Studies On Structure Of Anthocyanins From Blood Orange And Related Degradation Mechanism During Processing

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.