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Phytate negatively influences wheat dough and bread characteristics by interfering with cross-linking of glutenin molecules
Journal of Cereal Science 70 (2016) 199e206
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Phytate negatively influences wheat dough and bread characteristics by interfering with cross-linking of glutenin molecules Eun Young Park a, 1, E. Patrick Fuerst b, Byung-Kee Baik c, * a
School of Food Science, Washington State University, Pullman, WA 99164, USA Department of Crop and Soil Sciences, Western Wheat Quality Laboratory, Washington State University, Pullman, WA 99164-6420, USA c United States Department of Agriculture (USDA), Agricultural Research Service (ARS)-CSWQRU, Wooster, OH 44691, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 March 2016 Received in revised form 17 June 2016 Accepted 20 June 2016 Available online 21 June 2016
The influence of added phytate on dough properties and bread baking quality was studied to determine the role of phytate in the impaired functional properties of whole grain wheat flour for baking bread. Phytate addition to refined flour at a 1% level substantially increased mixograph mixing time, generally increased mixograph water absorption, and reduced the SDS-unextractable protein content of dough before and after fermentation as well as the loaf volume of bread. The added phytate also shifted unextractable glutenins toward a lower molecular weight form and increased the iron-chelating activity of dough. It appears that phytate negatively affects gluten development and loaf volume by chelating iron and/or binding glutenins, and consequently interfering with the oxidative cross-linking of glutenin molecules during dough mixing. Phytate could be at least partially responsible for the weak gluten network and decreased loaf volume of whole wheat flour bread as compared to refined flour bread. Published by Elsevier Ltd.
Keywords: Phytate Chelating capacity Dough property Bread quality
1. Introduction Phytic acid (referred to here as phytate or IP6) is myo-inositol hexakisphosphate. Phytate constitutes 1e2% of whole wheat by weight, is primarily located in the aleurone layer, and serves as a storage form of phosphorus (Harland and Morris, 1995). Phytate is negatively charged with six phosphate groups extending from a central inositol ring structure, enabling them to complex with positively charged molecules (Feil, 2001). Phytate is naturally complexed with divalent cationic minerals and proteins, and this complex formation depends on pH and concentration (Cheryan, 1980; Harland and Morris, 1995). The pKa values of twelve exchangeable hydrogen atoms on phytate range from 1.9 to 9.5, thus the formation of complexes between phytate and positively charged molecules (minerals and proteins) is possible in a broad pH range (Evans et al., 1982). The chelating ability of phytate for divalent mineral cations can decrease mineral absorption and availability, leading to nutrient deficiencies in humans, but the iron chelating capacity of phytate inhibits iron-catalyzed hydroxyl
* Corresponding author. E-mail address: [email protected] (B.-K. Baik). 1 Current address: Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea. http://dx.doi.org/10.1016/j.jcs.2016.06.012 0733-5210/Published by Elsevier Ltd.
radical formation and as such suppresses lipid peroxidation; thus, phytate also has potential nutritional benefits as an antioxidant (Kumar et al., 2010). In addition, phytate binds with proteins at both low and high pH values (Kumar et al., 2010). At low pH, negatively charged phytate can complex with positively charged amino acids on proteins, and at higher pH, complex formation of negatively charged phytate and proteins can be mediated by multivalent cations such as calcium and magnesium (Cheryan, 1980). The phytate-protein complexes modify protein structure causing aggregation of protein molecules around phytate, thus resulting in decreased protein solubility, enzymatic activity and proteolytic digestibility (Cheryan, 1980; Schlemmer et al., 2009). Thus phytate has two activities that might influence glutenin polymerization during wheat processing: the protein complexing activity and the antioxidant activity. Phytic acid (IP6) in wheat bran is degraded by phytase to inositol phosphates with one to five ester-linked phosphates (IP1 to IP5) and inositol (Reale et al., 2004). The content of myo-inositol pentakisphosphate (IP5) ranges from 5 to 10% of that of IP6, and only trace amounts of IP4 and lower forms have been observed in wheat bran (Dintzis et al., 1992). Endogenous phytases in wheat and yeast hydrolyze phytate during the breadmaking process (Türk et al., 1996). The addition of fungal phytase to whole wheat flour improved breadmaking performance, resulting in an increased
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specific volume of bread and decreased firmness of bread crumb (Haros et al., 2001a). Bran, where most wheat phytate is located, is responsible for the reduced product quality and sensory acceptance of whole wheat bread as compared to that made from refined wheat flour. We hypothesized that phytate is detrimental to dough processing and bread baking quality by functioning as i) an iron chelatingantioxidant, interfering with sulfhydryl crossing-linking of glutenins and/or ii) a protein complexing agent, reducing free interactions of glutenin proteins. The objective of this study was to determine the influence of added phytate on i) dough and bread characteristics of wheat flour, ii) glutenin characteristics, and iii) iron-chelating activity of dough. 2. Materials and methods 2.1. Materials Hard white spring and hard red spring wheat cultivars, Macon and Tara2002, respectively, were purchased from the Washington State Crop Improvement Association (Pullman, WA). Wheat was milled using a Bühler experimental mill (MLU-202, Bühler AG, Uzwil, Switzerland). Bran of Macon was ground using an Udy sample mill fitted with screen with 0.5 mm openings and blended with refined flour of Macon to prepare whole grain wheat flour. The refined wheat flours of Macon and Tara2002 were used for preparation of dough and bread with addition of phytate from three sources. Two forms of phytate were purchased from Sigma-Aldrich: phytic acid sodium salt hydrate (referred to as ‘sodium salt of PA’) and phytic acid solution. The latter was neutralized to pH 7 using sodium hydroxide to exclude the effects of low pH on dough (referred to as ‘neutralized PA’). In addition, phytate was extracted from wheat bran, purified and neutralized (referred to as ‘extracted-neutralized PA’) according to the following method. Wheat bran (15 g) of Macon and Tara2002 was extracted with 0.5 N HCl solution (150 mL) for 4 h with constant stirring (Kozlowska et al., 1996). The resulting extract was centrifuged at 2500g for 10 min. The supernatant was transferred to a column filled with 7.5 g of anion-exchange resin (Dowex 1 8, 100e200 mesh, Sigma-Aldrich Corp., St. Louis, MO). The column was washed with distilled water and loaded with phytate extract. The column retaining phytate was washed with distilled water and 0.1 M NaCl solution to remove inorganic phosphate. The resin-bound phytate was eluted with a 2 N HCl solution (10 mL), and the eluted solution was lyophilized (Park et al., 2006). The extracted phytate was then dissolved in water and neutralized to pH 7 with NaOH, yielding the extractedneutralized PA. 2.2. Dough properties Sodium salt of PA, neutralized PA or extracted-neutralized PA were added to refined flour from Macon and Tara2002 at a 1% level (flour dry weight basis), which is comparable to the phytate content of whole wheat flour, before dough mixing. The control consisted of refined wheat flour of the same variety without phytate addition. The water absorption and optimum mixing time of dough with and without added phytate were determined using a mixograph (National Mfg., Lincoln, NE) according to Approved Method 54e40.02 (AACCI, 2010). The mixograph test of dough without added phytate was run for 7 and 9 min for Macon and Tara, respectively. The mixograph test of dough added with phytate was run for 7 min for MaconþPAS, 10 min for MaconþEPAN, 11 min for MaconþPAN, 12 min for TaraþPAS, 19 min for TaraþEPAN, and 20 min for TaraþPAN based on the preliminary test. The dough was mixed for the optimum mixing time and fermented for 90 min at 30 C. Both the
initial “mixed dough” and subsequent “fermented dough” were lyophilized. The dried dough was ground in a cyclone mill (Udy Corp., Fort Collins, CO) with a 0.25 mm screen for analyses of SDSunextractable protein, iron chelating activity, and phytate content. 2.3. Baking properties The straight-dough bread baking test method (Approved Method 10e10.03) (AACCI, 2010) was used to bake pan bread. The bread formula consisted of refined flour (100 g) with 1% phytate (flour dry weight basis), non-fat dry milk (4 g), shortening (3 g), yeast (1.8 g), sugar (6 g), salt (1.5 g) and malt extract (5 mL). All ingredients were optimally mixed using a 100 g dough mixer (National Mfg., Lincoln, NE), and the dough was fermented for 90 min and proofed for 30 min at 30 C. Dough was baked for 21 min at 218 C in an oven (National Mfg., Lincoln, NE). The loaf volume of bread was immediately measured by rape seed displacement (Approved Method 10e05.01) (AACCI, 2010). The texture of bread was determined using a QTS-25 texture analyzer (Stable Micro Systems, Haslemere, England) with a cylindrical plastic probe of 25 mm diameter. Slices of bread (2 cm thick) from the center of the bread were compressed twice to 50% of original thickness at a speed of 1.0 mm/s. Bread crumb was immediately frozen, freeze-dried and ground for use in the analysis of phytate content. 2.4. Content and composition of SDS-unextractable proteins of dough before and after fermentation The SDS-unextractable protein content of dough before and after fermentation was quantified according to the method of Aussenac et al. (2001). Freeze-dried dough (0.24 g) was dispersed in 30 mL of 0.1 M sodium phosphate buffer (pH 6.9) containing 2% sodium dodecyl sulphate (SDS) (w/v) and then stirred for 2 h at 60 C. The extract was centrifuged for 30 min at 12,500g and the supernatant was removed. The remaining pellets were freezedried, and the nitrogen concentration of the freeze-dried pellets was used to estimate the SDS-unextractable protein content. Nitrogen content was determined using a combustion nitrogen analyzer (FP 528, LECO Corp., St. Joseph, MI). A factor of 5.7 was used to calculate protein content. The molecular weight distribution of SDS-unextractable proteins was determined using size exclusion HPLC (Redl et al., 1999). Freeze-dried dough (0.16 g) of Tara2002 was dispersed in 0.1 M sodium phosphate buffer (pH 6.9, 20 mL) containing 1% SDS, and then stirred for 80 min at 60 C. The extract was centrifuged for 30 min at 37,000g and the supernatant was discarded. The residue was suspended in 0.1 M sodium phosphate buffer (pH 6.9, 5 mL) containing 1% SDS, and sonicated (Sonic Dismembrator Model 500, Fisher Scientific, Pittsburgh, PA) to solubilize the insoluble protein fraction in the residue (Singh et al., 1990). After centrifugation for 30 min at 37,000g, the supernatant (20 mL) was filtered (0.45 mm, Millex LCR [PTFE], Millipore, Billerica, MA). Size exclusion HPLC was carried out using a Waters 2695 Separations Module and a Waters 996 Photodiode Array Detector (Waters, Milliford, MA), quantifying absorbance at 214 nm. The size exclusion column was 8.0 300 mm (OHpak SB-804 HQ, Shodex, New York, NY). The column was eluted with 0.1 M sodium phosphate buffer (pH 6.9) containing 0.1% SDS at a flow rate of 0.7 mL/min. The column was calibrated with thyroglobulin (MW 669,000) and alcohol dehydrogenase (MW 150,000) (Koh and Ng, 2008). The molecular weight distribution patterns of SDS-unextractable proteins in the dough of Tara2002 with and without added phytate was determined.
E.Y. Park et al. / Journal of Cereal Science 70 (2016) 199e206
2.5. Iron chelating activity of dough before and after fermentation For determination of iron chelating activity, freeze-dried dough (0.2 g) before and after fermentation was suspended in 4 mL acidified methanol (HCl/methanol/water, 1:80:10, v/v/v) for 2 h at ambient temperature with constant shaking (Mpofu et al., 2006). The suspension was centrifuged at 2500g for 10 min, and the supernatant was collected for determination of iron chelating activity. Iron chelating activity was determined using the 2, 2’-bipyridyl competition assay. The reaction mixture contained 1 mM FeSO4 solution (0.1 mL), dough extract (50 mL), Tris-HCl buffer (pH 7.4, 0.8 mL), 2, 2’-bipyridyl solution (0.1% in 0.2 M HCl, 0.4 mL) and 10% hydroxylamine-HCl (0.3 mL). The absorbance of the reaction mixture was measured at 522 nm with a spectrophotometer (UV1601, Shimadzu, Kyoto, Japan) and used to calculate iron chelating activity using the following equation (Wang et al., 2009):
. o n 100 Iron chelating activity ð%Þ ¼ 1 Asample Acontrol where A sample was the absorbance of the test sample and A control was the absorbance of the control (without dough extract, 0% activity).
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portion of the supernatant (4 mL) was mixed with 30 mg/mL EDTA (8 mL) and adjusted to pH 6 with 2 N NaOH. The phytate extract was then freeze-dried and dispersed in distilled water (5 mL). The solution was passed through a 0.45 mm filter, adjusted to pH 12.6 with NaOH and lyophilized. Dried samples (100 mg) were dissolved in 600 mL D2O and transferred to a 5 mM NMR tube. 1H and 31P NMR spectra were acquired at 295 K (Varian Model 400, Varian NMR Instruments, Palo Alto, CA), and 399.76 MHz and 161.83 MHz, respectively. 1H decoupled 31P NMR spectra were performed with a 7 s relaxation delay, 15,060 Hz spectral width, 7.3 ms 90 pulse on 31 P and 103.5 ms 90 pulse for 1H decoupling, and 128 scans. Exponential multiplication (2 Hz line broadening) and Fourier transformation were used for NMR data processing. Chemical shifts for 31P spectra are given in parts per million (ppm) with 85% H3PO4 as an external standard. 2.7. Statistical analysis All experiments were conducted in duplicate and statistical analyses of the data were performed using SAS version 9.1 (SAS Institute Inc., Cary, NC, USA). Differences among experimental mean values were tested for significance at p < 0.05 using Tukey’s studentized range (HSD) test. 3. Results
2.6. Phytate content and composition of dough after mixing, fermentation and baking
3.1. Influence of added phytate on dough properties Phytate of flour, of freeze-dried dough before and after fermentation and of freeze-dried bread (1.5 g) was extracted and purified as previously described for bran, but quantities and volumes were scaled to one-tenth those previously described. The phytate content was measured using Wade reagent (0.03% FeCl3$6H2O and 0.3% sulfosalicylic acid in water) (Latta and Eskin, 1980). The absorbance at 500 nm was measured immediately. A standard curve was developed using phytic acid sodium salt hydrate (2e16 mg). Phytate was also extracted for determination of composition by NMR (Reale et al., 2004). Phytate of freeze-dried dough (6 g) of refined flour, whole grain wheat flour and refined flour added with phytates before and after fermentation and of bread (6 g) from Macon were extracted with 0.75 M HCl solution (30 mL) for 2 h with constant shaking. The extract was centrifuged at 1800g for 15 min, and 6 mL of supernatant was incubated for 15 min at 100 C. After cooling, the solution was centrifuged at 1800g for 15 min. A
Flour
+ Sodium salt of PA
The mixograms of refined wheat flours with added phytate are shown in Fig. 1. There were clear changes in the mixogram patterns when phytate was added to refined wheat flour. Dough mixing time increased with the addition of phytate in both Macon and Tara2002. Significantly longer dough mixing times were observed with neutralized PA and extracted-neutralized PA than with sodium salt of PA. Water absorption of the dough with sodium salt of PA (64e65%) was similar to that of refined wheat flour (64e65%), but increased to 70% with the addition of neutralized PA and extracted-neutralized PA. These results indicate that the addition of phytate (especially neutralized PA and extracted-neutralized PA) to wheat flour delays gluten network formation and dough development with increased water absorption, while sodium salt of PA delayed gluten development but did not influence the water absorption of dough. Quantity and quality of protein are the main factors affecting the dough mixing properties of flour (Finney and
+ Neutralized PA
+ Extracted-neutralized PA
Macon
Tara2002
Fig. 1. Mixograms of Macon and Tara2002 refined flours with added sodium salt of phytic acid (PA), neutralized PA and extracted-neutralized PA.
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Table 1 SDS-unextractable protein content and iron chelating activity (%) of Macon and Tara2002 refined flour doughs with the addition of three types of phytate (PA) before and after fermentation.a Addition to flour
Macon
Tara2002
a b
SDS-unextractable protein of dough (%)b
Iron chelating Activity of dough (%)
Before fermentation
Before fermentation
After fermentation
a
No added PA Sodium salt of PA Neutralized PA Extracted-neutralized PA No added PA Sodium salt of PA Neutralized PA Extracted-neutralized PA
1.43 1.00 0.85 1.02 1.31 0.90 1.15 1.12
1.07 0.79 b 0.87 b 0.91 b 1.28a 1.02 b 1.03 b 0.99 b
a
After fermentation
b
0.58 b 5.93a 4.36a 5.42a 0.85 b 4.08a 5.13a 3.85a
2.87 10.36a 12.56a 13.58a 1.32c 9.23 b 14.91a 14.09a
ab b ab a c b b
Mean values with different letters within each column of each wheat variety are significantly different (p < 0.05). % of flour dry weight.
after fermentation, although the reduction in the SDSunextractable protein content of fermented dough was not significant when sodium salt of PA or extracted-neutralized PA was added to Macon flour. In previous studies, the amount of unextractable polymeric protein was positively correlated with improved dough properties and loaf volume, because polymerization of gluten proteins is a critical determinant of wheat breadmaking quality (Aussenac et al., 2001; Weegels et al., 1996). A reduction in the SDS-unextractable protein content of dough with added phytate was likely the result of reduced glutenin polymerization. The dough with added phytate appears to have a less extensive gluten network, and a correspondingly weaker dough than dough without added phytate. Fig. 2 shows the molecular weight distribution of SDS-
Shogren, 1972), but results show that dough mixing properties were affected by the addition of phytate even though there was no change in protein content. It appears that when phytate is added to refined flour, in quantities similar to that of whole wheat flour, the effect is sufficient to impart substantial influences on gluten development and dough mixing properties. 3.2. Influence of added phytate on sds-unextractable protein content of dough before and after fermentation The SDS-unextractable protein content of dough before and after fermentation is summarized in Table 1. In both Macon and Tara2002, the SDS-unextractable protein content of dough was reduced significantly with the addition of phytate both before and
a)
0.03
0.02
AU
AU
0.02
0.01
0.00 5
10
15
20
0
Time (min)
c)
5
10
15
20
15
20
Time (min)
d) 0.03
0.02
0.02
AU
AU
0.01
0.00 0
0.03
b)
0.03
Mw Mw 669,000 150,000
0.01
0.00
0.01
0.00 0
5
10 Time (min)
15
20
0
5
10 Time (min)
Fig. 2. Molecular weight profiles of SDS-unextractable proteins separated by size-exclusion HPLC. Shown are glutenins extracted from mixed doughs from Tara2002 with a) no added phytate (control), b) sodium salt of phytic acid (PA), c) neutralized PA and d) extracted-neutralized PA. AU ¼ absorbance units at 214 nm. The two dots represent molecular weight standards as identified in Fig. part ‘a’.
E.Y. Park et al. / Journal of Cereal Science 70 (2016) 199e206
a)
b) 1200
1100
2
2
900
0 b
b
b
-2
800
700
-4
1100 a
1
1000
b
c
Firmness (N)
a
Firmness (N)
1000
L o af V o lum e o f B re ad (m L )
L o af V o lum e o f B re ad (m L )
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c
900
0
800
-1 700
600
600 No added PA
+ Sodium salt of PA
-2
+ Neutralized + ExtractedPA neutralized PA
No added PA
+ Sodium salt of PA
+ Neutralized + ExtractedPA neutralized PA
Fig. 3. Bread loaf volume and firmness of a) Macon and b) Tara2002 baked with the addition of sodium salt of phytic acid (PA), neutralized PA and extracted-neutralized PA.
unextractable proteins in the dough of Tara2002 with added phytate. The refined flour dough with no added phytate exhibited a prominent glutenin peak near the 669 K marker and a much smaller glutenin peak near the 150 K marker. With the addition of phytate, there was a reduction in the first peak (near 669 K) and an increase in the second peak (near 150 K). The reduction of the first peak was greatest with added extracted-neutralized PA (Fig. 2d) and least with sodium salt of PA (Fig. 2b). These results clearly indicate that the added phytate decreases the proportion of the largest glutenin polymers (near 669 K) and increase the proportion of the smaller glutenin polymers (near 150 K) and thus phytate appears to reduce glutenin polymerization during dough mixing.
activity of phytases from both wheat and yeast (Türk et al., 1996).
3.3. Iron chelating activity of doughs before and after fermentation
3.5. Phytate content and composition
With the addition of phytate, the iron chelating activity of doughs increased several fold relative to the dough without added phytate both before and after fermentation (Table 1). Furthermore, the iron chelating activity of dough was reduced by fermentation, but iron chelating capacity was still substantially greater in the doughs with added phytate than in the refined wheat flour dough. The reduction of phytate during processing can be attributed to the
The changes in phytate content during dough mixing, fermentation and baking are depicted in Fig. 4. During the process of dough mixing, fermentation and baking, phytate content steadily decreased regardless of phytate type, but the degree of decrease differed among the phytate types. The decrease in phytate content during processing was greatest with sodium salt of PA in both Macon and Tara2002. A decrease in phytate content during bread
3.4. Baking properties The addition of all three types of phytate resulted in decreased bread loaf volumes (Fig. 3). With the addition of phytate, loaf volume of bread was reduced from 925 mL to 848e875 mL in Macon, and from 993 mL to 900e943 mL in Tara2002. Bread loaf volumes with added phytate were similar among the three types of phytate. Crumb texture was firmer in bread with added phytate (Fig. 3) possibly due to the smaller loaf volume of bread with added phytate.
a)
b) No added PA + Sodium salt of PA + Neutralized PA + Extracted-neutralized PA
Phytate (mg/g dry wt)
20
15
20
15
10
10
5
5
0
0
Flour
Mixed dough
Fermented dough
Bread
Flour
Mixed dough
Fermented dough
Bread
Fig. 4. Phytate content of flour, mixed dough, fermented dough and bread with no added phytate, or with phytate added as the sodium salt of phytic acid (PA), neutralized PA and extracted-neutralized PA in a) Macon and b) Tara2002. Units are mg phytate per gram dry weight of flour dough or bread.
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baking was also observed by Haros et al. (2001a). During baking, the phytate content of whole wheat bread was reduced by 50e60%, and the decrease was due to degradation of phytate by both endogenous phytase and yeast phytase (Türk et al., 1996). Endogenous phytase of wheat grain catalyzes the stepwise hydrolysis of phytate ultimately leading to inositol and monophosphates. Fig. 5 shows 1H-decoupled 31P NMR spectra of the doughs of cv. Macon. NMR analyses were conducted for refined flour dough (Fig. 5a), whole grain flour dough (Fig. 5b), and refined flour doughs with added 1% sodium salt of PA (Fig. 5c), neutralized PA (Fig. 5d) or
a)
extracted-neutralized PA (Fig. 5e). IP6 (four peaks) and orthophosphate (Pi) were the prominent peaks observed in the spectra. The NMR spectra confirmed much lower phytate levels in refined than in whole grain flour dough (Fig. 5a and b). The refined flour doughs containing added phytate types showed, in addition to the IP6 and Pi peaks, many smaller peaks (Fig. 5c, d, and e) likely representative of inositol phosphates IP1 to IP5. The appearance of inositol phosphates with a smaller number of phosphates in the spectra are due to the stepwise hydrolytic breakdown of phytate during processing, which corresponds to the decreasing phytate
b)
IP6
IP6
IP6
Pi IP6
Pi IP6
IP6 IP6
7.0
6.5
6.0
5.5
IP6
5.0
4.5
4.0
3.5
3.0
2.5
7.0
6.5
6.0
5.5
5.0
ppm
c)
4.5
4.0
3.5
3.0
2.5
3.0
2.5
ppm
Pi
d)
IP6
IP6
IP6
IP6
Pi
IP6 IP6
7.0
6.5
6.0
5.5
IP6
IP6
5.0
4.5
4.0
3.5
3.0
2.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
ppm
ppm
e) IP6
Pi
7.0
6.5
6.0
IP6
IP6
IP6
5.5
5.0
4.5
4.0
3.5
3.0
2.5
ppm Fig. 5. 1H-decoupled 31P NMR spectra of doughs prepared from a) refined flour (control), b) whole grain wheat flour, and refined flours with the addition of c) sodium salt of phytic acid (PA), d) neutralized PA and e) extracted-neutralized PA before fermentation. Doughs were prepared from cultivar Macon.
E.Y. Park et al. / Journal of Cereal Science 70 (2016) 199e206
shown previously (Fig. 4); however the peaks were too small to allow quantification. NMR spectra were also conducted for fermented dough and for bread; as expected, these spectra showed substantially decreased IP6 peaks and increased Pi peak (E.Y. Park, Ph. D dissertation, 2014, unpublished). The dough with added phytate extracted from wheat bran (extracted-neutralized PA, Fig. 5e) exhibited an NMR spectrum that was most similar to the spectrum of whole grain flour dough (Fig. 5b), suggesting, as expected, comparable compositions of phytate isolated from wheat bran and whole grain flour endogenous phytate. 4. Discussion Haros et al. (2001a, 2001b) proposed that phytate chelation of calcium is responsible for the negative influence of phytate on bread processing. In their work, they found increased specific volume index of whole wheat bread by up to 21% by the addition of fungal phytase, a finding consistent with our observations that adding phytate decreases bread loaf volume. They attributed the increased bread loaf volume to the activation of endogenous aamylase by phytase, since the hydrolysis of phytate by phytase increases free calcium ions that are necessary for a-amylase activity. However, our research suggests a more direct mechanism for the adverse effects of phytate on baking quality, by interference with glutenin polymerization. We observed reduction in both the overall quantity of SDS-unextractable protein (Table 1) and the ratio of large/small glutenin polymers (Fig. 2). Consistent with our observations, Tronsmo et al. (2002) reported that a lower ratio of large/small glutenin polymers corresponds to weaker dough and reduced bread baking quality. Reduction in SDS-unextractable protein content (Table 1) and glutenin molecular weight (Fig. 2) with the addition of phytate is consistent with our hypothesis that phytate inhibits strong gluten network formation. There appear to be two mechanisms by which phytate could interfere with glutenin polymerization: i) complexing of proteins with phytate, and ii) iron chelating-antioxidant activity of phytate. These mechanisms, which are not mutually exclusive, are discussed in more detail, below. Phytate forms complexes with proteins (Cheryan, 1980), and protein-phytate interactions could interfere with glutenin polymerization. It was reported that the strong polyanionic character of phytate enables it to bind with positively charged amino acid residues on proteins, such as lysine and arginine (de Rham and Jost, 1979), so it is possible that electrostatic interactions between added phytate and positively charged glutenin amino acid residues may interfere with gluten network development in dough during mixing. Our results do not clearly either support or refute this explanation. The other mechanism by which phytate may interfere with glutenin polymerization is via phytate-mediated iron chelation, which suppresses oxidative reactions by inhibiting iron-catalyzed hydroxyl radical formation (Fardet et al., 2008). By chelating iron, phytate acts as an antioxidant and may prevent the formation of free radicals in food (Kumar et al., 2010). Oxidation induces the covalent crosslinking of proteins (including glutenins) through the formation of disulfide bonds and dityrosine crosslinks, which are largely responsible for the viscoelastic properties of the gluten network, and ultimately improves bread quality (Rasiah et al., 2005). The disulfide cross-linkings of glutenin molecules could be disrupted by the antioxidant activity of phytate, attributed to iron chelation, resulting in dough breakdown and reduced dough stability (Koh and Ng, 2008). The reducing action of antioxidants, therefore, adversely affects the rheological properties of dough, such as rapid dough breakdown immediately after optimum mixing time (Kerr et al., 1993). Conversely, oxidants are often added to
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bread formulations to improve gluten strength and loaf volume (Goesaert et al., 2005). Therefore we suggest that the strong iron chelating-antioxidant activity of phytate interferes with oxidative cross-linking of glutenins, which then leads to the observed reduced levels of SDS-unextractable protein content (Table 1) and reduced molecular weight of SDS-unextractable glutenins (Fig. 2). 5. Conclusions The addition of phytate to refined wheat flour at a 1% level approximated the in situ phytate content of whole wheat flour and induced deleterious effects on dough properties and bread baking quality. The addition of phytate increased mixograph mixing time and water absorption with delayed dough development. Loaf volume of bread was reduced with the addition of phytate. Phytate addition was also associated with a decreased SDS-unextractable protein content and a reduced proportion of higher molecular weight SDS-unextractable glutenin, which explains the weakened gluten and dough strengths and reduced bread loaf volume. The negative influences of added phytate on gluten and dough development may be due to their iron chelating-mediated antioxidant activity, which reduces oxidative cross linking among glutenin molecules during dough mixing; formation of protein complexes by phytate may also interfere with this cross-linking. It is possible, therefore, that phytate is a factor in the weak gluten development and the decreased bread loaf volume of whole wheat flour. This assumption also agrees with the speculation by Noort et al. (2010) that liberation of reactive components due to cell breakage was primarily responsible for the detrimental effects of bran rather than bran physically interfering with gluten development. Accordingly, the hydrolytic breakdown of phytate in whole wheat flour processing, as reported by Haros et al. (2001b) may be an important approach for enhancing whole wheat bread processing. Acknowledgments NMR analysis was performed at the center for NMR spectroscopy at Washington State University. We appreciate Dr. Craig Morris and Dr. Dan Skinner for access to facilities and materials at USDA-ARS Western Wheat Quality Lab. References AACCI, 2010. Approved Methods of the American Association of Cereal Chemists, eleventh ed. AACC International, St. Paul, MN. Aussenac, T., Carceller, J.L., Kleiber, D., 2001. Changes in SDS solubility of glutenin polymers during dough mixing and resting. Cereal Chem. 78, 39e45. Cheryan, M., 1980. Phytic acid interactions in food systems. Crit. Rev. Food Sci. Nutr. 13, 297e335. de Rham, O., Jost, T., 1979. Phytate-protein interactions in soybean extracts and lowphytate soy protein products. J. Food Sci. 44, 596e600. Dintzis, F.R., Lehrfeld, J., Nelsen, T.C., Finney, P.L., 1992. Phytate content of soft wheat brans as related to kernel size, cultivar, location, and milling and flour quality parameters. Cereal Chem. 69, 577e577. Evans, W., McCourtney, E., Shrager, R., 1982. Titration studies of phytic acid. J. Am. Oil Chemists’ Soc. 59, 189e191. me sy, C., 2008. Is the in vitro antioxidant potential of wholeFardet, A., Rock, E., Re grain cereals and cereal products well reflected in vivo? J. Cereal Sci. 48, 258e276. Feil, B., 2001. Phytic acid. J. New Seeds 3, 1e35. Finney, K.F., Shogren, M.D., 1972. A ten-gram mixograph for determining and predicting functional properties of wheat flours. Bak. Dig. 46, 32e35, 38, 42, 77. Goesaert, H., Brijs, K., Veraverbeke, W.S., Courtin, C.M., Gebruers, K., Delcour, J.A., 2005. Wheat flour constituents: how they impact bread quality, and how to impact their functionality. Trends Food Sci. Technol. 16, 12e30. Harland, B.F., Morris, E.R., 1995. Phytate: a good or a bad food component? Nutr. Res. 15, 733e754. Haros, M., Rosell, C., Benedito, C., 2001a. Fungal phytase as a potential breadmaking additive. Eur. Food Res. Technol. 213, 317e322. Haros, M., Rosell, C.M., Benedito, C., 2001b. Use of fungal phytase to improve breadmaking performance of whole wheat bread. J. Agric. Food Chem. 49,
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5450e5454. Kerr, C., Hoseney, R., Faubion, J., 1993. Mixograph studies. VI. Combined effects of charge (pH), activated double-bond compounds, and oxidants on dough mixing properties. Cereal Chem. 70, 633e636. Koh, B.-K., Ng, P.K.W., 2008. Effects of ferulic acid and transglutaminase on hard wheat flour dough and bread. Cereal Chem. 86, 18e22. Kozlowska, H., Honke, J., Sadowska, J., Frias, J., Vidal-Valverde, C., 1996. Natural fermentation of lentils: influence of time, concentration and temperature on the kinetics of hydrolysis of inositol phosphates. J. Sci. Food Agric. 71, 367e375. Kumar, V., Sinha, A.K., Makkar, H.P.S., Becker, K., 2010. Dietary roles of phytate and phytase in human nutrition: a review. Food Chem. 120, 945e959. Latta, M., Eskin, M., 1980. A simple and rapid colorimetric method for phytate determination. J. Agric. Food Chem. 28, 1313e1315. Mpofu, A., Sapirstein, H.D., Beta, T., 2006. Genotype and environmental variation in phenolic content, phenolic acid composition, and antioxidant activity of hard spring wheat. J. Agric. Food Chem. 54, 1265e1270. Noort, M.W.J., van Haaster, D., Hemery, Y., Schols, H.A., Hamer, R.J., 2010. The effect of particle size of wheat bran fractions on bread quality-Evidence for fibreprotein interactions. J. Cereal Sci. 52, 59e64. Park, H.-R., Ahn, H.-J., Kim, S.-H., Lee, C.-H., Byun, M.-W., Lee, G.-W., 2006. Determination of the phytic acid levels in infant foods using different analytical methods. Food Control 17, 727e732. Rasiah, I.A., Sutton, K.H., Low, F.L., Lin, H.M., Gerrard, J.A., 2005. Crosslinking of wheat dough proteins by glucose oxidase and the resulting effects on bread and croissants. Food Chem. 89, 325e332. Reale, A., Mannina, L., Tremonte, P., Sobolev, A.P., Succi, M., Sorrentino, E.,
Coppola, R., 2004. Phytate degradation by lactic acid bacteria and yeasts during the wholemeal dough fermentation: a 31P NMR study. J. Agric. Food Chem. 52, 6300e6305. Redl, A., Morel, M.H., Bonicel, J., Guilbert, S., Vergnes, B., 1999. Rheological properties of gluten plasticized with glycerol: dependence on temperature, glycerol content and mixing conditions. Rheol. Acta 38, 311e320. Schlemmer, U., Frølich, W., Prieto, R.M., Grases, F., 2009. Phytate in foods and significance for humans: food sources, intake, processing, bioavailability, protective role and analysis. Mol. Nutr. Food Res. 53, S330eS375. Singh, N., Donovan, G., Batey, I., MacRitchie, F., 1990. Use of sonication and sizeexclusion high-performance liquid chromatography in the study of wheat flour proteins. I. Dissolution of total proteins in the absence of reducing agents. Cereal Chem. 67, 150e161. Türk, M., Carlsson, N.-G., Sandberg, A.-S., 1996. Reduction in the levels of phytate during wholemeal bread making; Effect of yeast and wheat phytases. J. Cereal Sci. 23, 257e264. Tronsmo, K.M., Færgestad, E.M., Longva, Å., Schofield, J.D., Magnus, E.M., 2002. A study of how size distribution of gluten proteins, surface properties of gluten and dough mixing properties relate to baking properties of wheat flours. J. Cereal Sci. 35, 201e214. Wang, J., Yuan, X., Sun, B., Tian, Y., Cao, Y., 2009. Scavenging activity of enzymatic hydrolysates from wheat bran. Food Technol. Biotechnol. 47, 39e46. Weegels, P.L., van de Pijpekamp, A.M., Graveland, A., Hamer, R.J., Schofield, J.D., 1996. Depolymerisation and re-polymerisation of wheat glutenin during dough processing. I. Relationships between glutenin macropolymer content and quality parameters. J. Cereal Sci. 23, 103e111.
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Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Phytate negatively influences wheat dough and bread characteristics by interfering with cross-linking of glutenin molecules Eun Young Park a, 1, E. Patrick Fuerst b, Byung-Kee Baik c, * a
School of Food Science, Washington State University, Pullman, WA 99164, USA Department of Crop and Soil Sciences, Western Wheat Quality Laboratory, Washington State University, Pullman, WA 99164-6420, USA c United States Department of Agriculture (USDA), Agricultural Research Service (ARS)-CSWQRU, Wooster, OH 44691, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 March 2016 Received in revised form 17 June 2016 Accepted 20 June 2016 Available online 21 June 2016
The influence of added phytate on dough properties and bread baking quality was studied to determine the role of phytate in the impaired functional properties of whole grain wheat flour for baking bread. Phytate addition to refined flour at a 1% level substantially increased mixograph mixing time, generally increased mixograph water absorption, and reduced the SDS-unextractable protein content of dough before and after fermentation as well as the loaf volume of bread. The added phytate also shifted unextractable glutenins toward a lower molecular weight form and increased the iron-chelating activity of dough. It appears that phytate negatively affects gluten development and loaf volume by chelating iron and/or binding glutenins, and consequently interfering with the oxidative cross-linking of glutenin molecules during dough mixing. Phytate could be at least partially responsible for the weak gluten network and decreased loaf volume of whole wheat flour bread as compared to refined flour bread. Published by Elsevier Ltd.
Keywords: Phytate Chelating capacity Dough property Bread quality
1. Introduction Phytic acid (referred to here as phytate or IP6) is myo-inositol hexakisphosphate. Phytate constitutes 1e2% of whole wheat by weight, is primarily located in the aleurone layer, and serves as a storage form of phosphorus (Harland and Morris, 1995). Phytate is negatively charged with six phosphate groups extending from a central inositol ring structure, enabling them to complex with positively charged molecules (Feil, 2001). Phytate is naturally complexed with divalent cationic minerals and proteins, and this complex formation depends on pH and concentration (Cheryan, 1980; Harland and Morris, 1995). The pKa values of twelve exchangeable hydrogen atoms on phytate range from 1.9 to 9.5, thus the formation of complexes between phytate and positively charged molecules (minerals and proteins) is possible in a broad pH range (Evans et al., 1982). The chelating ability of phytate for divalent mineral cations can decrease mineral absorption and availability, leading to nutrient deficiencies in humans, but the iron chelating capacity of phytate inhibits iron-catalyzed hydroxyl
* Corresponding author. E-mail address: [email protected] (B.-K. Baik). 1 Current address: Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea. http://dx.doi.org/10.1016/j.jcs.2016.06.012 0733-5210/Published by Elsevier Ltd.
radical formation and as such suppresses lipid peroxidation; thus, phytate also has potential nutritional benefits as an antioxidant (Kumar et al., 2010). In addition, phytate binds with proteins at both low and high pH values (Kumar et al., 2010). At low pH, negatively charged phytate can complex with positively charged amino acids on proteins, and at higher pH, complex formation of negatively charged phytate and proteins can be mediated by multivalent cations such as calcium and magnesium (Cheryan, 1980). The phytate-protein complexes modify protein structure causing aggregation of protein molecules around phytate, thus resulting in decreased protein solubility, enzymatic activity and proteolytic digestibility (Cheryan, 1980; Schlemmer et al., 2009). Thus phytate has two activities that might influence glutenin polymerization during wheat processing: the protein complexing activity and the antioxidant activity. Phytic acid (IP6) in wheat bran is degraded by phytase to inositol phosphates with one to five ester-linked phosphates (IP1 to IP5) and inositol (Reale et al., 2004). The content of myo-inositol pentakisphosphate (IP5) ranges from 5 to 10% of that of IP6, and only trace amounts of IP4 and lower forms have been observed in wheat bran (Dintzis et al., 1992). Endogenous phytases in wheat and yeast hydrolyze phytate during the breadmaking process (Türk et al., 1996). The addition of fungal phytase to whole wheat flour improved breadmaking performance, resulting in an increased
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specific volume of bread and decreased firmness of bread crumb (Haros et al., 2001a). Bran, where most wheat phytate is located, is responsible for the reduced product quality and sensory acceptance of whole wheat bread as compared to that made from refined wheat flour. We hypothesized that phytate is detrimental to dough processing and bread baking quality by functioning as i) an iron chelatingantioxidant, interfering with sulfhydryl crossing-linking of glutenins and/or ii) a protein complexing agent, reducing free interactions of glutenin proteins. The objective of this study was to determine the influence of added phytate on i) dough and bread characteristics of wheat flour, ii) glutenin characteristics, and iii) iron-chelating activity of dough. 2. Materials and methods 2.1. Materials Hard white spring and hard red spring wheat cultivars, Macon and Tara2002, respectively, were purchased from the Washington State Crop Improvement Association (Pullman, WA). Wheat was milled using a Bühler experimental mill (MLU-202, Bühler AG, Uzwil, Switzerland). Bran of Macon was ground using an Udy sample mill fitted with screen with 0.5 mm openings and blended with refined flour of Macon to prepare whole grain wheat flour. The refined wheat flours of Macon and Tara2002 were used for preparation of dough and bread with addition of phytate from three sources. Two forms of phytate were purchased from Sigma-Aldrich: phytic acid sodium salt hydrate (referred to as ‘sodium salt of PA’) and phytic acid solution. The latter was neutralized to pH 7 using sodium hydroxide to exclude the effects of low pH on dough (referred to as ‘neutralized PA’). In addition, phytate was extracted from wheat bran, purified and neutralized (referred to as ‘extracted-neutralized PA’) according to the following method. Wheat bran (15 g) of Macon and Tara2002 was extracted with 0.5 N HCl solution (150 mL) for 4 h with constant stirring (Kozlowska et al., 1996). The resulting extract was centrifuged at 2500g for 10 min. The supernatant was transferred to a column filled with 7.5 g of anion-exchange resin (Dowex 1 8, 100e200 mesh, Sigma-Aldrich Corp., St. Louis, MO). The column was washed with distilled water and loaded with phytate extract. The column retaining phytate was washed with distilled water and 0.1 M NaCl solution to remove inorganic phosphate. The resin-bound phytate was eluted with a 2 N HCl solution (10 mL), and the eluted solution was lyophilized (Park et al., 2006). The extracted phytate was then dissolved in water and neutralized to pH 7 with NaOH, yielding the extractedneutralized PA. 2.2. Dough properties Sodium salt of PA, neutralized PA or extracted-neutralized PA were added to refined flour from Macon and Tara2002 at a 1% level (flour dry weight basis), which is comparable to the phytate content of whole wheat flour, before dough mixing. The control consisted of refined wheat flour of the same variety without phytate addition. The water absorption and optimum mixing time of dough with and without added phytate were determined using a mixograph (National Mfg., Lincoln, NE) according to Approved Method 54e40.02 (AACCI, 2010). The mixograph test of dough without added phytate was run for 7 and 9 min for Macon and Tara, respectively. The mixograph test of dough added with phytate was run for 7 min for MaconþPAS, 10 min for MaconþEPAN, 11 min for MaconþPAN, 12 min for TaraþPAS, 19 min for TaraþEPAN, and 20 min for TaraþPAN based on the preliminary test. The dough was mixed for the optimum mixing time and fermented for 90 min at 30 C. Both the
initial “mixed dough” and subsequent “fermented dough” were lyophilized. The dried dough was ground in a cyclone mill (Udy Corp., Fort Collins, CO) with a 0.25 mm screen for analyses of SDSunextractable protein, iron chelating activity, and phytate content. 2.3. Baking properties The straight-dough bread baking test method (Approved Method 10e10.03) (AACCI, 2010) was used to bake pan bread. The bread formula consisted of refined flour (100 g) with 1% phytate (flour dry weight basis), non-fat dry milk (4 g), shortening (3 g), yeast (1.8 g), sugar (6 g), salt (1.5 g) and malt extract (5 mL). All ingredients were optimally mixed using a 100 g dough mixer (National Mfg., Lincoln, NE), and the dough was fermented for 90 min and proofed for 30 min at 30 C. Dough was baked for 21 min at 218 C in an oven (National Mfg., Lincoln, NE). The loaf volume of bread was immediately measured by rape seed displacement (Approved Method 10e05.01) (AACCI, 2010). The texture of bread was determined using a QTS-25 texture analyzer (Stable Micro Systems, Haslemere, England) with a cylindrical plastic probe of 25 mm diameter. Slices of bread (2 cm thick) from the center of the bread were compressed twice to 50% of original thickness at a speed of 1.0 mm/s. Bread crumb was immediately frozen, freeze-dried and ground for use in the analysis of phytate content. 2.4. Content and composition of SDS-unextractable proteins of dough before and after fermentation The SDS-unextractable protein content of dough before and after fermentation was quantified according to the method of Aussenac et al. (2001). Freeze-dried dough (0.24 g) was dispersed in 30 mL of 0.1 M sodium phosphate buffer (pH 6.9) containing 2% sodium dodecyl sulphate (SDS) (w/v) and then stirred for 2 h at 60 C. The extract was centrifuged for 30 min at 12,500g and the supernatant was removed. The remaining pellets were freezedried, and the nitrogen concentration of the freeze-dried pellets was used to estimate the SDS-unextractable protein content. Nitrogen content was determined using a combustion nitrogen analyzer (FP 528, LECO Corp., St. Joseph, MI). A factor of 5.7 was used to calculate protein content. The molecular weight distribution of SDS-unextractable proteins was determined using size exclusion HPLC (Redl et al., 1999). Freeze-dried dough (0.16 g) of Tara2002 was dispersed in 0.1 M sodium phosphate buffer (pH 6.9, 20 mL) containing 1% SDS, and then stirred for 80 min at 60 C. The extract was centrifuged for 30 min at 37,000g and the supernatant was discarded. The residue was suspended in 0.1 M sodium phosphate buffer (pH 6.9, 5 mL) containing 1% SDS, and sonicated (Sonic Dismembrator Model 500, Fisher Scientific, Pittsburgh, PA) to solubilize the insoluble protein fraction in the residue (Singh et al., 1990). After centrifugation for 30 min at 37,000g, the supernatant (20 mL) was filtered (0.45 mm, Millex LCR [PTFE], Millipore, Billerica, MA). Size exclusion HPLC was carried out using a Waters 2695 Separations Module and a Waters 996 Photodiode Array Detector (Waters, Milliford, MA), quantifying absorbance at 214 nm. The size exclusion column was 8.0 300 mm (OHpak SB-804 HQ, Shodex, New York, NY). The column was eluted with 0.1 M sodium phosphate buffer (pH 6.9) containing 0.1% SDS at a flow rate of 0.7 mL/min. The column was calibrated with thyroglobulin (MW 669,000) and alcohol dehydrogenase (MW 150,000) (Koh and Ng, 2008). The molecular weight distribution patterns of SDS-unextractable proteins in the dough of Tara2002 with and without added phytate was determined.
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2.5. Iron chelating activity of dough before and after fermentation For determination of iron chelating activity, freeze-dried dough (0.2 g) before and after fermentation was suspended in 4 mL acidified methanol (HCl/methanol/water, 1:80:10, v/v/v) for 2 h at ambient temperature with constant shaking (Mpofu et al., 2006). The suspension was centrifuged at 2500g for 10 min, and the supernatant was collected for determination of iron chelating activity. Iron chelating activity was determined using the 2, 2’-bipyridyl competition assay. The reaction mixture contained 1 mM FeSO4 solution (0.1 mL), dough extract (50 mL), Tris-HCl buffer (pH 7.4, 0.8 mL), 2, 2’-bipyridyl solution (0.1% in 0.2 M HCl, 0.4 mL) and 10% hydroxylamine-HCl (0.3 mL). The absorbance of the reaction mixture was measured at 522 nm with a spectrophotometer (UV1601, Shimadzu, Kyoto, Japan) and used to calculate iron chelating activity using the following equation (Wang et al., 2009):
. o n 100 Iron chelating activity ð%Þ ¼ 1 Asample Acontrol where A sample was the absorbance of the test sample and A control was the absorbance of the control (without dough extract, 0% activity).
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portion of the supernatant (4 mL) was mixed with 30 mg/mL EDTA (8 mL) and adjusted to pH 6 with 2 N NaOH. The phytate extract was then freeze-dried and dispersed in distilled water (5 mL). The solution was passed through a 0.45 mm filter, adjusted to pH 12.6 with NaOH and lyophilized. Dried samples (100 mg) were dissolved in 600 mL D2O and transferred to a 5 mM NMR tube. 1H and 31P NMR spectra were acquired at 295 K (Varian Model 400, Varian NMR Instruments, Palo Alto, CA), and 399.76 MHz and 161.83 MHz, respectively. 1H decoupled 31P NMR spectra were performed with a 7 s relaxation delay, 15,060 Hz spectral width, 7.3 ms 90 pulse on 31 P and 103.5 ms 90 pulse for 1H decoupling, and 128 scans. Exponential multiplication (2 Hz line broadening) and Fourier transformation were used for NMR data processing. Chemical shifts for 31P spectra are given in parts per million (ppm) with 85% H3PO4 as an external standard. 2.7. Statistical analysis All experiments were conducted in duplicate and statistical analyses of the data were performed using SAS version 9.1 (SAS Institute Inc., Cary, NC, USA). Differences among experimental mean values were tested for significance at p < 0.05 using Tukey’s studentized range (HSD) test. 3. Results
2.6. Phytate content and composition of dough after mixing, fermentation and baking
3.1. Influence of added phytate on dough properties Phytate of flour, of freeze-dried dough before and after fermentation and of freeze-dried bread (1.5 g) was extracted and purified as previously described for bran, but quantities and volumes were scaled to one-tenth those previously described. The phytate content was measured using Wade reagent (0.03% FeCl3$6H2O and 0.3% sulfosalicylic acid in water) (Latta and Eskin, 1980). The absorbance at 500 nm was measured immediately. A standard curve was developed using phytic acid sodium salt hydrate (2e16 mg). Phytate was also extracted for determination of composition by NMR (Reale et al., 2004). Phytate of freeze-dried dough (6 g) of refined flour, whole grain wheat flour and refined flour added with phytates before and after fermentation and of bread (6 g) from Macon were extracted with 0.75 M HCl solution (30 mL) for 2 h with constant shaking. The extract was centrifuged at 1800g for 15 min, and 6 mL of supernatant was incubated for 15 min at 100 C. After cooling, the solution was centrifuged at 1800g for 15 min. A
Flour
+ Sodium salt of PA
The mixograms of refined wheat flours with added phytate are shown in Fig. 1. There were clear changes in the mixogram patterns when phytate was added to refined wheat flour. Dough mixing time increased with the addition of phytate in both Macon and Tara2002. Significantly longer dough mixing times were observed with neutralized PA and extracted-neutralized PA than with sodium salt of PA. Water absorption of the dough with sodium salt of PA (64e65%) was similar to that of refined wheat flour (64e65%), but increased to 70% with the addition of neutralized PA and extracted-neutralized PA. These results indicate that the addition of phytate (especially neutralized PA and extracted-neutralized PA) to wheat flour delays gluten network formation and dough development with increased water absorption, while sodium salt of PA delayed gluten development but did not influence the water absorption of dough. Quantity and quality of protein are the main factors affecting the dough mixing properties of flour (Finney and
+ Neutralized PA
+ Extracted-neutralized PA
Macon
Tara2002
Fig. 1. Mixograms of Macon and Tara2002 refined flours with added sodium salt of phytic acid (PA), neutralized PA and extracted-neutralized PA.
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Table 1 SDS-unextractable protein content and iron chelating activity (%) of Macon and Tara2002 refined flour doughs with the addition of three types of phytate (PA) before and after fermentation.a Addition to flour
Macon
Tara2002
a b
SDS-unextractable protein of dough (%)b
Iron chelating Activity of dough (%)
Before fermentation
Before fermentation
After fermentation
a
No added PA Sodium salt of PA Neutralized PA Extracted-neutralized PA No added PA Sodium salt of PA Neutralized PA Extracted-neutralized PA
1.43 1.00 0.85 1.02 1.31 0.90 1.15 1.12
1.07 0.79 b 0.87 b 0.91 b 1.28a 1.02 b 1.03 b 0.99 b
a
After fermentation
b
0.58 b 5.93a 4.36a 5.42a 0.85 b 4.08a 5.13a 3.85a
2.87 10.36a 12.56a 13.58a 1.32c 9.23 b 14.91a 14.09a
ab b ab a c b b
Mean values with different letters within each column of each wheat variety are significantly different (p < 0.05). % of flour dry weight.
after fermentation, although the reduction in the SDSunextractable protein content of fermented dough was not significant when sodium salt of PA or extracted-neutralized PA was added to Macon flour. In previous studies, the amount of unextractable polymeric protein was positively correlated with improved dough properties and loaf volume, because polymerization of gluten proteins is a critical determinant of wheat breadmaking quality (Aussenac et al., 2001; Weegels et al., 1996). A reduction in the SDS-unextractable protein content of dough with added phytate was likely the result of reduced glutenin polymerization. The dough with added phytate appears to have a less extensive gluten network, and a correspondingly weaker dough than dough without added phytate. Fig. 2 shows the molecular weight distribution of SDS-
Shogren, 1972), but results show that dough mixing properties were affected by the addition of phytate even though there was no change in protein content. It appears that when phytate is added to refined flour, in quantities similar to that of whole wheat flour, the effect is sufficient to impart substantial influences on gluten development and dough mixing properties. 3.2. Influence of added phytate on sds-unextractable protein content of dough before and after fermentation The SDS-unextractable protein content of dough before and after fermentation is summarized in Table 1. In both Macon and Tara2002, the SDS-unextractable protein content of dough was reduced significantly with the addition of phytate both before and
a)
0.03
0.02
AU
AU
0.02
0.01
0.00 5
10
15
20
0
Time (min)
c)
5
10
15
20
15
20
Time (min)
d) 0.03
0.02
0.02
AU
AU
0.01
0.00 0
0.03
b)
0.03
Mw Mw 669,000 150,000
0.01
0.00
0.01
0.00 0
5
10 Time (min)
15
20
0
5
10 Time (min)
Fig. 2. Molecular weight profiles of SDS-unextractable proteins separated by size-exclusion HPLC. Shown are glutenins extracted from mixed doughs from Tara2002 with a) no added phytate (control), b) sodium salt of phytic acid (PA), c) neutralized PA and d) extracted-neutralized PA. AU ¼ absorbance units at 214 nm. The two dots represent molecular weight standards as identified in Fig. part ‘a’.
E.Y. Park et al. / Journal of Cereal Science 70 (2016) 199e206
a)
b) 1200
1100
2
2
900
0 b
b
b
-2
800
700
-4
1100 a
1
1000
b
c
Firmness (N)
a
Firmness (N)
1000
L o af V o lum e o f B re ad (m L )
L o af V o lum e o f B re ad (m L )
203
c
900
0
800
-1 700
600
600 No added PA
+ Sodium salt of PA
-2
+ Neutralized + ExtractedPA neutralized PA
No added PA
+ Sodium salt of PA
+ Neutralized + ExtractedPA neutralized PA
Fig. 3. Bread loaf volume and firmness of a) Macon and b) Tara2002 baked with the addition of sodium salt of phytic acid (PA), neutralized PA and extracted-neutralized PA.
unextractable proteins in the dough of Tara2002 with added phytate. The refined flour dough with no added phytate exhibited a prominent glutenin peak near the 669 K marker and a much smaller glutenin peak near the 150 K marker. With the addition of phytate, there was a reduction in the first peak (near 669 K) and an increase in the second peak (near 150 K). The reduction of the first peak was greatest with added extracted-neutralized PA (Fig. 2d) and least with sodium salt of PA (Fig. 2b). These results clearly indicate that the added phytate decreases the proportion of the largest glutenin polymers (near 669 K) and increase the proportion of the smaller glutenin polymers (near 150 K) and thus phytate appears to reduce glutenin polymerization during dough mixing.
activity of phytases from both wheat and yeast (Türk et al., 1996).
3.3. Iron chelating activity of doughs before and after fermentation
3.5. Phytate content and composition
With the addition of phytate, the iron chelating activity of doughs increased several fold relative to the dough without added phytate both before and after fermentation (Table 1). Furthermore, the iron chelating activity of dough was reduced by fermentation, but iron chelating capacity was still substantially greater in the doughs with added phytate than in the refined wheat flour dough. The reduction of phytate during processing can be attributed to the
The changes in phytate content during dough mixing, fermentation and baking are depicted in Fig. 4. During the process of dough mixing, fermentation and baking, phytate content steadily decreased regardless of phytate type, but the degree of decrease differed among the phytate types. The decrease in phytate content during processing was greatest with sodium salt of PA in both Macon and Tara2002. A decrease in phytate content during bread
3.4. Baking properties The addition of all three types of phytate resulted in decreased bread loaf volumes (Fig. 3). With the addition of phytate, loaf volume of bread was reduced from 925 mL to 848e875 mL in Macon, and from 993 mL to 900e943 mL in Tara2002. Bread loaf volumes with added phytate were similar among the three types of phytate. Crumb texture was firmer in bread with added phytate (Fig. 3) possibly due to the smaller loaf volume of bread with added phytate.
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Fig. 4. Phytate content of flour, mixed dough, fermented dough and bread with no added phytate, or with phytate added as the sodium salt of phytic acid (PA), neutralized PA and extracted-neutralized PA in a) Macon and b) Tara2002. Units are mg phytate per gram dry weight of flour dough or bread.
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baking was also observed by Haros et al. (2001a). During baking, the phytate content of whole wheat bread was reduced by 50e60%, and the decrease was due to degradation of phytate by both endogenous phytase and yeast phytase (Türk et al., 1996). Endogenous phytase of wheat grain catalyzes the stepwise hydrolysis of phytate ultimately leading to inositol and monophosphates. Fig. 5 shows 1H-decoupled 31P NMR spectra of the doughs of cv. Macon. NMR analyses were conducted for refined flour dough (Fig. 5a), whole grain flour dough (Fig. 5b), and refined flour doughs with added 1% sodium salt of PA (Fig. 5c), neutralized PA (Fig. 5d) or
a)
extracted-neutralized PA (Fig. 5e). IP6 (four peaks) and orthophosphate (Pi) were the prominent peaks observed in the spectra. The NMR spectra confirmed much lower phytate levels in refined than in whole grain flour dough (Fig. 5a and b). The refined flour doughs containing added phytate types showed, in addition to the IP6 and Pi peaks, many smaller peaks (Fig. 5c, d, and e) likely representative of inositol phosphates IP1 to IP5. The appearance of inositol phosphates with a smaller number of phosphates in the spectra are due to the stepwise hydrolytic breakdown of phytate during processing, which corresponds to the decreasing phytate
b)
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ppm Fig. 5. 1H-decoupled 31P NMR spectra of doughs prepared from a) refined flour (control), b) whole grain wheat flour, and refined flours with the addition of c) sodium salt of phytic acid (PA), d) neutralized PA and e) extracted-neutralized PA before fermentation. Doughs were prepared from cultivar Macon.
E.Y. Park et al. / Journal of Cereal Science 70 (2016) 199e206
shown previously (Fig. 4); however the peaks were too small to allow quantification. NMR spectra were also conducted for fermented dough and for bread; as expected, these spectra showed substantially decreased IP6 peaks and increased Pi peak (E.Y. Park, Ph. D dissertation, 2014, unpublished). The dough with added phytate extracted from wheat bran (extracted-neutralized PA, Fig. 5e) exhibited an NMR spectrum that was most similar to the spectrum of whole grain flour dough (Fig. 5b), suggesting, as expected, comparable compositions of phytate isolated from wheat bran and whole grain flour endogenous phytate. 4. Discussion Haros et al. (2001a, 2001b) proposed that phytate chelation of calcium is responsible for the negative influence of phytate on bread processing. In their work, they found increased specific volume index of whole wheat bread by up to 21% by the addition of fungal phytase, a finding consistent with our observations that adding phytate decreases bread loaf volume. They attributed the increased bread loaf volume to the activation of endogenous aamylase by phytase, since the hydrolysis of phytate by phytase increases free calcium ions that are necessary for a-amylase activity. However, our research suggests a more direct mechanism for the adverse effects of phytate on baking quality, by interference with glutenin polymerization. We observed reduction in both the overall quantity of SDS-unextractable protein (Table 1) and the ratio of large/small glutenin polymers (Fig. 2). Consistent with our observations, Tronsmo et al. (2002) reported that a lower ratio of large/small glutenin polymers corresponds to weaker dough and reduced bread baking quality. Reduction in SDS-unextractable protein content (Table 1) and glutenin molecular weight (Fig. 2) with the addition of phytate is consistent with our hypothesis that phytate inhibits strong gluten network formation. There appear to be two mechanisms by which phytate could interfere with glutenin polymerization: i) complexing of proteins with phytate, and ii) iron chelating-antioxidant activity of phytate. These mechanisms, which are not mutually exclusive, are discussed in more detail, below. Phytate forms complexes with proteins (Cheryan, 1980), and protein-phytate interactions could interfere with glutenin polymerization. It was reported that the strong polyanionic character of phytate enables it to bind with positively charged amino acid residues on proteins, such as lysine and arginine (de Rham and Jost, 1979), so it is possible that electrostatic interactions between added phytate and positively charged glutenin amino acid residues may interfere with gluten network development in dough during mixing. Our results do not clearly either support or refute this explanation. The other mechanism by which phytate may interfere with glutenin polymerization is via phytate-mediated iron chelation, which suppresses oxidative reactions by inhibiting iron-catalyzed hydroxyl radical formation (Fardet et al., 2008). By chelating iron, phytate acts as an antioxidant and may prevent the formation of free radicals in food (Kumar et al., 2010). Oxidation induces the covalent crosslinking of proteins (including glutenins) through the formation of disulfide bonds and dityrosine crosslinks, which are largely responsible for the viscoelastic properties of the gluten network, and ultimately improves bread quality (Rasiah et al., 2005). The disulfide cross-linkings of glutenin molecules could be disrupted by the antioxidant activity of phytate, attributed to iron chelation, resulting in dough breakdown and reduced dough stability (Koh and Ng, 2008). The reducing action of antioxidants, therefore, adversely affects the rheological properties of dough, such as rapid dough breakdown immediately after optimum mixing time (Kerr et al., 1993). Conversely, oxidants are often added to
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bread formulations to improve gluten strength and loaf volume (Goesaert et al., 2005). Therefore we suggest that the strong iron chelating-antioxidant activity of phytate interferes with oxidative cross-linking of glutenins, which then leads to the observed reduced levels of SDS-unextractable protein content (Table 1) and reduced molecular weight of SDS-unextractable glutenins (Fig. 2). 5. Conclusions The addition of phytate to refined wheat flour at a 1% level approximated the in situ phytate content of whole wheat flour and induced deleterious effects on dough properties and bread baking quality. The addition of phytate increased mixograph mixing time and water absorption with delayed dough development. Loaf volume of bread was reduced with the addition of phytate. Phytate addition was also associated with a decreased SDS-unextractable protein content and a reduced proportion of higher molecular weight SDS-unextractable glutenin, which explains the weakened gluten and dough strengths and reduced bread loaf volume. The negative influences of added phytate on gluten and dough development may be due to their iron chelating-mediated antioxidant activity, which reduces oxidative cross linking among glutenin molecules during dough mixing; formation of protein complexes by phytate may also interfere with this cross-linking. It is possible, therefore, that phytate is a factor in the weak gluten development and the decreased bread loaf volume of whole wheat flour. This assumption also agrees with the speculation by Noort et al. (2010) that liberation of reactive components due to cell breakage was primarily responsible for the detrimental effects of bran rather than bran physically interfering with gluten development. Accordingly, the hydrolytic breakdown of phytate in whole wheat flour processing, as reported by Haros et al. (2001b) may be an important approach for enhancing whole wheat bread processing. Acknowledgments NMR analysis was performed at the center for NMR spectroscopy at Washington State University. We appreciate Dr. Craig Morris and Dr. Dan Skinner for access to facilities and materials at USDA-ARS Western Wheat Quality Lab. References AACCI, 2010. Approved Methods of the American Association of Cereal Chemists, eleventh ed. AACC International, St. Paul, MN. Aussenac, T., Carceller, J.L., Kleiber, D., 2001. Changes in SDS solubility of glutenin polymers during dough mixing and resting. Cereal Chem. 78, 39e45. Cheryan, M., 1980. Phytic acid interactions in food systems. Crit. Rev. Food Sci. Nutr. 13, 297e335. de Rham, O., Jost, T., 1979. Phytate-protein interactions in soybean extracts and lowphytate soy protein products. J. Food Sci. 44, 596e600. Dintzis, F.R., Lehrfeld, J., Nelsen, T.C., Finney, P.L., 1992. Phytate content of soft wheat brans as related to kernel size, cultivar, location, and milling and flour quality parameters. Cereal Chem. 69, 577e577. Evans, W., McCourtney, E., Shrager, R., 1982. Titration studies of phytic acid. J. Am. Oil Chemists’ Soc. 59, 189e191. me sy, C., 2008. Is the in vitro antioxidant potential of wholeFardet, A., Rock, E., Re grain cereals and cereal products well reflected in vivo? J. Cereal Sci. 48, 258e276. Feil, B., 2001. Phytic acid. J. New Seeds 3, 1e35. Finney, K.F., Shogren, M.D., 1972. A ten-gram mixograph for determining and predicting functional properties of wheat flours. Bak. Dig. 46, 32e35, 38, 42, 77. Goesaert, H., Brijs, K., Veraverbeke, W.S., Courtin, C.M., Gebruers, K., Delcour, J.A., 2005. Wheat flour constituents: how they impact bread quality, and how to impact their functionality. Trends Food Sci. Technol. 16, 12e30. Harland, B.F., Morris, E.R., 1995. Phytate: a good or a bad food component? Nutr. Res. 15, 733e754. Haros, M., Rosell, C., Benedito, C., 2001a. Fungal phytase as a potential breadmaking additive. Eur. Food Res. Technol. 213, 317e322. Haros, M., Rosell, C.M., Benedito, C., 2001b. Use of fungal phytase to improve breadmaking performance of whole wheat bread. J. Agric. Food Chem. 49,
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