Characterization of a Potato Starch-digesting Bacterium and Its Production of Amylase

A bacterium which can utilize potato starch granules as sole carbon source was isolated and identified as Bacillus circulans from its physiological and biochemical properties. Scanning electron microscopic observation of potato starch granules recovered from the culture broth revealed that granules were degraded gradually from their surface resulting in elongated granules with layered structures on their surface. This bacterium produced extracellular amylase which can digest potato starch granules in vitro. The amylase has a unique property in that it produces only maltohexaose from gelatinized starch in the early stage of the reaction. For the production of this amylase potato starch was found to be most effective while soluble sugars including gelatinized starch and maltose had little effect.     

Enzymatic Improvement of the Cooking Quality of Aged Rice: A Main Mode of Protease Action

This study aims to improve the stickiness and brightness of cooked rice from aged rice grains by removing its albumins and globulins with proteases. In newly–harvested grains, the proteins were easily removed by extraction with water which contained minerals from rice. In aged grains, however, the proteins were already denatured and extracted with difficulty. Proteases hydrolyzed even the denatured proteins to make them extractable. As a result, starch granules on the surface of grains were liberated. Some proteases also hydrolyzed a starch granule-associated protein, and consequently, made the starch highly gelatinizable by heating. The protease treatment of aged grains was effective for two main reasons: liberation of starch granules and removal of the granule-associated protein. During cooking of rice the liberated starch without the associated proteins gelatinized to give rise to stickiness. The gelatinized starch is considered to form a more isotropic film on the surface of every cooked rice grain to give brightness as well.     

Internal structure and physicochemical properties of corn starches as revealed by chemical surface gelatinization

The organization of amylose and amylopectin within starch granules is still not well elucidated. This study investigates the radial distribution of amylose and amylopectin in different corn starches varying in amylose content (waxy corn starch (WC), common corn starch (CC), and 50% and 70% amylose corn starches (AMC)). Corn starches were surface gelatinized by 13 M LiCl at room temperature to different extents (approximately 10%, 20%, 30%, and 40%). The gelatinized surface starch and remaining granules were characterized for amylose content, amylopectin chain-length distribution, thermal properties, swelling power (SP), and water solubility index (WSI). Except for the outmost 10% layer, the amylose content in CC increased slightly with increasing surface removal. In contrast, amylose was more concentrated at the periphery than at the core for 50% and 70% AMC. The proportion of amylopectin A chains generally decreased while that of B1 chains generally increased with increasing surface removal for all corn starches. The gelatinization enthalpy usually decreased, except for 70% AMC, whereas the retrogradation enthalpy relatively remained unchanged for CC but increased for WC, 50% and 70% AMC with increasing surface removal. The SP and WSI increased with increasing surface removal for all corn starches, with WC showing a significant increase in SP after the removal of the outmost 10% layer. The results of this study indicated that there were similarities and differences in the distribution of amylose and amylopectin chains along the radial location of corn starch granules with varying amylose contents. More amylose-lipid complex and amylopectin long chains were present at the periphery than at the core for amylose-containing corn starches.     

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.     

Interrelationships between Water and cellular metabolism in Artemia cysts. VIII Sorption isotherms and derived thermodynamic quantities

An analysis of water vapor sorption by cysts of the brine shrimp, Artemia salina, has shown that at environmental water activities (aw) of 0.95 or less, the cysts equilibrate with the aw of their environment. Above this aw the metabolic activity of the cysts participates directly in their water content, and equilibration does not occur. In contrast, dried cysts killed by heat treatment or exposure to ammonia fumes equilibrated with all values of aw examined. Analysis of the temperature dependence of sorption isotherms revealed that below cyst hydrations of about 0.3 g H2O/g dried weight the temperature coefficient for water sorption was negative, but became positive at hydrations appreciably in excess of this value. Estimates for the differential and integral net enthalpy and entropy changes accompanying the sorption of water have been calculated from isotherms. These results have been interpreted and integrated with those from previous work on the hydration-dependence of metabolic activity. All of the examined hydration properties of the cysts have been shown to be due chiefly to the cellular component, and not the acellular shell. Analysis of the data by the Bradley equation has shown that the hydration behavior of the shell obeys this relationship, whereas that of the cellular component does not.     

Locations of hypochlorite oxidation in corn starches varying in amylose content

The general oxidation mechanism by hypochlorite on starch has been well studied, but the information on the distribution of the oxidation sites within starch granules is limited. This study investigated the locations where the oxidation occurred within corn starch granules varying in amylose content, including waxy corn starch (WC), common corn starch (CC), and 50% and 70% high-amylose corn starch (AMC). Oxidized corn starches were surface gelatinized by 13 M LiCl at room temperature to different extents (approximately 10%, 20%, 30%, and 40%). The surface-gelatinized remaining granules were separated and studied for structural characteristics including carboxyl content, amylose content, amylopectin chain-length distribution, thermal properties, and swelling properties. Oxidation occurred mostly at the amorphous lamellae. More carboxyl groups were found at the periphery than at the core of starch granules, which was more pronounced in oxidized 70% AMC. More amylose depolymerization from oxidation occurred at the periphery of CC. For WC and CC, amylopectin long chains (>DP 36) were more prone to depolymerization by oxidation. The gelatinization properties as measured by differential scanning calorimetry also supported the changes in amylopectin fine structure from oxidation. Oxidized starches swelled to a greater extent than their unmodified counterparts at all levels of surface removal. This study demonstrates that the locations of oxidation and physicochemical properties of oxidized starches are affected by the molecular arrangement within starch granules.     

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.     

Starch gelatinization under pressure studied by high pressure DSC

The gelatinization process of waxy corn starch under different pressures up to 10.0 MPa was investigated using a high pressure DSC. Compressed air and carbon dioxide were used as pressure resources. Effect of pressure and annealing under pressure on gelatinization of waxy corn starch was systematically studied, in particular on the gelatinization temperature and enthalpy. The results show that the peak temperature of gelatinization was increased slightly initially then remained stable with increasing pressure. The gelatinization enthalpy was decreased under pressure processing. Annealing the starch under pressure condition, just below its gelatinization temperature, increased gelatinization temperature but kept gelatinized enthalpy constant. Morphologies of starch granules treated under pressure were studied using an optical microscope and SEM. There is no discernable difference of starch granules treated with and without pressure, which indicates the pressures are not high enough to destroy crystalline structure. The intensity of the pressure acts as a key factor to influence the gelatinization of starch rather than the nature of the gas. Effect of pressure on the multi-endotherm detected by DSC for starch with intermediate water is used to study the mechanisms. The effect of pressure can be explained by the enhancement of water diffusion in the amorphous range.     

Sorption by cornstarch of components of a mixture of volatile organic substances from aqueous solutions

Effects of the composition of a mixture of organic substances on the binding of individual components in aqueous dispersions of native and gelatinized cornstarches with different contents of amylose was studied by capillary gas chromatography. Sorption was more active when alcohols were exposed to cornstarch as mixtures rather than individual compounds. In a multiple-component mixture, compounds belonging to distinct classes competed with each other for binding sites. Odorant sorption by aqueous starch dispersions was more sensitive to the composition of the mixture and the nature of starch when studied in aqueous dispersions of native starches (compared to those of gelatinized starches).     

In vitro utilization of amylopectin and high-amylose maize (Amylomaize) starch granules by human colonic bacteria.

It has been well established that a certain amount of ingested starch can escape digestion in the human small intestine and consequently enters the large intestine, where it may serve as a carbon source for bacterial fermentation. Thirty-eight types of human colonic bacteria were screened for their capacity to utilize soluble starch, gelatinized amylopectin maize starch, and high-amylose maize starch granules by measuring the clear zones on starch agar plates. The six cultures which produced clear zones on amylopectin maize starch- containing plates were selected for further studies for utilization of amylopectin maize starch and high-amylose maize starch granules A (amylose; Sigma) and B (Culture Pro 958N). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect bacterial starch-degrading enzymes. It was demonstrated that Bifidobacterium spp., Bacteroides spp., Fusobacterium spp., and strains of Eubacterium, Clostridium, Streptococcus, and Propionibacterium could hydrolyze the gelatinized amylopectin maize starch, while only Bifidobacterium spp. and Clostridium butyricum could efficiently utilize high-amylose maize starch granules. In fact, C. butyricum and Bifidobacterium spp. had higher specific growth rates in the autoclaved medium containing high-amylose maize starch granules and hydrolyzed 80 and 40% of the amylose, respectively. Starch-degrading enzymes were cell bound on Bifidobacterium and Bacteroides cells and were extracellular for C. butyricum. Active staining for starch-degrading enzymes on SDS-PAGE gels showed that the Bifidobacterium cells produced several starch-degrading enzymes with high relative molecular (M(r)) weights (>160,000), medium-sized relative molecular weights (>66,000), and low relative molecular weights (<66,000). It was concluded that Bifidobacterium spp. and C. butyricum degraded and utilized granules of amylomaize starch.     

Increased Hydrophobicity of Carboxymethyl Starch Film by Conjugation with Zein

A carboxymethyl starch (CMS) film was prepared by a process in which gelatinized CMS was dried, and subsequently treated with water-soluble carbodiimide in the presence of zein in 70% ethanol or 70% acetone to form acid-amide cross-linkages in order to increase the hydrophobicity of the surface of the film. A small amount of zein protein was found to be present on the surface of the zein-CMS conjugate (Zein-CMS) film, resulting in its insolubility in hot water, low water vapor permeability, and resistance to digestion with α-amylase and β-amylase. Digestion of the Zein-CMS film with protease rendered the film readily water-soluble, suggesting conjugation with zein as an effective means of increasing the hydrophobicity of biodegradable starch-based articles.     

Increased hydrophobicity of carboxymethyl starch film by conjugation with zein

A carboxymethyl starch (CMS) film was prepared by a process in which gelatinized CMS was dried, and subsequently treated with water-soluble carbodiimide in the presence of zein in 70% ethanol or 70% acetone to form acid-amide cross-linkages in order to increase the hydrophobicity of the surface of the film. A small amount of zein protein was found to be present on the surface of the zein-CMS conjugate (Zein-CMS) film, resulting in its insolubility in hot water, low water vapor permeability, and resistance to digestion with alpha-amylase and beta-amylase. Digestion of the Zein-CMS film with protease rendered the film readily water-soluble, suggesting conjugation with zein as an effective means of increasing the hydrophobicity of biodegradable starch-based articles.     

高水分、富含淀粉植物组织的扫描电镜制备技术优化

应用常规高真空扫描电子显微镜观察生物样品必须经过脱水和干燥处理,但无论采用临界点干燥还是冷冻干燥方法,都存在样品表面不同程度失真的问题。植物高水分、富含淀粉组织样品经处理后,容易出现淀粉流失、细胞壁变形等现象,从而造成扫描图像粗糙,无法获得真实的细胞内部结构。本文通过对CO_2临界点干燥、化学固定样品冷冻干燥和新鲜样品冷冻干燥3种扫描电镜样品制备技术中后期制样进行机械断裂和液氮脆断改进,优化出两种植物高水分、富含淀粉组织的扫描电镜样品制备方法:(1)样品首先进行FAA化学固定,经冷冻干燥后用液氮脆断,对断面喷金镀膜和扫描电镜观察。利用该方法所得细胞结构完整,细胞壁整齐,淀粉粒和蛋白轮廓明确,可用于分析淀粉粒和蛋白颗粒在细胞内的分布。(2)新鲜样品直接进行冷冻干燥,经液氮脆断后对断面喷金镀膜和扫描电镜观察。利用该方法所得细胞壁整齐,淀粉粒轮廓更清晰,并且无蛋白颗粒干扰,用于分析淀粉粒在细胞内的分布更加理想。     

Surface Localization of Zein Storage Proteins in Starch Granules from Maize Endosperm : Proteolytic Removal by Thermolysin and in Vitro Cross-Linking of Granule-Associated Polypeptides

Starch granules from maize (Zea mays) contain a characteristic group of polypeptides that are tightly associated with the starch matrix (C. Mu-Forster, R. Huang, J.R. Powers, R.W. Harriman, M. Knight, G.W. Singletary, P.L. Keeling, B.P. Wasserman [1996] Plant Physiol 111: 821–829). Zeins comprise about 50% of the granule-associated proteins, and in this study their spatial distribution within the starch granule was determined. Proteolysis of starch granules at subgelatinization temperatures using the thermophilic protease thermolysin led to selective removal of the zeins, whereas granule-associated proteins of 32 kD or above, including the waxy protein, starch synthase I, and starch-branching enzyme IIb, remained refractory to proteolysis. Granule-associated proteins from maize are therefore composed of two distinct classes, the surface-localized zeins of 10 to 27 kD and the granule-intrinsic proteins of 32 kD or higher. The origin of surface-localized δ-zein was probed by comparing δ-zein levels of starch granules obtained from homogenized whole endosperm with granules isolated from amyloplasts. Starch granules from amyloplasts contained markedly lower levels of δ-zein relative to granules prepared from whole endosperm, thus indicating that δ-zein adheres to granule surfaces after disruption of the amyloplast envelope. Cross-linking experiments show that the zeins are deposited on the granule surface as aggregates. In contrast, the granule-intrinsic proteins are prone to covalent modification, but do not form intermolecular cross-links. We conclude that individual granule intrinsic proteins exist as monomers and are not deposited in the form of multimeric clusters within the starch matrix.It has long been known that starch granules contain bound polypeptides, with protein levels of isolated starch granules from maize (Zea mays) ranging from 0.3 to 1.0% based upon measurement of N₂ (May, 1987). A recent study by our laboratory demonstrates that isolated starch granules from maize contain several dozen strongly bound polypeptides (Mu-Forster et al., 1996). The granule-associated proteins include starch-biosynthetic enzymes such as the waxy protein, SSI, and SBEIIb. These polypeptides are not removed from intact starch granules by protease treatment or detergent washing; therefore, they are believed to bind to the starch and to become irreversibly entrapped within the starch matrix.Based upon staining intensities of polypeptides extracted from the starch granule (Mu-Forster et al., 1996), approximately one-half of the granule-associated proteins in maize consist of low-molecular-mass polypeptides ranging between 10 and 27 kD. These bands fall within the size range displayed by the zein storage proteins, however, the spatial distribution of these polypeptides within the starch granule is unknown. Zeins have been defined as alcohol-soluble proteins that occur principally in protein bodies of maize endosperm and that may or may not require reduction before extraction (Wilson, 1991). The association of zeins with starch granules during endosperm development would not be expected because zein genes do not contain transit peptides that would target these proteins through the amyloplast envelope into the amyloplast stroma.The objective of this study was to establish the topology of granule-associated zeins in starch granules from maize endosperm. To accomplish this, it was necessary to distinguish between surface-localized and internalized polypeptides. Our working hypothesis defines polypeptides localized at the starch granule surface as those that are susceptible to hydrolysis upon treatment of intact granules with exogenous proteases. Conversely, internal granule proteins are defined as those that (a) become susceptible to proteolysis only following thermal disruption of the starch matrix, and (b) resist extraction by 2% SDS at room temperatures (Denyer et al., 1993; Rahman et al., 1995; Mu-Forster et al., 1996).In this study we were able to distinguish between surface-localized and internalized granule-associated polypeptides in starch granules from maize endosperm by use of the thermophilic protease thermolysin. Thermolysin is well suited for this purpose because it is highly active at starch-gelatinization temperatures, and has also been shown to effectively hydrolyze hydrophobic proteins located at the surfaces of chloroplasts and other subcellular organelles (Cline et al., 1984; Xu and Chitnis, 1995). Upon extended incubation of intact starch granules with thermolysin at subgelatinization temperatures, we found that zeins were selectively removed from the starch granule surface. All other granule-associated polypeptides remained inaccessible to proteolytic attack or to extraction by 2% SDS, unless the starch matrix was first disrupted by gelatinization. Our results distinguish between the surface-localized and granule-intrinsic proteins of maize endosperm, and establish that zeins are localized at the starch-granule surface. In addition, cross-linking experiments were conducted to determine nearest-neighbor relationships among zein subunits localized at the granule surface and granule intrinsic polypeptides localized within the starch matrix.     

Potato starch granule nanostructure studied by high resolution non-contact AFM

Surface studies at ambient conditions of potato starch granules subjected to multiple freezing and thawing, performed by a high resolution non-contact atomic force microscopy (nc-AFM), revealed some details of the starch granule nanostructure. After the treatment, a significant separation and a chain-like organisation of the granule surface elements have been observed. An accurate analysis of the granule surface nanostructure with a single amylopectine cluster resolution could be carried out. The oblong nodules of approximately 20-50 nm in diameter have been observed at the surface of the potato starch granules. The same size particles were precipitated by ethanol from gelatinized potato starch suspensions. They were also detected at the surface of oat and wheat starch granules. After multiple freezing and thawing, the eroded potato granule surface revealed a lamellar structure of its interior. The 30-40 nm inter-lamellar distances were estimated by means of nc-AFM. These findings fit previously proposed dimensions of the structural elements in the crystalline region of the starch granule. The observed surface sub-particles might correspond to the single amylopectine side chain clusters bundled into larger blocklets packed in the lamellae within the starch granule. The results supported the blocklet model of the starch granule structure.     

Extrusion of pectin/starch blends plasticized with glycerol

The microstructural and thermal dynamic mechanical properties of extruded pectin/starch/glycerol (PSG) edible and biodegradable films were measured by scanning electron microscopy (SEM) and thermal dynamic mechanical analysis (TDMA). SEM revealed that the temperature profile (TP) in the extruder and the amount of water present during extrusion could be used to control the degree to which the starch was gelatinized. TDMA revealed that moisture and TP during extrusion and by inference the amount of starch gelatinization had little effect on the mechanical properties of PSG films. Furthermore, TDMA revealed that PSG films underwent a glass transition commencing at about −50°C and two other thermal transitions above room temperature. Finally, it was concluded that the properties of extruded PSG films were comparable to those cast from solution.     

Influence of cross-linked potato starch treated with POCl3 on DSC, rheological properties and granule size

Differential scanning calorimetry (DSC), rheological measurements and granule size analyses were performed to characterize the influence of phosphorylation substitution levels on the properties of cross-linked potato starch. Phosphorus oxychloride (POCl₃) was used to produce the cross-linked potato starch. The levels of the reagent used for the reaction ranged between 40 and 5000 ppm (dwb). Storage (G′) and loss (G″) moduli were measured for a 5% (w/w) gelatinized starch dispersion stored at 20 °C for 24 h after heating at 85 °C for 30 min. The samples from 80 to 500 ppm were recognized as ‘strong gel'systems, whereas native potato starch showed ‘weak gel'behavior. Steady shear and dynamic viscoelastic properties of gelatinized starch dispersion were compared. Furthermore, granule mean diameter was measured by laser scattering for a 1% (w/w) dispersion heated at 85 °C for 30 min. The granules in the 100 ppm sample swelled to a maximum of about 2.6 times the native starch granule mean diameter.     

Controlled peeling of the surfaces of starch granules by gelatinization in aqueous dimethyl sulfoxide at selected temperatures

Microscopic examination of starch granules in 90:10 (v/v) Me(2)SO-H(2)O indicated that the granules were slowly being gelatinized from their surfaces. The rate of gelatinization was dependent on two variables: (1) the amount of water in Me(2)SO and (2) the temperature. An increase of water in Me(2)SO and/or an increase in temperature increased the rate of gelatinization and vice versa. Specific ratios of Me(2)SO and H(2)O (85:15-95:5) and temperatures (0-15 degrees C) were found to give controlled sequential peeling/gelatinization of eight kinds of starch granules in 1-12h, with amounts of 10-25% gelatinization per hour. It was observed that the percent of starch granule remaining versus time gave curves that were linear and others that had linear parts separated by one or more abrupt changes. No two starches had a similar gelatinization curve for the same two conditions of the amount of water and the temperature. It is hypothesized that these curves reflect different structural characteristics for the individual kinds of starch granules.     

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.     

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.