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Starch and protein analysis of wheat bread enriched with phenolics-rich sprouted wheat flour
Accepted Manuscript Starch and protein analysis of wheat bread enriched with phenolics-rich sprouted wheat flour Michał Świeca, Dariusz Dziki, Urszula Gawlik-Dziki PII: DOI: Reference:
S0308-8146(17)30242-X http://dx.doi.org/10.1016/j.foodchem.2017.02.052 FOCH 20605
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
2 September 2016 12 January 2017 11 February 2017
Please cite this article as: Świeca, M., Dziki, D., Gawlik-Dziki, U., Starch and protein analysis of wheat bread enriched with phenolics-rich sprouted wheat flour, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/ j.foodchem.2017.02.052
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Starch and protein analysis of wheat bread enriched with phenolics-rich sprouted wheat flour Running title: Bread with sprouted wheat flour Michał Świeca*1, Dariusz Dziki2, Urszula Gawlik-Dziki1 1
Department of Biochemistry and Food Chemistry, University of Life Sciences, Skromna Str.
8, 20-704 Lublin, Poland 2
Department of Thermal Technology, University of Life Sciences, Doświadczalna Str. 44,
20-280, Lublin, Poland. *Corresponding author: Department of Biochemistry and Food Chemistry, University of Life Sciences, Skromna Str. 8, 20-704 Lublin, Poland; Tel.: +48-81-4623327; fax: +48-814623324; email: [email protected]
Abstract Wheat flour in the bread formula was replaced with sprouted wheat flour (SF) characterized by enhanced nutraceutical properties, at 5%, 10%, 15% and 20% levels. The addition of SF slightly increased the total protein content; however, it decreased their digestibility. Some qualitative and quantitative changes in the electrophoretic pattern of proteins were also observed; especially, in the bands corresponding with 27 kDa and 15–17 kDa proteins. These results were also confirmed by SE-HPLC technique, where a significant increase in the content of proteins and peptides (molecular masses <20 kDa) was determined for breads with 20% of SF. Bread enriched with sprouted wheat flour had more resistant starch, but less total starch, compared to control bread. The highest in vitro starch digestibility was determined for the control bread. The studied bread with lowered nutritional value but increased nutritional quality can be used for special groups of consumers (obese, diabetic).
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Keywords: sprouted flour; bread; in vitro digestibility; nutritional quality; protein; starch; wheat. 1. Introduction Bread, pasta, and other wheat-based food products are commonly consumed in welldeveloped countries (Rebello, Greenway, & Finley, 2014). These products are of high nutritional value (60%–75% of starch, 10%–15% of proteins) (Troccoli, Borrelli, De Vita, Fares, & Di Fonzo, 2000) and also contain some pro-health components such as phenolics, phytic acid, and dietary fiber. However, their content is rather low compared to fruits, nuts, and vegetables (Yu, Haley, Perret, & Harris, 2002). Attributing to its widespread consumption, wheat bread could be improved as an excellent carrier of pro-health constituents. So far, wheat bread was improved by the introduction of phenolic-rich materials (Dziki, Różyło, Gawlik-Dziki, & Świeca, 2014; Andersen, Koehler, & Somoza, 2011), dietary fiber (Dhingra & Jood, 2002), or exogenous proteins (Dhingra & Jood, 2002) into its formula. Besides desirable pro-health effect, such components may influence on the bioaccessibility and bioavailability of nutrients. Functional ingredients (e.g. phenolics) may limit the digestibility of nutrients by interacting with bread matrix and digestive enzymes. Such effect was described for wheat products enriched with onion skin (Swieca, Gawlik-Dziki, Dziki, Baraniak, & Czyż, 2013) and parsley leaves (Łukasz Sęczyk, Świeca, Gawlik-Dziki, Luty, & Czyż, 2016), and isoflavone-rich soybean flour (Dhingra & Jood, 2002). On the other hand, supplements may enhance the nutritional value of the products, which was observed in the case of addition of carob flour (Łukasz Sęczyk, Świeca, & Gawlik-Dziki, 2016) or acha and bambara nut flour (Chinma et al., 2016). The quality of wheat flour is strongly determined by genetic factors (variety) and breeding conditions (fertilization and weather conditions) (Troccoli et al., 2000). Some studies indicate that quality of wheat flour can be improved by sprouting (Žilić et al., 2014). It
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is important when the wheat grains are of low quality and if they cannot be directly used for milling (Dziki, Gawlik-Dziki, Różyło, & Miś, 2015). Compared to normal wheat flour, sprouted flour contains higher amounts of free sugars, amino acids and peptides, and minerals; bioaccessibility is improved and carbohydrate fraction contains much more resistant starch (Noda et al., 2004; Žilić et al., 2014; van Hung, Maeda, Yamamoto, & Morita, 2012; Andersen et al., 2011). Sprouted flour also consists of higher amount of phenolics, especially those present in free fraction (Žilić et al., 2014). Additionally, low-molecular antioxidants, mainly responsible for antioxidant properties, in sprouted wheat flour may be significantly increased by elicitation (Świeca & Dziki, 2015). In the previous screening studies the conditions for wheat sprouting (Dziki, Gawlik-Dziki, Kordowska-Wiater, & Domań-Pytka, 2015; Gawlik-Dziki et al., 2016), which allowed to optimally increase nutraceutical potential of sprouted flour, were selected. The addition of that sprouted wheat flour significantly improved phenolics content (an increase by about 20% compared to the control) and antioxidant potential (especially free radical scavenging and lipids protecting capacities and chelating power) of bread without any negative influence on consumer acceptance and physical quality of loaf (Gawlik-Dziki, Dziki, Świeca, & Sęczyk, 2015). This study was aimed to check if the addition of sprouted wheat flour, obtained after induction of natural mechanisms of seedling resistance with willow bark infusion, to the wheat bread flour has any negative effect on the nutritional quality of product. A special emphasis was placed on the changes in proteins pattern and nutrients digestibility.
2. Materials and methods 2.1.
Chemicals used Amyloglucosidase, invertase, dinitrosalicylic acid, α-amylase, pancreatin, pepsin,
bile extract, Bradford reagent and TNBS (2,4,6-trinitrobenzenesulfonic acid) were
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purchased from Sigma-Aldrich (Poznan, Poland). All other chemicals were of analytical grade. 2.2.
Preparation of sprouted wheat flour Wheat seeds (Triticum aestivum, ssp. vulgare var. Bogatka) were purchased from
Lublin Agricultural Advisory Center in Końskowola. Seeds were sterilized in 1% (v/v) sodium hypochlorite for 10 min, then drained and washed with distilled water until they reached neutral pH. Then, the seeds were placed in 0.1% (v/v) Salix daphnoides bark infusion (SD). Bark of a willow Salix daphnoides, obtained from an ecological farm in Poland, was dried and pulverized in the laboratory mill and extracted with boiling water. Seeds were germinated for 4 days in dark, in a growth chamber on Petri dishes (φ 125 mm) lined with absorbent paper (approximately 400 seeds per dish) at 20°C. Seedlings were watered daily with 5 mL of Milli-Q water (Świeca & Dziki, 2015). The seedlings were dried at 80°C up to moisture level 14% on wet basis (wb), ground (particles below 0.2 mm), and stored for further analysis and bread supplementation. 2.3.
Bread making and sample preparation The flour used in the formula of control bread (C) was wheat flour (600 g), type 750
(average 0.75 g/100 g ash content, humidity 14%). The regular flour was replaced with sprouted wheat flour (SF) at 5, 10, 15, and 20 g/100 g levels (B1–B4, respectively). Apart from this 6 g of instant yeast and 12 g of salt were used for dough preparation. The general quantity of water necessary for the preparation of the dough was established through the marking of water absorption properties in the flour at the consistency of 350 Brabender units. The batches of dough were mixed in a spiral mixer for 6 min. The fermentation was performed at 30° C and 75% RH for 60 min (with 1 min transfixion after 30 min). Pieces of dough were molded by hand, panned, and proofed at 30°C and 75% RH over the period required for optimal dough development. After fermentation, the pieces of dough (300 g) were kept in the
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oven at 230°C for 30 min. The bread after baking was left to stand for 24 hours at room temperature. The breads were sliced (slices of about 1.5 cm thick). The crust was removed aseptically and kept frozen (at –20°C) until analysis. After thawing, the slices were dried and then manually crumbed, grounded in a mill, and screened through 0.5 mm sieve to obtain bread powder. 2.4.
In vitro digestion In vitro digestion was performed as described by Świeca, Baraniak, & Gawlik-Dziki
(2013). 2.5. Analysis of starch 2.5.1. Total and potentially bioavailable starch Total starch (TS) content in flour and bread was determined after dispersion of the starch granules in 2 M KOH according to Goñi, Garcia-Alonso, & Saura-Calixto (1997). Glucose content was determined by using the standard dinitrosalicylic acid (DNSA) method (Miller, 1959). Total starch was calculated as glucose × 0.9. The free reducing sugar and sucrose contents of the samples were determined to correct the obtained total starch values. For sucrose assay, the samples dispersed in sodium acetate buffer at pH 5.0 were treated with 200 µL of (10 mg in 1 mL of 0.4 M sodium acetate buffer, pH 5.0) invertase (EC 3.2.1.26; 300 U/ mg) for 30 min at 37°C. After centrifugation, reducing sugars were analyzed in the supernatants, using the DNSA reagent (Miller, 1959). The resistant starch (RS) and potentially bioavailable (AS) starch content were analyzed on the basis of the results obtained after simulated gastrointestinal digestion (Świeca et al., 2013). 2.5.2. In vitro starch digestibility The in vitro digestibility of starch was evaluated on the basis of total starch content (TS) and resistant starch (RS) determined after in vitro digestion (Świeca et al., 2013). 2.5.3. Relative digestibility of starch
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Relative digestibility of starch was determined according to the methods previously reported by Sęczyk, Świeca, & Gawlik-Dziki (2016). Results were expressed as fold change with respect to regular wheat flour (WF). 2.6. Analysis of protein 2.6.1. Protein content The protein content of the fractions was determined by the Bradford method (1976), using bovine serum albumin or wheat gluten as standard proteins. 2.6.2. Protein isolation and fractionation Different protein fractions (glutelins + gliadins, albumins + globulins, CM-proteins) and total proteins were isolated according to the procedure described by Hurkman and Tanaka (2007). 2.6.3. In vitro protein digestibility The in vitro digestibility of protein (PD) was evaluated on the basis of protein content before digestion (TP—sum of protein fractions determined according to the procedure described in the 2.6.2.) and after digestion in vitro (RP). RP PD[%] = 100% − × 100% TP For the determination of content of proteins after digestion procedure, pellets were
isolated according to procedure described by Hurkman & Tanaka (2007). Albumins and globulins content was described as the proteins present in supernatants obtained after in vitro digestion (correction for components of digestive system was performed). Proteins after in vitro digestion (RP) were determined as the sum of all fractions. 2.6.4. Relative digestibility of protein Relative digestibility of protein was determined according to the methods previously reported by Sęczyk, Świeca, & Gawlik-Dziki (2016). Results were expressed as fold change with respect to regular wheat flour (WF). 6
2.6.5. Electrophoresis Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using 12.5% acrylamide (w/v) (Laemlli, 1970). Samples were treated in denaturing electrophoretic buffer with SDS and β-mercaptoethanol (60 mM Tris-HCl pH 6.8, 10% glycerol (v/v), 0.025% Coomassie Brilliant Blue R (w/v); 2% SDS (w/v), 0.1 M βmercaptoethanol) and heated before SDS-PAGE. In total, 25 µL of protein extract was loaded on the gel. The run was performed in 1x TBE (Tris-borate-EDTA) buffer at a constant voltage of 200 V. Gels were stained with 0.2% Coomassie Brilliant Blue R (w/v) and destained in 50% methanol/10% acetic acid (v/v). Electropherograms analysis and quantification of the bands was carried out with Polydoc, Molecular Imaging System, Vilber Lourmat supplied with software PhotoCapt. 2.6.6. Size exclusion high performance liquid chromatography Flour and bread protein were characterized with size exclusion high performance liquid chromatography (SE-HPLC) according to the method of Swieca et al. (2013), by using a Varian ProStar HPLC System separation module (Varian, Palo Alto, USA) equipped with a column (COSMOSIL 5Diol-20-II Packed Column 7.5 mm ID x 300 mm) and ProStar DAD detector. The column thermostat was set at 30°C. Fifty microliters of each sample solution was loaded on the column, and proteins were eluted using a PBS buffer (phosphate buffered saline), pH 7.4. The flow rate was 0.8 mL/min. Ultraviolet detection was performed at a wavelength of 280 nm. 2.7.
Statistical analysis
All experimental results were mean ± SD of three independent experiments (n = 9). One-way analysis of variance (ANOVA) and Tukey’s post-hoc test were used to compare groups. (STATISTICA 6, StatSoft Inc., Tulsa, USA). Differences were considered significant at α<0.05.
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3.
Results and discussion Sprouting did not influence significantly on the protein content of the obtained flours;
however, small differences were found in the glutelins/gliadins fraction (Table 1). Electrophoretic profiles of total protein extracted from wheat flour and sprouted wheat flour showed that the high molecular weight glutenin subunits and the α- and β-gliadins were effectively mobilized during germination (Fig. 1). Compared to wheat flours, the increase in relative volume of band corresponding to protein with molecular masses of about 29–38 kDa and 15–17 kDa was also determined in sprouted flour. Main qualitative differences included the 27 kDa band - in the case of wheat flour it was undetectable. After the addition of sprouted wheat flour into breads, some qualitative and quantitative changes in the electrophoretic pattern of proteins, resulting from the composition of protein fraction of sprouted wheat flour, were observed. It was especially visible for bands corresponding with proteins of molecular masses 27 kDa and 15–17 kDa. It is difficult to compare the wheat flour/bread susceptibility to digestive enzymes (digestibility), due to differences in enzyme concentration and the time of hydrolysis in the subsequent steps of simulated digestion. Results obtained in this study, are in agreement with those presented by Abdel-Aal (2008) for spelt flour and spelt bread obtained after two-step digestion with pepsin and pancreatin. The addition of sprouted wheat flour into bread formula slightly increased the total protein content and decreased protein digestibility by about 13% and 5%, respectively when C and B4 breads were compared (Table 1). These results were partially confirmed from the study of relative protein digestibility (Fig. 2). Sprouted wheat flour, containing higher levels of phenolics (Dziki, Gawlik-Dziki, Kordowska-Wiater, et al., 2015), decreased the digestibility of proteins in supplemented breads. It may be speculated that this phenomenon was caused by the interactions of phenolics with digestive enzymes and/or other proteins (Sęczyk, Świeca, & Gawlik-Dziki, 2015; Labuckas, Maestri, Perelló,
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Martínez, & Lamarque, 2008), which was also reported for wheat bread enriched with phenolic-rich materials, e.g. onion skin (Swieca et al., 2013) and quinoa leaves (Swieca et al., 2014). Changes in the protein digestibility, regardless of the method used for its determination, did not exceed 10%. According to SE-HPLC analysis of samples obtained after digestion in vitro it was found that total area of chromatograms (compared to the control) obtained for B2 and B4 samples were bigger by 1.6% and 11.2%, respectively (Fig. 3). These samples were also characterized by higher content of proteins and peptides with molecular masses <20 kDa (see III, Fig. 3). The addition of sprouted wheat flour to bread formula significantly increased the content of low molecular masses proteins (20–6.5 kDa) and peptides (<6.5 kDa). These observations are supported by the studies of Pasini et al. (2001), which explains that high molecular weight wheat proteins were effectively digested by pepsin and pancreatin. On the other hand, the chromatogram obtained for the control bread showed a clearly visible increase of area <6.5 kDa (RT 34 min.). It may be suggested that high molecular weight wheat proteins were most effectively digested in the control bread, according to the digestibility studies that bread was characterized as highly digestible. Sprouting improves protein digestibility, but growing plant utilize most of the available proteins and those that are still present in sprouts can be less susceptible to the action of digestive enzymes (Świeca & Baraniak, 2014). This effect may also be enhanced by their interaction with phenolics (Swieca et al., 2013). Bread from wheat flour (C) had more total starch (TS), but less resistant starch (RS) compared to the bread enriched with sprouted wheat flour (Table 2). The difference might be because the material used for bread enrichment contained significantly more (by about 2 times more) resistant starch than wheat flour. During sprouting, starch is effectively mobilized and its content is usually significantly lower compared to the dormant seeds (Noda et al., 2004). These metabolic changes, occurring in germinated grains, are also responsible
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for the significant increase of free sugars content (Marton, Mandoki, Csapo-Kiss, & Csapo, 2010). Compared to wheat flour, in vitro starch digestibility of sprouted wheat flour was significantly lower (Table 2). The RS content obtained from the bread enriched with sprouted wheat flours showed that these products are excellent sources of RS. The highest in vitro starch digestibility was determined for the control bread. Breads with the addition of sprouted wheat flour were characterized with lowered digestibility, as was to be expected. These results were confirmed by the relative digestibility of starch - the highest digestibility was also determined for the control bread and further replacement of wheat flour with functional flour caused the decrease of this parameter (Fig. 2). There are many evidences that sprouting improves the bioaccessibility of sugars (Marton et al., 2010), but paradoxically starches are less susceptible to enzymes. This usually arises from the increased content of RS. Additionally, it may be suggested that interaction of phenolics with the enzymes of digestive tract and starch may play an important role, as was previously reported by Rohn, Rawel, & Kroll (2002) and Mkandawire et al. (2013). Sprouted wheat flour contained about 9 times more free reducing sugars (compared to wheat flour), and its addition to bread formula significantly increased the reducing sugar content of the functional bread. Conclusion In sum, the partial replacement (5%–20%) of wheat flour with functional sprouted wheat flour containing increased content of phenolics slightly increased the protein content and significantly decreased the starch digestibility. According to the results it may be stated that the studied bread with lowered nutritional value and increased nutraceutical quality may be of use for special groups of consumers (obese, and diabetic).
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Acknowledgments This study was partially financed by the Polish National Science Centre (grant 2012/07/B/NZ9/02463).
Conflict of interest statement The authors declare no conflict of interest.
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Figure captions Fig. 1. Electrophoretic analysis of proteins from flours and enriched bread Numbered brackets indicate region A, high molecular weight-glutenin subunits; region B, ωgliadins; region C, the α- and β-gliadins and low molecular weight-glutenin subunits; and region D, low molecular weight-albumins and globulins. WF- wheat flour, SF- sprouted wheat flour, C- control bread, B1-B4 - breads with 5-20% sprouted wheat flour; MM-molecular mass [kDa]. Fig. 2. Free reducing sugars, amino acids and peptides and relative digestibility of nutrients in flours and breads WF- wheat flour, SF- sprouted wheat flour, C- control bread, B1-B4- breads with 5%-20% sprouted wheat flour. The values are expressed as mean ± SD (n=9). The values within the same parameter designated by different letters are significantly different (α < 0.05). Fig. 3. Absorbance profiles of the eluates for the extracts after in vitro digestion of control and enriched bread obtained from size-exclusion chromatography. The absorbance profiles of the eluates for the extracts after in vitro digestion of control 624 and enriched bread obtained for size-exclusion chromatography. C – control bread, B2- bread with 10% of sprouted wheat flour ; B4- bread with 20% of sprouted wheat flour; DTC-components of digestive tract. Molecular mass markers (kDa): a. 102; b. 42; c. 35; d. 22; e. 18; f. 6.5; g. 3.
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10 11 16 500
B4 14 500
Fig. 1. Electrophoretic analysis of proteins from flours and enriched bread Numbered brackets indicate region A, high molecular weight-glutenin subunits; region B, ωgliadins; region C, the α- and β-gliadins and low molecular weight-glutenin subunits; and region D, low molecular weight-albumins and globulins. WF- wheat flour, SF- sprouted wheat flour, C- control bread, B1-B4 - breads with 5%-20% sprouted wheat flour; MM-molecular mass [kDa].
17
20
Relative starch digestibility Relative proteins digestibility Free reducing sugars Free amino acids and peptides
[fold change in respect to WF]
18 16
e
e e d
14 12 c
10 8
b
f
e
6 4 2
d b aa
c
e
b
a
d
de b
c
cd
c b
b c
b b
a
0 WF
SF
C
B1
B2
B3
B4
Sample Fig. 2. Free reducing sugars, amino acids and peptides and relative digestibility of nutrients in flours and breads WF- wheat flour, SF- sprouted wheat flour, C- control bread, B1-B4 - breads with 5%-20% sprouted wheat flour. The values are expressed as mean ± SD (n=9). The values within the same parameter designated by different letters are significantly different (α < 0.05).
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Fig. 3. Absorbance profiles of the eluates for the extracts after in vitro digestion of control and enriched bread obtained from size-exclusion chromatography. C – control bread, B2- bread with 10% of sprouted wheat flour ; B4- bread with 20% of sprouted wheat flour; DTC-components of digestive tract. Molecular mass markers (kDa): a. 102; b. 42; c. 35; d. 22; e. 18; f. 6.5; g. 3. Regions: I: < 25 kDa; II: >25 - <20 kDa; III: < 20 kDa.
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Table 1. Content and digestibility of proteins from flours and breads
Sample
Glutelins+ Gliadyns [mg/ g DW]
Albumins+ Globulins [mg/ g DW]
CM-proteins [mg/ g DW]
Total Proteins [mg/ g DW]
In vitro protein digestibility [%]
WF
86.63±1.51b
20.73±0.31c
19.43±0.23a
126.79±1.82bc
51.74±1.33abc
SF
90.68±2.06c
20.84±0.15c
18.89±0.22a
130.40±1.20cd
51.06±0.44b
C
79.73±2.39a
20.05±0.17b
19.01±0.17a
118.79±2.47a
52.55±0.36c
B1
86.35±2.95b
19.56±0.22ab
18.74±0.24a
124.66±2.85ab
52.31±0.26c
B2
94.73±3.03bc
19.22±0.37ab
19.45±0.14a
133.40±2.89d
50.03±0.43a
B3
96.43±2.47c
19.86±0.44ab
18.93±0.20a
135.22±2.72d
49.82±0.26a
B4
96.79±1.91c
19.49±0.16a
18.59±0.18a
134.87±1.84d
50.05±0.27a
WF- wheat flour, SF- sprouted wheat flour, C- control bread, B1-B4 - breads with 5%-20% sprouted wheat flour. The values are expressed as mean ± SD (n= 9). The values designated by the different letters are significantly different (α< 0.05).
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Table 2. Content and digestibility of starch from flours and breads Potentially Potentially In vitro bioavailable resistant starch Sample starch starch digestibility [mg/ g DW] [% of TS] [%] 33.5±0.56c WF 651.8±11.32c 433.2±17.1e 66.46±2.63c 68.3±0.26e SF 588.4±7.66b 186.6±2.50a 31.72±1.43a 27.0±0.58a C 594.0±10.43b 433.9±11.8e 73.05±1.99c 30.8±0.05b B1 491.4±8.99a 340.0±6.49d 69.19±2.32c 30.8±0.91bc B2 477.8±10.15a 300.5±15.5c 67.10±3.46c 33.7±1.02c B3 489.9±2.09a 264.6±3.5b 54.42±5.72b 36.6±1.12d B4 487.4±2.35a 269.0±10.1b 55.19±4.08b WF- wheat flour, SF- sprouted wheat flour, C- control bread, B1-B4 - breads with 5%-20% Total starch [mg/ g DW]
sprouted wheat flour. The values are expressed as mean ± SD (n= 9). The values designated by the different letters are significantly different (α< 0.05).
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1. Sprouted wheat flour (SF) was introduced to the wheat bread formula 2. The introduction of SF slightly increased the total protein content. 3. The addition of SF increased peptides and small proteins (<20 kDa) in breads. 4. Enriched breads were characterized by higher content of resistant starch. 5. Partial replacement of wheat flour with SF decreased the starch digestibility.
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S0308-8146(17)30242-X http://dx.doi.org/10.1016/j.foodchem.2017.02.052 FOCH 20605
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
2 September 2016 12 January 2017 11 February 2017
Please cite this article as: Świeca, M., Dziki, D., Gawlik-Dziki, U., Starch and protein analysis of wheat bread enriched with phenolics-rich sprouted wheat flour, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/ j.foodchem.2017.02.052
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Starch and protein analysis of wheat bread enriched with phenolics-rich sprouted wheat flour Running title: Bread with sprouted wheat flour Michał Świeca*1, Dariusz Dziki2, Urszula Gawlik-Dziki1 1
Department of Biochemistry and Food Chemistry, University of Life Sciences, Skromna Str.
8, 20-704 Lublin, Poland 2
Department of Thermal Technology, University of Life Sciences, Doświadczalna Str. 44,
20-280, Lublin, Poland. *Corresponding author: Department of Biochemistry and Food Chemistry, University of Life Sciences, Skromna Str. 8, 20-704 Lublin, Poland; Tel.: +48-81-4623327; fax: +48-814623324; email: [email protected]
Abstract Wheat flour in the bread formula was replaced with sprouted wheat flour (SF) characterized by enhanced nutraceutical properties, at 5%, 10%, 15% and 20% levels. The addition of SF slightly increased the total protein content; however, it decreased their digestibility. Some qualitative and quantitative changes in the electrophoretic pattern of proteins were also observed; especially, in the bands corresponding with 27 kDa and 15–17 kDa proteins. These results were also confirmed by SE-HPLC technique, where a significant increase in the content of proteins and peptides (molecular masses <20 kDa) was determined for breads with 20% of SF. Bread enriched with sprouted wheat flour had more resistant starch, but less total starch, compared to control bread. The highest in vitro starch digestibility was determined for the control bread. The studied bread with lowered nutritional value but increased nutritional quality can be used for special groups of consumers (obese, diabetic).
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Keywords: sprouted flour; bread; in vitro digestibility; nutritional quality; protein; starch; wheat. 1. Introduction Bread, pasta, and other wheat-based food products are commonly consumed in welldeveloped countries (Rebello, Greenway, & Finley, 2014). These products are of high nutritional value (60%–75% of starch, 10%–15% of proteins) (Troccoli, Borrelli, De Vita, Fares, & Di Fonzo, 2000) and also contain some pro-health components such as phenolics, phytic acid, and dietary fiber. However, their content is rather low compared to fruits, nuts, and vegetables (Yu, Haley, Perret, & Harris, 2002). Attributing to its widespread consumption, wheat bread could be improved as an excellent carrier of pro-health constituents. So far, wheat bread was improved by the introduction of phenolic-rich materials (Dziki, Różyło, Gawlik-Dziki, & Świeca, 2014; Andersen, Koehler, & Somoza, 2011), dietary fiber (Dhingra & Jood, 2002), or exogenous proteins (Dhingra & Jood, 2002) into its formula. Besides desirable pro-health effect, such components may influence on the bioaccessibility and bioavailability of nutrients. Functional ingredients (e.g. phenolics) may limit the digestibility of nutrients by interacting with bread matrix and digestive enzymes. Such effect was described for wheat products enriched with onion skin (Swieca, Gawlik-Dziki, Dziki, Baraniak, & Czyż, 2013) and parsley leaves (Łukasz Sęczyk, Świeca, Gawlik-Dziki, Luty, & Czyż, 2016), and isoflavone-rich soybean flour (Dhingra & Jood, 2002). On the other hand, supplements may enhance the nutritional value of the products, which was observed in the case of addition of carob flour (Łukasz Sęczyk, Świeca, & Gawlik-Dziki, 2016) or acha and bambara nut flour (Chinma et al., 2016). The quality of wheat flour is strongly determined by genetic factors (variety) and breeding conditions (fertilization and weather conditions) (Troccoli et al., 2000). Some studies indicate that quality of wheat flour can be improved by sprouting (Žilić et al., 2014). It
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is important when the wheat grains are of low quality and if they cannot be directly used for milling (Dziki, Gawlik-Dziki, Różyło, & Miś, 2015). Compared to normal wheat flour, sprouted flour contains higher amounts of free sugars, amino acids and peptides, and minerals; bioaccessibility is improved and carbohydrate fraction contains much more resistant starch (Noda et al., 2004; Žilić et al., 2014; van Hung, Maeda, Yamamoto, & Morita, 2012; Andersen et al., 2011). Sprouted flour also consists of higher amount of phenolics, especially those present in free fraction (Žilić et al., 2014). Additionally, low-molecular antioxidants, mainly responsible for antioxidant properties, in sprouted wheat flour may be significantly increased by elicitation (Świeca & Dziki, 2015). In the previous screening studies the conditions for wheat sprouting (Dziki, Gawlik-Dziki, Kordowska-Wiater, & Domań-Pytka, 2015; Gawlik-Dziki et al., 2016), which allowed to optimally increase nutraceutical potential of sprouted flour, were selected. The addition of that sprouted wheat flour significantly improved phenolics content (an increase by about 20% compared to the control) and antioxidant potential (especially free radical scavenging and lipids protecting capacities and chelating power) of bread without any negative influence on consumer acceptance and physical quality of loaf (Gawlik-Dziki, Dziki, Świeca, & Sęczyk, 2015). This study was aimed to check if the addition of sprouted wheat flour, obtained after induction of natural mechanisms of seedling resistance with willow bark infusion, to the wheat bread flour has any negative effect on the nutritional quality of product. A special emphasis was placed on the changes in proteins pattern and nutrients digestibility.
2. Materials and methods 2.1.
Chemicals used Amyloglucosidase, invertase, dinitrosalicylic acid, α-amylase, pancreatin, pepsin,
bile extract, Bradford reagent and TNBS (2,4,6-trinitrobenzenesulfonic acid) were
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purchased from Sigma-Aldrich (Poznan, Poland). All other chemicals were of analytical grade. 2.2.
Preparation of sprouted wheat flour Wheat seeds (Triticum aestivum, ssp. vulgare var. Bogatka) were purchased from
Lublin Agricultural Advisory Center in Końskowola. Seeds were sterilized in 1% (v/v) sodium hypochlorite for 10 min, then drained and washed with distilled water until they reached neutral pH. Then, the seeds were placed in 0.1% (v/v) Salix daphnoides bark infusion (SD). Bark of a willow Salix daphnoides, obtained from an ecological farm in Poland, was dried and pulverized in the laboratory mill and extracted with boiling water. Seeds were germinated for 4 days in dark, in a growth chamber on Petri dishes (φ 125 mm) lined with absorbent paper (approximately 400 seeds per dish) at 20°C. Seedlings were watered daily with 5 mL of Milli-Q water (Świeca & Dziki, 2015). The seedlings were dried at 80°C up to moisture level 14% on wet basis (wb), ground (particles below 0.2 mm), and stored for further analysis and bread supplementation. 2.3.
Bread making and sample preparation The flour used in the formula of control bread (C) was wheat flour (600 g), type 750
(average 0.75 g/100 g ash content, humidity 14%). The regular flour was replaced with sprouted wheat flour (SF) at 5, 10, 15, and 20 g/100 g levels (B1–B4, respectively). Apart from this 6 g of instant yeast and 12 g of salt were used for dough preparation. The general quantity of water necessary for the preparation of the dough was established through the marking of water absorption properties in the flour at the consistency of 350 Brabender units. The batches of dough were mixed in a spiral mixer for 6 min. The fermentation was performed at 30° C and 75% RH for 60 min (with 1 min transfixion after 30 min). Pieces of dough were molded by hand, panned, and proofed at 30°C and 75% RH over the period required for optimal dough development. After fermentation, the pieces of dough (300 g) were kept in the
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oven at 230°C for 30 min. The bread after baking was left to stand for 24 hours at room temperature. The breads were sliced (slices of about 1.5 cm thick). The crust was removed aseptically and kept frozen (at –20°C) until analysis. After thawing, the slices were dried and then manually crumbed, grounded in a mill, and screened through 0.5 mm sieve to obtain bread powder. 2.4.
In vitro digestion In vitro digestion was performed as described by Świeca, Baraniak, & Gawlik-Dziki
(2013). 2.5. Analysis of starch 2.5.1. Total and potentially bioavailable starch Total starch (TS) content in flour and bread was determined after dispersion of the starch granules in 2 M KOH according to Goñi, Garcia-Alonso, & Saura-Calixto (1997). Glucose content was determined by using the standard dinitrosalicylic acid (DNSA) method (Miller, 1959). Total starch was calculated as glucose × 0.9. The free reducing sugar and sucrose contents of the samples were determined to correct the obtained total starch values. For sucrose assay, the samples dispersed in sodium acetate buffer at pH 5.0 were treated with 200 µL of (10 mg in 1 mL of 0.4 M sodium acetate buffer, pH 5.0) invertase (EC 3.2.1.26; 300 U/ mg) for 30 min at 37°C. After centrifugation, reducing sugars were analyzed in the supernatants, using the DNSA reagent (Miller, 1959). The resistant starch (RS) and potentially bioavailable (AS) starch content were analyzed on the basis of the results obtained after simulated gastrointestinal digestion (Świeca et al., 2013). 2.5.2. In vitro starch digestibility The in vitro digestibility of starch was evaluated on the basis of total starch content (TS) and resistant starch (RS) determined after in vitro digestion (Świeca et al., 2013). 2.5.3. Relative digestibility of starch
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Relative digestibility of starch was determined according to the methods previously reported by Sęczyk, Świeca, & Gawlik-Dziki (2016). Results were expressed as fold change with respect to regular wheat flour (WF). 2.6. Analysis of protein 2.6.1. Protein content The protein content of the fractions was determined by the Bradford method (1976), using bovine serum albumin or wheat gluten as standard proteins. 2.6.2. Protein isolation and fractionation Different protein fractions (glutelins + gliadins, albumins + globulins, CM-proteins) and total proteins were isolated according to the procedure described by Hurkman and Tanaka (2007). 2.6.3. In vitro protein digestibility The in vitro digestibility of protein (PD) was evaluated on the basis of protein content before digestion (TP—sum of protein fractions determined according to the procedure described in the 2.6.2.) and after digestion in vitro (RP). RP PD[%] = 100% − × 100% TP For the determination of content of proteins after digestion procedure, pellets were
isolated according to procedure described by Hurkman & Tanaka (2007). Albumins and globulins content was described as the proteins present in supernatants obtained after in vitro digestion (correction for components of digestive system was performed). Proteins after in vitro digestion (RP) were determined as the sum of all fractions. 2.6.4. Relative digestibility of protein Relative digestibility of protein was determined according to the methods previously reported by Sęczyk, Świeca, & Gawlik-Dziki (2016). Results were expressed as fold change with respect to regular wheat flour (WF). 6
2.6.5. Electrophoresis Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using 12.5% acrylamide (w/v) (Laemlli, 1970). Samples were treated in denaturing electrophoretic buffer with SDS and β-mercaptoethanol (60 mM Tris-HCl pH 6.8, 10% glycerol (v/v), 0.025% Coomassie Brilliant Blue R (w/v); 2% SDS (w/v), 0.1 M βmercaptoethanol) and heated before SDS-PAGE. In total, 25 µL of protein extract was loaded on the gel. The run was performed in 1x TBE (Tris-borate-EDTA) buffer at a constant voltage of 200 V. Gels were stained with 0.2% Coomassie Brilliant Blue R (w/v) and destained in 50% methanol/10% acetic acid (v/v). Electropherograms analysis and quantification of the bands was carried out with Polydoc, Molecular Imaging System, Vilber Lourmat supplied with software PhotoCapt. 2.6.6. Size exclusion high performance liquid chromatography Flour and bread protein were characterized with size exclusion high performance liquid chromatography (SE-HPLC) according to the method of Swieca et al. (2013), by using a Varian ProStar HPLC System separation module (Varian, Palo Alto, USA) equipped with a column (COSMOSIL 5Diol-20-II Packed Column 7.5 mm ID x 300 mm) and ProStar DAD detector. The column thermostat was set at 30°C. Fifty microliters of each sample solution was loaded on the column, and proteins were eluted using a PBS buffer (phosphate buffered saline), pH 7.4. The flow rate was 0.8 mL/min. Ultraviolet detection was performed at a wavelength of 280 nm. 2.7.
Statistical analysis
All experimental results were mean ± SD of three independent experiments (n = 9). One-way analysis of variance (ANOVA) and Tukey’s post-hoc test were used to compare groups. (STATISTICA 6, StatSoft Inc., Tulsa, USA). Differences were considered significant at α<0.05.
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3.
Results and discussion Sprouting did not influence significantly on the protein content of the obtained flours;
however, small differences were found in the glutelins/gliadins fraction (Table 1). Electrophoretic profiles of total protein extracted from wheat flour and sprouted wheat flour showed that the high molecular weight glutenin subunits and the α- and β-gliadins were effectively mobilized during germination (Fig. 1). Compared to wheat flours, the increase in relative volume of band corresponding to protein with molecular masses of about 29–38 kDa and 15–17 kDa was also determined in sprouted flour. Main qualitative differences included the 27 kDa band - in the case of wheat flour it was undetectable. After the addition of sprouted wheat flour into breads, some qualitative and quantitative changes in the electrophoretic pattern of proteins, resulting from the composition of protein fraction of sprouted wheat flour, were observed. It was especially visible for bands corresponding with proteins of molecular masses 27 kDa and 15–17 kDa. It is difficult to compare the wheat flour/bread susceptibility to digestive enzymes (digestibility), due to differences in enzyme concentration and the time of hydrolysis in the subsequent steps of simulated digestion. Results obtained in this study, are in agreement with those presented by Abdel-Aal (2008) for spelt flour and spelt bread obtained after two-step digestion with pepsin and pancreatin. The addition of sprouted wheat flour into bread formula slightly increased the total protein content and decreased protein digestibility by about 13% and 5%, respectively when C and B4 breads were compared (Table 1). These results were partially confirmed from the study of relative protein digestibility (Fig. 2). Sprouted wheat flour, containing higher levels of phenolics (Dziki, Gawlik-Dziki, Kordowska-Wiater, et al., 2015), decreased the digestibility of proteins in supplemented breads. It may be speculated that this phenomenon was caused by the interactions of phenolics with digestive enzymes and/or other proteins (Sęczyk, Świeca, & Gawlik-Dziki, 2015; Labuckas, Maestri, Perelló,
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Martínez, & Lamarque, 2008), which was also reported for wheat bread enriched with phenolic-rich materials, e.g. onion skin (Swieca et al., 2013) and quinoa leaves (Swieca et al., 2014). Changes in the protein digestibility, regardless of the method used for its determination, did not exceed 10%. According to SE-HPLC analysis of samples obtained after digestion in vitro it was found that total area of chromatograms (compared to the control) obtained for B2 and B4 samples were bigger by 1.6% and 11.2%, respectively (Fig. 3). These samples were also characterized by higher content of proteins and peptides with molecular masses <20 kDa (see III, Fig. 3). The addition of sprouted wheat flour to bread formula significantly increased the content of low molecular masses proteins (20–6.5 kDa) and peptides (<6.5 kDa). These observations are supported by the studies of Pasini et al. (2001), which explains that high molecular weight wheat proteins were effectively digested by pepsin and pancreatin. On the other hand, the chromatogram obtained for the control bread showed a clearly visible increase of area <6.5 kDa (RT 34 min.). It may be suggested that high molecular weight wheat proteins were most effectively digested in the control bread, according to the digestibility studies that bread was characterized as highly digestible. Sprouting improves protein digestibility, but growing plant utilize most of the available proteins and those that are still present in sprouts can be less susceptible to the action of digestive enzymes (Świeca & Baraniak, 2014). This effect may also be enhanced by their interaction with phenolics (Swieca et al., 2013). Bread from wheat flour (C) had more total starch (TS), but less resistant starch (RS) compared to the bread enriched with sprouted wheat flour (Table 2). The difference might be because the material used for bread enrichment contained significantly more (by about 2 times more) resistant starch than wheat flour. During sprouting, starch is effectively mobilized and its content is usually significantly lower compared to the dormant seeds (Noda et al., 2004). These metabolic changes, occurring in germinated grains, are also responsible
9
for the significant increase of free sugars content (Marton, Mandoki, Csapo-Kiss, & Csapo, 2010). Compared to wheat flour, in vitro starch digestibility of sprouted wheat flour was significantly lower (Table 2). The RS content obtained from the bread enriched with sprouted wheat flours showed that these products are excellent sources of RS. The highest in vitro starch digestibility was determined for the control bread. Breads with the addition of sprouted wheat flour were characterized with lowered digestibility, as was to be expected. These results were confirmed by the relative digestibility of starch - the highest digestibility was also determined for the control bread and further replacement of wheat flour with functional flour caused the decrease of this parameter (Fig. 2). There are many evidences that sprouting improves the bioaccessibility of sugars (Marton et al., 2010), but paradoxically starches are less susceptible to enzymes. This usually arises from the increased content of RS. Additionally, it may be suggested that interaction of phenolics with the enzymes of digestive tract and starch may play an important role, as was previously reported by Rohn, Rawel, & Kroll (2002) and Mkandawire et al. (2013). Sprouted wheat flour contained about 9 times more free reducing sugars (compared to wheat flour), and its addition to bread formula significantly increased the reducing sugar content of the functional bread. Conclusion In sum, the partial replacement (5%–20%) of wheat flour with functional sprouted wheat flour containing increased content of phenolics slightly increased the protein content and significantly decreased the starch digestibility. According to the results it may be stated that the studied bread with lowered nutritional value and increased nutraceutical quality may be of use for special groups of consumers (obese, and diabetic).
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Acknowledgments This study was partially financed by the Polish National Science Centre (grant 2012/07/B/NZ9/02463).
Conflict of interest statement The authors declare no conflict of interest.
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Figure captions Fig. 1. Electrophoretic analysis of proteins from flours and enriched bread Numbered brackets indicate region A, high molecular weight-glutenin subunits; region B, ωgliadins; region C, the α- and β-gliadins and low molecular weight-glutenin subunits; and region D, low molecular weight-albumins and globulins. WF- wheat flour, SF- sprouted wheat flour, C- control bread, B1-B4 - breads with 5-20% sprouted wheat flour; MM-molecular mass [kDa]. Fig. 2. Free reducing sugars, amino acids and peptides and relative digestibility of nutrients in flours and breads WF- wheat flour, SF- sprouted wheat flour, C- control bread, B1-B4- breads with 5%-20% sprouted wheat flour. The values are expressed as mean ± SD (n=9). The values within the same parameter designated by different letters are significantly different (α < 0.05). Fig. 3. Absorbance profiles of the eluates for the extracts after in vitro digestion of control and enriched bread obtained from size-exclusion chromatography. The absorbance profiles of the eluates for the extracts after in vitro digestion of control 624 and enriched bread obtained for size-exclusion chromatography. C – control bread, B2- bread with 10% of sprouted wheat flour ; B4- bread with 20% of sprouted wheat flour; DTC-components of digestive tract. Molecular mass markers (kDa): a. 102; b. 42; c. 35; d. 22; e. 18; f. 6.5; g. 3.
16
1
2
34 5 6 7
8
9
10 11
WF
12 13
SF
C
B1
B2
B3
B4 MM
WF 1 2
3 5
4
6
7 8 9 10
116 000
11 12
A SF 66 500
1
2 345 6
7 8 9
10 11
12
B
13 14 C
1
2 3456
7
8
9 10
45 000
11 12
C
B1 1
2 3 4 56
7
8
9
10
11
35 000
12 13 B2
1
2
34 5
6
7
8
9
10
11 12 B3 25 000
1
2
34 5
6
7
8
9
D
10 11 16 500
B4 14 500
Fig. 1. Electrophoretic analysis of proteins from flours and enriched bread Numbered brackets indicate region A, high molecular weight-glutenin subunits; region B, ωgliadins; region C, the α- and β-gliadins and low molecular weight-glutenin subunits; and region D, low molecular weight-albumins and globulins. WF- wheat flour, SF- sprouted wheat flour, C- control bread, B1-B4 - breads with 5%-20% sprouted wheat flour; MM-molecular mass [kDa].
17
20
Relative starch digestibility Relative proteins digestibility Free reducing sugars Free amino acids and peptides
[fold change in respect to WF]
18 16
e
e e d
14 12 c
10 8
b
f
e
6 4 2
d b aa
c
e
b
a
d
de b
c
cd
c b
b c
b b
a
0 WF
SF
C
B1
B2
B3
B4
Sample Fig. 2. Free reducing sugars, amino acids and peptides and relative digestibility of nutrients in flours and breads WF- wheat flour, SF- sprouted wheat flour, C- control bread, B1-B4 - breads with 5%-20% sprouted wheat flour. The values are expressed as mean ± SD (n=9). The values within the same parameter designated by different letters are significantly different (α < 0.05).
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Fig. 3. Absorbance profiles of the eluates for the extracts after in vitro digestion of control and enriched bread obtained from size-exclusion chromatography. C – control bread, B2- bread with 10% of sprouted wheat flour ; B4- bread with 20% of sprouted wheat flour; DTC-components of digestive tract. Molecular mass markers (kDa): a. 102; b. 42; c. 35; d. 22; e. 18; f. 6.5; g. 3. Regions: I: < 25 kDa; II: >25 - <20 kDa; III: < 20 kDa.
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Table 1. Content and digestibility of proteins from flours and breads
Sample
Glutelins+ Gliadyns [mg/ g DW]
Albumins+ Globulins [mg/ g DW]
CM-proteins [mg/ g DW]
Total Proteins [mg/ g DW]
In vitro protein digestibility [%]
WF
86.63±1.51b
20.73±0.31c
19.43±0.23a
126.79±1.82bc
51.74±1.33abc
SF
90.68±2.06c
20.84±0.15c
18.89±0.22a
130.40±1.20cd
51.06±0.44b
C
79.73±2.39a
20.05±0.17b
19.01±0.17a
118.79±2.47a
52.55±0.36c
B1
86.35±2.95b
19.56±0.22ab
18.74±0.24a
124.66±2.85ab
52.31±0.26c
B2
94.73±3.03bc
19.22±0.37ab
19.45±0.14a
133.40±2.89d
50.03±0.43a
B3
96.43±2.47c
19.86±0.44ab
18.93±0.20a
135.22±2.72d
49.82±0.26a
B4
96.79±1.91c
19.49±0.16a
18.59±0.18a
134.87±1.84d
50.05±0.27a
WF- wheat flour, SF- sprouted wheat flour, C- control bread, B1-B4 - breads with 5%-20% sprouted wheat flour. The values are expressed as mean ± SD (n= 9). The values designated by the different letters are significantly different (α< 0.05).
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Table 2. Content and digestibility of starch from flours and breads Potentially Potentially In vitro bioavailable resistant starch Sample starch starch digestibility [mg/ g DW] [% of TS] [%] 33.5±0.56c WF 651.8±11.32c 433.2±17.1e 66.46±2.63c 68.3±0.26e SF 588.4±7.66b 186.6±2.50a 31.72±1.43a 27.0±0.58a C 594.0±10.43b 433.9±11.8e 73.05±1.99c 30.8±0.05b B1 491.4±8.99a 340.0±6.49d 69.19±2.32c 30.8±0.91bc B2 477.8±10.15a 300.5±15.5c 67.10±3.46c 33.7±1.02c B3 489.9±2.09a 264.6±3.5b 54.42±5.72b 36.6±1.12d B4 487.4±2.35a 269.0±10.1b 55.19±4.08b WF- wheat flour, SF- sprouted wheat flour, C- control bread, B1-B4 - breads with 5%-20% Total starch [mg/ g DW]
sprouted wheat flour. The values are expressed as mean ± SD (n= 9). The values designated by the different letters are significantly different (α< 0.05).
21
1. Sprouted wheat flour (SF) was introduced to the wheat bread formula 2. The introduction of SF slightly increased the total protein content. 3. The addition of SF increased peptides and small proteins (<20 kDa) in breads. 4. Enriched breads were characterized by higher content of resistant starch. 5. Partial replacement of wheat flour with SF decreased the starch digestibility.
22