Comparison of Amylose Determination Methods and the Development of a Dual Wavelength Iodine Binding Technique

It has long been recognized that limitations exist in the analytical methodology for amylose determination. This study was conducted to evaluate various amylose determination methods. Purified amylose and amylopectin fractions were obtained from corn, rice, wheat, and potato and then mixed in proportion to make 10, 20, 30, 50, and 80% amylose content starch samples for each source. These samples, considered amylose standards, were analyzed using differential scanning calorimetry (DSC), high-performance size-exclusion chromatography (HPSEC), and iodine binding procedures to generate standard curves for each of the methods. A single DSC standard equation for cereal starches was developed. The standard curve of potato starch was significantly different. Amylose standard curves prepared using the iodine binding method were also similar for the cereal starches, but different for potato starch. An iodine binding procedure using wavelengths at 620 nm and 510 nm increased the precision of the method. When HPSEC was used to determine % amylose, calculations based on dividing the injected starch mass by amylose peak mass, rather than calculations based on the apparent amylose/amylopectin ratio, decreased the inaccuracies associated with sample dispersion and made the generation of a cereal amylose standard curve possible. Amylose contents of pure starch, starch mixtures from different sources with different amylose ranges, and tortillas were measured using DSC, HPSEC, iodine binding, and the Megazyme amylose/amylopectin kit. All the methods were reproducible (±3.0%). Amylose contents measured by these methods were significantly different (P < 0.05). Amylose measurements using iodine binding, DSC, and Megazyme procedures were highly correlated (correlation coefficient >0.95). DSC and traditional iodine binding procedures likely overestimated true amylose contents as residual butanol in the amylose standards caused interference. The modified two-wavelength iodine binding procedure seemed to be the most precise and generally applicable method. Each amylose determination method has its benefits and limitations.     

Starch-Lipid Interactions in Common,Waxy, ae du,and ae su2 Maize Starches Examined by Differential Scanning Calorimetry

Four maize genotypes (common, waxy, ae du, and ae su2) were examined by differential scanning calorimetry (DSC) in the presence of four surfactants (sodium dodecyl sulfate [SDS], dodecyltrimethylammonium bromide [DOTAB], sodium hexadecyl sulfate [SHS], 1-monolauroyl-rac-glycerol [ML]) to characterize the starch-lipid complexes produced and provide evidence of differences in starch structure. The ionic surfactants with a C₁₂ hydrocarbon tail reduced the gelatinization onset temperature, a phenomenon that does not occur typically with neutral surfactants or with surfactants with longer hydrocarbon tails. Subtracting the DSC curves, the exotherm we suspected was caused by starch-lipid complexation, which occurs concomitantly with the gelatinization endotherm, was identified and provided evidence of the existence of amylopectin-lipid interactions. Apparent starch amylose content correlated well with enthalpies of amylose-lipid complexes. Complexes formed from DOTAB produced DSC endotherms that were broad and shallow and that shifted to lower temperatures as the DOTAB concentration increased. This was in contrast to other surfactants that normally produce amylose-lipid complex endotherms at temperatures independent of surfactant concentration.     

Morphological Changes of Granules of Different Starches by Surface Gelatinization with Calcium Chloride

Native starch granules of 11 selected cultivars (potato, waxy potato, sweet potato, normal maize, high‐amylose maize, waxy maize, wheat, normal barley, high‐amylose barley, waxy barley, and rice) were treated with a calcium chloride solution (4M) for surface gelatinization. The surface‐gelatinized starch granules were investigated using light microscopy and scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). In general, those starches with larger granule sizes required longer treatment time to complete the gelatinization. The salt solution treatment of starch was monitored by light microscopy and stopped when the outer layer of the granule was gelatinized. The surface gelatinized starch granules were studied using scanning electron microscopy. On the basis of the gelatinization pattern from calcium chloride treatments, the starches could be divided into three groups: 1) starches with evenly gelatinized granule surface, such as normal potato, waxy potato, sweet potato, maize, and high‐amylose maize; 2) starches with salt gelatinization concentrated on specific sites of the granule (i.e., equatorial groove), such as wheat, barley, and high‐amylose barley; and 3) starches that, after surface gelatinization, can no longer be separated to individual granules for SEM studies, such as waxy barley, waxy maize, and normal rice. The morphology of the surface gelatinized starch resembled that of enzyme‐hydrolyzed starch granules.     

Enzymatic Hydrolysis of Barley Starch Lipids

The efficiency of phospholipase and lipase preparations in the hydrolysis of lysophospholipids of native and gelatinized barley starch was examined. The degree of hydrolysis was analyzed by determination of the amount of released fatty acids by an enzymatic method. Thermal and structural properties of the enzyme-treated starch were studied by differential scanning calorimetry and light microscopy. Lysophospholipids of the gelatinized barley starch were easily hydrolyzed, in contrast to the lipids of the granular starch. The maximum degree of hydrolysis achieved for the gelatinized starch was 80% and for the native starch ≈20%. Gelatinization enthalpies and micrographs indicated that even though the amount of the released fatty acids from the native starch was small, formation of free fatty acids inhibited swelling and gelatinization of starch granules.     

Association of Starch Granule Proteins with Starch Ghosts and Remnants Revealed by Confocal Laser Scanning Microscopy

Starch suspensions (0.25%) were gelatinized to 70 and 100°C, and starch ghosts (defined as gelatinized starch granule envelopes after the majority of internal starch polymers have been released) and remnants were collected by centrifugation and washed with water. Protein was revealed in isolated gelatinized normal starch ghosts using confocal laser scanning microscopy and a protein‐specific dye that fluoresces only after reaction with primary amines in protein. This technique eliminates background interference from residual dye. Observation of fluorescent‐labeled protein in the starch ghosts at different optical depths of field revealed that protein was concentrated in the envelopes of swollen, gelatinized potato, maize, and wheat starch ghosts. Only traces of protein were found in gelatinized starch granule remnants of waxy maize and amylose‐free potato starches after they were heated to 100°C, indicating that the proteins observed in gelatinized normal maize starch were largely granule‐bound starch synthase (GBSS). Moreover, fragility of the gelatinized waxy and amylose‐free starch granule remnants might be caused in part by the lack of GBSS. Gel electrophoresis of proteins in starch ghosts confirmed that GBSS in potato and maize was tightly associated with the starch ghosts. The study provides a structural explanation for a role of granule‐associated proteins in maintaining the integrity of starch ghosts and remnant structures, and their consequent effect on paste rheology.     

Effects of Heat-Moisture Treatment and Lipids on Gelatinization and Retrogradation of Maize and Potato Starches

Effects of heat-moisture treatment (HMT) and lipids on the structure and gelatinization of maize and potato starches were studied, and the retrogradation process of 20% HMT starch gels was also investigated. Maize starch was physically modified by HMT or by defatting. Potato starch was physically modified by HMT or by adding monoglycerides. The X-ray pattern of the HMT maize starch was assigned to a combination of A and V patterns, which indicated that HMT formed crystallized amylose complexes and recrystallized amylose in maize starch granules. However, the X-ray pattern of defatted maize starch did not change for HMT, so the lipids originally existing in starch granules were important to the formation of new crystallites during this treatment. Differential scanning calorimetry (DSC) results suggested that weaker structures in amylopectin crystallites were more susceptible to degradation after HMT, while crystallized amylose complexes developed thermal stability after treatment. The amylose contents increased with increasing degree of HMT, which suggested that the newly created amylose arose from exterior linear chains of amylopectin degraded by the treatment. Investigation of retrogradation process showed that HMT significantly promoted retrogradation of starch gels, especially the initiation of recrystallization.     

Gelatinizing,Pasting, and Gelling Properties of Potato and Amaranth Starch Mixtures

Physicochemical properties of mixtures of native potato and native amaranth (Amaranthus cruentus), heat‐moisture treated (HMT) potato and heat‐moisture treated amaranth, cross‐linked potato and cross‐linked amaranth, native potato and heat‐moisture treated amaranth, and heat‐moisture treated potato, and native amaranth were tested at different ratios. Two peaks were noticed in the pasting curves when large differences of swelling factor and amylose leaching existed between individual components in the mixture. It seems that amylose leaching from one starch in a mixture may affect the swelling and much of the granular break down of the other. The mixtures showed stabilities in hot pastes that were higher than the less stable components in a mixture. Some mixtures such as HMT potato and native amaranth showed very specific nonadditive pasting behavior. Mixing 10% of native amaranth to HMT potato starch caused a large reduction of peak viscosity and cold paste viscosity, resulting in a very soft gel. In the differential scanning calorimeter, each component of a mixture gelatinized independently, showing two peaks corresponding to the individual components. When transition temperatures of both components were similar in DSC, the result was a single endotherm. Dramatic changes of pasting and subsequent gel properties resulted when thermal transition of the two components occurred in the same temperature range. Retrogradation enthalpies as measured by DSC were between the two individual components in all tested mixtures.     

Inhibition of buckwheat starch digestion by the formation of starch/bile salt complexes: possibility of its occurrence in the intestine

During the digestion of starch in foods, starch is mixed with bile in the duodenum. Because fatty acids and some kinds of polyphenols could bind to starch, it was postulated that bile salts might also bind to starch. The purpose of this paper is to study the effects of bile and bile salts on starch/iodine complex formation and pancreatin-induced starch digestion. Bile suppressed starch/iodine complex formation and inhibited pancreatin-induced starch digestion slightly in control buckwheat starch, but did so significantly in buckwheat starch from which fatty acids and polyphenols had been extracted. Such significant suppression and inhibition by bile were also observed in a reagent soluble starch. The effects of cholate and taurocholate on the starch/iodine complex formation and the pancreatin-induced starch digestion were essentially the same as those of bile. Bile, cholate, and taurocholate suppressed amylose/iodine complex formation more significantly than amylopectin/iodine complex formation and inhibited pancreatin-induced amylose digestion more effectively than the digestion of amylopectin. It is concluded from the results that bile salts could bind to starch, especially amylose, the helical structures of which were not occupied by other molecules such as fatty acids and polyphenols, and that the binding resulted in the inhibition of starch digestion by pancreatin. The conclusion suggests that the function of bile salts can be discussed from the point of not only lipid digestion but also starch digestion.     

Effect of Rice Starch-Lipid Complexes on In Vitro Digestibility,Complexing Index,and Viscosity

Effects of nonwaxy (21% amylose, 79% amylopectin) and waxy (100% amylopectin) rice starch-lipid complexes on the rate of in vitro digestibility were determined. Long-chain (≥C:18) saturated emulsifiers reduced digestibility more than short-chain (<C:18) saturated and unsaturated emulsifiers when complexed with nonwaxy and waxy rice starch. The largest decrease in digestibility (33%) was achieved with Polyaldo 10-1-2 (100% C18:0 with decaglyceryl monostearate modification) for nonwaxy rice. Waxy rice starch did not complex with most of the emulsifiers, in contrast to nonwaxy rice starch. Most of the emulsifiers that reduced digestibility by 10% or less were composed of unsaturated monoglycerides, including some acetylated and succinylated monoglycerides. The fluid behavior of nonwaxy rice starch-emulsifier solutions was more pseudoplastic than waxy rice starch-emulsifier solutions. The consistency index varied with emulsifiers. The nonwaxy rice starchemulsifier solutions and some of those prepared using waxy rice starch would be suitable for semisolid food applications. The waxy rice starchemulsifier solutions with low consistency (0.4–0.7) and high-flow behavior (0.7–0.8) indices would be suitable for beverage applications.     

Influence of physicochemical interactions between amylose and aroma compounds on the retention of aroma in food-like matrices

In food matrices, where starch is often used as a gelling or texturing agent, the occurrence of amylose-aroma complexes and their effect on the release of aroma compounds are difficult to determine. Indeed, thick or gelled systems are known to reduce the diffusion rate of flavor molecules, resulting in an increase of retention. Moreover, interactions between aroma compounds and matrix components might increase the retention of aroma compounds. The complexing behavior of three aroma compounds with amylose was studied by DSC and X-ray diffraction to determine the relative importance of these two factors. Their interaction properties were different: two of them formed complexes, and the third did not. These aroma compounds were added in food matrices containing different starches that induced different textures. Their retention was studied by static headspace analysis. The retention of aroma compounds appeared to depend on the amylose/amylopectin ratio of starch, both from the formation of complexes and by a viscosity effect.     

Characterization of water binding and dehydration in gelatinized starch

Using near-infrared spectroscopy and thermogravimetry coupled with differential scanning calorimetry (TG-DSC), we investigated the characteristics of water in starch and the effects of the inner structure of starch on dehydration. The results directly show that the dehydration process is significantly more favorable in native starch than in gelatinized starch. When the starch was heated to 100 °C, the water retention in gelatinized starch was 22.35 per total water content, much greater than that in native starch (4.3%). The hydrogen bond network that changes from native starch to gelatinized starch was simultaneously explored, and the weaker hydrogen bonds were found to be predominant in the hydrogen bond network of gelatinized starch.     

Morphologies and Thermal Properties of Hydroxypropylated High‐Amylose Corn Starch

High‐amylose (80%) corn starch was modified by hydroxypropylation with different molar substitution (MS). The unique microstructure of high‐amylose starch keeps its granules intact after hydroxypropylation. However, the microstructures and thermal properties strongly depend on the MS of hydroxypropylation. With increasing MS, the granule size was increased, which is partly due to disrupted granule structure, particularly in the amorphous region. Unlike normal starch, the modified high‐amylose corn starch showed a narrow gelatinization range measured by differential scanning calorimetry (DSC), which can be explained by destruction of amylose‐lipid complex. Internal microstructures and morphologies of hydroxypropylated starch were investigated using confocal laser scanning microscopy and to further explore the mechanism of chemical reaction and phase transitions.     

Influence of storage conditions on the structure, thermal behavior, and formation of enzyme-resistant starch in extruded starches

Starch structures from an extrusion process were stored at different temperatures to allow for molecular rearrangement (retrogradation); their thermal characteristics (DSC) and resistance to amylase digestion were measured and compared. The structure of four native and processed starches containing different amylose/amylopectin compositions (3.5, 30.8, 32, and 80% amylose content, respectively) before and after digestion was studied with small-angle X-ray scattering (SAXS) and X-ray diffraction (XRD). Rearrangement of the amylose molecules was observed for each storage condition as measured by the DSC endotherm at around 145 degrees C. The crystalline organization of the starches after processing and storage was qualitatively different to that of the native starches. However, there was no direct correlation between the initial crystallinity and the amount of enzyme-resistant starch (ERS) measured after in vitro digestion, and only in the case of high-amylose starch did the postprocess conditioning used lead to a small increase in the amount of starch remaining after the enzymatic treatment. From the results obtained, it can be concluded that retrograded amylose is not directly correlated with ERS and alternative mechanisms must be responsible for ERS formation.     

Amylopectin Staling of Cooked Milled Rices and Properties of Amylopectin and Amylose

Starches of waxy rices that showed varietal differences in hardness testing of cooked rice after amylopectin staling and high-amylose content (AC) rices differing in gel consistency (GC) and starch gelatinization temperature (GT) were studied to determine the factors related to varietal differences in amylopectin staling of cooked rice. Intermediate- and high-GT starches showed greater amylopectin staling of gelatinized rice by hardness testing values or differential scanning calorimetry (DSC) endotherm than did low-GT starches in both waxy and nonwaxy rices. Isoamylase-debranched amylopectins of waxy rices differed in the ratio of weight-average degree of polymerization (DP_w) fractions, but these fraction ratios were not simply related to differences in amylopectin staling of cooked rice. Among high-AC rices, amylopectin from low-GT starch was confirmed to have higher iodine affinity (2.3–2.5%) than amylopectin from intermediate-GT starches (1.7–1.8%), regardless of GC. Within high-AC starch of the same GT type, soft-GC rice corresponded with more A + B₁ DP_w 16–18 and less B₃ DP_w 150–200 fractions of debranched amylopectin and low DP_w of amylose. Amylopectin of amylose extender mutant of IR36 was confirmed to have a longer chain length than ordinary rice amylopectin: the debranched amylopectin has more B₂ DP_w 47–51 fraction, less A + B₁ DP_w fraction, but no B₄ fraction with DP_w > 200. Only high-AC amylopectin had debranched fraction with DP_w >120.     

冻结-高压湿热解冻对多种淀粉中支链淀粉断裂的影响

冻结和高压湿热解冻是含淀粉面团在食品加工过程的必要工艺,在此过程中淀粉球破裂、部分支链淀粉断裂成为直链淀粉,给面团和食品的物理性状控制带来许多不可预见的结果。该文通过研究加热时间、加热温度、冷冻时间和解冻时间对糊化后的小麦、甘薯、马铃薯和玉米淀粉中直链淀粉含量的影响,间接反映支链淀粉断裂情况,结合冻结解冻前后淀粉分子量分布、链长分布、光学和电子显微镜图谱提出了冻结-解冻过程不同淀粉中支链淀粉可能断裂方式。结果表明,4种淀粉中的直链淀粉含量先升高后下降,直链淀粉含量在4种淀粉的中达到峰值的时间分别为48,48,48,72 h。光学显微照片观察表明,冻融处理会导致更多凝胶化淀粉球的破裂。对于小麦支链淀粉,冻融解冻过程支链淀粉中侧链长度为5、6、7个葡萄糖残基的侧链对应3种可能的断裂方式:2+2+1、2+2+2及2+2+2+1;对于甘薯支链淀粉,支链淀粉中侧链长度为10、11和13个葡萄糖残基的侧链对应3种可能的断裂方式:3+3+4、2+2+3+4及2+2+2+3+4;对于马铃薯支链淀粉,支链淀粉中侧链长度为5和6个葡萄糖残基的侧链对应3种可能的断裂方式:2+3、2+4、3+3;而玉米支链淀粉中,支链淀粉中侧链长度为7、8、9个葡萄糖残基的侧链对应3种可能的断裂方式:2+5,3+5,和3+3+3(其中1表示1个葡萄糖;2表示含2个葡萄糖的麦芽糖、3表示含3个葡萄糖的麦芽多糖、4表示含4个葡萄糖的麦芽多糖和5表示含5个葡萄糖的麦芽多糖)。该论文结果为培育具有冻融稳定性的淀粉种子提供一种全新的思路,即通过基因方法控制植物减少容易断裂淀粉侧链的合成。     

Evidence of starch inclusion complexation with lactones

Starch, in particular the linear amylose, is able to form inclusion complexes with a wide spectrum of ligand molecules, among them flavor compounds. The complexing ability of a homologous series of gamma- and delta-lactones with potato starch was followed by amperometric iodine titration, differential scanning calorimetry, and wide-angle X-ray diffraction measurements. Lactones with a linear chain of a size > or = C(5) form inclusion complexes with starch, whereas lactones with a short linear chain, such as gamma-heptalactone, show poor complexing ability. The thermal stability of starch-lactone complexes increases with increasing chain length of the lactone. In general, lactones induce the formation of V(h) helices. Only delta-decalactone complexes with starch were not definitely identified as V(h) amylose helices. Complexation of starch dispersions with lactones induce turbidity and gelation or phase separation, both phenomena being the result of microphase separation.     

Granular Cold Water Gelling Starch Prepared from Chickpea Starch Using Liquid Ammonia and Ethanol

Granular cold water gelling (GCWG) starch was obtained by treatment of native chickpea starch with liquid ammonia at low temperature and atmospheric pressure. A free‐flowing powder of the gelatinized starch granules was produced by adding ethanol to the mixture of starch and liquid ammonia. Four ratios of liquid ammonia to starch (A/S) (2:1, 4:1, 6:1 and 8:1) and four ratios of starch ethanol (S/E) (1:1, 1:2, 1:3 and 1:4) were tested for production of GCWG starch. Homogenous mixtures of liquid ammonia and starch granules appeared when the A/S ratio was >4:1. Treating starch with a 4:1:3 ammonia‐starch‐ethanol (A/S/E) ratio on a w/w/v basis resulted in a free‐flowing powder of gelatinized starch, that formed a gel on addition of water at 23°C. Differential scanning calorimetry (DSC) thermograms of the GCWG starch, regardless of A/S/E ratio, displayed enthalpy values of 2.2–3.1 J/g compared with 15.5 J/g for native starch, indicating disappearance of crystallinity due to modification.     

Gelatinization,Pasting, and Gelling Properties of Sweetpotato and Wheat Starch Blends

Sweetpotato starch is high yielding but has very limited uses. It is possible to expand its application by blending it with other starches to obtain novel properties. In this study, functional properties of the blends of native sweetpotato starch with native, acid‐thinned, and hydroxypropylated wheat starch were studied at different ratios (75:25, 50:50, 25:75). The swelling factor, extent of amylose leaching, pasting, and gel textural properties of the blends were nonadditive of their individual components, and could be mathematically modeled by quadratic equations in relation to the ratios. Two peaks during pasting were observed for some starch mixtures studied by Rapid ViscoAnalyser (RVA). The gelatinization and retrogradation enthalpies (ΔH) of the blends were additive of their individual components and could be modeled by linear equations. All starch mixtures exhibited two peaks during differential scanning calorimetry (DSC) scan for gelatinization, but a single peak for retrograded starches. This study may provide basis for formulation of mixtures using starch from diverse sources to develop more natural starch systems with a range of physicochemical properties.     

Rheological Properties of Dispersions of Spherulites from Jet‐Cooked High‐Amylose Corn Starch and Fatty Acids

High‐amylose corn starch was cooked in an excess‐steam jet cooker in the presence of 5% oleic or palmitic acid, based on amylose. The cooked product was rapidly cooled in an ice bath and then freeze‐dried or drum‐dried. Amylose was removed from solution by forming helical inclusion complexes with the fatty acid, and the inclusion complexes formed submicron spherical particles upon cooling. The dried material was reconstituted to form a paste that exhibited gel‐like properties upon standing, but that flowed readily when shear was applied. The rheological properties of these pastes were measured to determine the effects on the flow properties of 1) the solids concentration in the reconstituted paste, 2) the method of sample drying and reconstitution, and 3) the fatty acid used. The materials were very spreadable, and at the highest concentrations their flow properties were similar to a commercial shortening. The pasting properties of the dried solids were also examined.     

Discovery of an amylose-free starch mutant in cassava (Manihot esculenta Crantz)

One of the objectives of the cassava-breeding project at CIAT is the identification of clones with special root quality characteristics. A large number of self-pollinations have been made in search of useful recessive traits. During 2006 harvests an S1 plant produced roots that stained brownish-red when treated with an iodine solution, suggesting that it had lower-than-normal levels of amylose in its starch. Colorimetric and DSC measurements indicated low levels (3.4%) and an absence of amylose in the starch, respectively. SDS-PAGE demonstrated the absence of GBSS enzyme in the starch from these roots. Pasting behavior was analyzed with a rapid visco-analyzer and resulted in larger values for peak viscosity, gel breakdown, and setback in the mutant compared with normal cassava starch. Solubility was considerably reduced, while the swelling index and volume fraction of the dispersed phase were higher in the mutant. No change in starch granule size or shape was observed. This is the first report of a natural mutation in cassava that drastically reduces amylose content in root starch.