Kinetic analysis of glucoamylase-catalyzed hydrolysis of starch granules from various botanical sources

The kinetics of glucoamylase-catalyzed hydrolysis of starch granules from six different botanical sources (rice, wheat, maize, cassava, sweet potato, and potato) was studied by the use of an electrochemical glucose sensor. A higher rate of hydrolysis was obtained as a smaller size of starch granules was used. The adsorbed amount of glucoamylase on the granule surface per unit area did not vary very much with the type of starch granules examined, while the catalytic constants of the adsorbed enzyme (k(0)) were determined to be 23.3+/-4.4, 14.8+/-6.0, 6.2+/-1.8, 7.1+/-4.1, 4.6+/-3.0, and 1.6+/-0.6 s(-1) for rice, wheat, maize, cassava, sweet potato, and potato respectively, showing that k(0) was largely influenced by the type of starch granules. A comparison of the k(0)-values in relation to the crystalline structure of the starch granules suggested that k(0) increases as the crystalline structure becomes dense.     

Kinetic Analysis of Glucoamylase-Catalyzed Hydrolysis of Starch Granules from Various Botanical Sources

The kinetics of glucoamylase-catalyzed hydrolysis of starch granules from six different botanical sources (rice, wheat, maize, cassava, sweet potato, and potato) was studied by the use of an electrochemical glucose sensor. A higher rate of hydrolysis was obtained as a smaller size of starch granules was used. The adsorbed amount of glucoamylase on the granule surface per unit area did not vary very much with the type of starch granules examined, while the catalytic constants of the adsorbed enzyme (k ₀) were determined to be 23.3±4.4, 14.8±6.0, 6.2±1.8, 7.1±4.1, 4.6±3.0, and 1.6±0.6 s^?1 for rice, wheat, maize, cassava, sweet potato, and potato respectively, showing that k ₀ was largely influenced by the type of starch granules. A comparison of the k ₀-values in relation to the crystalline structure of the starch granules suggested that k ₀ increases as the crystalline structure becomes dense.     

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.     

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.     

Determination of the maximum water solubility of eight native starches and the solubility of their acidic-methanol and -ethanol modified analogues

The maximum water solubilities of eight native starches from potato, shoti, tapioca, maize, waxy maize, amylomaize-7, wheat, and rice and their acid-methanol and acid-ethanol modified analogues have been determined. Maximum solubilities of 18.7 and 17.4 mg/mL were obtained for waxy maize and tapioca and 12.4 mg/mL for potato and maize starches by autoclaving 220 mg/10 mL at 121 degrees C; 8.7 mg/mL was obtained for shoti starch by stirring in 85:15 (v/v) Me(2)SO-H(2)O at 20 degrees C; and 7.0 and 5.2mg/mL for rice and amylomaize-7 starches by stirring in 1M NaOH at 20 degrees C. The acid-alcohol treated starches were 4-9 times more soluble than their native starches. The compositions of the solubilized starches had, in general, much higher ratios of amylose to amylopectin than the ratios in their native granules. A major exception to this was the acid-methanol treated potato, shoti, and rice starches that had much lower ratios of amylose to amylopectin than the ratios in their granules.     

Slow digestion property of native cereal starches

The slow digestion property of native cereal starches, represented by normal maize starch, was investigated. The in vitro Englyst test showed that 53.0% of the maize starch is slowly digestible starch (SDS), and scanning electron microscopy (SEM) revealed that SDS starts from an increase of pore size until almost complete fragmentation of starch granules. However, similar amounts of SDS ( approximately 50%) were shown for partially digested fragmented starch residuals, which would normally be considered resistant to digestion based on the Englyst assay. Molecularly, both amylopectin (AP) and amylose (AM) contributed to the amount of SDS as evidenced by a similar ratio of AP to AM at different digestion times. Consistently, similar degrees of crystallinity, comparable gelatinization behavior, and similar debranched profiles of starch residuals following different digestion times indicated that the crystalline and amorphous regions of starch granules were evenly digested through a mechanism of side-by-side digestion of concentric layers of semicrystalline shells of native starch granules.     

The substitution pattern in cationised and oxidised potato starch granules

The distribution pattern of substituents within the granules and the components of two cationised and two oxidised potato starches was studied. The level of crystallinity in wet-cationised (WC) and hypochlorite oxidised (HO) starch granules was similar to that of native starch granules but lower in dry-cationised (DC) and peroxide oxidised (PO) granules. However, the melting temperature of DC granules remained similar to native granules but was decreased in the other samples. With all modified starches, the initial rate of acid hydrolysis (lintnerisation) was increased compared to native granules. The degree of substitution decreased only slightly in WC granules after the lintnerisation, whereas virtually all the substituted glucosyl units in DC starch were hydrolysed already at initial stages. The decrease of substituents in the HO and PO starches was intermediate. The starches were partly resistant to the action of isoamylase and the successive beta-amylolysis, suggesting that substituents were found both close to the branches and near the nonreducing ends in the amylopectin component. It is suggested that the DC starch was preferentially cationised at the surface of the granules, whereas WC and oxidised starches were modified throughout the granules.     

双酶法水解辐照改性马铃薯淀粉动力学研究

为了解辐照改性马铃薯淀粉的酶解特性,用α-淀粉酶和糖化酶同时作用于马铃薯原淀粉和经400 kGy剂量辐照处理后淀粉,考察了pH值、酶解温度、α-淀粉酶用量、糖化酶用量对反应速率的影响.以米氏方程为基础,用Lineweaver-Burk法求解动力学参数.结果表明,辐照后马铃薯淀粉的酶解反应速率明显高于马铃薯原淀粉.在单一水解体系中,α-淀粉酶和糖化酶对辐照前后马铃薯淀粉的降解都遵循Michaelis-Menten方程,α-淀粉酶的Km分别为11.343 mg· mL-1和9.386 mg· mL-1,Vmax分别为0.406 mg（mL·min）-1和1.079 mg（mL·min）-1;糖化酶的Km分别为10.307 mg· mL-1和8.905 mg·mL-1,Vmax分别为0.338 mg（mL·min）-1和0.821mg（mL·min）-1;水解产物葡萄糖对反应体系具有竞争性抑制剂的作用,其抑制常数Ki分别为1.298 mg·mL-1和0.934 mg·mL-1.研究结果表明辐照有效提高了马铃薯淀粉的酶解反应活性.     

Evidence of native starch degradation with human small intestinal maltase-glucoamylase (recombinant)

Action of human small intestinal brush border carbohydrate digesting enzymes is thought to involve only final hydrolysis reactions of oligosaccharides to monosaccharides. In vitro starch digestibility assays use fungal amyloglucosidase to provide this function. In this study, recombinant N-terminal subunit enzyme of human small intestinal maltase-glucoamylase (rhMGAM-N) was used to explore digestion of native starches from different botanical sources. The susceptibilities to enzyme hydrolysis varied among the starches. The rate and extent of hydrolysis of amylomaize-5 and amylomaize-7 into glucose were greater than for other starches. Such was not observed with fungal amyloglucosidase or pancreatic alpha-amylase. The degradation of native starch granules showed a surface furrowed pattern in random, radial, or tree-like arrangements that differed substantially from the erosion patterns of amyloglucosidase or alpha-amylase. The evidence of raw starch granule degradation with rhMGAM-N indicates that pancreatic alpha-amylase hydrolysis is not a requirement for native starch digestion in the human small intestine.     

Adhesion of bifidobacteria to granular starch and its implications in probiotic technologies

Adhesion of 19 Bifidobacterium strains to native maize, potato, oat, and barley starch granules was examined to investigate links between adhesion and substrate utilization and to determine if adhesion to starch could be exploited in probiotic food technologies. Starch adhesion was not characteristic of all the bifidobacteria tested. Adherent bacteria bound similarly to the different types of starch, and the binding capacity of the starch (number of bacteria per gram) correlated to the surface area of the granules. Highly adherent strains were able to hydrolyze the granular starches, but not all amylolytic strains were adherent, indicating that starch adhesion is not a prerequisite for efficient substrate utilization for all bifidobacteria. Adhesion was mediated by a cell surface protein(s). For the model organisms tested (Bifidobacterium adolescentis VTT E-001561 and Bifidobacterium pseudolongum ATCC 25526), adhesion appeared to be specific for alpha-1,4-linked glucose sugars, since adhesion was inhibited by maltose, maltodextrin, amylose, and soluble starch but not by trehalose, cellobiose, or lactose. In an in vitro gastric model, adhesion was inhibited both by the action of protease and at pH values of < or =3. Adhesion was not affected by bile, but the binding capacity of the starch was reduced by exposure to pancreatin. It may be possible to exploit adhesion of probiotic bifidobacteria to starch granules in microencapsulation technology and for synbiotic food applications.     

Structural basis for the slow digestion property of native cereal starches

Native cereal starches are ideal slowly digestible starches (SDS), and the structural basis for their slow digestion property was investigated. The shape, size, surface pores and channels, and degree of crystallinity of starch granules were not related to the proportion of SDS, while semicrystalline structure was critical to the slow digestion property as evidenced by loss of SDS after cooking. The high proportion of SDS in cereal starches, as compared to potato starch, was related to their A-type crystalline structure with a lower degree of perfection as indicated by a higher amount of shortest A chains with a degree of polymerization (DP) of 5-10. The A-type amorphous lamellae, an important component of crystalline regions of native cereal starches, also affect the amount of SDS as shown by a reduction of SDS in lintnerized maize starches. These observations demonstrate that the supramolecular A-type crystalline structure, including the distribution and perfection of crystalline regions (both crystalline and amorphous lamellae), determines the slow digestion property of native cereal starches.     

Adhesion of Bifidobacteria to Granular Starch and Its Implications in Probiotic Technologies

Adhesion of 19 Bifidobacterium strains to native maize, potato, oat, and barley starch granules was examined to investigate links between adhesion and substrate utilization and to determine if adhesion to starch could be exploited in probiotic food technologies. Starch adhesion was not characteristic of all the bifidobacteria tested. Adherent bacteria bound similarly to the different types of starch, and the binding capacity of the starch (number of bacteria per gram) correlated to the surface area of the granules. Highly adherent strains were able to hydrolyze the granular starches, but not all amylolytic strains were adherent, indicating that starch adhesion is not a prerequisite for efficient substrate utilization for all bifidobacteria. Adhesion was mediated by a cell surface protein(s). For the model organisms tested (Bifidobacterium adolescentis VTT E-001561 and Bifidobacterium pseudolongum ATCC 25526), adhesion appeared to be specific for α-1,4-linked glucose sugars, since adhesion was inhibited by maltose, maltodextrin, amylose, and soluble starch but not by trehalose, cellobiose, or lactose. In an in vitro gastric model, adhesion was inhibited both by the action of protease and at pH values of ≤3. Adhesion was not affected by bile, but the binding capacity of the starch was reduced by exposure to pancreatin. It may be possible to exploit adhesion of probiotic bifidobacteria to starch granules in microencapsulation technology and for synbiotic food applications.     

Organisation of the external region of the starch granule as determined by infrared spectroscopy

Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) was used to study the external regions of starch granules. Native starches (wheat, potato, maize, waxy maize and amylomaize) were analysed and compared to gelatinised and acid-hydrolysed starches. The IR spectra of potato and amylomaize starches were closer to that of highly ordered acid-hydrolysed starch than the other starches. FTIR was not able to differentiate between A- and B-type crystallinity so the difference observed between starches was not related to this factor. The variation between starch varieties was interpreted in terms of the level of ordered structure present on the edge of starch granules with potato and amylomaize being more ordered on their outer regions. This could explain the high resistance of both these starches to enzyme hydrolysis.     

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.     

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.     

An investigation of the action of porcine pancreatic alpha-amylase on native and gelatinised starches

The action of pancreatic alpha-amylase (EC 3.2.1.1) on various starches has been studied in order to achieve better understanding of how starch structural properties influence enzyme kinetic parameters. Such studies are important in seeking explanations for the wide differences reported in postprandial glycaemic and insulinaemic indices associated with different starchy foodstuffs. Using starches from a number of different sources, in both native and gelatinised forms, as substrates for porcine alpha-amylase, we showed by enzyme kinetic studies that adsorption of amylase to starch is of kinetic importance in the reaction mechanism, so that the relationship between reaction velocity and enzyme concentration [E0] is logarithmic and described by the Freundlich equation. Estimations of catalytic efficiencies were derived from measurements of kcat/Km performed with constant enzyme concentration so that comparisons between different starches were not complicated by the logarithmic relationship between E0 and reaction velocity. Such studies reveal that native starches from normal and waxy rice are slightly better substrates than those from wheat and potato. After gelatinisation at 100 degrees C, kcat/Km values increased by 13-fold (waxy rice) to 239-fold (potato). Phosphate present in potato starch may aid the swelling process during heating of suspensions; this seems to produce a very favourable substrate for the enzyme. Investigation of pre-heat treatment effects on wheat starch shows that the relationship between treatment and kcat/Km is not a simple one. The value of kcat/Km rises to reach a maximum at a pre-treatment temperature of 75 degrees C and then falls sharply if the treatment is conducted at higher temperatures. It is known that amylose is leached from starch granules during heating and dissolves. On cooling, the dissolved starch is likely to retrograde and become resistant to amylolysis. Thus the catalytic efficiency tends to fall. In addition, we find that the catalytic efficiency on the different starches varies inversely with their solubility and we interpret this finding on the assumption that the greater the solubility, the greater is the likelihood of retrogradation. We conclude that although alpha-amylase is present in high activity in digestive fluid, the enzymic hydrolysis of starch may be a limiting factor in carbohydrate digestion because of factors related to the physico-chemical properties of starchy foods.     

A quantitative assessment of the importance of barley seed alpha-amylase, beta-amylase, debranching enzyme, and alpha-glucosidase in starch degradation.

Extracts of germinated barley (Hordeum vulgare L.) seeds of 41 different genotypes were analyzed for their activities of alpha-amylase, beta-amylase, alpha-glucosidase, and debranching enzyme and for their abilities to hydrolyze boiled soluble starch, nonboiled soluble starch, and starch granules extracted from barley seeds with water. Linear correlation analysis, used to quantitate the interactions between the seven parameters, revealed that boiled soluble starch was not a good substrate for predicting activities of enzymes functioning in in vivo starch hydrolysis as the extracts' abilities to hydrolyze boiled soluble starch was not correlated with their abilities to hydrolyze native starch granules. Activities of alpha-amylase and alpha-glucosidase were positively and significantly correlated with the seed extracts' abilities to hydrolyze all three starches. beta-Amylase was only significantly correlated with hydrolysis of boiled soluble starch. No significant correlations existed between debranching enzyme activity and hydrolysis of any of the three starches. Interactions between the four enzymes as they functioned together to hydrolyze the three types of starch were evaluated by path coefficient analysis. alpha-Amylase contributed to hydrolyses of all three starches primarily by its direct effect (noninteractive component). This direct contribution increased as the substrate progressed from the completely artificial boiled soluble starch, to the most physiologically significant substrate, native starch granules. alpha-Glucosidase contributed to the hydrolysis of boiled soluble starch primarily by its direct effect (noninteractive) yet contributed to starch granule hydrolysis primarily via its interaction with alpha-amylase (indirect effect). The contribution of beta-amylase to hydrolysis of boiled soluble starch was direct and it did not contribute significantly to hydrolysis of native starch granules.     

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.     

In depth study of a new highly efficient raw starch hydrolyzing α-amylase from Rhizomucor sp

A new α-amylase from Rhizomucor sp. (RA) was studied in detail due to its very efficient hydrolysis of raw starch granules at low temperature (32 °C). RA contains a starch binding domain (SBD) connected to the core amylase catalytic domain by a O-glycosylated linker. The mode of degradation of native maize starch granules and, in particular, the changes in the starch structure during the hydrolysis, was monitored for hydrolysis of raw starch at concentrations varying between 0.1 and 31%. RA was compared to porcine pancreatic α-amylase (PPA), which has been widely studied either on resistant starch or as a model enzyme in solid starch hydrolysis studies. RA is particularly efficient on native maize starch and release glucose only. The hydrolysis rate reaches 75% for a 31% starch solution and is complete at 0.1% starch concentration. The final hydrolysis rate was dependent on both starch concentration and enzyme amount applied. RA is also very efficient in hydrolyzing the crystalline domains in the maize starch granule. The major A-type crystalline structure is more rapidly degraded than amorphous domains in the first stages of hydrolysis. This is in agreement with the observed preferential hydrolysis of amylopectin, the starch constituent that forms the backbone of the crystalline part of the granule. Amylose-lipid complexes present in most cereal starches are degraded in a second stage, yielding amylose fragments that then reassociate into B-type crystalline structures, forming the final resistant fraction.     

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.