KR101130860B1 - A Method for determining Manufacturing type and post-fermentation year of Pu-erh tea and a Composition thereof - Google Patents

Abstract

The present invention relates to a method for distinguishing green tea from ripe tea and ripe tea, by comparing the amount of the specific biomarker compound contained in the tea, the composition for distinguishing it, and a method for distinguishing the post-fermentation period of the green tea and the composition thereof. .

Black tea, biomarker

Description

Method for determining manufacturing type and post-fermentation year of Pu-erh tea and a Composition

The present invention relates to a method for distinguishing green tea from ripe tea and ripe tea, by comparing the amount of the specific biomarker compound contained in the tea, the composition for distinguishing it, and a method for distinguishing the post-fermentation period of the green tea and the composition thereof. .

Green tea is largely divided into non-fermented tea and fermented tea, and fermented tea is classified into weakly fermented tea, semi-fermented tea (black tea, yellow tea, etc.), and post-fermented tea (boiled tea, black tea, etc.) according to the degree of fermentation.

The world's leading producers of post fermented tea are China and Japan. China's aerobic fermented tea uses fungi (Fungi) as a fermentation strain, the main production area is Yunnan Province Ssangfanfan. Japan's aerobic fermented tea uses fungi as the fermentation monarch and Toyama is a major producer of black tea, and anaerobic fermented tea uses anaerobic Lactobacillus Anaerobe as the fermentation strain. There is a difference of ((阿波). Also, anaerobic and aerobic fermented teas include anaerobic fungi (Fungi Anaerobe) as fermented strains and black tea whose main production area is Ishizuchi (石 鎚).

Traditional Chinese tea, derived from Boi County in China, is intentionally wounded when harvesting green tea leaves, roasted by heating, adds an appropriate amount of moisture, and fermented naturally using microorganisms in the air. However, it is inconvenient to separate the leaves from the ivory round agglomerate, and it is not easy to ingest, and because it is fermented by falling fungi in the air, it smells like a fungus or a germ, and may contain a pathogenic microorganism. In addition, the first taste has a strong astringent taste and bitter taste, so the preference is low.

In Korea, fermented tea is manufactured and consumed in the form of home-made industry intermittently at Jirisan, Boryeong, and Buddhist temples, and there are no specialized manufacturing facilities to secure accurate microbial bacteria.

Pu-erh is a kind of post-fermented tea made from green tea. It is Camelia sinensis O. ktze. Var. Assamica kitamura is a tree plant that grows wild in Yunnan Province, China. The fermentation by microorganisms in the air belongs to a class similar to black tea, which is a representative process of post-fermentation, meaning that fermentation does not stop even after a certain manufacturing process is finished.

These teas are largely divided into raw teas, which are natural fermented teas, and ripened teas, which are artificial fermented teas, according to the Jeddah method. Green tea, which has a tradition of hundreds of years, is characterized by the fact that it takes a long time to be dried and finished until it is widely consumed, and to compensate for this disadvantage, artificially inoculate microorganisms at the optimum temperature for popularizing the tea. There is a fermented tea which is a shortened fermented fermented tea. However, since the quality of tea is very different and only genuine tea is recognized according to the fermentation method, among the experts, it is essential for the tea discrimination method. In the case of Korea, it is known that in the domestic consumption of vogue tea, which is totally dependent on imports, vogue tea is priced in connection with the manufacturing process and the number of years. There is no evidence. Therefore, the purpose of this study is to provide accurate information on the classification method of tea leaf growth and grade, and to provide the criteria for classification according to the manufacturing process of tea and classification by fermentation year.

Voy tea is typically produced through two methods.

Raw Pu-erh tea is traditionally produced by pressing large, non-oxidized tea leaves and fermenting them at room temperature for several years.

Ripped Pu-erh teas are “ripened” for several months by microorganisms under moderate conditions before being pressed (Chen, H.-Y .; Lin-Shiau, S.-Y .; Lin, J Pu-erh tea; Its manufacturing and health benefits.In Tea and tea products: Chemistry and health - promoting properties . Ho, C.-T .; Lin, J.-K .; Shahidi, F., Eds .; CRC Press: Boca Raton, 2009; pp 9-15).

Aspergillus in Boicha from previous studies niger , Aspergillus gloucu , several species of Penicillium , Rhizopus , Saccharomyces , and Bacterium have been found.

In general, they have been found to be of better quality or flavor with their long-term fermentation process. Thus, connoisseurs and investors are willing to pay a high price of thousands of dollars per cake for old boy tea.

However, there are many fake counterfeit products on the market because it is hard to find and identify reliable old tea. Therefore, an accurate and systematic method of predicting the age of the boy tea and identifying the type of manufacturing process is necessary not only for consumers but also for producers in terms of quality assurance.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned problems and has been devised by the above necessity, and an object of the present invention is to provide a biomarker for distinguishing the type of manufacturing used for the production of a tea.

It is another object of the present invention to provide a method of distinguishing the type of manufacture used for the production of black tea.

In order to achieve the above object, the present invention is stricitinin, trigalloylglucose (trigalloylglucose), caffeoylquinic acid (epigallocatechin), epigallocatechin, epicatechin- Provides a method for distinguishing green tea from ripe tea by comparing the amounts of one or more compounds selected from the group consisting of 3-gallate, epigallocatechin-3-gallate, and galloylquinicacid .

In one embodiment of the present invention, the compound is characterized in that the green tea is higher in content than the ripe tea.

In another aspect, the present invention provides a method for distinguishing the green tea and aging tea of black tea by comparing the amount of gallate compound contained in the tea.

In one embodiment of the present invention, the compound is characterized in that the tea is higher in content than raw tea.

In another aspect, the present invention is stricitinin, trigalloylglucose, caffeoylquinic acid (caffeoylquinicacid), epigallocatechin, epicatechin-3-gallate, epigallocatechin-3- Provided is a biomarker composition for distinguishing green tea from matured tea and ripening tea comprising at least one compound selected from the group consisting of gallate, galloylquinicacid and gallate.

In addition, the present invention is stricitinin, caffeoylauinic acid, epigallocatechin, epicatechin-3-gallate, and epigallocatechin-3- contained in the raw tea of Boicha. Provided is a method for analyzing the post-fermentation period of Bochacha green tea by measuring a decrease in the amount of one or more compounds selected from the group consisting of gallates.

The present invention is also selected from the group consisting of stricitinin, caffeoylauinic acid, epigallocatechin, epicatechin-3-gallate, and epigallocatechin-3-gallate Provided is a marker composition for analyzing the post-fermentation period of a tea tea containing at least one compound.

The method of distinguishing the green tea and the ripe tea of the ivory tea of the present invention, or the method of analyzing the post-fermentation period of the ivory tea, is a method of measuring the relative change based on the intensity of the ionized peak appearing on the mass spectroscopy.

Therefore, the green tea is divided into the fresh tea based on the difference of the relative range of the mass peak and based on the distribution map located on the PCA drawing.

Hereinafter, the present invention will be described.

It is an object of the present invention to identify biomarkers for differentiating the type of preparation used for the production of tea.

We analyzed how the chemical composition of Voj tea changes during post-fermentation using liquid chromatography-mass spectrometry (LC-MS) and multivariate analysis.

These analyzes allow to accurately know the compositional change of Voj tea during post-fermentation. The correlation between the antioxidant activity of Boycha and its composition and the year of post-fermentation were determined.

In this approach, the metabolomics approach through liquid chromatography mass spectrometry is used to understand the metabolites of VO tea according to various manufacturing processes and to determine the biomarkers that determine the manufacturing process and aging stage. The difference in manufacturing process and degree of aging can be determined.

(+)-Catechin, (-)-Epicatechin-3-gallate, NaOH, AlCl _{3 ,} sodium carbonate, gallic acid, Folin Ciocalteu's phenol reagent, potassium persulfate, 2,2'-azinobis (3-) ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-s-triazine (TPTZ), 3- (4,5-dimethylthiazol- 2-yl) -2,5-diphenyltetrazolium bromide (MTT), FeCl ₃ H ₂ O, C ₂ H ₃ NaO ₂ H ₂ O, sulfanilamide, N- (1-naphthyl) ethylenediamine dihydrochloride, lipopolysaccharide ( E. coli , Serotype 0.55: B5), phosphoric acid, pyrrolidine dithiocarbamate, dimethyl sulfoxide (DMSO), And formic acid were purchased from Sigma Chemical (St. Louis, Mo.). Acetonitrile and water used for LC / MS were purchased in optimal grade from Fisher Scientific (Pittsburgh, Pa.).

Hereinafter, the present invention will be described.

In order to search for identification according to the manufacturing process of the tea and the year of maturation, the present inventor purchases 30 teas (18 fresh teas and 12 fresh teas) grown in Yunnan, China, which are imported and distributed in Korea. Table 1) was extracted under reflux for 2 hours at 70 degrees with 60% acetone and filtered under reduced pressure, the filtrate was 0.2uM PTFE filter. The acetone soluble portion was analyzed by liquid chromatography / mass spectrometry (LC-ESI MS (Variant Chromatograph Electrospray Ionization Mass Spectrometry, Varian 500MS, Varian, Inc., Palo Alto, CA)) Profiling through structural information. 100 * 2.0 mm filled with C18 resin at a flow rate of 0.2 mL / min using water (A) with 0.1% formic acid and acetonitrile (B) with 0.1% formic acid as mobile phase solvents for liquid chromatography. Material separation was performed by length and diameter columns,

Table 1 is a sample list of green tea purchased from Yunnan, China.

The Vojcha sample eluate with liquid chromatography is injected into the mass spectrometer.The ionization method is a small energy impact that can efficiently ionize various molecular weights, polarities and thermally unstable components of Vojka by ionization under atmospheric pressure. Was performed. The ionization of the sample spray was carried out at 30 psi pressure and 50 psi pressure sprayed with nitrogen gas for drying the sample at a heating zone temperature of 350 ° C. The mass range was 50-1000 m / z in anion mode using 70 V ionization energy. The signal was detected.

In order to normalize the mechanical error signals and the values of the respective sample signals in the data of each VAT sample component mass spectra, the data processing process was performed through XCMS software. The processed data sheets were analyzed for the manufacturing process and age of aging using the principal component analysis and partial least squares regression method for statistically significant analysis.

It was confirmed that 30 fractions of Voycha samples obtained in the above analysis were divided into two groups, ripening tea and fresh tea, according to the physicochemical properties and contents of their metabolites. (Fig. 1) Principal component analysis loading plot As a result, the main variables that act as biomarkers to distinguish green tea are stricitinin ( m / z 633), trigalloylglucose ( m / z 635), caffeoylquinicacid ( m / z 353), epigallocatechin ( m / z 305), epipicatechin- 3-gallate ( m / z 441), epigallocatechin-3-gallate ( m / z 457), and galloylquinic acid ( m / z 343) were found to be more plentiful compared to tea. On the other hand, gallic acid was found to be the main variable in the case of ripe tea, which has a higher content than raw tea (FIG. 2).

As a result of examining the discrimination method according to the maturation year that greatly distinguishes the superiority of Boi tea, it is classified by year and divided into less than 4 years of fermentation and more than 5 years. On the other hand, in the case of aging tea made by boiling by artificial process instead of natural fermentation, the identification by year was not clear.

In addition, when comparing the information of the current maturation year and the year observed in this experiment through the change of metabolite according to the fermentation time of raw tea and ripe tea, the difference between fresh tea and 2.1 year old tea was 2.9 years. (FIGS. 4 and 5)

In conclusion, the present invention details the difference in chemical composition of several vogue teas in terms of production process and post-fermentation period. As a result of the PCA analysis, two different types of Voycha were clearly separated by PC1. Fresh teas with high levels of phenol and flavonoids showed higher bioactivity than ripe teas. Using LC-MS and multivariate analyses, we gained broad characterization of the compositional changes seen in fresh tea during post-fermentation. Compounds such as epigallocatechin, epicatechin-3-gallate, strictinin, caffeoylauinic acid, and epigallocatechin-3-gallate decreased as the post-fermentation period increased. Thus, these compounds naturally explain why the fermented long-term fermented tea has low antioxidant activity. The post fermentation prediction model is designed through PLS analysis based on the metabolites of Voj tea. As a result of the PLS regression analysis, several commercially available green teas showed a pattern of specific composition change in post-fermentation time-dependent form. The inventors have also shown that metabolomics can be useful for analyzing composition patterns and illuminating the correlation between the bioactivity of Voycha and their compositional changes.

In the case of green tea, which is distinguished by the superiority of tea leaves, the above analysis method and bio-tea markers are very important judgment criteria for manufacturing process verification and quality control of aging.

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. However, the following examples are described for the purpose of illustrating the present invention and the scope of the present invention is not to be construed as limited by the following examples.

Example  1: manufacture of samples

Boy teas with different post-fermentation years were purchased from the market in Korea (Table 1).

All samples were grown in Yunnan, China. The samples were extracted three times with 60% aqueous acetone for two hours at 70 ° C. in a water bath. The mixtures were filtered through a 0.2 μm PTFE filter for LC / MS analysis.

Example  2: liquid chromatography Photodiode  Array Electrospray  Ionization-mass spectrometry LC - PDA - ESI Of MS ) Condition:

We used a 212-LC Binary Solvent Delivery System, a MetaThermTM HPLC column heater, a ProstarTM 410 AutoSampler, a ProstarTM 335 Photodiode Array Detector, and a 500-ion trap mass spectrometer (Varian Technologies (Palo Alto, Calif.) The system was run using the MS workstation software (version 6.9.2, Varian, Inc., Palo Alto, Calif.) LC-MS conditions were described in the existing paper (Nishitani, E .; Sagesaka, YMJ Food Compost). Anal. 2004, 17, 675-685; Lin, LZ; Chen, P .; Harnly, JMJ Agric.Food Chem. 2008, 56, 8130-8140; Wang, D .; Lu, J .; Miao, A. Xie, Z .; Yang, DJ Food Compost.Anal. 2008, 21, 361-369) Chromatographic analysis was performed on a ChromSep Gart column PurSuit XRs 3-C18 (Varian Inc.) at 45 ° C. column oven temperature. , ChromSep HPLC column SS 100 × 2.0 mm id, 3 μm particle size (Varian Inc., Lake Forest, CA) with Lake Forest, CA. A flow rate of 0.2 kmL / min was used The mobile phase consisted of a combination of A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) The gradient was 15 to 30% B (v / 15 at 15 minutes). v), linearly increased to 100% B at 30 minutes, maintained at 100% B until 35 minutes, then linearly reduced to 15% at 35.01 minutes and 15% for 40 minutes until injection of the next assay sample. The UV spectrum was set at 280 nm for real time monitoring of peak intensity. Photodiode analysis was used to continuously record absorbance from 200-600 GHz for difference component identification. Mass spectra were simultaneously obtained using electrospray ionization at negative ionization (NI) at 70 V for the range m / z 50-1000. For both NIs dry gas pressures and temperatures of 30 kPa and 350 ° C., nebulizer pressure (50 kPa), and a capital voltage of 70 V were used.

Example  3: Trolox Equivalent Antioxidant Capacity  ( TEAC )

TEAC analysis was performed by Re et al. (Re, R .; Pellegrini, N .; Proteggente, A .; Pannala, A .; Yang, M .; Rice-Evans, C. Free Radic. Biol. Med. 1999, 26, 1231- 1237). According to this paper, 7 μm of ABTS ammonium was dissolved in Potassium phosphate buffer (pH 7.4) and treated with 2.45 mM of potassium persulfate. The mixture was then left at room temperature for 12-16 hours until it turned dark blue. The solution was then diluted with Potassium phosphate buffer until absorbance reached 0.7 ± 0.02 at 734 nm. It was measured using a BioTek EL 808 microplate reader (Bio-tek Instruments Inc., Power Wave XS, Winooski, VT). Then 190 μL of the solution was mixed into 10 μL of sample or blank (60% aqueous acetone). The absorbance was recorded after 6 minutes at room temperature. The results are expressed as mg Trolox equivalent concentration per mg Boy tea. The concentration of the standard solution ranged from 0.25-4 μmM. The experiment was conducted three times.

Example  4: DPPH  Determination of Antioxidant Activity by Free radical Scavenging Assay

DPPH analysis was published in Dietz et al. (Dietz, BM; Kang, YH; Liu, G .; Eggler, AL; Yao, P .; Chadwick, LR; Pauli, GF; Farnsworth, NR; Mesecar, AD; van Breemen, RB Bolton, JL Chem. Res. Toxicol. 2005, 18, 1296-1305). A test sample or blank of 60% aqueous acetone (10 μL) and 190 μL of a 200 μM DPPH ethanol solution was incubated for 30 minutes at room temperature in a 96 well plate. The absorbance of DPPH free radicals was measured at 515 nm using a microplate reader. The results are expressed as mg Trolox equivalent concentration per mg Boy tea. The concentration of the standard solution was in the range of 0.156-2.5 mM. The experiment was conducted three times.

Example  5: Ferric Reducing Of Antioxidant Power  ( FRAP ) analysis

Antioxidant capacity of each standard and sample was performed with minor improvements to Benzie and Strain (Benzie, I. F .; Strain, J. J. Anal. Biochem. 1996, 239, 70-76). Briefly, 300 μl of FRAP reagent, fresh and warmed at 37 ° C., was mixed with 10 μl of test sample solution or blank (60% aqueous acetone). The absorbance was measured at 593 5nm after 6 minutes using a microplate reader. The FRAP reagent contains 10 μm / L TPTZ solution (in 40 μm / L HCl) in 2.5 μL distilled water, 20 μm / L FeCl3–H2O (in distilled water) in 2.5 μL, and 0.3 μM / L acetic acid buffer (pH in 25 mL). 3.6). The results are expressed as mg Trolox equivalent concentration per mg Boy tea. The concentration of standard solution was in the range of 0.25-2 mM. The experiment was conducted three times.

Example  6: Determination of total phenols and flavonoids

The total phenol amount was determined using the method of Singleton et al. (Singleton, VL; Orthofer, R .; Lamuela-Ravent, RM; Lester, P. In Methods in Enzymology. Academic Press: 1999; Vol. Volume 299, pp 152-178). Decided. The assay conditions were as follows: 10 [mu] l of sample was added to 0.2 [mu] N Folin-Ciocalteu's phenolic reagent (160 [mu] l) in 96 wells. After 3 minutes, 30 μl of saturated sodium carbonate solution was added to the mixture, followed by incubation for 1 hour at room temperature. The resulting absorbance of the mixture was measured at 750 nm using a microplate reader. Total phenolic content was calculated based on the standard curve with gallic acid. The standard solution concentrations ranged from 12.5-400 μg / mL. The results are expressed as mg gallic acid equivalent (GAE) per mg Boycha. The experiment was conducted three times.

Total flavonoid content was analyzed by slightly modifying the method described in Yoo et al. (Yoo, K. M .; Lee, C. H .; Lee, H .; Moon, B .; Lee, C. Y. Food Chem. 2008, 106, 929-936). 20 μl of sample or standard solution of (+)-catechin was added to each well in a 96 well plate. The standard solution concentrations ranged from 100-800 μg / mL. Distilled water (40 μL) and 6 μL of 5% (w / v) sodium nitrate were added to each well. After 5 minutes, 12 μL of 10% (w / v) AlCl 3 was added. After 6 minutes, 40 μl of 1 μM NaOH was added to the mixture and 42 μl of distilled water was added. Absorbance was measured at 515 nm using a microplate reader and its flavonoid content was expressed as mg (+)-catechin equivalent of mg per vogue tea. The experiment was conducted three times.

Example  7: Data processing

Data preprocessing was performed using MS Workstation 6.9 software (Palo Alto, Calif.). LC-MS spectra were converted to NetCDF format using Vx Capture (version 2.1, Laporte, MN). After conversion, LC-MS spectra were processed by XCMS. The XCMS parameters for the R language were performed by following simple commands such as XCMS's default settings below (http://masspec.scripps.edu/xcms/documentation.php). As a result, the data was sent to Excel (Microsoft, Redmond, WA) and statistical analysis was performed using another statistical program.

Example  8: Statistics and Multivariate ( Multivariate ) analysis

Statistical analysis was performed on all continuous variables using SIMCA-P + (version 11.5, Umetrics, Umea Sweden). Univariate statistics for multiple classes were performed by breakdown and one-way ANOVA using STATISTICA (version 7.0, StatSoft Inc., Tulsa, OK). Multivariate statistics were performed by unsupervised PCA (PCA) to obtain a general overview of the diversification of metabolites in the present invention. Supervised partial least square (PLS) statistics were also performed, which needed information about the assigned class of variables (X variable; LC-MS peak and Y variable; post fermentation period). All variables were Paletto scaling with mean-centering at column-wise status before PCA and PLS analysis.

The result of the above example is as follows.

Comparison of Antioxidant Activity of Two Types of Boy Tea

TEAC, DPPH and FARP assays were performed to compare the antioxidant activity of Boicha produced using the two preparation methods. Antioxidant activity of fresh tea was significantly superior to that of ripe tea in three different antioxidant assays. Ripe tea showed antioxidant activity of Trolox equivalent (per gram of Boycha) of 1170 (DPPH), 810 (FRAP) and 1090 (TEAC), whereas raw Pu-erh tea had 1770 (DPPH), 2780 (FRAP). ) And 2020 (TEAC) mg of Trolox equivalent (per gram of Boycha) antioxidant activity (Table 2). In summary, green tea showed higher antioxidant activity than tea.

Comparison of Total Phenol and Total Flavonoid Amounts

It has been found that green tea has more phenol and flavonoid compounds than green tea. As can be seen in Figure 1b, the total amount of phenols (182 mg gallic acid equivalent / grams of grams) of fresh tea was 1.65 times higher than that of ripe tea (110 mg gallic acid equivalents / grams of voic teas). The total flavonoid amount of fresh tea (98 μg gallic acid equivalent / boigram gram) was twice higher than that of ripe tea (49 μg gallic acid equivalent / boigram gram) (Table 2).

Vojcha  Post-fermentation period ( post - fermentation year ) And the relationship between total compound composition and bioactivity

In order to investigate the correlation between the post-fermentation year of Bojcha and total compound composition and bioactivity, the correlation coefficient was calculated using STATISTICA. The bioactivity of green tea was significantly correlated with their post fermentation period, while there was no significant correlation between ripening tea and their post fermentation period. In more detail, the post-fermentation period of fresh tea was negatively correlated with FRAP (r = -0.60, p <0.05) and total polyphenol amount (r = -0.65, p <0.05). The total flavonoid content of green tea correlated positively with FRAP (r = 0.75, p <0.05) and TEAC (r = 0.71, p <0.05), whereas the total phenolic amount of ripe tea was DPPH (r = 0.87, p <0.05) and TEAC ( r = 0.91, p <0.05). Considering the total phenol content of the green tea and the sensitivity of the post fermentation and the sensitivity of the FRAP assay, the FRAP assay may be useful for the analysis of the green tea.

Vojcha  Analysis according to different manufacturing methods

In order to find significantly different biomarkers between fresh and ripe tea, multivariate statistical analysis was performed. Multivariate statistics using unsupervised principal components analysis (PCA) indicate that the main factor separating metabolic profiles is the type of manufacture, not the post-fermentation period. The PCA score plot showed a clear separation of ripe and fresh tea based on the 377 main peak by PC1 (R ² X = 75.6%). The relatively large variation observed in fresh tea is the result of different post-fermentation periods. On the other hand, its raw materials seem to be similar to each other during rapid fermentation. This indicates that although the effect takes several years, post-fermentation of fresh tea has a significant effect on their composition, but the composition pattern of ripe tea is not significantly affected by their post-fermentation period.

The PCA loading plot was obtained using Pareto scaling with mean-centering from the LC-MS peak intensities of each peak. Some salient peaks were observed in the PCA loading plot. Peak identification is based on UV spectral data of reliable compounds, in-house database (Lee, JS; Kim, DH; Liu, K.-H .; Oh, TK; Lee, CH Rapid). Commun . Mass Spectrom . 2005, 19, 3539-3548) and co-chromatography, MS / MS spectral data. Remarkably different peaks were identified using this method. Some peaks were detected in the form of isotopes with dimers and their equivalents ( m / z 883 (441), 884 (441), 916 (457), 915 (457), 634 (633)). As shown in Figure 2, green tea is stricitinin ( m / z 633), trigalloylglucose ( m / z 635), caffeoylquinic acid ( m / z 353), epigallocatechin ( m / z 305), epicatechin-3-gallate ( m / z 441), epigallocatechin-3-gallate ( m / z 457), and galloylquinic acid ( m / z 343) contained significantly higher levels than tea (t-test, p <0.001). However, gallic acid was significantly lower in fresh tea (t-test, p <0.001). Overall, tea has low levels of catechins and high levels of gallic acid. Thus, antioxidant activities such as DPPH and TEAC of ethanol have a high correlation with total phenolic content rather than total flavonoid content.

377 peaks were extracted by XCMS from 90 samples of various Voycha. Among them, 343 peaks showed markedly different mass intensities between fresh and ripe teas ( p <0.001).

In the present invention, it was shown that green tea has higher bioactivity than ripe tea in antioxidant, NO inhibition and cell protective activity.

Raw tea  Various Post fermentation Pls analysis

In order to determine the correlation between the compound composition of the green tea and the post-fermentation period, PLS analysis using SIMCA-P was performed. As shown in FIG. 4, Aged and young (over 5 years) and young (less than 4 years) are distinguished by PLS component 1 as well as its PCA score plot. PLS analysis can be used as an algorithm to create a quality prediction model for a car. As shown in FIG. 4, the fermentation period prediction model after creation from the PLS regression represents the root mean square error of the fit for observation in the 2.08 year workset (RMSEE). Using this analysis, we found that the composition of the green tea gradually changed with their post-fermentation period (R ² = 78.5, Q ² = 73.8). This post fermentation prediction model can be used to predict the post fermentation period of fresh tea.

In order to identify some compounds that are particularly correlated with fermentation, correlation coefficients were calculated using STATISTICA. Remarkable compounds were identified in the same manner as described above. quinic acid (r = -0.688) (not shown), epigallocatechin-3-gallate (r = -0.567), caffeoylquinic acid (r = -0.565), epigallocatechin (r = -0.505), strictinin (r = -0.495), Compounds such as epicatechin-3-gallate (r = -0.435), and gallic acid (r = 0.546) are highly correlated with post fermentation duration ( p <0.05), and biomarkers can be used to determine the age of fresh tea. (FIG. 4). As mentioned above, the green tea's TPC and FRAP also correlate strongly with their post-fermentation period. These biomarkers can explain why older green teas have lower total phenol levels and lower antioxidant activity.

Chaotic  Various Post fermentation  Term Pls  analysis

As shown in FIG. 5, the old and young leans are not clearly separated by the PLS factor 1 or 2 as well as the PCA score plot. As mentioned earlier, the composition of ripe tea is markedly changed by rapid fermentation. This indicates that secondary metabolites of ripe tea are not severely affected by their post-fermentation period. Unlike the green tea, the ripe tea does not show a high crystal coefficient (R ² = 60.9, Q ² = 40.1) (FIG. 5).

Figure 1 (A) is a plot showing the PCA score plot of the Voycha (■, green tea; red is a tea), (B) PCA loading plot obtained using Pareto scaling with mean centering.

FIG. 2 (A) is a whisker plot and box of compounds that differ markedly between fresh and ripe teas. (B) is a diagram showing important compounds in relation to post-fermentation.

3 is a PCA score plot derived from Voycha with different post-fermentation periods. a is green tea, b is tea.

Figure 4 (a) is a PLS score plot of the green tea (red circle is 5 years or more; ■, 4 years or less), (b) PLS regression model for the prediction of the post-fermentation period of the green tea.

Figure 5 (a) is a PLS score plot of the tea (red circle is more than 5 years; ■, 4 years or less), (b) PLS regression model for the prediction of the post-fermentation period of the tea.

Claims (7)

Stricitinin and trigalloyl were compared by comparing the amounts of stricitinin and trigalloylglucose compounds contained in Bojcha and the amounts of gallate compounds contained in Bojcha. A method of distinguishing green tea from ripe tea, which distinguishes tea having a relatively high content of glucose (trigalloylglucose) and a relatively low content of gallate compound as a fresh tea. delete delete delete delete delete delete

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