| 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.
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