【作者】 马士成；

【导师】 梁月荣；

【作者基本信息】 浙江大学 ， 茶学， 2012， 博士

【摘要】 茶树作为聚氟植物对氟具有极强的耐性和累积特性,其老叶中氟含量比普通植物高2-3个数量级。尽管适量摄取氟有利于人体预防龋齿等健康功能,但长期大量饮用高氟茶叶可引发“饮茶型氟中毒”。近年来,茶叶降氟成为研究的热点,众多研究者围绕低氟品种选育、栽培技术改进、加工工艺改良和降氟剂开发等方面探讨降低茶叶中氟含量的方法。本文主要通过茶树氟累积条件下形态生理特性变化,研究茶树的耐氟生理特性及其机理；通过HPLC法分析氟铝处理下水培茶苗有机酸分泌特性,研究氟铝对茶树有机酸合成、分泌影响以及与氟吸收累积的关系。通过比较不同品种、不同器官和亚细胞结构氟含量差异,研究茶树氟累积的品种和器官特性；通过检测氟铝处理对茶树氟吸收速率、迁移率和富集系数的影响,分析铝对茶树氟吸收累积和迁移的影响及其机理；利用亚细胞结构分离和透射电镜结合能谱分析研究茶树叶片中氟的亚细胞分布；利用化学试剂浸提法和亚细胞器差速离心法研究茶树叶片中铝的形态比例及亚细胞分布。应用Fick第二定律研究茶汤中氟的浸提规律及其动力学模型,并根据动力学模型探讨了两步浸提法脱除茶叶中超标氟的可行性。结果表明：1、低浓度氟处理可促进茶树的生理代谢,有利于根尖伸长区生长；而较高浓度的氟对根系伸长具有一定的抑制作用,从而引起茶树逆境胁迫,导致根系细胞膜损伤、通透性增加,茶树叶片中自由基清除能力下降,进而引起过量的自由基攻击叶绿体类囊体膜上的大分子造成膜的裂解,导致叶绿体膜结构破坏,叶绿素含量降低。2、茶树体内可检测到的有机酸主要有草酸、丙酮酸、苹果酸、抗坏血酸、α-酮戊二酸和琥珀酸,而茶树根系分泌物中有机酸主要为草酸和苹果酸。研究结果表明茶树根部有机酸含量与根系分泌物中有机酸浓度均随氟处理浓度的增加而上升。氟处理浓度与根系有机酸含量和分泌量均有正相关关系,这可能与根际环境氟对茶树根系的接触和刺激引起有机酸含量的增加,从而提高根际环境氟的生物有效性,提高氟的吸收和转运。氟铝组合处理中,随铝浓度的增加,根系有机酸含量及分泌物中有机酸浓度均表现出一定程度的上升,这可能与铝胁迫诱导体内有机酸合成量增加。氟铝处理引起的根系分泌有机酸的变化可能是茶树对氟铝处理的一种应激反应,根系有机酸分泌物中草酸和柠檬酸等与茶树根际氟铝形态及其耐性有密切关系。3、高浓度氟处理促进茶树对氟的吸收,且氟的吸收速率存在阶段性,表现出起始阶段快速吸收,随时间延长而逐渐降低直至趋于平衡。在试验氟浓度下,茶树对氟的吸收主要以F-形态被动吸收,尤其是在高氟浓度情况下。随着处理氟浓度的提高,茶树根系向茎部、茎部向叶片以及由地下部向地上部的迁移率均明显增加。但各器官氟富集系数随着处理氟浓度的增加明显降低；在3个月处理时间里,茶树氟累积量与处理浓度相比具有一定的滞后性,表明茶树氟的累积是一个长期逐渐累积的过程,且茶树对氟的累积量可能存在一定的累积上限。茶树累积的总氟量一定程度受到低浓度铝的抑制作用；但高浓度铝处理,对新梢及第3-5位叶片的氟富集系数有明显提高作用,表明铝促进氟向新梢等新生叶片中的累积。4、氟在茶树嫩梢及成熟叶各亚细胞组分及细胞溶质中含量比例表现为：细胞壁>原生质体>叶绿体。叶片亚细胞结构中,细胞壁的氟含量比例最高,而作为重要元素储存部位的液泡中氟累积却非常有限,说明细胞壁是氟聚集的主要部位。随着处理氟浓度的提高,叶片各亚细胞结构中氟含量均显著上升。而且,细胞壁中和细胞溶质中氟上升比例大于叶绿体和原生质体。氟铝组合处理实验表明,高氟低铝处理(F24A16)下,氟向成熟叶细胞壁中累积量最大。有研究表明,细胞壁中淀粉酶和蛋白质等生物大分子含有的大量金属离子结合的基团结构和位点,如羟化物、羧基、醛基、氨基和磷酸盐等,氟可能与这些化学基团或位点结合而促使氟铝在细胞壁中富集；同时,在茶树在缺氟状态时,这些累积在细胞壁中的氟可以解离成游离状态转运到新生叶片、根系等器官。5、铝在茶树叶片化学形态及亚细胞分布：有机态铝占总铝的86.88%-88.11%,其中稳定有机态含量为36.87%-56.82%,不稳定有机态铝占31.28%-51.08%；无机态铝占铝总量的11.89%-13.12%,其化学形式有Al3+,Al(OH)2+和Al(OH)2+。不同茶树品种叶片之间的铝总含量存在显著差异,但有机态铝与无机态铝的比例差异不显著。茶树新梢及成熟叶中的铝主要分布在细胞壁中,分别占64.40%和83.24%,不同成熟度间含铝比例存在显著性差异。茶叶中铝主要以有机态形式存在,而且随着成熟度的增加分布于细胞壁上的铝比例增加。氟铝在细胞壁中可能形成某种稳定态化学基团结合。结合态铝的形成,可能具有降低铝的毒性。6、茶叶中的氟在浸泡过程的溶出过程表现为两个阶段,即起始的快速洗脱阶段和后续的慢速溶出阶段。In[c∞/(c∞-c)]与时间变化曲线显示,氟在茶叶颗粒内扩散行为是氟浸提的控速步骤。茶叶颗粒度大小对氟浸出动力学特性有显著影响,其作用大于温度的作用。在不同浸提条件下,扩散系数D1和D2可根据Fick第二定律计算得出,并据此构建了茶叶氟溶出模型,该模型可以用来阐述和预测茶汤中氟的溶出规律和溶出机理。将茶叶进行两步冲泡法浸提表明,第一次在50℃冲泡获得茶汤中氟的溶出比例高于茶多酚类和咖啡因的溶出比例。显示高氟茶叶可以通过两步冲泡法控制茶汤氟浓度。更多还原

【Abstract】 Tea(Camellia sinensis), as a fluorine (F) accumulator, accumulates F in mature tea leaves, which was100-1000times higher than other plant species. F is an important element to human health and a moderate intake of F is confirmed to be beneficial to alleviation of dental caries. However, excessive accumulation of F in human body can cause diseases such as fluorosis. The drinking-tea born fluorosis was reported to be caused by long-term consumption of a large quantities of tea containing extremely high level of F. Many researchers attempted to exploit measures to control F concentration in tea and tea liquor, including breeding cultivars with low-F level, improving cultivation methods, optimizing processing procedures and developing defluoride agent. This works studied F accumulation characteristics by investigating root morphology parameters, root vitality, cell membrane penetration, and antioxidase activities and malondialdehyde (MDA) content in tea leaves under F stress; The relationship between F accumulation and organic acid secretion was studied using high performance liquid chromatography (HPLC). F distribution differences between cultivars, organs and subcellular fractions under various treatment combination of F-and Al3+were investigated by isolating of cell fraction using differential centrifugation and transmission electron microscopy-energy dispersive X-ray (TEM-EDX). Various chemical species of aluminum and their subcellular distribution in tea leaves were also studied by using differential centrifugation technique and chemical extraction method; Kinetic and extraction model of F leaching from tea were studied using the second-order Fickian equation based on the kinetic behaviors and extraction model and a two-step infusion method was developed to control F level in tea liquor.1. Low level of F-promoted roots tips prolongation of tea plant. However, when tea plant was exposed to a high concentration of F-such as24mg·L-1, the root tip prolongation was inhibited, and root cell membrane was damage, which resulted in the cell membrane penetration increased. Level of free radical scarvenge activity decreased under F-stress. In this case, thylakoid membrane of the chloroplast was attacked and chloroplast membrane was injured, resulting in devrease in chlorophyll content.2. The major organic acids in tea root exudates were malic and oxalic acid, but malic, oxalic acid, pyruvic acid, ascorbic acid and a-ketoglutaric acid in tea leaf. Organic acids of tea plant under solution cultivation increased with increase in F concentration. These characteristics of tea root exudates might affect rhizosphere F chemical species and bioavailability. Al3+promoted the secretion of root exudate organic acids when tea plant was co-treated with F-and Al3+. The changes of organic acids might be a physiological response of tea plant to the stress of F-and Al3+.3. When tea plants were cultivated under high level F solution conditions, the F uptake rate by tea plant reached the highest at early24th h, and then declined. F might be passively absorbed in the form of F-when the tea plant was exposed to high concentration of F-upto24mg·L-1. F translocation rate in tea plant (from roots to stems and leaves) increased with increase in solution F-concentration. However, the F bioenrichment coefficients decreased with the increase in F concentration in culture solution. It is assumed that F accumulation in tea leaves was increasingly processed and it was inhibited by low concentration of Al. However, F accumulation in shoots and tender leaves (3rd,4th and5th) was enhanced by high concentration of Al3+in culture solution.4. Studies on subcellular distribution of F in tea plant leaves by isolation of cell fraction using differential centrifugation and TEM-EDX showed the following sequence:cell wall faction> protoplast fraction> chloroplast fraction in both tender shoots and mature leaves. Capacity of F accumulation in vacuole was quite limited. F distributed in subcellular fractions increased with the increase in F concentration of culture solution. The increase in F accumulation in cell wall was higher than in protoplast and chloroplast. In F" and A13+combination treatments, the accumulation of F in cell wall of mature leaves was the highest when tea plant was exposed to high F and low Al level. The absorbed F in tea leaves might be aggregated with Al in the cell wall where there are amylase and protein which are abundant in metal ion coordination groups such as hydroxide, carboxyl, aldehyde, amidogen and phosphate. These inhibited F-and Al3+cross-membrane transport, and decreased the F concentration in the protoplast. These might be a mechanism of tea plant tolerance to F. Under F starving condition, F in cell wall can be dissociated into free species which will be transported to young growing shoots and leaves of tea plants.5. Chemical species of aluminum and their subcellular distribution in tea leaves was studied by the differential centrifugation technique and chemical extraction method. It showed that organic aluminum amounted86.88%-88.11%of total aluminum in tea leaves, among which36.87%-56.82%was stable form and31.28%-51.08%unstable one. Inorganic aluminum was11.89%-13.12%of total aluminum in tea leaves, including species Al3-, Al(OH)2+and Al(OH)2+. There was significant difference in total aluminum level but no significant difference in ratio of organic aluminum to inorganic aluminum between various tea cultivars. The absorbed aluminum was mainly distributed in cell wall fraction which accounted for64.4%and83.24%of the total aluminum in the tender shoots and the matured leaves, respectively. The aluminum species of tea leaves is majorly in the organic forms and aluminum concentration in the cell wall fraction increased with the maturity of leaves. These might be contributed to lowering toxicity of aluminum and the aluminum tolerance of tea plant.6. Kinetics of F leaching from tea were studied. The leaching process of F could be divided into initial fast stage and later slow stage. The duration of initial fast stage depended on leaf particle size and temperature. The higher the temperature and the smaller the leaf particle size, the shorter the duration of the initial fast stage. Based on the test results, a two-step infusion method was developed to control F level in tea liquor. When the tea was infused in50℃water for20s (first infusion) and then the residue leaf was re-infused in boiling water for5min (second infusion), more than26percent of total F was removed during first infusion, but more than80percent of catechins and caffeine of the teas were remained in the second infusions. The F level in the second infusion was0.70mg·L-1or less. It is considered that the two-step infusion method is an alternative to control excessive F exposure for tea drinkers.更多还原