Food Chemistry 161 (2014) 1–7
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Preparation of immobilized glucose oxidase and its application in improving breadmaking quality of commercial wheat flour Lele Tang a,b, Ruijin Yang a,b,⇑, Xiao Hua b, Chaohua Yu a, Wenbin Zhang b, Wei Zhao a a b
State Key Laboratory of Food Science and Technology, Jiangnan University, 214122 Wuxi, China School of Food Science and Technology, Jiangnan University, 214122 Wuxi, China
a r t i c l e
i n f o
Article history: Received 6 December 2013 Received in revised form 20 March 2014 Accepted 22 March 2014 Available online 1 April 2014 Keywords: Glucose oxidase Immobilization Chitosan Sodium tripolyphosphate Breadmaking quality Commercial wheat flour
a b s t r a c t Preparation of immobilized glucose oxidase (GO) on chitosan (CS)-sodium tripolyphosphate (TPP) and its application in improving breadmaking quality of commercial wheat flour were investigated. The optimum conditions for GO immobilization were: viscosity of CS: 700 cP, ratio of CS to TPP (w/w): 5 to 1, and GO concentration 100 U/mL. The obtained CSTPP-GO was 5 lm-diameter particle with a pseudo-spherical shape. By addition of CSTPP-GO (400 U/kg flour) and fungal a-amylase (62.5 U/kg flour), bread springiness slightly increased from 0.923 to 0.940, specific volume of crumb increased by 13.48% and hardness decreased by 19.22%, compared to addition of KBrO3 (60 mg/kg flour). It could be concluded that CSTPP-GO combined with fungal a-amylase had potential application in improving breadmaking quality of commercial wheat flour. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Bread is an important food in human diet in many countries and contributes substantially to the daily intake of carbohydrates, dietary fibre, protein, minerals and B vitamins (Belitz, Grosch, & Schieberle, 2004, chap. 15). The functional breadmaking properties of wheat greatly depend on the gluten proteins (Wieser & Kieffer, 2001). However, cultivar differences, insect damage and environmental, genetic and post-harvest conditions of wheat may affect gluten properties and wheat processing quality (Georget, Underwood-Toscano, Powers, Shewry, & Belton, 2008; Lukow, White, & Sinha, 1995; Wrigley & Bietz, 1988). In order to overcome deficiencies in breadmaking quality (loaf volume, crumb structure, shelf-life, flavour and colour) of wheat flour, various bread improvers, such as oxidants, reductants, emulsifiers and enzymes, have been added (Haros, Rosell, & Benedito, 2002; Martinez-Anaya, 1996; Moayedallaie, Mirzaei, & Paterson, 2010). Potassium bromate (KBrO3) has been widely used in the baking industry for a long time as an effective improver for improving loaf volume, crumb structure and texture (Kohman, Hoffman, Godfrey, Ashe, & Blake, 1916). KBrO3, a slow acting oxidant, oxidizes free thiol groups generating small –S–S– compounds and bromide. It is active during later stages of fermentation and baking. However, ⇑ Corresponding author at: School of Food Science and Technology, Jiangnan University, 214122 Wuxi, China. Tel./fax: +86 510 85919150. E-mail address: [email protected] (R. Yang). http://dx.doi.org/10.1016/j.foodchem.2014.03.104 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
its application has been gradually restricted and even forbidden in many countries because of the potential relationship between bromate intake and cancer (Kurokawa et al., 1986; Ranum, 1992). Therefore, it is an urgent challenge to find the alternatives for KBrO3. Enzymes are attractive, safe alternatives to KBrO3. Trials of enzymes such as transglutaminase (EC 2.3.2.13) (Beck, Jekle, Selmair, Koehler, & Becker, 2011), laccase (EC 1.10.3.2) (Selinheimo, Autio, Kruus, & Buchert, 2007), hexose oxidase (EC 1.1.3.5) (Gül, Özer, & Dizlek, 2009) and glucose oxidase (EC 1.1.3.4) (Bonet et al., 2006; Rasiah, Sutton, Low, Lin, & Gerrard, 2005) have been studied in breadmaking. They are able to improve different aspects of bread qualities such as reinforcing gluten structure, strengthening dough stability, increasing specific volume, texture, elasticity and water holding capacity (Martínez-Anaya & Jiménez, 1997; Miller & Hoseney, 1999; Zhu, Rinzema, Tramper, & Bol, 1995). Fungal (Aspergillus niger) Glucose oxidase (GO) is one of the most effective alternative to KBrO3 (Hanft & Koehler, 2006; Vemulapalli, Miller, & Hoseney, 1998). In the presence of oxygen, GO catalyses the oxidation of b-D-glucose to D-gluconic acid with the release of hydrogen peroxide (H2O2). The H2O2 promotes the formation of disulphide linkages in the gluten networks and consequently improves dough handling properties, better gas retention capability and bread quality, especially for commercial wheat flour (Poulsen & Høstrup, 1998; Steffolani, Ribotta, Pérez, & León, 2010; Vemulapalli & Hoseney, 1998). However, GO is unable to replace KBrO3 due to some deficiencies including fast
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oxidation at the initial stage in dough development, low stability in flour and thermal-inactivation in the baking stage. Fast oxidation will cause rapid release of H2O2 at the beginning stage, resulting in over-developed dough and low volume and poor crumb of bread (Primo-Martín, Wang, Lichtendonk, Plijter, & Hamer, 2005; Rakotozafy et al., 1999). Immobilization is an effective method to control the release of H2O2 and improve the stability of GO. According to Tzanov, Costa, Gubitz, and Cavaco-Paulo (2002), immobilized GO can control the release of H2O2 and be used for at least 5 times for textile bleaching. But there are few people applying the immobilized GO to control the release of H2O2 in bakery. Wang, Zhu, and Zhou (2011) has proved that GO immobilized in alginate– chitosan microcapsules has good storage stabilities with 70.4% GO activity remained after over 2 months. This investigation has suggested a promising way to prepare potential breadmaking improver by immobilization of GO. Many researchers (Dash, Chiellini, Ottenbrite, & Chiellini, 2011; Park, Saravanakumar, Kim, & Kwon, 2010) have focused on drug delivery by Chitosan-TPP nanoparticles. In the present study, CSTPP-immobilized GO was prepared as the breadmaking improver. Immobilization conditions including the viscosity of chitosan, ratio of chitosan to TPP (w/w) and the concentration of GO were optimized. The H2O2-release profile and appearance of immobilized GO were characterized. Finally, the improvement of bread quality with the addition of CSTPP-GO was examined.
2.3. Enzyme assay The activities of free GO and CSTPP-GO were measured using indirect oxidation of o-dianisidine by horseradish peroxidase (HRP). The assays were performed according to the method developed by Bergmeyer (Bergmeyer & Grabi, 1988, chap. 2; Simpson, Jordaan, Gardiner, & Whiteley, 2007). Reaction mixtures contained 0.006% (w/v) o-dianisidine2HCl in 0.1 mol/L pH 7.0 potassium phosphate buffer, 10% (w/v) b-D-glucose in distilled water and 60 U/mL HRP in 0.1 mol/L pH 7.0 potassium phosphate buffer. They were mixed immediately prior to assaying for GO in the ratio 2.4:0.5:0.1, respectively. The reaction was initiated by the addition of 0.1 mL of GO (0.15–0.2 U/mL), and ended with 3 mL of the final reagent (0.5 mol/L H2SO4) 5 min later. The absorbance of the reaction was read at 436 nm against a blank consisting 0.1 mL pH 7.0 potassium phosphate buffer replace GO. One unit of GO activity was defined as the amount of enzyme that catalyses the conversion of 1 lmol b-D-glucose to D-gluconic acid and H2O2 per minute at 25 °C and pH 7.0. The enzyme activity recovery was calculated using Eq. (1). Enzyme activity recovery ð%Þ ¼
Calculated CSTPP-GO concentration 100 Theoretical GO concentration ð1Þ
2.4. Characterization of CS-TPP GO 2. Materials and methods 2.1. Materials Commercial wheat flour (moisture 13.8%, ash 0.42%, protein 10.2% w/w) was obtained from Wuxi Rongguo Flour Mill (Wuxi, Jiangsu, China). Glucose oxidase (10,000 U/g) Gluzyme mono 10000 BG was generously gifted by Novozymes (Bagsvaerd, Denmark). Butter, active dry yeast, commercial sugar and salt were obtained from the local market (Wuxi, Jiangsu, China). Food-grade chitosans (deacetylation degree 90%) with different viscosity (100, 700, 1200, 2200 cP, respectively) were purchased from Pan’an Chitosan Product Co. (Pan’an, Zhejiang, China). Sodium tripolyphosphate (analytical grade) was obtained from Sinopharm Chemical Reagent (Beijing, China). Potassium bromate was obtained from Shantou Xilong Chemical Factory (Shantou, Guangdong, China). All the other chemicals were of analytical grade. All solutions were prepared with distilled water. 2.2. Immobilization of GO on CS-TPP microparticles Immobilization of GO on CS-TPP microparticles was performed by ionotropic gelation according to the reported method (Jonassen, Kjøniksen, & Hiorth, 2012; Shu & Zhu, 2002) with slight modifications. Chitosan (0.5% w/v) was dissolved in 1% (w/v) acetic acid aqueous solution and insoluble impurities were removed by filtration. TPP aqueous solution (2.5 mg/mL) was filtered to remove impurities by 0.45 lm membrane before it was added dropwise into the chitosan solution by a constant flow pump with thorough stirring at room temperature. Subsequently, GO in 0.1 mol/L pH 5.6 phosphate buffer was added into the chitosan-TPP solution and the mixture solution was stirred for another 1 h. At last, the chitosanTPP-GO suspension was then spray dried to obtain the TPP crosslinked chitosan microspheres loaded with GO. The spray drying conditions were: 180 ± 5 °C inlet temperature, 70 ± 5 °C outlet temperature and 10 mL/min liquid flow rate. The dried product was finally collected and stored in a desiccator with silica gel at room temperature.
2.4.1. Microstucture of CS-TPP GO Scanning electron microscopy (SEM) was used to characterize the surface and shape of the spray-dried CSTPP-GO microparticles. The samples were coated for 70 s under an argon atmosphere with gold–palladium (sputter-coater) and examined with a scanning electron microscope (Quanta 200F, FEI, Hillsboro, OR, USA).Photographs were taken at a high voltage of 15.0 kv and a magnification of 1.2 or 2 or 5 or 10 or 15 k. The working distance was 15.9 nm. 2.4.2. Stability of CS-TPP GO The stability of the CS-TPP GO was determined by the enzyme activity which was investigated every week during 3 months. 2.4.3. In vitro release of hydrogen peroxide The hydrogen peroxide (H2O2) released from enzymatic catalysis was analyzed by colorimetric method (Graf & Penniston, 1980). 2.5. Breadmaking procedure and bread analysis 2.5.1. Breadmaking The breadmaking basic formulation used included: 100 g commercial wheat flour, 15 g commercial sugar, 1 g salt, 1.5 g active dry yeast, 57.3 g water (optimum level), 8 g butter. And other additives such as free GO 400 U/kg wheat flour, or KBrO3 60 mg/kg wheat flour, or CSTPP-GO 400 U/kg wheat flour were added in the formulation according to the different experiments, respectively. These additives were added before the mixing step. The ingredients (wheat flour, sugar, salt, yeast and additives) were blended for 5 min in a mixer and then water was added and mixed with all the ingredients to develop dough. Afterwards, butter was added into the dough and mixed until dough development under the optimal conditions. The resulting dough was allowed to rest for 15 min for the first fermentation at 25 °C and 75% relative humidity. After first fermentation, the dough was divided into 100 g pieces, rounded, moulded and placed in baking pans. The pieces were then proofed for 90 min at 38 °C and 85% relative humidity and baked at 210 °C for 15 min. Bread was removed from the pans and cooled at room temperature, then stored at room temperature in plastic bags for measurement and further analysis.
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2.5.2. Dough rheological properties The dough was prepared in a 50 g-Farinograph (Farinograph-E, Brabender, Germany). Dough basic formulation was: 100 g flour, 1 g NaCl, water according to farinographic absorption. And KBrO3 6 mg/100 g flour, or free GO 40 U/100 g flour, or CSTPP-GO 40 U/ 100 g flour was added in the formulation, respectively. Dough was then covered with a plastic film, to avoid dehydration, and left to rest for 15 min at room temperature. Dynamic oscillatory tests were performed in a rheometer (AR1000, TA Instruments, UK). The assay was executed at 25 ± 0.1 °C, using a plate–plate sensor system with a 2.0 mm gap between plates. To prevent sample dehydration during the assay, semisolid Vaseline oil was applied. Deformation and frequency sweeps were carried out. Before measurements, samples were rest for 30 min between plates to relax. Stress sweeps were performed at a constant frequency (1 Hz) in order to determine the linear viscoelastic range of each sample (data not shown); frequency sweeps (from 0.1 to 40 Hz) were performed at a constant stress within the linear viscoelastic range. Dynamic moduli G0 (elastic or storage modulus), G00 (viscous or loss modulus) and tan d (G00 /G0 ) were obtained as a function of frequency. 2.5.3. Bread quality Bread quality evaluation was carried out by determining the weight, loaf volume, bread specific volume (BSV), springiness and crumb hardness. The loaves were weighed and loaf volume was determined by rapeseed displacement just two hours after baking. The bread specific volume was expressed as the ratio of volume to weight of baked bread. Texture profile analysis (TPA) was performed by using a TA-XT2i Texture Analyser (Stable Micro Systems, Surrey, UK) equipped with an aluminium (25 mm in diameter) cylinder probe. Two hours after baking, the loaves were cut into 20 mm thick bread slices and subjected to a double cycle of compression under the following conditions: 1.0 mm/s pre-test speed, 3.0 mm/s test speed, 2.0 mm/s post-test speed. The crumb was compressed to 50% of the original height. The texture profile parameters of springiness were determined using the Texture Expert 1.22 software (Stable Micro Systems, Surrey, UK). The resulting peak force of compression was reported as bread hardness. 2.6. Fungal a-amylase compound with CSTPP-GO in improving bread texture Different dosages of fungal a-amylase were employed compound with CSTPP-GO in bread formulas to improve bread texture. The breadmaking formulation used was: 1000 g commercial wheat flour, 150 g commercial sugar, 10 g salt, 15 g active dry yeast, 573 g water (optimum level), 80 g butter, 400 U CSTPP-GO and fungal
a-amylase: 0, 31.25, 62.5 and 93.75 U. KBrO3 (60 mg/kg flour optimum quantity) was taken as a control. Then the breadmaking procedure was the same as the previous methods (Section 2.5.1). Bread quality was determined by previous methods (Section 2.5.3). 2.7. Statistical analysis SPSS statistical software 17.0 was used to perform the statistical analysis. A Fisher’s test (LSD) was made in order to evaluate differences among samples, while the relationship between measured parameters was assessed by Pearson’s test (significant level at p < 0.05). 3. Results and discussion 3.1. Preparation of CSTPP-GO The optimization for preparation of CSTPP-GO was based on the consideration of GO activity recovery and the main quality characteristics of bread with addition of CSTPP-GO. The viscosity of chitosan, ratio of CS to TPP and concentration of GO were varied, and the preparation parameters such as stirring speed and batch size were kept constant. 3.1.1. Effect of viscosity of chitosan on CSTPP-GO Chitosan solutions, 0.5 (w/v) were prepared by dissolving chitosan with different viscosity of 100, 700, 1200, 2200 cP in 1% (v/v) in acetic acid solution. GO was immobilized as described in Section 2.2 at the same conditions: weight ratio of CS to TPP 8:1, concentration of GO 50 U/mL. Enzyme activity recovery of immobilized GO prepared using chitosan with different viscosity is presented in Table 1. The GO activity recovery was achieved 77.9% with the chitosan of 700 cP, followed by 54% with 200 cP, 76.2% with 1200 cP and 61.1% with 2200 cP. Xu and Du (2003) proved that as the molecular weight of chitosan increased, the encapsulation efficiency of BSA was increased directly. As we all know, the molecular weight of chitosan increases with the viscosity. So we assumed that the ionic bonds of CSTPP-GO microparticles prepared with low viscosity chitosan were not strong and led to enzyme leakage. Sedimentation was observed during the preparation of CSTPP-GO microparticles with high viscosity chitosan and GO could not be immobilized well. Springiness, hardness and specific volume of bread prepared with CSTPP-GO 400 U/kg commercial wheat flour are shown in Table 1. The results are consistent with enzyme activity recovery (Table 1). The difference in springiness (0.92–0.94) was not significant. It indicated that oxidation did not have obvious effects on the structure of the gluten network. Specific volume was
Table 1 Different immobilized conditions of GO on enzyme activity recovery and bread quality (specific volume, hardness, springiness). Factors
Hardness (g)
Springiness
Viscosity (cP)
100 700 1200 2200
Activity recovery (%) 54.0 ± 1.03a 77.9 ± 0.89c 76.2 ± 0.75c 61.1 ± 0.92b
4.0 ± 0.10a 4.7 ± 0.04c 4.4 ± 0.06b 4.6 ± 0.05c
398.2 ± 7.4c 339.4 ± 8.8a 360.5 ± 6.7b 358.3 ± 5.6b
0.925 ± 0.03a 0.948 ± 0.015a 0.94 ± 0.01a 0.938 ± 0.025a
Weight ratio of CS/TPP
3:1 5:1 8:1 10:1
76.1 ± 1.11c 81.5 ± 0.83d 67.0 ± 0.64b 52.4 ± 0.73a
4.24 ± 0.08a 4.68 ± 0.05c 4.61 ± 0.04c 4.38 ± 0.06b
375.8 ± 2.5c 349.3 ± 1.6b 338.5 ± 5.5a 351.6 ± 3.2b
0.935 ± 0.015ab 0.955 ± 0.012b 0.942 ± 0.014b 0.91 ± 0.016a
Concentration of GO (U/mL)
50 100 150 200
81.1 ± 0.78c 85.1 ± 0.56d 74.0 ± 1.02b 62.0 ± 0.69a
4.7 ± 0.02b 4.75 ± 0.03b 4.48 ± 0.04a 4.48 ± 0.03a
349.1 ± 5.4b 312.7 ± 4.8a 342.4 ± 4.7b 365.2 ± 5.6c
0.955 ± 0.012a 0.925 ± 0.02a 0.932 ± 0.018a 0.923 ± 0.015a
Different letters within a column indicate significantly different values (p < 0.05).
Specific volume (mL/g)
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significantly increased from 4.0 mL/g (at 200 cP) to 4.7 mL/g (at 700, 1200 and 2200 cP) (p < 0.05), suggesting a limited improvement in gas retention capability. Hardness was markedly reduced and reached the lowest level at 700 cP among the four different viscosity of chitosan. According to the data in Table 1, chitosan with 700 cP was chosen for further experiments.
3.1.2. Effect of weight ratio of CS to TPP on CSTPP-GO The effect of weight ratio of CS to TPP on the enzyme activity recovery was shown in Table 1. The ratios were 3:1, 5:1, 8:1 and 10:1, respectively. GO activity recovery was the highest (78.3%) when the ratio of CS to TPP was 5:1. As the ratio of CS to TPP increased, the activity recovery decreased. It could be explained that when the weight of CS was large, TPP was not enough to form gelation with CS by ionic bonding. CSTPP-GO slightly increased bread specific volume as the enzyme activity recovery increased (Table 1). And the hardness decreased as the CSTPP-GO activity increased. The weight ratio of CS to TPP 5:1 was chosen for further experiment.
3.1.3. Effect of GO concentration on CSTPP-GO After chitosan-TPP solutions were formed, different concentration of GO (50, 100, 150 and 200 U/mL) were added. Table 1 showed that the concentration of GO had certain effect on GO activity recovery and bread quality during microparticle preparation. Enzyme activity recovery increased initially and then dropped with increase of GO concentration. GO could not be loaded well by CS-TPP solution because of the high concentration of GO. Enzyme activity was highest (85.1%) when GO concentration was 100 U/ mL. The baking test also confirmed this conclusion. Based on enzyme activity recovery and bread quality the optimum condition for preparation of CSTPP-GO was chitosan with 700 cP viscosity, CS to TPP (w/w): 5 to 1 and 100 U/mL GO.
3.2.2. In vitro release of H2O2 The release of H2O2 was investigated in phosphate buffer solutions (PBS) containing glucose as substrate for 12 h. At similar amounts and concentrations of enzyme and substrate, the release of H2O2 was different in free GO and CSTPP-GO samples (Fig. 2). During the first 6 h, CSTPP-GO samples released H2O2 more slowly than those with free GO. This might be due to CSTPP-GO limiting the diffusion of the substrate (glucose). After 6 h, the release of H2O2 continued rising steadily. The microparticles loaded with enzyme acted as micro-reactors, thereby impeding the diffusion of glucose and preventing product inhibition. As a result, the catalytic reaction was scarcely affected and H2O2 release was almost constantly rising throughout the reaction period. Conversely, for free GO samples, the release of H2O2 was initially rapid and became almost constant after 6 h. There was no significant release of H2O2 because of lack of substrate and reaction inhibition by the product. Therefore, controlled release of H2O2 by immobilized enzyme avoids fast oxidation of glucose oxidase during dough preparation and breadmaking. 3.2.3. Stability of CSTPP-GO The stability and shelf-life of immobilized enzyme is very important for industrial application. Hence, the stability of CSTPPGO was investigated by storing CSTPP-GO at 4 and 25 °C and measuring the residual GO activity every week for 3 months. The loss of GO activity of CSTPP-GO both at 4 °C and 25 °C was below 8%.
3.2. Characterization of CSTPP-GO 3.2.1. Morphology The morphology of CSTPP-GO microparticles was shown in Fig. 1. It was observed that the CSTPP-GO microparticles had pseudo-spherical shape with 5 lm diameter from SEM microphotographs. The particles produced at 180 ± 5 °C inlet air temperature and feed flow rate at 10 mL/min. When the CSTPP-GO solution was feed into the drying chamber by a rotary atomizer, the fluid meet the high temperature, and the water in the fluid evaporated instantly. Due to the instant evaporation of water, most of the microparticles had irregular and hollow surfaces.
Fig. 2. Release profiles of H2O2 produced by free GO and CSTPP-GO in PBS (0.1 M, pH 5.6).
Fig. 1. SEM microphotographs of CSTPP-GO microparticles prepared by spray drying: (a) 2500 magnification and (b) 15,000 magnification.
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L. Tang et al. / Food Chemistry 161 (2014) 1–7 Table 2 Effect of KBrO3, free GO and CSTPP-GO on bread quality. Sample
Specific volume (mL/g) A
Control KBrO3B Free GOC CSTPP-GOC
a
3.92 ± 0.01 4.26 ± 0.03c 4.18 ± 0.02b 4.44 ± 0.04d
Hardness (g) c
420.5 ± 4.4 340.1 ± 10.9a 387.2 ± 4.7b 353.3 ± 6.8a
Springiness 0.919 ± 0.031a 0.926 ± 0.053a 0.920 ± 0.022a 0.934 ± 0.034a
Different letters within a column indicate significantly different values (p < 0.05). A During breadmaking procedure, without any addition of additives was control. B The addition of KBrO3 60 mg/kg flour. C The addition of free GO and CSTPP-GO were 400 U/kg flour.
Fig. 3. Effect of KBrO3, free GO and CSTPP-GO on dough rheology: (a) G0 , (b) G00 and (c) tan d.
3.3. Effect of CSTPP-GO on breadmaking quality The rheological properties of doughs made with KBrO3, free GO, CSTPP-GO and control (without GO and KBrO3) were determined. In Fig. 3 is shown the typical frequency sweep obtained for doughs by rheometric assays. It can be seen that G0 > G00 in all the tested
range, and both moduli are frequency dependent, showing a solid viscoelastic behaviour of the commercial flour dough. This experiment confirmed that dough containing glucose oxidase or KBrO3 had more elastic properties relative to its viscous properties than dough without adding oxidant. It further indicated that the H2O2 was responsible for this effect on dough. H2O2 promoted the formation of disulphide linkages in the gluten network and consequently improved dough handling properties and bread quality (Poulsen & Høstrup, 1998; Steffolani et al., 2010; Vemulapalli et al., 1998). Both G0 and G00 values (Fig. 3a and b) for doughs made with CSTPP-GO were significantly higher than similar values for free GO, KBrO3 or control doughs, which indicates that more force was needed to deform doughs containing CSTPP-GO (Gujral & Rosell, 2004). Also, the tan d values (G00 /G0 ) for doughs made with CSTPP-GO were significantly lower than those doughs made with free GO or KBrO3. Thus, doughs containing CSTPP-GO had relatively more elastic properties relative to its viscous properties than doughs made with free GO or KBrO3. According to the results (Fig. 3c), dough made with free GO had lower tan d values than that with KBrO3, indicating that during dough development, free GO helped produce more cross-linking than KBrO3. The effect of the addition of KBrO3 (60 mg/kg flour), free GO (400 U/kg flour) and CSTPP-GO (400 U/kg flour) on bread quality was shown in Table 2. According to our results, although free GO significantly improved specific volume than the control, it was lower than the specific volume of bread made with KBrO3. And free GO significantly decreased hardness than the control, but it was higher than the hardness of bread made with KBrO3. This could partially be ascribed to fast oxidation of gluten. Rakotozafy et al. (1999) reported that fast oxidation caused excessive gluten networks and resulted in negative effect on gas retention ability. So, we can get the conclusion that GO is unable to replace KBrO3 fully. The result is consistent with the conclusion of Vemulapalli et al. (1998). In the case of bread with CSTPP-GO, the hardness and springiness was not obviously different from that with KBrO3, whereas the specific volume significantly increased from 4.26 to 4.44 in comparison with bread with KBrO3. These positive effects can be due to the slow release of H2O2. Fast release of H2O2 by free GO can oxidize thiolate anion (RS) resulted from breaking of –S–S– during the mixing process to form new disulphide bonds randomly, which is not good for the formation of glutenin macropolymer (Pescador-Piedra, Farrera-Rebollo, & CalderónDomínguez, 2010; Weegels, Pijpekamp, Graveland, Hamer, & Schofield, 1996). On the contrary, CSTPP-GO was proposed to promote the formation of more regular and stable disulphide linkages. As mentioned above, the formation of gluten network could be benefit from the slow oxidation of thiol group and the formation of –S–S– caused by the slow release of H2O2 during the dough development and baking. CS-TPP as a carrier not only prevented GO inactivation, but also provide a barrier to decelerate the diffusion of glucose substrate and the release of H2O2.
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Table 3 Effect of CSTPP-GO combined with FAA on bread quality. Sample Specific volume (mL/g) Hardness (g) Springiness
CSTPP-GOA (400) b
4.54 ± 0.04 348.9 ± 15.1c 0.924 ± 0.079a
CSTPP-GO(400)+FAA (31.25) b
4.58 ± 0.06 311.0 ± 10.4b 0.930 ± 0.10a
CSTPP-GO(400)+FAA (62.5) c
4.80 ± 0.08 276.1 ± 11.6a 0.940 ± 0.092a
CSTPP-GO(400)+FAA (93.75) b
4.61 ± 0.05 346.2 ± 12.3c 0.934 ± 0.087a
KBrO3B (60) 4.23 ± 0.04a 341.8 ± 17.9c 0.923 ± 0.063a
Different letters within a line indicate significantly different values (p < 0.05). A Unit of CSTPP-GO and FAA is U/kg flour. B Unit of KBrO3 is mg/kg flour.
3.4. Effect of addition of fungal a-amylase and CSTPP-GO on bread quality For investigating the effect of CSTPP-GO on bread quality, different dosages of fungal a-amylase (FAA) (31.25, 62.5 and 93.75 U/ kg flour) together with CSTPP-GO (400 U/kg flour) were used in breadmaking. KBrO3 (60 mg/kg flour optimum quantity) was taken as a positive control. Table 3 presented that bread quality was significantly improved with the addition of CSTPP-GO (400 U/kg flour) and 62.5 U/kg flour FAA. Springiness slightly increased from 0.923 to 0.940 and the specific volume of crumb also increased by 13.48%. The hardness decreased by 19.22%. This meant that bread with CSTPP-GO was softer and more elastic, and even larger than that with KBrO3. It could be concluded that CSTPP-GO was a good replacer of KBrO3 in breadmaking of commercial wheat flour. 4. Conclusion Glucose oxidase was effectively immobilized on chitosan-sodium tripolyphosphate, with a high enzyme activity recovery (85.1%). The optimum immobilization conditions were: 700 cP viscosity of chitosan, 5:1 weight ratio of CS to TPP and 100 U/mL GO. During the breadmaking of commercial wheat flour, CSTPP-GO shows a slower release of H2O2 than free GO and prevents GO from denaturing in dough. It also increases gas retention properties, thereby preventing fast oxidation of glucose oxidase during dough preparation. The addition of 400 U/kg flour CSTPP-GO and 62.5 U/ kg flour FAA at the beginning of flour mixing greatly improves dough rheological properties and bread quality, making CSTPPGO a feasible alternative to KBrO3. This study revealed that incorporation of CSTPP-GO into bread formula is a promising method for improvement of breadmaking quality of commercial wheat flour. Acknowledgements We are grateful to the National Key Technology R&D Program in the 12th Five year Plan of China (2011BAD23B03), the Natural Science Fund of Jiangsu Province (BK2011149) and the Project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions for financial support. References Beck, M., Jekle, M., Selmair, P. L., Koehler, P., & Becker, T. (2011). Rheological properties and baking performance of rye dough as affected by transglutaminase. Journal of Cereal Science, 54, 29–36. Belitz, H. D., Grosch, W., & Schieberle, P. (2004). Food chemistry (3rd revised ed.). Berlin: Springer-Verlag. Bergmeyer, J., & Grabi, M. (1988). Methods of enzymatic analysis: vol. II samples, reagents, assessment of results (3rd ed., ). Germany: Verlag chemie. Bonet, A., Rosell, C. M., Caballero, P. A., Gómez, M., Pérez-Munuera, I., & Lluch, M. A. (2006). Glucose oxidase effect on dough rheology and bread quality: A study from macroscopic to molecular level. Food Chemistry, 99, 408–415. Dash, M., Chiellini, F., Ottenbrite, R. M., & Chiellini, E. (2011). Chitosan–A versatile semi-synthetic polymer in biomedical applications. Progress in Polymer Science, 36, 981–1014. Georget, D. M. R., Underwood-Toscano, C., Powers, S. J., Shewry, P. R., & Belton, P. S. (2008). Effect of variety and environmental factors on gluten proteins: An
analytical, spectroscopic, and rheological study. Journal of Agricultural and Food Chemistry, 56, 172–1179. Graf, E., & Penniston, J. T. (1980). Method for determination of hydrogen peroxide, with its application illustrated by glucose oxidase. Clinical Chemistry, 26, 658–660. Gujral, H. S., & Rosell, C. M. (2004). Improvement of the breadmaking quality of rice flour by glucose oxidase. Food Research International, 37, 75–81. _ H. (2009). Improvement of the wheat and corn bran GÜL, H., ÖZer, M. S., & DIZlek, bread quality by using glucose oxidase and hexose oxidase. Journal of Food Quality, 32, 209–223. Hanft, F., & Koehler, P. (2006). Studies on the effect of glucose oxidase in bread making. Journal of the Science of Food and Agriculture, 86, 1699–1704. Haros, M., Rosell, C. M., & Benedito, C. (2002). Improvement of flour quality through carbohydrases treatment during wheat tempering. Journal of Agricultural and Food Chemistry, 50, 4126–4130. Jonassen, H., Kjøniksen, A. L., & Hiorth, M. (2012). Stability of chitosan nanoparticles cross-linked with tripolyphosphate. Biomacromolecules, 13, 3747–3756. Kohman, H. A., Hoffman, C., Godfrey, T. M., Ashe, L. H., & Blake, A. E. (1916). On the use of certain yeast nutriments in bread-making. The Journal of Industrial and Engineering Chemistry, 8, 781–789. Kurokawa, Y., Aoki, S., Matsushima, Y., Takamura, N., Imazawa, T., & Hayashi, Y. (1986). Dose–response studies on the carcinogenicity of potassium bromate in F344 rats after long term oral administration. Journal of the National Cancer Institute, 77, 977–982. Lukow, O. M., White, N. D. G., & Sinha, R. N. (1995). Influence of ambient storage conditions on the breadmaking quality of two hard red spring wheats. Journal of Stored Products Research, 31, 279–289. Martinez-Anaya, M. A. (1996). Enzymes and bread flavor. Journal of Agricultural and Food Chemistry, 44, 2469–2480. Martínez-Anaya, M. A., & Jiménez, Teresa (1997). Functionality of enzymes that hydrolyse starch and non-starch polysaccharide in breadmaking. European Food Research and Technology A, 205, 209–214. Miller, K. A., & Hoseney, R. C. (1999). Effect of oxidation on the dynamic rheological properties of wheat flour–water doughs. Cereal Chemistry, 76, 100–104. Moayedallaie, S., Mirzaei, M., & Paterson, J. (2010). Bread improvers: Comparison of a range of lipases with a traditional emulsifier. Food Chemistry, 122, 495–499. Park, J. H., Saravanakumar, G., Kim, K., & Kwon, I. C. (2010). Targeted delivery of low molecular drugs using chitosan and its derivatives. Advanced Drug Delivery Reviews, 62, 28–41. Pescador-Piedra, J. C., Farrera-Rebollo, R. R., & Calderón-Domínguez, G. (2010). Effect of glucose oxidase and mixing time on soluble and insoluble wheat flour protein fractions: Changes on SH groups and H2O2 consumption. Food Science and Biotechnology, 19, 1485–1491. Poulsen, C., & Høstrup, P. B. (1998). Purification and characterization of a hexose oxidase with excellent strengthening effects in bread. Cereal Chemistry, 75, 51–57. Primo-Martín, C., Wang, M. W., Lichtendonk, W. J., Plijter, J. J., & Hamer, R. J. (2005). An explanation for the combined effect of xylanase–glucose oxidase in dough systems. Journal of the Science of Food and Agriculture, 85, 1186–1196. Rakotozafy, L., Mackova, B., Delcros, J. F., Boussard, A., Davidou, S., Potus, J., et al. (1999). Effect of adding exogenous oxidative enzymes on the activity of three endogenous oxidoreductases during mixing of wheat flour dough. Cereal Chemistry, 76, 213–218. Ranum, P. (1992). Potassium bromate in bread baking. Cereal Foods World, 37, 253–258. Rasiah, I. A., Sutton, K. H., Low, F. L., Lin, H. M., & Gerrard, J. A. (2005). Crosslinking of wheat dough proteins by glucose oxidase and the resulting effects on bread and croissants. Food Chemistry, 89, 325–332. Selinheimo, E., Autio, K., Kruus, K., & Buchert, J. (2007). Elucidating the mechanism of laccase and tyrosinase in wheat breadmaking. Journal of Agricultural and Food Chemistry, 55, 6357–6365. Shu, X. Z., & Zhu, K. J. (2002). The influence of multivalent phosphate structure on the properties of ionically cross-linked chitosan films for controlled drug release. European Journal of Pharmaceutics and Biopharmaceutics, 54, 235–243. Simpson, C., Jordaan, J., Gardiner, N. S., & Whiteley, C. (2007). Isolation, purification and characterization of a novel glucose oxidase from Penicillium sp. CBS 120262 optimally active at neutral pH. Protein Expression and Purification, 51, 260–266. Steffolani, M. E., Ribotta, P. D., Pérez, G. T., & León, A. E. (2010). Effect of glucose oxidase, transglutaminase, and pentosanase on wheat proteins: Relationship with dough properties and bread-making quality. Journal of Cereal Science, 51, 366–373. Tzanov, T., Costa, S. A., Gubitz, G. M., & Cavaco-Paulo, A. (2002). Hydrogen peroxide generation with immobilized glucose oxidase for textile bleaching. Journal of Biotechnology, 93, 87–94.
L. Tang et al. / Food Chemistry 161 (2014) 1–7 Vemulapalli, V., & Hoseney, R. C. (1998). Glucose oxidase effects on gluten and water solubles. Cereal Chemistry, 75, 859–862. Vemulapalli, V., Miller, K. A., & Hoseney, R. C. (1998). Glucose oxidase in breadmaking systems. Cereal Chemistry, 75, 439–442. Wang, X., Zhu, K. X., & Zhou, H. M. (2011). Immobilization of glucose oxidase in alginate–chitosan microcapsules. International Journal of Molecular Sciences, 12, 3042–3054. Weegels, P. L., Pijpekamp, A. M., Graveland, A., Hamer, R. J., & Schofield, J. D. (1996). Depolymerisation and re-polymerisation of wheat glutenin during dough processing. I. Relationships between glutenin macropolymer content and quality parameter. Journal of Cereal Science, 1996(23), 103–111.
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Wieser, H., & Kieffer, R. (2001). Correlations of the amount of gluten protein types to the technological properties of wheat flours determined on a micro-scale. Journal of Cereal Science, 34, 19–27. Wrigley, C. W., & Bietz, J. A. (1988). Proteins and amino acids. In Y. Pomeranz (Ed.). Wheat: Chemistry and technology (Vol. 1, pp. 159–275). St. Paul, MN, U.S.A: American Association of Cereal Chemists. Xu, Y., & Du, Y. (2003). Effect of molecular structure of chitosan on protein delivery properties of chitosan nanoparticles. International Journal of Pharmaceutics, 250, 215–226. Zhu, Y., Rinzema, A., Tramper, J., & Bol, J. (1995). Microbial transglutaminase–A review of its production and application in food processing. Applied Microbiology and Biotechnology, 44, 277–282.
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Preparation of immobilized glucose oxidase and its application in improving breadmaking quality of commercial wheat flour Lele Tang a,b, Ruijin Yang a,b,⇑, Xiao Hua b, Chaohua Yu a, Wenbin Zhang b, Wei Zhao a a b
State Key Laboratory of Food Science and Technology, Jiangnan University, 214122 Wuxi, China School of Food Science and Technology, Jiangnan University, 214122 Wuxi, China
a r t i c l e
i n f o
Article history: Received 6 December 2013 Received in revised form 20 March 2014 Accepted 22 March 2014 Available online 1 April 2014 Keywords: Glucose oxidase Immobilization Chitosan Sodium tripolyphosphate Breadmaking quality Commercial wheat flour
a b s t r a c t Preparation of immobilized glucose oxidase (GO) on chitosan (CS)-sodium tripolyphosphate (TPP) and its application in improving breadmaking quality of commercial wheat flour were investigated. The optimum conditions for GO immobilization were: viscosity of CS: 700 cP, ratio of CS to TPP (w/w): 5 to 1, and GO concentration 100 U/mL. The obtained CSTPP-GO was 5 lm-diameter particle with a pseudo-spherical shape. By addition of CSTPP-GO (400 U/kg flour) and fungal a-amylase (62.5 U/kg flour), bread springiness slightly increased from 0.923 to 0.940, specific volume of crumb increased by 13.48% and hardness decreased by 19.22%, compared to addition of KBrO3 (60 mg/kg flour). It could be concluded that CSTPP-GO combined with fungal a-amylase had potential application in improving breadmaking quality of commercial wheat flour. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Bread is an important food in human diet in many countries and contributes substantially to the daily intake of carbohydrates, dietary fibre, protein, minerals and B vitamins (Belitz, Grosch, & Schieberle, 2004, chap. 15). The functional breadmaking properties of wheat greatly depend on the gluten proteins (Wieser & Kieffer, 2001). However, cultivar differences, insect damage and environmental, genetic and post-harvest conditions of wheat may affect gluten properties and wheat processing quality (Georget, Underwood-Toscano, Powers, Shewry, & Belton, 2008; Lukow, White, & Sinha, 1995; Wrigley & Bietz, 1988). In order to overcome deficiencies in breadmaking quality (loaf volume, crumb structure, shelf-life, flavour and colour) of wheat flour, various bread improvers, such as oxidants, reductants, emulsifiers and enzymes, have been added (Haros, Rosell, & Benedito, 2002; Martinez-Anaya, 1996; Moayedallaie, Mirzaei, & Paterson, 2010). Potassium bromate (KBrO3) has been widely used in the baking industry for a long time as an effective improver for improving loaf volume, crumb structure and texture (Kohman, Hoffman, Godfrey, Ashe, & Blake, 1916). KBrO3, a slow acting oxidant, oxidizes free thiol groups generating small –S–S– compounds and bromide. It is active during later stages of fermentation and baking. However, ⇑ Corresponding author at: School of Food Science and Technology, Jiangnan University, 214122 Wuxi, China. Tel./fax: +86 510 85919150. E-mail address: [email protected] (R. Yang). http://dx.doi.org/10.1016/j.foodchem.2014.03.104 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
its application has been gradually restricted and even forbidden in many countries because of the potential relationship between bromate intake and cancer (Kurokawa et al., 1986; Ranum, 1992). Therefore, it is an urgent challenge to find the alternatives for KBrO3. Enzymes are attractive, safe alternatives to KBrO3. Trials of enzymes such as transglutaminase (EC 2.3.2.13) (Beck, Jekle, Selmair, Koehler, & Becker, 2011), laccase (EC 1.10.3.2) (Selinheimo, Autio, Kruus, & Buchert, 2007), hexose oxidase (EC 1.1.3.5) (Gül, Özer, & Dizlek, 2009) and glucose oxidase (EC 1.1.3.4) (Bonet et al., 2006; Rasiah, Sutton, Low, Lin, & Gerrard, 2005) have been studied in breadmaking. They are able to improve different aspects of bread qualities such as reinforcing gluten structure, strengthening dough stability, increasing specific volume, texture, elasticity and water holding capacity (Martínez-Anaya & Jiménez, 1997; Miller & Hoseney, 1999; Zhu, Rinzema, Tramper, & Bol, 1995). Fungal (Aspergillus niger) Glucose oxidase (GO) is one of the most effective alternative to KBrO3 (Hanft & Koehler, 2006; Vemulapalli, Miller, & Hoseney, 1998). In the presence of oxygen, GO catalyses the oxidation of b-D-glucose to D-gluconic acid with the release of hydrogen peroxide (H2O2). The H2O2 promotes the formation of disulphide linkages in the gluten networks and consequently improves dough handling properties, better gas retention capability and bread quality, especially for commercial wheat flour (Poulsen & Høstrup, 1998; Steffolani, Ribotta, Pérez, & León, 2010; Vemulapalli & Hoseney, 1998). However, GO is unable to replace KBrO3 due to some deficiencies including fast
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oxidation at the initial stage in dough development, low stability in flour and thermal-inactivation in the baking stage. Fast oxidation will cause rapid release of H2O2 at the beginning stage, resulting in over-developed dough and low volume and poor crumb of bread (Primo-Martín, Wang, Lichtendonk, Plijter, & Hamer, 2005; Rakotozafy et al., 1999). Immobilization is an effective method to control the release of H2O2 and improve the stability of GO. According to Tzanov, Costa, Gubitz, and Cavaco-Paulo (2002), immobilized GO can control the release of H2O2 and be used for at least 5 times for textile bleaching. But there are few people applying the immobilized GO to control the release of H2O2 in bakery. Wang, Zhu, and Zhou (2011) has proved that GO immobilized in alginate– chitosan microcapsules has good storage stabilities with 70.4% GO activity remained after over 2 months. This investigation has suggested a promising way to prepare potential breadmaking improver by immobilization of GO. Many researchers (Dash, Chiellini, Ottenbrite, & Chiellini, 2011; Park, Saravanakumar, Kim, & Kwon, 2010) have focused on drug delivery by Chitosan-TPP nanoparticles. In the present study, CSTPP-immobilized GO was prepared as the breadmaking improver. Immobilization conditions including the viscosity of chitosan, ratio of chitosan to TPP (w/w) and the concentration of GO were optimized. The H2O2-release profile and appearance of immobilized GO were characterized. Finally, the improvement of bread quality with the addition of CSTPP-GO was examined.
2.3. Enzyme assay The activities of free GO and CSTPP-GO were measured using indirect oxidation of o-dianisidine by horseradish peroxidase (HRP). The assays were performed according to the method developed by Bergmeyer (Bergmeyer & Grabi, 1988, chap. 2; Simpson, Jordaan, Gardiner, & Whiteley, 2007). Reaction mixtures contained 0.006% (w/v) o-dianisidine2HCl in 0.1 mol/L pH 7.0 potassium phosphate buffer, 10% (w/v) b-D-glucose in distilled water and 60 U/mL HRP in 0.1 mol/L pH 7.0 potassium phosphate buffer. They were mixed immediately prior to assaying for GO in the ratio 2.4:0.5:0.1, respectively. The reaction was initiated by the addition of 0.1 mL of GO (0.15–0.2 U/mL), and ended with 3 mL of the final reagent (0.5 mol/L H2SO4) 5 min later. The absorbance of the reaction was read at 436 nm against a blank consisting 0.1 mL pH 7.0 potassium phosphate buffer replace GO. One unit of GO activity was defined as the amount of enzyme that catalyses the conversion of 1 lmol b-D-glucose to D-gluconic acid and H2O2 per minute at 25 °C and pH 7.0. The enzyme activity recovery was calculated using Eq. (1). Enzyme activity recovery ð%Þ ¼
Calculated CSTPP-GO concentration 100 Theoretical GO concentration ð1Þ
2.4. Characterization of CS-TPP GO 2. Materials and methods 2.1. Materials Commercial wheat flour (moisture 13.8%, ash 0.42%, protein 10.2% w/w) was obtained from Wuxi Rongguo Flour Mill (Wuxi, Jiangsu, China). Glucose oxidase (10,000 U/g) Gluzyme mono 10000 BG was generously gifted by Novozymes (Bagsvaerd, Denmark). Butter, active dry yeast, commercial sugar and salt were obtained from the local market (Wuxi, Jiangsu, China). Food-grade chitosans (deacetylation degree 90%) with different viscosity (100, 700, 1200, 2200 cP, respectively) were purchased from Pan’an Chitosan Product Co. (Pan’an, Zhejiang, China). Sodium tripolyphosphate (analytical grade) was obtained from Sinopharm Chemical Reagent (Beijing, China). Potassium bromate was obtained from Shantou Xilong Chemical Factory (Shantou, Guangdong, China). All the other chemicals were of analytical grade. All solutions were prepared with distilled water. 2.2. Immobilization of GO on CS-TPP microparticles Immobilization of GO on CS-TPP microparticles was performed by ionotropic gelation according to the reported method (Jonassen, Kjøniksen, & Hiorth, 2012; Shu & Zhu, 2002) with slight modifications. Chitosan (0.5% w/v) was dissolved in 1% (w/v) acetic acid aqueous solution and insoluble impurities were removed by filtration. TPP aqueous solution (2.5 mg/mL) was filtered to remove impurities by 0.45 lm membrane before it was added dropwise into the chitosan solution by a constant flow pump with thorough stirring at room temperature. Subsequently, GO in 0.1 mol/L pH 5.6 phosphate buffer was added into the chitosan-TPP solution and the mixture solution was stirred for another 1 h. At last, the chitosanTPP-GO suspension was then spray dried to obtain the TPP crosslinked chitosan microspheres loaded with GO. The spray drying conditions were: 180 ± 5 °C inlet temperature, 70 ± 5 °C outlet temperature and 10 mL/min liquid flow rate. The dried product was finally collected and stored in a desiccator with silica gel at room temperature.
2.4.1. Microstucture of CS-TPP GO Scanning electron microscopy (SEM) was used to characterize the surface and shape of the spray-dried CSTPP-GO microparticles. The samples were coated for 70 s under an argon atmosphere with gold–palladium (sputter-coater) and examined with a scanning electron microscope (Quanta 200F, FEI, Hillsboro, OR, USA).Photographs were taken at a high voltage of 15.0 kv and a magnification of 1.2 or 2 or 5 or 10 or 15 k. The working distance was 15.9 nm. 2.4.2. Stability of CS-TPP GO The stability of the CS-TPP GO was determined by the enzyme activity which was investigated every week during 3 months. 2.4.3. In vitro release of hydrogen peroxide The hydrogen peroxide (H2O2) released from enzymatic catalysis was analyzed by colorimetric method (Graf & Penniston, 1980). 2.5. Breadmaking procedure and bread analysis 2.5.1. Breadmaking The breadmaking basic formulation used included: 100 g commercial wheat flour, 15 g commercial sugar, 1 g salt, 1.5 g active dry yeast, 57.3 g water (optimum level), 8 g butter. And other additives such as free GO 400 U/kg wheat flour, or KBrO3 60 mg/kg wheat flour, or CSTPP-GO 400 U/kg wheat flour were added in the formulation according to the different experiments, respectively. These additives were added before the mixing step. The ingredients (wheat flour, sugar, salt, yeast and additives) were blended for 5 min in a mixer and then water was added and mixed with all the ingredients to develop dough. Afterwards, butter was added into the dough and mixed until dough development under the optimal conditions. The resulting dough was allowed to rest for 15 min for the first fermentation at 25 °C and 75% relative humidity. After first fermentation, the dough was divided into 100 g pieces, rounded, moulded and placed in baking pans. The pieces were then proofed for 90 min at 38 °C and 85% relative humidity and baked at 210 °C for 15 min. Bread was removed from the pans and cooled at room temperature, then stored at room temperature in plastic bags for measurement and further analysis.
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2.5.2. Dough rheological properties The dough was prepared in a 50 g-Farinograph (Farinograph-E, Brabender, Germany). Dough basic formulation was: 100 g flour, 1 g NaCl, water according to farinographic absorption. And KBrO3 6 mg/100 g flour, or free GO 40 U/100 g flour, or CSTPP-GO 40 U/ 100 g flour was added in the formulation, respectively. Dough was then covered with a plastic film, to avoid dehydration, and left to rest for 15 min at room temperature. Dynamic oscillatory tests were performed in a rheometer (AR1000, TA Instruments, UK). The assay was executed at 25 ± 0.1 °C, using a plate–plate sensor system with a 2.0 mm gap between plates. To prevent sample dehydration during the assay, semisolid Vaseline oil was applied. Deformation and frequency sweeps were carried out. Before measurements, samples were rest for 30 min between plates to relax. Stress sweeps were performed at a constant frequency (1 Hz) in order to determine the linear viscoelastic range of each sample (data not shown); frequency sweeps (from 0.1 to 40 Hz) were performed at a constant stress within the linear viscoelastic range. Dynamic moduli G0 (elastic or storage modulus), G00 (viscous or loss modulus) and tan d (G00 /G0 ) were obtained as a function of frequency. 2.5.3. Bread quality Bread quality evaluation was carried out by determining the weight, loaf volume, bread specific volume (BSV), springiness and crumb hardness. The loaves were weighed and loaf volume was determined by rapeseed displacement just two hours after baking. The bread specific volume was expressed as the ratio of volume to weight of baked bread. Texture profile analysis (TPA) was performed by using a TA-XT2i Texture Analyser (Stable Micro Systems, Surrey, UK) equipped with an aluminium (25 mm in diameter) cylinder probe. Two hours after baking, the loaves were cut into 20 mm thick bread slices and subjected to a double cycle of compression under the following conditions: 1.0 mm/s pre-test speed, 3.0 mm/s test speed, 2.0 mm/s post-test speed. The crumb was compressed to 50% of the original height. The texture profile parameters of springiness were determined using the Texture Expert 1.22 software (Stable Micro Systems, Surrey, UK). The resulting peak force of compression was reported as bread hardness. 2.6. Fungal a-amylase compound with CSTPP-GO in improving bread texture Different dosages of fungal a-amylase were employed compound with CSTPP-GO in bread formulas to improve bread texture. The breadmaking formulation used was: 1000 g commercial wheat flour, 150 g commercial sugar, 10 g salt, 15 g active dry yeast, 573 g water (optimum level), 80 g butter, 400 U CSTPP-GO and fungal
a-amylase: 0, 31.25, 62.5 and 93.75 U. KBrO3 (60 mg/kg flour optimum quantity) was taken as a control. Then the breadmaking procedure was the same as the previous methods (Section 2.5.1). Bread quality was determined by previous methods (Section 2.5.3). 2.7. Statistical analysis SPSS statistical software 17.0 was used to perform the statistical analysis. A Fisher’s test (LSD) was made in order to evaluate differences among samples, while the relationship between measured parameters was assessed by Pearson’s test (significant level at p < 0.05). 3. Results and discussion 3.1. Preparation of CSTPP-GO The optimization for preparation of CSTPP-GO was based on the consideration of GO activity recovery and the main quality characteristics of bread with addition of CSTPP-GO. The viscosity of chitosan, ratio of CS to TPP and concentration of GO were varied, and the preparation parameters such as stirring speed and batch size were kept constant. 3.1.1. Effect of viscosity of chitosan on CSTPP-GO Chitosan solutions, 0.5 (w/v) were prepared by dissolving chitosan with different viscosity of 100, 700, 1200, 2200 cP in 1% (v/v) in acetic acid solution. GO was immobilized as described in Section 2.2 at the same conditions: weight ratio of CS to TPP 8:1, concentration of GO 50 U/mL. Enzyme activity recovery of immobilized GO prepared using chitosan with different viscosity is presented in Table 1. The GO activity recovery was achieved 77.9% with the chitosan of 700 cP, followed by 54% with 200 cP, 76.2% with 1200 cP and 61.1% with 2200 cP. Xu and Du (2003) proved that as the molecular weight of chitosan increased, the encapsulation efficiency of BSA was increased directly. As we all know, the molecular weight of chitosan increases with the viscosity. So we assumed that the ionic bonds of CSTPP-GO microparticles prepared with low viscosity chitosan were not strong and led to enzyme leakage. Sedimentation was observed during the preparation of CSTPP-GO microparticles with high viscosity chitosan and GO could not be immobilized well. Springiness, hardness and specific volume of bread prepared with CSTPP-GO 400 U/kg commercial wheat flour are shown in Table 1. The results are consistent with enzyme activity recovery (Table 1). The difference in springiness (0.92–0.94) was not significant. It indicated that oxidation did not have obvious effects on the structure of the gluten network. Specific volume was
Table 1 Different immobilized conditions of GO on enzyme activity recovery and bread quality (specific volume, hardness, springiness). Factors
Hardness (g)
Springiness
Viscosity (cP)
100 700 1200 2200
Activity recovery (%) 54.0 ± 1.03a 77.9 ± 0.89c 76.2 ± 0.75c 61.1 ± 0.92b
4.0 ± 0.10a 4.7 ± 0.04c 4.4 ± 0.06b 4.6 ± 0.05c
398.2 ± 7.4c 339.4 ± 8.8a 360.5 ± 6.7b 358.3 ± 5.6b
0.925 ± 0.03a 0.948 ± 0.015a 0.94 ± 0.01a 0.938 ± 0.025a
Weight ratio of CS/TPP
3:1 5:1 8:1 10:1
76.1 ± 1.11c 81.5 ± 0.83d 67.0 ± 0.64b 52.4 ± 0.73a
4.24 ± 0.08a 4.68 ± 0.05c 4.61 ± 0.04c 4.38 ± 0.06b
375.8 ± 2.5c 349.3 ± 1.6b 338.5 ± 5.5a 351.6 ± 3.2b
0.935 ± 0.015ab 0.955 ± 0.012b 0.942 ± 0.014b 0.91 ± 0.016a
Concentration of GO (U/mL)
50 100 150 200
81.1 ± 0.78c 85.1 ± 0.56d 74.0 ± 1.02b 62.0 ± 0.69a
4.7 ± 0.02b 4.75 ± 0.03b 4.48 ± 0.04a 4.48 ± 0.03a
349.1 ± 5.4b 312.7 ± 4.8a 342.4 ± 4.7b 365.2 ± 5.6c
0.955 ± 0.012a 0.925 ± 0.02a 0.932 ± 0.018a 0.923 ± 0.015a
Different letters within a column indicate significantly different values (p < 0.05).
Specific volume (mL/g)
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significantly increased from 4.0 mL/g (at 200 cP) to 4.7 mL/g (at 700, 1200 and 2200 cP) (p < 0.05), suggesting a limited improvement in gas retention capability. Hardness was markedly reduced and reached the lowest level at 700 cP among the four different viscosity of chitosan. According to the data in Table 1, chitosan with 700 cP was chosen for further experiments.
3.1.2. Effect of weight ratio of CS to TPP on CSTPP-GO The effect of weight ratio of CS to TPP on the enzyme activity recovery was shown in Table 1. The ratios were 3:1, 5:1, 8:1 and 10:1, respectively. GO activity recovery was the highest (78.3%) when the ratio of CS to TPP was 5:1. As the ratio of CS to TPP increased, the activity recovery decreased. It could be explained that when the weight of CS was large, TPP was not enough to form gelation with CS by ionic bonding. CSTPP-GO slightly increased bread specific volume as the enzyme activity recovery increased (Table 1). And the hardness decreased as the CSTPP-GO activity increased. The weight ratio of CS to TPP 5:1 was chosen for further experiment.
3.1.3. Effect of GO concentration on CSTPP-GO After chitosan-TPP solutions were formed, different concentration of GO (50, 100, 150 and 200 U/mL) were added. Table 1 showed that the concentration of GO had certain effect on GO activity recovery and bread quality during microparticle preparation. Enzyme activity recovery increased initially and then dropped with increase of GO concentration. GO could not be loaded well by CS-TPP solution because of the high concentration of GO. Enzyme activity was highest (85.1%) when GO concentration was 100 U/ mL. The baking test also confirmed this conclusion. Based on enzyme activity recovery and bread quality the optimum condition for preparation of CSTPP-GO was chitosan with 700 cP viscosity, CS to TPP (w/w): 5 to 1 and 100 U/mL GO.
3.2.2. In vitro release of H2O2 The release of H2O2 was investigated in phosphate buffer solutions (PBS) containing glucose as substrate for 12 h. At similar amounts and concentrations of enzyme and substrate, the release of H2O2 was different in free GO and CSTPP-GO samples (Fig. 2). During the first 6 h, CSTPP-GO samples released H2O2 more slowly than those with free GO. This might be due to CSTPP-GO limiting the diffusion of the substrate (glucose). After 6 h, the release of H2O2 continued rising steadily. The microparticles loaded with enzyme acted as micro-reactors, thereby impeding the diffusion of glucose and preventing product inhibition. As a result, the catalytic reaction was scarcely affected and H2O2 release was almost constantly rising throughout the reaction period. Conversely, for free GO samples, the release of H2O2 was initially rapid and became almost constant after 6 h. There was no significant release of H2O2 because of lack of substrate and reaction inhibition by the product. Therefore, controlled release of H2O2 by immobilized enzyme avoids fast oxidation of glucose oxidase during dough preparation and breadmaking. 3.2.3. Stability of CSTPP-GO The stability and shelf-life of immobilized enzyme is very important for industrial application. Hence, the stability of CSTPPGO was investigated by storing CSTPP-GO at 4 and 25 °C and measuring the residual GO activity every week for 3 months. The loss of GO activity of CSTPP-GO both at 4 °C and 25 °C was below 8%.
3.2. Characterization of CSTPP-GO 3.2.1. Morphology The morphology of CSTPP-GO microparticles was shown in Fig. 1. It was observed that the CSTPP-GO microparticles had pseudo-spherical shape with 5 lm diameter from SEM microphotographs. The particles produced at 180 ± 5 °C inlet air temperature and feed flow rate at 10 mL/min. When the CSTPP-GO solution was feed into the drying chamber by a rotary atomizer, the fluid meet the high temperature, and the water in the fluid evaporated instantly. Due to the instant evaporation of water, most of the microparticles had irregular and hollow surfaces.
Fig. 2. Release profiles of H2O2 produced by free GO and CSTPP-GO in PBS (0.1 M, pH 5.6).
Fig. 1. SEM microphotographs of CSTPP-GO microparticles prepared by spray drying: (a) 2500 magnification and (b) 15,000 magnification.
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L. Tang et al. / Food Chemistry 161 (2014) 1–7 Table 2 Effect of KBrO3, free GO and CSTPP-GO on bread quality. Sample
Specific volume (mL/g) A
Control KBrO3B Free GOC CSTPP-GOC
a
3.92 ± 0.01 4.26 ± 0.03c 4.18 ± 0.02b 4.44 ± 0.04d
Hardness (g) c
420.5 ± 4.4 340.1 ± 10.9a 387.2 ± 4.7b 353.3 ± 6.8a
Springiness 0.919 ± 0.031a 0.926 ± 0.053a 0.920 ± 0.022a 0.934 ± 0.034a
Different letters within a column indicate significantly different values (p < 0.05). A During breadmaking procedure, without any addition of additives was control. B The addition of KBrO3 60 mg/kg flour. C The addition of free GO and CSTPP-GO were 400 U/kg flour.
Fig. 3. Effect of KBrO3, free GO and CSTPP-GO on dough rheology: (a) G0 , (b) G00 and (c) tan d.
3.3. Effect of CSTPP-GO on breadmaking quality The rheological properties of doughs made with KBrO3, free GO, CSTPP-GO and control (without GO and KBrO3) were determined. In Fig. 3 is shown the typical frequency sweep obtained for doughs by rheometric assays. It can be seen that G0 > G00 in all the tested
range, and both moduli are frequency dependent, showing a solid viscoelastic behaviour of the commercial flour dough. This experiment confirmed that dough containing glucose oxidase or KBrO3 had more elastic properties relative to its viscous properties than dough without adding oxidant. It further indicated that the H2O2 was responsible for this effect on dough. H2O2 promoted the formation of disulphide linkages in the gluten network and consequently improved dough handling properties and bread quality (Poulsen & Høstrup, 1998; Steffolani et al., 2010; Vemulapalli et al., 1998). Both G0 and G00 values (Fig. 3a and b) for doughs made with CSTPP-GO were significantly higher than similar values for free GO, KBrO3 or control doughs, which indicates that more force was needed to deform doughs containing CSTPP-GO (Gujral & Rosell, 2004). Also, the tan d values (G00 /G0 ) for doughs made with CSTPP-GO were significantly lower than those doughs made with free GO or KBrO3. Thus, doughs containing CSTPP-GO had relatively more elastic properties relative to its viscous properties than doughs made with free GO or KBrO3. According to the results (Fig. 3c), dough made with free GO had lower tan d values than that with KBrO3, indicating that during dough development, free GO helped produce more cross-linking than KBrO3. The effect of the addition of KBrO3 (60 mg/kg flour), free GO (400 U/kg flour) and CSTPP-GO (400 U/kg flour) on bread quality was shown in Table 2. According to our results, although free GO significantly improved specific volume than the control, it was lower than the specific volume of bread made with KBrO3. And free GO significantly decreased hardness than the control, but it was higher than the hardness of bread made with KBrO3. This could partially be ascribed to fast oxidation of gluten. Rakotozafy et al. (1999) reported that fast oxidation caused excessive gluten networks and resulted in negative effect on gas retention ability. So, we can get the conclusion that GO is unable to replace KBrO3 fully. The result is consistent with the conclusion of Vemulapalli et al. (1998). In the case of bread with CSTPP-GO, the hardness and springiness was not obviously different from that with KBrO3, whereas the specific volume significantly increased from 4.26 to 4.44 in comparison with bread with KBrO3. These positive effects can be due to the slow release of H2O2. Fast release of H2O2 by free GO can oxidize thiolate anion (RS) resulted from breaking of –S–S– during the mixing process to form new disulphide bonds randomly, which is not good for the formation of glutenin macropolymer (Pescador-Piedra, Farrera-Rebollo, & CalderónDomínguez, 2010; Weegels, Pijpekamp, Graveland, Hamer, & Schofield, 1996). On the contrary, CSTPP-GO was proposed to promote the formation of more regular and stable disulphide linkages. As mentioned above, the formation of gluten network could be benefit from the slow oxidation of thiol group and the formation of –S–S– caused by the slow release of H2O2 during the dough development and baking. CS-TPP as a carrier not only prevented GO inactivation, but also provide a barrier to decelerate the diffusion of glucose substrate and the release of H2O2.
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Table 3 Effect of CSTPP-GO combined with FAA on bread quality. Sample Specific volume (mL/g) Hardness (g) Springiness
CSTPP-GOA (400) b
4.54 ± 0.04 348.9 ± 15.1c 0.924 ± 0.079a
CSTPP-GO(400)+FAA (31.25) b
4.58 ± 0.06 311.0 ± 10.4b 0.930 ± 0.10a
CSTPP-GO(400)+FAA (62.5) c
4.80 ± 0.08 276.1 ± 11.6a 0.940 ± 0.092a
CSTPP-GO(400)+FAA (93.75) b
4.61 ± 0.05 346.2 ± 12.3c 0.934 ± 0.087a
KBrO3B (60) 4.23 ± 0.04a 341.8 ± 17.9c 0.923 ± 0.063a
Different letters within a line indicate significantly different values (p < 0.05). A Unit of CSTPP-GO and FAA is U/kg flour. B Unit of KBrO3 is mg/kg flour.
3.4. Effect of addition of fungal a-amylase and CSTPP-GO on bread quality For investigating the effect of CSTPP-GO on bread quality, different dosages of fungal a-amylase (FAA) (31.25, 62.5 and 93.75 U/ kg flour) together with CSTPP-GO (400 U/kg flour) were used in breadmaking. KBrO3 (60 mg/kg flour optimum quantity) was taken as a positive control. Table 3 presented that bread quality was significantly improved with the addition of CSTPP-GO (400 U/kg flour) and 62.5 U/kg flour FAA. Springiness slightly increased from 0.923 to 0.940 and the specific volume of crumb also increased by 13.48%. The hardness decreased by 19.22%. This meant that bread with CSTPP-GO was softer and more elastic, and even larger than that with KBrO3. It could be concluded that CSTPP-GO was a good replacer of KBrO3 in breadmaking of commercial wheat flour. 4. Conclusion Glucose oxidase was effectively immobilized on chitosan-sodium tripolyphosphate, with a high enzyme activity recovery (85.1%). The optimum immobilization conditions were: 700 cP viscosity of chitosan, 5:1 weight ratio of CS to TPP and 100 U/mL GO. During the breadmaking of commercial wheat flour, CSTPP-GO shows a slower release of H2O2 than free GO and prevents GO from denaturing in dough. It also increases gas retention properties, thereby preventing fast oxidation of glucose oxidase during dough preparation. The addition of 400 U/kg flour CSTPP-GO and 62.5 U/ kg flour FAA at the beginning of flour mixing greatly improves dough rheological properties and bread quality, making CSTPPGO a feasible alternative to KBrO3. This study revealed that incorporation of CSTPP-GO into bread formula is a promising method for improvement of breadmaking quality of commercial wheat flour. Acknowledgements We are grateful to the National Key Technology R&D Program in the 12th Five year Plan of China (2011BAD23B03), the Natural Science Fund of Jiangsu Province (BK2011149) and the Project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions for financial support. References Beck, M., Jekle, M., Selmair, P. L., Koehler, P., & Becker, T. (2011). Rheological properties and baking performance of rye dough as affected by transglutaminase. Journal of Cereal Science, 54, 29–36. Belitz, H. D., Grosch, W., & Schieberle, P. (2004). Food chemistry (3rd revised ed.). Berlin: Springer-Verlag. Bergmeyer, J., & Grabi, M. (1988). Methods of enzymatic analysis: vol. II samples, reagents, assessment of results (3rd ed., ). Germany: Verlag chemie. Bonet, A., Rosell, C. M., Caballero, P. A., Gómez, M., Pérez-Munuera, I., & Lluch, M. A. (2006). Glucose oxidase effect on dough rheology and bread quality: A study from macroscopic to molecular level. Food Chemistry, 99, 408–415. Dash, M., Chiellini, F., Ottenbrite, R. M., & Chiellini, E. (2011). Chitosan–A versatile semi-synthetic polymer in biomedical applications. Progress in Polymer Science, 36, 981–1014. Georget, D. M. R., Underwood-Toscano, C., Powers, S. J., Shewry, P. R., & Belton, P. S. (2008). Effect of variety and environmental factors on gluten proteins: An
analytical, spectroscopic, and rheological study. Journal of Agricultural and Food Chemistry, 56, 172–1179. Graf, E., & Penniston, J. T. (1980). Method for determination of hydrogen peroxide, with its application illustrated by glucose oxidase. Clinical Chemistry, 26, 658–660. Gujral, H. S., & Rosell, C. M. (2004). Improvement of the breadmaking quality of rice flour by glucose oxidase. Food Research International, 37, 75–81. _ H. (2009). Improvement of the wheat and corn bran GÜL, H., ÖZer, M. S., & DIZlek, bread quality by using glucose oxidase and hexose oxidase. Journal of Food Quality, 32, 209–223. Hanft, F., & Koehler, P. (2006). Studies on the effect of glucose oxidase in bread making. Journal of the Science of Food and Agriculture, 86, 1699–1704. Haros, M., Rosell, C. M., & Benedito, C. (2002). Improvement of flour quality through carbohydrases treatment during wheat tempering. Journal of Agricultural and Food Chemistry, 50, 4126–4130. Jonassen, H., Kjøniksen, A. L., & Hiorth, M. (2012). Stability of chitosan nanoparticles cross-linked with tripolyphosphate. Biomacromolecules, 13, 3747–3756. Kohman, H. A., Hoffman, C., Godfrey, T. M., Ashe, L. H., & Blake, A. E. (1916). On the use of certain yeast nutriments in bread-making. The Journal of Industrial and Engineering Chemistry, 8, 781–789. Kurokawa, Y., Aoki, S., Matsushima, Y., Takamura, N., Imazawa, T., & Hayashi, Y. (1986). Dose–response studies on the carcinogenicity of potassium bromate in F344 rats after long term oral administration. Journal of the National Cancer Institute, 77, 977–982. Lukow, O. M., White, N. D. G., & Sinha, R. N. (1995). Influence of ambient storage conditions on the breadmaking quality of two hard red spring wheats. Journal of Stored Products Research, 31, 279–289. Martinez-Anaya, M. A. (1996). Enzymes and bread flavor. Journal of Agricultural and Food Chemistry, 44, 2469–2480. Martínez-Anaya, M. A., & Jiménez, Teresa (1997). Functionality of enzymes that hydrolyse starch and non-starch polysaccharide in breadmaking. European Food Research and Technology A, 205, 209–214. Miller, K. A., & Hoseney, R. C. (1999). Effect of oxidation on the dynamic rheological properties of wheat flour–water doughs. Cereal Chemistry, 76, 100–104. Moayedallaie, S., Mirzaei, M., & Paterson, J. (2010). Bread improvers: Comparison of a range of lipases with a traditional emulsifier. Food Chemistry, 122, 495–499. Park, J. H., Saravanakumar, G., Kim, K., & Kwon, I. C. (2010). Targeted delivery of low molecular drugs using chitosan and its derivatives. Advanced Drug Delivery Reviews, 62, 28–41. Pescador-Piedra, J. C., Farrera-Rebollo, R. R., & Calderón-Domínguez, G. (2010). Effect of glucose oxidase and mixing time on soluble and insoluble wheat flour protein fractions: Changes on SH groups and H2O2 consumption. Food Science and Biotechnology, 19, 1485–1491. Poulsen, C., & Høstrup, P. B. (1998). Purification and characterization of a hexose oxidase with excellent strengthening effects in bread. Cereal Chemistry, 75, 51–57. Primo-Martín, C., Wang, M. W., Lichtendonk, W. J., Plijter, J. J., & Hamer, R. J. (2005). An explanation for the combined effect of xylanase–glucose oxidase in dough systems. Journal of the Science of Food and Agriculture, 85, 1186–1196. Rakotozafy, L., Mackova, B., Delcros, J. F., Boussard, A., Davidou, S., Potus, J., et al. (1999). Effect of adding exogenous oxidative enzymes on the activity of three endogenous oxidoreductases during mixing of wheat flour dough. Cereal Chemistry, 76, 213–218. Ranum, P. (1992). Potassium bromate in bread baking. Cereal Foods World, 37, 253–258. Rasiah, I. A., Sutton, K. H., Low, F. L., Lin, H. M., & Gerrard, J. A. (2005). Crosslinking of wheat dough proteins by glucose oxidase and the resulting effects on bread and croissants. Food Chemistry, 89, 325–332. Selinheimo, E., Autio, K., Kruus, K., & Buchert, J. (2007). Elucidating the mechanism of laccase and tyrosinase in wheat breadmaking. Journal of Agricultural and Food Chemistry, 55, 6357–6365. Shu, X. Z., & Zhu, K. J. (2002). The influence of multivalent phosphate structure on the properties of ionically cross-linked chitosan films for controlled drug release. European Journal of Pharmaceutics and Biopharmaceutics, 54, 235–243. Simpson, C., Jordaan, J., Gardiner, N. S., & Whiteley, C. (2007). Isolation, purification and characterization of a novel glucose oxidase from Penicillium sp. CBS 120262 optimally active at neutral pH. Protein Expression and Purification, 51, 260–266. Steffolani, M. E., Ribotta, P. D., Pérez, G. T., & León, A. E. (2010). Effect of glucose oxidase, transglutaminase, and pentosanase on wheat proteins: Relationship with dough properties and bread-making quality. Journal of Cereal Science, 51, 366–373. Tzanov, T., Costa, S. A., Gubitz, G. M., & Cavaco-Paulo, A. (2002). Hydrogen peroxide generation with immobilized glucose oxidase for textile bleaching. Journal of Biotechnology, 93, 87–94.
L. Tang et al. / Food Chemistry 161 (2014) 1–7 Vemulapalli, V., & Hoseney, R. C. (1998). Glucose oxidase effects on gluten and water solubles. Cereal Chemistry, 75, 859–862. Vemulapalli, V., Miller, K. A., & Hoseney, R. C. (1998). Glucose oxidase in breadmaking systems. Cereal Chemistry, 75, 439–442. Wang, X., Zhu, K. X., & Zhou, H. M. (2011). Immobilization of glucose oxidase in alginate–chitosan microcapsules. International Journal of Molecular Sciences, 12, 3042–3054. Weegels, P. L., Pijpekamp, A. M., Graveland, A., Hamer, R. J., & Schofield, J. D. (1996). Depolymerisation and re-polymerisation of wheat glutenin during dough processing. I. Relationships between glutenin macropolymer content and quality parameter. Journal of Cereal Science, 1996(23), 103–111.
7
Wieser, H., & Kieffer, R. (2001). Correlations of the amount of gluten protein types to the technological properties of wheat flours determined on a micro-scale. Journal of Cereal Science, 34, 19–27. Wrigley, C. W., & Bietz, J. A. (1988). Proteins and amino acids. In Y. Pomeranz (Ed.). Wheat: Chemistry and technology (Vol. 1, pp. 159–275). St. Paul, MN, U.S.A: American Association of Cereal Chemists. Xu, Y., & Du, Y. (2003). Effect of molecular structure of chitosan on protein delivery properties of chitosan nanoparticles. International Journal of Pharmaceutics, 250, 215–226. Zhu, Y., Rinzema, A., Tramper, J., & Bol, J. (1995). Microbial transglutaminase–A review of its production and application in food processing. Applied Microbiology and Biotechnology, 44, 277–282.
