慢消化淀粉的特性及形成机理研究
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摘要
慢消化淀粉(slowly digestible starch, SDS)是指那些在小肠中被完全消化吸收但速度较慢的淀粉。根据淀粉生物可利用性分类,可将其定义为在体外模拟消化条件下(pH 5.2、37℃) 20～120 min内被混酶(胰α-淀粉酶、糖化酶与转化酶)消化的淀粉营养片断。作为一种新型的功能性食品,SDS具有缓慢消化吸收、持续释放能量、维持餐后血糖稳态、预防和治疗各种与饮食相关慢性疾病的特殊生理学功能,因而日益成为食品科学和现代营养学领域的一个研究热点。目前,SDS的研究机构主要集中在欧美国家,相关报道仅局限于制备方法与生理功能研究;国内在此领域研究并不多见。本课题通过研究明确SDS的特性和其形成机理,这对食品加工行业开发利用我国丰富的淀粉资源,增加食品中SDS比例,提高食品营养品质具有重要的意义。
本文首先建立一种简单快捷的SDS体外测定方法,这是分析淀粉的消化性能、分子结构特征及SDS形成机理的前提。其次,利用快速黏度仪(RVA)、高效排阻色谱(HPSEC)、X-射线衍射(XRD)、差示扫描量热仪(DSC)、扫描电镜(SEM)等现代分析手段,对SDS特性与结构特征进行了研究。最后,探讨了蜡质玉米淀粉经普鲁兰酶部分脱支-重结晶修饰或高水分限制糊化处理后SDS的变化机制。其主要研究内容如下:
通过Englyst法、Guraya法和Shin法对普通玉米淀粉、蜡质玉米淀粉及马铃薯淀粉中SDS测定并与体内法相比较,在此基础上采用单因素与正交试验优化,得到一种新的SDS体外分析方法。最佳测定方法为:200 mg淀粉溶于15 mL pH 5.2的醋酸钠缓冲液,添加10 mL含胰α-淀粉酶(290 U/mL)与糖化酶(15 U/mL)的混酶液在37℃下水解,能满足SDS的体外简单快速测定。
采用改进Englyst法对不同品种淀粉中营养片断分析,得出天然的普通玉米、蜡质玉米、小麦、糯米及大米等A型谷物淀粉含SDS较高,B型马铃薯淀粉主要由抗性淀粉(resistant starch,RS)组成,这表明淀粉的营养片断与其结晶类型有关,谷物淀粉属于理想的SDS原料;经过蒸煮糊化处理后SDS与RS差异基本消失并大部分转变成易消化淀粉(rapidly digestible starch,RDS),因为加热破坏了淀粉颗粒的半结晶结构。通过体外模拟消化动力学曲线探讨了淀粉的慢消化性能与酶水解速率的关系,从酶水解动力学方程可推算出SDS(谷物淀粉)的动力学速率小于1 h-1。水解反应动力学速率比Englyst法更能准确反映淀粉水解过程的消化性能。采用体内实验研究天然淀粉的餐后血糖应答,得出不同谷物淀粉的血糖生成指数(glycemic index,GI)值均大于90,属于高GI食品,但相比食用葡萄糖的血糖应答曲线,淀粉的血糖曲线峰值减少,曲线下面积往后延伸扩展,即对应出现增加血糖生成指数(extended glycemic index,EGI)。EGI值代表淀粉消化后葡萄糖缓慢持续释放的性质,可作为SDS数量或质量的体内评价指标。采用RVA分析淀粉的黏滞性谱,发现淀粉热糊的崩解值与SDS含量呈显著负相关(r=-0.89,p<0.05)。通过凝胶色谱技术分析淀粉精细结构,发现支链淀粉的链长分布也与SDS密切相关,即长链FrⅠ(DP>30)、中长链FrⅡ(13 在蜡质玉米淀粉体外模拟消化过程中,随着水解时间的延长,残余淀粉样品中SDS含量几乎保持不变,RDS含量增加而RS含量减少;通过扫描电镜照片可以观察到,淀粉酶水解模式为由内向外逐层消化;天然淀粉经消化120min,Mw减小了约3倍,To、Tp、R(1047/1022)增加,而△H、晶体类型及Xc未发生改变,这证实在酶消化过程中淀粉无定型区和结晶区同时被水解;SDS由无定片层和结晶片层构成,并且大部分位于接近颗粒表面的外围区域。蜡质玉米淀粉经温和酸解处理后,淀粉颗粒被破坏,形成小片断聚集体,晶体结构仍保持A型,但Xc、R(1047/1022)增加,除To降低外,Tp、Tc、△H均提高,淀粉的链长分布曲线峰值发生改变,说明淀粉颗粒中无定型层和无定型背景发生酸降解消失。酸解处理后不同营养片断的比例发生变化,RDS增加,SDS与RS均降低;这表明无定型层和无定型背景对SDS有重要影响。
普鲁兰酶脱支-重结晶处理糊化蜡质玉米淀粉后,淀粉营养片断的比例与结构发生了改变。增加普鲁兰酶液浓度与减少酶脱支时间可得到高含量SDS;在4℃储藏有利于晶核形成,SDS含量则增多。因此,蜡质玉米淀粉经高浓度酶液(20或40 ASPU/g)脱支处理3～6 h,在4℃储藏2 d后可获得较多SDS。富含SDS的淀粉样品X-衍射图谱显示为B型晶体结构,增加储藏时间与温度使晶体结构转变为B+V型。相比糊化淀粉,经酶脱支-重结晶处理后高密度、表面多孔的不规则、多棱角状淀粉碎片被观察到;在DSC图谱上也显示出To、Tp、Tc与△H逐渐增加。依据这些变化可推断出SDS由一小部分双螺旋部分堆积有序结晶区和大部分无定型区构成。
蜡质玉米淀粉在过量水分中通过不同加热处理限制糊化,保持淀粉的慢消化特性。在淀粉未完全糊化发生的温度范围(<60℃) ,SDS含量超过40%,此时淀粉颗粒的外形与结构未发生明显变化。溶胀因子在50～60℃范围内增加缓慢,温度继续升高则快速增大,并在80℃时达到最大值。在65～80℃淀粉的△H、Xc、R(1047/1022)明显降低,这说明结晶簇结构和双螺旋结构的部分发生解聚。上述变化表明糊化处理淀粉的高SDS含量可通过在淀粉颗粒破坏前的结构重排过程达到,另外也验证了SDS由无定型结构和有序双螺旋结晶结构组成的结论。
Slowly digestible starch (SDS) is slowly digested throughout the small intestine resulting in a slow and prolonged release of glucose into the blood stream, coupled to a low glycemic response. Based on the rate and extent of digestibility using the in vitro Englyst assay, SDS is also the starch fraction digested between 20 and 120 min. As a new functional ingredient, SDS is becoming the research focus in food science and modern nutriology for its unique low glycemic index property. Through lessening the stress on regulatory systems related to glucose homeostasis, this starch type may be helpful in controlling and preventing dietary management of metabolic disorders, including diabetes, prediabetes, glycogen storage disease, cardiovascular disease, and obesity. Currently, there are only some reports about preparation method and physiological effect of SDS in occident countries, such as America and Switzerland. Also, SDS-based product is not commercially available in the current food marke. Thus, elucidating the characteristic and formation mechanism of SDS is important for food industry to utilize the abundant starch resource, make tailor-made carbohydrate foods with rich-SDS, and improve the nutritional property of food.
In this study, the method of in vitro analysis was firstly discussed and has unified standardization, which was important for underatanding the digestibility, molecular structure feature and formation mechanism of SDS. The aim of this project was to investigate the slow digestion property, structure basis by using the Rapid viscosity analyzer, High performance size-exclusion chromatography, X-ray diffraction, Differential scanning calorimeter, Scanning electron microscopy, etc., and to probe into the effect partially debraching and recrystallization or controlled gelatinization on SDS in waxy maize starch, which may provide the knowledge for research and development of starchy food with low glycemic index (GI). The main work was listed as follows:
The three in vitro methods of Englyst, Guraya and Shin were used to determine and compare the content of SDS of normal maize starch, waxy maize starch and potato starch with the value of in vivo test. The results showed that the method of Englyst with an optimal modification by one factor and orthogonal experiments was the most suitable one for the determination of SDS. The starch samples (200 mg) were added to the acetate buffer (15 mL, pH 5.2) and mixed thoroughly. The enzyme solution (10 mL, porcine pancreaticα-amylase 290 U/mL and amyloglucosidase 15 U/mL) was then added to the substrate, followed by incubation in a water bath (37 oC) with agitation.
Based on the modified Englyst test to measure the nutritionally important starch fractions, there is higher SDS (about 50%) in starch form common maize, waxy maize, wheat, sticky rice or rice compared with potato starch (16.9%). Therefore, cereal starches containing a large portion of SDS are considerd as ideal SDS materials while potato starch is the typical resistant starch (RS). When the native cereal starch was cooked in the boiling water bath, the slow digestion property was lost with a huge increase of rapidly digeatible starch (RDS). The inheret layer structure of crystalline and amorphous regions is likely the fundamental structure basis for slowly digestion property of starch. The relationship between slow digestion property and enzymatic hydrolysis rate of starches was investigated by establishment of in vitro hydrolysis kinetics. The starch digestion rate calculated using the exponential curve equation C=C∞×(l-e-kt) was less than 1 h-1 for cereal starches. Starch digestibility was reflected exactly by digestion rate of hydrolysis kinetics other than in vitro Englyst method. The glycemic response profile (the shape of the glycemic response curve) of cereal starch was significantly different from glucose powder with a delayed blood glucose peak and a prolonged and moderate elevation of glucose after the peak. The GI value of cereal starch was also more than 90% and belonged to high GI food, but extended GI (EGI) representing the slow glucose release property was positive and might be used as an in vivo indicator of the amount and/or quality of the SDS in foods. RVA was used to investigate viscosity profiles of different starch and the correlation between starch digestibility and RVA profile characteristics. The breakdown of different varieties starch was negatively correlated with SDS (r=-0.89, p<0.05). The RVA method potentially could be used as a screening tool for starch digestion properties. Six starches from different varieties were used as materials and the chromatogram of debranched amylopection were analyzed by SEC system. The correlation between starch digestibility and fine structure of amylopectin was also investigated. SDS fraction was positively correlated with FrⅠ(DP>30) and FrⅡ(13 The Englyst testing on partially hydrolysis residual starches showed an increase of RDS accompanied a reduction of RS with increasing digestion time, while SDS was an almost constant. Scanning electron micrographs showed that the pattern of enzymatic hydrolysis was inside-out layer-by-layer digestion. A relatively threefold decrease in the average molecular weight of starch components were observed afterα-amylolysis for 120 min. There were increases in the onset temperature, peak temperature and ratio of absorbance 1047/1022 cm-1, while the enthalpy of gelatinization, crystal structure, and crystallinity invaried, which attributed to simultaneously enzymatic hydrolysis of both crystalline and amorphous regions. These changes suggest that SDS may be consists of layered structure of amorphous and crystallite regions and located periphery of starch granule.
In order to study the slow digestion of starch, the changes of structure and in vitro digestibility of waxy maize starch after lintnerization using mild acid (2.2 M hydrochloric acid) were investigated. Results show that the granular appearance of waxy maize starch was destroyed and small fractions formed congeries. Lintnerization increased the X-ray intensities of major d-spacings and crystallinity of starches, but the x-ray diffraction patterns remained A-type. The ratios of ordered starch to amorphous starch also increased. The rise in the gelatinization transition temperature, the gelatinization temperature range and the enthalpy of gelatinization of starches were observed and a transformation in the chain length distribution profiles occured after lintnerization. The amount of RDS increased whereas SDS and RS content decreased. The amorphous regions of starch granules including both the amorphous background and the amorphous lamellae were degraded after lintnerization and affected the slow digestion property of native waxy maize starch.
The effects of debranching time, debranching enzyme concentration, storage time and temperature on digestibility and structural properties of waxy maize starch samples were investigated. When gelatinized starch was treated with higher enzyme concentration and less debranching time, higher SDS was formed while RS increased with time. The maximum SDS content was obtained by debranching for 3-6 h with higher concentration of pullulanase (20 or 40 ASPU/g) then storing at 4 oC for 2 days. X-ray diffraction pattern of treated starch at optimal conditions was similar to the B-type in which illuminated treated starches contain partially ordered crystalline structures. Scanning electron micrographs showed the treated starch had more irregular angular shapes with a higher crystallinity structure and pitted surface as debranching and recrystallization time increased. In differential scanning calorimetry themograms, the melting temperature and enthalpy of treated starches were gradually enhanced, resulting from reforming of double helix structure by low temperature recrystallization following debranching. These changes showed that SDS mainly consists of a partially ordered double helix crystallite structure with short amylopectin chains and amorphous regions.
An aqueous dispersion of waxy maize starch (5%, w/w) was partially gelatinized by heating at various temperatures for 5 min. When heated, SDS and RS levels were decreased inversely with RDS. A high SDS content (> 40%) was kept prior to the visible morphological and structural changes (at 60°C). Swelling factor began to increase slightly at 50-60°C and continued to maximum value at 80°C. A large decrease in the melting enthalpy, crystallinity, and ratio of 1047/1022 cm-1 attributed to partially dissociation of crystalline clusters and double helices occurred at 65-80°C. These changes showed that controlled gelatinized starch with slow digestion property occurred in the molecular rearrangement process before granule breakdown and SDS mainly consists of amorphous regions and a small portion of less perfect crystallites.
本文首先建立一种简单快捷的SDS体外测定方法,这是分析淀粉的消化性能、分子结构特征及SDS形成机理的前提。其次,利用快速黏度仪(RVA)、高效排阻色谱(HPSEC)、X-射线衍射(XRD)、差示扫描量热仪(DSC)、扫描电镜(SEM)等现代分析手段,对SDS特性与结构特征进行了研究。最后,探讨了蜡质玉米淀粉经普鲁兰酶部分脱支-重结晶修饰或高水分限制糊化处理后SDS的变化机制。其主要研究内容如下:
通过Englyst法、Guraya法和Shin法对普通玉米淀粉、蜡质玉米淀粉及马铃薯淀粉中SDS测定并与体内法相比较,在此基础上采用单因素与正交试验优化,得到一种新的SDS体外分析方法。最佳测定方法为:200 mg淀粉溶于15 mL pH 5.2的醋酸钠缓冲液,添加10 mL含胰α-淀粉酶(290 U/mL)与糖化酶(15 U/mL)的混酶液在37℃下水解,能满足SDS的体外简单快速测定。
采用改进Englyst法对不同品种淀粉中营养片断分析,得出天然的普通玉米、蜡质玉米、小麦、糯米及大米等A型谷物淀粉含SDS较高,B型马铃薯淀粉主要由抗性淀粉(resistant starch,RS)组成,这表明淀粉的营养片断与其结晶类型有关,谷物淀粉属于理想的SDS原料;经过蒸煮糊化处理后SDS与RS差异基本消失并大部分转变成易消化淀粉(rapidly digestible starch,RDS),因为加热破坏了淀粉颗粒的半结晶结构。通过体外模拟消化动力学曲线探讨了淀粉的慢消化性能与酶水解速率的关系,从酶水解动力学方程可推算出SDS(谷物淀粉)的动力学速率小于1 h-1。水解反应动力学速率比Englyst法更能准确反映淀粉水解过程的消化性能。采用体内实验研究天然淀粉的餐后血糖应答,得出不同谷物淀粉的血糖生成指数(glycemic index,GI)值均大于90,属于高GI食品,但相比食用葡萄糖的血糖应答曲线,淀粉的血糖曲线峰值减少,曲线下面积往后延伸扩展,即对应出现增加血糖生成指数(extended glycemic index,EGI)。EGI值代表淀粉消化后葡萄糖缓慢持续释放的性质,可作为SDS数量或质量的体内评价指标。采用RVA分析淀粉的黏滞性谱,发现淀粉热糊的崩解值与SDS含量呈显著负相关(r=-0.89,p<0.05)。通过凝胶色谱技术分析淀粉精细结构,发现支链淀粉的链长分布也与SDS密切相关,即长链FrⅠ(DP>30)、中长链FrⅡ(13
普鲁兰酶脱支-重结晶处理糊化蜡质玉米淀粉后,淀粉营养片断的比例与结构发生了改变。增加普鲁兰酶液浓度与减少酶脱支时间可得到高含量SDS;在4℃储藏有利于晶核形成,SDS含量则增多。因此,蜡质玉米淀粉经高浓度酶液(20或40 ASPU/g)脱支处理3～6 h,在4℃储藏2 d后可获得较多SDS。富含SDS的淀粉样品X-衍射图谱显示为B型晶体结构,增加储藏时间与温度使晶体结构转变为B+V型。相比糊化淀粉,经酶脱支-重结晶处理后高密度、表面多孔的不规则、多棱角状淀粉碎片被观察到;在DSC图谱上也显示出To、Tp、Tc与△H逐渐增加。依据这些变化可推断出SDS由一小部分双螺旋部分堆积有序结晶区和大部分无定型区构成。
蜡质玉米淀粉在过量水分中通过不同加热处理限制糊化,保持淀粉的慢消化特性。在淀粉未完全糊化发生的温度范围(<60℃) ,SDS含量超过40%,此时淀粉颗粒的外形与结构未发生明显变化。溶胀因子在50～60℃范围内增加缓慢,温度继续升高则快速增大,并在80℃时达到最大值。在65～80℃淀粉的△H、Xc、R(1047/1022)明显降低,这说明结晶簇结构和双螺旋结构的部分发生解聚。上述变化表明糊化处理淀粉的高SDS含量可通过在淀粉颗粒破坏前的结构重排过程达到,另外也验证了SDS由无定型结构和有序双螺旋结晶结构组成的结论。
Slowly digestible starch (SDS) is slowly digested throughout the small intestine resulting in a slow and prolonged release of glucose into the blood stream, coupled to a low glycemic response. Based on the rate and extent of digestibility using the in vitro Englyst assay, SDS is also the starch fraction digested between 20 and 120 min. As a new functional ingredient, SDS is becoming the research focus in food science and modern nutriology for its unique low glycemic index property. Through lessening the stress on regulatory systems related to glucose homeostasis, this starch type may be helpful in controlling and preventing dietary management of metabolic disorders, including diabetes, prediabetes, glycogen storage disease, cardiovascular disease, and obesity. Currently, there are only some reports about preparation method and physiological effect of SDS in occident countries, such as America and Switzerland. Also, SDS-based product is not commercially available in the current food marke. Thus, elucidating the characteristic and formation mechanism of SDS is important for food industry to utilize the abundant starch resource, make tailor-made carbohydrate foods with rich-SDS, and improve the nutritional property of food.
In this study, the method of in vitro analysis was firstly discussed and has unified standardization, which was important for underatanding the digestibility, molecular structure feature and formation mechanism of SDS. The aim of this project was to investigate the slow digestion property, structure basis by using the Rapid viscosity analyzer, High performance size-exclusion chromatography, X-ray diffraction, Differential scanning calorimeter, Scanning electron microscopy, etc., and to probe into the effect partially debraching and recrystallization or controlled gelatinization on SDS in waxy maize starch, which may provide the knowledge for research and development of starchy food with low glycemic index (GI). The main work was listed as follows:
The three in vitro methods of Englyst, Guraya and Shin were used to determine and compare the content of SDS of normal maize starch, waxy maize starch and potato starch with the value of in vivo test. The results showed that the method of Englyst with an optimal modification by one factor and orthogonal experiments was the most suitable one for the determination of SDS. The starch samples (200 mg) were added to the acetate buffer (15 mL, pH 5.2) and mixed thoroughly. The enzyme solution (10 mL, porcine pancreaticα-amylase 290 U/mL and amyloglucosidase 15 U/mL) was then added to the substrate, followed by incubation in a water bath (37 oC) with agitation.
Based on the modified Englyst test to measure the nutritionally important starch fractions, there is higher SDS (about 50%) in starch form common maize, waxy maize, wheat, sticky rice or rice compared with potato starch (16.9%). Therefore, cereal starches containing a large portion of SDS are considerd as ideal SDS materials while potato starch is the typical resistant starch (RS). When the native cereal starch was cooked in the boiling water bath, the slow digestion property was lost with a huge increase of rapidly digeatible starch (RDS). The inheret layer structure of crystalline and amorphous regions is likely the fundamental structure basis for slowly digestion property of starch. The relationship between slow digestion property and enzymatic hydrolysis rate of starches was investigated by establishment of in vitro hydrolysis kinetics. The starch digestion rate calculated using the exponential curve equation C=C∞×(l-e-kt) was less than 1 h-1 for cereal starches. Starch digestibility was reflected exactly by digestion rate of hydrolysis kinetics other than in vitro Englyst method. The glycemic response profile (the shape of the glycemic response curve) of cereal starch was significantly different from glucose powder with a delayed blood glucose peak and a prolonged and moderate elevation of glucose after the peak. The GI value of cereal starch was also more than 90% and belonged to high GI food, but extended GI (EGI) representing the slow glucose release property was positive and might be used as an in vivo indicator of the amount and/or quality of the SDS in foods. RVA was used to investigate viscosity profiles of different starch and the correlation between starch digestibility and RVA profile characteristics. The breakdown of different varieties starch was negatively correlated with SDS (r=-0.89, p<0.05). The RVA method potentially could be used as a screening tool for starch digestion properties. Six starches from different varieties were used as materials and the chromatogram of debranched amylopection were analyzed by SEC system. The correlation between starch digestibility and fine structure of amylopectin was also investigated. SDS fraction was positively correlated with FrⅠ(DP>30) and FrⅡ(13
In order to study the slow digestion of starch, the changes of structure and in vitro digestibility of waxy maize starch after lintnerization using mild acid (2.2 M hydrochloric acid) were investigated. Results show that the granular appearance of waxy maize starch was destroyed and small fractions formed congeries. Lintnerization increased the X-ray intensities of major d-spacings and crystallinity of starches, but the x-ray diffraction patterns remained A-type. The ratios of ordered starch to amorphous starch also increased. The rise in the gelatinization transition temperature, the gelatinization temperature range and the enthalpy of gelatinization of starches were observed and a transformation in the chain length distribution profiles occured after lintnerization. The amount of RDS increased whereas SDS and RS content decreased. The amorphous regions of starch granules including both the amorphous background and the amorphous lamellae were degraded after lintnerization and affected the slow digestion property of native waxy maize starch.
The effects of debranching time, debranching enzyme concentration, storage time and temperature on digestibility and structural properties of waxy maize starch samples were investigated. When gelatinized starch was treated with higher enzyme concentration and less debranching time, higher SDS was formed while RS increased with time. The maximum SDS content was obtained by debranching for 3-6 h with higher concentration of pullulanase (20 or 40 ASPU/g) then storing at 4 oC for 2 days. X-ray diffraction pattern of treated starch at optimal conditions was similar to the B-type in which illuminated treated starches contain partially ordered crystalline structures. Scanning electron micrographs showed the treated starch had more irregular angular shapes with a higher crystallinity structure and pitted surface as debranching and recrystallization time increased. In differential scanning calorimetry themograms, the melting temperature and enthalpy of treated starches were gradually enhanced, resulting from reforming of double helix structure by low temperature recrystallization following debranching. These changes showed that SDS mainly consists of a partially ordered double helix crystallite structure with short amylopectin chains and amorphous regions.
An aqueous dispersion of waxy maize starch (5%, w/w) was partially gelatinized by heating at various temperatures for 5 min. When heated, SDS and RS levels were decreased inversely with RDS. A high SDS content (> 40%) was kept prior to the visible morphological and structural changes (at 60°C). Swelling factor began to increase slightly at 50-60°C and continued to maximum value at 80°C. A large decrease in the melting enthalpy, crystallinity, and ratio of 1047/1022 cm-1 attributed to partially dissociation of crystalline clusters and double helices occurred at 65-80°C. These changes showed that controlled gelatinized starch with slow digestion property occurred in the molecular rearrangement process before granule breakdown and SDS mainly consists of amorphous regions and a small portion of less perfect crystallites.
引文
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