Method for preparing solid-state fermented loose black tea through mixed bacteria co-fermentation and prepared solid-state fermented loose black tea

Abstract

The invention discloses a method for preparing solid-state fermented loose dark tea through mixed bacterium co-fermentation and the solid-state fermented loose dark tea prepared thereof.The method for preparing the solid-state fermented loose dark tea through mixed bacterium co-fermentation comprises the following steps that 1, water is added into a tea leaf raw material, then high-temperature sterilization is conducted, and sterilized tea is obtained; (2) adding a compound bacterium suspension into the sterilized tea, uniformly mixing, and culturing and fermenting to obtain mixed bacterium fermented loose black tea; the compound bacterium suspension is prepared from aspergillus niger spore liquid and bacillus subtilis bacterium suspension; and (3) drying the mixed fermentation loose dark green tea to obtain the solid fermentation loose dark green tea. According to the invention, the aspergillus niger strain and the bacillus subtilis strain are mixed and fermented to prepare the solid-state fermented loose black tea, so that the theabrownin content and the antioxidant activity of the fermented tea can be improved. The fungus and bacteria co-fermentation can synergistically improve the growth speed of microorganisms, can shorten the fermentation period, and solves the problems of long pile fermentation time and non-uniform fermentation in the natural environment in the traditional dark tea production.

Description

Technical Field

[0001] The invention relates to the field of microbial fermentation, and in particular to a method for preparing solid-state fermented loose dark tea by mixed bacteria co-fermentation and the prepared solid-state fermented loose dark tea. Background Art

[0002] The growth, metabolism and succession of microbial communities during the dark tea processing ultimately form the unique physical and chemical composition, sensory quality and biological efficacy of dark tea. Studies have shown that the main dominant microorganisms in dark tea are Aspergillus niger, Penicillium, yeast and Bacillus. The fermentation metabolites of functional microorganism Aspergillus niger are diverse, including organic acids, vitamins, pesticides, valuable proteases and therapeutic agents, etc., which have potential applications in various fields such as agriculture, food industry and medicine.

[0003] Chinese patent CN116076589A discloses a method for efficiently producing Pu'er cooked tea by sequentially controlling fermentation, wherein Aspergillus oryzae is first inoculated and then Aspergillus niger is inoculated, and sequential fermentation significantly improves the quality of Pu'er cooked tea, and effectively shortens the fermentation cycle and improves production efficiency. Chinese patent CN115044485A discloses a Pu'er tea fermentation koji and a preparation method thereof, wherein dominant microorganisms such as Aspergillus niger, Penicillium and yeast are provided during the fermentation of Pu'er tea, thereby inhibiting the growth of miscellaneous bacteria during the fermentation process, and the fermented Pu'er tea has good quality. Chinese patent CN114128779A discloses a mixed fungus liquid fermented tea and its preparation method. Inoculating Eurotium rostratum and Aspergillus niger into the liquid fermentation matrix can effectively improve the bitterness and astringency of summer and autumn tea and black tea, realize the high-value utilization of Fucha, adjust and enrich the aroma, effectively improve the senses, realize the prevention and protection of alcoholic liver damage, and deeply control the fermentation process, control the metabolic components and the microenvironment, and propose the optimal process parameters for summer and autumn tea water extract, black tea water extract and Fucha water extract. Chinese patent CN118406617A discloses the application of a microbial composition in the fermentation extraction of Schisandra chinensis effective ingredients, Aspergillus niger and Bacillus subtilis, which can effectively improve the lignan, flavonoid and polysaccharide yields of the extract, and improve the antioxidant activity of flavonoid and polysaccharide components. However, there is no application of Aspergillus niger and Bacillus subtilis on the aroma of tea in the existing invention patents. Summary of the invention

[0004] The main purpose of the present invention is to provide a method for preparing solid-state fermented loose dark tea by mixed bacteria co-fermentation, which can shorten the fermentation time and improve the quality of fermented tea, and the solid-state fermented loose dark tea prepared by the method.

[0005] To achieve the above object, the present invention provides a method for preparing solid-state fermented loose dark tea by mixed bacteria co-fermentation, comprising the following steps:

[0006] (1) Sterilization

[0007] Adding water to the tea raw material, and then sterilizing it at high temperature to obtain sterilized tea;

[0008] (2) Fermentation

[0009] Adding a composite bacterial suspension to the sterilized tea, mixing evenly and then culturing and fermenting to obtain mixed bacterial fermented loose black tea; the composite bacterial suspension is composed of Aspergillus niger spore liquid and Bacillus subtilis suspension;

[0010] (3) Drying

[0011] The mixed bacteria fermented loose dark tea is dried to obtain the solid-state fermented loose dark tea.

[0012] Furthermore, in step (1), the tea raw material is any one of summer and autumn green tea, black tea, oolong tea, white tea and yellow tea.

[0013] Furthermore, in step (2), the Aspergillus niger spore liquid is obtained by culturing an Aspergillus niger strain deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241727.

[0014] The Aspergillus niger strain AN20240729, classified and named Aspergillus niger, is deposited in the China Center for Type Culture Collection (CCTCC), with a deposit number of CCTCCNO: M 20241727 and a deposit date of August 2, 2024. The colony morphology of the Aspergillus niger strain CCTCC NO: M 20241727 is characterized by a colony of 20 to 30 mm in length, in a concentric ring pattern, black on the front, white secondary hyphae on the edge, and gray-black on the back. The Aspergillus niger strain CCTCC NO: M 20241727 was isolated from the finished Ya'an Tibetan tea and sent to Shanghai Shenggong Biological Co., Ltd. for strain identification, and the identification result was Aspergillus niger.

[0015] Furthermore, in step (2), the Bacillus subtilis suspension is obtained by culturing a Bacillus subtilis strain deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241726.

[0016] The Bacillus subtilis strain BS20240729, classified as Bacillus subtilis, was sent to the "China Center for Type Culture Collection" for preservation on August 2, 2024 and showed survival. The preservation number is CCTCC NO: M 20241726. The preservation address is: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The colony morphology is characterized by: the colony is between 2 and 5 mm, grayish white or slightly yellow, and the surface is typically rough and irregular, with many ridges and wrinkles; the colony surface is opaque and rough. Bacillus subtilis strain CCTCC NO: M20241726 was isolated from the finished Ya'an Tibetan tea and sent to Shanghai Shenggong Biological Co., Ltd. for strain identification, and the identification result was Bacillus subtilis.

[0017] Furthermore, in step (2), the concentrations of the Aspergillus niger spore solution and the Bacillus subtilis suspension are both 1×10 6 The Aspergillus niger spore liquid and the Bacillus subtilis suspension are configured into a composite bacterial suspension in a volume ratio of 1:1, and 1 ml of the composite bacterial suspension is used for every 38 g of tea raw material.

[0018] Furthermore, in step (1), the ratio of tea material to water is 19 g:6 ml.

[0019] Furthermore, in step (1), the high temperature sterilization conditions are a temperature of 121° C. and a time of 10 min.

[0020] Furthermore, in step (2), the culture and fermentation conditions are a temperature of 22 to 35° C., a humidity of 70%, and a time of 2 to 8 days.

[0021] Furthermore, in step (3), the drying method is freeze drying, hot air drying, heat pump drying or room temperature drying.

[0022] The invention also provides solid-state fermented loose black tea, which is prepared according to the method.

[0023] The beneficial effects of the present invention are embodied in:

[0024] The invention uses Aspergillus niger CCTCC NO: M 20241727 and Bacillus subtilis CCTCC NO: M 20241726 for mixed fermentation to prepare solid-state fermented loose black tea, which can increase the theabrownin content and antioxidant activity of the fermented tea. This mixed fermentation has the effect of accelerating the growth of microorganisms and shortening the fermentation time, and solves the problems of long pile fermentation time and uneven fermentation in the natural environment during the traditional black tea fermentation process.

[0025] The invention can be applied to the solid-state fermentation of low-grade green tea, rough and old tea leaves and broken tea to prepare black tea products with high sweet, floral and woody aromas, thereby improving the economic value of low-grade green tea, rough and old tea leaves and broken tea; the Aspergillus niger and Bacillus subtilis of the invention can improve the safety of the product, the production process is simple, the fermentation is fast and the cost is low, and the product is suitable for industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The morphological images of Aspergillus niger colonies (left), Bacillus subtilis colonies (middle) and mixed colonies (right).

[0027] Figure 2 The following are actual photos of the raw material roasted green tea sample (left) and the solid-state fermented loose black tea sample (right) prepared in Example 3.

[0028] Figure 3 This is a diagram showing the color change of tea soup at different fermentation times according to the present invention.

[0029] Figure 4 This is the phylogenetic tree diagram of Aspergillus niger strain CCTCC NO: M 20241727.

[0030] Figure 5 This is the phylogenetic tree diagram of Bacillus subtilis strain CCTCC NO: M 20241726. DETAILED DESCRIPTION

[0031] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0032] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods; unless otherwise specified, the methods used in the examples of the present invention are methods known to those skilled in the art. Among them:

[0033] Roasted green tea was purchased from Huangshan Shuigong Tea Co., Ltd.

[0034] Common Aspergillus niger No. 1 is an Aspergillus niger strain deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with the deposit number CGMCC No. 11276 (disclosed in patent application 201610662623.4).

[0035] Common Bacillus subtilis No. 1 is a Bacillus subtilis strain deposited in the China Center for Type Culture Collection with a deposit number of CCTCC M2023165 (disclosed in patent application 202310287445.1).

[0036] Example 1

[0037] Preparation of Aspergillus niger spore solution

[0038] The Aspergillus niger strain CCTCC NO: M 20241727 used in this example was isolated from the finished product of Ya'an Tibetan tea and sent to Shanghai Shenggong Biological Co., Ltd. for strain identification. Its ITS gene sequence is shown in SEQ ID NO.1. The sequencing results were compared with the NCBI database, and the phylogenetic tree constructed is shown in Figure 4 As shown; combined with the strain morphology and ITS gene sequence analysis, it was preliminarily determined to be Aspergillus niger.

[0039] The preparation process of spore solution is as follows:

[0040] (1) Preparation of activation culture medium: Take 200 g of peeled potatoes, cut them into pieces and cook them for 20 min, then filter them with gauze to collect the filtrate, then add 20 g of agar powder to the filtrate at 60°C, stir until the agar powder is dissolved, add 20 g of glucose, stir until dissolved, add purified water to make the volume 1000 ml, sterilize in a high-temperature sterilizer at 121°C for 20 min, cool to 50°C, and pour into a plate;

[0041] (2) Activation of the strain: In a sterile operating environment, the Aspergillus niger strain with a deposit number of CCTCC NO: M20241727 was streaked onto the plate containing the culture medium obtained in step (1), and then the plate was inverted in a constant temperature incubator at 37° C. and cultured for 4 days;

[0042] like Figure 1 As shown in the figure, the morphological characteristics of Aspergillus niger colonies are as follows: the colonies are between 20 and 30 mm in size, in the shape of concentric rings, the front of the colonies are black, white secondary hyphae are produced on the edges, and the back is gray-black;

[0043] (3) Spore collection: Scrape the Aspergillus niger spores from the culture medium, dissolve them in 100 ml of water, place them on a shaker and mix them evenly, then adjust the spore concentration to 1×10 6 / ml to obtain Aspergillus niger spore liquid.

[0044] Example 2

[0045] Preparation of Bacillus subtilis suspension

[0046] The Bacillus subtilis strain CCTCC NO: M 20241726 used in this example was isolated from the finished product of Ya'an Tibetan tea and sent to Shanghai Shenggong Biological Co., Ltd. for strain identification. Its 16S rDNA gene sequence is shown in SEQ ID NO.2. The sequencing results were compared with the NCBI database, and the phylogenetic tree constructed is shown in Figure 5As shown; combined with the morphology and 16S rRNA gene sequence analysis of strain BS20240729, it was preliminarily determined to be Bacillus subtilis.

[0047] (1) Preparation of activation culture medium: Take 200 g of peeled potatoes, cut them into pieces and cook them for 20 min, then filter them with gauze to collect the filtrate, then add 20 g of agar powder to the filtrate at 60°C, stir until the agar powder is dissolved, add 20 g of glucose, stir until dissolved, add purified water to make the volume 1000 ml, sterilize in a high-temperature sterilizer at 121°C for 20 min, cool to 50°C, and pour into a plate;

[0048] (2) Activation of the strain: In a sterile operating environment, the Bacillus subtilis strain with the deposit number of CCTCC NO: M20241726 was streaked onto the plate containing the culture medium obtained in step (1), and then the plate was inverted in a constant temperature incubator at 37° C. and cultured for 2 days;

[0049] like Figure 1 As shown in the figure, the morphological characteristics of Bacillus subtilis colonies are as follows: the colonies are between 2 and 5 mm, grayish white or slightly yellowish, and the surface is typically rough and irregular, with many ridges and wrinkles; the surface of the colonies is opaque and rough;

[0050] (3) Bacteria collection: scrape the Bacillus subtilis cells on the culture medium, dissolve them in 100 ml of water, place them on a shaker and mix them evenly, then adjust the cell concentration to 1×10 6 / ml to obtain Bacillus subtilis suspension.

[0051] According to the method of Example 1, the Aspergillus niger strain with a preservation number of CCTCC NO: M 20241727 and the Bacillus subtilis strain with a preservation number of CCTCC NO: M 20241726 were streaked and inoculated at the same time, and the mixed colonies obtained by culture were as follows: Figure 1 shown.

[0052] Example 3

[0053] Preparation of solid-state fermented loose dark tea by mixed bacteria co-fermentation

[0054] (1) Sterilization: Take 38 g of roasted green tea, add 12 ml of purified water, mix well, put into a sealed conical bottle, and sterilize in a high-temperature sterilizer at 121° C. for 10 min to obtain a sterilized green tea raw material;

[0055] (2) Fermentation: The Aspergillus niger spore liquid prepared in Example 1 and the Bacillus subtilis suspension prepared in Example 2 were mixed in a volume ratio of 1:1 to prepare a composite bacterial suspension. In a sterile operating environment, 1 ml of the composite bacterial suspension was inoculated into the sterilized green tea raw material, mixed evenly, and placed in an environment with a temperature of 28° C. and a humidity of 70% for fermentation for 72 hours to obtain mixed bacterial fermented loose black tea;

[0056] (3) Freeze-drying: freeze-dry the mixed bacteria fermented loose dark tea at a vacuum degree of 5 Pa and a temperature of -50°C for 3 days to obtain the solid-state fermented loose dark tea.

[0057] Experimental Example 1

[0058] Detection of aroma components of solid-state fermented loose black tea

[0059] On the basis of Example 3, the composite bacterial suspension was replaced with the Aspergillus niger spore liquid prepared in Example 1. As Comparative Example 1, on the basis of Example 3, the composite bacterial suspension was replaced with the Bacillus subtilis suspension prepared in Example 2. As Comparative Example 2, on the basis of Example 3, the Aspergillus niger spore liquid in the composite bacterial suspension was replaced with the spore liquid of common Aspergillus niger No. 1 (prepared according to the method of Example 1). As Comparative Example 3, on the basis of Example 3, the Bacillus subtilis suspension in the composite bacterial suspension was replaced with the bacterial suspension of common Bacillus subtilis No. 1 (prepared according to the method of Example 2). As Comparative Example 4, the aroma components of the roasted green tea raw material, the solid-state fermented loose black tea prepared in Example 3 and Comparative Examples 1 to 4 were detected, and the detection method was as follows:

[0060] SPME extraction: Weigh 10μl of ethyl caprate, dilute to 10ml with ethanol and make up to volume (prepare a concentration of 1000ppm as the internal standard mother liquor), and store at -20℃ for later use. Dilute the mother liquor to 5ppm when using. Weigh a certain amount of tea sample to be tested, brew the tea sample to be tested in boiling water at a ratio of 1:20 for 5 minutes, filter the tea soup, and quickly cool it to room temperature in an ice water bath. Measure 10ml of filtered tea soup into a headspace bottle, add 3.3g of sodium chloride and 5μl of internal standard, and shake well. Place the headspace bottle in a 40℃ water bath, balance for 15 minutes, insert the extraction needle into the headspace bottle, push out the extraction fiber head, and let it adsorb 1cm from the tea soup surface for 30 minutes.

[0061] GC-MS analysis: injection port temperature 250°C, chromatographic column DB-5MS (30m×0.25mm, film thickness 0.25μm), carrier gas is 99.999% pure helium, average linear velocity 36cm / sec. Ion source is EI, temperature is set to 230°C. Heating program: initial temperature is 40°C, maintained for 3min; increased to 80°C at a rate of 2°C / min; increased to 140°C at a rate of 3°C / min; increased to 250°C at a rate of 14°C / min, maintained for 2min.

[0062] The test results are as follows: A total of 156 volatile compounds were identified by HS-SPME. Volatile compounds were divided into 7 categories according to different chemical structures, namely 45 higher alcohols, 24 ketones, 21 esters, 21 hydrocarbons, 18 aldehydes, 17 organic heterocyclic compounds, and 11 olefins. Among them, the number of alcohols, ketones and esters identified accounted for more than 70% of the total number of identified compounds, and they are the main components of volatile compounds in fermented tea. Ester compounds are an important class of tea aroma compounds and are crucial in shaping the aroma and flavor of tea. There are 12 ester compounds in mixed fermented tea and the aroma compound content is relatively high, reaching 52.42 mg / L. The aroma compound content in the fermentation raw material is only 20.67 mg / L. In order to further reveal the influence of volatile compounds in loose black tea fermented by different microorganisms, 38 key volatile compounds were visualized using heat maps. It is generally believed that substances with VIP values ​​greater than 1 and P less than 0.05 contribute to the differences in samples. Table 1 shows the 25 aroma substances with relatively large aroma contributions. And 18 aroma compounds with rOAV greater than 1 were screened, including geraniol, β-pinene, naphthalene, α-terpineol, citral, β-myrcene, valeraldehyde, linalool, 3-carene, 4-terpene alcohol, lily aldehyde, phenylacetaldehyde, L-β-pinene, cis-oxidized linalool, nerol, and geranylacetone; through aroma sensory QDA, it was found that the intensity of floral, woody and sweet aromas of CF samples increased significantly, and further orthogonal partial least squares discriminant analysis was used to believe that the floral, woody and sweet aroma attributes in fermented black tea were related to several alcohols and terpene compounds such as phenylacetaldehyde, α-terpineol, nerol, geraniol, naphthalene and 3-carene.

[0063] Table 1

[0064]

[0065]

[0066] Detection of theabrownin content and antioxidant activity of solid-state fermented loose dark tea

[0067] The theabrownin content and antioxidant activity of the solid-state fermented loose dark tea obtained in Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4 were tested.

[0068] The theabrownin content detection method is as follows:

[0069] (1) Test solution: Add 3 g of tea powder into 125 ml of boiling water for 10 min, and filter to obtain the test solution.

[0070] (2) 25 ml of the test solution plus 25 ml of n-butanol were placed in a 1000 ml funnel and shaken for 3 min. After standing, 2 ml of the lower layer solution was taken out and placed in a 25 ml volumetric flask. 2 ml of saturated acetic acid and 6 ml of water were added to the 25 ml volumetric flask and then the volume was fixed with 95% ethanol.

[0071] (3) Measure the absorbance at 380 nm.

[0072] Theabrownin (%)=7.06*2A / w*m*100

[0073] w: dry matter content of the sample; m: mass of the sample; A: absorbance

[0074] The antioxidant activity test method is as follows:

[0075] Mix 2 ml tea soup (6.7 μg / ml), 2 ml DPPH solution and 2 ml ethanol to prepare test solution (T) and control solution (C), respectively. The blank solution (B) is a mixture of DPPH solution (2 mL) and ethanol (2 mL). The reaction solution is reacted at room temperature for 30 minutes, and the absorbance is measured at 517 nm. The DPPH free radical scavenging rate (RD) is shown in the formula:

[0076]

[0077] AT: absorbance of the reaction between sample and DPPH solution; AC: absorbance of the reaction between sample and ethanol solution; AB: absorbance of the reaction between DPPH solution and ethanol solution.

[0078] Mix 1 ml ABTS solution with 0.5 mL tea soup, react in the dark for 10 minutes, and measure the absorbance at 734 nm. Prepare Trolox at concentrations of 0.1 mM, 0.2 mM, 0.4 mM, 0.8 mM, and 1.0 mM to react with ABTS solution, record the absorbance, and make a standard curve of ABTS scavenging ability with absorbance as the horizontal axis and Trolox concentration as the vertical axis. The ABTS scavenging ability of the sample is expressed by the standard concentration of Trolox.

[0079] The test results are as follows: the theabrownin content of the roasted green tea raw material is 73.89, and the antioxidant activity is 87.40. Mixed fermentation accelerates the fermentation rate of microorganisms. The theabrownin content produced by the mixed fermentation of Aspergillus niger strain CCTCC NO: M 20241727 and Bacillus subtilis strain CCTCCNO: M 20241726 is higher than that of the single bacteria fermentation group and the combination of existing strains, and the antioxidant activity is higher than that of the single bacteria fermentation group and the combination of existing strains.

[0080] Table 2

[0081]

[0082] Observation on the color of tea soup of solid-state fermented loose black tea

[0083] On the basis of Example 3, Comparative Example 1 and Comparative Example 2, different fermentation times were set respectively, and the prepared tea was brewed for 5 minutes at a tea-water ratio of 1:20, and the color of the tea soup was observed. The results were as follows: Figure 3 As shown in the figure (from top to bottom, the first row is the result of fermentation of Aspergillus niger alone, the second row is the result of fermentation of Bacillus subtilis alone, and the third row is the result of mixed fermentation of Aspergillus niger and Bacillus subtilis). It can be seen that the color of the tea soup changes significantly after mixed fermentation of Aspergillus niger strain CCTCC NO: M 20241727 and Bacillus subtilis strain CCTCC NO: M 20241726. The mixed fermentation speeds up the fermentation of loose black tea, making the tea soup red and thick.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing solid-state fermented loose dark tea by mixed bacteria co-fermentation, characterized in that: The following steps are involved: (1) Sterilization Adding water to the tea raw material, and then sterilizing it at high temperature to obtain sterilized tea; (2) Fermentation Adding a composite bacterial suspension to the sterilized tea, mixing evenly and then culturing and fermenting to obtain mixed bacterial fermented loose black tea; the composite bacterial suspension is composed of Aspergillus niger spore liquid and Bacillus subtilis suspension; (3) Drying The mixed bacteria fermented loose dark tea is dried to obtain the solid-state fermented loose dark tea.

2. The method for preparing solid-state fermented loose dark tea by mixed bacteria co-fermentation according to claim 1, characterized in that: In step (1), the tea raw material is any one of summer and autumn green tea, black tea, oolong tea, white tea and yellow tea.

3. The method for preparing solid-state fermented loose dark tea by mixed bacteria co-fermentation according to claim 1, characterized in that: In step (2), the Aspergillus niger spore liquid is obtained by culturing an Aspergillus niger strain deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M20241727.

4. The method for preparing solid-state fermented loose dark tea by mixed bacteria co-fermentation according to claim 1, characterized in that: In step (2), the Bacillus subtilis suspension is obtained by culturing a Bacillus subtilis strain deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M20241726.

5. The method for preparing solid-state fermented loose dark tea by mixed bacteria co-fermentation according to any one of claims 1 to 4, characterized in that: In step (2), the concentrations of the Aspergillus niger spore solution and the Bacillus subtilis suspension are both 1×10 6 The Aspergillus niger spore liquid and the Bacillus subtilis suspension are configured into a composite bacterial suspension in a volume ratio of 1:1, and 1 ml of the composite bacterial suspension is used for every 38 g of tea raw material.

6. The method for preparing solid-state fermented loose dark tea by mixed bacteria co-fermentation according to any one of claims 1 to 4, characterized in that: In step (1), the ratio of tea material to water is 19 g: 6 ml.

7. The method for preparing solid-state fermented loose dark tea by mixed bacteria co-fermentation according to any one of claims 1 to 4, characterized in that: In step (1), the high temperature sterilization conditions are a temperature of 121° C. and a time of 10 min.

8. The method for preparing solid-state fermented loose dark tea by mixed bacteria co-fermentation according to any one of claims 1 to 4, characterized in that: In step (2), the culture and fermentation conditions are a temperature of 22 to 35° C., a humidity of 70%, and a time of 2 to 8 days.

9. The method for preparing solid-state fermented loose dark tea by mixed bacteria co-fermentation according to any one of claims 1 to 4, characterized in that: In step (3), the drying method is freeze drying, hot air drying, heat pump drying or room temperature drying.

10. A solid-state fermented loose dark tea, characterized in that: Prepared according to the method according to any one of claims 1 to 9.

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