AU2020100810A4

AU2020100810A4 - A controllable method for processing duyun dark tea

Info

Publication number: AU2020100810A4
Application number: AU2020100810A
Authority: AU
Inventors: Xuejing GAO; Xiying GUO; Ping Li; Yaling LI; Canbiao LUO; Zhirui WEN; Jiagan YANG; Zhenjun ZHAO; Caibi ZHOU; Caiyuan ZHOU; Xiaolu ZHOU
Original assignee: Guizhou Bishu Science And Technology Service Co Ltd; Qiannan Normal University for Nationalities
Current assignee: Guizhou Bishu Science And Technology Service Co Ltd; Qiannan Normal University for Nationalities
Priority date: 2019-05-06
Filing date: 2020-05-21
Publication date: 2020-07-09
Anticipated expiration: 2028-05-06
Also published as: CN110771698A

Abstract

The invention discloses a controllable method for processing Duyun dark tea, which comprises the steps of picking fresh leaves, spreading, fixation, rolling, drying, sterilizing, preparing bacteria, inoculating, piling and drying. The processing method of the invention is stable, can effectively improve the quality of the Duyun dark tea, and improve the aromas and the mouthfeel of the Duyun dark tea. The Duyun dark tea produced by the method of the invention is rich in nutrient components, stable in tea leaf quality, good in color and luster, and free of T2, HT2 and four aflatoxins. The tea leaves of the invention are uniform and black brown in appearance, pure in fragrance, red brown in soup color, mellow in taste and dark brown in leaf bottom. 16 Drawings Si -1B .1B ||C AFG2 T2 HT2 1/2

Description

Drawings

Si

-1B .1B ||C

AFG2 T2 HT2

1/2

A controllable method for processing Duyun dark tea

TECHNICAL FIELD The present invention relates to a method of making tea, and more particularly, to a controllable method of processing Duyun dark tea.

BACKGROUND OF THE INVENTION Dark tea is named because of the black appearance of the finished tea. Dark tea belongs to one of the six major tea species and is a post-fermented tea. The main producing areas are Sichuan, Yunnan, Hubei, Hunan, Shanxi, Anhui and so on. The black hair tea raw material used in traditional dark tea has a high degree of maturity and is the main raw material for pressing the compressed tea. According to regional distribution, dark tea is mainly classified into Hunan dark tea (Poria tea, Qianliang tea, dark brick tea, Sanjian tea, etc.), Hubei old dark tea, Sichuan Tibetan tea (side tea), Anhui ancient Yi dark tea (An tea), Yunnan dark tea (Pu'er tea), Guangxi Liubao tea and Shanxi dark tea (Poria tea). The key step of processing dark tea is piling, which is a process in which ingredients comprised in the tea are subjected to a series of deep oxidization and decomposion under the coaction of the internal damp heat and the microorganisms, thereby forming dark teas with unique quality.

Dark tea is a popular tea variety, and there is a special process during the manufacture of dark tea, i.e. "piling", in which microorganisms play an important role. The piling fermentation process of dark tea is suitable for the growth of mycotoxins, and therefore, there is a possibility of mycotoxin contamination. The processing of the traditional dark tea is susceptible to the contamination of the foreign bacteria during the piling, so that the quality of the finished tea is not easily controlled. The main microorganisms involved in the dark tea piling process are Aspergillus niger, Penicillium spp., Yeast spp., Rhizopus spp., in addition to pathogenic bacteria and some toxin-producing fungi such as Aspergillus flavus, Penicillium citreo-viride, Penicillium islandicum, Penicillium citrinum, Aspergillus flavus Bi (AFB1), Aspergillus ochraceus A (OTA), Deoxynivalenol (DON), Penicillium patulum(PAT), etc.. Mycotoxins are biological toxins produced by some fungi during their growth. There are more than 200 mycotoxins known to date, and mycotoxins have a wide

I spectrum of toxicities, such as inhibition of the immune system and hematopoietic system, leading to neuroendocrine disorder, and damage to the liver and kidney and the like. Sometimes their toxic effects are manifested as endemic diseases, and more seriously, some mycotoxins have been shown to have carcinogenic, teratogenic and mutagenic effects. At present, Guizhou tea is too simplex, mainly including green tea and black tea. Therefore, the invention discloses a method for preparing dark tea from tea leaves produced in Duyun, Guizhou, which can effectively control the growth of microorganisms in the piling process, and has important significance for promoting the diversified development of the tea industry.

SUMMARY OF THE INVENTION The object of the present invention is to provide a controllable method for processing Duyun dark tea. The processing method of the invention is stable, can effectively improve the quality of Duyun dark tea, and improve the aromas and the mouthfeel of Duyun dark tea. The Duyun dark tea produced by the method of the invention is rich in nutrient components, stable in quality, good in color and luster, and free of T2, HT2 and four aflatoxins. The tea leaves of the invention are dark brown and uniform in appearance, pure in fragrance, red brown in soup color, mellow in taste, and dark in leaf bottom. The technical solution of the present invention lies in a controllable method for processing Duyun dark tea which comprises the following steps: (a) picking fresh leaves: picking fresh leaves of native local varieties in Duyun; (b) spreading: spreading the fresh leaves indoors at room temperature of °C-30°C for 1-4 hours to make them lose water to 60 to 80%; (c) fixation: putting leaves of 2-3 kg in a Cylinder type fixation machine at 180-220°C until the green odor disappears, the tea aromas is exposed, the leaf color changes from fresh green to dark green, and the leaves are sticky and conglomerate when pinched; (d) rolling: rolling for 20-30 min while hot, lightly pressing for one fourth of the time, moderately pressing for one half of the time and lightly pressing for one fourth of the time; (e) drying: deblocking and drying at room temperature of 25°C for 5 h in the sun to form a sun smell, light rolling for 5 min, and drying at 40°C to obtain a sun-dried green crude tea; (f) sterilizing: sterilizing the sun-dried green crude tea with moisture of 35% at 115°C for 30 min, and cooling for further use; (g) preparing bacteria: preparing the spore suspension of Trichosporon FD; (h) inoculating: singly inoculating at a ratio of 40 g of sterilized tea leaves: 0.1 mL of FD spore suspension; (i) piling: the inoculated tea leaves were subjected to pile fermentation for 25 days at a temperature of 30°C and a humidity of 80%; () drying: taking out the fermented tea leaves, and drying at 45°C to obtain the Duyun dark tea. In the above controllable method of processing the dark tea, the fresh leaves in the step (a) are three or four leaves on one bud. In the above controllable method of processing the Duyun dark tea, the spreading in the step (b) is performed by spreading the fresh leaves indoors for 3 hours at room temperature of 25°C until the water loss is up to 70%. In the above controllable method of processing the Duyun dark tea, the fixation in the step (c) is performed with a leaf amount of 2.5 kg at 200°C using a Cylinder type fixation machine. In the above controllable method of processing the Duyun dark tea, the spore suspension of Trichosporon FD in the step (g) is prepared as follows. Trichosporon FD are inoculated onto PDA slant culture medium and cultured at 20-40°C for 3-9 days. After the strains are mature, the spores are washed off with 0.3-1.5% normal saline and transferred into a triangular conical flask filled with glass beads. The spores are evenly distributed in the suspension by shaking sufficiently, and filtered through defatted cotton to obtain a single spore suspension. A coverslip is placed on a dry and clean blood count plate, and one drop of the spore suspension is added to the edge of the coverslip and is allowed to penetrate into the counting chamber by capillary action. When the spores sink to the bottom of the plate, they are counted and adjusted to the spore concentration of 107 cfu/mL. In the above controllable method of processing the Duyun dark tea, the spore suspension of Trichosporon FD in the step (g) is prepared as follows.Trichosporon FD are inoculated. After the strains are mature, the spores are washed off with 0.7-1.1% normal saline and transferred into a triangular conical flask filled with glass beads. The spores are evenly distributed in the suspension by shaking sufficiently, and filtered through defatted cotton to obtain a single spore suspension. A coverslip is placed on a dry and clean blood count plate, and one drop of the spore suspension is added to the edge of the coverslip, and is allowed to penetrate into the counting chamber by capillary action. When the spores sink to the bottom of the plate, they are counted and adjusted to the spore concentration of 107c fu/mL. In the above controllable method of processing the Duyun dark tea, the spore suspension of Trichosporon FD in the step (g) is prepared as follows. Trichosporon FD are inoculated onto PDA slant culture medium and cultured at 28°C for 5-7 days. After the strains are mature, the spores are washed off with 0.9% normal saline and transferred into a triangular conical flask filled with glass beads. The spores are evenly distributed in the suspension by shaking sufficiently, and filtered through defatted cotton to obtain a single spore suspension. A coverslip is placed on a dry and clean blood count plate, and one drop of the spore suspension is added to the edge of the coverslip, and is allowed to penetrate into the counting chamber by capillary action. When the spores sink to the bottom of the plate, they are counted and adjusted to the spore concentration of 10 7 cfu/mL.

In the above controllable method of processing the Duyun dark tea, Trichosporon FD are obtained by streaking and separating with gradient concentration of aged Pu'er tea as a raw material, and analyzing ITS sequences. Compared with the prior art, the pile-fermentation Duyun dark tea is post -fermented tea. The Duyun dark teas of the present invention are prepared from the sun-dried green crude tea made by the native local varieties in Duyun by pile fermentation. Piling under humidification condition is a key process for forming the quality of the dark tea. Microbial fermentation plays a decisive role in the quality formation of the Duyun dark tea and is essential for the quality formation of the Duyun dark tea. According to the invention, fresh leaves from three or four leaves on one bud of a native variety in the Duyun area are picked to prepare sun-dried crude tea, which is subjected to sterilization and then is singly inoculated with Trichosporon FD to prepare Duyun dark tea through pile fermentation. To verify the effect of the present invention, the inventors carried out the following experiments.

Experimental Examples: I. The preparation of sun-dried green crude tea from native local varieties in Duyun. (1) fresh leaves: three or four leaves on one bud,from native local varieties in Duyun; (2) spreading: spreading the fresh leaves indoors for 3 hours at room temperature of 25°C to make them lose water to 70%; (3) fixation: putting the leaves of 2.5kg in a Cylinder type fixation machine at 200°C until the green odor disappears, the tea aromas is exposed, the leaf color changes from fresh green to dark green, and the leaves are sticky and conglomerate when pinched; (4) rolling: rolling for a time period of 25 min while hot, lightly pressing for one fourth of the time, moderately pressing for one half of the time and lightly pressing for one fourth of the time; (5) drying: deblocking and drying at room temperature of 25°C for 5 h in the sun to form a sun smell, light rolling for 5 min, and drying at 40°C to obtain the sun-dried crude tea. II. Isolation and Identification of Strains 2.1 Strain isolation and morphological identification

Cooked Pu'er tea of high quality in 2010 (Brand: Hung Run Lai ) was screened. A dilution gradients of 10-2-10-7 were prepared, and plated onto culture medium for isolation. The cultured strains were purified and stored. Morphology of the obtained strains were preliminarily identified according to the morphological characteristics such as the shape, size, color and surface characteristics of the primary colonies in combination with the descriptions in the book "Mycology of China". 2.2 Molecular Identification First, strain DNA was extracted using a modified CTAB method. Next, the fragment of interest was PCR-amplified using KOD high-fidelity DNA polymerase, followed by 3730XL conventional sequencing reaction, and the whole-sequence genes were sequenced according to the ABI standard procedure. Finally, the sequences were aligned in the NCBI database to determine the classification status of the strain and construct an evolution tree. The strain was identified as Trichosporon FD by ITS sequence analysis. 2.3 Fermentation Sample Preparation (S3) Group: preparation of 107 cfu/mL spore suspension of Trichosporon FD: 40 g of the prepared sun-dried crude tea with moisture of 35% were placed in a 500 mL triangular flask, and sterilized at 115°C for 30min. After being cooled, the crude tea were inoculated with 0.1ml of the spore suspension of Trichosporon FD, and incubated in an artificial climate chamber for 25 days at a temperature of 30°C and a humidity of 80%, with shaking the flask once every 5 days. After the fermentation finished, the tea was taken out and dried at 45°C; (SI) Group: 40 g of the prepared sun-dried crude tea was subjected to natural fermentation (i.e., 35% moisture, without sterilization and bacteria added, incubated in natural environment of preparing sun-dried crude tea of native local varieties in Duyun at a temperature of 30°C and a humidity of 80% for 25 days with shaking once every 5 day. After the fermentation, the teas were taken out and dried at 45°C) as a positive control (SI); 40 g of the prepared sun-dried crude tea was sterilized at 115°C for 30min without adding the bacteria strain as a negative control (S2). 9 pile-fermentation samples were prepared using the three treatments, each with three replicates .

2.4 Fermentation Sample Detection

Water extracts---GB/T 8305-2002, tea polyphenols---GB/T 8313- 2002, amino acids---GB/T 8314-2002, soluble sugars---anthracenone colorimetry, tea pigments---system analysis, color difference---color difference meter, pH value---pH meter; T2, HT2, and 4 aflatoxins---NY/T 2071-2011.

III. Results and Analysis 3.1 Sensory Analysis of pile-Fermentation Samples The three groups of pile-fermentation samples S, S2 and S3 are as shown in FIG. 1. Table 1 is a sensory analysis table of the three groups of pile-fermentation samples S, S2 and S3. Table 2 is the sensory scores, physicochemical indices and safety of the three groups of pile-fermentation samples S1, S2 and S3.

Table 1 Sensory analysis of the pile-fermentation samples Si, S2 and S3 Group Appearance Aromas Soup color Taste Leaf bottom

Si black brown pure relatively red relatively relatively dark

brown mellow brown

S2 black brown fragrant yellow mellow yellow brown

S3 uniform black fragrant and red brown pretty mellow dark brown brown pure

Table 2 The sensory scores, physicochemical indices and safety of the pile

fermentation samples Sl, S2 and S3

Sample Index S1 S2 S3 sensory score 67.58 46.28 75.23 water extracts% 47.06 49.91 51.43 soluble sugars% 3.98 3.80 4.21 tea polysaccharides% 2.80 2.69 2.98 caffeine% 4.13 4.38 4.01 catechins% 6.83 15.67 7.39 theaflavins% 0.07 0.08 0.13 thearubigins% 0.14 4.01 0.12 theabrownins% 5.91 2.02 10.01 flavonoids% 1.18 1.58 1.37 tea polyphenols% 10.48 27.73 9.81 aa% 1.44 2.15 1.80 L 12.51 29.47 14.14 a 27.48 18.00 20.82 b 5.48 1.43 7.13 pH 5.7 4.4 5.1 T2, HT2 and 4 aflatoxins 0 0 0

S3 showed the best treatment effects. S3 samples were obtained by isolating and purifying the dominant strain FD of Pu'er tea and inoculating them into the sun-dried green crude tea prepared from the native local varieties in Duyun, followed by pile fermentation. At this time, the water extracts were 51.43%, the soluble sugars were 4.21%, the tea polysaccharides were 2.98%, the caffeine was 4.01%, the catechins were 7.39%, theaflavins were 0.13%, thearubigins were 0.12%, theabrownins were 10.01%, the flavonoids were 1.37%, and the tea polyphenols were 9.81%. 3.2 Toxicological Analysis of Pile-Fermentation Samples 3.2.1 Standard curve preparation (1) Optimization of Instrument Analysis Conditions Under optimized instrument conditions, extraction ion chromatograms of compound standard solutions of T2, HT2 and 4 aflatoxins (10 g/L) are shown in FIG. 2; (2) Linear relationship of detection methods, detection limits and quantification limits The instrument limit of quantitation (ILOQ) of the six targets was between 0.1 ig/L and 0.5 g/L, and the method limit of quantitation (MLOQ) was between 0.5 pg/L and 2.5 pg/L, calculated as S/N = 10 (see Table 3), indicating a high sensitivity ofthe method.

Table 3 Correlation coefficients of various standard curves, instrument detection limits and method quantification limit Analyte Linear equation R2 ILOQ ( pg/L) MLOQ (pg/L) AFB1 y=1.19e+0.05x+-6.1le+0.05 0.9973 0.5 2.5 AFB2 y=9.32e+0.04x+-5.lle+0.05 0.997 0.2 1.0 AFG1 y=2.47e+0.05+-1.08e+0.06 0.9933 0.1 0.5 AFG2 y=6.99e+0.04x+-3.31e+0.05 0.9962 0.2 1.0 HT2 y=1.67e+0.03x+-536 0.9917 0.2 1.0 T2 y=5.46e+0.03x+-1.82e+0.04 0.9951 0.5 2.5

3.2.2 Detection of Pile-Fermentation Samples The nine fermentation samples were detected according to the above method. None of them was observed to be positive, and the total ion current of the nine fermentation samples was plotted in FIG. 3.

IV. Discussion and Conclusion 4.1 Safety Analysis of Pile-Fermentation Samples of Duyun Dark tea The Duyun dark tea prepared by pile-fermentation has basic characteristics of red brown in color, brown red in soup color, thick taste and remarkable fragrance, which are the results of the enzymatic action of microorganisms and damp heat action, causing a series of reactions such as oxidation, degradation and condensation to the contents of the tea leaves. The safety of Duyun dark tea during the pile-fermentation is of concerns. The results of Mogensen J.M. et al were similar, namely the mycotoxins ochratoxin A, Fumonisins B2 and B4 were not detected in the Duyun dark tea, thereby confirming the safety of the Duyun dark tea. Nine fermentation samples were detected by ultra-high performance liquid chromatography-mass spectrometry/mass spectrometry detection, none of which were positive samples. The results showed that the dominant strain Trichosporon, when singly subjected to fermentation under controlled temperature and humidity (30°C, %), produced no T2, HT2 and 4 aflatoxins. Moreover, the natural strains, when subjected to fermentation under controlled temperature and humidity (30°C and 80%), produced no T2, HT2 and 4 aflatoxins either. 4.2 Conclusion The pile-fermentation Duyun dark tea is post-fermented tea. It is prepared from the sun-dried green crude tea made by the native local varieties in Duyun by pile

fermentation. Piling under humidification condition is a key process for forming the quality of the dark tea. Microbial fermentation plays a decisive role in the quality formation of the Duyun dark tea and is essential for the quality formation of the Duyun dark tea. The dominant strain FD of Pu'er tea of this study was obtained by streaking and separating with gradient concentration of aged Pu'er tea as a raw material, and was found to be Trichosporon by analyzing ITS sequences. The sun-dried crude tea of the native local varieties in Duyun as raw material was subjected to pile fermentation to obtain a pile-fermentation sample by using the above isolated FD and controlling temperature, humidity, moisture and fermentation time. The T2, HT2 and 4 aflatoxins in the pile fermentation samples were detected using an ultra-high performance liquid chromatography-mass spectrometry/mass spectrometry detection method. The results showed that the safety of the dominant strain Trichosporon was relatively high, when the Duyun dark tea was subjected to pile fermentation under the conditions of 30°C and 80% humidity. That is, S3 showed the best treatment effects. S3 samples were obtained by isolating and purifying the dominant strain FD of the Duyun dark tea and inoculating them into the sun-dried green crude tea prepared from the native local varieties in Duyun, followed by pile fermentation, and exhibited the best qualities. In summary, the processing method of the invention is stable, can effectively improve the quality of the Duyun dark tea, and improve the aromas and the mouthfeel of the Duyun dark tea. The Duyun dark teas produced by the method of the invention are rich in nutrient components, stable in tea leaf quality, good in color and luster, and free of T2, HT2 and four aflatoxins. The tea leaves of the invention are uniform and black brown in appearance, pure in fragrance, red brown in soup color, mellow in taste and dark in leaf bottom.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows photographs of appearance, leaf bottoms and soup colors of three groups of pile fermentation samples in the experimental examples; FIG. 2 shows MRM chromatograms of T2, HT2 and 4 aflatoxin compound standard solutions in the experimental examples; FIG.3 shows a graph of total ion current of T2, HT2 and 4 aflatoxins. (Note: Since the drawings provided in the patent application document can not be colored, FIG. 1 does not reflect the characteristics such as soup color, and the color picture can be provided if the examiner needs to do so.)

DETAILED EMBODIMENTS The present invention will be further described in combination with the following examples, but should not be construed as a limitation to the invention.

Example 1. A controllable method for processing Duyun dark tea, which comprises the following steps: (a) picking fresh leaves: picking fresh leaves (three or four leaves on one bud) of native local varieties in Duyun ; (b) spreading: spreading the fresh leaves indoors at room temperature of 25°C for 3 hours to make them lose water to 70%;

(c) fixation: putting leaves of 2.5kg in a Cylinder type fixation machine at 200°C until the green odor disappears, the tea aromas is exposed, the leaf color changes from fresh green to dark green, and the leaves are sticky and conglomerate when pinched; (d) rolling: rolling for 25 min while hot, lightly pressing for 5min, moderately pressing 10min and lightly pressing for 5min; (e) drying: deblocking and drying for 5 h at room temperature of 25°C in the sun to form a sun smell, light rolling for 5 min, and drying at 40°C to obtain a sun-dried green crude tea; (f) sterilizing: sterilizing the sun-dried green crude teas with moisture of 35% at 115°C for 30 min, and cooling for further use; (g) preparing bacteria: Trichosporon FD are plated onto PDA slant culture medium and cultured at 28°C for 6 days. After the strains are mature, the spores are washed off with 0.9% normal saline and transferred into a triangular conical flask filled with glass beads. The spores are evenly distributed in the suspension by shaking sufficiently, and filtered through defatted cotton to obtain a single spore suspension. A coverslip is placed on a dry and clean blood count plate, and 1 drop of the spore suspension is added to the edge of the coverslip, and allowed to penetrate into the counting chamber by capillary action. When the spores sink to the bottom of the plate, they are counted and adjusted to the spore concentration of 107 cfu/mL; (h) inoculating: singly inoculating at a ratio of 40 g of sterilized tea leaves: 0.1 mL of FD spore suspension; (i) piling: the inoculated tea leaves are subjected to pile fermentation for 25 days at a temperature of 30°C and a humidity of 80%; (j) drying: taking out the fermented tea leaves, and drying at 45°C to obtain the Duyun dark tea.

Example 2. A controllable method for processing Duyun dark tea which comprises the following steps: (a) picking fresh leaves: picking fresh leaves (three or four leaves on one bud) of native local varieties in Duyun; (b) spreading: spreading the fresh leaves indoors for 1.5 hours at room temperature of 20°C to make them lose water to 64%; (c) fixation: putting leaves of 2kg in a Cylinder type fixation machine at 180°C until the green odor disappears, the tea aromas is exposed, the leaf color changes from fresh green to dark green, and the leaves are sticky and conglomerate when pinched; (d) rolling: rolling for 20 min while hot, lightly pressing for one fourth of the time, moderately pressing for one half of the time and lightly pressing for one fourth of the time; (e) drying: deblocking and drying for 5 h at room temperature of 25°C in the sun to form a sun smell, light rolling for 5 min, and drying at 40°C to obtain a sun-dried green crude tea; (f) sterilizing: sterilizing the sun-dried green crude tea with moisture of 35% at 115°C for 30 min, and cooling for further use; (g) preparing bacteria: Trichosporon FD are plated onto PDA slant culture medium and cultured at 28°C for 5 days. After the strains are mature, the spores are washed off with 0.9% normal saline and transferred into a triangular conical flask filled with glass beads. The spores are evenly distributed in the suspension by shaking sufficiently, and filtered through defatted cotton to obtain a single spore suspension. A coverslip is placed on a dry and clean blood count plate, and 1 drop of the spore suspension is added to the edge of the coverslip, and is allowed to penetrate into the counting chamber by capillary action. When the spores sink to the bottom of the plate, they are counted and adjusted to the spore concentration of 107 cfu/mL; (h) inoculating: singly inoculating at a ratio of 40 g of sterilized tea leaves: 0.1 mL of FD spore suspension; (i) piling: the inoculated tea leaves are subjected to pile fermentation for 25 days at a temperature of 30°C and a humidity of 80%; (j) drying: taking out the fermented tea leaves, and drying at 45°C to obtain the Duyun dark tea.

Example 3. A controllable method for processing Duyun dark tea which comprises the following steps: (a) picking fresh leaves: picking fresh leaves (three or four leaves on one bud) of native local varieties in Duyun; (b) spreading: spreading the fresh leaves indoors for 4 hours at room temperature of 30°C to make them lose water to 78%; (c) fixation: putting leaves of 3kg in a Cylinder type fixation machine at 220°C until the green odor disappears, the tea aromas is exposed, the leaf color changes from fresh green to dark green, and the leaves are sticky and conglomerate when pinched; (d) rolling: rolling for 30 min while hot, lightly pressing for one fourth of the time, moderately pressing for one half of the time and lightly pressing for one fourth of the time; (e) drying: deblocking and drying for 5 h at room temperature of 25°C in the sun to form a sun smell, light rolling for 5 min, and drying at 40°C to obtain a sun-dried green crude tea; (f) sterilizing: sterilizing the sun-dried green crude teas with moisture of 35% at 115°C for 30 min, and cooling for further use; (g) preparing bacteria: Trichosporon FD are plated onto PDA slant culture medium and cultured at 28°C for 7 days. After the strains are mature, the spores are washed off with 0.9% normal saline and transferred into a triangular conical flask filled with glass beads. The spores are evenly distributed in the suspension by shaking sufficiently, and filtered through defatted cotton to obtain a single spore suspension. A coverslip is placed on a dry and clean blood count plate, and 1 drop of the spore suspension is added to the edge of the coverslip, and allowed to penetrate into the counting chamber by capillary action. When the spores sink to the bottom of the plate, they are counted and adjusted to the spore concentration of 107 cfu/mL; (h) inoculating: singly inoculating at a ratio of 40 g of sterilized tea leaves: 0.1 mL of FD spore suspension; (i) piling: the inoculated tea leaves are subjected to pile fermentation for 25 days at a temperature of 30°C and a humidity of 80%; (j) drying: taking out the fermented tea leaves, and drying at 45°C to obtain the Duyun dark tea.

Claims

What is claimed is: 1. A controllable method for processing Duyun dark tea comprising: (a) picking fresh leaves: picking fresh leaves of native local varieties in Duyun; (b) spreading: spreading the fresh leaves indoors for 1-4 hours at room temperature of 20°C-30°C to make them lose water to 60-80%; (c) fixation: putting leaves of 2-3 kg in a Cylinder type fixation machine at 180-220 0C until the green odor disappears, the tea aromas is exposed, the leaf color changes from fresh green to dark green, and the leaves are sticky and conglomerate when pinched;

(d) rolling: rolling for 20-30 min while hot, lightly pressing for one fourth of the time, moderately pressing for one half of the time and lightly pressing for one fourth of the time; (e) drying: deblocking and drying for 5 h at room temperature of 25 0 C in the sun to form a sun smell, light rolling for 5 min, and drying at 400 C to obtain a sun-dried green crude tea; (f) sterilizing: sterilizing the sun-dried green crude teas with moisture of 35% at 115 0C for 30 min, and cooling for further use; (g) preparing bacteria: preparing the spore suspension of Trichosporon FD; (h) inoculating: singly inoculating at a ratio of 40 g of sterilized tea leaves: 0.1 mL of FD spore suspension; (i) piling: the inoculated tea leaves are subjected to pile fermentation for 25 days at a temperature of 30 0 C and a humidity of 80%; 0 (j) drying: taking out the fermented tea leaves, and drying at 45 C to obtain the Duyun dark tea.
2. The method of claim 1, wherein the spreading in the step (b) comprises spreading the fresh leaves indoors for 3 hours at room temperature of 250 C until the water loss is up to 70%.
3. The method of claim 1, wherein the fixation in the step (c) is performed with a leaf amount of 2.5 kg at 2000 C using a Cylinder type water-removing machine.
4. The method of claim 1, wherein the spore suspension of Trichosporon FD in the step (g) is prepared by plating Trichosporon FD onto PDA slant culture medium and culturing at 20-40 0 C for 3-9 days; washing off the spores with 0.3-1.5% normal saline after the strains are mature, and transferring them into a triangular conical flask filled with glass beads; shaking sufficiently to allow the spores evenly distribute in the suspension, and filtering through defatted cotton to obtain a single spore suspension; placing a coverslip on a dry and clean blood count plate, adding 1 drop of the spore suspension to the edge of the coverslip to allow it to penetrate into the counting chamber by capillary action; and counting and adjusting the spore concentration to 107 cfu/mL when the spores sink to the bottom of the plate.
5. The method of claim 4, wherein the spore suspension of Trichosporon FD in the step (g) is prepared by plating Trichosporon FD onto PDA slant culture medium and culturing at 28°C for 5-7 days; washing off the spores with 0.9% normal saline after the strains are mature, and transferring them into a triangular conical flask filled with glass beads; shaking sufficiently to allow the spores evenly distribute in the suspension,and filtering through defatted cotton to obtain a single spore suspension; placing a coverslip on a dry and clean blood count plate, adding 1 drop of the spore suspension to the edge of the coverslip to allow it to penetrate into the counting chamber by capillary action; and counting and adjusting the spore concentration to 107 cfu/mL when the spores sink to the bottom of the plate.