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.     

Determination of the botanical origin of starch using direct potentiometry and PCA

A new approach for the determination of the botanical origin of starch is presented based on the formation of starch-triiodide complexes. The starch samples were extracted from wheat (Srpanjka), potato, maize, rye (Barun), barley (Conduct), rice, tapioca and a commercial modified starch. The amylose/amylopectin ratios of starches, among various other properties, differ between starches of different botanical origins. Triiodide ions bind characteristically to the amylose and amylopectin of the starch depending on the starch's origin. The new technique includes direct potentiometric measurements of the response of free triiodide ions in starch-triiodide solutions where the data is analysed by principal component analysis (PCA). PCA gave graphical results for statistical differentiation between starches of different botanical origins.     

A modified method of determining amylase activity in grain by falling number

The standard method for determination of amylase activity by the falling number was modified by us to study the carbohydrate-amylase complex and time course of starch hydrolysis by amylases from rye grain. The method is based on the use of potato starch as a standard substrate and aqueous extract of grain amylases as an enzyme source.     

Modified Method for the Determination of Grain Amylase Activity by the Falling Number

The standard method for determination of amylase activity by the falling number was modified by us to study the carbohydrate–amylase complex and time course of starch hydrolysis by amylases from rye grain. The method is based on the use of potato starch as a standard substrate and aqueous extract of grain amylases as an enzyme source.     

Selectivity and Thermal Properties of Various Starches Incorporating Free Fatty Acids

The effects of free fatty acids on the selectivity and thermal properties of starch samples incorporating free fatty acids were examined by DSC. An analysis of the free fatty acid values incorporated into cassava starch and potato starch shows that myristic acid was the highest and linoleic acid was the lowest, while the free fatty acid values of corn starch were significantly higher than those of the other starches. DSC measurements on corn starch show an initial peak and another peak in a higher-temperature region, this second peak differing according to the incorporated free fatty acid. It is thus considered that the state of the complex of each free fatty acid with amylose might be better understood by observing the respective DSC characteristics.     

Alteration of the properties of Aspergillus sp. K-27 glucoamylase on limited proteolysis with subtilisin

An active derivative (mol. wt. 48,000) of Aspergillus sp. K-27 glucoamylase (mol. wt. 76,000) was obtained by limited proteolysis with subtilisin. The amino acid sequences of native and modified enzymes at the N-termini were Ala-Gly-Gly-Thr-Leu-Asp and Ala-Val-Leu, respectively. The proteolysis greatly decreased the affinity of the enzyme for amylopectin and glycogen, but not for oligosaccharides. It also reduced the ability of the enzyme to degrade raw starch, abolished the ability of the enzyme to adsorb onto starch granules, and eliminated the synergistic action of the enzyme in the hydrolysis of starch granules with alpha-amylase. These findings imply that the enzyme has a specific affinity site for polysaccharide substrates besides the catalytic site, i.e., a starch-binding site, and that the former is removed by proteolysis. The extent of the reduction in the activity for raw starches caused by the modification varied with the starch source, as the modified enzyme digested raw potato starch better than either raw corn or sweet potato starches. A new method for evaluation of the raw starch-digesting activity of glucoamylase is described.     

Estimation of the in situ degradation of the washout fraction of starch by using a modified in situ protocol and in vitro measurements

The in situ degradation of the washout fraction of starch in six feed ingredients (i.e. barley, faba beans, maize, oats, peas and wheat) was studied by using a modified in situ protocol and in vitro measurements. In comparison with the washing machine method, the modified protocol comprises a milder rinsing method to reduce particulate loss during rinsing. The modified method markedly reduced the average washout fraction of starch in these products from 0.333 to 0.042 g/g. Applying the modified rinsing method, the fractional degradation rate (k _d) of starch in barley, oats and wheat decreased from on average 0.327 to 0.144 h⁻¹ whereas for faba beans, peas and maize no differences in k _d were observed compared with the traditional washing machine rinsing. For barley, maize and wheat, the difference in non-fermented starch in the residue between both rinsing methods during the first 4 h of incubation increased, which indicates secondary particle loss. The average effective degradation of starch decreased from 0.761 to 0.572 g/g when using the new rinsing method and to 0.494 g/g when applying a correction for particulate matter loss during incubation. The in vitro k _d of starch in the non-washout fraction did not differ from that in the total product. The calculated ratio between the k _d of starch in the washout and non-washout fraction was on average 1.59 and varied between 0.96 for oats and 2.39 for maize. The fractional rate of gas production was significantly different between the total product and the non-washout fraction. For all products, except oats, this rate of gas production was larger for the total product compared with the non-washout fraction whereas for oats the opposite was observed. The rate of increase in gas production was, especially for grains, strongly correlated with the in vitro k _d of starch. The results of the present study do not support the assumption used in several feed evaluation systems that the degradation of the washout fraction of starch in the rumen is much faster than that of the non-washout fraction.     

Changes in color and texture of wheat noodles during chilled storage

Wheat noodles cooked for different periods of time were stored at 5 °C, and color changes in their cross sections were quantitatively assessed by digital image analysis. The color of noodles with flattened moisture distributions whitened greatly during the early stages of chilled storage due to the retrogradation of starch, with the color change showing a significant correlation with the changes in noodle fragility. Color changes were also measured for wheat noodles and noodles containing modified starch with internal moisture distributions, and local changes within the noodles were kinetically analyzed. The addition of modified starch significantly reduced the color change in the noodle interior, where the moisture content was relatively low. Scanning calorimetric measurements indicated differences in the gelatinized state of modified starch and original wheat starch at low moisture contents, which affected the rate of color change in the interior of noodles containing modified starch.     

Sequential staining to assess viability and starch content in individual pollen grains.

Individual pollen grains may be assessed for viability and starch content by dusting a sample onto drops of an aqueous medium containing fluorescein diacetate and potassium iodide, and allowing them to accumulate free fluorescein for ten minutes. They are then illuminated with ultraviolet or blue light and photographed to record the proportion that fluoresce, as an index of viability. The preparation is exposed to iodine vapor and the same field of view rephotographed in bright field illumination to record starch content. Iodine vapor avoids disturbing the grains by adding further liquid, so that the same pollen grains may be classified by fluorescence and starch content. The method makes it possible to test whether viability and starch content are associated or depend on other variables, such as pollen-grain diameter. Iodine-potassium iodide is shown to be inadequate as a test for pollen viability. The method is quick and easy and provides data not otherwise available.     

Synthesis and characterization of starch-modified polyurethane

Corn starch was reacted with urethane prepolymer in order to modifying starch and preparing new hydrophobic copolymers. These copolymers were prepared by two-step reactions. The polycaprolactone terminated hexamethylene diisocyanate (HDI) (as prepolymer) was prepared by introducing diisocyanate on both ends of PCL at a molar ratio of 1:2 (PCL:HDI). The grafting was performed by addition of polycaprolactone based prepolymer to starch solution of DMSO with different weight ratio of starch and prepolymer. The samples were characterized and examined by FTIR and ¹H NMR spectroscopy, DSC analysis, and scanning electron microscopy (SEM). By introducing NCO groups onto the PCL terminals, the FTIR spectrum shows a new sharp peak, representing the NCO groups and formation of prepolymer. By grafting this prepolymer onto starch a NH and urethane band were appeared. The effect of prepolymer percentage on hydrophobicity was measured through contact angle and it was found that increases with increasing amount of prepolymer. Glass transition temperature (T_g) is also affected with increasing amount of urethane linkages. Surface morphology of modified starch was studied by SEM. It was observed that the surfaces of modified starch are rougher and disordered than the surface of unmodified starch particles. This confirms the grafting and modification of starch. This modified starch can be used as filler in biodegradable starch based polymers.     

Quantification of starch in plant tissues

This protocol describes a simple means of measuring the starch content of plant tissues by solubilizing the starch, converting it quantitatively to glucose and assaying the glucose. Plant tissue must initially be frozen rapidly to stop metabolism, then extracted to remove free glucose. Starch is solubilized by heating, then digested to glucose by adding glucan hydrolases. Glucose is assayed enzymatically. The method is more sensitive and accurate than iodine-based protocols, and is suitable for tissues that have a wide range of starch contents. Measurements on multiple samples can be completed within a day.     

Starch modification by iterated syneresis

Potato, tapioca, corn and wheat starches were solubilised in water and isolated from the solution by iterated syneresis and the effect of this physical modification on the physicochemical properties and structure was studied. The experimental starches were examined by chemical analysis, the Brabender rheological method, scanning electron or light microscopy, X-ray diffractometry, infrared spectroscopy, and differential scanning calorimetry. Physical modification was evidenced to the change starch–water interaction and the structure of starches. Pasting temperature shifted to lower values and the gelatinisation mechanism changed. All modified starches had a B-type of X-ray diffraction pattern. The melting temperature of starch crystallites was typical of retrograded starch, but the enthalpy change was higher. The correlation between the resistant starch content of modified starches and their crystal structure was discussed together with the thermal properties.     

Chemical modification of papain for use in alkaline medium

Chemical modification is a useful method to recognize and modify functional determinants of enzymes. Papain, an endolytic cysteine protease (EC3.4.22.2) from Carica papaya latex has been chemically modified using different dicarboxylic anhydrides of citraconic, phthalic, maleic and succinic acids. These anhydrides reacted with five to six amino groups of the lysine residues in the enzyme, thereby changing the net charge of the enzyme from positive to negative. The resultant enzyme had its optimum pH shifted from 7 to 9 and change in temperature optima from 60 to 80 °C. The modified papain also had a higher thermostability. Stability of the modified papain was further increased by immobilization of the enzyme either by adsorption onto inert matrix or by entrapment in polysaccharide polymeric gels. Entrapment in starch gel showed better retention of enzyme activity. Incorporation of modified and immobilized enzymes to branded domestic detergent powders was found to have very good activity retention. The papain entrapped in starch gel showed better stability and activity retention than in other carbohydrate polymers when added to domestic detergent powders.     

Enzymatic modification of cassava starch by bacterial lipase

Enzymatic modification of starch using long chain fatty acid makes it thermoplastic suitable for a myriad of industrial applications. An industrial lipase preparation produced by Burkholderia cepacia (lipase PS) was used for modification of cassava starch with two acyl donors, lauric acid and palmitic acid. Reactions performed with palmitic acid by liquid-state and microwave esterification gave a degree of substitution (DS) of 62.08% (DS 1.45) and 42.06% (DS 0.98), respectively. Thermogravimetric analysis showed that onset of decomposition is at a higher temperature (above 600°C) for modified starch than the unmodified starch (280°C). Modified starch showed reduction in α-amylase digestibility compared to native starch (76.5–18%). Swelling power lowered for modified starch as esterification renders starch more hydrophobic, making it suitable for biomedical applications as materials for bone fixation and replacements, carriers for controlled release of drugs and bioactive agents. Thus enzymatic esterification is ecofriendly.     

葡萄糖淀粉酶色氨酸残基及底物结合位点的研究

 本文用N-溴代琥珀酰亚胺(NBS)对葡萄糖淀粉酶进行特异性修饰,当酶分子表面有3个色氨酸残基被修饰后,酶活力完全丧失。用邹氏图解法测得酶活性中心有一个色氨酸残基是必需的。如果在酶液中加入不同的底物再用NBS氧化,用荧光发射和荧光猝灭光谱检测表明,底物对酶分子有不同程度的保护作用。在被测试的三种底物中,这种保护能力依为糊精>淀粉>麦芽糖。     

Process development of starch hydrolysis using mixing characteristics of Taylor vortices

In food industries, enzymatic starch hydrolysis is an important process that consists of two steps: gelatinization and saccharification. One of the major difficulties in designing the starch hydrolysis process is the sharp change in its rheological properties. In this study, Taylor–Couette flow reactor was applied to continuous starch hydrolysis process. The concentration of reducing sugar produced via enzymatic hydrolysis was evaluated by varying operational variables: rotational speed of the inner cylinder, axial velocity (reaction time), amount of enzyme, and initial starch content in the slurry. When Taylor vortices were formed in the annular space, efficient hydrolysis occurred because Taylor vortices improved the mixing of gelatinized starch with enzyme. Furthermore, a modified inner cylinder was proposed, and its mixing performance was numerically investigated. The modified inner cylinder showed higher potential for enhanced mixing of gelatinized starch and the enzyme than the conventional cylinder.     

Quantitative determination of alpha-amylase in glucoamylase preparations.

The use of modified starch substrates for the measurement of α-amylase activity is described. In the presence of excess glucoamylase, the amount of glucose released from substrates containing blockages to exo-enzyme action is proportional to the amount of α-amylase present. The levels of α-amylase in a number of commercial glucoamylase preparations have been determined by this method. The importance of α-amylase in achieving efficient conversion of starch into glucose is illustrated by a comparison of the time courses of degradation of amylopectin by glucoamylase preparations containing low and high levels of α-amylase.     

Solid-state and mechanical properties of aqueous chitosan-amylose starch films plasticized with polyols

The film-forming ability of chitosan and binary mixtures of chitosan and native amylose corn starch (Hylon VII) was evaluated with free films prepared by a casting/solvent evaporation method. Unplasticized and plasticized free chitosan films in aqueous acetic acid and respective films containing a mixture of chitosan and native amylose starch in acetic acid were prepared. Glycerol, sorbitol, and i-erythritol were used as plasticizers. Solid-state and mechanical properties of the films were studied by powder x-ray diffractometry (XPRD), differential scanning calorimetry (DSC), and a materials testing machine. The films composed of a mixture of chitosan and native amylose starch in acetic acid were clear and colorless. A plasticizer concentration of 20% wt/wt (of the polymer weight) ws sufficient to obtain flexible films with all samples tested. X-ray diffraction patterns and DSC thermograms indicated an amorphous state of the films independent of the type of plasticizer used. In conclusion, incorporation of native amylose com starch into chitosan films improves the consistency and the mechanical properties of the films.     

Rheological and gelation properties of rice starch modified with 4-alpha-glucanotransferase

Rheological measurements were performed to characterize rice starch modified with 4-alpha-glucanotransferase (4alphaGTase) isolated from Thermus scotoductus, in terms of effects of the enzyme and starch concentration on flow behavior, gel strength, and melting and gelling kinetics of the modified rice starch. Consistency index decreased and flow behavior index increased with the level of enzyme treatment, and at high level of enzyme treatment, it demonstrated Bingham plastic behavior. As the level of enzyme decreased and the starch concentration increased, gelation time decreased and the final gel strength increased significantly. Regardless of treatment variables, all the modified starch gels melted at similar temperature.     

Preparation and characterization of soluble starches having different molecular sizes and composition,by acid hydrolysis in different alcohols

Potato and waxy-maize starches were separately modified for 1 h at 65° with 0.36% hydrochloric acid in methanol, ethanol, 2-propanol, and 1-butanol. All of the modified starches were readily soluble in hot water, to give crystal-clear solutions up to a concentration of at least 20% (w/v). The modified granules were studied by light-microscopy and iodine-iodide staining. All of the modified starches retained their granule appearance, although with various degrees of damage that progressively increased from methanol to 1-butanol. Both hydrolysis and alcoholysis occurred, but to different extents in the different alcohols. The highest proportion of alcoholysis occurred in methanol where 50% of the resulting molecules were glycosides, the lowest in 1-butanol where 6% were glycosides. The number-average molecular weights of the modified starches also progressively decreased from 126,670 for the methanol-modified waxy-maize starch to 4,750 for the 1-butanol-modified potato starch. The methanol- and ethanol-modified potato starches were fractionated into amylose and amylopectin components. The 2-propanol- and 1-butanol-modified potato starches gave only an amylopectin component. The amylose components were characterized by gel-permeation chromatography on Bio-Gel A-5m, and the amylopectin components, on Bio-Gels A-150m and A-0.5m. The molecular sizes of the amylose and amylopectin components progressively decreased from methanol- to 1-butanol-modified starches. Furthermore, the polymodal composition of the amylopectin component was decreased to give a more homogeneous product. Waxy-maize starch was modified in methanol and 2-propanol and gave products that were of lower molecular size and more homogeneous than the polymodal native starch. It is shown that the differential effect of the different alcohols on the modification of the starch granules is produced by effecting different concentrations of acid inside the granule, where hydrolysis occurs in the 10–12% of water contained in the granule. It is postulated that 2-propanol and 1-butanol dissolve the double-helical, crystalline regions in the starch granule to give different types of products under otherwise identical conditions of modification.