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PubMed1983

Quantitative determination of starch, amylose, and amylopectin in plant tissues using glass fiber paper.

Methods for accurate and rapid determination of starch, amylose, and amylopectin in plant tissues are described. They are based on simplified extraction of starch with 32% perchloric acid and selective retention of the starch-iodine complex on a glass fiber disk (Whatman GF/A). The starch on the disk is dissolved in 0.75 M sulfuric acid and estimated with phenol. For amylose and amylopectin determination the starch on the disk is dissolved in perchloric acid, precipitated with ethanol, and retained on a 10-cm glass fiber strip. Both polysaccharides are separated by a chromatographic procedure involving development of the strip in a mixture of ethanol and dimethyl sulfoxide and in dimethyl sulfoxide. The strip is washed in ethanol and stained with iodine or used for polysaccharide quantitation. As little as 5 micrograms of starch or its components present in different amounts of plant material can be estimated.

Amylopectin↗
PubMed1977

The action pattern of human salivary alpha-amylase in the vicinity of the branch points of amylopectin.

Salivary alpha-amylase hydrolyses amylopectin in stages. At the end of the so-called second stage, there are present glucose, maltose, and a series of alpha-limit dextrins containing (1 leads to 4)- and (1 leads to 6)-alpha-D-glucosidic bonds. The structures of the limit dextrins containing a single (1 leads to 6)-bond were examined. Six such dextrins were found. Of these, two were capable of being further hydrolysed by alpha-amylase, whereas the remaining four were true, amylase-resistant alpha-limit dextrins. The structures of the limit dextrins afforded information about those (1 leads to 4)-alpha-D-glucosidic bonds of amylopectin that are capable of being cleaved by salivary alpha-amylase and those that are resistant. In order to define further the action of alpha-amylase, the alpha-amylolytic products of 6-alpha-maltotriosyl-D-glucose, 6(3)-alpha-maltotriosylmaltotriose, and 6(3)-alpha-maltotriosylmaltotetraose were examined.

Amylases↗
PubMed2003

Examination of the structure of amylopectin molecules by fluorescent labeling.

Amylopectin molecules from rice, maize, sweet potato and potato were examined by fluorescent labeling followed by gel-permeation HPLC. The number-average degree of polymerization (dp(n)) was determined to be in range of 9600-15,900. The molar-based distribution revealed the presence of three molecular species, large (dp(n) 13,400-26,500), medium (4400-8400) and small (700-2100). Their molar proportions differed by plant origin. The large species was a major component (43-63% by mole). A relatively large amount of the medium (16-28% by mole) and small (19-38%) species was found although their weight proportion was small (8-15, 1-4%, respectively). The three species from waxy rice amylopectin had a similar chain-length distribution and also a similar size-distribution of C chains. These results suggested that the three species were basically similar in cluster structure but different in number of clusters per molecule.

Amylopectin↗
PubMed1996

Structure of the cyclic glucan produced from amylopectin by Bacillus stearothermophilus branching enzyme.

The thermostable branching enzyme (BE, EC 2.4.1.18) from Bacillus stearothermophilus TRBE14 produces large cyclic glucans from waxy rice amylopectin similar to those obtained from amylose as described elsewhere [H. Takata, T. Takaha, S. Okada. M. Takagi, and T. Imanaka, J. Bacteriol., 178 (1996) 1600-1606]. The structure of the product (P-1) from the late-stage reaction was analyzed in detail. The weight-average degree of polymerization (dpw) of P-1 was 900. Its chain-length distribution was not significantly changed compared with that of amylopectin, although the amount of long chains (dp > 38) was slightly decreased. The cyclic component of P-1, which was isolated by the extensive action of glucoamylase, had dpw of 49. Three point five alpha-1,6 linkages were directly involved in the formation of the ring structure with several non-cyclic side chains linked to the ring. Based on these results, the action and new roles of BE are discussed.

1,4-alpha-Glucan Branching Enzyme↗
PubMed2001

High-performance anion-exchange chromatography-electrospray mass spectrometry for investigation of the substituent distribution in hydroxypropylated potato amylopectin starch.

The use of high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) coupled on-line with electrospray mass spectrometry (ESI-MS) for analysis of the substitution pattern in chemically modified starch, has been investigated. In order to characterise the distribution of substitution groups along the polymer chain, hydroxypropylated potato amylopectin starch (HPPAP) was subjected to enzymic hydrolysis, followed by analysis of the degradation products by HPAEC-PAD-MS. When using conventional chromatographic techniques for characterisation of enzymic hydrolysates, standard compounds are required for identification of the hydrolysis products. However, the on-line coupling with ESI-MS allowed identification of all products obtained, substituted as well as unsubstituted, and also of those compounds that co-eluted, without the need for standards. Further, HPAEC-PAD-MS was shown to be useful for analysis of the substitution pattern in modified starch; from results obtained it was suggested that the hydroxypropyl groups were homogeneously distributed in the amylopectin molecule. It was also shown that the starch hydrolysing enzymes were hindered by the hydroxypropyl groups and preferentially cleaved glucosidic linkages between unsubstituted glucose units.

Amylopectin↗
PubMed1996

Diffusion of mixed micelles of bile salt-lecithin in amylopectin gels: a Fourier transform infrared microspectroscopy approach.

Using fourier transform infrared (FTIR) microspectroscopy the average translational diffusion coefficients of bile salt-lecithin mixed micelles diffusing in amylopectin gels of varying concentration were measured, on the assumption that the diffusion coefficient was constant during diffusion. This assumption was examined based on quasielastic light scattering (QLS) measurements of the change in the mixed micellar size on dilution. The size increased significantly on dilution with a buffer without the bile salt, whereas the size was almost constant on dilution with a solution of the bile salt in the same buffer. Because bile salt molecules in intermicellar solution (being much smaller) diffuse much faster than the mixed micelles, the mixed micelles in the diffusing front can be treated as if diluted with the bile salt solution. Therefore their size will be constant during diffusion, validating the assumption of a constant diffusion coefficient. Based on this it is possible to show that the micelles diffuse with a diffusion coefficient of approximately 10(-11) m2 s-1, independent (within experimental error) of the amylopectin gel concentration over the range 5%-10% w/w.

Amylopectin↗
PubMed2003

Enzyme-aided investigation of the substituent distribution in cationic potato amylopectin starch.

The distribution of substituents along the polymer chain in cationic potato amylopectin starch, modified in solution, granular slurry, or dry state, was investigated. The starch derivatives were successively hydrolyzed by different enzymes, followed by characterization of the hydrolysis products obtained by means of electrospray mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). ESI-MS and MALDI-MS were proved to be appropriate techniques for identification of the substituted hydrolysis products, for which there are no standard compounds available. No highly substituted oligomers were found in the hydrolysates, which was taken as an indication of a more or less homogeneous distribution of cationic groups in the amylopectin molecules. Furthermore, from the results obtained it was suggested that the enzymes cleave glucosidic linkages only between unsubstituted glucose units and, preferentially, linkages in sequences containing more than two adjacent unsubstituted units. The determination of the amount of unsubstituted glucose produced from every successive hydrolysis step revealed slight differences between the different starch samples with respect to the homogeneity of the substitution pattern. Among the three samples under investigation, starch cationized in solution was found to have the most and dry-cationized starch the least homogeneous distribution of substituents.

Amylopectin↗
PubMedDate not supplied

Surface composition and morphology of starch, amylose, and amylopectin films.

The surfaces of solution-cast films of starch, amylose, and amylopectin were examined with scanning electron microscopy (SEM), atomic force microscopy (AFM), electron spectroscopy for chemical analysis (ESCA), and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The surface topography visualized by SEM showed that amylopectin films were very smooth whereas amylose and starch films were rougher. It appears that crystallinity or phase separation in the bulk of the film affects the surface topography. AFM showed that the outmost surfaces of all films were covered with small protrusions, 15-35 nm wide and 1-4 nm high. Studies with ESCA revealed the presence of 3-8% nitrogen on the surfaces. ToF-SIMS indicated that the nitrogen originates from protein because ionic fragments from amino acids and the peptide backbone were found. Extracts from the top surface layer of the starch film showed protein bands in gel electrophoresis (SDS-PAGE) around 60 kDa, which is in the same molecular weight range as the biosynthesizing enzyme GBSS I present in starch granules. The proteins apparently phase separated during film formation and migrated to the surface, resulting in an extensive enrichment of proteins in the film surface, where about 8% of the protein is present in the top 0.01% of the film. We believe that the protrusions observed with AFM could be one or a few proteins aggregated side by side.

Amylopectin↗
PubMed2004

Hydration of the amylopectin branch point. Evidence of restricted conformational diversity of the alpha-(1-->6) linkage.

The hydration behavior of a model compound for the amylopectin branch point, methyl 6'-alpha-maltosyl-alpha-maltotrioside, was investigated by combining molecular dynamics simulations in explicit water, 500 MHz NMR spectroscopy, including pulsed field gradient diffusion measurements, and exploratory multivariate data analysis. In comparison with results on a tetrasaccharide analogue, the study reveals that the conformational diversity of the three-bond alpha-(1-->6) linkage becomes quite limited in aqueous solution upon the addition of a fifth glucose residue that elongates the alpha-(1-->6) branch. This investigation reveals two plausible starch branch point structures, one that permits the formation of double helices and one that is adapted for interconnection of double helices. The apparent rigidity of the former is explained by the presence of water pockets/bridges in the vicinity of the branch point that lock the pentasaccharide structure into one conformational family that is able to accommodate the creation of the double-helical amylopectin structure.

Amylopectin↗
PubMed2002

Retention of aroma compounds in starch matrices: competitions between aroma compounds toward amylose and amylopectin.

The retention of three aroma compounds-isoamyl acetate, ethyl hexanoate, and linalool--from starch-containing model food matrices was measured by headspace analysis, under equilibrium conditions. We studied systems containing standard or waxy corn starch with one or two aroma compounds. The three studied aroma compounds interact differently: ethyl hexanoate and linalool form complexes with amylose, and isoamyl acetate cannot. However, in systems containing one aroma compound, we observed with both starches a significant retention of the three molecules. These results indicate that amylopectin could play a role in the retention of aroma. In systems containing two aroma compounds in a blend, the retentions measured for isoamyl acetate and for linalool were either equal to or less than those in systems where they were added alone. This phenomenon was attributed to competition between aroma compounds to bind with starch. The retention of aroma compounds blended in starch-based systems gave us additional information which confirmed that interactions occur not only with amylose but also with amylopectin.

Acyclic Monoterpenes↗
PubMed2001

The action of starch synthase II on 6"'-alpha-maltotriosyl-maltohexaose comprising the branch point of amylopectin.

The principle of using a chemically synthesized, well-defined branched oligosaccharide to provide a more detailed knowledge of the substrate specificity of starch synthase II (SSII) is demonstrated. The branched nonasaccharide, 6"'-alpha-maltotriosyl-maltohexaose, was investigated as a primer for particulate SSII using starch granules prepared from the low-amylose pea mutant lam as the enzyme source. The starch granule preparation from the lam pea mutant contains no starch synthases other than SSII and is devoid of alpha-amylase, beta-amylase and phosphorylase activity. SSII was demonstrated to catalyse a specific nonprocessive elongation of the nonreducing end of the shortest unit chain of 6"'-alpha-maltotriosyl-maltohexaose, i.e. the maltotriose chain. Maltotriose and maltohexaose, representing the two linear building units of the branched nonasaccharide, were also tested as primers for SSII. Maltotriose was elongated more efficiently than 6"'-alpha-maltotriosyl-maltohexaose and maltohexaose was used less efficiently. Compared to the surface exposed alpha-glucan chains of the granule bound amylopectin molecules, all three soluble oligosaccharides tested were poor primers for SSII. This indicates that in vivo, the soluble oligosaccharides supposedly released as result of amylopectin trimming reactions are not re-introduced into starch biosynthetic reactions via the action of the granule bound fraction of SSII.

Amylopectin↗
PubMed2002

Role of granule-bound starch synthase in determination of amylopectin structure and starch granule morphology in potato.

Reductions in activity of SSIII, the major isoform of starch synthase responsible for amylopectin synthesis in the potato tuber, result in fissuring of the starch granules. To discover the causes of the fissuring, and thus to shed light on factors that influence starch granule morphology in general, SSIII antisense lines were compared with lines with reductions in the major granule-bound isoform of starch synthase (GBSS) and lines with reductions in activity of both SSIII and GBSS (SSIII/GBSS antisense lines). This revealed that fissuring resulted from the activity of GBSS in the SSIII antisense background. Control (untransformed) lines and GBSS and SSIII/GBSS antisense lines had unfissured granules. Starch analyses showed that granules from SSIII antisense tubers had a greater number of long glucan chains than did granules from the other lines, in the form of larger amylose molecules and a unique fraction of very long amylopectin chains. These are likely to result from increased flux through GBSS in SSIII antisense tubers, in response to the elevated content of ADP-glucose in these tubers. It is proposed that the long glucan chains disrupt organization of the semi-crystalline parts of the matrix, setting up stresses in the matrix that lead to fissuring.

Amylopectin↗
PubMed1994

Glucose and insulin responses to barley products: influence of food structure and amylose-amylopectin ratio.

Postprandial glycemic and insulinemic responses and satiety with various barley products were evaluated in normal subjects. Also studied were the rate of in vitro starch digestion and the content of in vitro resistant starch (RS). Products tested were boiled intact (rice extender) and milled kernels (porridge) from four barley genotypes of Glacier with different amylose-amylopectin ratios (7-44% amylose). All barley products elicited lower metabolic responses and higher satiety scores when compared with white wheat bread. The lente behavior of the boiled flours was probably due to the viscous properties of the beta-glucans. However, the boiled flours produced higher glucose and insulin responses than did the corresponding boiled kernels. The impact of amylose: amylopectin on the metabolic responses was marginal. The high-amylose products released starch more slowly from a dialysis tubing during enzymic incubation of chewed samples compared with the corresponding products with less amylose. The RS content ranged from 0.4% in waxy to 5.6% in the high-amylose flour product (starch basis).

Adult↗
PubMed1995

Effect of long-term consumption of amylose vs amylopectin starch on metabolic variables in human subjects.

Long-term consumption of high-amylose starch on insulin and glucose response was investigated in 24 men: 10 control and 14 hyperinsulinemic (HI) subjects. Subjects consumed products made with standard (70% amylopectin, 30% amylose) or high-amylose (70% amylose, 30% amylopectin) cornstarch for two 14-wk periods in a crossover pattern. Starch products replaced usual starches in the self-selected diet for 10 wk followed by 4 wk of a controlled diet. After a starch-tolerance test with bread made from the starch consumed during that period, the insulin response curve area was significantly lower in all subjects after amylose consumption (P < 0.002). Glucose responses in HI and control subjects were similar and did not vary with the type of starch. Fasting triglyceride concentrations were significantly lower in subjects who consumed the high-amylose compared with the standard-starch diet throughout the study. Chronic consumption of high-amylose foods normalized the insulin response of hyperinsulinemic subjects and showed a potential benefit for diabetic subjects.

Adult↗
PubMed2004

Antisense downregulation of the barley limit dextrinase inhibitor modulates starch granule size distribution, starch composition and amylopectin structure.

The barley protein limit dextrinase inhibitor (LDI), structurally related to the alpha-amylase/trypsin inhibitor family, is an inhibitor of the starch debranching enzyme limit dextrinase (LD). In order to investigate the function of LDI, and the consequences for starch metabolism of reduced LDI activity, transgenic barley plants designed to downregulate LDI by antisense were generated. Homozygous antisense lines with reduced LDI protein level and activity were analysed and found to have enhanced free LD activity in both developing and germinating grains. In addition the antisense lines showed unpredicted pleiotropic effects on numerous enzyme activities, for example, alpha- and beta-amylases and starch synthases. Analysis of the starch showed much reduced numbers of the small B-type starch granules, as well as reduced amylose relative to amylopectin levels and reduced total starch. The chain length distribution of the amylopectin was modified with less of the longer chains (>25 units) and enhanced number of medium chains (10-15 units). These results suggest an important role for LDI and LD during starch synthesis as well as during starch breakdown.

Amylopectin↗
PubMed2005

Arabidopsis mutants Atisa1 and Atisa2 have identical phenotypes and lack the same multimeric isoamylase, which influences the branch point distribution of amylopectin during starch synthesis.

The aim of this work was to evaluate the function of isoamylase in starch granule biosynthesis in Arabidopsis leaves. A reverse-genetic approach was used to knockout AtISA1, one of three genes in Arabidopsis encoding isoamylase-type debranching enzymes. The mutant (Atisa1-1) lacks functional AtISA1 transcript and the major isoamylase activity (detected by native gels) in crude extracts of leaves. The same activity is abolished by mutation at the DBE1 locus, which encodes a second isoamylase-type protein, AtISA2. This is consistent with the idea that ISA1 and ISA2 proteins are subunits of the same enzyme in vivo. Atisa1-1, Atisa2-1 (dbe1), and the Atisa1-1/Atisa2-1 double mutant all have identical phenotypes. Starch content is reduced compared with the wild type but substantial quantities of the soluble glucan phytoglycogen are produced. The amylopectin of the remaining starch and the phytoglycogen in the mutants are structurally related to each other and differ from wild-type amylopectin. Electron micrographs reveal that the phytoglycogen-accumulating phenotype is highly tissue-specific. Phytoglycogen accumulates primarily in the plastids of the palisade and spongy mesophyll cells. Remarkably, other cell types appear to accumulate only starch, which is normal in appearance but is altered in structure. As phytoglycogen accumulates during the day, its rate of accumulation decreases, its structure changes and intermediates of glucan breakdown accumulate, suggesting that degradation occurs simultaneously with synthesis. We conclude that the AtISA1/AtISA2 isoamylase influences glucan branching pattern, but that this may not be the primary determinant of partitioning between crystalline starch and soluble phytoglycogen.

Amylopectin↗
PubMed2006

Screening for and identification of starch-, amylopectin-, and pullulan-degrading activities in bifidobacterial strains.

Forty-two bifidobacterial strains were screened for alpha-amylase and/or pullulanase activity by investigating their capacities to utilize starch, amylopectin, or pullulan. Of the 42 bifidobacterial strains tested, 19 were capable of degrading potato starch. Of these 19 strains, 11 were able to degrade starch and amylopectin, as well as pullulan. These 11 strains, which were shown to produce extracellular starch-degrading activities, included 5 strains of Bifidobacterium breve, 1 B. dentium strain, 1 B. infantis strain, 3 strains of B. pseudolongum, and 1 strain of B. thermophilum. Quantitative and qualitative enzyme activities were determined by measuring the concentrations of released reducing sugars and by high-performance thin-layer chromatography, respectively. These analyses confirmed both the inducible nature and the extracellular nature of the starch- and pullulan-degrading enzyme activities and showed that the five B. breve strains produced an activity that is consistent with type II pullulanase (amylopullulanase) activity, while the remaining six strains produced an activity with properties that resemble those of type III pullulan hydrolase.

Amino Acid Sequence↗
PubMed2006

Effect of barley and its amylopectin content on ruminal fermentation and nitrogen utilization in lactating dairy cows.

The effect of type of grain (corn vs. barley) and amylopectin content of barley grain (normal vs. waxy) on ruminal fermentation, digestibility, and utilization of ruminal ammonia nitrogen for milk protein synthesis was studied in a replicated 3 x 3 Latin square design trial with 6 lactating dairy cows. The experimental treatments were (proportion of dietary dry matter): CORN, 40% corn grain, NBAR, 30% normal Baronesse barley:10% corn grain, and WBAR, 30% high-amylopectin (waxy) Baronesse barley:10% corn grain. All grains were steam-rolled and fed as part of a total mixed ration. The NBAR and WBAR diets resulted in increased ruminal ammonia concentrations compared with CORN (8.2, 7.4, and 5.6 mM, respectively), but other ruminal fermentation parameters were not affected. Ruminal digestibility of dietary nutrients and microbial protein synthesis in the rumen were also not affected by diet. Corn grain had greater in situ effective ruminal dry matter degradability (62.8%) than the barley grains (58.2 and 50.7%, respectively), and degradability of the normal barley starch was greater than that of the waxy barley (69.3 and 58.9%, respectively). A greater percentage of relative starch crystallinity was observed for the waxy compared with the normal barley grain. Total tract apparent digestibility of dry matter and organic matter were decreased by WBAR compared with CORN and NBAR. Total tract starch digestibility was greater and milk urea nitrogen content was lower for CORN compared with the 2 barley diets. In this study, the extent of processing of the grain component of the diet was most likely the factor that determined the diet responses. Minimal processing of barley grain (processing indexes of 79.2 to 87.9%) reduced its total tract digestibility of starch compared with steam-rolled corn (processing index of 58.8%). As a result of the increased ammonia concentration and reduced degradability of barley dry matter in the rumen, the utilization of ruminal ammonia nitrogen for microbial protein synthesis was decreased with the barley diets compared with the corn-based diet. In this study, waxy Baronesse barley was less degradable in the rumen and the total digestive tract than its normal counterpart. The most likely reasons for these effects were the differences in starch characteristics and chemical composition, and perhaps the different response to processing between the 2 barleys.

Amylopectin↗