A kind of sphingomonas and preparation method of fermented black tea thereof

Abstract

The present invention provides a method for preparing sphingomonas and fermented black tea thereof, and belongs to the field of microbial technology. The sphingomonas is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, and the deposit number is CGMCC No.29158; the screened sphingomonas is applied to black tea fermentation, which can enhance the aroma of fermented black tea, utilize the unique metabolic ability of sphingomonas to aromatic compounds, promote the maturation of tea aroma, and improve the taste and flavor of black tea. The sphingomonas of the present invention can effectively increase the sweet and mellow aroma of fermented black tea, improve the quality of fermented black tea, increase the theanine content of black tea, and increase the efficacy of black tea.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a sphingomonas bacterium and an application and a preparation method thereof in fermenting black tea. Background Art

[0002] Black tea is one of the six major types of tea in my country. It is a fully fermented tea that is made through a series of processes including withering, rolling, fermentation and drying. Studies have shown that black tea is rich in functional ingredients such as theanine, theaflavins and thearubigins.

[0003] Theanine is a non-protein free amino acid unique to tea. It is the main substance that forms the aroma and freshness of black tea. It can reduce the bitterness of tea and increase the delicious taste of tea. Studies have shown that theanine has many important physiological functions. It can regulate the concentration of neurotransmitters in the brain and lower blood pressure. It can be used in combination with anti-tumor drugs to enhance the anti-cancer effect of anti-tumor drugs and reduce the side effects of anti-tumor drugs. Theanine can also be used as a moisturizer and nutrient in cosmetics. It can enhance the activity of skin cells, promote the production of collagen, maintain skin elasticity, and can also be used as an anti-wrinkle agent. At the same time, theanine has anti-inflammatory properties and can promote the healing of skin injuries.

[0004] Studies have shown that the use of microorganisms to ferment natural raw materials can produce extracts of fermented products that regulate the microecology of the skin surface and the physiological activity of the skin itself. At the same time, the fermentation method reduces the molecular weight of ingredients in natural raw materials that have good moisturizing, whitening, soothing and anti-aging effects, making them easier to be absorbed by the skin. At the same time, the toxic side effects are greatly reduced through structural modification.

[0005] The current technology of using microorganisms to ferment black tea has certain limitations in improving the effective ingredients such as theanine, and the quality of the fermented black tea, such as the smell, is still insufficient. Therefore, it is necessary to study more new microorganisms to improve the quality of black tea.

[0006] Through searching, there is no published literature report on using Sphingomonas to improve the quality of black tea in black tea production. Summary of the invention

[0007] In view of the above problems existing in the prior art, the present invention provides a method for preparing black tea fermented by sphingomonas and the same. The sphingomonas of the present invention can effectively increase the sweet and mellow aroma of fermented black tea and improve the quality of fermented black tea; after fermentation by the bacteria, the theanine content of black tea can be increased, thereby increasing the efficacy of black tea.

[0008] The technical solution of the present invention is as follows:

[0009] The present invention first provides a sphingomonas (Sphingobuim sp.), which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on November 27, 2023, with a deposit number of CGMCC No. 29158. The deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The present invention names the sphingomonas as γ-tea fermentation bacteria.

[0010] The present invention also provides a method for preparing the black tea fermented by Sphingomonas, which comprises the following steps:

[0011] S1. Preparing a sphingomonas seed solution: culturing the sphingomonas into a seed solution;

[0012] S2, withering fresh tea leaves: withering fresh tea leaves by natural withering indoors or by sunlight combined withering to obtain withered tea leaves;

[0013] S3, rolling: spraying 1-5wt% of mixed enzyme solution on the withered tea leaves, fermenting at low temperature, standing for 10-50min, and then sending them to a rolling machine for rolling to obtain rolled leaves;

[0014] S4, fermentation: adding 1-2 wt% of Sphingomonas to the rolled leaves for low temperature fermentation;

[0015] S5, drying: after the fermentation is completed, drying is performed to obtain the fermented black tea;

[0016] S6. Microwave sterilization: sending the dried fermented black tea leaves into a microwave machine for microwave sterilization.

[0017] Preferably, the method for culturing the seed solution in step S1 is: inoculating Sphingomonas in a test tube containing TPG seed culture medium, and shaking and culturing at 28-32°C for 18-20 hours to prepare primary seeds; then inoculating the primary seeds in TPG seed culture medium with an inoculation amount of 5-10%, and shaking and culturing for 16-20 hours to obtain Sphingomonas seed solution.

[0018] More preferably, the formula of the TPG seed culture medium is: sucrose 20 g / L, peptone 2.5 g / L, potassium dihydrogen phosphate 5 g / L, potassium sulfate 0.6 g / L, disodium hydrogen phosphate 1.5 g / L, calcium carbonate 0.1 g / L, yeast powder 2 g / L, and the pH is adjusted to 7.0-7.4.

[0019] Preferably, the moisture content of the withered tea leaves obtained in step S2 is 55%-65%.

[0020] Preferably, the mixed enzyme solution in step S3 consists of cellulase solution and aminopeptidase solution, the mass fractions of the cellulase solution and the aminopeptidase solution are 4-10% respectively; wherein the mass ratio of the cellulase solution to the aminopeptidase solution is 3-6:1, more preferably 4:1.

[0021] More preferably, the kneading speed in step S3 is 28-35 rpm / min, and the time is 38-50 min.

[0022] Preferably, the temperature of the low-temperature fermentation in step S4 is 28-30° C., the air humidity is 80-85%, and the fermentation time is 3-8 hours.

[0023] Preferably, the moisture content of the fermented black tea obtained after drying in step S5 is 5-10%.

[0024] Preferably, the sterilization temperature in step S6 is 90-105° C. and the time is 6-10 min.

[0025] The beneficial technical effects of the present invention are:

[0026] 1. The inventors found in their research that applying the selected Sphingomonas to black tea fermentation can enhance the aroma of fermented black tea, utilize the unique metabolic ability of Sphingomonas to aromatic compounds, promote the maturation of tea aroma, and improve the taste and flavor of black tea.

[0027] 2. When making fermented black tea, the present invention adds cellulase and aminopeptidase to increase the cell damage rate, significantly shorten the fermentation time, promote the production of theanine, enhance the color and aroma of the tea soup, and effectively improve the nutrients of the black tea.

[0028] 3. The process flow created by the present invention is simple, easy to operate and suitable for industrial production. DETAILED DESCRIPTION

[0029] The present invention is described in detail below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1: Screening and identification method of Sphingomonas

[0031] (1) Strain screening

[0032] First, add 10g of Keemun black tea to 90g of sterile water, shake and culture at 30℃ for 1h, filter to obtain the black tea extract, dilute the extract and spread it on improved Gao's No. 1 medium, culture it aerobically at 30℃ for 24h, pick the dominant bacteria on the plate, and perform three-zone purification culture on the dominant bacteria three times to obtain purified bacteria.

[0033] (2) Strain identification

[0034] Colony morphology: The colonies of the dominant strains are round, bright yellow, opaque, moist and smooth on the surface, without wrinkles, and about 1-2 mm in diameter.

[0035] The genomic DNA of the dominant strain was extracted using a kit, and its 16SrRNA sequence was amplified using PCR technology.

[0036] Through BLAST comparison analysis, it was found that the 16SrRNA sequence similarity between the above dominant strains and Sphingomonas was the highest, and the above analysis results were consistent with the biochemical identification results.

[0037] The modified Gao's No. 1 medium is composed of: 1.0 g potassium nitrate, 0.5 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 0.01 g ferrous sulfate, 0.5 g sodium chloride, 20.0 g soluble starch, 15.0 g agar, and a pH value of 7.2-7.4.

[0038] Example 2: Natural fermentation process of black tea

[0039] Select fresh tea leaves that have just unfolded, perform hot air withering on the fresh leaves, and wither the fresh tea leaves until the moisture content is 60%, thereby obtaining withered leaves.

[0040] The withered tea leaves were sent to a rolling machine for rolling. The speed of the rolling machine was controlled to be 33 rpm / min for 40 min. The rolled leaves were fermented at a low temperature of 30°C and the air humidity was maintained at 85% for 6 h.

[0041] After fermentation, the fermented tea leaves were dried at 110°C to a moisture content of 30%, and then dried at 70°C to a moisture content of 6%. The dried black tea was microwave sterilized at a temperature of 100°C for 8 minutes.

[0042] Theanine was extracted and its content was determined in fermented black tea.

[0043] The determination of theanine adopts the national standard GB / T23193-2017 detection method. According to the experimental requirements, 1.0g is weighed, ground into a sample, added into a 250mL conical flask, and 50mL of ultrapure water is added. The temperature is 80℃ to simulate the common water temperature for daily tea brewing. The mixture is soaked for 45min. After passing through a 0.45μm filter membrane, it is centrifuged at 13000r / min for 10min. 1mL of the sample is taken and analyzed by liquid chromatography.

[0044] The flow rate was 1.0 mL / min, the column temperature was 35°C, and the detection wavelength was 210 nm. The gradient elution conditions were 0-10 min, 100% ultrapure water phase; 10-20 min, 20% ultrapure water phase, 80% acetonitrile phase, and 20-40 min, 100% ultrapure water phase. Among them, 0-10 min was the analysis period of the sample to be tested, 10-20 min was the column washing period, and 20-40 min was the equilibrium period.

[0045] Example 3: Sphingomonas fermentation process of black tea

[0046] The purified Sphingomonas obtained in Example 1 was inoculated into a test tube containing a TPG seed culture medium, wherein the TPG seed culture medium was: 20 g / L sucrose, 2.5 g / L peptone, 5 g / L potassium dihydrogen phosphate, 0.6 g / L potassium sulfate, 1.5 g / L disodium hydrogen phosphate, 0.1 g / L calcium carbonate, 2 g / L yeast powder, and the pH was adjusted to 7.0-7.4; the culture was shaken at 30° C. for 18 h to prepare primary seeds, and then the primary seeds were inoculated into the TPG seed culture medium with an inoculation amount of 5%, and the culture was shaken for 18 h to obtain a secondary seed solution of Sphingomonas.

[0047] Select fresh tea leaves that have just unfolded, perform hot air withering on the fresh leaves, and wither the fresh tea leaves until the moisture content is 60%, thereby obtaining withered leaves.

[0048] The withered tea leaves were sent to a rolling machine for rolling, and the speed of the rolling machine was controlled to be 33 rpm / min for 40 min.

[0049] Spray 1.5wt% of Sphingomonas seed solution on the rolled leaves, stir evenly, and perform low-temperature fermentation at a temperature of 30°C and an air humidity of 85% for 6 hours. After the fermentation, dry the fermented tea leaves at 110°C to a moisture content of 30%, and then dry them at 70°C to a moisture content of 6%. The dried black tea is microwave sterilized at 100°C for 8 minutes.

[0050] Theanine was extracted and its content was determined in fermented black tea.

[0051] Example 4: Black tea fermentation process with Sphingomonas, cellulase and aminopeptidase

[0052] Select fresh tea leaves that have just unfolded, perform hot air withering on the fresh leaves, and wither the fresh tea leaves until the moisture content is 60%, thereby obtaining withered leaves.

[0053] The withered tea leaves were sent to a rolling machine for rolling, and the speed of the rolling machine was controlled to be 33 rpm / min for 40 min.

[0054] The withered tea leaves are sprayed with 3wt% of mixed enzyme solution, left to stand for 30 minutes, and then sent to a rolling machine for rolling, the rolling machine speed is controlled to be 33rpm / min, the rolling time is 40 minutes, and rolled leaves are obtained. The mixed enzyme solution is a mixture of cellulase solution and aminopeptidase solution with a mass ratio of 4:1, the mass fractions of the cellulase solution and the aminopeptidase solution are 5% respectively, and both the cellulase and the aminopeptidase are commercially available products.

[0055] Spray 1.5wt% of Sphingomonas seed solution on the rolled leaves, stir evenly, and perform low-temperature fermentation at a temperature of 30°C and an air humidity of 85% for 6 hours. After fermentation, dry the fermented tea leaves at 110°C to a moisture content of 30%, and then dry them at 70°C to a moisture content of 6%. The dried black tea is microwave sterilized at 100°C for 8 minutes. Theanine in the fermented black tea is extracted and the content is determined.

[0056] Test Example 1:

[0057] The fermented black tea obtained in Examples 2, 3 and 4 was compared in terms of black tea sensory quality. The results are shown in Table 1, mainly comparing the tea soup color, aroma, taste and tea base. The evaluation was conducted in accordance with the GB / T23776-2018 tea sensory evaluation method, using a combination of a percentage system and comments to evaluate the appearance, aroma, tea soup color, taste and tea base of the tea samples.

[0058] Take 3g of dry tea, with a tea-to-water ratio of 1:50, and brew with boiling water for 5 minutes. The evaluation team consists of 10 reviewers (6 females, 4 males, aged 20-40) with national middle and senior tea appraiser qualifications.

[0059] Table 1 Comparison of sensory qualities of different fermented black teas

[0060]

[0061]

[0062] Test Example 2:

[0063] The theanine content of the fermented black tea obtained in Examples 2-4 was compared, and the results are shown in Table 2 below.

[0064] The black tea was fermented by using the Sphingomonas selected from the fermented black tea. According to the comparison of theanine content in Example 3 and Example 4, it can be clearly seen that the content of theanine was significantly increased after adding cellulase and aminopeptidase during the fermentation of black tea. The addition of cellulase and aminopeptidase promoted the formation of theanine, effectively improving the quality and nutrients of black tea. This greatly affects the flavor, aroma, color and quality of black tea.

[0065] Table 2 Comparison of theanine content in different fermented black teas

[0066] Theanine % Example 2 2.45 Example 3 4.12 Example 4 5.05

[0067] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and implementation modes, and can be fully applicable to various fields suitable for the present invention. For those familiar with the art, for those of ordinary skill in the art, various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principle and spirit of the present invention. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A Sphingomonas species ( Sphingobuim s p .), characterized in that, The Sphingomonas is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC No.29158.

2. The method for preparing black tea fermented by Sphingomonas according to claim 1, characterized in that: The method comprises the following steps: S1. Preparing a sphingomonas seed solution: culturing the sphingomonas into a seed solution; S2, withering fresh tea leaves: withering fresh tea leaves by natural withering indoors or by sunlight combined withering to obtain withered tea leaves; S3, rolling: spraying 1-5wt% of the mixed enzyme solution on the withered tea leaves, fermenting at low temperature, standing for 10-50min, and then sending them to a rolling machine for rolling to obtain rolled leaves; S4, fermentation: adding 1-2 wt% of Sphingomonas to the rolled leaves for low temperature fermentation; S5, drying: after the fermentation is completed, drying is performed to obtain the fermented black tea; S6, microwave sterilization: sending the dried fermented black tea leaves into a microwave machine for microwave sterilization; In step S3, the mixed enzyme solution consists of cellulase solution and aminopeptidase solution, wherein the mass ratio of cellulase solution to aminopeptidase solution is 3-6:1, and the mass fractions of cellulase solution and aminopeptidase solution are 4-10%, respectively.

3. The preparation method according to claim 2, characterized in that: The method for culturing the seed solution in step S1 is: inoculating Sphingomonas in a test tube containing TPG seed culture medium, shaking and culturing at 28-32°C for 18-20 hours to prepare primary seeds; then inoculating the primary seeds in TPG seed culture medium with an inoculation amount of 5-10%, shaking and culturing for 16-20 hours to obtain Sphingomonas seed solution.

4. The preparation method according to claim 3, characterized in that: The formula of the TPG seed culture medium is: sucrose 20g / L, peptone 2.5g / L, potassium dihydrogen phosphate 5g / L, potassium sulfate 0.6g / L, disodium hydrogen phosphate 1.5g / L, calcium carbonate 0.1g / L, yeast powder 2g / L, and the pH is adjusted to 7.0-7.

4.

5. The preparation method according to claim 2, characterized in that: The moisture content of the withered tea leaves obtained in step S2 is 55%-65%.

6. The preparation method according to claim 2, characterized in that: The kneading speed in step S3 is 28-35 rpm / min, and the time is 38-50 min.

7. The preparation method according to claim 2, characterized in that: The temperature of the low-temperature fermentation in step S4 is 28-30° C., the air humidity is 80-85%, and the fermentation time is 3-8 hours.

8. The preparation method according to claim 2, characterized in that: The moisture content of the fermented black tea obtained after drying in step S5 is 5-10%.

9. The preparation method according to claim 2, characterized in that: The sterilization temperature in step S6 is 90-105° C. and the time is 6-10 minutes.

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