采前喷布壳聚糖处理和采后适度失水处理对红地球葡萄保鲜效应研究
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摘要
葡萄是营养价值和经济价值都很高的浆果之一。但是，由于葡萄含糖量高、水分多、果肉柔软而极易受机械损伤和病原菌的侵染，严重影响了葡萄的物流和周年供应。随着鲜食葡萄产量和国内国际市场需求量的不断扩大，葡萄的贮藏保鲜技术研究越来越受到人们的重视。目前国内外普遍使用SO2制剂保鲜葡萄，但是由于SO2残留会对人体健康造成威胁，现在已被许多国家限制使用。
壳聚糖具有安全无毒、抑菌、成膜、可降解等特性，近年来人们已经开始研究其在诱导果蔬抗病性方面的机制。本论文以红地球葡萄为材料，采前喷布0.1%、0.5%和1.0%壳聚糖，研究其对果实贮藏品质、质地特性、超微结构和采后生理等性质的影响，探讨采前喷布壳聚糖处理在葡萄保鲜中产生抗病反应的机制和通过诱导果实产生抗病性而控制采后病害发生的可行性；试验发现并验证了“淋激效应”现象，提出了“淋激效应”概念，并对其损伤机理进行了初步研究；同时为了探讨新的无硫保鲜方法，研究了采后适度失水处理对葡萄品质、电学特性和相关酶活性的影响，旨在为葡萄采后绿色保鲜提供理论和实践依据。研究主要取得以下结果：
（1）采前对红地球葡萄喷布浓度为0.1%、0.5%和1.0%的壳聚糖溶液，可以显著抑制果实冷藏中SSC和TA含量的下降，保持了果实品质。采前壳聚糖处理有助于红地球葡萄果粒质地的保持，显著抑制了果粒硬度、咀嚼性和凝聚性的下降，保持了果实弹性值；在贮藏后期，处理抑制了果实黏着性绝对值的上升，有助于果实回复性的保持。
（2）采前壳聚糖处理使红地球葡萄果肉具有较低的ABA积累，保持了较高的IAA、GA3、ZR含量及ZR/ABA和GA3/ABA比值；同时显著抑制了果皮中ABA含量的积累，使果皮中具有较高的IAA/ABA和ZR/ABA比值。
（3）采前0.1%、0.5%和1.0%壳聚糖处理提高了红地球葡萄中的总酚和类黄酮含量，显著提高了果实中的可溶性蛋白质含量。壳聚糖处理显著提高了果实中POD、PPO、SOD、PAL和GLU活性，诱导了果实的抗病性。
（4）通过透射电镜观察了采前喷布壳聚糖处理对果实果皮超微结构的影响：贮藏第1d，处理和对照果实结构相似，壳聚糖处理果实果皮细胞中可见大分子沉积物，细胞壁有增厚现象，0.5%处理果皮细胞壁中可见纹孔；贮藏第75d，壳聚糖处理葡萄果皮的细胞壁、中胶层、叶绿体和线粒体结构基本完整，纹孔多，细胞中可见大分子沉积物；对照果皮细胞壁松弛，中胶层大量溶解、消失，叶绿体逐渐解体，线粒体数目增多，纹孔较少，细胞中未见沉积物。
（5）在进行采前喷布壳聚糖处理保鲜葡萄试验中，摘袋后立即对葡萄喷淋壳聚糖溶液，发现果实冷藏至20d～30d时开始出现果皮开裂、果肉组织塌陷、汁液外渗等异常症状，由此提出了葡萄“淋激效应”概念。随后对“淋激效应”现象进行了验证，并对其损伤机理进行了初步研究。壳聚糖“淋激”处理果实的SSC高于其他处理，SSC和TA含量出现波动，在第45d达到高峰，而后下降。“淋激效应”促进了果实POD活性的升高，导致SOD、CAT和PPO活性下降，使果实发生损伤并在贮藏中快速腐烂。
（6）对红地球葡萄采收后冷藏过程中的电学参数进行了研究。结果表明，第一类电学参数Z、Lp、X、Rp、Y和B与测试频率之间存在着明显的指数关系；第二类参数θ、tanδ、Cp和Q因子与频率之间则不存在这种关系。在0.1kHz～3980kHz所测频率范围内，葡萄冷藏中Z、Lp、X和Rp随测试频率升高呈直线下降，Y和B呈直线上升；在同一频率下，果实Z、Lp、X和Rp随着贮藏时间的增加呈线性下降趋势。
（7）在0d～45d冷藏时间内，葡萄质地参数硬度和咀嚼性随着果实的衰老逐渐下降，黏着性绝对值和弹性呈上升趋势。在最佳测试频率0.1kHz下，电学参数与质地参数呈现较高相关性，并建立了X与质地参数硬度、黏着性、弹性和咀嚼性之间的回归方程，为实现葡萄无损检测提供了依据。
（8）葡萄在采后贮藏前进行失水处理是一种安全无硫的保鲜方法，适度失水处理有助于果实品质的保持，其中失水3%处理果实硬度和PPO活性显著（P<0.05）高于对照，POD活性极显著（P<0.01）高于对照。在0d～45d贮藏期内，随着葡萄的衰老，失水3%处理使葡萄保持了较高的Z、Lp、X、Rp和较低的ξ'和θ。
Grapes are one kind of fruits which have high nutritional and economic value, but theyare so easily mechanical damaged and pathogen infected because of their soft pulp, high sugarand moisture content that the logistics and anniversary supply are seriously affected. Nowthey are often combated by application of sulfur dioxide (SO2) to maintain quality. However,SO2is dangerous to people who are allergic to sulfites and its applications have beenrestricted in many countries.
Chitosan is non-toxic, safe, antibacterial, film, and degradable, so people have becomestudy the mechanism of its induction of disease resistance in fruit and vegetable. Toinvestigate the effect of chitosan preharvest sprays on quality, texture, ultrastructure andpostharvest physiology of Red globe grape, the fruits were treated with0.1%,0.5%and1.0%chitosan solution before harvest and then stored at (0±1)℃. On the basis of preharvesttreatment, the role of chitosan in grape fresh-keeping was explored; the mechanism ofchitosan induced disease resistance of grape was studied. In the end, the feasibility wasdiscussed if it is possible to control postharvest diseases of grapes by preharvest chitosansprays. At the same time, the effect of moderate dehydration treatment on grape was studiedin order to explore a new method no sulfur dioxide of fresh-keeping. The objective oftreatment was to study the effect of dehydration on quality, dielectrical parameters andphysiology of grape. Through these studies, it is possible to provide theoretical and practicalbasis for green method. All the results were showed below:
1. The fruits were treated with0.1%,0.5%and1.0%chitosan solution before harvest andthen stored at (0±1)℃. The results indicated that preharvest sprays with chitosan couldsignificantly keep the soluble solids and titratable acid content, the chitosan treatment washelpful on keeping the quality of grapes. The chitosan sprays could inhibit the decline ofhardness, chewiness and cohesiveness, keep the springiness of grape. In the late storage, thechitosan sprays were helpful of keeping resilience and inhibition adhesiveness rise of grape fruit.
2. The ABA content of pulp in chitosan treated group was lower than control, but thecontent of IAA, GA3, ZR, and the value of ZR/ABA and GA3/ABA were higher than control.Preharvest sprays with chitosan could also maintain the value of IAA/ABA and ZR/ABA,inhibit the ABA content in pericarp at the same time.
3. The content of total phenolics, flavonoids and soluble protein in pulp in treated groupwas higher than control, the same as the activity of POD, PPO, SOD, PAL and GLU, all ofthese indicated that chitosan sprays induced the disease resistance of grape.
4. Furthermore, transmission electron microscope was used to observe the ultrastructureof the grape. The results showed that in the first day of storage, there was no significantdifference between chitosan treated grape and control except for macromolecular sedimentand thicker cell wall in chitosan treated grape cell. There were some pits in cell wall in grapesprayed with0.5%chitosan. After seventy-five days, the middle lamella of preharvest treatedgrape was not completely dissolved; leaving a lot of small microfibrils, and the primary cellwall was integrated. Chloroplast and mitochondria of preharvest-treated grape were integrated;there were pits in cell wall, and macromolecular sediment in the cell. But the cell wallstructure of control grape swell and flab, the primary cell became crooked and thicker; mostof the middle lamella was dissolved and disappear. Chloroplast gradually becamedisorganization, the brink of membrane and grana lamella was unclear. Mitochondriaremained numerous, mitochondrial crista disappeared. There had little pits and nomacromolecular sediment in the cell.
5. In the paper, the effect of spray-shock was found and put forward for the first time inthe process of preharvet treatment, the possible mechanism was also been studied about howthe spray-shock led grape injury in the storage. The grape damaged in cold storage wouldappear such phenomenon as peel cracking, pulp collapse and juice outflow from20d to30d,all of these made fruit quality decrease rapidly and grape became rot quickly. Fruitspray-shocked with1.0%chitosan had higher soluble solid content than control fruit. Solublesolid and titratable acid content fluctuated and appeared a pear in45d. The spray-shockedfruit had higher POD activity than un-shocked fruit. Compared with the un-shocked grape, theshocked grape had lower SOD, CAT and PPO activity. The spray-shock led to the injury andfast decay of grape in the storage.
6. Dielectric parameters of Red globe grape during cold storage at0℃werenon-destructively measured over frequency range from0.1kHz to3980kHz. The resultsshowed that there are significant exponential relationship between impedance, resistance,inductance reactance, admittance, susceptance and frequency, but no exponential relationship for other dielectric parameters. Fruit impedance, resistance, inductance and reactancedecreased gradually by linear form with the increase of frequency, admittance andsusceptance increased linearly. At the optimum test frequency, impedance, resistance,inductance and reactance decreased linearly with the extension of storage time.
7. Firmness, chewiness, springiness and adhesiveness of grape were measured as textureattributes. In the storage of45d, fruit firmness and chewiness decreased gradually with thetime went on, springiness and the absolute value of adhesiveness went up contrarily. At0.1kHz frequency, the high correlation was found between dielectric and texture parameters,such as reactance with texture parameters (P<0.05). At last, the regression equation was builtfor dielectric and texture parameters, it was possible for the realization of grapenondestructive testing.
8. Postharvest dehydration treatment was a safe fresh-keeping method without sulfur. Allthe test results showed that postharvest moderate dehydration was one more effective way inkeeping the quality of grape during cold storage. Grape dehydrated3%has significant higherfirmness, PPO and POD activity than control. Compared with the un-dehydrated grape, thedehydrated one has higher impedance, reactance, inductance, resistance and lower dielectricconstant, impendence phase angle during45days storage.
壳聚糖具有安全无毒、抑菌、成膜、可降解等特性，近年来人们已经开始研究其在诱导果蔬抗病性方面的机制。本论文以红地球葡萄为材料，采前喷布0.1%、0.5%和1.0%壳聚糖，研究其对果实贮藏品质、质地特性、超微结构和采后生理等性质的影响，探讨采前喷布壳聚糖处理在葡萄保鲜中产生抗病反应的机制和通过诱导果实产生抗病性而控制采后病害发生的可行性；试验发现并验证了“淋激效应”现象，提出了“淋激效应”概念，并对其损伤机理进行了初步研究；同时为了探讨新的无硫保鲜方法，研究了采后适度失水处理对葡萄品质、电学特性和相关酶活性的影响，旨在为葡萄采后绿色保鲜提供理论和实践依据。研究主要取得以下结果：
（1）采前对红地球葡萄喷布浓度为0.1%、0.5%和1.0%的壳聚糖溶液，可以显著抑制果实冷藏中SSC和TA含量的下降，保持了果实品质。采前壳聚糖处理有助于红地球葡萄果粒质地的保持，显著抑制了果粒硬度、咀嚼性和凝聚性的下降，保持了果实弹性值；在贮藏后期，处理抑制了果实黏着性绝对值的上升，有助于果实回复性的保持。
（2）采前壳聚糖处理使红地球葡萄果肉具有较低的ABA积累，保持了较高的IAA、GA3、ZR含量及ZR/ABA和GA3/ABA比值；同时显著抑制了果皮中ABA含量的积累，使果皮中具有较高的IAA/ABA和ZR/ABA比值。
（3）采前0.1%、0.5%和1.0%壳聚糖处理提高了红地球葡萄中的总酚和类黄酮含量，显著提高了果实中的可溶性蛋白质含量。壳聚糖处理显著提高了果实中POD、PPO、SOD、PAL和GLU活性，诱导了果实的抗病性。
（4）通过透射电镜观察了采前喷布壳聚糖处理对果实果皮超微结构的影响：贮藏第1d，处理和对照果实结构相似，壳聚糖处理果实果皮细胞中可见大分子沉积物，细胞壁有增厚现象，0.5%处理果皮细胞壁中可见纹孔；贮藏第75d，壳聚糖处理葡萄果皮的细胞壁、中胶层、叶绿体和线粒体结构基本完整，纹孔多，细胞中可见大分子沉积物；对照果皮细胞壁松弛，中胶层大量溶解、消失，叶绿体逐渐解体，线粒体数目增多，纹孔较少，细胞中未见沉积物。
（5）在进行采前喷布壳聚糖处理保鲜葡萄试验中，摘袋后立即对葡萄喷淋壳聚糖溶液，发现果实冷藏至20d～30d时开始出现果皮开裂、果肉组织塌陷、汁液外渗等异常症状，由此提出了葡萄“淋激效应”概念。随后对“淋激效应”现象进行了验证，并对其损伤机理进行了初步研究。壳聚糖“淋激”处理果实的SSC高于其他处理，SSC和TA含量出现波动，在第45d达到高峰，而后下降。“淋激效应”促进了果实POD活性的升高，导致SOD、CAT和PPO活性下降，使果实发生损伤并在贮藏中快速腐烂。
（6）对红地球葡萄采收后冷藏过程中的电学参数进行了研究。结果表明，第一类电学参数Z、Lp、X、Rp、Y和B与测试频率之间存在着明显的指数关系；第二类参数θ、tanδ、Cp和Q因子与频率之间则不存在这种关系。在0.1kHz～3980kHz所测频率范围内，葡萄冷藏中Z、Lp、X和Rp随测试频率升高呈直线下降，Y和B呈直线上升；在同一频率下，果实Z、Lp、X和Rp随着贮藏时间的增加呈线性下降趋势。
（7）在0d～45d冷藏时间内，葡萄质地参数硬度和咀嚼性随着果实的衰老逐渐下降，黏着性绝对值和弹性呈上升趋势。在最佳测试频率0.1kHz下，电学参数与质地参数呈现较高相关性，并建立了X与质地参数硬度、黏着性、弹性和咀嚼性之间的回归方程，为实现葡萄无损检测提供了依据。
（8）葡萄在采后贮藏前进行失水处理是一种安全无硫的保鲜方法，适度失水处理有助于果实品质的保持，其中失水3%处理果实硬度和PPO活性显著（P<0.05）高于对照，POD活性极显著（P<0.01）高于对照。在0d～45d贮藏期内，随着葡萄的衰老，失水3%处理使葡萄保持了较高的Z、Lp、X、Rp和较低的ξ'和θ。
Grapes are one kind of fruits which have high nutritional and economic value, but theyare so easily mechanical damaged and pathogen infected because of their soft pulp, high sugarand moisture content that the logistics and anniversary supply are seriously affected. Nowthey are often combated by application of sulfur dioxide (SO2) to maintain quality. However,SO2is dangerous to people who are allergic to sulfites and its applications have beenrestricted in many countries.
Chitosan is non-toxic, safe, antibacterial, film, and degradable, so people have becomestudy the mechanism of its induction of disease resistance in fruit and vegetable. Toinvestigate the effect of chitosan preharvest sprays on quality, texture, ultrastructure andpostharvest physiology of Red globe grape, the fruits were treated with0.1%,0.5%and1.0%chitosan solution before harvest and then stored at (0±1)℃. On the basis of preharvesttreatment, the role of chitosan in grape fresh-keeping was explored; the mechanism ofchitosan induced disease resistance of grape was studied. In the end, the feasibility wasdiscussed if it is possible to control postharvest diseases of grapes by preharvest chitosansprays. At the same time, the effect of moderate dehydration treatment on grape was studiedin order to explore a new method no sulfur dioxide of fresh-keeping. The objective oftreatment was to study the effect of dehydration on quality, dielectrical parameters andphysiology of grape. Through these studies, it is possible to provide theoretical and practicalbasis for green method. All the results were showed below:
1. The fruits were treated with0.1%,0.5%and1.0%chitosan solution before harvest andthen stored at (0±1)℃. The results indicated that preharvest sprays with chitosan couldsignificantly keep the soluble solids and titratable acid content, the chitosan treatment washelpful on keeping the quality of grapes. The chitosan sprays could inhibit the decline ofhardness, chewiness and cohesiveness, keep the springiness of grape. In the late storage, thechitosan sprays were helpful of keeping resilience and inhibition adhesiveness rise of grape fruit.
2. The ABA content of pulp in chitosan treated group was lower than control, but thecontent of IAA, GA3, ZR, and the value of ZR/ABA and GA3/ABA were higher than control.Preharvest sprays with chitosan could also maintain the value of IAA/ABA and ZR/ABA,inhibit the ABA content in pericarp at the same time.
3. The content of total phenolics, flavonoids and soluble protein in pulp in treated groupwas higher than control, the same as the activity of POD, PPO, SOD, PAL and GLU, all ofthese indicated that chitosan sprays induced the disease resistance of grape.
4. Furthermore, transmission electron microscope was used to observe the ultrastructureof the grape. The results showed that in the first day of storage, there was no significantdifference between chitosan treated grape and control except for macromolecular sedimentand thicker cell wall in chitosan treated grape cell. There were some pits in cell wall in grapesprayed with0.5%chitosan. After seventy-five days, the middle lamella of preharvest treatedgrape was not completely dissolved; leaving a lot of small microfibrils, and the primary cellwall was integrated. Chloroplast and mitochondria of preharvest-treated grape were integrated;there were pits in cell wall, and macromolecular sediment in the cell. But the cell wallstructure of control grape swell and flab, the primary cell became crooked and thicker; mostof the middle lamella was dissolved and disappear. Chloroplast gradually becamedisorganization, the brink of membrane and grana lamella was unclear. Mitochondriaremained numerous, mitochondrial crista disappeared. There had little pits and nomacromolecular sediment in the cell.
5. In the paper, the effect of spray-shock was found and put forward for the first time inthe process of preharvet treatment, the possible mechanism was also been studied about howthe spray-shock led grape injury in the storage. The grape damaged in cold storage wouldappear such phenomenon as peel cracking, pulp collapse and juice outflow from20d to30d,all of these made fruit quality decrease rapidly and grape became rot quickly. Fruitspray-shocked with1.0%chitosan had higher soluble solid content than control fruit. Solublesolid and titratable acid content fluctuated and appeared a pear in45d. The spray-shockedfruit had higher POD activity than un-shocked fruit. Compared with the un-shocked grape, theshocked grape had lower SOD, CAT and PPO activity. The spray-shock led to the injury andfast decay of grape in the storage.
6. Dielectric parameters of Red globe grape during cold storage at0℃werenon-destructively measured over frequency range from0.1kHz to3980kHz. The resultsshowed that there are significant exponential relationship between impedance, resistance,inductance reactance, admittance, susceptance and frequency, but no exponential relationship for other dielectric parameters. Fruit impedance, resistance, inductance and reactancedecreased gradually by linear form with the increase of frequency, admittance andsusceptance increased linearly. At the optimum test frequency, impedance, resistance,inductance and reactance decreased linearly with the extension of storage time.
7. Firmness, chewiness, springiness and adhesiveness of grape were measured as textureattributes. In the storage of45d, fruit firmness and chewiness decreased gradually with thetime went on, springiness and the absolute value of adhesiveness went up contrarily. At0.1kHz frequency, the high correlation was found between dielectric and texture parameters,such as reactance with texture parameters (P<0.05). At last, the regression equation was builtfor dielectric and texture parameters, it was possible for the realization of grapenondestructive testing.
8. Postharvest dehydration treatment was a safe fresh-keeping method without sulfur. Allthe test results showed that postharvest moderate dehydration was one more effective way inkeeping the quality of grape during cold storage. Grape dehydrated3%has significant higherfirmness, PPO and POD activity than control. Compared with the un-dehydrated grape, thedehydrated one has higher impedance, reactance, inductance, resistance and lower dielectricconstant, impendence phase angle during45days storage.
引文
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