Structural and physicochemical characterisation of rye starch

Carbohydrate Research 346 (2011) 2727–2735

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Structural and physicochemical characterisation of rye starch S.V. Gomand ⇑, T. Verwimp  , H. Goesaert, J.A. Delcour Laboratory of Food Chemistry and Biochemistry, Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium

a r t i c l e

i n f o

Article history: Received 23 May 2011 Received in revised form 20 September 2011 Accepted 22 September 2011 Available online 29 September 2011 Keywords: Rye starch Wheat starch Amylose/amylopectin fractionation Amylopectin structure Gelatinisation Pasting

a b s t r a c t The gelatinisation, pasting and retrogradation properties of three rye starches isolated using a proteinasebased procedure were investigated and compared to those of wheat starch isolated in a comparable way. On an average, the rye starch granules were larger than those of wheat starch. The former had very comparable gelatinisation temperatures and enthalpies, but slightly lower gelatinisation temperatures than wheat starch. Under standardised conditions, they retrograded to a lesser extent than wheat starch. The lower gelatinisation temperatures and tendencies of the rye starches to retrograde originated probably from their higher levels of short amylopectin (AP) chains [degree of polymerisation (DP) 6–12] and their lower levels of longer chains (DP 13–24) than observed for wheat starch. The rapid visco analysis differences in peak and end viscosities between the rye starches as well as between rye and wheat starches were at least partly attributable to differences in the levels of AP short chains and in average amylose molecular weight. The AP average chain lengths and exterior chain lengths were slightly lower for rye starches, while the interior chain lengths were slightly higher than those for wheat starch. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Rye products have a high nutritional value, especially when they contain wholemeal rye flour because of their considerable levels of dietary fibre, vitamins, minerals and phytoestrogens.1 Starch is the major rye reserve carbohydrate, but only limited information is available on either its physicochemical properties or its structural characteristics. Rye starch can be isolated from rye flour by alkaline extraction or Pronase-based procedures.2 Wheat, maize, potato, cassava and rice starches are industrially isolated from their parent cereals and function as thickening, stabilising or filling agent. Their composition and physicochemical properties are

Abbreviations: AP, amylopectin; AM, amylose; CL, average chain length; BV, blue value; Tc, conclusion temperature; Tc,retro, conclusion temperature of retrograded amylopectin; DP, degree of polymerisation; dm, dry matter; DSC, differential scanning calorimetry; DHretro, enthalpy of retrograded amylopectin; ECL, exterior chain length; HPAEC, high-performance anion-exchange chromatography; HPSEC, high-performance size-exclusion chromatography; DH, gelatinisation enthalpy; ICL, interior chain length; IM, intermediate material; kmax, iodine binding wavelength maximum; BL, long B chain residues; DHaml, melting enthalpy amylose–lipid complexes; MW, molecular weight; To, onset temperature; To,retro, onset temperature of retrograded amylopectin; Tp, peak temperature; Tp,retro, peak temperature of retrograded amylopectin; PAD, pulsed amperometric detection; rt, room temperature; RVA, Rapid Visco Analyser; BS, short B chain residues; SEC, sizeexclusion chromatography; WAXD, wide angle X-ray diffraction. ⇑ Corresponding author. Tel.: +32 16 321582; fax: +32 16 321997. E-mail address: [email protected] (S.V. Gomand).   Present address: Natra Allcrump, Nijverheidsstraat 13, B-2390 Malle, Belgium. 0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2011.09.024

therefore well documented. In contrast, relatively little has been published on the isolation and properties of rye starch. Starch consists of a mixture of amylose (AM) and amylopectin (AP). Typical levels of AM and AP in cereal starches are 22–28% and 72–78%, respectively, although, for starches of some botanical sources, also high AM (up to 70% AM) and waxy (<1% AM) genotypes exist.3 Some minor components commonly associated with rye starch include lipids (0.4–1.7%), proteins (0.1–1.3%),4 phosphorus (0.03%) and other minerals (0.4%).5 AM is an essentially linear polymer consisting of a-D-glucopyranosyl residues linked by a-(1?4)-bonds with few (<1%) a-(1?6)-bonds.6 It has an average degree of polymerisation (DP) in the range of 500–6000.3 AM can form helical inclusion complexes with lipids and has a reported molecular weight (MW) of about 2.2  105.5 AP is a highly branched polymer consisting of a-D-glucopyranosyl residues, linked by a-(1?4)-linkages with 5–6% a-(1?6)-linkages.3 It has a DP ranging from 3  105 to 3  106 (Ref. 7) and a MW of 1  107 to 1  109.3 Several authors reported the presence of molecules intermediate between AM and AP in starches from botanical origins including wheat,8 potato,9 wrinkled pea,10 rice,11 high AM maize12,13 and oat.14 In rye starch, intermediate material (IM) also occurs.8,15 Several methods have been used to fractionate starch into AM, AP and, in some cases, IM. Two main methods currently used are aqueous leaching and selective precipitation. In the first method, AM can be selectively leached from starch granules during heating in aqueous media slightly above the gelatinisation temperature.16,17 At higher temperatures, in addition to AM, AP is extracted

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and an additional purification step is required. Pretreatment of starch with hot aqueous n-butanol before aqueous leaching makes AP less soluble and increases AM yields.16,17 A second long and tedious procedure gives more representative fractions. In this method, the starch granules are first completely dispersed in hot water or aqueous dimethyl sulfoxide. AM is then precipitated with a polar organic compound (such as thymol or n-butanol), which forms an insoluble complex with AM. AP can be recovered from the supernatant by freeze drying or by precipitation with alcohol. AM is then further purified by repeated recrystallisation from nbutanol–saturated water. The remaining supernatant may contain IM, which can be recovered by precipitation with alcohol.12,14 Another approach to fractionate starch is the use of concanavalin A, a protein that binds to the non-reducing ends in the AP molecules which hence precipitate. The concanavalin A–AP complex is then destroyed by hydrolysis with a protease. The AM binds very little concanavalin A and remains in solution.18 A PronaseÒ-based isolation procedure has been successful for isolating starch from rye flour in high yields.2 However, as is the case for its physicochemical properties, to date, only limited information is available on its structural features. Knowledge of the structure of starch is important for explaining differences in physical behaviour of the starch and hence was an objective of the present study. In addition, we evaluated differences in physicochemical and structural properties of starch of rye from different origins. To this end, starch was isolated from three rye samples of different origin by the above-mentioned PronaseÒ-based isolation procedure. The physicochemical properties of the rye starches were examined and compared with those of a wheat starch. The starches were then fractionated into AM, IM and AP. Structural features of the rye AM and AP were compared with those of the corresponding wheat polymers. This setup provided information on the relationship between the structural aspects and the physicochemical properties of rye starch. 2. Materials and methods 2.1. Materials Three rye samples of different cultivars (Avanti, Nikita and Treviso) were provided by Prof. Dr. W. Bergthaller (Institute for Cereal, Potato and Starch Technology, Detmold, Germany). Wheat (variety Zohra) was obtained from AVEVE (Landen, Belgium). Flour was produced by milling rye or wheat at a moisture level of 15% or 16%, respectively, with a Chopin (Villeneuve-la-Garenne, France) laboratory mill CD1. Extraction rates were 52%, 55%, 52% and 63% for Avanti, Nikita, Treviso and Zohra, respectively. Pronase E (Streptomyces griseus) and isoamylase (Pseudomonas amyloderamosa) were purchased from Sigma–Aldrich (Bornem, Belgium). Barley b-amylase and pullulanase were obtained from Megazyme (Bray, Ireland). All reagents, chemicals and enzymes used were of at least analytical grade and obtained from Sigma–Aldrich unless indicated otherwise. Where enzymes were used, their units (U) were as defined by the respective suppliers. 2.2. Starch isolation procedure The isolation procedure was based on the method described by Morrison et al.19 and Verwimp et al.2 It was performed without prior defatting. Rye or wheat flour was suspended in 0.1 M Tris– HCl buffer [pH 7.6, containing 0.5% (w/v) NaHSO3, 0.02% NaN3 and 0.01 M disodium EDTA] (1:10 w/v) and stirred for 1 h at 6 °C. Pronase E (0.5 mg/g original flour) was added, and the suspension was digested for 44 h at 6 °C under constant stirring. The digested material was passed through a 38-lm sieve with additional

deionized water until no more starch was extracted. The combined washings were centrifuged (1000g, 15 min, 6 °C). The crude starch obtained was suspended in water and centrifuged (1000g, 15 min, 6 °C). A proteinaceous layer was scraped off from the starch surface and discarded. This step was repeated until the starch was visually clean. It was then air-dried overnight and gently passed through a 250-lm sieve using a mortar and pestle. Yields of starch are expressed as percentage of the total starch content of flour.2 2.3. Starch characterisation Moisture and ash contents were determined based on AACC Methods 44-19 and 08-01,20 respectively. Protein contents were determined using the Dumas method, an adaptation of the AOAC Official Method21 to an automated Dumas protein analysis system (EAS varioMax N/CN, Elt, Gouda, The Netherlands). Total lipid content was determined based on Morrison et al.22 Monosaccharide compositions were determined by gas chromatography of the alditol acetates obtained after acid hydrolysis (120 min at 110 °C).23 Total starch content was determined using a total starch test kit (Megazyme, Bray, Ireland). AM content was determined by the method of Chrastil,24 using a commercial wheat starch (Meriwit I, Amylum, Aalst, Belgium) with known AM content (26.0%) for calibration. The AM contents are expressed as percentages of total starch contents. Granule size distributions, wide-angle X-ray diffraction (WAXD) and viscosity behaviour of the rye and wheat starches were studied using the methods in Verwimp et al.2 To calculate the relative crystallinity, Nikita served as reference starch because it had the highest crystallinity. Its relative crystallinity was taken as 100%. 2.4. Starch gelatinisation and retrogradation Differential scanning calorimetry (DSC) was carried out with a Q1000 DSC (TA Instruments, New Castle, DE, USA). Starch (4.00– 6.00 mg) was accurately weighed in an aluminum pan and water was added [1:2 w/w starch dm:water (dm: dry matter)]. The pans were hermetically sealed and equilibrated for at least 20 min. They were heated from 20 °C to 120 °C at 4 °C/min and an empty pan was used as reference. Calibration was with indium. The onset (To), peak (Tp) and conclusion temperatures (Tc), gelatinisation enthalpies (DH) and AM–lipid complex enthalpies (DHaml) were determined using TA Universal Analysis software. Temperature was expressed in °C and enthalpy in J/g. The gelatinisation temperature range was calculated as Tc To. The gelatinised samples were stored for 14 days at 20 °C, followed by a second heating to determine the level of starch retrogradation. Heating of the stored samples was done under the experimental conditions also used during the first heating. Analyses were performed at least in triplicate. The onset (To,retro), peak (Tp,retro) and conclusion (Tc,retro) temperatures, and the enthalpy (DHretro) of retrograded AP were determined under the same conditions as those of starch gelatinisation. 2.5. Rapid viscosity analysis Swelling and pasting behaviour were investigated with a Rapid Visco Analyser (RVA-4) (Newport Scientific, Sydney, Australia). Starch suspensions (10% dm, total weight 25.0 g) were subjected to the following time–temperature profile: equilibration for 2 min at 50 °C, a linear temperature increase to 95 °C in 9 min, holding at 95 °C for 15 min, a cooling step (9 min) with a linear temperature decrease to 50 °C and a final isothermal step at 50 °C (10 min). The peak viscosity (the maximum viscosity during pasting), breakdown (the difference between the peak viscosity and the viscosity at the end of the holding phase), setback (the difference between the viscosity at the end of cooling and the

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viscosity at the end of the holding phase) and final viscosity (the viscosity at the end of the RVA run) [all expressed in centiPoise (cP)] of 10.0% dm suspensions were determined. The pasting temperature (°C), that is, the temperature at which the derivative [d (viscosity) / d (time in min)] increased, was also determined. Viscosity measurements were carried out in duplicate and the difference between two measurements was less than 31 cP along the whole temperature profile of the analysis.

mixture was brought to 100 mL with deionized water and mixed immediately. Control solutions were made in the same way but without sample. All samples were scanned from 500 to 800 nm with an Ultraspec III UV–visible spectrophotometer (Amersham Pharmacia Biotech, Uppsala, Sweden). The BV was the extinction at 635 nm under the above experimental conditions. The kmax was the wavelength (in nm) at which the extinction was the highest over the range of the wavelengths.

2.6. Starch fractionation

2.8. Size-exclusion chromatography of non-granular starches and starch fractions

2.6.1. Preparation of non-granular starch Non-granular rye and wheat starches were prepared according to Klucinec and Thompson.12 Granular starch (10.0 g, dry weight) was dispersed in 200 mL 90% (v/v) DMSO by heating the mixture in boiling water for 180 min with frequent hand shaking. Following dispersion, 280 mL 95% EtOH was added, and the mixture was centrifuged (6500g, 15 min, 4 °C). The supernatant was discarded, and the pellet was washed by suspending it in 50 mL 95% EtOH, followed by centrifugation (6500g, 15 min, 4 °C). The washing procedure was repeated once. The precipitate was then dried at 50 °C for 24 h. 2.6.2. Starch fractionation Non-granular rye and wheat starches were fractionated based on the method of Klucinec and Thompson12 with slight modifications. An aliquot (6.0 g) was dispersed in 168 mL of 90% (v/v) DMSO. A solution containing 6.0% of both n-BuOH and isoamyl alcohol (1176 mL) was added. The mixture was stirred, incubated at 95 °C for 60 min and cooled to room temperature (RT) over a period of 18 h. After cooling, the mixtures were resuspended by shaking, and then they were centrifuged (10,000g, 15 min, 4 °C). The supernatant (AP I) was saved, and the residue was redispersed in a solution containing 5.2% of both n-BuOH and isoamyl alcohol (300 mL). This mixture was heated, cooled and centrifuged as above. The supernatant (AP II) was combined with the first one. The residue was redispersed and centrifuged in the same way. The supernatant (AP III) was combined with the first two supernatants. The residue was redispersed in 168 mL 90% (v/v) DMSO and 1176 mL of 6.0% n-BuOH was added. The mixture was heated, cooled and centrifuged as described above. The supernatant is referred to as the IM fraction. The residue (AM) was redispersed in 120 mL 90% (v/v) DMSO and AM was precipitated with 280 mL of 95% EtOH. After centrifugation (10,000g, 15 min, 4 °C), the AM pellet was washed with 50 mL of 95% EtOH and again centrifuged (6500g, 15 min, 4 °C). The AM was suspended in deionized water and lyophilized. The combined AP containing supernatants and the IM containing supernatant were each concentrated by rotary evaporation to ca. 300 mL. AP and IM were then precipitated with 350 mL and 250 mL of 95% EtOH, respectively. After centrifugation (10,000g, 15 min, 4 °C), the pellets were washed with 150 mL of 95% EtOH followed by centrifugation (6500g, 15 min, 4 °C). The AP and IM residues were suspended in deionized water and lyophilized. 2.7. Blue value and iodine binding wavelength maximum of non-granular starches and starch fractions The blue value (BV) and iodine binding wavelength maximum (kmax) of the non-granular rye and wheat starches and of their AM, IM and AP fractions were determined in triplicate according to the method of Klucinec and Thompson.12 A sample (40 mg) was dispersed in 10.0 mL of DMSO containing 10% 6.0 M urea. An aliquot (1.0 mL) was then placed in a 100-mL volumetric flask along with 95 mL of deionized water and 2.0 mL of an aqueous I2–KI solution (2.0 mg I2/mL and 20.0 mg KI/mL) was added. The

Size-exclusion chromatography (SEC) of non-granular rye and wheat starches and of their AM, IM and AP fractions was performed as described by Klucinec and Thompson12 with modifications. A sample (15 mg) was dispersed in 1.0 mL of concentrated mobile phase (1.0 M KOH) for 8 h under mild magnetic stirring and then diluted to 10.0 mL with deionized water. After filtration (0.45 lm), 5.0 mL of the filtrate was injected. Fractionation was with a Sepharose CL-2B column (74 cm  1.6 cm, Amersham Pharmacia Biotech) in an ascending mode (flow rate 0.5 mL/min) using degassed 0.1 M KOH. Detection was with a differential refractive index detector (RID-10A, Shimadzu, Kyoto, Japan). Fractions (5.0 mL) were collected and their iodine binding was examined. To this end, an aliquot (1.0 mL) of each SEC fraction was neutralized with 25 lL of 1.0 M HCl and mixed with 5.0 mL of iodine solution (0.38 mg of I2/mL and 0.90 mg of KI/mL). The extinction was measured at 620 nm. The column was calibrated with Shodex pullulan standards (Showa Denko, Tokyo, Japan) with peak MWs of 160.0  104, 78.8  104, 40.4  104, 21.2  104, 11.2  104, 4.73  104, 2.28  104 and 1.18  104 and corresponding to approximate DPs of 9875, 4875, 2500, 1300, 700, 300, 150 and 75. 2.9. Preparation of debranched amylopectins Debranching of rye and wheat AP was performed by the method of Umemoto and co-workers25 with slight modifications. AP (10.0 mg) was suspended in 3.0 mL of 100% MeOH in a screw cap tube and incubated in boiling water for 10 min. After centrifugation (1000g, 10 min, 18 °C), the residue was washed twice with 5.0 mL of deionized water with intermediate and final centrifugation steps as above. The sample was resuspended in 5.0 mL of deionized water and incubated in boiling water for 60 min. An aliquot (1.0 mL) of the gelatinized sample was added to 10 lL of 2.0% (w/v) sodium azide and hydrolysed with Pseudomonas amyloderamosa isoamylase (1250 U added in 50 lL of 600 mM NaOAc buffer pH 4.6) at 37 °C for 24 h. The enzyme was inactivated by heating (100 °C, 10 min). Next, the sample was incubated again with P. amyloderamosa isoamylase (625 U/50 lL of 600 mM NaOAc buffer pH 4.6) at 37 °C for 24 h. Finally, the enzyme was inactivated by heating (100 °C, 10 min), and the sample was cooled to RT. 2.10. Amylopectin average chain length and chain length distribution The average chain length (CL) of the isoamylase debranched rye and wheat amylopectin AP was determined by calculating the ratio of the total carbohydrate concentration to the concentration of reducing ends. Total carbohydrate level and content of reducing ends were analysed by the phenol–sulfuric acid method26 and the method described by Somogyi,27 respectively. The branch chain length distributions of the rye and wheat AP were determined by high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). HPAEC–PAD was performed with an ED-40 electrochemical

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detector (Dionex, Sunnyvale, CA, USA), a P-4000 gradient pump (Thermo Electron Corporation, Waltham, MA, USA) and an AS3000 autosampler (Thermo Electron Corporation). Samples were prepared by adding 0.25 mL of 1.5 M NaOH to the isoamylase debranched AP. The solution was vortexed, filtered (0.25 lm) and injected (20 lL) onto a CarboPac PA-100 anion-exchange column (250 mm  4 mm) in combination with a CarboPac PA-100 guard column (Dionex). The pulse potentials and durations, the eluents and the eluent gradient programme were as described by Jacobs et al.28 Individual peaks in the chromatograms were corrected for molar PAD detector responses.29 The relative amounts of AP chains were normalized and divided into four fractions (F1 with DP 6–12, F2 with DP 13–24, F3 with DP 25–36 and F4 with DP >36) according to Hanashiro and co-workers.30 2.11. Preparation of debranched b-limit dextrins of amylopectins The b-limit dextrins of rye and wheat AP were prepared using the method by Klucinec and Thompson31 with slight modifications. AP (12.0 mg) was dispersed in 120 lL of 90% (v/v) DMSO by heating in boiling water for 10 min. Warm NaOAc buffer (50 °C, 880 lL, 0.02 M, pH 6.0) was added. The mixture was mixed, incubated in boiling water for 10 min and cooled to 50 °C. Barley b-amylase (50 l lL, 250 U/mL 0.02 M NaOAc buffer, pH 6.0) was added, and the sample incubated at 50 °C for 48 h with constant agitation. The sample was then incubated in boiling water for 10 min and returned to a water bath at 50 °C, at which time an additional 50 lL of the above b-amylase buffered solution was added. The sample was incubated at 50 °C for 22 h with constant agitation. It was then heated in boiling water for 10 min and cooled to RT. An aliquot (0.2 mL) was saved for b-amylolysis limit determination based on the total carbohydrate26 and the reducing power27 of the solutions [b-amylolysis limit (%) = 100  reducing maltose/total glucose expressed as maltose]. A second aliquot (0.5 mL) was mixed with 1.5 mL of 95% EtOH, held overnight at 4 °C and subsequently centrifuged (250g, 10 min, 20 °C). The precipitated b-limit dextrin was washed twice with 0.5 mL of 95% EtOH and once with 0.5 mL acetone. Centrifugation after each washing step was at 1000g (10 min, 20 °C). The recovered b-limit dextrin was air-dried. The b-limit dextrin (3.0 mg) was mixed with 50 lL of 90% (v/v) DMSO and heated in boiling water for 10 min. Warm NaOAc buffer (50 °C, 350 lL, 0.02 M, pH 4.75) was added. The mixture was mixed, incubated in boiling water for 10 min and cooled to 37 °C. Isoamylase solution (40 lL, 25 U/mL in 0.02 M NaOAc buffer, pH 4.75) was added and the sample was incubated at 37 °C for 24 h with constant agitation. It was then incubated in boiling water for 10 min and returned to a water bath at 37 °C. To this solution of partially debranched b-limit dextrin, pullulanase (40 lL, 0.43 U/mL in 0.02 M NaOAc buffer, pH 4.75) was added. The sample was incubated at 37 °C for 24 h with constant agitation and then heated in boiling water for 10 min, cooled to RT and centrifuged (5000g, 10 min, 20 °C). An aliquot (0.1 mL) of each completely debranched b-limit dextrin was added to 200 lL of 100% DMSO and saved for analysis by high-performance size-exclusion chromatography (HPSEC). 2.12. High-performance size-exclusion chromatography of debranched b-limit dextrins of amylopectins HPSEC of debranched b-limit dextrins of rye and wheat AP was performed based on the procedure of Klucinec and Thompson.31 The HPSEC system consisted of a pump (LC-20AD/LC-20AT, Shimadzu) connected in series to an injector (SIL-20A autoinjector, Shimadzu, injected volumes 50 lL) and a differential refractive index detector (RID-10A, Shimadzu) which was operated at 40 °C.

Two 30-cm columns packed with 6-lm porous silica microspheres (Zorbax PSM 60S, Agilent Technologies, Wilmington, DE, USA) connected in series were used for the separations. They were maintained at 50 °C using a column oven (CTO-20AC, Shimadzu). The mobile phase (flow rate 0.5 mL/min) was an on-line degassed (DGU-20 A5, Shimadzu) 90% (v/v) DMSO. Detector responses and the resultant chromatograms were analyzed using LC Solution software (Version 1.21, Shimadzu). The system was calibrated using separate 50 lL injections of 8 separate solutions: glucose (MW = 180, DP 1), maltose (MW = 342, DP 2), maltopentaose (MW = 829, DP 5), maltoheptaose (MW = 1153, DP 7) and 4 Shodex pullulan standards (Showa Denko) with MWs of 11.2  104, 4.73  104, 1.18  104 and 0.59  104 and corresponding DPs of ca. 700, 300, 75 and 36. In order to obtain molar-based chromatograms, the differential refractive index response at each time was divided by the MW of the carbohydrate eluting at that time. The chromatograms of the debranched b-limit dextrins were divided into seven regions (I– VII) based on the retention times of the minima (between regions I–II, II–III, III–IV, V–VI and VI–VII) and the inflection (between IV– V) observed for rye cultivar Avanti. Regions I, II and III were considered to represent long B chain residues (BL), regions IV and V short B chain residues (BS) and regions VI and VII residues of A chains. DP areas of the different regions were: I (DP 400–2650), II (DP 50– 400), III (DP 22–50), IV (DP 7–22), V (DP 4–7), VI (DP 3) and VII (DP 2). The exterior chain length [ECL = CL  (b-amylolysis limit/ 100) + 2] and interior chain length (ICL = CL ECL 1) of AP were also calculated according to Klucinec and Thompson.31 2.13. Statistical analyses Pearson’s correlation coefficient analyses were performed (significance level P <0.05). Statistical analyses were conducted using the Statistical Analysis System software 8.1 (SAS Institute, Cary, NC, USA). 3. Results and discussion 3.1. Starch yield and chemical composition Table 1 lists the yields of the starches isolated from the rye and wheat flours and their chemical composition. The starch yields obtained for the rye samples were similar and in accordance with that described by Verwimp et al.2 For wheat, the starch yield was comparable to those obtained for the rye starches (Table 1). This is in contrast to earlier findings2 where the PronaseÒ-based isolation procedure resulted in a higher starch yield for wheat (90.0%) than for rye (80.9%). The rye and wheat starches were of high purity as shown by the low ash, protein, lipid and monosaccharide contents (Table 1). The AM content of the rye starches ranged between 22.4 and 24.9%. The wheat starch contained 23.5% AM. Table 1 Yield and chemical composition (%dm) of the starches isolated from flour of the rye cultivars Avanti, Nikita and Treviso and flour of the wheat cultivar Zohra Rye starch

Yield Ash Protein Total lipids Monosaccharides Arabinose Xylose Galactose Amylose

Wheat starch

Avanti

Nikita

Treviso

Zohra

85.5 0.12 0.04 0.36

83.0 0.10 0.03 0.33

81.5 0.11 0.01 0.34

82.4 0.13 0.12 0.43

0.07 0.06 0.02 22.4

0.06 0.04 0.02 24.2

0.06 0.04 0.02 24.9

0.05 0.03 0.02 23.5

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3.2. Granule size distribution All starches showed a bimodal particle size distribution (Fig. 1). The volume percentage of B-granules was 26%, 21% and 17% for rye cultivars Avanti, Nikita and Treviso, respectively. These figures are higher than the volume percentage of B-granules (14%) found for rye starches.2 Differences in starch granule size distribution between rye cultivars were also reported by Stoddard,32 who examined 50 rye cultivars and found B-granule content ranges between 21 and 39%. The wheat starch contained 20% B-granules, in line with the value for PronaseÒ-isolated wheat starch described in Verwimp et al.2 The A-type granules of the rye starches had an average particle size of 30–31 lm, while those of the wheat starch had an average particle size of 26 lm (Fig. 1). These results are in line with average particle sizes found for PronaseÒ-isolated rye and wheat starches described previously.2

Table 2 Average gelatinisation onset (To), peak (Tp) and conclusion (Tc) temperatures, gelatinisation intervals (Tc–To), gelatinisation enthalpy changes (DH), enthalpy changes of the melting of amylose–lipid complexes (DHaml), average onset (To,retro), peak (Tp,retro), conclusion (Tc,retro) temperatures of retrograded amylopectin and enthalpy changes of retrograded amylopectin (DHretro) of the starches isolated from rye and wheat flour as measured by DSCa Rye starch

To (°C) Tp (°C) Tc (°C) Tc–To (°C) DH (J/g) DHaml (J/g) To,retro (°C) Tp,retro (°C) Tc,retro (°C) DHretro (J/g)

Wheat starch

Avanti

Nikita

Treviso

Zohra

49.0 53.5 63.5 14.5 13.8 0.9 45.7 56.3 67.7 2.4

48.6 53.4 64.1 15.6 14.2 0.7 45.3 56.4 68.4 2.3

48.7 52.8 62.9 14.2 13.0 0.6 45.7 56.6 67.7 2.4

50.8 55.9 65.8 15.0 13.4 1.1 45.7 56.6 68.3 3.8

(0.8) (0.7) (0.6) (0.1) (0.1) (0.4) (0.2) (0.2) (0.4)

(0.1) (0.2) (0.4) (0.2) (0.2) (0.6) (0.2) (0.8) (0.3)

(0.2) (0.0) (0.2) (0.1) (0.2) (0.5) (0.4) (0.9) (0.3)

(0.2) (0.2) (0.2) (0.0) (0.0) (0.2) (0.1) (0.1) (0.2)

a Averages of at least triplicate measurements, with standard deviations between parenthesis.

The rye and wheat starches all showed an A-type polymorph with a small proportion of B-type polymorph. Relative crystallinities (i.e., as percentage of the crystallinity of Nikita) of 92%, 100% and 93% were found for the starches from rye cultivars Avanti, Nikita and Treviso, respectively. The starch from wheat cultivar Zohra had a relative crystallinity of 88%. A lower relative crystallinity for wheat starch than for rye starch, both isolated by the PronaseÒbased isolation procedure was observed earlier.2 3.4. DSC gelatinisation and retrogradation properties Table 2 represents the DSC gelatinisation temperatures and enthalpy changes of the rye and wheat starches. The starches from the different rye samples showed similar gelatinisation temperatures. In line with earlier results2,33, the rye starches had lower gelatinisation temperatures than the wheat starch. DH did not differ much among the starches. Somewhat lower DHaml values were observed for the rye starches than for the wheat starch, which can be explained by the lower lipid contents in the rye starches (Table 1). Table 2 lists To,retro, Tp,retro, Tc,retro and DHretro following storage of starches at 20 °C for 2 weeks. No differences in these temperatures of retrograded AP were observed between the investigated starches. To,retro and Tc,retro were ca. 45.5 °C and 68.0 °C, respectively. The melting enthalpy temperature ranges of retrograded AP of rye and wheat starches are comparable to those obtained by Fredriksson et al. when gelatinised starches were stored at 6 °C and rather small when compared to those of starches of other botanical sources (barley, wheat and potato).33 A small enthalpy temperature range of retrograded AP pointed to a more uniform quality of the crystals. While no differences in To,retro, Tp,retro and

Tc,retro were observed, DHretro is noticeably higher for the wheat cultivar Zohra than for the rye starches and is in agreement with Fredriksson et al.33 3.5. RVA viscosity behaviour Figure 2 shows RVA viscograms of the starches isolated from the rye and wheat flours and Table 3 summarizes their characteristics. Under the experimental conditions, the three rye starches had a pasting temperature of approximately 72 °C and exhibited similar viscosity behaviour, except for the starch from cultivar Treviso, which had lower breakdown and higher setback and end viscosities. The wheat starch had a higher pasting temperature (±78 °C), higher peak and breakdown viscosities, a lower setback and lower end viscosities than the rye starches (Fig. 2 and Table 3). The differences in gelatinisation and pasting behaviour between the starches may well be explained by differences in structure of AM and AP, as discussed below. 3.6. Properties of the non-granular starches and their starch fractions Fractionation of the rye and wheat non-granular starches resulted in three fractions, that is, AM, IM and AP (Table 4). The IM was indeed ‘intermediate’ in the sense that it behaved neither as regular AM (it did not precipitate with n-BuOH reagent) nor as regular AP (part of the material eluted after the void volume in HPSEC,

100 90 80 70 60 50 40 30 20 10 0

6000 5000

Volume percentage (%)

Nikita Treviso Zohra

10

5

Viscosity (cP)

Avanti

15

4000 3000 2000 1000 0 0

0 0

10

20

30

40 50 60 70 Granule size (µm)

80

90

100

Figure 1. Granule size distribution of the starches isolated from rye (Avanti, Nikita, Treviso) and wheat (Zohra) flour.

5

10

15

20 25 Time (min)

30

35

40

Temperature (°C )

3.3. WAXD pattern and relative crystallinity

45

Figure 2. RVA viscograms of the starches (10% dm) isolated from rye ( , Avanti; , Nikita; , Treviso) and wheat ( , Zohra) flour ( , temperature profile).

S. V. Gomand et al. / Carbohydrate Research 346 (2011) 2727–2735

Nikita

Treviso

Zohra

71.9 2650 790 1910 4280

72.2 2880 930 1990 4430

71.9 2900 690 2330 5220

77.7 3500 1890 1750 3830

0.60

2500 2000

0.40

1500 1000

0.20

500 0

0.00 50

NG

AM

IM

AP

Yield (%) Avanti Nikita Treviso Zohra

n.a. n.a. n.a. n.a.

22.9 24.5 23.4 24.5

3.9 3.2 6.2 3.8

73.2 72.3 70.4 71.7

BV Avanti Nikita Treviso Zohra

0.385 0.390 0.375 0.370

1.155 1.170 1.105 1.125

0.705 0.680 0.625 0.565

0.070 0.060 0.080 0.080

100

150 Time (min)

200

250

0.80

AM

3000 0.60

2500 2000

0.40

1500 1000

0.20

500 0

656 669 671 654

652 655 655 646

538 523 538 538

n.a.: not applicable

cf. infra). The existence of an IM fraction in rye starch was also observed by Banks and Greenwood8 and Hew and Unrau.15 Fractionation did not reveal significant differences among the rye starches and between the rye and wheat starches as the levels of AM, IM and AP recovered were similar. Only for rye cultivar Treviso a higher proportion of IM was obtained. The yields of IM were in line with those reported earlier for rye and wheat starches.8 Table 4 presents BV and kmax. For each starch, the non-granular starting material and the three fractions had a different iodine binding behaviour. The AM fraction had higher extinctions at all wavelengths (500–800 nm) (results not shown), higher BV and higher kmax than the IM fraction, which in its turn had higher extinctions at all wavelengths (500–800 nm), higher BV and higher kmax than the AP fraction. Iodine binding of the non-granular starches and of their AM and AP fractions was nearly identical for the rye and wheat starches. In contrast, the IM of the rye and wheat starches showed different iodine binding properties. Extinctions at all wavelengths (500–800 nm) and BV of the IM decreased in the order: Avanti, Nikita, Treviso and finally Zohra. The very low extinctions at all wavelengths (500–800 nm), low BV and low kmax of the AP fractions indicated that highly pure AP fractions were obtained. Figure 3 shows the SEC profiles of the non-granular rye and wheat starches and of their fractions. For rye, to the best of our knowledge, such profiles have not been reported earlier. The chromatograms of the non-granular starches show a fraction eluting at the void volume, consisting of very high MW molecules and considered to be AP, and a broad fraction eluting after the void volume containing molecules of lower MW and considered to be AM as demonstrated by their high iodine binding capacity. The overall distribution profiles were rather similar for the rye and wheat starches.

3500 RI (arbitrary units) .

629 628 631 629

0.00 50

100

150 Time (min)

200

250

0.80

IM

3000 0.60

2500 2000

0.40

1500 1000

0.20

500 0

0.00 50

3500 RI (arbitrary units) .

kmax (nm) Avanti Nikita Treviso Zohra

3500 RI (arbitrary units) .

Table 4 Yield (%) of the starch fractions, that is, amylose (AM), intermediate material (IM) and amylopectin (AP) and their corresponding blue value (BV) and kmax together with BV and kmax of non-granular (NG) starch from rye (Avanti, Nikita, Treviso) and wheat (Zohra)

Extinction (620 nm) .

Avanti

Extinction (620 nm) .

Pasting temperature (°C) Peak viscosity (cP) Breakdown viscosity (cP) Setback viscosity (cP) End viscosity (cP)

Wheat starch

0.80

NG

3000

Extinction (620 nm) .

Rye starch

3500

100

150 Time (min)

200

250 0.80

AP

3000 0.60

2500 2000

0.40

1500 1000

0.20

500 0

Extinction (620 nm) .

Table 3 RVA characteristics of the starches (10% dm) isolated from rye and wheat flour

RI (arbitrary units) .

2732

0.00 50

100

150 Time (min)

200

250

Figure 3. Size-exclusion chromatography (SEC) (Sepharose CL-2B) elution profiles of non-granular (NG) rye and wheat starches and their starch fractions, i.e. amylose (AM), intermediate material (IM) and amylopectin (AP). Total carbohydrate ( , Avanti; , Nikita; , Treviso; , Zohra) and iodine binding ( , Avanti; , 4 Nikita; , Treviso; , Zohra). MW markers (+) from left to right are 160.0  10 , 78.8  104, 40.4  104, 21.2  104, 11.2  104, 4.73  104, 2.28  104 and 1.18  104.

The AM fractions of the different starches showed similar, broad MW distributions, but some differences in peak DP could be observed. Among the three rye samples, the AM molecules from Treviso had the highest peak DP (2400), followed by those of Nikita (2150) and Avanti (1850). The wheat starch AM fraction contained molecules with a lower peak DP (1750) than the rye starch AM fractions. The iodine-binding values of the IM eluting at the void volume were higher than those of the AP fraction, suggesting that the void

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S. V. Gomand et al. / Carbohydrate Research 346 (2011) 2727–2735

volume material in the IM contained molecules with branch chains longer than those of AP. Banks and Greenwood8 also found IM in rye and wheat starches containing AP-like molecules with somewhat longer chain lengths than those of the AP fraction. The IM eluting after the void volume appeared to contain AM-like molecules, but of higher MW than those of the AM fraction. The very low iodine binding of the AP fractions confirmed their purity and suggested the presence of short branch chains.

Table 6 Molar chain length distribution (%) of isoamylase debranched amylopectin from rye and wheat starches as determined by HPAEC-PAD Rye amylopectin

F1 F2 F3 F4

(DP 6–12) (DP13–24) (DP 25–36) (DP >36)

Wheat amylopectin

Avanti

Nikita

Treviso

Zohra

57.5 37.5 4.5 0.5

57.5 37.5 4.5 0.5

56.5 38.5 4.5 0.5

54.0 41.0 4.5 0.5

3.7. Structural aspects of amylopectin Table 5 lists the b-amylolysis limits and average CL, ECL and ICL of the AP from rye and wheat starches. The b-amylolysis limits of the AP from the three rye samples were similar to each other (ca. 48–49%) but lower than those reported by Banks and Greenwood8 (58%) and Hew and Unrau15 (57–61%). The wheat AP had a higher b-amylolysis limit than the rye AP, suggesting some structural differences between wheat and rye AP. The b-amylolysis limit of the wheat AP was in line with earlier reported values.8,15,17,34,35 The average CL, ECL and ICL of the AP from the different rye samples were nearly identical (21–22, 12–13 and 8, respectively) (Table 4). The wheat AP had a somewhat higher average CL (23) and ECL (14) and a somewhat lower ICL (7) than the rye amylopectin. The average CL of the rye and wheat AP were in good agreement with those found in earlier studies. An average CL of 20 and 20–21 for rye and wheat AP, respectively, has been reported.8 Berry and co-workers5 found values of 21 and 23 for rye and wheat AP, respectively. Lii and Lineback,36 however, found a higher average CL for rye AP (26) than for wheat AP (17–20). Table 6 shows molar percentages of groups of unit chains in rye and wheat amylopectin determined by HPAEC–PAD. AP branch chain length distributions of the three rye samples were similar and showed a peak at DP 10. The latter is at variance with findings by Silverio and co-workers,37 who studied the chain length distribution of rye AP using HPSEC and HPAEC–PAD, respectively. The former authors found chain length distributions with peak maxima at DP 11, 18 and 47, while the latter found distribution patterns which showed peaks at DP 12, 15 and 20 and a larger proportion of material with a DP of approximately 45. The wheat AP, like the rye AP, displayed a peak at DP 10. Similar chain length distribution profiles for wheat AP with a peak at DP 10–11 were reported by Koizumi and Fukuda,38 Vermeylen et al.39 and Wickramasinghe et al.40 The chain length distribution profiles of the rye and wheat AP showed some differences (Table 6). The wheat AP had a lower level of chains with DP 6–12, especially of those with DP 6–9, and a higher level of chains with DP 13–24 than rye AP. No differences were found in the distribution of chains with DP >24. While the HPAEC-PAD results yielded information on the chain length distribution of the shorter chains of AP (DP <60), information on the ICL distribution of the AP, which, to the best of our

Table 5 b-Amylolysis limit, average chain lengths (CL), exterior chain lengths (ECL) and interior chain lengths (ICL), of rye (Avanti, Nikita, Treviso) and wheat amylopectins (Zohra) and molar chain ratios of A:B and short B chains to long B chains (BS:BL) of their debranched b-limit dextrins Rye amylopectin

b-Amylolysis limit (%) CL (DP) ECL (DP) ICL (DP) A:B BS:BL

Wheat amylopectin

Avanti

Nikita

Treviso

Zohra

48.3 22 13 8 0.8 2.8

49.1 21 12 8 0.8 2.9

48.7 22 13 8 0.8 2.7

53.9 23 14 7 0.8 2.5

knowledge, has not been reported before for rye, was obtained by HPSEC of the debranched AP b-limit dextrins. Figure 4 shows the mass and molar based chromatograms of the debranched blimit dextrins of the rye and wheat starch AP. Table 5 lists weight and molar percentages of A and B (BS and BL) chains as well as the molar ratios of A:B chains and of BS:BL chains. The AP from the different rye samples has similar distributions of A, BS and BL chains. The interior chain length distribution of the wheat AP, however, was somewhat different from that of the rye AP. The wheat AP contained a larger proportion of long B chains (regions I and II) and a lower proportion of shorter B chains (region 4). The ratio of BS:BL chains was also somewhat lower for the wheat AP than for the rye AP. The A:B chain ratios determined from the chromatograms were nearly identical for all rye and wheat AP (Table 5). The A:B chain ratios (0.8) were, however, lower than previously reported. Lii and Lineback36 found an A:B chain ratio of 1.8 for rye AP and 1.2–1.7 for wheat AP. It should be noted, however, that these values were determined by debranching b-limit dextrins with isoamylase and pullulanase and determining reducing groups released by the debranching enzymes. The HPSEC method used in this study is more reliable for determination of the A:B chain ratio than for the measurement of reducing power. 3.8. Structural aspects in relation to physicochemical properties Some structural differences observed between the rye and wheat AM and AP molecules may explain the differences in gelatinisation and pasting behaviour of the rye and wheat starches. The higher gelatinisation temperatures found for the wheat starch than for the rye starches (Table 2) might be due to the lower proportion of chains with DP 6–12 and the higher proportion of chains with DP 13–24 in the wheat AP (Table 6). It was reported earlier for starches from different botanical sources that the distribution of AP unit chains with DP 6–12 and DP 13–24 correlates negatively and positively, respectively, with gelatinisation temperature.41–48 In addition, the higher level of BL chains in wheat AP (Fig. 4 and Table 5) might also contribute to the higher gelatinisation temperatures found for the wheat than for the rye starches. In this respect, Asaoka and co-workers49 reported that rice starches containing a higher proportion of long inner B-chains had higher gelatinisation temperatures. Wheat starch tended to retrograde to a larger extent than rye starches, which is probably due to its lower level of chains with DP 6–12 and higher level of chains with DP 13–24 (Table 6). Gidley and Bulpin50 described that at least 10 glucose units are needed for double helix formation of maltooligosaccharides. That short chains (DP <12) inhibit AP retrogradation while longer chains (DP 15–24) promote AP retrogradation, was already earlier described for starches of different botanical sources.48,51,52 Logically, no relations existed between structural properties and enthalpic temperatures of retrograded AP as the latter did not notably differ. The higher pasting temperature and peak and breakdown viscosities were observed for the wheat than for rye starches (Fig. 2 and Table 3) might also be explained by the lower proportion of

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S. V. Gomand et al. / Carbohydrate Research 346 (2011) 2727–2735

Avanti Relative moles .

RI (arbitrary units) .

50 40 30 20 10

I

0 10

12

II

III

14

IV V

16

VIVII

18

20

22

wheat AM molecules (Fig. 3) might explain the differences in setback and end viscosities of the rye and wheat starches (Fig. 2 and Table 3). Starches containing AM molecules with a higher DP exhibited higher setback and end viscosities. Mua and Jackson56 also reported a positive correlation between AM DP and setback and final viscosity of maize starch. Furthermore, the higher proportion of IM in rye cultivar Treviso than in the other rye cultivars (Table 4), containing molecules with long branch chains, might also contribute to the higher setback and end viscosities found for this cereal (Fig. 2 and Table 3).

Time (min)

4. Conclusions

Nikita Relative moles .

RI (arbitrary units) .

50 40 30 20 10 0

10

12

14

16

18

20

22

18

20

22

Time (min)

Treviso Relative moles .

RI (arbitrary units) .

50 40 30 20 10 0

10

12

14

16 Time (min)

Zohra Relative moles .

RI (arbitrary units) .

50 40 30 20 10 0

10

12

14

16

18

20

22

Time (min) Figure 4. High-performance size exclusion chromatography (HPSEC) (Zorbax PSM 60S columns) elution profiles of debranched b-limit dextrins from rye (Avanti, Nikita, Treviso) and wheat (Zohra) amylopectin. Mass-basis ( ) and molar-basis ( ) chromatograms. MW markers (+) from left to right are 11.2  104, 4 4 4 4.73  10 , 1.18  10 , 0.59  10 , maltoheptaose, maltopentaose, maltose and glucose.

chains with DP 6-12 and the higher proportion of chains with DP 13–24 in the wheat AP (Table 6). For starches from different botanical sources, pasting temperature correlated negatively and positively with the level of AP unit chains with DP 6–12 and DP 13–24, respectively.53–55 In some studies, negative correlations were found between the content of AP short chains (DP 6–12) and the peak and breakdown viscosities of starch,53,54 whereas, in other studies, no significant correlations between AP chain length distribution and peak and breakdown viscosity of starch were found.43,55 The different peak DPs observed for the rye and

Starches were isolated from three rye flour samples and one wheat flour sample by a PronaseÒ-based isolation procedure, and their physicochemical and structural properties were analysed. Little if any differences in physicochemical properties between the rye samples of different origin were observed. However, the rye cultivar Treviso had a somewhat lower breakdown and higher setback and end viscosities than the starches from the other rye cultivars. Gelatinisation and AP retrogradation behaviour of rye and wheat starches did not differ. The rye starches differed from the wheat starch in showing a somewhat higher relative crystallinity, lower gelatinisation and pasting temperatures, lower peak and breakdown viscosities and higher setback and end viscosities, and a lower tendency to retrograde. In the second part, the rye and wheat starches were fractionated into their AM, IM and AP components. Some differences in AM peak DP were found among the rye samples and between the rye and wheat samples. Rye AP showed some minor differences in bamylolysis limit, average CL, ECL and ICL and had nearly identical chain length distributions and similar interior chain length distributions, indicating that different rye AP had similar structures. The wheat AP, however, had a somewhat higher b-amylolysis limit, average CL and ECL and a somewhat lower ICL than the rye AP. Besides, wheat AP had a lower proportion of chains with DP 6–12 and a higher proportion of chains with DP 13–24 than rye AP. These results demonstrate that the structure of the wheat AP differs from that of the rye AP. The differences in AM and AP structure may explain the differences in gelatinisation, retrogradation and pasting behaviour between the rye and wheat starches. Acknowledgements We thank Hilde Van den Broeck, Barbara Vangeneugden and Luc Van Den Ende for technical assistance. This study is part of the Methusalem programme ‘Food for the Future’ at the K.U. Leuven. References 1. Poutanen, K. Cereal Foods World 1997, 42, 682–683. 2. Verwimp, T.; Vandeputte, G. E.; Marrant, K.; Delcour, J. A. J. Cereal Sci. 2004, 39, 85–90. 3. Colonna, P.; Buléon, A. New Insights on Starch Structure and Properties. In: Cereal Chemistry and Technology: A Long Past and A Bright Future— Proceedings; 3rd ed., 1992, 9th International Cereal and Bread Congress. pp 25–42. 4. Schierbaum, F.; Radosta, S.; Richter, M.; Kettlitz, B.; Gernat, C. Starch/Stärke 1991, 43, 331–339. 5. Berry, C. P.; D’Appolonia, B. L.; Gilles, K. A. Cereal Chem. 1971, 48, 415–427. 6. Ball, S.; Guan, H. P.; James, M.; Myers, A.; Keeling, P.; Mouille, G.; Buléon, A.; Colonna, P.; Preiss, H. Cell 1996, 86, 349–352. 7. Zobel, H. F. Starch/Stärke 1988, 40, 44–50. 8. Banks, W.; Greenwood, C. T. Starch/Stärke 1967, 19, 394–398. 9. Yoon, J.-W.; Lim, S.-T. Carbohydr. Res. 2003, 338, 611–617. 10. Bertoft, E.; Qin, Z.; Manelius, R. Starch/Stärke 1993, 45, 420–425. 11. Takeda, Y.; Tomooka, S.; Hizukuri, S. Carbohydr. Res. 1993, 246, 267–272. 12. Klucinec, J. D.; Thompson, D. B. Cereal Chem. 1998, 75, 887–896. 13. Wang, Y. J.; White, P.; Pollak, L.; Jane, J. Cereal Chem. 1993, 70, 521–525.

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