Serratia nematphila and prodigiosin as well as production method and application of serratia nematphila and prodigiosin

Abstract

The invention provides serratia nematophila, prodigiosin as well as a production method and application of the serratia nematophila and the prodigiosin. The serratia nematophila is serratia nematophila, the strain number is S207, the serratia nematophila is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 2025727. The serratia nematophagi can also be a mutant strain which is formed by the serratia nematophagi with the strain number of S207 after the serratia nematophagi is irradiated by ultraviolet rays. The production method of the prodigiosin comprises the following steps: carrying out fermentation culture on the serratia nematophila provided by the invention, and separating the prodigiosin from a fermentation culture product. The serratia nematophila has the characteristic of high yield of the prodigiosin, the prodigiosin produced by using the serratia nematophila has the characteristic of high yield, and the produced prodigiosin has a good application prospect in the aspects of bacteriostasis and preparation of bacteriostatic agents.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically a nematode Serratia marcescens, styraxin, its production method, and its application. Background Technology

[0002] Viscopolamine is a natural pigment produced by microbial secondary metabolism. It typically has a tripyrrole ring skeleton and belongs to the alkaloid family, possessing various biological activities such as antioxidant, antitumor, and antibacterial activity. Currently, *Serratia* spp. are the main microorganisms producing viscopolamine and its analogues. *Serratia* is a Gram-negative bacillus with extremely strong survival capabilities, able to survive in various environments, such as plant roots, water, insects, and mammals. Viscopolamine currently has broad application prospects in the food, cosmetics, and biopharmaceutical fields. With continuous technological development, the demand for viscopolamine is increasing, indicating a huge market potential. Therefore, screening new strains from nature, and even further mutagenesis breeding to obtain mutant strains, followed by fermentation culture of these strains, is an important approach to obtaining viscopolamine. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the purpose of this invention is to provide a *Serratia nematode* strain, squalene, its production method, and its applications. The *Serratia nematode* strain of this invention has the characteristic of high squalene production. The production of squalene using the *Serratia nematode* strain of this invention has the characteristic of high yield, and the squalene produced has good application prospects in antibacterial activity and the preparation of antibacterial agents.

[0004] The above objectives of this invention are achieved through the following technical solutions:

[0005] In a first aspect, the present invention provides a Serratia nematotophila strain, strain number S207, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 2025727 and accession date of April 8, 2025.

[0006] In a second aspect, the present invention provides a Serratia nematode strain that produces high levels of serotonin. The Serratia nematode strain that produces high levels of serotonin is a mutant strain formed by irradiating the Serratia nematode strain described in the first aspect under ultraviolet light for a duration of 1 to 7 minutes, preferably 1 to 6 minutes, more preferably 1 to 5 minutes, for example, any of the following durations or any two of the following durations: 60s, 90s, 120s, 150s, 180s, 210s, 240s, 280s, 300s.

[0007] In some embodiments of the present invention, irradiation for 1 to 7 minutes is continuous irradiation for 1 to 7 minutes.

[0008] In some embodiments of the present invention, the mutant strain is a stable, high-yielding Serratia nematodes strain obtained by genetic stability screening of live Serratia nematodes obtained after ultraviolet light irradiation.

[0009] In some embodiments of the present invention, the mutant strain satisfies the following conditions: 1) High squalene production: Under the same fermentation culture conditions, the squalene production of the mutant strain is higher than that of Serratia marcescens described in the first aspect; 2) Stable squalene production: The mutant strain is passaged and the strain obtained from the passaged culture is fermented to produce squalene, and the squalene production of adjacent generations of strains is not significantly different. For example, the difference in squalene production is less than the value of the lower squalene production. Preferably, the absolute value of the percentage increase in squalene production (percentage increase in squalene production = (squalene production of the next generation strain - squalene production of the previous generation strain) is less than 100%, preferably less than 80%, more preferably less than 50%, further preferably less than 30%, and even more preferably less than 20%.

[0010] In some embodiments of the present invention, during the screening of mutant strains, the yield of styraxin can be determined by the OD of the bacterial solution obtained after the strain has been fermented for 24–72 hours. 535 Value representation.

[0011] In some embodiments of the present invention, the specific process for selecting a stable, high-yielding strain of styraxin includes: inoculating the mutant strain onto a solid culture medium (e.g., beef extract peptone solid medium), subculturing once every 18–48 hours, for a total of 4–10 subculturings; taking the strain obtained from each subculturing as the inoculum and inoculating it into a liquid culture medium (e.g., beef extract peptone liquid medium) for shake culture for 24–72 hours; and measuring the OD of the bacterial solution obtained from the shake culture. 535 Then select strains that meet the following conditions: the OD value of the bacterial suspension obtained from two adjacent generations of strains cultured on a shaker. 535 The values ​​are not significantly different; for example, the OD values ​​of the two bacterial cultures are similar. 535 The difference in value is less than OD 535 OD of small bacterial cultures 535 The values, preferably, are the OD values ​​of the two bacterial cultures. 535 Percentage increase in value (OD) 535 Percentage increase in value = (OD value of the bacterial culture of the next generation strain) 535 Value - OD of the previous generation strain's bacterial culture 535 (value) / OD of the previous generation strain 535The absolute value of the value is less than 100%, preferably less than 80%, more preferably less than 50%, even more preferably less than 30%, and even more preferably less than 20%.

[0012] In some embodiments of the present invention, the solid culture medium is LB agar or beef extract peptone solid culture medium.

[0013] In some embodiments of the present invention, the culture medium used for fermentation culture is LB liquid medium.

[0014] In some embodiments of the present invention, the culture medium used for fermentation is a beef extract peptone liquid culture medium comprising the following components:

[0015] 0–2 wt% carbon source, 0–2 wt% nitrogen source, 0–1 wt% inorganic salt.

[0016] In some embodiments of the present invention, the carbon source is selected from one or more of starch, lactose, glucose, ethanol, maltose, glycerol and sucrose; preferably, the carbon source is starch and / or glycerol; more preferably, the carbon source is starch and glycerol; and even more preferably, the mass ratio of starch to glycerol is 1:(1-3).

[0017] In some embodiments of the present invention, the nitrogen source is selected from one or more of corn steep liquor, urea, ammonium sulfate, and yeast; preferably, the nitrogen source is ammonium sulfate.

[0018] In some embodiments of the present invention, the inorganic salt is selected from one or more of MgCl2, CaCl2, NaCl, KCl, ZnCl2, MgSO4, etc.

[0019] In some embodiments of the present invention, the culture medium used for fermentation is a beef extract peptone liquid culture medium comprising the following components: starch 0.1-0.5 wt%, glycerol 0.5-1 wt%, and ammonium sulfate 1-2 wt%. Preferably, the culture medium used for fermentation further comprises 0.2 wt% inorganic salt, wherein the inorganic salt is one or more of KCl, ZnCl2, and ZnSO4. More preferably, the pH value of the culture medium used for fermentation is 5-8, preferably 6-7.

[0020] In some embodiments of the present invention, the beef extract peptone liquid culture medium comprises the following components: 2-4 g / L beef extract, 5-15 g / L peptone, and 2-8 g / L NaCl; preferably, the solvent of the beef extract peptone liquid culture medium is water.

[0021] In some embodiments of the present invention, the culture medium used for fermentation culture comprises the following components: 0.25 wt% starch, 0.75 wt% glycerol, 1.5 wt% ammonium sulfate, 0.3 wt% beef extract, 1 wt% peptone, and 0.5 wt% sodium chloride; preferably, the pH value of the culture medium used for fermentation culture is 5 to 8, and more preferably 6 to 7.

[0022] In some embodiments of the present invention, the fermentation culture conditions include: a temperature of 26–30°C and a time of 20–72 h.

[0023] Thirdly, the present invention provides the use of *Serratia nematodes* as described in the first aspect or the second aspect in the production of styraxone.

[0024] Fourthly, the present invention provides a method for producing squalene, the method comprising fermenting and culturing either the *Serratia nematodes* described in the first aspect or the *Serratia nematodes* described in the second aspect, and separating squalene from the fermentation culture product.

[0025] In some embodiments of the present invention, the culture medium used for fermentation culture is LB liquid medium.

[0026] In some embodiments of the present invention, the culture medium used for fermentation is a beef extract peptone liquid culture medium comprising the following components:

[0027] 0–2 wt% carbon source, 0–2 wt% nitrogen source, 0–1 wt% inorganic salt.

[0028] In some embodiments of the present invention, the carbon source is selected from one or more of starch, lactose, glucose, ethanol, maltose, glycerol and sucrose; preferably, the carbon source is starch and / or glycerol; more preferably, the carbon source is starch and glycerol; and even more preferably, the mass ratio of starch to glycerol is 1:(1-3).

[0029] In some embodiments of the present invention, the nitrogen source is selected from one or more of corn steep liquor, urea, ammonium sulfate, and yeast; preferably, the nitrogen source is ammonium sulfate.

[0030] In some embodiments of the present invention, the inorganic salt is selected from one or more of FeCl3, CaCl2, KCl, ZnCl2, ZnSO4, MgSO4, etc.

[0031] In some embodiments of the present invention, the culture medium used for fermentation is a beef extract peptone liquid culture medium comprising the following components: starch 0.1-0.5 wt%, glycerol 0.5-1 wt%, and ammonium sulfate 1-2 wt%. Preferably, the culture medium used for fermentation further comprises 0.2 wt% inorganic salt, wherein the inorganic salt is one or more of KCl, ZnCl2, and ZnSO4. More preferably, the pH value of the culture medium used for fermentation is 5-8, preferably 6-7.

[0032] In some embodiments of the present invention, the beef extract peptone liquid culture medium comprises the following components: 2-4 g / L beef extract, 5-15 g / L peptone, and 2-8 g / L NaCl; preferably, the solvent of the beef extract peptone liquid culture medium is water.

[0033] In some embodiments of the present invention, the culture medium used for fermentation culture comprises the following components: 0.25 wt% starch, 0.75 wt% glycerol, 1.5 wt% ammonium sulfate, 0.3 wt% beef extract, 1 wt% peptone, and 0.5 wt% sodium chloride; preferably, the pH value of the culture medium used for fermentation culture is 5 to 8, and more preferably 6 to 7.

[0034] In some embodiments of the present invention, the fermentation culture conditions include: a temperature of 26–30°C and a time of 20–72 h.

[0035] In some embodiments of the present invention, the method for separating styraxin from fermentation culture products includes extracting the cells in the fermentation culture products using an organic solvent and / or breaking down the cells in the fermentation culture products using ultrasound, and then recovering the pigment-containing liquid portion.

[0036] In some embodiments of the present invention, the bacterial cells are first dispersed in an organic solvent and then crushed in the organic solvent before the crushing process.

[0037] In some embodiments of the present invention, the organic solvent includes one or more of methanol, ethanol, chloroform, ethyl acetate, etc.

[0038] In some embodiments of the present invention, the method further includes sequentially subjecting the recovered pigment-containing liquid to rotary evaporation and chromatography purification.

[0039] In some embodiments of the present invention, the rotary evaporation is carried out at 30–50°C.

[0040] In some embodiments of the invention, the chromatographic purification includes elution using a mixture of petroleum ether (PE) and ethyl acetate (EA).

[0041] Fifthly, the present invention provides a strychnine produced by the production method described in the fourth aspect.

[0042] In a sixth aspect, the present invention provides the use of the styraxin described in the fifth aspect in the antibacterial or preparation of antioxidant or antitumor drugs.

[0043] In some embodiments of the present invention, the antibacterial effect includes inhibiting the growth and reproduction of bacteria and / or fungi.

[0044] In some embodiments of the present invention, the bacteria include Staphylococcus aureus, Bacillus megaterium, Bacillus subtilis, Vibrio parahaemolyticus, Bacillus amyloliquefaciens, Bacillus licheniformis, Escherichia coli, etc.

[0045] In some embodiments of the present invention, the fungus includes Rhizopus oryzae, Alternaria alternata, etc.

[0046] In a seventh aspect, the present invention provides an antibacterial agent comprising the styraxin described in the fifth aspect.

[0047] In some embodiments of the present invention, the antibacterial agent further includes a metal salt, wherein the metal salt includes one or more of potassium salt, manganese salt, and zinc salt; preferably, the potassium salt includes potassium chloride, potassium sulfate, potassium nitrate, potassium acetate, and / or, the manganese salt includes potassium permanganate, manganese dioxide, manganese sulfate, manganese citrate, manganese acetate, and / or, the zinc salt includes zinc chloride, zinc nitrate, and zinc sulfate.

[0048] In some embodiments of the present invention, the concentration of styraxin in the antibacterial agent is 0.1 to 2 mg / mL.

[0049] In some embodiments of the present invention, the concentration of the metal salt in the antibacterial agent is 0.05 to 0.2 mol / L, preferably 0.08 to 0.15 mol / L.

[0050] In some embodiments of the present invention, the antibacterial agent comprises a potassium salt and / or a manganese salt; preferably, the potassium salt is potassium chloride, and / or the manganese salt is manganese sulfate.

[0051] The beneficial effects of this invention are as follows:

[0052] This invention provides a novel *Serratia nematode* strain and its mutant strain. The *Serratia nematode* strain and its mutant strain of this invention have the characteristic of high production of styraxin. The production of styraxin using the *Serratia nematode* strain and its mutant strain of this invention has the characteristic of high yield. The styraxin produced has good application prospects in anti-oxidation, anti-tumor or antibacterial, as well as in the preparation of antibacterial agents or anti-oxidation or anti-tumor drugs. Attached Figure Description

[0053] Figure 1 This indicates the results of isolated and purified red single colonies, as well as streaks on plates and slants.

[0054] Figure 2 This indicates the results of microscopic morphological observation of the strain.

[0055] Figure 3 This represents the results of strain genomic DNA and PCR agarose gel electrophoresis.

[0056] Figure 4 This represents a phylogenetic tree of strains constructed based on 16S rDNA sequencing.

[0057] Figure 5 This indicates the red pigment extract sample obtained by the present invention.

[0058] Figure 6 This represents the full-wavelength ultraviolet-visible scan of the red pigment obtained by this invention.

[0059] Figure 7 This shows the LC-MS spectrum of the red pigment obtained in this invention.

[0060] Figure 8 This shows the FT-IR spectrum of the red pigment obtained by the present invention.

[0061] Figure 9 This indicates the effect of different carbon sources on the production of lecithin by *Serratia nematotophila* (strain number S207).

[0062] Figure 10 This indicates the effect of different nitrogen sources on the production of lecithin by *Serratia nematotophila* (strain number S207).

[0063] Figure 11 This study demonstrates the effect of different inorganic salts on the production of lecithin by *Serratia nematotophila* (strain number S207).

[0064] Figure 12 This indicates the use of beef extract peptone liquid medium as the base and the use of medium number 20 to obtain styrax.

[0065] Figure 13The figure shows the antibacterial effects of styraxin against Staphylococcus aureus, Bacillus megaterium, Bacillus subtilis, Vibrio parahaemolyticus, Bacillus amyloliquefaciens, Bacillus licheniformis, and Escherichia coli. In the figure, a represents NA medium inoculated with Staphylococcus aureus, b represents NA medium inoculated with Bacillus megaterium, c represents NA medium inoculated with Bacillus subtilis, d represents NA medium inoculated with Vibrio parahaemolyticus, e represents NA medium inoculated with Bacillus amyloliquefaciens, f represents NA medium inoculated with Bacillus licheniformis, and g represents NA medium inoculated with Escherichia coli.

[0066] Figure 14 The figure shows the antibacterial effect of styraxone on Rhizopus oryzae and Streptomyces chromis. In the figure, h represents a PDA plate inoculated with Rhizopus oryzae, and i represents a PDA plate inoculated with Streptomyces chromis. Detailed Implementation

[0067] The following examples further illustrate the technology of the present invention. These examples are illustrative and exemplary of the present invention and do not limit the scope of the invention in any way.

[0068] Unless otherwise specified, the culture media used below were prepared with reference to the 4th edition of "Experimental Tutorial of Microbiology" by Xu Deqiang et al. and "Principles and Methods of Food Microbiology Testing" by He Zhifei et al.

[0069] I. Acquisition and Identification of Serratia nematotophila

[0070] 1.1 Strains Isolation and Purification

[0071] After diluting and spreading the bamboo shoot shell waste compost sample onto LB agar plates, red bacterial strains were selected and spread onto LB agar plates. The plates were then incubated at 28°C for 24 hours to obtain single red colonies. Figure 1 As shown in Figure a. Single red colonies were picked and streaked onto LB agar plates and LB slant, and incubated at 28°C for 24 hours to obtain single red colonies. This streaking process was repeated multiple times to isolate and purify the strain, resulting in a purified strain, as shown in Figure a. Figure 1 As shown in b, the purified strain can finally be preserved in glycerol.

[0072] 1.2 Strain Identification

[0073] (1) Morphological observation

[0074] The strains isolated and purified above were inoculated into 100 mL of beef extract peptone liquid medium at an inoculation rate of 5%, and cultured in a constant temperature incubator at 28℃ for 48 h. The bacterial culture was then spread onto beef extract peptone solid medium and cultured in a constant temperature incubator at 28℃ for 48 h. The colony morphology characteristics were then observed.

[0075] The isolated and purified bacterial strains were inoculated at a rate of 5% into 100 mL of beef extract peptone liquid medium and cultured at 28℃ for 48 h. The bacterial suspension was then inoculated at a rate of 5% into 100 mL of beef extract peptone liquid medium and cultured at 28℃ for another 48 h. Gram staining and electron microscopy were performed to observe the bacterial morphology. The Gram staining results are shown below. Figure 2 As shown in Figure a, the electron microscope scanning image is as follows: Figure 2 As shown in b.

[0076] (2) Physiological and biochemical characteristics analysis

[0077] The isolated and purified strains were inoculated at a rate of 5% into 100 mL of beef extract peptone liquid medium and cultured at 28℃ for 48 h. Preliminary physiological and biochemical characteristics of the strains were identified according to the 4th edition of *Microbiology Experimental Tutorial* by Xu Deqiang et al. and *Principles and Methods of Food Microbiology Testing* by He Zhifei et al. The main tests performed included: fermentation of different sugar alcohols, indole test, MR test, VP test, starch hydrolysis test, citrate utilization test, gelatin liquefaction test, H2S test, urease test, phenylalanine deaminase test, nitrate reduction test, lipase test, salt tolerance test, and pH tolerance test. Some results are shown in Table 1. Table 1 shows that this strain can utilize glucose, sucrose, maltose, lactose, galactose, rhamnose, D-mannose, trehalose, D-sorbitol, and inositol; but cannot utilize D-arabinose and mannitol. The VP test, starch hydrolysis test, citrate utilization test, gelatin liquefaction test, nitrate reduction test, and lipase test are positive; the indole test, MR test, H2S test, urease test, and phenylalanine deaminase test are negative. Furthermore, salt tolerance tests indicate that this strain can adapt to salt concentrations ranging from 0% to 8%, with an optimal growth concentration of 1% to 2%. pH tolerance tests indicate that this strain can adapt to pH ranges from 5 to 10, with an optimal growth pH of 5 to 8. Based on the morphological characteristics of the strain, it is preliminarily identified as a bacterium belonging to the genus *Serratia*.

[0078] Table 1 Physiological and biochemical characteristics of the strains

[0079]

[0080]

[0081] (3) 16S rDNA sequencing

[0082] The isolated and purified strains were inoculated at a rate of 5% into 100 mL of beef extract peptone liquid medium and cultured at 28°C for 48 h. Genomic DNA was extracted from the strains using a bacterial genomic DNA extraction kit, and conserved 16S rDNA sequences were selected for PCR amplification to obtain the desired target gene fragment. Specifically, two pairs of universal primers were used: 27F (5'-AGAGTTT-GATCCTGGCTCAG-3') and 1429R (5'-GGTTACCTT-GTTACGACTT-3'), and the PCR amplification reaction was performed according to the following program: 94°C pre-denaturation for 300 s; 94°C denaturation for 30 s, 58°C annealing for 45 s, 72°C extension for 60 s, for a total of 32 cycles; and a final extension at 72°C for 600 s. The PCR products were examined by 1.0% agarose gel electrophoresis and then sequenced. The sequence results were compared with the NCBI database using BLAST, and a phylogenetic tree was constructed using the neighbor-joining method. The results of the strain genomic DNA extraction are shown below. Figure 3 As shown in Figure a, the electrophoretic analysis results of the PCR products are as follows: Figure 3 As shown in Figure b, PCR amplification using universal primers for bacterial 16S rDNA yielded a 1500 bp 16S rDNA gene sequence. Multiple sequence alignment of the strain's 16S rDNA gene sequence with known closely related strains in GenBank was performed, and the results are shown below. Figure 4 As shown, from Figure 4 It can be seen that this strain is closely related to Serratia nematodes, with a similarity of 100%.

[0083] Based on the combined results of morphological observation, physiological and biochemical analysis, and 16S rDNA sequencing, the strain was identified as *Serratia nematodes* and named *Serratia nematodes* S207.

[0084] II. Production and Identification of Pyruvic Red

[0085] (1) Take the strain obtained from the isolation and purification in 1.1 above and inoculate it into LB liquid medium at an inoculation rate of 5%. Incubate in a constant temperature incubator at 28℃ for 48h. Centrifuge the cultured bacterial solution at 8000r / min for 10min at 4℃, discard the supernatant, wash it 3 times with distilled water, and collect the precipitate.

[0086] (2) Mix the precipitate collected in step (1) with acidic methanol (adjust the pH of the methanol to 3 with 0.1 mol / L hydrochloric acid) at a ratio of 1 g: 20 mL, sonicate at 30 °C for 5 min, centrifuge at 6000 r / min for 10 min, filter and discard the bacterial residue, collect the liquid part and evaporate to dryness at 40 °C to obtain the crude red pigment product.

[0087] (3) Dissolve the crude red pigment obtained in step (2) in acidic methanol (adjust the pH of methanol to 3 with 0.1 mol / L hydrochloric acid), add a small amount of silica gel and stir evenly. First, elute with a PE:EA ratio of 5:1 for 3 hours, then elute with a PE:EA ratio of 3:1 for 5 hours. Collect the concentrated pigment eluent with a large proportion and consistent color to obtain the purified red pigment product. The sample is shown below. Figure 5 As shown.

[0088] The red pigment product obtained in step (3) was sampled and analyzed by UV-Vis full-wavelength scanning, liquid chromatography-mass spectrometry, and Fourier transform infrared spectroscopy. The UV-Vis full-wavelength scanning results are as follows: Figure 6 As shown, the red pigment has a specific absorption peak in the wavelength range of 200–800 nm, with a peak value at 535 nm. This is basically consistent with the reported UV absorption spectrum of styraxin. The results of liquid chromatography-mass spectrometry analysis are as follows: Figure 7 As shown, Figure 7 The major fragment ion is 324.20405 m / z, and it is [M+H]. + Appearing in the form of Figure 7 The fragment ion peaks are consistent with those of pyruvic erythrin in existing literature, both being 324.20 m / z, indicating that the erythrin molecule obtained in this invention also has a relatively low molecular weight of 323, consistent with the reported molecular weight of pyruvic erythrin. Fourier transform infrared spectroscopy analysis results are as follows: Figure 8 As shown, the dominant absorption wavenumber in the FT-IR spectrum is 3297 cm⁻¹. -1 2926cm -1 2857cm -1 2360cm -1 1728cm -1 and 1663cm -1 3297cm -1 The peak representing the "-NH-" stretching in the pyrrole ring is 2926 cm⁻¹. -1 The antisymmetric stretching peak representing the methylene group "-CH2-" is at 2857 cm⁻¹. -1 The peak representing the symmetric stretching of the methylene group "-CH2-" is 1728 cm⁻¹. -1 Represents the ketone stretching peak, 1663 cm⁻¹ -1 The peaks represent carbon-carbon double bond stretching; these peaks are consistent with the main characteristic peaks of the Fourier transform infrared spectrum of styraxone reported in existing literature. After comparison with the known structure of styraxone, these characteristic peaks can be identified as the functional groups contained in styraxone.

[0089] III. Selection and breeding of Serratia marcescens, a nematode that produces high levels of styraxone.

[0090] The strains isolated and purified in 1.1 were inoculated into beef extract peptone liquid medium at a 5% inoculation rate and cultured in a constant temperature incubator at 28℃ for 24 hours. The cultured bacterial solution was then transferred to six sterile culture dishes numbered 1–6, with 5 mL in each dish. Five sterile culture dishes numbered 2–6 were then placed under a UV lamp (25 cm away) for 90 s, 150 s, 210 s, 270 s, and 330 s, respectively. The sterile culture dish numbered 1 was placed in the dark. Subsequently, the contents of the six sterile culture dishes were collected... Take 10 μL of bacterial suspension from each petri dish and dilute it with 29.99 mL of sterile water in a sterile centrifuge tube. Then, spread the diluted bacterial suspension onto beef extract peptone solid medium plates numbered 1–6 (wherein, the bacterial suspensions from the six sterile petri dishes numbered 1–6 correspond sequentially to the six beef extract peptone solid medium plates numbered 1–6). Spread 100 μL of bacterial suspension onto each beef extract peptone solid medium plate. Incubate at 28℃ in the dark for 24 h, observing and recording the number of colonies on each beef extract peptone solid medium plate. Simultaneously, collect the inoculum from the darkest red single colonies on the beef extract peptone solid medium and inoculate it at a 5% inoculum rate into centrifuge tubes containing 8 mL of beef extract peptone liquid medium. Incubate at 28℃ and 140 rpm for 36 h on a shaker. Measure the OD of each bacterial suspension using a UV spectrophotometer. 535 The values ​​are shown in Table 2.

[0091] Table 2. OD values ​​of bacterial cultures after irradiation with UV lamps for different durations. 535 value

[0092] Mutagenesis time 0s 90s 150s 210s 270s 330s <![CDATA[OD 535 Value 1.741 2.748 2.286 2.398 3.000 1.907

[0093] In addition, based on the recorded colony counts, the number of colonies on beef extract peptone solid culture media numbered 1 to 6 showed a decreasing trend, indicating that the lethality of the strains increased with the extension of irradiation time.

[0094] Genetic stability tests were performed on the strains obtained after different mutagenesis times: Using sterile toothpicks, bacterial cultures were collected from single, dark red colonies numbered 1–5 on beef extract peptone solid medium. These cultures were then spot-inoculated onto five new beef extract peptone solid mediums. Subculture was performed every 1 day for 0, 1, 2, 3, 4, 5, and 6 subcultures. After each subculture, the strains from the beef extract peptone solid medium were used as inoculum (the beef extract peptone solid mediums from subcultures 0, 1, 2, 3, 4, and 5 were first stored at 4°C and then collected uniformly after the 6th subculture). These inoculum were then inoculated into beef extract peptone liquid medium at a 5% inoculum rate and cultured on a shaker at 28°C and 140 rpm for 24 hours. The OD values ​​of the culture solution were measured using a UV spectrophotometer. 535Values, for each treatment with 3 replicates, obtained through the OD of the 3 replicates. 535 The average values ​​were calculated, and the results are shown in Table 2. Table 2 shows the comparison results of different strains at the same generation: OD values ​​of the 3rd, 4th, 5th, and 6th generation bacterial cultures of strains S207-A, S207-B, and S207-D. 535 The values ​​were all higher than the OD of strain S207. 535 Values; compared with S207-A, the OD values ​​of the 3rd, 4th, 5th, and 6th generation bacterial cultures of S207-B and S207-D strains were... 535 Value and OD of strain S207 535 The differences in values ​​were generally large, with the OD values ​​of the 3rd, 4th, 5th, and 6th generations of S207-B strain being particularly high. 535 Value and OD of strain S207 535 The overall difference in values ​​was the largest. Table 2 shows the comparison results of different generations of the same strain: compared with S207-B, the OD values ​​of any two adjacent generations of S207-A or S207-D strains were significantly higher. 535 The relatively small difference in values ​​indicates that the strain has relatively good genetic stability. In conclusion, strain S207-D can be selected as the optimal strain with high and stable production of styraxone.

[0095] Table 3. OD values ​​of bacterial cultures obtained from shaker culture 535 value

[0096]

[0097]

[0098] In Table 3, the generation number refers to the number of generations passed through the beef extract peptone solid medium.

[0099] IV. Selection of Culture Media for Serratia nematodes

[0100] (1) Take the strain obtained from the isolation and purification in 1.1 above and inoculate it into liquid culture medium at an inoculation rate of 5%. Incubate in a constant temperature incubator at 28℃ for 72h. Take a sample of the cultured bacterial solution for OD analysis. 535 Value determination was performed using three replicates for each numbered culture medium, and the OD values ​​of the three replicates were used for determination. 535 The average value was calculated, and the results are shown in Table 4. The liquid culture medium was obtained by adding specific amounts of components according to the carbon source, nitrogen source, inorganic salt composition and content shown in Table 4, based on beef extract peptone liquid culture medium. The pH value of the liquid culture medium was 7, and it was used after autoclaving at 121℃ for 20 min. The components of the basic beef extract peptone liquid culture medium were: 3.0 g beef extract, 10.0 g peptone, 5.0 g NaCl, and 1000 mL water.

[0101] Table 4. Effects of different culture media on the production of pyruvicin by Serratia nematodes

[0102]

[0103]

[0104] from Figure 9 It can be seen that the OD of bacterial cultures obtained using starch or glycerol as carbon sources is higher than that of bacterial cultures obtained using starch or glycerol as carbon sources. 535 The values ​​are relatively high, with the OD values ​​of bacterial cultures obtained using starch as a carbon source being particularly high. 535 The value is 2.167. From Figure 10 It can be seen that the OD of the bacterial culture obtained using ammonium sulfate as a nitrogen source is... 535 The highest value is 2.283. From Figure 11 It can be seen that inorganic salts have no significant effect on the accumulation of erythromycin produced by this strain.

[0105] As shown in Table 4, the OD of the bacterial culture obtained by fermentation using culture medium number 20 was... 535 Based on the highest value, the inventors again inoculated the strain isolated and purified in 1.1 above into beef extract peptone liquid medium and medium number 20 at an inoculation rate of 5%, respectively, and cultured them in a constant temperature incubator at 28°C for 72 hours. Then, the yield of styracil-containing rubigin obtained from the two culture media was obtained by the following method:

[0106] 1) Centrifuge the cultured bacterial solution at 8000 r / min for 10 min at 4℃, discard the supernatant, wash three times with distilled water, and collect the precipitate. The precipitate weight obtained using beef extract peptone liquid medium as the base was 1.3 g, and the precipitate weight obtained using medium number 20 was 2.13 g.

[0107] 2) The collected precipitate was extracted with 2.5 times the weight of the bacterial cell in anhydrous ethanol for 12 hours. The extracted product was ultrasonically disrupted at 30°C for 5 minutes, and then centrifuged at 6000 rpm for 5 minutes to separate the supernatant. The lower layer of bacteria was extracted again with 2.5 times the weight of the bacterial cell in anhydrous ethanol for 10 minutes. The extracted product was ultrasonically disrupted at 30°C for 5 minutes, and then centrifuged at 6000 rpm for 5 minutes to separate the supernatant. The extraction, ultrasonic disruption, and centrifugation steps were repeated until the bacteria lost their color. All supernatants were then combined and rotary evaporated at 40°C to constant weight to obtain the crude pigment product. The product was weighed and the yield was calculated. The results showed that the yield of squalene using beef extract peptone liquid medium as the base was 40 mg, and the yield of squalene using medium number 20 was 130 mg. The styraxin obtained using beef extract peptone liquid culture medium as the base is as follows: Figure 12 As shown in the middle left figure, the styraxin obtained using culture medium number 20 is as follows: Figure 12 As shown in the middle right figure.

[0108] V. Antibacterial effect of styraxin

[0109] 1. Antibacterial activity of styracil against bacteria (antibacterial activity was determined using the filter paper disc method)

[0110] 1) Prepare 7 centrifuge tubes of 50mL each, add 5mL of NB medium to each tube, autoclave and sterilize, then inoculate the 7 types of bacteria shown in Table 5 into the 7 centrifuge tubes, label them, and incubate them in a shaker at 37℃ for 24h to obtain activated bacterial culture.

[0111] 2) Cool the NA medium, which has been sterilized by moist heat at 121°C for 20 min, to about 46°C and pour it into a glass culture dish that has been sterilized by dry heat at 160°C for 2 h. Place the dish horizontally on a laminar flow hood and wait for it to solidify. Then, use a pipette to take 0.2 mL of the activated bacterial culture solution obtained in step 1) and add it to the glass culture dish on the laminar flow hood. Finally, use a sterile spreader to spread the bacterial solution evenly.

[0112] 3) Obtain purified squalene product according to the production and identification steps (1)-(3) of squalene. Prepare a squalene solution with a concentration of 0.4 mg / mL using the squalene product. Immerse a 6 mm diameter filter paper into the squalene solution, remove the filter paper and drain off the excess solution. Then, attach 3 filter paper pieces clockwise and at a certain distance to the glass culture dish on which the bacterial solution was just coated in step 2). Place the glass culture dish in a 37℃ constant temperature incubator for 24 h and observe whether there is an inhibition zone. Measure the diameter of the inhibition zone (mm) with a vernier caliper. The antibacterial effect is represented by the ratio of the diameter D of the inhibition zone to the diameter d of the filter paper. The results are shown in Table 5 and Figure 13 As shown in Table 5 and Figure 13 It can be seen that lecithin has varying degrees of inhibitory effects on Staphylococcus aureus, Bacillus megaterium, Bacillus subtilis, Vibrio parahaemolyticus, Bacillus amyloliquefaciens, Bacillus licheniformis, and Escherichia coli. It has a strong inhibitory effect on Staphylococcus aureus, Bacillus subtilis, Vibrio parahaemolyticus, Bacillus amyloliquefaciens, and Bacillus licheniformis, but a weaker inhibitory effect on Bacillus megaterium and Escherichia coli.

[0113] Table 5. Antibacterial activity of styraxin against bacteria

[0114]

[0115] 2. Antifungal activity of styracil against fungi (antifungal activity was determined using the plate confrontation method).

[0116] 1) Prepare 6 PDA culture media, inoculate the 6 fungi shown in Table 6 into the 6 PDA culture media respectively, label them, and incubate them in a constant temperature incubator at 28℃ for 3 days to obtain activated fungi.

[0117] 2) Cool the PDA medium, which has been moist-heat sterilized at 121℃ for 20 minutes, to about 46℃, and pour it into a glass petri dish that has been dry-heat sterilized at 160℃ for 2 hours. Place the dish horizontally on a laminar flow hood and wait for it to solidify. Use a sterile punch (6 mm in diameter) to make holes in the PDA medium containing the activated fungi, and then insert the fungal block into the center of the glass petri dish on the laminar flow hood.

[0118] 3) Obtain purified squalene product according to the production and identification steps (1)-(3) of squalene. Prepare a squalene solution with a concentration of 0.4 mg / mL using the squalene product. Immerse a 6 mm diameter filter paper into the squalene solution, remove the filter paper and drain off the excess solution. Then, attach two filter paper pieces to the glass culture dish from step 2), symmetrically attaching them 3 cm away from the center of the glass culture dish. After incubating the glass culture dish at 28℃ for 4 days, use vernier calipers to measure the width of the inhibition zone of squalene against 6 fungi. The width of the inhibition zone is the distance (mm) between the edge of the fungal colony and the edge of the fungal moss. The antibacterial effect is represented by the ratio of the diameter D of the inhibition zone to the diameter d of the filter paper. The results are shown in Table 6 and Figure 14 As shown in Table 6 and Figure 14 It can be seen that levofloxacin has a significant inhibitory effect on Rhizopus oryzae and Streptomyces chromis, but no significant inhibitory effect on Aspergillus niger, Stem blight pathogen, Morchella esculenta, and Morchella esculenta.

[0119] Table 6. Antifungal activity of styraxin against fungi

[0120]

[0121] Note: In Table 6, "-" indicates that the antibacterial effect is not obvious.

[0122] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to embodiments, but it should be understood that the terms used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications having the same function.

Claims

1. A type of Serratia nematode, characterized in that, The nematotophilia mentioned is Serratia nematotophila, strain number S207, which is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO:M 2025727.

2. A Serratia marcescens nematode that produces high levels of sclerotin, characterized in that, The high-yield serrata nematode Serratia is a mutant strain formed by irradiating the Serratia nematode Serratia of claim 1 under ultraviolet light for 1-7 min, preferably 1-6 min, more preferably 1-5 min; preferably, the mutant strain is a stable high-yield serratia nematode Serratia obtained by genetic stability screening of live Serratia nematode Serratia obtained after ultraviolet light irradiation.

3. The use of Serratia nematodes as described in claim 1 or 2 in the production of strychnine.

4. A method for producing styrax erythrin, characterized in that, The production method includes fermenting and culturing *Serratia nematodes* as described in claim 1 or 2 and isolating serotonin from the fermentation culture product.

5. The production method according to claim 4, characterized in that, The culture medium used for fermentation is LB liquid medium or beef extract peptone liquid medium containing the following components: 0–2 wt% carbon source, 0–2 wt% nitrogen source, 0–1 wt% inorganic salt, of which, The carbon source is selected from one or more of starch, lactose, glucose, ethanol, maltose, glycerol, and sucrose; preferably, the carbon source is starch and / or glycerol. The nitrogen source is selected from one or more of corn steep liquor, urea, ammonium sulfate, and yeast; preferably, the nitrogen source is ammonium sulfate. The inorganic salt is selected from one or more of FeCl3, CaCl2, KCl, ZnCl2, ZnSO4, and MgSO4.

6. The production method according to claim 4 or 5, characterized in that, The culture medium used for fermentation is a beef extract peptone liquid culture medium comprising the following components: starch 0.1-0.5 wt%, glycerol 0.5-1 wt%, and ammonium sulfate 1-2 wt%. Preferably, the culture medium also comprises 0.2 wt% inorganic salt, wherein the inorganic salt is one or more of KCl, ZnCl2, and ZnSO4. Alternatively, the culture medium comprises the following components: starch 0.25 wt%, glycerol 0.75 wt%, ammonium sulfate 1.5 wt%, beef extract 0.3 wt%, peptone 1 wt%, and sodium chloride 0.5 wt%. More preferably, the pH of the culture medium is 5-8, preferably 6-7.

7. The production method according to any one of claims 4 to 6, characterized in that, Fermentation culture conditions include: temperature of 26–30℃ and time of 20–72 h; And / or, the method for separating styraxin from fermentation culture products includes extracting the cells in the fermentation culture products using an organic solvent and / or breaking the cells in the fermentation culture products using ultrasound, and then recovering the pigmented liquid portion; preferably, the organic solvent includes one or more of methanol, ethanol, chloroform, and ethyl acetate, and / or, the method further includes sequentially subjecting the recovered pigmented liquid to rotary evaporation and chromatography purification.

8. A type of styraxin produced by the production method according to any one of claims 4 to 7.

9. The use of the lecithin of claim 8 in the inhibition of bacteria or in the preparation of antioxidant or antitumor drugs; preferably, the inhibition of bacteria includes inhibiting the growth and reproduction of bacteria and / or fungi; more preferably, the bacteria include Staphylococcus aureus, Bacillus megaterium, Bacillus subtilis, Vibrio parahaemolyticus, Bacillus amyloliquefaciens, Bacillus licheniformis, Escherichia coli, and / or, the fungi include Rhizopus oryzae and Alternaria alternata.

10. An antibacterial agent, characterized in that, The antibacterial agent includes the styraxin as described in claim 8; preferably, the antibacterial agent further includes a metal salt, wherein the metal salt includes one or more of potassium salt, manganese salt, and zinc salt; more preferably, the potassium salt includes potassium chloride, potassium sulfate, potassium nitrate, potassium acetate, and / or, the manganese salt includes potassium permanganate, manganese dioxide, manganese sulfate, manganese citrate, manganese acetate, and / or, the zinc salt includes zinc chloride, zinc nitrate, and zinc sulfate.

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