Preparation and characterization of new and improved soluble-starches, -amylose, and -amylopectin by reaction with benzaldehyde/zinc chloride

Seven different starches from potato, rice, maize, waxymaize, amylomaize-VII, shoti, and tapioca, and potato amylose and potato amylopectin have been reacted with benzaldehyde, catalyzed by ZnCl₂, to give new water-soluble starches and water soluble-amylose and soluble-amylopectin. In contrast to the native starches, aqueous solutions of the modified starches could not be precipitated with 2-, 3-, or 4-volumes of ethanol. β-Amylase gave no reaction with the modified starches, in contrast to the native starches, indicating that the modification occurred exclusively at the nonreducing-ends, giving 4,6-benzylidene-d-glucopyranose at the nonreducing-ends. Reactions of α-amylase with native and modified potato and rice starches gave a decrease in the triiodide blue color and an increase in the reducing-value that were similar for the native- and modified-starches, indicating the modified starches had not been significantly altered by the modification. The benzaldehyde-modified starches and benzaldehyde-modified potato amylose and potato amylopectin components, therefore, have a starch structure very much like their native counterparts, in contrast to the Lintner, Small, and the alcohol/acid-hydrolyzed soluble-starches that have undergone acid hydrolysis. The benzaldehyde-modified starches and starch components have significantly higher water solubility than their native counterparts even though the structures of the modified starches had only been slightly altered from the structures of their native counterparts. They all gave crystal-clear solutions that did not retrograde.     

Preparation and characterization of gelatinized granular starches from aqueous ethanol treatments

In order to modify the properties of native starch granules, the formation of gelatinized granular forms (GGS) from normal, waxy, and high amylose maize, as well as potato and tapioca starches was investigated by treating granules with aqueous ethanol at varying starch:water:ethanol ratios and then heating in a rotary evaporator to remove ethanol. The modified starches were characterized using bright field, polarized and electron microscopy. Short/long range molecular order and enthalpic transitions on heating were also studied using infrared spectroscopy, X-ray diffractometry and differential scanning calorimetry respectively. A diffuse birefringence pattern without Maltese cross was observed for most GGS samples. Treatment with aqueous ethanol resulted in starch-specific changes in the surface of granules, most noticeably swelling and disintegration in waxy maize, surface wrinkling in normal maize and tapioca, swelling and opening-up in potato starches, and swelling and bursting in high amylose maize. The ratio of ethanol to water at which original granular order was disrupted also varied with starch type. GGS had less short range molecular order than native granules as inferred by comparing 1047/1022 wave number ratio from infrared spectroscopy. Similarly, A- and B-type diffraction reflections were either reduced or completely lost with evolution of V-type patterns in GGS.     

Reactions of alpha amylases with starch granules in aqueous suspension giving products in solution and in a minimum amount of water giving products inside the granule

Porcine pancreatic alpha amylase (PPA) and Bacillus amyloliquefaciens alpha amylase (BAA) were allowed to react with starch granules from maize, waxy maize, amylomaize-7, and potato in an aqueous suspension with a starch to water ratio of 1:10 and in a minimum of water with a starch to water ratio of 1:1. Quantitative amounts of the maltodextrin products were determined by TLC and scanning densitometry. The two alpha amylases gave different products that were characteristic of their unique action patterns. The percent conversion differed for the different kinds of starches and for the two kinds of reaction conditions. Maize and waxy maize starches were converted into about twice as much maltodextrins than were amylomaize-7 and potato starches by both enzymes and under both reaction conditions. The aqueous suspension gave much greater conversion into maltodextrins than did the minimum water condition. BAA gave 3-14% greater conversion of the granules into maltodextrins than did PPA, with the exception of potato starch.     

Change of granular and molecular structures of waxy maize and potato starches after treated in alcohols with or without hydrochloric acid

Starches from waxy maize and potato were treated in methanol and 2-propanol either with or without 0.36% hydrochloric acid at 65 °C for 1 h. The granule morphology, molecular structure and pasting properties of the starches were determined and the effects of treatments on the granule and molecular structures of starch were investigated. Starch treated in alcohols without acid showed loss of native order through the hilum of granules, and no obvious molecular degradation was found. However, acid–alcohol treated starch showed many cracks inside granules, and both waxy maize and potato starches showed obvious molecular degradation after treated. Furthermore, the amylose chains and long chains of amylopectin of starch were more easily degraded with acid–alcohol treatment. The pasting viscosity of acid–alcohol treated starches were also obviously less than that of their counterpart native starch and starch after alcohol treatment. The extent of degradation of molecules and the decrease of pasting viscosity on potato starch after acid–alcohol treated were more obvious than that of waxy maize starch. The result indicates that the degradation preferentially occur in the amorphous region when starch treated by acid–alcohol, and the degradation of starch molecules enhances the amorphous excretion and the occurrence of cracks inside the granules.     

Enzyme modification of starch granules: formation and retention of cyclomaltodextrins inside starch granules by reaction of cyclomaltodextrin glucanosyltransferase with solid granules

Cyclomaltodextrin glucanosyltransferase (CGTase) was adsorbed into starch granules and allowed to react at 37 degrees C. The reaction was conducted with the granules removed from an aqueous environment, but containing 50% w/w water inside the granule. Reaction for 20 h gave a maximum of 1.4%, w/w of cyclodextrins (CDs) inside the granule. Waxy maize and maize starches gave the highest amounts of CDs (1.3 and 1.4%, respectively), with tapioca and amylomaize-7 starches giving about 50% less (0.9 and 0.6%, respectively). Reaction of a combination of CGTase and isoamylase with solid starch granules gave a 2.6-fold increase in the formation of CDs, with a maximum yield of 3.4 and 100% retention inside waxy maize starch granules.     

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.     

Damaged starch characterisation by ultracentrifugation

The relative molecular size distributions of a selection of starches (waxy maize, pea and maize) that had received differing amounts of damage from ball milling (as quantified by susceptibility to alpha-amylase) were compared using analytical ultracentrifugation. Starch samples were solubilised in 90% dimethyl sulfoxide, and relative size distributions were determined in terms of the apparent distribution of sedimentation coefficients g*(s) versus s(20,w). For comparison purposes, the sedimentation coefficients were normalised to standard conditions of density and viscosity of water at 20 degrees C, and measurements were made with a standard starch loading concentration of 8 mg/mL. The modal molecular size of the native unmilled alpha-glucans were found to be approximately 50S, 51S and 79S for the waxy maize, pea and maize amylopectin molecules, respectively, whilst the pea and maize amylose modal molecular sizes were approximately 14S and approximately 12S, respectively. As the amount of damaged starch increased, the amylopectin molecules were eventually fragmented, and several components appeared, with the smallest fractions approaching the sedimentation coefficient values of amylose. For the waxy maize starch, the 50S material (amylopectin) was gradually converted to 14S, and the degradation process included the appearance of 24S material. For the pea starch, the situation was more complicated than the waxy maize due to the presence of amylose. As the amylopectin molecules (51S) were depolymerised by damage within this starch, low-molecular-weight fragments added to the proportion of the amylose fraction (14S)--although tending towards the high-molecular-weight region of this fraction. As normal maize starch was progressively damaged, a greater number of fragments appeared to be generated compared to the other two starches. Here, the 79S amylopectin peak (native starch) was gradually converted into 61 and 46S material and eventually to 11S material with a molecular size comparable to amylose. Amylose did not appear to be degraded, implying that all the damage was focused on the amylopectin fraction in all three cases. Specific differences in the damage profiles for the pea and maize starches may reflect the effect of lipid-complexed amylose in the maize starch.     

Starch biosynthesis: sucrose as a substrate for the synthesis of a highly branched component found in 12 varieties of starches

D-[14C]glucose was incorporated into starch when 12 varieties of starch granules were incubated with [14C]sucrose. Digestion of the 14C-labeled starches with porcine pancreatic alpha amylase showed that a high percentage (16.1-84.1%) of the synthesized starch gave a relatively high molecular weight alpha-limit dextrin. Hydrolysis of the 12 varieties of starch granules by alpha amylase, without sucrose treatment, also gave an alpha-limit dextrin, ranging in amounts from 0.51% (w/w) for amylomaize-7 starch to 8.47% (w/w) for rice starch. These alpha-limit dextrins had relatively high molecular weights, 2.47 kDa for amylomaize-7 starch to 5.75 kDa for waxy maize starch, and a high degree of alpha-(1-->6) branching, ranging from 15.6% for rice starch to 41.1% for shoti starch. ADPGlc and UDPGlc did not synthesize a significant amount (1-2%) of the branched component, suggesting that sucrose is the probable substrate for the in vivo synthesis of the component and that sucrose is not first converted into a nucleotide-glucose diphosphate intermediate.     

Structural and thermodynamic properties of rice starches with different genetic background Part 2. Defectiveness of different supramolecular structures in starch granules

High-sensitivity differential scanning microcalorimetry (HSDSC), small-angle X-ray scattering (SAXS), light (LM) and scanning electronic (SEM) microscopy techniques were used to study the defectiveness of different supramolecular structures in starches extracted from 11 Thai cultivars of rice differing in level of amylose and amylopectin defects in starch crystalline lamellae. Despite differences in chain-length distribution of amylopectin macromolecules and amylose level in starches, the invariance in the sizes of crystalline lamellae, amylopectin clusters and granules was established. The combined analysis of DSC, SAXS, LM and SEM data for native starches, as well as the comparison of the thermodynamic data for native and annealed starches, allowed to determine the structure of defects and the localization of amylose chains in crystalline and amorphous lamellae, defectiveness of lamellae, clusters and granules. It was shown that amylose “tie chains”, amylose–lipid complexes located in crystalline lamellae, defective ends of double helical chains dangling from crystallites inside amorphous lamellae (“dangling” chains), as well as amylopectin chains with DP 6–12 and 25–36 could be considered as defects. Their accumulation can lead to a formation of remnant granules. The changes observed in the structure of amylopectin chains and amylose content in starches are reflected in the interconnected alterations of structural organization on the lamellar, cluster and granule levels.     

Quantification of total iodine in intact granular starches of different botanical origin exposed to iodine vapor at various water activities

Iodine has been used as an effective tool for studying both the structure and composition of dispersed starch and starch granules. In addition to being employed to assess relative amylose contents for starch samples, it has been used to look at the molecular mobility of the glucose polymers within intact starch granules based on exposure to iodine vapor equilibrated at different water activities. Starches of different botanical origin including corn, high amylose corn, waxy corn, potato, waxy potato, tapioca, wheat, rice, waxy rice, chick pea and mung bean were equilibrated to 0.33, 0.75, 0.97 water activities, exposed to iodine vapor and then absorbance spectra and LAB color were determined. In addition, a new iodine quantification method sensitive to <0.1% iodine (w/w) was employed to measure bound iodine within intact granular starch. Amylose content, particle size distribution of granules, and the density of the starch were also determined to explore whether high levels of long linear glucose chains and the surface area-to-volume ratio were important factors relating to the granular iodine binding. Results showed, in all cases, starches complexed more iodine as water content increased and waxy starches bound less iodine than their normal starch counterparts. However, much more bound iodine could be measured chemically with waxy starches than was expected based on colorimetric determination. Surface area appeared to be a factor as smaller rice and waxy rice starch granules complexed more iodine, while the larger potato and waxy potato granules complexed less than would be expected based on measured amylose contents. Corn, high amylose corn, and wheat, known to have starch granules with extensive surface pores, bound higher levels of iodine suggesting pores and channels may be an important factor giving iodine vapor greater access to bind within the granules. Exposing iodine vapor to moisture-equilibrated native starches is an effective tool to explore starch granule architecture.     

Characterization of barley starches of waxy, normal, and high amylose varieties

Four varieties of barley starches, W.B. Merlin, glacier, high amylose glacier, and high amylose hull-less glacier, were isolated from barley seeds. Apparent and absolute amylose contents, molecular size distributions of amylose and amylopectin, amylopectin branch-chain-length distributions, and Naegeli dextrin structures of the starches were analyzed. W.B. Merlin amylopectin had the longest detectable chain length of DP 67, whereas glacier, high amylose glacier and high amylose hull-less glacier amylopectins had the longest detectable chain length of DP 82, 79, and 78, respectively. All the four starches displayed a substantially reduced proportion of chains at DP 18–21. Amylopectins of high amylose varieties did not show significantly larger proportions of long chains than that of normal and waxy barley starch. Onset gelatinization temperatures of all four barley starches ranged from 55.0 to 56.5°C. Absolute amylose contents of W.B. Merlin, glacier, high amylose glacier, and high amylose hull-less glacier were 9.1, 29.5, 44.7, and 43.4%, respectively; phospholipid contents were 0.36, 0.78, 0.79, and 0.97%, respectively.     

Starch crystal solubility and starch granule gelatinisation

The solubility and dissolution behaviour of A- and B-type crystals of short chain amylose were measured both directly and using differential scanning calorimetry in the temperature range 30-110 degrees C. Dissolution in the calorimeter was affected by super-heating to the extent of 24-28 degrees C. Following trends previously found by calorimetry the B-type crystal polymorph was more soluble than the A-type. Analysis of the chain composition of the dissolved material revealed a preferential solubilisation of the short chains at the lower temperatures. The solubility of both crystal polymorphs and the magnitude of the preferential solubilisation effect was reduced in the presence of 30% w/w sucrose. A comparison of calorimetric measurements of crystal dissolution and the gelatinisation of native granular waxy maize and potato starches found some broad similarities, such as transition temperatures and their composition dependence, and some differences, such as the relatively narrow temperature range of granular gelatinisation, which reflects its cooperative nature.     

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.     

Sorption of mono- and sesquiterpenes by natural polysaccharides

Sorption of terpenoids (essential oil components) from aqueous solutions by six types of native food starches was studied by capillary gas chromatography. Sorption of volatile substances did not depend on amylose content in starch and specific surface of its granules. The degree of sorption was maximum (86%) for corn starch containing 25-28% amylose and decreased in the following order: tapioca starch (77%) > potato starch (74%) > wheat starch (70%) > high-amylose corn starch (58%) > amylopectin corn starch (57%). Amylopectin corn starch differed from other starches in the mechanism of sorption and selectivity to compounds with various functional groups.     

Sorption of Monoterpenoids and Sesquiterpenoids by Natural Polysaccharides

Sorption of terpenoids (essential oil components) from aqueous solutions by six types of native food starches was studied by capillary gas chromatography. Sorption of volatile substances did not depend on amylose content in starch and specific surface of its granules. The degree of sorption was maximum (86%) for corn starch containing 25–28% amylose and decreased in the following order: tapioca starch (77%) > potato starch (74%) > wheat starch (70%) > high-amylose corn starch (58%) > amylopectin corn starch (57%). Amylopectin corn starch differed from other starches in the mechanism of sorption and selectivity to compounds with various functional groups.     

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.     

The influence of amylose and amylopectin characteristics on gelatinization and retrogradation properties of different starches

Physico-chemical properties of starch from wheat, rye, barley (waxy, high-amylose and normal-amylose), waxy maize, pea and potato (normal-amylose and high-amylopectin) were studied. Emphasis was given to the amylose (total, apparent and lipid-complexed) and amylopectin characteristics as well as to the gelatinization and retrogradation properties measured using differential scanning calorimetry. The total amylose content varied from ca. 1 % for waxy maize to 37% for high-amylose barley. The amylopectin characteristics were determined by high-performance size-exclusion chromatography after debranching with isoamylase. The weight-average degree of polymerization ( _w) was 26, 33 and 27 for the A-, B-, and C-type starches, respectively. In general, the potato starches exhibited the highest retrogradation enthalpies and the cereal starches the lowest, while the pea starch showed an intermediate retrogradation enthalpy. The data were analysed by principal component analysis (PCA). The _w showed positive correlation to the melting interval, the peak minimum, the offset temperatures of the retrogradation-related endotherm as well as to the gelatinization and retrogradation enthalpies. However, the high-amylose barley retrograded to a greater extent than the other cereal starches, despite low _w (24). The amylose content was negatively correlated to the onset and the peak minimum temperatures of gelatinization.     

Effects of an added inclusion-amylose complex on the retrogradation of some starches and amylopectin

The effects of added cetyltrimethylammonium bromide (CTAB)-amylose complex on retrogradation of some starches (waxy-maize, maize, and potato starch) and on amylopectin from potato have been studied by differential scanning calorimetry (DSC). The starches and amylopectin samples with added CTAB-amylose complex received four different heat treatments prior to storage and DSC measurements that either melted the complex or left the complex intact. The calorimetry measurements showed that intact CTAB-amylose complex had much less effect on decreasing the retrogradation of the starches and the amylopectin than samples with melted complex prior to measurements. This is discussed in relation to possible complex formation of amylopectin and lipids and the effects of adding uncomplexed lipids on the retrogradation of waxy starches and amylopectin.     

Preparation of clear noodles with mixtures of tapioca and high-amylose starches

Ways to simulate the making of clear noodles from mung bran starch were investigated by studying the molecular structures of mung bean and tapioca starches. Scanning electron micrographs showed that tapioca starch granules were smaller than those of mung bean starch. X-ray diffraction patterns of mung bean and tapioca starch were A- and C_A-patterns, respectively. Iodine affinity studies indicated that mung bean starch contained 37% of apparent amylose and tapioca starch contained 24%. Gel permeation chromatograms showed that mung bean amylopectin had longer peak chain-length of long-branch chains (DP 40) than that of tapioca starch (DP 35) but shorter peak chain-length of short-branch chains (DP 16) than that of tapioca starch (DP 21). P-31 n.m.r. spectroscopy showed that both starches contained phosphate monoesters, but only mung bean starch contained phospholipids. Physical properties, including pasting viscosity, gel strength, and thermal properties (gelatinization), were determined. The results of the molecular structure study and physical properties were used to develop acceptable products using mixtures of cross-linked tapioca and high-amylose maize starches. Tapioca starch was cross-linked by sodium trimetaphosphate (STMP) with various reaction times, pH values, and temperatures. The correlation between those parameters and the pasting viscosity were studied using a visco/amylograph. Starches, cross-linked with 0.1% STMP, pH 11.0, 3.5 h reaction time at 25, 35, and 45°C (reaction temperature), were used for making noodles. High-amylose maize starch (70% amylose) was mixed at varying ratios (9, 13, 17, 28, 37, and 44%) with the cross-linked tapioca starches. Analysis of the noodles included: tensile strength, water absorption, and soluble loss. Noodle sensory properties were evaluated using trained panelists. Noodles made from a mixture of cross-linked tapioca starch and 17% of a high-amylose starch were comparable to the clear noodles made from mung bean starch.     

Genotype-specific spatial distribution of starch molecules in the starch granule: a combined CLSM and SEM approach

Starch granule types from a variety of botanical sources were selected to represent differences in crystalline polymorph, amylose and phosphate content, and amylopectin chain length distribution. Equimolar labeling of starch molecules with the fluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) was used to construct a detailed map of the distribution of amylose and amylopectin within the granule by confocal laser scanning microscopy (CLSM) analysis. Medium- and high-resolution scanning electron microscopy (SEM) were used to provide detailed images of granule surface structures. By using a combined surface and internal imaging approach, interpretations of a number of previous structural observations is presented. In particular, internal images of high amylose maize and potato suggest that multiple initiations of new granules are responsible for the compound or elongated structures observed in these starches. CLSM optical sections of rice granules revealed an apparent altered distribution of amylose in relation to the proposed growth ring structure, hinting at a novel mechanism of starch molecule deposition. Well-described granule features, such as equatorial grooves, channels, cracks, and growth rings were documented and related to both the internal and external observations. A new method for probing the phosphate distribution in native granules was developed using a phosphate-binding fluorescent dye and CLSM.