Screening and application of streptomyces
By screening and applying Streptomyces sp. G57, the problem of insufficient aroma of tea was solved. By fermenting tea, the content of reducing sugar and volatile aroma substances was increased, and the sweetness and aroma of tea was significantly improved.
Patent Information
- Application Number
- CN202411558583.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-04
- Publication Date
- 2025-02-14
AI Technical Summary
In the prior art, the effect of Streptomyces on the aroma and taste of tea leaves is unknown, and a microorganism that can enhance the sweetness of tea soup and the aroma of tea leaves is lacking.
A strain of Streptomyces sp. G57 was screened and used. This strain has high pectinase activity. By fermenting tea leaves, it promotes pectin hydrolysis, increases the reduced sugar content of tea leaves and the production of volatile aroma substances.
Through the fermentation of Streptomyces G57, the reduced sugar content of tea increased by 28.53%, and volatile substances with sweet and floral properties were produced, such as levon-β-pinene, ethyl hexanoate and β-cyclolemon, which had sweet and floral properties. Aldehyde significantly improves the sweetness and aroma of tea.
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Abstract
Description
Technical Field
[0001] The invention relates to the screening and application of a Streptomyces strain, and belongs to the technical field of microorganisms. Background Art
[0002] Pectin in tea is mainly found in plant cell walls and is the main substance that constitutes the intercellular layer of tea. Low-quality Wuyi rock tea has the disadvantage of insufficient aroma. By adding exogenous enzymes to ferment tea, the release of aroma substances can be promoted. Studies have shown that the aroma quality of aged Wuyi rock tea has been significantly improved by fermenting it with cellulase, pectinase, laccase, and β-glucosidase (Xie Liling, Xue Wanru, Li Danyang, et al. Effect of exogenous enzymes on the aroma quality of aged Wuyi rock tea [J]. Food Research and Development, 2023, 44(05): 155-164.).
[0003] Streptomyces has the ability to produce pectinase. Researchers have isolated several strains of Streptomyces from humus-rich soils that have the ability to produce pectinase, which they have applied to the fermentation of potato starch byproducts (Qu Ziyue. Construction of a Pectinase-Producing Bacterial Community and Its Effect on the Conversion of Potato Starch Byproducts [D]. Harbin Institute of Technology, 2019). However, it is unknown whether Streptomyces can affect the aroma of tea, and its effect on the taste of tea is also unclear. Therefore, there is an urgent need to develop microbial agents that can not only promote the breakdown of tea plant cell walls, produce reducing sugars, and enhance the sweetness of tea, but also provide tea with other secondary metabolites, thereby enhancing the tea's aroma. Summary of the Invention
[0004] In response to the above-mentioned deficiencies in the existing technology, the present invention provides the screening and application of a strain of Streptomyces. The purpose is to solve the technical problem of the lack of a microorganism that can enhance the sweetness of tea soup and increase the aroma of tea, as it is currently unknown whether Streptomyces can affect the aroma of tea, and its effect on the taste of tea is also unclear.
[0005] The first technical solution provided by the present invention is a strain of Streptomyces sp. G57, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 23, 2024, with a deposit number of GDMCC No: 65047.
[0006] The second technical solution provided by the present invention is a microbial preparation containing the Streptomyces G57 described in the first technical solution.
[0007] In certain embodiments, the amount of Streptomyces G57 added to the microbial preparation is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.
[0008] The third technical solution provided by the present invention is a starter culture, which contains the Streptomyces G57 described in the first technical solution or the microbial preparation described in the second technical solution.
[0009] In certain embodiments, the amount of Streptomyces G57 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 Streptomyces G57 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] The fifth technical solution provided by the present invention is a method for fermenting tea leaves, which comprises fermenting tea leaves with the Streptomyces G57 described in the first technical solution, the microbial preparation described in the second technical solution, or the starter described in the third technical solution.
[0012] In certain embodiments, the method comprises spraying the culture solution of Streptomyces G57 on the surface of tea leaves for fermentation for 2 to 3 days, at a fermentation temperature of 35 to 40° C., and at a fermentation humidity of 80 to 85%. Preferably, the method comprises spraying the culture solution of Streptomyces G57 on the surface of tea leaves for fermentation for 3 days, at a fermentation temperature of 37° C., and at a fermentation humidity of 85%.
[0013] In certain embodiments, the preparation method of Streptomyces G57 culture solution is as follows: Streptomyces G57 is cultured in LB liquid medium at 37°C and 220 r·min -1 The activated culture was cultured for 24 h, and then transferred to the fermentation medium at a 2% (v / v) inoculum, and incubated at 37°C and 220 r·min. -1 Fermentation culture for 72 hours.
[0014] The sixth technical solution provided by the present invention is the use of Streptomyces G57 described in the first technical solution, or the microbial preparation described in the second technical solution, in improving the sweetness and aroma of tea.
[0015] In certain embodiments, the substance emitting the aroma includes L-β-pinene, ethyl hexanoate, and β-cyclocitral.
[0016] The seventh technical solution provided by the present invention is a method for improving the sweetness and aroma of tea, wherein the method comprises fermenting tea leaves using the Streptomyces G57 described in the first technical solution or the microbial preparation described in the second technical solution.
[0017] In certain embodiments, the method comprises spraying the culture solution of Streptomyces G57 on the surface of tea leaves and fermenting for 2 to 3 days at a fermentation temperature of 35 to 40° C. and a fermentation humidity of 80 to 85%. Preferably, the method comprises spraying the culture solution of Streptomyces G57 on the surface of tea leaves and fermenting for 3 days at 37° C. and a humidity of 85%.
[0018] In certain embodiments, the substance emitting the aroma includes L-β-pinene, ethyl hexanoate, and β-cyclocitral.
[0019] The technical effects of the present invention are as follows:
[0020] The present invention uses a specific plate screening method with pectin as a substrate to obtain a strain Streptomyces sp. G57 with the ability to produce pectinase, thereby increasing the reducing sugar content and aroma content of tea. The pectinase activity in the fermentation broth of the strain is 1.82 U·L -1 When Streptomyces sp.-G57 was used to ferment low-quality tea, pectin was hydrolyzed, reducing sugar content increased by 28.53%, and volatile compounds with floral and fruity aromas, such as L-β-pinene (resin aroma), ethyl hexanoate (fruity aroma), and β-cyclocitral (herbal, rose aroma), were produced, with concentrations of 3.86±0.31μg·kg -1 , 1.40±0.27μg·kg -1 and 1.37±0.29μg·kg -1 . The purpose of enhancing the sweetness and aroma of tea is achieved.
[0021] Biomaterial Deposit
[0022] The Streptomyces G57 of the present invention has a taxonomic name of Streptomyces sp. and was deposited in the Guangdong Provincial Microbial Culture Collection Center on August 23, 2024. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 65047. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 Pectinase activity of the strains. A. Pectinase activity bar graph; B. Transparent circle on the identification plate of G57 strain.
[0024] Figure 2 Reducing sugar content in fermented low-quality tea. A: Standard curve for glucose standard solution; B: Reducing sugar content in tea leaves. Note: Group P represents untreated low-quality tea; Group CK represents a control group with an equal amount of water added; Group G57 represents an experimental group fermented with the strain.
[0025] Figure 3The volatile compounds added to low-quality rock tea after fermentation. Note: Group P is low-quality tea that has not undergone any treatment; Group C is the control group with an equal amount of water added; Group S is the experimental group with the addition of a strain for fermentation. DETAILED DESCRIPTION
[0026] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.
[0027] The culture medium used in the following examples is:
[0028] Enrichment medium: LB liquid medium, tryptone (10g L -1 ), yeast extract (5g·L -1 )、NaCl(10g·L -1 );
[0029] Pectinase screening and identification culture medium: K2HPO4 (1g·L -1 )、MgSO4·7H2O(0.5g·L -1 )、NaNO3(3g·L -1 )、FeSO4·7H2O(0.01g·L -1 ), pectin (2g·L -1 ), agar (20 g·L -1 );
[0030] Fermentation medium: pectin (10g·L -1 ), peptone (10 g·L -1 )、NaCl(5g·L -1 )、K2HPO4(1g·L -1 ).
[0031] Among them, tryptone and yeast extract were purchased from Thermo Fisher Scientific, agar was purchased from Sangon Biotechnology Co., Ltd., and pectin, NaNO3, FeSO4·7H2O, MgSO4·7H2O, NaCl and K2HPO4 were purchased from Sinopharm Chemical Reagent Co., Ltd.
[0032] Example 1 Screening and identification of strains.
[0033] 1. Initial screening
[0034] Soil was collected from a tea garden in Wuyishan Scenic Area, Wuyishan City, Fujian Province. Rhizosphere soil was collected at a depth of 20–40 cm, 20 cm from the main trunk of the tea tree, using a five-point sampling method. Loose soil associated with the roots was shaken off, and the remaining soil and roots were placed in a sterile, sealed plastic bag. The bag was immediately brought back to the laboratory, mixed thoroughly, and passed through a 2 mm sieve. Soil samples were stored at -80°C for subsequent experiments.
[0035] 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 stirred at 10 °C and 220 r·min. -1 The total microorganisms in the soil were eluted by shaking for 3 hours under the same conditions. The solid-liquid mixture was then filtered through 4 layers of sterile gauze, and the filtrate was centrifuged in a 50 mL centrifuge tube at 8000 rpm for 15 minutes at 10°C. The operation was repeated and the precipitates were combined to obtain the bacterial cells, which were the total microorganisms in the soil. The eluted bacterial liquid was inoculated into LB liquid 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 a pectinase primary screening identification plate and cultured in a 37°C constant temperature incubator. Colonies growing on the plate and having a transparent circle were selected and further streaked and isolated to obtain single colonies.
[0036] LB culture medium and pectinase primary screening identification plates were used to screen strains capable of decomposing and producing pectinase. After enrichment culture and multiple purifications, the strains were cultured at 37°C for 3 days. Strains with D / d (transparent zone diameter / bacterial diameter) greater than 3 were selected, and finally 12 strains with the ability to produce pectinase were isolated.
[0037] 2. Rescreening
[0038] The pectinase-producing strains screened were first inoculated into LB liquid medium and incubated at 37°C and 220 r·min. -1 Activate for 48 hours and adjust OD 600 At 0.4-0.8, transfer 3% of the inoculum to the fermentation medium, 28℃, 160r·min -1 After 72 h of incubation, the mixture was centrifuged at 7000 rpm and 4°C for 10 min, and the supernatant was obtained as the crude enzyme solution, which was then used to test the pectinase activity.
[0039] The specific experimental procedure is as follows: Using 0.25% pectin solution as the reaction substrate, pectinase is used to hydrolyze pectin to generate galacturonic acid, which can react with DNS solution to form a colorimetric reaction, thereby determining the pectinase activity. The specific procedure is as follows: 0.25% pectin solution is preheated in a 40℃ water bath, 0.2mL of crude enzyme solution is added to the preheated 0.2mL pectin solution, and after reacting at 40℃ for 1h, 0.2mL of 0.4mol·L -1 After the reaction is completed, mix with 0.4 mL of DNS solution and react in a boiling water bath for 5 minutes to develop color. After 1-2 minutes in an ice water bath, measure the OD 540 . With 0.4 mol·L - 1 The reaction system in which crude enzyme solution was inactivated by NaOH was used as a control.
[0040] Prepare different concentrations of D-galacturonic acid standard solutions (0, 1.0, 2.0, 3.0, 4.0, 5.0 mg mL -1 ), take 600 μL of different concentrations of standard solution, mix with 400 μL DNS solution, make three parallels for each standard, boil in water bath for 5 minutes, cool in ice water bath for 1 minute, and detect OD 540 .
[0041] Definition of pectinase activity: The amount of enzyme required to decompose pectin to produce 1 mg of galacturonic acid in 1 min at 40°C and pH 4.6 is defined as 1 enzyme activity unit (U).
[0042] The calculation formula of pectinase activity is as follows:
[0043]
[0044] Where:
[0045] A is the OD of the experimental group and the control group 540 Difference;
[0046] K is the absorption constant;
[0047] T is the reaction time, in min;
[0048] V is the total volume of the reaction reagents in mL.
[0049] The purified strain was inoculated into the fermentation medium and the pectinase activity was measured after 72 h of fermentation. Figure 1 As shown in the figure, the regression equation of the galacturonic acid standard curve is y = 0.7735x + 0.187, where x is the concentration of galacturonic acid (in mg mL -1 ), y is the absorbance at 540 nm, and the coefficient of certainty R 2 =0.9932, K value is the concentration of galacturonic acid when the absorbance value is 1, and the regression equation shows K = 1.051. All the strains screened have the ability to produce protease, among which the G57 strain has the highest pectinase activity in the fermentation broth, which is 1.82U·L -1 .
[0050] 3. Identification
[0051] Strain G57 16S rDNA was amplified using universal primers for bacterial identification. The upstream primer was 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and the downstream primer was 1492R (5′-GGTTACCTTGTTACGACTT-3′). The PCR amplification system is shown in Table 1. The PCR amplification program was as follows: 95°C for 3 minutes (pre-denaturation), 95°C for 15 seconds (denaturation), 45°C for 15 seconds (annealing), and 72°C for 30 seconds (extension). The denaturation-annealing-extension step was repeated for 34 cycles, followed by extension at 72°C for 5 minutes to terminate the amplification.
[0052] Table 1 16S rDNA amplification system
[0053]
[0054] PCR amplification products were verified using 2% agarose gels. Amplification products with the correct band size were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing using the same primers as the amplification primers. Sequencing results were bidirectionally spliced and then BLAST alignment was performed on NCBI to determine bacterial genus identity.
[0055] 16S rDNA identification results indicate that strain G57 is a Streptomyces sp. strain. It has been named Streptomyces sp. G57 and is currently deposited with the Guangdong Provincial Center for Microbiological Culture Collection, with the strain collection number GDMCC No. 65047.
[0056] Example 2: Fermentation of tea leaves using Streptomyces sp. G57
[0057] Streptomyces sp. G57 was cultured in LB liquid medium at 37°C and 220 r·min -1 The activated culture was cultured for 24 h, and then transferred to the fermentation medium at a 2% (v / v) inoculum, and incubated at 37°C and 220 r·min. -1 Fermentation was carried out for 72 hours. 600 Adjust to about 1, with a 20% inoculation amount (2 mL of strain fermentation liquid is added to every 10 g of low-quality tea, i.e., the bacterial concentration is 1×10 6CFU / mL) is evenly sprayed onto the surface of low-quality rock tea, and the tea leaves are evenly rolled so that the tea leaves are covered with fermentation liquid. Low-quality tea with 20% sterile water added (2mL sterile water is added for every 10g of low-quality tea) is used as the control group. The above steps are all performed in a sterilized self-sealing bag and placed in a constant temperature and humidity incubator for fermentation for 3 days at a temperature of 37°C and a humidity of 85%. After fermentation, the tea leaves are spread flat on a metal tray and dried at 70°C. The reducing sugar content of the tea leaves is detected, and the volatile substances are determined by headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC / MS).
[0058] Reducing sugars are detected using the DNS assay, which uses a colorimetric reaction between reducing sugars and a DNS solution to determine their content. The procedure is as follows: Weigh 0.6g of tea leaves into a 50mL centrifuge tube, add 30mL of boiling water, and incubate in a boiling water bath for 45 minutes to fully extract the tea leaves. After extraction, cool to room temperature, centrifuge at 7000rpm for 10 minutes, and retain the supernatant. Mix 1.0mL of the supernatant with 2.0mL of the DNS solution. Perform triplicate analysis for each standard. Boil in water for 2 minutes, cool on ice for 1 minute, and measure absorbance at 540nm.
[0059] Prepare glucose standard solutions of different concentrations (0, 0.2, 0.4, 0.6, 0.8, 1.0 mg mL -1 ), take 1 mL of standard solution of different concentrations, mix evenly with 2 mL of DNS solution, boil in a boiling water bath for 2 min, cool in an ice water bath for 1 min, and measure the absorbance at 540 nm.
[0060] The fermentation liquid of strain Streptomyces sp. G57 was inoculated on the surface of low-quality tea, and the reducing sugar and volatile substances were determined after 3 days of fermentation. Figure 2 As shown, the regression equation of the glucose standard curve is y = 2.6334x + 0.078, where x is the concentration of the glucose standard solution (in mg mL -1 ), y is the absorbance at 540 nm, and the coefficient of certainty R 2 =0.9966. The reducing sugar content of tea leaves fermented for 3 days after inoculation with Streptomyces sp. G57 was 20.61 mg·g -1 Compared with untreated tea leaves, the reducing sugar content increased by 28.53%, and compared with water-treated tea leaves, the reducing sugar content increased by 26.09%. This indicates that after the strain is inoculated into tea leaves, it promotes the hydrolysis of pectin into reducing sugars by producing pectinase.
[0061] The volatile substances were detected as follows: 2 g of tea powder was weighed into a 20 mL headspace extraction bottle, and 10 μL of 0.2155 g·L was added to the center of the powder solid.-1 Ethyl decanoate was used as an internal standard to prevent the influence of dripping onto the bottle wall and affecting the semi-quantitative detection. Each sample was measured in triplicate. 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 tip was inserted into a TSQ8000 triple quadrupole gas chromatography-mass spectrometer (Thermo Fisher Scientific, USA). The analytes were thermally desorbed at 250°C for 4 min before analysis. Volatile metabolites were then identified using a DB-5MS (60 m × 0.25 mm ID × 0.25 μm thickness) 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 temperature was adjusted to 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) ionization mode was used with an EI voltage of 70 eV. The full scan mode used a mass scan range of 33-400 m / z with an acquisition rate of 10 scans / s. -1 .
[0062] For the qualitative analysis of volatile metabolites, a comparison was performed between the computer spectral library WILEY8.0 and the data system library (NIST14). Substances with similarities less than 800 and lacking CAS numbers were removed. Volatile metabolites with forward and reverse matches of 800 or more and aroma characteristics described in the Pubchem database were screened. The internal standard method was used to calculate the ratio of the peak area of the aroma volatile substance to the peak area of the internal standard ethyl decanoate, and the content of the volatile metabolite was semi-quantitatively analyzed. The results were expressed as micrograms of volatile metabolites per kilogram of dry weight of tea sample (μg·kg -1 tea leaves dry weight).
[0063] like Figure 3 As shown in Table 3, fermentation with the strain Streptomyces sp. G57 produces cis-4-heptenal, β-cyclocitral, hexyl hexanoate, 2-butyl-1-octanol, styrene, terpinolene, and L-β-pinene compared to unfermented tea leaves. Most of these substances have sweet and floral aromas, as shown in Table 3. Semi-quantitative analysis revealed that the content of L-β-pinene (resin aroma) was 3.86 ± 0.31 μg kg -1 The content of ethyl hexanoate (fruity aroma) was 1.40±0.27μg·kg -1 β-cyclocitral (herbal, rose aroma) content was 1.37±0.29μg·kg -1Sweet and fruity aromas are the aroma attributes of high-quality rock tea. Therefore, the aroma quality of tea fermented with this strain is improved to a certain extent.
[0064] Table 2 Aroma characteristics of volatile substances added to low-quality rock tea after fermentation
[0065]
[0066] Although the present invention has been disclosed above in terms of preferred embodiments, 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 Streptomyces sp. G57, characterized in that: The Streptomyces G57 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 23, 2024, with the deposit number GDMCC No: 65047.
2. A microbial preparation containing the Streptomyces G57 according to claim 1.
3. The preparation according to claim 2, characterized in that The amount of Streptomyces G57 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 Streptomyces G57 according to claim 1 or the microbial preparation according to claim 2 or 3.
5. Use of the Streptomyces G57 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 method for fermenting tea leaves, characterized in that: The method comprises fermenting tea leaves with the Streptomyces G57 according to claim 1, the microbial preparation according to claim 2 or 3, or the starter according to claim 4.
7. The method according to claim 6, characterized in that The method comprises the following steps: spraying the culture solution of Streptomyces G57 on the surface of tea leaves for fermentation for 2-3 days, the fermentation temperature being 35-40° C., and the fermentation humidity being 80-85%.
8. Use of the Streptomyces G57 according to claim 1, or the microbial preparation according to claim 2 or 3, in improving the sweetness and aroma of tea.
9. A method for improving the sweetness and aroma of tea leaves, the method comprising fermenting tea leaves using the Streptomyces G57 described in claim 1 or the microbial preparation described in claim 2 or 3.
10. The method according to claim 9, characterized in that The method comprises the steps of spraying the culture solution of Streptomyces G57 on the surface of tea leaves, and fermenting the tea leaves for 2 to 3 days at a temperature of 35 to 40° C. and a humidity of 80 to 85%.