Article pubs.acs.org/JAFC
Effect of Processing on Phenolic Composition of Dough and Bread Fractions Made from Refined and Whole Wheat Flour of Three Wheat Varieties Yingjian Lu,†,‡ Devanand Luthria,*,‡ E. Patrick Fuerst,# Alecia M. Kiszonas,# Liangli Yu,† and Craig F. Morris#,⊥ †
Department of Nutrition and Food Science, University of Maryland, College Park, Maryland 20742, United States Beltsville Human Nutrition Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705, United States # Department of Crop and Soil Sciences, Washington State University, Pullman, Washington 99164, United States ⊥ Western Wheat Quality Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Pullman, Washington 99164, United States ‡
ABSTRACT: This study investigated the effect of breadmaking on the assay of phenolic acids from flour, dough, and bread fractions of three whole and refined wheat varieties. Comparison of the efficacy of two commonly used methods for hydrolysis and extraction of phenoilc acids showed that yields of total phenolic acids (TPA) were 5−17% higher among all varieties and flour types when samples were directly hydrolyzed in the presence of ascorbate and EDTA as compared to the method separating free, soluble conjugates and bound, insoluble phenolic acids. Ferulic acid (FA) was the predominant phenolic acid, accounting for means of 59 and 81% of TPA among all refined and whole wheat fractions, respectively. All phenolic acids measured were more abundant in whole wheat than in refined samples. Results indicated that the total quantified phenolic acids did not change significantly when breads were prepared from refined and whole wheat flour. Thus, the potential phytochemical health benefits of total phenolic acids appear to be preserved during bread baking. KEYWORDS: whole and refined wheat, phenolic acids analysis, three bread fractions, flour and two dough fractions, two base hydrolysis and extraction methods
■
INTRODUCTION Epidemiological studies have shown that consumption of whole grains provides a protective effect against chronic health conditions such as cardiovascular disease, type 2 diabetes, and certain forms of cancers.1 The exact mechanisms for the health beneficial properties of the whole grains have not been completely determined; however, it is thought that the higher concentration of the bioactive phytochemicals, such as vitamins, dietary fiber, minerals, and phenolic acids, in whole grains may play an important role in these benefits.2 Furthermore, the U.S. Department of Agriculture has recommended the consumption of three or more ounce-equivalents of whole-grain products per day.3 Wheat (Triticum aestivum) is one of the oldest crops and has been cultivated since 10000−8000 BCE. Wheat-based food products are consumed globally and are important sources of energy, proteins, carbohydrates, fiber, minerals, phenolic acids, vitamins, and other bioactive phytochemicals. Whole wheat flour includes the bran, germ, and endosperm, whereas refined wheat flour is rich in the endosperm, a carbohydrate-enriched fraction that is deficient in the above-mentioned nutritionally significant compounds. Phenolic acids are widely distributed in grains and are present in high concentrations in whole wheat grains. Phenolic acids are present in comparatively higher concentrations in the aleurone cell wall and seed coat as compared to the endosperm.4 © 2014 American Chemical Society
Thermal processing is often assumed to reduce the concentration of bioactive phytochemicals compared to fresh foods. There have been a number of recent studies evaluating the effect of baking on the concentration of bioactive phytochemicals. In support of the possible decrease in phytonutrients during cereal processing, Hensen et al.5 showed that the content of total ester-bound phenolic acids and ferulic acid dehydrodimers decreased in whole rye bread during baking. However, the antioxidant activity of whole rye bread fractions as measured by three different procedures, namely, Folin−Ciocalteu, oxygen radical antioxidant capacity, and trolox equivalent antioxidant capacity, by Michalska et al.6 increased during baking. In another study, increasing baking time and temperature increased antioxidant activity in whole wheat pizza crust made from two wheat varieties.7 However, no significant change in antioxidant activity was observed with variations in bran particle size and fermentation time.7 Additionally, baking increased the free phenolic acid, whereas bound phenoilc acids decreased or changed slightly in three whole grain bakery products (bread, cookies, and muffins).8 Received: Revised: Accepted: Published: 10431
April 25, 2014 October 2, 2014 October 6, 2014 October 6, 2014 dx.doi.org/10.1021/jf501941r | J. Agric. Food Chem. 2014, 62, 10431−10436
Journal of Agricultural and Food Chemistry
Article
sodium hydroxide and acidified with 6 N HCl. Free phenolic acids were extracted with diethyl ether and ethyl acetate (1:1, v/v). The residue was stored overnight at room temperature and hydrolyzed with 2 N sodium hydroxide, acidified by hydrochloric acid, and then extracted with ethyl ether and ethyl acetate (1:1, v/v) for analysis of insoluble bound phenolic acid. After evaporation of the organic phase under nitrogen, the residue was redissolved in methanol/water (75:25, v/v) and filtered through a 0.45 μm membrane filter. The filtered extract was analyzed for phenolic acid quantification by HPLC. In method B, the ground flour sample was mixed with 2 N NaOH containing 10 mM EDTA and 1% ascorbic acid for 30 min at 40−45 °C.15 The reaction mixture was acidified with 6 N HCl and then extracted with ethyl acetate. The organic phase was dried, and the residue was redissolved in methanol/water (75:25, v/v). Then, the extract was filtered through a 0.45 μm membrane filter and analyzed by HPLC. Four replicate base hydrolyses, extractions, and analyses of each fraction were carried out. Analysis of Phenolic Acids by HPLC-ESI-MS. Quantification of phenolic acids from all samples was carried out using an Agilent 1290 Infinity LC system coupled to diode array (DA) and mass spectrometry (MS) detectors from Agilent Technologies (Palo Alto, CA, USA). Identification of phenolic acids was achieved by comparison of mass and ultraviolet spectral data as previously reported and by comparison of retention time with authentic commercial phenolic acid standards.9,14 Briefly, separation of phenolic acids was achieved using a reversed phase C18 Luna column (150 × 4.6 mm; particle size = 5 μm) from Phenomenex (Torrance, CA, USA) at ambient temperature (between 22 and 27 °C) as previously described.9 Separation was achieved using a binary gradient with two solvents. Solvent A was 0.1% (v/v) formic acid in water, and solvent B was 0.1% (v/v) formic acid in methanol. The flow rate was set to 1 mL min−1. Twenty microliters was used for injection. The linear gradient consisted of 5−30% B for 25 min, followed by 30% B for 35 min, then ramped from 30 to 100% B for 10 min, and held at 100% B for 5 min. The mobile phase concentration was returned to 5% B and held for 10 min before the next injection. The mass spectral data were collected in both positive and negative ion modes at low and high fragment voltages (70 and 250 V). The instrument was set to scan from 100 to 750 mass units. The temperature of the drying gas was 300 °C at a flow rate of 12 L min−1; the capillary voltage for both positive and negative ion modes was adjusted at 3000 V and a nebulizer pressure of 50 psi. The LC system was directly connected to a mass spectrometer with no stream splitting. Statistical Analysis. Data were reported as means ± standard deviations (SD) for four replicate treatments. One-way analysis of variance (ANOVA) and Tukey’s tests were performed using SPSS (SPSS for Windows, version 10.0.5, SPSS Inc., Chicago, IL, USA). Correlation analyses were performed using a two-tailed Pearson correlation test. Statistical significance was declared at P < 0.05.
Two of the most commonly used methods for the determination of phenolic acids are (1) a total phenolic acids method in which the substrate is subjected directly to base hydrolysis in the presence of EDTA and ascorbic acid9 and (2) extraction and separation of soluble (free, conjugated) and insoluble bound phenolic acids followed by hydrolysis in the absence of absorbate and EDTA.10 In the present study, we carried out a direct comparison of these two methods in refined and whole wheat flours of three wheat varieties. We subsequently investigated the effects of the major processes in refined and whole wheat breadmaking by evaluating the phenolic acid and composition in each of the three wheat varieties utilizing the total phenolic acids method. Phytochemicals were analyzed in flour (F), mixed dough (MD), proofed dough (PD), bottom crust (BCT), crumb (CM), and upper crust (UCT) fractions of all wheat varieties.
■
MATERIALS AND METHODS
Wheat and Bread Samples. Three spring wheat cultivars representing three wheat classes, ‘Westbred 936’ (hard red), ‘Alpowa’ (soft white), and ‘Blanca Grande’ (hard white), were grown as previously described.11 Grain was tempered and milled to approximately 70% extraction (refined white flour) on a Bühler MLU-202 flour mill. The particle size of bran and shorts mill streams was reduced with a pin mill so that ≥70% passed through a 180 μm sieve; reduced fractions were then blended with the refined wheat flour to produce whole wheat flour. Flour samples were stored at −20 °C prior to processing. Bread was prepared from refined and whole wheat flours of each variety following the 100 g straight-dough pan bread test (90 min fermentation and 33 min proof are used, then doughs are baked for 21 min at 218 °C).12,13 Dough and bread fractions were made as follows: a dough sample was frozen immediately after mixing, referred to as “mixed dough”, and another dough sample was frozen after proofing, referred to as “proofed dough”. After baking, the loaves were divided into the following three components: “upper crust”, which represented that part of the loaf exposed directly to oven temperatures; “bottom crust”, representing that part of the crust in contact with the loaf pan; and “crumb”, which was everything except crust fractions. The crust components were scraped to remove the adhering crumb component. Each fraction was lyophilized and stored at −20 °C. Each flour sample was also tested as the “starting point”. Dried samples were broken up with a mortar and pestle and ground in a Tecator Cemotec 1090 burr mill set at the finest setting and stored at −20 °C. Prior to analysis, each fraction was further ground to a particle size of 40 mesh using a Micromill manufactured by Bel Art Products (Pequannock, NJ, USA). Chemicals and Reagents. Phenolic acid (vanillic, caffeic, syringic, p-coumaric, ferulic, and sinapic acids) standards and ascorbic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium hydroxide and all solvents (ethyl ether, ethyl acetate, and methanol) were obtained from Fisher Chemicals (Fair Lawn, NJ, USA). All chemicals and solvents were of either analytical grade or HPLC grade and were used directly without further purification. Ethylenediaminetetraacetic acid (EDTA) was purchased from EMD Chemicals (Gibbstown, NJ, USA). Polyvinylidene difluoride (PVDF) syringe filters with a pore size of 0.45 μm were purchased from National Scientific Co. (Duluth, GA, USA). Comparison of Two Phenolic Acid Methods. Flour samples from the three wheat varieties were hydrolyzed separately by two common methods. Method A allowed separation of soluble (free and conjugated) and bound insoluble phenolic acids,10 and method B involved direct hydrolysis of the total phenolic acids in the sample in the presence of EDTA and ascorbic acid.14 Briefly, the ground sample was extracted with acetone/methanol/water (7:7:6, v/v/v) first to obtain the soluble supernatant and residue. Soluble free and conjugated phenolics were first acidified (pH 2) by hydrochloric acid; free phenolics were extracted by ethyl ether and ethyl acetate (1:1, v/v), and then conjugated phenolics were hydrolyzed with 2 N
■
RESULTS AND DISCUSSION Comparison of Two Methods for Phenolic Acid Hydrolysis and Extraction. There are two chemical methods commonly used for extraction of phenolic acids. Method A allowed the separation of soluble (free and conjugated) and insoluble bound phenolic acids, whereas method B directly extracted phenolic acids after base hydrolysis of the plant matrix in the presence of EDTA and ascorbic acid.15 The results using method A (Figure 1A,B) showed that most (>82%) of the phenolic acids in all three varieties exist in insoluble bound form. These results were similar to those previously published in a study from our group and by other researchers.10,11,16 Ferulic acid was the most abundant phenolic acid present in soluble and insoluble bound forms for all three wheat varieties. It accounted for about 64.1−67.9% of total soluble phenolic acids and for between 84.4 and 85.6% of total insoluble bound phenolic acids (Figure 1C,D). Whent et al.11 also found that ferulic and p-coumaric acids were primarily in the insoluble 10432
dx.doi.org/10.1021/jf501941r | J. Agric. Food Chem. 2014, 62, 10431−10436
Journal of Agricultural and Food Chemistry
Article
of ferulic acid has been reported in a previous study by Moore et al.10 The amounts of total phenolic acids quantified in all three wheat varieties using method B hydrolysis and extraction were significantly higher than those of method A (P < 0.05). This is primarily due to the presence of additional antioxidant ascorbic acid and chelating agent EDTA. However, method A provided useful information on the soluble and insoluble bound phenolic acids that may also have importance in the determination of the bioavailability of the bioactive phenolic acids. In the present study, method B (base hydrolysis in the presence of EDTA and ascorbic acid) was used for comparison of phenolic acids content in refined and whole wheat fractions. Comparison of Phenolic Acids in Refined versus Whole Wheat Fractions. The quantities of total phenolic acids extracted from the refined flour samples were significantly lower than those from the whole wheat flour samples (Figure 2). This is in agreement with previously published papers in
Figure 2. Total phenolic acids of (A) refined wheat (RF) and (B) whole wheat (WW) flours of three wheat varieties by using two extraction methods. Method A involves separation of soluble (free and conjugated) and insoluble bound phenolic acids, whereas method B allows only determination of total phenolic acids in the presence of EDTA and ascorbic acid. The same letter indicates no statistical difference, whereas different letters stand for significant statistical difference (P > 0.05). Figure 1. Soluble (free and conjugated) and insoluble bound phenolic acids of (A) refined wheat (RF) and (B) whole wheat (WW) flours of three wheat varieties. Soluble (free and conjugated) and insoluble bound ferulic acid of (C) refined wheat (RF) and (D) whole wheat (WW) flours of three wheat varieties. The same letter indicates no statistical difference, whereas different letters stand for significant statistical difference (P > 0.05).
which authors observed only about 1/10 of the total phenolic acids content in wheat flour as compared to the whole grain flour.17−19 The phenolic acid composition and content in refined (RF) and whole wheat (WW) flour (F), mixed dough, proofed dough, bottom crust, crumb, and upper crust are presented in Tables 1, 2, and 3. In terms of overall abundance, FA was the most abundant phenolic acid, accounting for a mean of 59% (range from 51 to 68%) of TPA among RF fractions and a
bound form in flour samples from five wheat varieties. A similar high concentration (89.2−94.6%) of the insoluble bound form 10433
dx.doi.org/10.1021/jf501941r | J. Agric. Food Chem. 2014, 62, 10431−10436
Journal of Agricultural and Food Chemistry
Article
Table 1. Total Phenolic Acids (TPA) of the Flour, Dough, and Bread Fractions from ‘WB936’ (Hard Red Wheat) Varietya type
fraction
VA
CA
p-CA
SyA
SiA
FA
total
RF RF RF RF RF RF
flour MD PD CM BCT UCT
1.0 1.5 1.7 4.4 7.4 4.2
± ± ± ± ± ±
0.0f 0.1e 0.1d 0.2b 0.1a 0.1c
8.3 8.8 8.8 8.9 9.4 8.9
± ± ± ± ± ±
0.0c 0.1b 0.0b 0.1b 0.3a 0.6b
2.6 5.2 6.7 6.3 7.6 6.0
± ± ± ± ± ±
0.1e 0.6d 0.3ab 0.4bc 0.9a 0.5cd
4.5 4.5 4.4 6.4 5.6 7.0
± ± ± ± ± ±
0.0d 0.3d 0.3d 0.1b 0.2c 0.5a
15.0 14.3 14.1 14.8 14.1 17.1
± ± ± ± ± ±
0.1b 1.0b 0.3b 0.1b 0.3b 0.8a
65.6 55.0 52.7 68.7 66.3 70.6
± ± ± ± ± ±
0.9b 4.9c 3.2c 1.1ab 1.4b 2.5a
97.0 89.3 88.6 109.6 110.5 113.8
± ± ± ± ± ±
0.9b 6.5c 2.2c 3.5a 1.3a 1.4a
WW WW WW WW WW WW
flour MD PD CM BCT UCT
24.7 18.2 13.7 10.9 23.5 11.6
± ± ± ± ± ±
1.7a 1.5b 0.1c 0.4d 0.2a 0.4d
12.5 17.6 15.9 15.1 19.4 17.9
± ± ± ± ± ±
0.4e 0.5bc 0.7cd 2.5d 0.7a 1.7ab
27.5 21.1 25.7 22.9 29.1 17.4
± ± ± ± ± ±
0.4ab 0.5c 0.5b 0.8c 1.8a 1.9d
21.0 17.5 15.5 18.5 24.9 20.2
± ± ± ± ± ±
0.4b 0.1 cd 1.1d 3.6bcd 0.6a 3.3bc
57.7 56.2 49.0 52.0 42.7 37.8
± ± ± ± ± ±
2.4a 4.1ab 6.5c 3.2bc 5.4d 2.8d
602.2 563.6 534.7 534.4 569.8 631.9
± ± ± ± ± ±
11.3a 33.6ab 43.4bc 48.9bc 8.9ab 94.1a
745.6 694.3 654.4 650.5 709.5 736.9
± ± ± ± ± ±
20.1a 29.0ab 50.2bc 51.1bc 12.0ab 97.0a
a
RF, refined wheat; WW, whole wheat; CM, crumb; UCT, upper crust; BCT, bottom crust; MD, mixed dough; PD, proofed dough. VA, CA, SyA, pCA, FA, and SiA stand for vanillic, caffeic, syringic, p-coumaric, ferulic, and sinapic acids, respectively. Results are expressed as μg g−1 of flour (F), dough (MD or PD), or bread (BCT, CM, or UCT) on a dry sample weight basis. The results are expressed as the mean ± SD of four replicate extractions and analysis followed by a letter. The same letter indicates no statistical difference, whereas different letters stand for significant statistical difference (P > 0.05).
Table 2. Total Phenolic Acids (TPA) of the Flour, Dough, and Bread Fractions from ‘Alpowa’ (Soft White Wheat) Varietya type
fraction
VA
CA
p-CA
SyA
SiA
FA
total
RF RF RF RF RF RF
flour MD PD CM BCT UCT
3.8 4.5 4.2 5.2 5.1 9.0
± ± ± ± ± ±
0.2d 0.2bc 0.4cd 0.5b 0.7b 0.5a
2.8 2.9 3.3 3.3 3.5 3.8
± ± ± ± ± ±
0.2d 0.0d 0.2c 0.1c 0.0b 0.2a
6.3 6.8 9.6 13.0 7.4 9.6
± ± ± ± ± ±
0.2c 0.2c 0.5b 1.6a 1.0c 1.4b
5.1 5.7 5.6 5.9 5.6 5.8
± ± ± ± ± ±
0.1d 0.1b 0.1c 0.1a 0.1c 0.1b
10.0 9.1 8.9 9.2 7.7 11.2
± ± ± ± ± ±
0.1b 0.1bc 0.1c 1.0bc 1.0d 0.2a
38.1 35.6 33.3 39.2 38.2 41.7
± ± ± ± ± ±
0.6bc 0.4cd 1.9d 2.5ab 3.6bc 1.2a
66.0 64.9 64.9 75.7 67.5 81.0
± ± ± ± ± ±
0.6c 0.4c 2.2c 1.8b 5.19c 6.0a
WW WW WW WW WW WW
flour MD PD CM BCT UCT
38.3 10.0 40.0 13.8 13.8 20.3
± ± ± ± ± ±
1.3a 0.8d 1.5a 2.0c 2.3c 1.1b
9.3 9.2 4.8 7.4 11.0 12.7
± ± ± ± ± ±
0.4c 0.1c 0.1e 0.7d 0.2b 0.2a
14.1 13.3 14.1 15.8 19.7 14.9
± ± ± ± ± ±
0.2b 2.2b 1.5b 2.3b 0.4a 2.2b
13.3 14.0 14.6 14.3 14.3 13.3
± ± ± ± ± ±
0.5b 1.4ab 0.7a 0.3ab 0.6ab 0.4b
63.1 60.2 75.2 35.0 49.7 70.7
± ± ± ± ± ±
1.1b 3.0b 3.0a 2.1d 3.0c 3.3a
489.8 462.8 458.7 521.6 504.4 547.8
± ± ± ± ± ±
13.9cd 11.7de 8.4e 10.2ab 40.1bc 3.9a
627.8 569.5 607.3 607.9 608.1 679.9
± ± ± ± ± ±
12.8b 18.0c 7.3b 13.5b 25.2b 4.3a
a
RF, refined wheat; WW, whole wheat; CM, crumb; UCT, upper crust; BCT, bottom crust; MD, mixed dough; PD, proofed dough. VA, CA, SyA, pCA, FA, and SiA stand for vanillic, caffeic, syringic, p-coumaric, ferulic, and sinapic acids, respectively. Results are expressed as μg g−1 of flour (F), dough (MD or PD), or bread (BCT, CM, or UCT) on a dry sample weight basis. The results are expressed as the mean ± SD of four replicate extractions and analysis followed by a letter. The same letter indicates no statistical difference, whereas different letters stand for significant statistical difference (P > 0.05).
outer tissues of the wheat kernel, including aleurone and pericarp, compared to the starchy endosperm tissue; ferulate ester and ether cross-linkages were most abundant in the outer pericarp.4 The latter observations indicate that FA is far lower in the endosperm, consistent with our observation that FA is far lower in RF compared to WW fractions. Comparison of Processing Fractions within Each Variety. In comparison of the phenolic acid compositions among the six RF fractions, the upper crust fraction had the highest TPA of all fractions in all three varieties. RF upper crust also had the highest FA content in ‘WB936’ and ‘Alpowa’ (Tables 1−3). However, in ‘Blanca Grande’, FA content in the upper crust did not stand out among the fractions. Among the three RF bread fractions, the crumb fraction had the lowest TPA levels in ‘WB936’ and ‘Blanca Grande’, but in ‘Alpowa’ the bottom crust TPA was lower than that of the crumb. Both mixed and proofed dough fractions generally had slightly lower TPA and FA than bread fractions. However, the two dough fractions were similar, suggesting that fermentation had little effect on FA and TPA contents. When the effect of processing
mean of 81% (range from 76 to 86%) among WW fractions. Sinapic acid was the second most abundant phenolic acid, accounting for means of 13 and 8.5% of TPA in RF and WW fractions, respectively. Among the fractions evaluated, TPA ranged from 5.5- to 9.8-fold greater in WW fractions than in RF fractions (Tables 1−3). All individual phenolic acids were also higher in WW than in RF fractions (Tables 1−3). The greatest increases came from FA and sinapic acid, which had 11.5- and 5.5-fold increases, respectively, in WW compared to RF when averaged across fractions and varieties. Our results are consistent with previous studies demonstrating that total phenoilc acids were around 10-fold greater in whole wheat than in refined wheat products and are also consistent with our additional work on two other varieties.19,20 In addition, Baublis et al.21 reported that antioxidant activity was better in whole wheat than in refined wheat products. The dramatic increase in FA in WW compared to RF fractions was correlated with previously demonstrated higher levels (8.4−15.5-fold) of FA in rye bran than in RF flour.22 Ferulates were also far more abundant (ca. 100-fold) in the 10434
dx.doi.org/10.1021/jf501941r | J. Agric. Food Chem. 2014, 62, 10431−10436
Journal of Agricultural and Food Chemistry
Article
Table 3. Total Phenolic Acids (TPA) of the Flour, Dough, and Bread Fractions from ‘Blanca Grande’ (Hard White Wheat) Varietya type
fraction
VA
CA
p-CA
SyA
SiA
FA
total
RF RF RF RF RF RF
flour MD PD CM BCT UCT
3.8 7.3 6.7 4.5 11.2 15.2
± ± ± ± ± ±
0.3d 0.7c 0.4c 0.2d 0.8b 1.0a
2.8 3.4 3.3 3.3 3.2 4.1
± ± ± ± ± ±
0.0d 0.1b 0.1b 0.1b 0.0c 0.1a
12.9 6.8 5.6 7.2 5.8 6.7
± ± ± ± ± ±
0.2a 0.7b 0.3c 1.2b 0.2c 0.2b
5.0 5.8 5.9 5.9 5.8 5.8
± ± ± ± ± ±
0.1c 0.1ab 0.1a 0.1a 0.0b 0.0b
8.7 7.1 11.7 9.3 9.1 9.2
± ± ± ± ± ±
0.5b 0.7c 0.4a 0.6b 0.3b 0.1b
50.2 42.2 47.9 48.3 49.6 48.6
± ± ± ± ± ±
0.6a 2.4c 1.4b 0.6b 0.6ab 1.1ab
83.4 72.6 81.2 78.6 84.5 89.6
± ± ± ± ± ±
1.3bc 3.0e 2.0 cd 1.8d 1.0b 2.2a
WW WW WW WW WW WW
flour MD PD CM BCT UCT
26.8 36.5 18.6 12.4 30.1 31.9
± ± ± ± ± ±
0.7c 1.4a 1.2d 1.7e 4.4b 3.9b
5.8 7.3 8.9 8.7 8.9 9.3
± ± ± ± ± ±
0.7c 0.4b 0.2a 0.1a 0.5a 0.9a
11.9 18.7 18.8 18.3 17.6 18.8
± ± ± ± ± ±
2.1b 3.2a 1.1a 0.7a 1.6a 1.1a
17.4 17.6 18.6 18.5 17.2 18.7
± ± ± ± ± ±
0.4b 0.2b 0.4a 0.4a 0.5b 0.6a
66.2 49.8 84.6 84.7 49.9 49.9
± ± ± ± ± ±
1.7b 2.7c 3.2a 1.5a 4.1c 1.5c
593.8 580.5 598.4 566.0 567.0 619.4
± ± ± ± ± ±
11.4ab 16.7bc 20.9ab 12.2c 18.1c 13.9a
721.8 710.6 747.9 708.6 690.8 748.0
± ± ± ± ± ±
8.6ab 15.6bc 26.3a 15.5bc 20.3c 19.0a
a RF, refined wheat; WW, whole wheat; CM, crumb; UCT, upper crust; BCT, bottom crust; MD, mixed dough; PD, proofed dough. VA, CA, SyA, pCA, FA, and SiA stand for vanillic, caffeic, syringic, p-coumaric, ferulic, and sinapic acids, respectively. Results are expressed as μg g−1 of flour (F), dough (MD or PD), or bread (BCT, CM, or UCT) on a dry sample weight basis. The results are expressed as the mean ± SD of four replicate extractions and analysis followed by a letter. The same letter indicates no statistical difference, whereas different letters stand for significant statistical difference (P > 0.05).
Comparison of Wheat Varieties. When RF fractions for the three varieties were compared, TPA and FA contents were always highest in ‘WB936’, intermediate in ‘Blanca Grande’, and lowest in ‘Alpowa’ (Tables 1−3). In the WW fractions, TPA and FA contents for ‘Alpowa’ were consistently lowest as expected, but there was no consistent trend in the relative levels of TPA or FA when ‘WB936’ and ‘Blanca Grande’ were compared. Specifically for the WW flour fraction, however, FA content was greatest in ‘WB936’, intermediate in ‘Blanca Grande’, and lowest in ‘Alpowa’ (Tables 1−3), as previously reported.11 Conclusions. We investigated the effect of the breadmaking process and wheat varieties on the quantities of phenolic acids of refined and whole wheat fractions. Two commonly used base hydrolysis methods for the analysis of phenolic acids were tested. Method A provided information on the soluble (free and conjugated) and insoluble bound phenolic acids, which may have significantly different bioavailabilities and nutritional properties. Using method A, insoluble phenolics were far more abundant than soluble formsm but both soluble and insoluble phenolics were several times more abundant in WW flour than in RF flour (Figure 1A,B). Using both methods A and B, FA was the predominant phenolic acid present in both soluble and bound insoluble forms for all three wheat varieties. In method B, the whole sample was directly hydrolyzed in the presence of ascorbic acid and EDTA and showed 5−17% higher yields of TPA than method A (Figure 2). The effects of processing and varieties on phenolic composition in RF and WW fractions were evaluated using method B. FA was the most abundant phenolic acid, accounting for means of 59 and 81% of TPA among all RF and WW fractions, respectively. All phenolic acids measured were more abundant in WW than in RF fractions (Tables 1−3). The greatest increase came from FA, which had an 11.5-fold increase in WW compared to RF when averaged across fractions and varieties. Evaluation of the effects of the processing steps revealed that TPA and FA tended to decrease slightly in both RF and WW doughs compared to flour. However, the two dough fractions generally had similar TPA and FA levels, indicating that
of RF fractions on the minor phenolic acids, that is, those other than FA, was examined, some trends were noted. In two of three varieties, the highest levels of vanillic, caffeic, and sinapic acids were seen in the RF upper crust, similar to the trends noted above for FA and TPA. Also, the lowest levels of vanillic, caffeic, and p-coumaric acids were seen in the RF flour of all three varieties, suggesting the possibility that the availability to hydrolysis or chemistry of these phenolic acids may be altered during processing. The TPA trends among WW and RF fractions were similar as the upper crust, bottom crust, and flour fractions were generally higher than crumb, mixed dough, and proof dough fractions. TPA and FA in the dough fractions were, on average, similar to those in the crumb fraction. As in RF fractions, the TPA and FA levels were generally similar in the two WW dough fractions. No consistent trends were observed in examination of the effect of processing on the minor phenolic acids of WW fractions. It is a safe generalization that, in our study, the total process of preparing bread did not decrease TPA, FA, and other phenolic acids. Similarly, Mattila et al.19 found that baking did not significantly reduce the concentration of phenolic acids. In both RF and WW fractions we generally observed a slight decrease in TPA and FA in preparing dough fractions from flour. Accordingly, decreases in individual insoluble bound phenolic acids during dough preparation have also been reported by Abdel-Aal and Rabalski.8 Similarly, Han and Koh23 found an approximately 40% decrease in antioxidant activity in dough compared to flour samples. The decrease generally observed in dough preparation was more than compensated for by an increase in TPA and FA in baked bread fractions. The increase during baking appeared to be correlated with temperature, because the upper crust (highest temperature) most often had the highest TPA and FA levels. It is possible that intense heat rendered phenolic acids more physically accessible or chemically more susceptible to the hydrolysis process. Similar increases in phenolic acid levels and antioxidant activity during baking were reported in two other studies8,22 and in our own evaluations in two other wheat varieties.20 10435
dx.doi.org/10.1021/jf501941r | J. Agric. Food Chem. 2014, 62, 10431−10436
Journal of Agricultural and Food Chemistry
Article
(6) Michalska, A.; Amigo-Benavent, M.; Zielinski, H.; Del Castillo, M. D. Effect of bread making on formation of Maillard reaction products contributing to the overall antioxidant activity of rye bread. J. Cereal Sci. 2008, 48, 123−132. (7) Moore, J.; Luther, M.; Cheng, Z.; Yu, L. Effects of baking conditions, dough fermentation, and bran particle size on antioxidant properties of whole-wheat pizza crusts. J. Agric. Food Chem. 2009, 57, 832−839. (8) Abdel-Aal, E. S. M.; Rabalski, I. Effect of baking on free and bound phenolic acids in whole grain bakery products. J. Cereal Sci. 2013, 57, 312−318. (9) Luthria, D. L.; Liu, K.; Memon, A. A. Phenolic acids and antioxidant capacity of distillers dried grains with solubles (DDGS) as compared with corn. J. Am. Oil Chem. Soc. 2012, 89, 1297−1304. (10) Moore, J.; Hao, Z.; Zhou, K.; Luther, M.; Costa, J.; Yu, L. Carotenoid, tocopherol, phenolic acid, and antioxidant properties of Maryland-grown soft wheat. J. Agric. Food Chem. 2005, 53, 6649− 6657. (11) Whent, M.; Huang, H.; Xie, Z.; Lutterodt, H.; Yu, L.; Fuerst, E.; Morris, C.; Yu, L.; Luthria, D. Phytochemical composition, antiinflammatory, and antiproliferative activity of whole wheat flour. J. Agric. Food Chem. 2012, 60, 2129−2135. (12) AACC. Methods 10-10B and 46-30. In Approved Methods of the American Association of Cereal Chemists, 10th ed.; AACC: St. Paul, MN, USA, 2000. (13) Yu, L.; Haley, S.; Perret, J.; Harris, M. Antioxidant properties of hard winter wheat extracts. Food Chem. 2002, 78, 457−461. (14) Luthria, D. L.; Mukhopadhyay, S.; Krizek, D. T. Content of total phenolics and phenolic acids in tomato (Lycopersicon esculentum Mill.) fruits as influenced by cultivar and solar UV radiation. J. Food Compos. Anal. 2006, 19, 771−777. (15) Nardini, M.; Cirillo, E.; Natella, F.; Mencarelli, D.; Comisso, A.; Scaccini, C. Detection of bound phenolic acids: prevention by ascorbic acid and ethylenediaminetetraacetic acid of degradation of phenolic acids during alkaline hydrolysis. Food Chem. 2002, 79, 119−124. (16) Adom, K.; Sorrells, M.; Liu, R. Phytochemical profiles and antioxidant activity of wheat varieties. J. Agric. Food Chem. 2003, 51, 7825−7834. (17) Beta, T.; Shin, N.; Dexter, J. E.; Sapirstein, H. D. Phenolic content and antioxidant activity of pearled wheat and roller-milled fractions. Cereal Chem. 2005, 82, 390−393. (18) Hatcher, D. W.; Kruger, J. E. Simple phenol acids in flours prepared from Canadian wheat: relationship to ash content, color, and polyphenol oxidase activity. Cereal Chem. 1997, 74, 337−343. (19) Mattila, P.; Pihlava, J.; Hellström, J. Contents of phenolic acids, alkyl-and alkenylresorcinols, and avenanthramides in commercial grain products. J. Agric. Food Chem. 2005, 53, 8290−8295. (20) Lu, Y.; Lv, J.; Bayard, A.; Yu, L.; Fuerst, E. P.; Morris, C. F.; Yu, L.; Luthria, D. Effect of processing on phytochemical profile and antiproliferative activity of dough and bread fractions made from refined and whole wheat flours. J. Food Sci. (submitted for publication). (21) Baublis, A.; Decker, E. A.; Clydesdale, F. M. Antioxidant effect of aqueous extracts from wheat based read-to-eat breakfast cereals. Food Chem. 2000, 68, 1−6. (22) Andreasen, M. F.; Christensen, L. P.; Meyer, A. S.; Hansen, Å. Ferulic acid dehydrodimers in rye (Secale cereal L.). J. Cereal Sci. 2000, 31, 303−307. (23) Han, H.; Koh, B. Antioxidant activity of hard wheat flour, dough and bread prepared using various processes with the addition of different phenolic acids. J. Sci. Food Agric. 2011, 91, 604−608.
fermentation had little effect. The decrease generally observed in preparing dough was more than compensated for by an increase in TPA and FA in both RF and WW bread fractions. The increase during baking appeared to be related to temperature, because the upper crust, exposed to the greatest heat, generally had the highest TPA and FA levels. It is possible that intense heat rendered phenolic acids more physically accessible or chemically more susceptible to the hydrolysis process. When RF fractions for the three varieties were compared, TPA and FA contents were always highest in ‘WB936’, intermediate in ‘Blanca Grande’, and lowest in ‘Alpowa’ (Tables 1−3). In the WW fractions, TPA and FA contents for ‘Alpowa’ were consistently lowest as expected, but there was no consistent trend in the relative levels of TPA or FA when ‘WB936’ and ‘Blanca Grande’ were compared. In summary, we observed slightly higher TPA yields when samples were directly hydrolyzed in the presence of ascorbate and EDTA (method B) than when a procedure that omitted these reagents (method A) was used. Among the three varieties evaluated, ‘WB936’ and ‘Blanca Grande’ consistently had higher TPA levels than ‘Alpowa’. The effects of processing on phenolic acid composition were evaluated using direct hydrolysis (method B). TPA generally decreased slightly in dough compared to flour, but increased during baking, which was especially notable in the upper crust fraction, which was exposed to the most intense heat. Results clearly indicated that the phenolic acids measured do not decrease, and may even increase slightly, when bread is prepared from RF and WW flour.
■
AUTHOR INFORMATION
Corresponding Author
*(D.L.) Phone: (301) 504-7247. Fax: (301) 504-8314. E-mail:
[email protected]. Funding
This project was supported by Agriculture and Food Research Initiative Grant 2009-02347 from the USDA National Institute of Food and Agriculture. Notes
The authors declare no competing financial interest.
■ ■
ACKNOWLEDGMENTS We acknowledge Samina Shami of USDA-ARS for her assistance in carrying out experimentation. REFERENCES
(1) Jones, J. M. Grain-based foods and health. Cereal Foods World 2006, 51, 108−113. (2) Fardet, A. New hypotheses for the health-protective mechanisms of whole-grain cereals: what is beyond fibre? Nutr. Res. Rev. 2010, 23, 65−134. (3) USDA. Dietary guidelines for Americans 2010, http://www. health.gov/dietaryguidelines/dga2010/document/pdf/DGA2010.pdf (accessed July 24, 2013). (4) Barron, C.; Surget, A.; Rouau, X. Relative amounts of tissue in mature wheat (Triticum aestivum L.) grain and their carbohydrate and phenolic acid composition. J. Cereal Sci. 2007, 45, 88−96. (5) Hansen, H. B.; Andreasen, M. F.; Nielsen, M. M.; Larsen, L. M.; Knudsen, K. E. B.; Meyer, A. S.; Christensen, L. P.; Hansen, A. Changes in dietary fibre, phenolic acids and activity of endogenous enzymes during rye bread-making. Eur. Food Res.Technol. 2002, 214, 33−42. 10436
dx.doi.org/10.1021/jf501941r | J. Agric. Food Chem. 2014, 62, 10431−10436
