A bacillus subtilis strain and application thereof

Abstract

The application provides a bacillus subtilis strain, which is classified as Bacillus subtilis, is preserved in the China Center for Type Culture Collection, and has a preservation number of CCTCC NO: M 20241726. The application also provides a culture method of the above-mentioned strain and application of the strain in fermenting preparation of loose black tea. Meanwhile, the application also provides a preparation method of loose black tea. The advantages of the application are as follows: (1) the theabromine content of the black tea product prepared by using the strain as a fermentation strain is significantly higher than that of products prepared by using other strains, and the product has prominent floral and woody fragrance; (2) the application can be applied to the preparation of a fermentation loose black tea product with high theabromine content and prominent floral and woody fragrance through solid-state fermentation of summer and autumn green tea, so that the economic value of the summer and autumn green tea is improved; and (3) the production process is simple, the fermentation is fast and the cost is low, and the application is suitable for industrial large-scale production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial fermentation, in particular to a bacillus subtilis strain and application thereof. BACKGROUND

[0002] Black tea is one of the six tea categories, and is a post-fermented tea unique to China. It has a long history of production and is produced in Yunnan, Hunan, Hubei, Guangxi and other places in China. Because the raw material is relatively old, it is piled and fermented for a long time during processing, resulting in dry tea with oil black or black brown color, hence the name black tea.

[0003] Bacillus has been reported to exist in the fermentation process of black tea, and is the dominant bacteria in the fermentation process of black tea. In the fermentation of black tea, bacillus subtilis can grow and metabolize on the tea matrix, promote the biosynthesis of L-theanine, and help form the flavor of tea aroma characteristics.

[0004] However, there is currently less overall research on bacillus strains for black tea fermentation, and the existing bacillus strains have some shortcomings in improving the quality of black tea. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a new bacillus subtilis strain and its application. The use of the strain in the fermentation process of black tea can effectively increase the content of theabrownine, while making the product's floral and woody aroma prominent, and improving the product quality.

[0006] The present application solves the above technical problems by adopting the following technical solutions:

[0007] A bacillus subtilis strain BS20240729, classified and named as Bacillus subtilis, was sent to the China Center for Type Culture Collection on August 2, 2024 and showed survival, with the preservation number CCTCC NO: M20241726 and the preservation address Wuhan University, China Center for Type Culture Collection, Wuhan, Wuchang District, Bayi Road, Hubei Province, China.

[0008] A culture method of the above-mentioned bacillus subtilis strain, comprising the following steps:

[0009] (1) Preparation of culture medium: peeled potatoes are boiled with water and then filtered with 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 adjusted, high-temperature sterilization is performed, and after cooling, it is poured into a flat plate;

[0010] (2) Activation culture of the strain: under sterile conditions, the target strain is inoculated into a flat plate containing the culture medium, and then activated culture is performed;

[0011] (3) Bacterial suspension preparation: in a sterile operating environment, the activated target strain is scraped into sterile water, mixed uniformly with glass beads, and a bacterial suspension of Bacillus subtilis is obtained.

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

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

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

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

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

[0017] The use of the above-mentioned Bacillus subtilis strain in the fermentation preparation of loose black tea.

[0018] A preparation method of loose black tea, which uses the above-mentioned Bacillus subtilis strain as a target strain for fermentation, comprising the following steps:

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

[0020] (2) Solid-state fermentation: inoculate the obtained sterilized tea raw material with a bacterial suspension of the target strain for fermentation, mix uniformly, and then culture and ferment;

[0021] (3) Drying: dry to obtain loose black tea.

[0022] As one of the preferred modes of the present application, in step (1), the ratio of the amount of tea raw material to water is 38g:12mL.

[0023] As one of the preferred modes of the present application, in step (2), the concentration of the bacterial suspension is 1.0x10 5 ~ 1.0x10 7 / mL; the inoculation ratio is 1mL of bacterial suspension per 38g of tea.

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

[0025] As one of the preferred modes of the present application, in the step (3), the drying mode is selected from one of vacuum freeze drying, hot air drying, heat pump drying or normal temperature drying mode.

[0026] As one of the preferred modes of the present application, the vacuum freeze drying conditions are: vacuum degree-10pa, temperature-50℃, time 3 days; the hot air drying conditions are: drying box 70℃, time 2h; normal temperature drying: sterile normal temperature environment, time 1 day.

[0027] The present application has the following advantages compared with the prior art:

[0028] (1) The present application screens and separates a Bacillus subtilis BS20240729 from Sichuan Ya'an Tibetan tea, which is used as a strain for fermenting loose black tea, and has the following advantages: ① The fermented loose black tea product based on the strain has a significantly higher content of theabrownine than other strain products; ② The product has prominent floral and woody aroma;

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

[0030] (3) The production process of the black tea product of the present application is simple, fast and low in cost, and is suitable for large-scale industrial production. BRIEF DESCRIPTION OF DRAWINGS

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

[0032] Figure 2 is a 16S rDNA phylogenetic tree of the strain BS20240729 of the present application. DETAILED DESCRIPTION

[0033] The embodiments of the present application are described in detail below, and the embodiments are implemented on the premise of the technical scheme of the present application, and detailed implementation modes and specific operation processes are given, but the protection scope of the present application is not limited to the following embodiments. Meanwhile, unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the technical field.

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

[0035] PDA flat plate medium: 200 g peeled potatoes were weighed, cut into pieces, and boiled in 1000 mL water for 20 min, filtered with gauze, then 20 g agar powder was added under heating, heating and stirring were continued, after the agar powder was completely dissolved, 20 g glucose was added, stirred evenly, and after cooling, the water was made up to 1000 mL, wrapped, sterilized at 121°C for 20 min, and cooled to 50°C, then poured into a flat plate.

[0036] Chase's selective medium: 30 g sucrose, 3 g sodium nitrate, 1 g potassium phosphate dibasic, 0.5 g magnesium sulfate, 0.01 g ferrous sulfate, and 0.5 g potassium chloride were weighed, mixed evenly with distilled water, then 20 g agar powder was added under heating, heating and stirring were continued, and after the agar powder was completely dissolved, the water was made up to 1000 mL, wrapped, sterilized at 121°C for 20 min, and cooled, then poured into a flat plate.

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

[0038] 1. Screening and isolation of the strain

[0039] In a clean bench, an appropriate amount of high-quality Tibetan tea produced in Ya'an, Sichuan was dissolved in 100 mL sterile water, and placed in a shaking bed at a temperature of 37°C and a rotation speed of 150 r / min for 1 day of shaking culture. Then 200 microliters of the culture solution was inoculated on PDA flat plate medium, and placed in a constant temperature incubator at 37°C for 4 days of culture. Single colonies were picked and inoculated on Chase's selective medium for repeated streaking and separation culture until single colonies appeared. The single colonies were inoculated on PDA flat plate medium, and placed in a constant temperature incubator at 37°C for 2 days of culture, then stored at 4°C for standby use, and named BS20240729.

[0040] 2. Identification of the strain

[0041] (1) Morphological identification

[0042] Strain BS20240729 was inoculated on PDA flat plate medium, and the colony morphology was observed.

[0043] As shown in Figure 1 , the colony morphology characteristics were as follows: the colony was between 2-5 mm, grayish white or slightly yellow, the surface showed a typical rough and irregular morphology with many elevations and wrinkles; the colony surface was opaque and rough.

[0044] (2) 16S rDNA sequence identification

[0045] Strain BS20240729 was sent to Shanghai Shengong Biotechnology Co., Ltd. for sequencing, and the 16S rDNA gene sequence is shown as SEQ ID NO. 1; the sequencing results were compared in the NCBI database, and the constructed phylogenetic tree is shown asFigure 2 The strain BS20240729 was identified as Bacillus subtilis by combining its morphology and 16S rRNA gene sequence analysis.

[0046] (3) Preservation and identification of the strain in the preservation center

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

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

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

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

[0051] (1) Sterilization

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

[0053] (2) Preparation of bacterial suspension

[0054] Under sterile operating conditions, the Bacillus subtilis activated for 4 days (Example 2) was scraped into sterile water, and the concentration of the bacterial suspension was adjusted to 1.0 x 10 6

[0055] (3) Fermentation

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

[0057] (4) Freeze-drying

[0058] The fermented tea leaves were placed in a vacuum freeze dryer, and were freeze-dried under the conditions of -10 pa and -50°C for 3 days to obtain the target black tea.

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

[0060] (1) Sterilization

[0061] ​To 38 g of green tea raw material (Huangshan Shui Gong Tea Industry Co., Ltd.) add 12 mL of water, then place in an autoclave and sterilize at 121 °C for 20 min to obtain sterilized raw material for fermentation of the target fermented loose black tea.

[0062] (2) Preparation of bacterial suspension

[0063] Under aseptic conditions, scrape Bacillus subtilis (Example 2) after 2 days of activation culture into sterile water, and adjust the concentration of the bacterial suspension to 1.0 x 10 5 cells / mL.

[0064] (3) Fermentation

[0065] Under aseptic conditions, add 1 mL of the Bacillus subtilis bacterial suspension of step (2) to the sterilized tea leaf raw material, mix well, and then solid-state ferment for 8 days under the following conditions: temperature 22 °C, humidity 70%.

[0066] (4) Hot air drying

[0067] Place the fermented product in a drying oven and dry at 70 °C for 2 h to obtain the target fermented loose black tea.

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

[0069] (1) Sterilization

[0070] To 38 g of green tea raw material (Huangshan Shui Gong Tea Industry Co., Ltd.) add 12 mL of water, then place in an autoclave and sterilize at 121 °C for 20 min to obtain sterilized raw material for fermentation of the target fermented loose black tea.

[0071] (2) Preparation of bacterial suspension

[0072] Under aseptic conditions, scrape Bacillus subtilis (Example 2) after 5 days of activation culture into sterile water, and adjust the concentration of the bacterial suspension to 1.0 x 10 7 cells / mL.

[0073] (3) Fermentation

[0074] Under aseptic conditions, add 1 mL of the Bacillus subtilis bacterial suspension of step (2) to the sterilized tea leaf raw material, mix well, and then solid-state ferment for 2 days under the following conditions: temperature 37 °C, humidity 70%.

[0075] (4) Natural drying

[0076] Place the fermented product in a natural environment and dry for 1 day to obtain the target fermented loose black tea.

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

[0078] The preparation method of the comparative example scattered black tea is basically the same as that of example 3, and the main difference is that "ordinary bacillus subtilis (China typical culture preservation center, preservation number CCTCC M 2023165)" is used instead of "bacillus subtilis strain BS20240729".

[0079] Test example 1: aroma detection of solid-state fermented scattered black tea

[0080] The aroma substances of green tea raw materials and the scattered black tea prepared by the present application (example 3 is taken as an example) and comparative example 1 are detected respectively.

[0081] I. Detection method:

[0082] (1) SPME extraction

[0083] 10 μL of ethyl caprate was weighed, diluted to 10 mL with ethanol and made up to volume (a concentration of 1000 ppm was prepared as an internal standard stock solution), and stored at -20℃ for standby. When used, the stock solution was diluted to 5 ppm. A certain amount of tea sample to be tested was weighed, brewed according to the tea-water ratio of 1:20 for 5 min, and then the tea soup was filtered and rapidly cooled to room temperature in an ice water bath. 10 mL of filtered tea soup was taken into a headspace bottle, 3.3 g of sodium chloride solid and 5 μL of internal standard were added, and then shaken well. The headspace bottle was placed in a 40℃ water bath, and balanced for 15 min. After balancing, the extraction needle was inserted into the headspace bottle, and the extraction fiber head was pushed out to a distance of 1 cm from the tea soup liquid surface for adsorption for 30 min.

[0084] (2) GC-MS analysis

[0085] The inlet temperature was 250℃, the chromatographic column was DB-5MS (30m x 0.25mm, film thickness 0.25μm), the carrier gas was pure 99.999% helium, and the average linear velocity was 36cm / sec. The ion source was EI, and the temperature was set to 230℃. The temperature rising program was as follows: the initial temperature was 40℃, maintained for 3 min; increased to 80℃ at a rate of 2℃ / min; increased to 140℃ at a rate of 3℃ / min; increased to 250℃ at a rate of 14℃ / min, maintained for 2 min.

[0086] II. Test results:

[0087] The scattered black tea prepared by the present application was co-detected by HS-SPME to identify 77 kinds of volatile compounds, including 22 kinds of higher alcohols, 10 kinds of esters, 13 kinds of ketones, 8 kinds of benzene, 10 kinds of aldehydes, 13 kinds of terpenes and 1 kind of other. Among them, the number of identified alcohols, aldehydes and terpenes accounts for more than 70% of the total number of identified compounds, which is the main component of volatile compounds in fermented tea.

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

[0089] The present application screens 9 aroma compounds with relatively high contribution degree and rOAV greater than 1, including linalool oxide, beta-cyclocitral, beta-ionone, nonanal, alpha-terpineol, beta-pinene, alpha-ionone, geraniol, and galactolactone, as shown in Table 1; through aroma sensory QDA, it is found that the flower and woody aroma intensity of the Bacillus subtilis fermented loose black tea sample is significantly increased; further orthogonal partial least squares discriminant analysis shows that the flower and woody aroma properties in the fermented loose black tea are related to several alcohol and ketone compounds such as “beta-ionone, geraniol, linalool oxide”.

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

[0091]

[0092]

[0093] Meanwhile, combined with the results of the 9 aroma compounds with rOAV greater than 1 of the green tea raw material and the loose black tea of Comparative Example 1 (Table 1), it can be seen that the strain of the present application is used as a black tea fermentation strain, and the flower and woody aroma are prominent, and the content is significantly higher than that of the green tea raw material and the products of other strains.

[0094] Test Example 2: Detection of the content of theabromine in the solid-state fermented loose black tea

[0095] The content of theabromine in the green tea raw material and the loose black tea fermented by the present application (Example 3 is taken as an example) and Comparative Example 1 was detected, respectively.

[0096] I. Detection method:

[0097] (1) 3 g of crushed and sieved tea leaves were added to 125 mL of boiling water and placed in a boiling water bath for 10 min of heat preservation and extraction, and then filtered to obtain a test solution.

[0098] (2) 25 mL of the test solution and 25 mL of n-butanol were placed in a 1000 mL funnel, mixed by shaking for 3 min, and then separated by layering, and 2 mL of the lower layer solution was taken 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 95% ethanol was added to constant volume.

[0099] (3) The absorbance was measured at 380 nm, and the content of theabromine was calculated according to the following formula:

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

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

[0102] II. Test results:

[0103] The results are shown in Table 2.

[0104] Table 2: Theabromine content test results

[0105]

[0106]

[0107] From the results in Table 2, it can be seen that the strain of the present application as a black tea fermentation strain has a theabromine content significantly higher than that of green tea raw materials and other strain fermentation products.

[0108] In summary, the Bacillus subtilis strain BS20240729 of the present application can effectively increase the theabromine content, at the same time, make the product have prominent floral and woody aroma, and improve the product quality.

[0109] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A strain of Bacillus subtilis, characterized in that, Category naming Bacillus subtilis It is deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20241726.

2. A method for culturing the Bacillus subtilis strain as described in claim 1, characterized in that, Includes the following steps: (1) Culture medium preparation: Take peeled potatoes, boil them in water and filter them with gauze; collect the filtrate, and then add agar powder and glucose to the filtrate under heating conditions to dissolve them completely; then make up the volume, sterilize at high temperature, cool and pour into plates; (2) Activation culture of strain: Under aseptic conditions, the target strain is inoculated onto a plate containing culture medium and then activated culture is carried out; (3) Preparation of bacterial suspension: Under aseptic conditions, the above-activated target strain was scraped into sterile water, and mixed evenly by shaking with glass beads to obtain Bacillus subtilis bacterial suspension.

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

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

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

6. The application of a Bacillus subtilis strain as described in claim 1 in the fermentation preparation of loose black tea.

7. A method for preparing loose-leaf dark tea, characterized in that, Using the Bacillus subtilis strain of claim 1 as the target strain for fermentation includes the following steps: (1) Sterilization: Add water to the tea raw material and then sterilize it at high temperature to obtain sterilized tea raw material; (2) Solid-state fermentation: The sterilized tea raw materials were inoculated with a bacterial suspension of the target strain for fermentation, mixed evenly, and then cultured for fermentation; wherein, the concentration of the bacterial suspension was 1.0 × 10⁻⁶. 5 ~1.0×10 7 The inoculation ratio was 1 mL of bacterial suspension per 38 g of tea leaves; the culture and fermentation conditions were: temperature 22-37℃, humidity 70%, and time 2-8 days. (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 step (1), the ratio of tea leaves to water is 38g:12mL.

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