Amylose was probably the first biopolymer for which a helical structure was proposed (Hanes, 1937). The well-known deep-blue complex formed with iodine was later proved to involve amylose in a helical conformation (Rundle and French, 1943), details of which have been a matter of dispute until the present time (Nimz et al., 2003; Calabrese and Khan, 1999; Minick et al., 1991; Ziegast and Pfannemuller, 1982). The color and intensity of the complex depending on the chain length (CL) of the amylose (Bailey and Whelan, 1961; Banks et al., 1971; Manners and Stark, 1974; John et al., 1983). At CL > 80 the wavelength maximum (λmax) of the absorption of light is >610 nm and is typical for amylose. λmax shifts to lower wavelengths and the color shifts to red for shorter chains (Bailey and Whelan, 1961). Thus, the short chains of amylopectin possess a λmax at 530-575 nm (Archibald et al., 1961; Shibanuma et al., 1994; Takeda et al., 1999; Hung and Morita, 2007; Annor et al., 2014b; Waduge et al., 2014; Gayin et al., 2016b). The blue value (Gilbert and Spragg, 1964) (BV) is defined as the absorbance at 680 nm (though sometimes measured at 640 nm) of 1 mg starch in 100 mL of a mixture containing 2 mg I2 and 20 mg KI. For a “true” BV, the absorbance should be multiplied with the factor 4 (because old colorimeters used 4-cm cuvettes rather than 1-cm cuvettes in modern instruments). BV for amylose (Takeda et al., 1984, 1986, 1999; Takeda and Preiss, 1993; Shibanuma et al., 1994; Nilsson et al., 1996; Schulman et al., 1995) is 1.01-1.63, whereas that for amylopectin (Takeda and Preiss, 1993; Shibanuma et al., 1994; Takeda et al., 1999; Nilsson et al., 1996; Schulman et al., 1995) is 0.08e0.38. Though BV is easy to measure, it should be considered mainly as a qualitative test for amylose.
The most frequently used quantitative test for amylose is to measure the iodine affinity (IA) potentiometrically (Schoch, 1964). An automated amperometric titration method was also described (Takeda et al., 1987b). In most cases, approximately 20 g of iodine is bound per 100 g amylose, in contrast to only 0.5-1.1 g iodine/100 g amylopectin (Takeda et al., 1986, 1987b, 1999; Takeda and Preiss, 1993; Schulman et al., 1995). Assuming the IA value of amylose is 20, the amylose content is calculated from IA of a defatted starch sample as (Takeda et al., 1987b):
% Apparent amylose = % IAstarch/20 X 100
It should be noted that the amylopectin component in some starches, notably high-amylose starches, possesses unusually long chains (Klucinec and Thompson, 1998; Baba and Arai, 1984; Montgomery et al., 1964; Takeda et al., 1993c; Bradbury and Bello, 1993), which increases IA for the starch and leads to an overestimation of the amylose content. Also, the amylopectin of indica varieties of rice contains unusually high levels of long chains (Takeda et al., 1987b). It is, however, possible to correct the obtained apparent amylose values to true values if the IA of the purified amylopectin is taken into account (Takeda et al., 1987b):
% Amylose = (IAstarch -IAamylopectin)(IAamylose – IAamylopectin)x100
A considerable part of the amylose in many starches, especially cereal starches, is complexed with lipids, mainly lysophospholipids (Fredriksson et al., 1998; Morrison et al., 1984, 1993; Andersson et al., 1999). These lipide-amylose complexes (LAM) have no affinity for iodine. A colorimetric determination of LAM and the total (true) amylose content was described by Morrison and Laignelet (1983). In this, starch granules are dissolved in hot 90% DMSO-10% 6 M urea, treated with a mixture of I2 and KI, and the absorbance at 635 nm is measured from which the fraction of apparent amylose (corresponding to free amylose, FAM) is obtained. A part of the dissolved sample is defatted in ethanol before the measurement is repeated to obtain the total amylose. In both cases the content is calculated from the general equation:
% Amylose = (28.414 X absorbance) – 6.218
LAM is obtained as the difference between the total amylose and FAM (Morrison and Laignelet, 1983).
Chrastil (1987) developed an iodine-binding method suitable for both defatted starch and flour. A stable absorbance at 620 nm was obtained by developing the amylose-iodine complex in acidic conditions using trichloroacetic acid. A rapid method for the estimation of amylose in maize starches, including high-amylose starch, was developed by Knutson and Grove (1994). The starch granules are gelatinized without heat in 3 M CaCl2 and then further sonicated at 60-70°C in an iodine-DMSO mixture. After dilution in water, the absorbance is read at 600 nm and the amylose content is determined from a standard curve. A microscale method was described by Mohammadkhani et al. (1998), in which only 2-3 cereal seeds are needed for the amylose determination. Zhu et al. (2008) recommended a method in which the absorbance of the amylose-iodine complex is measured at both 620 and 510 nm. More recently, Kaufman et al. (2015) developed a 96-well plate IA assay suitable for screening large sets of samples. Campbell et al. (2002) suggested that near-infrared transmittance spectroscopy could partly replace the iodine-staining methods and enable rapid screening of apparent amylose contents in breeding programs.
Amylose can also be quantified using the lectin concanavalin A. As mentioned above, the lectin forms an insoluble complex with amylopectin and possible intermediate, branched molecules. The amylose content in starch is obtained as the difference between the carbohydrate content in the solution before and after precipitation of the amylopectine-concanavalin A complex (Gibson et al., 1997). This method is also applicable to flour samples if the total starch and amylose concentrations are measured enzymatically using α-amylase and amyloglucosidase (Gibson et al., 1997). The released glucose is then usually measured with a glucose oxidase-peroxidase method (commonly known as GOPOD) (Gibson et al., 1997; Karkalas and Tester, 1992).
Amylose contents were also estimated by size-fractionation of starches using GPC (Wang et al., 1993a; Blennow et al., 2001; Boyer et al., 1980) or high-performance size-exclusion chromatography (HPSEC) (Bradbury and Bello, 1993; Kobayashi et al., 1985; Mua and Jackson, 1995; Simsek et al., 2013). There is a risk, however, that solubilization problems related to the starch components affect the result (Chen et al., 1997; Gidley et al., 2010). Other problems may arise from the shear scission of amylopectin in the column (Gidley et al., 2010; Cave et al., 2009). Though no baseline separation of amylose and amylopectin normally is obtained, GPC on a column packed with TSK HW75 S was found to be the most accurate among several different amylose determination methods (Gerard et al., 2001). The fraction of amylose in GPC is identified by measuring the BV (Krisman, 1962) of the collected fractions (Wang et al., 1993a; Gayin et al., 2016b; Manelius and Bertoft, 1996; Vamadevan et al., 2014), or online by postcolumn iodine injection in the case of HPSEC (Suortti and Pessa, 1991; Pessa et al., 1992; Autio et al., 1992). An alternative method is to enzymatically debranch the starch sample before GPC (Fredriksson et al., 1998; Bertoft et al., 2008; Wang et al., 1993a; Sargeant, 1982) or HPSEC (Bradbury and Bello, 1993; Wang and White, 1994a). The long amylose chains are eluted from the column before the short chains of the amylopectin (Karkalas and Tester, 1992). Also in this case, however, a baseline separation between the two fractions is difficult to achieve.