摘要
莲藕(Nelumbo nucifera)是中国重要的特色水生蔬菜和出口创汇蔬菜,其营养丰富,具有清热生津、滋补胃阴等药用价值。莲藕含水量丰富、皮薄,外观洁白脆嫩,表皮保护性差、易破损,贮藏期间易失水、干缩和变色。莲藕适合鲜切加工,但切分形成的机械损伤极易导致表皮和肉质部褐变,影响莲藕感官品质和内在质量。本试验的前期研究结果表明,4℃条件下鲜切莲藕经褐变抑制剂处理后最长可贮藏15天,且明确莲藕的褐变主要是酶促褐变。本试验比较了江苏种植最多“美人红”和南京地区销售最普遍“3735”两品种莲藕鲜切后的品质及生理生化变化,并以“3735”为试材进一步探索常温贮藏条件下鲜切莲藕褐变模型建立、酚类定性及褐变前后蛋白差异表达,以深入研究鲜切莲藕褐变的机制。本研究的主要结果如下：
1.“美人红”和“3735”贮藏过程中品质和生理特性的比较
以“美人红”和“3735”为材料,比较了鲜切后4℃贮藏期间两品种品质和生理特性的变化。结果显示,“美人红”可溶性蛋白质、Vc及总酚含量较高,褐变相关酶POD和SOD的活性较强,而“3735”呼吸强度大、丙二醛生成量多。“美人红”和“3735”两品种莲藕鲜切后低温贮藏期间的品质和生理特性变化虽然在大多数指标上出现较为一致的趋势,如褐变加重、呼吸增强、还原糖增加、可溶性蛋白和总酚减少、Vc减少、O2·-和MDA增加、POD活力降低和SOD活力增加等,但“3735”褐变更快,呼吸增强更多,还原糖增加更迅速,O2·-含量上升较慢,MDA增加较多。因此,“美人红”品种更合适鲜切加工。
2.鲜切莲藕贮藏期间褐变数学模型的建立
选用极易褐变的“3735”作为材料,以清水处理为对照(CK),比较了五种保鲜剂0.1%NaHSO3、0.2%柠檬酸(CA)、0.01%抗坏血酸(AA)、0.1%L-半胱氨酸(Cys)以及复合保鲜剂(CP:0.1%、CA、0.005%AA和0.05%Cys)处理对低温条件下鲜切莲藕品质和生理生化变化的影响。结果显示,相同条件下,CP处理组褐变较轻,MDA、呼吸强度、O2·-及H202含量较低,SOD、POD、CAT活性较高。
鲜切莲藕贮藏过程中褐变主因子分析显示,褐变与品质和生理的指标如L*、电导率、呼吸强度、MDA含量、活性氧含量及POD活性有关,其中L*贡献率高达64%；次因子分析褐变与SOD、CAT有关。以直观反映褐变度的L*值作为因变量,其它9个指标作为自变量,对L*作线性回归分析,经逐步回归后,建立鲜切莲藕L*的回归模型预测方程为：L*=65.784-11.062x3-0.101x5+0.003x6(其中X3为MDA,X5为O2·-,X6为POD)。线性回归分析可见,鲜切莲藕贮藏过程中L*与MDA含量、O2·-含量呈显著负相关,与POD活性呈显著正相关。
3.鲜切莲藕褐变过程中酚类与褐变关系及清除自由基的能力
试验选取不同部位的莲藕比较酚类物质含量及其清除自由基能力,并用HPLC方法定性和定量酚类物质。结果表明,莲藕不同部位酚类物质含量存在显著差异：藕皮7.19mg/g,藕节7.61mg/g,藕尖2.54mg/g,藕片2.10mg/g。藕皮中含有焦性没食子酸、羟酪胺和儿茶酚三种酚,且以焦性没食子酸为主；藕节中的主要酚类物质是羟酪胺,藕尖中只有没食子酸,藕片中则含有焦性没食子酸、羟酪胺、儿茶酚和咖啡酸四种。等量酚类物质条件下,莲藕不同部位清除率的情况为：藕片>藕尖>藕皮>藕节,四个部位酚类提取物的抗氧化能力的大小并不与其总酚含量的多少成正比,鲜切莲藕中总酚含量的高低与清除自由基的能力没有显著相关。鲜切莲藕贮藏过程中总酚含量呈现下降趋势,单位质量酚清除自由基能力则呈现先升后降最后略有上升的趋势。这些都说明酚类物质含量和组成的不同与其抗氧化能力存在密切联系。
常温贮藏期间,鲜切藕片中的酚类物质种类不变,但含量会改变,其中与莲藕褐变关系密切的为焦性没食子酸、儿茶酚和咖啡酸,其中焦性没食子酸与莲藕清除自由基能力关系密切。
4.鲜切莲藕褐变前后蛋白表达差异研究
采用酚抽法和改良后的TCA-丙酮法提取莲藕中总蛋白,运用双向电泳和MALDI-TOF-TOF/MS分析及数据库检索鉴定鲜切莲藕褐变前后的差异蛋白种类及功能。试验确立了适合鲜切莲藕蛋白差异分析的双向电泳条件为：酚抽法提取总蛋白,1500μ的蛋白上样量,选用17cm,pH4-7胶条,12%胶浓度,G-250考染。
通过比对褐变前后鲜切莲藕蛋白2-DE图谱,发现40个具有统计学意义且差异大于2倍或者小于0.5的蛋白点,其中5个褐变前存在而褐变后消失,5个褐变后新出现,8个随着褐变发生表达量上调、22个表达下调。
40个差异蛋白点共鉴定成功38个,鉴定成功率为95%,包括4个贮藏蛋白和34个功能蛋白。34个功能蛋白中4个蛋白(L1、L3、L15、L20)只在褐变前表达,2个蛋白(L5、L12)只在褐变后表达,7个蛋白(L11、L16、L17、L21、L28、L32、L33)褐变后上调表达,其余21个差异蛋白褐变后下调表达。
对鲜切莲藕褐变前后发生变化的蛋白进行鉴定后按功能分类,这些蛋白涉及到应激反应(7个)、细胞结构(4个)、物质和能量代谢(12个)、呼吸(4个)、信号转导(2个)、基因表达(2个),未知功能(3个),其中变化最多的差异蛋白与物质代谢调控、活性氧代谢以及呼吸调控等相关。
鲜切莲藕褐变前后蛋白表达差异的比较显示,与褐变相关的蛋白主要功能集中在植物应激反应、细胞呼吸及物质代谢和能量代谢等方面,具体体现在通用应激蛋白USP、活性氧相关蛋白SOD、POD、铁蛋白以及细胞内能量转换核心酶ATPase等的差异表达上,这些蛋白褐变后减量表达,显示莲藕褐变后细胞代谢调控途径和方向发生改变,对抗外界环境的胁迫反应能力下降,分解代谢加强,能量利用率降低,Cu/Zn-SOD和硫氧还蛋白过氧化物酶(TPx)表达量降低,氧自由基清除能力减弱,细胞结构破坏剧烈。
Lotus root (Nelumbo nucifera) is one of the important hydrophytic perennial and export vegetable in China with rich nutrients and medical value. Lotus root has the characteristics of high water, thin skin, white color and crisp texture, but is easy to suffer water loss, shrinkage and color change during storage period. Although lotus root is suitable for fresh-cut use, the mechanical injury due to cutting process would result in serious browning thereafter quality deterioration both sensory and objective. The previous experiment showed that the maximum storage life of fresh-cut lotus root at4℃is about15days, and the browning is mainly enzymatic. This study was focused on "Meirenhong"(most important cultivar planted in Jiangsu province) and "3735"(most commonly consumered in Nanjing markets) to determine the physiological and biochemical changes after fresh-cut during storage, and further to investigate the browning model, changes of phenols and differentiate of protein expression during browning process at room temperature by taking "3735" as experimental material. The main results are as follows:
1. Comparison of quality and physiological characteristics of "Meirenhong" and "3735"
Quality and physiological characteristics of "Meirenhong" and "3735" during storage at4℃were measured and compared. The results showed that the higher contents of soluble protein, Vc and total phenol (TP), and the greater peroxidase (POD) and superoxide dismutase (SOD) activities were observed in "Meirenhong", while "3735" had higher respiratory rate and malondialdehyde (MDA) content. Although the change trends of most measured indexes were similar between "Meirenhong" and "3735" during storage, such as becoming more servere browning, enhancing the respiration intensity, increased in reducing sugar, O2·-and MDA, reduced in losses of soluble protein, TP and Vc, decreased in POD activity and increased in SOD activity, etc."3735" was easier to brown and had a greater respiratory rate, a greater increase in reducing sugar and MDA content, and slower O2·-increase. Overall,"Meirenhong" is more appropriate for fresh-cut use.
2. Mathematical Model on browning of fresh-cut lotus root during storage
Six treatments of water (CK),0.1%NaHSO3,0.2%citric acid (CA),0.01%ascorbic acid (AA),0.1%L-cysteine (Cys) and composite preservative (CP:0.1%CA,0.005%AA and0.05%Cys) were used to determine the effect of each treatment on quality and physiology of fresh-cut lotus root during storage at low temperature. Compared to all treatments, CP treatment had less browning, lower levels of MDA, respiration, O2·-and H2O2and higher activities of SOD, POD and CAT.
The primary factor on the browning of fresh-cut lotus root was mainly related to the quality and physiological indicators such as L*, conductivity, respiration, MDA content, reactive oxygen and POD activity, and the contribution rate of L*was64%. The secondary factor was mainly related to SOD and CAT activities. As the visual reflect of browning degree, L*was used for dependent variable, other nine indexes were used for independent variables, and linear regressive analysis was presented. The simplified regression model was L*=65.784-11.062x3-0.101x5+0.003x6(x3:MDA, x5:O2·-,X6:POD). The regression analysis showed that browning degree had significant negative correlation with MDA and O2·-content, but had significant positive correlation with POD activity.
3. Relationship between phenol and browning of fresh-cut lotus root and free radical scavenging capacity in during browning
Different parts of lotus root were chosen to compare phenol content and free radical scavenging capacity. HPLC method was used to detect the components and quantitative phenol. The results show that the total phenol contents in different parts of lotus root were different:peel7.19mg/g, node7.61mg/g, tip2.54mg/g and root flesh2.10mg/g. The components of phenols in peel were pyrogallic acid, catechol and hydroxyltyramine, and mainly was gallic acid. The main phenol in node was hydroxyltyramine, gallic acid in tip. The flesh of lotus root contained gallic acid, hydroxyltyramine, catechin and caffeic acid. Under the same phenol condition, free radical scavenging capacities in different parts of lotus root were also different:root>tip>peel>node, and this was not in direct proportion to phenol content. There was no significant relationship between phenol contents and free radical scavenging capacity. The phenol content in fresh-cut lotus root present a down trend during storage, while free radical scavenging capacities showed an ascending-descending-ascending trend.There was close relationship between the difference of phenol contents and composition and antioxidant capacity.
Phenol composition in fresh-cut lotus root was the same during storage at room temperature, and pyrogallic acid, catechol and caffeic acid were closely related to browning, especially the amount of pyrogallic acid.
4. Differential expression of proteins before and after browning in fresh-cut lotus root
Trichloroacetic acid/acetone precipitation and phenol extraction were used to determine their efficiency on extracting total proteins from fresh-cut lotus root. Differential expression of proteins was analyzed by2-DE and MALDI-TOF-TOF. The suitable2-DE analysis conditions for lotus root were:phenol extraction for protein extraction,1500ug sample handling, IPG for17cm with pH4-7,12%gel concentration and G-250staining.
By comparing2-DE protein patterns of fresh-cut lotus root before and after the browning,40protein spots were detected which had statistically significant and the difference was greater than2times or less than0.5. Among them,5spots only exited before browning, and5new spots appeared after browning,8spots increased and22spots decreased with the progress of browning.
38spots were successfully identified based on all available information, including4storage and34functional proteins, and successful identification rate was95%. There were4proteins (L10, L3, L15and L20) expressed only before browning,2proteins (L5and LI2) expressed only after browning,7proteins (L11, L16, L17, L21, L28, L32and L33) up-expressed after browning, and the last21proteins down-expressed.
The34functional proteins were classified into following groups:stress response (7), cell structure (4), material and energy metabolism (12), respiration (4), signal transduction (2), gene expression (2) and unknown protein (3). The group with greatest difference in protein expression was related to material metabolism and regulation, reactive oxygen species metabolism and respiratory control.
Differential expression showed that function of distinct proteins was focut on stress response, respiratory, material and energy metabolism, etc. The distinct proteins included USP, SOD, POD, ferrin, ATPase and so on. The down-regulation of these proteins after brown showed that the way and direction of metabolic control and regulation changed, stress response activity declined, catabolism strengthened, energy utilization decreased, expression of Cu/Zn-SOD and TPx decreased, activity of cleaning reactive oxygen system weakened, and destruction of cell structure got intense after brown.
引文
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