酶解结合高剪切破壁技术对蜂花粉酚类物质及抗氧化活性的影响

为了提高蜂花粉体外抗氧化活性及其对质粒DNA氧化损伤的保护作用,该研究以荷花、枣花、茶花、油菜、玫瑰、五味子6种蜂花粉为对象,研究纤维素酶酶解结合高剪切破壁技术对蜂花粉酚类物质溶出的影响。破壁试验结果表明,采用质量分数1%~4%纤维素酶(荷花1%;枣花4%;茶花2%;油菜1%;玫瑰4%;五味子3%)结合30 s 10000~15000 r/min高剪切,6种蜂花粉均可达到90%以上破壁率。高效液相色谱-二极管阵列检测法(High Performance Liquid Chromatography-Diode Array Detection,HPLC-DAD)分析结果表明6种蜂花粉破壁后酚类物质的溶出量和种类均有不同程度增加,荷花蜂花粉没食子酸含量较破壁前增加6倍,枣花蜂花粉经破壁后山奈酚溶出,且含量较高,为10.31 mg/g,破壁后油菜蜂花粉没食子酸含量为11.59 mg/g,较破壁前提高59%。油菜蜂花粉总黄酮含量为29.6mg/g(以芦丁当量计),相较破壁前提高6.4倍,总酚含量为21.60 mg/g(以没食子酸当量计),相较未破壁提高1.5倍;体外抗氧化试验表明,破壁能够将枣花蜂花粉Fe²⁺络合力提高2.2倍,油菜蜂花粉Fe³⁺还原力提高8倍。破壁后荷花、枣花、茶花、油菜、玫瑰、五味子6种蜂花粉对pBR322质粒DNA氧化损伤的保护作用分别提高了60.5%、12.4%、287.7%、442.1%、82.5%、4.8%。结果表明,纤维素酶酶解结合高剪切破壁方法,能够促进蜂花粉酚类物质的溶出,增强体外抗氧化活性,有效预防DNA氧化损伤。研究结果为蜂花粉资源的开发与利用提供理论依据。

Effects of enzymatic hydrolysis combined with high-shear wall breaking technology on phenolic compounds and antioxidant activity of bee pollen

Abstract: Bee pollen is collected by honeybees from the pollen of flowers, with the addition of the nectar and their own secretions to form grains. Approximately 70% of the substances in bee pollen have biological activities, such as antioxidant, liver protection, anti-inflammatory, antibacterial and anti-cancer. However, the outer wall of pollen spores has the characteristics of acid resistance, alkali resistance, and anti-microbial decomposition. Without breaking the wall of spores, phenolic compounds can only be released from the germination pores, and thus the digestibility of bee pollen is only 52%-59% in vivo, meaning that most of them cannot be digested by human. Therefore, the wall breaking of spores has become necessary to improve the bioavailability of bee pollen. In this study, taking six kinds of bee pollen as research objects, including lotus, jujube, camellia, rape, rose, and schisandra, a wall breaking method of enzymatic hydrolysis combined with high-shear technology was used to reveal the effect of wall breaking on the phenolic compounds and antioxidant activities of bee pollen. An attempt was made to explore the protective effect of bee pollen, with or without wall breaking, on the OH-mediated oxidative damage of plasmid DNA for bee-pollen functional foods. An investigation was also made to find the effects of cellulase enzymatic hydrolysis on the wall breaking rates of different kinds of bee pollen. Different kinds of bee pollen exhibited different degrees of tolerance to the cellulase. The wall breaking rate of bee pollen increased with the increase of cellulase concentration. After six kinds of bee pollen enzymatically hydrolyzed, the wall breaking rate was significantly improved with high shear at 10 000-15 000 r/min for 30 s. More than 90% of wall breaking rate was achieved with less cellulase in the combined methods. The release of phenolic compounds from rape bee pollen was the most significant after the wall was broken (P<0.05). The total flavonoid content increased by 6.4 times, and the total phenol content increased from 8.6 mg/g to 21.6 mg/g, as well as the total phenol content of camellia bee pollen was 15.7 mg/g, an increase of 11.3% after the wall was broken. The phenolic compounds in six kinds of bee pollen were analyzed using high-performance liquid chromatography method with diode array detection (HPLC-DAD). The results showed that the types and content of phenolic compounds in bee pollen increased after the wall was broken. The concentration of jujube bee pollen to scavenge 50% DPPH free radicals is reduced by about 95%, and rape bee pollen is reduced by about 92%, the Fe2+ complex ability of jujube bee pollen increased from 0.36 mg/g to 1.16 mg/g, increased by about 2.2 times. The Fe3+ reducing power in the six types of bee pollen increased after the walls were broken. The Fe3+ reducing power of rape bee pollen increased from 6.2 mg/g to 55.8 mg/g, increased 8 times. After walls breaking, the protective abilities against DNA oxidative damage increased by 60.5%, 12.4%, 287.7%, 82.5%, 442.7% and4.8% in the six kinds of bee pollen( lotus, jujube, camellia, rape, rose and schisandra), respectively, indicating that the wall breaking made phenolic compounds of bee pollen fully be released, which can effectively improve the protective ability of OH-induced pBR322 plasmid DNA oxidative damage. This findings can provide a theoretical basis and strategy for the development of bee-pollen functional foods.