导致柑橘果实油胞病的橘油挥发组分分析

谢 姣，邓丽莉，明 建，姚世响，曾凯芳※



导致柑橘果实油胞病的橘油挥发组分分析

谢 姣1,2，邓丽莉1,3，明 建1,3，姚世响1,3，曾凯芳1,3※

（1. 西南大学食品科学学院，重庆 400715；2. 贵州医科大学公共卫生学院，贵州 550025； 3. 西南大学食品贮藏与物流研究中心，重庆 400715）

油胞病是一种主要的柑橘生理性病害，其典型特征是病斑区油胞凸起、油胞周围组织坏死并形成绿色、黄色甚至褐色病斑，这种症状不仅引起果皮外观品质下降，还严重影响了柑橘果实的商业价值。目前研究认为油胞病发病与橘油泄露有关。因此，该研究分别采用市售橘油、提取橘油、橘油非挥发性成分及26种挥发性成分单品分别处理锦橙、脐橙和椪柑果实，结果发现市售橘油和提取橘油处理油胞病发病率均为100.00%；橘油非挥发性成分及单品-蒎烯、石竹烯、朱栾倍半萜、-金合欢烯和-金合欢烯处理其发病率均为0；而其余21种挥发性成分单品处理均出现油胞病症状，且发病率≥73.33%。研究橘油成分对柑橘果实油胞病的影响将为进一步探索油胞病的防治措施提供理论基础。

果实；有机挥发性成分；病害；锦橙；脐橙；椪柑；油胞病；橘油

0 引 言

柑橘因其外观、风味以及营养价值而深受消费者的青睐，是世界上最受欢迎的亚热带水果之一[1-3]。然而由于柑橘果实油胞病产生的果皮外观缺陷不仅大大降低柑橘果实外观品质，还对采后贮藏造成影响，往往引起严重的经济损失[4-6]。柑橘油胞病，又称油斑病，是一种常见的柑橘果皮生理性病害，常见于果实成熟期以及采后贮藏期[7]。目前大多数柑橘品种均发现有油胞病的产生，且不同品种柑橘对油胞病的敏感性不同[8]。油胞病典型发病症状为发病区整体果皮下陷、油胞周围组织细胞坏死以及发病区油胞凸起甚至出现油胞破裂现象，而发病区色泽呈现绿色、黄色甚至褐色[9-11]。

据报道，柑橘果实在发育、成熟以及贮藏期产生油胞病的主要原因为虫害、机械损伤以及气候变化[6]。其中，对模拟机械损伤而引起油胞病的研究表明其引起的油胞病产生的原因可能是机械损伤引起位于果皮黄皮层中的油胞破裂而释放出橘油所致[12]。另外，刘丽丹等[13-15]的研究证明了使用市售橘油能诱导柑橘果皮油胞病的产生。此外，利用-柠檬烯处理同样能诱导油胞病的产生[16]。以上研究结果表明油胞病的产生与橘油存在一定的关联性[17]。

目前关于柑橘果实油胞病主要致力于果实油胞病发生后果皮组织的变化机理研究[12-14,16]以及果皮色素变化的研究[4,18]，而具体哪些橘油成分能引起柑橘果实油胞病的发生的研究鲜有报道。柑橘精油主要由85%~99%的挥发性成分和1%~15%的非挥发性成分组成[19-21]。其中柑橘精油中挥发性成分主要是单萜（包括-侧柏烯、-蒎烯、桧烯、-月桂烯以及-柠檬烯）、倍半萜烯（包括顺式--罗勒烯、甘香烯、-石竹烯和-金合欢烯）、醛类（包括辛醛、香茅醛、橙花醛和香叶醛）、醇类（包括松油醇、芳樟醇、香茅醇和香叶醇）、酯类（包括乙酸香茅酯、乙酸橙花酯和乙酸香叶酯）、氧化物类（包括石竹烯氧化物、顺式-柠檬烯氧化物和反式-柠檬烯氧化物）以及酮类（包括诺卡酮、长叶薄荷酮和香芹酮）等氧化衍生物的混合物[22-26]，而精油中非挥发性成分主要由碳氢化合物、固醇类、脂肪酸、类胡萝卜素、香豆素、补骨脂素和黄酮类等组成[20-21]。

因此，本研究以锦橙、脐橙和椪柑为研究对象，分别利用市售橘油和提取橘油（分别从锦橙、脐橙和椪柑果皮中提取）处理以上3个品种柑橘果实来确定油胞病的产生与橘油的关联性。另外，采用橘油非挥发性成分分别处理3个品种柑橘果实，从而明确橘油中对柑橘油胞病起诱导作用的成分。此外，前期试验利用固相微萃取结合GC-MS分析市售橘油和提取橘油发现，2类橘油中挥发性成分同样是由萜烯烃类（包括单萜和倍半萜烯类）、醛类、醇类、酯类、氧化物类以及酮类组成，因此，Xie等[5]通过研究锦橙、脐橙和椪柑健康果皮和油胞病果皮中的挥发性成分，确定了3个品种柑橘油胞病果皮中发生主要变化的是26种挥发性成分即10种萜烯烃类化合物即-蒎烯、-石竹烯、朱栾倍半萜、-金合欢烯、-金合欢烯、-柠檬烯、月桂烯、桧烯、-萜品油烯和-萜品油烯，5种萜醇类化合物芳樟醇、-松油醇、-松油醇、香叶醇和香茅醇，6种醛类化合物壬醛、癸醛、十一醛、十二醛、香茅醛和香叶醛，2种氧化物类化合物顺-柠檬烯氧化物和反-柠檬烯氧化物，2种酯类乙酸橙花酯和乙酸香叶酯以及酮类化合物香芹酮。因而，本研究最后利用这26种挥发性成分分别处理3个品种柑橘果实，从而明确橘油中具体挥发性成分对3个品种柑橘果实油胞病的影响，这为进一步研究油胞病的发病机制及控制措施提供理论指导。

1 材料与方法

1.1 材料与试剂

试验柑橘品种为花期220 d左右的锦橙（Osbeck cv.#447#），脐橙（Osbeck cv. Fengji）和椪柑（Blanco cv. Ponkan），采自重庆市北碚区歇马镇柑橘果园，挑选无物理损伤、无病虫害、大小均匀一致的健康果实以及油胞病果实，室温下洗净，晾干，备用。

柑橘精油（纯度为100%，由挤压法获得），英国植优雅香精油公司；无水硫酸钠，分析纯，成都市科龙化工试剂厂。标品：-柠檬烯、-萜品油烯、-萜品油烯、-石竹烯、壬醛、癸醛、香叶醛、十二醛、十一醛、芳樟醇、-松油醇、-松油醇、香茅醇、香叶醇、橙花醇乙酸酯、乙酸香叶酯和香芹酮购自中国上海TCI公司；-蒎烯、香桧烯、-月桂烯、-金合欢烯、-金合欢烯、瓦伦西亚橘烯、香茅醛购自美国sigma公司；顺-柠檬烯氧化物，反-柠檬烯氧化物购自日本WAKO公司。以上标品除月桂烯（溶于甲基叔丁基醚中保持稳定）和乙酸香叶酯外其他标品纯度均大于90%。

1.2 方 法

1.2.1 冷压法柑橘果皮橘油收集

参照Sawamura等[27-28]的方法并加以改进。分别将3个品种柑橘切成8等份，去除果肉部分，仔细去除果皮中白皮层，保留黄皮层。使用装有饱和氯化钠溶液的离心管收集利用枪头压出的油滴5 mL，整个收集工作在冰上操作完成；收集液于4 ℃、4 000×离心15 min后，再用无水硫酸钠于5 ℃条件下干燥以上离心后的上层油状液24 h，过滤，所得3个品种柑橘橘油于-20 ℃保存，待用。

1.2.2 市售橘油处理

参照Knight等[16,29-30]方法并加以改进。处理组采用直接涂抹方法，将15L纯度为100%的橘油直接涂抹至果实赤道周围约3 cm2面积，晾干；对照果实未用橘油处理；将晾干的处理组和对照组果实用厚度为0.015 mm的聚乙烯袋单果包装，黑暗处理，并于25 ℃、相对湿度为85%条件下贮藏。样品每组10个果实，每组设3个平行，试验重复2次。

1.2.3 提取橘油处理

将从1.2.1中分别制取的锦橙、脐橙和椪柑果皮中的精油分别涂抹至相应品种柑橘果皮上，其后参照1.2.2处理方式处理。样品每组10个果实，每组设3个平行，试验重复2次。

1.2.4 橘油非挥发性性成分处理

将从1.2.1制取的3个品种柑橘橘油5 mL置于10 mL离心管中，于室温条件下敞口放置（黑暗处理）以使橘油中挥发性成分挥发完全，周期约20 d；分别用双重蒸馏水和乙醇溶液溶解挥发后的橘油干物质（体积溶解比例为1∶1），过滤，参照1.2.2处理方法处理对应的柑橘品种，对照组只使用相应的溶剂（双重蒸馏水和乙醇溶液）进行处理。样品每组10个果实，每组设3个平行，试验重复2次。

1.2.5 橘油挥发性成分处理

利用1.1所列橘油挥发性成分的标品处理柑橘果实，处理方法参照1.2.2。样品每组10个果实，每组设3个平行，试验重复2次。

1.2.6 发病率测定

以上柑橘果实处理后72 h进行观察，柑橘果实上出现油胞病症状认定为发病，其油胞病发病率参参照Garcia-Martin等[31]的方法，其计算公式如式（1）所示。

1.3 数据分析

采用IBM SPSS Statistics 22.0软件对实验数据进行分析，用-test方法进行差异显著性分析；采用GraphPad Prism 7以及Photoshop 6.0进行图片的绘制。

2 结果与分析

2.1 市售橘油和提取橘油处理对柑橘果实油胞病发病率及发病症状的影响

如表1所示，市售橘油处理后的锦橙、脐橙和椪柑分别于25 ℃、相对湿度85%条件下贮藏72 h后，3个品种的柑橘果实油胞病发病率均为100.00%，且相对对照组在相同的贮藏条件下的油胞病发病率具有显著性差异（<0.05）。另外，利用1.2.1冷压法制取的锦橙、脐橙和椪柑果皮橘油即提取橘油分别处理对应柑橘果实并贮藏72 h后，发现3个品种柑橘果实油胞病发病率均为100.00%，且相对对照组具有显著性差异（<0.05）。

如图1所示，市售橘油分别处理锦橙、脐橙和椪柑果实后，3个品种柑橘果实产生的油胞病具有相似的发病症状，即发病区整体塌陷，油胞凸起，油胞周围组织下陷，发病区颜色呈现褐色。另外，观察到3个品种柑橘油胞病发病面积与处理面积即3 cm2基本相同。以上结果揭示了市售橘油处理其果皮出现了油胞病的典型症状，结合市售橘油处理3个品种柑橘果实统计的油胞病发病率均为100.00%，说明市售橘油处理能诱导3个品种柑橘油胞病的产生。

表1 市售橘油和提取橘油处理对柑橘果实油胞病发病率的影响（平均值±标准差）

注：数字的统计学差异（<0.05）用不同的字母表示，下同。

Note: Statistics differences (<0.05) of number are indicated by different letters, the same as below.

图1 市售橘油处理对柑橘果实油胞病发病症状的影响

如图2所示，分别利用1.2.1中制取的3个品种柑橘橘油处理对应柑橘品种并贮藏72 h, 结果发现其诱导3个品种柑橘果皮油胞病的发病症状与市售橘油处理引起的油胞病有相似的症状即发病区整体塌陷，油胞凸起，油胞周围组织下陷，发病区颜色为浅褐色。另外，提取橘油处理3个品种柑橘同样观察到其发病面积与处理面积相同。以上结果揭示了提取橘油处理其果皮同样出现了油胞病的典型症状，结合提取橘油处理其果实油胞病发病率，说明提取橘油能诱导3个品种柑橘油胞病的产生。据报道，油胞病的形成是位于柑橘果皮有色层中的油胞所含橘油泄露从而造成果皮的光毒性损伤而引起的果皮生理性失调[32-33]。这种果皮损伤主要表现为发病区整体组织下陷，油胞相对油胞间组织凸起，油胞周围组织出现坏死，形成绿色、黄色甚至褐色病斑。基于橘油的泄露引起油胞病的产生这一结论[32-33]以及刘丽丹等利用市售橘油诱导脐橙油胞病的产生[13-15]，说明橘油本身与油胞病可能存在一定的关联性[17]。目前，对于橘油诱导油胞病主要以脐橙为研究对象并以合成橘油研究其诱导作用，但并不明确市售橘油对其他柑橘品种以及柑橘所制取的橘油（提取橘油）对相应柑橘品种果实油胞病的诱导作用。而结合以上市售橘油及提取橘油诱导3个品种柑橘果实出现油胞病的症状符合以上油胞病典型症状，且其发病率均为100.00%，这可进一步说明橘油与油胞病的形成存在一定的关联性。

图2 提取橘油处理对柑橘果实油胞病发病症状的影响

2.2 橘油非挥发性物质处理对柑橘果实油胞病发病率及发病症状的影响

据报道，冷压法收集的橘油由非挥发性成分和挥发性成分组成，而橘油中这2类成分对柑橘果实油胞病的诱导作用未有报道。此外，橘油非挥发性成分有脂溶性和水溶性2类化合物。因此，本试验分别利用蒸馏水和乙醇溶解非挥发性物质处理3个品种柑橘来确非挥发性成分对油胞病的诱导作用。从图3可知，3个品种柑橘果皮橘油中的非挥发性物质分别使用蒸馏水和乙醇溶解，利用其溶解物分别处理3个品种柑橘果实，结果发现2种溶解物处理未出现油胞病典型症状，且其发病率均为0并相对对照组无显著性差异（>0.05）。以上结果阐明了非挥发性成分的溶解物（脂溶性和水溶性溶解物）对3个品种的柑橘果实油胞病均不具诱导作用，这进一步说明橘油中对3个品种柑橘具有诱导作用的是橘油中的挥发性成分而不是其非挥发性成分。

2.3 橘油挥发性成分处理对柑橘果实油胞病发病率及发病症状的影响

由于橘油非挥发性成分对柑橘没有诱导作用，因此可以推测橘油中的挥发性成分是诱导柑橘油胞病发生的主要原因。此外，根据柑橘果皮释放具有光毒性的橘油是导致油胞病的主要原因[28-29]，这一理论进一步说明柑橘果皮油胞病发生后其发病区挥发性成分变化显著的物质可能是诱导油胞病产生的原因。Xie等[5]研究油胞病和健康果皮精油中挥发性成分发现在油胞病果皮中的10种萜烯烃类化合物，6种醛类化合物，2种氧化物类化合物，2种酯类以及酮类化合物含量相对健康果皮具有显著性的差异。因此，本研究利用以上26种挥发性成分分别处理3个品种柑橘来确定具体成分对果实油胞病的诱导作用。从表2可知，橘油挥发性性成分中的10种萜烯烃类化合物除-蒎烯、-石竹烯、朱栾倍半萜、-金合欢烯、-金合欢烯不能引起柑橘果实发生油胞病以外，其余5种萜烯烃类化合物均能引起柑橘果实发生油胞病且相对对照组均有显著性差异（<0.05），其中，-柠檬烯和月桂烯处理分别诱导脐橙果实的油胞病发病率为93.33%与73.33%，桧烯、-萜品油烯和-萜品油烯诱导3个品种柑橘果实的油胞病发病率均为100.00%。另外，橘油挥发性性成分中5种萜醇类化合物即芳樟醇、-松油醇、-松油醇、香叶醇和香茅醇，6种醛类化合物即壬醛、癸醛、十一醛、十二醛、香茅醛和香叶醛，2种氧化物类化合物即顺-柠檬烯氧化物和反-柠檬烯氧化物诱，2种酯类化合物即乙酸香叶酯和乙酸橙花酯以及酮类化合物香芹酮诱导3个品种柑橘果实的油胞病发病率均为100.00%且相对对照组均有显著性差异（<0.05）。

注：图3a和3b中从左至右的图层分布分别为锦橙、脐橙和椪柑果实，从上至下分别为对照组和处理组。

表2 橘油挥发性成分处理对柑橘果实油胞病发病率的影响（平均值±标准差）

从图4a、4b和4e可知，5种萜烯烃类化合物、5种萜醇类化合物和香芹酮引起3个品种柑橘果实发生油胞病的症状大致相似，即部分油胞在发病区凸起，油胞周围组织向下有轻微塌陷。另外，发病区域部分油胞出现塌陷现象，这种现象在萜烯烃类化合物诱导锦橙和椪柑果皮发生的油胞病中较为明显，而在萜醇类和酮类化合物诱导脐橙果皮发生的油胞病中较为明显。此外，5种萜烯烃类化合物诱导锦橙和椪柑发病区域颜色为浅褐色，而脐橙发病区域有紫褐色病斑产生；5种萜醇类化合物诱导3个品种柑橘发病区域颜色均为深褐色；香芹酮诱导锦橙和椪柑发病区域颜色为深褐色，而脐橙发病区域颜色为浅褐色。从图4c可知，6种醛类化合物引起3个品种柑橘果实发生油胞病的症状大致相似，即发病区油胞均出现塌陷现象，而油胞周围组织向下严重下陷，且3个品种柑橘发病区域颜色均为深褐色。从图4d和4e可知，2种氧化物类化合物和2种酯类化合物引起3个品种柑橘果实发生油胞病的症状大致相似，即发病区油胞凸起，油胞周围组织向下有轻微下陷。另外，与以上挥发性物质不同的是2种氧化物引起的脐橙和椪柑油胞病发病区域颜色均为红褐色，而锦橙发病区域颜色为浅黄色；2种酯类化合物引起的锦橙和椪柑油胞病发病区域颜色为深褐色，而脐橙发病区域颜色为浅褐色。以上结果说明萜烯烃类化合物中-柠檬烯、月桂烯、桧烯、-萜品油烯和-萜品油烯等处理3个品种柑橘具有油胞病的典型症状且发病率显著高于对照组，揭示这5种萜烯烃类化合物能诱导果实油胞病的形成，这一结果同-柠檬烯能诱导油胞病的产生的结果一致[30]，而其他5种萜烯烃类化合物处理因不具油胞病典型症状说明其不能引起柑橘果实发生油胞病。5种萜醇类、6种醛类、2种氧化物类、2种酯类和1种酮类化合物均能引起柑橘果实油胞病的发生。综上，橘油挥发性成分引起的柑橘果实油胞病均具有各自典型特征，3个品种柑橘果实油胞病发病症状略有差异，这可能是品种的敏感性不同所致。

注：图4a、4b、4c、4d、4e和4f中从上至下图层分布为上层为锦橙、中间层为脐橙以及下层为椪柑果实。

3 结 论

通过研究市售橘油、提取橘油以及橘油非挥发性成分对柑橘果实油胞病的诱导作用，明确了橘油中能诱导柑橘果实油胞病的成分为挥发性成分。因此，本研究利用油胞病果皮中发生主要变化的26种挥发性成分来确定橘油具体成分对柑橘果实的诱导作用。结果发现，这些成分中除-蒎烯、-石竹烯、朱栾倍半萜、-金合欢烯和-金合欢烯不能诱导柑橘果实油胞病，其余21种挥发性物质均能诱导油胞病并呈现出油胞病的典型症状。以上研究为阐述柑橘果皮油胞病的发生机制提供了理论基础，并为进一步研究油胞病的防治措施提供理论指导。

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Analysis of volatile components of citrus oil causing oleocellosis in citrus fruits

Xie Jiao1,2, Deng Lili1,3, Ming Jian1,3, Yao Shixiang1,3, Zeng Kaifang1,3※

(1.400715,; 2.550025,; 3.400715,)

Citrus, consumed widely throughout the world since its appearance, flavor and nutritional value, is one of the most popular subtropical fruits in the world. However, oleocellosis that often occurs at harvest time and during postharvest storage hasa negative effect on the external quality of citrus fruits due to the appearance defects formed on the surface of the pericarp, eventually causes extensive economic damage to citrus industry. Oleocellosis, a major physiology disorder in citrus, is characterized by the appearance of outstanding oil glands of blemish area, tissue necrosis occurred in the adjacent epidermis of oil glands, and formation of green, yellow even brown spots in the disorder area. It was reported thatthe occurrence of oleocellosis is highly correlated with the release of peel oil from the glands located in citrus pericarp; oleocellosis results from various injuries such as friction from hail and the mechanical damage, and the injuries will induce the breakdown of glands to form oleocellosis development. Those studies about oleocellosis development revealed that there was a certain correlation between oleocellosis and citrus oil during the oleocellosis occurrance. At present, little information was available on which citrus oil components could cause the occurrence of oleocellosis in citrus fruits. In generally, the citrus oil obtained by the cold compression method consists of non-volatile components (including two kinds of compounds: lipo-soluble and water-soluble) and volatile components. The induction of these two components in citrus oil on oleocellosis development of citrus fruit has not been reported. In terms of that, the fruits of Jincheng, Navel orange and Ponkan mandarin fruits were used as raw material, and the commercial citrus oil and extracted citrus oils from the three citrus varieties were used to treat the three types of citrus fruit to confirm whether the oleocellosis could be induced by the two types of oils, respectively. The results showed that the oleocellosis symptoms both occurred in the three citrus types after the treatment of commercial citrus oil and extracted citrus oils, respectively, which indicated that the citrus oil could induce the oleocellosis. Additionally, non-volatile components in citrus oil which were treated with the distilled water- and ethanol-soluble also were used to induce the three citrus types, respectively. The results showed that the treatment of dissolving substances (including liposoluble and water-soluble) of non-volatile compositions in citrus oil on citrus fruits did not induce the occurrence of oleocellosis. From those results above, it could be concluded that the volatile components in the citrus oil were the main reason for oleocellosis development. Therefore, the twenty-six kinds of volatile componends that that significantly changed in oleocellosis peels compared to the healthy ones were applied to induce the three citrus types of fruits to study the oleocellosis incidence and symptoms based on this conclusion, respectively. The results showed that twenty-one volatile components contained five terpene hydrocarbons (including d-limonene, β-myrcene, sabinese, γ-terpinolene and α-terpinolene), five terpene alcohols (including linalool, β-terpineol, α-terpineol, geraniol and citronellol), six aldehydes (including nonanal, decanal, undecanal, dodecanal, citronellal and geranial), two oxides (including (E)-limonene oxide and (Z)-limonene oxide), two esters (including geranyl acetate and neryl acetate) and one ketone (including (-)-carvone) compounds could induce oleocellosis development of Jincheng, Navel orange and Ponkan fruits, however, the volatile compositions of α-pinene, β-caryophyllene, valencene, β-farnesene and α-farnesene could not incuce the oleocellosis development of the three citrus types, respectively. In summary, the volatile components in citrus oil which could induce oleocellosis development of citrus fruit were identified by studying the induction of commercial citrus oil, extracted citrus oils and non-volatile compositions in citrus oil on oleocellosis development of citrus fruit. In addition, oleocellosis development in Jincheng, Navel orange and Ponkan induced by the twenty-one volatile components of citrus oil has their own characteristics, for example, the phenomena that parts of oil glands in disease areas collapsed were more obvious in Jincheng and Ponkan oleocellosis fruits induced by terpene hydrocarbons compared to Navel orange, and the color of disease areas showed pale brown in the two types of oleocellosis fruits, however, Navel orange oleocellosis fruits showed purple brown.. However, the symptoms of oleocellosis in three varieties of citrus fruit had slightly different, which might be due to different varieties of sensitivity in citrus fruit. Studying the effect of specific components in citrus oil on the oleocellosis of citrus peels will provide a theoretical basis for explaining the mechanism of oleocellosis in citrus peel and theoretical guidance for further exploration on the prevention and control measures.

fruit; volatile organic components; disease; Jincheng; Navel orange; Ponkan mandarin; oleocellosis; citrus oil

2018-05-30

2018-09-11

国家自然科学基金面上项目：采后柑橘果实磷脂酶D对橘油的响应及油胞病的调控机制（31471631）

谢 姣，博士生，从事农产品加工及贮藏工程方面的研究。Email：lxj4516@126.com

曾凯芳，博士，博士生导师，教授，主要从事果蔬贮藏与保鲜的教学与研究工作。Email：zengkaifang@hotmail.com

10.11975/j.issn.1002-6819.2019.01.038

S609+.3; S666.2

A

1002-6819(2019)-01-0310-07

谢 姣，邓丽莉，明 建，姚世响，曾凯芳. 导致柑橘果实油胞病的橘油挥发组分分析[J]. 农业工程学报，2019，35(1)：310－316. doi：10.11975/j.issn.1002-6819.2019.01.038 http://www.tcsae.org

Xie Jiao, Deng Lili, Ming Jian, Yao Shixiang, Zeng Kaifang. Analysis of volatile components of citrus oil causing oleocellosis in citrus fruits[J]. Transactions of the Chinese Society of Agricultural Engineering (Transactions of the CSAE), 2019, 35(1): 310－316. (in Chinese with English abstract) doi：10.11975/j.issn.1002-6819.2019.01.038 http://www.tcsae.org