Ratio of A Chains to B Chains in Rice Amylopectins

Cereal Chem. 65(5):424-427 The ratio of A chains to B chains of rice amylopectins from two pairs of action of pullulanase on the /3-limit dextrin and from A and B chains by the near isogenic lines differing in the waxy gene was determined by the action combined action of /3-amylase and pullulanase. Waxy and nonwaxy (lowof pullulanase and of /3-amylase plus pullulanase on the fl-limit dextrins amylose) rice amylopectin were shown to have similar ratios of A to B followed by the determination of maltotriose in the digests by quantitative chains of 1. 11.5, despite differences in [-j] and freeze-thaw stability. gel filtration chromatography. Maltotriose is derived from A chains by the A number of revisions have been proposed for the fine structure of amylopectin (Enevoldsen 1985, Manners 1985). In most of these, an important parameter is the ratio of A chains to B chains (Marshall and Whelan 1974). The A chains are unsubstituted chains linked to the rest of the amylopectin molecule through an a-D-(1-6)-glucosidic linkage; B chains are linked through an ac-D(1-6)-glucosidic linkage but also carry one or more substituent chains joined to primary (C-6) hydroxyl groups. Amylopectins from waxy varieties of cereals are frequently used for structural analysis. This is a somewhat risky shortcut when the results are extrapolated to encompass nonwaxy amylopectin without reservation. For example, amylopectin from waxy sorghum and waxy corn has an A chain-B chain ratio of 2.6 as compared to a ratio of 1.7 for nonwaxy corn amylopectin (Marshall and Whelan 1974), suggesting that waxy amylopectin may have a different branching pattern to nonwaxy amylopectin. An A chain-B chain ratio of 1.5 is reported for nonwaxy rice amylopectin (Marshall and Whelan 1974) and a ratio of 1.3-1.5 was obtained for waxy rice starch fl-limit dextrin (Umeki and Yamamoto 1977, Asaoka et al 1985). The measurement of A chain-B chain ratio reported by Marshall and Whelan (1974) is based on the comparison of reducing power generated when amylopectin fl-limit dextrin is digested with isoamylase or a mixture of isoamylase and pullulanase. When digested with isoamylase, maltotriosyl (derived from oddnumbered glucose unit A chains) should be released but not maltosyl stubs (from even-numbered glucose unit A chains), whereas the mixture of enzymes should debranch all of the A chains. The difference in reducing power should be equivalent to one-half of the A chains. Although the method was novel, small errors in measuring reducing sugar led to large errors in the final A chain-B chain ratio (Altwell et al 1980). Moreover, the A chain-B chain ratio appears to be affected by isoamylase concentration (Manners and Matheson 1981). Alternately, the maltose and maltotriose released may be estimated after separation by chromatographic methods (Manners 1985). Amylopectins of three pairs of isogenic lines from brown rices differing in the waxy gene have been isolated and compared (Vidal and Juliano 1967). In the present study, the A chain-B chain ratios of two of the three pairs of rice amylopectin were determined from the amount of maltotriose released by subjecting their fl-limit dextrins to the action of pullulanase and to the combined action of pullulanase plus 0i-amylase. Quantitative gel filtration chromatography was used to measure maltotriose in the digests because of its built-in controls (Enevoldsen 1978). A preliminary communication on this study has been presented (Enevoldsen 1980, Juliano 1985). 'Department of Brewing Chemistry, Carlsberg Research Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby, Copenhagen, Denmark. 2 Cereal Chemistry Department, International Rice Research Institute, Los Bahos, Laguna, Philippines. © 1988 American Association of Cereal Chemists, Inc. MATERIALS AND METHODS Grains of two pairs of near isogenic lines, Caloro and its near isogenic waxy line Cal 5563A1 and Century Patna 231 and Waxy Century Patna 231, were obtained from the multiplication plots of the International Rice Research Institute farm, Los Bahos, Philippines, during the 1965 drying season (Vidal and Juliano 1967). Rough rice was dehulled with a McGill sheller, and contaminant translucent grains were removed from the waxy brown rices. Starch was prepared from brown rice by soaking in 1.2% sodium dodecylbenzene sulfonate/0.12% sodium sulfite solution, homogenizing, and passing through a 160-mesh sieve followed by several protein extractions with the same detergent solution (Reyes et al 1965). The purified starch was washed repeatedly with water, air-dried at 350 C, ground to a fine powder with mortar and pestle, and defatted with refluxing 95% ethanol for 24 hr in a Soxhlet apparatus. Purified starches (25 g) were fractionated by autoclaving for 2 hr at 1250 C in 1,250 ml of water and 125 ml of a 1:1 (v/v) mixture of Pentasol 27 (mixed amyl alcohols, Pennsalt) and 1-butanol; the dispersion was filtered through glass wool, reheated to boiling, and 100 ml more Pentasol/butanol mixture was added (Reyes et al 1965). The mixture was cooled to room temperature in Dewar flasks for one to two days and for another day at 40 C. Amylose-alcohol complex was removed by centrifuging at 1,000 X g at 0° C for 20-30 min. The amylopectin supernatant was carefully decanted and precipitated by pouring into 5 vol of 95% ethanol with stirring and allowing the precipitate to settle for at least two days, triturated into a powder, washed with 95% ethanol, and dried. Intrinsic viscosity of amylopectins was measured at 300 C in 1. ON KOH using no. 50 Cannon-Fenske viscometers (Reyes et al 1965). Mean chain length was estimated by periodate oxidation (Reyes et al 1965). Freeze-thaw stability of 5% rice starch gels stored at -20° C and thawed alternately at room temperature was adapted from Schoch (1967). Starch gel was prepared by heating duplicate 5% starch dispersions in water in plastic test tubes for 10 min and cooling. The gels were initially placed at -200C for 15 hr. then thawed by immersing in tap water for 1.5 hr. The tube was then tested for water separation or syneresis by centrifuging at 760 X g for 10 min and watching for free water. Stable gels were refrozen for at least 5 hr at -20° C and then rethawed and tested until syneresis was observed. The number of freeze-thaw cycles required for syneresis was recorded. At Copenhagen, fl-limit dextrin was prepared from 200 mg of rice amylopectin and waxy rice starch boiled in 20 ml 0.1 N NaOH for several minutes, cooled, and neutralized with 2 ml of LON H2SO4 to pH 5.0-6.5, and 2 ml of 0.2M acetate buffer (pH 5.07) was added. Then, 0.4 ml of the sweet potato ,B-amylase (Boehringer) solution (500 units per milliliter, 1 U/mg of amylopectin) and 0.5 ml toluene were added to the polysaccharide dispersion, and the mixture was incubated 24 hr at 300C. The digests (about 25 ml) were boiled for 5-10 min to inactivate the ,B-amylase. An aliquot of 2.0 ml was taken for total carbohydrate by phenol-H2SO4 (Dubois et al 1956), and for reducing sugar (Somogyi 1952) and quantitative gel filtration chromatography. The main digest (about 20 ml) was dialyzed for several days against 424 CEREAL CHEMISTRY deionized water and freeze-dried. The yield was 65-73 mg. ,/-Limit dextrin (20 mg) was dispersed in 0.90 ml of water and 0. 10 ml of 0. 2M acetate buffer pH 5, and mixed with 20 ,ul of Aerobacter aerogenes pullulanase (Boehringer) (28 U/ ml, 0.56 U). A 100-,ul aliquot was diluted with 400 ml of water, and 1 pl of /3-amylase (0. 5 U) was added. Toluene was added to the remaining 920 ,l of /3-limit dextrin plus pullulanase and to the aliquot with added /8-amylase and incubated at 30°C for 24 hr. Aliquots were also analyzed for total carbohydrate by phenol-H 2 SO4 (Dubois et al 1956) and for reducing sugars (Somogyi 1952). Quantitative gel filtration chromatography used Bio-Gel P-2 (Bio-Rad) lot no. 11324 as described by Enevoldsen (1978). Sample size injected was 10-,ul digests for amylopectin + ,/-amylase, 16 ,l for /3-limit dextrin + pullulanase, and 20 ,1 for ,/-limit dextrin + /3-amylase + pullulanase. Carbohydrate concentration was estimated at 420 nm by orcinol-sulfuric acid reagent. One of every 10 runs was a calibration mixture of dextran, maltose, and glucose. Quantification was based on the area of the orcinol-sulfuric acid curve for 40 ,ug of glucose. The void volume peak eluted in about 3 hr and elution time for total volume was about 8 hr. Results were expressed as weight percent of total carbohydrates (as glucose) in the digest. The coefficient of variation of the method was 1-2%. RESULTS AND DISCUSSION ,/-Amylolysis limits, which were calculated from maltose estimated by quantitative gel filtration chromatography, overlapped for waxy rice starch and amylopectin and nonwaxy rice amylopectin for the two pairs of isogenic lines (Table I). The other major fraction was the ,/-limit dextrin plus trace amount of glucose (Fig. 1). Maltose (M2) and maltotriose (M3) recoveries from the action of pullulanase on amylopectin ,8-limit dextrin to hydrolyze A chains showed the expected 1:1 molar ratio (Table I). Higher maltooligosaccharides recovered were 0.9-1.8% M4, 2.3-3.8% M5, 2.9-3.6% M6, 2.2--2.6% M7, 2.4-3.2% M8, 2.8-3.3% Mg, and 64.8-67.9% > M1o. The highest value for M4 (1.85%) coincided with the lowest degree of /3-amylolysis (53.5%), and thus perhaps reflects that some even-numbered A chains were not completely degraded to maltose stubs, which in turn could explain the comparatively low molar ratio of M2/ M3 (0.94) in this case (Cal 5563A 1 amylopectin). Combined action of /3-amylase and pullulanase on /3-limit dextrin yielded mainly M2 and M3 (Table I) and some glucose (0 to trace), except for 0.91% glucose for Century Patna 231 waxy starch, and 1.00-3.06% high molecular weight fraction in all except Amylopectin + /3-amylase