Optimization of experimental conditions for the separation of small and large starch granules by gravitational field-flow fractionation

Separation of small and large barley starch granules by gravitational field-flow fractionation was investigated from the point of view of sample pre-treatment, amount of injected sample, and elution conditions. The sample pre-treatment study resulted in the conclusion that it is reasonable to soak the starch granules for at least 24 h prior to separation. The experiments with different amounts of injected sample show that it is possible to increase as well as decrease twofold the sample amount usually used without any change in retention ratios. The implementation of flow-rate gradients for elution of the starch granules reduced total separation time. However, the applied flow-rate gradients did not improve the resolution of peaks A and B compared with the generally used constant flow-rate. Thus, for barley starch granules, the constant flow-rates within the range from 0.8 to 1.0 ml/min seem to provide the best compromise of total separation time, peak resolution and instrumental expense.     

非对称场流分离系统的构建及其在淀粉颗粒粒径表征中的应用

淀粉颗粒粒径与分子尺寸分别在1~100 μm和20~250 nm之间,是影响淀粉功能特性的重要因素之一。非对称场流分离(AF4)是一种基于样品与外力场相互作用机制的分离技术,已应用于表征淀粉分子尺寸分布。商品化的AF4系统的粒径检测范围为1 nm~10 μm,对于淀粉颗粒粒径表征具有一定的局限性。该文研制了AF4分离系统;考察了其在微米尺度下对红薯、莲子和大米淀粉颗粒粒径表征的性能;采用微米尺寸的聚苯乙烯乳化球(PS)标准样品验证了构建的AF4系统的分离性能。实验结果显示,构建的AF4系统对PS混合样品(粒径2、6、12、20 μm)实现了基线分离,同商品化AF4相比提高了检测上线,具有分离表征淀粉颗粒的潜力。此外,该文研究了载液组成对淀粉颗粒分离表征的影响;通过光学显微镜验证了构建的AF4系统在微米尺度上对淀粉颗粒粒径分布的表征能力。最后,采用商品化的AF4系统串联多角度激光光散射检测器和示差折光检测器对3种淀粉分子进行了分离表征,考察了淀粉的溶解温度对其表征结果的影响。在摩尔质量10 ⁶~10⁸ g/mol范围内,红薯和莲子淀粉的回转半径和水合半径的比值(R_g/R_h)在0.9~1.1之间,大米淀粉的R_g/R_h在1.2~1.4之间。实验结果证明构建的AF4系统是一种快速、准确的淀粉颗粒粒径表征方法,与商品化的AF4系统结合可为研究淀粉尺寸分布与其功能性质之间的关系提供技术支持。     

Determination of particle size distribution by gravitational field-flow fractionation: Dimensional characterization of silica particles

Summary Gravitational field-flow fractionation (GFFF) is the simplest and cheapest of field-flow fractionation (FFF) techniques, although it is still at an early development stage. The application of GFFF to the determination of particle size distribution (PSD) of silica particles used as chromatographic supports is described. The accuracy of the method is evaluated by comparing PSDs obtained by GFFF with those obtained by laser diffraction, a non-separative technique widely applied to particle characterization. It is ultimately demonstrated that a low-cost GFFF channel can simply replace the column of a standard HPLC system, allowing laboratories that are not specialized in size analysis to perform accurate PSD studies with standard HPLC expertise.     

Capillary liquid chromatographic-high-resolution mass spectrometric analysis of ribonucleotides

Performance of gravitational field-flow fractionation (GFFF) is improved here with respect to the ability to fractionate and distinguish different varieties of wine-making yeast from Saccharomyces cerevisiae. A new GFFF channel with non-polar walls has been employed to enhance fractionation selectivity and reproducibility. Since GFFF retention depends from first principles on particle size, Coulter counter measurements were performed in order to compare size distribution profiles with GFFF profiles. From such a comparison, GFFF was shown to be able to reveal differences in yeast cells other than size. This could make use of GFFF for screening different varieties of wine-making yeast towards future quality assessment procedures based on a possible correlation between yeast cell morphology indexes and quality indexes.     

Different elution modes and field programming in gravitational field-flow fractionation. IV. Field programming achieved with channels of non-constant cross-sections

Force field programming provided increased speed of separation with an improved resolution and detection capability in many field-flow fractionation (FFF) techniques. Gravitational field-flow fractionation (GFFF) uses the Earth's gravitational field to cause the settlement of particles towards the channel accumulation wall. Although this field is constant and relatively weak, there are different ways to implement force field programming in GFFF. Because hydrodynamic lift forces (HLF) participate in the separation process in focusing (hyperlayer) elution mode, they can control the resulting force field acting on particles via changes in flow-velocity. These changes can be accomplished by a programmable pump or with channels of non-constant cross-sections. This work is focused on flow-velocity programming accomplished with channels of non-constant cross-sections. Three trapezoidal channels of decreasing breadth and two channels of decreasing height (along the longitudinal axis) are tested as tools for optimization of the separation of a model silica gel particle mixture. The trapezoidal channels yielded reduced separation times. However, taking into account both speed of separation and resolution, the optimization effect was lower compared with the flow-rate gradients generated by a programmable pump. The channels of non-constant height did not yield advantageous separations.     

Different elution modes and field programming in gravitational field-flow fractionation. III. Field programming by flow-rate gradient generated by a programmable pump.

Gravitational field-flow fractionation (GFFF) utilizes the Earth's gravitational field as an external force that causes the settlement of particles towards the channel accumulation wall. Hydrodynamic lift forces oppose this action by elevating particles away from the channel accumulation wall. These two counteracting forces enable modulation of the resulting force field acting on particles in GFFF. In this work, force-field programming based on modulating the magnitude of hydrodynamic lift forces was implemented via changes of flow-rate, which was accomplished by a programmable pump. Several flow-rate gradients (step gradients, linear gradients, parabolic, and combined gradients) were tested and evaluated as tools for optimization of the separation of a silica gel particle mixture. The influence of increasing amount of sample injected on the peak resolution under flow-rate gradient conditions was also investigated. This is the first time that flow-rate gradients have been implemented for programming of the resulting force field acting on particles in GFFF.     

In vitro and in vivo digestibility of native maize starch granules varying in amylose contents

Digestibility of maize starch granules with different amylose content (AL-0, 22, 54, 68, 80, or 90%) was investigated. Measurement of the in vivo resistant starch (RS) content of the starches was performed using surgically prepared ileorectostomized rats. The rats were fed a purified diet containing one of the starches at 652.5 g/kg diet. The in vivo RS content was determined based on the fecal starch excretion. The dietary fiber (DF) value increased as a function of the amylose content in the starch and showed a positive linear correlation with the gelatinization temperature of the granules. In contrast, the in vitro RS content was likely to depend on both the surface area and amylose contents of the starch granules. The maximum in vitro RS content was obtained with AL-68 (54.4%). In vivo RS content showed a significant correlation with the amount of in vitro RS but not in respect to the DF detected. The in vivo RS content of AL-68 (43.4%) was higher than that found in AL-90 (37.8%). A profound gap was observed for AL-54 between the amount of DF (6.4%) and RS (in vitro = 46.6% and in vivo = 40.9%) present. The results suggest that both in vitro and in vivo digestibility of maize starch is affected by the amylose content and surface area of the granules. The current evaluation suggests that the physiological occurrence of RS from maize starch might be predictable by reference to the in vitro RS value.     

Fractionation and characterization of starch granules using field-flow fractionation (FFF) and differential scanning calorimetry (DSC)

Starch is one of the main carbohydrates in food; it is formed by two polysaccharides: amylose and amylopectin. The granule size of starch varies with different botanical origins and ranges from less than 1 μm to more than 100 μm. Some physicochemical and functional properties vary with the size of the granule, which makes it of great interest to find an efficient and accurate size-based separation method. In this study, the full-feed depletion mode of split-flow thin cell fractionation (FFD-SF) was employed for a size-based fractionation of two types of starch granules (corn and potato) on a large scale. The fractionation efficiency (FE) of fraction-a for corn and potato granules was 98.4 and 99.4%, respectively. The FFD-SF fractions were analyzed using optical microscopy (OM) and gravitational field-flow fractionation (GrFFF). The respective size distribution results were in close agreement for the corn starch fractions, while they were slightly different for the potato starch fractions. The thermal properties of FFD-SF fractions were analyzed, and the results for the potato starch showed that the peak temperature of gelatinization (T_p) slightly decreases as the size of the granules increases. Additionally, the enthalpy of gelatinization (ΔH) increases when the granule size increases and shows negative correlation with the gelatinization range (ΔT).

     

Consolidated Conversion of Hulled Barley into Fermentable Sugars Using Chemical,Thermal, and Enzymatic (CTE) Treatment

A novel process using chemical, thermal, and enzymatic treatment for conversion of hulled barley into fermentable sugars was developed. The purpose of this process is to convert both lignocellulosic polysaccharides and starch in hulled barley grains into fermentable sugars simultaneously without a need for grinding and hull separation. In this study, hulled barley grains were treated with 0.1 and 1.0 wt.-% sulfuric acid at various temperatures ranging from 110 to 170 °C in a 63-ml flow-through packed-bed stainless steel reactor. After sulfuric acid pretreatment, simultaneous conversion of lignocellulose and starch in the barley grains into fermentable sugars was performed using an enzyme cocktail, which included α-amylase, glucoamylase, cellulase, and β-glucosidase. Both starch and non-starch polysaccharides in the pre-treated barley grains were readily converted to fermentable sugars. The treated hulled barley grains, including their hull, were completely hydrolyzed to fermentable sugars with recovery of almost 100% of the available glucose and xylose. The pretreatment conditions of this chemical, thermal, and enzymatic (CTE) process for achieving maximum yield of fermentable sugars were 1.0 wt.% sulfuric acid and 110 °C. In addition to starch, the acid pretreatment also retained most of the available proteins in solid form, which is essential for subsequent production of fuel ethanol and high protein distiller’s dried grains with solubles co-product.     

Experimental study on the retention of silica particles in gravitational field-flow fractionation effects of the mobile phase composition

Effects of mobile phase composition can play an effective role in modulating the retention of particles in gravitational field-flow fractionation (GFFF), the simplest and cheapest among field-flow fractionation (FFF) techniques. In the framework of an optimized procedure for the GFFF characterization of particulate systems, an experimental approach to the effects of the mobile phase composition on the retention of silica particles retention is presented. The role of the ionic strength and the presence of surfactant are emphasized, with special regards to the shape of the particles. Moreover, the first experimental evidence of potential-barrier GFFF is reported.     

Impacts of Hydrothermal Treatments on the Morphology,Structural Characteristics,and In Vitro Digestibility of Water Caltrop Starch

The influence of hydrothermal treatments on the structural properties and digestibility of water caltrop starch was investigated. Scanning electron microscopy (SEM) showed some small dents on the surface of starch granules for samples treated with heat moisture treatment (HMT), but not for samples treated with annealing (ANN) which generally showed smoother surfaces. The gelatinization temperature of starch was generally increased by hydrothermal treatments, accompanied by a trend of decreasing breakdown viscosity. These results implied the improvement of thermal and shearing stability, particularly for HMT in comparison to ANN. After being cooked, the native and ANN-modified water caltrop starch granules were essentially burst or destroyed. On the other hand, the margin of starch granules modified by HMT and dual hydrothermal treatments remained clear with some channels inside the starch granules. X-ray diffraction revealed that the crystalline pattern of water caltrop starch changed from the C_A-type to the A-type and the relative crystallinity reduced with increasing moisture levels of HMT. Results of ANN-modified water caltrop starch were mostly similar to those of the native one. Moreover, water caltrop starch modified with HMT20 and dual modification contained a pronouncedly higher resistant starch content. These results suggested that HMT, ANN, and dual modification effectively modified the functional properties of water caltrop starch.     

Sedimentation field flow fractionation monitoring of rice starch amylolysis

Enzymatic starch granule hydrolysis is one of the most important reactions in many industrial processes. In this work, we investigated the capacity of SdFFF to monitor the native rice starch amylolysis. In order to determine if fractogram changes observed were correlated to granule biophysical modifications which occurred during amylolysis, SdFFF separation was associated with particle size distribution analysis. The results showed that SdFFF is an effective tool to monitor amylolysis of native rice starch. SdFFF analysis was a rapid (less than 10 min), simple and specific method to follow biophysical modifications of starch granules. These results suggested many different applications such as testing series of enzymes and starches. By using sub-population sorting, SdFFF could be also used to better understand starch hydrolysis mechanisms or starch granule structure.     

Copolymers of modified starches with polyacrylonitrile

A study was made of the ceric ammonium nitrate-initiated graft polymerization of acrylonitrile (AN) onto a number of modified starches that had been reduced in molecular weight by either acid, hypochlorite, or enzyme treatment. With highly soluble starches, much of the starting material was recovered as ungrafted carbohydrate, and the reaction product was largely dimethylformamide-soluble polymer with a high polyacrylonitrile (PAN) content. The molecular weight of grafted PAN was lower when the modified starches existed as granules in water dispersion; however, heating (60°C) an aqueous slurry of an acid-modified corn starch (with intact granules) before the reaction had relatively little effect on the composition of the copolymer. Decreasing the concentrations in water of modified starch and AN resulted in more frequent and lower molecular weight grafts of PAN. Aqueous methanol as a reaction medium for an acid-modified starch with intact granules led to more frequent grafting of lower molecular weight PAN than when water alone was used. The number of grafted chains, however, was fewer than found with unmodified wheat starch under comparable conditions. A modified starch with the granule structure completely broken down gave no detectable reaction in aqueous methanol.     

药物控释载体醋酸酯淀粉的消化性能研究

采用生物体外(in-vitro)消化模型模拟人体消化道环境，对不同取代度的醋酸酯化木薯淀粉的消化速率进行了研究；用微生物酶对醋酸酯化木薯淀粉进行生物降解并测定各个样品的抗消化淀粉含量。结果表明醋酸酯化会增大淀粉颗粒的消化速率，但随取代度的提高消化速率呈下降趋势。同样随取代度的提高，醋酸酯化也会降低淀粉糊的消化速率。醋酸酯淀粉卡抗消化淀粉含量低于原淀粉，且取代度越高含量越低。醋酸酯化会破坏和抑制淀粉中抗消化淀粉的形成。     

Effects of NaOH treatment of cereal starch granules on the extent of granular starch hydrolysis

Effect of NaOH treatment on granular hydrolysis of cereal starches was studied and granular starch hydrolyzing enzyme is used to hydrolyze native and NaOH-treated starch for 24?h. The dextrose equivalent value of NaOH-treated starch increased significantly compared to native starch, i.e., 28–38?% for corn, 7–37?% for rice, but no significant increase for corn starch. Scanning electron microscopy micrographs showed that NaOH treatment caused an enlargement of pores and degrades the surface of starch granules. Hydrolyzed-treated starch exhibited rougher surface and more porous granules compared to native starch. The swelling power and pasting properties of NaOH-treated starches were markedly altered after hydrolysis. X-ray pattern of all starches showed no changes and the amylose content decrease significantly after hydrolysis, which could due to extensive degradation of amorphous region. Evidently, NaOH treatment below gelatinization temperature was effective in enhancing the degree of granular starch hydrolysis.     

Structure of starch-bentonite gels

Mixed gels of starch and bentonite are investigated in the interval 0.056–0.089 of total solids/water ratio and 0-100% starch in the solids. The bentonite was a sodium calcium bentonite with a Na/Ca ratio of 1.76. In water it forms gels consisting of a network of band-type structures. Starch forms gels through hydrogen bonds between granules and/or amylose and amylopectin present on the external surfaces of granules and/or in fully stretched form. Mixed gels of bentonite and starch were obtained by adding corn starch granules to the already formed bentonite gels and heating the mixture above the Kofler gelatinization temperature. Amylose and amylopectin were adsorbed on strands of band-type structures of mont-morillonite lamellae. Starch gellation, e.g. diffusion of amylose out of the granule, was facilitated in the presence of bentonite. On the other hand, the presence of starch favored delamination of the montmorillonite particle into thinner lamellae. Maximum gelatinization and polymer adsorption were observed for gels with 20% starch and 80% bentonite. Montmorillonite networks formed the continuous phase for 0-80% starch. At higher starch concentrations, montmorillonite flakes were dispersed within the polymer network. Increase in the water content of the gels caused segregation of the bentonite and starch.     

大米淀粉糊化过程的光谱分析

采用衰减全反射傅立叶变换红外光谱仪跟踪测定了不同品种大米淀粉的糊化过程,同时与X-射线衍射仪测定的淀粉结晶度相对比,研究了淀粉颗粒内结晶结构在糊化过程中变化的详细情况.利用红外光谱仪计算出天然大米淀粉及其在糊化过程中各个阶段代表结晶区特征的1047cm-1和代表非晶区特征的1022cm-1两处红外吸收峰强度的比值.结果表明,天然淀粉的结晶区主要由支链淀粉侧链的双螺旋结构所形成;在加热过程中淀粉的结晶结构被破坏,并且直链淀粉含量越高,其结晶结构在糊化过程中破坏越慢,说明直链淀粉能抑制淀粉结晶结构的破坏.利用X-射线衍射仪测定了大米淀粉糊化过程各个阶段的结晶度,进一步验证了淀粉的结晶结构在糊化过程中的损失.虽然,两种测定方法对"结晶度"的定义不同,但对于淀粉结晶程度的测定具有相关性和可比性,能为研究淀粉的糊化行为提供有利的补充信息.     

Isolation, identification and characterisation of starch-interacting proteins by 2-D affinity electrophoresis

A 2-D affinity electrophoretic technique (2-DAE) has been used to isolate proteins that interact with various starch components from total barley endosperm extracts. In the first dimension, proteins are separated by native PAGE. The second-dimensional gel contains polysaccharides such as amylopectin and glycogen. The migration of starch-interacting proteins in this dimension is determined by their affinity towards a particular polysaccharide and these proteins are therefore spatially separated from the bulk of proteins in the crude extract. Four distinct proteins demonstrate significant affinity for amylopectin and have been identified as starch branching enzyme I (SBEI), starch branching enzyme IIa (SBEIIa), SBEIIb and starch phosphorylase using polyclonal antibodies and zymogram activity analysis. In the case of starch phosphorylase, a protein spot was excised from a 2-DAE polyacrylamide gel and analysed using Q-TOF MS/MS, resulting in the alignment of three internal peptide sequences with the known sequence of the wheat plastidic starch phosphorylase isoform. This assignment was confirmed by the determination of the enzyme's function using zymogram analysis. Dissociation constants (Kd) were calculated for the three enzymes at 4 degrees C and values of 0.20, 0.21 and 1.3 g/L were determined for SBEI, SBEIIa and starch phosphorylase, respectively. Starch synthase I could also be resolved from the other proteins in the presence of glycogen and its identity was confirmed using a polyclonal antibody and by activity analysis. The 2-DAE method described here is simple, though powerful, enabling protein separation from crude extracts on the basis of function.     

Starch Granule Size and Morphology of Arabidopsis thaliana Starch-Related Mutants Analyzed during Diurnal Rhythm and Development

Transitory starch plays a central role in the life cycle of plants. Many aspects of this important metabolism remain unknown; however, starch granules provide insight into this persistent metabolic process. Therefore, monitoring alterations in starch granules with high temporal resolution provides one significant avenue to improve understanding. Here, a previously established method that combines LCSM and safranin-O staining for in vivo imaging of transitory starch granules in leaves of Arabidopsis thaliana was employed to demonstrate, for the first time, the alterations in starch granule size and morphology that occur both throughout the day and during leaf aging. Several starch-related mutants were included, which revealed differences among the generated granules. In ptst2 and sex1-8, the starch granules in old leaves were much larger than those in young leaves; however, the typical flattened discoid morphology was maintained. In ss4 and dpe2/phs1/ss4, the morphology of starch granules in young leaves was altered, with a more rounded shape observed. With leaf development, the starch granules became spherical exclusively in dpe2/phs1/ss4. Thus, the presented data provide new insights to contribute to the understanding of starch granule morphogenesis.     

The rheology of concentrated suspensions of deformable particles

The rheology of slightly deformable particles, in particular their dilatant behaviour, has been investigated using waxy maize starch as a ‘model’ suspension. Suspensions of such large particles present considerable experimental problems in rheological terms in that they exhibit phenomena such as wall slip and sedimentation. These are discussed in some detail, along with the methodologies developed to overcome them. Comparisons of the results with hard sphere data in the literature indicate that whilst the deformability of the starch granules does not play a significant role in the high shear limiting viscosity, the dilatant transition does appear to be strongly affected.