Journal of Tea Science

Progress on the Preparation Technologies and the Active Improvement of EGCG Nano-carriers

QI Jie, XU Yinglei, LIANG Wenyi, FEI Duo, WU Xiner, JIN Jianchang, DU Qizhen, XU Yongquan, GAO Ying

Journal of Tea Science. 2017, 37(2): 119-129.

Abstract ( 530 )

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(-)-Epigallocatechin gallate (EGCG) is one of the most important bioactive compounds in green tea, which has shown antitumor activity in various stages of tumor formation. Loading EGCG in nano-carriers is an effective technique to improve the stability and bioavailability of EGCG. Many studies had shown that various kinds of materials can be used for the preparation of nanoparticles as a EGCG nano-carrier, which significantly improved the biological activity of EGCG. The preparation methods, characteristics and the biological activity of EGCG nano-carrier were reviewed, and the improvement of effectiveness by EGCG nanoparticles formulation was also dicussed.

Molecular Cloning, Bioinformatics and Expression Analysis of GGPS Gene Family in Tea Plant

FANG Jie, LI Chunfang, MA Chunlei, CHEN Liang

Journal of Tea Science. 2017, 37(2): 130-138.

Abstract ( 542 )

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Geranylgeranyl diphosphate synthase (GGPS) is a constitutive enzyme in the terpenoid biosynthesis pathway and plays an important role in plant growth. Five putative gene sequences of GGPS were cloned by RACE and RT-RCR, and named as CsGGPS1-4 and CsGGPS9, respectively. Three allelic variants (CsGGPS9-1, CsGGPS9-2 and CsGGPS9-3) were detected for CsGGPS9, and phylogenetic analysis indicated CsGGPS9 was different from the others. Protein sequence analysis revealed that all CsGGPSs had a conserved polyprenyl_synt domain but no N-terminal signal peptide. Subcellular location predication showed that CsGGPS1, CsGGPS2 and CsGGPS4 might be located in chloroplast while CsGGPS2 and CsGGPS4 might be located in mitochondria. The 3D model of CsGGPSs were predicted by Swiss Model and combined with the ‘three floor’ model indicated that CsGGPS1, CsGGPS2 and CsGGPS4 were bona fide GGPS. CsGGPS3 was the small subunit of heterodimer GPS. Although CsGGPS9 shared a similar structure with the others, the main product of it could be isopentenyl pyrophosphate with chain length longer than 30. The qRT-PCR analysis showed that CsGGPS1 had low expression levels in all tissues except the ‘two and a bud’. By contrast, CsGGPS2 was highly expressed in all tissues with the highest levels in flowers, where in the expression levels increased first and then decreased during flower blooming. CsGGPS3 exhibited higher expression levels in the leaves and tender root than the flower. The expression levels of CsGGPS4 were similar in different tissues, with the same pattern as CsGGPS2 during flower blooming. CsGGPS9 was significantly higher expressed in the young leaves than the mature leaves.

Genetic Analysis of Full- and Half-sib Families of Tea Cultivar Jinguanyin Based on SSR Molecular Markers

WANG Rangjian, YANG Jun, KONG Xiangrui, GAO Xiangfeng

Journal of Tea Science. 2017, 37(2): 139-148.

Abstract ( 369 )

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Accurate identification of the genetic differences among tea cultivars is the prerequisite for conservation and utilization. Genetic differences of the full- and half-sib families of tea cultivar Jinguanyin were analyzed based on SSR molecular markers. The results showed that the genotyping data of the tested 31 SSR markers were highly stable, with totally 117 alleles and averagely 3.77 alleles per marker (ranging from 2 to 8). The number of genotypes, gene diversity index, gene heterozygosity, polymorphism information content (PIC) and discrimination power (PID), genetic distance ranged from 2 to 11, 0.15 to 0.8, 0.17 to 0.94, 0.14 to 0.77, 0.07 to 0.73 and 0.10 to 0.52, averagely 5.16, 0.54, 0.61, 0.48, 0.29 and 0.35, respectively. The genetic diversity followed the order that full-sib tea cultivars ˂ half-sib tea cultivars ˂ non-sib tea cultivars. The tested tea cultivars could be divided into 5 categories by the phylogenetic tree, which was closely correlated with leaf colour and tea manufacture characteristics. It’s a low probability (3.85×10^-5) to identify the wrong cultivar using 6 core SSR markers, which were highly accurate in identification and could be used for fingerprinting analysis in the tested clonal tea cultivars.

Cloning and Expression Analysis of Phosphoenolpyruvate Transporter Gene CsPPT2 in Tea Plant(Camellia sinensis)

JI Zhifang, GAN Yudi, CHEN Changsong, YANG Dingjun, SUN Kang, LI Xinghui, CHEN Xuan

Journal of Tea Science. 2017, 37(2): 149-159.

Abstract ( 388 )

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A full length cDNA sequence of phosphoenolpyruvate transporter gene (CsPPT2) was obtained from tea plant(Camellia sinensis) cultivar ‘Baiye 1’ by polymerase chain reaction (PCR) and rapid amplification of cDNA ends PCR(RACE-PCR). The nucleotide sequence length of this gene was 1469 bp, containing a complete open reading frame (1218 bp) encoding 406 amino acids. Bioinformatic analysis showed that the predicted molecular weight of the protein was 44.6 kD, and the theoretic isoelectric point was 9.90. CsPPT2 has 6 trans-membrane regions, which may belong to the hydrophobic chloroplast trans-membrane protein. Phylogenetic tree analysis showed the phosphoenolpyruvate transporter family in tea plant could be divided into two subgroups, with CsPPT1 and CsPPT2 belonged to different subgroups. The expression of CsPPT1 and CsPPT2 were compared in different periods and plant organs. It showed that CsPPT2 was expressed in all tested tissues, with higher expression level in roots and mature leaves than those of CsPPT1. But the expression level of CsPPT1 in young shoots was higher than CsPPT2. CsPPT2 was transiently elevated at the beginning of the albino, but inhibited during the development of albino, indicates that the inhibition of the expression of CsPPT2 may act as a key factor for low-catechins and high-amino acids in ‘Baiye 1’.

Establishment of Precise Color Measuring Device for Tea (Camellia sinesis L.) Leaf and Its Application in Identification of Rare Germplasm

KONG Xiangrui, YANG Jun, WANG Rangjian

Journal of Tea Science. 2017, 37(2): 160-166.

Abstract ( 330 )

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A tea leaf color measurement system was established by using the scanner, color picker tool ColorPix and RGB color rules. The value of color difference was converted into CIELab color system parameters by the online tool ColorHexa and then the leaf colors of Typical Green, Yellow and Purple tea Germplasms were comparative analyzed through circular direction and radial direction methods. The results showed that the single leaf multi - point repeat value in radial measurement and the mid-ring position value in circular method had similar leaf color characterization. The 120 tea germplasms were randomly selected and measured for the brightness L^* and chromatic components a^* and b^*. The values of lightness L^* and color difference a^* and b^* between the two methods were significantly and positively correlated. However, when the facilitation and the efficiency of analysis were taken into consideration, it is better to use the color value in the mid-ring position by circular direction method. When the leaf color values of 120 germplasms were measured in the mid-ring position to draw the three-dimensional scatter plots, the mauve color group was effectively separated from the purple germplasms, which further proved the efficiency of the method to accurately identify specific leaf color germplasm.

Effects of Black Tea Extract on Serum Uric Acid Levels in Hyperuricemic Mice

ZHU Chuang, LIU Zenghui, ZHAO Yongqing, TAI Lingling, XU Yan

Journal of Tea Science. 2017, 37(2): 167-172.

Abstract ( 394 )

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This study was aimed to investigate the effect of black tea extract on the level of serum uric acid in hyperuricemic mice. Thirty six KM male mice were randomly divided into the control group, model group, tea-treated low dose group, middle dose group, high dose group and allopurinol group. Mice in the control group and tea-treated groups were treated with saline and black tea extract respectively for consecutive 7 days. The later were given black tea extract 1 hour after modeling on the seventh day. The model group was given PO through intraperitoneal injection and yeast extract intragastrically to establish model on the seventh day. The results showed that compared with the model group, the levels of serum UA and BUN (blood urea nitrogen) were decreased in tea-treated groups, with significant decreases in the middle (P<0.05) and high dose groups (P<0.01). The serum Cr level (creatinine) of tea-treated groups were decreased and the XOD activity in the high dose group was significantly lower than those in the model group (P<0.05). there were slight but not significantly inhibitory effect in the low and middle dose groups. These date suggest that black tea extract possesses significant anti-hyperuricemic effect on hyperuricemic mice induced by PO and yeast extract.

Research on Acute and Subacute Toxicity Evaluation of Liupao Tea

WU Wenliang, LIN Yong, LIU Zhonghua, HUANG Jian'an, LONG Zhirong, TENG Cuiqing, MA Shicheng, QIU Ruijin, CAO Zhonghuan

Journal of Tea Science. 2017, 37(2): 173-181.

Abstract ( 805 )

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Liupao tea has a long history and is characterized as famous agricultural products of Guangxi, but the study of its safety is not enough. In order to verify the safety, the main physicochemical components of Liupao tea from Wuzhou were measured by conventional test method of national standard, then, the acute and subacute toxicities of Liupao tea were systemically studied in accordance with the procedures for toxicological assessment on food safety. The experimental results showed that, the physicochemical indexes of Liupao tea were repeatable, which were followed with Guangxi local standards in the acute toxicity test. Using bliss method, the calculated median lethal dose (LD₅₀) of Liupao tea was 9.38 g·kg^-1 with 95% confidence interval (8.43~10.44 g·kg^-1), which was higher than acute toxicity criteria (5 g·kg^-1). In the subacute study, compared with the control group, no death or clinical signs, or abnormally hematological, biochemical and histopathological changes were found in either groups of rats receiving daily oral gavage of Liupao tea. The three dose treatments were 0.47, 0.94 and 1.88 g·kg^-1·d^-1 for 30 days (P>0.05). Thus, Liupao tea products belong to the actual non-toxic grade and fulfill the requirements of food safety according to the food toxicology standards.

Grading of Machine Picked Tea Based on Image Processing Technology and Neural Network

WU Zhengmin, CAO Chengmao, XIE Chengjian, WU Jiasheng, HU Wangyang, WANG Tianyu

Journal of Tea Science. 2017, 37(2): 182-190.

Abstract ( 403 )

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To solve the picking problem of famous green tea, a new technology was proposed to classify machine picked tea in this paper. Fresh tea leaves plucked by machine are often mixed, with tea stalks and have a high broken rate and thereby only suitable for making general green tea. The convex hull area, convex hull perimeter, long axial length, short axial length and other morphological features of machine plucked tea leaves were analyzed by Labview vision, image processing technology and neural network to screen high quality tea. Industrial CCD camera with appropriate optical system was used to collect object classification features. User interface was developed by Labview, which can realize the data interaction, convenient operation, short development cycle and meet different users’ requirements. Finally, sample test showed that the correct rate of tea classification could reach about 90%, which provides a good theoretical basis for further research of tea grading equipment.

Residue Pattern of Polycyclic Aromatic Hydrocarbons in Tea Leaves during Green Tea Manufacturing Process

GAO Guanwei, CHEN Hongping, LIU Pingxiang, MA Guicen, HAO Zhenxia, WANG Chen, CHAI Yunfeng, LU Chengyin

Journal of Tea Science. 2017, 37(2): 191-200.

Abstract ( 380 )

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Residues of polycyclic aromatic hydrocarbons (PAHs) in fresh tea leaves and green tea during manufacturing process were determined by gas chromatography-tandem mass spectrometry. Results showed that considerable variations of PAHs were identified in tea samples (a bud and two leaves) from different locations, indicating the contamination levels in the tea producing areas might significantly affect PAH residues in fresh tea leaves. The variations of PAHs contents in tea buds, tender leaves and mature leaves were 40.5-52.8βμg·kg^-1, 50.0-67.4βμg·kg^-1 and 91.5-97.6βμg·kg^-1, respectively. The order of PAHs contents follows as mature leaves>tender leaves>tea buds. Vehicle exhaust was hypothesized to have a direct effect on PAH residues in tea buds and tender leaves, as their levels were extremely high within 50βm around the roads. During the green tea manufacturing process, evaporation of moisture and PAHs by electric heating were the key factors affecting PAHs residues, which increased from 61.0βμg·kg^-1 to 166.1βμg·kg^-1 in tea samples. Moreover, PAH residues might evaporate during the spreading and drying processes, which decreased from 292.0βμg·kg^-1 to 171.9βμg·kg^-1.

Separation and Stability Analysis of Thearubigins

LI Shan, CHEN Xiaoqiang, HUA Jinjie, LI Jia, DONG Chunwang, WANG Yincheng, JIANG Yongwen, YUAN Haibo

Journal of Tea Science. 2017, 37(2): 201-210.

Abstract ( 578 )

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In this study, thearubigins were extracted by organic extraction, and purified by reversed-phase C₁₈column chromatography to obtain three types of thearubigins, namely ethyl acetate, n-butanol and acid n-butanol extracts. The stability of thearubigins under different conditions was analyzed by scanning UV-Vis absorption wavelength based on color difference. The results showed that there was no caffeine and theaflavins, but only a small amount of catechins were detected in the three kinds of thearubigins after 30%, 50% and 70% methanol gradient elution. The color values of thearubigins after purification were 128.04 for ethyl acetate extracts, 91.04 for n-butanol extracts and 76.16 for acidic n-butanol extracts. Shading treatment, pH=3 acidic conditions and temperature below 45℃will facilitate the stability of thearubigins.

Screen and Identification of Fungi Strain Degrading Caffeine in Pu-erh Tea during Solid-state Fermentation

MA Cunqiang, ZHOU Binxing, WANG Hongzhen, WANG Pan

Journal of Tea Science. 2017, 37(2): 211-219.

Abstract ( 521 )

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To identify fungi strain involved in caffeine degradation in Pu-erh tea, caffeine liquid medium was used to screen target strain in Pu-erh tea of different stages during solid-state fermentation. The candidate strains were then analyzed for colony morphology, morphological characteristics and 18βS rDNA. The target strain was inoculated into tea for fermentation. Caffeine, theophylline, theobromine and 3-methylxanthine were determined by HPLC during the fermentation process. Results showed that the most effective strain was isolated from Pu-erh tea during solid-state fermentation, which could degrade 87.70% caffeine within 48 hours and was identified as Aspergillus sydowii with similarity of 99.8%. During the inoculated fermentation with Aspergillus sydowii, caffeine declined sharply and only 0.414%±0.077% (w/w) was reserved. Theophylline increased significantly from less than 0.1% (w/w) to 2.129% ±0.246% (w/w) and became the major purine alkaloid. Theobromine was relatively stable without significant change (P<0.05). The 3-Methylxanthine content had an obvious increase by 130%. The fungi stain was firstly isolated and identified to be involved in caffeine degradation, which confirmed fungus will facilitate the production of decaffeinated Pu-erh tea. In addition, caffeine biotransformation products were developed.

Feeding Selection of Tea Cultivars by the Tea Weevil and the Four Induced Terpenoids

HAN Juanjuan, LI Xiwang, LIU Fengjing, XIN Zhaojun, ZHANG Jin, ZHANG Xin, SUN Xiaoling

Journal of Tea Science. 2017, 37(2): 220-227.

Abstract ( 396 )

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The discrepancies of host selection and food consumption of tea weevil in Longjing 43 and three other tea cultivars (namely Longjing 43, Huangdan, Fuyun 6 and Dangui) were investigated in the present study. The emission discrepancies of the four induced terpenoids were also determined in the four cultivars. The results showed that the landing rate and food consumption of tea weevil female adults in Fuyun 6 were significantly higher than those in Longjing 43 when there had food choice. While the food consumptions of both male and female tea weevil adults in Longjing 43 were significantly higher than those in Huangdan. However, when there had no food choice, food consumption of the same gender had no significant difference with each tea cultivar. When compared with the controls, the infestation of tea weevils dramatically induced the emission level of the four terpenoids in each tea cultivar. When the tea plants were infested by the tea weevils for 12 h, the amount of linalool in Dangui, the amount of (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), and farnesene in Fuyun 6 were significantly higher than those in LongJing 43. When the tea plants were infested by the tea weevils for 24 h, the amount of ocimene in Huangdan and Dangui, the amount of linalool in Fuyun 6, the amount of DMNT and the amount of farnesene in the three tea cultivars, Huangdan, Dangui and Fuyun 6, were significantly higher than those in Longjing 43.

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