Article pubs.acs.org/JAFC
Activation of Endogenous Phytase and Degradation of Phytate in Wheat Bran Jia Guo, Yuan-Yuan Bian, Ke-Xue Zhu,* Xiao-Na Guo, Wei Peng, and Hui-Ming Zhou* State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jiangsu Province, People’s Republic of China ABSTRACT: Wheat bran contains a significant amount of the anti-nutritional factor phytate. This study is the first to explore the effectiveness of activating endogenous phytase and further reducing phytate content through resulting programmed cell death (PCD). Effects of solid−liquid ratio (1:1, 1:2, 1:3, and 1:6), incubation temperature (4, 20, 38, 55, and 70 °C), metal ions (Na+, K+, Ca2+, and Mg2+), gibberellin concentration (0, 5, 50, 500, 2000, and 5000 mg/L), hydrogen peroxide concentration (0, 0.2, 0.4, 0.6, 0.8, and 1.0%), and incubation time (30, 80, 180, and 360 min) on activation of endogenous phytase activity and phytate degradation in wheat bran samples are discussed in this study. It was found that when the wheat bran was incubated with distilled water at 55 °C for 80 min, its endogenous phytase activity was dramatically increased 4-fold from 12.96 to 53.54 FTU/g, whereas the phytate content was reduced by about 70% from 45.20 to 13.52 mg/g. By comparison of photomicrographs of raw wheat bran sample and sample incubated with distilled water for 360 min at 55 °C, a conclusion could be drawn that PCD in aleurone cells had occurred. KEYWORDS: endogenous phytase, phytate, wheat bran, solid−liquid ratio, gibberellin, hydrogen peroxide, metal ions
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INTRODUCTION In recent decades, whole grain consumption has attracted wide interest due to its benefits of improving serum lipid concentrations,1 lowering blood pressure and blood glucose,2−4 and reducing risk for diabetes.5 It also plays a positive role in avoidance of obesity,6 stroke,7 coronary heart disease,8 and certain gastrointestinal disorders.9 In terms of nutrition, whole grains are rich in fiber, starch, vitamins, minerals, and other bioactive components.10 However, many studies have indicated that whole grains also contain a significant amount of phytate (myo-inositol hexakisphosphate, IP6). Phytate, also known as phytic acid, is a natural plant compound comprising a simple ringed carbohydrate with six phosphate groups attached to each carbon and is a major storage form of phosphorus in cereals, legumes, nuts, etc.11 Historically, because phytate can bind essential dietary minerals such as Ca, Fe, Zn, and Mg, and thus decrease their bioavailability in humans, it was considered solely as an antinutrient.12−14 In worst cases, phytate restrains the activity of some digestive enzymes (such as protease, amylase, and trypsin) and consequently influences the utilization of protein, fat, and starch.15−19 Therefore, degradation of plant phytate content is necessary and important. At present, the conventional methods of phytate degradation in whole grains are heating, soaking, germination, fermentation, and enzymatic hydrolysis. Khan demonstrated that the reduction in phytate could reach 46.7% for fresh maize and 52.6% for dry maize by heating.20 In the study of Skoglund, soaking for 9 h at room temperature reduced the phytate content by 45% in a whole-grain diet.21 However, Egli made an opposite observation that soaking was not effective on wheat grain, whereas germination for 72 h could reduce around 33.0% of the phytate content.22 During the process of fermentation, as stated by Buddrick et al., the phytate content showed a pronounced drop by 81.1%.23 Endogenous wheat phytase © 2014 American Chemical Society
effectively degraded wheat IP6 + IP5 at pH 4 and 5, microbial phytase was efficient toward IP6 + IP5 at pH 3−5, and a recombinant wheat phytase degraded IP6 + IP5 in different phytate samples in the range of 12−70% degradation.24 Compared with other cereals, wheat is reported to show a high phytase activity (915−1561 FTU/kg) and a high level of phytates (whole wheat, 0.39−1.35 g/100 g; wheat bran, 2.1− 7.3 g/100 g),25,26 which were mainly located in the aleurone layer of wheat bran.25,27,28 Consequently, reducing phytate content in wheat bran by activation of endogenous phytase is feasible. Besides, this method has many advantages that include simple operation, low cost, big capacity, etc., which are good for the industrial production processes. It also involves using mild conditions, with no damage induced to grain quality, and produces hydrolysates containing low inositol phosphates, inositol, and phosphates, which in turn have a high commercial value. Unfortunately, few related studies on this topic were found. According to the research of Swanson et al., in the process of programmed cell death (PCD) acidic hydrolases (including phytases) were continuously accumulated in protein storage vacuoles (PSVs), where the storage materials were continuously metabolized and depleted with those hydrolases.29 Therefore, the endogenous phytase could be secreted by wheat aleurone cells through the PCD and hydrolyze phytate under appropriate conditions. Therefore, this paper will focus on the activation of endogenous phytase and further reducing phytate levels in wheat bran. Received: Revised: Accepted: Published: 1082
September 11, 2014 December 15, 2014 December 16, 2014 December 16, 2014 DOI: 10.1021/jf504319t J. Agric. Food Chem. 2015, 63, 1082−1087
Article
Journal of Agricultural and Food Chemistry In general, multiple metal ions, such as Na+, K+, Ca2+, and Mg2+, are considered to change some zymogens into active enzymes or accelerate the enzymatic reaction rate. Gibberellin, a type of plant hormone, controls many plant developmental processes, such as germination, growth, and flowering.30,31 Many studies suggest that aleurone cells could synthesize and secrete a range of hydrolytic enzymes in response to exogenous gibberellin through initiating PCD.32 This process of PCD can also be initiated by all types of reactive oxygen species (ROS) including hydrogen peroxide.33−36 Moreover, our previous experiments and previous report37 revealed that solid−liquid ratio, incubation temperature, and incubation time also have obvious effects on endogenous phytase activity and phytate content in wheat bran. Hence, we attempted to evaluate the influence of solid− liquid ratio, incubation temperature, and incubation time in this study. Meanwhile, gibberellin and hydrogen peroxide were used to induce PCD and then activate endogenous phytase and reduce phytate level in wheat bran.
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Optical Microscope Observations. The raw wheat bran and incubated wheat bran were stained with hematoxylin−eosin staining solution and iodine solution and rinsed with distilled water. The samples were subsequently observed with an optical microscope (Nikon, T1-SAM, Japan). Statistical Analysis. All determinations were performed in sextuplicate. The data obtained are expressed as the mean ± standard deviation. The data obtained in this study were analyzed by one-way analysis of variance (ANOVA), and the differences among means were determined through Dunnet T3 test by using the software SPSS 16.0 for Windows. p < 0.05 was considered to be significant.
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RESULTS AND DISCUSSION Solid−Liquid Ratio. Endogenous phytase activity and phytate content of wheat bran samples under various solid− liquid ratios (1:1, 1:2, 1:3, and 1:6) at 55 °C during 360 min of incubation with distilled water are presented in Figure 1. The
MATERIALS AND METHODS
Materials and Reagents. Wheat bran (Xinong 979, hard wheat, harvested in 2014 June, Henan, and stored for 2 months) was purchased from COFCO (Zhengzhou, China). Experiments were performed immediately after milling. Phytic acid (>95%) was obtained from Sigma-Aladdin (Shanghai, China). Standard sodium phytate were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Other chemicals were of analytical grade unless stated otherwise. Incubation. Wheat bran samples (1 g) were incubated in 1−6 mL of working solution of different gibberellin concentrations (0−5000 mg/L), different hydrogen peroxide concentrations (0−1.0%), and different metal ions (0.1 mol/L Na+, K+, Ca2+, and Mg2+) in beakers at 4, 20, 38, 55, and 70 °C for 360 min. After 30, 80, 180, and 360 min, the mixtures were used to determine endogenous phytase activity and phytate content. The levels of gibberellin concentration, hydrogen peroxide concentration, and metal ion were chosen according to the previous studies with some modifications.38−41 Phytic Acid. Phytic acid was measured according to the bipyridine colorimetric method reported by Buddrick and Jones et al.23 Phytic acid was used as internal standard. All analyses were carried out in sextuplicate. Phytase Activity. Phytase activity was measured by determination the inorganic orthophosphate released from the phytic acid by phytase. Standard phytase activity assay was performed at 37 °C according to the method of Huang.42 Half a gram of wheat bran sample was mixed with 15 mL of acetate buffer A (20.52 g of anhydrous sodium acetate, 0.5 g of Triton X-100, and 0.5 g of bovine serum albumin in 1000 mL of distilled water, pH 5.5 ± 0.01), extracted for 15 min under ultrasonic treatment, and stirred with a magnetic stirrer for 30 min at room temperature. Then the mixture was centrifuged (centrifuge TGL-16G, Shanghai Anting Scientific Instrument Factory, Shanghai, China) at 5000g for 4 min. The supernatant (0.2 mL) was incubated at 37 °C with 1.8 mL of buffer A for 5 min and added to 4 mL of 7.5 mmol/L standard sodium phytate. After mixing, the solution was incubated at 37 °C for 30 min, and the enzyme reaction was stopped by the addition of 4 mL of 5% TCA. Inorganic phosphate (Pi) was determined colorimetrically.43 Two hundred microliters of the resulting solution was added to 5 mL of acetate buffer B (20.52 g of anhydrous sodium acetate in 1000 mL of distilled water, pH 5.5 ± 0.01), 200 μL of 10% ascorbic acid, and 200 μL of 10% ammonium molybdate. Its absorbance was measured at 700 nm after 30 min by a SpectraMax M5Multifunctional microplate reader (Molecular Devices, Sunnyvale, CA, USA). All determinations were performed in sextuplicate. One unit of phytase activity (1 FTU) was defined as the amount of enzyme that liberates 1 μmol of orthophosphate per minute from phytate at 37 °C and pH 5.5.44
Figure 1. Effect of solid−liquid ratio (1:1, 1:2, 1:3, and 1:6) at 55 °C on endogenous phytase activity and phytate content of wheat bran samples: (a) effect of solid−liquid ratio on endogenous phytase activity; (b) effect of solid−liquid ratio on phytate content. Data values are based on sextuplicate measurements and shown as the mean ± SD.
results show that the solid−liquid ratio had an extremely significant (p < 0.0001) effect on endogenous phytase activity and phytate content of wheat bran, which respectively demonstrated an upward or a downward trend with the rising solid−liquid ratio. During incubation, every condition could obtain a better point with a maximum endogenous phytase activity (U*) and a minimum phytate content (P*). At high solid−liquid ratio (1:1), U* (53.54 FTU/g) in wheat bran was higher than those incubated at the other lower solid−liquid ratios, which was about 4.13 times greater than raw material. U* at solid−liquid ratios of 1:2, 1:3, and 1:6 were 3.28, 2.11, and 1.54 times, respectively, as high as raw material. In addition, P* (13.52 mg/g) at a solid−liquid ratio of 1:1 was 29.91% of raw material, whereas at solid−liquid ratios of 1:2, 1:3, and 1:6 the values were 40.93, 70.01, and 95.13%, respectively. Phytase was reported to be inactive in dry conditions, whereas it was activated in wet conditions to degrade phytate.45 A moderate amount of water could well activate the endogenous phytase activity and reduce phytate, yet a high amount of water probably promoted aleurone cell swelling and rupturing to death. Therefore, a solid−liquid ratio of 1:1 was 1083
DOI: 10.1021/jf504319t J. Agric. Food Chem. 2015, 63, 1082−1087
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Journal of Agricultural and Food Chemistry selected in follow-up experiments. In addition, this ratio is favorable for subsequent drying. Incubation Temperature. Endogenous phytase activity and phytate content of wheat bran samples under various temperatures (4, 20, 38, 55, and 70 °C) during 360 min of incubation with distilled water (solid−liquid ratio of 1:1) are shown in Figure 2. It could be found that there was a steadily
Figure 3. Effect of metal ions on endogenous phytase activity and phytate content of wheat bran samples: (a) effect of metal ions on endogenous phytase activity; (b) effect of metal ions on phytate content. Data values are based on sextuplicate measurements and shown as the mean ± SD.
(157.23%). Accordingly, it decreased the P* by 13.27%, followed by Ca+ (29.11%), Na2+ (30.78%), and K+ (54.80%). Phytate in wheat exists mainly in the form of phytin (calcium magnesium phytate). Thus, adding Ca2+ and Mg2+ would increase the concentration of enzymatic hydrolysates to inhibit the enzymatic reaction. Moreover, Ca2+ competed with phytate for the active site of endogenous phytase and inhibited its activity or formed Ca−phytate complexes that were insoluble and undegradable.47 The results of this experiment are inconsistent with those from Zhang48 and Yao,41 in which the activity of β-propeller phytases (BPPs) were activated by a reasonable concentration of Ca2+. This could be because BPPs were mainly from Bacillus, and their degradation mechanism was different from that of wheat endogenous phytase. Gibberellin Concentration. Under the conditions of incubation temperature at 55 °C, a solid−liquid ratio of 1:1, and in the incubation solution, the effect of gibberellin concentration (0, 5, 50, 500, 2000, and 5000 mg/L) on the endogenous phytase activity and phytate content in wheat bran samples was measured, and the results are displayed in Figure 4. Generally, gibberellin was counterproductive on activation of endogenous phytase and phytate degradation. It was shown that gibberellin had little effect at low concentration (5 mg/L). However, with the increase of the concentration, the deleterious effect of gibberellin began to be revealed. When the gibberellin concentration was 50 and 500 mg/L, U* fell to around 30 FTU/g and P* rose to >40 mg/g. When the gibberellin concentration reached 2000 mg/L, U* dropped to 20.60 FTU/g and P* went up to 40.67 mg/g. When the gibberellin concentration reached 5000 mg/L, the results improved (U* was 29.59 FTU/g, and P* was 25.16 mg/g) but were still worse than those from incubation with distilled water. Gibberellin was generally regarded as having positive effects on the PCD.49 The barley aleurone layer and its protoplast treated by gibberellin would secrete large amounts of protease, nuclease, α-amylase, and other hydrolases.50 However, our results indicated that secretion of endogenous phytase activity by aleurone had nothing to do with gibberellin’s induction. This
Figure 2. Effect of incubation temperature on endogenous phytase activity and phytate content of wheat bran samples: (a) effect of incubation temperature on endogenous phytase activity; (b) effect of incubation temperature on phytate content. Data values are based on sextuplicate measurements and shown as the mean ± SD.
increasing tendency of U* from 4 °C (23.15 FTU/g) to 55 °C. However, at 70 °C, U* revealed a sudden drop (42.51 FTU/g), only 79.40% of that at 55 °C but a little higher than that at 38 °C (38.92 FTU/g). P*, as well, was extremely significantly (p < 0.0001) influenced by incubation temperature. When compared with raw wheat bran, P* was decreased by 8.74% (41.25 mg/g), 34.60% (29.56 mg/g), 53.50% (21.02 mg/g), 70.09% (13.52 mg/g), and 47.96% (23.52 mg/g) at incubation temperatures of 4, 20, 38, 55, and 70 °C respectively, in comparison with that in raw wheat bran (45.20 mg/g). Obviously, higher incubation temperatures could decrease P*, but extremely high incubation temperature (70 °C) induced a negative effect. Appropriate increase in incubation temperature was helpful to promote phytase reaction in aleurone cells, but when the temperature was much higher than ambient temperature, aleurone cell vitality was subject to inhibition and a serious decline in endogenous phytase activity occurred. Loss of endogenous phytase activity at high temperature (70 °C) may be explained by conformational change and irreversible inactivation.46 Metal Ions. Four commonly used metal ion activators (Na+, K+, Ca2+, and Mg2+) were used to investigate their influence on endogenous phytase activity and phytate content in wheat bran samples during incubation at a solid−liquid ratio of 1:1 and 55 °C (Figure 3). Obviously, all of the metal ions had a negative impact on U* and P*. The most significant (p < 0.0001) factor was Mg2+ (U*, 19.39 FTU/g; P*, 39.20 mg/g), which when compared with raw wheat bran just increased the U* by 49.58%, followed by Na+ (78.34%), Ca2+ (82.19%), and K+ 1084
DOI: 10.1021/jf504319t J. Agric. Food Chem. 2015, 63, 1082−1087
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U* and P* was significantly negative (p < 0.0001). With the rising hydrogen peroxide concentration, U* gradually declined and P* ascended when compared with distilled water. Notably, 1.0% hydrogen peroxide severely inhibited the phytase activity, which decreased rapidly to zero and induced almost no change in phytate content. According to Golovina’s report, reactive oxygen species (ROS) provided by moderate concentration of hydrogen peroxide could induce the PCD process,52 whereas research carried out in this paper pointed the other way. This may be attributed to the selection of raw materials. In their previous study, wheat kernel was selected, whereas wheat bran was selected in this study. Incubation Time. From Figures 1−5, it could be found that, in most cases, the endogenous phytase activity rose rapidly and reached the peak at about 80 min of incubation and then steadily declined to zero while the phytate content continuously decreased during the first 80 min of incubation and then slightly increased. The exceptions were the incubations at 4, 20, 38, and 70 °C or the incubation with 1.0% hydrogen peroxide. The lower the temperature was, the later the U* and P* occurred. In addition, a high concentration of hydrogen peroxide (1.0%) led to rapid inactivation of endogenous phytase; hence, phytate content had little change with the incubation time. This study also highlighted that incubation time was a limiting factor for degradation of phytate in wheat bran during incubation. A short period of time might not achieve the peak of endogenous phytase secreted and synthesized by aleurone cells, and there was insufficient phytase in cells, whereas a long period of time would cause the enzyme activity to decrease gradually and lose its utilization value. Research showed that phytase in wheat belongs to 6-purple acid phosphatase (6PAPhy) and its final enzyme hydrolysates are diphosinositide and phosphate,53 which would not disturb results of phytate content by bipyridine colorimetric method.54 Nevertheless, after 80 min of incubation and with the reduction of phytase activity, some substances such as IP3, IP4, and other complexes might be generated and would result in high phytate content. Optical Microscope Observations. The photomicrographs of raw wheat bran sample and sample incubated with distilled water for 360 min at 55 °C are displayed in Figure 6. As shown in Figure 6a,b, the aleurone cell content had been stained crimson, whereas the cell walls were extremely thick and presented a transparent color. After incubation (Figure 6c− e), the aleurone cells almost disappeared (leaving only a thin layer of pink cell content and a thinner cell wall, Figure 6d), or aleurone cells disappeared completely (Figure 6e), where only neatly arranged long cylindrical transparent cells of intermediate layer were observed. In cereal aleurone tissue, PCD preceded extensive vacuolation of PSVs. At the time of death, a highly vacuolated status occurred showing a single large vacuole occupied virtually all of the cytoplasm.55 Then the cell wall became thinner or partial autolysis occurred, plasma membrane integrity was abruptly lost, and cellular autolysis occurred.56,57 Although there was no vacuolization phenomenon found in this study, the incubated sample still indicated that the PCD and cell autolysis occurred in aleurone cells. This work showed that endogenous phytase activity could be effectively activated to reduce phytate level in wheat bran during the process of activation for 80 min at 50 °C with a solid−liquid ratio of 1:1. Under this experimental condition,
Figure 4. Effect of gibberellin concentration on endogenous phytase activity and phytate content of wheat bran samples: (a) effect of gibberellin concentration on endogenous phytase activity; (b) effect of gibberellin concentration on phytate content. Data values are based on sextuplicate measurements and shown as the mean ± SD.
may be because the main role of gibberellin was to awaken the dormant seed, whereas the selected raw material was fresh and had not yet entered the dormant period or generated adequate germination inhibitors. Thus, the aleurone cells were vital and could produce endogenous phytase by moderate water without gibberellin.51 Hydrogen Peroxide Concentration. Under the conditions of incubation temperature at 37 °C and a solid−liquid ratio of 1:1, the effect of hydrogen peroxide concentration (0, 0.2, 0.4, 0.6, 0.8, and 1.0%) on the endogenous phytase activity and phytate content in wheat bran samples was determined (Figure 5). It was found that the effect of hydrogen peroxide on
Figure 5. Effect of hydrogen peroxide concentration on endogenous phytase activity and phytate content of wheat bran samples: (a) effect of hydrogen peroxide concentration on endogenous phytase activity; (b) effect of hydrogen peroxide concentration on phytate content. Data values are based on sextuplicate measurements and shown as the mean ± SD. 1085
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Figure 6. Photomicrographs of raw wheat bran sample and sample incubated with distilled water for 360 min at 55 °C: (a) raw wheat bran; (b) enlarged map of white arrow; (c) incubated sample; (d) enlarged map of black arrow; (e) enlarged map of red arrow.
gibberellin, hydrogen peroxide, and metal ions showed undesirable results. Compared with traditional phytate degradation, the method described in this paper is more effective, efficient, and suitable for industrial production.
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AUTHOR INFORMATION
Corresponding Authors
*(K.-X.Z.) Phone/fax: +(86) 510 85329037. E-mail: kxzhu@ jiangnan.edu.cn. *(H.-M.Z.) Phone/fax: +(86) 510 85809610. E-mail:
[email protected]. Funding
This work was sponsored by Qing Lan Project, China Postdoctoral Science Foundation (Grant 2014M560396), Jiangsu Planned Projects for Postdoctoral Research Funds (Grant 1402072C), and the National Key Technology R&D Program (Grant 2012BAD34B01). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. Ben-Guo Liu from Henan Institute of Science and Technology for his corrections. 1086
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Journal of Agricultural and Food Chemistry
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DOI: 10.1021/jf504319t J. Agric. Food Chem. 2015, 63, 1082−1087
