Article pubs.acs.org/JAFC
Effects of Recombinated Anabaena sp. Lipoxygenase on the Protein Component and Dough Property of Wheat Flour Xiaoming Wang,† Fengxia Lu,† Chong Zhang,† Yingjian Lu,‡ Xiaomei Bie,† Yajuan Xie,† and Zhaoxin Lu*,† †
College of Food Science and Technology, Nanjing Agriculture University, Nanjing 210095, China Department of Nutrition and Food Sciences, University of Maryland, College Park, Maryland 20742, United States
‡
ABSTRACT: The improvement effect of recombinated Anabaena sp. lipoxygenase (ana-rLOX) on the rheological property of dough was investigated with a farinograph and an extensograph. When 30 U/g ana-rLOX was added to wheat flour, the dough stability time extended from 7 to 9.5 min, the degree of softening increased about 31.1%, and the farinograph index also ascended. The dough with added ana-rLOX showed stronger resistance to extension throughout 135 min of resting time as compared to the dough without ana-rLOX. In addition, the protein component in the dough was varied with ana-rLOX. The glutenin in the dough was increased, whereas the gliadin, albumin, and globulin were decreased after the additino of ana-rLOX to the flours. Ana-rLOX could make globulin-3A, globulin 1a, and S48186 grain softness protein cross-link with gliadin and lowmolecular-weight (LMW) glutenin, leading to the formation of the protein polymer. These results based on proteomic analysis might provide evidence that ana-rLOX could affect the gluten protein component and explain why it improved the farinograph and extensograph parameters of wheat flour. KEYWORDS: recombinated Anabaena sp. lipoxygenase, rheological property, extensograph, wheat flour, protein component
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As a new flour improver, lipoxygenase (LOX, EC 1.13.11.12) is natural and safe in food. This enzyme has a number of effects on the quality of wheat dough such as bleaching and quality improving.10 The effects may involve the oxidation of pigments and unsaturated fatty acids. It has also been reported that LOX increases the mixing tolerance of dough and generally enhances dough rheological properties.11 Although some studies have pointed out that the hydrogen peroxide radicals produced by LOX would possibly influence dough rheology,12 the mechanism of effects on the protein structure and dough rheology by LOX is generally unknown. LOX promotes the formation of covalent bonds among polypeptide chains either by oxidative cross-linking of −SH groups, by cross-linking of tyrosine residues, or by acyl-transfer reactions among amino acid residues.13 However, it was not clear how the proteins would be cross-linked with other proteins when their structures were affected by radicals induced by LOX. Wheat proteins have been widely detected using HPLC and mass spectrometry techniques alone or in combination with other chromatographic techniques.14 The use of matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) for mapping tryptic peptides of wheat protein demonstrated good agreement between MS measured molecular weights and those derived from the gene sequences.15 Thus, it is possible to determine the change of wheat protein structure with LOX catalysis by means of HPLC and MALDI-MS.
INTRODUCTION It is well-known that the proteins in wheat flour are responsible for the dough rheological properties and the product quality.1 The major types of flour protein could be classified according to their solubility in different solvents.2 These proteins are the water-soluble albumins, salt-soluble globulins, acid- or alcoholsoluble gliadins, and a glutenin polymer that is partially soluble in acetic acid or alcohol.3 The gliadin and glutenin of wheat storage proteins, collectively called gluten,4 and the wheat gluten network are made up of high-molecular-weight (HMW) and low-molecular-weight (LMW) glutenin subunits stabilized by intermolecular disulfide bonds.5 In the dough preparation process, water was first mixed with wheat flour, and then the hydrated proteins started to become interconnected during kneading, forming a continuous spatial network that was classically described as the continuous gluten phase.6 Many studies have dealt with the rheological properties of dough and its improvers, which showed that the incorporation of improver altered the properties of dough by increasing the water absorption and lengthening the development time. Also, it has been found to alter the extension properties of wheat dough during fermentation.7,8 Incorporation of improver compounds into flour products has been widely practiced for the purpose of enhancing dough quality. Synthetic improvers such as potassium bromate, azo potassium amide, and calcium peroxide have been commercially used in numerous products for this purpose.9 However, their safety issues, particularly their potential carcinogenicity and genotoxicity effects on the human body, have raised many concerns. The fact that consumers prefer naturally occurring bioactive components encouraged researchers to identify and develop novel natural and cost-effective improvers. © 2014 American Chemical Society
Received: Revised: Accepted: Published: 9885
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Table 1. Ana-rLOX Effect on Farinograph Characteristics of Wheat Floura ana-rLOX (U/g) 0 5 10 20 30 40 a
water absorption (%) 57.70 58.00 59.70 65.00 65.20 57.60
± ± ± ± ± ±
0.10a 0.20b 0.10c 0.20d 0.30d 0.10a
development time (min) 3.70 4.20 4.70 3.80 3.20 2.40
± ± ± ± ± ±
0.10c 0.10d 0.10e 0.10c 0.10b 0.10a
stability time (min) 7.00 7.70 8.80 9.50 9.50 7.10
± ± ± ± ± ±
0.20a 0.10b 0.20c 0.20d 0.30d 0.10a
degree of softening (ICC) 74.50 78.60 93.20 97.10 97.50 71.30
± ± ± ± ± ±
1.30b 1.40c 1.70d 1.50e 1.70e 1.10a
Data are expressed as means ± SD, n = 3, p < 0.05. 200 mg dry KBr). A blank KBr disk was used as background. The FTIR spectrum was smoothed and the baseline was corrected automatically by using the built-in software of the spectrophotometer. Detection of Total Proteins in the Dough by HPLC. The total protein from 100 mg of dough was extracted stepwise three times with 1.5 mL of Tris-HCl (pH 8.5), vortexed for 2 min at room temperature, and incubated for 60 min at 40 °C with shaking. Samples were centrifuged at 10000g for 3 min. Supernatants were collected, mixed all together, and filtered through a 0.45 μm nylon filter. The protein extracts were analyzed on a Waters chromatography system (600S controller, 616 pumps, and 717 autosampler) (Waters Corp., New Castle, DE, USA), controlled with Millennium software by using a procedure similar to that of Marchylo et al. Twenty microliters of the filtered extract was injected into a 300SB-C8 reverse phase analytical column (4.6 × 250 mm, 5 μm particle size, 300 Å pore size; Agilent Technologies), and separation was carried out at 60 °C by using a linear gradient of aqueous acetonitrile containing 0.1% TFA at a flow rate of 1 mL/min. The acetonitrile concentration was increased from 24 to 50% over 80 min and held constant thereafter. Absorbance was monitored at 210 nm. To determine protein concentration, the standard curve based on BSA was made (y = 0.492x; R2 = 0.9966). Determination of Protein Component in the Dough by MS/ MS. One hundred microliters from the HPLC fraction was vacuum concentrated (Speed Vac) to complete dryness and redissolved in 5 μL of 50% acetonitrile containing 0.1% TFA. Then, 5 μL of 100 mM ammonium hydrogen carbonate was added into the solution to optimize conditions for tryptic digestion. The proteins were reduced by adding 1.1 μL of 100 mM dithiothreitol (DTT), followed by incubation in a water bath at 60 °C for 45 min with intermittent vortex every 10 min. The reduced proteins were alkylated by adding 11.1 μL of 100 mM iodoacetamide and keeping the solution in the dark for 30 min with intermittent vortex every 10 min. The reduced and alkylated proteins were incubated with 1.5 μL of trypsin (100 ng/ul) at 37 °C for 20 h. Tryptic digests (1 μL) were deposited on a metal surface sample plate, and 160 mg/mL 2,5-dihydroxybenzoic acid (DHB) (1 μL) was added on the top with air-drying.20 Samples were then analyzed in positive ion mode by using a MALDI with hybrid quadruple time-of-flight tandem mass spectrometry (QqToF-MS) prototype instrument constructed in collaboration with MDS SCIEX (model PBS-II, Ciphergen Biosystems Inc., Fremont, CA, USA). Peptide spectra were obtained using 600−1200 shots, whereas MS/ MS spectra were obtained using 4000−6000 shots. Statistical Analysis. Data were reported as means ± standard deviations (SD) for triplicate treatments. One-way analysis of variance (ANOVA) was performed using SPSS for Windows (version 10.0.5; SPSS Inc., Chicago, IL, USA).
It was found that the C-terminus of the allene oxide synthase (AOS)−LOX fusion gene from Anabaena sp. PCC 7120 belonged to the LOX family.16 Previously, we successfully cloned, expressed, and secreted an ana-rLOX from Anabaena sp. PCC 7120 in Bacillus subtilis.17 The recombined ana-rLOX exhibited ability as a flour improver to form peroxidation radical in the presence of polyunsaturated fatty acid12 and improve the quality of wheat flour.10 However, the mechanism of improving the quality of wheat flour by adding ana-rLOX was not clear. Therefore, the protein component and dough properties of wheat flour were investigated by measuring their farinograph and extensograph parameters and analyzed by HPLC-MS/MS in this study.
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MATERIALS AND METHODS
Raw Materials. Wan 44 wheat used in this study was a mediumgluten variety bred in Anhui province of China, and it was suitable for noodle-making (protein content, 11.7%; wet gluten, 36.5%; gluten index, 75.7). Measurement methods are described in detailed in the next paragraph. These cultivars were milled into flour by using a Brabender Laboratory Mill (Brabender, Duisburg, Germany) according to the AACC-approved method 26-21A (AACC 2000).18 The flours were stored at 4 °C until further use. Purified ana-rLOX was used in the work, and the enzyme activity was 30000 U/mg. Measurement of Dough Property. The farinograph parameters of flour were measured by using a Brabender Farinograph (mixer bowl, 300 g; Brabender), according to the standard method. Ana-rLOX was added into 300 g of wheat flour (Wan 44), reaching final enzyme activities of 10, 20, 30, and 40 U/g flour, respectively. Then, these flours were mixed in the farinograph bowl at 63 rpm and 30 °C for 20 min. The parameters of water absorption and stability time were automatically exported from the farinograph device. The extensograph parameters of flour were measured by using a Brabender extensograph. A piece of dough (150 g) was cut into a standard cylindrical shape by using the extensograph. The test piece was allowed to rest for 45 min in the extensograph rest cabinet at 30 °C. After that, the dough was stretched by the extensograph hook until rupture occurred. The characteristics were described as proposed by the extensograph, according to the standard method (AACC, 2000). Resistance to extension (R, mm), dough extensibility (E, mm), and R/ E values at 45, 90, and 135 min were indicated. Protein Extraction and Concentration Measurements. Protein was extracted from wheat flour on the basis of its solubility using a modified Osborne procedure.19 The protein extracts were further purified through dialysis and lyophilization and stored at 4 °C until use. Protein content was assayed by the colorimetric Bradford method using bovine serum albumin (BSA) as standard. Analyses were carried out at wavelength λ= 595 nm (UV−vis spectrophotometer (Shimadzu, UV-2450, Japan). To determine protein concentration, the standard curve based on BSA was made (y = 0.492x; R2 = 0.9966). FT-IR Measurements. An FT-IR spectrum of glutenin was collected between 4000 and 500 cm−1(mid-infrared region) on a PerkinElmer Spectrum 100 FTIR spectrometer (Thermo Nicolet Corp., Avatar 360, USA) with 256 scans at a resolution of 4 cm−1. The sample was ground with spectroscopic grade potassium bromide (KBr) powder and then pressed into 1 mm pellets (2 mg of sample per
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RESULTS AND DISCUSSION Effects of Ana-rLOX on the Index of a Flour’s Quality Property. The dough rheological properties are considered to predict the processing behavior and control the quality of food products. The farinograph is used to estimate the water absorption of flour, the formation time, the stability time, and the degree of softening of dough during mixing. These parameters are often used as indices to evaluate flour quality. The improvement effects of ana-rLOX on flour quality were 9886
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Table 2. Ana-rLOX Effect on Extensogram Characteristics of Wheat Dougha 0 U/g ana-rLOX index 2
extension area (cm ) resistance (BU) extensibility (mm) ratio (R/E) a
30 U/g ana-rLOX
45 min
90 min
135 min
45 min
90 min
135 min
110.0 ± 0.8 471.0 ± 2.3 155.0 ± 2.4 2.5
122.0 ± 0.8 648.0 ± 5.8 150.0 ± 1.5 3.1
117.0 ± 0.5 401.0 ± 2.0 136.0 ± 2.1 3.7
112.0 ± 0.9 500.0 ± 5.5 137.0 ± 2.9 3.7
132.0 ± 1.3 712.0 ± 2.1 122.0 ± 2.5 5.8
118.0 ± 1.1 749.0 ± 6.7 107.0 ± 3.3 7.0
Data are expressed as means ± SD, n = 3. BU, Brabender unit; ratio R/E, resistance/extensibility.
R/E of the dough with ana-rLOX was larger than that without enzyme and approached around 7.0 at 135 min, which indicated that the strength of the dough was significantly improved. It is generally known that the strength of dough and its three-dimensional network depends on the arrangement and number of disulfide bonds and sulfhydryl groups of the proteins, and most of the changes in dough-mixing properties are caused by thiol−disulfide interchange reactions.24 It has been reported that LOX reacted with the molecular oxygen and polyunsaturated fatty acids and formed the peroxides and hydroperoxides, which could oxidize the thiol groups of the proteins to form inter- and intramolecular disulfide bonds.12 Thus, the improvement of stretching ability of the dough could be because of cross-linking between the flour proteins by anarLOX in the work. Ana-rLOX Effect on Protein Component in the Dough. Various kinds of proteins in dough were extracted to investigate the change of quality properties of the flour by adding anarLOX. As shown in Figure 1, it was found that the glutenin in
investigated by using the farinograph, and the results are shown in Table 1. The water absorption of the dough was significantly increased after the addition of 30 U/g of ana-rLOX to wheat flour, reaching the maximum of 65.2%. However, the water absorption decreased when 40 U/g ana-rLOX was added to the flour. Flour water absorption is the percent of weight gain after the flour soaked in water under 30 °C. Higher water absorption reflected higher production rate. The increase of water absorption in wheat food could soften the core of the food and prolong the shelf life.21 The dough development time (DDT) reached the longest at 10 U/g enzyme, and then it decreased by 13% with increasing ana-rLOX to 40 U/g. For pure wheat flour, DDT reflects the total energy amount that is needed to develop the gluten network.22 The increase in the DDT showed that the dough became a more compact gluten network. However, DDT has an effect on the productivity; therefore, longer DDT for the dough should be avoided to improve output.23 The dough stability time was an indicator of the strength of flour. A higher value represents a stronger dough property. The dough stability time increased from 7 to 9.5 min when anarLOX was added to wheat flours from 0 to 30 U/g. This showed that the addition of ana-rLOX increased the stability time of the dough. In addition, the softening degree, also called mixing tolerance index, was applied to determine the degree to which dough would soften over a period of mixing. In this study, the softening degree of dough increased with the addition of ana-rLOX and reached the maximum of 97 at 30 U/ g, an increase of 31.1% compared to that without ana-rLOX. The result suggested that the gluten network was strengthened with the addition of ana-rLOX, which led to cross-linking of inter/intra-proteins of the rough. From the above result, it was found that the indices of the quality property of flour were improved and so the strength of the dough increased when a certain range of ana-rLOX was added to the flour. However, an overdose of ana-rLOX would shorten the development time and stability time and yield a poor-quality dough because overadding ana-rLOX could cause excessive cross-linking of the wheat protein and prevent the flour from absorbing water. Effects of Ana-rLOX on Extensograph Parameters of Flour Dough. The extensograph measures the extensibility and resistance of dough to stretch. A higher resistance to extension indicates that the dough requires a larger force to stretch. Table 2 presents the ana-rLOX effect on the extensograph parameters of the dough at 45, 90, and 135 min of fermentation time. The extension area and maximum resistance of the dough with addition of ana-rLOX were larger than those without enzyme. The maximum resistance reached 749.0 ± 6.7 BU at 135 min, which was a 34.73% improvement as compared to no enzyme. However, the extensibility of the dough with ana-rLOX was lower than that without enzyme.
Figure 1. Effect of ana-rLOX on the protein component of the dough: (▲) albumin globulin; (■) gliadin; (●) gluten.
the dough was increased, whereas the levels of gliadin, albumin, and globulin were decreased, with the increase of ana-rLOX activity from 5 to 40 U/g, indicating that the protein component was changed after the addition of ana-rLOX. Part of albumin and globulin was combined with glutenin turned into glutenin copolymers; therefore, the resistance of dough was improved. The protein component of wheat flour influenced mixing and baking properties. Gliadins and glutenins were the two primary storage proteins in wheat. Glutenin shows elastic property and enhanced cohesive strength of flour dough, and gliadin exhibits a viscous property and increases the extensibility of the gluten phase.25 This could also demonstrate that ana-rLOX could improve the rheological properties of wheat flour dough. Ana-rLOX Effect on FT-IR Spectroscopy of Glutenin. FT-IR spectra of the glutenin have several bands due to 9887
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hydration glutenin.26 The observed result could explain the enhancement of the water absorption in wheat flour modified with ana-rLOX as being due to the increased flexibility of the glutenin molecule by the shift of amide I and III bands. A similar effect of NaCl on β-sheet conformations was observed by FT-IR in glutenin proteins.29 Analysis of Protein Component of Dough Modified with Ana-rLOX by HPLC and MS/MS. HPLC coupled with mass spectrometry is a very powerful analytical technique due to its high sensitivity and accuracy. In HPLC, the proteins are eluted according to different surface hydrophobicities. Total protein in wheat dough before and after the addition of anarLOX was extracted and determined by HPLC to understand the change of the protein components. Figure 3 shows that
different vibrations of the peptide moiety. Amide I and amide III bands are the most frequently used to assign secondary structures of proteins. Amide I originates from CO stretching vibration of the amide group, whereas amide III comes from CN stretching and NH bending.26 In this work, the effect of ana-rLOX on the glutenin structure was evaluated through the two bands around 1660 and 1231 cm−1 and assigned to amides I and III, respectively. Figure2 shows that the amide I band at around 1660 cm−1 correlated to the α-helix conformation shifted to 1653 cm−1,27
Figure 3. HPLC chromatograms of the protein extracts from wheat dough before and after the addition of ana-rLOX: (A) non-ana-rLOX; (B) 30 U/g ana-rLOX.
there were four protein peak regions in the dough without the addition of ana-rLOX. However, the F1 peak at 14 min was not shown in the dough with ana-rLOX, indicating that the protein component could be altered with the addition of ana-rLOX. Generally, as the elution time increased, the polarity of the substances eluted out gradually decreased in gradient elution. Thus, the protein at 14 min could be considered as a polar and hydrophilic substance, such as albumins or globins with better water solubility. Then, each fraction was analyzed by MS/MS to determine the protein component to investigate why the protein at F1 peak did not show in the dough with ana-rLOX. The protein kinds, their molecular weights, and the characteristics of these fractions are summarized in Tables 3 and 4. The fractions at 14 min (F1), 20 min (F2), and 55 min (F4) peaks were analyzed by MS/MS. Seventeen peptide adductions [M + H]+ (m/z 1189.6, 1189.6, 1064.6, 2426.1, 1360.7, 1906.0, 1822.9, 1189.6, 1012.4, 991.5, 1280.6, 1028.6, 3164.4, 1813.9, 1522.8, 1311.6, 1362.5, and 1346.5) for fraction F1 were
Figure 2. FT-IR spectra of glutenin from wheat dough before and after the addition of ana-rLOX: (A) glutenin with ana-rLOX; (B) glutenin without ana-rLOX.
indicating that the α-helix conformation in the glutenin of the dough with ana-rLOX was decreased as compared to that without ana-rLOX. However, the amide III band (1200−1300 cm−1) at around 1231 cm−1 correlating to the β-sheet conformation was shifted to 1237 cm−1,28 which indicated that the β-sheet conformation in the dough glutenin modified by ana-rLOX tended to increase. The α-helix conformation of the protein has a more compact and stable structure, whereas the structure of the β-sheet conformation of protein is less compact and stable.27 Belton et al. reported that β-sheet was related to high chain mobility produced by an increase of 9888
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Table 3. Identification of Wheat Proteins for F1, F2, and F4 Peaks by MS/MS accessionb
protein reference scan(s) F1 peak 1. globulin-3A (Triticum aestivum) 7623 7629 7520 6720 8769 9309 9793 4758 6959 6410 7775 2. globulin 1 (Triticum aestivum) 7069 10720 3. S48186 grain softness 11732 12343 6735 6734 F2 peak 1. γ-gliadin (Triticum aestivum) 9657 11457 8748 8759 9145 2. gliadin/avenin-like 6955 8759 9145 3. gliadin/avenin-like 7847 7848 7258 7347 F4 peak 1. glutenin (wheat), low molecular weight 9277 9419 2. low-molecular-weight glutenin subunit (Triticum aestivum) 8707 8694 9568 9558
peptide sequence
a
M/H
+
Rspc 390979705
K.ALRPFDEVSR.L K.ALRPFDEVSR.L K.ILHTISVPGK.F R.AKDQQDEGFVAGPEQQQEHER.G R.DTFNLLEQRPK.I R.GSAFVVPPGHPVVEIASSR.G R.GSSNLQVVCFEINAER.N R.HEQEEQGR.G R.RPYVFGPR.S R.SFHALAQHDVR.V R.VAIMEVNPR.A
1189.6 1189.6 1064.6 2426.1 1360.7 1906.0 1822.9 1012.4 991.5 1280.6 1028.6
4 5 1 1 1 1 3 1 1 1 1 110341790
R.QEVQGGQYGSETGGSQQQQQGGGYHGVTVGR.G YHGVTVGR.G R.AGEGAVGVPLFQAQWGAR.E
3164.4
1
1813.9
K.AIWTSIQGDLSGFK.G K.DM*PLSWFFPR.T K.VDSCSDYVM*DR.C K.VDSCSDYVMDR.C
1522.8 1311.6 1362.5 1346.5
1 1086237 1 1 1 1 133741924
R.DALLQQCSPVADM*SFLR.S R.DALLQQCSPVADMSFLR.S R.SQAVQPRSCLVM*WEQCCQQLK.A R.SQAVQPRSCLVM*WEQCCQQLK.A R.SQAVQPRSCLVMWEQCCQQLK.A
1966.9 1950.9 2652.2 2652.2 2636.2
R.SAWEPQHPSSPEHQPTPQPQEHPVPHQK.L R.SQVVQHSSCLVM*WEQCCQQLK.A R.SQVVQHSSCLVMWEQCCQQLK.A
3217.5 2651.2 2635.2
R.CQAIHNVVESIR.Q R.CQAIHNVVESIR.Q R.M*SLHTLPSMCK.I R.QQQHHQPQQEVQLEGLR.M
1425.7 1425.7 1320.6 2083.0
1 1 1 2 2 281335538 1 1 1 281335544 1 1 1 1 121455
K.VFLQQQCIPVAMQR.C R.SQMLQQSICHVMQQQCCQQLR.Q
1717.9 2691.2
1 1 336092119
K.VFLQQQCSPVAM*PQSLAR.S K.VFLQQQCSPVAM*PQSLAR.S K.VFLQQQCSPVAMPQSLAR.S K.VFLQQQCSPVAMPQSLAR.S
2076.0 2076.0 2060.0 2060.0
1 1 1 1
a Italic and bold indicate the occurrence in wheat proteins before and after the addition of ana-rLOX. bGenBank accession number. cRank of preliminary score.
obtained. The peptides were matched with those in Triticum aestivum database and were identified as globulin-3A, globulin
1, and grain softness protein of T. aestivum, respectively (Table 3). 9889
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Table 4. Identification of Wheat Protein with Added Ana-rLOX for F2′ and F4′ Peaks by MS/MS accessionb
protein reference scan(s) F2′ peak 1. globulin 3A (Triticum aestivum) 6579 8535 9018 9639 4445 4798 6342 2. S48186 grain softness 11732 12343 6735 6734 3. γ-gliadin 9657 11457 8748 8759 9145 4. gliadin/avenin-like seed protein (Triticum aestivum) 6955 8759 9145 5. gliadin/avenin-like seed protein (Triticum aestivum) 7847 7848 7258 7347 6. globulin 1 (Triticum aestivum) 7102 7777 7. globulin 1 (Triticum aestivum) 7069 10720 8. A27319 gliadin- wheat 6638 8222 F4′ peak 1. S48186 wheat softness protein 1a, 15K 11902 12484 12977 6860 2. wheat softness protein-1B2 12093 6483 7243
peptide sequence
a
+
M/H
Rspc 390979705
R.AKDQQDEGFVAGPEQQQEHER.G R.DTFNLLEQRPK.I R.GSAFVVPPGHPVVEIASSR.G R.GSSNLQVVCFEINAER.N R.HEQEEQGR.G R.RGSGSESEEEQDQQR.Y R.SFHALAQHDVR.V
2426.1 1360.7 1906.0 1822.9 1012.4 1721.7 1280.6
K.AIWTSIQGDLSGFK.G K.DM*PLSWFFPR.T K.VDSCSDYVM*DR.C K.VDSCSDYVMDR.C
1522.8 1311.6 1362.5 1346.5
R.DALLQQCSPVADM*SFLR.S R.DALLQQCSPVADMSFLR.S R.SQAVQPRSCLVM*WEQCCQQLK.A R.SQAVQPRSCLVM*WEQCCQQLK.A R.SQAVQPRSCLVMWEQCCQQLK.A
1966.9 1950.9 2652.2 2652.2 2636.2
R.SAWEPQHPSSPEHQPTPQPQEHPVPHQK.L R.SQVVQHSSCLVM*WEQCCQQLK.A R.SQVVQHSSCLVMWEQCCQQLK.A
3217.5 2651.1 2635.2
1 1 1 281335544
R.CQAIHNVVESIR.Q R.CQAIHNVVESIR.Q R.M*SLHTLPSMCK.I R.QQQHHQPQQEVQLEGLR.M
1425.7 1425.7 1320.6 2083.0
1 1 1 1 110341801
R.QEVQGGQYGSETGGGQQQGGGYHGVTVGR.G HGVTVGR.G R.DYEQSMPPLGEGR.H
2878.3
1
1478.7
1 110341790
R.QEVQGGQYGSETGGSQQQQQGGGYHGVTVGR.G GYHGVTVGR.G R.AGEGAVGVPLFQAQWGAR.E
3164.4
1
1813.9
1 100783
R.DVVLQQHNIAHAR.S R.NLALQTLPR.M
1500.8 1025.6
1 1
1 1 1 1 1 1 1 1086237 1 1 1 1 133741924 1 1 1 2 2 281335538
1086237 K.AIWTSIQGDLSGFK.G K.DM*PLSWFFPR.T K.DMPLSWFFPR.T K.VDSCSDYVMDR.C
1522.8 1311.6 1295.6 1346.5
1 1 1 1 60652218
K.DM*PLSWIFPR.T K.LDSCSDYVM*DR.C K.LDSCSDYVMDR.C
1277.6 1376.5 1360.5
1 1 1
9890
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Table 4. continued accessionb
protein reference scan(s) 3. LMW wheat glutenin subunit 9277 9419 4. LMW glutenin 8707 8694 9568 9558 5. globulin-3A (Triticum aestivum) 7665 8743
peptide sequence
a
+
M/H
K.VFLQQQCIPVAMQR.C R.SQMLQQSICHVMQQQCCQQLR.Q
1717.9 2691.2
K.VFLQQQCSPVAM*PQSLAR.S K.VFLQQQCSPVAM*PQSLAR.S K.VFLQQQCSPVAMPQSLAR.S K.VFLQQQCSPVAMPQSLAR.S
2076.0 2076.0 2060.0 2060.0
K.ALRPFDEVSR.L R.DTFNLLEQRPK.I
1189.6 1360.7
Rspc 121455 1 1 336092119 1 1 1 1 390979705 12 1
a
Italic and bold indicate the occurrence in wheat proteins before and after the addition of ana-rLOX. bGenBank accession number. cRrank of preliminary score.
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Twelve peaks at m/z 1966.9, 1950.9, 2652.2, 2636.2, 2652.2, 3217.5, 2651.2, 2635.2, 1425.7, 1425.7, 1320.6, and 2083.0 were assigned to the peptide adduction [M + H]+ in fraction F2 at the 20 min peak. The compounds of the fraction F2 were identified as three major proteins, namely, γ-gliadin and two gliadin/avenin-like seed proteins (Table 3). However, the protein component was changed in the protein fraction F2′ by adding ana-rLOX (Table 4), and the new protein moleculed such as globulin 3A, globulin 1, and grain softness protein besides γ-gliadin and two gliadin/avenin-like seed proteins were found in fraction F2′. This observation indicated that the hydrophilic globulin could be integrated into the gliadin owing to the peroxidation radicals initialized by ana-rLOX. In the protein fraction F4 at the 55 min peak, six of the peptides adductions (m/z 1717.9, 2691.2, 2076.0, 2676.0, 2060.0, and 2060.0) were found and were identified as lowmolecular-weight glutenin and GLTA-wheat glutenin from T. aestivum. In fraction F4′, new protein molecules, such as S48186 grain softness protein 1a, grain softness protein 1B2, and globulin 3A, were also observed. This result demonstrated that the hydrophilic protein component in wheat flour was combined into glutenin by enzymatic reaction after the addition of ana-rLOX. The oxidative polymerization of the proteins to HMW fractions was an important prerequisite to obtain strong gluten. In our previous study, we had already observed that the flour treated with ana-rLOX improved the dough quality in terms of higher strength and tenacity, which have shown an increase of disulfide bond content because of the oxidation of free −SH groups.17 Moreover, these results provided clear evidence that ana-rLOX from Anabaena sp. PCC 7120 could make globulin cross-link with gliadin and LMW glutenin, which improved the rheologcal properties of the dough. Some studies have shown that LOX affected the flour quality by inducing S−S bond formation of glutenin,30 but this study demonstrated that anarLOX could make glutenin cross-link with other kinds of proteins, such as globulin-3A, globulin 1, and S48186 grain softness protein 1a, and 15K, forming HMW glutenin. These results not only provided clear evidence that ana-rLOX influenced the protein component of wheat flour but also explained the mechanism of its improving effect on the farinograph and extensograph parameters of wheat flour.
AUTHOR INFORMATION
Corresponding Author
*(Z.L.) Phone/fax: +86-25-84396583. E-mail:
[email protected]. cn. Funding
This work was funded by the National Natural Science Foundation of China (31071605; 31470095), the Chinese “863 High-Tech” Program (2012AA022207), the Science and Technology Support Program of Jiangsu Province (BE2011390), and the project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Notes
The authors declare no competing financial interest.
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