摘要
猕猴桃果肉的鲜绿色主要由其叶绿素决定，是猕猴桃果实的重要商品性状之一，也使其成为成熟后果肉仍保持绿色的少数水果种类之一。然而，叶绿素很不稳定，在加工和贮藏中容易发生降解而失绿，导致一些猕猴桃加工制品呈现黄褐色或淡黄色。因此，研究猕猴桃制浆中叶绿素的降解机理及其护绿方法对于改善以果浆为原料加工的猕猴桃制品的质量具有重要的理论价值和现实意义。
本文以“秦美”猕猴桃为试材，研究采后用化学催熟剂乙烯利处理对其叶绿素及其品质的影响，猕猴桃果肉叶绿素的提取及抗氧化活性，以及制浆中引起猕猴桃果肉中叶绿素降解的因素和降解规律，并对其降解产物进行鉴定，以期提出针对制浆中叶绿素降解的护绿方法，为改善猕猴桃加工制品的质量提供理论依据和技术支持。研究取得以下主要结果：
（1）乙烯利处理显著增强了猕猴桃果实的呼吸速率，提高了可溶性固形物和总酚含量，降低了果实硬度和可滴定酸含量，对猕猴桃果肉的绿色值、抗坏血酸含量和叶绿素含量没有显著影响。乙烯利处理提高了叶绿素酶、脱镁螯合酶、SOD和POD的活性，对APX活性没有显著影响。相关性分析表明，猕猴桃叶绿素的降解与叶绿素酶活性呈显著的正相关。乙烯利处理缩短了猕猴桃果实的成熟时间，约4d即可达到成熟状态，对照则需7d。
（2）通过CCD试验建立了猕猴桃果肉Chl a、Chl b提取的匀浆时间、液固比和提取时间3因素的回归方程，此模型在试验范围内能较准确地预测Chl a、Chl b的提取得率。得到猕猴桃果肉Chl a的最优提取工艺为：匀浆时间9.93min，液固比8.63﹕1，提取时间115min，Chl a的得率为14.22μg·g~(-1)FW；Chl b的最优提取工艺为：匀浆时间11.26min，液固比7.83﹕1，提取时间114.71min，Chl b的得率为12.13μg·g~(-1)FW。与人工合成的抗氧化剂BHA相比，猕猴桃果肉叶绿素提取液对ABTS+和·OH的清除率以及还原能力高于BHA，清除O—
2的活性低于BHA。（3）猕猴桃果浆的叶绿素含量和绿色易受温度及pH值的影响，其热降解反应符合一级反应动力学规律。在相同pH值条件下，随温度升高，Chl a、Chl b和-a*的k降低，t1/2缩短。随pH值增加，Chl a的Ea为14.69～66.02kJ mol-1，Chl b为40.88～54.64kJ mol-1，-a*为48.55～64.14kJ mol~(-1)。Chl a、Chl b降解和-a*损失的相关性在pH值3.3时较好。猕猴桃果实制浆中提高pH值至中性有助于保护果浆的叶绿素含量和绿色。
（4）采用甲醇-水-乙酸乙酯的梯度洗脱系统，可在35min内实现对猕猴桃果浆中叶绿素及其8种衍生物的分析。猕猴桃果浆热处理过程中，果浆的鲜绿色逐渐转变为浅黄绿色，叶绿素主要降解为脱镁叶绿素。热处理3min可杀灭POD，15min可使猕猴桃果浆中叶绿素酶活性下降93%。
（5）在加热条件下，猕猴桃果浆中的Chl b主要降解为脱镁叶绿素b，Chl a则主要降解为焦脱镁叶绿素a。提高猕猴桃果浆的pH值降低了脱镁叶绿素和焦脱镁叶绿素的相对生成率，从而提高了叶绿素的相对保存率。
（6）在pH值5.0以下时，在猕猴桃果浆中添加Zn~(2+)溶液，对Chl a和Chl b的保存率没有显著影响。当调节猕猴桃果浆的pH值为5.0时，加Zn~(2+)溶液的果浆中Chl a和Chl b的相对保存率分别为6.44%和11.04%，而未加Zn~(2+)溶液的果浆中，其Chl a和Chl b的相对保存率则分别为5.45%和9.20%，存在显著差异。继续提高果浆的pH值，则这种显著差异仍然存在，表明在高pH值条件下，加入一定浓度的Zn~(2+)溶液可使Chl a和Chl b的相对保存率得以提高，向果浆中添加Ca~(2+)也有同样的效果。调节猕猴桃果浆的pH值为5.0时，在果浆中添加Zn~(2+)对果浆的a*值的效果优于添加Ca~(2+)，但两者没有显著差异。
（7）与未添加Zn~(2+)或Ca~(2+)的猕猴桃果浆相比，加入Zn~(2+)或Ca~(2+)的果浆中，均能检测到更高含量的脱镁叶绿素和焦脱镁叶绿素。在pH值为5.0的热处理后果浆中，脱镁叶绿素a和b的相对生成率分别为81%和553%，在相同pH值条件下的果浆中添加Ca~(2+)后，脱镁叶绿素a和b的相对生成率分别显著提高至117%和832%，显著高于添加Zn~(2+)的果浆中脱镁叶绿素a和b的相对生成率（100%和671%）。这可能与Zn~(2+)-叶绿素复合物和Ca~(2+)-叶绿素复合物的形成有关。
The attractive green flesh color of kiwifruit is the important commercial character anddetermined by its chlorophylls, and kiwifruit is also among the very few fruit that are stillgreen when they are ready-to-eat. Chlorophylls which are unsteady and easy to degrade andde-greening in producing and storage, which lead to some of kiwifruit products, appearyellow-brown or light yellow color. It has important realistic meaning and theory value forkiwifruit products with kiwifruit puree as raw materials to study breakdown mechanism andgreen color preserving methods of kiwifruit puree during break processing.
Using green-fleshed kiwifruit (Actinidia deliciosa cv. Qinmei) as an experimentalmaterial, this study tested that the effects of ethephon treatment on chlorophylls degradationand quality properties of kiwifruit during ripening, the chlorophylls extraction condition ofkiwifruit flesh and its antioxidant activity in vitro, chrorophylls degradation kinetics andgreen color loss in heated kiwifruit puree and the main degradation derivatives were identified,with a view to preserving green color of puree and to improve the quality of kiwifruitprocessing products. The main results were exhibited as follows:
(1) Ethephon treatment could increase respiration rates significantly and decreasefirmness and titratable acidity of kiwifruit, concomitant with higher soluble solidsconcentrations and total phenol contents. Moreover, ethephon treatment had no significanteffects on green color, ascorbic acid contents or chlorophyll contents. The activities ofchlorophyllase, Mgdechelatase, SOD and POD were enhanced gradually during ripeningcompared to the controls, no significant effects on APX. And a significant positive correlationbetween chlorophyll degradation and chlorophyllase activity was also observed. Ripenedkiwifruit can be obtained through ethephon treatment after4d while controls need7d.
(2) The optimum extraction conditions for highest recovery of chlorophyll a (Chl a) andchlorophyll b (Chl b) were carried out using central composite design (CCD). The optimumextraction conditions for Chl a were9.93min，8.63﹕1，115min，and Chl b were11.26min，7.83﹕1and114.71min，respectively. Predicted values for extraction of Chl a and Chl bagreed well with the experimental values. Antioxidant capabilities of the optimally obtained chlorophylls extracts from kiwifruit revealed better scavenging abilities on ABTS·+and·OH,and reducing power as compared to BHA, while they were weaker than BHA inO2-· scavenging ability.
(3) The chlorophylls and green color of kiwifruit puree were easily influenced bytemperature and pH value. The breakdown of chlorophyll a, chlorophyll b and green colorloss (-a*) values followed a first-order reaction. With the increase of temperature and pHvalue, the rate constant and half-life value of chlorophyll a, chlorophyll b and green color loss(-a*) values decreased. The activation energies ranged from14.69～66.02kJ mol~(-1),40.88～54.64kJ mol~(-1)and48.55～64.14kJ mol-1for chlorophyll a, chlorophyll b and green colorloss (-a*) values, respectively. In addition, significant correlation between chlorophylls andgreen color loss (-a*) values were found for kiwifruit puree at pH3.3. Higher pH value(neutral condition) would benefit for preserving chlorophylls and green color in heatedkiwifruit puree.
(4) Chlorophylls and their derivative were separated within35min using a gradientmobile phase of methanol, water and ethyl acetate. During heating treatment, the bright greencolor of kiwifruit puree turn to light yellow-green color, the main degradation derivatives ofchlorophylls were pheophytins. Heating treatment of3min produced complete PODinactivation in puree when93%inactivation of chlorophyllase activity after15min heatingtime.
(5) Under heating treatment, chlorophyll b degraded to pheophytin b when chlorophyll adegraded to pyropheophytin a. With the pH value of kiwifruit puree enhanced, the relativepheophytins concentration and pyropheophytins concentration declined gradually, resulting inrelative chlorophylls concentration increased.
(6) The has no significant effect on the relative chlorophyll a and chlorophyll bconcentrations when added to the kiwifruit puree with Zn~(2+)solution below pH5.0. When pHvalue was5.0for the kiwifruit puree, the relative chlorophyll a and chlorophyll bconcentration were6.44%and11.04%for pulp with Zn~(2+)solution, while that of the pulp ofno Zn~(2+)solution were5.45%and9.20%respectively, there has significant differencesbetween both. Continue to raise the pH value of the pulp, this significant difference still exists,indicating that the relative chlorophyll a and chlorophyll b concentration can be enhanced atthe high pH value conditions by adding a certain concentration of Zn~(2+)solution. Added to thekiwifruit puree with Ca~(2+)solution also has the same effect. The addition of Zn~(2+)was better fora*value of kiwifruit puree than that of Ca~(2+)when the pH value of kiwifruit puree was5.0,but there has no significant difference between both.
(7) Compared with kiwifruit puree with no added Zn~(2+)or Ca~(2+), there have more relative pheophytins and pyropheophytins concentration were detected in kiwifruit puree with addingZn~(2+)or Ca~(2+)under the different pH values.. The relative pheophytin a and pheophytin bconcentrations of puree were81%and553%at pH5.0after heating treatment, while theirconcentration of puree with added Ca~(2+)significant increased to117%and832%under samepH value, and significant higher than that of added Zn~(2+)puree(100%and671%). This may beexplained that there have Zn~(2+)-chlorophyll complexes and Ca~(2+)-chlorophyll complexformations.
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