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.     

Determination of hydrophobicity of dry-heated wheat starch granules using sucrose fatty acid esters (SFAE)

Sucrose fatty acid esters (SFAE) were adsorbed onto dry-heated (120?°C for 10, 20, 40, 60, and 120?min) wheat starch granules and extracted with ethyl ether in a Soxhlet apparatus without gelatinization of the starch granules. The amount of sucrose in the extracted SFAE was determined by the phenol sulfate method. A gradual increase of the sucrose from 159 to 712?μg, in SFAE per gram of starch, occurred with increasing dry-heating time and demonstrated the increased hydrophobicity of the starch granules. Increase of the SFAE was highly correlated (r?=?0.9816) to increase of the oil-binding capacity of the dry-heated wheat starch granules. Non-waxy rice, waxy rice, sweet potato, and potato starch granules also showed higher hydrophobicity after dry-heating by this method.     

Effects of structural imperfection on gelatinization characteristics of amylopectin starches with A- and B-type crystallinity

The aim of the present work was to investigate the effect of physical structures on the properties of starch granules. Starches with a high amylopectin content possessing A- and B-type crystallinity were chosen for the study. The gelatinization temperature decreased in the following order: maize (A) > potato (B) > wheat (A) > barley (A), which did not reflect a correlation with the type of crystallinity. Low values of gelatinization temperature were accompanied with high free surface energy of the crystallites. It is proposed that these data are caused by different types of imperfections in starch crystals. Annealing resulted in an enhancement of the gelatinization temperature and a decrease of the free surface energy of the crystallites for all starches reflecting a partial improvement of crystalline perfection. A limited acid hydrolysis (lintnerization) of the starches decreased the gelatinization temperature because of a partial disruption of the crystalline lamellae and an increase of the amount of defects on the edges of the crystallites. Annealing of the lintnerized starches improved the structure of maize and potato starch, giving them similar structural and physicochemical parameters, which was opposite the behavior of the annealed sample from wheat. The possible nature of removable and nonremovable defects inside the crystalline region of the starch granules is discussed. It is concluded that, besides the allomorphic A- and B-types of crystal packing, physical defects in the crystals possess a major impact on starch gelatinization.     

Porcine pancreatic alpha-amylase hydrolysis of native starch granules as a function of granule surface area

Porcine pancreatic alpha-amylase activity on native starch granules is more accurately described as a function of surface area of the granules rather than of substrate concentration. The apparent K(m) of alpha-amylolysis of native starch from potato, maize, and rice expressed as a function of substrate concentration was largest for potato with a single value of V(max). However, the ratio of the slope of a Lineweaver-Burk plot to that of rice for enzymatic hydrolysis of native potato and maize starch were 7.78 and 2.58, respectively, which were very close to the ratio of surface area per mass of the two starch granules to that of rice. Therefore, the reciprocal of initial velocity was a linear function of the reciprocal of surface area for each starch granule. Surface area was calculated assuming the starch granules were spherical. The values obtained by this calculation were in good agreement with the value obtained by the photomicrographic method. By comparing enzymatic digestion of native maize granules to that of rice granules, it was concluded that the presence of pores in maize granules appeared to significantly affect overall rate of digestion after sufficient reaction time, but not at the very initial stage of hydrolysis.     

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.     

Itaconic acid production by Aspergillus terreus on raw starchy materials

Starchy materials such as corn starch, soft wheat flour, potato flour, cassava flour, sorghum starch, sweet potato and industrial potato flours, either acid or enzymatically hydrolysed, were used as substrates for itaconic acid production by Aspergillus terreus NRRL 1960. Both production and yield were highest on corn starch (18·4 g l⁻¹ and 34·0%, respectively). The degree of hydrolysis had a great influence on acid production which was highest when corn starch was saccharified at 85 DE (dextrose equivalent). In a 3 litre benchtop fermenter, itaconic acid production and productivity were 19·8 g l⁻¹ and 0·13 g l⁻¹ h⁻¹, respectively.     

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.     

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.     

Glucoamylase production by the marine yeast Aureobasidium pullulans N13d and hydrolysis of potato starch granules by the enzyme

Under the optimal conditions, 10 U/ml of glucoamylase was produced by the marine yeast Aureobasidium pullulans N13d. It was noticed that the crude glucoamylase actively hydrolyzed potato starch granules, but poorly digested raw corn starch and sweet potato starch, resulting in conversion of 68.5, 19 and 22% of them into glucose within 6 h of incubation in the presence of 40 g/l of potato starch granules and 20 U/ml of the crude enzyme. When potato starch granules concentration was increased from 10 to 80 g/l, hydrolysis extent was decreased from 85.6 to 60%, while potato starch granules concentration was increased from 80 to 360 g/l, hydrolysis extent was decreased from 60 to 56%. Ratio of hydrolysis extent of potato starch granules to hydrolysis extent of gelatinized potato starch was 86.0% and the hydrolysis extent of potato starch granules by action of the crude glucoamylase (1.0 U/ml) was 18.5% within 30 min at 60 °C. Only glucose was detected during the hydrolysis, indicating that the crude enzyme could hydrolyze both α-1,4 and α-1,6 linkages of starch molecule in the potato starch.     

Production of native-starch-degrading enzymes by a Bacillus firmus/lentus strain

Summary A bacterium belonging to the Bacillus firmus/lentus-complex and capable of growth on native potato starch was isolated from sludge of a pilot plant unit for potato-starch production. Utilization of a crude enzyme preparation obtained from the culture fluid after growth of the microorganism on native starch, resulted in complete degradation of native starch granules from potato, maize and wheat at a temperature of 37°C. Glucose was found as a major product. Production of maltose, maltotriose and maltotetraose was also observed. Native-starch-degrading activity (NSDA) could be selectively adsorbed on potato-starch granules, whereas soluble-starch-degrading activity (SSDA) remained mainly in solution. The use of such a starch-adsorbed enzyme preparation on native starch resulted in a completely changed product pattern. An increase in oligosaccharides concomitant with less glucose formation was observed. An increased conversion of soluble starch to maltopentaose was possible with this starch-adsorbed enzyme preparation. It is concluded that NSDA comes from -amylase(s) and SSDA from glucoamylase(s) and/or -glucosidase(s). Cultivation of B. firmus/lentus on glucose, maltose, or soluble starch resulted in substantially smaller quantities of (native) starch-degrading activity.Offprint requests to: D. J. Wijbenga     

气候变化背景下中国南方地区季节性干旱特征与适应Ⅵ.防旱避灾种植模式优化布局

南方地区是我国重要的农业种植区,季节性干旱严重影响该地区的农业生产.本文基于南方地区不同干旱分区中选取的13个典型地区1981-2007年气象资料和作物生育期、产量等资料,依据各地逐年降水量将其分为干旱年、正常年和丰水年3种不同降水年型,利用作物水分临界期需水量与降水量的耦合度、气象产量、单位面积产值以及全生育期的水分利用效率和降水量5个指标,对典型地区种植模式的综合效益进行评价,得到南方不同区域不同降水年型下的优化种植模式.结果表明: 半干旱区在干旱年型下,宜采取2种抗旱种植模式：马铃-玉米-甘薯和冬小麦-中稻-甘薯.半湿润区在干旱年型下,种植模式以冬小麦-中稻-甘薯最优,油菜-中稻-甘薯次之.在温润区(即典型的季节性干旱区),江南地区在3种年型下均以马铃薯-双季稻最优;西南地区宜搭配抗旱作物进行三熟制种植,如冬小麦-中稻-甘薯、冬小麦-玉米-甘薯、马铃薯-双季稻等.从最大程度利用水热资源角度考虑,三熟种植模式最优,以水旱轮作为主,丰水年型宜搭配水稻.     

Effect of waxy rice flour and cassava starch on freeze-thaw stability of rice starch gels

Repeatedly frozen and thawed rice starch gel affects quality. This study investigated how incorporating waxy rice flour (WF) and cassava starch (CS) in rice starch gel affects factors used to measure quality. When rice starch gels containing 0-2% WF and CS were subjected to 5 freeze-thaw cycles, both WF and CS reduced the syneresis in first few cycles. However CS was more effective in reducing syneresis than WF. The different composite arrangement of rice starch with WF or CS caused different mechanisms associated with the rice starch gel retardation of retrogradation, reduced the spongy structure and lowered syneresis. Both swollen granules of rice starch and CS caused an increase in the hardness of the unfrozen and freeze-thawed starch gel while highly swollen WF granules caused softer gels. These results suggested that WF and CS were effective in preserving quality in frozen rice starch based products.     

High alcohol production by solid substrate fermentation from starchy substrates using thermotolerant Saccharomyces cerevisiae

Solid Substrate Fermentation system (SSF) was used to produce ethanol from various starchy substrates like sweet sorghum, sweet potato, wheat flour, rice starch, soluble starch and potato starch using thermotolerant yeast isolate (VS₃) by simultaneous saccharification and fermentation process. Alcohol produced was estimated by gas chromatography after an incubation time of 96 hrs at 37v°C and 42v°C. More ethanol was produced from rice starch and sweet sorghum. The maximum amount of ethanol produced from these substrates using VS₃ was 10 g/100 g and 3.5 g/100 g substrate (rice starch) and 8.2 g and 7.5 g/100 g substrate (sweet sorghum) at 37v°C and 42v°C respectively.     

Pretreatment of starch raw materials and their enzymatic hydrolysis by immobilized glucoamylase

The pretreatment of starch raw materials such as sweet potato, potato and cassava has been carried out using various types of crusher, viz juice mixer, homogenizer and high-speed planetary mill. The effect of pretreatment of the materials on their enzymatic hydrolysis was studied. High-speed planetary mill treatment was the most effective and comparable with heat treatment (pasting). Various crushing times were used to examine the effect of crushing by mill treatment on the enzymatic hydrolysis. In the enzymatic hydrolysis of cassava, the use of both cellulase [1,4-(1,3; 1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and glucoamylase [1,4-α-d-glucan glucohydrolase, EC 3.2.1.3] enhanced the d-glucose yield. The immobilization of glucoamylase was studied by radiation polymerization of hydrophilic monomers at low temperature, and it was found that enzymatic activity of the immobilized glucoamylase particles varied with monomer concentration and particle size. Starchy raw materials pretreated with the mill can be efficiently hydrolysed by immobilized glucoamylase.     

Preparation of Koji from corn hulls for alcoholic fermentation without cooking

Corn hulls, the outer peel covering the corn grain, were used for preparation of koji, which was applied to alcoholic fermentation without cooking of raw strachy materials. Corn hull koji had lower saccharifying power, α-amylase, CMCase and xylanase than wheat bran koji, but higher protease and pectinase activities, Its alcoholic fermentations of cassava starch and sweet potato were also superior to those of wheat bran koji: corn hull koji gave 10.3% (v/v) of alcohol with 93% yield from 20 g of cassava starch, while wheat bran koji gave 9.4% (v/v) of alcohol with 90.4% yeild; and corn hull koji gave 9.1% (v/v) of alcohol with 92.6% yielded from 50 g of sweet potato, while wheatbran koji gave 8.1 (v/v) of alcohol with 88.6% yield.     

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.     

Production of an amylase-degrading raw starch by Gibberella pulicaris

An endophytic fungus, Gibberella pulicaris, produced an amylase which degraded raw starches from cereals and other crops including raw potato, sago, tapioca, corn, wheat and rice starch. In each case, glucose was the main product. Among the raw starches used, raw potato starch gave the highest enzyme activity (85 units mg^–1 protein) and raw wheat starch the lowest (49 units mg^–1 protein). The highest amylase production (260 units mg^–1 protein) was achieved when the concentration of raw potato starch was increased to 60 g l^–1. Optimum hydrolysis was at 40°C and pH 5.5.     

Effect of adsorption of an inhibitory factor on raw starch hydrolysis by glucoamylase

An inhibitory factor (IF) produced byAspergillus niger strain 19, and which inhibits the action of glucoamylase on starch, has the ability to be tightly adsorbed on to various raw starches, though the amount differs from starch to starch. Based on the hydrolysis of the IF-starch complex by glucoamylase, the inhibitions per unit IF adsorbed are similar for some varieties of starch. The effectiveness ratio of IF (% hydrolysis inhibition per % IF adsorbed on raw starch) for corn, sweet potato, waxy rice and waxy corn starches are 1.1, 1.0, 0.85 and 0.96, respectively. These results support the hypothesis that both glucoamylase and IF are adsorbed on to a common binding site on raw starch. However, the effectiveness ratio of IF for cassava and wheat starches are 0.71 and 1.65, respectively, which differ significantly from other varieties of starch.

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Résumé Un inhibiteur (facieur IF) produit par la souche 19 d'Aspergillus niger et qui inhibe l'action de la glucoamylase sur l'amidon a la propriété d'être fortement adsorbé sur dives amidons, bien que la quantité varie d'amidon à amidon. Sur la base de l'hydrolyse du complexe amidon-IF par la glucoamylase, les inhibitions par unité d'IF adsorbé sont sembiables pour quelques variétés d'amidon. Le rapport d'efficience de IF (% d'inhibition de l'hydrolyse par % de IF adsorbé sur l'amidon cru), pour le maïs, la patate douce, et les amidons de riz cireux et de maîs cireux vaut respectivement 1.1, 1.0, 0.85 et 0.96. Ces résultats soutiennent l'hypothèse que tant la glucoamylase que le lacteur IF sont adsorbés sur un site commun de liaison de l'amidon cru. Toutefois, le rapport d'efficience du facteur IF pour les amidons de manioc et de froment valent respectivement 0.71 et 1.65, valeurs significativement différentes de celles pour les aufres variétés d'amidon.

     

Characterization of a Granule-Bound Starch Synthase Isoform Found in the Pericarp of Wheat

Waxy wheat (Triticum aestivum L.) lacks the waxy protein, which is also known as granule-bound starch synthase I (GBSSI). The starch granules of waxy wheat endosperm and pollen do not contain amylose and therefore stain red-brown with iodine. However, we observed that starch from pericarp tissue of waxy wheat stained blue-black and contained amylose. Significantly higher starch synthase activity was detected in pericarp starch granules than in endosperm starch granules. A granule-bound protein that differed from GBSSI in molecular mass and isoelectric point was detected in the pericarp starch granules but not in granules from endosperm. This protein was designated GBSSII. The N-terminal amino acid sequence of GBSSII, although not identical to wheat GBSSI, showed strong homology to waxy proteins or GBSSIs of cereals and potato, and contained the motif KTGGL, which is the putative substrate-binding site of GBSSI of plants and of glycogen synthase of Escherichia coli. GBSSII cross-reacted specifically with antisera raised against potato and maize GBSSI. This study indicates that GBSSI and GBSSII are expressed in a tissue-specific manner in different organs, with GBSSII having an important function in amylose synthesis in the pericarp.     

绣球菌生物学特性若干问题的研究

绣球菌菌丝在蛋白胨培养基中生长最佳,硫酸铵、尿素、复合肥对菌丝生长有抑制作用。选用基础培养基或在基础培养基中添加0.3%蛋白胨作为绣球菌母种培养基较为合适。栽培料中添加淀粉可促进绣球菌的生长,接种50d后观察发现,添加新鲜去皮马铃薯块比马铃薯淀粉效果好,前者100%菌袋出现原基,后者只有50%菌袋出现原基,且原基正处在胶质片时期。添加大米淀粉,小麦淀粉效果与马铃薯块接近,甘薯淀粉与马铃薯淀粉效果接近。