抗性淀粉酶法制备及其特性与应用的研究
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摘要
RS3（Resistant Starch Ⅲ）是膳食中抗性淀粉的主要组成部分，对人体具有重要的生理功能。作为一种新型的功能性食品原料，RS3制备方法和应用的研究成为近年来碳水化合物科学的热点课题。采用酶法制备抗性淀粉，不仅简化了工艺对设备和劳动强度的要求，而且无化学残留隐患，具有较高的安全性，符合当今食品加工发展的趋势，便于实现工业化生产。目前，就抗性淀粉制备方法而言，急需一种以低直链淀粉为原料制备高含量RS3的方法。本研究以我国丰富的淀粉资源——普通玉米淀粉为原料，利用α-淀粉酶内切淀粉长链、普鲁兰酶脱支的特性，对淀粉进行连续处理，通过对反应条件的选择和控制，不仅提高了抗性淀粉的得率，而且所制备的产品展现出良好的加工特性和功能特性。在此基础上，研制了感官特性良好的抗性淀粉饼干产品。
采用用普鲁兰酶对普通玉米淀粉进行脱支处理，在单因素试验基础上，采用响应面法研究pH值、反应温度、反应时间和普鲁兰酶添加量对抗性淀粉得率的影响，试验显著性分析表明：各因素对玉米抗性淀粉得率的影响大小不同，依次为：普鲁兰酶添加量＞反应时间＞反应温度＞pH值。其中普鲁兰酶添加量、反应时间、反应温度对反应的影响达到极显著程度，pH值在试验范围内对抗性淀粉得率也具有显著影响，且普鲁兰酶添加量与反应温度、反应温度与时间、反应温度与pH值有交互作用。普鲁兰酶法制备抗性淀粉的最优条件为：普鲁兰酶添加量12.8ASPU/g、反应时间32h、反应温度46.2℃、pH5.0。在此条件下，抗性淀粉得率为46.20%。与相关文献报道（32.4%）对比发现，利用优化后的工艺条件有效的提高了抗性淀粉的得率。
在普鲁兰酶脱支处理前，用α-淀粉酶对玉米淀粉进行适当的水解，以提高普鲁兰酶脱支效率，增加抗性淀粉得率。研究结果表明：在温度90℃，pH5.5，α-淀粉酶添加量4.0u/g，水解15min后，用普鲁兰酶连续处理（12.8ASPU/g、反应时间32h、反应温度46.2℃、pH5.0），可以制备得到抗性淀粉含量约58.87%的产品。
以玉米原淀粉、压热/冷却循环所制备的抗性淀粉做对照，对制备所得抗性淀粉的黏度、吸水性、和抗酶解性能进行测定，RS3显示出较低的黏度和吸水性，且具有良好抵抗胃酸和胰淀粉酶水解作用的特性。采用电镜扫描和差示扫描量热（DSC）法观察复合酶法制备玉米抗性淀粉的颗粒形态和热力学性质，结果表明：玉米淀粉经复合酶法处理后，形成更加致密层状晶体结构，表面形态结构与压热/冷却循环所得抗性淀粉差异较大；DSC分析表明：玉米淀粉经复合酶处理后，原有的晶体结构被破坏，直链分子重新排布，形成新的更为稳定的晶体结构；相变起始温度TO，峰值温度TP，终了温度TC和相变焓分别为110.9、143.5、171.9℃、132.3J/g，显著高于由压热/冷却循环所得抗性淀粉，表明复合酶所制备的抗性淀粉具有更高的热稳定性。
采用紫外—可见吸收光谱、FT-IR、 HPSEC、X-RD）等分析技术对抗性淀粉形成的机理进行研究，结果表明：普通玉米淀粉经复合酶处理后，淀粉中直链淀粉比例明显提高，链长显著降低，重均分子量4983，重均聚合度31，分散度1.08，表明分子量分布具有较好的均匀性，高比例的直链淀粉、适当的链长和聚合度是抗性淀粉得率提高的主要原因。复合酶法制备所得抗性淀粉的晶型处于由B+V型向V型发生转变的状态，晶体结构的改变导致了RS3抗酶解性能的增加。
模拟健康成人及婴儿肠道环境，对抗性淀粉在人体内的发酵特性进行研究，结果表明：抗性淀粉在人体肠道微生物作用下降解的主要产物为甲酸、乙酸、丙酸、丁酸等短链脂肪酸，丙酸含量最高；在两种不同的发酵环境中具有相同的产酸规律变化，即大多数短链脂肪酸是在发酵时间12h内产生的，产物中短链脂肪酸的含量随发酵时间延长和发酵液中抗性淀粉含量的增加而增加，在发酵时间6h～12h内，丁酸的含量增加的速率尤为显著。比较相同浓度的抗性淀粉在健康成人及婴儿肠道环境中同种脂肪酸的含量可以发现：由于肠道菌群种类和数量的变化，使得抗性淀粉在婴儿肠道环境中比成人具有更高发酵效率，各短链脂肪酸浓度明显增大。由此可知，健康婴儿肠道的微生物菌群的构成更有利于玉米抗性淀粉发酵产生短链脂肪酸。抗性淀粉在人体肠道环境中不仅具有较高的产生短链脂肪酸的性能，而且代谢产物中丁酸占短链脂肪酸的比例（16.7～30.0%）是传统的膳食纤维（5.0～8.0%）2～6倍。因此，RS3可以作为人体肠道菌生产丁酸的良好来源。
以感官评分为指标，通过单因素和正交试验设计优化抗性淀粉饼干的最佳配方为：以低筋粉面粉100%为基准，抗性淀粉25.0%、植物油24.0%、蔗糖32.0%、膨松剂1.6%，全蛋液2.0%，食盐1.0%，水10.0%。在此配方条件下，对抗性饼干进行感官、理化和卫生指标测定，结果表明：抗性淀粉饼干色泽均匀、呈金黄色；外形完整，花纹清晰，厚薄基本均匀，无收缩、变形、起泡、裂痕现象；断面结构呈多孔状，细密，无大孔洞；香味纯正，口感松脆细腻、不黏牙、无颗粒感。其各项理化指标和卫生指标均符合饼干国家相应标准；抗性淀粉饼干的水解指数和血糖指数与对照组差异显著，属于中等血糖指数食品。
RS3, a major type of resistant starch in food, has important physiological functions in human body andexcellent food processing characteristics. As a new functional food ingredient, the study on RS3preparation and application has become a hot topic of carbohydrate science. Enzymatic preparation ofresistant starch reduces the requirements of traditional technology for temperature, pressure, equipment,and other conditions, leaves no chemical residues, and has higher safety, so it is in line with thedevelopment trend of food processing nowadays and can be industrialized easily. Currently, there is anurgent need for a method to prepare RS3from low amylose, and the prepared RS3should have goodprocessing characteristics and functions. In this study, starch was extracted from the corn that is commonlyseen in China as a raw material for starch and was then continuously processed using α-amylase (forincision of the long chains of starch) and pullulanase (for debranching). By selection and control of thereaction conditions, the yield of resistant starch was increased, and the prepared product showed goodprocessing characteristics and functional features. On this basis, the resistant starch product with goodorganoleptic properties and certain physiological effects on human body were developed.
Pullulanase was used for debranching common corn starch, and on the basis of single-factor test, theresponse surface method was adopted to investigate the effects of pH, reaction temperature, reaction time,and pullulanase amount on the yield of resistant starch. The significance analysis for this test showed thatthe effects of these factors on the yield of resistant corn starch were different, i.e. pullulanase amount>reaction time> reaction temperature> pH, wherein the effects of pullulanase amount, reaction time, andreaction temperature on the reaction reached a very significant degree, pH also had a significant effectwithin the test range, and there were interactions between pullulanase amount and reaction temperature,between reaction temperature and reaction time, and between reaction temperature and pH. The optimumconditions for resistant starch preparation with pullulanase were as follows: amount of pullulanase12.8ASPU/g, reaction temperature46.2℃, reaction time32h, pH5.0. Under the conditions, the yield ofresistant starch reached46.20%, increased as compared with the value recorded in the relevant literature(32.4%).
Before being debranched with pullulanase, corn starch was properly hydrolyzed with α-amylase, topromote the efficiency of debranching and increase the yield of resistant starch. The results of this studyshowed that: the highest formation of RS (58.87%) was obtained under the following conditions: reactiontemperature90℃, pH5.5, reaction time15min, and amount of α-amylase4.0U/g.
With the resistant starch prepared from native corn starch by hot pressing/cooling circulation methodas the control, the viscosity, water absorbability, and anti-enzymatic properties of the prepared resistantstarch were measured, and RS3showed relatively low viscosity and water absorbability and highresistances to gastric acid and pancreatic amylase. Electron microscope scanning and differential scanningcalorimetry (DSC) were employed to observe the particle morphology and thermodynamic properties of theresistant corn starch prepared with complex enzymes, and the results showed that after being processedwith complex enzymes, corn starch lost the original particle morphology and smoothness and formed alayered crystal structure that is more compact than before, displaying a significant difference from theresistant starch prepared by hot pressing/cooling circulation method with respect to surface morphology;DSC analysis showed that after being processed with complex enzymes, the original crystal structure ofcorn starch was destroyed, and the straight chain molecules were rearranged, to form a new crystalstructure that is more stable; the initial temperature TO, peak temperature TP, final temperature TC, andenthalpy of phase change were110.9℃,143.5℃,171.9℃, and132.3J/g, respectively, significantly higherthan those of the resistant starch prepared by hot pressing/cooling circulation method, indicating that theresistant starch prepared with complex enzymes has higher thermal stability.
Visible absorption spectroscopy, infrared spectroscopy, X-ray diffraction high performance sizeexclusion chromatography (HPSEC), and other techniques were adopted to investigate the mechanism ofthe increases in the yield and resistance of resistant starch. The results showed that after common corn starch was processed with complex enzymes, the proportion of straight chain starch in the starch increasedsignificantly, and the chain length decreased significantly, with a weight-average molecular weight of4983,a weight-average degree of polymerization of31, and a dispersity of1.08, suggesting a narrow molecularweight distribution. High proportion of straight chain starch as well as appropriate chain length and degreeof polymerization constitute one of the important reasons for the increases in the yield and resistance ofresistant starch. In addition, the crystal of the resistant starch prepared with complex enzymes was in thetransformation from B+V form to V form, so the crystal structure change leads to improved anti-enzymaticproperties of RS3.
The intestinal environments of healthy adults and infants were simulated to investigate thecharacteristics of fermentation of resistant starch in human body. The results showed that the maindegradation products of resistant starch under the effects of human gut microbes were formic acid, aceticacid, propionic acid, butyric acid, and other short-chain fatty acids, with propionic acid accounting for thehighest proportion; fatty acids were produced in the same way in the two different fermentationenvironments: most of the short-chain fatty acids were produced within12h of fermentation, the contentsof short-chain fatty acids increased as fermentation proceeded and the content of resistant starch in thefermentation liquid rose, and butyric acid content increased at a particularly significant rate. Aftercomparison between the contents of the same fatty acids produced by the same concentration of resistantstarch in the intestinal environments of healthy adults and infants, it is found that due to the changes in thetypes and quantities of gut microbes, the resistant starch was fermented more efficiently in infants than inadults, leading to significantly higher concentrations of short-chain fatty acids in infants. Therefore, thecomposition of gut microbes in healthy infants is more conducive to fermentation of resistant corn starchand production of short-chain fatty acids. Resistant starch in human intestinal environment can produceshort-chain fatty acids more efficiently, and of the short-chain fatty acids, butyric acid accounted for16.7-30.0%,2-6times that of butyric acid from conventional dietary fibers (5.0-8.0%). Therefore, resistantstarch can serve as a good source for butyric acid produced by human gut microbes.
As a new functional food ingredient, the amount of resistant starch may have different effects on theorganoleptic properties of food and the addition of other ingredients. With the organoleptic score as theindex, single-factor and orthogonal experiments were carried out to design the optimum formula ofresistant starch biscuits: self-raising flour100%, RS25.0%, vegetable oil24.0%sugar32.0%leaveningagent1.6%, whole egg2.0%, salt1.0%, water10.0%. The organoleptic, physicochemical, and healthindices for resistant starch biscuits produced using the optimum formula were measured. The resultsshowed that the resistant starch biscuits had the advantages of uniform color (golden yellow), intactappearance, clear patterns, uniform thickness, no shrinkage, no deformation, no blistering, no cracking, fineinternal pores, pure flavor, crisp and delicate taste, no sticking to teeth, and no grainy taste. All thephysicochemical and health indices met national standards. The in vitro hydrolysis index and glycemicindex of resistant starch biscuits were measured, and the results showed significant differences from thecontrol group in terms of the hydrolysis index and glycemic index, so resistant starch biscuits are the foodwith moderate glycemic index.
采用用普鲁兰酶对普通玉米淀粉进行脱支处理，在单因素试验基础上，采用响应面法研究pH值、反应温度、反应时间和普鲁兰酶添加量对抗性淀粉得率的影响，试验显著性分析表明：各因素对玉米抗性淀粉得率的影响大小不同，依次为：普鲁兰酶添加量＞反应时间＞反应温度＞pH值。其中普鲁兰酶添加量、反应时间、反应温度对反应的影响达到极显著程度，pH值在试验范围内对抗性淀粉得率也具有显著影响，且普鲁兰酶添加量与反应温度、反应温度与时间、反应温度与pH值有交互作用。普鲁兰酶法制备抗性淀粉的最优条件为：普鲁兰酶添加量12.8ASPU/g、反应时间32h、反应温度46.2℃、pH5.0。在此条件下，抗性淀粉得率为46.20%。与相关文献报道（32.4%）对比发现，利用优化后的工艺条件有效的提高了抗性淀粉的得率。
在普鲁兰酶脱支处理前，用α-淀粉酶对玉米淀粉进行适当的水解，以提高普鲁兰酶脱支效率，增加抗性淀粉得率。研究结果表明：在温度90℃，pH5.5，α-淀粉酶添加量4.0u/g，水解15min后，用普鲁兰酶连续处理（12.8ASPU/g、反应时间32h、反应温度46.2℃、pH5.0），可以制备得到抗性淀粉含量约58.87%的产品。
以玉米原淀粉、压热/冷却循环所制备的抗性淀粉做对照，对制备所得抗性淀粉的黏度、吸水性、和抗酶解性能进行测定，RS3显示出较低的黏度和吸水性，且具有良好抵抗胃酸和胰淀粉酶水解作用的特性。采用电镜扫描和差示扫描量热（DSC）法观察复合酶法制备玉米抗性淀粉的颗粒形态和热力学性质，结果表明：玉米淀粉经复合酶法处理后，形成更加致密层状晶体结构，表面形态结构与压热/冷却循环所得抗性淀粉差异较大；DSC分析表明：玉米淀粉经复合酶处理后，原有的晶体结构被破坏，直链分子重新排布，形成新的更为稳定的晶体结构；相变起始温度TO，峰值温度TP，终了温度TC和相变焓分别为110.9、143.5、171.9℃、132.3J/g，显著高于由压热/冷却循环所得抗性淀粉，表明复合酶所制备的抗性淀粉具有更高的热稳定性。
采用紫外—可见吸收光谱、FT-IR、 HPSEC、X-RD）等分析技术对抗性淀粉形成的机理进行研究，结果表明：普通玉米淀粉经复合酶处理后，淀粉中直链淀粉比例明显提高，链长显著降低，重均分子量4983，重均聚合度31，分散度1.08，表明分子量分布具有较好的均匀性，高比例的直链淀粉、适当的链长和聚合度是抗性淀粉得率提高的主要原因。复合酶法制备所得抗性淀粉的晶型处于由B+V型向V型发生转变的状态，晶体结构的改变导致了RS3抗酶解性能的增加。
模拟健康成人及婴儿肠道环境，对抗性淀粉在人体内的发酵特性进行研究，结果表明：抗性淀粉在人体肠道微生物作用下降解的主要产物为甲酸、乙酸、丙酸、丁酸等短链脂肪酸，丙酸含量最高；在两种不同的发酵环境中具有相同的产酸规律变化，即大多数短链脂肪酸是在发酵时间12h内产生的，产物中短链脂肪酸的含量随发酵时间延长和发酵液中抗性淀粉含量的增加而增加，在发酵时间6h～12h内，丁酸的含量增加的速率尤为显著。比较相同浓度的抗性淀粉在健康成人及婴儿肠道环境中同种脂肪酸的含量可以发现：由于肠道菌群种类和数量的变化，使得抗性淀粉在婴儿肠道环境中比成人具有更高发酵效率，各短链脂肪酸浓度明显增大。由此可知，健康婴儿肠道的微生物菌群的构成更有利于玉米抗性淀粉发酵产生短链脂肪酸。抗性淀粉在人体肠道环境中不仅具有较高的产生短链脂肪酸的性能，而且代谢产物中丁酸占短链脂肪酸的比例（16.7～30.0%）是传统的膳食纤维（5.0～8.0%）2～6倍。因此，RS3可以作为人体肠道菌生产丁酸的良好来源。
以感官评分为指标，通过单因素和正交试验设计优化抗性淀粉饼干的最佳配方为：以低筋粉面粉100%为基准，抗性淀粉25.0%、植物油24.0%、蔗糖32.0%、膨松剂1.6%，全蛋液2.0%，食盐1.0%，水10.0%。在此配方条件下，对抗性饼干进行感官、理化和卫生指标测定，结果表明：抗性淀粉饼干色泽均匀、呈金黄色；外形完整，花纹清晰，厚薄基本均匀，无收缩、变形、起泡、裂痕现象；断面结构呈多孔状，细密，无大孔洞；香味纯正，口感松脆细腻、不黏牙、无颗粒感。其各项理化指标和卫生指标均符合饼干国家相应标准；抗性淀粉饼干的水解指数和血糖指数与对照组差异显著，属于中等血糖指数食品。
RS3, a major type of resistant starch in food, has important physiological functions in human body andexcellent food processing characteristics. As a new functional food ingredient, the study on RS3preparation and application has become a hot topic of carbohydrate science. Enzymatic preparation ofresistant starch reduces the requirements of traditional technology for temperature, pressure, equipment,and other conditions, leaves no chemical residues, and has higher safety, so it is in line with thedevelopment trend of food processing nowadays and can be industrialized easily. Currently, there is anurgent need for a method to prepare RS3from low amylose, and the prepared RS3should have goodprocessing characteristics and functions. In this study, starch was extracted from the corn that is commonlyseen in China as a raw material for starch and was then continuously processed using α-amylase (forincision of the long chains of starch) and pullulanase (for debranching). By selection and control of thereaction conditions, the yield of resistant starch was increased, and the prepared product showed goodprocessing characteristics and functional features. On this basis, the resistant starch product with goodorganoleptic properties and certain physiological effects on human body were developed.
Pullulanase was used for debranching common corn starch, and on the basis of single-factor test, theresponse surface method was adopted to investigate the effects of pH, reaction temperature, reaction time,and pullulanase amount on the yield of resistant starch. The significance analysis for this test showed thatthe effects of these factors on the yield of resistant corn starch were different, i.e. pullulanase amount>reaction time> reaction temperature> pH, wherein the effects of pullulanase amount, reaction time, andreaction temperature on the reaction reached a very significant degree, pH also had a significant effectwithin the test range, and there were interactions between pullulanase amount and reaction temperature,between reaction temperature and reaction time, and between reaction temperature and pH. The optimumconditions for resistant starch preparation with pullulanase were as follows: amount of pullulanase12.8ASPU/g, reaction temperature46.2℃, reaction time32h, pH5.0. Under the conditions, the yield ofresistant starch reached46.20%, increased as compared with the value recorded in the relevant literature(32.4%).
Before being debranched with pullulanase, corn starch was properly hydrolyzed with α-amylase, topromote the efficiency of debranching and increase the yield of resistant starch. The results of this studyshowed that: the highest formation of RS (58.87%) was obtained under the following conditions: reactiontemperature90℃, pH5.5, reaction time15min, and amount of α-amylase4.0U/g.
With the resistant starch prepared from native corn starch by hot pressing/cooling circulation methodas the control, the viscosity, water absorbability, and anti-enzymatic properties of the prepared resistantstarch were measured, and RS3showed relatively low viscosity and water absorbability and highresistances to gastric acid and pancreatic amylase. Electron microscope scanning and differential scanningcalorimetry (DSC) were employed to observe the particle morphology and thermodynamic properties of theresistant corn starch prepared with complex enzymes, and the results showed that after being processedwith complex enzymes, corn starch lost the original particle morphology and smoothness and formed alayered crystal structure that is more compact than before, displaying a significant difference from theresistant starch prepared by hot pressing/cooling circulation method with respect to surface morphology;DSC analysis showed that after being processed with complex enzymes, the original crystal structure ofcorn starch was destroyed, and the straight chain molecules were rearranged, to form a new crystalstructure that is more stable; the initial temperature TO, peak temperature TP, final temperature TC, andenthalpy of phase change were110.9℃,143.5℃,171.9℃, and132.3J/g, respectively, significantly higherthan those of the resistant starch prepared by hot pressing/cooling circulation method, indicating that theresistant starch prepared with complex enzymes has higher thermal stability.
Visible absorption spectroscopy, infrared spectroscopy, X-ray diffraction high performance sizeexclusion chromatography (HPSEC), and other techniques were adopted to investigate the mechanism ofthe increases in the yield and resistance of resistant starch. The results showed that after common corn starch was processed with complex enzymes, the proportion of straight chain starch in the starch increasedsignificantly, and the chain length decreased significantly, with a weight-average molecular weight of4983,a weight-average degree of polymerization of31, and a dispersity of1.08, suggesting a narrow molecularweight distribution. High proportion of straight chain starch as well as appropriate chain length and degreeof polymerization constitute one of the important reasons for the increases in the yield and resistance ofresistant starch. In addition, the crystal of the resistant starch prepared with complex enzymes was in thetransformation from B+V form to V form, so the crystal structure change leads to improved anti-enzymaticproperties of RS3.
The intestinal environments of healthy adults and infants were simulated to investigate thecharacteristics of fermentation of resistant starch in human body. The results showed that the maindegradation products of resistant starch under the effects of human gut microbes were formic acid, aceticacid, propionic acid, butyric acid, and other short-chain fatty acids, with propionic acid accounting for thehighest proportion; fatty acids were produced in the same way in the two different fermentationenvironments: most of the short-chain fatty acids were produced within12h of fermentation, the contentsof short-chain fatty acids increased as fermentation proceeded and the content of resistant starch in thefermentation liquid rose, and butyric acid content increased at a particularly significant rate. Aftercomparison between the contents of the same fatty acids produced by the same concentration of resistantstarch in the intestinal environments of healthy adults and infants, it is found that due to the changes in thetypes and quantities of gut microbes, the resistant starch was fermented more efficiently in infants than inadults, leading to significantly higher concentrations of short-chain fatty acids in infants. Therefore, thecomposition of gut microbes in healthy infants is more conducive to fermentation of resistant corn starchand production of short-chain fatty acids. Resistant starch in human intestinal environment can produceshort-chain fatty acids more efficiently, and of the short-chain fatty acids, butyric acid accounted for16.7-30.0%,2-6times that of butyric acid from conventional dietary fibers (5.0-8.0%). Therefore, resistantstarch can serve as a good source for butyric acid produced by human gut microbes.
As a new functional food ingredient, the amount of resistant starch may have different effects on theorganoleptic properties of food and the addition of other ingredients. With the organoleptic score as theindex, single-factor and orthogonal experiments were carried out to design the optimum formula ofresistant starch biscuits: self-raising flour100%, RS25.0%, vegetable oil24.0%sugar32.0%leaveningagent1.6%, whole egg2.0%, salt1.0%, water10.0%. The organoleptic, physicochemical, and healthindices for resistant starch biscuits produced using the optimum formula were measured. The resultsshowed that the resistant starch biscuits had the advantages of uniform color (golden yellow), intactappearance, clear patterns, uniform thickness, no shrinkage, no deformation, no blistering, no cracking, fineinternal pores, pure flavor, crisp and delicate taste, no sticking to teeth, and no grainy taste. All thephysicochemical and health indices met national standards. The in vitro hydrolysis index and glycemicindex of resistant starch biscuits were measured, and the results showed significant differences from thecontrol group in terms of the hydrolysis index and glycemic index, so resistant starch biscuits are the foodwith moderate glycemic index.
引文
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