those of Vogrin ci c et al. (2010), and showed that in sourdough bread, the transformation of rutin and quercetin proceeds similarly to that in yeasted bread. A similar concentration of quercetin (5.08 mg/g) remained in the sourdough bread as in their yeasted bread (5.00 mg/g). During sourdough fermentation and the baking process, the rutin disappeared completely and none was found in the bread. This was different from yeasted bread, where about 0.44 mg/ g rutin remained in the bread (Vogrin ci c et al., 2010). Surprisingly, the sourdough starter showed high concentrations of rutin (1.54 mg/g DM) and quercetin (12.53 mg/g DM) (Table 2). There was much more quercetin in the TB sourdough starter than in the TB flour. Moreover, the total concentration of quercetin contained in the TB sourdough starter (quercetin plus the quercetin part of the rutin molecule) was higher than the total quercetin in the dry matter of the TB flour. This indicates that microorganisms mainly used substances other than these flavonoids for their metabolic processes. Through degradation of other substances as energy sources for bacterial metabolism, the concentrations of quercetin (bound and free) increased (Table 2). However, it is also possible that the bacteria used the sugars released after the degradation of the rutin molecules. Rutin molecules were degraded during the sourdough fermentation (Table 4). It is not known if this is an effect of the rutinase enzyme in the TB flour (Yasuda and Nakagawa, 1994; Suzuki et al., 2002), or if there are enzymes with the same effects that are produced by the sourdough microorganisms. If the degradation of the rutin molecules during the sourdough fermentation was a result of the TB rutinase, this means that the TB rutinase molecules persisted and remained active for at least 10 h under the conditions of sourdough fermentation. Because all of the rutin molecules were degraded during the sourdough and bread dough fermentation and rising, we cannot establish what occurs to the rutin molecules during the baking. This is different to TB yeasted bread, where some rutin persists in the dough during rising and appears in the bread even after baking (Vogrin ci c et al., 2010). The type of sugar moiety is an important determinant for the intestinal uptake of dietary quercetin glycosides (Arts et al., 2004). It has also been reported that quercetin from the diet is widely distributed in animal tissues (De Boer et al., 2005). As far as is known, quercetin is an anti-oxidant with similar bioavailability and similar health-maintaining effects to rutin (Sikder et al., 2014).
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From this standpoint, the transition of all of the rutin and the appearance of this as quercetin in sourdough bread are acceptable. Indeed, the improved content of quercetin is a welcome addition to the other substances in sourdough bread that have healthpromoting effects. 4. Conclusions We have shown that the combined effects of sourdough fermentation and the baking process preserve quercetin, but not rutin, in CB and TB bread doughs and the sourdough bread. During sourdough fermentation, there was conversion of rutin to quercetin. In the CB samples, the anti-oxidant activity increased from flour to sourdough, and then further to bread dough and sourdough bread. In contrast, in the TB samples, the highest anti-oxidant activity was for the flour and sourdough, and the lowest was for the sourdough bread. Despite its quercetin content, the TB sourdough bread had a lower anti-oxidant activity than the CB bread. The finding that quercetin remains in the TB sourdough bread is important for consideration of the potential importance of TB sourdough bread products in health-preserving nutrition. The molecular and structural bases of the differences in the specific loaf volumes of the CB and TB sourdough breads, and the possible impact of the quercetin content on loaf volume, and antioxidant activity, are suggested lines for further research. Acknowledgements This study was financed by the Slovenian Research Agency, through programmes P1-0143 “Biology of Plants” (P1-0212) and P3-0395 “Nutrition and Public Health”, and projects L4-7552 and J4-5524, supported by EUFORINNO 7th FP EU Infrastructure Programme (RegPot No. 315982). The research leading to these results has received funding from the European Community under project ITEM 26220220180: Building Research Centre “AgroBioTech”. The authors are grateful to Dragan Abram for skilful technical assistance. References AACC International, 2008. Approved Methods of the American Association of Cereal Chemistry, eleventh ed. American Association of Cereal Chemists, St Paul. Arts, I.C.W., Sesink, A.L.A., Faassen-Petersa, M., Hollman, P.C.H., 2004. The type of sugar moiety is a major determinant of the small intestinal uptake and subsequent biliary excretion of dietary quercetin glycosides. Brit. J. Nutr. 91 (6), 841e847. Bonafaccia, G., Marocchini, M., Kreft, I., 2003. Composition and technological properties of the flour and bran from common and tartary buckwheat. Food Chem. 80, 9e15. Cao, W., Chen, W.J., Suo, Z.R., Yao, Y.P., 2008. Protective effect of ethanolic extracts of buckwheat groats on DNA damage caused by hydroxyl radicals. Food Res. Int. 41, 924e929. CIE International Commission on Illumination, 1986. Publication CIE, n.15.2 Wien, Austria. De Boer, V.C.J., Dihal, A.A., Van der Woude, H., Arts, I.C.W., Wolffram, S., Alink, G.M., Rietjens, I.M., Keijer, J., Hollman, P.C., 2005. Tissue distribution of quercetin in rats and pigs. J. Nutr. 135 (7), 1718e1725. Fabjan, N., Rode, J., Kosir, I.J., Wang, Z., Zhang, Z., Kreft, I., 2003. Tartary buckwheat (Fagopyrum tataricum Gaertn.) as a source of dietary rutin and quercitrin. J. Agric. Food Chem. 51 (22), 6452e6455. Goderis, B., Putseys, A., Gommes, C.J., Bosmans, G.M., Delcour, J.A., 2014. The structure and thermal stability of amylose-lipid complexes: a case study on amylose-glycerol monostearate. Crys. Grow. Des. 14 (7), 3221e3233. Holasova, M., Fiedlerova, V., Smrcinova, H., Orsak, M., Lachman, J., Vavreinova, S., 2002. Buckwheat e the source of antioxidant activity in functional foods. Food Res. Int. 35, 207e211. Houben, A., Hochstotter, A., Becker, T., 2012. Possibilities to increase the quality in gluten-free bread production: an overview. Eur. Food Res. Tech. 235 (2), 195e208. Michalska, A., Amigo Benavent, M., Zielinski, H., Dolores del Castillo, M., 2008. Effect of bread making on formation of Maillard reaction products contributing to the overall antioxidant activity of rye bread. J. Cereal Sci. 48 (1), 123e132.
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