An actinomycete with the ability to produce odor and its application

Abstract

The present invention belongs to the field of microorganisms, and specifically relates to an actinomycete with the ability to produce odors and its application. This actinomycete strain has a strong ability to produce odors. During the germination period of this strain, the FSC increases from 2594 at 0 h to 3644 at 5 h, and the concentration of 2-methylisoborneol increases from 6.3144 ng / L at 0 h to 30.4838 ng / L at 5 h, showing excellent ability to produce 2-methylisoborneol. This strain can be used to determine the source of the odor problem in water sources.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and particularly to an actinomycete with the ability to produce odors and its application. Background Art

[0002] The problem of odors, especially the earthy and musty odors in water source reservoirs, has become a serious challenge to drinking water safety. Geosmin and 2-methylisoborneol are the main odor-causing substances, with extremely low human odor thresholds. According to the national standard "Hygienic Standard for Drinking Water" GB 5749-2022, the drinking water limit values of these substances are only 10 ng / L, and the standard stipulates that the sensory properties of drinking water are good, without abnormal odors or tastes. During the seasonal reproduction of aquatic microorganisms such as cyanobacteria, actinomycetes, myxobacteria, and fungi in natural water bodies, a series of secondary metabolites will be released, including these two substances. Cyanobacteria are considered the main source of these two odor-causing compounds, which has attracted the attention of scholars. However, during the non-cyanobacteria bloom stage, the research on odor-causing substances mainly secreted by actinomycetes is often overlooked. Determining the source of the odor problem is of great significance for targeted prevention and control of the odor problem. The odor production by actinomycetes requires less light than that by algae and has a wide spatial distribution, not limited to the shallow layer of the reservoir. Moreover, actinomycetes in natural water bodies can be divided into active hyphae and dormant spores. Actinomycete spores have strong adaptability and tolerance to extreme environments and need to germinate into hyphae and produce odor-causing substances under suitable conditions. This process is usually random and regulated by various objective factors such as temperature, nutrient salts, and pH. Limited by this, the odor events caused by the germination of actinomycete spores will lose the opportunity for early intervention. Therefore, it is necessary to provide actinomycetes with strong odor-producing ability as detection targets to determine the source of the odor problem in water sources. Summary of the Invention

[0003] To solve the above technical problems, the present invention provides an actinomycete with the ability to produce odors and its application.

[0004] An actinomycete with the ability to produce odors, the actinomycete is Streptomyces antibioticus LJH21, which is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 20241163, the deposit date is June 6, 2024, and the deposit address is Wuhan University, Wuhan, China.

[0005] The application of the actinomycete in determining the source of the odor problem in water sources.

[0006] Preferably, the water to be tested is incubated at a constant temperature of 30 °C in a solid microbial medium containing potassium dichromate to obtain a pre-test substance. The pre-test substance is incubated at a constant temperature of 30 °C in a spore-producing medium to obtain a pre-germinated substance. The pre-germinated substance is germinated and cultured at 30 °C in a liquid microbial medium. During the germination culture process, germination determination is carried out to determine the germination of the actinomycetes. At the same time, the content of 2-methylisoborneol is detected during the germination process. If the content of 2-methylisoborneol increases, the actinomycetes are one of the sources of water odor initiation;

[0007] The formula of the solid microbial medium is: 20 g of soluble starch, 1 g of potassium nitrate, 0.5 g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate, 0.5 g of sodium chloride, 0.01 g of ferrous sulfate, 15 g of agar, and water is made up to 1 L;

[0008] 80 mg of potassium dichromate is added to each liter of the solid microbial medium;

[0009] The formula of the liquid microbial medium is: 20 g of soluble starch, 1 g of potassium nitrate, 0.5 g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate, 0.5 g of sodium chloride, 0.01 g of ferrous sulfate, and water is made up to 1 L;

[0010] The formula of the spore-producing medium is: 20 g of oatmeal, 0.001 g of ferrous sulfate, 0.001 g of manganese chloride, 0.001 g - 0.0012 g of zinc sulfate, 1.29 g of calcium chloride, 0.95 g of magnesium chloride, and water is made up to 1 L.

[0011] Preferably, the method for germination determination includes the following steps: The pre-germinated substance is germinated and cultured at 30 °C in the liquid microbial medium to obtain a mixed actinomycete solution. The mixed actinomycete solution is mixed with the staining agent of a flow cytometer, and after heating in the dark, a bacterial solution is obtained. The bacterial solution is measured by a flow cytometer to obtain the germination rate of the spores. In the prior art, the determination method of spore germination still mainly relies on traditional optical microscope observation, with low sample measurement efficiency and strong subjectivity, which will greatly extend the detection time and cause the odor events caused by the germination of actinomycete spores to lose the opportunity for early intervention. The method and actinomycetes used in the application of the present invention simply and effectively identify the germination state of actinomycete spores.

[0012] Preferably, the volume ratio of the mixed actinomycete solution to the staining agent is 195:5.

[0013] Preferably, the staining agent is a mixed solution of SYBR Green I and dimethyl sulfoxide, and the volume ratio of SYBR Green I to dimethyl sulfoxide is 5:495.

[0014] Preferably, the heating condition is heating at 35 °C for 15 min.

[0015] Preferably, a gas chromatography-mass spectrometer is used to detect the content of 2-methylisoborneol.

[0016] Preferably, the detection conditions are as follows: the chromatographic column is DB-WAX, 30.0 m × 250 μm, 0.25 μm; the initial temperature is 40 °C and is maintained for 2 min, then raised to 240 °C at a rate of 10 °C / min and maintained for 3 min; the injection port temperature is 250 °C; the transfer line temperature is 240 °C; the carrier gas is He; the carrier gas flow rate is 1.0 mL / min; splitless; mass spectrometry conditions: EI source; electron energy 70 eV; ion source temperature 230 °C; quadrupole 150 °C; the scan mode is Scan; the scan mass range is 35 u to 550 u.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The actinomycetes of the present invention are Streptomyces antibioticus LJH21, which is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 20241163. Its metabolites contain geosmin and 2-methylisocamphenol. During the germination period of this strain, the cell particle size increases from 2594 at 0 h to 3644 at 5 h, and the relative growth rate reaches 40.5%. The concentration of 2-methylisocamphenol increases from 6.3144 ng / L at 0 h to 30.4838 ng / L at 5 h, showing excellent ability to produce 2-methylisocamphenol.

[0019] Using the actinomycetes with the ability to produce 2-methylisocamphenol of the present invention as the detection target can determine the source of the odor in the water source, which is conducive to seizing the opportunity for early intervention, so as to effectively control the water source odor problem as early as possible and ensure the safety of the water source. Description of the Drawings

[0020] Figure 1 It is a phylogenetic tree diagram of the actinomycetes strain.

[0021] Figure 2 It is a diagram for measuring the germination of actinomycetes spores. Among them, (a) is the analysis of the particle size range of actinomycetes spores by flow cytometry, (b) is the comparison of the cell particle sizes of actinomycetes spores at 0 h and 5 h of germination, and (c) is the change trend of the cell particle size and the relative growth rate of actinomycetes spores from 0 h to 5 h of germination. In the figure, SSC represents the cell granularity, FSC represents the cell particle size, and the A connected to SSC and FSC is the abbreviation of area, representing a way of reading data.

[0022] Figure 3It is a comparison chart of the cell granularity range of actinomycete spores at 0 h and 5 h of germination. Among them, (a) is the cell granularity range chart of actinomycete spores at 0 h of germination, and (b) is the cell granularity range chart of actinomycete spores at 5 h of germination. In the figure, SSC represents cell granularity, and A connected after SSC is the abbreviation of area, representing a way of reading data.

[0023] Figure 4 It is a line chart of the production of 2-methylisoborneol by actinomycetes during spore germination.

[0024] Figure 5 It is a comparison chart of the cell morphology of actinomycete spores at 0 h and 5 h of germination based on scanning electron microscopy. Among them, (a) is the cell morphology chart of actinomycete spores at 0 h of germination, and (b) is the cell morphology chart of actinomycete spores at 5 h of germination. Specific implementation mode

[0025] The following describes the specific implementation mode of the present invention in detail, but it should be understood that the protection scope of the present invention is not limited by the specific implementation mode. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the protection scope of the present invention. The experimental methods described in each embodiment of the present invention are all conventional methods unless otherwise specified.

[0026] Example 1

[0027] A method for screening odor-producing actinomycetes includes the following steps:

[0028] Step (1), preparation of a solid medium for enriching culturable actinomycetes: Each liter of the solid medium includes 20 grams of soluble starch, 1 gram of potassium nitrate, 0.5 gram of dipotassium hydrogen phosphate, 0.5 gram of magnesium sulfate, 0.5 gram of sodium chloride, 0.01 gram of ferrous sulfate, and 15 grams of agar. Mix the above substances in a 1000 mL volumetric flask, add ultrapure water to make up the volume, pour it into a 1 L sterile conical flask, and adjust the pH to 7.4. Put the above medium into an autoclave and sterilize it at 121 °C for 30 min, and add 80 mg of potassium dichromate to prevent the growth of bacteria and fungi. Wait until it cools to 55 °C and prepare it into a solid plate.

[0029] Preparation of a liquid medium for enriching culturable actinomycetes: The difference from the solid medium is that agar and potassium dichromate are not added, and the remaining steps and conditions are the same as those for the preparation of the solid medium.

[0030] Step (2), enrichment culture: Take 20 mL from the water sample collected from the water source reservoir.

[0031] Filter a 0.22μm polyester fiber membrane. After the filtration is completed, use sterile forceps to remove the filter membrane and attach it to the solid plate of actinomycetes. Wrap it with sealing film to ensure sufficient oxygen in the constant temperature incubator. Invert the plate and place it in a 30°C constant temperature incubator for 8 days until the colony size is uniform and can be picked.

[0032] Step (3): Pick the white bacterial cells in the culture in step (2) and perform streak isolation on the solid medium prepared in (1). Ensure sufficient oxygen in the constant temperature incubator. Invert the plate and place it in a 30°C constant temperature incubator for 8 days. Under a sterile environment, perform odor recognition on the screened actinomycetes. Repeat the above steps until the actinomycetes on the solid plate have a single color, consistent appearance size, and no associated other actinomycetes.

[0033] Step (4): Prepare the spore-producing medium for actinomycetes: The formula of the spore-producing medium is 20 grams of oatmeal per liter, 0.001 grams of ferrous sulfate, 0.001 grams of manganese chloride, 0.001 grams of zinc sulfate, 1.29 grams of calcium chloride, 0.95 grams of magnesium chloride, and the solvent is ultrapure water. Put the above medium into an autoclave and sterilize it at 121°C for 30 minutes. Wait until it cools to 55°C and prepare it into a solid plate.

[0034] Calcium and magnesium ions are key trace elements in the growth of actinomycetes. Calcium ions are crucial for maintaining the stability of the spore wall, which will improve the environmental adaptability of spores. Moreover, magnesium ions are related to the protein synthesis of actinomycetes. The lack of magnesium ions will lead to a decrease in spore production. Therefore, in the present invention, by adding these two elements additionally, the purpose of generating high-quality actinomycete spores is achieved.

[0035] Step (5): Pick the well-screened actinomycetes in step (3) into the spore-producing medium for actinomycetes in step (4) and perform streaking. Wrap it with sealing film. Invert the plate and place it in a 30°C constant temperature incubator for 12 days.

[0036] Step (6): Use a sterile cotton swab to scrape the actinomycete spores cultured in step (5) to make the spores suspended in a sterile phosphate buffer solution. Use an oscillator to shake vigorously, and then filter the spore suspension through three layers of sterile lens paper. Repeat the shaking and filtering three times. Determine the final concentration of the spore suspension by the dilution plating method. The final concentration of the spore suspension is 10 8 CFU / mL. Finally, mix the spore suspension of actinomycetes with sterile glycerol at a ratio of 1:1 in a 50 mL centrifuge tube and store it in a -20°C refrigerator.

[0037] Identify the odor-producing actinomycetes, including odor-producing ability identification, molecular biology identification, spore germination determination, and morphological determination.

[0038] I. Identification of the odor-producing ability of odor-producing actinomycetes

[0039] Prepare the actinomycete culture medium in Step (1) of Preparation Example 1. Sterilize the culture medium together with a 250 mL conical flask. After sterilization, take 100 mL of the culture medium and add it to the 250 mL conical flask. Take 1 mL of the spore suspension and add it to the 250 mL conical flask. Place the above conical flask in an oscillating incubator for cultivation at a rotation speed of 130 rpm and a temperature of 30 °C. After culturing in the dark for 10 days, extract 10 mL of the bacterial liquid for odor measurement.

[0040] For odor measurement, pretreatment of the bacterial liquid is required. The specific operation is as follows: Take 8 mL of the bacterial liquid and transfer it into a 15 mL extraction bottle. Take 2.5 g of NaCl and a magnetic stir bar, and then quickly seal it. Age the SPME extraction fiber head at 250 °C in the GC-MS injection port until there are no impurity peaks. Place the sample bottle on the solid-phase microextraction device, set the temperature to 70 °C, and the stirring speed to 500 rpm; preheat the sample bottle on the extraction device for 15 min; insert the SPME extraction head through the bottle cap into the headspace part of the sample, push out the fiber head, with the extraction head about 1.0 cm above the upper surface of the sample, and perform headspace extraction for 20 min; withdraw the fiber head and pull out the extraction head from the sample bottle; then insert the extraction head into the GC-MS injection port, push out the fiber head, and desorb at 250 °C for 3 min for injection analysis.

[0041] The setting method of GCMS is as follows: The chromatographic column is DB-WAX, 30.0 m × 250 μm, 0.25 μm; the initial temperature is maintained at 40 °C for 2 min, and then increased to 240 °C at a rate of 10 °C / min and maintained for 3 min; the vaporization chamber temperature is 250 °C; the transfer line temperature is 240 °C; the carrier gas is He; the carrier gas flow rate is 1.0 mL / min; without splitting. Mass spectrometry conditions: EI source; electron energy 70 eV; ion source temperature 230 °C; quadrupole 150 °C; the scanning mode is Scan; the scanning mass range is 35 u to 550 u.

[0042] Perform qualitative analysis on the measurement results, and use the MS database NIST11 and retention time for qualitative analysis; column bleed peaks, etc. should be deducted from the results of database screening. According to database comparison, all actinomycetes produced geosmin and 2-methylisoborneol, and the abbreviation of 2-methylisoborneol is 2-MIB. The results show that this actinomycete is an odor-producing actinomycete.

[0043] II. Molecular biological identification of odor-producing actinomycetes

[0044] Extract DNA and perform PCR amplification and sequencing on the actinomycetes separated and purified in Step (3) of Example 1; the specific steps are as follows: Use the Ezup column-type bacterial genomic DNA extraction kit to extract the DNA of actinomycetes according to the instructions, and use the 16S rDNA amplification sequence to perform PCR amplification on the extracted DNA to obtain an amplification fragment, and the PCR length is about 1500 bp. The PCR amplification primer sequences are:

[0045] 27F: AGTTTGATCMTGGCTCAG, designated as SEQ ID NO.2;

[0046] 1492R: GGTTACCTTGTTACGACTT, designated as SEQ ID NO.3.

[0047] The specific PCR amplification system is as follows: 0.5 μL of Template, 2.5 μL of 10× Buffer, 1 μL of dNTP with a concentration of 2.5 mM, 0.2 μL of polymerase, 0.5 μL of the forward primer 27F with a concentration of 10 μM, 0.5 μL of the reverse primer 1492R with a concentration of 10 μM, and double-distilled water is added to make up to 25 μL. Among them, Template is genomic DNA with a concentration of 20 - 50 ng / μL. During amplification, the concentration of genomic DNA is 30 ng / μL, and the 10× Buffer contains Mg 2+ 。

[0048] The PCR cycling conditions are: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 1 min for 30 cycles, repair extension at 72°C for 10 min, and termination of the reaction at 4°C.

[0049] The amplified fragments were subjected to gel electrophoresis. 1% agarose electrophoresis was set up, and electrophoresis was observed at a voltage of 150 V, current of 100 mA, and time of 20 min. The actinomycete DNA target band required for cutting the electrophoresis band was cut, and the PCR product was directly sequenced using the PCR amplification primers. The sequence length of the odor-producing actinomycete strain is 1466 bp.

[0050]

[0051] After uploading the sequencing results to NCBI, an accession number was obtained, which was OQ525968. The comparison with the actinomycete sequences in the GenBank database showed that Figure 1 as shown, indicating that the actinomycete to be tested was Streptomyces antibioticus 100% similar to JCM, NR112299.1. Therefore, the identified strain was Streptomyces antibioticus.

[0052] III. Determination of the spore germination and odor production ability of odor-producing actinomycetes

[0053] Based on the steps (1) to (6) of Example 1, 1 mL of the spore solution of actinomycetes was added to a 250 mL sterile conical flask containing 100 mL of the liquid medium described in step (1). The above conical flask was placed in an oscillating incubator for cultivation, with a rotation speed of 130 rpm and a temperature of 30 °C. Figure 4 During the period of 0 h to 5 h of the germination of actinomycete spores, 10 mL of the uniformly mixed actinomycete bacterial liquid was taken from the conical flask every 1 hour into a sterile centrifuge tube to obtain the mixed actinomycete bacterial liquid at different times.

[0054] 195 μL of the above mixed actinomycete bacterial liquid at different times was respectively mixed with 5 μL of the flow cytometer staining agent in a 2 mL sterile centrifuge tube and shaken well. The centrifuge tube was placed in a metal bath at 35 °C and heated in the dark for 15 minutes to allow the staining agent to fully bind to the actinomycetes.

[0055] Configuration of the flow cytometer staining agent: Each 500 μL of the staining agent included 5 μL of SYBR Green I nucleic acid dye stock solution dissolved in 495 μL of dimethyl sulfoxide. The above dimethyl sulfoxide was all filtered through a 0.2 μm PTFE membrane. The outer wall and the cap of the 2 mL centrifuge tube containing the staining agent were wrapped with adhesive tape for light shielding treatment and stored in a refrigerator at -20 °C.

[0056] The flow cytometer aspirated the above-mentioned heated bacterial liquid, and specific gates were selected according to the sampling results to distinguish broken particles from the target strain, as shown in Figure 2 . At the same time, the number of sampling particles in each gate was set to 10,000 each time, the sampling speed was medium speed, and the fluorescence intensity FL1-H was less than 600. Among them, H was the electrical pulse signal intensity, which was the abbreviation of height and represented the peak value of the pulse signal. Finally, the flow cytometer parameters FSC and SSC were analyzed to judge the spore germination state of actinomycetes. FSC reflects the size of the cell diameter, SSC reflects the change of cell granularity, and the overall spore germination rate of actinomycetes was reflected by the relative growth rate, that is, the ratio of FSC in the late germination stage to FSC in the initial stage. The change results of FSC at 0 h and 5 h are shown in Figure 2 , and the change results of SSC are shown in Figure 3 .

[0057] Before measuring the concentration of odorant substances, the sample was pretreated. The specific operation was to take 8 mL of spore solution into a 15 mL extraction bottle, add 2.5 g of dried NaCl, and then quickly seal it. The SPME extraction fiber head was aged at 250 °C in the GC-MS injection port until there were no impurity peaks. The sample bottle was placed on the solid-phase microextraction device, the temperature was set at 70 °C, and the stirring speed was 500 rpm; the sample bottle was placed on the extraction device and preheated for 15 min; the SPME extraction head was inserted through the bottle cap into the headspace part of the sample, the fiber head was pushed out, and the extraction head was about 1.0 cm above the upper surface of the sample, and headspace extraction was carried out for 20 min; the fiber head was withdrawn, and the extraction head was pulled out from the sample bottle; then the extraction head was inserted into the GC-MS injection port, the fiber head was pushed out, and desorption was carried out at 250 °C for 3 min, and gas chromatography-mass spectrometry analysis was performed. The results of the change in 2-MIB concentration during the period of 0 h to 5 h of actinomycete spore germination are shown in Figure 4 .

[0058] Figure 2 Among them, the target actinomycete spores and debris were distinguished by a specific gate, and the fluorescence intensity FL1-A of the spores at different times changed within the gate. Among them, A is the abbreviation of area, representing a way of reading data. After the actinomycete spores germinated for 5 h, the overall FSC curve shifted to the right, indicating that the particle size of the dormant spores in the culture medium increased, and the spores changed from the dormant state to the resuscitated state. The FSC increased from 2594 at 0 h to 3644 at 5 h, and the relative growth rate also reached 40.5%.

[0059] Figure 3 Among them, the SSC distribution of actinomycete spores showed differences. The intracellular metabolism of some resuscitated spores began to be active, and the cell organelles became complex, resulting in an increase in FSC. On the contrary, the dormant spores remained unchanged. In addition, the fluorescence intensity FL1-A of the germinated spores also increased with time.

[0060] Figure 4 Among them, actinomycetes continuously produced 2-MIB during spore germination. The 2-MIB concentration increased from 6.3144 ng / L at 0 h to 30.4838 ng / L at 5 h. It shows that this actinomycete has a strong ability to produce 2-MIB during the spore germination period.

[0061] There are a series of disadvantages in the traditional microscopic counting for measuring actinomycete spore germination, such as uneven spore distribution, difficult to distinguish, large result error, old equipment, etc. However, the above method can efficiently judge the germination status of actinomycete spores in water by the specific binding of spores and staining agents, high-throughput screening by flow cytometry, rapid determination and analysis, relative growth rate, and combined with the changes in SSC and FL1-A.

[0062] IV. Spore morphology determination

[0063] Centrifuge the actinomycete spore culture solution at 0 h and 5 h at 8000 rpm for 10 min. After discarding the supernatant, add 1 mL of 2.5% glutaraldehyde by volume to a 1.5 mL centrifuge tube and let it stand in a refrigerator at 4°C for 8 h. Soak the spores in the centrifuge tube with sterile PBS to remove the excess glutaraldehyde, repeat 3 times, each time for 15 min, and remove the supernatant by centrifugation. Use anhydrous ethanol with different concentrations to remove the excess water in the system. The different concentrations of anhydrous ethanol are anhydrous ethanol with a volume fraction of 30%, 50%, 70%, 90%, 95% and 100% respectively. After the soaking, centrifuge. Finally, add 0.5 mL of isoamyl acetate and let it dry naturally in a fume hood. Scrape the spores on the centrifuge tube wall, fix them with conductive glue and spray gold for scanning electron microscopy shooting.

[0064] Figure 5 Among them, the morphological differences before and after the germination of actinomycete spores are significant. At 0 h, it was clearly observed that the actinomycete spores were arranged in sequence, showing a cylindrical shape with a wrinkled surface. At 5 h after germination, some spores had already completed germination to form hyphae and adhered to the dormant spores.

[0065] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. To prevent repetition, the present invention describes preferred embodiments.

[0066] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0067] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. An actinomycete with olfactory ability, characterized in that: The actinomycetes are antibiotic Streptomyces ( Streptomyces antibioticus )LJH21, deposited in China Center for Type Culture Collection, with the accession number CCTCC NO: M 20241163.

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