Bacillus licheniformis and application thereof in tea aroma enhancement

Abstract

The invention discloses screening and application of bacillus licheniformis and belongs to the technical field of microorganisms. According to the invention, a specific plate with casein as a substrate is used for screening to obtain a strain Bacillus licheniformis-4B with protease production capacity, so that accumulation of pyrazine substance precursor amino acid is increased. The enzyme activity of the protease is 1164.86 U.L <-1 >, and the content of 2, 5-dimethylpyrazine in fermentation liquor is 10 times that of a sterile culture medium. After the bacillus licheniformas-4B strain is applied to fermentation of low-quality tea, the content of 2, 5-dimethylpyrazine and the content of 2-ethyl-3, 5-dimethylpyrazine are remarkably increased, and the types of pyrazine are increased. And the purpose of tea aroma enhancement is achieved.

Description

Technical Field

[0001] The invention relates to the screening and application of a strain of Bacillus licheniformis, and belongs to the technical field of microorganisms. Background Art

[0002] Wuyi rock tea is a type of oolong tea, which is a semi-fermented tea. In the process of making tea, it goes through the steps of picking, withering, making green, killing green, rolling, baking, etc. to make dry tea. The dry tea is then piled, screened, and baked to finally make tea. In the entire processing of tea, the enzymes in tea play an important role in the transformation of its components, including the degradation of macromolecules and the mutual transformation of small molecules. Therefore, enzymes can be added to tea to achieve the effect of improving quality and enhancing aroma. Proteases can hydrolyze protein peptide bonds to generate amino acids with different taste characteristics, and then amino acids can produce pyrazines through Maillard reaction.

[0003] High-quality Wuyi rock tea has a roasted and woody aroma, which can be provided by pyrazines. At present, microbial fermentation is mostly used in the black tea pile and tea soup processing in the field of tea processing, and is less used in oolong tea. The quality of Wuyi rock tea is directly related to the geographical environment, in which the microorganisms in the environment play a role in the material metabolism of tea. However, it is unknown which specific microorganisms have a key impact on the aroma of tea, and the role of microorganisms in the aroma of Wuyi rock tea is also unclear. Therefore, it is urgent to develop a microorganism that can be used in low-quality Wuyi rock tea, in order to achieve the purpose of improving the quality of tea and increasing the aroma of tea. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides the screening and application of a strain of Bacillus licheniformis, with the purpose of solving the technical problem of the lack of a microorganism that can improve the quality of tea and increase the aroma of tea as it is currently unknown which specific microorganism has a key impact on the aroma of tea, and the role of microorganisms in the aroma of Wuyi Rock Tea is also unclear.

[0005] The first technical solution provided by the present invention is a strain of Bacillus licheniformis 4B, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 23, 2024, with a deposit number of GDMCC No: 65046.

[0006] The second technical solution provided by the present invention is a microbial preparation containing the Bacillus licheniformis 4B described in the first technical solution.

[0007] In certain embodiments, the amount of Bacillus licheniformis 4B added to the microbial preparation is at least 1×106 CFU / mL or 1×10 6 CFU / g.

[0008] The third technical solution provided by the present invention is a fermentation agent, which contains the Bacillus licheniformis 4B described in the first technical solution or the microbial preparation described in the second technical solution.

[0009] In certain embodiments, the amount of Bacillus licheniformis 4B added to the starter is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0010] The fourth technical solution provided by the present invention is the use of the Bacillus licheniformis 4B described in the first technical solution, or the microbial preparation described in the second technical solution, or the starter described in the third technical solution in fermenting tea leaves.

[0011] In certain embodiments, the tea leaves are Wuyi rock tea leaves.

[0012] The fifth technical solution provided by the present invention is a method for fermenting tea leaves, which comprises fermenting tea leaves with the Bacillus licheniformis 4B described in the first technical solution, the microbial preparation described in the second technical solution, or the starter described in the third technical solution.

[0013] In certain embodiments, the tea leaves are Wuyi rock tea leaves.

[0014] In certain embodiments, the method comprises spraying the culture solution of Bacillus licheniformis 4B on the surface of tea leaves for fermentation for 2 to 3 days, the fermentation temperature is 35 to 40° C., and the fermentation humidity is 20 to 30%. Preferably, the method comprises spraying the culture solution of Bacillus licheniformis 4B on the surface of tea leaves for fermentation for 2 days, the fermentation temperature is 37° C., and the fermentation humidity is 20%.

[0015] In certain embodiments, the preparation method of the culture solution of Bacillus licheniformis 4B is as follows: Bacillus licheniformis 4B is cultured in LB liquid medium at 37°C and 220 r·min -1 After activation culture for 24 h, the inoculum was transferred to the fermentation medium at 2% (v / v) at 37°C and 220 r / min. -1 Fermentation culture for 48h.

[0016] The sixth technical solution provided by the present invention is the use of the Bacillus licheniformis 4B described in the first technical solution, or the microbial preparation described in the second technical solution, in improving the content of pyrazines in tea leaves.

[0017] In certain embodiments, the pyrazines include 2,5-dimethylpyrazine and 2-ethyl-3,5-dimethylpyrazine.

[0018] The seventh technical solution provided by the present invention is a method for increasing the amount of pyrazines in tea leaves, wherein the method comprises fermenting tea leaves with the Bacillus licheniformis 4B described in the first technical solution or the microbial preparation described in the second technical solution.

[0019] In certain embodiments, the method comprises spraying the culture solution of Bacillus licheniformis 4B on the surface of tea leaves for fermentation for 2 to 3 days, the fermentation temperature is 35 to 40° C., and the fermentation humidity is 20 to 30%. Preferably, the method comprises spraying the culture solution of Bacillus licheniformis 4B on the surface of tea leaves for fermentation at 37° C. and 20% humidity for 2 days.

[0020] The technical effects of the present invention are as follows:

[0021] The strain Bacillus licheniformis-4B with protease production ability was obtained by specific plate screening with casein as substrate to increase the accumulation of amino acids, the precursor of pyrazine substances. Its protease activity was 1164.86 U·L -1 , and the content of 2,5-dimethylpyrazine in the fermentation broth is 10 times that of the sterile culture medium. After Bacillus licheniformis-4B strain was used for fermentation of low-quality tea, 2,5-dimethylpyrazine and 2-ethyl-3,5-dimethylpyrazine increased significantly, and the types of pyrazine increased. The purpose of enhancing the aroma of tea was achieved.

[0022] Biomaterial Deposit

[0023] The licheniformis 4B of the present invention has a taxonomic name of Bacillus licheniformis. It has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 23, 2024, with the deposit address being the 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, and the deposit number is GDMCC No: 65046. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a transparent circle diagram of the identification plate of the protease-producing strain of the present invention.

[0025] Figure 2 The diagram shows the enzyme activity of the protease-producing strains of the present invention; (a) casein standard curve; (b) protease activities of four strains.

[0026] Figure 3 This is a composition diagram of aroma volatile substances in the fermentation broth of strain 4B of the present invention.

[0027] Figure 4The free amino acid content and pyrazine composition of the fermented low-quality tea of ​​the present invention are shown in Figure 2; (a) free amino acid content of fermented low-quality tea; (b) pyrazine composition of fermented low-quality tea. Group P is the original untreated low-quality tea; Group C is the fermented tea with the addition of sterile fermentation medium; Group 4B is the fermented tea with the addition of strains; Group 4BR is the dried fermented tea with the addition of strains. DETAILED DESCRIPTION

[0028] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0029] Test method:

[0030] 1. Definition of protease activity: Under the conditions of 40°C and pH 7.0, the amount of enzyme required to decompose casein to produce 1 μg of tyrosine in 1 min is defined as 1 enzyme activity unit (U).

[0031] The calculation formula of protease activity is as follows:

[0032]

[0033] Where:

[0034] A is the OD of the experimental group and the control group 680 Difference;

[0035] K is the absorption constant;

[0036] T is the reaction time, in min;

[0037] V is the total volume of the reaction reagents in mL.

[0038] 2. The measurement method of total free amino acid content refers to GB / T 8314-2013.

[0039] The raw materials used in the embodiment:

[0040] Enrichment medium: LB liquid medium, tryptone (10g L -1 )、yeast extract (5g·L -1 )、NaCl(10g·L -1 );

[0041] Protease screening and identification culture medium:

[0042] Casein (10g·L -1 ), beef extract (3g·L -1 ), NaCl (5 g·L -1 ), K2HPO4(2g·L -1 ), agar (20 g·L-1 ), pH 7.4;

[0043] Fermentation medium: casein (10 g L -1 ), beef extract (3g·L -1 ), NaCl (5 g·L -1 ), K2HPO4(2g·L -1 ), pH 7.4.

[0044] Among them, tryptone and yeast extract were purchased from Thermo Fisher Scientific, agar was purchased from Sangon Biotechnology Co., Ltd., beef extract was purchased from Beijing Hongrun Baoshun Technology Co., Ltd., and casein, NaCl and K2HPO4 were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0045] Example 1 Screening and identification of strains

[0046] 1. Initial screening

[0047] The soil was collected from the tea garden in Wuyishan Scenic Area, Wuyishan City, Fujian Province. The rhizosphere soil was collected 20 cm from the main trunk of the tea tree and 20-40 cm vertically using the five-point sampling method. The loose soil combined with the root system was shaken off, and the remaining soil and the root system were placed in a sterile sealed plastic bag, immediately brought back to the laboratory for mixing, and sieved through a 2 mm sieve. The soil samples were stored in a -80 ° C refrigerator for subsequent experiments.

[0048] 5 g of the soil sample and 100 mL of sterile saline (0.9% NaCl solution, pH 7.0) were added to a 500 mL conical flask with baffles and heated at 10 °C and 220 r / min. -1 The total soil microorganisms were eluted by shaking for 3 hours under the conditions. Then, the solid-liquid mixture was filtered with 4 layers of sterile gauze, and the filtrate was centrifuged in a 50 mL centrifuge tube at 10 ° C and 8000 rpm for 15 minutes. The operation was repeated, and the precipitates were combined to obtain the bacteria, which were the total soil microorganisms. The eluted bacterial solution was inoculated into LB liquid culture medium and incubated at 37 ° C and 220 r·min -1 The enrichment culture was carried out for 48 hours. The enrichment culture solution was further spread on the protease primary screening identification plate and placed in a 37°C constant temperature incubator for culture. The colonies growing on the plate and having transparent circles were selected and further streaked and isolated to obtain single colonies.

[0049] LB medium and protease primary screening plate were used to screen strains that could decompose and produce proteases. After enrichment culture and multiple purifications, four strains with different colony morphologies were finally isolated and named 2B, 4B, 7B and 8B. Figure 1As shown, the 2B strain is white with a smooth surface; the 4B strain colonies are light yellow with a smooth surface; the 7B strain colonies are white with a sticky surface; and the 8B strain colonies are milky white with wrinkled surfaces.

[0050] 2. Rescreening

[0051] The functional strains that produced protease were screened and inoculated into the fermentation medium. -1 After 48 h of fermentation, the cells were centrifuged at 7000 rpm and 4 °C for 10 min, and the supernatant was collected as the crude enzyme solution to measure the protease activity of the strain. -1 The casein with a concentration of 100 μg / ml was used as the reaction substrate. The casein substrate was hydrolyzed by protease to generate amino acids containing phenolic groups. The protease activity was determined by the characteristic that the protease could generate molybdenum blue and tungsten blue products with the reduced Folin reagent under alkaline conditions. The specific operation of the enzyme activity determination was as follows: 0.8 mL of fermentation crude enzyme solution and 0.2 mL of casein were preheated at 40 °C for 2 min, mixed evenly and reacted at 40 °C for 1 h, 1 mL of TCA was added to terminate the reaction, precipitated for 10 min, centrifuged, 0.2 mL of the supernatant was taken, 1 mL of sodium carbonate was added and mixed, and then 0.2 mL of Folin phenol was added and the mixture was bathed in a 40 °C water bath for 20 min, and the OD was determined by colorimetric analysis. 680 The reaction system in which the crude enzyme solution was fermented after the activity was terminated by TCA was used as the control group.

[0052] 0.2 mL of tyrosine solution with different concentrations (0, 20, 40, 60, 80, 100, 150, 200 μg mL -1 ) was mixed with 1 mL sodium carbonate solution and 0.2 mL foline phenol in a 40°C water bath for 20 min, and the OD680 value was measured by color development. -1 ) as the abscissa and the absorbance value at 680 nm as the ordinate to draw a tyrosine standard curve.

[0053] The purified strain was inoculated into the fermentation medium and the protease activity was measured after 48 h of fermentation. Figure 2 As shown, the regression equation of the tyrosine standard curve is y=0.00648x+0.06809, and the coefficient of certainty R 2 =0.99831, K value is the concentration of tyrosine when the absorbance value is 1, and K = 143.81 can be obtained from the regression equation. The four strains screened all have the ability to produce protease, among which the protease activity in the fermentation broth of strain 4B is relatively high, which is 1164.86U·L -1 .

[0054] During the fermentation process of protease-producing functional strains, it was found that strain 4B produced a burnt smell, and then the fermentation broth was used for qualitative and quantitative detection of volatile substances using headspace solid phase microextraction-gas chromatography and mass spectrometry (HS-SPME-GC / MS), with the sterilized LB liquid culture medium as the blank control. The determination method is as follows.

[0055] 8 mL of fermentation liquid after passing through a 0.22 μm filter membrane was taken into a 20 mL headspace extraction bottle, and 2 g of NaCl was added to make it supersaturated. Then 10 μL of 0.2155 g·L -1 Concentration of ethyl decanoate was used as an internal standard. Three technical parallels were determined for each sample. Extraction was performed by headspace solid phase microextraction (50 / 30 μm DVB / CAR / PDMS fiber, Supelco, Bellefonte, PA, USA) at 60°C for 30 min. After extraction, the analyte-rich SPME extraction head was inserted into the GC system of a TSQ8000 triple quadrupole gas spectrometer (Thermo Fisher Scientific, USA), and the analytes were thermally desorbed at 250°C for 4 min before analysis. Secondly, a DB-5MS (60 m × 0.25 mm inner diameter × 0.25 μm thickness) was used in combination to identify volatile metabolites, with helium C-60 as the carrier gas at a flow rate of 1 mL min -1 The injection port was heated to 250°C. The oven temperature was fixed at 40°C for 2 min and the heating rate was 10°C min -1 The temperature was raised to 250°C at a rate of 100 °C and then maintained for 5 min. The transfer line and ion source temperatures were maintained at 280°C and 210°C, respectively. Electron impact (EI) was used as the ionization mode, and the EI voltage was 70 eV. The full scan mode used a mass scan range of 33-400 m / z, and the acquisition rate was 10 times·s -1 .

[0056] For the qualitative analysis of volatile metabolites, the computer spectral library WILEY8.0 and the data system library (NIST14) were compared, and the substances with similarity less than 800 and lacking CAS numbers were removed. Volatile metabolites with forward and reverse matching degrees above 800 and fragrance characteristics described in the Pubchem database were screened out. The internal standard method was used for calculation, and the content of the volatile metabolite was semi-quantitatively analyzed based on the ratio of the peak area of ​​the aroma volatile substance to the peak area of ​​the internal standard ethyl decanoate. The results were expressed in micrograms of volatile metabolites per milliliter of fermentation broth (μg·mL -1 Fermentation broth) indicated.

[0057] The fermentation liquid of strain 4B has a strong caramel aroma, similar to the aroma of roasted tea leaves, and it is speculated that the strain also has the function of producing aroma. Therefore, the volatile substances in the fermentation liquid after 48 hours of fermentation were measured, and the sterile fermentation medium under the same fermentation time was used as the control group. The content of substances with aroma attributes was semi-quantitatively calculated and a bar graph was drawn. The results are shown in the figure. Figure 3 As shown. Among them, the content of aldehydes and alcohols with sweet floral aroma in the fermentation broth of strain 4B decreased, or even disappeared, while ketones with a pleasant fruity aroma were produced, indicating that the alcohols and aldehydes in the culture medium were further converted into ketones during the aerobic growth of the strain. At the same time, pyrazines such as 2,5-dimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, and trimethylpyrazine in the fermentation broth of strain 4B increased significantly, especially the content of 2,5-dimethylpyrazine (semi-quantitative 380μg·L -1 ) is the control group (semi-quantitative 38.90 μg·L -1 These substances have pungent odors, mainly roasted nuts, roasted potatoes, and sweet woody aromas, which are the reason why the fermentation liquid of the 4B strain has a burnt aroma.

[0058] 3. Identification

[0059] The 16S rDNA of the strain was amplified using universal primers for bacterial identification, with the upstream primer being 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and the downstream primer being 1492R (5′-GGTTACCTTGTTACGACTT-3′). The PCR amplification system is shown in Table 1. The PCR amplification program was: 95°C for 3 min (pre-denaturation), 95°C for 15 s (denaturation), 55°C for 15 s (annealing), 72°C for 30 s (extension), and the denaturation-annealing-extension steps were repeated for 34 cycles, followed by extension at 72°C for 5 min to terminate the amplification.

[0060] Table 1 16S rDNA amplification system

[0061]

[0062] The gryA gene of the strain was then identified. Bacterial universal primers were used for amplification. The upstream primer was 42F (5′-CAGTCAGGAAATGCGTACGTCCTT-3′), and the downstream primer was 1066R (5′-CAAGGTAATGCTCCAGGCATTGCT-3′). The PCR amplification system is shown in Table 2. The PCR amplification program was: 95°C for 3 min (pre-denaturation), 95°C for 15 s (denaturation), 55°C for 15 s (annealing), 72°C for 15 s (extension), the denaturation-annealing-extension steps were repeated for 35 cycles, and then the amplification was terminated by extension at 72°C for 5 min.

[0063] Table 2gryA gene amplification system

[0064]

[0065] The PCR amplification products were verified by 2% agarose gel, and the amplification products with the correct band size were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing primers were the same as the amplification primers. After bidirectional splicing, the sequencing results were compared with BLAST on NCBI to determine the strain type.

[0066] The combined identification results of 16S rDNA and gyrA genes indicated that the 4B strain was a Bacillus licheniformis strain. It was named Bacillus licheniformis 4B. The strain has been deposited in the Guangdong Microbiological Culture Collection Center, and the strain collection number is GDMCC No: 65046.

[0067] Example 2: Fermentation of tea leaves using Bacillus licheniformis 4B

[0068] Bacillus licheniformis 4B was cultured in LB liquid medium at 37°C and 220 r·min -1 After activation culture for 24 h, the inoculum was transferred to the fermentation medium at 2% (v / v) at 37°C and 220 r / min. -1 The fermentation was carried out for 48 hours. 600 Adjust to about 1, spray 20% inoculum (add 2mL of strain fermentation liquid for every 10g of low-quality tea) evenly on the surface of low-quality tea, roll the tea leaves evenly so that the tea leaves are covered with fermentation liquid, and add 20% sterile fermentation medium (add 2mL of sterile fermentation medium for every 10g of low-quality tea, that is, the bacterial concentration is 1×10 6 CFU / mL) of low-quality rock tea was used as the control group. The above steps were all performed in sterilized self-sealing bags and placed in a constant temperature and humidity incubator for fermentation for 2 days at a temperature of 37°C and a humidity of 20%. After the fermentation, the total free amino acid content of some tea leaves was determined according to GB / T 8314-2013. The remaining tea leaves were spread on a metal tray, dried at 70°C, and then volatile substances were determined by HS-SPME-GC / MS. The method is as follows: weigh 2g of tea powder in a 20mL headspace extraction bottle, and add 10μL 0.2155g·L -1Concentration of ethyl decanoate was used as an internal standard to avoid the influence of dripping on the bottle wall on the semi-quantitative detection. Three techniques were used in parallel for each sample. The gas chromatography and mass spectrometry conditions were the same as in Example 1. The qualitative and semi-quantitative analysis methods of metabolites were the same as in Example 1. The results were expressed in micrograms of volatile metabolites per kilogram of dry weight of tea sample (μg·kg -1 Tea leaves dry weight).

[0069] The fermentation liquid of strain Bacillus licheniformis-4B was inoculated on the surface of low-quality tea. After 2 days of fermentation, free amino acids and volatile substances (pyrazines) were determined. The results are as follows: Figure 4 As shown. The free amino acid content in tea leaves increased by 17% after fermentation with the addition of strains, and decreased by 8% after drying. In addition, the content of pyrazines in tea fermented with strains was at least 1 times higher than that in original low-quality tea and tea fermented with sterile fermentation medium, which were 1.9 times that of the control group and 2.6 times that of the original tea, respectively. Among them, 2,5-dimethylpyrazine and 2-methylpyrazine were prominent in tea fermented with strains, and the contents after semi-quantitative determination were 133.33±3.23μg·kg -1 and 214.21±5.58μg·kg -1 , while 2-ethyl-5-methylpyrazine and 2-ethyl-3,5-dimethylpyrazine are almost unique to the strain fermented tea. The production pathway of pyrazine compounds is reductive ketone and dehydrogenative ketone, which are formed by heterocyclization of amino ketone and Strecker aldehyde in the presence of amino acids. The higher the concentration of amino acids provided, the greater the proportion of pyrazine production, and the production of pyrazine increases rapidly from 70°C, and the production rate reaches a maximum at 120°C. Therefore, in addition to the strain itself producing high-content pyrazine substances (2,5-dimethylpyrazine and 2-ethyl-3,5-dimethylpyrazine), it can degrade tea proteins to produce amino acids, providing precursors for other pyrazine substances with a roasted nut smell (such as 2-methylpyrazine, 2-ethyl-5-methylpyrazine, etc.) added to the fermented tea with added functional strains, thereby optimizing the quality of low-quality tea.

[0070] Table 3 Pyrazine content of fermented tea leaves

[0071]

[0072] Note: “-” indicates substances that could not be detected or whose content was too low to be detected.

[0073] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Bacillus licheniformis 4B, characterized in that: The Bacillus licheniformis 4B was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 23, 2024, with the deposit number GDMCC No: 65046.

2. A microbial preparation containing the Bacillus licheniformis 4B according to claim 1.

3. The preparation according to claim 2, characterized in that The amount of Bacillus licheniformis 4B added to the microbial preparation is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. A leavening agent, characterized in that The fermentation agent contains the Bacillus licheniformis 4B according to claim 1 or the microbial preparation according to claim 2 or 3.

5. Use of the Bacillus licheniformis 4B according to claim 1, or the microbial preparation according to claim 2 or 3, or the starter according to claim 4 in fermenting tea leaves.

6. A fermentation method for Wuyi rock tea, characterized in that: The method comprises fermenting tea leaves with the Bacillus licheniformis 4B of claim 1, the microbial preparation of claim 2 or 3 or the starter of claim 4.

7. The method according to claim 6, characterized in that The method comprises the following steps: spraying the culture solution of Bacillus licheniformis 4B on the surface of tea leaves for fermentation for 2 to 3 days, the fermentation temperature being 35 to 40° C., and the fermentation humidity being 20 to 30%.

8. Use of the Bacillus licheniformis 4B according to claim 1, or the microbial preparation according to claim 2 or 3, in increasing the yield of pyrazines in tea leaves.

9. The use according to claim 8, characterized in that: The pyrazine substances include 2,5-dimethylpyrazine and 2-ethyl-3,5-dimethylpyrazine.

10. A method for increasing the content of pyrazines in tea, characterized in that: The method comprises fermenting tea leaves using the Bacillus licheniformis 4B described in claim 1 or the microbial preparation described in claim 2 or 3.

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