不同保鲜处理对紫背天葵品质和生理生化的影响及其POD特性研究
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摘要
紫背天葵(Gynura bicolor D.C)富含多种纤维素及多种微量元素、氨基酸和蛋白质、花色苷、黄酮类物质和挥发油等,是药食同源的特色蔬菜。因其具有良好的预防和保健效果,受到愈来愈多消费者的喜爱。紫背天葵蒸腾和呼吸作用旺盛,贮藏过程中感官和食用品质下降迅速,营养物质流失严重,增加了损失率。前期研究结果表明,0℃条件下紫背天葵没有冷害症状,表明其对贮藏低温不敏感且0℃适合紫背天葵的保鲜贮藏。本试验研究了采后植酸、1-MCP单独处理、纳米材料与气调相结合对贮藏期间紫背天葵采后品质和生理生化的变化及POD的酶学特性,主要结果如下：
1.植酸处理对冷藏紫背天葵品质和生理特性变化的影响
以清水处理为对照,研究0,0.1 mM,0.5 mM和1.0 mM浓度的植酸处理对冷藏(0℃)期间紫背天葵品质和生理变化的影响。0.5 mM植酸处理有效抑制了贮藏期间紫背天葵的腐烂指数和细胞膜渗透率的上升,显著保留了叶绿素、花青素、游离酚、总酚、还原糖、总糖、游离氨基酸和可溶性蛋白的含量,较好地保持了紫背天葵的色泽和品质；呼吸强度显著低于对照组(P<0.05),差值最大可达54mgCO2kg-1·h-1;保持了较高的FP和TP含量；PPO活性为对照的84.5%,SOD和POD活性为对照的1.1倍和1.2倍,保持了较高的自由基清除酶的活性,抑制了O2-含量的增加/积累。所有植酸处理中,0.5m M PA对于紫背天葵保鲜效果最好,延长贮藏期至15天；但1.0 mMPA处理使紫背天葵产生了灼烧现象。
相关性分析显示,O2-与POD和SOD均呈负相关(P<0.05),表明植酸处理可推迟紫背天葵的衰老,有利于保鲜期的延长。
2.1-MCP处理对冷藏紫背天葵品质和生理特性变化的影响
分别以0,0.25 ppm,0.50 ppm和1.00 ppm浓度的1-MCP处理紫背天葵0,6h,12h和24 h研究冷藏期间紫背天葵品质和生理生化变化。试验结果表明,1-MCP处理浓度和时间对紫背天葵腐烂指数有显著作用,提高1-MCP处理浓度和时间,可以减少腐烂的发生,0.50 ppm与1.00 ppm处理组差异不显著,24 h处理较12h加速了紫背天葵组织的崩溃；1-MCP处理的所有浓度和时间,均对紫背天葵呼吸强度有抑制效率,以中高等剂量和时间的处理效果良好,但12h与24 h、0.50 ppm与1.00 ppm处理组之间的差异并不显著；1-MCP处理可显著抑制紫背天葵叶绿素和花青素的降解,使其保持较好的色泽和感官品质；1-MCP可显著延缓贮藏期间紫背天葵还原糖、总糖、游离氨基酸和可溶性蛋白含量的减少,可减少MDA和O2-的生成与积累,保持较高的自由基清除酶的活性,降低细胞膜渗透率,保持较好的细胞膜完整性。
试验结果表明,相同处理时问条件下,0.50 ppm处理效果最优；相同处理浓度条件下,12h处理效果最优；综合所有试验结果,紫背天葵保鲜的最适处理条件为0.50ppm 1-MCP处理12h。
3.气调结合纳米材料包装处理对冷藏紫背天葵品质和生理特性变化的影响
比较了纳米包装袋(NP)、CA(023%,C02<5%)和NP+CA处理对冷藏紫背天葵品质和生理生化变化的影响。相同贮藏时间条件下,紫背天葵的腐烂率都表现为CA>NP>CA+NP,贮藏结束时NP和CA+NP处理紫背天葵腐烂率均显著低于CA处理组(7.64%),且CA+NP处理腐烂率最低(6.97%)；相关性分析显示,CA+NP处理对紫背天葵腐烂率的抑制效果具有显著差异(P<0.05)。贮藏期间3处理紫背天葵的呼吸差异与腐烂率变化情况一致,即CA+NP处理紫背天葵的呼吸强度为3处理中最低的,且贮藏结束时仅为CA处理的56.6%；与CA和NP处理比较,CA+NP减少了紫背天葵组织中O2-与MDA的积累,保持了细胞膜完整性,且相对提高了活性氧清除酶SOD和CAT的活性。综合以上结果,3处理中气调(3%O2,CO2<5%)结合纳米材料包装(NP)用于紫背天葵的保鲜效果最好。
4.不同采收期紫背天葵品质和生理特性比较
试验比较了不同采收时间紫背天葵物质组成和生理生化的差异,并对其贮藏性进行了评价。紫背天葵的物质成分分析显示,6月份采收的紫背天葵的叶绿素、花青素、游离氨基酸和可溶性可溶性蛋白含量均显著高于5月与4月份采收紫背天葵的含量,特别是花青素是5月的近2倍、4月的3倍以上。生理生化分析显示,6月份采收的紫背天葵具有最低的呼吸速率、细胞膜渗透率,最少的O2-和MDA含量；较高的可溶性固形物含量,相对较强的POD、CAT和SOD活性,提示此时的紫背天葵不仅自由基生成少且自我清除能力强,具有较好的潜在贮藏性。
比较贮藏期间物质和生理生化变化的情况可见,6月份采收的紫背天葵贮藏期间,叶绿素、总糖和可溶性蛋白含量下降趋势最缓,呼吸强度、O2-和MDA含量变化幅度最小。以上结果证明,6月份采收的紫背天葵耐贮性最好。
5.紫背天葵POD酶学特性的初步研究
对紫背天葵酚类提取物采用紫外扫描和HPLC等技术进行分析,确定紫背天葵叶和茎中均含有绿原酸、咖啡酸；叶中还含有肉桂酸,以绿原酸最多；茎中还含有表儿茶酚,以绿原酸与表儿茶酚的含量最多。
紫背天葵叶与茎的POD最适底物均为焦性没食子酸,最适pH值分别为pH 5.0和pH 6.0,且在pH 5.0和pH 5.4时酶活性的保留率最高。紫背天葵POD具有较强的耐热性,在80℃保温3 min后才能完全失活；紫背天葵叶POD最适温度为60℃,茎最适温度在50～70℃之间；紫背天葵叶的POD初始失活常数K为25.5×10-2min-1,其Z值为14.2℃。1 mmol·L-14-HR和2 mmol·L-1植酸处理能够较好的抑制POD的活性,EDTA-2Na盐与Zn(Ac) 2对紫背天葵POD有促进作用。Cu2+对紫背天葵POD有促进作用,Al3+则对POD有显著的抑制作用。
本研究中检测到紫背天葵POD同工酶谱共有10条带。条带1在紫背天葵发育不同阶段的茎叶中均检测到,存在具有普遍性；条带2为茎所特有,条带6、7、8为叶片所特有；条带3、4与紫背天葵采后衰老相关；条带5与组织病症表现密切联系,而条带9、10与腐烂相关。对紫背天葵叶中POD条带1采用加热、硫酸铵分级沉淀、DEAE-Sepharose离子交换层析进行分级纯化,获得了POD条带1单一酶带,经检测蛋白纯度提高了8.1倍,回收率为26.5%。
Gynura bicolor D.C (Gynura), rich in protein and amino acids, edible fiber, minerals, anthocyanin, flavonoid, and essential oil, is concerned as multi-functional plant such as vegetable and medical use. Gynura becomes more and more popular nowdays as a vital force of vegetable market. Like most leaf vegetable, the high levels of transpiration and respiration of Gynura result in the decline of sensory and eating qualities, reduction of nutritious substances and increase of post-harvest loss. The results showed that there was no chilling injury symptom occurred in Gynura stored at 0℃, indicating Gynura is not sensitivie to low temperature and 0℃is appropriate for Gynura storage. This thesis studied the effects of PA,1-MCP and controlled atmosphere (CA) combined nano-packaging on postharvest quality, physiology of Gynura D.C and POD characteristics. The main results are as following:
1. Effect of PA on the changes of quality and physiology of Gynura during refrigerated storage
0,0.1,0.5 and 1.0 mM phytic acid (PA) was applied to Gynura to investigate the effect of PA on changes of quality and physiology of Gynura during refrigerated storage. Compared with control,0.5 mM PA treatment significantly delayed the decay index, maintained cell membrane integrity, slowed down the loss of anthocyanin, chlorophyll, total carbohydrate, reducing sugar, protein and amino acid, and then better color and quality of Gynura after 20 days storage.0.5 mM PA also inhibited significantly respiration compared with control (P<0.05) (the maximum difference could be as much as 54 mg CO2 kg-1.h-1). On the other hand,0.5 mM PA could maintained total phenolics and free phenol at high level, and higher activities of PPO, SOD and POD (84.5%,110% and 120% respectively of that in control) and then greater ability of removing free radical and retarding O2- accumulation. Among all PA treatments,0.5 mM was most effective, and this treatment could extend the storage life to 15 days. It should mention that 1.0 mM treatment could lead to burning symptom on leaves of Gynura.
Correlative analysis reveals significant negative correlation of O2- to POD and SOD, which indicated PA could delay the senscence and therefore prolong the storage period of Gynura.
2. Effect of 1-MCP on the changes of quality and physiology of Gynura during refrigerated storage
0,0.25 ppm,0.50 ppm,1.00 ppm 1-MCP was use to treat Gynura for 0,6h,12 h, and 24 h respectively to investigate the effect of 1-MCP on changes of quality and physiology of Gynura during refrigerated storage. The results showed that the treating concentration and duration of 1-MCP had a significant influence on decay index of Gynura, and less decay was found in higher concentration or/and longer period of 1-MCP treatment and there was no significant difference between 0.50 ppm and 1.00 ppm 1-MCP treatment. However, both 0.50 ppm and 1.00 ppm 1-MCP treated for 24 h enhanced decay of Gynura. All 1-MCP treatment could inhibit the respiration of Gynura, and the higher concentration or/and longer duration had better result, but treatments with 12 h and 24 h,0.50 ppm and 1.00 ppm 1-MCP were no significant difference.1-MCP could siginificantly inhibit the reduction of anthocyanin, chlorophyll and then maintain the better product color and sensory quality.1-MCP could also maintain total carbohydrate, reducing sugar, protein and amino acid at higher levels; reduce O2- and MDA accumulation, keeping higher enzyme activity for removing free radicals and then cell membrane integrity.
Comparisons showed 0.50 ppm 1-MCP was the best among all treated concentrations, and 12 h treatment was the best among all treated duration. Overall, the combination of 0.50 ppm 1-MCP treated 12 h was most suitable treatment for Gynura storage.
3. Effect of CA combined with nano-material packaging on the changes of quality and physiology of Gynura during refrigerated storage
Effect of NP (nano-material packaging), CA (3% O2, CO2≤5%) and CA+NP (CA combined with nano-material packaging) on the changes of quality and physiology of Gynura during refrigerated storage was investigated. The value of Gynura decay index was in the order of CA>NP>CA+NP at the same storage duration, and significantly smaller for the treatments of NP and CA+NP than that of control (P<0.05) at the end of storage with the smallest for CA+NP (6.97%). Respiratory rate of Gynura showed the same change as the tendency of decay index and CA+NP had the lowest respiratory intensity during storage period (only 56.6% of CA at day 20). Compared with CA and NP, CA+NP was more effective to retard the accumulation of O2- and MDA in Gynura tissue, to maintain cell membrane integrity, and had higher activity of enzymes (SOD, CAT) and then removed free radical. Overall, CA(O23%, CO2≤5%)+NP had the best preservation effect on Gynura among 3 treatments during 20 days storage period.
4. Comoarison of harvest time on the quality and physiology of Gynura
Comparison of different harvested time on the quality and physiology of Gynura was made. According to the analysis of substances composition, Gynura harvested in Jun had the highest contents of anthocyanin, chlorophyll, AAs and soluble protein, compared to that of Gynura harvested in April and May. What is especial is that the content of anthocyanin was respectively 2 times and 3 times in that of May and Apr. Gynura had lower respiration and relative leakage rate, lower content of O2.- and MDA production, higher TSS content, and higher relative activities of POD, CAT and SOD. All these indicate Gynura harvested in June produced not only less oxygen free radical but also had strong scavenging ability. This means that Gynura harvested in Jun had better potential to be stored.
According to the analysis of physiological and biochemical changes, Gynura harvested in Jun had the slowest downward trend of chlorophyll, total carbohydrate and protein, and the smallest change of respiratory rate, content of O2.- and MDA. Above results indicated Gynura harvested in Jun had better storage capability.
5. Basic investigation on POD characteristics of Gynura
The phenols extracted from fresh Gynura were investigated by ultraviolet scanning and HPLC. The results showed that there were chlorogenic acid and caffeic acid in both leaf and stem of Gynura. Moreover, cinnamic acid was also existed in leaf and (-)-Catechin in stem. The quantity analysis showed that the main phenol in the leaf and stem of Gynura was chlorogenic acid, chlorogenic acid and (-)-Catechin, respectively.
Pyrogallic acid was the most suitable substrate of POD both from leaves and stems of Gynura and its optimal pH was 5.0 and 6.0, respectively, and had the highest activity at pH 5.0 from leaves, and 5.4 from stems. The optimal temperature of POD from leaves was 60℃,50～70℃from stem. The inactivation of Gynura POD required more than 3 min at 80℃, indicating the strong heat resistance. The calculated K was 25.5×10-2 min-1 and Z was 14.2℃in leaf of Gynura.1 mmol.L-1 4-HR and 2 mmol.L-1 PA had the best inhibition on POD activity of Gynura; EDTA-2Na, Zn(Ac)2 and Cu2+ could enhance the activity, while Al3+ could significangtly inhibit POD activity.
The electrophoresis test showed that the pattern of POD isozymes had 10 bands. Band 1 was catholicity, band 2 was stem specialized, and band 6,7,8 were related to leaves; band 3,4 were closely linked to senescence, band 5 to disease, band 9,10 to decay. Gynura POD band 1 was separated and purified from abstracting liquor of Gynura leaves through salting precipitation, dialysis and DEAE-Sepharose column chromatography. Purfied times of POD were 8.1 and recovery was 26.5%.
1.植酸处理对冷藏紫背天葵品质和生理特性变化的影响
以清水处理为对照,研究0,0.1 mM,0.5 mM和1.0 mM浓度的植酸处理对冷藏(0℃)期间紫背天葵品质和生理变化的影响。0.5 mM植酸处理有效抑制了贮藏期间紫背天葵的腐烂指数和细胞膜渗透率的上升,显著保留了叶绿素、花青素、游离酚、总酚、还原糖、总糖、游离氨基酸和可溶性蛋白的含量,较好地保持了紫背天葵的色泽和品质；呼吸强度显著低于对照组(P<0.05),差值最大可达54mgCO2kg-1·h-1;保持了较高的FP和TP含量；PPO活性为对照的84.5%,SOD和POD活性为对照的1.1倍和1.2倍,保持了较高的自由基清除酶的活性,抑制了O2-含量的增加/积累。所有植酸处理中,0.5m M PA对于紫背天葵保鲜效果最好,延长贮藏期至15天；但1.0 mMPA处理使紫背天葵产生了灼烧现象。
相关性分析显示,O2-与POD和SOD均呈负相关(P<0.05),表明植酸处理可推迟紫背天葵的衰老,有利于保鲜期的延长。
2.1-MCP处理对冷藏紫背天葵品质和生理特性变化的影响
分别以0,0.25 ppm,0.50 ppm和1.00 ppm浓度的1-MCP处理紫背天葵0,6h,12h和24 h研究冷藏期间紫背天葵品质和生理生化变化。试验结果表明,1-MCP处理浓度和时间对紫背天葵腐烂指数有显著作用,提高1-MCP处理浓度和时间,可以减少腐烂的发生,0.50 ppm与1.00 ppm处理组差异不显著,24 h处理较12h加速了紫背天葵组织的崩溃；1-MCP处理的所有浓度和时间,均对紫背天葵呼吸强度有抑制效率,以中高等剂量和时间的处理效果良好,但12h与24 h、0.50 ppm与1.00 ppm处理组之间的差异并不显著；1-MCP处理可显著抑制紫背天葵叶绿素和花青素的降解,使其保持较好的色泽和感官品质；1-MCP可显著延缓贮藏期间紫背天葵还原糖、总糖、游离氨基酸和可溶性蛋白含量的减少,可减少MDA和O2-的生成与积累,保持较高的自由基清除酶的活性,降低细胞膜渗透率,保持较好的细胞膜完整性。
试验结果表明,相同处理时问条件下,0.50 ppm处理效果最优；相同处理浓度条件下,12h处理效果最优；综合所有试验结果,紫背天葵保鲜的最适处理条件为0.50ppm 1-MCP处理12h。
3.气调结合纳米材料包装处理对冷藏紫背天葵品质和生理特性变化的影响
比较了纳米包装袋(NP)、CA(023%,C02<5%)和NP+CA处理对冷藏紫背天葵品质和生理生化变化的影响。相同贮藏时间条件下,紫背天葵的腐烂率都表现为CA>NP>CA+NP,贮藏结束时NP和CA+NP处理紫背天葵腐烂率均显著低于CA处理组(7.64%),且CA+NP处理腐烂率最低(6.97%)；相关性分析显示,CA+NP处理对紫背天葵腐烂率的抑制效果具有显著差异(P<0.05)。贮藏期间3处理紫背天葵的呼吸差异与腐烂率变化情况一致,即CA+NP处理紫背天葵的呼吸强度为3处理中最低的,且贮藏结束时仅为CA处理的56.6%；与CA和NP处理比较,CA+NP减少了紫背天葵组织中O2-与MDA的积累,保持了细胞膜完整性,且相对提高了活性氧清除酶SOD和CAT的活性。综合以上结果,3处理中气调(3%O2,CO2<5%)结合纳米材料包装(NP)用于紫背天葵的保鲜效果最好。
4.不同采收期紫背天葵品质和生理特性比较
试验比较了不同采收时间紫背天葵物质组成和生理生化的差异,并对其贮藏性进行了评价。紫背天葵的物质成分分析显示,6月份采收的紫背天葵的叶绿素、花青素、游离氨基酸和可溶性可溶性蛋白含量均显著高于5月与4月份采收紫背天葵的含量,特别是花青素是5月的近2倍、4月的3倍以上。生理生化分析显示,6月份采收的紫背天葵具有最低的呼吸速率、细胞膜渗透率,最少的O2-和MDA含量；较高的可溶性固形物含量,相对较强的POD、CAT和SOD活性,提示此时的紫背天葵不仅自由基生成少且自我清除能力强,具有较好的潜在贮藏性。
比较贮藏期间物质和生理生化变化的情况可见,6月份采收的紫背天葵贮藏期间,叶绿素、总糖和可溶性蛋白含量下降趋势最缓,呼吸强度、O2-和MDA含量变化幅度最小。以上结果证明,6月份采收的紫背天葵耐贮性最好。
5.紫背天葵POD酶学特性的初步研究
对紫背天葵酚类提取物采用紫外扫描和HPLC等技术进行分析,确定紫背天葵叶和茎中均含有绿原酸、咖啡酸；叶中还含有肉桂酸,以绿原酸最多；茎中还含有表儿茶酚,以绿原酸与表儿茶酚的含量最多。
紫背天葵叶与茎的POD最适底物均为焦性没食子酸,最适pH值分别为pH 5.0和pH 6.0,且在pH 5.0和pH 5.4时酶活性的保留率最高。紫背天葵POD具有较强的耐热性,在80℃保温3 min后才能完全失活；紫背天葵叶POD最适温度为60℃,茎最适温度在50～70℃之间；紫背天葵叶的POD初始失活常数K为25.5×10-2min-1,其Z值为14.2℃。1 mmol·L-14-HR和2 mmol·L-1植酸处理能够较好的抑制POD的活性,EDTA-2Na盐与Zn(Ac) 2对紫背天葵POD有促进作用。Cu2+对紫背天葵POD有促进作用,Al3+则对POD有显著的抑制作用。
本研究中检测到紫背天葵POD同工酶谱共有10条带。条带1在紫背天葵发育不同阶段的茎叶中均检测到,存在具有普遍性；条带2为茎所特有,条带6、7、8为叶片所特有；条带3、4与紫背天葵采后衰老相关；条带5与组织病症表现密切联系,而条带9、10与腐烂相关。对紫背天葵叶中POD条带1采用加热、硫酸铵分级沉淀、DEAE-Sepharose离子交换层析进行分级纯化,获得了POD条带1单一酶带,经检测蛋白纯度提高了8.1倍,回收率为26.5%。
Gynura bicolor D.C (Gynura), rich in protein and amino acids, edible fiber, minerals, anthocyanin, flavonoid, and essential oil, is concerned as multi-functional plant such as vegetable and medical use. Gynura becomes more and more popular nowdays as a vital force of vegetable market. Like most leaf vegetable, the high levels of transpiration and respiration of Gynura result in the decline of sensory and eating qualities, reduction of nutritious substances and increase of post-harvest loss. The results showed that there was no chilling injury symptom occurred in Gynura stored at 0℃, indicating Gynura is not sensitivie to low temperature and 0℃is appropriate for Gynura storage. This thesis studied the effects of PA,1-MCP and controlled atmosphere (CA) combined nano-packaging on postharvest quality, physiology of Gynura D.C and POD characteristics. The main results are as following:
1. Effect of PA on the changes of quality and physiology of Gynura during refrigerated storage
0,0.1,0.5 and 1.0 mM phytic acid (PA) was applied to Gynura to investigate the effect of PA on changes of quality and physiology of Gynura during refrigerated storage. Compared with control,0.5 mM PA treatment significantly delayed the decay index, maintained cell membrane integrity, slowed down the loss of anthocyanin, chlorophyll, total carbohydrate, reducing sugar, protein and amino acid, and then better color and quality of Gynura after 20 days storage.0.5 mM PA also inhibited significantly respiration compared with control (P<0.05) (the maximum difference could be as much as 54 mg CO2 kg-1.h-1). On the other hand,0.5 mM PA could maintained total phenolics and free phenol at high level, and higher activities of PPO, SOD and POD (84.5%,110% and 120% respectively of that in control) and then greater ability of removing free radical and retarding O2- accumulation. Among all PA treatments,0.5 mM was most effective, and this treatment could extend the storage life to 15 days. It should mention that 1.0 mM treatment could lead to burning symptom on leaves of Gynura.
Correlative analysis reveals significant negative correlation of O2- to POD and SOD, which indicated PA could delay the senscence and therefore prolong the storage period of Gynura.
2. Effect of 1-MCP on the changes of quality and physiology of Gynura during refrigerated storage
0,0.25 ppm,0.50 ppm,1.00 ppm 1-MCP was use to treat Gynura for 0,6h,12 h, and 24 h respectively to investigate the effect of 1-MCP on changes of quality and physiology of Gynura during refrigerated storage. The results showed that the treating concentration and duration of 1-MCP had a significant influence on decay index of Gynura, and less decay was found in higher concentration or/and longer period of 1-MCP treatment and there was no significant difference between 0.50 ppm and 1.00 ppm 1-MCP treatment. However, both 0.50 ppm and 1.00 ppm 1-MCP treated for 24 h enhanced decay of Gynura. All 1-MCP treatment could inhibit the respiration of Gynura, and the higher concentration or/and longer duration had better result, but treatments with 12 h and 24 h,0.50 ppm and 1.00 ppm 1-MCP were no significant difference.1-MCP could siginificantly inhibit the reduction of anthocyanin, chlorophyll and then maintain the better product color and sensory quality.1-MCP could also maintain total carbohydrate, reducing sugar, protein and amino acid at higher levels; reduce O2- and MDA accumulation, keeping higher enzyme activity for removing free radicals and then cell membrane integrity.
Comparisons showed 0.50 ppm 1-MCP was the best among all treated concentrations, and 12 h treatment was the best among all treated duration. Overall, the combination of 0.50 ppm 1-MCP treated 12 h was most suitable treatment for Gynura storage.
3. Effect of CA combined with nano-material packaging on the changes of quality and physiology of Gynura during refrigerated storage
Effect of NP (nano-material packaging), CA (3% O2, CO2≤5%) and CA+NP (CA combined with nano-material packaging) on the changes of quality and physiology of Gynura during refrigerated storage was investigated. The value of Gynura decay index was in the order of CA>NP>CA+NP at the same storage duration, and significantly smaller for the treatments of NP and CA+NP than that of control (P<0.05) at the end of storage with the smallest for CA+NP (6.97%). Respiratory rate of Gynura showed the same change as the tendency of decay index and CA+NP had the lowest respiratory intensity during storage period (only 56.6% of CA at day 20). Compared with CA and NP, CA+NP was more effective to retard the accumulation of O2- and MDA in Gynura tissue, to maintain cell membrane integrity, and had higher activity of enzymes (SOD, CAT) and then removed free radical. Overall, CA(O23%, CO2≤5%)+NP had the best preservation effect on Gynura among 3 treatments during 20 days storage period.
4. Comoarison of harvest time on the quality and physiology of Gynura
Comparison of different harvested time on the quality and physiology of Gynura was made. According to the analysis of substances composition, Gynura harvested in Jun had the highest contents of anthocyanin, chlorophyll, AAs and soluble protein, compared to that of Gynura harvested in April and May. What is especial is that the content of anthocyanin was respectively 2 times and 3 times in that of May and Apr. Gynura had lower respiration and relative leakage rate, lower content of O2.- and MDA production, higher TSS content, and higher relative activities of POD, CAT and SOD. All these indicate Gynura harvested in June produced not only less oxygen free radical but also had strong scavenging ability. This means that Gynura harvested in Jun had better potential to be stored.
According to the analysis of physiological and biochemical changes, Gynura harvested in Jun had the slowest downward trend of chlorophyll, total carbohydrate and protein, and the smallest change of respiratory rate, content of O2.- and MDA. Above results indicated Gynura harvested in Jun had better storage capability.
5. Basic investigation on POD characteristics of Gynura
The phenols extracted from fresh Gynura were investigated by ultraviolet scanning and HPLC. The results showed that there were chlorogenic acid and caffeic acid in both leaf and stem of Gynura. Moreover, cinnamic acid was also existed in leaf and (-)-Catechin in stem. The quantity analysis showed that the main phenol in the leaf and stem of Gynura was chlorogenic acid, chlorogenic acid and (-)-Catechin, respectively.
Pyrogallic acid was the most suitable substrate of POD both from leaves and stems of Gynura and its optimal pH was 5.0 and 6.0, respectively, and had the highest activity at pH 5.0 from leaves, and 5.4 from stems. The optimal temperature of POD from leaves was 60℃,50～70℃from stem. The inactivation of Gynura POD required more than 3 min at 80℃, indicating the strong heat resistance. The calculated K was 25.5×10-2 min-1 and Z was 14.2℃in leaf of Gynura.1 mmol.L-1 4-HR and 2 mmol.L-1 PA had the best inhibition on POD activity of Gynura; EDTA-2Na, Zn(Ac)2 and Cu2+ could enhance the activity, while Al3+ could significangtly inhibit POD activity.
The electrophoresis test showed that the pattern of POD isozymes had 10 bands. Band 1 was catholicity, band 2 was stem specialized, and band 6,7,8 were related to leaves; band 3,4 were closely linked to senescence, band 5 to disease, band 9,10 to decay. Gynura POD band 1 was separated and purified from abstracting liquor of Gynura leaves through salting precipitation, dialysis and DEAE-Sepharose column chromatography. Purfied times of POD were 8.1 and recovery was 26.5%.
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