Structure and Physicochemical Properties of Starches from Sieve Fractions of Oat Flour Compared with Whole and Pin-Milled Flour

Cereal Chem. 84(6):533-539 One Oat cultivar grown in Idaho (three field sites) was pin-milled and separated by sieving to investigate whether starch from oat bran differs from the remainder of kernel. Ground oat particles were classified into three sieve fractions: 300-850 pm, 150-300 om and <150 Om). Il-Glucan content in sieve fractions was analyzed and starch was extracted from kernels without milling and from kernels of each sieve fraction. -Glucan contents of 300-850. 150-300. and <150 pin fractions were 4.2, 2.3, and 0.8%, respectively. Therefore, starch in bran (300-850 pin fraction) and endosperm (< 150 om sieve fraction) were separated. Starch isolated from entire kernels had significantly higher apparent and absolute amylose content than starch from the 300-850 .tm sieve fraction. Starch from different sieve fractions was not significantly different in the apparent amylose, absolute amylose, amylopectin molecular weight, gyration radii, starch gelatinization, and amylose-lipid complex thermal transition temperatures. Starch from the 150-300 pin fraction had significantly lower peak, final, and setback viscosity compared with the starch isolated from the 300-850 om and 53 pin higher enthalpy change of starch gelatinization, but no difference in gelatinization temperatures, than those with particles <53 jtm (Scanlon et al 1988). Marshall (1992) showed that milled brown Cereal Products & Food Science Research Unit, National Center for Agricultural Utilization Research, ARS, USDA, 1815 N. University Street, Peoria. IL 61604. Names are necessary to report factually on available data: however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable. 2 Corresponding author. Phone: 309-688-6447. Fax: 309-681-6685. E-mail address: [email protected] Department of Food Science and Human Nutrition. 2312 Food Sciences Building, Iowa State University, Ames, IA 50011. doi:l 0.1 094/CCHEM-84-6-0533 This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. AACC International, Inc., 2007. rice with particle size <250 .im had lower onset gelatinization temperature compared with milled rice with particle size 250-1,400 jim. Milled rice particles <500 pin lower peak gelatinization temperature than particle size 500-1,400 pm, and particles <355 pin lower enthalpy change of gelatinization than particle size 355-1,400 p.m. Chen et al (1999) studying different methods of milling waxy rice found dry hammer-milled flour with particles of 197-215 p.m had the highest pasting temperature and semi-dry ground flour with particle sizes of 126-145 p.m had lowest pasting temperature and setback viscosity. Levels of 3-glucan in sieved fractions of barley have been studied (Yoon et a! 1995). They found finely ground barley had the highest 3-glucan content in flour with particle sizes 103-149 p.m and considerably lower content for flour particles >250 pin <103 Vim. Isolating starch from milled cereal sieve fractions and characterizing the structure and functional starch properties has had little investigation. Vasanthan and Bhatty (1995) purified starch from waxy, normal, and high-amylose barley after pin-milling and found fine sieve screens were successful at separating the small and large granules exhibited as the bimodal granule size distribution of barley starch. The most extensive studies of starch properties from sieved fractions have been conducted for corn (Dowd et al 1999) or wheat (Tang et al 2005) kernels. Gelatinization temperature and enthalpy change decreased for starch isolated from corn fibers (pericarp) compared with starch obtained from corn slurry (endosperm) during corn wet-milling. No differences in pasting viscosities were observed for all starch sources, but corn fiber and washed corn fiber starch pasted at higher temperature than starch from the corn wetmilling slurry. Apparent amylose content of starch from wheat fractions was similar, but gelatinization temperature varied. In this study, we investigate the structure and functional properties of starch separated in the oat bran fraction during milling, in which we expect to find different characteristics compared with starch found in the other mill fractions. Pin-milling oat kernels without sieving was included as a control treatment which, based on lack of reports, we expected would not alter starch structure or functional properties. However, alteration in starch structure was observed after pin-milling and these findings are also discussed. MATERIALS AND METHODS Plant Material, Milling, and Starch Isolation Oats (Avena sativa L. cv. Ajay), grown in 2003 at three different field sites (replicates) near Aberdeen, ID, were stored one to Vol. 84, No. 6, 2007 533 two months in a dry environment at 12°C, then ground three times separately for each replicate (1 kg of sample/milling) using a pin mill (Alpine, Kolloplex, Augsburg, Germany) for 6 mm, with no repeated milling. Each milled sample was sifted using a Rototap shaker developed by the USDA using sieve squares (40-cm dimension) shaken by a 3/4 horsepower motor from General Electric (Fort Wayne, IN) for 1 hr (3 x 20 mm, top fraction sieved 2nd and 3rd time) with a 20-min interruption involving cleaning of sieves and allowing sample to cool in between in a series of sieves consisting of 850-jim mesh (No. 20) on top, followed by a 300-jim mesh (No. 50), 150-pm mesh (No. 100), and a 75-pm mesh (No. 200). Starch was then isolated from sieved fractions, from pin-milled oats without sieving, and from intact kernels using the method by Kasemsuwan et al (1995) with further modification (Stevenson 2003) in which sieve fractions (150 g) or oat kernels (500 g) were initially ground in 1.5 L of 0.3% (w/v) sodium metabisulfite using a commercial blender (Waring, New Hartford. CT, high mode used) and filtered through a screen of 106-pm mesh. Filtrate was centrifuged at 10,400 x g for 40 mm, then the pellet was washed five to eight times (until supernatant was clear) with 1.5 L of 10% toluene (v/v) in 0.IM sodium chloride to remove protein and lipids, with at least 4 hr between washes. Starch was then washed three times with deionized water, then washed twice with ethanol, and recovered by filtration with Whatman No. 4 filter paper. Purified starch cake was dried in a convection oven at 35°C for 48 hr; final moisture of starch was 7-8%. All reagents used were obtained from Fisher Scientific (Pittsburgh, PA). Properties of Oat Starches 13-Glucan content of oat sieve fractions was measured using a 3-glucan diagnostic kit (Megazyme International Ireland Ltd., Wicklow, Ireland) based on Approved Method 32-23 (AACC International 2000) in which sieved fractions were hydrolyzed by lichenase and 3-glucosidase and resulting glucose measured using glucose oxidase/peroxidase reagent. Scanning electron microscopy (JOEL model 6400V, Tokyo. Japan) was used to observe oat starches at 1,500x magnification. Oat starch powders were spread on silver tape and mounted on a brass disk, then coated with gold/palladium (60:40) for all three replicates. Weight-average molecular weight (Mw) and z-average gyration radius (Ri) of amylopectin from oat starches were determined using high-performance size-exclusion chromatography equipped with multi-angle laser-light scattering and refractive index detectors (HPSEC-MALLS-RI). Oat starch (duplicate measurements of each of three ground samples for each replicate) was prepared as described by Yoo and Jane (2002a). The HPSEC system consisted of HP 1050 series isocratic pump (Hewlett Packard, Valley Forge, PA), multiangle laser-light scattering detector (Dawn DSP-F, Wyatt Technology, Santa Barbara, CA) and HP 1047A refractive index detector. To separate amylopectin from amylose, a Shodex OH pak SB-G guard column and SB-804 and SB-806 analytical columns (JM Science, Grand Island, NY) were used. Operating conditions and data analysis are described by Yoo and Jane (2002b), except that flow rate was 0.6 mL/min and sample injection concentration was 0.5 mg/mL. Apparent and absolute amylose contents of oat starches were determined following the procedure of Lu et al (1996). Analysis was based on iodine affinities of defatted whole starch and amylopectin fraction using a potentiometric autotitrator (702 SM Titrino, Brinkmann Instrument, Westbury, NY). Starch samples were defatted using a 90% dimethyl sulfoxide (DMSO) solution, followed by alcohol precipitation. Determination of amylose content was duplicated for starches of each of three ground samples of each oat replicate. Amylopectin was fractionated by the selective precipitation of amylose with n-butanol as described by Schoch (1942). Amylopectin (2 mg/mL) was defatted in 90% DMSO at 100°C for I hr, followed by stirring for 24 hr, and then debranched using isoamylase (EC 3.2.1.68 from Pseudomonas amyloderamosa) (EN 102, Hayashibara Biochemical Laboratories, Okayama, Japan) as described by Jane and Chen (1992). Branch chain length distribution of amylopectin was determined using an HPAEC system (Dionex300 and Dionex-GP50 gradient pump, Sunnyvale, CA) equipped with an amyloglucosidase (EC 3.2.1.3, from Rhizopus mold, A7255, Sigma Chemical, St. Louis, MO) postcolumn, online reactor and a pulsed amperometric detector (Dionex-ED50) (HPAEC-ENZPAD) (Wong and Jane 1997). PA-100 anion exchange analytical column (250 x 4 mm, Dionex) and a guard column were used for separating debranched amylopectin samples. Gradient profile of the eluents and operating conditions were described previously (McPherson and Jane 1999), except Chromeleon v.6.50 software was used. HPAEC-ENZ-PAD analysis was duplicated for starches from each of three ground samples of each oat replicate. Thermal properties of oat starches were determined using differential scanning calorimetry (DSC 2920 modulated, TA Instruments, New Castle, DE). Approximately 6 mg of oat product powder was weighed in a stainless steel pan, mixed with 18 mg of deionized water, and sealed. Sample was allowed to equilibrate for 2 hr and scanned at a rate of I O'C/min over a temperature range of 0-120°C. An empty pan was used as reference. Rate of starch retrogradation was determined using the same gelatinized samples, stored at 4°C for seven days, and analyzed using DSC as described previously (White et al 1989). All thermal properties were determined in triplicate for starches from each of three ground samples of each oat replicate. The pasting properties of oat starches were analyzed using a Rapid Visco-Analyser (RVA-4, Foss North America, Eden Prairie, MN) (Jane et al 1999). Oat starch suspension (8%, w/w), in duplicate for each of three ground samples of each oat replicate, was prepared by weighing oat starch (2.24 g, dsb) into an RVA canister and making up the total weight to 28 g with deionized water. Oat starch suspension was equilibrated at 30°C for I mm, heated at a rate of 6.0°C/mm to 95°C, maintained at 95°C for 5.5 mm, cooled to 50°C at a rate of 6.0°C/mm, and then maintained at 50°C for S mm. Constant paddle rotating speed (160 rpm) was used throughout the entire analysis except for a speed of 960 rpm for the first 10 sec to disperse sample. The standard error of the mean (RVU) for 300-850 jim, 150-300 jim, and <150 jim sieve fractions was 2.0, 1.1, and 1.5, respectively, for peak viscosity; 5.1, 2.9, and 2.4, respectively, for final viscosity; and 5.0, 3.0, and 2. 1, respectively, for setback viscosity. Statistical Analysis All statistical significance tests were calculated using SAS methods (SAS Institute, Cary, NC) and applying the Tukey difference test (Ramsey and Schafer 1996). RESULTS AND DISCUSSION Oat flour fractions separated by sifting were primarily collected using No. 50, 100, and 200 sieves. Therefore, only particles with diameter ranges of 300-850 jim. 150-300 pm, and <150 pm were studied. The percentage of collected flour fractions based on initial kernel weight for the 300-850 jim. 150-300 pm, and <150 pm sieve fractions collected were 34.1. 26.5. and 24.5%, respectively. Very few particles (<0.1%) passed through the No. 200 sieve. Percentage dry weight 3-glucan content for the oat sieve fractions 300-850 jim. 150-300 jim. and <150 pm were 4.2, 2.3, and 0.8%, respectively (3-glucan content is low because hull fragments are included). Therefore the sieves were successful in separating a considerably higher proportion of the 0-glucan-rich bran, allowing us to investigate whether starch from the oat bran (endosperm exterior) differs in structure and physicochemical properties when compared with starch from the remaining endosperm. 534 CEREAL CHEMISTRY Starch Granule Morphology Scanning electron micrography (SEM) of oat starch showed starch from entire kernels, pin-milled oats, and three sieve fractions had granule morphology consisting of larger spherical granules with diameter ranges of 7-9 gm, smaller spherical granules with a range of 3.5-5 j.tm, and many highly irregular granules (7:1 proportion of irregular to speherical granules). Some granules were dome-shaped, which combined with the highly irregular shaped granules, is the typical morphology of compound starches. Oat starch has previously been reported to be a compound (Jane et al 1994). Starch granules from all sieve fractions and entire kernels showed no difference. Amylose