【作者】 邢志强；

【导师】 李立祥；

【作者基本信息】 安徽农业大学 ， 茶学， 2009， 硕士

【摘要】 γ-氨基丁酸(又称4-氨基丁酸,γ-aminobutyric acid,简称GABA)是一种非蛋白质天然氨基酸,具有良好的保健功效。茶鲜叶具有GABA生物合成基础,是天然GABA的良好来源。目前,GABA在茶叶中的富集方法较为单一,成本相对较高,本研究以茶鲜叶为材料,按普通烘青绿茶生产工艺,采用多种逆境处理方式研究富集GABA的方法,旨在探索新的GABA富集方式;并采用ODS-C18色谱柱HPLC法,利用2、4-二硝基氟苯(2,4-Dinitro-1-fluorobenzene,简称DNFB)柱前衍生,进行茶叶中的GABA与L-谷氨酸(L-Glutamic acid,简称L-Glu)的精确定量方法研究,为茶叶源GABA的检测提供一种准确的、成本相对低廉的分析手段。采用DNFB柱前衍生,以0.05M乙酸钠缓冲液(pH=6.5,含10mL/L N,N-二甲基甲酰胺)和50%乙腈(V/V)为流动相,建立了茶叶源GABA和L-Glu的HPLC检测方法,并与Waters AccQ·Tag法进行了对比。同时考察了DNFB试剂的用量,衍生产物的稳定性及检测波长。结果表明:GABA与L-Glu浓度在0.03mM～2.0mM范围内,峰面积与浓度之间有良好的线性关系,相关系数R2均为1;各样品γ-氨基丁酸、L-谷氨酸含量RSD(%)分别为0.91%～2.01%和0.94%～1.91%,加标回收率分别为95.82%～99.33%和95.83%～99.88%;10mL/LDNFB乙腈溶液添加量1mL;避光保存一周内,衍生物稳定性良好;衍生物检测波长为360nm。获得HPLC色谱条件:SHIMADZU LC-20AD液相色谱仪,LCsolution液相色谱工作站;色谱柱:大连Elite Hypersil ODS-C18柱(250mm×4.6mm i.d.,5μm);流动相A:0.05M乙酸钠缓冲液(pH=6.5,含10mL/L N,N-二甲基甲酰胺);流动相B:乙腈-水(V/V=1:1);流速:1.0mL/min;柱温:28℃;进样量:5μL;检测波长:360nm。采用充CO2、充N2、真空、水浸渍、冷冻、紫外线照射等方式对茶鲜叶进行GABA富集处理,并以常规绿茶加工为对照,对处理过程中GABA和L-Glu的含量变化进行考察。结果表明:常规的绿茶加工过程中,GABA含量介于0.04mg/g ~0.05mg/g,GABA不会富集;CO2厌氧处理GABA在茶鲜叶中富集,其含量从0.04mg/g升至2.90mg/g,并GABA与L-Glu呈现出明显的消长关系,L-Glu含量从起始的1.57 mg/g降至0.42 mg/g;充N2厌氧处理过程中,亦呈现出类似规律,GABA含量从0.04mg/g升至2.43mg/g,L-Glu则从起始的1.57 mg/g降至0.43 mg/g;真空度0.04-0.1MPa范围内,鲜叶中GABA的富集并不明显,GABA含量峰值仅为0.47mg/g,而L-Glu在处理始末含量却有所增加;水浸渍处理过程中,GABA与L-Glu也呈现消长关系,但不明显,GABA峰值仅为0.81mg/g,L-Glu最低值为1.22mg/g;冷冻和紫外线照射处理均未能使得鲜叶中GABA的总量有所增加,L-Glu含量始末变化不大。为了更好的明确逆境处理条件,本研究考察了光照条件对CO2厌氧处理和水浸渍处理的影响。结果发现:光照条件对不同逆境处理介质的影响不相同。光照处理GABA峰值分别比暗处理条件下高出0.68mg/g(CO2厌氧处理)和0.52 mg/g(水浸渍处理)。CO2厌氧处理光照条件下,GABA在厌氧处理阶段富集,在有氧阶段呈下降;水浸渍处理光照条件下,GABA在有氧阶段富集,在水浸渍阶段下降。CO2厌氧光照处理使得L-Glu消耗的更多;水浸渍光照处理对L-Glu的变化趋势有影响,但L-Glu最终含量在增加。各处理GABA富集效果依次为:光照CO2厌氧处理(峰值2.90mg/g)、光照N2厌氧处理(峰值2.43mg/g)、遮光CO2厌氧处理(峰值2.22mg/g)、光照水浸渍处理(峰值1.33mg/g)、遮光水浸渍处理(峰值0.81mg/g)、真空处理(峰值0.47mg/g)、冷冻处理(峰值0.11mg/g)和紫外线照射处理(峰值0.07mg/g)。通过分析检测、香气成分分析和感官审评,考察了富集GABA处理对茶样主要化学成分和品质的影响。结果表明:咖啡碱和儿茶素总量与对照组相比,变化不大;对照样茶多酚含量总体变化不大,充CO2、充N2、真空、紫外线照射处理对茶多酚含量影响不大,水浸渍处理使得茶多酚总量有所增加,含量达到了30%以上,冷冻处理过程中,茶多酚总量减少了5.47%。除了冷冻处理含量基本不变,其余各处理氨基酸总量均有不同程度的增加:对照组氨基酸总量增幅达27.8%,紫外照射处理结果与对照组相近,充CO2、充N2也有13.4%～16.5%的增幅,真空处理增幅达到25.2%,水浸泡处理也有较小增幅。各处理可溶性糖呈现无规律变化。处理间香精油组分差异较明显,主要香气物质种类和数量不尽一致,但同一处理介质(A、B,G、H)间无明显差异。茶样感官审评结果表明,GABA茶的加工方式对于成茶的品质有很大的影响,GABA的提高伴随着成茶品质的下降。更多还原

【Abstract】 γ-Aminobutyric acid (referred to as GABA) is a natural nonprotein amino acid which has good performance at health care. And tea leaves, which have biosynthesis ability of GABA, are natural source of GABA. But there are only few enriching ways of GABA in tea leaves, and the cost is high. This work mainly focused on the study of GABA enriching ways in tea leaves, and then processed the leaves to be baked green tea. In order to quantify GABA and L-Glutamic acid (referred to as L-Glu) of tea samples precisely, a quantification method of GABA and L-Glu in tea with HPLC was established. In the method, the ODS-C18 column and 2,4-Dinitro-1-fluorobenzene(referred to as DNFB) pre-column derivatization were involved.Compared with Waters AccQ·Tag, a method for the determination of GABA and L-Glu in tea by using HPLC was developed. DNFB was taken as the derivative reagent, while 0.05M sodium acetate buffer solution (pH=6.5, with 10mL/L N,N-dimethylformamide) and 50% acetonitrile solution (V/V) were used as the mobile phase. And we compared the results with. The factors such as detection wavelength, amount of DNFB and stability of derivatived amino acids were studied. Results showed that the peak area of GABA and L-Glu had a good linear relationship with their concentrations range from 0.03mM to 2.0mM and the correlation coefficients were both 1. The RSD(%) of GABA and L-Glu in each sample were 0.91% to 2.01% and 0.94% to 1.91%, and the recoveries were 95.82% to 99.33% and 95.83% to 99.88%, respectively. Other results are: the addition volume of 10mL/L DNFB in acetonitrile solution to tea infusion is 1mL; preserved in dark, within one week, derivatived amino acids had good stability; the detection wavelength of derivatives is 360nm.The optimal HPLC chromatographic conditions are as follows: SHIMADZU LC-20AD HPLC with LCsolution workstation; Dalian Elite Hypersil ODS-C18 column (250mm×4.6mm id, 5μm); mobile phase A is 0.05M sodium acetate buffer (pH = 6.5, containing 10mL/L N, N-dimethylformamide); mobile phase B is acetonitrile-water (V/V=1:1); flow rate is 1.0 mL/min; column temperature: 28℃; injection volume is 5μL; detection wavelength is 360nm.The enrichment of GABA in tea leaves was conducted by the treatments of CO2, N2, vacuum, water soaking, freezing and UV-irradiation, respectively. The conventional processing of green tea was taken as control to monitor the changes of GABA and L-Glu content. The results showed that GABA was less enriched and its content was in range of 0.04 mg/g~0.05 mg/g during the conventional green tea processing. GABA was enriched in the fresh tea leaves treated by CO2 and its content was increased from 0.04 mg/g to 2.90 mg/g. L-Glu content was declined from 1.57 mg/g to 0.42 mg/g in the fresh tea leaves treated by CO2 and showed a clear relation of growth and decline with GABA. A similar result was detected in the anaerobic treatment by N2. GABA content was increased from 0.04 mg/g up to 2.43 mg/g and L-Glu content was declined from 1.57 mg/g to 0.43 mg/g. The enrichment of GABA was not apparent during the vacuum range of 0.04-0.1MPa, its content is only 0.47 mg/g at the peak and L-Glu had a little increase in the whole processing. The contents of GABA and L-Glu also showed a relationship of increase and decline, although not obviously in the water soaking process. GABA content was 0.81mg/g at the peak while L-Glu content was 1.22 mg/g at the bottom. The freezing and UV-irradiation treatment did not increased the total GABA content in fresh tea leaves and did not changed the L-Glu content either.In order to understand the effect of the adverse environment better, we investigated the impact of light conditions on CO2 and water impregnation treatment. The results showed that light conditions had different impacts on different adverse environments. The GABA content was increased by 0.68 mg/g (in CO2 anaerobic treatment) and 0.52 mg/g (in water soaking treatment) compared with dark conditions. In the CO2 anaerobic treatment under light conditions, GABA was concentrated in the anaerobic treatment stage and decreasing in the aerobic phase; in the water soaking treatment under light conditions, GABA was concentrated in the aerobic stage and decreasing in the water soaking phase. L-Glu was consumed more in the illumination CO2 anaerobic treatment and its content was also influenced in the water impregnation with light treatment, but the final content of L-Glu was on the rise.The effect of the treatments in GABA enriching as follows: anaerobic treatment by CO2 under light(peak at 2.90mg/g), anaerobic treatment by N2 under light (peak at 2.43mg/g), anaerobic treatment by CO2 under shading light (peak at 2.22mg/g), water soaking under light (peak at 1.33mg/g), water soaking under shading light (peak at 0.81mg/g), vacuum processing (peak at 0.47mg/g), frozen (peak at 0.11mg/g) and UV-irradiation treatment (peak at 0.07mg/g).This paper explored the impact of GABA enriching processing on the quality and chemical composition of tea samples by analyzing the components of aroma, and sensory evaluation. The results show that total amount of caffeine and catechins have little difference compared with the control group; the total amount of tea polyphenols has no obvious change within the control group, added treatments such as filling CO2, charging N2, vacuum and UV-irradiation processing to tea samples have little effect on amounts of tea polyphenols. But water soaking makes an increase in total polyphenols content (more than 30%). At the same time, the amount of tea polyphenols reduced 5.47% after refrigeration process. The intervention measures mentioned above make the total amount of amino acid for tea samples increasing to some extent except refrigeration process with little change, for example, total amount of amino acid with the increase of 27.8% in the control group and with similar increase in UV-irradiation, with growth of 13.4%-16.5% by filling CO2 and charging N2, 25.2% increase after vacuum process, by comparison, water soaking makes increase a little. Measures above to water-soluble sugar did not show any regular change. In the processing of each intervention measures, components of theol have obvious differences, because the main type and quantity of fragrance material are not consistent, which do not show any difference in the same measures intervened (A、B,G、H). Sensory evaluation of the tea samples shows that GABA tea processing method has a great impact on quality of the tea, especially, making the quality of tea declined.更多还原