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牛蒡低聚果糖诱导水果采后保鲜的机制研究
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摘要
水果采后保鲜是水果生产中的重要问题,也是储藏生理学领域的研究热点,其中涉及到微生物侵染引起的水果腐烂和代谢作用的继续引起的水果营养成分变化、变质。基于采后保鲜的两个要点,开发了包括生物物理技术(低温、低压、气调、电场、套袋和包膜等)和生物化学技术(化学杀菌剂、拮抗酵母菌和诱导子等)的采后保鲜技术。其中,天然诱导子具有抗病效果显著、食品安全性高、成本低和保持采后品质的优势。牛蒡低聚果糖(Burdock Fructooligosaccharide,BFO)是从牛蒡根中分离得到的一种诱导子,已有的研究证明BFO能够降低烟草、番茄和黄瓜等植物的病害,并初步探索了其作为诱导子诱导系统获得抗病性的机制,对番茄采后自然感病和灰霉病具有较好的防治效果。但是对采后果蔬的抗病机理尚缺少系统的研究。
     本文的研究内容包括:
     1.BFO诱导不同水果采后抗病性的特性,并初步探索BFO诱导抗病的机制。表型观察结果表明,BFO能够激活葡萄对灰霉病、苹果对青霉病、香蕉对炭疽病、猕猴桃对灰霉病、桔子对青霉病、草莓和巴梨对自然发病病原菌的抗病性。为了研究BFO对不同水果采后抗病性的特性,用浓度为0.01%-1.5%(w/v)具有梯度BFO溶液处理葡萄,接种相应病原菌,调查处理后3d的病情指数,测得了葡萄(0.5%)、苹果(0.5%)、香蕉(0.75%)、猕猴桃(0.5%)、桔子(0.75%)、草莓(0.25%)和巴梨(0.25%)的最佳处理浓度。用最佳BFO处理水果分别测定发病率,结果表明,BFO能够降低葡萄(最大降幅78.9%)、苹果的发病率(最大降幅幅34.4%)、香蕉(最大降幅39.1%)、猕猴桃(最大降幅36.4%)和桔子(最大降幅22.4%)的发病率,但是对于发病较快的草莓(最大降幅15.4%)和巴梨(最大降幅5.9%)效果不显著。BFO处理对七种水果采后病害的病情指数,并通过病情指数对时间作图的曲线,积分计算了病程曲线面积(AUDPC),根据处理组曲线下面积较对照组减少的百分比计算了防效。结果表明,BFO在不同水果中的防效分别为葡萄29.1%、苹果26.2%、香蕉14.9%、猕猴桃106%、桔子14%、草莓14.3%和巴梨11.4%。对BFO诱导的七种水果采后抗病性的机理进行了初步的探索。研究了BFO对七种水果后短时间内H2O2含量和表皮组织总酚的影响,结果表明BFO处理水果3h,葡萄、香蕉和草莓出现H2O2迅速提高的峰值,6h苹果和巴梨出现H2O2迅速提高的峰值,BFO对猕猴桃和桔子H2O2激活作用并不显著;BFO处理能够显著减缓葡萄皮总酚含量的下降、提高苹果表皮的总酚含量,能够提高香蕉和草莓的总酚含量,但是对猕猴桃、桔子和巴梨的总酚含量的影响较小。
     BFO能够降低七种水果的发病率和病情指数,在不同水果中存在差异。BFO对葡萄灰霉病和苹果青霉病有显著的诱导抗病作用。总盼和H2O2的测定结果暗示BFO对葡萄、苹果、香蕉、草莓和巴梨具有相似诱导抗病机制,BFO诱导猕猴桃和桔子抗病性的机制可能不同于前述五类水果。
     2BFO诱导葡萄采后抗病性的机制。以葡萄作为研究材料,研究了BFO诱导水果采后抗病性的机理。BFO处理葡萄后,3h H2O2水平提高J了2.7倍(P<0.05),细胞内H2O2作为信号物质与SA信号通常为协同效应,共同激活SAR。,SA能够激细胞内病程相关基因非表达子(nonexpressor of pathogenesis-related genes1, NPR1),通常对NPR1的表达也有诱导上调作用。我们的结果表明BFO处理葡萄皮细胞中与基础抗性相关的NPR1.1上调表达了3.1倍(P<005),与SAR相关的NPR1.2上调了19.8倍(P<0.05)。NPR1蛋白可以与基因的启动子W-Box结合,激活SA通路相关基因表达,其中SA通路的标志性基因PR1上调表达了3.1倍(P<0.05)。具有直接杀菌活性的病程相关蛋白几丁质酶和β-1,3-葡聚糖酶的酶活力分别提高了47%(P<0.05)和29.1%(P<005)。葡萄皮中典型的植保素反式白藜芦醇的合成通路被激活,关键基因芪合酶(Stilbene synthase, STS)基因上调表达2.8倍(P<0.05)。反式白藜芦醇含量提高了28倍(P<0.05)。水杨酸信号通路的信号转导激活了水杨酸的进一步合成,结果表明,BFO诱导苯丙氨酸解氨酶基因(PAL)表达上调了2.6倍(P<0.05),PAL酶活力提高了99%(P<0.05)。SA在BFO处理后第二天开始在葡萄皮内显著高于对照组水平。但是对糖基化水杨酸(SAG)影响不大。积累的SA可以进一步合成水杨酸酯作为SAR的信号物质在组织间传递。
     综合上述结果,BFO通过激活水杨酸依赖的信号通路诱导葡萄的采后抗病性。
     3BFO对葡萄采后生理的影响。The effect of BFO on postharvest physiology in grape葡萄细胞内的的活性氧清除体系的关键酶包含了SOD和CAT,维持着细胞的氧化还原平衡。采后对照组SOD酶活力逐渐下降。BFO处理组诱导SOD酶活力提高,并在整个保藏期保持了高于对照组的水平,其中最大增幅达到75%。CAT酶活力在保藏过程中呈现上升的趋势,BFO处理的葡萄中的CAT酶活力较对照组提高了54%(P<0.05)。丙二醛是细胞膜被氧化后释放的化合物,其含量能够反映细胞膜的完整性和细胞的氧化水平。BFO处理降低了葡萄皮中的MDA含量,其中最大降幅达到59%(P<0.05)。巨峰葡萄在采摘后很快发生褐变,影响食用品质。褐变一方面是POD将酚类和黄酮类氧化聚合形成有色化合物,另一方面PPO催化酚类转化为醌类引起的。对照组在第6天就有超过90%葡萄发生褐变,而BFO处理组第10天褐变率才达到90%。测定了POD和PPO的活力,结果表明,BFO处理组较对照组POD活力降低了28%(P<0.05),PPO酶活力降低了47%(P<0.05)。本研究测定了葡萄的失重率、呼吸速率、可溶性固形物、可滴定酸和维生素C含量等品质相关指标在BFO处理后的变化。BFO处理对可溶性固形物的含量没有显著的影响,但是保持了较高的可滴定酸的含量,BFO处理组的可滴定酸含量较对照组提高了20%(P<0.05)。维生索C含量在整个保藏过程中逐渐下降,BFO处理能够延缓维生素C的下降速度。BFO处理后第五天,葡萄处理组Vc含量较对照组高9.4%(P<0.05)。
     综上所述,BFO是一种有效的诱导子,能够作为天然果蔬保鲜剂应用于多种水果,尤其对葡萄有非常显著的效果,具有巨大的应用潜力。
Postharvest process is an important step in fruit industry and also the research hotspot of Storage Physiology. Postharvest decay caused by microorganism and the change/loss of nutrition caused by continued metabolism are both involved in postharvest process. It is based on the two above key point that a series of postharvest technology have been developed Postharvest technologies include biophysics technology (low temperature, low pressure, dynamic controlled atmosphere, electric field, packaging and eatable film etc.) and biochemistry technology (synthetic fungicides, antagonistic yeasts and elicitor etc.). Nature elicitors have many advantages, such as remarkable effect in postharvest disease control, edible safety, low cost and nutrition protection. Burdock fructooligosaccharide (BFO) is an elicitor isolated from Arctium lappa root. Further studies have showed that BFO induced resistance in Nicoliana tabacum, Cucumis salivas and Lycopcrsicon esculenlum Other study have proved BFO act as an elicitor to induce systemic acquired resistance in plant. Also, Nature diseased and Botrylis cinerea infected tomato can be controlled by BFO. However there is no systemic research in the mechanism of BFO induced postharvest resistance.
     These studies include:
     1.The characteristic of BFO induced resistance in different fruits and its preliminary mechanism study. The BFO treatment concentration have been optimized among fruits, grape against Holiylis cinerca, apple agaist Penicillium expansum, banana agaist Calletotrrichum musae, kiwi fruit agaist Botrytis cinerea, citrus agaist Penicillium expansum, strawberry and Bartlett pear agaist nature disease. In banana and citrus, higher concentration (0.75%, w/v) is needed. In grape, apple and kiwifruit, medium concentration (0.5%, w/v) is needed. In strawberry and Bartlett pear, lower concentration (0.25%, w/v) is needed. Decay percentage has been analyzed, the results indicated that:The decay percentages of grape and apple have been notably decreased by BFO treatment. The decay percentages of banana, kiwifruit and citrus was also been decresed by BFO treatment. BFO treatment has little effect on strawberry and Bartlett pear. Disease index also been tested. We used the disease index to calculate the area under disease progress curve (AUDPC) and the control effect. These results indicated that BFO has high control effect (p<0.05) in grape (29.1%) and apple (26.2%) and has low control effect (p<0.05) in banana (14.9%), kiwifruit (10.6%), citrus (14.0%), strawberry (14.3%) and Bartlett pear (11.4%). Total phenol of7kinds of fruits post BFO treatment has been analyzed. The results indicated that total phenol of grape and apple has notably increased after BFO treatment. The total phenol of banana and strawberry was also increased after BFO treatment. BFO treatment has little effect on kiwifruit, citrus and Bartlett pear. Hydrogen peroxide (H2O2) content also been tested in a short time period after BFO treatment. A3h H2O2-peak has been observed in grape, banana and strawberry after BFO treatment. A6h H2O2-peak has been observed in apple and Bartlett pear. No H2O2-peak has been observed in kiwifruit and citrus after BFO treatment. There is a synergistic effect between H2O2signal molecular and SA signal molecular. The H2O2-peak indicated induction of SA-dependent pathway.
     2. The mechanism of BFO induced resistance in grape. Grape has been chosen as a model to investigate the underlying mechanism of BFO induced resistance. Gene expression, ezyme activity and important compound in grape skin associated with SA-dependent signaling pathway have been analyzed after BFO treatment. The results indicated that the H2O2content increased2.7folds (p<0.05) after BFO treatment. There is a synergistic effect between H2O2signal molecular and SA signal molecular. Nonexpressor of pathogenesis-related genes1(NPR1) can be activated by SA signal and then combined to the gene promoter. SA signal can also induce the NPR1expression. Our results indicated that, a3.1folds upregulation (p<0.05) of NPR1.1has been observed which associated with the basic resistance. A19.8folds upregulation (p<0.05) of NPR1.2have been observed after BFO treatment which associated with induced resisitance. PR1is the maker gene of SA induced resistance A3.1folds upregulation (p<0.05) of PRI have been tested The activity of chitinase and β-1,3-glucanase increased47%(p<0.05) and29.1%(p<0.05) after BFO treatment. These two enzyme are pathogenesis-related proteins can directly inhibit and kill fungi pathogen The most important phytoalexins is trans-resveratrol. Stilbene synthase (STS) is a key enzyme in the biosynthesis pathway Our results indicated that STS gene upregulated for2.8folds (p<0.05). Trans-resveratrol increased28folds (p <0.05). The SA-dependent signaling pathway further activated the biosynthesis of SA. Our results indicated that, BFO induced SA accumulation in grape skin but has little effect on SAG PAL gene upregulated for2.6folds (p<0.05), while PAL enzyme activity increased99%(p<0.05). The SA product can be further been synthetized into MeSA, which act as a long distance signal to activat SAR in other tissue.
     3. The effect of BFO on postharvest physiology in grape. There is a balance between reactive oxygen species (ROS) generation and elimination by active oxygen scavenging system to maintain a stable balance in plant cell. The active oxygen scavenging system includes SOD and CAT. Our results indicated that after BFO treatment delayed the decrease of SOD activity in grape and increased the activity of CAT and the CAT activity increased54%(p<0.05) than control. MDA has also been tested which was used as a marker of cell membrane integrity. Our results indicated that, after BFO treatment MDA decresed59%(p<0.05)compared with the control Kyoho grapes changes color rapidly after harvest, with close to90%of fruit showing color change6days post treatment. However, in the BFO-treated group,90%color change occurred10days post treatment, indicating a slower color change rate. In the control group, the POD enzyme activity increased gradually in postharvest grapes; however, BFO treatment repressed the POD enzyme activity, so that on the third and fourth day post treatment, the POD activity was lower than that of the control by28.3%(p<0.05) and25.7%(p<0.05), respectively (Fig.3B). BFO also slowed the dramatic increase in PPO enzyme activity compared to the control grape skins, showing42%(p<0.05) lower activity on the second day post harvest. The respiration rate in the control grapes elevated during storage. The respiration rate was restricted after BFO application. During storage, weight loss increased in both the control and BFO-treated grapes. A suppression of weight loss was observed in the BFO-treated grapes compared with the control. the TSS in grapes declined during the storage period in both the control and BFO-treated groups. There was a slight difference in TSS activity between BFO-treated grapes and the control. A notable TA decrease was observed in control groups, compared to the preserved high levels of TA in BFO treated groups. The difference reached20%(p<0.05). Vitamin C decresed after harvest. BFO treatment could inhibit the decrease of Vitamin C. The Vc content was9.4%(p<0.05) higher in BFO treated grape than control.
     In conclusion, BFO is an efficient elicitor can be widly used in postharvest fruits. It has great application potential.
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