单宁酶水解石榴皮多酚的工艺优化及酶解产物鉴定

Process Optimization of Hydrolysis of Pomegranate Peel Polyphenols by Tannase and Hydrolysates Identification

从石榴皮中提取的多酚具有抗炎、抗氧化、抗癌等多种生物活性,但是其中的大分子的鞣花单宁如安石榴苷(PG)不能被动物胃肠道直接吸收,需要先水解成小分子物质如鞣花酸(EA)才能被吸收进入血液来发挥其生物功能。本试验利用单宁酶将石榴皮多酚中的大分子单宁(相对分子质量>500)水解为小分子酚酸,旨在优化酶解工艺及产物纯化工艺,阐明酶解前后石榴皮多酚在成分上的变化,为石榴皮多酚作为促进动物健康的功能性饲料添加剂的开发利用提供基础数据。通过比较单宁酶水解前后石榴皮多酚的高效液相色谱指纹图谱,确定PG降解率和EA生成率为水解程度检测指标后,利用单因素试验和正交试验优化酶解条件。通过静态吸附和动态吸附试验确定大孔树脂纯化条件,并用液相色谱-质谱联用技术鉴定酶解前后石榴皮多酚在成分上的变化。结果显示:最佳酶解条件为pH 5.2、温度45℃、加酶量30 mL/g底物、底物浓度4 g/L,时间16 h。最佳酶解产物纯化条件为D101型大孔树脂,95%乙醇为洗脱剂,上样体积为300 mL,洗脱体积为120 mL。高效液相色谱检测显示原石榴皮多酚中含有43.64%的PG(相对分子质量1 084)、4.85%的EA(相对分子质量302),酶解产物中PG含量为0.00%、EA含量为45.73%。原石榴皮多酚中分析出7种主要多酚化合物,质荷比(m/z)分别为1 083、1 083、783、757、633、401、300,相对分子质量分布在302~1 084;酶解产物中分析出4个主要多酚化合物,m/z分别为600、300、291、247,相对分子质量分布在248~602。本试验利用单宁酶将含大量以PG为主的鞣花单宁的石榴皮多酚水解成富含小分子多酚EA的酶解产物,并优化建立了最佳酶解条件及酶解产物纯化条件,可为石榴皮多酚的开发及其在饲料生产中的应用提供基础数据。

Polyphenols from pomegranate peel has multiple biological activities such as anti-inflammatory,antioxidant and anti-cancer activities. However,the high molecular ellagitannins such as punicalagin in pomegranate peel polyphenols can not be absorbed by animal gastrointestine directly,but have to be hydrolyzed into small molecules such as ellagic acid first and then be absorbed into bloodstream and exerts physiological effects.This study was conducted to hydrolyze the high molecule tannins（ relative molecular mass500） in pomegranate peel polyphenols into small molecule phenolic acids by using tannase,optimize the hydrolysis process and hydrolysates purification process,and clarify the composition changes before and after the hydrolysis. By doing this,we hope it could provide basic data for the exploration of pomegranate peel polyphenols as functional feed additives promoting animal health. By comparing the high performance liquid chromatography fingerprints before and after the hydrolysis,we determined ellagic acid increasing rate and punicalagin degradation rate as indicators of enzymolysis degree. Using the single factor design and orthogonal combination design,we optimized the enzymolysis conditions,and established the macroporous resin purification conditions of hydrolysates by using the methods of static adsorption and dynamic adsorption. Moreover,the composition changes before and after hydrolysis of pomegranate peel polyphenols were determined by a liquid chromatography-mass spectrometry.The results showed that the optimum reaction conditions for the enzymolysis were pH 5.2,45 ℃,enzyme volume 30 mL/g substrate,substrate concentration 4 g/L,16 h. For enzymatic products purification parameters,D101 macroporous resin was tested to be the best absorbing material,the best elution was 95% ethanol,the best loading volume was 300 mL,and the best collecting volume of elution was 120 mL. The contents of punicalagin（ relative molecular mass was 1 084） and ellagic acid（ relative molecular mass was 302） in the pomegranate peel polyphenols were 43.64% and 4.85%,respectively. Under the optimal enzymatic hydrolysis conditions and the optimal purification conditions,the enzymatic products of pomegranate peel polyphenols was obtained,the punicalagin content of which was 0. 00%,and the ellagic acid content of which was 45. 73%.There were seven kinds of main polyphenols in the pomegranate peel polyphenols had been determined,the mass-to-charge ratio（ m/z） of them were 1 083,1 083,783,757,633,401 and 300,respectively,and the relative molecular mass was from 302 to 1 084. There were four kinds of main polyphenols in the enzymatic products pomegranate peel polyphenols had been determined,the m/z of them were 600,300,291 and 247,respectively,and the relative molecular mass was from 248 to 602. This experiment optimized the reaction conditions of tannase hydrolyzing pomegranate peel polyphenols and the hydrolysates purification conditions of the enzymatic products of pomegranate peel polyphenols,obtained hydrolysates rich in small molecular polyphenols,and this work could provide basic data for the development and utilization of pomegranate peel polyphenols as new functional animal feed additives.