啤酒中多酚物质的研究
摘要
原花青素低聚物是迄今最引人注目的抗氧化物质,而啤酒酿造用原料酒花是原花青素类物质的一个很好的来源。酒花原花青素对啤酒酿造过程中的非生物稳定性及抗氧化性能及人类保健均产生极大影响。
本文利用RP-HPLC及UPLC-MS法分析市售葡萄籽原花青素对照品。RP-HPLC采用Zorbax Eclipse XDB-C_(18)(4.6×250 mm,5μm,美国Agilent公司)为色谱柱,以甲醇-水(含0.05%三氟乙酸)为流动相,流速0.4 mL/min,柱温25℃,DAD检测波长280 nm,进样量10μL,梯度洗脱分离后得到8种组份。依据ESI-MS选择性,采用UPLC-MS鉴定各化合物组成。共分析得到(±)-儿茶素、(-)-表儿茶素没食子酸酯3种单体,原花青素B2、B3、B4三种二聚体,原花青素C1和1种原花青素二聚体单酯。
对酒花中原花青素进行有机溶剂萃取、大孔树脂粗分离和Sephadex LH-20层析分离纯化。以乙醇为萃取剂的有机溶剂萃取最佳条件为:pH为4.0-4.2,萃取剂乙醇浓度为50%,萃取温度85℃(沸腾),萃取时间2h,料液比为1∶20。采用已确立的最佳酒花浸提条件浸提酒花,得到酒花中原花青素粗提液,上以大孔吸附树脂为填料的层析柱进一步分离提纯。从国产ADS-8、ADS-17、ADS-21、AB-8和D101五种不同大孔吸附树脂中选择分离效果最好的AB-8树脂,进行原花青素粗提液的层析柱动态吸附实验,得到最佳大孔树脂吸附分离条件为:柱体积为14.5mL时,酒花粗提液原花青素浓度为1.8 mg/mL,上样量为30 mL,上样流速为20 mL/h,洗脱剂乙醇浓度为100%,洗脱流速为60 mL/h,洗脱剂用量为70 mL。该方法对于提纯酒花中原花青素具有较高普适性。经此三步提纯,最终分离得到包括儿茶素、原翠雀素在内的多种化合物。
研究中对酒花原花青素进行分级,得到不同平均聚合度的七种原花青素。对这七种原花青素聚合物进行抗氧化实验,同时以(+)-儿茶素和抗坏血酸作对照,实验内容包括还原能力的检测、抑制脂质体过氧化能力测定、在亚油酸体系中的抗氧化能力检测、羟基自由基清除能力测定以及麦汁煮沸过程中对TBA产生的影响。实验结果表明,各物质还原能力由强到弱依次为:mDP为1.5-4的原花青素﹥(+)-儿茶素﹥抗坏血酸﹥mDP=5.55的原花青素;不同聚合度的酒花原花青素对脂质体的氧化过程的抑制作用具有一定的浓度效应且与聚合度关联不大;原花青素低聚物和抗坏血酸抑制亚油酸的自氧化作用随着添加量的增大而增强;随着样品浓度的增加,清除羟基自由基[HO·]效果逐渐加强;麦汁煮沸过程中TBA的产生与原花青素聚合度大小有较大关联性,聚合度为2左右的原花青素抗氧化能力最强,能够抑制老化前驱物质的产生。
Proanthocyanidins oligomers are the most interesting antioxidant around the world, which could easily be found in hops. Hop proanthocyanidins have a great impact non-biological stability and oxidant resistance in beer brewing, and it is good for human health too.
In this study, the commercial proanthocyanidins from grape seed extracts which were used as reference substance were analyzed by RP-HPLC and UPLC-MS. Target compounds were separated by Zorbax Eclipse XDB-C18(4.6×250 mm,5μm) using methanol-water (including 0.05% TFA) as mobile phase. The flow rate was 0.4 mL min-1, column temperature was 25℃, and injection volume was 10μL. 8 compounds were obtained at OD280, and they were identified using UPLC-MS. There were three monomers, three dimers, one trimer and one dimer-monoester. Afterwards, the proanthocyanidins from hop were purified and analyzed. The hops were processed by organic solvent extraction, macroporous resin adsorption, and sephadex LH-20 chromatography. The optimum organic solvents extraction conditions were established as follows:50% ethanol(v/v) was used as solvent, and the extraction was taken for 2 h at pH4.0-4.2 and 85℃with the ratio of solvent to material at 20:1(v/w). Then the crude proanthocyanidins from hop were purified by using column chromatography (φ10×200 mm). AB-8 resin was chosen in this study from 5 kinds of macroporous resins (ADS-8, ADS-17, ADS-21, AB-8, and D101) for its excellent adsorption and desorption properties. The parameters were set as follows: the column volume, 14.5 mL; sample concentration, 1.8 mg/mL; sample volume, 3 mL; sample flow velocity, 20 mL/h. The adsorbed hop proanthocyanidins could be well eluted by 70 mL 100% ethanol with a elution rate of 60 mL/h. This method also has a good applicability in purification of hop proanthocyanidins. Finally various chemicals were purified including (±)-catechin and prodelphinidin.
Seven proanthocyanidins with different degree of polymerization were obtained by the classification of average degree of polymerization. At the same time, the antioxidative activities of these polymers were measured using (+)- catechin and Vitamin C(Vc) as control. The tests included reducing capability, ability of inhibiting lipid peroxidation, ability of antioxidant in linoleic acid system, ability of scavenging hydroxyl free radical, and impact on TBA changes during wort boiling. The results of reducing ability were as follows: proanthocyanidins of mDP=1.5-4 > (+)- catechin> Vc > proanthocyanidins of mDP=5.55. Hop proanthocyanidins with any mDPs could inhibit the peroxidation of lipid, and the strength of inhibition effect has nothing to do with mDP. Besides, the inhibition had a certain concentration effect. In the linoleic acid system, the oligomeric procyanidins and Vc had the same effect in terms of inhibiting the oxidation of linoleic acid, and the inhibiting effect enhanced with increased dosage. The ability of scavenging [HO·] gradually strengthened with concentration increased. During wort boiling there was an obvious relationship with TBA changes and the mDP of procyanidins. Proanthocyanidins of mDP=2 had the most powerful antioxidative ability, which could significantly inhibited the forming of precursors.
本文利用RP-HPLC及UPLC-MS法分析市售葡萄籽原花青素对照品。RP-HPLC采用Zorbax Eclipse XDB-C_(18)(4.6×250 mm,5μm,美国Agilent公司)为色谱柱,以甲醇-水(含0.05%三氟乙酸)为流动相,流速0.4 mL/min,柱温25℃,DAD检测波长280 nm,进样量10μL,梯度洗脱分离后得到8种组份。依据ESI-MS选择性,采用UPLC-MS鉴定各化合物组成。共分析得到(±)-儿茶素、(-)-表儿茶素没食子酸酯3种单体,原花青素B2、B3、B4三种二聚体,原花青素C1和1种原花青素二聚体单酯。
对酒花中原花青素进行有机溶剂萃取、大孔树脂粗分离和Sephadex LH-20层析分离纯化。以乙醇为萃取剂的有机溶剂萃取最佳条件为:pH为4.0-4.2,萃取剂乙醇浓度为50%,萃取温度85℃(沸腾),萃取时间2h,料液比为1∶20。采用已确立的最佳酒花浸提条件浸提酒花,得到酒花中原花青素粗提液,上以大孔吸附树脂为填料的层析柱进一步分离提纯。从国产ADS-8、ADS-17、ADS-21、AB-8和D101五种不同大孔吸附树脂中选择分离效果最好的AB-8树脂,进行原花青素粗提液的层析柱动态吸附实验,得到最佳大孔树脂吸附分离条件为:柱体积为14.5mL时,酒花粗提液原花青素浓度为1.8 mg/mL,上样量为30 mL,上样流速为20 mL/h,洗脱剂乙醇浓度为100%,洗脱流速为60 mL/h,洗脱剂用量为70 mL。该方法对于提纯酒花中原花青素具有较高普适性。经此三步提纯,最终分离得到包括儿茶素、原翠雀素在内的多种化合物。
研究中对酒花原花青素进行分级,得到不同平均聚合度的七种原花青素。对这七种原花青素聚合物进行抗氧化实验,同时以(+)-儿茶素和抗坏血酸作对照,实验内容包括还原能力的检测、抑制脂质体过氧化能力测定、在亚油酸体系中的抗氧化能力检测、羟基自由基清除能力测定以及麦汁煮沸过程中对TBA产生的影响。实验结果表明,各物质还原能力由强到弱依次为:mDP为1.5-4的原花青素﹥(+)-儿茶素﹥抗坏血酸﹥mDP=5.55的原花青素;不同聚合度的酒花原花青素对脂质体的氧化过程的抑制作用具有一定的浓度效应且与聚合度关联不大;原花青素低聚物和抗坏血酸抑制亚油酸的自氧化作用随着添加量的增大而增强;随着样品浓度的增加,清除羟基自由基[HO·]效果逐渐加强;麦汁煮沸过程中TBA的产生与原花青素聚合度大小有较大关联性,聚合度为2左右的原花青素抗氧化能力最强,能够抑制老化前驱物质的产生。
Proanthocyanidins oligomers are the most interesting antioxidant around the world, which could easily be found in hops. Hop proanthocyanidins have a great impact non-biological stability and oxidant resistance in beer brewing, and it is good for human health too.
In this study, the commercial proanthocyanidins from grape seed extracts which were used as reference substance were analyzed by RP-HPLC and UPLC-MS. Target compounds were separated by Zorbax Eclipse XDB-C18(4.6×250 mm,5μm) using methanol-water (including 0.05% TFA) as mobile phase. The flow rate was 0.4 mL min-1, column temperature was 25℃, and injection volume was 10μL. 8 compounds were obtained at OD280, and they were identified using UPLC-MS. There were three monomers, three dimers, one trimer and one dimer-monoester. Afterwards, the proanthocyanidins from hop were purified and analyzed. The hops were processed by organic solvent extraction, macroporous resin adsorption, and sephadex LH-20 chromatography. The optimum organic solvents extraction conditions were established as follows:50% ethanol(v/v) was used as solvent, and the extraction was taken for 2 h at pH4.0-4.2 and 85℃with the ratio of solvent to material at 20:1(v/w). Then the crude proanthocyanidins from hop were purified by using column chromatography (φ10×200 mm). AB-8 resin was chosen in this study from 5 kinds of macroporous resins (ADS-8, ADS-17, ADS-21, AB-8, and D101) for its excellent adsorption and desorption properties. The parameters were set as follows: the column volume, 14.5 mL; sample concentration, 1.8 mg/mL; sample volume, 3 mL; sample flow velocity, 20 mL/h. The adsorbed hop proanthocyanidins could be well eluted by 70 mL 100% ethanol with a elution rate of 60 mL/h. This method also has a good applicability in purification of hop proanthocyanidins. Finally various chemicals were purified including (±)-catechin and prodelphinidin.
Seven proanthocyanidins with different degree of polymerization were obtained by the classification of average degree of polymerization. At the same time, the antioxidative activities of these polymers were measured using (+)- catechin and Vitamin C(Vc) as control. The tests included reducing capability, ability of inhibiting lipid peroxidation, ability of antioxidant in linoleic acid system, ability of scavenging hydroxyl free radical, and impact on TBA changes during wort boiling. The results of reducing ability were as follows: proanthocyanidins of mDP=1.5-4 > (+)- catechin> Vc > proanthocyanidins of mDP=5.55. Hop proanthocyanidins with any mDPs could inhibit the peroxidation of lipid, and the strength of inhibition effect has nothing to do with mDP. Besides, the inhibition had a certain concentration effect. In the linoleic acid system, the oligomeric procyanidins and Vc had the same effect in terms of inhibiting the oxidation of linoleic acid, and the inhibiting effect enhanced with increased dosage. The ability of scavenging [HO·] gradually strengthened with concentration increased. During wort boiling there was an obvious relationship with TBA changes and the mDP of procyanidins. Proanthocyanidins of mDP=2 had the most powerful antioxidative ability, which could significantly inhibited the forming of precursors.
引文
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