【作者】 王仁才；

【导师】 熊兴耀；

【作者基本信息】 湖南农业大学 ， 果树学， 2006， 博士

【摘要】 刺葡萄（Vitis davidii Foěx.）为葡萄科葡萄属东亚种群下的一个种，在湖南西部和南部的山区广泛分布，资源丰富。其果实酸甜、风味浓。目前多作为鲜食品种栽培。但果粒小，果皮厚，种子多，其加工制汁以及从果皮、种子提取天然活性物质的前景可能更为广阔。目前关于刺葡萄果实加工及其综合利用方面的研究较少。本论文对刺葡萄籽原花青素的提取与分离纯化技术，刺葡萄皮色素的提取及其理化性能，刺葡萄果汁的加工特性及其加工技术，以及刺葡萄籽油的提取与功效成分及药理作用等方面进行了研究，主要研究结果如下：（1）超临界CO₂萃取刺葡萄籽油工艺的主要影响因素为种子粉碎粒径、萃取压力、萃取温度、分离压力、分离温度。正交试验结果显示，超临界CO₂萃取刺葡萄籽油的实验室最佳工艺条件为：刺葡萄种籽粉碎粒径0.53mm，萃取压力30MPa，萃取温度35℃，分离温度55℃，分离压力12MPa。在此最佳萃取条件下，萃取刺葡萄籽油的产率为13.5％。通过对实验室最佳工艺的放大试验，其放大试验结果与实验室小试结果基本一致，说明了实验室小试实验数据和工艺参数具有可靠性和稳定性。超临界CO₂萃取技术所提取的刺葡萄籽油与溶剂提取法相比，其籽油澄清、无沉淀、无异味，加热不变色，不饱和脂肪酸含量高（87.71％）、碘价高(134.5gI₂·（100g）^-1)、皂化价高（195mg KOH·g-1）、酸价低(2.08mg KOH·g^-1)、游离脂肪酸少、油脂质量好，无溶剂残留，能满足食品、药品等行业对产品高质量的要求。而溶剂法提取的刺葡萄籽油虽产率高（15％），但萃取工艺复杂，所得籽油油质浑浊，具异味，酸价高(5.02 KOH mg·g^-1)，质量差，且产品中溶剂残留量高(石油醚残留量18690mg·kg^-1)，还残留砷(0.18mg·kg^-1)等有害物质。因此，超临界CO₂萃取技术是提取刺葡萄籽油的最佳方法。同时，刺葡萄种子小（平均单粒重0.03g），但相对于赤霞珠、解百纳等品种，其种子含油量高，籽油碘价高，酸价及不皂化物低，其籽油质量更好，刺葡萄籽适于提取葡萄籽油。（2）在刺葡萄籽原花青素的提取中，采用溶剂提取刺葡萄原花青素的适宜工艺条件为70％乙醇溶剂、温度60℃、提取时间120min。在此条件下，刺葡萄籽原花青素的萃取率为51.2％，纯度43.8％。溶剂提取法虽然可获得较高的萃取率，但萃取工艺复杂，萃取时间长，产品纯度低。超临界CO₂萃取刺葡萄籽原花青素的主要影响工艺因素为萃取压力、萃取温度、萃取时间、料液比、夹带剂浓度。其实验室最佳萃取工艺条件是萃取压力为30MPa，萃取温度为55℃，料液比为1∶0.8，60％乙醇夹带剂。以该最佳萃取组合为试验条件，萃取刺葡萄籽原花青素的平均产率达17.33mg·（100g）^-1，纯度为89.7％。同样组合条件萃取赤霞珠种子原花青素的产率为34.20mg·（100g）^-1，纯度为70.1％。说明该最佳萃取组合条件为萃取刺葡萄籽原花青素的适宜条件。同时，超临界CO₂萃取的刺葡萄籽原花青素的纯度比溶剂提取的高很多，超临界CO₂萃取技术是提取葡萄籽原花青素很有发展潜力的方法。对超临界CO₂提取的原花青素产品采用高效液相制备色谱进行分离提纯，制备高纯度刺葡萄籽原花青素。其主要影响因素为流动相与流动相流速。以色谱分离峰高、峰面积、保留时间为分离效果衡量指标，确定以水∶甲醇∶正丁醇（86∶18∶5）为流动相，流速50mL·min^-1为比较理想的分离制备条件。在此条件下，通过分离制备，获得纯度为96.6％的高纯度原花青素。（3）刺葡萄果皮色素溶剂提取的适宜条件是：80％乙醇水溶液为提取剂，料液比为1∶5，在80℃温度下提取1.5h，以该工艺提取色素产率为2.2％，色价22.77，色素含量高。对所提取色素的性能分析表明，该色素色价高，性质稳定，耐酸性、耐光性好，常温下不易变质；蔗糖及V_c对其稳定性影响较小，可用于食品及化妆品的添加剂，是具有较好开发前景的天然色素。（4）刺葡萄果汁风味浓，营养价值较高，单宁含量低，涩味较低，外观色泽较好，适合加工成果汁饮料，而且其果汁富含原花青素(22.76mg·mL^-1)，具有开发功能性饮料的潜力。刺葡萄汁加工工艺中，最佳护色工艺为在果汁打浆后采用短时热处理，以破坏氧化酶活性，防止果汁发生酶促褐变；最佳护色方案是70℃保温5min后，立即冷却；果汁加工的最佳澄清工艺为粗滤后的刺葡萄汁采用高速离心机在6000r·min^-1的转速下离心10min。在此基础上通过配方试验，得到较好的刺葡萄果汁饮料配方，即白砂糖6％、柠檬酸0.05％、葡萄香精0.01％、苋菜红0.0001％、刺葡萄原汁25％。在此基础上，研制出刺葡萄果汁饮料，该饮料较市售康师傅葡萄汁饮料的感官评分更高。通过主要营养及功效成分分析发现，该饮料原花青素含量高达5.71mg·mL^-1，而康师傅葡萄汁饮料中未检测到原花青素，说明刺葡萄汁饮料比康师傅葡萄汁饮料更具保健功效。（5）通过对超临界CO₂萃取的刺葡萄籽油进行GC／MS成分分析研究表明，刺葡萄籽油的成分以脂肪酸为主，其中不饱和脂肪酸含量占87％，不饱和脂肪酸主要是亚油酸，含量为82.32％，明显高于赤霞珠籽油（65％），也高于国内外已有报道的葡萄籽油亚油酸含量。通过未经甲酯化刺葡萄籽油样品的GC／MS分析，在葡萄中首次发现刺葡萄籽油中含有较高的角鲨烯成分，在葡萄属植物尚未见含有角鲨烯成分的研究报道。刺葡萄籽油动物学药理研究表明，在高血脂动物模型成立的前提下，刺葡萄籽油在血清总胆固醇（TC）、HDL／LDL比率及甘油三酯（TG）检测中均呈阳性指标，证明刺葡萄籽油具有调节动物血脂的作用，且其效果优于山葡萄籽油，差异均达极显著水平。临床学研究表明，服用刺葡萄籽油软胶囊其血脂中TG、TC、LDL-C明显低于对照组，而HDL-C及治疗有效率明显高于对照，说明刺葡萄籽油在治疗人体高血脂症方面有明显效果。通过服用刺葡萄籽油软胶囊的各试验组血清SOD、GSH-Px活力和总抗氧化能力比较观察，服用刺葡萄籽油软胶囊后指标比服用前明显提高，而且显著高于其它试验组，其抗氧化疗效明确。因此，刺葡萄籽油具有良好的降血脂及提高人体免疫力等保健功效。综上所述，刺葡萄果实不仅可作鲜食，而且更适合于加工制汁，以及从果皮、种子提取天然功效成分。本研究结果不仅为刺葡萄功效成分的开发利用提供了依据，而且有利于促进刺葡萄的综合利用与产业化生产。更多还原

【Abstract】 Spine grape （Vitis davidii Foěx.） is one of the fruit crops species, distributeswidely in the western and northern of Hunan province. The grape has favorite flavorand taste. But the fruit is small, contained thick peel and large amount of seed. It issuitable for processing. Now there are few reports about studies on spine grape. Theresearch used Ziqiu （a new cultivar of spine grape） as experiment material, mainlyfocused on extracting of seed oil and procyanidins in spine grape seed by supercriticalcarbon dioxide extraction（SCF-CO₂）, isolating and purifying of procyanidins, thepharmacological evaluation of functional compositions in the seed oil, extraction andthe physical and chemical properties of natural pigment in spine grape peel, as well asthe processing characteristics and the processing technique of fruit juice. The mainresults are following:1. The result of single factor test showed that the main influence factors ofextracting oil from spine grape seed by SCF-CO₂ were granule diameter of grindingseed, extraction pressure, extraction temperature, separation pressure, separationtemperature. The results of orthogonal layout experiment showed that the bestlaboratory technique was 0.53 mm in granule diameter of grinding seed, 30MPa inextraction pressure, 35℃at extraction temperature, 12MPa in separation pressure,55℃at separation temperature. The optimal extraction process in laboratory-scale wasblown up to pilot plant scale successfully and results accorded with laboratory-scale. Itwas proved that the extraction process in laboratory-scale was reliable and stable.Compared with solvent extraction method, the technique of extraction bySCF-CO₂ was simple. The seed oil by this method was clarifying, did not change colorwhen it were heated, and had no peculiar smell. The content of unsaturated fatty acids in the seed oil was higher. Its iodine value was higher (134.5gI₂·（100g）^-1), acid valuewas lower (2.08 KOH mg·g^-1) and saponification value was higher (195 mg KOH·g^-1).On the other hand the yield of seed oil by solvent extraction method was higher thanthat of SCF-CO₂ method, its technique was complex. The seed oil by this method wasfeculent, unstable, had peculiar smell, and there was some solvent and noxiouschemical substances such as arsenic in the oil. So SCF-CO₂ extraction was a goodmethod for extracting oil from spine grape seed.cThe average seed weight of spine grape was 0.03g, and the content of seed oilwas 15.6%, which more than the seed of Carbernet Sauvignon （13.4%）. The seed oiliodine value of spine grape was higher than Carbernet Sauvignon seed oil, acid valueand unsaponifiables was lower than it. Therefore the seed of spine grape waspreferable raw material for extracting grape seed oil.2. The optimum technique for extracting procyanidins from spine grape seed bysolvent extraction was that it conducted at 60℃for 120min by using 70% ethanol.Under this condition, the extraction yield rate of procyanidins was 51.2%, andpurification degree was 43.8%. The solvent extraction method was complex,time-consuming and purification degree low in despite of higher yield rate. Theresult of single factor test showed that the main influence factors for extractingprocyanidins from spine grape seed by SCF-CO₂ were extraction pressure, extractiontemperature, extraction time, the ratio of material to liquid and entrainer. The results oforthogonal layout experiment showed that the best laboratory technique for extractingprocyanidins was 30MPa in extraction pressure, 55℃at extraction temperature,1:0.8 in the ratio of materiel to liquid, and 60% ethanol in entrainer. Under thiscondition, the average extraction yield rate of procyanidins reached to 17.33mg·（100g）^-1 and average purification degree reached to 89.7%. By the same extractiontechnique, extraction yield rate and purification degree of Carbernet Sauvignon canreach to 34.20mg·（100g）^-1 and 70.1%, respectively. Thus the technique obtained in theexperiment was the best condition for extracting procyanidins of spine grape seed, andsupercritical carbon dioxide extraction method was of developmental potential inextracting of grape seed procyanidins.Flowing phase and flow velocity of flowing phase were the main factors that influence isolation and purification of spine grape seed procyanidins with PHPLC.Perfect isolation and purification conditions were obtained: flowing phase was water,methanol,n-butyl alcohol （86:18:5）, and velocity of flow was 50mL·min^-1. PHPLCcondition obtained in the test was applied to isolate and purified the grape seedprocyanidins which was prepared by supercritical carbon dioxide extraction, andobtained the procyanidins product which purification degree reached to 96.6%.3. The best conditions of pigment extraction from spine grape peel with solventextraction method were the following: solvent was 80% ethanol, the ratio of materialto ethanol was 1:5, extraction temperature was 80℃and the duration was 1.5h. Theyield rate of pigment was 2.2%, color value was 22.77 and the content of spine grapepeel pigment was higher. The extraction method was cheaper and safer because thesolvent was ethanol instead of poisonous solvent. The property analysis of spine grapepeel pigment showed that the color value was high, the pigment was endurable to acidand light, and not likely to metamorphose at normal temperature. The pigment fromspine grape peel was a kind of very important natural pigment and can be applied asadditive in food and cosmetic because of being stable to sucrose and vitamin C.4. Spine grape fruit juice had higher nutrition value, stronger flavor, lowercontent of tannin and acerbity, better color and luster. Especially that the procyanidinscontent of spine grape (22.76mg·mL^-1) was much higher than that of Kyoho(2.06mg·mL^-1). So the spine grape juice was potential to be functionality juice. Inorder to protect bright rose color, the grape juice must be heated for a short time aftercrushing to destroy the oxidase and prevent enzymatic browning. The best method ofprotecting color was cooling immediately after juice being maintained at 70℃for 5minute. The grape juice should be clarified after crude filtration in order to guaranteethe juice stability. Centrifugation at 6000r·min^-1 for 10 minute was the best method.Spine grape juice directions was obtained in the test: sugar 6%, citric acid 0.05%,grape essence 0.01%, amaranth red 0.0001%, grape fumet 25%. Spine grape juiceproduced according to the directions was more receptible than the normal grape juice.The nutriments and functionality components of finished products were analyzed. Theprocyanidins content of finished product reached to 5.71 mg·mL^-1, but no procyanidinscan be detected in the control group. So spine grape juice was healthier than the common grape juice.5. Spine grape seed oil by SCF-CO₂ extraction was analyzed by Gaschromatography-mass spectrometry （GC/MS） method. The result indicated that theseed oil contained 87% unsaturated fatty acids, with 82.32% linoleic acid in it, which ishigher than other grape seed oil had reported. Squalene was found firstly in the spinegrape seed oil which wasn’t methy lesterize by GC/MS method.Experiment hyperllpernla model was established with rats to observe the changeof biochemistry data of plasma lipid. The results showed that rats’ TC and TG weresignificantly decreased and ratio of HDL/LDL was increased after feeding with spinegrape seed oil. These proved that spine grape seed oil possessed marked therapeutic onexperimental hyperllpernla. The study result on the clinical effect of spine grape seedoil to hyperlipidemia showed that the TC content of treatment group were markedlydecreased and HDL were increased as compared with the control group. Spine grapeseed oil capsule had certain effect for treating hyperlipidemia. The observation of theantioxygen effect in aged people showed that the value of SOD enzyme activity,GSH-PX enzyme activity, T-AOC in patients which took spine grape oil capsule wereincreased evidently. Spine grape oil capsule had stronger antioxidative effect, and itsantioxidativel therapeutic effect was identified.In conclusion, Vitis davidii Foex is not only fit for fresh-eating, but also suit forjuice processing, and extracting functional compositions from its peel and seed. Thestudies can provide reference for improving research, exploiture of spine grape更多还原