Characterization of maize amylose-extender (ae) mutant starches. Part I: Relationship between resistant starch contents and molecular structures

Abstract

Endosperm starches were isolated from kernels of seven maize amylose-extender (ae) lines: three new ae-lines, derived from a Guatemalan breeding cross with pedigrees of GUAT209:S13 × (OH43ae × H99ae) B-B-4-1-2-1-1, GUAT209:S13 × (OH43ae × H99ae) B-B-4-4-2-1-1, and GUAT209:S13 × (OH43ae × H99ae) B-B-4-4-2-1-2, designated as GSOH1, GSOH2, and GSOH3, respectively, were developed by the USDA-ARS Germplasm Enhancement of Maize (GEM) Project, and four existing inbred lines, H99ae, OH43ae, B89ae, and B84ae. The resistant starch (RS) contents, measured using AOAC method 991.43 for total dietary fiber, showed that the three new-line starches had larger RS contents (39.4–43.2%) than the four inbred lines (11.5–19.1%). This study was conducted to understand relationship between the RS content and molecular structure of the maize ae-mutant starch. Analytical results showed that the three new-line starches had larger apparent (83.1–85.6%) and absolute amylose-contents (57.4–62.6%) than the starches of the inbred ae-lines (61.7–67.7% and 35.5–44.7%, respectively). The RS content of the ae-mutant starch was positively correlated with both the apparent and absolute amylose-contents of the starch with correlation coefficients of 0.99 and 0.96, respectively. Gel permeation chromatograms revealed that all seven ae starches contained large proportions of intermediate components (IC), 22.4%–52.0%. All seven ae starches displayed similar onset gelatinization temperatures (64.5–65.8 °C), but the three new-line starches displayed higher conclusion temperatures (122.0–130.0 °C) than the four inbred-line starches (100.5–105.3 °C). These results indicated that the crystalline structure of the three new ae-line starches was retained after boiling at ∼100 °C. The crystalline structure was resistant to enzyme hydrolysis and resulted in greater RS contents.

Introduction

Starch is the major energy reserve in higher plants and is the second largest biomass on earth next to cellulose. Starch is an important ingredient for food and nonfood applications; it serves not only as the major energy source in food and feed, but also as a thickener, a binding agent, a texturizer, a filler, a film forming agent, and feedstock for fermentation of biomaterial and fuel. Starch consists of two major components: amylose and amylopectin. Amylose has mainly linear molecules with α-1,4 linked d-glucosyl units and a few branches of α-1,6 linkages (French, 1973, Hizukuri et al., 1981). Amylopectin is a highly branched molecule, consisting of about 5% α-1,6 linkages (French, 1973).

Normal maize starch consists of 20–30% amylose. One of the maize mutants, amylose-extender (ae) mutant, produces starch with a much larger amylose-content and amylopectin with significantly longer branch-chains than the normal maize starch (Baba and Arai, 1984, Baba et al., 1982, Jane and Chen, 1992, Jane et al., 1999, Kasemsuwan et al., 1995, Shi and Seib, 1995, Takeda et al., 1993, Yuan et al., 1993). Because the long branch-chains of the amylopectin also bind iodine, form helical complex, and develop dark blue-color, which inflate the value of amylose-content. Thus, the apparent amylose-content of the maize ae-mutant starch is substantially larger than the absolute amylose-content. For the determination of absolute amylose-content, the iodine bound by amylopectin is subtracted from the total iodine bound by the starch (Jane et al., 1999). The very long branch-chains of the ae-mutant amylopectin are also known for developing the B-type crystallinity of the starch (Hizukuri et al., 1983, Kasemsuwan et al., 1995). These ae-mutant starches contain intermediate components (IC) that consist of branched molecules with molecular weights smaller than amylopectin but similar to amylose (Baba and Arai, 1984, Baba et al., 1982, Kasemsuwan et al., 1995, Wang et al., 1993). Studies have shown that starch with a larger amylose-content has less susceptibility to enzyme hydrolysis (Jane et al., 2003, Okuda et al., 2005).

In the human digestive system, a portion of starch cannot be digested and absorbed in the small intestine and is passed to the large intestine for bacteria fermentation. This starch is known as resistant starch (RS) (Englyst & Macfarlane, 1986). RS is classified into four types. Type I RS is starch that is entrapped in plant tissue and not susceptible for enzyme hydrolysis. Type II RS is native raw starch granules having the B-type polymorphism, such as potato, wrinkle pea, and high-amylose maize starches, which are resistant to enzyme hydrolysis. Type III RS is retrograded amylose (Englyst, Kingman, & Cummings, 1992), and Type IV RS is chemically modified starch (Woo & Seib, 2002). Studies have suggested that consumption of RS made from high-amylose maize starch brings a wide range of health benefits, such as lowering the glycemic index and promoting colon health (Sajilata, Singhal, & Kulkarni, 2006). Thus, it is important to understand the molecular structures and properties of the RS in the high-amylose maize starch.

Many methods have been reported in the literature for analyzing RS contents of different foods or raw material. Examples include Englyst’s method (Englyst et al., 1992) for prepared or ready-to-eat food products, and AOAC 2002.02/AACC 32–40 method (AOAC, 2005) for analysis of RS contents in raw starch and plant material. For human consumption, starch is used as an ingredient and is subjected to cooking. The resistant starch content of raw starch without cooking, therefore, is misleading and not meaningful. For example, the resistant starch content of raw potato starch is 63.39% (AOAC, 2005), but in practice, people do not eat raw potato. After cooking, the resistant starch content of freshly cooked potato is 3% (Englyst & Cummings, 1987). Because of this misleading result generated from the AOAC 2002.02 method, AOAC 991.43 method (AOAC, 2003) for total dietary fiber analysis is a preferred method for determination of RS content of starch samples (Seib and Woo, 1999, Shin and Seib, 2004, Shin et al., 2004). When isolated starch samples (purity >99%) were subjected to the AOAC 991.43 analysis, residues remained after thermal stable α-amylase hydrolysis at ∼100 °C for 30 min and subsequent glucoamylase hydrolysis are considered true resistant starch according to the AACC Dietary Fiber Definition Committee Report (AOAC, 2001) and other studies (Champ et al., 2003, Gray, 2006).

The objective of this study was to understand relationships between RS contents and structures of maize ae-mutant starches. Starches were isolated from kernels of four existing maize ae-mutant inbred lines and three new ae-lines that contained pedigrees of a Guatemalan line (Campbell, Jane, Pollak, Blanco, & O’Brien, 2007) and were developed from the USDA-ARS GEM project at Truman State University. The RS, amylose, amylopectin, and IC-contents of the starches, the molecular weights and the branch structures of the amylopectin and IC, and the thermal properties of the mutant starches were analyzed. The relationship between the RS contents and the starch structures were proposed and discussed.