甜菊糖苷中莱鲍迪A苷的研究
|
摘要
甜味剂对食品的感官品质起非常重要的作用,为满足现代生活对食品风味、营养、安全的要求,有必要研究新型的甜味剂。莱鲍迪A苷作为天然甜味剂甜菊糖苷中的主要成分,甜度高,余味小,是甜菊糖苷的高端产品,可被欧美国家认可作为食品添加剂。本论文通过制备色谱制备得到了莱鲍迪A苷的高纯产品,建立了莱鲍迪A苷的分析方法；摸索改进和优化了天然甜味剂莱鲍迪A苷的提取纯化工艺；考察了提纯所得的莱鲍迪A苷作为甜味剂对小鼠的毒性和血糖影响；研究了莱鲍迪A苷的甜味性质,并探索将莱鲍迪A苷替代蔗糖应用于食品,考察其对食品品质的影响。
首先用高效液相制备色谱制备莱鲍迪A苷的高纯产品,探讨了分离甜菊糖苷中莱鲍迪A苷的高效液相色谱条件,在制备条件(采用Lichrospher NH210mmX250mm制备柱,以乙腈水(35+65,v/v)溶液为流动相,4mL/min流速,进样量200μL)下制备得到了相对纯度为99%的高纯样品,通过样品的质谱、核磁共振、红外、紫外图谱的综合分析及与资料报导数据的比对,确认高纯样品为莱鲍迪A苷。在此基础上建立了检测甜菊糖苷混合物中莱鲍迪A含量的HPLC方法,在莱鲍迪A苷量在3-100μg范围之间呈良好的线性关系,用于甜菊糖苷提取物中莱鲍迪A苷的检测,测定的RSD为2.78%,回收率在91.97%～98.42%之间。
为了工业化提纯莱鲍迪A苷,有必要改进甜菊糖苷的加工质量控制方法。为此,建立了甜菊总苷测定的紫外快速方法,在4-100μg/mL范围之内,总苷标准溶液的浓度与紫外205nm下的吸光度之间呈良好的线性关系,相应的线性回归方程为Y=0.0095+7.9623X(R=0.9994,n=9),方法的RSD在1.40-2.38%之间,测定甜叶菊提取物中总糖苷,回收率在95.9%～100.2%之间,与容量法(GB2870第二法,工厂质量控制方法)相比快速、简便,稳定性、精密度、准确度良好。
摸索改进了莱鲍迪A苷的提取方法。以甜叶菊为原料,通过超声波辅助提取法与传统水浴浸提法进行对比,结果表明：超声波辅助提取法不仅可以缩短时间,而且甜菊糖苷的得率提高,尤其是提取液中莱鲍迪A苷含量相对提高。故提取甜菊糖苷时,超声波提取法优于传统水浴浸提法。通过响应面分析得到超声提取甜菊糖苷最佳的工艺条件为：提取温度68℃,超声功率60W,提取时间32min。
以甜菊糖苷混合物为原料,摸索纯化莱鲍迪A苷的方法。采用重结晶法一次纯化得到莱鲍迪A苷的相对纯度93%(总苷含量95%)的产品,结晶产率60.7g/100g。重结晶的条件：以甲醇+异丙醇(99：1)复合溶剂作重结晶溶剂、固液比(W/V)1：8,在-10℃保温放置48h。通过因素实验得到溶剂选择是提高结晶莱鲍迪A苷纯度的关键。结晶温度和时间、溶剂固液比、是否加入晶种是提高结晶产率的影响因素。多次重结晶纯化得到莱鲍迪A苷的相对纯度大于95.8%的产品,但结晶产率明显降低。
采用改良Karber法研究了莱鲍迪A苷对于小鼠的急性毒性,测得莱鲍迪A苷对小鼠的口服急性毒性LDso为22284mg/kg,可认为莱鲍迪A苷基本无毒；亚急性毒性实验结果显示莱鲍迪A苷对小鼠的体重、脏器重量、脏器指数没有直接影响。小鼠的餐后血糖反应实验结果显示摄入莱鲍迪A苷30min时血糖值出现峰值,较摄入葡萄糖慢了15min,并且较摄入相当量葡萄糖的血糖峰值略低。同时,构建四氧嘧啶型糖尿病小鼠模型与正常小鼠对照,经连续10天灌胃莱鲍迪A苷溶液,测得正常小鼠空腹血糖与灌胃生理盐水的空白小鼠相比无显著性差异,四氧嘧啶型糖尿病模型组小鼠空腹血糖与灌胃生理盐水的四氧嘧啶型糖尿病模型对照小鼠相比也无显著性差异,证明莱鲍迪A苷对小鼠空腹血糖基本没有影响。
对采用重结晶法从甜菊糖苷混合物中提纯获得莱鲍迪A苷(93%)产品的甜味性质进行研究,莱鲍迪A苷(93%)的甜度是2%蔗糖溶液的400倍以上,但甜味不如蔗糖持久、甜味圆润质感差,高浓度有余味,随莱鲍迪A苷浓度降低,余味效果变好。与相应浓度的甜菊糖苷混合物(莱鲍迪A苷60%)对比,甜度提高了40%以上,余味明显减小,甜味味质有了明显的改善和提高。莱鲍迪A苷(93%)的相对甜度经验公式为RS=482.10—24.95×ES。在等甜2%蔗糖浓度时,在品尝温度4℃下相对甜度可达540倍。莱鲍迪A苷溶液的相对甜度随pH变化而变化,在pH6.0时相对甜度最低。pH相同,莱鲍迪A苷溶液的相对甜度随酸种类的变化而变化,苹果酸会提高相对甜度,而酒石酸降低相对甜度。
采用HPLC测定各溶液莱鲍迪A苷含量和测定相对甜度的方法进行了莱鲍迪A苷溶液的热、酸、光稳定性的研究。莱鲍迪A苷(pH7)溶液在100℃水浴条件下煮沸60min,相对甜度没有发生变化,莱鲍迪A苷的含量基本不变,可认为莱鲍迪A苷溶液的热稳定性良好。但莱鲍迪A苷(pH4)溶液在100℃水浴条件下煮沸60min,相对甜度下降,莱鲍迪A苷保留率60min时下降5.2%,说明在一定的酸度下加热,莱鲍迪A苷会降解。莱鲍迪A苷(pH7.0)的溶液暴露在阳光下1周、2周、3周、4周后相对甜度没有发生变化,莱鲍迪A苷的含量不变,说明光对莱鲍迪A苷溶液的稳定性的影响很小。
最后,将莱鲍迪A苷替代部分蔗糖应用于三类典型食品：固体、半固体、液体。用莱鲍迪A苷替代部分蔗糖配制的果味饮料,随蔗糖替代度的升高,果味饮料的粘稠度、可溶性固形物降低,感官评定结果,蔗糖替代度以20%最好。用莱鲍迪A苷替代部分蔗糖制备的冰淇淋,随蔗糖替代度的升高,总固形物逐渐降低,膨胀率基本不变,感官评定结果,蔗糖替代度以20%最好。用莱鲍迪A苷替代部分蔗糖制备的面包测定结果,随蔗糖替代度的升高,面包的水分含量基本不变,面包的比容和面包芯硬度有所下降,感官品质评定中,10%的蔗糖替代量制备的面包与对照样品感官评定总分较接近。
Sweetener, as one important food ingredient with a huge market demand, is an essential part of the sensory quality of foods, and plays an important role in the food industry. Sucrose as one typical sweetener has been used widely in food industry, and in order to meet the continuously increasing demand of nutrition, flavor, and security of foods, there needs to explore new sweeteners that are of natural resources. A sweetener of Rebaudioside A glucoside from stevia extract was investigated in the curent study. The analytical methods and purification process of Rebaudioside A with a high efficiency of purification was established, and its physicochemical properties, particularly its sweetness, were also studied. Rebaudioside A has a great potential as a natural sweetener in food applications.
The separation method of Rebaudioside A glycoside and its molecular structure were investigated. Rebaudioside A glucoside was first purified by a high-performance liquid chromatographic (HPLC) method to a relative purity of 99% using Lichrospher NH2 10mmⅩ250mm preparation column with acetonitrile water (35+65, v/v) solution as the mobile phase in a flow rate of 4mL/min. Rebaudioside A glucoside was confirmed by a comprehensive analysis using data from Mass spectrum, NMR, IR, and UV. Based on the above procedure, a method to quantify Rebaudioside A glucoside was also established in a linear range of 3-100μg of Rebaudioside A glucoside with a RSD of 2.78% and recovery rate of 91.97%-98.42%.
A quick method to measure the concentration of total glycosides was also established based on UV spectrophotometer in a linear range of 4-100μg/mL, and the concentration could be calculated using the linear regression equation of Y=0.0095+7.9623X (R 0.9994, n=9) in which Y is the absorbance and X is the concentration (μg/mL). The variation coefficient of the method is 1.40-2.38%, and the recovery rate is 95.9%-100.2% with a detection limit of 0.8μg/mL. This method can be used for on-line quality control with its simplicity, stability and high precision.
In order to maximize the yield of extracts from Stevia rebaudiana Bertoni, Response surface methodology (RSM) was employed to optimize the ultrasound-assisted extraction conditions. The results indicated optimum extraction conditions were an extraction temperature of 68℃, sonic power of 60 W and time of 32 min. Using a short application of ultrasound, the yield of extracts increased by a factor of 1.5 at lower extraction temperature (68℃) and substantially shorter extraction time (32 min) compared with that of classical extraction. The components analysis of crude extracts revealed that the relative amount of rebaudioside A increased in the ultrasound-assisted extracts as compared with extracts obtained by classical process, and the ultrasound-assisted extract had better quality.
The procedure for a bench top-scale purification of Rebaudioside A from raw material of stevia extract was explored to improve its production throughput. A purity of 93.1% Rebaudioside A was obtained by re-crystallization after the raw material was dissolved (1:8, w/v) in a solvent of methanol/isopropanol (99:1), and insulatedly crystallized at-10℃for 48 hrs. The above condition for re-crystallization was obtained using a series of single-factor experiments, and the solvent selection is the utmost important factor affecting the purity of the Rebaudioside A in the crystallized product while the initial formation process of crystal is the second important factor. The ratio of solvent to solid, the container size and shape, the crystallization temperature and time, and the existence of crystallization seed only improve the crystallization yield.
The physiological effects after consuming Rebaudioside A were studied. A value of 22284mg/kg as the LD50 for mice was obtained using modified Karber method measuring the acute toxicity in mice, indicating that the Rebaudioside A is non-toxic for consumption, which is further supported by the sub-acute toxicity test showing that mice body weight, organ weight, and organ index are not directly affected after feeding Rebaudioside A (1-1.5g/day/kg) for10 days. The effect on postprandial glucose response in mice after Rebaudioside A intake was also examined. For normal mice, no significant difference was found in the peak blood glucose level between feeding glucose (1g/kg) and glucose-equivalent Rebaudioside A (1.4g/kg) although the peak time delayed 15 min after feeding Rebaudioside A. The interference experiment showed that feeding Rebaudioside A (in the same dosage as above) for 10 days has no effect on the fasting blood glucose level for both normal and alloxan-induced diabetic mice, however the body weight of Rebaudioside A-treated mice decreased compared to control group, which together suggest that Rebaudioside A might affect the energy intake that is likely beneficial to health.
The sweetness of purified Rebaudioside A was examined compared to sucrose and stevia extract. The relative sweetness of Rebaudioside A is 300-500 times of sucrose based on sensory test result, but the sweetness of Rebaudioside A feels sharper compared to sucrose under normal dosage although there is aftertaste that is dosage-dependent. Compared with the corresponding concentration of stevia extract, sweetness increased more than 40% but its aftertaste reduced, which showed that the Rebaudioside A is much sweeter than commonly used stevia extract. Further study showed the sweetness of Rebaudioside A is concentration-dependent in a manner of RS=482.10-24.95×ES in which ES represents the percentage of Rebaudioside A. In the meantime, the relative sweetness is affected by temperature, and a 540-fold RS was achieved at 4℃and 2%(ES) concentration. The RS also showed a bell-shaped relationship with pH (4.0-8.0) with a lowest RS at pH 6.0. The stability of Rebaudioside A solution was mainly affected by pH, and a low pH (e.g.4.0) tends to decrease the stability of Rebaudioside A with a corresponding decrease of sweetness. Rebaudioside A is also stable to sunlight, and in a solution of pH7.0, no significant changes were found in terms of its sweetness under sunlight for 4 weeks.
The food application of Rebaudioside A was studied as the last part of the project. Three types of food product were select representing solid, semi-solid and liquid foods. In the fruit drink, only 10-30% sucrose could be replaced by Rebaudioside A without noticeable difference in sensory properties, however, the viscosity of the drink is decreased. In semi-solid ice cream, replacing sucrose decreased the total solid material content, and sensory test showed 20% replacement is the best. For the solid food of bread, substitution of sucrose with Rebaudioside A decreased the core hardness, and only 10% can be used to satisfy the sensory quality of the bread.
首先用高效液相制备色谱制备莱鲍迪A苷的高纯产品,探讨了分离甜菊糖苷中莱鲍迪A苷的高效液相色谱条件,在制备条件(采用Lichrospher NH210mmX250mm制备柱,以乙腈水(35+65,v/v)溶液为流动相,4mL/min流速,进样量200μL)下制备得到了相对纯度为99%的高纯样品,通过样品的质谱、核磁共振、红外、紫外图谱的综合分析及与资料报导数据的比对,确认高纯样品为莱鲍迪A苷。在此基础上建立了检测甜菊糖苷混合物中莱鲍迪A含量的HPLC方法,在莱鲍迪A苷量在3-100μg范围之间呈良好的线性关系,用于甜菊糖苷提取物中莱鲍迪A苷的检测,测定的RSD为2.78%,回收率在91.97%～98.42%之间。
为了工业化提纯莱鲍迪A苷,有必要改进甜菊糖苷的加工质量控制方法。为此,建立了甜菊总苷测定的紫外快速方法,在4-100μg/mL范围之内,总苷标准溶液的浓度与紫外205nm下的吸光度之间呈良好的线性关系,相应的线性回归方程为Y=0.0095+7.9623X(R=0.9994,n=9),方法的RSD在1.40-2.38%之间,测定甜叶菊提取物中总糖苷,回收率在95.9%～100.2%之间,与容量法(GB2870第二法,工厂质量控制方法)相比快速、简便,稳定性、精密度、准确度良好。
摸索改进了莱鲍迪A苷的提取方法。以甜叶菊为原料,通过超声波辅助提取法与传统水浴浸提法进行对比,结果表明：超声波辅助提取法不仅可以缩短时间,而且甜菊糖苷的得率提高,尤其是提取液中莱鲍迪A苷含量相对提高。故提取甜菊糖苷时,超声波提取法优于传统水浴浸提法。通过响应面分析得到超声提取甜菊糖苷最佳的工艺条件为：提取温度68℃,超声功率60W,提取时间32min。
以甜菊糖苷混合物为原料,摸索纯化莱鲍迪A苷的方法。采用重结晶法一次纯化得到莱鲍迪A苷的相对纯度93%(总苷含量95%)的产品,结晶产率60.7g/100g。重结晶的条件：以甲醇+异丙醇(99：1)复合溶剂作重结晶溶剂、固液比(W/V)1：8,在-10℃保温放置48h。通过因素实验得到溶剂选择是提高结晶莱鲍迪A苷纯度的关键。结晶温度和时间、溶剂固液比、是否加入晶种是提高结晶产率的影响因素。多次重结晶纯化得到莱鲍迪A苷的相对纯度大于95.8%的产品,但结晶产率明显降低。
采用改良Karber法研究了莱鲍迪A苷对于小鼠的急性毒性,测得莱鲍迪A苷对小鼠的口服急性毒性LDso为22284mg/kg,可认为莱鲍迪A苷基本无毒；亚急性毒性实验结果显示莱鲍迪A苷对小鼠的体重、脏器重量、脏器指数没有直接影响。小鼠的餐后血糖反应实验结果显示摄入莱鲍迪A苷30min时血糖值出现峰值,较摄入葡萄糖慢了15min,并且较摄入相当量葡萄糖的血糖峰值略低。同时,构建四氧嘧啶型糖尿病小鼠模型与正常小鼠对照,经连续10天灌胃莱鲍迪A苷溶液,测得正常小鼠空腹血糖与灌胃生理盐水的空白小鼠相比无显著性差异,四氧嘧啶型糖尿病模型组小鼠空腹血糖与灌胃生理盐水的四氧嘧啶型糖尿病模型对照小鼠相比也无显著性差异,证明莱鲍迪A苷对小鼠空腹血糖基本没有影响。
对采用重结晶法从甜菊糖苷混合物中提纯获得莱鲍迪A苷(93%)产品的甜味性质进行研究,莱鲍迪A苷(93%)的甜度是2%蔗糖溶液的400倍以上,但甜味不如蔗糖持久、甜味圆润质感差,高浓度有余味,随莱鲍迪A苷浓度降低,余味效果变好。与相应浓度的甜菊糖苷混合物(莱鲍迪A苷60%)对比,甜度提高了40%以上,余味明显减小,甜味味质有了明显的改善和提高。莱鲍迪A苷(93%)的相对甜度经验公式为RS=482.10—24.95×ES。在等甜2%蔗糖浓度时,在品尝温度4℃下相对甜度可达540倍。莱鲍迪A苷溶液的相对甜度随pH变化而变化,在pH6.0时相对甜度最低。pH相同,莱鲍迪A苷溶液的相对甜度随酸种类的变化而变化,苹果酸会提高相对甜度,而酒石酸降低相对甜度。
采用HPLC测定各溶液莱鲍迪A苷含量和测定相对甜度的方法进行了莱鲍迪A苷溶液的热、酸、光稳定性的研究。莱鲍迪A苷(pH7)溶液在100℃水浴条件下煮沸60min,相对甜度没有发生变化,莱鲍迪A苷的含量基本不变,可认为莱鲍迪A苷溶液的热稳定性良好。但莱鲍迪A苷(pH4)溶液在100℃水浴条件下煮沸60min,相对甜度下降,莱鲍迪A苷保留率60min时下降5.2%,说明在一定的酸度下加热,莱鲍迪A苷会降解。莱鲍迪A苷(pH7.0)的溶液暴露在阳光下1周、2周、3周、4周后相对甜度没有发生变化,莱鲍迪A苷的含量不变,说明光对莱鲍迪A苷溶液的稳定性的影响很小。
最后,将莱鲍迪A苷替代部分蔗糖应用于三类典型食品：固体、半固体、液体。用莱鲍迪A苷替代部分蔗糖配制的果味饮料,随蔗糖替代度的升高,果味饮料的粘稠度、可溶性固形物降低,感官评定结果,蔗糖替代度以20%最好。用莱鲍迪A苷替代部分蔗糖制备的冰淇淋,随蔗糖替代度的升高,总固形物逐渐降低,膨胀率基本不变,感官评定结果,蔗糖替代度以20%最好。用莱鲍迪A苷替代部分蔗糖制备的面包测定结果,随蔗糖替代度的升高,面包的水分含量基本不变,面包的比容和面包芯硬度有所下降,感官品质评定中,10%的蔗糖替代量制备的面包与对照样品感官评定总分较接近。
Sweetener, as one important food ingredient with a huge market demand, is an essential part of the sensory quality of foods, and plays an important role in the food industry. Sucrose as one typical sweetener has been used widely in food industry, and in order to meet the continuously increasing demand of nutrition, flavor, and security of foods, there needs to explore new sweeteners that are of natural resources. A sweetener of Rebaudioside A glucoside from stevia extract was investigated in the curent study. The analytical methods and purification process of Rebaudioside A with a high efficiency of purification was established, and its physicochemical properties, particularly its sweetness, were also studied. Rebaudioside A has a great potential as a natural sweetener in food applications.
The separation method of Rebaudioside A glycoside and its molecular structure were investigated. Rebaudioside A glucoside was first purified by a high-performance liquid chromatographic (HPLC) method to a relative purity of 99% using Lichrospher NH2 10mmⅩ250mm preparation column with acetonitrile water (35+65, v/v) solution as the mobile phase in a flow rate of 4mL/min. Rebaudioside A glucoside was confirmed by a comprehensive analysis using data from Mass spectrum, NMR, IR, and UV. Based on the above procedure, a method to quantify Rebaudioside A glucoside was also established in a linear range of 3-100μg of Rebaudioside A glucoside with a RSD of 2.78% and recovery rate of 91.97%-98.42%.
A quick method to measure the concentration of total glycosides was also established based on UV spectrophotometer in a linear range of 4-100μg/mL, and the concentration could be calculated using the linear regression equation of Y=0.0095+7.9623X (R 0.9994, n=9) in which Y is the absorbance and X is the concentration (μg/mL). The variation coefficient of the method is 1.40-2.38%, and the recovery rate is 95.9%-100.2% with a detection limit of 0.8μg/mL. This method can be used for on-line quality control with its simplicity, stability and high precision.
In order to maximize the yield of extracts from Stevia rebaudiana Bertoni, Response surface methodology (RSM) was employed to optimize the ultrasound-assisted extraction conditions. The results indicated optimum extraction conditions were an extraction temperature of 68℃, sonic power of 60 W and time of 32 min. Using a short application of ultrasound, the yield of extracts increased by a factor of 1.5 at lower extraction temperature (68℃) and substantially shorter extraction time (32 min) compared with that of classical extraction. The components analysis of crude extracts revealed that the relative amount of rebaudioside A increased in the ultrasound-assisted extracts as compared with extracts obtained by classical process, and the ultrasound-assisted extract had better quality.
The procedure for a bench top-scale purification of Rebaudioside A from raw material of stevia extract was explored to improve its production throughput. A purity of 93.1% Rebaudioside A was obtained by re-crystallization after the raw material was dissolved (1:8, w/v) in a solvent of methanol/isopropanol (99:1), and insulatedly crystallized at-10℃for 48 hrs. The above condition for re-crystallization was obtained using a series of single-factor experiments, and the solvent selection is the utmost important factor affecting the purity of the Rebaudioside A in the crystallized product while the initial formation process of crystal is the second important factor. The ratio of solvent to solid, the container size and shape, the crystallization temperature and time, and the existence of crystallization seed only improve the crystallization yield.
The physiological effects after consuming Rebaudioside A were studied. A value of 22284mg/kg as the LD50 for mice was obtained using modified Karber method measuring the acute toxicity in mice, indicating that the Rebaudioside A is non-toxic for consumption, which is further supported by the sub-acute toxicity test showing that mice body weight, organ weight, and organ index are not directly affected after feeding Rebaudioside A (1-1.5g/day/kg) for10 days. The effect on postprandial glucose response in mice after Rebaudioside A intake was also examined. For normal mice, no significant difference was found in the peak blood glucose level between feeding glucose (1g/kg) and glucose-equivalent Rebaudioside A (1.4g/kg) although the peak time delayed 15 min after feeding Rebaudioside A. The interference experiment showed that feeding Rebaudioside A (in the same dosage as above) for 10 days has no effect on the fasting blood glucose level for both normal and alloxan-induced diabetic mice, however the body weight of Rebaudioside A-treated mice decreased compared to control group, which together suggest that Rebaudioside A might affect the energy intake that is likely beneficial to health.
The sweetness of purified Rebaudioside A was examined compared to sucrose and stevia extract. The relative sweetness of Rebaudioside A is 300-500 times of sucrose based on sensory test result, but the sweetness of Rebaudioside A feels sharper compared to sucrose under normal dosage although there is aftertaste that is dosage-dependent. Compared with the corresponding concentration of stevia extract, sweetness increased more than 40% but its aftertaste reduced, which showed that the Rebaudioside A is much sweeter than commonly used stevia extract. Further study showed the sweetness of Rebaudioside A is concentration-dependent in a manner of RS=482.10-24.95×ES in which ES represents the percentage of Rebaudioside A. In the meantime, the relative sweetness is affected by temperature, and a 540-fold RS was achieved at 4℃and 2%(ES) concentration. The RS also showed a bell-shaped relationship with pH (4.0-8.0) with a lowest RS at pH 6.0. The stability of Rebaudioside A solution was mainly affected by pH, and a low pH (e.g.4.0) tends to decrease the stability of Rebaudioside A with a corresponding decrease of sweetness. Rebaudioside A is also stable to sunlight, and in a solution of pH7.0, no significant changes were found in terms of its sweetness under sunlight for 4 weeks.
The food application of Rebaudioside A was studied as the last part of the project. Three types of food product were select representing solid, semi-solid and liquid foods. In the fruit drink, only 10-30% sucrose could be replaced by Rebaudioside A without noticeable difference in sensory properties, however, the viscosity of the drink is decreased. In semi-solid ice cream, replacing sucrose decreased the total solid material content, and sensory test showed 20% replacement is the best. For the solid food of bread, substitution of sucrose with Rebaudioside A decreased the core hardness, and only 10% can be used to satisfy the sensory quality of the bread.
引文
1.胡国华,马正智.我国功能性高倍甜味剂的发展现状与前景[J].中国食品添加剂,2008,6：68-71
2.保国裕.蔗糖、淀粉糖、化学合成高倍甜味剂三分糖市的趋势与对策[J].广西蔗糖,2006,4：42-43
3.中国甜菊信息网http://icgr.caas.net.cn/csa。
4.孙玉华,于喜水.甜菊苷类的研究与开发[J].黑龙江医药,2003,16(1)：41-42.
5.胡献丽,董文宾,郑丹,杨兆艳.甜菊及甜菊糖研究进展[J].食品研究与开发,2005,26(1)：36-38.
6 Hanson J. R.,de Oliveira B. H. Stevioside and related sweet diterpenoid glycosides[J]. Natural Product Reports,1993,10 (3):301-309.
7. Brandle, J. E., Rosa, N. Heritability for yield, leaf stem ratio and stevioside from landrace cultivar of Stevia rebaudiana[J]. Canadian Journal of Plant Science,1992,72,1263-1266.
8. L. O. Nabors. Alternative Sweeteners[M].1991. New York:Marcel Dekker Inc.
9. Grenby, T.H.. Developments in Sweeteners[M].1989,3. Elsevier, London.
10. Soejarto D. D., Kinghorn A. D., Farnsworth N. R.. Potential sweetening agents of plant rigin Organoleptic evaluation of Stevia leaf herbarium samples for sweetness [J].Nat Prod, 1982,45:590-599.
11. Toskulkao,C, Chaturat,L, Temcharoen P. Acute toxicity of stevioside, a natural sweetener, and its metabolite, steviol, in several animal specie [J].Drug Chem Toxicol,1997,20:31-44.
12. Kamath S.K., Kinghorn A. D. Safety assessment of some stevia rebaudiana sweet principles [J].Fed Proc,1982,41:1568.
13. Krejci M. E., Koechel D. A. Acute effects of carboxyatrac tyloside and stevioside,inhibitors of mitochondrial ADP/ATP translocation, on renal function and ultrastructure in pentobar tal-anesthetized dogs [10].Toxicology,1992,72:299-313.
14. Aze Y, Toyoda K, Imaida K,. Subchronic oral toxicity study of stevioside in F344 rats [J].Bull Natl Inst Hyg Sci,1991:48-54.
15. Xili L., Chengjiany B., Eryi X. Chronic oral toxicity and carcinogenicity study of stevioside in rats[J]. Food Chem Toxicol,1992,30:957-965.
16. Toyoda K, Matsui H, Shoda T, etal.Asessment of the carcinogenicity of stevioside in F344 rats[J].Food Chem Toxicol,1997,35:597-603.
17. Hagiwara A, Fukushima S, Kitaori M. Effects of threesweeteners on rat urinary bladder carcinogenesis initiated by N-butyl-N-(4-hydroxybutyl) nitrosamine [J].Gann,1984, 75:763-768.
18. Suttajit M, Vinitketkaumnuen U, Meevatee U, et al. Mutagenicity and human chromosomal effect of stevioside, a sweetener fromStevia rebaudiana Bertoni [J].Environ Health Perspectives,1993,101:53-56.
19. Matsui M, Matsui K, Kawasaki Y. Evaluation of the genotoxicity of stevioside and steviol using six in vitro and one in vivo mutagenicity assays[J].Mutagenesis,1996,11:573-579.
20. Ishidate M, Sofuni T, Yoshikawa K. Primary muta genicity screening of food additives currently used in Japan [J]. Food Chem Toxicol,1984,22:623-636.
21.Yodyingyuard V.,Bunyawong S. Effect of stevioside on growth and reproduction[J].Hum Reprod,1991,6:158-165.
22. Mazzei-Planas G & Kuc J Contraceptive properties of Stevia rebaudiana[J].Science,1968, 162:1007.
23. Oliverira-Filho R M, Uehara O A, Minetti C A, etal. Chronic administration of aqueous extract of Stevia rebaudiana Bertoniin rats:Endocrine effects [J].GenPharmacol,1989,20:187-191.
24. Melis M S-Effects of chronic administration of Stevia rebaudianaon fertility in rats[J]. Ethnopharmacology,1999,67(2):157-161.
25. Usami M, Sakemi K, Kawashima K, etal. Yeratogenicity study of stevioside in rats[J].BullNatl InstHygSci,1995,113:31-35.
26. Federal Food, Drug and Cosmetic Act of 1994(revised April 2000)[Z]. Section 402, Section 409 and 21 CFR 170 and 21 CFR 189-1.
27.李晓瑜.甜菊糖苷的安全性研究进展[J].中国食品添加剂,2003,2(5)：11-14.
28. Henkel, J. Sugar substitutes:American opt for sweetness and lite. FDA Consumer[M]. 1999,33(6). Available:http://www.fda.gov/fdac/699_toc. html.
29. WHO. Toxicological evaluation of certain food additives and contaminants[M]. WHO food additives series 42.Geneva:World Health Organization.1999:138.
30. Scientific Committee on Food. Opinion on Stevioside as a Sweetener. CS/ADD/EDUL/167 final 17 June 1999.
31. Scientific Committee on Food. Opinion on Stevia Rebaudiana Bertoni plants and leaves. CS/NF/STEV/3 final 17 June 1999.
32.张亚雄,胡为民,胡滨.甜菊糖中A3苷的分离方法初探[J].中国食品添加剂,1998,4：49-51.
33. Truong, T. T., Valicek, P., Nepovim, A., Vanek, T. Correlation between stevioside content-in leaves, their area and the number of roots in plant[J]. Sci. Agr. Biohemica,1999, 30:249-255
34. Brandle, J. E.. Genetic control of rebaudioside A and C concentration in leaves of the sweet herb, Stevia rebaudiana[J]. Canadian Journal of Plant Science,1999,79,85-92.
35. Nikolai Bondarev, Oxana Reshetnyak, Alexander Nosov. Effects of nutrient medium composition on development of Stevia rebaudiana shoots cultivated in the roller bioreactor and their production of steviol glycosides[J].Plant Science,2003,165:845-850
36. Nikolai Bondarev, Oxana Reshetnyak, Alexander Nosov. Peculiarities of diterpenoid steviol glycoside production in in vitro cultures of Stevia rebaudiana Bertoni[J]. Plant Science,2001,161:155-163.
37. Nikolai Bondarev, Oxana Reshetnyak, Alexander Nosov.. Features of development of Stevia rebaudiana shoots cultivated in the roller bioreactor and their production of steviol glycoside[J]. Planta Med,2002,68:757-759.
38.黄应森.中国甜菊和甜菊糖苷的分型研究[J].中国糖料.1999,4：26-29
39. Lyakhovkin A.G., Long T.D., Titov D.A., Anh M.P.. Cultivation and Utilization of Stevia (Stevia rebaudiana Bertoni) [M]. Agricultural Publishing House, Hanoi,1993.
40. Zhang, S. Q., Kumar, A.,& Kutowy, O. Membrane-based separation scheme for processing sweeteners from stevia leaves[J]. Food Research International,2000, 33:617-620.
41. Takahashi, M., Matsuda, M., Ohahi, K., etal. Analysis of anti-rotavirus activity of extract from Stevia rebaudiana[J]. Antiviral Research,2001,49:15-24.
42. Simone K.Yoda,Marcia O.M.Marques,etal. Supercritical fluid extraction from Stevia rebaudiana Bertoni using CO2 and CO2 twater:extraction kinetics and identification of extracted components [J]. Journal of Food Engineering,2003,57:125-134
43. Liu, J. and Li, S. F. Y. Separation and determination of stevia sweeteners by capillary lectrophoresis and high performance liquid chromatography[J]. Liq. Chromarogr, 1995,18:1703-1719.
44. Liu, J., Ong, C. P., Li, S. F. Y. Subcritical fluid extraction of stevia sweeteners from Stevia rebaudiana[J]. Journal of Chromatographic Science,1997,35:446-450.
45. Payzant JD, Laidler JK, Ippolito RM, Method of extracting selected sweet glycosides from the Stevia rebaudiana plant[P]. US Patent 5,962,678,1998.
46. Brandle JE, Starratt AN, Kirby CW, Pocs R. Rebaudioside-F, a diterpenen glycoside from Stevia rebaudiana[J]. Phytochemistry,2002,59:367-370.
47.孙玉华,于喜水.甜菊式类的研究与开发[J].黑龙江医药.2003,16(1)：41-42.
48.赵永金,孙信庆.从甜叶菊中提取甜菊苷的工艺研究[J].西北农业大学学报,1999,27：80-83
49.刘宗林,彭义交,郭洋等.甜叶菊苷的提取与结晶工艺研究[J].食品科学,2002,23(8)：99-100
50. YamasakiK,KohdaH,KobayashiT,KasaiR,TanakaO. Structuresof 13 Stevia diterpene-glucosides:application of C NMR[J]. Tetrahedron Lett,1976;13:1005-1008.
51. Sakamoto I.,Yamasaki K.,Tanaka O. Applicationof CNMRspectro-scopy to chemistry of plant glycosides:rebaudiosides-D and-E, new sweet diterpene-glucosides of Stevia rebaudiana Bertoni[J]. Chem Pharm Bull,1977,25(12):3437-3439.
52. Compadre, C.M., Hussain, R.A., etal. Mass spectral analysis of some derivatives and in vitro metabolites of steviol, the aglycone of the natural sweeteners, stevioside, rebaudioside A and rubusoside[J]. Biomed. Environ. Mass Spectrom,1988,15:211-222.
53. Brandle, J.E., Starratt, A.N., Gijzen, M.. Stevia rebaudiana:its agricultural, biological, and chemical properties [J]. Canadian Journal of Plant Science,1998,78:527-536.
54. Sakamoto, I., Yamasaki, K., Tanaka, O.. Application of 13C NMR spectroscopy to chemistry of natural glycosides:Rebaudioside-C, a new sweet diterpene glycoside of Stevia rebaudiana[J]. Chemical and Pharmaceutical Bulletin,1977,25:844-846.
55. Jan M.C. Geuns, Molecules of Interest Stevioside[J]. Phytochemistry,2003,64:913-921
56.陈天红,张杨,刘晓航,史作清等.高分子吸附剂对甜叶菊中各糖苷吸附选择性的研究[J].中国科学(B),1998,28(5)：460-465.
57.施荣富,史作清,王春红等.吸附树脂的色层吸附及其在甜菊苷分离纯化中的作用[J].离子交换与吸附,2001,17(1)：23-30.
58.张杨,陈天红等.重结晶法分离精制莱鲍迪A苷的研究[J].离子交换与吸 附,1998,16(4)：515-520.
59. Tomas Vanek, etal. Determination of stevioside in plant material and fruit teas[J]. Food composition and analysis.2001,14:383-388
60. Hutapea, A.M., Toskulkao, C., Wilairat, P., etal. High-performance liquid chromatographic separation and quantitation of stevioside and its metabolites[J]. Liq. Chrom. Rel. Technol,1999,22:1161-1170.
61. Mauri P, Catalano G, Gardana C, etal. Analysis of Stevia glycosides by capillary electrophoresis[J]. Electrophoresis,1996;17:367-71.
62. Ahmed, M.S., Dobberstein, R.H. Use of p-bromophenacyl bromide to enhance ultraviolet detection of water-soluble organic acids (steviolbioside and rebaudioside B) in high-performance liquid hromatographic analysis[J]. Chromatogr,1980,192:387-393.
63. Fujinuma, K., Saito, K.,Nakazato, etal. Thin layer chromatographic detection and liquid chromatographic determination of stevioside and rebaudioside A in beverages and foods following reversed phase column chromatography[J]. Assoc. Off. Anal. Chem,1986,69:799-802.
64. N. Kolb, J. L. Herrera, D. J. Ferreyra, and R. F. Uliana. Analysis of Sweet Diterpene Glycosides from Stevia rebaudiana:Improved HPLC Method[J]. Agric. Food Chem,2001, 49,4538-4541.
65. Nikolova-Damyanova, B., Bankova, V., and Popov, S. Separation and quantitation of stevioside and rebaudioside A in plant extracts by normal-phase HPLC and TLC:A comparison[J]. Phytochemical analysis,1994,5:81-85.
66. Makapugay, H. C., Nanayakkara, N. P. D., Kinghorn, A. D. Improved high-performance liquid chromatographic separation of the Stevia rebaudiana sweet diterpene glycosides using linear gradient elution[J]. Chromatogr,1984,283:390-395.
67.刘超,李来生等.高效液相色谱法测定甜叶菊糖中的甜菊苷和莱鲍迪苷A[J].分析试验室,2007,26(7)：23-26
68. Jan M.C. Geuns, Patrick Augustijns, Raf Mols, Johan G. Buyse, Bert Driessen. Metabolism of stevioside in pigs and intestinal absorption characteristics of stevioside, rebaudioside A and steviol[J].Food and Chemical Toxicology,2003,41:1599-1607
69. Bearder, J.R., MacMillan, J., Wels, C.M.. The metabolism of steviol to 13-hydroxylated ent-gibberellanes and ent-keuranes[J]. Phytochemistry,1975,14:1741-1748.
70. Hagiwara, A., Fukushima, S., Kitaori, M.. Effects of the three sweeteners on rats urinary bladder carcinogenesis initiated by Nbutyl-N-(4-hydroxybutyl)-nitrosamine[J]. Gann, 1984,75:763-768.
71. Geuns, J.M.C., Malheiros, R.D., etal. Metabolism of stevioside by chickens. Agric. Food Chem.2003b,51:1095-1101.
72. Hutapea, A.M., Toskulkao, C., etal. Digestion of Stevioside, a Natural Sweetener, by Various Digestive Enzymes. Clin. Biochem. Nutr.,1997,23,177-186.
73. Koyama, E., Kitazawa, K., Ohori, Y, Sakai, N., Izawa, O., Kakegawa, K., Fujino, A., Ui, M., Intestinal degradation, absorption, and hepatic metabolism of the glycosidic sweeteners, Stevia mixture[M]. In:Proceedings second IUPA-International Symposium on Sweeteners, Hiroshima,2001.12.
74. Koyama, E., Kitazawa, K, Ohori, Y, Izawa, O., Kakegawa, K., Fujino, A., Ui, M., In vitro metabolism of the glycosidic sweeteners, Stevia mixture and enzymatically modified Stevia in human intestinal microflora[J]. Food Chem. Toxicol,2003a,41:359-374.
75. Liu, J.-C, Kao, P.-K., Chan, P., etal. Mechanismof the antihypertensive effect of stevioside in anesthesized dogs[J]. Pharmacology,2003,67:14-20.
76. Temcharoen, P., Pimbua, J., Glinsukon, T., Rojanapo, W., Apibal, S. Mutagenic activity of steviol to Salmonella typhimurium TM 677:Comparison of the activity of S9 liver fractions from five laboratory animal species[J]. Bull. Health Sci.& Tech,1998,1:38-45.
77. Temcharoen, P., Suwannatrai, M., Klongpanichpak, S., etal. Mutagenicity of steviol and its oxidative derivatives in Salmonella typhimurium TM677[J]. Chem. Pharm. Bull, 2002,50:1007-1010.
78. Toskulkao, C., Chaturat, L., Temcharoen, P., etal. Acute toxicity of stevioside, a natural sweetener, and its metabolite, steviol, in several animal species [J]. Drug Chem. Toxicol, 1997.20,31-44.
79. Totte, N., Charon, L., Rohmer, M., Compernolle, F., etal. Biosynthesis of the diterpenoid steviol, an entkaurene derivative from Stevia rebaudiana Bertoni, via the methylerythritol phosphate pathway[J]. Tetrahedron Letters,2000,41:6407-6410.
80. Wingard, R.E., Brown, J.P., Enderlin, F.E., etal. Intestinal degradation and absorption of the glycosidic sweeteners stevioside and rebaudioside A[J]. Experientia,1980,36:519-520.
81. Wood, D.J., Lirette, A., Crober, D.C., Ju-Hy,. The effect of stevia as a feed sweetener on weight gain and feed consumption of broiler chickens[J]. Animal Sci,1996,76 (2):267-269.
82. Yamamoto, N.S., Bracht, K., Ishii, A.M., etal. Effect of steviol and its structural analogues on glucose production and oxygen uptake in rat renal tubules[J]. Experientia,1985,41:55-57.
83. Yasukawa, K., Kitanaka, S., Seo, S.. Inhibitory effect of stevioside on tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse skin[J]. Biol. Pharm. Bull,2002,25:1488-1490.
84. Xili, L., Chengjiany, B., Eryi, X., etal. Chronic oral toxicity and carcinogenicity study of stevioside in rats[J]. Food Chem. Toxicol,1992,30,957-965.
85. Das, S., Das, A.K., Murphy, R.A., etal. Evaluation of the Cariogenic Potential of the Intense Natural Sweeteners Stevioside and Rebaudioside A. Caries Res,1992 26,363-366.
86.班敬民,许德义,雏蓬轶.甜菊苷对大鼠及犬的降血压作用[J].医药导报,2000,19(3)：205-206.
87. Chan, P., Xu, D.-Y, Liu, J.C.,etal. The effect of stevioside on blood pressure and plasma catecholamines in spontaneously hypertensive rats[J]. Life Sciences,1998 63,1679-1684.
88. Chan, P., Tomlinson, B., Chen, Y.J.,etal. A double-blind placebo-controlled study pf the effectiveness and tolerability of oral stevioside in human hypertension[J]. Br. J. Clin. Pharmacol,2000,50:215-220.
89.赵永焕.甜叶菊苷在面包中的应用研究[J].中国畜产与食品,1999,6(1)：9-10
90. Chang S.S., Cook J.M.. Stability studies of stevioside and rebaudioside A in carbonated beverages[J]. Agric Food Chem,1983,31(2):409-12.
91. Ishikawa H,Kitahata S,OthaniK,etal.Production of stevioside and rubusoside derivatives by transfructosylation of b-fructofuranosidase[J]. Agric Biol hem,1990,54(12):3137-3143.
92.常丽娟,王竹天.甜菊糖苷化学分析法研究概况[J].国外医学卫生学分册,2007,34(3)：197-200.
93.陈慕英,于喜水,孙玉华.甜菊苷类的研究[J].中医药信息,2001,18(3)：23-24.
94.施荣富,史作清,冯君谦.食品饮料中甜菊苷的检测方法[J].中国食品添加剂,1994,3：19-23.
95.项秀珠,郭秀珠,黄品湖.甜菊苷含量的测定方法研究[J].浙江农业科学,1996,2：97-99.
96.黄海水,林静.甜菊糖苷总苷量的快速测定[J].厦门大学学报(自然科学 版),1990,29(3)：354-355.
97.邵寒娟,胡涌刚,丁亮.毛细管电泳有效分离测定甜菊糖苷新方法的研究[J].分析科学学报,2001,17(2)：127-130.
98.王德骥.关于甜菊糖苷的甜度、甜味和苦涩后味的成因机理[J].中国食品添加剂,2007,3：41-44
99.C.J.克利斯威尔主编.有机化合物的光谱分析[M].周黛玲,李光英译.上海：上海科学技术出版社,1985,152-155.
100.李占朝,陈光,喻长远.十二碳二元酸的重结晶精制工艺研究[J].合成纤维工业,2009,32(2)：31-33
101.张桂玲,温四民.甜味植物研究进展[J].安徽农业科学,2006,34(18)：4712～4713.
102. Roberts, A, Renwick, A.G. Comparative toxicokinetics and metabolism of rebaudioside A, stevioside, and steviol in rats[J]. Food and Chemical Toxicology,2008.05.006
103. J.H. Costa-Silva,C.R. Lima,E.J.R.Silva,etal. Acute and subacute toxicity of the Carapa guianensis Aublet (Meliaceae) seed oil[J]. Journal of Ethnopharmacology, 2008,116,495-500
104.李建科主编.食品毒理学[M].北京：中国计量出版社,2007,234-238
105.袁晓晴,顾小红,汤坚,蒋再良.水溶性癞葡萄降血糖多肽MC22125的分离纯化[J].Journal of Anhui Agri. Sci,2007,35(31):9819-9821.
106. Stig E.U. Dyrskog,Per B. Jeppesen,Jianguo Chen,etal. The Diterpene Glycoside, Rebaudioside A, Does not Improve Glycemic Control or Affect Blood Pressure After Eight Weeks Treatment in the Goto-Kakizaki Rat[J]. The Review of Diabetic Studies,2005,2(2):84-91
107.汪文陆.AK糖与其他甜味剂混合使用时甜度和风味的评价[J].食品科学,1994.10：9-12.
108.钱镭.老化均质条件对冰淇淋膨胀率和抗融性的影响[J].食品科技,2008,8：88-91.
109.刘爱国,杨明主编.冰淇淋配方设计与加工技术[M].北京,化学工业出版社,2008.73-79
110.袁钻如.差示扫描量热法(DSC)定量测试阿德福韦酯晶型的研究[J].分析测试技术与仪器,2008,6：105-108.
111.郭祀远.蔗糖的使用特性与糖业的发展[J].中国甜菜糖业,1998,6：27-30.
112.赵安庆,张晓宇.淀粉粘度测定方法综述[J].甘肃联合大学学报.2005,4：87-88.
113.汪海波.β-葡萄糖苷酶水解分离纯化大豆异黄酮苷元研究[J].食品科技,2007；114-118.
114.张水华.食品分析[M].北京：中国轻工业出版社.2008：124-125.
115.黄建蓉,崔炳群,梁莹.甜味剂的感官评价[J].中国食品添加剂.2007,1：114-117.
116.凌关庭.天然食品添加剂手册[M].北京：化学工业出版社.2000.172-175.
117.梁智.糖醇与低聚糖[J].中国食品添加剂.2004,1：96-99.
118.郑建仙主编.功能性食品[M].北京：中国轻工业出版社.1995.325-333.
119.刘学梅,刘传富,王冰等.麦芽糖醇在面包中的应用研究[J].食品科技,2009,34(5)：174-177.
120.杨海军.功能性添加剂在冰淇淋生产中的应用[J].中国乳品,2007,7：44-46.
2.保国裕.蔗糖、淀粉糖、化学合成高倍甜味剂三分糖市的趋势与对策[J].广西蔗糖,2006,4：42-43
3.中国甜菊信息网http://icgr.caas.net.cn/csa。
4.孙玉华,于喜水.甜菊苷类的研究与开发[J].黑龙江医药,2003,16(1)：41-42.
5.胡献丽,董文宾,郑丹,杨兆艳.甜菊及甜菊糖研究进展[J].食品研究与开发,2005,26(1)：36-38.
6 Hanson J. R.,de Oliveira B. H. Stevioside and related sweet diterpenoid glycosides[J]. Natural Product Reports,1993,10 (3):301-309.
7. Brandle, J. E., Rosa, N. Heritability for yield, leaf stem ratio and stevioside from landrace cultivar of Stevia rebaudiana[J]. Canadian Journal of Plant Science,1992,72,1263-1266.
8. L. O. Nabors. Alternative Sweeteners[M].1991. New York:Marcel Dekker Inc.
9. Grenby, T.H.. Developments in Sweeteners[M].1989,3. Elsevier, London.
10. Soejarto D. D., Kinghorn A. D., Farnsworth N. R.. Potential sweetening agents of plant rigin Organoleptic evaluation of Stevia leaf herbarium samples for sweetness [J].Nat Prod, 1982,45:590-599.
11. Toskulkao,C, Chaturat,L, Temcharoen P. Acute toxicity of stevioside, a natural sweetener, and its metabolite, steviol, in several animal specie [J].Drug Chem Toxicol,1997,20:31-44.
12. Kamath S.K., Kinghorn A. D. Safety assessment of some stevia rebaudiana sweet principles [J].Fed Proc,1982,41:1568.
13. Krejci M. E., Koechel D. A. Acute effects of carboxyatrac tyloside and stevioside,inhibitors of mitochondrial ADP/ATP translocation, on renal function and ultrastructure in pentobar tal-anesthetized dogs [10].Toxicology,1992,72:299-313.
14. Aze Y, Toyoda K, Imaida K,. Subchronic oral toxicity study of stevioside in F344 rats [J].Bull Natl Inst Hyg Sci,1991:48-54.
15. Xili L., Chengjiany B., Eryi X. Chronic oral toxicity and carcinogenicity study of stevioside in rats[J]. Food Chem Toxicol,1992,30:957-965.
16. Toyoda K, Matsui H, Shoda T, etal.Asessment of the carcinogenicity of stevioside in F344 rats[J].Food Chem Toxicol,1997,35:597-603.
17. Hagiwara A, Fukushima S, Kitaori M. Effects of threesweeteners on rat urinary bladder carcinogenesis initiated by N-butyl-N-(4-hydroxybutyl) nitrosamine [J].Gann,1984, 75:763-768.
18. Suttajit M, Vinitketkaumnuen U, Meevatee U, et al. Mutagenicity and human chromosomal effect of stevioside, a sweetener fromStevia rebaudiana Bertoni [J].Environ Health Perspectives,1993,101:53-56.
19. Matsui M, Matsui K, Kawasaki Y. Evaluation of the genotoxicity of stevioside and steviol using six in vitro and one in vivo mutagenicity assays[J].Mutagenesis,1996,11:573-579.
20. Ishidate M, Sofuni T, Yoshikawa K. Primary muta genicity screening of food additives currently used in Japan [J]. Food Chem Toxicol,1984,22:623-636.
21.Yodyingyuard V.,Bunyawong S. Effect of stevioside on growth and reproduction[J].Hum Reprod,1991,6:158-165.
22. Mazzei-Planas G & Kuc J Contraceptive properties of Stevia rebaudiana[J].Science,1968, 162:1007.
23. Oliverira-Filho R M, Uehara O A, Minetti C A, etal. Chronic administration of aqueous extract of Stevia rebaudiana Bertoniin rats:Endocrine effects [J].GenPharmacol,1989,20:187-191.
24. Melis M S-Effects of chronic administration of Stevia rebaudianaon fertility in rats[J]. Ethnopharmacology,1999,67(2):157-161.
25. Usami M, Sakemi K, Kawashima K, etal. Yeratogenicity study of stevioside in rats[J].BullNatl InstHygSci,1995,113:31-35.
26. Federal Food, Drug and Cosmetic Act of 1994(revised April 2000)[Z]. Section 402, Section 409 and 21 CFR 170 and 21 CFR 189-1.
27.李晓瑜.甜菊糖苷的安全性研究进展[J].中国食品添加剂,2003,2(5)：11-14.
28. Henkel, J. Sugar substitutes:American opt for sweetness and lite. FDA Consumer[M]. 1999,33(6). Available:http://www.fda.gov/fdac/699_toc. html.
29. WHO. Toxicological evaluation of certain food additives and contaminants[M]. WHO food additives series 42.Geneva:World Health Organization.1999:138.
30. Scientific Committee on Food. Opinion on Stevioside as a Sweetener. CS/ADD/EDUL/167 final 17 June 1999.
31. Scientific Committee on Food. Opinion on Stevia Rebaudiana Bertoni plants and leaves. CS/NF/STEV/3 final 17 June 1999.
32.张亚雄,胡为民,胡滨.甜菊糖中A3苷的分离方法初探[J].中国食品添加剂,1998,4：49-51.
33. Truong, T. T., Valicek, P., Nepovim, A., Vanek, T. Correlation between stevioside content-in leaves, their area and the number of roots in plant[J]. Sci. Agr. Biohemica,1999, 30:249-255
34. Brandle, J. E.. Genetic control of rebaudioside A and C concentration in leaves of the sweet herb, Stevia rebaudiana[J]. Canadian Journal of Plant Science,1999,79,85-92.
35. Nikolai Bondarev, Oxana Reshetnyak, Alexander Nosov. Effects of nutrient medium composition on development of Stevia rebaudiana shoots cultivated in the roller bioreactor and their production of steviol glycosides[J].Plant Science,2003,165:845-850
36. Nikolai Bondarev, Oxana Reshetnyak, Alexander Nosov. Peculiarities of diterpenoid steviol glycoside production in in vitro cultures of Stevia rebaudiana Bertoni[J]. Plant Science,2001,161:155-163.
37. Nikolai Bondarev, Oxana Reshetnyak, Alexander Nosov.. Features of development of Stevia rebaudiana shoots cultivated in the roller bioreactor and their production of steviol glycoside[J]. Planta Med,2002,68:757-759.
38.黄应森.中国甜菊和甜菊糖苷的分型研究[J].中国糖料.1999,4：26-29
39. Lyakhovkin A.G., Long T.D., Titov D.A., Anh M.P.. Cultivation and Utilization of Stevia (Stevia rebaudiana Bertoni) [M]. Agricultural Publishing House, Hanoi,1993.
40. Zhang, S. Q., Kumar, A.,& Kutowy, O. Membrane-based separation scheme for processing sweeteners from stevia leaves[J]. Food Research International,2000, 33:617-620.
41. Takahashi, M., Matsuda, M., Ohahi, K., etal. Analysis of anti-rotavirus activity of extract from Stevia rebaudiana[J]. Antiviral Research,2001,49:15-24.
42. Simone K.Yoda,Marcia O.M.Marques,etal. Supercritical fluid extraction from Stevia rebaudiana Bertoni using CO2 and CO2 twater:extraction kinetics and identification of extracted components [J]. Journal of Food Engineering,2003,57:125-134
43. Liu, J. and Li, S. F. Y. Separation and determination of stevia sweeteners by capillary lectrophoresis and high performance liquid chromatography[J]. Liq. Chromarogr, 1995,18:1703-1719.
44. Liu, J., Ong, C. P., Li, S. F. Y. Subcritical fluid extraction of stevia sweeteners from Stevia rebaudiana[J]. Journal of Chromatographic Science,1997,35:446-450.
45. Payzant JD, Laidler JK, Ippolito RM, Method of extracting selected sweet glycosides from the Stevia rebaudiana plant[P]. US Patent 5,962,678,1998.
46. Brandle JE, Starratt AN, Kirby CW, Pocs R. Rebaudioside-F, a diterpenen glycoside from Stevia rebaudiana[J]. Phytochemistry,2002,59:367-370.
47.孙玉华,于喜水.甜菊式类的研究与开发[J].黑龙江医药.2003,16(1)：41-42.
48.赵永金,孙信庆.从甜叶菊中提取甜菊苷的工艺研究[J].西北农业大学学报,1999,27：80-83
49.刘宗林,彭义交,郭洋等.甜叶菊苷的提取与结晶工艺研究[J].食品科学,2002,23(8)：99-100
50. YamasakiK,KohdaH,KobayashiT,KasaiR,TanakaO. Structuresof 13 Stevia diterpene-glucosides:application of C NMR[J]. Tetrahedron Lett,1976;13:1005-1008.
51. Sakamoto I.,Yamasaki K.,Tanaka O. Applicationof CNMRspectro-scopy to chemistry of plant glycosides:rebaudiosides-D and-E, new sweet diterpene-glucosides of Stevia rebaudiana Bertoni[J]. Chem Pharm Bull,1977,25(12):3437-3439.
52. Compadre, C.M., Hussain, R.A., etal. Mass spectral analysis of some derivatives and in vitro metabolites of steviol, the aglycone of the natural sweeteners, stevioside, rebaudioside A and rubusoside[J]. Biomed. Environ. Mass Spectrom,1988,15:211-222.
53. Brandle, J.E., Starratt, A.N., Gijzen, M.. Stevia rebaudiana:its agricultural, biological, and chemical properties [J]. Canadian Journal of Plant Science,1998,78:527-536.
54. Sakamoto, I., Yamasaki, K., Tanaka, O.. Application of 13C NMR spectroscopy to chemistry of natural glycosides:Rebaudioside-C, a new sweet diterpene glycoside of Stevia rebaudiana[J]. Chemical and Pharmaceutical Bulletin,1977,25:844-846.
55. Jan M.C. Geuns, Molecules of Interest Stevioside[J]. Phytochemistry,2003,64:913-921
56.陈天红,张杨,刘晓航,史作清等.高分子吸附剂对甜叶菊中各糖苷吸附选择性的研究[J].中国科学(B),1998,28(5)：460-465.
57.施荣富,史作清,王春红等.吸附树脂的色层吸附及其在甜菊苷分离纯化中的作用[J].离子交换与吸附,2001,17(1)：23-30.
58.张杨,陈天红等.重结晶法分离精制莱鲍迪A苷的研究[J].离子交换与吸 附,1998,16(4)：515-520.
59. Tomas Vanek, etal. Determination of stevioside in plant material and fruit teas[J]. Food composition and analysis.2001,14:383-388
60. Hutapea, A.M., Toskulkao, C., Wilairat, P., etal. High-performance liquid chromatographic separation and quantitation of stevioside and its metabolites[J]. Liq. Chrom. Rel. Technol,1999,22:1161-1170.
61. Mauri P, Catalano G, Gardana C, etal. Analysis of Stevia glycosides by capillary electrophoresis[J]. Electrophoresis,1996;17:367-71.
62. Ahmed, M.S., Dobberstein, R.H. Use of p-bromophenacyl bromide to enhance ultraviolet detection of water-soluble organic acids (steviolbioside and rebaudioside B) in high-performance liquid hromatographic analysis[J]. Chromatogr,1980,192:387-393.
63. Fujinuma, K., Saito, K.,Nakazato, etal. Thin layer chromatographic detection and liquid chromatographic determination of stevioside and rebaudioside A in beverages and foods following reversed phase column chromatography[J]. Assoc. Off. Anal. Chem,1986,69:799-802.
64. N. Kolb, J. L. Herrera, D. J. Ferreyra, and R. F. Uliana. Analysis of Sweet Diterpene Glycosides from Stevia rebaudiana:Improved HPLC Method[J]. Agric. Food Chem,2001, 49,4538-4541.
65. Nikolova-Damyanova, B., Bankova, V., and Popov, S. Separation and quantitation of stevioside and rebaudioside A in plant extracts by normal-phase HPLC and TLC:A comparison[J]. Phytochemical analysis,1994,5:81-85.
66. Makapugay, H. C., Nanayakkara, N. P. D., Kinghorn, A. D. Improved high-performance liquid chromatographic separation of the Stevia rebaudiana sweet diterpene glycosides using linear gradient elution[J]. Chromatogr,1984,283:390-395.
67.刘超,李来生等.高效液相色谱法测定甜叶菊糖中的甜菊苷和莱鲍迪苷A[J].分析试验室,2007,26(7)：23-26
68. Jan M.C. Geuns, Patrick Augustijns, Raf Mols, Johan G. Buyse, Bert Driessen. Metabolism of stevioside in pigs and intestinal absorption characteristics of stevioside, rebaudioside A and steviol[J].Food and Chemical Toxicology,2003,41:1599-1607
69. Bearder, J.R., MacMillan, J., Wels, C.M.. The metabolism of steviol to 13-hydroxylated ent-gibberellanes and ent-keuranes[J]. Phytochemistry,1975,14:1741-1748.
70. Hagiwara, A., Fukushima, S., Kitaori, M.. Effects of the three sweeteners on rats urinary bladder carcinogenesis initiated by Nbutyl-N-(4-hydroxybutyl)-nitrosamine[J]. Gann, 1984,75:763-768.
71. Geuns, J.M.C., Malheiros, R.D., etal. Metabolism of stevioside by chickens. Agric. Food Chem.2003b,51:1095-1101.
72. Hutapea, A.M., Toskulkao, C., etal. Digestion of Stevioside, a Natural Sweetener, by Various Digestive Enzymes. Clin. Biochem. Nutr.,1997,23,177-186.
73. Koyama, E., Kitazawa, K., Ohori, Y, Sakai, N., Izawa, O., Kakegawa, K., Fujino, A., Ui, M., Intestinal degradation, absorption, and hepatic metabolism of the glycosidic sweeteners, Stevia mixture[M]. In:Proceedings second IUPA-International Symposium on Sweeteners, Hiroshima,2001.12.
74. Koyama, E., Kitazawa, K, Ohori, Y, Izawa, O., Kakegawa, K., Fujino, A., Ui, M., In vitro metabolism of the glycosidic sweeteners, Stevia mixture and enzymatically modified Stevia in human intestinal microflora[J]. Food Chem. Toxicol,2003a,41:359-374.
75. Liu, J.-C, Kao, P.-K., Chan, P., etal. Mechanismof the antihypertensive effect of stevioside in anesthesized dogs[J]. Pharmacology,2003,67:14-20.
76. Temcharoen, P., Pimbua, J., Glinsukon, T., Rojanapo, W., Apibal, S. Mutagenic activity of steviol to Salmonella typhimurium TM 677:Comparison of the activity of S9 liver fractions from five laboratory animal species[J]. Bull. Health Sci.& Tech,1998,1:38-45.
77. Temcharoen, P., Suwannatrai, M., Klongpanichpak, S., etal. Mutagenicity of steviol and its oxidative derivatives in Salmonella typhimurium TM677[J]. Chem. Pharm. Bull, 2002,50:1007-1010.
78. Toskulkao, C., Chaturat, L., Temcharoen, P., etal. Acute toxicity of stevioside, a natural sweetener, and its metabolite, steviol, in several animal species [J]. Drug Chem. Toxicol, 1997.20,31-44.
79. Totte, N., Charon, L., Rohmer, M., Compernolle, F., etal. Biosynthesis of the diterpenoid steviol, an entkaurene derivative from Stevia rebaudiana Bertoni, via the methylerythritol phosphate pathway[J]. Tetrahedron Letters,2000,41:6407-6410.
80. Wingard, R.E., Brown, J.P., Enderlin, F.E., etal. Intestinal degradation and absorption of the glycosidic sweeteners stevioside and rebaudioside A[J]. Experientia,1980,36:519-520.
81. Wood, D.J., Lirette, A., Crober, D.C., Ju-Hy,. The effect of stevia as a feed sweetener on weight gain and feed consumption of broiler chickens[J]. Animal Sci,1996,76 (2):267-269.
82. Yamamoto, N.S., Bracht, K., Ishii, A.M., etal. Effect of steviol and its structural analogues on glucose production and oxygen uptake in rat renal tubules[J]. Experientia,1985,41:55-57.
83. Yasukawa, K., Kitanaka, S., Seo, S.. Inhibitory effect of stevioside on tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse skin[J]. Biol. Pharm. Bull,2002,25:1488-1490.
84. Xili, L., Chengjiany, B., Eryi, X., etal. Chronic oral toxicity and carcinogenicity study of stevioside in rats[J]. Food Chem. Toxicol,1992,30,957-965.
85. Das, S., Das, A.K., Murphy, R.A., etal. Evaluation of the Cariogenic Potential of the Intense Natural Sweeteners Stevioside and Rebaudioside A. Caries Res,1992 26,363-366.
86.班敬民,许德义,雏蓬轶.甜菊苷对大鼠及犬的降血压作用[J].医药导报,2000,19(3)：205-206.
87. Chan, P., Xu, D.-Y, Liu, J.C.,etal. The effect of stevioside on blood pressure and plasma catecholamines in spontaneously hypertensive rats[J]. Life Sciences,1998 63,1679-1684.
88. Chan, P., Tomlinson, B., Chen, Y.J.,etal. A double-blind placebo-controlled study pf the effectiveness and tolerability of oral stevioside in human hypertension[J]. Br. J. Clin. Pharmacol,2000,50:215-220.
89.赵永焕.甜叶菊苷在面包中的应用研究[J].中国畜产与食品,1999,6(1)：9-10
90. Chang S.S., Cook J.M.. Stability studies of stevioside and rebaudioside A in carbonated beverages[J]. Agric Food Chem,1983,31(2):409-12.
91. Ishikawa H,Kitahata S,OthaniK,etal.Production of stevioside and rubusoside derivatives by transfructosylation of b-fructofuranosidase[J]. Agric Biol hem,1990,54(12):3137-3143.
92.常丽娟,王竹天.甜菊糖苷化学分析法研究概况[J].国外医学卫生学分册,2007,34(3)：197-200.
93.陈慕英,于喜水,孙玉华.甜菊苷类的研究[J].中医药信息,2001,18(3)：23-24.
94.施荣富,史作清,冯君谦.食品饮料中甜菊苷的检测方法[J].中国食品添加剂,1994,3：19-23.
95.项秀珠,郭秀珠,黄品湖.甜菊苷含量的测定方法研究[J].浙江农业科学,1996,2：97-99.
96.黄海水,林静.甜菊糖苷总苷量的快速测定[J].厦门大学学报(自然科学 版),1990,29(3)：354-355.
97.邵寒娟,胡涌刚,丁亮.毛细管电泳有效分离测定甜菊糖苷新方法的研究[J].分析科学学报,2001,17(2)：127-130.
98.王德骥.关于甜菊糖苷的甜度、甜味和苦涩后味的成因机理[J].中国食品添加剂,2007,3：41-44
99.C.J.克利斯威尔主编.有机化合物的光谱分析[M].周黛玲,李光英译.上海：上海科学技术出版社,1985,152-155.
100.李占朝,陈光,喻长远.十二碳二元酸的重结晶精制工艺研究[J].合成纤维工业,2009,32(2)：31-33
101.张桂玲,温四民.甜味植物研究进展[J].安徽农业科学,2006,34(18)：4712～4713.
102. Roberts, A, Renwick, A.G. Comparative toxicokinetics and metabolism of rebaudioside A, stevioside, and steviol in rats[J]. Food and Chemical Toxicology,2008.05.006
103. J.H. Costa-Silva,C.R. Lima,E.J.R.Silva,etal. Acute and subacute toxicity of the Carapa guianensis Aublet (Meliaceae) seed oil[J]. Journal of Ethnopharmacology, 2008,116,495-500
104.李建科主编.食品毒理学[M].北京：中国计量出版社,2007,234-238
105.袁晓晴,顾小红,汤坚,蒋再良.水溶性癞葡萄降血糖多肽MC22125的分离纯化[J].Journal of Anhui Agri. Sci,2007,35(31):9819-9821.
106. Stig E.U. Dyrskog,Per B. Jeppesen,Jianguo Chen,etal. The Diterpene Glycoside, Rebaudioside A, Does not Improve Glycemic Control or Affect Blood Pressure After Eight Weeks Treatment in the Goto-Kakizaki Rat[J]. The Review of Diabetic Studies,2005,2(2):84-91
107.汪文陆.AK糖与其他甜味剂混合使用时甜度和风味的评价[J].食品科学,1994.10：9-12.
108.钱镭.老化均质条件对冰淇淋膨胀率和抗融性的影响[J].食品科技,2008,8：88-91.
109.刘爱国,杨明主编.冰淇淋配方设计与加工技术[M].北京,化学工业出版社,2008.73-79
110.袁钻如.差示扫描量热法(DSC)定量测试阿德福韦酯晶型的研究[J].分析测试技术与仪器,2008,6：105-108.
111.郭祀远.蔗糖的使用特性与糖业的发展[J].中国甜菜糖业,1998,6：27-30.
112.赵安庆,张晓宇.淀粉粘度测定方法综述[J].甘肃联合大学学报.2005,4：87-88.
113.汪海波.β-葡萄糖苷酶水解分离纯化大豆异黄酮苷元研究[J].食品科技,2007；114-118.
114.张水华.食品分析[M].北京：中国轻工业出版社.2008：124-125.
115.黄建蓉,崔炳群,梁莹.甜味剂的感官评价[J].中国食品添加剂.2007,1：114-117.
116.凌关庭.天然食品添加剂手册[M].北京：化学工业出版社.2000.172-175.
117.梁智.糖醇与低聚糖[J].中国食品添加剂.2004,1：96-99.
118.郑建仙主编.功能性食品[M].北京：中国轻工业出版社.1995.325-333.
119.刘学梅,刘传富,王冰等.麦芽糖醇在面包中的应用研究[J].食品科技,2009,34(5)：174-177.
120.杨海军.功能性添加剂在冰淇淋生产中的应用[J].中国乳品,2007,7：44-46.