摘要
“健康”成为人们日益关注的问题。对天然植物生理活性成分展开研究,开发出为人类健康造福的天然植物功能产品,具有深远的现实意义和广阔的应用前景。其中,天然色素,尤其是植物花色苷色素的开发利用成为当今研究的热点之一。花色苷广泛存在于植物花、叶、果中,可作为天然食用色素和抗氧化剂。因为其安全、无毒且具有一定营养和药理作用,日益受到食品、化妆品、医药等行业及消费者青睐,具有巨大的应用潜力。
刺葡萄(Vitis davidii Fo(e|¨)x.)原产我国陕西、甘肃、华中、华南及西南等地,近年在湖南省湘西北山区、湘南等地种植量不断增加。刺葡萄果皮厚实、色泽深、富含花色苷,且作为鲜果不可食用部分及葡萄加工的副产物,来源广泛,价格低廉,实属可以开发利用的天然食用色素资源。但由于目前缺乏技术支撑,该资源尚未得到有效利用。对刺葡萄皮色素开展系统的研究,有助于该植物资源得到迅速、合理、有效、全面的开发与利用,同时有助于这一优良品种的进一步推广种植,改善当地农业产业结构,增加农民收入。
故此,本文针对刺葡萄皮色素开发利用中亟待解决的几个问题开展研究。确立了刺葡萄皮花色苷的测定方法,探明了刺葡萄皮花色苷的含量;运用三元二次正交旋转组合设计和多项式回归分析,优化了刺葡萄皮色素的提取工艺参数;研究了刺葡萄皮色素在食品环境中的应用稳定性,并研究了刺葡萄皮花色苷的热降解动力学规律;筛选了刺葡萄皮色素纯化用合适的大孔树脂,建立了静态、动态吸附及解吸的工艺条件;采用薄层层析(TLC)、系列柱层析(CC)、HPLC等方法探索了刺葡萄皮色素的分离工艺,同时运用UV-vis、HPLC-MS、IR、NMR等手段,对刺葡萄皮花色苷进行了结构鉴定。试验研究结果表明:
1、刺葡萄皮花色苷含量丰富,其色价值是赤霞珠葡萄皮的2.43倍,红提葡萄皮的2.87倍,巨峰葡萄皮的21.75倍。花色苷含量达到2.5227mg/gFW(以3'甲花翠素.3.葡萄糖苷计)。
2、以70%乙醇(含0.03%盐酸)作为刺葡萄皮粉色素的提取剂比较合适,料液比1:15,温度50℃,时间60min分别为单因素最优条件。三元二次正交旋转组合设计和多项式回归分析,建立了色素提取量回归模型,得到响应面最佳条件,根据生产实际情况调整为温度47℃,提取时间70min,料液比1:17。回归模型优化效果明显,预测精度高。以酸化水(含0.5%醋酸)作为新鲜刺葡萄皮色素的提取剂比较合适,料液比1:10,温度50℃,时间120min分别为单因素最优条件。回归模型得到响应面最佳条件,根据生产实际情况调整为温度45℃,提取时间128min,料液比1:13。
3、pH值对刺葡萄皮色素稳定性影响很大,应在酸性环境中使用;室外强光加速花色苷的降解;H_2O_2导致色素液迅速褪色,抗坏血酸降低刺葡萄花色苷的稳定性,亚硫酸钠使色素液颜色变淡,刺葡萄皮色素在低浓度的苯甲酸钠、山梨酸钾溶液(≤0.5g/L)中色泽稳定;Mg~(2+)、Mn~(2+)、K~+使色素液颜色稍减,Zn~(2+)、Ca~(2+)对色素液有一定辅色效果,Al~(3+)、Cu~(2+)使色素液吸光度明显增加,Fe(3+)使色素液迅速产生黑褐色沉淀,失去花色苷的特点:蔗糖、葡萄糖、食盐对刺葡萄皮色素均有辅色作用,辅色效果与浓度有关;有机酸对刺葡萄皮色素的增色效果为草酸>柠檬酸>乳酸>没食子酸>硼酸。
4、刺葡萄皮花色苷的热降解符合动力学一级反应方程。同一pH值条件下,降低温度可显著延长刺葡萄皮色素的半衰期;不同pH条件下,刺葡萄花色苷降解所需的活化能Ea(pH1.0)>Ea(pH3.0)>Ea(pH4.5),说明热处理时,低pH值有利于刺葡萄皮花色苷的保存,但须注意pHl.0高温下(≥80℃)的水解作用。
5、在供试树脂中,HP-20大孔树脂为刺葡萄皮色素纯化最适用的吸附剂。其静态吸附最优条件为温度40℃,pH3,饱和吸附量达到32.0796mg/g湿树脂(色素原液浓度1.2357mg/mL,以天然葡萄皮色素标样计);洗脱液80%乙醇(含0.05%盐酸)可得最佳洗脱效果;动态吸附、解吸工艺参数,选用上样液浓度1.5891～3.1377mg/mL,上样流速4.5BV/h,洗脱速度3BV/h,洗脱剂用量8BV为宜,树脂动态饱和吸附量达到39.00mg/mL湿树脂,解吸率92%以上,色素液纯化后色价为原来的5.93倍。
6、经系列柱层析(HP-20大孔树脂、聚酰胺、葡聚糖凝胶LH-20),从刺葡萄皮色素中分离出主要的花色苷单体.AN1,占花色苷总量的85.805%,纯度95%以上。运用UV-vis、HPLC-MS、IR、NMR等手段鉴定其结构,确立AN1为锦葵啶-[3-O-β-D-葡萄糖基-6”-香豆酰]-[5-O-β-D-葡萄糖基]。另两种花色苷AN2、AN3的结构,经UV-vis、HPLC-MS初步推断AN2为芍药啶-[3-O-葡萄糖基]-[5-O-葡萄糖基],AN3锦葵啶-[3-O-葡萄糖基]-[5-O-葡萄糖基]。
"Health" is becoming the most popular concern.So it has far-reaching significance in realism and expansive foreground in appliance to develop functional products from botanical resources for human being's health through the studing on physiologically active ingredient in natural plants.In this case,the developing and utilizing of natural pigments,especially anthocyanin pigments from plants is becoming one of the research hotspot at present.Being the natural edible pigments and antioxidants,anthocyanins is widely distributing in the flowers,leaves and fruits of plants.Because of its safety and nontoxicity,but of its nutritional and the pharmacological functions,anthocyanins is being paid more and more attention on by food industry,medical industry and consumers,thus it has huge potential in appliance.
Brier grape(Vitis davidii Fo(e|¨)x.)grew in China originally,and distributed in Shanxi, Gansu,the central China,southern,and south-western China.The yield is increasing in recent years from the north-western mountainous area and south area in Hunan province. Vitis skin has abundant anthocyanins which is very thick and deep in colour,and it's rich in resource and low in price as the inedible part of fruit and the by-poducts of Vitis.So,it's really a kind of natural edible pigments resource which is developable.This excellent resource hasn't been utilized yet,however,because of the lacking of enough technology.The systemic study on Vitis skin pigment will be helpful not only to the developing and utilizing of this resource more timely,reasonable,effective and comprehensive,but also to the enlaging cultivation and improving on the regional agriculture structure which results in the increase of farmers' income.
In this thesis,the content of Vitis skin anthocyanins(VSA)was determined on the basis of selecting of mensuration.Through the design of quadratic rotational combination test and Multiple regression analysis,the parameters of the extraction technics of Vitis skin pigment(VSP)were optimized.The stability of VSP in food circumstances was studied, and the thermal degradation kinetics of VSA was also investigated.The most suitable macroporous resin to purify the VSP was screened out,and on this basis,the parameters of static and dynamatic absorption/desorption technics for purifing VSP were established.This article also studied the isolation technics of VSA by using several methods such as TLC, CC,HPLC,etc.Ultraviolet-visible spectrum,HPLC-MS,IR,and NMR were applied to identify the molecular structure of VSA.
The results showed that:
1.Vitis skin contained high amount of anthocyanins(2.5227mg/g FW),its colour-value was 2.43,2.87,and 21.75 times to that of Chixiazhu,Hongti,and Jufeng grape respectively. 2.70%aqueous ethanol+0.03%HCl was suitable extractant of pigment from dry Vitis skin powder,and the single factors as followed were most suitable for extracting VSP:50℃as extraction temperature,1:15 as the ratio of Vitis skin to extractant,60min as extraction time. Through the design of quadratic rotational combination test and Multiple regression analysis,the established regression model relating to extraction yield of VSP indicated that the optimized condition of the response surface model(RSM)was ajustied as 47℃,70min, and 1:17 to practical production.The demonstration tests on optimum condition of RSM showed that the regression model had significant optimization effect and precise prediction. On the other hand,during extracting pigment from fresh Vitis skin,distilled water (+0.5%acetic acid)was suitable extractant,and the single factors as followed were most suitable:50℃as extraction temperature,1:15 as the ratio of Vitis skin to extractant, 60min as extraction time.The optimized condition of the RSM was ajustied as 45℃, 128min,and 1:13 to practical production.
3.Value of pH was a significant factor on the stability of VSP,and VSP should be used under acidic circumstances.The sunlight accelerated the degradation of VSA.Ascorbic Acid and H_2_O2 enhanced the fading of VSP solution;Sodium sulfite led to fade of colour of VSP solution;Low concentration of Benzoic Acid and Potassium sorbate(≤0.5g/L)had little influence on the stability of VSP.Mg~(2+),Mn~(2+)and K~+ lowed the absorbency of VSP slightly; Zn~(2+)and Ca~(2+)had copigmentation effect on VSP solution;Al~(3+)and Cu~(2+)added the absorbency of VSP;Fe~(3+)caused dark-brown precipitate in VSP solution and made it not be anthocyanin any more.Sucrose,glucose and salt had all copigmentation function,and the effects of copigmentation were related to the concentration of these copigment.The enhancement effects of organic acids on the absorbency of VSP were in following order: oxalic acid>citric acid>lactic acid>gallic acid>boric acid.
4.It was proved that the thermal degradation of VSA was accordant with the first-level reaction equation of kinetics.At the same pH,the half life of VSA could be prolonged remarkably with the falling of temperature;at different pH,the activation energy(E_a) needed for the degradation of VSA was ordered as E_a(pH1.0)>E_a(pH3.0)>E_a(pH4.5). This result explained that lower pH was more helpful to the preservation of VSA when heating,but the hydrolization of pH1.0 VSA which occurred at very high temperatures (>80℃)should be paid more attention to.
5.The macroporous resin HP-20 was the most suitable sorbent among the macroporous resins be tested. Under the optimum condition of static adsorption,that was 40℃and pH3.0,the saturation adsorption was 32.0796mg/g(pigment solution 1.2357mg/mL,in terms of GSP),and the best desorption solvent was 80%ethonal(+0.05%HC1).The optimized parameters of dynamic adsorption/desorption were chosen as follow:pigment solution 1.5891～3.1377mg/mL; velocity of pigment solution flow 4.5BV/h;the volume and velocity of desorption solvent 8 BV,3 BV/h.Under the condition mentioned above,the saturation adsorption of HP-20 to VSP was 39.00mg/mL wet resin,and the desorption ratio was more than 92%,the colour value of purified pigment was 5.93 times to that of unpurified.
6.After systemic column chromatography separation(HP-20 macroporous resin,polyamide, Sephadex LH-20),the leading anthocyanin monomer AN1 was obtained and its purity was over 95%(the content of AN1 in VSA was 85.805%).The result of structure identification combining UV-vis,HPLC-MS,IR and NMR indicated that AN1 is malvidin-3,5-glucose-coumarate.According to the results of UV-vis and HPLC-MS,the two other anthocyanins AN2 and AN3 from VSA were identified as peonidin-3,5-diglucose and malvidin-3,5-diglucose respectively.
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