W.R. Morrison

An improved colorimetric procedure for determining apparent and total amylose in cereal and other starches

An improved method for the colorimetric determination of amylose as its blue polyiodide complex is described. Starch is dissolved in urea—dimethylsulphoxide and aliquots of the solution are used to determine apparent amylose (measured in the presence of any amylose complexing monoacyl lipids which may be present) and total amylose (measured on lipid-free starch, precipitated from urea—dimethylsulphoxide solution with ethanol). When factors such as ionic concentration, sequence and timing of steps in the procedure and solution temperature are controlled, the coefficient of variation in replicate determinations ( n = 6) is 0·2–10·0%, except with waxy starches, where it is 10·6–10·8%. The limitations of the method and sources of error in published methods for the preparation of lipid-free starches and in the development of stable amylose-polyiodide colour complexes are discussed. In some wheat and non-waxy rice starches the difference between total and apparent amylose is 30·5–7·4% amylose.

Lipids in cereal starches: A review

Starches in the endosperm of cereals may be unique in having appreciable quantities of monoacyl lipids inside the starch granules. These lipids are almost exclusively lysophospholipids in wheat, barley, rye and triticale, but include substantial proportions of free fatty acids (FFA) in other cereals. In addition, the starch granules may acquire surface lipids, which are mostly FFA, from the endosperm non-starch lipids. Methods for isolating suitably purified starch are discussed and lipid composition data are given. The relationship between amylose content and lipid content in wheat, barley, maize and rice starches is described and the biological significance of the lipids is discussed. The question whether lipids exist as inclusion complexes with amylose in native starch granules or not is debated, but cannot be resolved conclusively with present experimental techniques.

A relationship between the amylose and lipid contents of starches from diploid cereals

Amylose and lipids were quantified in 29 maize, 31 barley, 29 rice, 6 oat, 12 millet and 8 sorghum samples. The low-amylose waxy maize, waxy barley and waxy rice samples had very low contents of lipids compared with the normal starches, whereas the high-amylose maize and barley starches had elevated levels of lipids. In barley starch these lipids were mostly lysophospholipids with traces of free fatty acids; more balanced proportions of free fatty acids and lysophospholipids were found in the other starches. The fatty acid compositions of the total lipids in all non-waxy starches (using mean values for each cereal) were 36–55% palmitic, 2–3% stearic, 4–28% oleic, 29–46% linoleic and 1–4% linolenic acids. There were significant positive correlations between amylose and lipid contents in starches from maize, barley or rice covering a large range of amylose content, and in oat starches. However, there were no correlations between amylose and lipid contents over the limited range of amylose contents found in the normal maize, barley, rice, or sorghum and millet starches, or within the high-amylose maize or barley starches. It is suggested that there is a fundamental relationship between amylose and lipids in starches from each of these cereal species that is subject to disturbance by various factors.

SOME FACTORS DETERMINING THE THERMAL PROPERTIES OF AMYLOSE INCLUSION COMPLEXES WITH FATTY ACIDS

Abstract The thermal properties of water-insoluble amylose-stearic acid (18:0) complexes prepared under various conditions were studied by differential scanning calorimetry (DSC). Complexes were studied normally at a concentration of 5% in water at pH ∼7. Type I complexes formed at ≤ 60°C had dissociation temperatures (Tm) in the range 96–104°C. Type IIa polymorphs formed at ≥ 90°C had Tm = 114–121°C. Various ratios of types I and IIa were formed at 80°C depending on the duration of heating, but no intermediate form was detected. Annealing of the type IIa complex at 105°C and at 115°C gave rise to increasing proportions of type IIb polymorphs with Tm = 121–125°C and dissociation enthalpies of 32–34 J/g of amylose, depending on the temperature and time of annealing. Conversion into the higher polymorphs was retarded at a higher concentration (10%) of the complex under identical conditions, and was delayed at pH ∼ 4.7. The dissociation temperatures of amylose complexes with the cis-unsaturated fatty acids oleic (18:1), linoleic (18:2), and linolenic (18:3) also depended on the temperature of formation, and three distinct types were obtained (I, IIa, and IIb). Significant decreases in the Tm of the three polymorphs were observed for each double bond in the fatty acid guest molecule. When type I and type II complexes were made using various proportions of 18:0 and 18:2, mixed acid complexes were obtained with Tm values intermediate between those of the monoacid complexes. The origin of the endothermic transitions on heating the three types of complexes is discussed.

The effects of ambient temperature during the grain-filling period on the composition and properties of starch from four barley genotypes

Starch was isolated from four genotypes of barley (one waxy, two normal and one high-amylose) grown at constant ambient temperatures of 10, 15, and 20 °C. There was evidence of physiological stress in grain grown at the higher temperatures, notably reduced starch accumulation, smaller A- and B-granules and fewer B-granules. In the waxy and normal genotypes amylose (AM) and amylopectin (AP) contents were little affected by increasing ambient temperature, but the lipid contents of the starches did show a strong response. No differences in the fine structure of AP in response to temperature were detected. Starch gelatinization temperatures (GT) were 50–55 °C, 52–62 °C and 60–63 °C in starches from grain grown at 10, 15 and 20 °C, respectively. Since the native starches could all be annealed to give similar elevated GT values (72–73 °C, the range of GT in the native starches was probably due to differences in the perfection of AP crystallinity which increased with barley growth temperature. All starches showed marked differences in their swelling curves which clearly related to ambient temperature. Swelling began at the same temperature as gelatinization (loss of crystalline order), At 80 °C, when there was no detectable order, swelling was determined primarily by AP content (45·3–97·6% in the 12 starches) but it was markedly inhibited by lipid (a temperature-dependent variable) probably through a mechanism which involved formation of AM-lipid inclusion complexes.

The amylose and lipid contents of starch granules in developing wheat endosperm

Starch was isolated from three varieties of wheat at various stages of grain development (endosperm filling) and separated by sedimentation into eight fractions according to granule size. Starch granule dimensions were measured using a Coulter Counter with 100-Channel analyser, and whole wheat and starch granule compositions were determined. At most stages of development the numbers of Agranules/endosperm remained constant while granule size increased, but the numbers of B-granules/endosperm increased throughout grain development. The amylose content (% dry weight) increased with granule size and with granule maturity, whereas lysophospholipid content (dry weight basis) generally decreased with granule size but increased with granule maturity. Starch deposited on individual large A-granules at successive stages of development contained higher proportions of amylose and lysophospholipids than initial deposits. Similarly, small B-granule starch synthesised at successive stages of grain development contained more amylose and lysophospholipids than the earlier B-granule starch. It was calculated that there should be gradients of amylose and lysophospholipid composition along the outer radii of A-granules, and similar changes in successive populations of B-granules. It is concluded that there was a close relationship between amylose content and lysophospholipid content in the developing A-granules and in the developing B-granules in the starch of each wheat variety, but that this relationship was always different for the A- and B-granules.

Effects of elevated growth temperature and carbon dioxide levels on some physicochemical properties of wheat starch

Abstract Crops of winter wheat (cv. Hereward) were grown in the field under double-skinned polyethylene tunnels in two consecutive seasons (1991–92 and 1992–93). Air containing ambient (350 ppm) or elevated (700 ppm) concentrations of carbon dioxide was circulated through the tunnels, and temperature gradients, typically from 1°C below ambient to 4–7°C above ambient, were maintained within each tunnel. Despite a shorter crop duration and warmer temperatures in the first season, most grain and starch properties showed a similar response to temperature between seasons. Thousand grain weight and grain starch content declined with increase in temperature (from 55±5 mg to 18±2 mg, and from 31±3 mg to 7±2 mg, respectively), the latter reflecting both decreases in granule sizes and fewer amyloplasts per endosperm. Contents of total amylose and lipid-free amylose increased with temperature (from 26±1% to 31±1%, and from 21±1% to 25±1%, respectively), but the contents of lipid-complexed amylose (5·2±1·5%) and lysophospholipids (0·9±0·2%) varied independently of temperature. Starch gelatinisation temperatures ranged from 57·5 to 64·5°C in the first season, and from 58·0 to 61·9°C in the second season, increasing with increase in temperature in both seasons, the data for the two seasons providing almost separate clusters. Gelatinisation enthalpy was constant in the first season (12·6±1 J/g amylopectin) and in the second season (15·5±0·5 J/g amylopectin) with no effect of temperature. The differences in carbon dioxide concentration had no consistent effects on the parameters measured, but small effects were discernible on thousand grain weight, starch content and lipid-free amylose content. There were also effects in certain treatment combinations, specifically at warmer temperatures in the first season and at cooler temperatures in the second season, on thousand grain weight, non-starch solids and lipid-complexed amylose contents.

Resistance to acid hydrolysis of lipid-complexed amylose and lipid-free amylose in lintnerised waxy and non-waxy barley starches

Abstract Waxy barley starches (0.8–4.0% lipid-complexed amylose = L·AM, 0.9–3.4% lipid-free amylose = F·AM) and non-waxy barley starches (6.1–7.2% L·AM, 23.1–25.9% F·AM) were lintnerised by steeping in 2 M HCl at 35°C for 140 h. Material solubilised from the waxy starches was estimated to be 70.7% of their amylopectin (AP) plus 3.7% of their L·AM and F·AM, and material solubilised from the non-waxy starches was estimated to be 70.7% of their AP plus 28.9% of their L·AM and F·AM. The polysaccharide components of the insoluble residue were characterised by HPLC, GPC, and λ max of the polyiodide complex. I was concluded that short chain-length (CL 16) material was from external chains of AP, intermediate material (modal CL 46) was from retrogated F·AM, and longer chain residues (CL 77, 120–130_ were from lipid-complexed segments of L·AM. The starch lysophospholipids were completely hydrolysed to free fatty acids which remained complexed with L·AM residues. This was shown by the 13 C CP/MAS-NMR spectrum which had a clear resonance at 31 ppm from mid-chin methylene carbons of fatty acids in complexes. The C-1 signal of the L·AM residues also included a feature at 104 ppm indicative of single V 6 AM helices. The wide-angle X-ray diffraction patterns of the residues of non-waxy starches were Cc-type ( = mixed A + B types), whereas the spectra of the original starches were A-type. It is suggested that, during the early stages of lintnerisation, amorphous (F·AM was partially hydrolysed into material (CL 13 C CP/MAS-NMR spectra, which were consistent with a mixture of double helices and V-type glycosidic conformations, with only a small proportion of non-ordered regions. Broad DSC endotherms were found for both waxy (50–110°C) and non-waxy (50–110°C) lintner residues, which were assigned to disordering of double helices from short chains (modal CL 16) for waxy residues, together with disordering of longer chains (modal CL 46) in double-helix residues of F·AM and also V-helix residues of L·AM for non-waxy starch residues.

Solid state13C NMR investigation of lipid ligands in V-amylose inclusion complexes

The characteristics of the ligands in inclusion complexes formed from stearic, palmitic, oleic, linoleic, linolenic and docosahexaenoic acids, glycerol monooleate (GMO), glycerol monopalmitate (GMP) and lysophosphatidylcholine (LPC) have been studied by13C NMR in dry and hydrated forms of the complexes, with13C labels being used for the car☐yl and C-1(3) glycerol carbons in stearic acid and GMO, respectively.13C NMR provides definitive proof that V-amylose inclusion complexes have been formed with the mono-car☐ylic fatty acids of varying degrees of unsaturation, GMO, GMP and LPC. The chemical shift of the mid-chain methylenes in stearic acid moves about 1.5 ppm upfield upon complexation with the1H rotating frame relaxation times becoming identical for the lipid and amylose. With the exception of docosahexaenoic acid, the mid-chain methylenes inside the V-helical segments have essentially the same chemical shift for all the other unsaturated fatty acids and lipids investigated. The cross-polarisation dynamics for the car☐yl and glycerol groups in stearic acid and GMO, respectively, have indicated that these bulky polar groups occupy highly mobile conformations in the hydrated complexes which must lie outside the V-helical segments adjacent to the amorphous domains.

Measurement of the dimensions of wheat starch granule populations using a Coulter Counter with 100-channel analyzer

A method is described for measuring the size distribution of wheat starch granules, using a Coulter Counter model ZM, 100-channel analyzer and a log range expander coupled to a computer. The raw data (counts per channel) showing the characteristic biomodal starch granule size distribution could be described by two overlapping normal distributions for the small B-granule and large A-granule populations respectively. Hydrated starch granule dimensions, including mean and modal volumes and diameters, specific surface areas, and volume or weight percentage A- and B-granules, were calculated from the raw data or from normalised data. The method is precise and reproducible, and it has been used to demonstrate varietal differences and edaphic and climatic effects on starch granule dimensions.