蜂花粉中有效成分的提取及功能性研究
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摘要
花粉是植物的雄性生殖细胞,含有丰富的各种营养成分,在国际上有“天然完全营养素”的美誉。玉米花粉多糖具有抗肿瘤、抗氧化、增强免疫力等功能,但坚硬的花粉壁阻碍了多糖成分的溶出,降低了提取效率。动态超高压微射流技术是一项新兴的高压均质技术,其剧烈的处理条件可导致植物细胞破碎,且对活性成分几乎无破坏。
本研究侧重于应用动态超高压微射流技术对玉米花粉进行破壁,并与干法粉碎破壁、复合酶酶解破壁比较破壁效果和多糖得率,在此基础上对玉米花粉多糖抗氧化活性功能进行了评价,为今后玉米花粉多糖更深入的研究与开发奠定基础。同时对花粉中有助于治疗前列腺疾病的功能因子--环氧肟酸进行了定性和定量分析。本论文的主要研究内容与结论如下：
1、以葡萄糖为对照,优选蒽酮-硫酸法测定玉米花粉多糖的含量。结果表明：玉米花粉多糖的吸收光谱与葡萄糖基本一致,标准曲线的线性关系良好(R2=0.9996),此法在回收率、精密度、稳定性方面均较好,可作为测定玉米花粉多糖含量较为理想的方法。
2、采用正交试验优化干法粉碎花粉并提取多糖的条件,结果表明提取溶液pH值对多糖得率有极显著的影响,最佳条件为：取粉碎并过120目筛分的花粉,调节花粉乳液pH值7.0,料液比1：15,提取温度70℃,提取时间3 h,提取两次,此时多糖得率为7.239%。
3、采用果胶酶和纤维素酶组成的复合酶对玉米花粉进行酶解破壁处理,通过正交试验优化酶解条件。结果表明果胶酶添加量是影响花粉多糖得率的最主要因素。最佳条件为：果胶酶添加量300U／g花粉,纤维素酶添加量150U／g花粉,20%花粉乳浓度,pH4.5,50℃酶解破壁5h,在此条件下多糖得率为7.194%。
4、采用动态超高压微射流技术对玉米花粉进行湿法破壁处理,Box-Benhnken响应面设计优化条件参数。结果表明均质压力和提取温度对多糖得率的线性效应和曲面效应均显著。最佳提取条件为：微射流均质压力127MPa,均质处理一次,料液比1：20,72.4℃下提取1.7 h,提取2次,此时玉米花粉多糖得率达到理论最大值8.086%,与验证试验结果一致。与干法粉碎和酶解破壁提取结果相比,提取时间缩短了1.3 h,多糖得率提高了0.847%。
5、红外光谱扫描的结果表明三种破壁方式提取的多糖的红外光谱图基本吻合,主要官能团没有差异,说明使用高压微射流技术不会破坏玉米花粉多糖的结构。高倍扫描电镜观察破壁效果的结果表明：干法粉碎和酶解破壁处理的花粉仍有少量未完全破壁的花粉颗粒,粒径不均匀,可见原材料的粉末特征；而经微射流均质处理的花粉均完全裂解为大量碎片,粒度明显变小,内容物全部溢出。因此,超高压微射流技术处理花粉的破壁效果最好。
6、初步探讨超高压微射流技术使花粉破壁的机理是：高压均质过程中剧烈的处理条件使花粉细胞的组织结构完全破碎,原来颗粒比较粗大的花粉乳液被加工成颗粒非常细微的乳浊液,使花粉细微颗粒充分与提取溶剂相接触,从而提高提取效率。
7、通过紫外扫描和HPLC-MS对舍尼通药片提取物进行定性分析,最终确定其为环氧肟酸,纯度约为94%。采用Sephadex G-25和G-10二者联用对玉米花粉DIBOA提取液进行纯化,经HPLC定量分析并计算得到玉米花粉中环氧肟酸的含量约为0.029%(290μg/g)。
8、玉米花粉粗多糖和精制多糖组分PPMC对DPPH、O2和.OH自由基均有一定的清除作用,并且随着多糖浓度的增大清除作用增强。二者对DPPH的清除作用最大,对·OH和O2·-自由基的清除作用都比较弱。在同一浓度下,精制多糖组分PPMC对这三种自由基的清除率都略高于粗多糖。
Pollen is male reproductive cells of plants. It is rich in various nutrients and hailed as natural complete nutrients internationally. Maize pollen polysaccharides are capable of antitumor, antioxidant, enhancing the immune function. But stiff pollen wall hindered dissolution of polysaccharides and lowered extraction efficiency. Dynamic high pressure micro-fluidization (DHPM) is a rising high pressure homogenization technique. Plant cells could be disrupted by violent treatment conditions, and the active ingredient was little damaged.
Dynamic high pressure micro-fluidization technology was applied on maize pollen wall-breaking in this study. Maize pollen wall-breaking effect and yield of polysaccharides were compared with dry grinding and enzymatic hydrolysis wall-breaking. Then anti-oxidative function of maize pollen polysaccharides was evaluated in order to prepare for future research and development of maize pollen polysaccharides from now on. At the same time, the pollen function factor cyclic hydroxamic acid which contributed to treatment of prostate diseases was done qualitative and quantitative analysis. The main contents and conclusions were as follows:
1. The content of maize pollen polysaccharides was determined by selected anthrone-sulfuric acid method with glucose as standard. The results were as follows: The absorption spectrum of maize pollen polysaccharides was consistent with glucose, and the linear relationship of standard curve was good (R2=0.9996). The recovery, precision and stability of this method were good, so anthrone-sulfuric acid method was ideal method which could be used to determine pollen polysaccharides content.
2. Polysaccharides were extracted from broken maize pollen by dry comminution, and optimum extract conditions were obtained by orthogonal experiment. Results showed that pH value of extract had most significant impact on yield of polysaccharides. The optimum conditions were as follows:pollen by dry comminution and got over 120 mesh, extract of pH 7.0, ratio of solid-liquid 1:15, 70℃of extraction temperature for 3h, and extraction two times. The yield of polysaccharides was 7.239% under these conditions.
3. Maize pollen wall was broken by enzymolysis with composition of pectinase and cellulase enzyme, and optimum enzymolysis conditions were obtained by orthogonal experiment. Results showed that pectinase addition was principal factor that affected yield of polysaccharides. The optimum conditions were as follows: pectinase addition of 300U/g pollen, cellulase addition of 150U/g pollen,20% of pollen concentration, pH4.5,50℃of enzymolysis temperature for 5h. The yield of polysaccharides was 7.194% under these conditions.
4. Maize pollen wall was disrupted with wet method using DHPM technology, and the parameters were optimized by Box-Benhnken response surface method. The results showed that the linear effect and surface effect of homogenization pressure and extraction temperature on polysaccharides yield were significant. The optimum conditions were as follows:homogenization pressure 127.35 MPa, homogenizing one time, ratio of solid-liquid 1:20,72.41℃of extraction temperature for 1.74 h and extraction two times. Under these conditions the yield of maize pollen polysaccharides was theoretical maximum of 8.086% which was consistent with validation experiment results. Compared with dry comminution and enzymolysis wall-breaking extraction, extraction time was shortened 1.26 h and the yield of polysaccharides increased 0.847%.
5. IR spectra scan results showed that infrared spectrum of maize pollen polysaccharides extracted by three wall-breaking methods was basically identical, and major functional groups had no differences. This indicated that the structure of pollen polysaccharides was not destroyed by DHPM. Pollen wall-breaking effect of three wall-breaking methods was observed by SEM. The results showed that:a few of incompletely broken pollen grains were still observed after dry comminution and enzymolysis treatment, and particles size was not uniform, the powder characteristics of raw materials were observed. However, pollen cells had been completely broken into lots of pieces after treatment by DHPM, and particles size was significantly smaller, and all of the contents overflowed. Therefore, wall-breaking effect of DHPM was best.
6. The mechanism of maize pollen broken after DHPM treatment was preliminarily discussed as follows:in the high pressure homogenization process, the organizational structure of pollen cells was completely broken as a result of violent treatment conditions, and the original coarse grain pollen emulsion was processed into particles fine emulsion. The surface area of pollen particles was increased so that particles could fully contact with extraction solvent, thereby the extraction efficiency was enhanced.
7. Cernilton extract was done qualitative analysis by UV spectral scan and HPLC-MS, and it was ultimately determined as cyclic hydroxamic acid whose purity was about 94%. Maize pollen DIBOA extract was purified by Sephadex G-25 and G-10 combined column. The content of maize pollen DIBOA was about 0.029% (290μg/g) after HPLC quantitative analysis and calculation.
8. Antioxidant abilities of crude and refined maize pollen polysaccharides PPMC were measured as DPPH scavenging activity, activity against hydroxyl radical and superoxide anion scavenging activity. The investigation demonstrated that they possessed free radical scavenging activity, and scavenging effect enhanced with increasing of polysaccharides concentration. Scavenging activity of crude and refined polysaccharides on DPPH was better than the scavenging activity of hydroxyl radical and superoxide anion. In the same concentration, scavenging activity of refined polysaccharides PPMC was slightly better than crude polysaccharides.
本研究侧重于应用动态超高压微射流技术对玉米花粉进行破壁,并与干法粉碎破壁、复合酶酶解破壁比较破壁效果和多糖得率,在此基础上对玉米花粉多糖抗氧化活性功能进行了评价,为今后玉米花粉多糖更深入的研究与开发奠定基础。同时对花粉中有助于治疗前列腺疾病的功能因子--环氧肟酸进行了定性和定量分析。本论文的主要研究内容与结论如下：
1、以葡萄糖为对照,优选蒽酮-硫酸法测定玉米花粉多糖的含量。结果表明：玉米花粉多糖的吸收光谱与葡萄糖基本一致,标准曲线的线性关系良好(R2=0.9996),此法在回收率、精密度、稳定性方面均较好,可作为测定玉米花粉多糖含量较为理想的方法。
2、采用正交试验优化干法粉碎花粉并提取多糖的条件,结果表明提取溶液pH值对多糖得率有极显著的影响,最佳条件为：取粉碎并过120目筛分的花粉,调节花粉乳液pH值7.0,料液比1：15,提取温度70℃,提取时间3 h,提取两次,此时多糖得率为7.239%。
3、采用果胶酶和纤维素酶组成的复合酶对玉米花粉进行酶解破壁处理,通过正交试验优化酶解条件。结果表明果胶酶添加量是影响花粉多糖得率的最主要因素。最佳条件为：果胶酶添加量300U／g花粉,纤维素酶添加量150U／g花粉,20%花粉乳浓度,pH4.5,50℃酶解破壁5h,在此条件下多糖得率为7.194%。
4、采用动态超高压微射流技术对玉米花粉进行湿法破壁处理,Box-Benhnken响应面设计优化条件参数。结果表明均质压力和提取温度对多糖得率的线性效应和曲面效应均显著。最佳提取条件为：微射流均质压力127MPa,均质处理一次,料液比1：20,72.4℃下提取1.7 h,提取2次,此时玉米花粉多糖得率达到理论最大值8.086%,与验证试验结果一致。与干法粉碎和酶解破壁提取结果相比,提取时间缩短了1.3 h,多糖得率提高了0.847%。
5、红外光谱扫描的结果表明三种破壁方式提取的多糖的红外光谱图基本吻合,主要官能团没有差异,说明使用高压微射流技术不会破坏玉米花粉多糖的结构。高倍扫描电镜观察破壁效果的结果表明：干法粉碎和酶解破壁处理的花粉仍有少量未完全破壁的花粉颗粒,粒径不均匀,可见原材料的粉末特征；而经微射流均质处理的花粉均完全裂解为大量碎片,粒度明显变小,内容物全部溢出。因此,超高压微射流技术处理花粉的破壁效果最好。
6、初步探讨超高压微射流技术使花粉破壁的机理是：高压均质过程中剧烈的处理条件使花粉细胞的组织结构完全破碎,原来颗粒比较粗大的花粉乳液被加工成颗粒非常细微的乳浊液,使花粉细微颗粒充分与提取溶剂相接触,从而提高提取效率。
7、通过紫外扫描和HPLC-MS对舍尼通药片提取物进行定性分析,最终确定其为环氧肟酸,纯度约为94%。采用Sephadex G-25和G-10二者联用对玉米花粉DIBOA提取液进行纯化,经HPLC定量分析并计算得到玉米花粉中环氧肟酸的含量约为0.029%(290μg/g)。
8、玉米花粉粗多糖和精制多糖组分PPMC对DPPH、O2和.OH自由基均有一定的清除作用,并且随着多糖浓度的增大清除作用增强。二者对DPPH的清除作用最大,对·OH和O2·-自由基的清除作用都比较弱。在同一浓度下,精制多糖组分PPMC对这三种自由基的清除率都略高于粗多糖。
Pollen is male reproductive cells of plants. It is rich in various nutrients and hailed as natural complete nutrients internationally. Maize pollen polysaccharides are capable of antitumor, antioxidant, enhancing the immune function. But stiff pollen wall hindered dissolution of polysaccharides and lowered extraction efficiency. Dynamic high pressure micro-fluidization (DHPM) is a rising high pressure homogenization technique. Plant cells could be disrupted by violent treatment conditions, and the active ingredient was little damaged.
Dynamic high pressure micro-fluidization technology was applied on maize pollen wall-breaking in this study. Maize pollen wall-breaking effect and yield of polysaccharides were compared with dry grinding and enzymatic hydrolysis wall-breaking. Then anti-oxidative function of maize pollen polysaccharides was evaluated in order to prepare for future research and development of maize pollen polysaccharides from now on. At the same time, the pollen function factor cyclic hydroxamic acid which contributed to treatment of prostate diseases was done qualitative and quantitative analysis. The main contents and conclusions were as follows:
1. The content of maize pollen polysaccharides was determined by selected anthrone-sulfuric acid method with glucose as standard. The results were as follows: The absorption spectrum of maize pollen polysaccharides was consistent with glucose, and the linear relationship of standard curve was good (R2=0.9996). The recovery, precision and stability of this method were good, so anthrone-sulfuric acid method was ideal method which could be used to determine pollen polysaccharides content.
2. Polysaccharides were extracted from broken maize pollen by dry comminution, and optimum extract conditions were obtained by orthogonal experiment. Results showed that pH value of extract had most significant impact on yield of polysaccharides. The optimum conditions were as follows:pollen by dry comminution and got over 120 mesh, extract of pH 7.0, ratio of solid-liquid 1:15, 70℃of extraction temperature for 3h, and extraction two times. The yield of polysaccharides was 7.239% under these conditions.
3. Maize pollen wall was broken by enzymolysis with composition of pectinase and cellulase enzyme, and optimum enzymolysis conditions were obtained by orthogonal experiment. Results showed that pectinase addition was principal factor that affected yield of polysaccharides. The optimum conditions were as follows: pectinase addition of 300U/g pollen, cellulase addition of 150U/g pollen,20% of pollen concentration, pH4.5,50℃of enzymolysis temperature for 5h. The yield of polysaccharides was 7.194% under these conditions.
4. Maize pollen wall was disrupted with wet method using DHPM technology, and the parameters were optimized by Box-Benhnken response surface method. The results showed that the linear effect and surface effect of homogenization pressure and extraction temperature on polysaccharides yield were significant. The optimum conditions were as follows:homogenization pressure 127.35 MPa, homogenizing one time, ratio of solid-liquid 1:20,72.41℃of extraction temperature for 1.74 h and extraction two times. Under these conditions the yield of maize pollen polysaccharides was theoretical maximum of 8.086% which was consistent with validation experiment results. Compared with dry comminution and enzymolysis wall-breaking extraction, extraction time was shortened 1.26 h and the yield of polysaccharides increased 0.847%.
5. IR spectra scan results showed that infrared spectrum of maize pollen polysaccharides extracted by three wall-breaking methods was basically identical, and major functional groups had no differences. This indicated that the structure of pollen polysaccharides was not destroyed by DHPM. Pollen wall-breaking effect of three wall-breaking methods was observed by SEM. The results showed that:a few of incompletely broken pollen grains were still observed after dry comminution and enzymolysis treatment, and particles size was not uniform, the powder characteristics of raw materials were observed. However, pollen cells had been completely broken into lots of pieces after treatment by DHPM, and particles size was significantly smaller, and all of the contents overflowed. Therefore, wall-breaking effect of DHPM was best.
6. The mechanism of maize pollen broken after DHPM treatment was preliminarily discussed as follows:in the high pressure homogenization process, the organizational structure of pollen cells was completely broken as a result of violent treatment conditions, and the original coarse grain pollen emulsion was processed into particles fine emulsion. The surface area of pollen particles was increased so that particles could fully contact with extraction solvent, thereby the extraction efficiency was enhanced.
7. Cernilton extract was done qualitative analysis by UV spectral scan and HPLC-MS, and it was ultimately determined as cyclic hydroxamic acid whose purity was about 94%. Maize pollen DIBOA extract was purified by Sephadex G-25 and G-10 combined column. The content of maize pollen DIBOA was about 0.029% (290μg/g) after HPLC quantitative analysis and calculation.
8. Antioxidant abilities of crude and refined maize pollen polysaccharides PPMC were measured as DPPH scavenging activity, activity against hydroxyl radical and superoxide anion scavenging activity. The investigation demonstrated that they possessed free radical scavenging activity, and scavenging effect enhanced with increasing of polysaccharides concentration. Scavenging activity of crude and refined polysaccharides on DPPH was better than the scavenging activity of hydroxyl radical and superoxide anion. In the same concentration, scavenging activity of refined polysaccharides PPMC was slightly better than crude polysaccharides.
引文
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