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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.
... 12,19 In addition, compared with other treatments, foliar spraying the dark and white tea infusions were more conducive to the synthesis of photosynthetic pigments, indicating that the different tea infusions had various effects, which may be related to the contents and compositions of tea. 23 Moreover, the different tea infusions also increased the biomass of C. betacea to some extent, which is also consistent with the previous studies of straw application. [39][40][41] So, the tea infusion could promote the growth of C. betacea in Se-contaminated soil, which may be related to the polyphenols in tea. ...
... [39][40][41] So, the tea infusion could promote the growth of C. betacea in Se-contaminated soil, which may be related to the polyphenols in tea. 23,24 However, further studies are needed to verify this hypothesis. ...
... Types and concentrations of polyphenols and amino acids in various tea infusions are different. 22,23 This might explain why various treatments had different effects on the antioxidant system of C. betacea. ...
Selenium (Se) is one of the essential trace elements for human, but the Se deficiency of most areas resulting in low a Se content in fruits. To increase the Se uptake in fruit trees, the effects of foliar spraying four types of tea (black, green, white, and dark) infusions on the growth and Se uptake of Cyphomandra betacea were studied using a pot experiment. The white and dark tea infusions increased the biomass, superoxide dismutase activity, and catalase activity of C. betacea, while the black and green tea infusions had no significant effects. The green, white, and dark tea infusions increased the contents of photosynthetic pigments in C. betacea, the while the black tea infusions had no significant effects. The black, white, and dark tea infusions increased the peroxidase activity of C. betacea, while the green tea infusion decreased. All tea infusions increased the potassium (K) content in aboveground part of C. betacea and phosphorus (P) content in leaves to some extent. The different tea infusions decreased the Se contents in roots and shoots of C. betacea. Compared to the control, the black, green, white, and dark tea infusions decreased the Se contents in shoots by 16.67%, 27.30%, 33.33%, and 40.07%, respectively. Correlation analysis showed that the contents of P and K were negatively correlated with the Se content. Therefore, foliar spraying tea infusion can decrease the Se uptake in C. betacea, and the white and dark tea infusions can promote C. betacea growth.
... 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.
... (0,2 g), MnSO4.4H2O (0,05 g), ammonium citrate (2 g), sodium acetate-3H2O (5 g), nước cất (đủ 1 lít), pH 6,2 [18,11] có bổ sung 1,5% monosodium glutamate (MSG), 2% saccharose, 2% peptone [12]. Trong nghiên cứu này, chúng tôi thử nghiệm thay thế hàm lượng saccharose bổ sung trong môi trường thích hợp bởi một số dịch trái cây có chứa hàm lượng saccharose cao như dịch thanh long đỏ [13], dịch dưa hấu [14] và dịch dứa [15]. ...
... Việc tạo dẫn xuất của GABA được thực hiện theo mô tả của Syu và cs. [18]. ...
Gamma-aminobutyric acid (GABA), chất ức chế dẫn truyền thần kinh, được vi khuẩn lactic (LAB), nhóm vi khuẩn được cho là có tính an toàn nhất trong thực phẩm và dinh dưỡng, sinh tổng hợp. Với mục đích làm giảm chi phí sản xuất GABA, chúng tôi nghiên cứu việc thay thế một số nguyên liệu tự nhiên và điều kiện nuôi cấy ảnh hưởng đến khả năng sinh tổng hợp GABA của chủng Pediococcus acidilactici TC7. Hàm lượng GABA trong môi trường nuôi cấy được xác định bằng phương pháp sắc ký lỏng hiệu năng cao (HPLC). Kết quả cho thấy hàm lượng GABA đạt cao nhất (30,784 ± 0,104 mM) khi chủng Pediococcus acidilactici TC7 được nuôi cấy trong môi trường các điều kiện thích hợp. Theo đó, môi trường thích hợp là MRS, bổ sung 50 mL dịch thanh long đỏ và 50 mL dịch sữa bò; pH ban đầu của dịch môi trường là 6; quá trình lên men sinh GABA được thực hiện ở 40 °C trong 72 giờ.
... 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.
... Amino acid dabsylation was performed based on a previous method (70,77) with a slight modification. Ten-microliter aliquot of free amino acids extracted from H. elongata cells or standard amino acids was mixed with 2 µL of 2.5 mM internal standard norvaline and 8 µL of 1 M NaHCO 3 to adjust the pH of the mixture to pH 9. ...
Halophilic bacteria have adapted to survive in high-salinity environments by accumulating amino acids and their derivatives as organic osmolytes. L-Proline (Pro) is one such osmolyte that is also being used as a feed stimulant in the aquaculture industry. Halomonas elongata OUT30018 is a moderately halophilic bacterium that accumulates ectoine (Ect), but not Pro, as an osmolyte. Due to its ability to utilize diverse biomass-derived carbon and nitrogen sources for growth, H. elongata OUT30018 is used in this work to create a strain that overproduces Pro, which could be used as a sustainable Pro-rich feed additive. To achieve this, we replaced the coding region of H. elongata OUT30018’s Ect biosynthetic operon with the artificial self-cloned proBm1AC gene cluster that encodes the Pro biosynthetic enzymes: feedback-inhibition insensitive mutant γ-glutamate kinase (γ-GKD118N/D119N), γ-glutamyl phosphate reductase, and pyrroline-5-carboxylate reductase. Additionally, the putA gene, which encodes the key enzyme of Pro catabolism, was deleted from the genome to generate H. elongata HN6. While the Ect-deficient H. elongata KA1 could not grow in minimal media containing more than 4% NaCl, H. elongata HN6 thrived in the medium containing 8% NaCl by accumulating Pro in the cell instead of Ect, reaching a concentration of 353.1 ± 40.5 µmol/g cell fresh weight, comparable to the Ect accumulated in H. elongata OUT30018 in response to salt stress. With its genetic background, H. elongata HN6 has the potential to be developed into a Pro-rich cell factory for upcycling biomass waste into single-cell feed additives, contributing to a more sustainable aquaculture industry.
IMPORTANCE
We report here the evidence for de novo biosynthesis of Pro to be used as a major osmolyte in an ectoine-deficient Halomonas elongata. Remarkably, the concentration of Pro accumulated in H. elongata HN6 (∆ectABC::mCherry-proBm1AC ∆putA) is comparable to that of ectoine accumulated in H. elongata OUT30018 in response to high-salinity stress. We also found that among the two γ-glutamate kinase mutants (γ-GKD118N/D119N and γ-GKD154A/E155A) designed to resemble the two known Escherichia coli feedback-inhibition insensitive γ-GKD107N and γ-GKE143A, the γ-GKD118N/D119N mutant is the only one that became insensitive to feedback inhibition by Pro in H. elongata. As Pro is one of the essential feed additives for the poultry and aquaculture industries, the genetic makeup of the engineered H. elongata HN6 would allow for the sustainable upcycling of high-salinity waste biomass into a Pro-rich single-cell eco-feed.
... The composition in amino acids in tea is characterized by high abundance of tea-specific theanine. Other amino acids reported are glutamic acid, asparagine serine and tryptophan (Alcázar et al., 2007;Horanni & Engelhardt, 2013;Syu et al., 2008). In all cases, sugared black tea possesses a high C:N ratio (carbon over nitrogen weight ratio), with a typical sugar addition ranges from 50 to 100 g/L (Tran et al., 2020b;Tran, et al., , 2022b. ...
Driving kombucha fermentation through microbial consortia remains based on scarce scientific elements, especially regarding the consumption of nitrogenous nutrients. This study aimed at analyzing quantitatively proteins and free amino nitrogen (FAN) concentrations, and qualitatively the soluble proteins profiles during a 12-day production process. The effect of microbial interactions was evaluated by comparing the growth of previously isolated major microorganisms in monoculture and cocultures to original kombucha. Protein and FAN relative concentrations kinetics depended on microbial compositions. These parameters were associated to less variations and a positive effect on total microbial population in original kombucha, underlining a complex yet better management of the nutrients. Analysis of extracellular proteins by electrophoresis showed the presence of a microbial 15 kDa fraction common to all cultures. In contrast, a 21 kDa protein fraction was associated with the presence of Acetobacter indonesiensis. Exploratory proteomics analysis revealed the presence of characteristic extracellular moonlighting proteins, originating from this acetic bacteria. Production of extracellular vesicles was successfully confirmed with the isolation and observation of vesicles in A. indonesiensis culture, as well as in kombucha. The results characterize the central role of microbial interactions on FAN consumption and open new perspectives on the function of the bacterial vesicles observed.
... Amino acids are the basic components of body proteins and serve as substrates for protein synthesis. Free amino acids also have 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 [3]. Nine amino acids are considered essential amino acids, which means that they cannot be synthesised de novo in the body or the rate of synthesis does not meet the body's needs, and they must be obtained from dietary protein [4]. ...
Fertilisation with ash from biomass combustion has a positive effect on the quality of nutrients in agrifood raw materials, improving their chemical composition and bioavailability. In the experiments carried out, the protein content and the profile of free amino acids in barley flour were examined from cultivation fertilised with biomass ash at various doses. Barley flour from Haplic Luvisol soil was characterised by a significantly higher (by 13.8% on average) total protein content compared to flour obtained from grains from Gleyic Chernozem soil. The highest protein content but a low content of free amino acids were found in the grains of plants fertilised with the mineral NPK (D1). An increase in the total pool of free amino acids in flour was observed, especially in the case of Haplic Luvisol soil. On average, after fertilising, significantly more ASP, ASN, GLU, GLY, ALA, and CYS were obtained in variant D4 (1.5 t·ha−1), and there were also significantly more TAU and GABA than in the control, up by 30.2% and 23%, respectively. A beneficial effect of fertilisation on the essential amino acid content in barley flour was found, but only up to the dose of D4, when it was significantly higher than in the control and under mineral fertilising (D1), up by 23.7% and 9.2%, respectively. High ash doses reduced the content of free amino acids in the tested barley flour. This study confirmed that using an alternative method of fertilising with plant biomass ash has a beneficial effect on protein quality and nutritional value.
... mg/100 g [16] , but it is not suitable for use in food processing. Plant and microbial sources of GABA mainly include cereals [17][18][19][20][21][22][23][24][25][26][27][28][29] , beans [30][31][32][33][34][35][36][37][38] , fruits [39][40][41][42][43][44][45][46] , vegetables [39,40,[47][48][49][50] , tea [51][52][53][54][55][56] and edible fungi [57][58][59][60] (as shown in Figure 1). Legume seeds, generally contain higher levels of GABA due to the presence of glutamic acid (Glu, the precursor of GABA). ...
γ-aminobutyric acid (GABA), a non-protein amino acid, plays an important role in the human nervous system. GABA has revealed numerous physiological benefits in the medical and pharmaceutical fields in humans. It is regarded as an edible alternative treatment for the prevention and treatment of many diseases. As a result, GABA-rich functional foods are highly favored by consumers. Therefore, in order to meet the demand for healthy food, the use of effective and safe enrichment methods to increase the content of GABA in food has received much attention. The content of GABA in natural foods, bioenrichment techniques, and the health benefits of GABA-rich foods are discussed. In addition, this paper also discusses the shortcomings and future challenges of GABA bioconcentration technology, in order to provide a new idea for the investigation of GABA enrichment technology.
... ，在调节突触传递、促进神经元发育、缓解压力以及预防抑郁等方面都发挥着重 要作用 [4][5][6] 。值得注意的是，GABA 的各种生物活性已成为医学和药学研究的热点，包括预防神经细 胞损伤、降血糖、降血压、抗炎、抗氧化、改善睡眠以及抗抑郁等 [7][8][9] 。此外，GABA 还可以保护肝 脏、肾脏和肠道，防止毒素引起的损伤 [10,11] 。 基于 GABA 众多的健康益处，富含 GABA 的保健用途食品得到大力开发，如大米、乳制品、茶 叶等 [12,13] ，且广受消费者追捧。然而，由于天然食品中的 GABA 含量普遍较低 [14] 。在正常的膳食摄 入量下，GABA 的摄入难以达到明显的健康益处。此外，尽管 GABA 具有良好的热稳定性，但在 105 ℃以上的热处理下仍会发生热分解，从而导致其含量下降 [15] [16] ，但不适宜用于食品加工中。GABA 的植物和微生物来源 主要包括谷类 [17][18][19][20][21][22][23][24][25][26][27][28][29] 、豆类 [30][31][32][33][34][35][36][37][38] 、水果 [39][40][41][42][43][44][45][46] 、蔬菜 [39,40,[47][48][49][50] 、茶叶 [51][52][53][54][55][56] 和食用菌 [57][58][59][60] (如图 1 所示)。 豆类种子，通常由于谷氨酸(Glu，GABA 的前体)的存在而含有较高含量的 GABA。其中，芸豆含 ⻝品营养化学 第 1 卷 第 3 期(2023) 2/18 量能达到 69.2 mg/100 g [36] 。在谷类中，大麦和红米的 GABA 含量较高 [25,61] 。在水果中，荔枝中的 GABA 含量能达到 139.0-350.0 mg/100 g [40,43] 。但苹果中的 GABA 含量仅有 0.0-0.5 mg/100 g [39,40] 。在 蔬菜中，菠菜和番茄具有较高的 GABA 含量。但番茄中 GABA 的含量受品种以及成熟期的影响较大 [47] 。洋葱中仅有 0.1 mg/100 g 的 GABA 含量。在茶叶中，乌龙茶的 GABA 含量最高可达到 97.0 mg/100 ...
γ-氨基丁酸（GABA）是一种非蛋白质氨基酸，在人类神经系统中起着重要作用。在医学和制药领域对GABA的研究已经揭示了GABA对人类众多的生理益处。因此，富含GABA的功能性食品备受消费者青睐。因此，为了满足人们对健康食品的需求，运用有效且安全的富集手段以增加GABA在食品中的含量受到广泛关注。本文主要综述了GABA在天然食品中的含量、生物富集技术以及富含GABA食品的健康益处。此外，本文还探讨了关于GABA生物富集技术存在的缺陷以及未来所面临的挑战，以期为GABA生物富集技术的研究提供一个新思路。
... Among the amino acids of tea, theanine accounts for over 40% of total free amino acids, and theanine itself has a fresh and mellow taste, thus able to alleviate bitterness and enhance the sweet note (Syu, Lin, Huang, & Lin, 2008). As shown in Table S3, the theanine content is relatively high in green tea and yellow tea, which could promote their fresh and mellow perception, but its content is relatively low in white tea. ...
This study fristly investigated the taste quality formation and leaf conducting tissue changes in six types of Chinese tea (green, black, oolong, yellow, white, and dark) made from Mingke No.1 variety. Non-targeted metabolomics showed the vital manufacturing processes (green tea-de-enzyming, black tea-fermenting, oolong tea-turning-over, yellow tea-yellowing, white tea-withering, and dark tea-pile-fermenting) were highly related to their unique taste formation, due to different fermentation degree in these processes. After drying, the retained phenolics, theanine, caffeine, and other substances significantly impacted each tea taste quality formation. Meanwhile, the tea leaf conducting tissue structure was significantly influenced by high processing temperature, and the change of its inner diameter was related to moisture loss during tea processing, as indicated by its significant different Raman characteristic peaks (mainly cellulose and lignin) in each key process. This study provides a reference for process optimization to improve tea quality.
... L-theanine is a nonproteinogenic amino acid derived from tea leaves (Camellia sinensis). 44 It is an analog of glutamate. Studies have found L-theanine to pass freely through the blood-brain barrier, reduce presynaptic glutamate release increase inhibitory neurotransmitter g-aminobutyric acid (GABA), and enhance glycine and serotonin and dopamine release. ...
An integrative approach to treating anxiety in children and adolescents takes a biopsychosocial-spiritual approach. Early life stress may translate into anxiety via epigenetic mechanisms, the adoption of maladaptive coping tendencies (poor eating, sedentary lifestyle, substance use), and dysregulation of central autonomic nervous system function. Each of these mechanisms may increase inflammatory markers. This article will explore the efficacy of CIM interventions that work on these mechanisms through mind-body-medicine, acupuncture, nutrition, and supplements.
... 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 (Pn), and stomatal conductance (gs), 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.
... μg g −1 . 30 On the other hand, γ-aminobutyric acid (GABA) also decreased after fermentation, because GABA was the most abundant amino acid in IF16d sample of 157.55 μg g −1 , which is about twice the content in the NF16d sample (75.25 μg g −1 ). γ-Aminobutyric acid and Thea are compounds imparting a mouth-drying sensation and velvety astringency, whereas Asp and Glu provide an umami-like taste. ...
BACKGROUND
Pingwu Fuzhuan brick tea is a type of post‐fermented tea manufactured from leaves of the tea plant, Camellia sinensis var. sinensis, the quality of which 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 the main fungus in this tea and its effect on the fermentation process and tea quality can provide insights into the manufacturing of ‘western road’ border‐selling tea and could lay the foundation for the 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 provided tea samples with better taste and tea infusion color. The proportions of velvety and sweet‐tasting amino acids increased after 16‐day fermentation with PW‐1. Alcohols were the most abundant volatiles, with 40.13% and 39.43% content in NF16d and IF16d tea samples, respectively. Orthogonal partial least‐squares discriminant analysis (OPLS‐DA) and hierarchical clustering analysis (HCA) further revealed that naturally fermented 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. © 2020 Society of Chemical Industry
... 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.
L-Proline (Pro) is an essential amino acid additive in livestock and aquaculture feeds. Previously, we created a Pro overproducing Halomonas elongata HN6 by introducing an engineered salt-inducible Pro biosynthetic mCherry-proBm1AC operon and deleting a putA gene that encoded a Pro catabolic enzyme in the genome of H. elongata OUT30018. Here, we report a generation of a novel Pro overproducing H. elongata HN10 strain with improved salt tolerance and higher Pro yield by expressing the mCherry-proBm1AC operon and deleting the putA gene in the genome of a spontaneous mutant H. elongata GOP, which overproduces glutamic acid (Glu) that is a precursor for Pro biosynthesis. The optimal salt concentration for growth of H. elongata HN10 was found to be 7% to 8% w/v NaCl, and the average Pro yield of 166 mg/L was achieved when H. elongata HN10 was cultivated in M63 minimal medium containing 4% w/v glucose and 8% w/v NaCl.
A moderately halophilic eubacterium, Halomonas elongata, has been used as cell factory to produce fine chemical 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine), which functions as a major osmolyte protecting the cells from high-salinity stress. To explore the possibility of using H. elongata to biosynthesize other valuable osmolytes, an ectoine-deficient salt-sensitive H. elongata deletion mutant strain KA1 (ΔectABC), which only grows well in minimal medium containing up to 3% NaCl, was subjected to an adaptive mutagenesis screening in search of mutants with restored salt tolerance. Consequently, we obtained a mutant, which tolerates 6% NaCl in minimal medium by overproducing L-glutamic acid (Glu). However, this Glu-overproducing (GOP) strain has a lower tolerance level than the wild-type H. elongata, possibly because the acidity of Glu interferes with the pH homeostasis of the cell and hinders its own cellular accumulation. Enzymatic decarboxylation of Glu to γ-aminobutyric acid (GABA) by a Glu decarboxylase (GAD) could restore cellular pH homeostasis; therefore, we introduced an engineered salt-inducible HopgadBmut gene, which encodes a wide pH-range GAD mutant, into the genome of the H. elongata GOP strain. We found that the resulting H. elongata GOP-Gad strain exhibits higher salt tolerance than the GOP strain by accumulating high concentration of GABA as an osmolyte in the cell (176.94 µmol/g cell dry weight in minimal medium containing 7% NaCl). With H. elongata OUT30018 genetic background, H. elongata GOP-Gad strain can utilize biomass-derived carbon and nitrogen compounds as its sole carbon and nitrogen sources, making it a good candidate for the development of GABA-producing cell factories.
IMPORTANCE
While the wild-type moderately halophilic H. elongata can synthesize ectoine as a high-value osmolyte via the aspartic acid metabolic pathway, a mutant H. elongata GOP strain identified in this work opens doors for the biosynthesis of alternative valuable osmolytes via glutamic acid metabolic pathway. Further metabolic engineering to install a GAD system into the H. elongata GOP strain successfully created a H. elongata GOP-Gad strain, which acquired higher tolerance to salt stress by accumulating GABA as a major osmolyte. With the ability to assimilate biomass-derived carbon and nitrogen sources and thrive in high-salinity environment, the H. elongata GOP-Gad strain can be used in the development of sustainable GABA-producing cell factories.
Gamma-aminobutyric acid (GABA) is a naturally occurring potential bioactive compound present in plants, microorganisms, animals, and humans. Especially, as a main inhibitory neurotransmitter in the central nervous system, GABA possesses a broad spectrum of promising bioactivities. Thus, functional foods enriched with GABA have been widely sought after by consumers. However, the GABA levels in natural foods are usually low, which cannot meet people's demand for health effects. With the increasing public awareness on the food securities and naturally occurring processes, using enrichment technologies to elevate the GABA contents in foods instead of exogenous addition can enhance the acceptability of health-conscious consumers. Herein, this review provides a comprehensive insight on the dietary sources, enrichment technologies, processing effects of GABA, and its applications in food industry. Furthermore, the various health benefits of GABA-enriched foods, mainly including neuroprotection, anti-insomnia, anti-depression, anti-hypertensive, anti-diabetes, and anti-inflammatory are also summarized. The main challenges for future research on GABA are related to exploring high GABA producing strains, enhancing the stability of GABA during storage, and developing emerging enrichment technologies without affecting food quality and other active ingredients. A better understanding of GABA may introduce new windows for its application in developing functional foods.
Chin-shin oolong tea is the most widely planted variety in Taiwan. This study fermented eight whole grains fermentation starter (EGS) with light (LOT), medium (MOT), and fully (FOT) oxidized Chin-shin oolong teas for ten weeks. Comparing the three fermentation beverages, it was found that LOT fermentation can obtain the highest catechins (1644.56 ± 60.15 ppm) among the functional and antioxidant components. MOT can obtain the highest glucuronic acid (19,040.29 ± 2903.91 ppm), tannins, total phenols, flavonoids, and angiotensin-converting enzyme (ACE) inhibitory activity. FOT can obtain the highest GABA (1360.92 ± 123.24 ppm). In addition, both the LOT and MOT showed a significant increase in their ability to scavenge DPPH radicals after fermentation. EGS fermented with lightly or moderately oxidized Chin-shin oolong tea may be considered a novel Kombucha.
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.
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.