【作者】 曹少谦；

【导师】 潘思轶；

【作者基本信息】 华中农业大学 ， 食品科学， 2009， 博士

【摘要】 血橙是重要的甜橙品种,是具有高营养价值、高商业价值的水果,但是其在加工中容易褐变而导致商业价值降低。本研究从花色苷降解的角度出发,在对血橙中主要花色苷的分离纯化及结构鉴定的基础上,通过研究血橙主要花色苷的降解途径,及血橙汁内源因子在花色苷降解过程中的作用,进一步阐明由花色苷降解引起的血橙汁褐变机制。同时,对血橙花色苷抗氧化活性及血橙汁加工过程中抗氧化的变化进行研究,对血橙花色苷的构效关系进行了探讨。主要研究结果如下:1、对血橙汁进行成分分析,并研究了血橙汁在贮藏过程中成分的变化。结果表明,血橙汁富含花色苷、黄酮类物质、抗坏血酸、氨基酸、糖类等物质,还含有Ca、Mg、Zn、Fe、Cu等丰富的人体必需微量元素。血橙汁含有的可溶性糖主要是蔗糖、果糖、葡萄糖。在血橙汁贮藏过程中,随着花色苷含量的下降橙汁鲜艳的红色也逐渐褪色成浅黄褐色,抗坏血酸含量显著下降,还原糖含量变化缓慢,总酸含量平缓增加。2、通过静态吸附解吸、动态吸附解吸试验对7种大孔吸附树脂（D3520、D4020、NKA-2、NKA-9、SIPI-DA201、D14、D16）和5种弱酸性阳离子交换树脂（D151、D152、110、HD-1、JK-110）进行筛选,找到对血橙花色苷特异性最好的树脂NKA-9,并就其对血橙花色苷的提取纯化进行了条件优化,结果表明,柠檬酸调pH为2-3的50%乙醇洗脱效果最好。再利用凝胶柱层析分离纯化血橙花色苷提取物,洗脱条件为:洗脱液采用甲醇/水/甲酸（50:48:2,v/v/v）,流速0.6mL/min。3、利用HPLC-MS、GC、FTIR等技术以及化学定性反应等方法对血橙中花色苷及其他黄酮类化合物进行了鉴定。结果表明,血橙中主要含有8种花色苷和4种黄酮类物质,花色苷分别为:矢车菊素-3-葡萄糖苷、矢车菊素-3（6”-丙二酰）葡萄糖苷、飞燕草素-3-芸香糖苷、矢车菊素-3-芸香糖苷、矢车菊素-3（3”-丙二酰）葡萄糖苷、矢车菊素-3（6”-二乙二酰）葡萄糖苷、矢车菊素-5-葡萄糖苷和矢车菊素-3-葡萄糖苷加成物:4-乙烯基儿茶酚,其中前2种花色苷是血橙中主要花色苷,含量分别占44.3%和36.5%；黄酮类物质为:芸香柚皮苷、橙皮苷、柚皮苷和新橙皮苷。另外,本研究成功分离到血橙中三种具有典型结构代表性的花色苷,即矢车菊素3-葡萄糖苷、矢车菊素-3-葡萄糖苷的丙二酰化衍生物及矢车菊素3-葡萄糖苷吡喃型衍生物。4、本研究确定了血橙花色苷的水解平衡位点pK为3.4,正处于橙汁的pH值附近,因此血橙汁中花色苷极不稳定,pH4.0时对热最为敏感。另外,血橙花色苷对热、光都很不稳定。金属离子都表现出对花色苷热降解的保护作用,Al³⁺对血橙花色苷有明显的增效作用,且随着浓度增加增色作用越强;Cu²⁺低浓度下,对花色苷热降解有一定保护作用,但是高浓度下有明显的促进花色苷降解的作用。葡萄糖对花色苷的降解有一定保护作用,而果糖和蔗糖会促进花色苷的降解,且果糖作用大于蔗糖。5、采用pH示差法和HPLC法对血橙花色苷热降解动力学进行了研究,两种方法测得的矢车菊素-3-葡萄糖苷的降解是一致的。但是,对于矢车菊素-3（6”-丙二酰）葡萄糖苷,两种方法得到的结果却不一致,HPLC分析显示矢车菊素-3（6”-丙二酰）葡萄糖苷不如矢车菊素-3-葡萄糖苷稳定,同时研究表明矢车菊素-3（6”-丙二酰）葡萄糖苷降解中易脱掉酰基生成中间产物矢车菊素-3-葡萄糖苷;而pH示差法得到的结果反映的是体系中总花色苷的降解情况,从而造成了对矢车菊素-3（6”-丙二酰）葡萄糖苷稳定性的高估。而吡喃型花色苷与矢车菊素-3-葡萄糖苷和矢车菊素-3（6”-丙二酰）葡萄糖苷相比,在所研究温度下,热稳定性较差。同时,研究了血橙花色苷热处理过程中色泽降解动力学,并确定血橙汁热处理过程中花色苷降解动力学及色泽降解动力学之间的线性关系可以用公式a/a₀=0.559（C/C₀）+0.43来表示。6、通过对模拟体系中花色苷的降解动力学的研究,确定了血橙汁中内源因子及其相互作用对花色苷降解的影响。结果显示,抗坏血酸和糖能显著促进花色苷的降解,并且两者表现出协同作用,而黄酮类物质则对花色苷的降解具有显著的保护作用,并且与前两者的促进作用相比,这种保护作用在血橙花色苷的降解过程中占主导地位。黄酮类物质和抗坏血酸能降低温度对花色苷降解速率的影响,但是糖促进花色苷降解却与温度密切相关。在糖存在的模拟体系中,花色苷的降解高温时（＞70℃）与其它模拟体系不同,不再符合一级动力学,且血橙中含有的可溶性糖对花色苷降解的促进作用依次为:葡萄糖＜蔗糖＜果糖。另外,在Cu²⁺与抗坏血酸的体系中发现,Cu²⁺本身不促进花色苷的降解,甚至还表现出一定的保护作用,但是当体系中Cu²⁺与抗坏血酸共存时,就能大大促进花色苷的降解,Cu²⁺对花色苷降解的作用存在一种耦合氧化机制,即Cu²⁺通过催化抗坏血酸的氧化来促进花色苷的氧化降解。7、采用化学发光法对血橙花色苷体外抗氧化活性及对DNA损伤的保护作用进行了评价,结果表明,血橙花色苷有很强的体外抗氧化活性及对DNA损伤的保护作用,但是血橙中其它黄酮类物质表现出比花色苷更强的作用。三种典型花色苷抗氧化活性强弱依次为:吡喃型花色苷＞矢车菊素-3（6”-丙二酰）葡萄糖苷＞矢车菊素-3-葡萄糖苷,与低温下三者的稳定性一致,说明三者抗氧化能力与结构稳定性成正相关;而三者对DNA损伤的保护作用大小与抗氧化活性大小相反。另外,还对血橙汁热处理过程中抗氧化能力及对DNA损伤保护能力的变化进行了研究,试验表明随着热处理时间的增长,血橙汁抗氧化活性及对DNA损伤的保护能力不断下降,当热处理1h左右,抗氧化能力及对DNA损伤保护能力会下降一半。更多还原

【Abstract】 Blood orange （Citrus sinensis） is an important kind of sweet orange with high nutritional value and high commercial value. The fruit rapidly lose its bright red colour and turn brown during processing, resulting in a reduced market value. This study originated from the degradation of blood orange anthocyanins. On the basis of isolation and identification of the major anthocyanins from blood orange, we investigated the degradation pathway of major anthocyanins obtained from blood orange, and the effect of intrinsic factors of blood orange juice on the degradation of anthocyanins. The results allow us further understand the browning mechanism of blood orange juice. In addition, we studied the antioxidant activity of blood orange anthocyanins, and the the antioxidant activity of blood orange juice during thermal processing. The structure-activity relationship of blood orange was investigated. The major results are as fallow:1.The compositions of blood orange were analyzed, and the changes of compisition of blood orange during storage were studied. Blood orange juice was abundant in anthocyanins, other flavonoids, ascorbic acid, amino acid and sugars etc. there were also rich trace elements such as Ca, Mg, Zn, Fe, Cu, which were necessary to human. The sugars in blood orange juice were glucose, sucrose and fructose. During the storage of blood orange juice, the attractive red color was lost along with the decreasing of anthocyanin content; the content of ascorbic acid declined significantly; reducing sugar content changed tardily; total acid content increased smoothly.2.According to the static adsorptive/desorptive capacity and dynamic adsorptive/desorptive capacity for blood orange anthocyanins of different resins （seven kinds of macroporous resin and five kinds of weakly acidic-cation exchange resin）, NKA-9 macroporous resin was the optimum for blood orange anthocyanins, and the optimal elution reagent was 50% ethanol with citric acid （pH 2-3）. The best separation of Toyopearl TSK HW-40S column was obtained using a mobile phase of 35% methanol with 2% formic acid at a flow-rate of 0.6 mL/min.3. The anthocyanins of blood orange were identified as cyanidin-3- glucoside, cyanidin-3-（6"-malonyl） glucoside, delphinidin-3-rutinoside, cyanidin-3-rutinoside, cyanidin-3-（3"-malonyl） glucoside, cyanidin-3-（6"-dioxalyl） glucoside, cyanidin-5-glucoside and cyanidin-3-glucoside-derived pyranoanthocyanins.Cyanidin-3-glucoside and cyanidin-3-（6"-malonyl） glucoside were the two major anthocyanins of blood orange, the contents of them were 44.3% and 36.5%, respectively. The flavonoids of blood orange were narirutin, hesperidin, naringin and neohesperidin. In addition, three kinds of representative anthocyanins of blood orange （cyanidin-3-glucoside, cyanidin-3-（6"-malonyl） glucoside and cyanidin-3-glucoside -derived pyranoanthocyanins） were purified.4.The pK of blood orange anthocyanins was about 3.4. It was similar with the pH value of blood orange juice, so the anthocyanins of blood orange were unstable in blood orange juice, and were the most sensitive at pH 4.0. In addition, the anthocyanins blood orange were unstable for heat and light. Metal ions had protective effect on the thermal degradation of anthocyanins. Al³⁺ had hyperchromic effect on anthocyanins, and the effect was enhanced with the increasing of Al³⁺ content. Cu²⁺ also had a protective effect on the thermal degradation of anthocyanins at lower content, but it had significant stimulative effect. Glucose had a protective effect on the thermal degradation of anthocyanins, whereas sucrose and fructose could promote the degradation of anthocyanins, and the effect of fructose was greater than sucrose.5.The thermal degradation kinetics of blood orange anthocyanins were investigated. The results indicated that cyanidin-3-（6"-malonyl） glucoside was less stable than cyanidin-3- glucoside. Acylation with malonic acid could enhance the color stability, but not structural stability. Compared with cyanidin-3- glucoside and cyanidin-3-（6"-malonyl） glucoside, the pyranoanthocyanin was less stable at selected temperature in the study. In addition, the color degradation kinetics during thermal processing was investigated. The relationships between visual color and anthocyanins conternt during thermal processing at selected temperatures could be expressed by the same equation: a/a₀=0.559（C/C₀）+0.43.6.Degradation kinetics of anthocyanins in model systems and the effect of intrinsic factors on anthocyanins degradation and were investigated. The results indicated that ascorbic acid and sugars significantly accelerated anthocyanins degradation, and they had a synergistic effect on the anthocyanins degradation. However, flavonoids had a protective effect on degradation of anthocyanins, and the preotective effect of flavonoids played a more important role in the degradation of anthocyanins comparing to the negative effect of ascorbic acid or sugars. The promoting effects of sugars on anthocyanins degradation correlated with the temperature, and the degradation of anthocyanins in the presence of sugars followed complex reaction kinetics at comparatively higher temperature （above 70℃）. The stimulative effects of sugars on anthocyanins degradation were according to the following descending order: fructose＞sucrose＞glucose. In addition, although Cu²⁺ showed a protective effect on anthocyanins degradation, it could significantly accelerate anthocyanins degradation in the presence of ascorbic acid. So the effect of Cu²⁺ on anthocyanins degradation was a coupled oxidation. 7. Antioxidant activity and preventing DNA damage effect of blood orange anthocyanins were evaluated by using chemiluminescence method. The results showed anthocyanins of blood orange had strong antioxidant activity and preventing DNA damage effect, while the antioxidant activity of other flavonoids in blood orange was stronger than anthocyanins. The antioxidant activity of the three representative anthocyanins of blood orange were according to the following descending order: cyanidin-3-glucoside -derived pyranoanthocyanins＞cyanidin-3-（6"-malonyl） glucoside＞cyanidin-3-glucoside, consenting to the stability of them at lower tempterature. Whlie the preventing DNA damage effects of them were according to the following order: cyanidin-3-glucoside > cyanidin-3-（6"-malonyl） glucoside＞cyanidin-3-glucoside -derived pyranoanthocyanins. In addition, the antioxidant activity and preventing DNA damage effect of blood orange juice during thermal processing were investigated. The results indicated that the antioxidant activity and preventing DNA damage effect of blood orange juice declined along with thermal processing. The antioxidant activity and preventing DNA damage effect would decrease to half when thermal processing for about 1h.更多还原