Effects of Heat Treatment of Barley Starches on In Vitro Digestibility and Glucose Responses in Rats'

Starches were purified from barley flours milled from Waxbar, Glacier, high-amylose Glacier (HAG) and hull-less high-amylose Glacier (HHAG) cultivars. Wheat starch, maize amylopectin, maize amylose, and normal maize starch were used for comparative controls. Starches were either boiled or moisture-autoclaved (3 or 12 times) with subsequent cooling overnight, after which enzyme-resistant starch (ERS) was measured. In vitro digestibility and hydrolysis rates over time were determined. Postprandial glucose responses in rats were investigated with starches from Waxbar, Glacier, HAG, and wheat. Production of ERS varied from 0.6% in waxy starches to 18.6% in the high-amylose barleys, compared to 44.2% in maize amylose starch. Boiling of starches produced only marginal effects on digestibility and hydrolysis rates, and no effects on blood Both rate and extent of starch hydrolysis in vitro are regarded as predictors of metabolic responses to complex carbohydrate in vitro (O'Dea and Holm 1985). It has been well established that starch hydrolysis differences correlate with postprandial blood glucose changes in humans under experimental conditions (Brand et al 1985, Bornet et al 1989, Lund and Johnson 1991). Amylomaize starch has been shown to be less susceptible to amylolysis than normal maize (Dreher et al 1984). High-amylose maize starch has been shown to have a positive lowering effect on blood glucose and insulin levels, as well as triglycerides and cholesterol in humans (Behall et al 1988, 1989; Amelsvoort and Weststrate 1992). Goddard et al (1984) observed similar responses from high-amylose rice. Behall et al (1989) concluded that longterm intake of a high-amylose starch may benefit individuals with elevated glucose and insulin levels and apparent insulin resistance, as in early adult-onset diabetes and for hyperlipidemic subjects. High-amylose barley starch purified from the high-amylose mutant of Glacier (HAG) was also found to be less susceptible to ox-amylase than was normal barley starch (Pomeranz et al 1972). Calvert et al (1976) reported that rats consumed less of a purified diet prepared with HAG starch than of a similar diet containing starch prepared from normal Glacier (NG) and, consequently, gained at a slower rate. In another study, Calvert et al (1981) fed diets prepared with HAG and NG to growing swine and found that barley starch type did not significantly affect gain or feed consumption. Rubin et al (1974) demonstrated HAG to be nutritionally superior to five other barleys, including Glacier, as measured by growth of weanling rats. Newman et al ( 1978) confirmed these findings and suggested that the superiority of the HAG was due to a higher lysine content of HAG protein as compared to NG protein. 'Contribution J-3080, Montana Agricultural Experiment Station. Partial funding for this research was supplied by the Montana Wheat and Barley Committee, Great Falls. 2 Metabolism and Nutrient Interactions Laboratory, Beltsville Human Nutrition Research Center, USDA-ARS, Beltsville, MD 20705. Department of Plant, Soil, and Environmental Sciences, Montana State University, Bozeman, MT 59717-0312. Corresponding author. Fax: 406/994-3933. 4 Montana Agricultural Experiment Station, Montana State University, Bozeman, MT 59717-03 12. Publication no. C-1996-0805-07R. © 1996 American Association of Cereal Chemists, Inc. 588 CEREAL CHEMISTRY glucose levels in rats. Autoclaving, however, produced significant differences in digestibility and blood glucose responses between starch types. Digestibility of waxy starches was not changed in vitro (P > 0.05), but blood glucose in rats was increased (P < 0.05) after ingestion of autoclaved Waxbar barley starch. In contrast, digestibilities of HAG and HHAG starches were reduced by 14 and 20% (P < 0.001) after 3 and 12 autoclaving-cooling cycles. Autoclaved HAG starch significantly lowered glucose peaks in rats compared to Waxbar and Glacier starches at 30 min (P < 0.01). The in vivo results corresponded to the in vitro study, which demonstrated that digestibilities of different cereal starches followed the pattern: waxy > normal > high-amylose starches after heat-moisture autoclaving, possibly due to the formation of ERS from the amylose component. Xue et al (1991) showed that blood glucose levels in broiler chicks fed two uncooked (raw) milling fractions (flour and red dog) from HAG and HHAG barleys were not significantly flattened when compared with that of NG barley milling fractions and were higher than that of the maize control. Plasma cholesterol, however, was significantly reduced in chicks fed the high-amylose flour and red dog fractions. Bjorck et al (1990) reported that the amylose-to-amylopectin ratio in different barley genotypes (waxy, normal, and high-amylose starch) produced no differences in enzymatic hydrolysis when the barley flour was boiled. Autoclaving the flour, however, produced a concomitant decrease for in vitro digestibility with increased amylose content. This was explained by an increased amount of ERS (3%) and the formation of amyloselipid complexes. The dietary fiber content of barley, largely in the form of (1->3),(1->4) mixed linked P-D-glucans (,B-glucans), may contribute to the glycemic effect. Dietary fiber is known to flatten glucose curves in humans (Jenkins and Jenkins 1985). Oat ,B-glucan has been reported to lower blood glucose and insulin in rats (Vachon et al 1988) and humans (Wood et al (1994 ). Barley [B-glucan has been shown to lower cholesterol in hypercholesterolemic individuals (Newman et al 1989). In addition to the high-amylose content, high-amylose Glacier barley is also relatively high in f-glucan (7%). Therefore, effects of DF and other components in the barley may confound starch effects on glucose responses when whole barley meal is fed. The objectives of these studies were to: 1) estimate the formation of ERS during the autoclaving process with different ratios of amylose and amylopectin in barley and maize starches; 2) determine in vitro digestibility and hydrolysis rate of barley and maize starches with different amylose and amylopectin content and heat treatments (autoclaved vs. nonautoclaved); and 3) evaluate the glucose responses of autoclaved barley starch compared to wheat starch in rats. MATERIALS AND METHODS Barley Flour Waxbar, a waxy hull-less barley, Glacier, a covered normal starch barley, and two high-amylose mutants of Glacier, covered and hull-less, were grown in Arizona in 1990. The barleys were Cereal Chem. 73(5):588-592 milled through a MIAG MULTOMAT eight-roll dry mill at the Western Wheat Quality Laboratory (Washington State University, Pullman, WA). Flour was made up of 2nd break (B), 3rd B. 1st middling (M), 2nd M, 3rd M, and 4th M streams (2B-4M, 2nd break flour to 4th middling) from each cultivar. Starch Isolation Starches were isolated from barley flours (2B-4M) by a modification of the method of Morrison and Laignelet (1983) and Szczodrak and Pomeranz (1991). After steeping briefly in 0.02M HCl and neutralizing with 0.02M NaOH to pH 7.6, the starch suspensions were incubated for 24 hr at 370C with protease (5 mg/g, Type XIV, Sigma) and 0.01% thimerosal (T512, Sigma) to free the starch. The suspension was filtered through 88and 61-[tm mesh sieves to remove the fiber. The residue was homogenized (homogenizor, Brinkmann Instruments, Co. Westbury, NY) with water and screened again. The filtered suspension was centrifuged at 2,000 rpm for 15 min, and the supernatant was discarded. The brown and white layers were combined and purified with an 8:1 mixture of water and toluene by a shaking procedure (McDonald and Stark 1988). As a result of this treatment, the denatured protein and dissolved fat were concentrated in the supernatant toluene layer, while the purified starch granules precipitated in the aqueous layer. The toluene layer was separated from the aqueous layer, and the procedure was repeated until a completely clear toluene layer was obtained. The wet starches were air-dried. TABLE I Yield and Chemical Composition of Starches Sample Yield Starcha Amyloseb Protein Lipid Ash Waxbar barley 51.3 94.1 7.3 0.05 0.13 0.09 Glacier barley 56.0 92.6 29.4 0.16 0.07 0.21 HAG barleyc 50.8 92.3 40.3 0.25 0.07 0.25 HHAG barleyd 43.3 92.6 40.5 0.19 0.04 0.22 Wheat (S5127)e ... 90.4 25.0 0.03 ... 0.18 Maize amylopectin * 93.6 ... 0.05 0.03 0.10 (A7780)e Maize normal ... 94.2 25.0 0.09 0.05 0.06 (S4126)e Maize amylose * 74.3 70.0 0.69 0.01 0.09 (A7043)e a Dry matter (%). bStarch (%) values provided by Sigma Chemical Co. (St. Louis, MO). c High-amylose Glacier. d Hull-less high-amylose Glacier. e Sigma product number in parenthesis. Wheat starch (unmodified), maize amylopectin, maize starch, and maize amylose were purchased from Sigma Chemical Co., St. Louis MO. Analyses of purified starches were made for protein, ether extract, ash (AOAC 1984) and starch (Aman and Hesselman 1984). Amylose percentage was determined with gel filtration chromatography (Torneport et al 1990). Starch Treatment (Formation of ERS) The formation and determination of ERS were according to the methods described by Szczodrak and Pomeranz (1991). The starch and water ratio used for ERS formation was 1:5. The suspension was autoclaved for 1 hr at 121'C using a thermostatically controlled autoclave and cooled overnight at 40C. Starches were subjected to 3 and 12 autoclaving-cooling cycles. The treated samples were vacuum-dried from -40'C to room temperature (freeze dryer, FTS Systems Co., Stone Ridge NY). All the materials were ground on a Wiley mill (A. H. Thomas Co., Philadelphia, PA) to pass a screen with 0.5-mm diameter openings. ERS Determination ERS was estimated by an enzymatic-gravimetric assay as described by Sievert and Pomeranz (1989). It was considered as the residue remaining after incubation of the sample with a heat-stable a-amylase (A-3306, Sigma) and amyloglucosidase (from Aspergillus niger, A-9913). This assay was a modification of the AOAC method for the determination of insoluble dietary fiber (AOAC 1984). TABLE II Yield of Enzyme Resistant Starch from Autoclaving-Cooling Cyclesa