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Molecular Characteristics of New Wheat Starch and Its Digestion Behaviours
Journal of Integrative Agriculture 2014, 13(5): 1146-1153
May 2014
RESEARCH ARTICLE
Molecular Characteristics of New Wheat Starch and Its Digestion Behaviours ZHOU Zhong-kai1, HUA Ze-tian1, 2, YANG Yan1, ZHENG Pai-yun1, ZHANG Yan1 and CHEN Xiaoshan1 1 2
Key Laboratory of Food Nutrition and Safety, Ministry of Education/Tianjin University of Science and Technology, Tianjin 300457, P.R.China China National Japonica Rice R&D Center, Tianjin 300457, P.R.China
Abstract In order to understand the effect of starch molecular characteristics on the gel structure, which subsequently influence the gel digestion behaviours, three wheat starches, control (conventional wheat starch), two new wheat cultivars with different genetic backgrounds (by knocking out SBE IIb and SBE IIa, respectively) were used in this study. In comparison with control, slight differences in the morphology of the starch granules of new wheat 1 were observed, whereas the starch granules of new wheat 2 had irregular shapes both for A-type granules and B-type granules. Starch molecular weight size was determined by SE-HPLC, and the results indicate that there was a subtle increase in the amylose content in the starch of new wheat 1 compared to that of control. The starch of new wheat 2 had the highest amylose content, and the molecular weight (MW) of its amylopectin was the lowest among the three starches. Fourier transform infrared spectroscopy (FTIR) was employed to investigate starch gel structure and the results suggest that the molecules of starch gel from new wheat 2 are more likely to re-associate to form an organized conformation. The digestion behaviours of the three starch gels were measured using a mixture of pancreatin α-amylase and amyloglucosidase. The results indicated that the starch gels of control and new wheat 1 had very high digestibility of 91.7 and 91.9%, respectively, whereas the digestibility of wheat 2 starch gel was only 36.2%. In comparison with the digestion curve patterns of control and new wheat 1 starch gels, the new wheat 2 exhibited a much lower initial velocity. These results indicated that the molecules in the starch of new wheat 2 are more readily to re-associate to form an organized structure during gel formation because of its unique molecular characteristics. Key words: wheat starch, starch gels, molecular characteristics, digestion
INTRODUCTION Besides being a major plant metabolite, starch provides the principal energy source in the diet of humans (Björck et al. 1994). It has been believed that the control of glucose releasing rate from starch in foods may play an important role in human health by maintaining a proper blood glucose level and providing
extended energy supply. Studies have shown that long-term consumption of starchy foods with high glycemic index (GI) has been associated with obesity, diabetes, and coronary heart disease (Skrabanja et al. 1999; Ludwig 2003). Thus, the property of starch is crucial in a balanced human diet. According to its digestion behaviour, starch can be divided into three groups, rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) to
Received 19 November, 2012 Accepted 28 January, 2013 Correspondence ZHOU Zhong-kai, Tel: +86-22-60601442, E-mail: [email protected]
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(13)60621-8
Molecular Characteristics of New Wheat Starch and Its Digestion Behaviours
specify the quality of starch in food products (Englyst et al. 1992; Ludwig 2003). In three starch groups, SDS is considered to be beneficial for the host to control glucose release; RS can reach the large bowel to act as a substrate for microbial fermentation to produce a number of active metabolites, such as shortchain fatty acids (SCFA). SCFA is important for maintenance of normal colonic function and health (Englyst et al. 1996; Bird et al. 2000). Among the various SCFA, butyrate appears to be particularly important in this regard, which is the preferred fuel for colonocytes, plays a role in preserving a normal cellular phenotype, and is involved in the maintenance of an epithelial barrier function and modulation of mucin secretion (Barcelo et al. 2000; Topping et al. 2001). Meanwhile, of the carbohydrates, starch is considered as the most buytyrogenic (Godet et al. 1993). Although the susceptibility of starch to hydrolysis by amylases has been shown to be influenced by many factors, e.g., processing method, food texture, and food compositions, etc. (Englyst et al. 1987; Tovar et al. 1992), hydrolysis of starch by α-amylases is certainly influenced by the physicochemical properties of the starch itself (Slaughter et al. 2001), and molecular structure is considered to be one of the most important factors influencing starch property in terms of digestion. Thus, in recent years, an increasing number of studies have focused on manipulating starch molecular structure to alter its digestion properties. For instance, clear-cut involvement of branching enzymes of the A family in amylopectin synthesis A
B
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has been demonstrated in pea (Tovar et al. 2002), rice (Bhattacharyya et al. 1990) and maize (Mizuno et al. 1993). In these cases, the mutants displayed a similar phenotype, i.e., a reduction in starch content correlated to a large increase in amylose content. In maize this is also accompanied by a switch from the A to the B type of diffraction pattern with a net loss in crystallinity. However, studies on manipulating molecular structure in wheat mutants are very rare and the information regarding the properties of starch in mutant wheats is also very limited. Nevertheless, starch assimilation in the human gastrointestinal tract is a complex process, and the knowledge of the specific molecular properties that are responsible for resistance to enzymatic digestion is still very limited. Thus, the objective of present study was to investigate how the molecular structure influences starch molecular re-association after cooking, and subsequently influences its digestion property. In this study, three wheat starches with different molecular characterizations were used.
RESULTS Starch morphology The morphologies of starches of the three wheat varieties were shown in Fig. 1. All the three starch varieties appeared to be a mixture of simple and compound granules, and most of the compound granules contained clusters of individual granules C
Fig. 1 Starch morphologies. A, control. B, new wheat 1 (SBE IIb knock out). C, new wheat 2 (SBE IIa knock out).
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ZHOU Zhong-kai et al.
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including both A-type and B-type (Fig. 1-A-C). In this study, starch granules of the control wheat (control) appeared to have two sizes, a large lenticular shaped granule (A-granules) and small spherical granules (B-granules), indicating its biosynthesis occurred at the two different development stages (Fig. 1-A). There was slight difference in the morphologies of the starches between control and new wheat 1, at which the starch of new wheat 1 had spherical disclike morphology. Starch granules of new wheat 2 had a totally different morphology compared to those of control and new wheat 1, which showed irregular shapes both for A-type granules and B-type granules (Fig. 1-C). Meanwhile, although surfaces of new wheat 1 starch granules still seemed to be smooth, the granules showed depressions with disc-like morphology.
content of amylose (both relatively high MW amylose fraction and relatively low MW amylose fraction). Meanwhile, another significant difference in the MW of amylopectin among the three starches was also noticed, at which the MW of amylopectin in the starch of new wheat 2 appears to be much lower than that of amylopectin in the starches of control and new wheat 1 (7 166 vs. 45 523, 43 646 kDa).
IR spectra of native starch and its corresponding gel The changes in the IR spectrum before and after cooking are recorded and demonstrated in Fig 3. In general, cooking changed the IR profile, at which the band at 1 022 cm-1 became absolutely dominant for cooked starch due to the loss of molecular order (Fig. 3), which may be associated with the transformation of double helices into single helices. Meanwhile, great reduction in the absorbance at 999 cm-1 was also noticed and a slight peak shifting from 999 to 1 002 cm-1 was observed as well after the cooking. These phenomena were found for all the three starches (Fig. 3). Furthermore, three starches showed different responses to the cooking treatment, e.g., the attitude of the reduction in the absorbance at 999 cm-1 for new wheat 2 starch after the cooking was the least among the three starches. In other words, cooking greatly reduced the absorbance at 999 cm-1 for both control and new wheat 1 starches, and the attitudes of the reduction in this band after the cooking were at a very similar level for the two starches. This IR similarity might be due to their very close molecular structures of these two starches.
Starch molecular characteristics
Detector response
The molecular profiles of the three kinds of starches are illustrated in Fig. 2. It shows that starch molecules in each starch can be separated into three fractions on the basis of their molecular weight (MW): high MW fraction (mainly represented by amylopectin), medium MW fraction (amylose with relatively higher MW), and low MW fraction (amylose with relatively lower MW). In comparison with the molecular profile of control starch, there was an increase in the amylose content in the starch of new wheat 1, but the increment did not seem to be outstanding. However, compared to that of control starch, a totally different molecular profile was obtained for the starch of new wheat 2. This sample was significantly lower in the content of amylopectin and higher in the 0.60 0.58 0.56 0.54 0.52 0.50 0.48 0.46 0.44 0.42 0.40 0.38 0.36 0.34 0.32
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Fig. 2 Molecular structure of the three wheat starches. Fr I, amylopectin; Fr II, amylose with relatively higher MW; Fr III, amylose with relatively lower MW.
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Molecular Characteristics of New Wheat Starch and Its Digestion Behaviours
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Fig. 3 Effect of cooking and storage on IR spectra of the three starches. A, control. B, new wheat 1. C, new wheat 2.
Hydrolysis kinetics The digestion rate of each starch gel is presented in Fig. 5, which is the measurement of the increase in glucose concentration in the incubation system at designated intervals. The digestion progress for starch gels of control and new wheat 1 showed a biphasic pattern, i.e., a rapid rate at the initial hydrolysis stage
IR 1022/999-1045/1022 (cm-1)
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Fig. 4 Changes in the gel structures, expressed as the difference in the IR relative absorbance during storage at room temperature.
3.5 Glucose content (mg mL-1)
The molecular re-association process of the three starches during the storage is monitored using IR spectrum and illustrated in Fig. 4. Two bands at 1 045 and 999 cm-1 in the strained region are associated with double helices although they possibly possess different packing. Absorbance corresponding to the bands around 1 045 and 999 cm-1 is a result of the more stable packing of the chain segments, which is better than that of the 1 022 cm-1 band. Thus, the molecular reassociation process during storage could be expressed as the difference in the IR absorption between 1 022/999-1 045/1 022 cm-1 (Fig. 4).
3.0 Control New wheat 1 New wheat 2
2.5 2.0 1.5 1.0 0.5 0.0
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Incubation time (min)
Fig. 5 Hydrolysis kinetics of the three starch gels by the mixture of pancreatic α-amylase and amyloglucosidase.
followed by a progressively decreased rate thereafter (Fig. 5). Both of the digestion curves exhibit a plateau during the time course of digestion. The overall digestion rate of starch gel of new wheat 2 was much lower than those of control and new wheat 1 throughout the designated course. A much lower initial velocity (2.1×10-2 ), expressed as the slope of digestion curve within the first 20 min incubation time) was found in the digestion of the starch gel of new wheat 2 compared with those of control and new wheat 1 (both for 13.0×10-2). These results indicated that the starch gel made from new wheat 2 had much slower glucose releasing kinetics due to its unique molecular structure. The digestion extent was also calculated, and the results showed that over 90% of the starch gels of control and new wheat 1 (91.7 and 91.9%, respectively) were digested after completion of the designated digestion course, whereas only 36.2% of starch gel from new wheat 2 was digested at the same time point.
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DISCUSSION The visualization in this study further confirmed that these granules are generally either small and spherical or large and ellipsoidal depending strongly on the botanical backgrounds. Much attention had recently been paid to the long-term form of storage present in the endosperm of cereals, but very little information is available concerning either the structure or the morphology of such granules. From another perspective, the irregular shapes observed in this study on the high amylose wheat starch variety (i.e., new wheat 2) further emphasized the importance of amylopectin as a parameter of granule morphogenesis during starch biosynthesis (Stinard et al. 1993). The difference in HPLC chromatograms between control starch and new wheat 1 starch was minor, indicating that there were no significant changes in the molecular profile, in particular in the amylose content by knocking out of SBE IIb during the starch biosynthesis. However, the significant difference in the molecular profiles between control starch and new wheat 2 indicated that the changes in molecular structure of starch could be achieved by mutations, in particular by knocking out of SBE IIa during the starch biosynthesis. The characteristics of starch molecular structure would subsequently influence its physicochemical properties, such as gelatinization and retrogradation during cooking process. In order to further understand how starch molecular re-association behaviour is manipulated by its molecular structure, starch was cooked in excess water and stored at room temperature to ensure it is gelatinized and retrograded. It is interesting to note that the starch gel of new wheat 2 showed the fastest reduction in this value during the whole period of gel storage, which indicates that the molecules in this starch gel are more likely to re-associate to form an organized conformation. It has been reported that the lower the value is, the faster the molecular reassociations process occurs and the recovering level to its previous conformation is a molecularly dependent phenomenon (Björck et al. 1994; Bernazzani et al. 2001; Gaudier et al. 2004). The FTIR results
ZHOU Zhong-kai et al.
further proved that molecular re-associations (i.e., retrogradation) of the starch are a multi-stage process with differing in the rate among the starches. It is interesting to note that much faster molecular reassociation process was found for the starch gel of new wheat 2 through all the designated storage intervals compared to those of control and new wheat 1. Waigh et al. (2000) has postulated that two stages are involved during starch gelatinization in excess water. The first stage involves a slow side by side dissociation of helices and the second stage involves a rapid helix coil transition. Fechner et al. (2005) also suggested that starch with rich-amylose had shown all stages of retrogradation (a fast retrogradation stage and a slow retrogradation stage along the storage) using a Raman spectroscopy. These significant differences in the digestion rate and extent among the three starch gels may be caused by their gel structures. As revealed by IR spectra in this study, the chain segments of starch molecules of cooked new wheat 2 packed in more stable conformation than those of the cooked control and new wheat 1. These different packing morphologies would subsequently influence starch digestion behaviours. Fechner et al. (2005) also applied TEM to visualize how molecules affect their packing properties. They found that configuration of amylopectin hinders the long-scale rearrangement of the crystallites, although it still can form parallel double helices by the associations of its short side branches after a long term storage. Consequently, the starch containing a higher proportion of amylopectin configuration would have a high sensitivity to the amylolysis. However, semicrystalline structure formed by the associations of amylose molecules by parallel double helices demonstrated fractal-like networks, and these networks substantially condensed, yielding thick aggregates (Putaux et al. 2000). Thus, starch with such a structure would show a high resistance to the amylolysis. From the molecularly structural point of view, the starch of new wheat 2 seems to prefer forming a condensed aggregate with a highly organized structure due to its molecular characterizations, i.e., a much higher in the content/proportion of amylose, and a lower MW of amylopectin.
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Molecular Characteristics of New Wheat Starch and Its Digestion Behaviours
CONCLUSION Starch molecular structure manipulates starch morphology, cooking propertiess (e.g., gel structure) and its subsequent digestion behaviours. Among the three tested starches in this study, control and new wheat 1 showed a very similar molecular profile, subsequently leading to a similarity in the granular morphology, starch gel structure, and even digestion properties. The starch of new wheat 2, characterized by a higher in amylose content and lower in the MW of amylopectin, showed totally different starch properties in the above described parameters. Thus, the manipulation of starch molecular structure could control starch digestion behaviours, which would benefit the development of new kinds of starches containing a higher amount of SDS and/or RS.
MATERIALS AND METHODS Enzymes and chemicals Alpha-amylase (type VI-B, from porcine pancreas) was purchased from Sigma Chemical Co. (St Louis, MO, USA), and amyloglucosidase (from A. niger, high purity) purchased from Megazyme Australia Pty Ltd. (Victoria, Australia). Chemicals and solvents were of ACS-certified grade.
Starches Three wheat starches, control, new wheat 1 and new wheat 2 with different genetic backgrounds obtained from Tianjin University of Science & Technology, China, were used in this study. Their pedigrees are Italian Funo and the derived lines/hybridizations. The control starch was isolated from the parent. Starch of new wheat 1 was developed by knocking out SBE IIb, and starch of new wheat 2 was developed by knocking out SBE IIa.
FTIR Starch cooking and gel storage 1 mL of reverse osmosis (RO) water was added to 60 mg of starches in separate 25-mL test tubes. Each tube was capped and placed in a boiling water bath for 10 min treatment and then cooled to room temperature before FTIR measurements. The changes in the gel structure were determined using a FTIR at designated time intervals. After each measurement, the tube
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was capped and stored at room temperature until the next measurement. FTIR measurements The FTIR spectra were recorded on a Varian spectrometer (Model: Excalibur 3100, Varian, Inc., USA) equipped with a cooled deuterated triglycine sulphate (DTGS) detector. The measurement was performed on a MIRacle TM attenuated total reflectance (ATR) crystal plate with Digital Readout High Pressure Clamp (Pike Technologies, USA). The starch precipitate was directly loaded on the plate and scanned in the range of 3 600-600 cm-1 at a resolution of 4 cm-1. Prior to recording, the spectra were transformed against an empty cell as background. Finally the spectra were ATR deconvoluted using Varian Resolutions Pro software. A half-bandwith of 15 cm-1 and a K factor of 1.5 with triangular apodization were applied. The IR absorbance values ranging 1 200 and 800 cm-1 for each starch gel at different storage intervals of 0, 2, 10, 20 h were obtained via each scanning.
Starch molecular characterization by SEHPLC An 8-mL aliquot of aqueous dimethylsulfoxide (90:10, DMSO/water, v/v) was added to 20 mg of starches in separate 25-mL test tubes. Each tube was capped and placed in a boiling water bath for 60 min and then cooled to room temperature. The mixture was centrifuged at 2 095×g for 10 min. The supernatant was aspirated into a separate 25-mL test tube and the dissolved starch was precipitated by adding 18 mL of 95% ethanol. The tube was kept at 4°C overnight and then centrifuged (2 095×g for 15 min). The starch precipitate was collected for subsequent analyses by SE-HPLC. The starch precipitate (around 20 mg), prepared as described earlier, was redissolved in 0.5 mL of 0.2 mol L-1 NaOH and mixed vigorously for approximately 5 s. The solution was neutralized by addition of sodium acetate buffer (0.5 mL; 0.05 mol L-1, pH 4.0) before adding ionexchange resin (0.20 g, BioRad AG ® 501-X8, Hercules, USA) and incubating at 50°C for 1.5 h with occasional shaking. After centrifuging at 10 000 r min -1 for 10 min, the clear supernatants were collected for SE-HPLC analysis. The HPLC system comprised a GBC pump (LC 1150, GBC Instruments, Vic, Australia) equipped with auto sampler (GBC, LC1610) and evaporative light scattering detector (ELSD) (ALLTech, Deerfield, USA). The UltrahydrogelTM 1000 column, Ultrahydrogel TM 250 column and guard column (7.8 mm×300 mm, Waters, Japan) were used and maintained at 35°C during HPLC operation. Ammonium acetate buffer (0.05 mol L-1; pH 5.2) was used as mobile phase at a flow rate of 0.8 mL min-1. A 50-mL aliquot of the supernatant was used for injection. Conditions for ELSD operation were: tube temperature, 115°C; N2 gas flow rate, 2.0 L min-1; gain, 16; impactor, on. Dextrans of various MW were used as standards for measuring the MW of amylose
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and amylopectin.
Starch gel digestion in vitro with pancreatin α-amylase and amyloglucosidase Starch gel preparation A 10-mL aliquot of aqueous dimethylsulfoxide (90:10, DMSO/water, v/v) was added to 50 mg of starches in separate 25-mL test tubes. Each tube was capped and placed in a boiling water bath for 60 min and then cooled to room temperature before centrifuging at 2 095×g for 10 min. The supernatant was collected into a separate 75-mL polypropylene centrifuge tube and the dissolved starch was precipitated by adding 30 mL of 95% ethanol. The tube was then centrifuged again (2 095×g for 10 min). The supernatant was aspirated to waste and the starch precipitate was re-dissolved in 20 mL of RO water by vortexing for 1 min. The starch solution was then stored at 4°C for 7 d to ensure the re-association of starch molecules. After the 7 d storage, the whole starch was recovered by the EtOH again, and the precipitate was freeze dried. 10 mL of pH 5.2 NaAc buffer (0.1 mol L-1) was added to 31.5 mg of the freeze dried starches in separate 25-mL test tubes, and vortexed for 2 min. The starch suspension was kept at 4°C for 2 d before digestion. Starch gel digestion Prior to the digestion, 0.44 mL of CaCl2 (0.1 mol L-1) was added into the starch suspension. The starch suspension was equilibrated at 37°C for 1 h with magnetic stirring, and then 0.15 mL of enzyme solution containing 2.3 U of porcine pancreatin α-amylase and 24 U of amyloglucosidase was added. Enzymatic digestion was carried out at 37°C with magnetic stirring, and 0.3 mL aliquots of hydrolysed solution were withdrawn at different time intervals. The aliquots were immediately put in a boiling water bath for 5 min to deactivate the enzymes. The glucose content in each hydrolyzate was determined using the Megazyme Glucose Assay Kit (GOPOD method). Each sample was analysed in duplicates.
Statistical analyses Values were expressed as means±SE for three starches. Digestion rate of each starch sample is the measurement of the increase in glucose concentration in the system at each designated time. Digestion extent of each starch gel was calculated as the percentage of digested starch in the total starch at the designated incubation intervals. The amount of released glucose from the digested starch was measured using Megazyme Glucose Assay Kit (GOPOD method), and the amount of digested starch was calculated after conversion of released glucose into starch by use of factor 0.9. Initial velocity of amylases hydrolysis of the starch gels was expressed as the slope of digestion curve within the first 20 min incubation time. Each sample was analysed at least in duplicates. Experimental data were subjected to analysis
of variance using Genstat 5 (Ver. 4.1, 1998, Clarendon, NY). Treatments were tested separately for least significant difference (LSD) at a 5% level of probability.
Acknowledgements This work was supported by the China-Euro Collaboration Funds from the Minister of Science and Technology of China (SQ2013ZOA100001).
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Molecular Characteristics of New Wheat Starch and Its Digestion Behaviours
starches-a review. Canadian Journal of Physiology and Pharmacology, 69, 79-92. Ludwig D S. 2002. The glycemic index: physiological mechanisms relating to obesity, diabetes, and cardiovascular disease. Journal of the American Medical Association, 287, 2414-2423. Ludwig D S. 2003. Glycemic load comes of age. The Journal of Nutrition, 133, 2695-2696. Mizuno K, Kawasaki T, Shimada H, Satoh H, Kobayashi E, Okumura S, Arai Y, Baba T. 1993. Alteration of the structural properties of starch components by the lack of an isoform of starch branching enzyme in rice seeds. The Journal of Biological Chemistry, 268, 19084-19091. Putaux J L, Buléon A, Chanzy H. 2000. Network formation in dilute amylose and amylopectin studied by TEM. Macromolecules, 33, 6416-6422. Skrabanja V, Liljeberg H G M, Hedley C L, Kreft I, Björck I M E. 1999. Influence of genotype and processing on the in vitro rate of starch hydrolysis and resistant starch hydrolysis in peas (Pisum sativum L.). Journal of Agricultural and Food Chemistry, 47, 2033-2039. Slaughter S L, Ellis P R, Butterworth P J. 2001. An
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investigation of the action of porcine pancreatic α-amylase on native and gelatinised starches. Biochimica et Biophysica Acta, 1525, 29-36. Stinard P S, Robertson D S, Schnable P S. 1993. Genetic isolation, cloning, and analysis of a mutator-induced, dominant antimorph of the maize amylose extender1 Locus. The Plant Cell, 5, 1555-1566. Topping D L, Clifton P M. 2001. Short-chain fatty acids and human colonic function: Roles of resistant starch and nonstarch polysaccharides. Physiological Reviews, 81, 1031-1064. Tovar J, Björck I M, Asp N G. 1992. Incomplete digestion of legume starches in rats: A study of precooked flours containing retrograded and physically inaccessible starch fractions. The Journal of Nutrition, 122, 1500-1507. Tovar J, Melito C C, Herrera E, Rascón A, Pérez E. 2002. Resistant starch formation does not parallel syneresis tendency in different starch gels. Food Chemistry, 76, 455-459. Waigh T A, Kato K L, Donald A M, Gidley M J, Clarke C J, Riekel C. 2000. Side-chain liquid crystalline model for starch. Starch/Stärke, 52, 450-460. (Managing editor WENG Ling-yun)
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May 2014
RESEARCH ARTICLE
Molecular Characteristics of New Wheat Starch and Its Digestion Behaviours ZHOU Zhong-kai1, HUA Ze-tian1, 2, YANG Yan1, ZHENG Pai-yun1, ZHANG Yan1 and CHEN Xiaoshan1 1 2
Key Laboratory of Food Nutrition and Safety, Ministry of Education/Tianjin University of Science and Technology, Tianjin 300457, P.R.China China National Japonica Rice R&D Center, Tianjin 300457, P.R.China
Abstract In order to understand the effect of starch molecular characteristics on the gel structure, which subsequently influence the gel digestion behaviours, three wheat starches, control (conventional wheat starch), two new wheat cultivars with different genetic backgrounds (by knocking out SBE IIb and SBE IIa, respectively) were used in this study. In comparison with control, slight differences in the morphology of the starch granules of new wheat 1 were observed, whereas the starch granules of new wheat 2 had irregular shapes both for A-type granules and B-type granules. Starch molecular weight size was determined by SE-HPLC, and the results indicate that there was a subtle increase in the amylose content in the starch of new wheat 1 compared to that of control. The starch of new wheat 2 had the highest amylose content, and the molecular weight (MW) of its amylopectin was the lowest among the three starches. Fourier transform infrared spectroscopy (FTIR) was employed to investigate starch gel structure and the results suggest that the molecules of starch gel from new wheat 2 are more likely to re-associate to form an organized conformation. The digestion behaviours of the three starch gels were measured using a mixture of pancreatin α-amylase and amyloglucosidase. The results indicated that the starch gels of control and new wheat 1 had very high digestibility of 91.7 and 91.9%, respectively, whereas the digestibility of wheat 2 starch gel was only 36.2%. In comparison with the digestion curve patterns of control and new wheat 1 starch gels, the new wheat 2 exhibited a much lower initial velocity. These results indicated that the molecules in the starch of new wheat 2 are more readily to re-associate to form an organized structure during gel formation because of its unique molecular characteristics. Key words: wheat starch, starch gels, molecular characteristics, digestion
INTRODUCTION Besides being a major plant metabolite, starch provides the principal energy source in the diet of humans (Björck et al. 1994). It has been believed that the control of glucose releasing rate from starch in foods may play an important role in human health by maintaining a proper blood glucose level and providing
extended energy supply. Studies have shown that long-term consumption of starchy foods with high glycemic index (GI) has been associated with obesity, diabetes, and coronary heart disease (Skrabanja et al. 1999; Ludwig 2003). Thus, the property of starch is crucial in a balanced human diet. According to its digestion behaviour, starch can be divided into three groups, rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) to
Received 19 November, 2012 Accepted 28 January, 2013 Correspondence ZHOU Zhong-kai, Tel: +86-22-60601442, E-mail: [email protected]
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(13)60621-8
Molecular Characteristics of New Wheat Starch and Its Digestion Behaviours
specify the quality of starch in food products (Englyst et al. 1992; Ludwig 2003). In three starch groups, SDS is considered to be beneficial for the host to control glucose release; RS can reach the large bowel to act as a substrate for microbial fermentation to produce a number of active metabolites, such as shortchain fatty acids (SCFA). SCFA is important for maintenance of normal colonic function and health (Englyst et al. 1996; Bird et al. 2000). Among the various SCFA, butyrate appears to be particularly important in this regard, which is the preferred fuel for colonocytes, plays a role in preserving a normal cellular phenotype, and is involved in the maintenance of an epithelial barrier function and modulation of mucin secretion (Barcelo et al. 2000; Topping et al. 2001). Meanwhile, of the carbohydrates, starch is considered as the most buytyrogenic (Godet et al. 1993). Although the susceptibility of starch to hydrolysis by amylases has been shown to be influenced by many factors, e.g., processing method, food texture, and food compositions, etc. (Englyst et al. 1987; Tovar et al. 1992), hydrolysis of starch by α-amylases is certainly influenced by the physicochemical properties of the starch itself (Slaughter et al. 2001), and molecular structure is considered to be one of the most important factors influencing starch property in terms of digestion. Thus, in recent years, an increasing number of studies have focused on manipulating starch molecular structure to alter its digestion properties. For instance, clear-cut involvement of branching enzymes of the A family in amylopectin synthesis A
B
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has been demonstrated in pea (Tovar et al. 2002), rice (Bhattacharyya et al. 1990) and maize (Mizuno et al. 1993). In these cases, the mutants displayed a similar phenotype, i.e., a reduction in starch content correlated to a large increase in amylose content. In maize this is also accompanied by a switch from the A to the B type of diffraction pattern with a net loss in crystallinity. However, studies on manipulating molecular structure in wheat mutants are very rare and the information regarding the properties of starch in mutant wheats is also very limited. Nevertheless, starch assimilation in the human gastrointestinal tract is a complex process, and the knowledge of the specific molecular properties that are responsible for resistance to enzymatic digestion is still very limited. Thus, the objective of present study was to investigate how the molecular structure influences starch molecular re-association after cooking, and subsequently influences its digestion property. In this study, three wheat starches with different molecular characterizations were used.
RESULTS Starch morphology The morphologies of starches of the three wheat varieties were shown in Fig. 1. All the three starch varieties appeared to be a mixture of simple and compound granules, and most of the compound granules contained clusters of individual granules C
Fig. 1 Starch morphologies. A, control. B, new wheat 1 (SBE IIb knock out). C, new wheat 2 (SBE IIa knock out).
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including both A-type and B-type (Fig. 1-A-C). In this study, starch granules of the control wheat (control) appeared to have two sizes, a large lenticular shaped granule (A-granules) and small spherical granules (B-granules), indicating its biosynthesis occurred at the two different development stages (Fig. 1-A). There was slight difference in the morphologies of the starches between control and new wheat 1, at which the starch of new wheat 1 had spherical disclike morphology. Starch granules of new wheat 2 had a totally different morphology compared to those of control and new wheat 1, which showed irregular shapes both for A-type granules and B-type granules (Fig. 1-C). Meanwhile, although surfaces of new wheat 1 starch granules still seemed to be smooth, the granules showed depressions with disc-like morphology.
content of amylose (both relatively high MW amylose fraction and relatively low MW amylose fraction). Meanwhile, another significant difference in the MW of amylopectin among the three starches was also noticed, at which the MW of amylopectin in the starch of new wheat 2 appears to be much lower than that of amylopectin in the starches of control and new wheat 1 (7 166 vs. 45 523, 43 646 kDa).
IR spectra of native starch and its corresponding gel The changes in the IR spectrum before and after cooking are recorded and demonstrated in Fig 3. In general, cooking changed the IR profile, at which the band at 1 022 cm-1 became absolutely dominant for cooked starch due to the loss of molecular order (Fig. 3), which may be associated with the transformation of double helices into single helices. Meanwhile, great reduction in the absorbance at 999 cm-1 was also noticed and a slight peak shifting from 999 to 1 002 cm-1 was observed as well after the cooking. These phenomena were found for all the three starches (Fig. 3). Furthermore, three starches showed different responses to the cooking treatment, e.g., the attitude of the reduction in the absorbance at 999 cm-1 for new wheat 2 starch after the cooking was the least among the three starches. In other words, cooking greatly reduced the absorbance at 999 cm-1 for both control and new wheat 1 starches, and the attitudes of the reduction in this band after the cooking were at a very similar level for the two starches. This IR similarity might be due to their very close molecular structures of these two starches.
Starch molecular characteristics
Detector response
The molecular profiles of the three kinds of starches are illustrated in Fig. 2. It shows that starch molecules in each starch can be separated into three fractions on the basis of their molecular weight (MW): high MW fraction (mainly represented by amylopectin), medium MW fraction (amylose with relatively higher MW), and low MW fraction (amylose with relatively lower MW). In comparison with the molecular profile of control starch, there was an increase in the amylose content in the starch of new wheat 1, but the increment did not seem to be outstanding. However, compared to that of control starch, a totally different molecular profile was obtained for the starch of new wheat 2. This sample was significantly lower in the content of amylopectin and higher in the 0.60 0.58 0.56 0.54 0.52 0.50 0.48 0.46 0.44 0.42 0.40 0.38 0.36 0.34 0.32
Control
Buffer
New wheat 1
4.55×107
4.10 ×105
4.20×105 8.56×10 Fr I Fr II 0
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15
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4.23×105 7.17×106 9.70×103
8.76 ×10
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New wheat 2
4.36×107
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0
5
10
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Fr I Fr II
Fr III 20
25
30
0
5
10
15
Fr III 20
25
30
Retention time (min)
Fig. 2 Molecular structure of the three wheat starches. Fr I, amylopectin; Fr II, amylose with relatively higher MW; Fr III, amylose with relatively lower MW.
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Molecular Characteristics of New Wheat Starch and Its Digestion Behaviours
0.4
0.3 0.2 0.1
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New wheat 1 Cooked
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Absorbance
Control Cooked Absorbance
Absorbance
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0.2 0.1
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900
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New wheat 2 Cooked
0.3 0.2 0.1
1 300 1 200 1 100 1 000 900
800
700
1 300 1 200 1 100 1 000 900
Wavenumber (cm-1)
Wavenumber (cm-1)
800
700
Wavenumber (cm-1)
Fig. 3 Effect of cooking and storage on IR spectra of the three starches. A, control. B, new wheat 1. C, new wheat 2.
Hydrolysis kinetics The digestion rate of each starch gel is presented in Fig. 5, which is the measurement of the increase in glucose concentration in the incubation system at designated intervals. The digestion progress for starch gels of control and new wheat 1 showed a biphasic pattern, i.e., a rapid rate at the initial hydrolysis stage
IR 1022/999-1045/1022 (cm-1)
1.8 1.5
Control
1.2
New wheat 1
0.9 0.6
New wheat 2
0.3 0.0
0
5
10
15
20
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Gel storage duration (h)
Fig. 4 Changes in the gel structures, expressed as the difference in the IR relative absorbance during storage at room temperature.
3.5 Glucose content (mg mL-1)
The molecular re-association process of the three starches during the storage is monitored using IR spectrum and illustrated in Fig. 4. Two bands at 1 045 and 999 cm-1 in the strained region are associated with double helices although they possibly possess different packing. Absorbance corresponding to the bands around 1 045 and 999 cm-1 is a result of the more stable packing of the chain segments, which is better than that of the 1 022 cm-1 band. Thus, the molecular reassociation process during storage could be expressed as the difference in the IR absorption between 1 022/999-1 045/1 022 cm-1 (Fig. 4).
3.0 Control New wheat 1 New wheat 2
2.5 2.0 1.5 1.0 0.5 0.0
0
20
40
60
80
100
Incubation time (min)
Fig. 5 Hydrolysis kinetics of the three starch gels by the mixture of pancreatic α-amylase and amyloglucosidase.
followed by a progressively decreased rate thereafter (Fig. 5). Both of the digestion curves exhibit a plateau during the time course of digestion. The overall digestion rate of starch gel of new wheat 2 was much lower than those of control and new wheat 1 throughout the designated course. A much lower initial velocity (2.1×10-2 ), expressed as the slope of digestion curve within the first 20 min incubation time) was found in the digestion of the starch gel of new wheat 2 compared with those of control and new wheat 1 (both for 13.0×10-2). These results indicated that the starch gel made from new wheat 2 had much slower glucose releasing kinetics due to its unique molecular structure. The digestion extent was also calculated, and the results showed that over 90% of the starch gels of control and new wheat 1 (91.7 and 91.9%, respectively) were digested after completion of the designated digestion course, whereas only 36.2% of starch gel from new wheat 2 was digested at the same time point.
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DISCUSSION The visualization in this study further confirmed that these granules are generally either small and spherical or large and ellipsoidal depending strongly on the botanical backgrounds. Much attention had recently been paid to the long-term form of storage present in the endosperm of cereals, but very little information is available concerning either the structure or the morphology of such granules. From another perspective, the irregular shapes observed in this study on the high amylose wheat starch variety (i.e., new wheat 2) further emphasized the importance of amylopectin as a parameter of granule morphogenesis during starch biosynthesis (Stinard et al. 1993). The difference in HPLC chromatograms between control starch and new wheat 1 starch was minor, indicating that there were no significant changes in the molecular profile, in particular in the amylose content by knocking out of SBE IIb during the starch biosynthesis. However, the significant difference in the molecular profiles between control starch and new wheat 2 indicated that the changes in molecular structure of starch could be achieved by mutations, in particular by knocking out of SBE IIa during the starch biosynthesis. The characteristics of starch molecular structure would subsequently influence its physicochemical properties, such as gelatinization and retrogradation during cooking process. In order to further understand how starch molecular re-association behaviour is manipulated by its molecular structure, starch was cooked in excess water and stored at room temperature to ensure it is gelatinized and retrograded. It is interesting to note that the starch gel of new wheat 2 showed the fastest reduction in this value during the whole period of gel storage, which indicates that the molecules in this starch gel are more likely to re-associate to form an organized conformation. It has been reported that the lower the value is, the faster the molecular reassociations process occurs and the recovering level to its previous conformation is a molecularly dependent phenomenon (Björck et al. 1994; Bernazzani et al. 2001; Gaudier et al. 2004). The FTIR results
ZHOU Zhong-kai et al.
further proved that molecular re-associations (i.e., retrogradation) of the starch are a multi-stage process with differing in the rate among the starches. It is interesting to note that much faster molecular reassociation process was found for the starch gel of new wheat 2 through all the designated storage intervals compared to those of control and new wheat 1. Waigh et al. (2000) has postulated that two stages are involved during starch gelatinization in excess water. The first stage involves a slow side by side dissociation of helices and the second stage involves a rapid helix coil transition. Fechner et al. (2005) also suggested that starch with rich-amylose had shown all stages of retrogradation (a fast retrogradation stage and a slow retrogradation stage along the storage) using a Raman spectroscopy. These significant differences in the digestion rate and extent among the three starch gels may be caused by their gel structures. As revealed by IR spectra in this study, the chain segments of starch molecules of cooked new wheat 2 packed in more stable conformation than those of the cooked control and new wheat 1. These different packing morphologies would subsequently influence starch digestion behaviours. Fechner et al. (2005) also applied TEM to visualize how molecules affect their packing properties. They found that configuration of amylopectin hinders the long-scale rearrangement of the crystallites, although it still can form parallel double helices by the associations of its short side branches after a long term storage. Consequently, the starch containing a higher proportion of amylopectin configuration would have a high sensitivity to the amylolysis. However, semicrystalline structure formed by the associations of amylose molecules by parallel double helices demonstrated fractal-like networks, and these networks substantially condensed, yielding thick aggregates (Putaux et al. 2000). Thus, starch with such a structure would show a high resistance to the amylolysis. From the molecularly structural point of view, the starch of new wheat 2 seems to prefer forming a condensed aggregate with a highly organized structure due to its molecular characterizations, i.e., a much higher in the content/proportion of amylose, and a lower MW of amylopectin.
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
Molecular Characteristics of New Wheat Starch and Its Digestion Behaviours
CONCLUSION Starch molecular structure manipulates starch morphology, cooking propertiess (e.g., gel structure) and its subsequent digestion behaviours. Among the three tested starches in this study, control and new wheat 1 showed a very similar molecular profile, subsequently leading to a similarity in the granular morphology, starch gel structure, and even digestion properties. The starch of new wheat 2, characterized by a higher in amylose content and lower in the MW of amylopectin, showed totally different starch properties in the above described parameters. Thus, the manipulation of starch molecular structure could control starch digestion behaviours, which would benefit the development of new kinds of starches containing a higher amount of SDS and/or RS.
MATERIALS AND METHODS Enzymes and chemicals Alpha-amylase (type VI-B, from porcine pancreas) was purchased from Sigma Chemical Co. (St Louis, MO, USA), and amyloglucosidase (from A. niger, high purity) purchased from Megazyme Australia Pty Ltd. (Victoria, Australia). Chemicals and solvents were of ACS-certified grade.
Starches Three wheat starches, control, new wheat 1 and new wheat 2 with different genetic backgrounds obtained from Tianjin University of Science & Technology, China, were used in this study. Their pedigrees are Italian Funo and the derived lines/hybridizations. The control starch was isolated from the parent. Starch of new wheat 1 was developed by knocking out SBE IIb, and starch of new wheat 2 was developed by knocking out SBE IIa.
FTIR Starch cooking and gel storage 1 mL of reverse osmosis (RO) water was added to 60 mg of starches in separate 25-mL test tubes. Each tube was capped and placed in a boiling water bath for 10 min treatment and then cooled to room temperature before FTIR measurements. The changes in the gel structure were determined using a FTIR at designated time intervals. After each measurement, the tube
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was capped and stored at room temperature until the next measurement. FTIR measurements The FTIR spectra were recorded on a Varian spectrometer (Model: Excalibur 3100, Varian, Inc., USA) equipped with a cooled deuterated triglycine sulphate (DTGS) detector. The measurement was performed on a MIRacle TM attenuated total reflectance (ATR) crystal plate with Digital Readout High Pressure Clamp (Pike Technologies, USA). The starch precipitate was directly loaded on the plate and scanned in the range of 3 600-600 cm-1 at a resolution of 4 cm-1. Prior to recording, the spectra were transformed against an empty cell as background. Finally the spectra were ATR deconvoluted using Varian Resolutions Pro software. A half-bandwith of 15 cm-1 and a K factor of 1.5 with triangular apodization were applied. The IR absorbance values ranging 1 200 and 800 cm-1 for each starch gel at different storage intervals of 0, 2, 10, 20 h were obtained via each scanning.
Starch molecular characterization by SEHPLC An 8-mL aliquot of aqueous dimethylsulfoxide (90:10, DMSO/water, v/v) was added to 20 mg of starches in separate 25-mL test tubes. Each tube was capped and placed in a boiling water bath for 60 min and then cooled to room temperature. The mixture was centrifuged at 2 095×g for 10 min. The supernatant was aspirated into a separate 25-mL test tube and the dissolved starch was precipitated by adding 18 mL of 95% ethanol. The tube was kept at 4°C overnight and then centrifuged (2 095×g for 15 min). The starch precipitate was collected for subsequent analyses by SE-HPLC. The starch precipitate (around 20 mg), prepared as described earlier, was redissolved in 0.5 mL of 0.2 mol L-1 NaOH and mixed vigorously for approximately 5 s. The solution was neutralized by addition of sodium acetate buffer (0.5 mL; 0.05 mol L-1, pH 4.0) before adding ionexchange resin (0.20 g, BioRad AG ® 501-X8, Hercules, USA) and incubating at 50°C for 1.5 h with occasional shaking. After centrifuging at 10 000 r min -1 for 10 min, the clear supernatants were collected for SE-HPLC analysis. The HPLC system comprised a GBC pump (LC 1150, GBC Instruments, Vic, Australia) equipped with auto sampler (GBC, LC1610) and evaporative light scattering detector (ELSD) (ALLTech, Deerfield, USA). The UltrahydrogelTM 1000 column, Ultrahydrogel TM 250 column and guard column (7.8 mm×300 mm, Waters, Japan) were used and maintained at 35°C during HPLC operation. Ammonium acetate buffer (0.05 mol L-1; pH 5.2) was used as mobile phase at a flow rate of 0.8 mL min-1. A 50-mL aliquot of the supernatant was used for injection. Conditions for ELSD operation were: tube temperature, 115°C; N2 gas flow rate, 2.0 L min-1; gain, 16; impactor, on. Dextrans of various MW were used as standards for measuring the MW of amylose
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and amylopectin.
Starch gel digestion in vitro with pancreatin α-amylase and amyloglucosidase Starch gel preparation A 10-mL aliquot of aqueous dimethylsulfoxide (90:10, DMSO/water, v/v) was added to 50 mg of starches in separate 25-mL test tubes. Each tube was capped and placed in a boiling water bath for 60 min and then cooled to room temperature before centrifuging at 2 095×g for 10 min. The supernatant was collected into a separate 75-mL polypropylene centrifuge tube and the dissolved starch was precipitated by adding 30 mL of 95% ethanol. The tube was then centrifuged again (2 095×g for 10 min). The supernatant was aspirated to waste and the starch precipitate was re-dissolved in 20 mL of RO water by vortexing for 1 min. The starch solution was then stored at 4°C for 7 d to ensure the re-association of starch molecules. After the 7 d storage, the whole starch was recovered by the EtOH again, and the precipitate was freeze dried. 10 mL of pH 5.2 NaAc buffer (0.1 mol L-1) was added to 31.5 mg of the freeze dried starches in separate 25-mL test tubes, and vortexed for 2 min. The starch suspension was kept at 4°C for 2 d before digestion. Starch gel digestion Prior to the digestion, 0.44 mL of CaCl2 (0.1 mol L-1) was added into the starch suspension. The starch suspension was equilibrated at 37°C for 1 h with magnetic stirring, and then 0.15 mL of enzyme solution containing 2.3 U of porcine pancreatin α-amylase and 24 U of amyloglucosidase was added. Enzymatic digestion was carried out at 37°C with magnetic stirring, and 0.3 mL aliquots of hydrolysed solution were withdrawn at different time intervals. The aliquots were immediately put in a boiling water bath for 5 min to deactivate the enzymes. The glucose content in each hydrolyzate was determined using the Megazyme Glucose Assay Kit (GOPOD method). Each sample was analysed in duplicates.
Statistical analyses Values were expressed as means±SE for three starches. Digestion rate of each starch sample is the measurement of the increase in glucose concentration in the system at each designated time. Digestion extent of each starch gel was calculated as the percentage of digested starch in the total starch at the designated incubation intervals. The amount of released glucose from the digested starch was measured using Megazyme Glucose Assay Kit (GOPOD method), and the amount of digested starch was calculated after conversion of released glucose into starch by use of factor 0.9. Initial velocity of amylases hydrolysis of the starch gels was expressed as the slope of digestion curve within the first 20 min incubation time. Each sample was analysed at least in duplicates. Experimental data were subjected to analysis
of variance using Genstat 5 (Ver. 4.1, 1998, Clarendon, NY). Treatments were tested separately for least significant difference (LSD) at a 5% level of probability.
Acknowledgements This work was supported by the China-Euro Collaboration Funds from the Minister of Science and Technology of China (SQ2013ZOA100001).
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Molecular Characteristics of New Wheat Starch and Its Digestion Behaviours
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