The azidation of starch

Starch is an inexpensive commodity that has been used for non-food purposes for many years. Some of these uses include cross-linked starches that are synthesized with a variety of multifunctional reagents. One unexplored possibility is the use of azides for cross-linking. To this end, azide derivatives of different starches have been synthesized, including the first reported synthesis of 6-deoxy-6-azido-amylopectin. Lithium salts, which were found to not be essential for the dissolution of starch in the reaction, were replaced with sodium azide. The time for this derivatization reaction to reach completion was determined to be 1 h. N,N-dimethylacetamide was also found to be a suitable solvent. Initial experiments suggest that the azide derivative does cross-link starch when activated by heat.     

Banana starch structure and digestibility

It is well known that raw banana starch is a good source of resistant starch. Less is known, however, regarding the digestion property of gelatinized banana starch. In this study, banana starch cooked for 20 min in excess water had a significant fraction of slowly digestible starch (19%), as well as resistant fraction (27%). Amylopectin is thought to be responsible for its slow digestion property, since banana starch studied here has a relatively low amylose content of 11.2%. Chain-length distribution analysis revealed that banana amylopectin has a significantly different structure from corn or potato amylopectin in that it has a higher proportion of very long chains. Retrogradation studies support the view that banana starch retrogrades at a substantially faster rate than corn or potato starch leading to less digestible cooked starch. Additionally, banana starch has unique pasting properties making it behave like a chemically lightly cross-linked starch. Banana starch is unique, both nutritionally and functionally, to warrant further investigation on potential commercial uses.     

Genetic elimination of a starch granule protein, SGP-1, of wheat generates an altered starch with apparent high amylose

A starch granule protein, SGP-1, is a starch synthase bound to starch granules in wheat endosperm. A wheat lacking SGP-1 was produced by crossing three variants each deficient in one of three SGP-1 classes, namely SGP-A1, -B1 or -D1. This deficient wheat (SGP–1 null wheat) showed some alterations in endosperm starch, meaning that SGP-1 is involved in starch synthesis. Electrophoretic experiments revealed that the levels of two starch granule proteins, SGP-2 and -3, decreased considerably in the SGP-1 null wheat though that of the waxy protein (granule-bound starch syn- thase I) did not. The A-type starch granules were deformed. Apparent high amylose level (30.8–37.4%) was indicated by colorimetric measurement, amperometric titration, and the concanavalin A method. The altered structure of amylopectin was detected by both high- performance size-exclusion chromatography and high-performance anion exchange chromatography. Levels of amylopectin chains with degrees of polymerization (DP) 6–10 increased, while DP 11–25 chains decreased. A low starch crystallinity was shown by both X-ray diffraction and differential scanning calorimetry (DSC) analyses because major peaks were absent. Abnormal crystallinity was also suggested by the lack of a polarized cross in SGP-1 null starch. The above results suggest that SGP-1 is responsible for amylopectin synthesis. Since the SGP-1 null wheat produced novel starch which has not been described before, it can be used to expand variation in wheat starch. Received: 30 April 1999 / Accepted: 9 November 1999     

植物体内淀粉磷酸化的生物学研究进展

植物中淀粉是主要储存碳水化合物形式,是食品和工业应用中最重要的植物原材料之一。而植物淀粉中惟一的取代是磷酸化作用,更体现了淀粉的独特性能。淀粉的品质和理化性质影响其应用。因此对淀粉的研究是有重要意义的。主要介绍了淀粉磷酸化作用机制,概述GWD功能与磷酸化作用和淀粉代谢的关系的生物学研究进展,并在此基础上讨论利用基因工程改良淀粉品质的可能途径以及今后的研究任务。     

Molecular fractionation of starch by density-gradient ultracentrifugation

Amylose and amylopectin in corn and potato starches were fractionated by centrifugation at 124,000g for 3-72 h at 40 degrees C in a gradient media, Nycodenz, based on their sedimentation rate differences. The fractions were collected from a centrifuge tube, and then analyzed by the phenol-sulfuric acid method and iodine-binding test. Amylopectin, a large and highly branched starch molecule, migrated faster than amylose and quickly reached its isopycnic point with a buoyant density of about 1.25 g/mL, exhibiting a sharp and stable carbohydrate peak. Amylose, which is a relatively small and linear molecule, however, migrated slowly in a broad density range and continued moving to higher density regions, eventually overlapping with amylopectin peak as the centrifugation continued. This could indicate that the buoyant density of amylose is similar to that of amylopectin. Under centrifugal conditions of 3 h and 124,000g, amylose and amylopectin molecules were clearly separated, and the presence of intermediate starch molecules (11.5 and 7.7% for corn and potato starch, respectively) was also observed between amylose and amylopectin fractions. The amylose content of corn and potato starches was 22.6 and 21.1%, respectively, based on the total carbohydrate analysis after the ultracentrifugation for 3 h. In alkaline gradients (pH 11 or 12.5), the sedimentation rate of starch molecules and the buoyant density of amylopectin were reduced, possibly due to the structural changes induced by alkali.     

Physico-chemical characterisation of sago starch

The physico-chemical characteristics of various sago starch samples from South East Asia were determined and compared to starches from other sources. X-ray diffraction studies showed that all the sago starches exhibited a C-type diffraction pattern. Scanning electron microscopy showed that they consist of oval granules with an average diameter around 30 μm. Proximate composition studies showed that the moisture content in the sago samples varied between 10.6% and 20.0%, ash between 0.06% and 0.43%, crude fat between 0.10% and 0.13%, fiber between 0.26% and 0.32% and crude protein between 0.19% and 0.25%. The amylose content varied between 24% and 31%. The percentage of amylose obtained by colourimetric determination agreed well with the values obtained by fractionation procedures and potentiometric titration. Intrinsic viscosities and weight average molecular weight were determined in 1M KOH. Intrinsic viscosity for amylose from sago starches varied between 310 and 460 ml/g while for amylopectin the values varied between 210 and 250 ml/g. The molecular weight for amylose was found to be in the range of 1.41×10⁶ to 2.23×10⁶ while for amylopectin it was in the range of 6.70×10⁶ to 9.23×10⁶. The gelatinisation temperature for the sago starches studied varied between 69.4°C and 70.1°C. The exponent ‘a’ in the Mark–Houwink equation and the exponent ‘’ in the equation R_g=kM was found to be 0.80 and 0.58, respectively for amylose separated from sago starch and these are indicative of a random coil conformation. Two types of pasting properties were observed. The first was characterised by a maximum consistency immediately followed by sharp decrease in consistency while the second type was characterised by a plateau when the maximum consistency was reached.     

Recent developments of analytical ultracentrifugation in biopolymer research

The development of the analytical ultracentrifuge as a full on-line computer controlled device enables an ever broader application of these ultracentrifuge methods for characterizing macromolecular substances. In this short review some of the numerous possibilities for analyzing biopolymers with respect to solution conformation, conformational changes, association behavior, and homologous and heterologous interactions (including the thermodynamics) are discussed. Most of the results presented here are from the last five years and should be helpful for researchers to get an insight into the structure function relationships of biopolymers. Accepted: 18 October 1996     

萌发中食松幼苗淀粉合酶同工酶与淀粉成分的关系

利用14C－ADPG标定法测定可溶性及与淀粉粒结合的淀粉合酶活性,采用过氯酸抽提、DMSO玻璃纤维纸层析、硫酸水解法定量测定各类淀粉成分,探讨了食松（PinusedulisEngelm）幼苗生长过程中淀粉合酶与淀粉成分间的关系。结果表明,在胚根出现以后,淀粉含量迅速增加,伴随着淀粉颗粒数目和质量的增加,两类淀粉合酶活性的增加以及淀粉合酶免疫印迹图谱的变化。支链淀粉是食松淀粉的主要成分,占总淀粉的84％。可溶性淀粉合酶峰值比淀粉粒结合的淀粉合酶活性峰值高1．3倍,与支链淀粉和直链淀粉的比例相对应。结果支持食松可溶性淀粉合酶是负责支链淀粉合成的主要酶的假说,同时表明淀粉粒结合的淀粉合酶在支链淀粉的合成中也有作用。     

基因工程改良淀粉品质

淀粉对人类生活十分重要,它不仅是人们的能量和营养来源,而且还是重要的工业原材料。对于淀粉合成过程及淀粉的加工、使用一直是淀粉研究的重点内容。淀粉的合成在最后阶段涉及到３个关键性的酶是：ＡＤＰＧ焦磷酸化酶、淀粉合成酸以及淀粉分支酶。它们分别催化ＡＤＰ－葡萄糖的形成、葡聚糖链的延伸以及分支链的形成。另外淀粉去分支酶对淀粉最终结构的形成也起到重要作用。本文将介绍上述４个酶近年来的生物化学和分子生物学研究     

Relationship Between Isozymes of Starch Synthases and Starch Composition in Germination Pinyon Seedlings

The relation between starch synthases and starch composition in the germinating pinyon ( Pinus edulis Engelm) seedlings was studied. Using the method of 14C-glucose transferred from 14C-ADPG in the assay of starch synthases activity. Starch was extracted with 32% HC1O4, separated on glass fiber with DMSO, and assayed with the sulfuric acid-phenol method. After the emergence of radicle, starch content increased rapidly accompanied with the increase of starch grains in number and size, the increase of both soluble and granulebound starch synthase activity and the change of the pattern of Western-blot. Amylopectin was the major composition in pinyon starch, accounted for 84% of the total starch. The activity of soluble starch synthase was 1.3 times higher than that of the granule-bound starch synthase, corresponding to the ratio of amylopectin to amylose. This result supports the conventional theory that soluble starch synthase is the major enzyme responsive for the synthesis of amylopectin, and also supports that granule-bound starch synthase is functional in the synthesis of amylopectin.     

The molecular deposition of transgenically modified starch in the starch granule as imaged by functional microscopy

The molecular deposition of starch extracted from normal plants and transgenically modified potato lines was investigated using a combination of light microscopy, environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy (CLSM). ESEM permitted the detailed (10 nm) topographical analysis of starch granules in their hydrated state. CLSM could reveal internal molar deposition patterns of starch molecules. This was achieved by equimolar labelling of each starch molecule using the aminofluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS). Starch extracted from tubers with low amylose contents (suppressed granule bound starch synthase, GBSS) showed very little APTS fluorescence and starch granules with low molecular weight amylopectin and/or high amylose contents showed high fluorescence. Growth ring structures were sharper in granules with normal or high amylose contents. High amylose granules showed a relatively even distribution in fluorescence while normal and low amylose granules had an intense fluorescence in the hilum indicating a high concentration of amylose in the centre of the granule. Antisense of the starch phosphorylating enzyme (GWD) resulted in low molecular weight amylopectin and small fissures in the granules. Starch granules with suppressed starch branching enzyme (SBE) had severe cracks and rough surfaces. Relationships between starch molecular structure, nano-scale crystalline arrangements and topographical-morphological features were estimated and discussed.     

Effect of high temperature on fine structure of amylopectin in rice endosperm by reducing the activity of the starch branching enzyme

Rice (Oryza sativa L.) grain quality is affected by the environmental temperature it experiences. To investigate the physiological molecular mechanisms of the effect of high temperatures on rice grain, a non-waxy indica rice was grown under two temperature conditions, (29/35 degrees C) and (22/28 degrees C), during the ripening stage in two phytotrons. The activities and gene expression of key enzymes for the biosynthesis of amylose and amylopectin were examined. The activity and expression levels of soluble endosperm starch synthase I were higher at 29/35 degrees C than that at 22/28 degrees C. In contrast, the activities and expression levels of the rice branching enzyme1, the branching enzyme3 and the granule bound starch synthase of the endosperm were lower at 29/35 degrees C than those at 22/28 degrees C. These results suggest that the decreased activity of starch branching enzyme reduces the branching frequency of the branches of amylopectin, which results in the increased amount of long chains of amylopectin of endosperm in rice grain at high temperature.     

Starch branching enzyme IIb in wheat is expressed at low levels in the endosperm compared to other cereals and encoded at a non-syntenic locus

Studies of maize starch branching enzyme mutants suggest that the amylose extender high amylose starch phenotype is a consequence of the lack of expression of the predominant starch branching enzyme II isoform expressed in the endosperm, SBEIIb. However, in wheat, the ratio of SBEIIb and SBEIIa expression are inversely related to the expression levels observed in maize and rice. Analysis of RNA at 15 days post anthesis suggests that there are about 4-fold more RNA for SBE IIa than for SBE IIb. The genes for SBE IIa and SBE IIb from wheat are distinguished in the size of the first three exons, allowing isoform-specific antibodies to be produced. These antibodies were used to demonstrate that in the soluble fraction, the amount of SBE IIa protein is two to three fold higher than SBIIb, whereas in the starch granule, there is two to three fold more SBE IIb protein amount than SBE IIa. In a further difference to maize and rice, the genes for SBE IIa and SBE IIb are both located on the long arm of chromosome 2 in wheat, in a position not expected from rice–maize–wheat synteny.     

Extrusion processing of high amylose potato starch materials

Thermoplastic starch was prepared by mixing native high amylose potato starch and normal potato starch in a Buss co-kneading extruder at starch to glycerol ratios of 100:45 and 100:30. The materials were also conditioned to different moisture contents at different relative humidities at 23 °C. After the mixing, the compounds were extruded into sheets with a Brabender laboratory extruder. The thermoplastic high amylose materials exhibited a higher melt viscosity than the normal potato starch materials when conditioned at 53% relative humidity. Increasing the moisture content in HAP from 27% to 30% (by weight) lowered the melt viscosity to the same level as that of normal potato starch with a moisture content of 28%. In general, the high amylose materials were more difficult to extrude than the thermoplastic material based on normal starch. The main extrusion problems encountered with the high amylose starch were unstable flow, insufficient melt tenacity and clogging of the die. By increasing the moisture content, increasing the compression ratio of the screw and increasing the rotation rate of the screw, the problems were reduced or eliminated. However, only with a starch to glycerol ratio of 100:45 was an acceptable extrusion result obtained. Extruded sheets of such high amylose materials had a stress at break of about 5 MPa at room temperature and 53% relative humidity, whereas the corresponding value for normal potato (thermoplastic) starch was 3 MPa. The elongation at break was also higher in the case of the high amylose material. The results are discussed in terms of residual crystallinity of the starch materials.     

The influence of alterations in ADP-glucose pyrophosphorylase activities on starch structure and composition in potato tubers

In order to examine whether alterations in the supply of precursor molecules into the starch biosynthetic pathway affected various characteristics of the starch, starch was isolated from potato (Solanum tuberosum L.) tubers containing reduced amounts of the enzyme ADP-glucose pyrophosphorylase (AGPase). It was found that although the type of crystalline polymorph in the starch was not altered, the amylose content was severely reduced. In addition, amylopectin from the transgenic plants accumulated more relatively short chains than that from control plants and the sizes of starch granules were reduced. The starch granules from the transgenic plants contained a greater amount of granule-bound starch synthase enzyme, which led to an increase in the maximum activity of the enzyme per unit starch tested. The K _m for ADP-glucose was, at most, only slightly altered in the transgenic lines. Potato plants containing reduced AGPase activity were also transformed with a bacterial gene coding for AGPase to test whether this enzyme can incorporate phosphate monoesters into amylopectin. A slight increase in phosphate contents in the starch in comparison with the untransformed control was found, but not in comparison with starch from the line with reduced AGPase activity into which the bacterial gene was transformed. Received: 2 February 1999 / Accepted: 25 March 1999     

A comparative study of edible canna (Canna edulis) starch from different cultivars. Part II. Molecular structure of amylose and amylopectin

Molecular structures of starches isolated from Japanese-green, Thai-green and Thai-purple cultivars of edible canna (Canna edulis Ker) were investigated. The absolute amylose content ranged from 19 to 25%. Degrees of polymerization (DPn) values of amylose determined by fluorescence-labeling method were 1590 for Thai-purple, 1620 for Japanese-green and 1650 for Thai-green cultivars. Mole% of branched fraction of amyloses from edible canna starches examined by a HPLC system after β-amylolysis of labeled amyloses was 13–16%. Branch chain-length distributions of amylopectin analyzed by HPSEC after debranching with isoamylase, followed by fluorescence-labeling of unit chain, showed bimodal distribution with the DPn range of 25–28. The amylopectin of edible canna starches contained high amounts of organic phosphorus (391–420 ppm). The distribution profile of phosphorylated chains, separated from non-phosphorylated chains by DEAE-Sephadex A-50 chromatography, indicated that the phosphate groups were located mostly in long B-chains of amylopectin molecules.     

Gel formation in mixtures of high amylopectin potato starch and potato starch

The effects of starch granules on the rheological behaviour of gels of native potato and high amylopectin potato (HAPP) starches have been studied with small deformation oscillatory rheometry. The influence of granule remnants on the rheological properties of samples treated at 90 °C was evident when compared with samples treated at 140 °C, where no granule remnants were found. The presence of amylose in native potato starch gave to stronger network formation since potato starch gave higher moduli values than HAPP, after both 90 and 140 °C treatments. In addition, amylose may have strengthened the network of HAPP because higher moduli values were obtained when native potato starch was added to the system. The moduli values of the mixtures also increased with increasing polysaccharide concentration in the system, which is due to an increment in the polysaccharide chain contacts and entanglements. Finally, it was found that a mixture of commercial amylose from potato starch and HAPP resulted in lower values of G′ compared to native potato starch. This indicates that the source of amylose is important for the properties in a blend with native amylopectin.     

Internal structure and physicochemical properties of corn starches as revealed by chemical surface gelatinization

The organization of amylose and amylopectin within starch granules is still not well elucidated. This study investigates the radial distribution of amylose and amylopectin in different corn starches varying in amylose content (waxy corn starch (WC), common corn starch (CC), and 50% and 70% amylose corn starches (AMC)). Corn starches were surface gelatinized by 13 M LiCl at room temperature to different extents (approximately 10%, 20%, 30%, and 40%). The gelatinized surface starch and remaining granules were characterized for amylose content, amylopectin chain-length distribution, thermal properties, swelling power (SP), and water solubility index (WSI). Except for the outmost 10% layer, the amylose content in CC increased slightly with increasing surface removal. In contrast, amylose was more concentrated at the periphery than at the core for 50% and 70% AMC. The proportion of amylopectin A chains generally decreased while that of B1 chains generally increased with increasing surface removal for all corn starches. The gelatinization enthalpy usually decreased, except for 70% AMC, whereas the retrogradation enthalpy relatively remained unchanged for CC but increased for WC, 50% and 70% AMC with increasing surface removal. The SP and WSI increased with increasing surface removal for all corn starches, with WC showing a significant increase in SP after the removal of the outmost 10% layer. The results of this study indicated that there were similarities and differences in the distribution of amylose and amylopectin chains along the radial location of corn starch granules with varying amylose contents. More amylose-lipid complex and amylopectin long chains were present at the periphery than at the core for amylose-containing corn starches.     

Formation and crystallization of amylose–monoglyceride complex in a starch matrix

The formation of amylose–lipid complexes in a gelatinized potato starch matrix was investigated using potato starch and glycerol monopalmitin. These complexes exist in two forms, with the amounts of each of the forms being dependent on the temperatures and durations of the pre-treatments.

Differential scanning calorimetry (DSC) was used to analyze transition temperatures and melting enthalpies, and thereby determine the amount of the complexes in the samples. X-ray diffraction analysis was used to investigate their crystallinity.

In measurements with DSC, form I started to melt at 88.5°C, and form II at 112.9°C. When complex form II was preheated at 100 or 110°C, its melting point rose to 116.3 and 119.7°C, respectively, because of an annealing effect. The same phenomenon occurred with complex form I: when preheated at 90°C, its melting point rose to 96.8°C. The crystal formation of form II appeared to be slower when treated at 110°C than at 100°C. Their maximum melting enthalpies were reached after about 24 h and 4 h of preheating, respectively. In X-ray diffraction analyses, form II showed a V-pattern, but form I did not. This indicates that form II is more crystalline than form I. It was possible to transform form I into form II when it was heat treated, because form I was then partially or totally melted.

As a comparison, the charged substance cetyltrimethylammonium bromide created complex form I with amylose in the starch matrix, but not form II.