Bacillus subtilis strain and application thereof

Abstract

The invention provides a bacillus subtilis strain, which is classified and named as bacillus subtilis, is preserved in the China Center for Type Culture Collection (CCTCC), and has the preservation number of CCTCC NO: M 20241726. The invention also provides a culture method of the strain and application of the strain in fermentation preparation of loose dark green tea. Meanwhile, the invention further provides a preparation method of the loose black tea. The strain has the advantages that (1) the strain is used as a strain for fermenting loose black tea, the theabrownin content of a black tea product is obviously higher than that of other strain products, and the flower fragrance and the costustoot are prominent; (2) the method can be applied to solid-state fermentation of summer and autumn green tea to prepare a fermented loose black tea product with high theabrownin and outstanding flower fragrance and elecampane, and the economic value of the summer and autumn green tea is improved; (3) the production process is simple, the fermentation is rapid, the cost is low, and the method is suitable for industrial large-scale production.

Description

Technical Field

[0001] The invention relates to the field of microbial fermentation, and in particular to a Bacillus subtilis strain and application thereof. Background Art

[0002] Dark tea is one of the six major types of tea. It is a post-fermented tea and a unique tea in China. It has a long production history and is produced in Yunnan, Hunan, Hubei, Guangxi and other places in China. Because the raw materials are relatively coarse and old, the fermentation time is long during the processing, resulting in the dry tea being oily black or dark brown in color, hence the name dark tea.

[0003] Bacillus has been reported to exist in the fermentation process of dark tea and is the dominant bacteria in the fermentation process of dark tea. In the fermentation of dark tea, Bacillus subtilis can grow and metabolize on the tea matrix, promote the biosynthesis of L-theanine, and contribute to the formation of the characteristic flavor of tea aroma.

[0004] However, there is little overall research on Bacillus strains for dark tea fermentation, and the research on the application of existing Bacillus strains in improving the quality of dark tea is also slightly insufficient. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a novel Bacillus subtilis strain and its application. The use of the strain in the fermentation process of dark tea can effectively increase the content of theabrownin, while making the flower fragrance and wood fragrance of the product prominent, thereby improving the product quality.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] A Bacillus subtilis strain BS20240729, classified and named Bacillus subtilis, was sent to the China Center for Type Culture Collection for preservation on August 2, 2024 and appeared to be alive. The preservation number is CCTCC NO: M20241726. The preservation address is: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0008] A method for culturing the above-mentioned Bacillus subtilis strain comprises the following steps:

[0009] (1) Preparation of culture medium: Peeled potatoes are boiled in water and filtered through gauze; the filtrate is collected, and then agar powder and glucose are added to the filtrate under heating conditions to dissolve them completely; then the volume is fixed, sterilized at high temperature, cooled, and poured into a plate;

[0010] (2) Activation culture of bacterial strains: Under sterile conditions, the target strain is inoculated on a plate containing culture medium and then activated;

[0011] (3) Preparation of bacterial suspension: In a sterile operating environment, the activated target strain was scraped into sterile water, and the mixture was shaken and mixed with glass beads to obtain a Bacillus subtilis suspension.

[0012] As one of the preferred embodiments of the present invention, in the step (1), water is added and boiled for 20 minutes and then filtered with gauze.

[0013] As one of the preferred embodiments of the present invention, in step (1), the sterilization conditions are a temperature of 121° C. and a time of 20 min.

[0014] As one of the preferred embodiments of the present invention, in the step (1), the mixture is poured into a flat plate when cooled to 50-55°C; more preferably, the mixture is poured into a flat plate when cooled to 50°C.

[0015] As one of the preferred embodiments of the present invention, in step (1), 200 g of peeled potatoes, 20 g of agar powder and 20 g of glucose are used per 1000 mL of culture medium.

[0016] As one of the preferred embodiments of the present invention, in step (2), the culture conditions are: temperature 22-37° C., time 2-5 days; more preferably 37° C., culture time 2-5 days.

[0017] An application of the above-mentioned Bacillus subtilis strain in fermenting and preparing loose black tea.

[0018] A method for preparing loose black tea, using the above-mentioned Bacillus subtilis strain as a target strain for fermentation, comprises the following steps:

[0019] (1) Sterilization: adding water to the tea raw material, and then sterilizing at high temperature to obtain sterilized tea raw material;

[0020] (2) solid-state fermentation: inoculating the obtained sterilized tea raw material with a bacterial suspension of the target strain for fermentation, mixing evenly, and then culturing and fermenting;

[0021] (3) Drying: After drying, loose black tea is obtained.

[0022] As one of the preferred embodiments of the present invention, in step (1), the ratio of tea leaves to water is 38 g:12 mL.

[0023] As one of the preferred embodiments of the present invention, in step (2), the concentration of the bacterial suspension is 1.0×10 5 ~1.0×10 7 / mL; the inoculation ratio is: 1mL of bacterial suspension for every 38g of tea leaves.

[0024] As one of the preferred embodiments of the present invention, in step (2), the culture and fermentation conditions are: temperature 22-37° C., humidity 70%, and time 2-8 days.

[0025] As one of the preferred embodiments of the present invention, in step (3), the drying method is selected from vacuum freeze drying, hot air drying, heat pump drying or room temperature drying.

[0026] As one of the preferred embodiments of the present invention, the vacuum freeze-drying conditions are: vacuum degree -10pa, temperature -50°C, time 3 days; hot air drying conditions are: drying oven 70°C, time 2h; room temperature drying: sterile room temperature environment, time 1 day.

[0027] The advantages of the present invention compared to the prior art are:

[0028] (1) The present invention screened and isolated a Bacillus subtilis BS20240729 from Tibetan tea in Ya'an, Sichuan. The strain was used as a fermentation strain for loose dark tea, which has the following advantages: ① The theabrownin content of the fermented loose dark tea product based on the strain is significantly higher than that of products from other strains; ② The floral and woody aromas are prominent;

[0029] (2) The Bacillus subtilis strain of the present invention can be applied to the solid-state fermentation of summer and autumn green tea to prepare fermented loose black tea products with high theabrownin content and prominent floral and woody aroma, thereby improving the economic value of summer and autumn green tea;

[0030] (3) The dark tea product of the present invention has a simple production process, fast fermentation and low cost, and is suitable for industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a colony morphology diagram of strain BS20240729 of the present invention;

[0032] Figure 2 The 16S rDNA phylogenetic tree of the strain BS20240729 of the present invention. DETAILED DESCRIPTION

[0033] The following is a detailed description of the embodiments of the present invention. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are provided, but the protection scope of the present invention is not limited to the following embodiments. Meanwhile, unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0034] The culture medium formula involved in the following examples is as follows:

[0035] PDA plate culture medium: weigh 200g of peeled potatoes, cut into pieces, add 1000mL of water and boil for 20min, filter with gauze, then add 20g of agar powder under heating conditions, continue heating and stirring, after the agar powder is dissolved, add 20g of glucose, stir evenly, add water to 1000mL after cooling, wrap, sterilize at 121℃ for 20min, cool to 50℃, and pour into the plate.

[0036] Czapek selection medium: weigh 30g sucrose, 3g sodium nitrate, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.01g ferrous sulfate, and 0.5g potassium chloride, mix well with distilled water, add 20g agar powder under heating conditions, continue heating and stirring, and add water to 1000mL after the agar powder is dissolved, wrap, sterilize at 121℃ for 20min, cool, and pour into a plate.

[0037] Example 1: Isolation and identification of Bacillus subtilis strain BS20240729

[0038] 1. Screening and isolation of strains

[0039] In the clean bench, take an appropriate amount of high-quality Tibetan tea produced in Ya'an, Sichuan, and dissolve it in 100 mL of sterile water. Place it in a shaker at a temperature of 37°C and a speed of 150 r / min for shaking culture for 1 day, then aspirate 200 μL of culture solution and inoculate it on PDA plate culture medium. After culturing it at a constant temperature of 37°C for 4 days, pick a single colony and repeat streaking and isolation culture in Cha's selective medium until a single colony appears; pick a single colony and inoculate it on PDA plate culture medium, place it in a constant temperature incubator at 37°C for 2 days, and then store it at 4°C for standby use, and name it BS20240729.

[0040] 2. Identification of strains

[0041] (1) Morphological identification

[0042] The strain BS20240729 was inoculated on PDA plate culture medium and its colony morphology was observed.

[0043] like Figure 1 As shown, the morphological characteristics of the colonies are: the colonies are between 2 and 5 mm, grayish white or slightly yellow, and the surface is typically rough and irregular with many ridges and wrinkles; the surface of the colonies is opaque and rough.

[0044] (2) 16S rDNA sequence identification

[0045] The strain BS20240729 was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Its 16S rDNA gene sequence is shown in SEQ ID NO.1. The sequencing results were compared with the NCBI database, and the phylogenetic tree was constructed as shown in Figure 2 As shown; combined with the morphology and 16S rRNA gene sequence analysis of strain BS20240729, it was preliminarily determined to be Bacillus subtilis.

[0046] (3) Strain preservation and identification in the collection center

[0047] The strain BS20240729 was sent to China Center for Type Culture Collection for preservation, and its classification was identified and named Bacillus subtilis, with the preservation number CCTCC NO: M 20241726.

[0048] Example 2: Activation culture of Bacillus subtilis strain BS20240729

[0049] Under sterile conditions, the Bacillus subtilis strain BS20240729 was inoculated and streaked on a PDA plate medium; after streaking, the plate was inverted and placed in a constant temperature incubator at 37° C. for activation for 2 to 5 days.

[0050] Example 3: Fermentation preparation of loose black tea

[0051] (1) Sterilization

[0052] 12 mL of water was added to 38 g of green tea raw material (Huangshan Shuigong Tea Industry Co., Ltd.), and then sterilized in a high pressure sterilizer at 121° C. for 20 min to obtain a sterilized raw material for post-sterilization fermentation of loose black tea.

[0053] (2) Preparation of bacterial suspension

[0054] In a sterile operating environment, scrape the activated Bacillus subtilis (Example 2) after 4 days of culture and add it to sterile water, and adjust the concentration of the bacterial suspension to 1.0×10 6 Pieces / mL.

[0055] (3) Fermentation

[0056] Under aseptic conditions, 1 mL of the Bacillus subtilis suspension of step (2) was added to the sterilized tea raw material, mixed evenly and then solid-state fermented for 5 days under the following fermentation conditions: temperature 28° C., humidity 70%.

[0057] (4) Freeze drying

[0058] The fermented tea leaves were placed in a vacuum freeze dryer and freeze-dried for 3 days at "-10 Pa, -50 ° C" to obtain the desired loose black tea.

[0059] Example 4: Fermentation preparation of loose black tea

[0060] (1) Sterilization

[0061] 12 mL of water was added to 38 g of green tea raw material (Huangshan Shuigong Tea Industry Co., Ltd.), and then sterilized in a high pressure sterilizer at 121° C. for 20 min to obtain a sterilized raw material for post-sterilization fermentation of loose black tea.

[0062] (2) Preparation of bacterial suspension

[0063] In a sterile operating environment, scrape the activated Bacillus subtilis cultured for 2 days (Example 2) into sterile water, and adjust the concentration of the bacterial suspension to 1.0×10 5 Pieces / mL,

[0064] (3) Fermentation

[0065] Under aseptic conditions, 1 mL of the Bacillus subtilis suspension of step (2) was added to the sterilized tea raw material, mixed evenly and then solid-state fermented for 8 days under the following fermentation conditions: temperature 22° C., humidity 70%.

[0066] (4) Hot air drying

[0067] The fermented product was placed in a drying oven and dried at 70°C for 2 hours to obtain the desired loose black tea.

[0068] Example 5: Fermentation preparation of loose black tea

[0069] (1) Sterilization

[0070] 12 mL of water was added to 38 g of green tea raw material (Huangshan Shuigong Tea Industry Co., Ltd.), and then sterilized in a high pressure sterilizer at 121° C. for 20 min to obtain a sterilized raw material for post-sterilization fermentation of loose black tea.

[0071] (2) Preparation of bacterial solution

[0072] In a sterile operating environment, scrape the activated Bacillus subtilis (Example 2) after 5 days of culture and add it to sterile water, and adjust the concentration of the bacterial suspension to 1.0×10 7 Pieces / mL.

[0073] (3) Fermentation

[0074] Under aseptic conditions, 1 mL of the Bacillus subtilis suspension of step (2) was added to the sterilized tea raw material, mixed evenly and then solid-state fermented for 2 days under the following fermentation conditions: temperature 37° C., humidity 70%.

[0075] (4) Natural drying

[0076] The fermented product is placed in a natural environment and dried for 1 day to obtain the desired loose black tea.

[0077] Comparative Example 1: Fermentation preparation of loose black tea (based on common Bacillus subtilis strains)

[0078] The preparation method of the loose black tea in this comparative example is basically the same as that in Example 3, the main difference being that “common Bacillus subtilis (China Center for Type Culture Collection, collection number CCTCC M 2023165)” is used instead of “Bacillus subtilis strain BS20240729”.

[0079] Test Example 1: Aroma Detection of Solid-State Fermented Loose Black Tea

[0080] Aroma substances were detected for green tea raw materials and loose black tea prepared by fermentation of the present invention (Example 3) and Comparative Example 1.

[0081] 1. Detection method:

[0082] (1) SPME extraction

[0083] Weigh 10μL of ethyl caprate, dilute it to 10mL with ethanol and make up to volume (prepare the concentration to 1000ppm as the internal standard mother liquor), and store it at -20℃ for later use. Dilute the mother liquor to 5ppm when using. Weigh a certain amount of tea sample to be tested, brew it for 5 minutes at a tea-to-water ratio of 1:20, 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 solid and 5μL of internal standard, and shake well. Place the headspace bottle in a 40℃ water bath and balance for 15min. After balance, insert the extraction needle into the headspace bottle, push out the extraction fiber head, and let it adsorb 1cm away from the tea soup surface for 30min.

[0084] (2) GC-MS analysis

[0085] The injection port temperature was 250°C, the chromatographic column was DB-5MS (30m×0.25mm, film thickness 0.25μm), the carrier gas was 99.999% pure helium, and the average linear velocity was 36cm / sec. The ion source was EI, and the temperature was set to 230°C. The heating program was as follows: the initial temperature was 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, and maintained for 2min.

[0086] 2. Test results:

[0087] A total of 77 volatile compounds were identified in the loose black tea prepared by the present invention by HS-SPME, including 22 higher alcohols, 10 esters, 13 ketones, 8 benzenes, 10 aldehydes, 13 terpenes, and 1 other. Among them, the number of alcohols, aldehydes and terpenes identified accounted for more than 70% of the total number of identified compounds, and they were the main components of volatile compounds in fermented tea.

[0088] Higher alcohol compounds are an important class of tea aroma compounds and are crucial in shaping the aroma and flavor of tea. The fermented loose black tea of ​​the present invention has 22 kinds of alcohol compounds, and the aroma compound content is relatively high, reaching 13.37 mg / L, accounting for more than 50% of the total aroma compound content. It is generally believed that substances with a VIP value greater than 1 and a P value less than 0.05 contribute to the difference of the samples.

[0089] The present invention screened out 9 aroma compounds with relatively high contribution degree and rOAV greater than 1, including linalool oxide, β-cyclocitral, β-ionone, nonanal, α-terpineol, β-pinene, α-ionone, geraniol, and galactolactone, as shown in Table 1; through aroma sensory QDA, it was found that the intensity of floral and woody aroma of loose black tea samples fermented by Bacillus subtilis was significantly increased; further orthogonal partial least squares discriminant analysis was used, and it was considered that the floral and woody aroma attributes in fermented loose black tea were related to several alcohol and ketone compounds such as "β-ionone, geraniol, linalool oxide".

[0090] Table 1 Contents of 9 aroma compounds with rOAV greater than 1

[0091]

[0092]

[0093] At the same time, combined with the aroma compound results of 9 aroma compounds with rOAV greater than 1 of green tea raw materials and comparative example 1 loose black tea (Table 1), it can be seen that when the strain of the present invention is used as a strain for black tea fermentation, its floral and woody aromas are prominent, and the content is significantly higher than that of green tea raw materials and other strain products.

[0094] Test Example 2: Detection of theabrownin content in solid-state fermented loose dark tea

[0095] The theabrownin content of green tea raw materials and the loose black tea prepared by fermentation of the present invention (Example 3) and Comparative Example 1 was detected respectively.

[0096] 1. Detection method:

[0097] (1) Add 3 g of crushed and sieved tea leaves into 125 mL of boiling water and place in a boiling water bath for 10 min. Filter to obtain the test solution.

[0098] (2) Place 25 mL of the test solution and 25 mL of n-butanol in a 1000 mL funnel, shake and mix thoroughly for 3 min, let stand to separate the layers, and take 2 mL of the lower layer into a 25 mL volumetric flask; add 2 mL of saturated acetic acid and 6 mL of water to the 25 mL volumetric flask, and then make up to volume with 95% ethanol.

[0099] (3) Measure the absorbance at 380 nm and calculate the theabrownin content according to the following formula:

[0100] Theabrownin (%)=7.06*2A / w*m*100;

[0101] Wherein, w: dry matter content of the sample; m: mass of the sample; A: absorbance.

[0102] 2. Test results:

[0103] The results are shown in Table 2.

[0104] Table 2 Theabrownin content test results

[0105]

[0106]

[0107] From the results in Table 2, it can be seen that when the strain of the present invention is used as a strain for dark tea fermentation, the theabrownin content is significantly higher than that of green tea raw materials and products fermented by other strains.

[0108] In summary, the Bacillus subtilis strain BS20240729 of the present invention can effectively increase the content of theabrownin, while making the flower fragrance and wood fragrance of the product prominent, thereby improving the product quality.

[0109] 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 and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A Bacillus subtilis strain, characterized in that It is classified and named Bacillus subtilis and deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M 20241726.

2. A method for culturing the Bacillus subtilis strain according to claim 1, characterized in that: The steps include: (1) Preparation of culture medium: Peeled potatoes are boiled in water and filtered through gauze; the filtrate is collected, and then agar powder and glucose are added to the filtrate under heating conditions to dissolve them completely; then the volume is fixed, sterilized at high temperature, cooled, and poured into a plate; (2) Activation culture of bacterial strains: Under sterile conditions, the target strain is inoculated on a plate containing culture medium and then activated; (3) Preparation of bacterial suspension: In a sterile operating environment, the activated target strain was scraped into sterile water, and the mixture was shaken and mixed with glass beads to obtain a Bacillus subtilis suspension.

3. The method for culturing the Bacillus subtilis strain according to claim 2, characterized in that: In the step (1), 200 g of peeled potatoes, 20 g of agar powder and 20 g of glucose are used per 1000 mL of culture medium.

4. The method for culturing the Bacillus subtilis strain according to claim 2, characterized in that: In the step (1), the sterilization conditions are a temperature of 121° C. and a time of 20 min.

5. The method for culturing the Bacillus subtilis strain according to claim 2, characterized in that: In the step (2), the culture conditions are: temperature 22-37° C., and time 2-5 days.

6. Use of the Bacillus subtilis strain as claimed in claim 1 in fermenting and preparing loose black tea.

7. A method for preparing loose black tea, characterized in that: Using the Bacillus subtilis strain of claim 1 as a target strain for fermentation comprises the following steps: (1) Sterilization: adding water to the tea raw material, and then sterilizing at high temperature to obtain sterilized tea raw material; (2) solid-state fermentation: inoculating the obtained sterilized tea raw material with a bacterial suspension of the target strain for fermentation, mixing evenly, and then culturing and fermenting; (3) Drying: After drying, loose black tea is obtained.

8. The method for preparing loose black tea according to claim 7, characterized in that: In the step (1), the ratio of tea material to water is 38 g:12 mL.

9. The method for preparing loose black tea according to claim 7, characterized in that: In step (2), the concentration of the bacterial suspension is 1.0×10 5 ~1.0×10 7 / mL; the inoculation ratio is: 1mL of bacterial suspension for every 38g of tea leaves.

10. The method for preparing loose black tea according to claim 7, characterized in that: In the step (2), the culture and fermentation conditions are: temperature 22-37° C., humidity 70%, and time 2-8 days.

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