茶叶γ-氨基丁酸富集方法及其检测方法的研究
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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。
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.
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。
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.
引文
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2.安藤干夫.発芽玄米が日本丸を救ラ[J].食の科学, 2001, 23(4): 66-73
3.龚加顺.γ-氨基丁酸茶(Gabaron)的药理作用[J].农牧产品开发, 1998, 6:16-17
4. Okada T, Sugishita T, Murakami T et al. Effect of the defatted rice germ enriched with GABA for sleeplessness, depression, autonomic disorder by oral administration [J]. Nippon Shokuhin Kagaku Kougaku Kaishi (In Japan), 2000, 47(8):596-603
5. Crawford LA, Bown AW, Breitkreuz KE et al. The Synthesis ofγ-Aminobutyric Acid in Response to Treatments Reducing Cytosolic pH [J]. Plant Physiology, 1994, 104(3):865-871
6. Effer WR, Ranson SL. Respiratory Metabolism in Buckwheat Seedlings [J]. Plant Physiology, 1967, 42:1042-1052
7. Guinn G, Brinkerhoff LA. Effect of Root Aeration on Amino Acid Levels in Cotton Plants [J]. Crop Science, 1970, 10:175-178
8. Thompson JF, Stewart CR, Morris CJ. Changes in Amino Acid Content of Excised Leaves During Incubation I. The Effect of Water Content of Leaves and Atmospheric Oxygen Level [J]. Plant Physiology, 1966, 41:1578-1584
9. Streeter JG, Thompson JF. Anaerobic accumulation ofγ-aminobutyric acid and alanine in radish leaves (Raphanus sativus L.) [J]. Plant Physiology, 1972, 49: 572-578
10. Lane TR, Stiller M. Glutamic Acid Decarboxylation in Chlorella [J]. Plant Physiology, 1970, 45:558-562
11. Wood JD, Watson WJ, Ducker AJ. The effect of hypoxia on brainγ-aminobutyric acid levels [J]. Journal of Neurochemistry, 1968, 15(7):603-608
12. Tsushida T, Murai T. Conversion of Glutamic Acid toγ-Aminobutyric Acid in Tea Leaves under Anaerobic Conditions [J]. Agriculture and Biological Chemistry, 1987, 51(11):2865-2871
13. Naidu BP, Paleg LG, Aspinall D et al. Amino acid and glycine betaine accumulation in cold-stressed wheat seedlings [J]. Phytochemistry, 1991, 30(2):407-409
14. Mayer RR, Cherry JH, Rhodes D. Effects of Heat Shock on Amino Acid Metabolism of Cowpea Cells [J]. Plant Physiology, 1990, 94:796-810
15.许建军,江波,许时婴.比色法快速测定乳酸菌谷氨酸脱羧酶活力及应用[J].微生物学通报, 2004, 31(2):66-71
16. Narayan VS, Nair PM. Metabolism, enzymology and possible roles of 4-aminobutyrate in higherplant [J]. Phytochemistry, 1990, 29(2):367-375
17. Naylor AW, Tolbert NE. Glutamic acid metabolism in green and etiolated barley leaves [J]. Physiologia Plantarum, 1956, 9(2):220-229
18. Dixon ROD, Fowden L.γ-Aminobutyric acid metabolism in plants [J]. Ann Bot, 1961, 25: 513-530
19. Streeter J.G., Thompson J.F. In vivo and in vitro studies onγ-Aminobutyric acid metabolism with the radish plant (Raphanus sativus L.). Plant Physiology,1972.49:579-584
20. Tokunaga M, Nakano Y, Kitaoka S. The GABA shunt in the callus cells derived from soybean cotyledon [J]. Agriculture and Biological Chemistry, 1976, 40:115-120
21.萧慧,萧伟祥.茶叶中γ-氨基丁酸的形成机理与富集技术[J].福建茶叶, 2003, 2:33-35
22.穆小民,杨晓贺,吴显荣等.林生黧豆谷氨酸脱羧酶的性质及其比较分析研究[J].中国农业大学学报, 1999, 4(3):29-34
23. Hiroshi Ueno. Enzymatic and structural aspects on glutamate decarboxylase [J]. Journal of Molecular Catalysis B: Enzymatic, 2000, 10(1-3):67–79
24. Baum G, Chen Y, Arazi T et al. A plant glutamate decarboxylase containing a calmodulin binding domain. Cloning, sequence, and functional analysis [J]. Journal of Biological Chemistry, 1993, 268(26):19610-19617
25. Snedden WA, Koutsia N, Baum G, et al. Activation of a recombinant petunia glutamate decarboxylase by calcium/calmodulin or by a monoclonal antibody which recognizes the calmodulin binding domain [J]. Journal of Biological Chemistry, 1996, 271(8): 4148-4153
26. Snedden WA, Arazi T, Fromm H et al. Calcium/calmodulin activation of soybean glutamate decarboxylase [J]. Plant Physiology, 1995, 108(2):543-549
27. Ling V, Snedden WA, Shelp BJ et al. Analysis of a soluble calmodulin binding protein from fava bean roots: identification of glutamate decarboxylase as a calmodulin-activated enzyme [J]. The Plant Cell, 1994, 6(8):1135-1143
28. Aurisano N, Bertani A, Reggiani R. Involvement of calcium and calmodulin in protein and amino acid metabolism in rice roots under anoxia [J]. Plant and Cell Physiology, 1995, 36(8):1525-1529
29. Cholewa E, Bown AW, Cholewinski AJ et al. Cold-shock-stimulatedγ-aminobutyric acid synthesis is mediated by an increase in cytosolic Ca2+, not by an increase in cytosolic H+ [J]. Canadian Journal of Botany, 1997, 75(3):375-382
30. Knight MR, Campbell AK, Smith MS et al. Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium [J]. Nature, 1991, 352(8):524-526
31. Sebastiani L, Lindberg S, Vitagliano C. Cytoplasmic free Ca2+ dynamics in single tomato (Lycopersicon esculentum) protoplast subjected to chilling temperatures [J]. Physiologia Plantrum,1999, 105(2):239-244
32. Roberts JKM, Hooks MA, Miaullis AP et al. Contribution of malate and amino acid metabolism to cytoplasmic pH regulation in hypoxic maize root tips studied using nuclear magnetic resonance spectroscopy [J]. Plant Physiology, 1992, 98: 480-487
33. Wallace W, Secor J, Schrader LE. Rapid accumulation ofγ-aminobutyric acid and alanine in soybean leaves in response to an abrupt transfer to lower temperature, darkness, or mechanical manipulation [J]. Plant Physiology, 1984, 75: 170-175
34. Ramputh AI, Bown AW. Rapidγ-aminobutyric acids synthesis and the inhibition of the growth and development of oblique-banded leaf-roller larvae [J]. Plant Physiology, 1996, 111(4):1349-1352
35. Snedden WA, Chung I, Pauls RH et al. Proton/L-glutamate symport and the regulation of intracellular pH in isolated mesophyll cells [J]. Plant Physiology, 1992, 99:665-671
36. Satyanarayan V, Nair PM. Purification and characterization of glutamate decarboxylase from Solanum tuberosum [J]. European Journal of Biochemistry, 1985, 150(1):53-60
37. Breitkreuz KE, Shelp BJ. Subcellular compartmentation of the 4-aminobutyrate shunt in protoplasts from developing soybean cotyledons [J]. Plant Physiology, 1995, 108(1):99-103
38. Carroll AD, Stewart GR, Phillips R. Dynamics of nitrogenous assimilate partitioning between cytoplasmic and vacuolar fractions in carrot cell suspension cultures [J]. Plant Physiology, 1992, 100: 1808-1814
39. Kurkdjian A, Guern J. Intracellular pH: Measurement and Importance in Cell Activity. Annual Review of Plant Physiology and Plant Molecular Biology [M]. 1989, 40:271-303
40. Carroll AD, Fox GG, Laurie S et al. Ammonium assimilation and the role ofγ-aminobutyric acid in pH homeostasis in carrot cell suspensions [J]. Plant Physiology, 1994, 106(2):513-520
41. Bosse Wigge, Silke Kr?mer, Per Gardestr?m. The redox levels and subcellular distribution of pyridine nucleotides in illuminated barley leaf protoplasts studied by rapid fractionation [J]. Physiologia Plantarum, 1993, 88(1):10-18
42. Shelp BJ, Walton CS, Snedden WA et al. Gaba shunt in developing soybean seeds is associated with hypoxia [J]. Physiologia Plantarum, 1995, 94(2):219-228
43. Bown AW, Shelp BJ. The Metabolism and Functions ofγ-Aminobutyric Acid [J]. Plant Physiology, 1997, 115(1):1-5
44.杨胜远,陆兆新,吕凤霞等.谷氨酸脱羧酶测定中GABA比色定量方法研究[J].食品科学, 2006, 27(07):205-209
45.杨广容,邵宛芳,蔡新等.摊放程度对云南大叶种扁形绿茶品质的影响[J].云南农业大学学报, 2007, 22(3):404-408
46.宁井铭,杨进华.鲜叶摊放与绿茶品质的综述[J].茶业通报, 2001, 23(4):30-32
47.宛晓春主编.茶叶生物化学(第三版)[M].北京:中国农业出版社,2003年版.pp:91-95, pp:132-144
48. Sawai Y, Yamaguchi Y, Miyama D et al. Cycling treatment of anaerobic and aerobic incubation increases the content ofγ-aminobutyric acid in tea shoots [J]. Amino Acids, 2001, 20(3):331-334
49.黄亚辉,郑红发等. Gabaron茶加工过程中γ-氨基丁酸和谷氨酸的动态变化研究[J].食品科学, 2005, 26(8):117-120
50.林智,林钟鸣,尹军峰等.厌氧处理对茶叶-氨基丁酸含量及其品质的影响[J].食品科学, 2004, 25(2):35-39
51.林智,大森正司等.γ-氨基丁酸茶(Gabaron Tea)降血压机理的研究.茶叶科学, 2001, 21(2):153-156
52.朱旗,施兆鹏等. HPLC检测分析速溶绿茶游离氨基酸[J].茶叶科学, 2001, 21(2): 134-136
53.房克敏,李再贵等. HPLC法测定发芽糙米中γ-氨基丁酸含量[J].食品科学, 2006, 27(4):208-211
54.李立祥,童梅英.固样方法对茶叶化学成分及品质的影响[J].安徽农业大学学报, 2000, 27(4):394-395
55.商业部茶叶畜产局,商业部杭州茶叶加工研究所.茶叶理化品质分析[M].上海:上海科学技术出版社, 1989
56. Thomas H. Schultz, Robert A. Flath, T. Richard Mon et al. Isolation of volatile components from a model system [J]. Agricultural and Food Chemistry, 1977, 25 (3): 446-449
57.王同和,胡敏,张久谦等.名优绿茶感官品质相关因子分析[J].茶叶科学, 2008, 28 (1):33-38
58.津志田藤二郎.强化降血压茶的研制[J].国外农学-茶叶, 1987, (4):36—37
59.津志田藤二郎等.γ-フミノ酪酸き蓄积だせた茶の制造とその特征[J].农化, 1987, 61:8l7-822
60. Tei Yamanish. Flavor of tea [J]. Food reviews international, special issue on tea, 1995, 11(3):587-506
61. Mosandl A. Capillary gas chromatography in quality assessment of flavours and fragrances: Applications of chromatography and electrophoresis in food science [J]. Journal of Chromatography, 1992, 624(2):267-292
62. Mitsuya Shimoda, Hiroko Shigematsu, Hudeki Shiratsuchi, et al. Comparison of the odor concentrates by SDE and adsorptive column method from green tea infusion [J]. Journal of Agricultural and Food Chemistry, 1995, 43(6):1616-1620
63.陈玉琼,倪德江.不同加工方法对名优绿茶香气成分差异性的研究[J].华中农业大学学报, 1997, 16(1):93-95
64.夏涛,童启庆.冷冻萎凋对茶叶多酚氧化酶和β-葡萄糖苷酶活性的影响初探[J].浙江农业大学学报, 1997, 23(1):108-110
65.黄建琴,王文杰,丁勇等.冷冻萎凋对工夫红茶品质的影响.中国茶叶, 2005, 27(2):18-19
66.沈佐君,李小鹏,孙曾培等.一种新的氨基酸测定衍生化方法[J].基础医学与临床, 1998, 18 (4):75-80
67.李平,宛晓春,李健等.茶氨酸的衍生化及毛细管电泳定量技术[J].茶叶科学, 2004,24(2):119-123
68.王镜岩,朱圣庚,徐长法.生物化学(第三版下册)[M].北京:高等教育出版社,2002年版. pp:303-310
2.安藤干夫.発芽玄米が日本丸を救ラ[J].食の科学, 2001, 23(4): 66-73
3.龚加顺.γ-氨基丁酸茶(Gabaron)的药理作用[J].农牧产品开发, 1998, 6:16-17
4. Okada T, Sugishita T, Murakami T et al. Effect of the defatted rice germ enriched with GABA for sleeplessness, depression, autonomic disorder by oral administration [J]. Nippon Shokuhin Kagaku Kougaku Kaishi (In Japan), 2000, 47(8):596-603
5. Crawford LA, Bown AW, Breitkreuz KE et al. The Synthesis ofγ-Aminobutyric Acid in Response to Treatments Reducing Cytosolic pH [J]. Plant Physiology, 1994, 104(3):865-871
6. Effer WR, Ranson SL. Respiratory Metabolism in Buckwheat Seedlings [J]. Plant Physiology, 1967, 42:1042-1052
7. Guinn G, Brinkerhoff LA. Effect of Root Aeration on Amino Acid Levels in Cotton Plants [J]. Crop Science, 1970, 10:175-178
8. Thompson JF, Stewart CR, Morris CJ. Changes in Amino Acid Content of Excised Leaves During Incubation I. The Effect of Water Content of Leaves and Atmospheric Oxygen Level [J]. Plant Physiology, 1966, 41:1578-1584
9. Streeter JG, Thompson JF. Anaerobic accumulation ofγ-aminobutyric acid and alanine in radish leaves (Raphanus sativus L.) [J]. Plant Physiology, 1972, 49: 572-578
10. Lane TR, Stiller M. Glutamic Acid Decarboxylation in Chlorella [J]. Plant Physiology, 1970, 45:558-562
11. Wood JD, Watson WJ, Ducker AJ. The effect of hypoxia on brainγ-aminobutyric acid levels [J]. Journal of Neurochemistry, 1968, 15(7):603-608
12. Tsushida T, Murai T. Conversion of Glutamic Acid toγ-Aminobutyric Acid in Tea Leaves under Anaerobic Conditions [J]. Agriculture and Biological Chemistry, 1987, 51(11):2865-2871
13. Naidu BP, Paleg LG, Aspinall D et al. Amino acid and glycine betaine accumulation in cold-stressed wheat seedlings [J]. Phytochemistry, 1991, 30(2):407-409
14. Mayer RR, Cherry JH, Rhodes D. Effects of Heat Shock on Amino Acid Metabolism of Cowpea Cells [J]. Plant Physiology, 1990, 94:796-810
15.许建军,江波,许时婴.比色法快速测定乳酸菌谷氨酸脱羧酶活力及应用[J].微生物学通报, 2004, 31(2):66-71
16. Narayan VS, Nair PM. Metabolism, enzymology and possible roles of 4-aminobutyrate in higherplant [J]. Phytochemistry, 1990, 29(2):367-375
17. Naylor AW, Tolbert NE. Glutamic acid metabolism in green and etiolated barley leaves [J]. Physiologia Plantarum, 1956, 9(2):220-229
18. Dixon ROD, Fowden L.γ-Aminobutyric acid metabolism in plants [J]. Ann Bot, 1961, 25: 513-530
19. Streeter J.G., Thompson J.F. In vivo and in vitro studies onγ-Aminobutyric acid metabolism with the radish plant (Raphanus sativus L.). Plant Physiology,1972.49:579-584
20. Tokunaga M, Nakano Y, Kitaoka S. The GABA shunt in the callus cells derived from soybean cotyledon [J]. Agriculture and Biological Chemistry, 1976, 40:115-120
21.萧慧,萧伟祥.茶叶中γ-氨基丁酸的形成机理与富集技术[J].福建茶叶, 2003, 2:33-35
22.穆小民,杨晓贺,吴显荣等.林生黧豆谷氨酸脱羧酶的性质及其比较分析研究[J].中国农业大学学报, 1999, 4(3):29-34
23. Hiroshi Ueno. Enzymatic and structural aspects on glutamate decarboxylase [J]. Journal of Molecular Catalysis B: Enzymatic, 2000, 10(1-3):67–79
24. Baum G, Chen Y, Arazi T et al. A plant glutamate decarboxylase containing a calmodulin binding domain. Cloning, sequence, and functional analysis [J]. Journal of Biological Chemistry, 1993, 268(26):19610-19617
25. Snedden WA, Koutsia N, Baum G, et al. Activation of a recombinant petunia glutamate decarboxylase by calcium/calmodulin or by a monoclonal antibody which recognizes the calmodulin binding domain [J]. Journal of Biological Chemistry, 1996, 271(8): 4148-4153
26. Snedden WA, Arazi T, Fromm H et al. Calcium/calmodulin activation of soybean glutamate decarboxylase [J]. Plant Physiology, 1995, 108(2):543-549
27. Ling V, Snedden WA, Shelp BJ et al. Analysis of a soluble calmodulin binding protein from fava bean roots: identification of glutamate decarboxylase as a calmodulin-activated enzyme [J]. The Plant Cell, 1994, 6(8):1135-1143
28. Aurisano N, Bertani A, Reggiani R. Involvement of calcium and calmodulin in protein and amino acid metabolism in rice roots under anoxia [J]. Plant and Cell Physiology, 1995, 36(8):1525-1529
29. Cholewa E, Bown AW, Cholewinski AJ et al. Cold-shock-stimulatedγ-aminobutyric acid synthesis is mediated by an increase in cytosolic Ca2+, not by an increase in cytosolic H+ [J]. Canadian Journal of Botany, 1997, 75(3):375-382
30. Knight MR, Campbell AK, Smith MS et al. Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium [J]. Nature, 1991, 352(8):524-526
31. Sebastiani L, Lindberg S, Vitagliano C. Cytoplasmic free Ca2+ dynamics in single tomato (Lycopersicon esculentum) protoplast subjected to chilling temperatures [J]. Physiologia Plantrum,1999, 105(2):239-244
32. Roberts JKM, Hooks MA, Miaullis AP et al. Contribution of malate and amino acid metabolism to cytoplasmic pH regulation in hypoxic maize root tips studied using nuclear magnetic resonance spectroscopy [J]. Plant Physiology, 1992, 98: 480-487
33. Wallace W, Secor J, Schrader LE. Rapid accumulation ofγ-aminobutyric acid and alanine in soybean leaves in response to an abrupt transfer to lower temperature, darkness, or mechanical manipulation [J]. Plant Physiology, 1984, 75: 170-175
34. Ramputh AI, Bown AW. Rapidγ-aminobutyric acids synthesis and the inhibition of the growth and development of oblique-banded leaf-roller larvae [J]. Plant Physiology, 1996, 111(4):1349-1352
35. Snedden WA, Chung I, Pauls RH et al. Proton/L-glutamate symport and the regulation of intracellular pH in isolated mesophyll cells [J]. Plant Physiology, 1992, 99:665-671
36. Satyanarayan V, Nair PM. Purification and characterization of glutamate decarboxylase from Solanum tuberosum [J]. European Journal of Biochemistry, 1985, 150(1):53-60
37. Breitkreuz KE, Shelp BJ. Subcellular compartmentation of the 4-aminobutyrate shunt in protoplasts from developing soybean cotyledons [J]. Plant Physiology, 1995, 108(1):99-103
38. Carroll AD, Stewart GR, Phillips R. Dynamics of nitrogenous assimilate partitioning between cytoplasmic and vacuolar fractions in carrot cell suspension cultures [J]. Plant Physiology, 1992, 100: 1808-1814
39. Kurkdjian A, Guern J. Intracellular pH: Measurement and Importance in Cell Activity. Annual Review of Plant Physiology and Plant Molecular Biology [M]. 1989, 40:271-303
40. Carroll AD, Fox GG, Laurie S et al. Ammonium assimilation and the role ofγ-aminobutyric acid in pH homeostasis in carrot cell suspensions [J]. Plant Physiology, 1994, 106(2):513-520
41. Bosse Wigge, Silke Kr?mer, Per Gardestr?m. The redox levels and subcellular distribution of pyridine nucleotides in illuminated barley leaf protoplasts studied by rapid fractionation [J]. Physiologia Plantarum, 1993, 88(1):10-18
42. Shelp BJ, Walton CS, Snedden WA et al. Gaba shunt in developing soybean seeds is associated with hypoxia [J]. Physiologia Plantarum, 1995, 94(2):219-228
43. Bown AW, Shelp BJ. The Metabolism and Functions ofγ-Aminobutyric Acid [J]. Plant Physiology, 1997, 115(1):1-5
44.杨胜远,陆兆新,吕凤霞等.谷氨酸脱羧酶测定中GABA比色定量方法研究[J].食品科学, 2006, 27(07):205-209
45.杨广容,邵宛芳,蔡新等.摊放程度对云南大叶种扁形绿茶品质的影响[J].云南农业大学学报, 2007, 22(3):404-408
46.宁井铭,杨进华.鲜叶摊放与绿茶品质的综述[J].茶业通报, 2001, 23(4):30-32
47.宛晓春主编.茶叶生物化学(第三版)[M].北京:中国农业出版社,2003年版.pp:91-95, pp:132-144
48. Sawai Y, Yamaguchi Y, Miyama D et al. Cycling treatment of anaerobic and aerobic incubation increases the content ofγ-aminobutyric acid in tea shoots [J]. Amino Acids, 2001, 20(3):331-334
49.黄亚辉,郑红发等. Gabaron茶加工过程中γ-氨基丁酸和谷氨酸的动态变化研究[J].食品科学, 2005, 26(8):117-120
50.林智,林钟鸣,尹军峰等.厌氧处理对茶叶-氨基丁酸含量及其品质的影响[J].食品科学, 2004, 25(2):35-39
51.林智,大森正司等.γ-氨基丁酸茶(Gabaron Tea)降血压机理的研究.茶叶科学, 2001, 21(2):153-156
52.朱旗,施兆鹏等. HPLC检测分析速溶绿茶游离氨基酸[J].茶叶科学, 2001, 21(2): 134-136
53.房克敏,李再贵等. HPLC法测定发芽糙米中γ-氨基丁酸含量[J].食品科学, 2006, 27(4):208-211
54.李立祥,童梅英.固样方法对茶叶化学成分及品质的影响[J].安徽农业大学学报, 2000, 27(4):394-395
55.商业部茶叶畜产局,商业部杭州茶叶加工研究所.茶叶理化品质分析[M].上海:上海科学技术出版社, 1989
56. Thomas H. Schultz, Robert A. Flath, T. Richard Mon et al. Isolation of volatile components from a model system [J]. Agricultural and Food Chemistry, 1977, 25 (3): 446-449
57.王同和,胡敏,张久谦等.名优绿茶感官品质相关因子分析[J].茶叶科学, 2008, 28 (1):33-38
58.津志田藤二郎.强化降血压茶的研制[J].国外农学-茶叶, 1987, (4):36—37
59.津志田藤二郎等.γ-フミノ酪酸き蓄积だせた茶の制造とその特征[J].农化, 1987, 61:8l7-822
60. Tei Yamanish. Flavor of tea [J]. Food reviews international, special issue on tea, 1995, 11(3):587-506
61. Mosandl A. Capillary gas chromatography in quality assessment of flavours and fragrances: Applications of chromatography and electrophoresis in food science [J]. Journal of Chromatography, 1992, 624(2):267-292
62. Mitsuya Shimoda, Hiroko Shigematsu, Hudeki Shiratsuchi, et al. Comparison of the odor concentrates by SDE and adsorptive column method from green tea infusion [J]. Journal of Agricultural and Food Chemistry, 1995, 43(6):1616-1620
63.陈玉琼,倪德江.不同加工方法对名优绿茶香气成分差异性的研究[J].华中农业大学学报, 1997, 16(1):93-95
64.夏涛,童启庆.冷冻萎凋对茶叶多酚氧化酶和β-葡萄糖苷酶活性的影响初探[J].浙江农业大学学报, 1997, 23(1):108-110
65.黄建琴,王文杰,丁勇等.冷冻萎凋对工夫红茶品质的影响.中国茶叶, 2005, 27(2):18-19
66.沈佐君,李小鹏,孙曾培等.一种新的氨基酸测定衍生化方法[J].基础医学与临床, 1998, 18 (4):75-80
67.李平,宛晓春,李健等.茶氨酸的衍生化及毛细管电泳定量技术[J].茶叶科学, 2004,24(2):119-123
68.王镜岩,朱圣庚,徐长法.生物化学(第三版下册)[M].北京:高等教育出版社,2002年版. pp:303-310