Determination of Optimum Conditions for Enzymatic Debranching of Cassava Starch and Synthesis of Resistant Starch Type III using Central Composite Rotatable Design

Cassava starch was debranched by treatment with isoamylase and pullulanase and the yield of resistant starch type III (RS III) optimized with respect to starch solids concentration (7.5‐15%, w/v), incubation time (8‐24 h) and enzyme concentration using central composite rotatable design. Higher concentrations of pullulanase (10‐35 U/g starch) compared to isoamylase (30–90 mU/g starch) were required to give a similar degree of starch hydrolysis within the experimental domain. A clear debranching end‐point was identifiable by following the reducing value, blue value and β‐hydrolysis limit of cassava starches debranched using isoamylase. It was difficult to define a debranching endpoint of pullulanase treatment by these parameters due to contaminating α‐D ‐(1→4) activity. The yield of RS III was significantly higher in isoamylolysates and increased steadily with increasing degree of hydrolysis to peak at 57.3%. Purification of the debranched material further increased the RS III yield to 64.1%. Prolonged (24 h) hydrolysis of cassava starch with high concentration of pullulanase (35 U/g) gave lower RS III contents in the purified (34.2%) and unpurified (36.2%) hydrolysates compared to 49.5 and 62.4%, respectively, at moderate pullulanase concentration (22.5 U/g) and incubation time (16 h).     

酶脱支处理对玉米缓慢消化淀粉形成的影响

以玉米原淀粉为原料,研究普鲁兰酶脱支处理糊化后制备缓慢消化淀粉(slowly digestible starch,SDS)过程中各影响因素(温度、p H值、酶用量、贮藏及干燥条件)对SDS形成的影响。结果表明,在57.5℃、p H 4.9、酶用量60 U/g的条件下脱支8 h,然后煮沸灭酶30 min,再经4℃冷藏、60℃干燥后,可得SDS含量为31.09%的产品。原淀粉、酶脱支处理样品及脱支并去除快速消化淀粉样品的X射线衍射图谱表明,脱支处理后,玉米淀粉结晶结构由A型向B型转变。因此,通过酶脱支处理提高SDS含量的可能原因是形成了新的结晶结构,SDS含量与结晶的数量和质量有关。采用酶法制备SDS具有较好的工业化应用前景。     

Resistant Starch Made from Banana Starch by Autoclaving and Debranching

Resistant starches (RS) were prepared from banana starch by debranching with pullulanase for different times and after autoclaving treatment. The different treatments produced seven RS products, which were tested with respect to available starch (AS), RS and in vitro hydrolysis rate. The control sample (without debranching) had the highest AS (80.5%) and the lowest RS content (9.1%). The samples debranched for 5 h and longer did not show significant differences (α = 0.05) in AS (approximately 72%) and RS (approximately 18%). The RS values obtained in the samples prepared were twice as high as that of the control sample. However, the sample debranched for the longest time had the highest hydrolysis rate, demonstrating that this product has a high digestion rate. Banana starch is a good source for RS preparation by autoclaving due to its high RS content and can be an alternative source in developing countries for obtaining a nutraceutic ingredient for functional food preparation.     

Structural characterizations and digestibility of debranched high-amylose maize starch complexed with lauric acid

Structural characterizations and digestibility of debranched high-amylose maize starch complexed with lauric acid (LA) were studied. The cooked starch was debranched by using pullulanase and then complexed. Light microscopy showed that the lipids complexed starches had irregularly-shaped particles with strong birefringence. Gel-permeation chromatograms revealed that amylopectin degraded to smaller molecules during increasing debranching time, and the debranch reaction was completed at 12 h. Debranching pretreatment and prolonged debranching time (from 2 h to 24 h) could improve the formation of starch lipids complex. X-ray diffraction pattern of the amylose–lipid complexes changed from V-type to a mixture of B- and V-type polymorphs and relative crystallinity increased as the debranching time increased from 0 to 24 h. In DSC thermograms, complexes from debranched starch displayed three separated endotherms: the melting of the free lauric acid, starch–lipid complexes and retrograded amylose, respectively. The melting temperature and enthalpy changes of starch–lipid complex were gradually enhanced with the increasing of debranching time. However, no significant enthalpy changes were observed from retrograded amylose during the starch–lipid complex formation. Rapidly digestible starch (RDS) content decreased and resistant starch (RS) content increased with the increasing of debranching time, while the highest slowly digestible starch (SDS) content was founded at less debranching time of 2 h. The crystalline structures with dense aggregation of helices from amylose-LA complex and retrograded amylose could be RS, while SDS mostly consisted of imperfect packing of helices between amylopectin residue and amylose or LA.     

酶解法制备荞麦抗性淀粉的工艺优化

为确定荞麦粉制备抗性淀粉的工艺条件,采用普鲁兰酶酶解脱支法,并通过单因素和正交试验研究了影响抗性淀粉得率的因素。结果表明：影响抗性淀粉得率的因素主次顺序依次为荞麦粉浓度、普鲁兰酶用量、酶解时间和酶解温度。酶解法制备荞麦抗性淀粉的适宜工艺条件为荞麦粉浓度5 g/（100 mL）、普鲁兰酶用量7.2 PUN/g、酶解温度45℃、酶解时间8 h,在此条件下测得的抗性淀粉含量为15.82%。与原粉相比,普鲁兰酶酶解脱支与湿热法相结合制备荞麦抗性淀粉使其抗性淀粉含量显著提高。     

Synthesis and in vitro digestion of resistant starch type III from enzymatically hydrolysed cassava starch

Resistant starch type III (RS III) was synthesised from cassava starch by autoclaving followed by debranching with pullulanase, at varied concentrations (0.4–12 U g^?1) and times (2–8 h), and recrystallisation (?18 to 90 °C for 1–16 h). The highest RS III yield (22 g/100 g) was obtained at an enzyme concentration of 4 U g^?1 after 8 h incubation, followed by recrystallisation at 25 °C for 16 h. Varying the recrystallisation conditions indicated that higher RS III yields (30–35 g/100 g) could be obtained at 90 °C within 2 h. Thinning cassava starch using α‐amylase prior to debranching using pullulanase did not further increase the RS III content. In vitro digestion data showed that whereas 44% RS III was digested after 6 h, the corresponding value for cassava starch was 89%.     

Effect of debranching and storage condition on crystallinity and functional properties of cassava and potato starches

Debranching starch by pullulanase is considered to improve the RS content of starch which is widely used to produce the starch‐based foods with high‐health benefit impacts. In this study, the cassava and potato starches were debranched by pullulanase, followed by an autoclave treatment and storage at −18°C, 4°C, or 25°C to investigate their crystallinity and functional properties. After debranching, the potato starch contained significantly higher CL (35.4 glucose units) than did the cassava starch (32.4 glucose units). The debranched cassava and potato starches after retrogradation at the storage temperatures had a typical B‐type crystalline structure although the native cassava and potato starches exhibited the different crystalline forms (A‐ and B‐type, respectively). The RS contents of the debranched cassava and potato starches significantly improved with higher RS content of the debranched potato starch than that of the debranched cassava starch at the same storage condition. The storage temperature significantly affected the RS formation of the debranched starches with the highest RS content at storage temperature of −18°C (35 and 48% for the debranched cassava and potato starches, respectively). The debranched starches had significantly lower viscosities and paste clarities but higher solubilities than did the native starches. As a result, the debranched cassava and potato starches can be considered for use not only in functional foods with enhanced health benefits but also in pharmaceutical and cosmetic industries.     

Preparation and properties of RS III from waxy maize starch with pullulanase

Waxy maize starch was treated by pullulanase debranching and retrogradation at room temperature to produce resistant starch (RS). Physicochemical properties, crystalline structure and in-vitro digestibility of starch samples with different RS content were investigated. Compared with native starch, apparent amylose content of RS products increased. Based on Gel Permeation Chromatography (GPC) the Molecular Weight Distribution (MWD) of resistant starches significantly changed. Scanning Electron Microscopy (SEM) showed that upon pullulanase debranching and retrogradation treatment the granular structure of native starch was destroyed and all RS samples exhibited irregular shaped fragments. Crystal structure of samples changed from A–type to a mixture of B and V–type. The crystallinity of resistant starch also improved as compared with native starch. Moreover, samples with higher resistant starch showed higher relative crystallinity. Differential Scanning Calorimetry (DSC) determination showed that To、Tp、Tc and ΔH all increased which was in agreement with RS content. The resistance of waxy maize starch with Pullulanase treatment to α-amylase digestibility also increased, while the in-vitro digestibility of products decreased.     

Crystallinity,Thermal and Morphological Characteristics of Resistant Starch Type III Produced by Hydrothermal Treatment of Debranched Cassava Starch

Cassava starch was debranched using pullulanase and the linear glucans recrystallized by incubation at 60°C or by temperature cycling at 120/60°C, and further subjected to heat‐moisture treatment (HMT). Resistant starch (RS III) contents increased from 21.4 g/100 g in the debranched starch (DS) to 67.3 g/100 g in the debranched starch incubated at 60°C (DRS) and 47.8 g/100 g in the debranched starch subjected to temperature cycling (DCS), and further to 84.8 g/100 g and 88.4% g/100 g in HMT‐DRS and HMT‐DCS, respectively. Total crystallinity varied between 31.4‐59.8% and the crystalline type was C in DS and DRS and A in DCS, HMT‐DRS and HMT‐DCS. The melting properties were characterized by broad endotherms, but the exact melting region and enthalpy were dependent on recrystallization method. The main endothermic peaks of DS and DRS occurred at 103.9 and 109.8°C, respectively, whereas DCS exhibited split endotherms at 113.6 and 138.1°C. Heat‐moisture treatment broadened the endotherms and increased their enthalpies. Scanning electron micrographs revealed surface topography differences related to size and aggregation of individual crystalline bodies.     

响应面法优化板栗抗性淀粉制备工艺

为了提高板栗抗性淀粉含量,并获得抗性淀粉制备方法的最适工艺参数,本研究优化了压热—普鲁兰酶法制备板栗抗性淀粉的工艺,在单因素试验基础上,采用响应面法研究淀粉悬浮液质量分数、普鲁兰酶添加量、酶解时间和冷凝时间对抗性淀粉得率的影响,建立各因素与抗性淀粉得率关系的数学回归模型。最终根据实际工艺操作确定最佳的制备工艺条件为淀粉悬浮液质量分数11.00%,酶添加量9 PUN/g、酶解时间10 h、冷凝时间15 h。在该制备条件下,测得抗性淀粉得率为64.90%,基本符合理论预测值(65.70%)。试验证明,响应面法能够提高板栗抗性淀粉的制备率。     

The impact of coupled acid or pullulanase debranching on the formation of resistant starch from maize starch with autoclaving–cooling cycles

Maize starch was treated by autoclaving–cooling cycles, coupled with acid or pullulanase hydrolysis to prepare resistant starch (RS). Debranching of retrograded or gelatinized maize starch with acid or pullulanase was studied to show the corresponding impact on RS formation. When maize starch was treated with three autoclaving–cooling cycles and retrograded maize starch was hydrolyzed at room temperature, with 0.1 mol L⁻¹ citric acid for 12 h, analysis results showed that debranching of citric acid was helpful in RS formation for RS yield increased from 8.5 to 11%. Debranching of gelatinized or retrograded maize starch at 60 °C with pullulanase at addition level of 3 PUN g⁻¹ starch showed a more favorable effect on RS formation. When gelatinized maize starch was hydrolyzed by pullulanase for 12 h and then treated with two autoclaving–cooling cycles, RS yield increased to 23.5%. If retrograded maize starch subjected to one autoclaving–cooling cycle was hydrolyzed by pullulanase for 10 h and then followed by two autoclaving–cooling cycles, RS yield elevated to 32.4%. The debranching effect of pullulanase on retrograded maize starch to help RS formation is obvious and most effective, indicating this treatment is applicable in RS preparation to increase the RS yield.     

微波-酶法制备马铃薯抗性淀粉工艺参数的优化

以马铃薯精制淀粉为原料,抗性淀粉得率为评价指标,通过单因素及正交试验确定了微波-酶解法制备马铃薯抗性淀粉的最佳工艺条件:在淀粉乳质量分数15%,微波作用时间90 s,微波作用功率800 W,耐高温α-淀粉酶添加量10 CU/g干淀粉,耐高温α-淀粉酶作用时间30 min,普鲁兰酶添加量0.10 PUN(G)/g干淀粉,普鲁兰酶酶解时间6 h,普鲁兰酶作用温度55℃的条件下,4℃老化24 h。经重复验证,RS得率最高达14.0%。     

Effect of debranching and heat treatments on formation and functional properties of resistant starch from high-amylose corn starches

High-amylose corn starches [(Hylon V (H5) and Hylon VII (H7)] were debranched with pullulanase, followed by autoclaving–storing cycles and drying in an oven (at 50 °C) or freeze-dryer. The samples were autoclaved at 123 and 133 °C and stored at 4 and 95 °C. Molecular weights of the samples decreased and resistant starch (RS) contents increased with increased debranching time. RS contents of H7 samples were higher than those of H5 samples. RS contents of oven-dried samples were higher than those of freeze-dried samples. Debranching caused decreases in DSC peak temperature (T _p) and increases in enthalpy (ΔH) values of H5 and H7. Autoclaving at 133 °C caused higher ΔH values as compared to autoclaving at 123 °C. The solubility and water-binding values of autoclaved-only (control) and autoclaved–debranched (3–48 h) samples and the samples treated with autoclaving–storing cycles after debranching of both H5 and H7 were higher than those of their respective native starches. Debranching of starch samples affected the emulsion capacity of albumin adversely, but improved the emulsion stability of albumin. Cold viscosity values of freeze-dried samples were higher than those of oven-dried samples. Autoclaving–storing cycles after debranching caused decreases in peak, breakdown and final viscosity values.     

Effect of Cooling,and Freezing on the Digestibility of Debranched Rice Starch and Physical Properties of the Resulting Material

Ten percent non‐waxy and waxy starch suspensions were debranched with pullulanase followed by heating and cooling (1 °C) to crystallize and/or gel. Products with a range of textures can be made depending on the type (waxy and non‐waxy) of starch used. The water holding capacity was 35% and 84% for waxy and non‐waxy cooled debranched starch, respectively, at 4 h of cooling and did not change. The hardness of the debranched waxy and non‐waxy starch continued to increase beyond 24 h up to 45 g and 245 g of force, respectively. The particle size of precipitates of non‐waxy and waxy debranched starch was 45 μm and 4 μm after 4 h of cooling and did not change. Cooling of debranched non‐waxy starch at 1 °C for 12 h without agitation decreased digestibility by 59%; with stirring digestibility decreased by 42% after 24 h of cooling. Freezing of debranched cooled waxy and non‐waxy starch does not effect the decreases in digestibility. Particle size of debranched, cooled/freeze‐thawed, dried, and milled starch affects digestibility.     

淀粉平均聚合度对抗性淀粉得率影响的研究

在对普鲁兰酶最佳酶解条件优化的基础上,采用普鲁兰酶与耐高温淀粉酶协同制备RS(resistantstarch),改变淀粉的聚合度,分析玉米淀粉聚合度与抗性淀粉得率的关系。结果表明:提高原料淀粉中直链淀粉含量并将其聚合度控制在一定程度,可有效的提高RS得率,同时,DSC(differentialscanningcalorimeter)和X-衍射图谱表明,抗性淀粉与原玉米淀粉在结构上有着显著差异。     

响应面试验优化晶种诱导-双酶复合法制备RS_3型籼米抗性淀粉工艺

以微波预糊化籼米淀粉为原料,自制RS_3型马铃薯抗性淀粉为晶种,研究RS_3型籼米抗性淀粉的晶种诱导-双酶复合法制备工艺。利用扫描电子显微镜对淀粉颗粒形貌进行表征并研究淀粉的抗酶解性。在单因素试验的基础上,固定其他酶解条件,以RS_3型籼米抗性淀粉产率为响应值,确定晶种添加量、异淀粉酶添加量、普鲁兰酶添加量和普鲁兰酶酶解时间作为影响产率的主要因素,进行Box-Behnken响应面优化试验。得到RS3型籼米抗性淀粉的最佳制备工艺条件为:晶种添加量5%、异淀粉酶添加量8 U/g、普鲁兰酶添加量8 U/g、普鲁兰酶酶解时间3.50 h。在此最佳制备工艺条件下,RS_3型籼米抗性淀粉产率为27.42%,RS3失去原有的淀粉颗粒形貌,表面变得粗糙,结晶结构致密,具有较强抗酶解能力。     

普鲁兰酶加酶量对蜡质玉米抗性淀粉影响及性质研究

选用蜡质玉米淀粉为原料,高温糊化后采用普鲁兰酶脱支,产生短直链淀粉,重新结晶制备抗性淀粉。结果表明,8%(w/w)淀粉乳添加20 ASPU/g(基于淀粉干基重)普鲁兰酶在58℃反应24 h,然后在20℃凝沉24 h产生样品抗性淀粉含量最高,达到27.69%。理化性质研究表明,所有抗性淀粉样品颗粒形貌遭到破坏,形成不规则碎片;X-射线衍射图谱均有新的结晶结构出现,显示为B+V型;DSC分析结果显示,随抗性淀粉含量增加,不同样品峰值温度和糊化焓也增加。     

响应面法优化玉米抗性淀粉制备工艺

研究普鲁兰酶法制备玉米抗性淀粉的工艺。在单因素试验基础上,采用响应曲面法研究pH值、反应温度、反应时间和加酶量对抗性淀粉得率的影响,优化玉米抗性淀粉制备工艺,建立各因素与抗性淀粉得率关系的数学回归模型。确定最佳的制备工艺条件为普鲁兰酶加酶量12.8ASPU/g、反应时间32h、反应温度46.2℃、pH5.0。在该制备条件下,抗性淀粉得率为46.2%。     

豌豆抗性淀粉的酶法制备及其性质研究

以豌豆淀粉为原料,经糊化、普鲁兰酶脱支和凝沉处理,使其分子结构发生改变,制备出高含量的抗性淀粉,并研究了其理化性质。结果表明,在加酶量为300 ASPU/g,脱支时间12 h,凝沉时间24 h时,抗性淀粉含量达到最高52.66%;经糊化、脱支和凝沉处理后的样品结晶结构由C型变为B+V型;随着抗性淀粉含量的增加,其溶解度逐渐降低且均高于原淀粉,但膨胀度均低于原淀粉;消化产物随抗性淀粉含量的增加而降低。     

3种大米淀粉脱支前后结构及流变特性

通过普鲁兰酶处理糯米、粳米和籼米淀粉,研究酶水解对3 种大米淀粉结构和流变特性的影响。结果表明,经普鲁兰酶处理后3 种大米淀粉的结晶度降低,无定型区域增加;链长分布结果表明3 种大米淀粉的精细结构相似,水解反应对较短的侧链更有效,糯米淀粉更易被酶解;脱支淀粉和天然大米淀粉的傅里叶变换红外光谱没有明显差异,—OH的伸缩振动吸收峰相对增强;添加普鲁兰酶后,淀粉糊黏度急剧下降,糯米淀粉黏度下降最快,较容易被水解;流变学特性表明淀粉颗粒分子间缔合、排列松散,运动性增强,溶解度和持水力有所增强。糯米淀粉对普鲁兰酶处理较其他两种大米淀粉更为敏感。结论：脱支处理改善了淀粉凝胶性能,增强了淀粉的流动性。