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Oxidative crosslinking of water-extractable wheat arabinoxylans by recombinant lipoxygenase and its effect on bread properties
LWT - Food Science and Technology 103 (2019) 1–7
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Oxidative crosslinking of water-extractable wheat arabinoxylans by recombinant lipoxygenase and its effect on bread properties
T
Chong Zhang1, Pei Wang1, Jie Yang, Di Ren, Zhaoxin Lu, Haizhen Zhao, Fengxia Lu∗ College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing, 210095, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Oxidative crosslinking WEAX Ana-rLOX Free radicals Bread staling
Water-extractable arabinoxylan (WEAX) is a pivotal component of wheat flour, which plays a positive role in bread making. In present study, the effect of recombinated Anabaena sp. Lipoxygenase (ana-rLOX) on the WEAX characteristics was evaluated. The results showed that ana-rLOX could effectively promote the oxidative crosslinking of the WEAX by acting as a free radical-generating agent and further elevated the particle size and viscosity of WEAX. The diferulic bridges induced by ana-rLOX could be formed through β-5'/α-O-4′, 5-5′ and 4O-5′ bonds. The crosslinked WEAX by ana-rLOX were superior in improving fresh bread quality and delaying the staling behavior as compared with the addition of ana-rLOX or WEAX alone. These results showed that ana-rLOX could improve the fresh and aged bread quality by modifying the WEAX fractions.
1. Introduction Arabinoxylan belongs to the nonstarch polysaccharide, which forms the cell wall of cereal endosperm and bran. About 25% of wheat arabinoxylan is water extractable (Mansberger et al., 2014). The conformation of water-extractable arabinoxylan (WEAX) is semi-flexible and regarded as dough improver, which can elevate the organoleptic quality of bread (Dervilly-Pinel, Thibault, & Saulnier, 2001). Ferulic acid (FA) is the predominant phenolic acid in wheat, and is mainly coupled to the C(O)-5 of arabinose through an ester linkage (Mendis & Simsek, 2014). Incorporation of FA into arabinose residues promotes the gel formation by developing the intermolecular crosslinks of WEAX. It has been demonstrated that some chemical or enzymatic free radicalgenerating agents like laccase, peroxidase and lipoxygenase can promote the oxidative crosslinking between AX molecules through the formation of dimeric- and trimeric-FA (Garcia, Rakotozafy, Telef, Potus, & Nicolas, 2002). As a new flour improver, lipoxygenase (LOX, EC 1.13.11.12) is green and safe in food industry. This enzyme can exert significant effect on dough quality, such as improving the microstructure and rheological properties of dough (Bahal, Sudha, & Ramasarma, 2013). The LOX gene from Anabaena sp. PCC 7120 had been successfully expressed in B. subtilis and the purified recombinant LOX was acquired in our laboratory (Zhang et al., 2012). The recombinated Anabaena sp. Lipoxygenase (ana-rLOX) catalyzes the oxidation of poly-unsaturated fatty acids to
form fatty acid hydroperoxides. This action was believed to produce fatty acid radicals during the intermediate steps of substrate peroxidation, which can crosslink the wheat protein fractions (Wang et al., 2014). However, the effect of ana-rLOX on the crosslinking of WEAX remains largely unelucidated. Polymerization of FA catalyzed by laccase and peroxidase had been widely investigated, and the storage stability of laccase-induced arabinoxylan gels was related to the generated radicals by laccase (Carvajal-Millan, Guigliarelli, Belle, Rouau, & Micard, 2005; Leskovac, Trivić, Wohlfahrt, Kandrac, & Pericin, 2005). Therefore, it was speculated that ana-rLOX might also promote the oxidative crosslinking between WEAX molecules by polymerizing FA regioselectively. Against this background, the main aim of this study was to investigate the rheological properties, molecular weight, particle size of WEAX gels and the polymerization of FA induced by the ana-rLOX, and to manifest the mechanism of oxidative crosslinking of WEAX catalyzed by ana-rLOX through electron spin-resonance spectroscopy (ESR) and high-performance liquid chromatography-mass spectrometry (HPLCMS). In addition, the effect of potential induced WEAX crosslinks by ana-rLOX on the bread aging behavior was also investigated.
∗
Corresponding author. E-mail address: [email protected] (F. Lu). 1 Co-first author, contributed equally to this work. https://doi.org/10.1016/j.lwt.2018.12.077 Received 6 October 2018; Received in revised form 29 December 2018; Accepted 30 December 2018 Available online 31 December 2018 0023-6438/ © 2018 Published by Elsevier Ltd.
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filtered through 0.45 μm cellulose membranes. The average particle size (hydrodynamic radius) was analyzed by an integrated-laser light scattering instrument (Zetasizer Nano particle size distribution analyzer).
2. Materials and methods 2.1. Materials
2.6. Viscosity measurement
Wheat bran was obtained from Jiangsu golden land seed industry Co., Ltd. (Yangzhou, Jiangsu, China), amyloglucosidase (AMG 300 L) and alpha-amylase (Termamyl 120 L) were purchased from Novozymes (Bagsvaerd, Denmark). All the used chemicals were of analytical grade.
The viscosity of 1% (w/v) WEAX solutions was measured by using Brookfield DV-II + Pro rotation viscometer (Brookfield Co., Ltd., USA) with a rotor SC4-18) at different shear rates at 20 °C.
2.2. Extraction and purification of WEAX from wheat bran 2.7. Measurement of radical formation with electron spin resonance (ESR) The extraction of WEAX was performed according to Wang, Tao, Jin, and Xu (2016) with some modifications. Wheat bran (100 g) was defatted with n-hexane and the sample (100 g) was heated at 130 °C for 90 min to inactivate the endogenous enzymes. After cooling to 50 °C, 100 g bran was added with 1 L distilled water with 0.02 mL α-amylase and stirred at 65 °C for 90 min. Then the solids were removed by centrifugation at 5000g for 10 min. The α-amylase was added and allowed to react at 95 °C for 1 h to degrade the starch contaminants. Afterwards, the pH was adjusted to 6.5 with 0.3 mL amyloglucosidase added and kept at 55 °C for 24 h. The enzymes were inactivated by heating at 120 °C for 30 min. The resulting solution was deproteinized by mixing with 10% bentonit solution, and the solution was cleared by centrifugation at 5000g for 10 min. The supernatant was collected and mixed with 4 times the volume of 95% aqueous ethanol. The mixtures were allowed to stand at 4 °C overnight. Finally, the crude WEAX was centrifuged (10,000×g, 4 °C, 10 min), followed by freeze-drying. The crude WEAX was further purified according to Vansteenkiste, Babot, Rouau, and Micard (2004). The protein content was assessed by the micro-Kjeldahl procedure with a nitrogen/protein conversion factor of 5.7.
The free radicals formed during the ana-rLOX action on LA with the addition of FA were detected by using ESR employing a spin trapping technique using α-(4-Pyridyl-1-oxide)-N-tert-butylnitrone (POBN). The reaction mixture contained 25 mM LA, ana-rLOX (72 IU/mg WEAX), 100 mM POBN, and 0.1 mM FA. After incubating in dark at 16 °C for 90 min, the mixture was loaded into a EPR Bruker EMX-10/12 spectrometer (Bruker EMX/plus, Germany) equipped with a high sensitivity cylindrical cavity operating at 9.77 GHz with 50 kHz modulation frequency to detect the radical intensity. ESR instrument settings for the spin trapping experiments were as follows: microwave power 20 mW; modulation amplitude 1.19 G; time constant 0.6 s; receiver gain 106; magnetic field, 70 G (Wang et al., 2014). 2.8. Reverse-phase (RP)-HPLC and mass spectrometer analysis of the oxidation products from FA and ana-rLOX The above mixture of the enzyme reaction was freeze-dried and redissolved in 300 mL of 50% (v/v) aqueous methanol. The RP-HPLC was conducted using an Agilent C18 column (4.6 mm × 250 mm, 0.5 μm). Elution was performed using a gradient system adapted from Piber and Koehler (2005). The gradient profile consisted of solvent A (10%, v/v, aqueous acetonitrile with 1 mM TFA), solvent B (80%, v/v, aqueous methanol with 1 mM TFA), and solvent C (80%, v/v, aqueous acetonitrile with 1 mM TFA) in the following program: 90% A, 5% B, and 5% C at the initial stage, with a linear gradient to 26% A, 37% B, and 37% C over the 25 min, then linear gradient to 0% A, 50% B, and 50% C over the 5 min, linear gradient to 90% A, 5% B, and 5% C over the 15 min, and held isocratically at 90% A, 5% B, and 5% C for 10 min, the flow rate was set at 1 mL/min. For MS analysis, the RP-HPLC fractions were collected and analyzed by the Xevo G2-S QTOF MS system (Waters Corp, Milford, USA) equipped with electrospray ionization (ESI) source. The capillary voltage and a cone voltage were set to 1000 and 35 V, respectively. The nebulization and gas was at 500 °C, with a flow rate at 1000 L/h. The source temperature of cone gas was 120 °C, with a flow rate at 150 L/h (Yang et al., 2016).
2.3. Determination of molecular weight (Mw), monosaccharides composition and FA content The Mw of WEAX was analyzed by an Agilent 1200 series high performance liquid chromatography (HPLC) system (Agilent Technologies, Santa Clara, CA, USA) according to Wang et al. (2019). For the monosaccharides composition analysis, 1 mg of WEAX was reacted with 300 μL of 4.0 M trifluoroacetic acid (TFA) at 120 °C for 2 h. The hydrolyzed WEAX was derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) and then analyzed by Agilent XDB-C18 column (Marcotuli et al., 2015). FA quantification was conducted following the method described by Hartmann, Piber, and Koehler (2005), p. 10 mg of WEAX was suspended in 2 mL of 4 M NaOH under nitrogen atmosphere and the hydrolysis was performed at 35 °C for 18 h after adjusting the pH to 2 with HCl. Then the solution was extracted with 5 mL of ethyl ether for three times, followed by the air-drying with nitrogen. The residue was redissolved in 1 mL of methanol, passed though the 0.45 μm filter, and separated by an Agilent Eclipse plus C18 column (4.6 × 250 mm, 5 μm). The elution was detected by an ultraviolet (UV) detector.
2.9. Effect of WEAX on the wheat bread quality A basic recipe consisted of wheat flour (200 g), instant dry yeast (3 g), salt (2 g), sugar (20 g) and water (90 g) and set as the control group. Ana-rLOX (20 U/g) and/or 1% (w/w, flour basis) WEAX were added. All ingredients were mixed in a laboratory mixer (Dongfangfude JHMZ-200, Beijing, China) to achieve the complete development. Then the dough was divided into 150 g, kneaded and mechanically sheeted and rolled. The dough was fermented at 30 °C for 45 min under the 85% relative humidity, and then baked at 215 °C for 15 min (Wang, Lee, Xu, & Jin, 2016). The loaf volume was determined by the rapeseed displacement method, and the specific volume was calculated by the loaf volume divided by the bread weight. The texture of bread including hardness, gumminess, chewiness and resilience were determined by a TA-XT2 texture analyzer (Stable Microsystems, Surrey, UK) equipped with Texture Expert v.1.20 software and calculated according to Huang, Guo, Wang, Ding, and Cui (2016). For the aging study, bread was cut
2.4. Preparation of the recombinated Anabaena sp. lipoxygenase (anarLOX) Recombinated Ana-rLOX was produced by extracellular production of B. subtilis recombined with Anabaena sp. PCC 7120 gene and purified by a nickel-charged NTA agarose column as described by Zhang et al. (2012). 2.5. Average particle size distribution Different dosage of Ana-rLOX was mixed with the 1% (w/v) WEAX solutions (pH 6.0, dissolved in 50 mM phosphate buffer) and 25 mM linoleic acid (LA), the mixtures were incubated at 25 °C for 2 h, and 2
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Table 1 Characteristics of WEAX extracted from wheat bran.
WEAX Ana-rLOX + WEAX
AX (%)
Protein (%)
Mw (kDa)
Ara (%)
Xyl (%)
Gal (%)
Glu (%)
A/X
FA (%)
85.46 ND
3.53 ± 0.08 ND
398.1 460.4
46.57 ± 0.39 ND
51.43 ± 0.42 ND
1.27 ± 0.09 ND
0.73 ± 0.05 ND
0.89 ND
0.436 ± 0.02 0.203 ± 0.02
AX = 0.88 × [(%Ara - 0.7 × %Gal) + %Xyl]; A/X= (%Ara-0.7 × %Gal)/%Xyl; ND, not determined; Ara, arabinose; Xyl, xylose; Gal, galactose; Glu, glucose; FA, ferulic acid.
from the center of each loaf (20 mm in thickness), packed in polypropylene bags and stored at 4 °C for 7 days. The water content of the bread crumb was measured gravimetrically by drying the sample at 105 °C to the constant weight. 2.10. Statistical analysis Data were expressed as means ± standard deviations (SD), and a one-way ANOVA was performed, followed by Duncan's multiple-range tests. P values lower than 0.05 were considered to be statistically significant. All statistical analyses were performed using SPSS software version 19.0 (Chicago, IL, USA). 3. Results and discussion 3.1. Characteristics of WEAX As shown in Table 1, the AX content of the purified WEAX was 85.46%, with a Mw of 398.1 kDa and protein content of 3.53%. Xylose (Xyl) and arabinose (Ara) were the major sugars, with minor contaminated galactose and glucose. The Ara to Xyl ratio (A/X) of WEAX was 0.89, which was higher than the previous results by Courtin and Delcour (1998). This indicated the obtained WEAX possessed a high substitution degree, which could increase the steric hindrance of rotation intramolecularly. This further led to a lowered molecular flexibility and aggregation rate of the molecular chain. As an important group for interacting inter- or intra-molecularly with other biomolecules, the ferulic acid (FA) content of WEAX was 0.436%. 3.2. Effect of ana-rLOX on the average particle size and viscosity of WEAX As shown in Fig. 1A, the hydrodynamic radius steadily elevated with the increased addition of ana-rLOX/LA in WEAX solutions. The average particle size of WEAX was increased by 42.68% under treatment of 72 IU/mg ana-rLOX/LA. The results might be due to the modified reaction by ana-rLOX/LA, which induced the formation of crosslinking structure in WEAX and further enhanced the average particle size (Primo-Martin, Wang, Lichtendonk, Plijter, & Hamer, 2005). Meanwhile, the viscosity of WEAX solution with and without enzyme at different shear rate were also detected (Fig. 1B). All the samples exhibited the shear-thinning behavior, since the viscosity decreased with the enhanced shear rate. The viscosity of WEAX solution with Ana-rLOX was higher than that of the control group. When adding 36 IU/mg ana-rLOX/LA, a slight increase in viscosity could be noticed. However, the viscosity had been remarkably improved by nearly 2.5 times than the control, when the dosage of ana-rLOX/LA increased up to 72 IU/mg. The enhanced viscosity was in accordance with the enlarged particle size and was probably originated from the crosslinking formation of WEAX.
Fig. 1. (A) Particle size of WEAX under the different addition levels of ana) and with the rLOX. (B) Viscosity versus shear rate of WEAX without ( ) and 72 IU/mg ( ) ana-rLOX/LA. addition of 36 IU/mg (
of WEAX catalyzed by ana-rLOX. The POBN was used to trap free radicals during the reaction, thus the intensity of POBN was the lowest. When incorporation with ana-rLOX and LA, six radical species in the ESR spectrum were presented regardless of the presence of FA (Fig. 2), indicating the generated adducts from the free radical of lipid oxidation and POBN. The intensity of POBN + Ana-rLOX + LA group was the highest, while the intensity of free radicals with the addition of FA decreased dramatically. Lipid peroxyl radicals were originated from the peroxidation of polyunsaturated fatty acids catalyzed by lipoxygenase, which could further react with POBN and generate the stable radicals detected by ESR (Wang et al., 2014). The decreased free radicals with the incorporation of FA might be attributed to that FA could compete with the POBN for the peroxyl radicals and formed the dimers or
3.3. ESR determinations LOX-catalyzed LA peroxidation is complex involving a series of free radicals. As the free radicals generated during LA peroxidation was short-lived, radical trap POBN and ESR spectroscopy approach was undertaken to study the dynamic behavior of the oxidative crosslinking 3
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Fig. 2. ESR spectra of POBN radical adducts from POBN (blue line), POBN + ana-rLOX + LA (black line) and POBN + ana-rLOX + LA + FA (red line). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3. RP-HPLC profile of the FA after reaction with ana-rLOX. Di-FA, dimeric ferulic acid. (B) ESI-MS spectra of Di-FA. Signals were assigned to the structure of the ions. Molecular mass of the identified Di-FA is 386 g/mol. (C) Speculative structures of the Di-FA related to those known from ferulate homocoupling.
4
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Fig. 4. The cross-section image (A), specific volume (B) and texture profile (C) of bread with or without the addition of ana-rLOX, WEAX and ana-rLOX + WEAX. The hardness, guminess, chewniess and resilence were indicated by black bar, dark grey bar, light grey bar and filled circle, respectively.
trimers of FA via the covalent crosslinking in the WEAX network as speculated.
formation of FA dimers or trimers from the FA group on the side chain. When oxidase was added, FA underwent oxidative crosslinking through different reaction systems, so that the FA monomer content attached to the AX side chain was significantly reduced by 53.4% (Table 1), indicating that FA was involved in the oxidation crosslinking process under the action of Ana-rLOX on WEAX, which further elevated the Mw
3.4. Oxidation of FA and fractionation of oxidation products The oxidation crosslinking reaction of WEAX occurred through the 5
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3.5. Effect of WEAX crosslinking on the breadmaking performances and storage characteristics of wheat bread The cross-section images of bread under the different treatment are shown in Fig. 4A. Specific volume and textural profile are the most important parameters for the bakery products. The combined incorporation of ana-rLOX and WEAX (ana-rLOX + WEAX) was the most effective on improving the specific volume of bread, followed by the addition of ana-rLOX and WEAX alone (Fig. 4B). The significant increase in specific volume contributed to the decrease in crumb hardness with ana-rLOX and/or WEAX addition. Meanwhile, the gumminess and chewiness of bread decreased while the resilience increased, with the most evident effect observed for the ana-rLOX + WEAX group (Fig. 4C). These results demonstrated that the combined incorporation of ana-rLOX and WEAX was superior in enhancing the bread quality. WEAX was generally regarded as a dough improver to enhance the organoleptic quality of bread. The improved effect of WEAX on bread quality was greatly dependant on the structure of WEAX, including the branched degree, Mw and the amount of bounded FA groups (Saeed, Pasha, Anjum, & Sultan, 2011). The improved effect of WEAX on bread properties was attributed to the molecular interactions between WEAX and dough components, as well as the protection of the foams during the heating of dough conferred by its viscous property (Biliaderis, Izydorczyk, & Rattan, 1995). Therefore, the enhanced viscosity and Mw of WEAX by ana-rLOX could contribute to its superior improved effect. During the storage of bread, the bread aged and resulted in the texture hardening, elasticity loss, dryness and loss of smell. In this study, hardness and moisture content were chosen as important indicators for bread aging (Nivelle, Bosmans, & Delcour, 2017). As shown in Fig. 5A, the hardness of the crumb increased during the storage at 4 °C, in which the hardness of the crumb was lowest for the anarLOX + WEAX group, followed by the WEAX, ana-rLOX and control group. Therefore, adding ana-rLOX and/or WEAX was also beneficial to the bread quality during storage. Moreover, the hardening rate was also suppressed by WEAX and/or ana-rLOX, with the most obvious effect for the ana-rLOX + WEAX group. The storage of bread led to the subsequent decrease in the water content of crumb. The moisture content of ana-rLOX + WEAX group was higher than the other groups, and the moisture loss rate was also reduced (Fig. 5B). The results of hardening rate and water retention property demonstrated that WEAX had the capability to suppress the bread staling behavior, and WEAX + anarLOX had a synergistic effect on enhancing the bread quality. Bread staling is mainly determined by the retrogradation behavior of starch, which involves the water redistribution and starch recrystallization (Delcour et al., 2010). Starch recrystallization is a complex behavior, accompanying with a series of physicochemical changes such as gel formation, exudation of water, and increased turbidity, viscosity and degree of crystallinity (Hoover, Hughes, Chung, & Liu, 2010). As a crucial factor, rate and extent of starch retrogradation is largely dependent on water content. During the storage of bread, moisture migrates from crumb to crust, and from gluten to starch, contributing to the starch recrystallization behavior (Nivelle et al., 2017). Therefore, the WEAX crosslinking induced by ana-rLOX formed the network structure, and consequently improved the water retention ability of WEAX and dough. Thus, the moisture content could be better preserved, and the bread staling was delayed.
Fig. 5. The hardness (A) and water content (B) of bread during the storage for ), ana-rLOX( ), WEAX ( ) and WEAX + ana-rLOX ( ) the control ( group.
of WEAX. Furthermore, the data available from the literature suggested that the dimerization of FA was influenced by many factors, the kinds of enzyme, the temperature and pH of the reaction system (Garcia, Lalatiana Rakotozafy, & Nicolas, 2004). Different enzymes catalyzed the formation of FA oxidation products in various ways, even the same enzymes like lipoxygenase from different sources could catalyze oxygenation at different sites. Therefore, it was essential to investigate the formation of FA oxidation products catalyzed by Ana-rLOX in order to explain the mechanism of crosslinking between WEAX molecules. As shown in Fig. 3, the peak around 14.0 min had the m/z of 387.1, corresponding to the dimeric species of FA. Therefore, the dimeric FA could be formed via the β-β′, α′-O-γ, β-β′, α-O-γ′, β-5'/α-O-4′, 5-5′ and 4-O-5′ bonds (Kupriyanovich, Medvedeva, Rokhin, & Kanitskaia, 2007). As a pivotal interaction moiety, FA was involved in the oxidative crosslinking reaction of individual AX to form a larger network, and a similar combination reaction could also occur between the bounded FA of AX and cysteine side-chains of gluten (Wang, Hamer, van Vliet, & Oudgenoeg, 2002). Moreover, since FA was esterfied at the C(O)-5 position of the arabinofuranose units, the diferulic bridges could be formed only through the β-5'/α-O-4′, 5-5′ and 4-O-5'. However, it should be pointed out that the detailed structural information needed further investigations to clearly elucidate the concise structure of the adducts.
4. Conclusion Ana-rLOX could promote the crosslinking formation of WEAX molecules, thus the viscosity and particle size increased significantly. FA polymerization catalyzed by ana-rLOX was evidenced by ESR and HPLC-MS. The Mw of the dimeric ferulic acid is 386, which might be in the form of β-5'/α-O-4′, 5-5′ and 4-O-5'. The results also showed that the presence of WEAX gels catalyzed by ana-rLOX could suppress the loss of water and hardening rate of the bread crumb. The results 6
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confirmed that the crosslinked WEAX by ana-rLOX were the most effective for improving fresh bread quality and delaying the staling, suggesting that ana-rLOX could act as an effective bread improver by inducing the crosslinking reaction of WEAX besides its oxidizing effect on the gluten proteins.
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Acknowledgement This work was financially supported by grants from the National Natural Science Foundation of China (No. 31671800, 31801550), Key Technology Research and Development Program of Jiangsu Province (NO. BE2018319), Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control. References Bahal, G., Sudha, M. L., & Ramasarma, P. R. (2013). Wheat germ lipoxygenase: Its effect on dough rheology, microstructure, and bread making quality. International Journal of Food Properties, 16, 1730–1739. https://doi.org/10.1080/10942912.2011.607932. Biliaderis, C. G., Izydorczyk, M. S., & Rattan, O. (1995). Effect of arabinoxylans on breadmaking quality of wheat flours. Food Chemistry, 53, 165–171. https://doi.org/10. 1016/0308-8146 (95)90783-4. Carvajal-Millan, E., Guigliarelli, B., Belle, V., Rouau, X., & Micard, V. (2005). Storage stability of laccase induced arabinoxylan gels. Carbohydrate Polymers, 59, 181–188. https://doi.org/10.1016/j.carbpol.2004.09.008. Courtin, C., & Delcour, J. (1998). Physicochemical and bread-making properties of low molecular weight wheat-derived arabinoxylans. Journal of Agricultural and Food Chemistry, 46, 4066–4073. https://doi.org/10.1021/jf980339t. Delcour, J. A., Bruneel, C., Derde, L. J., Gomand, S. V., Pareyt, B., Putseys, J. A., et al. (2010). Fate of starch in food processing: From raw materials to final food products. Annual Review of Food Science and Technology, 1, 87–111. https://doi.org/10.1146/ annurev.food. 102308.124211. Dervilly-Pinel, G., Thibault, J.-F., & Saulnier, L. (2001). Experimental evidence for a semiflexible conformation for arabinoxylans. Carbohydrate Research, 330, 365–372. https://doi.org/10.1016/S0008-6215(00)00300-1. Garcia, R., Lalatiana Rakotozafy, A., & Nicolas, J. (2004). Analysis and modeling of the ferulic acid oxidation by a glucose oxidase−peroxidase association. comparison with a hexose oxidase−peroxidase association. Journal of Agricultural and Food Chemistry, 52, 3946–3953. https://doi.org/10.1021/jf035113r. Garcia, R., Rakotozafy, L., Telef, N., Potus, J., & Nicolas, J. (2002). Oxidation of ferulic acid or arabinose-esterified ferulic acid by wheat germ peroxidase. Journal of Agricultural and Food Chemistry, 50, 3290–3298. https://doi.org/10.1021/jf011355k. Hartmann, G., Piber, M., & Koehler, P. (2005). Isolation and chemical characterisation of water-extractable arabinoxylans from wheat and rye during breadmaking. European Food Research and Technology, 221, 487–492. https://doi.org/10.1007/s00217-0051154-z. Hoover, R., Hughes, T., Chung, H. J., & Liu, Q. (2010). Composition, molecular structure, properties, and modification of pulse starches: A review. Food Research International, 43, 399–413. https://doi.org/10.1016/j.foodres.2009.09.001. Huang, G., Guo, Q., Wang, C., Ding, H. H., & Cui, S. W. (2016). Fenugreek fibre in bread: Effects on dough development and bread quality. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 71, 274–280. https://doi.org/10.1016/j. lwt.2016.03.040. Kupriyanovich, Y. N., Medvedeva, S. A., Rokhin, A. V., & Kanitskaia, L. V. (2007).
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Oxidative crosslinking of water-extractable wheat arabinoxylans by recombinant lipoxygenase and its effect on bread properties
T
Chong Zhang1, Pei Wang1, Jie Yang, Di Ren, Zhaoxin Lu, Haizhen Zhao, Fengxia Lu∗ College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing, 210095, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Oxidative crosslinking WEAX Ana-rLOX Free radicals Bread staling
Water-extractable arabinoxylan (WEAX) is a pivotal component of wheat flour, which plays a positive role in bread making. In present study, the effect of recombinated Anabaena sp. Lipoxygenase (ana-rLOX) on the WEAX characteristics was evaluated. The results showed that ana-rLOX could effectively promote the oxidative crosslinking of the WEAX by acting as a free radical-generating agent and further elevated the particle size and viscosity of WEAX. The diferulic bridges induced by ana-rLOX could be formed through β-5'/α-O-4′, 5-5′ and 4O-5′ bonds. The crosslinked WEAX by ana-rLOX were superior in improving fresh bread quality and delaying the staling behavior as compared with the addition of ana-rLOX or WEAX alone. These results showed that ana-rLOX could improve the fresh and aged bread quality by modifying the WEAX fractions.
1. Introduction Arabinoxylan belongs to the nonstarch polysaccharide, which forms the cell wall of cereal endosperm and bran. About 25% of wheat arabinoxylan is water extractable (Mansberger et al., 2014). The conformation of water-extractable arabinoxylan (WEAX) is semi-flexible and regarded as dough improver, which can elevate the organoleptic quality of bread (Dervilly-Pinel, Thibault, & Saulnier, 2001). Ferulic acid (FA) is the predominant phenolic acid in wheat, and is mainly coupled to the C(O)-5 of arabinose through an ester linkage (Mendis & Simsek, 2014). Incorporation of FA into arabinose residues promotes the gel formation by developing the intermolecular crosslinks of WEAX. It has been demonstrated that some chemical or enzymatic free radicalgenerating agents like laccase, peroxidase and lipoxygenase can promote the oxidative crosslinking between AX molecules through the formation of dimeric- and trimeric-FA (Garcia, Rakotozafy, Telef, Potus, & Nicolas, 2002). As a new flour improver, lipoxygenase (LOX, EC 1.13.11.12) is green and safe in food industry. This enzyme can exert significant effect on dough quality, such as improving the microstructure and rheological properties of dough (Bahal, Sudha, & Ramasarma, 2013). The LOX gene from Anabaena sp. PCC 7120 had been successfully expressed in B. subtilis and the purified recombinant LOX was acquired in our laboratory (Zhang et al., 2012). The recombinated Anabaena sp. Lipoxygenase (ana-rLOX) catalyzes the oxidation of poly-unsaturated fatty acids to
form fatty acid hydroperoxides. This action was believed to produce fatty acid radicals during the intermediate steps of substrate peroxidation, which can crosslink the wheat protein fractions (Wang et al., 2014). However, the effect of ana-rLOX on the crosslinking of WEAX remains largely unelucidated. Polymerization of FA catalyzed by laccase and peroxidase had been widely investigated, and the storage stability of laccase-induced arabinoxylan gels was related to the generated radicals by laccase (Carvajal-Millan, Guigliarelli, Belle, Rouau, & Micard, 2005; Leskovac, Trivić, Wohlfahrt, Kandrac, & Pericin, 2005). Therefore, it was speculated that ana-rLOX might also promote the oxidative crosslinking between WEAX molecules by polymerizing FA regioselectively. Against this background, the main aim of this study was to investigate the rheological properties, molecular weight, particle size of WEAX gels and the polymerization of FA induced by the ana-rLOX, and to manifest the mechanism of oxidative crosslinking of WEAX catalyzed by ana-rLOX through electron spin-resonance spectroscopy (ESR) and high-performance liquid chromatography-mass spectrometry (HPLCMS). In addition, the effect of potential induced WEAX crosslinks by ana-rLOX on the bread aging behavior was also investigated.
∗
Corresponding author. E-mail address: [email protected] (F. Lu). 1 Co-first author, contributed equally to this work. https://doi.org/10.1016/j.lwt.2018.12.077 Received 6 October 2018; Received in revised form 29 December 2018; Accepted 30 December 2018 Available online 31 December 2018 0023-6438/ © 2018 Published by Elsevier Ltd.
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filtered through 0.45 μm cellulose membranes. The average particle size (hydrodynamic radius) was analyzed by an integrated-laser light scattering instrument (Zetasizer Nano particle size distribution analyzer).
2. Materials and methods 2.1. Materials
2.6. Viscosity measurement
Wheat bran was obtained from Jiangsu golden land seed industry Co., Ltd. (Yangzhou, Jiangsu, China), amyloglucosidase (AMG 300 L) and alpha-amylase (Termamyl 120 L) were purchased from Novozymes (Bagsvaerd, Denmark). All the used chemicals were of analytical grade.
The viscosity of 1% (w/v) WEAX solutions was measured by using Brookfield DV-II + Pro rotation viscometer (Brookfield Co., Ltd., USA) with a rotor SC4-18) at different shear rates at 20 °C.
2.2. Extraction and purification of WEAX from wheat bran 2.7. Measurement of radical formation with electron spin resonance (ESR) The extraction of WEAX was performed according to Wang, Tao, Jin, and Xu (2016) with some modifications. Wheat bran (100 g) was defatted with n-hexane and the sample (100 g) was heated at 130 °C for 90 min to inactivate the endogenous enzymes. After cooling to 50 °C, 100 g bran was added with 1 L distilled water with 0.02 mL α-amylase and stirred at 65 °C for 90 min. Then the solids were removed by centrifugation at 5000g for 10 min. The α-amylase was added and allowed to react at 95 °C for 1 h to degrade the starch contaminants. Afterwards, the pH was adjusted to 6.5 with 0.3 mL amyloglucosidase added and kept at 55 °C for 24 h. The enzymes were inactivated by heating at 120 °C for 30 min. The resulting solution was deproteinized by mixing with 10% bentonit solution, and the solution was cleared by centrifugation at 5000g for 10 min. The supernatant was collected and mixed with 4 times the volume of 95% aqueous ethanol. The mixtures were allowed to stand at 4 °C overnight. Finally, the crude WEAX was centrifuged (10,000×g, 4 °C, 10 min), followed by freeze-drying. The crude WEAX was further purified according to Vansteenkiste, Babot, Rouau, and Micard (2004). The protein content was assessed by the micro-Kjeldahl procedure with a nitrogen/protein conversion factor of 5.7.
The free radicals formed during the ana-rLOX action on LA with the addition of FA were detected by using ESR employing a spin trapping technique using α-(4-Pyridyl-1-oxide)-N-tert-butylnitrone (POBN). The reaction mixture contained 25 mM LA, ana-rLOX (72 IU/mg WEAX), 100 mM POBN, and 0.1 mM FA. After incubating in dark at 16 °C for 90 min, the mixture was loaded into a EPR Bruker EMX-10/12 spectrometer (Bruker EMX/plus, Germany) equipped with a high sensitivity cylindrical cavity operating at 9.77 GHz with 50 kHz modulation frequency to detect the radical intensity. ESR instrument settings for the spin trapping experiments were as follows: microwave power 20 mW; modulation amplitude 1.19 G; time constant 0.6 s; receiver gain 106; magnetic field, 70 G (Wang et al., 2014). 2.8. Reverse-phase (RP)-HPLC and mass spectrometer analysis of the oxidation products from FA and ana-rLOX The above mixture of the enzyme reaction was freeze-dried and redissolved in 300 mL of 50% (v/v) aqueous methanol. The RP-HPLC was conducted using an Agilent C18 column (4.6 mm × 250 mm, 0.5 μm). Elution was performed using a gradient system adapted from Piber and Koehler (2005). The gradient profile consisted of solvent A (10%, v/v, aqueous acetonitrile with 1 mM TFA), solvent B (80%, v/v, aqueous methanol with 1 mM TFA), and solvent C (80%, v/v, aqueous acetonitrile with 1 mM TFA) in the following program: 90% A, 5% B, and 5% C at the initial stage, with a linear gradient to 26% A, 37% B, and 37% C over the 25 min, then linear gradient to 0% A, 50% B, and 50% C over the 5 min, linear gradient to 90% A, 5% B, and 5% C over the 15 min, and held isocratically at 90% A, 5% B, and 5% C for 10 min, the flow rate was set at 1 mL/min. For MS analysis, the RP-HPLC fractions were collected and analyzed by the Xevo G2-S QTOF MS system (Waters Corp, Milford, USA) equipped with electrospray ionization (ESI) source. The capillary voltage and a cone voltage were set to 1000 and 35 V, respectively. The nebulization and gas was at 500 °C, with a flow rate at 1000 L/h. The source temperature of cone gas was 120 °C, with a flow rate at 150 L/h (Yang et al., 2016).
2.3. Determination of molecular weight (Mw), monosaccharides composition and FA content The Mw of WEAX was analyzed by an Agilent 1200 series high performance liquid chromatography (HPLC) system (Agilent Technologies, Santa Clara, CA, USA) according to Wang et al. (2019). For the monosaccharides composition analysis, 1 mg of WEAX was reacted with 300 μL of 4.0 M trifluoroacetic acid (TFA) at 120 °C for 2 h. The hydrolyzed WEAX was derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) and then analyzed by Agilent XDB-C18 column (Marcotuli et al., 2015). FA quantification was conducted following the method described by Hartmann, Piber, and Koehler (2005), p. 10 mg of WEAX was suspended in 2 mL of 4 M NaOH under nitrogen atmosphere and the hydrolysis was performed at 35 °C for 18 h after adjusting the pH to 2 with HCl. Then the solution was extracted with 5 mL of ethyl ether for three times, followed by the air-drying with nitrogen. The residue was redissolved in 1 mL of methanol, passed though the 0.45 μm filter, and separated by an Agilent Eclipse plus C18 column (4.6 × 250 mm, 5 μm). The elution was detected by an ultraviolet (UV) detector.
2.9. Effect of WEAX on the wheat bread quality A basic recipe consisted of wheat flour (200 g), instant dry yeast (3 g), salt (2 g), sugar (20 g) and water (90 g) and set as the control group. Ana-rLOX (20 U/g) and/or 1% (w/w, flour basis) WEAX were added. All ingredients were mixed in a laboratory mixer (Dongfangfude JHMZ-200, Beijing, China) to achieve the complete development. Then the dough was divided into 150 g, kneaded and mechanically sheeted and rolled. The dough was fermented at 30 °C for 45 min under the 85% relative humidity, and then baked at 215 °C for 15 min (Wang, Lee, Xu, & Jin, 2016). The loaf volume was determined by the rapeseed displacement method, and the specific volume was calculated by the loaf volume divided by the bread weight. The texture of bread including hardness, gumminess, chewiness and resilience were determined by a TA-XT2 texture analyzer (Stable Microsystems, Surrey, UK) equipped with Texture Expert v.1.20 software and calculated according to Huang, Guo, Wang, Ding, and Cui (2016). For the aging study, bread was cut
2.4. Preparation of the recombinated Anabaena sp. lipoxygenase (anarLOX) Recombinated Ana-rLOX was produced by extracellular production of B. subtilis recombined with Anabaena sp. PCC 7120 gene and purified by a nickel-charged NTA agarose column as described by Zhang et al. (2012). 2.5. Average particle size distribution Different dosage of Ana-rLOX was mixed with the 1% (w/v) WEAX solutions (pH 6.0, dissolved in 50 mM phosphate buffer) and 25 mM linoleic acid (LA), the mixtures were incubated at 25 °C for 2 h, and 2
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Table 1 Characteristics of WEAX extracted from wheat bran.
WEAX Ana-rLOX + WEAX
AX (%)
Protein (%)
Mw (kDa)
Ara (%)
Xyl (%)
Gal (%)
Glu (%)
A/X
FA (%)
85.46 ND
3.53 ± 0.08 ND
398.1 460.4
46.57 ± 0.39 ND
51.43 ± 0.42 ND
1.27 ± 0.09 ND
0.73 ± 0.05 ND
0.89 ND
0.436 ± 0.02 0.203 ± 0.02
AX = 0.88 × [(%Ara - 0.7 × %Gal) + %Xyl]; A/X= (%Ara-0.7 × %Gal)/%Xyl; ND, not determined; Ara, arabinose; Xyl, xylose; Gal, galactose; Glu, glucose; FA, ferulic acid.
from the center of each loaf (20 mm in thickness), packed in polypropylene bags and stored at 4 °C for 7 days. The water content of the bread crumb was measured gravimetrically by drying the sample at 105 °C to the constant weight. 2.10. Statistical analysis Data were expressed as means ± standard deviations (SD), and a one-way ANOVA was performed, followed by Duncan's multiple-range tests. P values lower than 0.05 were considered to be statistically significant. All statistical analyses were performed using SPSS software version 19.0 (Chicago, IL, USA). 3. Results and discussion 3.1. Characteristics of WEAX As shown in Table 1, the AX content of the purified WEAX was 85.46%, with a Mw of 398.1 kDa and protein content of 3.53%. Xylose (Xyl) and arabinose (Ara) were the major sugars, with minor contaminated galactose and glucose. The Ara to Xyl ratio (A/X) of WEAX was 0.89, which was higher than the previous results by Courtin and Delcour (1998). This indicated the obtained WEAX possessed a high substitution degree, which could increase the steric hindrance of rotation intramolecularly. This further led to a lowered molecular flexibility and aggregation rate of the molecular chain. As an important group for interacting inter- or intra-molecularly with other biomolecules, the ferulic acid (FA) content of WEAX was 0.436%. 3.2. Effect of ana-rLOX on the average particle size and viscosity of WEAX As shown in Fig. 1A, the hydrodynamic radius steadily elevated with the increased addition of ana-rLOX/LA in WEAX solutions. The average particle size of WEAX was increased by 42.68% under treatment of 72 IU/mg ana-rLOX/LA. The results might be due to the modified reaction by ana-rLOX/LA, which induced the formation of crosslinking structure in WEAX and further enhanced the average particle size (Primo-Martin, Wang, Lichtendonk, Plijter, & Hamer, 2005). Meanwhile, the viscosity of WEAX solution with and without enzyme at different shear rate were also detected (Fig. 1B). All the samples exhibited the shear-thinning behavior, since the viscosity decreased with the enhanced shear rate. The viscosity of WEAX solution with Ana-rLOX was higher than that of the control group. When adding 36 IU/mg ana-rLOX/LA, a slight increase in viscosity could be noticed. However, the viscosity had been remarkably improved by nearly 2.5 times than the control, when the dosage of ana-rLOX/LA increased up to 72 IU/mg. The enhanced viscosity was in accordance with the enlarged particle size and was probably originated from the crosslinking formation of WEAX.
Fig. 1. (A) Particle size of WEAX under the different addition levels of ana) and with the rLOX. (B) Viscosity versus shear rate of WEAX without ( ) and 72 IU/mg ( ) ana-rLOX/LA. addition of 36 IU/mg (
of WEAX catalyzed by ana-rLOX. The POBN was used to trap free radicals during the reaction, thus the intensity of POBN was the lowest. When incorporation with ana-rLOX and LA, six radical species in the ESR spectrum were presented regardless of the presence of FA (Fig. 2), indicating the generated adducts from the free radical of lipid oxidation and POBN. The intensity of POBN + Ana-rLOX + LA group was the highest, while the intensity of free radicals with the addition of FA decreased dramatically. Lipid peroxyl radicals were originated from the peroxidation of polyunsaturated fatty acids catalyzed by lipoxygenase, which could further react with POBN and generate the stable radicals detected by ESR (Wang et al., 2014). The decreased free radicals with the incorporation of FA might be attributed to that FA could compete with the POBN for the peroxyl radicals and formed the dimers or
3.3. ESR determinations LOX-catalyzed LA peroxidation is complex involving a series of free radicals. As the free radicals generated during LA peroxidation was short-lived, radical trap POBN and ESR spectroscopy approach was undertaken to study the dynamic behavior of the oxidative crosslinking 3
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Fig. 2. ESR spectra of POBN radical adducts from POBN (blue line), POBN + ana-rLOX + LA (black line) and POBN + ana-rLOX + LA + FA (red line). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3. RP-HPLC profile of the FA after reaction with ana-rLOX. Di-FA, dimeric ferulic acid. (B) ESI-MS spectra of Di-FA. Signals were assigned to the structure of the ions. Molecular mass of the identified Di-FA is 386 g/mol. (C) Speculative structures of the Di-FA related to those known from ferulate homocoupling.
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Fig. 4. The cross-section image (A), specific volume (B) and texture profile (C) of bread with or without the addition of ana-rLOX, WEAX and ana-rLOX + WEAX. The hardness, guminess, chewniess and resilence were indicated by black bar, dark grey bar, light grey bar and filled circle, respectively.
trimers of FA via the covalent crosslinking in the WEAX network as speculated.
formation of FA dimers or trimers from the FA group on the side chain. When oxidase was added, FA underwent oxidative crosslinking through different reaction systems, so that the FA monomer content attached to the AX side chain was significantly reduced by 53.4% (Table 1), indicating that FA was involved in the oxidation crosslinking process under the action of Ana-rLOX on WEAX, which further elevated the Mw
3.4. Oxidation of FA and fractionation of oxidation products The oxidation crosslinking reaction of WEAX occurred through the 5
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3.5. Effect of WEAX crosslinking on the breadmaking performances and storage characteristics of wheat bread The cross-section images of bread under the different treatment are shown in Fig. 4A. Specific volume and textural profile are the most important parameters for the bakery products. The combined incorporation of ana-rLOX and WEAX (ana-rLOX + WEAX) was the most effective on improving the specific volume of bread, followed by the addition of ana-rLOX and WEAX alone (Fig. 4B). The significant increase in specific volume contributed to the decrease in crumb hardness with ana-rLOX and/or WEAX addition. Meanwhile, the gumminess and chewiness of bread decreased while the resilience increased, with the most evident effect observed for the ana-rLOX + WEAX group (Fig. 4C). These results demonstrated that the combined incorporation of ana-rLOX and WEAX was superior in enhancing the bread quality. WEAX was generally regarded as a dough improver to enhance the organoleptic quality of bread. The improved effect of WEAX on bread quality was greatly dependant on the structure of WEAX, including the branched degree, Mw and the amount of bounded FA groups (Saeed, Pasha, Anjum, & Sultan, 2011). The improved effect of WEAX on bread properties was attributed to the molecular interactions between WEAX and dough components, as well as the protection of the foams during the heating of dough conferred by its viscous property (Biliaderis, Izydorczyk, & Rattan, 1995). Therefore, the enhanced viscosity and Mw of WEAX by ana-rLOX could contribute to its superior improved effect. During the storage of bread, the bread aged and resulted in the texture hardening, elasticity loss, dryness and loss of smell. In this study, hardness and moisture content were chosen as important indicators for bread aging (Nivelle, Bosmans, & Delcour, 2017). As shown in Fig. 5A, the hardness of the crumb increased during the storage at 4 °C, in which the hardness of the crumb was lowest for the anarLOX + WEAX group, followed by the WEAX, ana-rLOX and control group. Therefore, adding ana-rLOX and/or WEAX was also beneficial to the bread quality during storage. Moreover, the hardening rate was also suppressed by WEAX and/or ana-rLOX, with the most obvious effect for the ana-rLOX + WEAX group. The storage of bread led to the subsequent decrease in the water content of crumb. The moisture content of ana-rLOX + WEAX group was higher than the other groups, and the moisture loss rate was also reduced (Fig. 5B). The results of hardening rate and water retention property demonstrated that WEAX had the capability to suppress the bread staling behavior, and WEAX + anarLOX had a synergistic effect on enhancing the bread quality. Bread staling is mainly determined by the retrogradation behavior of starch, which involves the water redistribution and starch recrystallization (Delcour et al., 2010). Starch recrystallization is a complex behavior, accompanying with a series of physicochemical changes such as gel formation, exudation of water, and increased turbidity, viscosity and degree of crystallinity (Hoover, Hughes, Chung, & Liu, 2010). As a crucial factor, rate and extent of starch retrogradation is largely dependent on water content. During the storage of bread, moisture migrates from crumb to crust, and from gluten to starch, contributing to the starch recrystallization behavior (Nivelle et al., 2017). Therefore, the WEAX crosslinking induced by ana-rLOX formed the network structure, and consequently improved the water retention ability of WEAX and dough. Thus, the moisture content could be better preserved, and the bread staling was delayed.
Fig. 5. The hardness (A) and water content (B) of bread during the storage for ), ana-rLOX( ), WEAX ( ) and WEAX + ana-rLOX ( ) the control ( group.
of WEAX. Furthermore, the data available from the literature suggested that the dimerization of FA was influenced by many factors, the kinds of enzyme, the temperature and pH of the reaction system (Garcia, Lalatiana Rakotozafy, & Nicolas, 2004). Different enzymes catalyzed the formation of FA oxidation products in various ways, even the same enzymes like lipoxygenase from different sources could catalyze oxygenation at different sites. Therefore, it was essential to investigate the formation of FA oxidation products catalyzed by Ana-rLOX in order to explain the mechanism of crosslinking between WEAX molecules. As shown in Fig. 3, the peak around 14.0 min had the m/z of 387.1, corresponding to the dimeric species of FA. Therefore, the dimeric FA could be formed via the β-β′, α′-O-γ, β-β′, α-O-γ′, β-5'/α-O-4′, 5-5′ and 4-O-5′ bonds (Kupriyanovich, Medvedeva, Rokhin, & Kanitskaia, 2007). As a pivotal interaction moiety, FA was involved in the oxidative crosslinking reaction of individual AX to form a larger network, and a similar combination reaction could also occur between the bounded FA of AX and cysteine side-chains of gluten (Wang, Hamer, van Vliet, & Oudgenoeg, 2002). Moreover, since FA was esterfied at the C(O)-5 position of the arabinofuranose units, the diferulic bridges could be formed only through the β-5'/α-O-4′, 5-5′ and 4-O-5'. However, it should be pointed out that the detailed structural information needed further investigations to clearly elucidate the concise structure of the adducts.
4. Conclusion Ana-rLOX could promote the crosslinking formation of WEAX molecules, thus the viscosity and particle size increased significantly. FA polymerization catalyzed by ana-rLOX was evidenced by ESR and HPLC-MS. The Mw of the dimeric ferulic acid is 386, which might be in the form of β-5'/α-O-4′, 5-5′ and 4-O-5'. The results also showed that the presence of WEAX gels catalyzed by ana-rLOX could suppress the loss of water and hardening rate of the bread crumb. The results 6
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confirmed that the crosslinked WEAX by ana-rLOX were the most effective for improving fresh bread quality and delaying the staling, suggesting that ana-rLOX could act as an effective bread improver by inducing the crosslinking reaction of WEAX besides its oxidizing effect on the gluten proteins.
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