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Biomacromolecules 2011, 12, 123–133

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Study of the Structure and Properties of Native and Hydrothermally Processed Wild-Type, lam and r Variant Pea Starches that Affect Amylolysis of These Starches Rumana Tahir,† Peter R. Ellis,† Tatiana Y. Bogracheva,‡ Cheryl Meares-Taylor,‡ and Peter J. Butterworth*,† Department of Biochemistry, Biopolymers Group, Diabetes and Nutritional Sciences Division, King’s College London, Franklin Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom, and John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, United Kingdom Received September 9, 2010; Revised Manuscript Received November 18, 2010

Starches from WT, lam, and r pea mutants differing in amylopectin/amylose contents (70, 90, and 28% amylopectin, respectively) were used in kinetic studies of pancreatic R-amylase action at 37 °C and for investigations of their supramolecular structure and physicochemical properties during heating. For WT and lam starches, amylase accessibility and catalytic efficiencies (CE) increased following hydrothermal processing up to 100 °C. Accessibility changed relatively less in r during heating with increasing Km between 60-90 °C. Limiting values of Km after gelatinization were very similar for all three mutants, indicating that relative proportions of amylose/amylopectin have little influence on amylase accessibility once ordered structures are lost. For WT and lam, increases in enzyme accessibility and CE paralleled a rise in amorphous content. It is suggested that the complex behavior for r resulted from amylose gel formation between 60-90 °C. Amorphous amylopectin seems a better substrate for amylase than amorphous amylose.

Introduction Starch is a main source of digestible carbohydrate in the human diet and is therefore the major source of glucose appearing in the blood circulation after digestion of a starchcontaining meal. Considerable differences, however, occur in postprandial blood glucose and insulin responses to ingestion of different foods containing identical amounts of starch.1 These differences are assumed to reflect large variations in the rate and extent of starch digestion in the gastrointestinal tract.2-5 Full explanations for the differences remain to be elucidated, but dissimilarities in the structure of starch granules of different botanical species and structural alterations subsequent to processing of foods are assumed to be significant factors.6 Certain fractions of starch granules are not hydrolyzed by R-amylase,7,8 and these reach the large intestine and become fermented by intestinal microorganisms to short chain fatty acids, for example, butyrate.9,10 “Resistant” starch present in animal and human foods can contain physically entrapped starch within cells of plant tissue. Predictions of susceptibility to amylolysis are therefore difficult. Limiting the degree of postprandial glycemia and insulinemia is important in the prevention and treatment of diabetes mellitus and cardiovascular disease and also has implications for obesity management.3,11-13 A rise in glucose concentration in the blood circulation occurs within 15-20 min of ingestion of a starchrich meal,4,14 indicating that initial stages of digestion are of physiological significance. Rapid rises in glycemia strongly influence the insulin response by a direct action on pancreatic islet cells. The insulinemic response is also modulated by gut hormones such as glucose-dependent insulinotropic polypeptide * To whom correspondence should be addressed. Tel.: +44 (0) 207 848 4592. Fax: +44 (0) 207 848 4500. E-mail: [email protected]. † King’s College London. ‡ John Innes Centre.

(GIP) and glucagon-like peptide (GLP-1), whose release are stimulated by the active absorption of glucose and other nutrients.5 In healthy human subjects, amylose-rich starches produce lower postprandial blood glucose and insulin responses than amylopectin-rich varieties.15-17 From greater understanding of the molecular basis for the variations in glycemic behavior of different starches, it may be possible to exploit genetic manipulation to produce foods in which the starch content is digested slowly and predictably, even after severe hydrothermal processing18 Many previous studies involved physical characterization of the variant starches and measurements of their rates of hydrolysis over several hours to estimate relative proportions of readily digested and resistant starch. Such studies do not address directly the physiologically important early stages of digestion. Native starch granules are semicrystalline with amorphous and ordered regions and it is generally accepted that the short amylopectin chains are arranged in double helices that give rise to crystallites. It is likely that, in the initial stages of amylase digestion, the target is the amorphous regions.6 In excess water, starch granules swell to a limited extent and, when heated, the ordered structures begin to break down at a particular temperature, and this is followed by amylose leaching and by a large increase in granular swelling. The sum of these processes is related to initial starch granular structure and called gelatinization. This present paper reports the results of a continuation of our investigations19-21 into the effects of starch granular structure on the initial phases of amylolysis. Three representative starches were chosen for the investigation. These starches differ in their molecular and supramolecular structure with respect to amylose/amylopectin ratio, to the type and proportion of ordered structures and in certain physicochemical characteristics including the response to hydrothermal processing and the degree of amylose leaching and granular swelling. These studies are

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expected to help our understanding of the links between R-amylase catalyzed digestion and starch crystalline structure and physicochemical properties.

Materials and Methods Pea Mutants and Biochemicals. Starch was extracted from wild type and mutant pea seeds developed at the John Innes Centre (Norwich).22 The mutants were essentially isogenic (except for mutations at either the lam or r loci) with the wild type (WT) parental line from which the mutants were derived. The r mutant is associated with decreased activity of starch branching enzyme 1 and results in a decrease of the overall rate of starch synthesis, an increase in the proportion of amylose and a fall in the proportion of amylopectin.23 In addition, the morphology of starch granules is affected. The lam mutant lacks granule-bound starch synthase 124 and produces starch with very low amylose content, hence the designation as lam. Phosphate buffered saline (PBS) tablets and porcine pancreatic R-amylase (type 1) were obtained from Sigma-Aldridge Co. Ltd. (Poole, Dorset, U.K.). The manufacturers state the activity of this preparation to be approximately 1000 units per mg protein, where 1 unit catalyzes the release of 1 mg of maltose from starch in 3 min. The purity of the preparation was checked by electrophoresis on denaturing polyacrylamide gels. For assays of activity, a stock solution of 572 nM in PBS containing 1 mg/mL of bovine serum albumin was prepared. The concentration was calculated by assuming a Mr of 56 kDa for amylase.21 All other reagents, in the best grade available, were purchased from the same company (i.e., Sigma-Aldridge). Extraction and Analysis of Pea Starches. Starch was extracted from the seeds of wild type parental line of pea and from mutants that are near isogenic to this WT except for the genes at the r or lam loci.25 The seeds were ground in a Cyclotec 1093 pin-mill (Foss Decator, Hoganas, Sweden), and the resulting meal was made into a slurry with water at a solid to liquid ratio of 1 to 10, (i.e., 300 g pea flour to 3 L of water), with adjustment of the pH to 8.0-8.5 with 0.1 M NaOH. The mixture was centrifuged at 2500 g and the supernatant then removed. The precipitate had a very viscous green/yellow top layer and a solid cream/white main part. The viscous top layer was removed and the rest of the precipitate was resuspended in distilled water. The centrifugation and separation procedure was repeated twice to ensure loss of the viscous layer. The resulting uniform white precipitate was resuspended in approximately 2.5 L of distilled water and filtered sequentially through two stainless steel sieves of 300 and 53 µm, respectively (Endecotts Ltd., London). The collected filtrate was poured into a large flask and stored overnight at 4 °C to allow precipitation of the starch. The precipitate was collected, resuspended in 2-3 L of distilled water and allowed to stand at 4 °C for 10 h. The procedure was repeated for a few times until the precipitate appeared white and the supernatant was no longer green in color. The starch precipitate was collected by filtering under vacuum then placed on a stainless steel tray to dry at a laboratory temperature of 20-22 °C and 33% humidity. This took 2-3 days but after 1 day of drying, any clumps of material were dispersed by gentle use of a spatula. Extracted starch was examined by light microscopy for contamination with plant cell walls, the main components of which are nonstarch polysaccharides (NSP). Only fragments of cell walls from ruptured cells of pea seeds were observed. Whole cells with intact cell walls were completely absent. Chemical analysis of the NSP content of starch samples confirmed the microscopic examinations. An assay performed by Lancashire County Laboratory (Pedders Way, Preston, Lancs., U.K.) gave respective NSP values of 0.5 and 0.2% for WT and lam starch samples (Table 1, Supporting Information). Samples of r starch were found to be nonsuitable for this analysis because of difficulty in dissolving r starch in dimethyl sulphoxide. The amounts of noncarbohydrate materials present were determined using established methods.26 The percentage of damaged granules was estimated by examination, under light microscopy, of the number of granules that took up Congo

Tahir et al. red dye from a bathing solution.19 A thousand granules of each starch type were counted. Light Microscopy of Native Starches. To examine the purity and morphology of the extracted starches, samples were mounted in water before examination using a Carl Zeiss Axiophot microscope with a JVC 3-CCD video camera attachment connected to a PC equipped with ImageProPus 4.0 Software. For examination under polarized light, starch samples were dispersed in 0.6 M KCl solution, and equilibrated while stirring at room temperature. A Gene frame (ABgene Ltd., Epsom, Surrey, U.K.) placed on a microscope slide was filled with starch dispersion and carefully covered with a glass coverslip, making sure that air bubbles were excluded. The slide, with sample, was placed on a Mettler FP 82 microscope hot stage and viewed under a Zeiss Axiophot microscope equipped with cross polarizers and λ-plate. The hot stage was heated from 30-98 °C at a heating rate of 1 °C/min. The behavior of individual starch granules during heating was viewed and photographed using a long focus Nikon objective (40×) and total magnification of 400×. Determination of Amylose/Amylopectin Ratios. An assay kit (AMYL 11/99 from Megazyme, Bray, Co. Wicklow, Ireland) was used for amylose determination. In brief, the method is based on removal of amylopectin using concanavalin A27 followed by hydrolysis of the remaining amylose using an amyloglucosidase/R-amylase mixture and the colorimetric determination of the resulting glucose. Amylopectin is determined by the difference from the total starch content determined by conversion of a complete starch sample to glucose by reaction with amylase and amyloglucosidase. Processing of Starch. Suspensions of starch granules (10-20 mg/ mL) were prepared in 20-30 mL of freshly prepared PBS (pH 7.4). The mixture was agitated gently for 20 min in a 150 mL conical flask. This was conducted at 25 °C. For preparation of the suspension of r pea starch, the starch-PBS mixture was gently homogenized (UltraTurrax homogenizer, Janke & Kunkel, IKA-Werk, Staufen, Germany) at the lowest speed using 4-6 pulses of 1-2 s duration to break up any lumps and produce a homogeneous suspension. For heat treatment, suspensions of starch (10 mg/mL) were prepared in 20-30 mL of PBS solution (pH 7.4) in a 150 mL conical flask and placed into either a boiling water bath (100 °C) or a water bath with a thermostat set to the required temperature. For temperatures of 55, 60, 65, and 75 °C, a thermocouple probe was securely placed inside the flask with its end immersed inside the suspension to monitor and regulate the temperature of treatment. The flask was agitated gently by swirling the mixture for 20 min in the water bath. Prior to the start of the treatment, the conical flask was weighed accurately and sealed with a large glass marble and metal foil to minimize evaporation during treatment. Following treatment, the flask was reweighed and any lost water was replaced with distilled-deionized water. When pea starch r was pretreated at 120 °C, a 10 mg/mL suspension in 50 mL of PBS was placed in a 250 mL Duran bottle and homogenized at low speed using 6-7 pulses of 1-2 s duration. The bottle and contents were weighed and then heated at 100 °C for 10 min with constant mixing by swirling. This produced a suspension of shallow depth within the bottle to minimize the effect of sedimentation during the lengthy autoclave cycle. The sample was heated at 120 °C for 20 min in an autoclave and then allowed to cool to 25 °C. Any loss of water was detected by reweighing the Duran bottle and the difference made up with distilled-deionized water. The heating and cooling cycle in the autoclave lasted for 90 min. All pretreated samples were used directly for the enzyme assay experiments. This protocol was adhered to rigorously. Assay of r-Amylase and Determination of Kinetic Parameters. The assay system has been described in detail elsewhere.19-21 The method, in outline, was performed as follows. Starch solutions ranging in concentration from 0.125-1.0% (1.25-10 mg/mL) in PBS were reacted with 1.43 nM R-amylase at 37 °C under constant mixing. Samples were withdrawn at timed intervals up to 12 min and transferred to ice cold 0.3 M Na2CO3 stop solution. After sedimentation of

Amylolysis of Variant Pea Starches nonreacted starch by centrifugation, the reducing sugar concentration of the supernatant was determined by a Prussian blue method and expressed as maltose equivalents by reference to a standard curve prepared for this sugar. Initial reaction rates were calculated from linear progress curves of the data obtained from the timed intervals up to 12 min and analyzed by weighted nonlinear regression fit to the Michaelis-Menten equation using Enzfitter software (Biosoft Great Shelford, Cambridgeshire, U.K.) to provide values for Km, kcat, and kcat/ Km. So-called rates of hydrolysis were calculated from the rate of release of maltose from 0.5% starch (5 mg/mL) during the initial 12 min of reaction. The enzyme concentration used for determining relative rates of hydrolysis was the same for each starch (1.43 nM). Reaction rates that were linear with time enabled data to be treated by Michaelis-Menten kinetics, where appropriate, rather than as a logarithmic first order reaction.28 Differential Scanning Calorimetry and Wide-Angle X-ray Diffraction. These methods were used to study the type and proportion of ordered structures. A Setaram Micro-DSC III (Seteram Instruments Ltd., Lyon, France) was used. Starch powders samples (33-35 mg) were mixed in a Setaram Micro DSCIII cuvette with 0.8-0.85 g deionized water and then equilibrated at 20 °C for 2 h. The suspension was then cooled to 10 °C, equilibrated for 1 h at this temperature and then heated to 120 °C at a heating rate of 0.5 °C/min. Subsequently it was cooled at a rate of 1 °C/min to 10 °C and reheated to 120 at 0.5 °C/min. The thermogram produced by the second heating demonstrated that no retrogradation occurred when the cooled starch suspension was stored for an hour. DSC thermograms were used to determine the total proportion of ordered structures in starches. Using previously described methodology,29 the specific enthalpy (∆Hsp) was determined and the total proportion of ordered structures (i.e., the proportion of double helices, DH%) was calculated from the following equation:

DH% ) (∆Hsp/∆HDH) × 100 where ∆Hsp is the specific enthalpy of DSC transition and ∆HDH is the enthalpy of DSC transition per dry weight of ordered structures. Depending on the type of crystallinity, 34 or 36 J/g values were used for ∆HDH.29,30 DH% for both WT and lam were found to be similar to values previously found for these starches using NMR and DSC methods.29,30 DH% for r starch was independently measured using 13C CP/MAS NMR29,30 and was calculated from a comparison of C4 resonances (peak at 81.5 ppm) for starch equilibrated at 40% relative humidity and for amorphous starch with similar dry weight. A good agreement between DSC and NMR methods for r starch was obtained (Table 2, Supporting Information). Wide angle X-ray diffraction data and polymorph compositions for r starch were produced as described in Bogracheva et al.31 The starch sample was equilibrated at 98% relative humidity before measurements were taken. Data analysis was performed using Origin software, (MicroCal Europe, Milton Keynes, Bucks, U.K.). Scanning Electron Microscopy. Scanning electron microscopy (SEM) was used to obtain morphological information of native and hydrothermally treated starches at 100 °C. Native starch samples were mounted on circular aluminum studs and then examined and photographed in a FEI QUANTA 200F SEM at an accelerating potential of 5 kV. For images of heat-treated starches at 100 °C, a cryo-SEM procedure was employed that was developed by Fannon and BeMiller.32 Swelling Power (SW). A combination of modified blue dextran33 and centrifugation based34 methods was used. The centrifugation method is reliable only for fully gelatinized starch where the swollen granule phase and the continuous water phase can be separated by centrifugation. On the other hand, the blue dextran method gives accurate results for starch in both its native state and when gelatinized to different extents, provided that the blue dextran concentration is accurately controlled. It has been shown, however, that at very high

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temperatures when swelling is extremely high, blue dextran can affect the water distribution between the swollen granules and the continuous water phase.35 In the present paper, such a condition only occurred for lam starch heated to 110 °C and higher. For such samples the centrifugation method was required, but in all other cases the blue dextran method was used. The blue dextran method is based on the incompatibility of high molecular weight blue dextran molecules and the phase of swollen granules (heated or unheated) in starch/water suspensions. It has been shown that under specific concentrations of blue dextran and starch in suspension, the blue dextran concentration within the dispersed phase of the swollen granules is negligible and that blue dextran is not absorbed on the phase separation surfaces.35 These concentrations were used in our measurements. Starch (10-100 mg depending on anticipated swelling) and approximately 5 g of deionized water were accurately weighed into 10 mL Pyrex tubes and closed with water tight caps and rubber seals. The tubes were heated in a temperature-controlled shaking water bath from 20-100 °C at a heating rate of 0.5 °C/min, and samples were removed for analysis at 5 °C intervals. Heating temperatures in excess of 100 °C were obtained by transferring samples from the water bath to a dry block preheated to 100 °C and the temperature raised to the required temperature. Intermittent mixing was performed by inversion of the tubes during heating. After heating to the required temperatures, the tubes were cooled to 20 °C for 10 min in a water bath. Approximately 0.5 g of very accurately weighed 0.5% (w/w) blue dextran solution (2 × 106 average molecular weight) was added and the tubes were recapped and gently inverted to distribute the dextran solution evenly throughout the starch suspension. The samples were centrifuged at 2000 × g for 10 min at 20 °C. The absorbance of the supernatant was measured using an Hitachi U-1100 spectrophotometer at 620 nm against a blank prepared in an identical fashion to the test sample except for the addition of blue dextran. The blue dextran concentration in the supernatant (BDsuper) was determined from the equation

BDsuper ) (AS - AR) × 0.1022 where AS and AR are the absorbance at 620 nm of the sample and reference, respectively. The multiplication factor (0.1022) was obtained from absorbance measurements made with blue dextran solutions.35 The weight of water (g) absorbed by the swollen starch granules (Wabs) was calculated using the equation

Wabs ) (W + WBD) - Wsuper where W is the weight of water added to the starch sample (in g), WBD is the water (in g) added when 0.5% blue dextran solution was added to the sample, and Wsuper is the water (in g) in the supernatant after heating. Wsuper was determined using the value of BDsuper and the weight of 0.5% blue dextran solution added to the starch sample. Taking into consideration that some amylose was solubilized during heating, the swelling power (SW) was determined as follows:

SW ) [(ST - AMY) + Wabs]/(ST - AMY) where ST is the total weight of starch and AMY is total weight of amylose (g) in the supernatant determined from measurements of leached amylose (see Amylose Leaching below). For the centrifugation method, samples were prepared similarly to those for the blue dextran method, except for the inclusion of blue dextran, and were then centrifuged at 2000 × g for 10 min at 20 °C. It was shown that for these samples, centrifugation resulted in full separation of swollen granules from bulk solution.42 SW was determined from the equation

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where STpt (dry weight in g of starch in the precipitate) and Wabs were determined by measuring both wet and dry weights of the two phases separated by centrifugation. Amylose Leaching. A modified36 iodine binding method37 was used to determine the concentration of amylose leached from starches swelled in excess water. After centrifugation to remove granular particulate matter, appropriate volumes of supernatant samples (0.1-2 mL) were treated with 5 mL of 0.5% trichloracetic acid solution then 0.05 mL of I2-KI solution (1.27 g I2 per 3 g of KI/100 mL) was added and the total volume made up with distilled water to 1 L with thorough mixing. After 30 min at 25 °C, the absorbance of the solution was determined at 620 nm against a reagent blank. A calibration curve was produced by using samples in which the ratio of amylose to amylopectin was varied from 10-100. For reliability of the assay method, calibration curves revealed that the total amylose concentration in test solutions should not exceed 1%. Above this level, calibration curves became nonlinear because of formation of stable suspensions of amylose aggregates and gels. The proportion of leached amylose was calculated taking into consideration the initial concentration of amylose in starch, the proportion of water in the initial starch suspension, and SW and the amylose concentration in the continuous water phase.

Results Composition and Properties of the Starches. Three different pea starches studied in this work were extracted from wild type peas (WT) and from lam and r mutants. These starches differ widely in molecular composition, starch granular structure, and physicochemical properties25,32-34,38 and were chosen for investigation of the relationships between amylase action and starch structure and properties. Data for the purity of the extracted starches are shown in Table 1 of the Supporting Information available for this paper. The contamination from ash (estimate of inorganic matter), lipid, and proteins in the pea starches was low. The amounts of crude protein (calculated from total N × 6.25) are similar to those reported previously.38,39 The total lipid content (obtained by acid hydrolysis) represents both free and bound lipids, but our values are lower than those previously reported for smooth and wrinkled pea by about 50%.40 The three pea starches did not differ greatly in lipid content, however, and thus the effect of lipid was not taken into account when considering the differences in rates of amylolysis and dissimilarities in enzyme kinetic behavior. The ash contents were fractionally higher than those reported previously.38,39 Because the protein and ash contents of pea starches were very low, however, their effects on most properties are likely to be very small or negligible, and therefore, their presence was overlooked when considering the relationships between starch structure and kinetics of R-amylase action. The degree of starch damage was very low (i.e., lam > r. The Km values for WT and lam decreased by factors of approximately 22 and 10, respectively, as the processing temperature was raised, to reach a limiting value of 0.9 ( 0.2 mg/mL (Figure 3b). The native r starch had a relatively low Km (2.8 mg/mL). In contrast with WT and lam, Km for r starch starts to increase when the proportion of amorphous material begins to rise with the accompanying heat treatment and reached a value of 5 mg/mL at 75 °C. At higher temperatures, Km fell to 0.6 mg/mL after processing at 100 °C and therefore became very similar to the value for WT and lam. Extending the heating of r to 120 °C brought about a small increase of Km to 0.8 mg/mL that is probably not significantly different from the value found for 100 °C (Figure 3b) and, in any case, is virtually identical to the limiting values observed for WT and lam. The lower Km value for native r suggests better

substrate accessibility in the native state and corresponds with the significantly higher proportion of amorphous material in r than in WT and lam (Table 2, Figure 3b). The fall in Km for WT and lam from 50 °C and above corresponds with the increase in the proportion of amorphous material. The initial increase in Km value for r starch, when the proportion of amorphous material starts to grow, is indicative of the different events that occur in this starch when subjected to heating compared with WT and lam starches. As expected, the relationship between Km and the amorphous content is not linear. The apparent Km value will decrease as the concentration of preferred substrate (i.e., amorphous starch) increases. The constant will eventually reach a minimum level reflecting an “absolute” rather than an “apparent” value when the reaction mixtures contain sufficient amorphous material. Values for kcat of WT starch rose with increases in the pretreatment temperature in correspondence with the increase in the proportion of amorphous starch (Figure 4a). A similar relationship was found for lam starch. The limiting value for kcat seen with WT and lam was 1.22 ( 0.12 × 105 min-1. In a previous paper,21 because of an unfortunate typographical error, we reported a value of 0.98 × 104 min-1 instead of 0.98 × 105 min-1 for amylase acting on wheat starch. With the correction of the factor of 10, it can be seen that the values are essentially identical. Similar values of (1.0-1.6) × 105 min-1 for kcat were reported by us for amylase acting on gelatinized starches from a number of different botanical species.19 The maximum in kcat (turnover number) reflects a condition in which the catalytic process is unimpeded by physical factors affecting the availability of R-glucan chains and of water for interaction at the catalytic site of the enzyme and, therefore, should be identical for all starch samples.21 The relationship between kcat and the content of amorphous starch is not linear, but the constant approaches its limiting turnover number as the various constraints on the catalytic event are removed by the rise in amorphous material. For r starch, kcat rises in proportion with

Amylolysis of Variant Pea Starches

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Figure 3. Effect of hydrothermal pretreatments of the WT, lam and r starches on the changes in proportion of amorphous material (as a percentage of total starch content) and enzyme kinetic parameters determined for the action of porcine pancreatic R-amylase at 37 °C. The starches were pretreated by heating suspensions of starch in excess aqueous solution for 20 min at the indicated temperatures. Samples were cooled to 37 °C before use as substrates for amylase. The kinetic data points for WT, lam, and r starches show the means of 3-5 independent duplicate determinations in each case: (a) hydrolysis rate (µM/min) (2); percentage of amorphous material (b); (b) Km (mg/mL) (2); percentage of amorphous material (b). The initial hydrolysis rate was determined at a starch concentration of 5 mg/mL.

the amorphous material at pretreatments above 55 °C but, surprisingly, appears to peak following 75 °C treatment and then decline somewhat at the higher temperatures. The catalytic efficiencies (CE), expressed as kcat/Km ratios, for amylase acting on the processed starches are shown in Figure 4b. CE values rose with increased processing temperature, and therefore amorphous content, to reach a common value of (122 ( 6) × 103 min-1(mg/mL)-1 at 100 °C. Thus, although the kcat values for the r mutant were relatively low (see above), the lower and, therefore, more favorable Km value compensated for the lowered kcat value, so that the CE of amylase action was essentially the same for all the starches after processing at 100 °C. Pretreatment of r starch at 120 °C, however, resulted in material that had a lower CE than the 100 °C value that was common for all the starches.

Discussion Hydrolysis rate data in the literature usually show starch digestibility curves obtained over lengthy time periods that can exceed 3 h.28,52 Given the relative rapidity, however, in which a peak of glucose concentration appears in portal and peripheral blood after ingestion of a starch-rich meal,4,14,53 the initial rates of hydrolysis may provide better guides to the speed at which various starches are digested in the intestine. A paper containing data from pigs appeared recently54 that compared the rates of product formation in vitro with those in vivo, and the authors show a strong correlation between a peak in the in vitro reaction rate occurring at 6 min with the peak in portal blood glucose concentration occurring at 69 min. It is also recognized that the peak rise in blood glucose is of clinical relevance. For

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Figure 4. Effect of hydrothermal pretreatments of the WT, lam, and r starches on the change in proportion of amorphous material (as a percentage of total starch content), and kcat and CE (kcat/Km) parameters for the action of porcine pancreatic R-amylase at 37 °C. The starches were pretreated by heating suspensions of starch in excess aqueous solution for 20 min at the indicated temperatures. Samples were cooled to 37 °C before use as substrates for amylase. The kinetic data points for WT, lam, and r starches show the means of 3-5 independent duplicate determinations in each case: (a) kcat (min-1; 2); percentage of amorphous material (b); (b) CE (min-1/mg/mL; 2); percentage of amorphous material (b).

example, Ceriello et al.55 have recently reported that the oscillation of blood glucose has deleterious effects on endothelial function and oxidative stress in type 2 diabetic patients, important risk factors in cardiovascular diabetes complications. Thus, the early stages in starch hydrolysis are likely to be important physiologically and potentially in relation to health and disease, notably diabetes management. We have analyzed hydrolysis rates and the kinetic parameters Km, kcat, and kcat/Km ratio (this latter term provides a measure of the catalytic efficiency). These various parameters characterize the amylolysis process from different perspectives. Starch, both native and heat-treated, is not soluble in water, unless the treatment temperature is very high, and its supramolecular structure is very complex; therefore, research into

amylase action can be a formidable challenge. Reports in the literature suggest that amylase attacks the amorphous regions of granules preferentially during early stages of starch digestion,40,51 although it has been claimed, from studies using high amylase concentrations, that both crystalline and noncrystalline regions of maize starch can be readily digested.52 How amylase digests crystalline areas is not clear. The most likely mechanism is that initially the amorphous areas are attacked. After the amorphous areas disappear, the double helices start to unwind from the ends; these ends then effectively become amorphous and consequently become subject to attack by amylase. Under the conditions of our experiments, however, it is likely that the enzyme reacts with amorphous starch that is relatively exposed and accessible at the surface of the granules.56

Amylolysis of Variant Pea Starches

Because the Km value represents the substrate concentration needed to produce a catalytic activity that is one-half of the maximum rate (i.e., Vmax/2), changes in Km as starch is processed can be regarded as directly related to the increase in the fraction of R-glucan chains that is accessible to amylase and able to be digested.56 Suspensions of untreated r starch contain a much higher proportion of amorphous material, possess a surface area that appears to be larger than WT and lam starches,56 and have similar water content within the granules. It is expected, therefore, that r starch should be easier to digest than WT or lam. Also, amylose seems usually to be enriched within the peripheral regions of starch granules and these “peripheral” molecules tend to have shorter chain lengths than more centrally placed amylose.57 If this material is amorphous, digestion should be facilitated. There seems to be little information, however, about the distribution pattern of amylose in high amylose starches such as r, although the average chain length has been shown to be slightly longer than in WT and lam pea.29,39 It is possible that because of its relative abundance, amylose is more evenly distributed throughout the r granule. Nevertheless, the expected ease of digestion was reflected in the hydrolysis rate and CE for native r starch that were significantly higher than for the other two types and Km was much lower showing good accessibility of substrate. WT contains a higher proportion of amorphous starch than lam, but the higher Km for native WT implies that the amorphous regions are less accessible to amylase than in lam starch. That the HR values for the native WT and lam starches are similar, in spite of the differences in Km, however, suggests that the greater proportion of amorphous material in WT and its composition compensates for its more limited availability. The onset of the notable increases in CE and HR that result from hydrothermal processing occur approximately 10 °C earlier in WT compared with lam. These results correlate well with the sharp increase in the proportion of amorphous material in these starches (Figures 3a and 4b). The amorphous regions in lam will mainly be formed from amylopectin, whereas in WT the amorphous material is largely amylose. The kinetic data suggest, therefore, that amorphous amylopectin is a better substrate for amylase than amorphous amylose. This result is in good agreement with the finding that, for mixtures of purified amylose and amylopectin, Km values fell as the proportion of amylopectin was increased in the mixture.58 When WT and lam starch suspensions are heated to temperatures where crystalline structures begin to break, all the kinetic parameters reveal that the starch becomes a better substrate for R-amylase. The increases in catalytic susceptibility correspond well with the increasing proportions of amorphous material as well as increasing amylose solubility. Our present results cannot fully distinguish between the relative importance of the increase in amorphous material within the granules and the rise in amounts of leached amylose in the surrounding bulk solution (Figures 1b, 3, and 4). The impact of these two potential effectors on the kinetics of amylolysis could be a useful area to explore in future investigations, but at present, we can only suggest that amylose in solution is much easier to digest than amorphous material within the granule. The behavior of r starch in response to heating is much more complex than for WT and lam. The hydrolysis rate increases with heat pretreatment in correspondence with an increase in amorphous material, but the rise in HR is not as great as that seen for WT and lam, even though the rate for native r starch is significantly higher than for the other types. Although r starch

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has a high proportion of amorphous material and its granular crystalline structure was completely disrupted after treatment in excess water at 110 °C, the granular swelling and solubilization of amylose were found to be very restricted in that only about 20% of this polysaccharide became solubilized after pretreatment at 120 °C. In contrast, for WT and lam, the proportion of amylose solubilized after 120 °C treatment was 80 and 56%, respectively (Figure 1b). Also, the total amount of water absorbed by the r granules was less than for WT and lam (Figure 1c). The limited solubilization of amylose and the lowered water uptake obviously impact on the relative ease of digestibility by R-amylase. The rise in Km of r starch with temperature treatment between 55-75 °C (Figure 3b) indicates that availability of substrate becomes less favorable in spite of the increase in the proportion of amorphous material that accompanies the rise in treatment temperature. Treatment at 100 °C or above, however, eventually brings about more favorable substrate availability. We suggest that the complex kinetic behavior associated with r starch results from a number of starch granular changes during heating, including the following. (a) As the starch is heated, crystalline structures within the granule begin to break down and this disruption allows more water to be absorbed (Figure 1a,c).30,59,60 (b) The amorphous regions take up water with the possibility of solubilization of some amylose. (c) At high temperatures and concentrations, amylose can form gels.30,33,59 Amylose is a high molecular weight, essentially linear, polysaccharide and therefore a low diffusion rate for amylose can be expected. Taking into consideration the combined effects of a very high proportion of amylose within r starch granules, restrictions in molecular diffusion rate and rapid water uptake when crystalline structure begins to break down during heating, the conditions for gel formation at 55-80 °C within the r granules are favored. The onset of gelation slows down both granular swelling and the solubilization of amylose resulting in cessation of release of the latter into the surrounding aqueous medium until the temperature is high enough to solubilize the gel material itself.60,61 This occurs at about 150 °C.35 Amylose gel within the granule is expected to restrict the diffusion of the enzyme to suitable reaction sites on amylose and also hinder the diffusion of reaction products away from the active site. Restricted diffusion of the enzyme would be expected to have a negative influence on amylose-amylase binding interactions and accompanying limits on the diffusion away from reaction products would lead to product inhibition. In vitro studies62 showed that the rate of diffusion of proteins in amylose gels and, as a consequence, the accessibility of protein to water spaces within the gel network are decreased as the gel concentration is raised. The concentration of any gel formed within or around the r starch granule would be relatively high given the limited uptake of water that is observed with this granule (Figure 1c). It is not unreasonable to believe, therefore, that formation of an amylose gel during heat pretreatments could account for the complex enzyme kinetic properties, particularly the fall in kcat observed when r starch is used as a substrate for R-amylase following starch pretreatment at high temperatures.

Conclusions In the present paper, the effect of amylase action during the hydrothermal processing of three types of pea starches (WT, lam, and r starches) was investigated. The starches differed in the proportion of amylose/amylopectin, the proportion of ordered structures (double helices and crystallites), and crystalline type.

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It was shown that during heating in excess water, the granular ordered structures are disrupted and amylose is leached. These processes are related to the initial granular structure and are greatly affected by the high proportion of amylose in r starch. Amylase attacks the amorphous area of starch during the initial stages of hydrolysis and it was shown that the proportion and availability of amorphous areas determines the kinetic parameters of amylase action. Changes in Km, kcat, and hydrolysis rate that are indicative of favorable conditions for catalysis are associated with the changes in the proportion of amorphous starch, indicating that the enzyme acts on amorphous material. Starch from the r line, however, exhibited more complex behavior in the way that kinetic parameters responded to hydrothermal processing. Starch from this line had a relatively slow rate of structural disruption, low water absorption, and low amylose leaching. Taking into consideration the very high amylose content of this starch, it was suggested that this behavior was the result of amylose gel formation within and around the granules during heating. This gel hinders enzyme-starch interaction and the diffusion away of reaction products. If temperature treatments are high enough, the amylose gel is solubilized and the kinetic parameters for r attain the same values as those seen for processed WT and lam. Acknowledgment. The authors thank Prof. Cliff Hedley of the John Innes Centre, Norwich, U.K., for the gift of the pea mutants and for expert advice on all aspects of their properties. Expert technical assistance was provided by Mr. Ian Topliff of the John Innes Centre, Norwich, U.K. Dr. Rumana Tahir was in receipt of a BBSRC Industrial CASE award in collaboration with RHM Technology, High Wycombe, U.K. We thank Mr. Frederick Warren and Mr. Jai David (King’s College London) for help with the preparation with the figures and estimation of starch damage. Mrs. Marjorie Sanders and Dr. Lee Hartley of RHM Technology are thanked for their interest and support of the project. Supporting Information Available. Tables containing details of chemical and structural properties of the starches used in this investigation. This material is available free of charge via the Internet at http://pubs.acs.org.

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