酯类物质代谢关键酶对冷藏南果梨香气变淡的影响及其调控

发布时间：2019-01-08 19:05

【摘要】：南果梨是辽宁省的特产水果,采收期为9月上、中旬。采收时的南果梨肉脆、汁少、无香气,但后熟后的南果梨多汁、果肉绵软、香气浓郁。南果梨贮藏期短,在常温贮藏半个月左右达到最佳食用期,随后果实会快速衰老。目前,国内普遍通过冷藏来延长其贮藏期。然而,生产中发现,经过冷藏的南果梨在后熟至最佳食用期时香气明显变淡,严重影响了冷藏南果梨的商品价值。因此,研究南果梨经冷藏后香气变淡的机制有重要的意义。本研究系统的分析了南果梨采后自然后熟及冷藏后常温货架期间酯类香气成分种类及含量的变化、酯类香气代谢合成途径关键酶活性及相应蛋白的表达。采用程序降温处理的方法,调控冷藏南果梨香气转淡的问题。主要研究结果如下:(1)优化western blot检测南果梨组织蛋白质表达的条件,得出优化条件为:选用Trizol法提取南果梨果实总蛋白,聚丙烯酰胺凝胶电泳分离总蛋白的最适上样量为50μg,抗体孵育最适时间为一抗室温、摇床条件下孵育2 h,二抗置于摇床上在室温条件下孵育1h,采用ECL化学发光分析试验结果,南果梨果实Actin、LOX及AAT蛋白一抗的最适稀释比分别为1:400、1:200和1:600。(2)冷藏90 d和180 d后的常温货架期间,酯类香气成分的种类和总酯含量均出现减少,且冷藏时间越长,酯类香气成分损失越严重。冷藏180d的南果梨果实在常温货架期间,脂氧合酶(lipoxidase,LOX)和醇酰基转移酶(alcohol acyltransferase,AAT)活性及LOX蛋白表达出现的高峰较冷藏90 d的处理提早3 d,LOX和AAT的蛋白表达及AAT活性在整个常温货架期间显著低于冷藏90 d的处理,冷藏后常温最佳食用期时LOX蛋白表达、AAT活性与对应蛋白表达量均受到抑制,且随冷藏时间延长,抑制现象越明显。说明AAT活性、AAT及LOX蛋白表达的下调可能影响了南果梨果实酯类香气物质的合成,可能与南果梨果实酯类香气的变淡相关。(3)程序降温处理的南果梨冷藏期间及冷藏90d后的常温货架期间,当果实后熟至最佳食用期时酯类香气的种类和总酯含量较常规冷藏组有大幅度提升,主要的酯类香气成分均显著高于常规冷藏组。程序降温处理提高了 LOX和AAT的活性和对应蛋白表达量,可能在一定程度上促进了南果梨酯类香气的生成。
[Abstract]:Nanguo Pear is a special fruit in Liaoning Province. The flesh of Nanguo pear is crisp, less juice, no aroma, but after ripening, Nanguo pear is juicy, soft and fragrant. Nanguo pear has a short storage period and reaches the optimum edible period at room temperature for about half a month, and then the fruit will senescence quickly. At present, cold storage is widely used in China to prolong its storage period. However, it was found in the production that the aroma of the refrigerated Nanguo pear decreased obviously from the later ripening to the optimum edible period, which seriously affected the commercial value of the refrigerated Nanguo pear. Therefore, it is of great significance to study the mechanism of aroma thinning after cold storage of Nanguo pear. This study systematically analyzed the changes of the kinds and contents of ester aroma components, the key enzyme activities and the expression of corresponding proteins in the metabolic synthesis pathway of Nanguo pear during the natural ripening after harvest and at room temperature after cold storage. The method of programmed cooling was used to control the problem of aroma thinning of refrigerated Nanguo pear. The main results are as follows: (1) the optimal conditions for detecting protein expression in Nanguo pear tissue by western blot were obtained as follows: extraction of total protein from Nanguo pear by Trizol method. The optimum sample amount for the separation of total protein by polyacrylamide gel electrophoresis was 50 渭 g, the optimum incubation time of antibody was room temperature of the first antibody, the second antibody was incubated in shaking bed for 2 hours, and the second antibody was incubated at room temperature for 1 hour. The results of ECL chemiluminescence analysis showed that the optimal dilution ratios of Actin,LOX and AAT proteins were 1: 400: 1 200 and 1: 600 respectively. (2) the shelf life at room temperature after 90 days and 180 days of cold storage. The species and total ester contents of ester aroma components decreased, and the longer the storage time was, the more serious the loss of ester aroma components was. The peak of lipoxygenase (lipoxidase,LOX) and alcohol acyltransferase (alcohol acyltransferase,AAT) activity and the expression of LOX protein in fruits of Nanguo pear were 3 days earlier than those of 90 days of cold storage. The protein expression and AAT activity of LOX and AAT were significantly lower than those of 90 d cold storage treatment during the whole shelf life at room temperature. The expression of LOX protein, AAT activity and corresponding protein expression were inhibited during the optimum eating period of normal temperature after cold storage, and the expression of AAT and corresponding protein were inhibited with the prolongation of cold storage time. The more obvious the inhibition was. These results suggested that the downregulation of AAT activity and the expression of AAT and LOX proteins might affect the synthesis of esters in the fruits of Nanguo pear. It may be related to the light aroma of fruit esters. (3) the cold storage period of Nanguo pear and the shelf period of normal temperature after 90 days of cold storage. When the fruit matured to the best edible stage, the species and total ester contents of esters increased significantly, and the main ester aroma components were significantly higher than those of the conventional cold storage group. The programmed cooling treatment increased the activity of LOX and AAT and the expression of corresponding proteins, which may promote the formation of esters in Nanguo pear to some extent.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS255.1

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