Peroxidation Radical Formation and Regiospecificity of Recombinated

Feb 4, 2014 - Peroxidation radical formation and the regiospecificity of recombinated lipoxygenase from Anabaena sp. PCC7120 (ana-rLOX) were ...
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Peroxidation Radical Formation and Regiospecificity of Recombinated Anabaena sp. Lipoxygenase and Its Effect on Modifying Wheat Proteins Xiaoming Wang,† Fengxia Lu,† Chong Zhang,† Yingjian Lu,§ Xiaomei Bie,† Di Ren,† 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: Peroxidation radical formation and the regiospecificity of recombinated lipoxygenase from Anabaena sp. PCC7120 (ana-rLOX) were characterized by using ESR and HPLC-MS. It was found that ana-rLOX oxygenated at the C-13 position of the substrate linoleic acid (LA); at C-13 and C-16 of α-linolenic acid (ALA); at C-9, C-12, and C-15 of arachidonic acid (AA); at C12, C-15, and C-18 of eicosapentaenoic acid (EPA); and at C-14 and C-16 of docosahexaenoic acid (DHA), respectively. A total of 7, 14, 30, 28, and 18 radical adducts for LA, ALA, AA, EPA, and DHA were respectively identified by HPLC-MS. The functional characteristics of wheat protein, such as foaming capacity (FC), foam stability (FS), emulsifying activity index (EAI), emulsifying stability index (ESI), increased with enzymatic reactions. However, the average particle size of wheat proteins decreased with addition of ana-rLOX/LA. The ana-rLOX was also positivele effective in improving dough properties. These results provided clear evidence that ana-rLOX from Anabaena sp. could effectively improve the quality of wheat flour, which suggested that the enzyme could be applied as flour improver. KEYWORDS: radical formation, recombinated lipoxygenase, wheat proteins, wheat flour, natural flour improver

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popular in food processing.15,16 Recently, the cloning, expression, and functional analysis of genes coding for LOXs shed new light on the function of specific LOXs. In addition to LOX enzyme-mechanistic studies, the impact of LOX oxidation on dough has been investigated. The main uses included bleaching flour pigments through oxidation of carotenoid pigments, releasing bound lipids, improving dough rheology, and increasing loaf volume through promoting the loss of flour sulfhydryl groups.14,17−20 However, none of these studies has investigated the impact of LOX on proteins in depth, especially flour composition of wheat protein. The recombinant LOX is a new resource in food enzymology. Nonetheless, until now, no studies have been reported on recombinant LOX (rLOX) for food applications because of its low activity by heterogeneous expression. Previously, we successfully cloned, expressed, and secreted ana-rLOX from Anabaena sp. PCC 7120 in Bacillus subtilis. The production of the recombinant Ana-rLOX reached 76 U/mL (171.9 μg/mL) in the supernatant.21 In the previous work, the capability of the lipoxygenase from the procaryotes Anabaena sp. PCC7120 in improving dough qualities as a flour improver was investigated.22 However, the regiospecificity of LOX from Anabaena sp. PCC7120 has not been studied, and its derivatives are still unknown. The lipid regiospecificity of anarLOX and formation of lipid peroxidation radicals were characterized with a method that combined spin trapping, electrospin resonance (ESR), and mass spectrometry (ESI/

INTRODUCTION Incorporation of flour improver compounds into food products has been widely practiced for the purpose of enhancing food quality. Synthetic improvers, such as potassium bromate, azopotassium amide, and calcium peroxide, have been commercially used in numerous products for this purpose.1,2 However, there is growing concern about their safety, particularly their potential carcinogenicity and genotoxicity. As naturally occurring bioactive components are preferred in the markets, researchers are focusing on identifying and developing novel natural and cost-effective improvers.3−5 Recent studies have suggested that lipoxygenase (LOX) has been used as a potential dietary source of natural improver. Lipoxygenase is an oxygenase that can catalyze polyunsaturated fatty acid (PUFA) with cis-1,4 and pentadiene structure. This action was believed to involve the oxidation of PUFA by oxygen. Fatty acid radicals produced during the intermediate steps of substrate peroxidation were responsible for crosslinking of protein.6−8 These reactions might be attributed to certain specific sequences in the products. For instance, flourprocessing companies in the United States and Europe have been using soy flour to improve wheat flour’s qualities for a long time because soy flour is rich in LOX. A great deal of evidence of LOX associated with specific effects has been reported, indicating that LOX could increase mixing tolerance and generally enhances dough rheological properties.9−12 LOX has food-related applications in breadmaking and aroma production.13,14 As a new flour improver, LOX is safe, nutritious, and healthy. However, it is very difficult to implement in large-scale production due to its low production rate and purity from plants such as soybean. Hughes and Casey proposed that recombinant lipoxygenase would become © 2014 American Chemical Society

Received: Revised: Accepted: Published: 1713

December 2, 2013 January 27, 2014 January 27, 2014 February 4, 2014 dx.doi.org/10.1021/jf405425c | J. Agric. Food Chem. 2014, 62, 1713−1719

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the emulsions at 500 nm was measured in a spectrophotometer with blank of 0.1% SDS solution. Average particle size and specific surface area of proteins (albumin-, globulin-, gluten-soluble proteins) before and after ana-rLOX/LA enzymatic reaction, were measured by an integrated-laser light scattering instrument (Mastersizer 2000, Malvern Instruments Co. Ltd., Worcestershire, UK). Relative refractive index and absorption were set at 1.414 and 0.001, respectively. The particle size and specific surface area of proteins were analyzed using Malvern Mastersizer software (version 5.12c, Malvern Instruments Co. Ltd.). The protein was frozen and lyophilized according to the same method as previously described. Protein texture was observed by using a scanning electron microscope (SEM). The images were taken by using the SEM with a 6 kV acceleration voltage. The micrographs were taken using the same magnification. Effect of ana-rLOX on Texture of Wheat Flours. The anarLOX was added into 300 g of three kinds of wheat flours (Annong0950, Yang13, and Yang15), reaching final enzyme activities of 10, 20, 30, and 40 IU/g flour, respectively. Then, they were mixed in a Brabender Farinograph (mixer bowl 300 g, Brabender OHG, Duisburg, Germany) at 63 rpm and 30 °C for 10 min. Texture parameters including springiness (SP), gumminess (GU), cohesiveness (CO), and resilience (RE) of dough were evaluated using a textural analyzer (TA-XT plus, Stable Micro System Ltd., UK) equipped with a cylindrical probe of P20 (20 mm in diameter). The tested parameters for the instrument were set as follows: pretest speed, 2.0 mm/s; crosshead speed, 1 mm/s; post-test speed, 10.0 mm/s; rupture test distance, 1%; distance, 50%; and time, 5.00 s. The loaves were cut into slices of 15 mm, and the ends were discarded. All tests were conducted in triplicate Statistical Analysis. Data were reported as means ± standard deviations (SD) for triplicate treatments. One-way analysis of variance (ANOVA) and orthogonal design were performed using SPSS (SPSS for Windows, version 10.0.5, SPSS Inc., Chicago, IL, USA). Statistical significance was declared at P < 0.05.

MS). Also, the modification effects of the enzyme on the wheat proteins were investigated in the current work.

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MATERIALS AND METHODS

Materials. Three wheat varieties (Annong0950, Yang13, and Yang15) bred in China were used in this study. These cultivars were milled into flour using a Brabender experimental mill (Brabender, Duisburg, Germany) according to AACC approved method 26-21A. The flours were stored at 4 °C until further use. High-purity α-[4-pyridyl 1-oxide]-N-tert-butyl nitrone (POBN) and high-quality linoleic acid (LA), linolenic acid (ALA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) were purchased from Sigma Chemical Co. Ethanol, acetonitrile (ACN), and tetrahydrofuran (THF) were of HPLC grade. Purified ana-rLOX (activity = 30000 IU/mg) was prepared in our laboratory. Measurement of Radical Formation with ESR. Enzyme reactions were performed in 1 mL of 50 mM Tris-HCl buffer (pH 8). The reaction mixture containing 1 mM linoleic acid (linolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid), 20 mM POBN, and 8 × 103 IU/mL ana-rLOX was incubated at 16 °C without light. After 30 min, the reaction product was injected for ESR to detect the peroxidation radical. ESR spectra were obtained with a Bruker ElexSys E500 spectrometer equipped with a super high Q cavity operating at 9.78 GHz and room temperature. The ESR spectrometer settings were as follow: modulation frequency, 100 kHz; modulation amplitude, 1.0 G; microwave power, 20 mW; receiver gain, 106; time constant, 0.6 s; and magnetic field scan, 70 G. Determination of Radical Species from ana-rLOX with MS. The above mixture of enzyme reaction was terminated by ACN after 30 min, and the reaction products were separated with a centrifuge (Thermo, USA) at 104 rpm. The supernatant was kept and concentrated by evaporation. Finally, the concentrated sample was filtered by using a 0.45 μL membrane filter and injected for MS analysis. MS detection of lipid peroxidation radicals was carried out with a Finnigan LCQ ion trap mass spectrometer (Thermo, USA) using an electrospray ionization (ESI) and Finnigan X calibur software (version 1.2). ESI in positive ion mode was employed. The conditions were as follows: sheath gas flow, 35 arbitrary units; capillary temperature, 80 °C; capillary voltage, 17 V; and spray voltage, 4.5 kV. Samples were infused into the mass spectrometer at 5 μL/min. Separation of Wheat Proteins and Their Modification with ana-rLOX/LA. Wheat protein (albumin, globulin, and gluten) was extracted from wheat flour using a classical Osborne procedure based on protein solubility with modification.22 The protein extracts were further purified through dialysis and lyophilization and stored at 4 °C for further use. Protein content was assayed according to the colorimetric Bradford method using BSA as a standard.23 Wheat proteins modified with ana-rLOX were dissolved in 1% protein solution (w/v) containing 0.05% LA in 200 mL at 16 °C, with thermostatic shaking at 180 rpm for 2 h. The wheat proteins after enzyme reaction were extracted with acetone, and the extracts were dried through vacuum evaporation. Determination of Functional Properties, Particle Size Distribution, and Morphological Observation of Wheat Proteins Modified with ana-rLOX/LA. Functional properties such as the foam capacity (FC) and foam stability (FS) of wheat proteins prepared were determined according to the following Makri method.24 FC was measured in terms of volume increase on whipping expressed as percentage of original volume of the liquid. FS was expressed as percentage of foam volume remaining in relation to initial foam volume at room temperature after 0, 5, 10, 20, and 30 min. The emulsifying activity index (EAI), and emulsifying stability index (ESI) were tested according to Dipak’s method.25 The mixture of 140 mL modified protein and 60 mL oil was homogenized at 10000 rpm for 1 min, room temperature A sample of 0.02 mL of the emulsion was respectively taken out from the bottom of the vessel at 0 and 20 min, then diluted with 1 mL of 0.1% SDS solution. The light absorption of

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RESULTS AND DISCUSSION Radical Formation of PUFA Peroxidation by anarLOX. LOX-catalyzed PUFA peroxidation is a complex

Figure 1. ESR spectra of POBN radical adducts from ana-rLOX/LA oxidation. The reaction mixture contained 20 mM POBN, 0.1 M LA, and 8 × 103 IU/mL ana-rLOX.

biochemical event that generate a series of free radicals.6,7 However, there is limited direct evidence about the reactive and short-lived PUFA-derived free radicals due to the lack of an appropriate methodology. An ESR spectroscopy approach, with spin trapping, was taken here to investigate the dynamic behavior of fatty acids catalyzed by ana-rLOX with POBN. To 1714

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Table 1. Identification of POBN Adducts and Oxygenated Positions in ana-rLOX/PUFA Reaction PUFA α-linolenic acid

linoleic acid 474.7 508.8 490.1 266.0 418.0

POBN-LA POBN-LA(OH)2 POBN-LAO POBN-C5H11 POBN-C13H19O3

473.3 489.3 502.7 506.9 443.5 459.9 475.8 477.6 420.0 436.0 437.6 378.5 289.9 322.0

C-13

arachidonic acid

POBN-LOA POBN-LOAO POBN-LOAO2 POBN-LOA(OH)2 POBN-C16H25O2 POBN-C16H25O2-O POBN-C16H25O2-O2 POBN-C16H25O2(OH)2 POBN-C13H21O2-O POBN-C13H21O2-O2 POBN-C13H21O2(OH)2 POBN-C11H19O2 POBN-C7H11 POBN-C7H11-O2

C-13, C-16

MS Identification (m/z) POBN-AA POBN-AAO POBN-AAO2 POBN-AA(OH)2 POBN-C5H11 POBN-C15H21O2 POBN-C15H21O2-O POBN-C15H21O2-O2 POBN-C15H21O2(OH)2 POBN-C8H15 POBN-C12H17O2-O POBN-C12H17O2-O2 POBN-C11H19 POBN-C9H13O2 POBN-C9H13O2-O POBN-C9H13O2-O2 POBN-C9H13O2(OH)2 POBN-C3H6O2 POBN-C17H16 POBN-C17H16-O POBN-C17H16-O2 POBN-C17H16(OH)2 POBN-C6H10O2 POBN-C14H22-O POBN-C14H22-O2 POBN-C14H22(OH)2 POBN-C11H18 POBN-C9H14O2 POBN-C11H18-O POBN-C11H18O2 POBN-C11H18(OH)2 Regiospecific C-9, C-12, C-15

499.3 515.1 531.2 532.9 265.8 427.6 443.8 459.8 461.9 306.2 404.0 420.2 346.2 348.1 364.3 379.7 380.5 269.4 415.0 431.0 446.8 449.1 309.1 400.9 417.0 419.4 345.0 349.1 360.7 377.0 379.0

albumin albumin (R) globulin globulin (R) gluten gluten (R)

FC (V0) 320 360 292 310 362 385

± ± ± ± ± ±

4a 5b 5a 2b 3a 4b

FS (V30) 149 181 171 188 125 190

± ± ± ± ± ±

3a 6b 4a 6b 5a 7b

EAI (m2g−1) 4948 5826 5838 6019 3336 3299

± ± ± ± ± ±

16a 23b 10a 5b 12a 19a

ESI (min) 22 26 26 30 25 34

± ± ± ± ± ±

496.6 512.8 528.9 531.3 224.2 467.7 484.3 502.1 263.7 427.6 444.3 460.1 461.9 304.5 403.7 420.0 321.9 401.9 403.7 361.7 330.0 364.3 361.7 401.9 289.8 306.4 321.9 324.2

POBN-EPA POBN-EPAO POBN-EPAO2 POBN-EPA(OH)2 POBN-C2H5 POBN-C18H25O2 POBN-C18H25O2O POBN-C18H25O2(OH)2 POBN-C5H9 POBN-C15H21O2 POBN-C15H21O2-O POBN-C15H21O2-O2 POBN-C15H21O2(OH)2 POBN-C8H13 POBN-C12H17O2-O POBN-C12H17O2-O2 POBN-C7H11O2 POBN-C13H19-O2 POBN-C13H19(OH)2 POBN-C10H15O2 POBN-C10H15 POBN-C10H15-O2 POBN-C10H15(OH)2 POBN-C13H19O2 POBN-C7H11 POBN-C7H11O POBN-C7H11O2 POBN-C7H11(OH)2

C-12, C-15, C-18

docosahexaenoic acid 522.7 538.9 557.0 555.0 303.6 413.8 446.1 449.4 383.6 333.9 268.1 466.0 481.5 308.1 426.9 444.0 389.9 362.1

POBN-DHA POBN-DHAO POBN-DHA(OH)2 POBN-DHAO2 POBN-C8H13 POBN-C14H19O2 POBN-C14H19O2-O2 POBN-C14H19O2(OH)2 POBN-C14H21 POBN-C8H11O2 POBN-C3H5O2 POBN-C19H23-O POBN-C19H23-O2 POBN-C6H9O2 POBN-C16H23O POBN-C16H23(OH)2 POBN-C12H19O2 POBN-C10H13(OH)2

C-14, C-16

POBN, generating the stable radicals displayed in the ESR spectrum.26 A similar six peaks were found in soybean LOX.27 In addition, it was found that the spectrum of adding anarLOX was significantly different from that of nonenzyme, and the intensity of the peak with ana-rLOX was markedly increased. In comparison, the intensity of the peak without ana-rLOX was much lower, which indicated that ana-rLOX promoted the formation of LA peroxidation free radicals (Figure 1). Lipid peroxyl radicals resulted from the peroxidation of polyunsaturated fatty acids by soybean lipoxygenase.28 The increase of the intensity of the peak with ana-rLOX might be because ana-rLOX promoted more free radicals during the lipid peroxidation. Identification of the Oxygenated Position and Peroxidation Species Catalyzed by ana-rLOX. ESR alone cannot provide molecular structures and identities from hyperfine coupling constants, particularly those from spin adducts. Here, the combination with spin trapping and mass spectrometry (MS) was utilized to characterize PUFA-derived peroxidation radicals generated by ana-rLOX and to provide direct proof of the formation of PUFA peroxidation. The position oxygenated and the radical species formed on five PUFA (LA, ALA, AA, EPA, and DHA) are shown in Table 1. By the ESI/MS analysis of oxidation ion fragments, the

Table 2. Effect of ana-rLox on Physical Properties of Wheat Proteinsa protein

eicosapentaenoic acid

1a 1b 0a 1b 0a 1b

a

FC, foaming capacity; FS, foam stability; EAI, emulsifying activity index; ESI, emulsifying stability index; V0, enzymatic reaction at 0 min; V30, enzymatic reaction for 30 min; (R), adding ana-rLOX/LA.

facilitate comparison, all samples were tested at the same condition (aN = 15.7G and aH = 2.8G). In Figure 1, six peaks of the radical species in the ESR spectrum are displayed in the reaction system by adding ana-rLOX with LA and POBN or LA with POBN, indicating that the free radical of lipid oxidation and POBN adducts existed. The POBN was used to trap free radicals during the reaction. It could be assumed that the oxidation of the LA fatty acid promoted the formation of transient unpaired-spin-density species, after reacting with 1715

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Table 3. Effect of ana-rLOX/LA on the Particle Size of Wheat Proteina protein

D[4,3] (μm)

D[3,2] (μm)

SSA (m2/mg)

albumin albumin (R) globulin globulin (R) gluten gluten (R)

18.20 9.91 23.16 9.14 136.34 79.12

5.79 3.92 2.10 1.75 31.74 15.62

1.04 1.53 2.86 3.43 0.19 0.38

a Volume mean diameter (D[4,3]); surface area mean diameter (D[3,2]); specific surface area (SSA) of protein. (R), adding anarLOX/LA.

15.30 The ana-rLOX did not oxygenate on the sites C-5, C-8, and C-11, but did on sites C-14, C-16, and C-18, which were not reported in latest published papers, neither from animals nor from plants or bacteria. We should note that the different positions LOXs acted on would create different novel compounds; this result may have a special application in the food and clinical fields. In the radical adducts and their m/z of PUFA with anarLOX, totals of 7, 14, 30, 28, and 18 radical adducts for LA, ALA, AA, EPA, and DHA were respectively identified by ESI/ MS. LOX-catalyzed PUFA peroxidation is a well-known free radical chain reaction. Peroxyl radical is generated from the reaction of oxygen and can also be converted into other types of free radicals through H abstraction; Fe2+ converted into Fe3+ through electron transfer.31 As the result of chain reaction with peroxyl radical, many secondary radicals were produced and further induced more reactions. These new radicals can be metabolized to form new leukotrienes, lipoxins, jasmonic acids, green leaf volatiles, and lactones.32 Effect of ana-rLOX/LA on Funtional Properties of Wheat Protein. The use of soybean flour for commercial production of white bread has been well documented, and its main uses include bleaching flour, improving dough rheology, and increasing loaf volume. However, the effect of ana-rLOX/ LA on wheat protein has not been well reported. Table 2 presents FC and FS of wheat proteins as albumin, globulin, and gluten with or without ana-rLOX/LA. By adding ana-rLOX/LA in wheat proteins, FC and FS were significantly increased. The FC of albumin, globulin, and gluten solution respectively increased from 320, 292, and 360 mL before the enzymatic reaction to 360, 310, and 385 mL, and the FS of albumin, globulin, and gluten solution respectively increased from 149, 170, and 120 mL before the enzymatic reaction to 189, 188, and 190 mL, which suggested that the structural modification of wheat proteins had occurred and the physical properties had been changed during ana-rLOX enzymatic reaction. The proteins might generate two disulfide bonds through thiol oxidation with radicals initialized from anarLOX/LA, from which protein aggregation was produced, and finally the FC and FS increased. As shown in Table 2, the EAI and ESI of wheat proteins were significantly enhanced with the ana-rLOX/LA reaction system. The EAI of albumin and globulin solution respectively increased from 4948 and 5838 to 5838 and 6019 m2 g−1, but there was no significant change in the EAI for gluten. The ESI of albumin, globulin, and gluten solution increased from 22, 26, and 25 to 26, 30, and 34 min, respectively. The increase of EAI and ESI may result in the change of hydrophilic groups and hydrophobic groups related to emulsibility of protein. In the

Figure 2. Effect of ana-rLOX on particle size distribution of wheat protein. (R), with ana-rLOX/LA.

oxygenation position on LA was C-13; C-13 and C-16 on ALA; C-9, C-13 and C-15 on AA; C-12, C-15 and C-18 on EPA; C14 and C-16 on DHA, respectively. These results indicated that the positions oxygenated by ana-rLOX could vary with different PUFA. LA, as a substrate molecular oxygen, can be introduced either at carbon 9 (9-LOX) or at carbon 13 (13-LOX) of the hydrocarbon backbone, which led to the formation of 9hydroperoxy and 13-hydroperoxy derivatives of LA (9- and 13HPODE), respectively.29 In mammals, LOXs are similarly classified according to their positional specificity of arachidonic acid oxygenation, which can take place either at position C-5, C-8, C-9, C-11, C-12, or C1716

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Figure 3. Effect of ana-rLOX on micrographic structure of wheat flour by scanning electron micrograph: (left) no ana-rLOX/LA; (right) adding anarLOX/LA.

Table 4. Modification Effects of ana-rLOX on Different Wheat Flours variety

ana-rLOX (U/g)

Annong0950

0 10 20 30 40

0.32 0.34 0.34 0.35 0.34

springiness ± ± ± ± ±

0.01a 0.01b 0.01b 0.02b 0.01 b

0.27 0.30 0.30 0.31 0.28

cohesiveness ± ± ± ± ±

0.01c 0.01b 0.01b 0.01b 0.01a

23.90 22.72 25.06 38.98 29.27

± ± ± ± ±

1.32a 1.45a 1.63b 2.25c 1.97b

0.073 0.068 0.065 0.066 0.063

± ± ± ± ±

0.011a 0.015b 0.012b 0.017b 0.012c

Yang13

0 10 20 30 40

0.35 0.36 0.39 0.39 0.37

± ± ± ± ±

0.01a 0.01a 0.01b 0.02b 0.01b

0.30 0.30 0.38 0.31 0.33

± ± ± ± ±

0.01a 0.01a 0.02c 0.01a 0.01b

29.17 30.10 58.65 31.54 32.41

± ± ± ± ±

1.92a 1.18a 2.73d 1.85a 1.76b

0.061 0.072 0.079 0.061 0.076

± ± ± ± ±

0.016a 0.014b 0.016c 0.011a 0.018b

Yang15

0 10 20 30 40

0.40 0.41 0.43 0.45 0.43

± ± ± ± ±

0.01a 0.01a 0.02b 0.02b 0.01b

0.30 0.33 0.32 0.40 0.41

± ± ± ± ±

0.01a 0.01d 0.01b 0.01c 0.01c

31.74 49.70 27.67 38.14 63.59

± ± ± ± ±

1.50a 3.05c 1.92a 2.86b 4.65d

0.061 0.071 0.075 0.095 0.078

± ± ± ± ±

0.012a 0.014b 0.016b 0.015d 0.015c

1717

gumminess

resilience

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trend was shown in weak-, medium-, and strong-gluten flours (Table 4). The ana-rLOX positively affected CO in three doughs, too (Table 4). For weak-gluten flour, the dough’s CO increased by 13.3% at 30 IU/g of ana-rLOX and then decreased after the addition of more ana-rLOX. The same trend was also shown in medium-gluten flour with a maximum of 26.7% increase at 20 IU/g ana-rLOX. However, the dough’s CO of strong-gluten flour increased with more addition of ana-rLOX, and the maximum value of 37.7% was observed at 40 U/g ana-rLOX. ana-rLOX enhanced the dough’s GU in three flours. For weak-gluten flour, the dough’s GU increased by 63.1% at 30 IU/g of ana-LOX, followed by a decrease after additional anaLOX. The same trend was also shown in medium-gluten flour with a maximum of 101% increase at 20 IU/g ana-LOX. For strong-gluten flour, dough GU increased with more addition of ana-LOX, and the maximum of 100% increase was observed at 40 IU/g ana-rLOX (Table 4). Table 4 also shows different effects of ana-LOX on the dough’s RE among three flours. The dough’s RE of weak-gluten flour significantly decreased by 13.7% at 40 IU/g ana-rLOX as ana-rLOX increased. For medium-gluten flour, RE increased by 29.5% at 20 IU/g, but decreased thereafter. For strong-gluten flour, the maximum of the dough’s RE is 55.7% increase at 30 IU/g ana-rLOX. The results supported that ana-LOX-treated bread volume improved by 16.8%, the loaf height increased by 10.3%, and the specific volume increased by 18.2%. When LOX was added into dough, the increased number of formed disulfide bonds enhanced the ability of dough to retain gas.36 In American baking practices, the main purpose for adding enzyme-active soy flour into wheat flour is to improve dough properties.37 Overall, the additional ana-rLOX in the flours was able to improve wheat dough’s processing characteristics including SP, CO, GU, and RE to different degrees except RE for low-gluten flour. Although the activity of adding anarLOX for reaching the best effect of improving dough quality was different, these results provided clear evidence that anarLOX from Anabaena sp. PCC7120 could effectively improve the quality of wheat flour and explained why the enzyme increased loaf height, bread volume, and specific volume,22 suggesting that it could be not only applied to baking practices but also used as a natural flour improver for wheat-based food.

work, the conformation of wheat protein was changed in the oxidation process, caused by enzymatic reaction, and hydrophobic groups were exposed, which improved the lipophilicity of protein. Therefore, the emulsifying properties of proteins were improved. Through increasing EAI and ESI of wheat protein, the original dough of incompatibility of dispersed phase system becomes more homogeneous, and all kinds of materials in the dough were mixed evenly. Therefore, the quality of products is even and stable.33 Effect of ana-rLOX/LA on Particle Size and Surface Structure of Wheat Proteins. The particle size of wheat proteins in solution ranged broadly from 1 to 1000 μm (Figure 2). It was found that there were significant differences in the volume mean diameter (D[4,3]), the surface area mean diameter (D[3,2]), and the specific surface area (SSA) by addition of ana-rLOX/LA in protein solutions. The average particle size D[3,2] of albumin, globulin, and gluten solution decreased from 5.79, 2.10, and 31.74 to 3.92, 1.75, and 15.62 μm after the enzymatic reaction, respectively (Table 3). These results might be attributed to the modified reaction that was caused by ana-rLOX/LA, which made the protein structure more compact and led to the decrease of average particle size of wheat proteins. In addition, lipid peroxidation radicals could also induce S−S bond formation. The results indicated that all kinds of wheat protein were cross-linked by lipid peroxidation radicals. To confirm the above explanation, wheat proteins with or without ana-rLOX/LA were observed by scanning electron microscopy (SEM). The outer topography and inner structure of protein modified with or without ana-rLOX/LA are presented in Figure 3. Proteins modified by enzymatic reaction were in regular shape with a denser and tighter surface in comparison with nonenzymatic reaction. The wheat albumin, globulin, and gluten appeared to be tighter network structures, and wheat albumin presented the membrane-like structure by cross-linking of the radicals reaction after the addition of anarLOX/LA. The results indicated that the structure of wheat proteins became tighter and more compact due to the oxidation of proteins after the addition of ana-rLOX/LA. Therefore, the particle size of wheat proteins became smaller. All of the above changes of wheat proteins showed that the free radical yielded from the lipid oxidation affect the formation of the network of wheat protein. These results suggested that ana-rLOX might be useful in food industries to improve textural and leavening characteristics of bread, cakes, or toppings and confectionery products in which foaming properties are important, similar to that reported by Mirsaeedghazi.34 Modification Effect of ana-rLOX on Different Wheat Flours. It has been reported that LOX increases mixing tolerance and generally enhances dough rheological properties,35 suggesting that LOX could be utilized as a natural food improver. In this study, three wheat flours, Annong0950, Yang13, and Yang15, which are weak-, medium-, and stronggluten wheat flours, were used to determine whether ana-rLOX could effectively improve dough quality. Table 4 shows that the dough processing characteristics of the three wheat flours, such as springiness (SP), cohesiveness (CO), gumminess (GU), and resilience (RE), were improved after the addition of ana-rLOX in flours. The ana-rLOX had a positive impact on SP for three doughs. SP of three doughs with 30 IU/g ana-rLOX increased significantly by 8.4, 9.9, and 13.1%, respectively. A similar

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AUTHOR INFORMATION

Corresponding Author

*(Z. Lu) Phone/fax: +86-25-84396583. E-mail: [email protected]. cn. Funding

This work was funded by the National Natural Science Foundation of China (31071605; 31201423), Chinese “863 High-Tech” Program (2012AA022207), Science and Technology Support Program of Jiangsu Province (BE2011390), and a 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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