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Determination of Theanine, GABA, and Other Amino Acids in Green, Oolong, Black, and Pu-erh Teas with Dabsylation and High-Performance Liquid Chromatography
Abstract
Dabsyl chloride (dimethylaminoazobenzene sulfonyl chloride), a useful chromophoric labeling reagent for amino acids and amines, was developed in this laboratory in 1975. Although several methods have been developed to determine various types of amino acids, a quick and easy method of determining theanine, GABA, and other amino acids has not been developed in one HPLC system. In this paper are analyzed the free amino acid contents of theanine and GABA in different teas (green tea, black tea, oolong tea, Pu-erh tea, and GABA tea) with a dabsylation and reverse phase high-performance liquid chromatography (HPLC) system coupled with a detector at 425 nm absorbance. Two reverse phase columns, Hypersil GOLD and Zorbax ODS, were used and gave different resolutions of dabsyl amino acids in the gradient elution program. The data suggest that the tea source or the steps of tea-making may contribute to the theanine contents variations. High theanine contents of high-mountain tea were observed in both green tea and oolong tea. Furthermore, the raw (natural fermented) Pu-erh tea contained more theanine than ripe (wet fermented) Pu-erh tea, and the GABA contents in normal teas were generally lower than that in GABA tea.
... These steps are frequently interrupted by the arduous task of analyzing numerous samples [13][14][15]. The prevailing methods for assaying GABA include an amino acid analyzer [16], high-performance liquid chromatography (HPLC) [17], and gas chromatography [18]. Although targeting for absolute quantitation, these methods require expensive apparatus or rigorous sample preprocessing steps [19]. ...
... Jones et al. [43] has established a spectrofluorometric method, based on o-phthalaldehyde, for rapidly and sensitively quantitating total free amino acids in soil. However, o-phthalaldehyde has been used more frequently to derivatize amino acids in the methods with separation step(s), such as HPLC or an amino acid analyzer [16,17,46,47], while 1,8-diazafluoren-9-one is a frequently-used visualizer of latent fingerprints in forensics [44,45]. When analyzing some biological samples enriched in amino acid(s), such as fermentative ones, ninhydrin-based colorimetry is still rather popular. ...
... The fermentation medium contained 0.2 M monosodium L-glutamate; and was added to solid L-glutamic acid at 650 g/L just before the fermentation. The added substrate mainly existed in the form of powder due to its low solubility (about 10 g/L) [17,48]. The detailed experimental operations for the fermentation are presented in Section 2.2.2. ...
Gamma-aminobutyric acid (GABA) is a functional metabolite in various organisms. Herein, a sensitivity intensified ninhydrin-based chromogenic system (SINICS), achieved by ethanol and ethyl acetate, is described for the reliable relative quantitation of GABA. A 2.9 mL SINICS kit comprises 1% ninhydrin, 40% ethanol, 25% ethyl acetate, and 35 μL 0.2 M sodium acetate buffer (pH 5.0). In practice, following the addition of a 0.1 mL sample to the kit, the chromogenic reaction is completed by heating at 70 °C for 30 min. The kit increased the color development sensitivity of L-glutamic acid and GABA, with the detection limits being reduced from 20 mM and 200 mM to 5 mM and 20 mM, respectively. The chromophore was stable for at least 2 h at room temperature, which was sufficient for a routine colorimetric analysis. The absorbance at 570 nm with the deduction of background directly represents the content of amino acid. For a proof-of-concept, the SINICS was adopted to optimize the GABA fermentation process of Levilactobacillus brevis CD0817. The results demonstrated that SINICS is an attractive alternative to the available ninhydrin-based colorimetric methods.
... Previous studies using HPLC have determined GABA to be present to varying extents in commercially prepared GABA-enriched teas, including 1560 mg/100 g [9]; 19 mg/100 g [6] and 272 mg/100 g [4]. The type of tea according to the degree of oxidation (e.g., oolong, black) was not specified in these studies. ...
... mg/100 g) > oolong (14.8-20.7 mg/100 g). On the other hand, Syu and colleagues [6] found the GABA content of different teas to be green (19.6-105 mg/100 g) equal to oolong (10-101 mg/100 g) > black (34-55 mg/100 g). In contrast to our findings of 5 mg GABA/100 g regular oolong tea, others have shown, on average, higher GABA concentrations in commercially prepared regular oolong teas. ...
... Like GABA, theanine content measured across a range of commercial tea types is found to vary in concentration, and even within the different tea types, there is significant variation [6,25,26]. For example, commercially prepared white teas measured between 53 and 3337 mg theanine/100 g w/w [25] while oolong teas ranged from 85 to 282 mg/100 g [25], averaging 101 mg/100 g [6]. ...
We have previously shown that the consumption of GABA-enriched oolong tea is effective in reducing stress in a student cohort. However, key constituent content has not been previously investigated, especially as applied to a standard cup of tea. Further, it has not been substantiated whether it is the suggested GABA content or other constituents that lead to these observed changes in stress behaviour. Using reverse-phase HPLC, we determined the actual content of four chemicals known to influence stress in 200 mL cups of regular or GABA-enriched oolong tea brewed to manufacturer’s instructions. We found eight times as much γ-aminobutyric acid (GABA) and 1.5 times as much caffeine in GABA-enriched oolong tea as in regular oolong tea. In contrast, there was 10 times less epigallocatechin gallate (EGCG), and half as much theanine in the GABA-enriched tea. Thus, there are changes in multiple constituents in GABA-enriched oolong tea that may contribute to the biological effects we observed in students consuming these teas.
... Two of these products, fermented radish and fermented eggplant also had the highest amount of Glu among the plant-based food products, with 17.56 mg/g dw and 19.85 mg/g dw, respectively. It is worth to note that the GABA content in these fermented foods was higher than the GABA content in other plant-based GABA-foods, such as GABA tea (0.197 mg/g) (Syu et al., 2008) or fermented black raspberry juice (0.028 mg/mL) (Kim et al., 2009). ...
... Fermentation is the key processing step to enrich GABA in food products, when started from raw materials owning high Glu concentration. Many studies were successful in enhancing the GABA content of fermented products such as cheese, fermented fish, yogurt and tea (Chun et al., 2007;Jannoey et al., 2010b;Jeng et al., 2007;Siragusa et al., 2007;Syu et al., 2008). Almost all these studies have focused on increasing the GABA content by screening microbial strains and studying fermentation conditions. ...
... Germinated legumes and fermented foods are rich sources of GABA due to the fact that GAD is activated during germination and it is produced by microorganisms during fermentation, providing that free Glu as precursor is present. Awide range of fermented foods rich in GABA content can be counted as fermented tea (Syu et al. 2008;Horanni and Engelhardt 2013), meat seasoning powder (Kantachote et al. 2016), cheeses (Diana et al. 2014), and tempeh (Aoki et al. 2003;Long 2013). ...
... Two of these products, fermented radish and fermented eggplant, also had the highest amount of Glu among the plantbased food products, with 17.56 mg/g DW and 19.85 mg/g DW, respectively. It is worth to note that the GABA content in these fermented foods was higher than the GABA content in other plant-based GABA foods, such as GABA tea (0.197 mg/ g) (Syu et al. 2008) or fermented black raspberry juice (0.028 mg/mL) (Kim et al. 2009). ...
In this study, a high-performance liquid chromatography (HPLC) method was adapted to measure the γ-aminobutyric acid (GABA) and glutamate (Glu) content in different fermented foods. The best solvent to extract GABA and Glu from the food matrices was 75% EtOH and water, combined with 4% SSA as posttreatment. The extracted amino acids were derivatized with o-phthalaldehyde (OPA) for 2 min at room temperature and analyzed by HPLC with fluorescence detector (λex = 340 nm and λem = 455 nm). Animal-based fermented products had a higher GABA content, compared with plant-based and soy-based fermented products. Among 17 fermented samples, fish sauce showed the highest amount of GABA (16.29 ± 0.53 mg/kg DW). Results obtained by the HPLC method were compared with those obtained by a spectrophotometric method, which is based on the reaction of ω-amino acids with phenol. All measured GABA contents, obtained by HPLC method, were much lower than those obtained by the spectrophotometric method, which was due to the nonspecific reaction of the coloring reagent with other amino acids than GABA. It can be concluded that GABA content in food products can only be measured by HPLC.
... Its chemical components and properties vary due to the time of year that the tealeaves are harvested and the fermentation methods used. The main components of Pu-erh tea extracts include tea polyphenol, tea pigment, tea polysaccharide and alkaloids (3). In previous years, studies on the potential health-beneficial effects of Pu-erh tea have identified a range of biological activities, including anti-oxidation (4), anti-obesity (5), anti-inflammation, anti-immunosenescence (6), anti-hyperlipidemic (7), antitumor (8), antiviral (9) and antibacterial (10) effects. ...
... The ingredients in fermented Pu-erh tea include caffeine, polyphenols, γ-aminobutyric acid, theanine, statin, polysaccharides (3,(20)(21)(22), theaflavins, thearubigins and theabrownins (23)(24)(25)(26). ...
Pu-erh tea has become a focus of research due to its reported biological activities, including anti-oxidation, anti-inflammation and anti-immunosenescence. The present study was performed to evaluate the potential gastroprotective function of Pu-erh tea extracts against ethanol-induced gastric mucosal damage in rats. Sprague Dawley rats were randomly divided into seven groups: A normal control, a model control, a cimetidine (0.08 g/kg) group, three Pu-erh tea extracts groups (low, moderate and high-dose; 0.50, 1.00 and 1.50 g/kg, respectively, and a green tea powder (1.00 g/kg) group. The normal and model groups were pre-treated with distilled water while the other groups were respectively administered cimetidine, Pu-erh tea extracts and green tea powder for 14 days. Then, absolute ethanol was orally administered to the rats of all groups excluding the normal controls. The effects of the pretreatments on gastric mucosal injury were evaluated by gross assessment of gastric lesions, examination of histopathology and determination of myeloperoxidase (MPO) activity and asymmetric arginine (ADMA) concentration in gastric mucosal homogenate. Pre-treatment with cimetidine or Pu-erh tea extracts markedly suppressed the formation of ethanol-induced gastric lesions. Furthermore, clear decreases in MPO activity and ADMA concentration in the gastric mucosal homogenate were observed following pretreatment with cimetidine or Pu-erh tea extracts. The anti-gastric ulcer activity of green tea was less than that of Pu-erh tea. Overall, these effects of Pu-erh tea extracts may be due to potential functions in protecting the gastric mucus layer and suppressing inflammation.
... Other amino acids reported are glutamic acid, asparagine serine, tryptophan (Alcázar et al., 2007;Horanni and Engelhardt, 2013;Syu et al., 2008). ...
Kombucha is a fermented beverage obtained from the transformation of sugared tea infusion through the activity of a consortium of yeasts and bacteria, during which a cellulosic biofilm is formed. The study of microbial interaction that occur in this matrix aims at improving the control over the production process at industrial scale. The methodology used in this work is mainly based on the isolation and selection of yeasts and acetic acid bacteria from a determined kombucha consortium.
The selected microorganisms are then used to inoculate monocultures and cocultures in sugared black tea. Different parameters were followed at the microbiological level (population counts, composition in genera and species), at the chemical level (composition in sugars, organic acids, amino acids, proteins, volatile compounds, oxygen) and at the sensory level (gustative and olfactive descriptions).
Results point towards the essential role of yeast-acetic acid bacteria association during the production process of kombucha, regarding both volatile and non-volatile compositions. They underline specifically the importance of yeast metabolism in those transformations. The matrix also plays a determining role in the chemical composition and organoleptic profile of kombucha because of its abundance in carbon substrates and its poorness in nitrogenous substrates. The kombucha biofilm possesses a strong relationship with microbial interactions, both regarding its formation and its function. However, it is not mandatory for the essential microbial activities involved in the beverage’s production to occur. Highlighted interactions, including commensalisms and competitions, form as a whole a mutualistic system of interactions between kombucha yeasts and acetic acid bacteria.
La kombucha est une boisson fermentée issues de la transformation d’une infusion de thé sucrée par l’activité d’un consortium microbien de levures et de bactéries donnant lieu à la production d’un biofilm cellulosique. L’étude des interactions microbiennes au sein de cette matrice a pour but d’améliorer la maîtrise du procédé de fabrication à échelle industrielle. La méthodologie employée dans ces travaux s’appuie principalement sur l’isolement et la sélection de levures et de bactéries acétiques d’un consortium donné, puis de leur mise en œuvre en monocultures et cocultures dans du thé noir sucré. Différents paramètres ont été suivi aux niveaux microbiologique (populations, composition en genres et espèces), chimique (composition en sucres, acides organiques, acides aminés, protéines, composés volatils, oxygène) et sensoriel (descriptions gustative et olfactive). Les résultats montrent le caractère essentiel de l’association levure-bactérie acétique dans le processus de fabrication de la kombucha au niveau des composés fixes et volatiles. Ils soulignent en particulier l’importance du métabolisme levurien dans ces transformations. La matrice joue également un rôle déterminant dans la composition chimique et le profil organoleptique de la kombucha de part son abondance en susbstrats carbonés et sa pauvreté en substrats azotés. Bien qu’étroitement lié aux interactions microbiennes tant au niveau de sa formation que de sa fonction, le biofilm de kombucha ne conditionne pas les activités microbiennes essentielles à la production de la boisson. L’ensemble des interactions mises en évidence, incluant commensalismes et compétitions, forment un système d’intéractions glogalement mutualiste entre les levures et les bactéries acétiques de la kombucha.
... 2.4.5. Determination of γ-Aminobutyric Acid (GABA) Content GABA in fermented samples was extracted according to the method of Bai et al. [25], and the samples were processed according to the method of Syu et al. [26]. Two milliliters of NaHCO3 solution was added to the fermented samples and centrifuged at 6000× g for 10 min. ...
The fruit and leaves of mulberry (Morus alba L.) contain a variety of health-beneficial components. At present, although both alcoholic and non-alcoholic mulberry beverages are commercially available, studies on fermented alcoholic beverages mixed with both mulberry fruit and mulberry leaf are rare. In this study, different amounts (1, 2 and 3%, g/mL dry weight) of mulberry leaves were added during the alcoholic fermentation of mulberry juice. After 9 days of fermentation, the 1-deoxynojirimycin (DNJ) content increased from 61.12 ± 3.10 to 153.39 ± 3.98 μg/mL, and the quercetin content increased from 0.45 ± 0.01 to 20.14 ± 0.08 mg/L in the mulberry alcoholic beverages with the addition of mulberry leaves at 2%. Moreover, the ABTS+ scavenging capacity at the end of fermentation for the same sample was enhanced by 40.9%. In addition, the total sugar, total phenols, total anthocyanins, and γ-aminobutyric acid (GABA) contents of the fermented samples all decreased significantly at the end of fermentation. A total of 33 volatile compounds and 17 free amino acids were detected in the fermented alcoholic beverages with mulberry leaves added. The total free amino acid content increased with the increase in mulberry leaf addition. Principal component analysis showed that the addition of mulberry leaves during fermentation increased the contents of GABA, DNJ, total flavonols and protocatechuic acid in mulberry alcoholic beverages. All these studies revealed the dynamic changes in functional components in the alcoholic fermentation of mulberry juices with the addition of mulberry leaves. Overall, the addition of mulberry leaf powder at 2% was selected as the appropriate addition for producing mulberry alcoholic beverages with enhanced nutritional value.
... Pu−erh tea is a famous dark tea originating in Yunnan Province and is divided into pu−erh ripe tea (PRT) and pu−erh raw tea [1]. As a post−fermented tea, PRT undergoes pile−fermentation using Yunnan sun−dried green tea (SGT) as the raw material. ...
The mellow and thick taste is a unique characteristic of pu−erh ripe tea infusion, and it is closely related to the chemical composition of pu−erh ripe tea, which is less studied. This paper clarifies and compares the chemical composition of pu−erh ripe tea to that of the raw materials of sun−dried green tea, and uses membrane separation technology to separate pu−erh ripe tea into the rejection liquid and the filtration liquid. The results show that microorganisms transformed most physicochemical components, except caffeine, during the pile fermentation. It was found that total tea polyphenols, soluble proteins, total soluble sugars, theabrownin, and galloylated catechins became enriched in the rejection liquid, and the rejection liquid showed a more obvious mellow and thick characteristic. Taste interactions between crude protein, crude polysaccharide, and theabrownin were determined. They illustrated that the mellow and thick taste of pu−erh ripe tea with the addition of theabrownin increased from 4.45 to 5.13. It is of great significance to explore the chemical basis of the mellow and thick taste in pu−erh tea for guiding the pu−erh tea production process and for improving the quality of pu−erh tea.
... Moreover, in all shooting periods, Thea concentration of black and oolong teas was lower than that of green tea. It was reported that Thea concentration varied from 687 to 3030 μg/g in green teas, 170-2831 μg/g in oolong teas, and 471-1070 μg/g in black teas (Syu, Lin, Huang, and Lin, 2008). ...
In this study, it was aimed to investigate the effect of fermentation degree on total phenolic content, individual catechins, gallic acid, theaflavins, caffeine content, antioxidant and antimicrobial activity in teas. Green tea, black tea and oolong tea (partially oxidized 7 different degrees) were produced from the same cultivar of tea leaves harvested in first shooting period. The higher degree of fermentation was resulted in higher theaflavins, however in lower individual catechins and total phenolic content. Standard catechins gradually decreased with extended fermentation duration and epigallocatechin gallate decreased 87% from green tea to oolong to black tea. Higher antioxidant activity was observed in green and lower fermented oolong teas. There were significant (p<0.01) positive correlations (r²>0.9) between DPPH, ABTS assays and total phenolic content, catechin, epigallocatechin gallate, epicatechin, epicatechin gallate and total catechins. Significant (p<0.05), positive correlations (r²>0.7) were also observed between ORAC and those catechins. Moreover, the tea samples especially green tea showed antibacterial effects against three of the tested pathogenic bacteria Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853 and antifungal effects against Candida albicans ATCC 10231. However, no antimicrobial activity was detected against Escherichia coli.
... It is well known that green tea contains many bioactive components. [3][4][5] In particular, the effect of green tea polyphenols (GTPs; Fig. 2) is in the limelight. GTPs having high bioactivity may interact with a drug taken at the same time. ...
The details of incompatibility between aripiprazole (ARIP) oral solution and green tea were examined. When the ARIP oral solution was mixed with a commercial PET bottled green tea beverage, the residual rate of ARIP in the mixed solution decreased to 15.7–17.6%. Mixing with ARIP reduced the content of gallate-type green tea polyphenols (GTPs) in the mixed solution but not the content of non-gallate-type GTPs. Furthermore, using pH 3.0 lactic acid buffer, 2.23 mM ARIP solution and 2.23 mM GTP solution were prepared, and the same volumes of ARIP solution and GTP solution were mixed. When the gallate-type GTP solution was mixed, the residual rate of ARIP in the mixed solution decreased. On the other hand, when the non-gallate-type GTP solution was mixed, the residual rate of ARIP in the mixed solution did not decrease. From the above results, it was found that the main reason for the incompatibility between ARIP oral solution and green tea was the formation of an insoluble substance composed of ARIP and gallate-type GTPs in green tea. Furthermore, experimental results using the continuous variation method revealed that ARIP and (−)-epigallocatechin gallate, which is the most representative gallate-type GTP, interact at a molar ratio of 3 : 2.
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... It has been reported that accumulation of carbohydrates in tea leaves may be related to plant adaptation to decreasing temperatures (77). AA is another major indicator for tea quality, which contributes brothy, sweet, and umami flavor to tea infusions (78). L-theanine is a unique nonproteinogenic AA, with health benefits such as reducing blood pressure, relieving stress, and anxiety (79,80). ...
Shading modifies the microenvironment and can provide plants with some protection from frequent heat, drought, frost, and hail induced by climate change and has the potential to improve plant growth, yield, and quality. Tea (Camellia sinensis) is an ancient plant originating from tropical and subtropical regions and prefers to grow in partial shade under the forest canopy. The emerging tea industry in the United States (US) requires research support on establishing tea fields in novel environmental conditions as well as on producing high-quality tea products. This study investigated the effects of black, blue, and red shade nets on tea plant growth and seasonal leaf qualities in the southeastern US with a humid subtropical climate. When compared to no-shade control, black, blue, and red shade nets increased plant growth index (PGI), net photosynthetic rate (P n), and stomatal conductance (g s), decreased air and leaf surface temperatures in summer, and reduced cold damage in winter. No significant difference was found among the black, blue, and red shade nets on tea plant growth. Varying contents of total polyphenols, carbohydrates, free amino acids, L-theanine, gallic acid, caffeine, and catechins in fresh tea leaves were observed among different shade treatments and harvesting seasons. 69.58% of the variations were depicted in a biplot by principal component analysis. Red shade was considered helpful for improving green tea quality by increasing the content of L-theanine and free amino acids in tea leaves collected in spring and fall when compared to no-shade control.
... Enzyme activity was determined by measuring GABA production rate using HPLC analysis, with some modifications [46,47]. The reaction mixture comprised 400 µL of Na 2 HPO 4 -citric acid buffer (80 mM, pH 6.0), 500 µL of l-Glu (50 mM), 50 µL of PLP (0.02 mM), and 50 µL of purified enzyme. ...
Background
Gamma-aminobutyric acid (GABA) is an important bio-product used in pharmaceuticals and functional foods and as a precursor of the biodegradable plastic polyamide 4. Glutamate decarboxylase (GAD) converts l -glutamate ( l -Glu) into GABA via decarboxylation. Compared with other methods, develop a bioconversion platform to produce GABA is of considerable interest for industrial use.
Results
Three GAD genes were identified from three Bacillus strains and heterologously expressed in Escherichia coli BL21 (DE3). The optimal reaction temperature and pH values for three enzymes were 40 °C and 5.0, respectively. Of the GADs, GADZ11 had the highest catalytic efficiency towards l -Glu (2.19 mM − 1 s − 1 ). The engineered E. coli strain that expressed GADZ11 was used as a whole-cell biocatalyst for the production of GABA. After repeated use 14 times, the cells produced GABA with an average molar conversion rate of 98.6% within 14 h.
Conclusions
Three recombinant GADs from Bacillus strains have been conducted functional identification. The engineered E. coli strain heterologous expressing GADZ1, GADZ11, and GADZ20 could accomplish the biosynthesis of l -Glu to GABA in a buffer-free reaction at a high l -Glu concentration. The novel engineered E. coli strain has the potential to be a cost-effective biotransformation platform for the industrial production of GABA.
... Pu-erh tea is a traditional Chinese tea that is particularly popular in Southwestern China and South Asian countries (Roda et al., 2019). Puerh tea includes two varieties known as ripe Pu-erh tea and raw Puerh tea (Syu et al., 2008). Both varieties have been proved to decrease the levels of cholesterol and triglycerides (Hou et al., 2009), prevent lipid-derived disorders (Cai et al., 2017), and alleviate obesity (Xia et al., 2019). ...
Pu‐erh tea is very popular in Southwestern China and South Asian countries and is now becoming increasingly popular in Europe due to its well‐documented beneficial effects on human health. Pu‐erh tea aqueous extracts can maintain intestinal homeostasis. However, the mechanism of its beneficial effects on intestinal flora disorder is not clear. In this study, we focused on the effects of ripe Pu‐erh tea aqueous extracts on the intestinal microbiota in an intestinal flora disorder mouse model. Physiological indexes and the tissue section staining results showed that feeding Pu‐erh tea extract could help mice regain weight and alleviate intestinal inflammation. Further assessment of the intestinal microflora found that Pu‐erh tea extract could promote the growth of intestinal probiotics and inhibit pathogenic bacteria, thereby achieving a treatment effect for enteritis. This study provides new evidence for the therapeutic effect of Pu‐erh tea. Pu‐erh tea is very popular in Southwestern China and South Asian countries, and now is becoming more and more popular in Europe because of its well‐documented beneficial effects on human health. This study explored the mechanism of Pu‐erh tea extract in promoting human health and improving intestinal flora disorder and found that Pu‐erh tea extract can alleviate the intestinal inflammation caused by antibiotics, reduce the degree of intestinal lesions, promote the growth of intestinal probiotics and inhibit pathogenic bacteria.
... Enzyme activity was determined by measuring GABA production using HPLC analysis, with some modi cations [40,41]. The reaction mixture comprised 400 μL Na 2 HPO 4 -citric acid buffer (80 mM, pH 6.0), 500 μL l-Glu (50 mM), 50 μL PLP (0.02 mM), and 50 μL puri ed enzyme. ...
Background
Gamma-aminobutyric acid (GABA) is an important bio-product used in pharmaceuticals and functional foods and as a precursor of the biodegradable plastic polyamide 4. Glutamate decarboxylase (GAD) converts l-glutamate (l-Glu) into GABA via decarboxylation. Compared with other methods, develop a bioconversion platform to produce GABA is of considerable interest for industrial use.ResultsThree GAD genes were identified from three Bacillus strains and heterologously expressed in Escherichia coli BL21 (DE3). The optimal reaction temperature and pH values for three enzymes were 40 °C and 5.0, respectively. Of the GADs, GADZ11 had the highest catalytic efficiency towards l-Glu (2.19 mM − 1 ·s − 1 ). The engineered E. coli strain that expressed GADZ11 was used as a whole-cell biocatalyst for the production of GABA. After repeated use 8 times, the cells produced GABA with an average molar conversion rate of 97% within 8 h.Conclusions
Three recombinant GADs from Bacillus strains have been conducted functional identification. And engineered E. coli strain heterologous expressing GADZ1, GADZ11, and GADZ20 could accomplish the biosynthesis of l-Glu to GABA in a buffer-free reaction at a high l-Glu concentration. The novel engineered E. coli strain has the potential to be a cost-effective biotransformation platform for the industrial production of GABA.
... L-Theanine, the primary form in green tea, comprises more than half of the total free amino acids in tea. 2 Recent research demonstrated the presence of D-theanine at much lower concentrations in some types of tea. 3 L-Theanine was first discovered from the leaves of green tea (Camellia sinensis) and identified as a unique taste constituent of the tea in the late 1940s. 4 Henceforth, growing studies suggest that Ltheanine can not only improve the flavor and quality of green tea but also possess a wide variety of beneficial effects across species and disease models, including protecting cardiovascular function, 5 reducing stress, 6 improving immunity, 7 and reducing tumors. ...
l-Theanine is the most popular nonprotein amino acid contained in tea leaves. It is one of the umami components of green tea, contributing to the unique flavor of tea. Because of its various health functions, l-theanine has been commercially developed as a valuable ingredient easily used for various applications in food and pharmaceutical industries. Nowadays, l-theanine is mass-produced by plant extraction, chemical synthesis, or enzymatic transformation in factories. This review embodies the available up to date information on the l-theanine synthesis metabolism in the tea plant as well as approaches to produce it, placing emphasis on the biotransformation of l-theanine. It also gives insight into the challenges of l-theanine production on a large scale, as well as directions for future research. This review comprehensively summarizes information on l-theanine to provide an approach for an in-depth study of l-theanine production.
... The derivatization of GABA was carried out as described by Syu et at. [21] with minor modification. Briefly, GABA in the supernatants was derivatized by dabsylation with 4 mM 4-dimethylaminoazobenzen-4-sulfonyl chloride at 70°C for 20 min. ...
This study was aimed to identify and optimize the culture conditions for gamma-aminobutyric acid (GABA) production by a lactic acid bacterium strain isolated from mam nem, a fermented fish sauce. Among the six isolates obtained from mam nem, the MN12 had the most potent GABA-producing capability. The strain was then identified to be Pedioccocus pentosaceus by employing MALDI-TOF-MS and phenylalanyl-tRNA synthase sequencing methods. The initial cell density of 5.10⁶ CFU/mL, monosodium glutamate concentration of 60 mM, initial pH of 7, temperature of 45°C and cultivation time of 72 h were found to be the optimal culture conditions for highest production of GABA, reaching 27.9 ± 0.42 mM, by this strain. The cultivation conditions for GABA production by P. pentosaceus MN12 have been successfully optimized, providing a foundation for the development of fermented foods enriched with GABA.
... It is the amino acid found in the highest concentration in tea, accounting for 1-2% of the dry weight and more than 50% of the amino acid content (Juneja et al., 1999). Theanine is more abundant in green tea than in black or oolong tea (Kim et al., 2019) and varies according to the method of production (Syu et al., 2008). Theanine crosses the blood brain barrier via the leucine-preferring transport system (Yokogoshi et al., 1998). ...
Teas enriched in GABA are consumed for their beneficial effects on blood pressure, stress and anxiety. These effects may involve actions of GABA on the central and peripheral nervous systems. The anaerobic procedures for the production of GABA-enriched teas increase GABA levels by 10–20 times. They also significantly alter the levels of other constituents that may interact with the actions of GABA. These include epigallocatechin gallate, caffeine and theanine. The possible interactions of these active constituents make the understanding of the effects of GABA-enriched teas complex. More data is needed to establish where and how GABA is acting following consumption of GABA-enriched teas. While there is considerable evidence that such GABA is acting on GABA receptors in the periphery, there is rather less evidence that is acting directly in the brain. Certainly, there is more to the action of GABA-enriched teas than GABA itself.
... Previous studies have analyzed L-theanine amount per gram of tea leaf (Thippeswamy et al. 2006;Alcázar et al. 2007;Syu et al. 2008) mentioned in the (Table 1, Table 2 and Table 3 respectively) and the amount of L-theanine at different stages of growing (At high higher stage its 0.55±0.26g/100g, at medium stage its 1.05±0.43g/100g ...
Tea is the second most consumed beverage around the world after water. Apart from other tea preparations, approximately 98% of peoples drink tea with milk. The essential bioactive component, i.e., L-theanine present in tea along with catechins, tannin, and polyphenols, which has prominent health benefits, including relaxation. L-theanine is proteionic amino acid present only in tea, and which is safe for daily intake. This review focused on L-theanine present in different types of tea, commercially available tea powders, extraction methods for L-theanine isolation, characterization, and health benefits of L-theanine.
... In qualitative analysis of components, many publications have reported the analysis of the chemical components of GT and PRT (Zhang, Li, Ma, & Tu, 2011;Zhang et al., 2019). However, most studies have focused on the analysis of a certain class of components, such as catechins and their derivatives (Zhang et al., 2011), flavonoids and their derivatives (Jiang, Engelhardt, Thräne, Maiwald, & Stark, 2015), amino acids (Syu, Lin, Huang, & Lin, 2008), or a small number of other components (Chen, Ye, Cheng, Yin, & Sun, 2007). For the quantitative analysis of components, a simple internal standard method was generally adopted (Xin et al., 2018), or few important components were quantified by an external standard method . ...
To reveal the characteristic chemical profiles of Pu-erh raw tea (PRT) and traditional green tea (TGT), a high-throughput analytical method based on UPLC-Q-Orbitrap-MS/MS was proposed. 145 components were characterized with a three-level qualitative strategy and the integrated filtering strategy combining nitrogen rule, mass defect, and diagnostic ions information. 124 components were quantified using an internal standard method. The total contents of flavan-3-ols and derivatives, phenolic acids and derivatives were higher in PRT than TGT, while flavonoids were reversed. Furthermore, partial least squares–discriminant analysis (PLS-DA) models were established to classify TGT and PRT. 23 characteristic components were revealed by variable importance in projection method. Their difference in content is between 1.5-7.3 times for PRT and TGT. The results showed the chemical characteristics of TGT and PRT clearly and comprehensively. The high-throughput method demonstrated considerable potential in the analysis of complex chemical system, such as food and herbs.
... www.nature.com/scientificreports/ in tea, has an umami taste. After black tea and dark tea making critical steps, fermentation or post fermentation, the content of L-theanine is dramatically decreased or not detectable 30,31 . Therefore, it is important to detect the changes in L-theanine and other amino acids content that occur during QZT manufacturing processes (Table 1). ...
Qingzhuan tea (QZT), a post-fermented tea, has been reported to have anti-obesity and anti-hyperglycemic effects, perhaps due to bioactive compounds that inhibit lipase and α-amylase. It is unknown what chemical constituents’ changes and what bioactive compounds occur during the manufacture of QZT. The aim of this study was to determine the secondary metabolites changes that occur during post-fermentation and how these changes affect the ability of QZT to inhibit the activities of lipase and α-amylase. During the processing steps, metabolites levels and their inhibitory effects on lipase and α-amylase were assessed. Changes in content and activities suggested that the first turn over or the second turn over was critical for the formation and conversion of bioactive compounds responsible for the anti-obesity and hypoglycemic effects. The relationship between constituents and activities was further evaluated by correlation analysis, which showed that amino acids and flavonoids might be responsible for the anti-obesity and anti-hyperglycemic effects of QZT. This study clarified that compounds were altered during pile fermentation of QZT and tentatively identified the bioactive compounds formed during QZT manufacture.
... Among these ingredients, free amino acids are in a prominent position, because they not only affect the flavor of tea infusions but also have various pharmacological actions Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1033 7-020-03864 -5) contains supplementary material, which is available to authorized users. 1 3 [10][11][12]. Previous studies have demonstrated that the amino acid content of tea has a significant influence on the quality of tea [3]. ...
An effective method for the determination of 20 free amino acids in tea samples using 4-(carbazole-9-yl)-benzyl chloroformate (CBBC-Cl) as a derivatization reagent by high-performance liquid chromatography combined with fluorescence detection (HPLC-FLD) has been established. CBBC-Cl quickly derivatized amino acids within 6 min in basic solution (pH 9.0) at room temperature (ca. 25 °C). The maximum excitation and emission wavelengths of the stable derivatives were 290 nm and 370 nm, respectively. The separation of 20 amino acids, including the theanine (THE) derivative, was optimized on a Hypersil BDS-C18 column. Identification of these derivatives was performed via liquid chromatography/electrospray ionization mass spectrometry (LC/ESI/MS) in positive-ion detection mode. Under optimal conditions, excellent linearity for all the amino acids was obtained, with determination coefficients of r² > 0.9986. The detection limits (LODs) and quantitation limits (LOQs) ranged from 0.13 to 1.5 μg·kg⁻¹ and 0.43 to 4.95 μg·kg⁻¹, respectively. Satisfactory recoveries, ranging from 86.7 to 106.4%, were obtained for all the amino acids in the tea samples. The developed method has been successfully applied to the quantification of 20 amino acids in tea samples.
... Fermentation is the key processing step to enrich GABA in food products, when started from raw materials owning high Glu concentration. Many studies were successful in enhancing the GABA content of fermented products such as cheese, fermented fish, yogurt, and tea (Chun et al., 2007;Jannoey et al., 2010;Jeng, Chen, Fang, Hou, & Chen, 2007;Siragusa et al., 2007;Syu, Lin, Huang, & Lin, 2008). Almost all these studies have focused on increasing the GABA content by screening microbial strains and studying fermentation conditions. ...
The effects of food processing on γ‐aminobutyric acid (GABA) losses in GABA solution and germinated soymilk (GSM) were investigated. The different processing steps included variation in pH (2.0–8.0), temperature (70 and 90°C), storage (8 days), and sugar addition (0% and 5%). Results showed that no significant effects of temperature and sugar addition were observed on the GABA content in a GABA solution in a wide pH range (2.0–8.0). However, the temperature caused the loss of GABA in GSM when no sugar was added in a pH range of 4.0–8.0, but this was not observed in GSM with 5% of sugar added at pH 6.5. GSM without sugar addition can be stored at 4°C for 8 days without a decrease in the GABA content. Similarly, GABA was almost stable in the experiment imitating the digestion in stomach in which GSM was adjusted to pH 2 and incubated for 2 hr at 37°C. Germinated soymilk is a healthy beverage rich in GABA content. The loss of GABA during heat treatment is inevitable due to the interaction between GABA with other soymilk components. Results of this research revealed that the stability of GABA is different in GABA solution and food matrices.
... Fermented milk was centrifuged and filtered through a Millex-HA 0.22-μm pore size filter to collect the supernatant. The concentration of GABA in the supernatant was analyzed using HPLC according to the dabsyl chloride (Sigma-Aldrich) derivatization method (Syu et al., 2008). The GABA reagent standard was purchased from Sigma-Aldrich. ...
γ-Aminobutyric acid (GABA) is a potentially bioactive ingredient with health-promoting properties that is added to functional foods. Streptococcus thermophilus was selected to produce naturally GABA-enriched fermented milk. This strain can yield a GABA concentration of 2.8 g/L after a 48-h fermentation. In the presence of 1 g/L food-grade casein hydrolysate as a nitrogen source, S. thermophilus yielded GABA concentrations as high as 5.4 g/L or even 8.3 g/L when cocultured with Lactobacillus rhamnosus. In other words, both of these added conditions promoted GABA enrichment. The GABA dose achieved with fermented milk was comparable to the doses of commercially available GABA supplements. Additionally, the in situ use of S. thermophilus to produce GABA-enriched fermented milk was cost effective. The complete genomic sequence of S. thermophilus GABA has been published and will be highly useful to other researchers studying the regulation of genes related to GABA accumulation. In conclusion, the S. thermophilus GABA-producing strain reported herein represents a natural method for the production of fermented milk containing high GABA concentrations.
... Tea has been traditionally used as a medicinal plant in Asian countries including India, China, and Japan [2], and it is believed to have potential effects on prevention or treatment of many diseases, including cancer, cardiovascular disease, and neurodegenerative disease [3]. e relevant active components include polyphenols (catechins and flavonoids), alkaloids (e.g., caffeine, theophylline, and theobromine), amino acids, and vitamins [4,5]. In particular, the polyphenols are primarily responsible for the beneficial effects of tea, and the flavonoids have antioxidant, antiinflammatory, antiallergic, and antimicrobial effects [3]. ...
Green tea is generally considered safe, but there have been concerns regarding side effects relating to the main component, catechins, especially hepatotoxicities. We have previously shown beneficial effects of fermented green tea with Aquilariae Lignum (fGT) via an oral route in diabetic and obese models. Thus, the toxicological safety of fGT was assessed at limited oral doses for a rodent. Mice or rats of both genders were orally administered distilled water as a control and fGT at 2.0, 1.0, and 0.5 g/kg. There were no mortalities or gross abnormalities in the fGT groups for 2 weeks following the single oral dose in mice. No fGT-relevant abnormalities were found in postmortem and histopathological examinations, suggesting LD 50 of fGT at more than 2.0 g/kg with no specific target organs. There were also no fGT-relevant mortalities or abnormal signs in the repeated oral dose for 13 weeks in rats. In the fGT groups, no body weight changes or daily metabolic changes were found, and hematological and serum biochemical ranges were normal. The postmortem and histopathological examinations revealed few fGT-related abnormalities in most of the organs including the liver, although slight lymphoid cell hyperplasia in the lymph node was observed in a few rats with fGT at 2.0 g/kg. This may be secondary to increased immune response to the highest dose because there were no histopathological lesions or organ weight changes. It suggests nontoxic safety of fGT at up to 2.0 g/kg, which provides useful information for clinical use.
... Matcha is thus rich in both theanine and caffeine together with a lower content of catechins than normal-grade green teas (Unno et al., 2018). Nevertheless, the reason why actual measured values of theanine do not always coincide well with the aforementioned theoretical abundance in green tea leaves (Ekborg-Ott et al., 1997;Syu et al., 2008;Unno et al., 2018) is not fully clarified so far. The possibility that the grade of green tea leaves used for measurement is fluctuating between studies is not ruled out. ...
The green tea amino acid theanine is abundant in green tea rather than black and oolong teas, which are all made of the identical tea plant “Chanoki” (Camellia sinensis). Theanine has a molecular structure close to glutamine (GLN) compared to glutamic acid (Glu), in terms of the absence of a free carboxylic acid moiety from the gamma carbon position. Theanine efficiently inhibits [3H]GLN uptake without affecting [3H]Glu uptake in rat brain synaptosomes. In contrast to GLN, however, theanine markedly stimulates the abilities to replicate and to commit to a neuronal lineage following prolonged exposure in cultured neural progenitor cells (NPCs) prepared from embryonic and adult rodent brains. Upregulation of transcript expression is found for one of the GLN transporter isoforms, Slc38a1, besides the promotion of both proliferation and neuronal commitment along with acceleration of the phosphorylation of mechanistic target of rapamycin (mTOR) and relevant downstream proteins, in murine NPCs cultured with theanine. Stable overexpression of Slc38a1 similarly facilitates both cellular replication and neuronal commitment in pluripotent embryonic carcinoma P19 cells. In P19 cells with
stable overexpression of Slc38a1, marked phosphorylation is seen for mTOR and downstream proteins in a manner insensitive to further additional phosphorylation by theanine. Taken together, theanine would exhibit a novel pharmacological property to up-regulate Slc38a1 expression for activation of the intracellular mTOR signaling pathway required for neurogenesis after sustained exposure in undifferentiated NPCs in the brain. In this review, a novel neurogenic property of the green tea amino acid theanine is summarized for embryonic and adult neurogenesis with a focus on the endogenous amino acid GLN on the basis of our accumulating evidence to date.
... The glutamate and GABA contents of the cultures were determined by high-performance liquid chromatography (HPLC) with a reversephase C18 column (Waters, 25 cm × 4.6 mm) and a photodiode array detector (Waters 996, USA) (Syu et al., 2008). For each sample, 100 mg of dry algal cells was ground thoroughly with quartz sand. ...
The primary goal of this study was to assess the roles of chemical factors and bioprocess strategies on a mixotrophic culture of the microalga Haematococcus pluvialis during γ-aminobutyric acid (GABA) production. A two-stage strategy was used to increase the biomass and GABA accumulation of H. pluvialis. In stage I, mixotrophic growth of H. pluvialis in the presence of fulvic acid (FA) produced a high biomass (1.84 g L-1) with a GABA content of 25.45 mg g-1. Furthermore, a maximum GABA content of 38.57 mg g-1 was obtained when cells were cultured with 0.4 g L-1 NaCl under photoautotrophic conditions in stage II, whereas the carbohydrate content of cells sharply decreased from 26.68 to 18.22 %. In addition, salt stress upregulated the expression of the gad and cam genes. The results of this study demonstrate an efficient strategy to produce GABA from the microalga H. pluvialis.
... This concentration of theanine was between that observed in green tea and black tea and varied dramatically between concentrations as high as in green tea to as low as in black tea. 35 Consistently, in this study, the CV for theanine (61.95%) was the highest CV observed among the nine tested metabolites (Table 1), suggesting a great diversity of umami flavor in tea leaves among sources, which may be dramatically affected by environmental factors. Taken together, the results in this study suggest that oolong tea bitterness and astringency are similar to those flavors in green tea, although diverse umami flavors are likely regulated by environmental factors. ...
Tea (Camellia sinensis) is the most widely consumed beverage crop in the world, with consumption consistently increasing in recent years. To increase productivity to match the rising demand, tea trees are often amply fertilized, which also alters nutrient concentrations in leaves. However, little information is available on how leaf nutrient concentrations affect tea quality. Oolong tea is a unique semifermented Chinese tea famous for its distinctive flavor and beneficial impacts on human health. In this study, 165 and 144 one-tip-three-leaf tissue samples were collected from two main oolong tea growing regions, Anxi and Wuyi, respectively. Concentrations of N, P, and K and nine abundant secondary metabolites were quantified in tea leaves. Results revealed substantial variation and close correlations among tested secondary metabolites, with the highest variation observed in the concentration of theanine, implying umami is the most varied flavor in tea leaves. Boundary-line analysis revealed that leaf N, P, and K concentrations significantly affected most secondary metabolites, with insufficient or excess leaf N, P, or K concentrations, leading to reductions in secondary metabolite contents and thereby affecting tea quality. The effects of each nutrient on secondary metabolism varied between the two studied tea-producing regions, suggesting the involvement of soil nutrient status. In short, N, P, and K concentrations significantly affect secondary metabolism in tea leaves from oolong tea producing areas, and therefore, determining the most effective nutrient management strategies for optimizing leaf nutrient status is of great importance for producing the highest quality oolong tea.
... L-theanine (n-ethylglutamic acid; LT) is a non-proteinogenic amino acid derived almost exclusively from tea leaves (Camellia sinensis), with the highest concentrations found in green, oolong, and Pu-erh tea ( Syu et al., 2008). A natural ethylamide analogue of glutamate, preclinical studies have found LT to pass freely through the blood-brain barrier, reduce presynaptic glutamate release ( Kakuda et al., 2008), increase inhibitory neurotransmitter GABA, and enhance glycine and dopamine release ( Kakuda et al., 2008;Kimura and Murata, 1971;Shen et al., 2011;Yamada et al., 2007Yamada et al., , 2005. ...
... Both protein amino acids, especially essential ones, as well as non-protein, such as theanine or GABA (gamma-aminobutyric) acid, present in tea fulfil a very important role as precursors of bioactive and health-promoting substances and components of polyphenol or glutathione biosynthesis [16]. Free amino acids also have a great nutritional value as easily available substrates for numerous biochemical and metabolic changes in the human body, therefore their presence in food is exceptionally valuable and desired [17,18]. ...
Tea is one of the most popular drinks in the world, commonly consumed by consumers from all age groups mainly due to its refreshing taste, attractive aroma, and potentially beneficial impact on health. The composition of a tea drink depends on numerous factors, such as time and brewing temperature, degree of crumbling of tea leaves, and degree of mixing. Diffusion of the polyphenolic compounds, minerals, caffeine or theanine typical of tea infusions have been the subject of studies conducted by numerous authors. Promoting the extraction of amino acids from tea leaves when preparing infusions through the induction of a magnetic field constitutes not only another step towards the optimisation of the extraction process, but is also one of the methods to improve the nutritional value of tea infusions. The purpose of this work was to verify a hypothesis concerning the improvement of the extraction of amino acids from dried tea during the preparation of infusions by applying a permanent or variable magnetic field induced under laboratory conditions. A variable magnetic field applied as a factor assisting extraction resulted in an increased concentration in the total number of amino acids in green and black tea infusions. A statistically significant improvement in the level of free amino acids was observed after application of extraction assisted by a variable magnetic field with induction at 100 mT and a frequency of 50 Hz. Extraction using a variable magnetic field for tea infusions may constitute a good solution to assist traditional water extraction methods for research purposes.
... Teas Tea is one of the most widely consumed beverages in the world made from the leaves and buds of Camellia sinensis (L.). Tea contains many chemical components such as amino acids, polyphenols (catechins and flavonoids), polysaccharides, volatile oils, vitamins, minerals and alkaloids (Syu, Lin, Huang, & Lin, 2008). Amino acids account for approximately 1-4% of the dry weight of fresh tea leaves, which mainly comprise theanine, glutamic acid (Glu), Asp, Arg and GABA (Zhao et al., 2013). ...
GABA has attracted great attention over the last several decades due to its ubiquity in life. It is an important molecule naturally present in considerable amounts in many feed and food matrices of vegetable and animal origin. GABA occurs naturally in plants, animals and microorganisms, having diverse physiological functions and great potential health benefits. Extensive data demonstrates that GABA content is usually higher in plants than in animals and its concentration is in the range of mg g−1 depending on plant matrix, development stage and postharvest processing conditions. In animals, GABA was found at significantly high levels in the brain and central nervous system and some specific peripheral tissues like livestock muscles in the range of μg g−1. Food items produced by different types of animals, such as eggs, milk or honey, also show remarkable GABA content without any processing steps. A healthy diet following the set of recommendations of WHO national food-based dietary guidelines (FBDG) or/and the Healthy Eating Plate (Harvard) will provide a considerable amount of GABA as a natural nutrient. Additionally, considering its potential health benefits, many efforts are being allocated to developing new technological processes for GABA enhancement in traditional foodstuffs or avoiding losses after processing treatments.
... As for the individual compounds determined in the experiments, GABA and Glu contributed the largest fractions to total FAAs. In particular, the non-protein amino acid GABA functions as an important neurotransmitter signal in humans, has blood pressure lowering activity, and has been used as a component of pharmaceuticals and functional foods (Syu et al. 2008). Except for GABA, the contents of three other non-protein amino acids in ginkgo seeds were at a trace level. ...
This work presented a new analytical methodology based on hydrophilic interaction ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry in multiple-reaction monitoring mode for analysis of 24 underivatized free amino acids (FAAs) in functional foods. The proposed method was first reported and validated by assessing the matrix effects, linearity, limit of detections and limit of quantifications, precision, repeatability, stability and recovery of all target compounds, and it was used to determine the nutritional substances of FAAs in ginkgo seeds and further elucidate the nutritional value of this functional food. The result showed that ginkgo seed turned out to be a good source of FAAs with high levels of several essential FAAs and to have a good nutritional value. Furthermore, the principal component analysis was performed to classify the ginkgo seed samples on the basis of 24 FAAs. As a result, the samples could be mainly clustered into three groups, which were similar to areas classification. Overall, the presented method would be useful for the investigation of amino acids in edible plants and agricultural products.
... The techniques for extraction of free polyamines and for HPLC analysis were described by Zhu et al. 28 Analysis of GABA and Glutamate. GABA and glutamate were extracted and purified according to the method of Bai et al. 29 and then analyzed by HPLC as described by Syu et al. 30 GAD and DAO Activity Assay. The tea leaves were homogenized in 70 mM potassium phosphate buffer (pH 5.8), containing 2 mM βmercaptoethanol, 2 mM EDTA, 0.2 mM pyridoxal phosphate, and 100 g L −1 glycerin. ...
Gamma-Aminobutyric acid (GABA) is an important bioactive component of tea (Camellia sinensis) providing various health benefits. We studied GABA accumulation via the GABA shunt and the polyamine degradation pathways under anoxia in tea leaves. Anoxia caused a ~ 20-fold increment on GABA concentration, relative to fresh tea leaves. This increment was due to the increase of glutamate decarboxylase and diamine oxidase activities. Genes involved in GABA formation, such as CsGAD1, and CsGAD2, were significantly up-regulated by anoxia. The concentration of putrescine and spermine, two substrates for GABA production, was also increased by anoxia. Treating tea leaves with aminoguanidine completely inhibited diamine oxidase activity during anoxia, but the concentration of GABA only decreased by ~ 25 %. We infer that about one fourth of GABA formed in tea leaves under anoxia comes from the polyamine degradation pathway, opening the possibility of producing GABA tea based through the regulation of metabolism.
... Due to the weak UV-visible absorption characteristics of GABA, it requires derivatization before analysis with agents such as o-phthalaldehyde, 2-hydroxynaphthaldehyde, dabsylchloride, or 9-fluorenylmethyl chloroformate. 66 For example, mung beans, black beans, and soybeans were analyzed for GABA content using a modified method from Srisang et al. 67 GABA was extracted with 3% ...
Beans, peas, and lentils are all types of pulses that are extensively used as foods around the world due to their beneficial effects on human health including their low glycemic index, cholesterol lowering effects, ability to decrease the risk of heart diseases and their protective effects against some cancers. These health benefits are a result of their components such as bioactive proteins, dietary fibers, slowly digested starches, minerals and vitamins, and bioactive compounds. Among these bioactive compounds, γ-aminobutyric acid (GABA), a non-proteinogenic amino acid with numerous reported health benefits (e.g. anti-diabetic and hypotensive effects, depression and anxiety reduction) is of particular interest. GABA is primarily synthesized in plant tissues by the decarboxylation of L-glutamic acid in the presence of glutamate decarboxylase (GAD). It is widely reported that during various processes including enzymatic treatment, gaseous treatment (e.g. with carbon dioxide), and fermentation (with lactic acid bacteria), GABA content increases in the plant matrix. The objective of this review paper is to highlight the current state of knowledge on the occurrence of GABA in pulses with special focus on mechanisms by which GABA levels are increased and the analytical extraction and estimation methods for this bioactive phytochemical.
This study aimed to improve γ-aminobutyric acid (GABA) content and sensory characteristics of brown rice (BR) cake by static magnetic field (SMF)-assisted germination. BR was pre-treated by SMF (10 mT, 60 min, 25 °C), germinated for 36 h, and then germinated BR (GBR) was used to prepare rice cake. The optimal formula was: 60% GBR, 40% white rice, 1% yeast, 20% sugar, and 55% water. SMF significantly increased the GABA content by stimulating glutamate decarboxylase, with the values increasing from 28.17 to 32.43 mg/100g and from 2.50 to 6.27 mg/100g in GBR (36 h) and GBR cake, respectively. SMF also altered the swelling power and water solubility of GBR flour by promoting the hydrolysis of starch, protein, and fiber, thus improving the texture, flavor, and storage stability of GBR cake. Overall, SMF could be a prospective technique for improving the nutritional and sensory qualities of whole-grain food.
An tea (AT) is a dark tea with a unique flavor that affects the quality of storage time. However, changes in the sensory quality and chemical composition of AT during storage are unknown. The present study performed sensory evaluation and metabolomic analysis of AT at seven storage time points. The results indicated that the tea liquor color changed from orange to dark red during storage, and the astringency intensity gradually decreased concomitant with the appearance of a stale taste. Seventy differential metabolites were identified by untargeted liquid chromatography coupled with mass spectrometry (LC–MS) including catechins, flavonols and their glycosides, free amino acids, alkaloids, and phenolic acids and their derivatives. Targeted analysis demonstrated that quercetin, quercetin 3-O-glucuronide, quercetin 3-O-rutinoside, glycine, aspartic acid, alanine, serine, arginine, threonine, tyrosine, theanine, GABA and isoleucine were highly correlated with storage time (r < −0.8 or r > 0.8). Furthermore, linear regression analysis confirmed that these 13 substances were good predictors of AT storage time (correlation coefficient of 0.8767). The present study reveals the changes in the components during storage of AT and provides a theoretical basis for quality improvement of stored dark tea.
A major challenge facing by astaxanthin industrialization is the low productivity and high production costs. This study established a two-stage cultivation strategy based on the application of NaCl to improve the production of biomass and astaxanthin by Haematococcus pluvialis. During the first growth stage, 12.5 mg L⁻¹ NaCl led to a remarkable enhancement in biomass, which was 1.28 times compared with the control. Moreover, 2 g L⁻¹ NaCl stimulated the astaxanthin content from 12.18 mg g⁻¹ to 25.92 mg g⁻¹ during the second induction stage. Simultaneously, salinity stress application increased the lipids and GABA contents, as well as the levels of Ca²⁺ and carotenogenic genes’ expression, but suppressed the contents of carbohydrate and protein and high-light induced-ROS. This study proposed a simple and convenient strategy for efficient coproduction of biomass and astaxanthin and provides insights into the underlying mechanism of astaxanthin biosynthesis in H. pluvialis induced by salinity stress.
Microbial fermentation is the critical step of Pu-erh tea manufacture, which will induce dramatic changes in the chemical composition and content of tea. In this research, we applied multi-methods based on UHPLC-Q-TOF/MS to profile the dynamic changes of oligopeptides, free amino acids, and derivatives (OPADs) during Pu-erh fermentation and predicted the potential bioactivities in silico. A total of 60 oligopeptides, 18 free amino acids, and 42 amino acid derivatives were identified, and the contents of most of them decreased after fermentation. But several N-acetyl amino acids increased 7-36 times after fermentation, and they might be the potential inhibitors of neurokinin-1 receptor. Moreover, the results of metamicrobiology showed Aspergillus niger and Aspergillus luchuensis were more prominent to metabolize protein, oligopeptides, and amino acids. Overall, these findings provide valuable insights about dynamic variations of OPADs during Pu-erh tea fermentation and are beneficial for guiding practical fermentation and quality control of Pu-erh tea.
Tea is the most consumed beverage worldwide, and l-theanine in tea leaves significantly affects their flavor and market quality. We have developed and validated a fast and reliable gas chromatographic method with flame ionization detection (GC-FID) to quantify l-theanine after its extraction from Camellia sinensis (tea plant) and derivatization. The procedure was completed in 40 min, from extraction to chromatographic analysis, with a recovery rate of more than 93% and allowing a high sample throughput. The GC-FID intraday precision was within 0.57-2.28%, while the interday precision ranged from 1.57 to 13.48%. The intraday accuracy ranged from -6.84 to 5.26%, while the interday accuracy ranged from -1.08 to 3.12%. The limit of detection was 2.28 μg/mL, and the limit of quantification was 6.47 μg/mL. The GC-FID method was validated by high-performance liquid chromatography with UV detection (HPLC-UV) and was used to investigate the biosynthesis and regulation of l-theanine in tea plants. We found that plants fed with ethylamine significantly increased l-theanine concentrations in roots, while exogenous supplementation of glutamic acid, carbamide, and glutamine did not significantly affect the l-theanine level in roots. Our results also indicated that roots were not indispensable for the biosynthesis of l-theanine, which was detected in undifferentiated embryonic calluses in concentrations (g/100 g dry weight) as high as in leaves of whole plants (1.67 and 1.57%, respectively) and without any exogenous theanine precursor supplementation.
Concentrations of total catechins and theanine are important indices for monitoring the degree of fermentation, a key factor in determining the types, qualities, and taste characteristics of tea, during manufacturing process. Until now, there has not been any near-infrared (NIR) study applying theanine as an index for monitoring the degree of fermentation of tea. It is necessary to understand and monitor the degree of fermentation according to the concentrations of total catechins and theanine in tea by using NIR spectroscopy, especially for manufacturers of tea products. NIR spectroscopy integrated with modified partial least-squares regression (MPLSR) method was used for spectral analysis of total catechins and theanine in 161 tea samples in the full visible NIR wavelength range of 400−2498 nm (25000−4003 cm⁻¹). Both the optimal calibration models of total catechins and theanine built by MPLSR showed satisfactory predictability, with coefficient of determination of calibration (Rc²) and residual predictive deviation (RPD) up to 0.94 and 3.38, respectively. High total accuracy rates of 100 % were also achieved for the discrimination of green tea, partially fermented tea, and black tea in the validation sets by the optimal MPLSR calibration model of theanine. The results indicated that NIR spectroscopy has the potential to be adopted as an effective method of rapid and accurate inspection of the degree of fermentation of tea. This technique could contribute substantially to quality assurance during fermentation process of tea.
In this study, near-infrared (NIR) spectroscopy was exploited for non-destructive determination of theanine content of oolong tea. The NIR spectral data (400-2500 nm) were correlated with the theanine level of 161 tea samples using partial least squares regression (PLSR) with different wavelengths selection methods, including the regression coefficient-based selection, uninformative variable elimination, variable importance in projection, selectivity ratio and flower pollination algorithm (FPA). The potential of using the FPA to select the discriminative wavelengths for PLSR was examined for the first time. The analysis showed that the PLSR with FPA method achieved better predictive results than the PLSR with full spectrum (PLSR-full). The developed simplified model using on FPA based on 12 latent variables and 89 selected wavelengths produced R-squared (R²) value and root mean squared error (RMSE) of 0.9542, 0.8794 and 0.2045, 0.3219 for calibration and prediction, respectively. For PLSR-full, the R² values of 0.9068, 0.8412 and RMSEs of 0.2916, 0.3693, were achieved for calibration and prediction. Also, the optimized model using FPA outperformed other wavelengths selection methods considered in this study. The obtained results indicated the feasibility of FPA to improve the predictability of the PLSR and reduce the model complexity. The nonlinear regression models of support vector machine regression and Gaussian process regression (GPR) were further utilized to evaluate the superiority of using the FPA in the wavelength selection. The results demonstrated that utilizing the wavelength selection method of FPA and nonlinear regression model of GPR could improve the predictive performance.
This study aimed to investigate the effect of oxidation on free amino acids and their derivatives in tea. Oolong tea samples, oxidized for 0, 10, 30, and 60 min, green tea, and black tea were produced from the same batch of fresh tea leaves. Effects of shooting periods on free amino acids and their derivatives were also evaluated in this study. Free amino acids and their derivatives were extracted with water and analyzed by LC-MS/MS. It was observed that total amino acid content of teas in the first shooting period (16231 ± 241 μg/g-31391 ± 2034 μg/g) was higher than the second (9635 ± 379 μg/g-11665 ± 426 μg/g) and third (13937 ± 360 μg/g-17661 ± 1359 μg/g) shooting periods. Content of most amino acids including gamma aminobutyric acid (GABA) in black and oolong teas were higher than those of green tea. However, theanine contents of black and oolong teas were lower than that of green tea. Kynurenine, kynurenic acid, quinolinic acid, dopamine, tyramine, phenylethylamine and tryptamine were found in all tea samples. Kynurenic acid content was the highest (1 ± 0.1 μg/g-3±0.4 μg/g) in green, oolong and black tea samples. Concentrations of kynurenine, kynurenic acid, quinolinic acid, and dopamine were mostly found to be higher in green tea than black tea.
L-theanine (γ-Glutamylethylamide) is a non-protein water soluble amino acid (AA) mostly occurred in leaves of Camellia sinensis (green tea). This is a key component of green tea, and is considered as the most abundant form of total amino acids in green tea (i.e. about 50%). L-theanine is an exclusive taste ingredient of tea producing an attractive flavor and aroma in tea. It has worth notice biological effects like antioxidant, growth promoter, immune booster, anti-stresser, hepactoprotective, antitumor, anti-aging, antimicrobial, anti-inflammatory and anti-anxiety activities. It could reduce the oxidative impairment by reducing the synthesis of reactive oxygen species (ROS), oxidative parameters and lipid damage as well as increasing the activity of antioxidant enzymes. The oral ingestion of L-theanine enhanced γδ T-cell proliferation. Therefore, it is being considered an essential compound of green tea’ that has ability to improve immune function. The L-theanine can be used as a potential treatment for hepatic injury and immune-related liver diseases via the downregulation of the inflammatory response through the initiation of nitric oxide (NO) synthesis and glutathione (GSH) production are likely to be critical for the control of hepatic diseases as well as for the improvement of immune function. In addition it could be used as a best natural feed additive with potent anti-stressor via decreasing the levels of corticosterone (CORT), dopamine (DA) and noradrenaline (NA). After systematically reviewed the literature it is noticed that, mostly studies done on mice, pig, human and butterfly; while dietary supplementation studies of L-theanine in animal and poultry especially among broilers is very limited due to less awareness of this amino acid. So, the aim of this review is to encourage the veterinarian and poultry researchers to conduct more research at the molecular level about this amino acid to expose its’ more beneficial effects and its’ mechanism of absorption for potential use of this unique green tea amino acid in poultry nutrition.
The study of the role of neurotransmitter systems in the pathogenesis of epilepsy is one of the priorities of epileptology. New data on the functions of free neurotransmitter-like amino acid in the central nervous system are of the greatest importance and determine the prospects for the development of novel effective anticonvulsants. It is widely believed in clinical medicine that epilepsy has distinct gender characteristics. The aim of this study was to investigate the gender peculiarities in the content of neurotransmitter amino acids in the brain of Krushinsky-Molodkina (KM) rats, which were used as model organisms for the study of genetically induced audiogenic epilepsy. The content of Asp, Glu, GABA, Gly, and Tau of the medulla oblongata, hippocampus and cerebral cortex were determined using high-performance liquid chromatography (HPLC) in intact KM rats, KM rats exposed to a series of epileptiform seizures, and Wistar rats (control group). Both the Wistar and KM rats had gender distinctions in the distribution of free amino acids among the investigated brain parts. The audiogenic epilepsy was characterized by smoothing gender differences as well as differences between the concentrations of free amino acids in the cortex and medulla oblongata, specific for Wistar rats. The changes observed in male rats after the set of seizures included the increase in GABA concentration and a decrease in the Gly level in all investigated brain parts, as well as the decrease of the Tau content in the cortex and hippocampus. At the same time, the Glu content in cortex increased, while the Asp level decreased. After 6 days of audiogenic stimulations the female KM rats demonstrated the increase in the Glu level in all investigated brain parts, the increase in Gly and Asp levels in hippocampus, and no changes in the GABA content. Thus, after the set of epileptiform seizures the KM rats achieved a new steady state of the studied amino acids pool, which differed in males and females. In this case, gender differences significantly changed after the seizures.
Laochuancha is a type of ancient tea plant originating from Mingshan district of Ya’an city, Sichuan province in China, which is used to produce tea products with excellent quality. Mingshan Laochuancha green tea (MLGT) is a type of green tea manufactured from Laochuancha tea leaves. Currently, much less is known regarding the chemical compositions of MLGT and its bioactivity. Herein, the present study explores, for the first time, the chemical compositions and hypolipidemic activity of MLGT. It was observed that K was the most abundant element of 26.58 mg/g, and contents of toxic As, Cd, Cr and Pb elements were all below concentration limits. Alcohols (55.65%) were the main volatiles, and numerous volatiles with chestnut-like aroma were detected. Total content of 21 amino acids was 28.61 mg/g, and amino acids with velvety-like and umami taste totally accounted for 65.39%. The high content of amino acids and low ratio of polyphenols to total amino acids were attributed to strong umami and mellow taste of MLGT. Moreover, catechins and alkaloids were abundant in MLGT, where EGCG (85.82 mg/g) and caffeine (33.78 mg/g) were at highest content. Analyses of chemical compositions revealed excellent quality of MLGT. Correspondingly, MLGT showed potent hypolipidemic activity, and water extract of MLGT at 200 μg/mL significantly reduced lipid level to 43.06% of high-fat zebrafish. Results firstly revealed the quality characteristics of MLGT and provided further insights into bioactivity of Laochuancha.
Background:
Pingwu Fuzhuan brick tea is a type of post-fermented tea manufactured from leaves of tea plant, Camellia sinensis var. sinensis, of which the quality is influenced by numerous factors, especially microorganisms. Currently, there is little research on the effect of microorganisms on the fermentation and quality characteristics of Pingwu Fuzhuan brick tea. Investigation of main fungus in Pingwu Fuzhuan brick tea and its effect on fermentation process and tea quality can provide insights into manufacturing of "western road" border-selling tea and lay a foundation for popularization of Pingwu Fuzhuan brick tea.
Results:
The main "golden flower fungus" in Pingwu Fuzhuan brick tea was isolated and identified as Eurotium cristatum (GenBank accession number: MF800948.1; strain PW-1). Compared with natural fermentation, PW-1 inoculated fermentation accelerated biotransformation of phenolic compounds, which imparted tea samples with better taste and tea infusion color. The proportions of velvety-like and sweet-tasting amino acids were increased after 16-day fermentation with PW-1. Alcohols were the most abundant volatiles, of which the contents were 40.13% and 39.43% in NF16d and IF16d tea samples. The orthogonal partial least squares discriminant analysis (OPLS-DA) and hierarchical clustering analysis (HCA) further revealed naturally and PW-1 fermented teas were significantly different.
Conclusion:
Strain PW-1 plays an important role in the fermentation process of Fuzhuan brick tea. Considering fermentation efficiency and tea quality, fermentation inoculated with E. cristatum PW-1 can be applied in the manufacturing of "western road" border-selling tea. This article is protected by copyright. All rights reserved.
γ-aminobutyric acid (GABA) is a key physiologically active molecule in organisms. Separation of glutamate from its decarboxylated product GABA has been vigorously pursued. The interaction between these two compounds severely hindered their disassociation. Herein, we present a new strategy, termed zinc acetate-assisted differential precipitation/dissolution (ZA-DPD), for the removal of glutamate by step by step recovering pure GABA solution and discarding pure glutamate pellet, essentially attributed to the use of two core reagents (zinc acetate-assisted glutamate-precipitating reagent, and glutamate-rejecting reagent). In each precipitation, the zinc acetate-assisted glutamate-precipitating reagent guaranteed most GABA still soluble although the rest co-precipitated with glutamate; in the coupled dissolution, the co-precipitated GABA was fully dissolved with or without (in the case of glutamate-rejecting reagent used in the final dissolution) co-dissolution of glutamate. The process was repeated twice until glutamate was thoroughly removed. An accurate quantitative method coupling ZA-DPD with colorimetry was thereafter established for the determination of GABA. This study may facilitate the areas associated with GABA or glutamate.
Large-leaf yellow tea (LYT) is made from mature tea leaves with stems and has unique sensory characteristics different from other teas. To study the chemical changes of LYT during processing, samples were collected from each step for quantitative and qualitative analyses by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). LC-MS based non-targeted and targeted metabolomics analyses revealed that the tea sample after roasting was markedly different from samples before roasting, with the levels of epi-catechins and free amino acids significantly decreased, but the epimerized catechins increased dramatically. After accounting for common compounds in tea, N-ethyl-2-pyrrolidinone-substituted flavan-3-ols were found to be the marker compounds responsible for the classification of all samples, as they rapidly rose with increasing processing temperature. These findings suggested that the predominant changes in the tea constituents during large-leaf yellow tea roasting were the thermally induced degradation and epimerization of catechins and the formation of N-ethyl-2-pyrrolidinone-substituted flavan-3-ols from L-theanine.
The crude tea polysaccharides (CTPS) from Qingzhuan brick tea(QZBT) were extracted and fractionated to afford two fractions, namely TPS-1 and TPS-2. Analyses were conducted concerning the structural characterization and antioxidant activities of these samples. Component analysis revealed that the carbohydrate, uronic acid, protein and polyphenol contents of these samples differed significantly. Fourier transform infrared analysis showed that these samples showed similar characteristic absorption peaks for polysaccharides. Ultraviolet-visible spectroscopy, circular dichroism, scanning electron microscopy and thermogravimetric analyses indicated that there were considerable differences in the presence of protein, surface features, conformational characteristics and thermodynamic behaviors. For antioxidant activities in vitro, CTPS, TPS-1 and TPS-2 exhibited concentration-dependent antioxidant activities, with TPS-2 showing significantly higher antioxidant activity than CTPS and TPS-1. These results provide a scientific and strong foundation for the use of tea polysaccharides(TPS) from QZBT and further research towards the relationships between the characteristics and antioxidant activities of TPS.
L-theanine (N-ethyl-L-glutamine) or theanine is a major amino acid uniquely found in green tea. L-theanine has been historically reported as a relaxing agent, prompting scientific research on its pharmacology. Animal neurochemistry studies suggest that L-theanine increases brain serotonin, dopamine, GABA levels and has micromolar affinities for AMPA, Kainate and NMDA receptors. In addition has been shown to exert neuroprotective effects in animal models possibly through its antagonistic effects on group 1 metabotrophic glutamate receptors. Behavioural studies in animals suggest improvement in learning and memory. Overall, L-theanine displays a neuropharmacology suggestive of a possible neuroprotective and cognitive enhancing agent and warrants further investigation in animals and humans.
Tea remains the most consumed drink in the world after water, well ahead of coffee, beer, wine and carbonated soft drinks. An accumulated number of population studies suggests that consumption of green and black tea beverages may bring positive health effects (1). One hypothesis explaining such effects is that the high levels of flavonoids in tea can protect cells and tissues from oxidative damage by scavenging oxygen-free radicals. Chemically, the flavonoids found in green and black tea are very effective radical scavengers. The tea flavonoids may therefore be active as antioxidants in the digestive tract or in other tissues after uptake. A substantial number of human intervention studies with green and black tea demonstrates a significant increase in plasma antioxidant capacity in humans approximately 1 h after consumption of moderate amounts of tea (1-6 cups/d). There are initial indications that the enhanced blood antioxidant potential leads to reduced oxidative damage to macromolecules such as DNA and lipids. However, the measurement of oxidative damage through biomarkers needs to be further established. In conclusion, tea flavonoids are potent antioxidants that are absorbed from the gut after consumption. Tea consumption consistently leads to a significant increase in the antioxidant capacity of the blood. Beneficial effects of increased antioxidant capacity in the body may be the reduction of oxidative damage to important biomolecules. The scientific support is strongest for the protection of DNA from oxidative damage after black or green tea consumption. However, the quality of the studies now available is insufficient to draw firm conclusions. Therefore, further evidence from human intervention studies is required.
L-theanine (N-ethyl-L-glutamine) or theanine is a major amino acid uniquely found in green tea. L-theanine has been historically reported as a relaxing agent, prompting scientific research on its pharmacology. Animal neurochemistry studies suggest that L-theanine increases brain serotonin, dopamine, GABA levels and has micromolar affinities for AMPA, Kainate and NMDA receptors. In addition has been shown to exert neuroprotective effects in animal models possibly through its antagonistic effects on group 1 metabotrophic glutamate receptors. Behavioural studies in animals suggest improvement in learning and memory. Overall, L-theanine displays a neuropharmacology suggestive of a possible neuroprotective and cognitive enhancing agent and warrants further investigation in animals and humans.
All the experiments were performed on rabbits.1. When γ-aminobutyric acid was injected intravenously, it caused a fall of blood pressure and some changes in respiration.The thresholdal dose of γ-aminobutyric acid affecting blood pressure was less than 70 γ/kg. of body weight.The effect of γ-aminobutyric acid on blood pressure increased linearly with the logarithm of its concentration. But, in very high concentrations, its effect decreased.The optimal dose was 8-12 mg/kg. of body weight.2.β-alanine and δ-aminovaleric acid also caused fall of blood pressure. But, their thresholdal doses were much larger than those of γ-aminobutyric acid.e-aminocaproic acid had only a slight effect and α-amino acids no effect on blood pressure.Glutamate always caused a rise of blood pressure.3. It was concluded that the depressive action of γ-aminobutyric acid on blood pressure is due to an optimal distance between the amino group and carboxylic group.
Tea is one of the most popular beverages in the world. The number of reports on the analysis of tea components, especially for catechins, has recently been increasing. We review the recent reports on the analysis of tea components using the analytical methods of high-performance liquid chromatography and high-performance capillary electrophoresis.
4-Dimethylaminoazobenzene-4′-sulfonyl chloride (DABS-Cl,dabsylchloride) was synthesized by reaction of sodium 4-dimethylaminoazobenzene-4′-sulfonate with phosphorus pentachloride. DABS-Cl formed deep red, plate-shaped crystals and was stable at room temperature. DABS-Cl reacted readily with all amino acids to form dabsyl amino acids. The dabsyl amino acids (10-11-10-10 mol) were visualized on thin layer plates and found to be photo-stable. DABS-Cl was shown to be a new chromophoric labeling reagent for amino acids, peptides, and proteins, and because of its stability and sensitivity, the reagent is useful for qualitative and quantitative analyses.
In this method for quantifying 4-hydroxyproline in human urine, 50 microL of urine is hydrolyzed, derivatized with phenylisothiocyanate (PITC), and then quantified by reversed-phase HPLC with ultraviolet detection. The detection limit in urine is 373 pg of hydroxyproline per 50-microL injection. The total CVs for high- and low-concentration pools are 5.3% and 3.9%, respectively (10 runs in 10 days). The standard curve of the assay is linear over a range of 0 to 22 nmol per injection. We estimated the normal range for hydroxyproline excretion in men on an unrestricted diet to be 123-308 mumol/24 h. We also report hydroxyproline concentrations in patients with metastatic bone disease and cirrhosis of the liver.
A new approach to the pre-column derivatization and analysis of amino acids is described. The method is based upon formation of a phenylthiocarbamyl derivative of the amino acids. The derivatization method is rapid, efficient, sensitive, and specific for the analysis of primary and secondary amino acids in protein hydrolyzates. The liquid chromatographic system allows for the rapid, bonded-phase separation with ultraviolet detection of the common amino acids with 12-min analysis time and a 1-pmol sensitivity.
The reaction between primary amines and o-phthaldialdehyde in the presence of a thiol was exploited to measure the concentrations of 21 amino acids in serum by high-performance liquid chromatography with fluorescent detection. The method described here includes an automatic on-line precolumn procedure for derivatizing the amino acids, permitting full automation and avoiding problems due to time-dependent decay of fluorescence of the derivatives. The total analysis time is less than 60 min and limits of sensitivity are about 100 fmol. Proline, hydroxy-proline, and cysteine are not detected. Comparison with data generated by a standard amino acid analyzer shows this technique to be reliable.
A high performance liquid chromatographic method has been developed for the determination of ammonia, methylamine, ethylamine, dimethylamine, and diethylamine in fish and shrimp samples. These aliphatic amines are modified by pre-column derivatization with dabsyl chloride which stabilizes the molecules, facilitates extraction, and improves detection of nanogram amounts. The levels of these amines in 18 kinds of dried fish and shrimp, 17 kinds of fresh fish, and 9 kinds of canned fish were analyzed. Dimethylamine was found to be abundantly distributed in various kinds of shrimp and fish. The complete assay procedure takes about 150 min. Recovery from the sample extract is varied between 94 to 98% depending on which of the four aliphatic amines or ammonia was studied. Crystalline dabsyl derivatives of 14 amines were synthesized and their physicochemical properties including colors, melting points, electronic absorption maxima, molar absorptivities, and infrared spectra are described.
The effect of theanine, one of the components of green tea, on the blood pressure and brain 5-hydroxyindoles in spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) was investigated by intraperitoneally administering theanine. The effect of glutamine, which is structurally similar to theanine, was also examined. When SHR were injected with various amounts of theanine (0, 500, 1000, 1500, and 2000 mg/kg), the change was dose-dependent, and a significant decrease in blood pressure was observed with the high doses (1500 and 2000 mg/kg). A dose of 2000 mg/kg of theanine did not alter the blood pressure of WKY, while the same dose to SHR decreased it significantly. On the other hand, glutamine administration to SHR did not change either the blood pressure or the heart rate. The brain 5-hydroxyindole level was significantly decreased by theanine administration to both WKY and SHR, the decrease being dose-dependent.
γ-Aminobutyric acid (GABA) is known to be involved in the regulation of blood pressure by modulating the neurotransmitter release in the central and peripheral sympathetic nervous systems. This study investigated the antihypertensive effect of green tea rich in GABA (GABA-rich tea) in young and old Dahl salt-sensitive (S) rats. GABA-rich tea was made by fermenting fresh green tea leaves under nitrogen gas. In experiment 1, 21 11-month-old rats, fed a 4% NaCl diet for 3 weeks, were given water (group W), an ordinary tea solution (group T), or a GABA-rich tea solution (group G) for 4 weeks. The average GABA intake was 4.0 mg/rat per day. After 4 weeks of the treatment, blood pressure was significantly decreased in group G (176 ± 4; P < .01) compared with group W (207 ± 9) or group T (193 ± 5 mm Hg). Plasma GABA levels were more elevated in group G (111 ± 54) than in group W (not detectable) or group T (14 ± 8 ng/mL; P < .01 v G). In experiment 2, 21 5-week-old rats, fed a 4% NaCl diet, were divided into groups W, T, and G. The average GABA intake was 1.8 mg/rat per day. Body weight or chow and beverage consumption did not differ significantly among the three groups. After 4 weeks of the treatment, although blood pressure was comparable in groups W and T (165 ± 3 v 164 ± 5 mm Hg, mean ± SE), it was significantly lower in group G (142 ± 3 mm Hg) than in the other groups (P < .01). Plasma aldosterone concentration was increased in group G compared to the other groups. Thus, GABA-rich tea seems not only to decrease the established high blood pressure but to prevent the development of hypertension in Dahl S rats fed a high salt diet.
Am J Hypertens (1995) 8, 74–79; doi: 10.1016/0895-7061(94)00141-W
The neuroprotective effects of theanine and catechins contained in green tea are discussed. Although the death of cultured rat cortical neurons was induced by the application of glutamic acid, this neuronal death was suppressed with exposure to theanine. The death of hippocampal CA1 pyramidal neurons caused by transient forebrain ischemia in the gerbil was inhibited with the ventricular preadministration of theanine. The neuronal death of the hippocampal CA3 region by kainate was also prevented by the administration of theanine. Theanine has a higher binding capacity for the AMPA/kainate receptors than for NMDA receptors, although the binding capacity in all cases is markedly less than that of glutamic acid. The results of the present study suggest that the mechanism of the neuroprotective effect of theanine is related not only to the glutamate receptor but also to other mechanisms such as the glutamate transporter, although further studies are needed. One of the onset mechanisms for arteriosclerosis, a major factor in ischemic cerebrovascular disease, is probably the oxidative alteration of low-density lipoprotein (LDL) by active oxygen species. The oxidative alterations of LDL were shown to be prevented by tea catechins. Scavenging of *O(2)(-) was also exhibited by tea catechins. The neuroprotective effects of theanine and catechins contained in green tea are a focus of considerable attention, and further studies are warranted.
The four major commercial teas, oolong, black, pu-erh, and green teas, have been manufactured in southeast Asia. In this study, we evaluated the growth suppressive and hypolipidemic effect of these four different tea leaves by oral feeding to male Sprague-Dawley rats for 30 weeks. The results showed that the suppression of body weights of tea leaves-fed groups were in the order: oolong tea > pu-erh tea > black tea > green tea. Pu-erh tea and oolong tea could lower the levels of triglyceride more significantly than that of green tea and black tea, but pu-erh tea and green tea were more efficient than oolong tea and black tea in lowering the level of total cholesterol. In lipoprotein, 4% pu-erh tea could increase the level of HDL-C and decrease the level of LDL-C, but other teas simply decrease the levels of both. The activity of antioxidant enzyme SOD is increased in all tea-fed groups as compared to the basal diet-fed group. Finally, relative weight ratios of liver to epididylmal adipose tissue were lower in feeding oolong tea and pu-erh tea groups. On the basis of these findings, it seemed that the fully fermented pu-erh and black tea leaves and partially fermented oolong tea leaves were more effective on their growth suppressive and hypolipidemic effects as compared to the nonfermented green tea leaves.
Aimed at defining the key drivers for the quality-determining umami taste of a high-grade powdered green tea, called mat-cha, a bioactivity-guided fractionation using solvent extraction, solvent precipitation, preparative chromatographic separations, and human psychophysical experiments was applied on freshly prepared mat-cha. Liquid chromatography-tandem mass spectrometry and one-/two-dimensional nuclear magnetic resonance studies on isolated fractions led to the identification of l-theanine, succinic acid, 3,4,5-trihydroxybenzoic acid (gallic acid), and (1R,2R,3R,5S)-5-carboxy-2,3,5-trihydroxycyclohexyl-3,4,5-trihydroxybenzoate (theogallin) as umami-enhancing compounds in the green tea beverage, and it can be shown by sensory studies that these compounds are able to raise the umami intensity of sodium l-glutamate proportionally.
Two liquid chromatographic methods that involve precolumn derivatization with o-phthaladehyde (OPA) and phenylisothiocyanate (PITC) with fluorescence and diode array UV detection for the determination of theanine have been developed. The chromatographic separations were achieved by reverse-phase high-performance liquid chromatography using octadecyl columns and gradient elution. The methods were applied to evaluate the theanine content of commercial tea leaves. The coefficient of variation of the peak area repeatability for within day (n = 8) and between day (n = 8 over 10 days) was lower than 3% for both of the methods. The estimated limit of detection (LOD) and limit of quantitation (LOQ) for the OPA method was 0.12 and 0.35 microg theanine, respectively. The PITC method was 500-fold more sensitive with LOD and LOQ values of 0.25 and 0.75 ng, respectively. The theanine content of the commercial tea samples varied from 2-5 mg/g leaf. The overall % recoveries for these methods ranged from 93-99.3. The sensitivity and simplicity of the method render them suitable for use in quality control laboratories.
An improved capillary electrophoretic (CE) separation and indirect ultraviolet (in-UV) detection system was proposed for the amino acid analysis in tea samples with a home-made partition cell and a background electrolyte (BGE) of p-aminobenzoic acid (PAB). PAB improved the separation efficiency and detection limits of the amino acids. The partition cell prevented PAB from chemical reaction at the electrode, reduced baseline noise and kept electric current inside the cell. The separation parameters of the amino acids, such as different BG-Es, BGE concentration, buffer pH and electroosmotic flow (EOF) modifiers, were investigated. The CE separation was carried out with the running buffer solution of pH 11. 2, 10 mmol/L PAB containing 0. 014 mmol/L cetyltrimethylamonium bromide (CTAB), an applied voltage of - 15 kV and a detection wavelength of 254 nm. Sixteen amino acids were separated within 14 min under the selected conditions. The linear ranges of the amino acids were 0. 02 - 0. 60 mmol/L except for theanine (0. 02 - 3. 80 mmol/L) and gamma-aminobutyric acid (0. 02 - 2. 00 mmol/L). The recoveries were in the range from 83. 0% to 106%. The relative standard deviations of peak area were less than 5% (n = 5) and the detection limits were in the range of 1. 7 -4. 5 gammamol/L. The method is fast, convenient and sensitive, and has been applied to the determination of 11 amino acids in tea samples satisfactorily.
In this paper, the differentiation of green, black, Oolong, white, and Pu-erh teas has been carried out according to their free amino acid contents. Alanine, arginine, asparagine, aspartic acid, glutamic acid, isoleucine, histidine, leucine, phenylalanine, serine, theanine, threonine, and tyrosine have been determined by liquid chromatography with derivatization with o-phthalaldehyde and fluorescence detection. The chromatographic separation was achieved with a Hypersil ODS column and gradient elution. The amino acid contents were used as chemometric descriptors for classification purposes of different tea varieties. Principal component analysis, k-nearest neighbors, linear discriminant analysis, and artificial neural networks were applied to differentiate tea varieties. Using back-propagation multilayer perceptron artificial neural networks, 100% success in the classification was obtained. The most differentiating amino acids were glutamic acid, asparagine, serine, alanine, leucine, and isoleucine.