莲原花青素的酶辅助提取技术及其在油脂中的抗氧化活性研究
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摘要
原花青素是广泛存在于植物中的一类多酚化合物,许多研究表明,原花青素具有很强的抗氧化能力、清除自由基能力和药理活性。
本研究以植物莲的成熟花托(莲房)为原料,尝试采用酶辅助提取的方法提高原花青素的提取率,将其应用于油脂中,研究其对不饱和脂肪酸、油脂、脂肪氧合酶等的氧化抑制作用,并将其添加在火腿中,研究莲原花青素在食品中的抗氧化作用。此外,采用等高线法研究了莲原花青素与其他抗氧化剂的相互作用。
本研究的主要成果如下：
(1)采用纤维素酶和果胶酶对莲房组织进行酶解,采用四因素三水平正交实验对酶解时间、加酶量和酶解温度等提取工艺参数进行优化,获得了最佳工艺参数为纤维素酶添加量为0.7%,果胶酶添加量0.1%,酶解温度55℃,酶解时间2.5 h。后又结合微波和超声的技术手段,发现与醇提法相比,优化后的提取工艺能将莲房原花青素的提取率提高约48%。
(2)采用液相、红外等方法对不同方法提取的原花青素进行了结构的对比,发现并没有区别,说明酶辅助提取的方法能显著提高原花青素的提取率。在此基础上采用DPPH法进行了抗氧化性的对比,结果表明酶辅助提取和醇提法提取的原花青素有着相同的抗氧化性。
(3)从大豆中提取脂肪氧合酶,采用亚油酸体系研究不同浓度莲原花青素对其活性的抑制作用,并与茶多酚进行比较。结果表明,原花青素与茶多酚在亚油酸体系中对脂肪氧合酶活性的抑制效果几乎一致,当溶液浓度为0.08mg·mL-1时,原花青素和茶多酚的抑制率分别为88.68%和91.77%,其IC50分别为：0.0470 mg·mL-1和0.0468 mg·mL-1。
(4)采用烘箱法研究莲原花青素在不饱和脂肪酸体系的抗氧化作用。结果表明,在油酸体系中,无论是单独使用不同浓度的原花青素,还是与其他抗氧化剂复配,均能显示较强的抗氧化性,其中原花青素与维生素E的复配效果最优。在亚油酸亚麻酸体系中,不同浓度的原花青素均显示一定的抗氧化性。
(5)采用烘箱法研究莲原花青素在各类动植物油脂中的抗氧化作用。结果表明,原花青素对多种油脂有良好的抗氧化活性。在茶籽油中少量的添加即可取得很好的效果。而与维生素E复配后,则能显著延长猪油的氧化诱导期。
(6)将Isobologram法与DPPH法结合,研究莲原花青素与其他抗氧化剂的相互作用。Isobologram分析图中莲房原花青素与VC、VE、半胱氨酸复合组的效应点都在相加线及95%可信限的下方,理论IC50,add值与实验IC50,mix值有显著性差异,并且相互作用指数γ都小于1,表明莲房原花青素与VC、VE、半胱氨酸复配后都有抗氧化协同性。
(7)按猪油中莲原花青素与维生素E的最佳配比浓度添加于火腿中,观察到此种复合抗氧化剂能显著提高肉类食品的抗氧化活性。
Procyanidins is a type of polyphenols compound, in recent years, much studies and agreat amount of work of procyanidins have made clear that procyanidins has many outstanding characteristics such as the ability of antioxidation、seavenging·OH, and excellent biological and pharmacologic activities.
The study has used the seedpod of the lotus of Nelumbo nucifera Gaertn, try to enhance the extraction ratio of procyanidins through enzyme assistanted. And study the antioxidation of procyanidins in unsaturated fatty acid、oils and lipoxygenase.And add it to hams to research its anoxitantion in food which contain oils.Furthermore, used the isobologram to study the interaction of procyanidins with other antioxidant.
My main research results are as follows:
1.The cellulase and pectase have been used in the extraction process.An orthogonal experiment is carried out to optimize the parameters of enzyme time, temperature and so on. The best technic parameters are like this:the amount of cellulase is 0.7%, the amount of pectase is 0.1%, enzyme temperature is 55℃, enzyme time is 2.5 hours. Then combine the methods of microwave and ultrasonic, it find that compare with the method of ethanol extraction,the technic after optimized can increase the extraction ratio greatly.
2.HPLC and IR have been used to compare the form of the samples, the result shows that enzyme assistant extraction can enhance the extraction ratio of procyanidins observably and the forms have no distinguish. On this basis I compared their antioxidation by DPPH and find that they have the same antioxidation.
3.Extract lipoxygenase from bean, linoleic acid system has been used to study the inhibit effect of different concentration procyanidins and tea polyphenol to it. The result shows that procyanidins and tea polyphenol have the same inhibit effect to lipoxygenase in linoleic acid system. When the concentration is 0.08mg-mL-1,the inhibition ratios of procyanidins and tea polyphenol are 88.68% and 91.77% and the IC50 are 0.0470 mg·mL-1 and 0.0468 mg·mL-1.
4. Study on the antioxidation of procyanidins in unsaturated fatty acid by oven. The result shows that:in oleic acid, no matter use procyanidins alone or with other antioxidants, they all showed excellent antioxidation, and the best is procyanidins compound with vitamin E.In linoleic acid and linolenic acid, procyanidins with different concentrations all has some antioxidation.
5.Study on the antioxidation of procyanidins in oils by oven. The results show that procyanidins has favorable antioxidation in many kinds of oils. In tea oil, only a little procyanidins shows excellent effect. And when it compounds with vitamin E, the oxidative induction time of lard oil has been prolonged remarkable.
6.Combine isobologram with DPPH, I studied the interaction of procyanidins with other antioxidants. The points of combinations were found to be lying below the additivity line and 95% confidence intervals in the Isobolographic graphs.There was a significant difference between the theoretical IC50,add values and the experimental IC50,mix values.The interaction index values of the combinations were less than 1.these results indicated that LSPC combined with VC、VE and cysteine had synergistic antioxidant properties.
7.The best matching of procyanidins with VE in lard oil has been used in harm, and it shows outstanding antioxidation.
本研究以植物莲的成熟花托(莲房)为原料,尝试采用酶辅助提取的方法提高原花青素的提取率,将其应用于油脂中,研究其对不饱和脂肪酸、油脂、脂肪氧合酶等的氧化抑制作用,并将其添加在火腿中,研究莲原花青素在食品中的抗氧化作用。此外,采用等高线法研究了莲原花青素与其他抗氧化剂的相互作用。
本研究的主要成果如下：
(1)采用纤维素酶和果胶酶对莲房组织进行酶解,采用四因素三水平正交实验对酶解时间、加酶量和酶解温度等提取工艺参数进行优化,获得了最佳工艺参数为纤维素酶添加量为0.7%,果胶酶添加量0.1%,酶解温度55℃,酶解时间2.5 h。后又结合微波和超声的技术手段,发现与醇提法相比,优化后的提取工艺能将莲房原花青素的提取率提高约48%。
(2)采用液相、红外等方法对不同方法提取的原花青素进行了结构的对比,发现并没有区别,说明酶辅助提取的方法能显著提高原花青素的提取率。在此基础上采用DPPH法进行了抗氧化性的对比,结果表明酶辅助提取和醇提法提取的原花青素有着相同的抗氧化性。
(3)从大豆中提取脂肪氧合酶,采用亚油酸体系研究不同浓度莲原花青素对其活性的抑制作用,并与茶多酚进行比较。结果表明,原花青素与茶多酚在亚油酸体系中对脂肪氧合酶活性的抑制效果几乎一致,当溶液浓度为0.08mg·mL-1时,原花青素和茶多酚的抑制率分别为88.68%和91.77%,其IC50分别为：0.0470 mg·mL-1和0.0468 mg·mL-1。
(4)采用烘箱法研究莲原花青素在不饱和脂肪酸体系的抗氧化作用。结果表明,在油酸体系中,无论是单独使用不同浓度的原花青素,还是与其他抗氧化剂复配,均能显示较强的抗氧化性,其中原花青素与维生素E的复配效果最优。在亚油酸亚麻酸体系中,不同浓度的原花青素均显示一定的抗氧化性。
(5)采用烘箱法研究莲原花青素在各类动植物油脂中的抗氧化作用。结果表明,原花青素对多种油脂有良好的抗氧化活性。在茶籽油中少量的添加即可取得很好的效果。而与维生素E复配后,则能显著延长猪油的氧化诱导期。
(6)将Isobologram法与DPPH法结合,研究莲原花青素与其他抗氧化剂的相互作用。Isobologram分析图中莲房原花青素与VC、VE、半胱氨酸复合组的效应点都在相加线及95%可信限的下方,理论IC50,add值与实验IC50,mix值有显著性差异,并且相互作用指数γ都小于1,表明莲房原花青素与VC、VE、半胱氨酸复配后都有抗氧化协同性。
(7)按猪油中莲原花青素与维生素E的最佳配比浓度添加于火腿中,观察到此种复合抗氧化剂能显著提高肉类食品的抗氧化活性。
Procyanidins is a type of polyphenols compound, in recent years, much studies and agreat amount of work of procyanidins have made clear that procyanidins has many outstanding characteristics such as the ability of antioxidation、seavenging·OH, and excellent biological and pharmacologic activities.
The study has used the seedpod of the lotus of Nelumbo nucifera Gaertn, try to enhance the extraction ratio of procyanidins through enzyme assistanted. And study the antioxidation of procyanidins in unsaturated fatty acid、oils and lipoxygenase.And add it to hams to research its anoxitantion in food which contain oils.Furthermore, used the isobologram to study the interaction of procyanidins with other antioxidant.
My main research results are as follows:
1.The cellulase and pectase have been used in the extraction process.An orthogonal experiment is carried out to optimize the parameters of enzyme time, temperature and so on. The best technic parameters are like this:the amount of cellulase is 0.7%, the amount of pectase is 0.1%, enzyme temperature is 55℃, enzyme time is 2.5 hours. Then combine the methods of microwave and ultrasonic, it find that compare with the method of ethanol extraction,the technic after optimized can increase the extraction ratio greatly.
2.HPLC and IR have been used to compare the form of the samples, the result shows that enzyme assistant extraction can enhance the extraction ratio of procyanidins observably and the forms have no distinguish. On this basis I compared their antioxidation by DPPH and find that they have the same antioxidation.
3.Extract lipoxygenase from bean, linoleic acid system has been used to study the inhibit effect of different concentration procyanidins and tea polyphenol to it. The result shows that procyanidins and tea polyphenol have the same inhibit effect to lipoxygenase in linoleic acid system. When the concentration is 0.08mg-mL-1,the inhibition ratios of procyanidins and tea polyphenol are 88.68% and 91.77% and the IC50 are 0.0470 mg·mL-1 and 0.0468 mg·mL-1.
4. Study on the antioxidation of procyanidins in unsaturated fatty acid by oven. The result shows that:in oleic acid, no matter use procyanidins alone or with other antioxidants, they all showed excellent antioxidation, and the best is procyanidins compound with vitamin E.In linoleic acid and linolenic acid, procyanidins with different concentrations all has some antioxidation.
5.Study on the antioxidation of procyanidins in oils by oven. The results show that procyanidins has favorable antioxidation in many kinds of oils. In tea oil, only a little procyanidins shows excellent effect. And when it compounds with vitamin E, the oxidative induction time of lard oil has been prolonged remarkable.
6.Combine isobologram with DPPH, I studied the interaction of procyanidins with other antioxidants. The points of combinations were found to be lying below the additivity line and 95% confidence intervals in the Isobolographic graphs.There was a significant difference between the theoretical IC50,add values and the experimental IC50,mix values.The interaction index values of the combinations were less than 1.these results indicated that LSPC combined with VC、VE and cysteine had synergistic antioxidant properties.
7.The best matching of procyanidins with VE in lard oil has been used in harm, and it shows outstanding antioxidation.
引文
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52.WHYSNER J,WANG C X,ZHANG E,et al.Dose response of promotion by butylated hydroxyanisole in chemically initiated tumours of the rat forestomach[J].Food and Chemical Toxicology,1994,32(3):215-222.
53.Brown AG,et al. Identification of the thearubigins as polymer proanthocyanidins. Nature.1969,221:742
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76. Li Xiu-Qin, Ji Chao, Sun Yan-Yan, Yang Min-Li, Chu Xiao-Gang.Analysis of synthetic antioxidants and preservatives in edible vegetable oil by HPLC/TOF-MS[J]. Food Chemistry,2009,113:692-700
77.Borowicz K K, Jaszczyk B, Luszczki J J, Czuczwar S J. Interactions between two enantiomers of losigamone and conventional antiepileptic drugs in the mouse maximal electroshock model—An isobolographic analysis[J]. European Journal of Pharmacology,2007,567:110-116.
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