石榴皮多酚提取纯化及纳米乳制备工艺研究
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摘要
本文以新疆石榴皮为研究对象,对石榴皮多酚提取纯化、纳米乳的制备工艺及纳米乳的稳定性进行了研究。分别采用溶剂提取法、超声波辅助提取法和酶提取法研究了提取溶剂、料液比、提取时间、提取温度、石榴皮颗粒度、提取次数、超声波功率、酶解液浓度、酶解液pH值等因素对石榴皮多酚得率的影响。通过石榴皮多酚粗提液的静态吸附和解吸试验,从9种大孔吸附树脂中筛选出石榴皮多酚纯化的最优树脂,考察了该树脂对石榴皮多酚的静态、动态吸附与解吸性能及主要影响因素(上样液的pH值、浓度、体积、流速,洗脱剂的选择、用量、流速等)。采用滴定法结合伪三元相图优选了表面活性剂、助表面活性剂、油相及Km值,在此基础上制备石榴皮多酚纳米乳,并对其形态特征、稳定性(高温试验、高压试验、强光照试验及高速离心试验)进行研究。结果表明：三种提取石榴皮多酚的最优工艺条件分别为,溶剂浸提法：乙醇浓度50%,料液比1：20(w/V),提取时间90min,提取温度40℃,选择过60目的石榴皮粉提取两次,多酚得率为(20.50+0.06)%(n=5)。超声波辅助提取法：乙醇浓度50%、料液比1∶25(w/V)、超声时间30min、超声波功率360W,石榴皮多酚得率为(21.22±0.06)%(n=5)。酶法提取：浓度为0.25mg/mL,质量比为2∶1的纤维素酶和果胶酶的复合酶,酶解时间1 50min、酶解温度50℃、酶解液pH值为6.0,石榴皮多酚得率为(23.87+0.08)%(n=5)。其中酶法提取多酚得率最高,其次是超声波辅助提取法,而溶剂浸提法得率最低。酶法所耗时间最长,超声波辅助法所需时间最短。综合考虑,选择超声波辅助法提取石榴皮多酚进行后续试验。SP-825树脂对石榴皮多酚有很好的吸附和解吸性能,其最优的动态吸附工艺条件为：上样液pH值为4.0,浓度10.25mg/mL,上样量为4BV,流速控制在2BV/h。最优的解吸工艺条件为：洗脱剂为80%乙醇溶液,洗脱液用量为3BV,洗脱流速控制在1BV/h。在此优化条件下,石榴皮多酚的吸附率、解析率、收率、纯度的平均值分别为(85.31+0.06)%,(81.14±0.11)%,(68.20+0.10)%和(32.12±0.12)%(n=3)。石榴皮多酚纳米乳的最优配方为：石榴皮多酚4.4%(w/w)、EL-4034.1%(w/w)、无水乙醇17.1%(w/w)、IPM为5.7%(w/w)、蒸馏水38.7%(w/w)。石榴皮多酚纳米乳澄清透明,在透射电镜下旱球体,粒径为(35.50±0.64)mm,且均匀稳定。石榴皮多酚纳米乳分别在90℃水浴放置10d,在121℃、1.0Mpa下20mmin,在3000～40001x的光照下10d,石榴皮多酚的损失率分别仅为0.77%、0.32%和1.33%。在相同条件下,对照液的多酚损火率分别达到19.45%、7.37%和28.52%。高速离心试验中石榴皮多酚纳米乳仍保持澄清透明,证明石榴皮多酚纳米乳有很好的稳定性。
Xinjiang pomegranate peel as the research object, the extraction, purification, nanoemulsion preparation technology and stability of polyphenols were studied. The extraction experiment of pomegranate peel polyphenols was conducted by solvent extraction, ultrasonic-assisted extraction and enzymatic treatment respectively. The optimal parameters, including extraction solvent, solid to liquid ratio, extraction time, extraction temperature, pomegranate peel particle size, extraction times, ultrasonic power, enzyme solution concentration, and enzyme solution pH, were selected. Static adsorption and desorption experiments were carried out and the suitable resin was screened from 9 macroporous absorbent resins for pomegranate peel polyphenols.The purification technology of the crude extracts by macroporous adsorbing resins were decided through the experiments, which included static, dynamic adsorption and desorption efficiency, the sample solution pH, concentration, volume, and the eluent type, dosage, flow rate, and so on. A nanoemulsion of pomegranate peel polyphenols was prepared on the base of optimization on surfactant, cosurfactant, oil and Km value by using titration with pseudo-ternary phase diagram. The morpohologic characteristics and stability of nanoemulsion with high temperature, high pressure, highlight exposure and high speed centrifugation were studied. The results showed that the three optimum extraction conditions for polyphenols from pomegranate peel were:the solvent extraction method for 50% ethanol concentration, solid to liquid ratio of 1:20 (w/V), extraction time 90min, extraction temperature 40℃, the 60 screen mesh pomegranate peel powder, extraction twice, polyphenols yield was (20.50±0.06)%(n=5); the ultrasonic extraction method:50% ethanol concentration, solid to liquid ratio 1:25(w/V), ultrasonic time 30 min, ultrasonic power 360W, polyphenols yield was (21.22±0.06)%(n=5); the enzymatic extraction:the concentration 0.25mg/mL of 2:1 cellulase and pectinase, treatment time 150min, treatment temperature 50℃, enzymolysis pH 6.0, polyphenols yield was (23.87±0.08)%(n=5). The extraction effect for enzymatic extraction was the highest, followed by ultrasonic extraction, and it was lowest for solvent extraction. However, the time consumed by the enzymatic method is the longest, and the ultrasonic method requires the least time. The ultrasonic assisted extraction of pomegranate peel polyphenols was best method. Macroporous resin SP-825 possessed the strongest absorbent ability and easier desorption property, and its optimal dynamic adsorption conditions were:the sample solution pH4.0, concentration 10.25mg/mL, sample volume 4BV, flow rate 2BV/h; the optimal desorption conditions were:eluent of 80% ethanol solution, the amount of eluent 3BV, elution flow rate 1BV/h. In optimum conditions, the pomegranate peel polyphenols average adsorption rate, desorption rate, yield and purity reached to (85.31±0.06)%, (81.14±0.11)%, (68.20±0.10)% and (32.12±0.12)%(n=3) respectively. The optimal formula of pomegranate peel polyphenols nanoemulsion is:pomegranate peel polyphenols 4.4%(w/w), EL-40 34.1%(w/w), ethanol 17.1%(w/w), IPM 5.7%(w/w), distilled water 38.7%(w/w). Pomegranate peel polyphenols nanoemulsion was clear and transparent, showed spherical (35.50±0.64) nm in diameter, uniform and stable under transmission electron microscopy (TEM). Pomegranate peel polyphenols nanoemulsion was dealt with 90℃for 10d,121℃, 1.0Mpa for 20min and 3000-40001x for 10d, the loss of pomegranate peel polyphenols were only 0.77%,0.32% and 1.33% respectively, while the loss of control were 19.45%,7.37% and 28.52%.The nanoemulsion was also limpid by high speed centrifugation. The pomegranate peel polyphenols nanoemulsion has good stability.
Xinjiang pomegranate peel as the research object, the extraction, purification, nanoemulsion preparation technology and stability of polyphenols were studied. The extraction experiment of pomegranate peel polyphenols was conducted by solvent extraction, ultrasonic-assisted extraction and enzymatic treatment respectively. The optimal parameters, including extraction solvent, solid to liquid ratio, extraction time, extraction temperature, pomegranate peel particle size, extraction times, ultrasonic power, enzyme solution concentration, and enzyme solution pH, were selected. Static adsorption and desorption experiments were carried out and the suitable resin was screened from 9 macroporous absorbent resins for pomegranate peel polyphenols.The purification technology of the crude extracts by macroporous adsorbing resins were decided through the experiments, which included static, dynamic adsorption and desorption efficiency, the sample solution pH, concentration, volume, and the eluent type, dosage, flow rate, and so on. A nanoemulsion of pomegranate peel polyphenols was prepared on the base of optimization on surfactant, cosurfactant, oil and Km value by using titration with pseudo-ternary phase diagram. The morpohologic characteristics and stability of nanoemulsion with high temperature, high pressure, highlight exposure and high speed centrifugation were studied. The results showed that the three optimum extraction conditions for polyphenols from pomegranate peel were:the solvent extraction method for 50% ethanol concentration, solid to liquid ratio of 1:20 (w/V), extraction time 90min, extraction temperature 40℃, the 60 screen mesh pomegranate peel powder, extraction twice, polyphenols yield was (20.50±0.06)%(n=5); the ultrasonic extraction method:50% ethanol concentration, solid to liquid ratio 1:25(w/V), ultrasonic time 30 min, ultrasonic power 360W, polyphenols yield was (21.22±0.06)%(n=5); the enzymatic extraction:the concentration 0.25mg/mL of 2:1 cellulase and pectinase, treatment time 150min, treatment temperature 50℃, enzymolysis pH 6.0, polyphenols yield was (23.87±0.08)%(n=5). The extraction effect for enzymatic extraction was the highest, followed by ultrasonic extraction, and it was lowest for solvent extraction. However, the time consumed by the enzymatic method is the longest, and the ultrasonic method requires the least time. The ultrasonic assisted extraction of pomegranate peel polyphenols was best method. Macroporous resin SP-825 possessed the strongest absorbent ability and easier desorption property, and its optimal dynamic adsorption conditions were:the sample solution pH4.0, concentration 10.25mg/mL, sample volume 4BV, flow rate 2BV/h; the optimal desorption conditions were:eluent of 80% ethanol solution, the amount of eluent 3BV, elution flow rate 1BV/h. In optimum conditions, the pomegranate peel polyphenols average adsorption rate, desorption rate, yield and purity reached to (85.31±0.06)%, (81.14±0.11)%, (68.20±0.10)% and (32.12±0.12)%(n=3) respectively. The optimal formula of pomegranate peel polyphenols nanoemulsion is:pomegranate peel polyphenols 4.4%(w/w), EL-40 34.1%(w/w), ethanol 17.1%(w/w), IPM 5.7%(w/w), distilled water 38.7%(w/w). Pomegranate peel polyphenols nanoemulsion was clear and transparent, showed spherical (35.50±0.64) nm in diameter, uniform and stable under transmission electron microscopy (TEM). Pomegranate peel polyphenols nanoemulsion was dealt with 90℃for 10d,121℃, 1.0Mpa for 20min and 3000-40001x for 10d, the loss of pomegranate peel polyphenols were only 0.77%,0.32% and 1.33% respectively, while the loss of control were 19.45%,7.37% and 28.52%.The nanoemulsion was also limpid by high speed centrifugation. The pomegranate peel polyphenols nanoemulsion has good stability.
引文
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