Method for producing prodigiosin at high yield, protection method of prodigiosin and application of prodigiosin

Abstract

The invention provides a high-yield prodigiosin method, a prodigiosin protection method and application. The method comprises the following steps: obtaining a serratia nematophagi mutant strain, screening serratia nematophagi with high and stable yield from the mutant strain, carrying out fermentation culture, and separating prodigiosin from a fermentation culture product, the serratia nematophila is serratia nematophila, the strain number of the serratia nematophila is S207, the serratia nematophila is preserved in the China Center for Type Culture Collection, and the preservation number of the serratia nematophila is CCTCC NO: M 2025727. The protection method of the prodigiosin comprises the following steps: storing the prodigiosin in a protective agent with the pH value of 2-6 at 4-60 DEG C, and avoiding the irradiation of ultraviolet light. A large amount of prodigiosin can be rapidly produced by using the method for producing the prodigiosin at high yield, the prodigiosin can be stably preserved by using the protection method for the prodigiosin, the decoloration rate of the prodigiosin can be reduced, and the growth and reproduction of bacteria and fungi can be effectively inhibited by using the bacteriostasis method for bacteriostasis.

Description

Technical Field

[0001] This invention belongs to the field of styraxin technology, specifically a method for high-yield styraxin production, a method for protecting styraxin, 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, viscopolamine can be prepared through chemical synthesis and microbial fermentation. Chemical synthesis is relatively difficult, involves many reaction steps, and has low yields, making large-scale production challenging. Microbial fermentation, on the other hand, offers advantages such as environmental friendliness, mild conditions, low cost, and ease of industrialization. Therefore, its production has become a research hotspot both domestically and internationally in recent years. 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 advancements, the demand for viscopolamine is increasing, indicating a huge market potential.

[0003] However, the production of squalene mainly relies on naturally occurring squalene-producing bacteria. These bacteria have low yields, complex production processes, and high costs, limiting their industrial application. Therefore, identifying high-yield squalene-producing bacterial strains through isolation, identification, and mutagenesis breeding is of significant theoretical and practical value. In recent years, extensive research has been conducted on squalene-producing Serratia marcescens. To obtain high-yield strains, physicochemical mutagenesis, protoplast fusion technology, and genetic engineering are commonly used to modify the strains. However, genetic engineering breeding is complex and requires stringent conditions, thus its application is limited. Consequently, traditional physical and chemical mutagenesis methods for selecting high-yield strains remain the preferred strategy for most microbial breeding. Furthermore, existing research indicates that squalene is susceptible to environmental influences; different external factors can lead to discoloration or decreased activity. Therefore, improving the preservation methods of squalene to enhance its stability during storage is also crucial.

[0004] Numerous studies have shown that squalene exhibits a variety of biological activities, including antibacterial, antifungal, anticancer, and antiviral properties. It can also be used to rapidly eliminate most plankton that cause red tides. Among these, its antibacterial activity has been the most studied. However, existing research also indicates that the antibacterial activity of squalene is easily affected by environmental factors. Therefore, studying the factors that influence the antibacterial activity of squalene is particularly important for improving its antibacterial effect. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for high-yield production of styraxone, a method for protecting styraxone, and its applications. The method for high-yield production of styraxone using this invention can rapidly produce large quantities of styraxone. The method for protecting styraxone using this invention can stably preserve styraxone and reduce its decolorization rate. The antibacterial method of this invention can effectively inhibit the growth and reproduction of bacteria and fungi.

[0006] The above objectives of this invention are achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a method for high-yield production of serratia rubigin, the method comprising obtaining a mutant strain of *Serratia nematotophila*, screening *Serratia nematotophila* strains with high and stable serratia rubigin production from the mutant strains for fermentation culture, and isolating serratia rubigin from the fermentation culture product; wherein the *Serratia nematotophila* strain is *Serratia nematotophila*, strain number S207, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 2025727, and accession date April 8, 2025.

[0008] In some embodiments of the present invention, the method of obtaining mutant strains of Serratia nematodes includes one or more of the following: ultraviolet mutagenesis, microwave mutagenesis, low-energy ion implantation, laser mutagenesis, chemical reagent mutagenesis, and genetic engineering.

[0009] In some embodiments of the present invention, the method for obtaining the mutant strain of Serratia nematodes is to irradiate the Serratia nematodes under ultraviolet light for 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 durations: 60s, 90s, 120s, 150s, 180s, 210s, 240s, 280s, 300s.

[0010] In some embodiments of the present invention, the irradiation of 1 to 7 minutes is continuous irradiation of 1 to 7 minutes.

[0011] In some embodiments of the present invention, the method for screening Serratia nematodes strains with high and stable squalene production from mutant strains is to select strains that meet 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 nematodes strain number S207; 2) Stable squalene production: The mutant strain is passaged and the strains obtained from the passaged culture are fermented to produce squalene. 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%.

[0012] In some embodiments of the present invention, during the screening of *Serratia nematodes* strains with high and stable production of sclerotin, the sclerotin production can be measured by the OD value of the bacterial solution obtained after fermentation culture of the strain for 24–72 h. 535 Value representation.

[0013] In some embodiments of the present invention, the specific process for selecting strains with stable vitriol production 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 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 535 The 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%.

[0014] In some embodiments of the present invention, the solid culture medium is LB agar or beef extract peptone solid culture medium.

[0015] In some embodiments of the present invention, the culture medium used for fermentation culture is LB liquid medium.

[0016] 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:

[0017] 0–2 wt% carbon source, 0–2 wt% nitrogen source, 0–1 wt% inorganic salt.

[0018] 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).

[0019] 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.

[0020] In some embodiments of the present invention, the inorganic salt is selected from one or more of MgCl2, CaCl2, NaCl, KCl, ZnCl2, MgSO4, etc.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] In some embodiments of the present invention, the organic solvent includes one or more of methanol, ethanol, chloroform, ethyl acetate, etc.

[0028] In some embodiments of the present invention, the method further includes sequentially subjecting the recovered pigment-containing liquid to rotary evaporation and chromatography purification.

[0029] In some embodiments of the present invention, the rotary evaporation is carried out at 30–50°C.

[0030] In some embodiments of the invention, the chromatographic purification includes elution using a mixture of petroleum ether (PE) and ethyl acetate (EA).

[0031] In a second aspect, the present invention provides a strychnine produced by the method described in the first aspect.

[0032] Thirdly, the present invention provides a method for protecting styraxin, the method comprising storing styraxin in a protective agent with a pH value of 2 to 6, storing it at 4 to 60°C and avoiding exposure to ultraviolet light; preferably, the protective agent comprises an organic solvent, a metal salt and / or an oxidizing agent, wherein the metal salt is selected from one or more of manganese salt, zinc salt, potassium salt, ferrous salt, sodium salt, magnesium salt, and calcium salt, and the oxidizing agent comprises one or more of H2O2 and HNO3.

[0033] In some embodiments of the present invention, the styraxin includes the styraxin described in the second aspect.

[0034] In some embodiments of the present invention, the organic solvent includes one or more of ethanol, methanol, petroleum ether, ethyl acetate, etc.

[0035] In some embodiments of the present invention, the protective agent may also contain a small amount of water, for example, the water accounts for no more than 40% of the total volume fraction of the protective agent, preferably no more than 30%, more preferably no more than 20%, and even more preferably no more than 10%.

[0036] In some embodiments of the present invention, the pH value of the protective agent is 3 to 5, preferably 3.5 to 4.5.

[0037] In some embodiments of the present invention, the metal salt is selected from one or more of manganese salt, zinc salt, and potassium salt; preferably, the manganese salt includes potassium permanganate, manganese dioxide, manganese sulfate, manganese citrate, and manganese acetate, and / or the zinc salt includes zinc chloride, zinc nitrate, and zinc sulfate, and / or the potassium salt includes potassium chloride, potassium sulfate, potassium nitrate, and potassium acetate.

[0038] In some embodiments of the present invention, the concentration of the metal salt in the protective agent is 0.00001 to 0.2 mol / L, preferably 0.0001 to 0.1 mol / L.

[0039] In some embodiments of the present invention, the protective agent includes HNO3; preferably, the concentration of HNO3 in the protective agent is 0.0002 to 0.001 mol / L, more preferably 0.0004 to 0.0008 mol / L.

[0040] In some embodiments of the present invention, the pH value of the protective agent is 2.0, the protective agent includes MnSO4 and HNO3, and the concentration of HNO3 in the protective agent is 0.0004 mol / L; preferably, the concentration of MnSO4 in the protective agent is 0.002 mol / L.

[0041] In some embodiments of the present invention, the pH value of the protective agent is 2 to 4, the protective agent includes ZnSO4 and HNO3, and the concentration of HNO3 in the protective agent is 0.0006 to 0.0008 mol / L; preferably, the concentration of ZnSO4 in the protective agent is 0.002 mol / L.

[0042] In some embodiments of the present invention, the pH value of the protective agent is 2.0, the protective agent includes KCl and HNO3, and the concentration of HNO3 in the protective agent is 0.0008 mol / L; preferably, the concentration of KCl in the protective agent is 0.002 mol / L.

[0043] Fourthly, the present invention provides a method for inhibiting bacteria, the method comprising contacting a bacterial strain with an antibacterial agent comprising the styraxin described in the second aspect, wherein the antibacterial conditions include pH ≤ 6 and temperature ≤ 40°C; preferably, the bacterial strain comprises 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 some embodiments of the present invention, the concentration of styraxin in the antibacterial agent is 0.1 to 2 mg / mL.

[0047] In some embodiments of the present invention, the antibacterial conditions include a pH value of 4 to 6 and a temperature of 0 to 40°C.

[0048] 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, zinc salt, etc.; 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, zinc sulfate.

[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 method for high-yield production of squalene, which enables rapid production of large quantities of squalene. This invention also provides a method for protecting squalene, which stably preserves it and reduces its decolorization rate. Finally, this invention provides an antibacterial method that effectively inhibits the growth and reproduction of bacteria and fungi. Attached Figure Description

[0053] Figure 1 Indicates the OD of lecithin under different light conditions. 535 A graph showing how the value changes over time. Detailed Implementation

[0054] 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.

[0055] 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.

[0056] I. Selection and breeding of strains producing high levels of erythromycin

[0057] 1. First irradiation:

[0058] Using *Serratia nematotophila* strain S207 as the bacterial strain, a 5% inoculum was added to beef extract peptone liquid medium and incubated at 28°C for 24 hours. The resulting bacterial culture was then transferred to six sterile culture dishes numbered 1–6, 5 mL per dish. Five of these sterile dishes (numbered 2–6) were then irradiated under a UV lamp (25 cm away) for 90 s, 150 s, 210 s, 270 s, and 330 s, respectively. The sterile dish numbered 1 was placed in the dark. Subsequently, the bacterial culture was collected from each of the six sterile dishes... Take 10 μL of bacterial suspension from each culture 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 culture 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 value.

[0059] 2. Second irradiation:

[0060] Collect the deep red single colonies from the beef extract peptone solid medium obtained after the first irradiation and inoculate them into beef extract peptone liquid medium at a 5% inoculum rate. Incubate at 28℃ for 24 hours. Transfer the resulting bacterial culture to six sterile culture dishes numbered 7–12 (wherein, the bacterial cultures obtained from the first irradiation, numbered 1–6, correspond to the six sterile culture dishes numbered 7–12 for the second irradiation), 5 mL in each sterile culture dish. Then place five sterile culture dishes numbered 8–12 under a UV lamp (25 cm away) for 60 s, 120 s, 180 s, etc., respectively. After 240s and 300s, the sterile culture dish numbered 7 was placed in the dark. 10μL of bacterial solution was taken from each of the six sterile culture dishes and diluted with 29.99mL of sterile water in a sterile centrifuge tube. The diluted bacterial solution was then spread onto beef extract peptone solid medium numbered 7–12 (wherein, the bacterial solution obtained from the six sterile culture dishes numbered 7–12 corresponds to the six beef extract peptone solid medium numbered 7–12 respectively). 100μL of bacterial solution was spread onto each beef extract peptone solid medium. The medium was incubated in a constant temperature incubator at 28℃ in the dark for 24h. The number of colonies on each beef extract peptone solid medium was observed and recorded. Simultaneously, a single colony of deep red color was taken from the beef extract peptone solid medium and inoculated into a centrifuge tube containing 8 mL of beef extract peptone liquid medium at an inoculation rate of 5%. After incubation at 28℃ and 140 rpm for 36 h on a constant temperature shaker, the OD of each bacterial culture was measured using a UV spectrophotometer. 535 value.

[0061] For each treatment in the first and second irradiations, three replicates were performed. The OD values ​​of the three replicates were then analyzed. 535 The average value was calculated, and the results are shown in Table 1. Table 1 shows that excessively long irradiation time affects the yield of styraxin from the strain, and the OD value of the fermentation product obtained after the second irradiation is lower. 535 The values ​​are generally low, indicating that the first-generation strains of the strain that have just undergone one irradiation have a low yield of styraxin after being irradiated again.

[0062] Table 1. OD values ​​of bacterial cultures after irradiation with UV lamps for different durations. 535 value

[0063]

[0064] Furthermore, based on the recorded colony counts, the number of colonies on beef extract peptone solid media numbered 1–6 showed a decreasing trend, indicating that the lethality of the strain increased with the duration of irradiation during the first irradiation. The number of colonies on beef extract peptone solid media numbered 7–12 also showed a decreasing trend, indicating that the lethality of the strain increased with the duration of irradiation during the second irradiation; moreover, the number of colonies on beef extract peptone solid media numbered 8–12 was even lower than that on beef extract peptone solid media numbered 2–6.

[0065] 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 and 8–10 on beef extract peptone solid medium. These cultures were then spot-inoculated onto eight 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 strains were 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. 535 Values, 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 was selected as the optimal strain for high and stable production of styraxone.

[0066] Table 2. OD values ​​of bacterial cultures obtained by shaking incubation 535 value

[0067]

[0068] In Table 2, the generation number refers to the number of generations passed through the beef extract peptone solid medium.

[0069] II. Methods for protecting styraxin

[0070] 1. Obtaining erythromycin

[0071] (1) The strain S207-D obtained above was inoculated into the culture medium (the culture medium includes the following components: starch 0.25wt%, glycerol 0.75wt%, ammonium sulfate 1.5wt%, beef extract 0.3wt%, peptone 1wt%, sodium chloride 0.5wt%) at an inoculation rate of 5% and cultured statically at 28℃ for 72h. After centrifugation at 8000r / min for 10min at 4℃, the supernatant was discarded and the lower layer of bacteria was washed three times with distilled water. The bacteria were then filtered and recovered.

[0072] (2) Mix the bacterial cells obtained 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 cell residue, collect the liquid part and evaporate to dryness at 40 °C to obtain crude styraxin.

[0073] (3) Dissolve the crude pigment product 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, and rotary evaporate it at 40°C to obtain concentrated pure styraxin.

[0074] 2. Preservation of erythromycin

[0075] The styraxin obtained in step 1 above was dissolved in methanol to obtain a styraxin solution (the concentration of styraxin was approximately 0.2 mg / mL). The effects of different storage conditions on the stability of styraxin were investigated using the following preservation methods:

[0076] Protection Method 1: Adjust the pH of the sphagnum molybdate solution to 2.0, 4.0, 6.0, 8.0, 10.0, and 12.0 using 0.1 mol / L hydrochloric acid and / or 0.1 mol / L sodium hydroxide, and then use a UV spectrophotometer to detect the OD of the sphagnum molybdate solution. 535 The OD value of the initial styrax red pigment solution was obtained. 535The OD value of the styraxin solution was then measured using a UV spectrophotometer after being stored at room temperature in the dark for 2 hours, 3 hours, 4 hours, and 5 hours. 535 The value was calculated, and the OD of the styrax red solution was determined at this time. 535 The value relative to the OD of the initial styrax red pigment solution 535 The percentage of the value is recorded as the residual rate of styraxin at this time; for each treatment, three replicates are used, and the OD of the three replicates is calculated. 535 The average value was used to calculate the residual rate, and the color of the styraxone solution was observed after 5 hours of storage. The results are shown in Table 3. Table 3 shows that styraxone exhibits high stability when the pH of the styraxone solution is 2.0, 4.0, and 6.0, indicating that storage under acidic conditions can maintain the high stability of styraxone.

[0077] Table 3. Effect of pH value of styraxone solution on the stability of styraxone.

[0078]

[0079] Protection method 2: First, use an ultraviolet spectrophotometer to detect the OD of the styrax red solution. 535 The OD value of the initial styrax red pigment solution was obtained. 535 The value was approximately 0.72. The styraxin solution was then placed under sunlight, fluorescent light, and ultraviolet light for 2 hours, 3 hours, 4 hours, and 5 hours respectively. The OD value of the styraxin solution was then measured using an ultraviolet spectrophotometer. 535 For each storage method and processing method, calculate the OD of the three replicates. 535 The average value, the result is as follows Figure 1 As shown in Figure a. From Figure 1 As shown in Figure a, fluorescent lamps have no significant effect on the stability of styraxone, while exposure to sunlight has a small effect on the stability of styraxone. However, ultraviolet light has a significant effect on the stability of styraxone, with the stability of styraxone decreasing rapidly within 2 hours.

[0080] To further understand the effect of ultraviolet light on the stability of sphagnum molybdate, fresh sphagnum molybdate solution was prepared and placed under an ultraviolet lamp for 10 min, 20 min, 30 min, 40 min, and 50 min. The OD value of the sphagnum molybdate solution was then measured using an ultraviolet spectrophotometer. 535 For each storage method and processing method, calculate the OD of the three replicates. 535 The average value, the result is as follows Figure 1 As shown in Figure b. From Figure 1 As shown in Figure b, the stability of styraxin decreases rapidly under ultraviolet light, especially within 40 minutes, when the OD of the styraxin solution... 535 The value decreased rapidly.

[0081] Protection Method 3: Add the following metal salt solutions (0.1 mol / L each) to the squalene solution at a volume ratio of 1:50: FeCl3, FeCl2, ZnSO4, KCl, NaCl, CdCl2, CaCl2, MnSO4, MgSO4, CuSO4, and Ba(OH)2. First, use a UV spectrophotometer to measure the OD of the squalene solution. 535 The OD value of the initial styrax red pigment solution was obtained. 535 The OD value of the styraxin solution was then measured using a UV spectrophotometer after being stored at room temperature in the dark for 2 hours and 3 hours. 535 The value was then calculated, and the OD of the styrax red solution was then determined. 535 The value relative to the OD of the initial styrax red pigment solution 535 The percentage of the value is recorded as the residual rate of styraxin at this time; for each preservation method and each treatment method, three replicates are used, and the OD of the three replicates is calculated. 535 The average value was used to calculate the residual rate, and the results are shown in Table 4. Table 4 shows that MnSO4, ZnSO4, and KCl have different degrees of significant color-enhancing effects on squalene, and the effect of metal salts on squalene solutions becomes more significant with increasing time. Squalene is relatively stable in solutions containing FeCl2, NaCl, MgSO4, and CaCl2, and its color remains purplish-red without significant change. However, CuSO4, Ba(OH)2, FeCl3, and CdCl2 cause discoloration of squalene, indicating that squalene has a significant effect on the color of CuSO4, Ba(OH)2, FeCl3, and CdCl2. 2+ Ba 2+ Fe 3+ Cd 2+ Metal ions are quite sensitive. In particular, the presence of FeCl3 in the solution significantly affects styraxin, turning its color orange-yellow, which is presumably due to Fe... 3+ Its inherent properties cause the solution to change color drastically.

[0082] Table 4. Effects of metal salts on the stability of styracin

[0083]

[0084] Protection Method 4: Add a 0.1 mol / L dilute nitric acid solution to the styraxin solution, and adjust the concentration of HNO3 in the solution to 0.0002 mol / L, 0.0004 mol / L, 0.0006 mol / L, 0.0008 mol / L, and 0.0010 mol / L respectively. First, use a UV spectrophotometer to detect the OD of the styraxin solution. 535 The OD value of the initial styrax red pigment solution was obtained. 535The OD value of the styraxin solution was then measured using a UV spectrophotometer after storing it at room temperature in the dark for 2 hours. 535 The value was then calculated, and the OD of the styrax red pigment solution at that time was then determined. 535 The value relative to the OD of the initial styrax red pigment solution 535 The percentage of the value is recorded as the residual rate of styraxin at this time; for each preservation method and each treatment method, three replicates are used, and the OD of the three replicates is calculated. 535 The average value was used to calculate the residual rate, and the results are shown in Table 5. Table 5 shows that after adding HNO3 to the styraxin solution, the OD of styraxin... 535 The residual rate remained above 98% within 2 hours, indicating that styracin can exist stably in solutions containing HNO3, that is, styracin has good antioxidant properties.

[0085] Table 5. Effects of HNO3 on the stability of styraxin

[0086]

[0087] Protection Method 5: Add the following metal salt aqueous solutions (MnSO4, ZnSO4, KCl) to the squalene solution at a volume ratio of 1:50 (metal salt aqueous solution to squalene solution). Then add a 0.1 mol / L dilute nitric acid aqueous solution to the solution, adjusting the HNO3 concentration to 0.0004 mol / L, 0.0006 mol / L, and 0.0008 mol / L, respectively. Adjust the pH of the squalene solution to 2.0, 4.0, and 6.0 using 0.1 mol / L hydrochloric acid and / or 0.1 mol / L sodium hydroxide. Store at room temperature in the dark for 5 hours. Then, use a UV spectrophotometer to detect the OD of the squalene solution. 535 For each storage method and processing method, calculate the OD of the three replicates. 535 The average values ​​are shown in Table 6. Table 6 shows that using composite protectants 1, 4, 5, and 7 to preserve styraxone can improve its stability.

[0088] Table 6. Effects of composite protective agents on the stability of styraxene.

[0089]

[0090] III. Antibacterial effect of styraxin

[0091] 1. Antibacterial activity of styracil against bacteria (antibacterial activity was determined using the filter paper disc method)

[0092] 1) Prepare 7 centrifuge tubes of 50 mL each, add 5 mL of NB medium to each tube, autoclave and sterilize, then inoculate the 7 types of bacteria shown in Table 8 into the 7 centrifuge tubes, label them, and incubate them in a shaker at 37°C for 24 h to obtain activated bacterial culture.

[0093] 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.

[0094] 3) Following step 1 of the method for protecting styraxone, a concentrated pure styraxone product was obtained. The pure styraxone product was then used to prepare a styraxone solution with a concentration of 0.4 mg / mL.

[0095] 4) Immerse 6mm diameter filter paper pieces in the styraxin solution from step 3), then remove the filter paper and drain off excess solution. Place three filter paper pieces clockwise at intervals into the glass culture dish from step 2) where the bacterial suspension was first applied. Incubate the glass culture dish at 37℃ for 24 hours, observing for the presence of inhibition zones. Measure the diameter (mm) of the inhibition zone using calipers. The antibacterial effect is represented by the ratio of the inhibition zone diameter D to the filter paper diameter d. The results are shown in Table 7. Table 7 shows that styraxin 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.

[0096] Table 7. Antibacterial activity of styracil against bacteria

[0097]

[0098] 2. Antibacterial activity of levofloxacin against Bacillus subtilis and Staphylococcus aureus under different conditions (antibacterial activity was determined using the filter paper disc method).

[0099] (1) Different pH values: Following the steps above for the antibacterial activity of squalene against bacteria, the difference lies in preparing a squalene solution with a concentration of 0.4 mg / mL, and adjusting the pH value of the squalene solution to 4.0, 6.0, and 8.0 respectively using 0.1 mol / L sodium hydroxide solution and / or 0.1 mol / L hydrochloric acid solution. The results are shown in Table 8. As can be seen from Table 8, when the pH value is 4.0, squalene has a good antibacterial effect against Bacillus subtilis; when the pH value is 4.0 or 6.0, squalene has a good antibacterial effect against Staphylococcus aureus.

[0100] (2) Different metal ions: Following the steps above for the antibacterial activity of squalene against bacteria, the difference lies in preparing a squalene solution with a concentration of 0.4 mg / mL, and adding zinc sulfate, manganese sulfate, and potassium chloride respectively to make the concentration of zinc sulfate, manganese sulfate, or potassium chloride in the squalene solution 0.1 mol / L. After mixing, the solution was stored at room temperature in the dark for 3 hours. The results are shown in Table 8. As can be seen from Table 8, when zinc sulfate, manganese sulfate, and potassium chloride are added to the squalene solution, squalene has a good antibacterial effect against Bacillus subtilis and Staphylococcus aureus.

[0101] (3) Different antibacterial temperatures: Following the steps above for the antibacterial activity of squalene against bacteria, the difference lies in preparing a squalene solution with a concentration of 0.4 mg / mL and placing it in a 4℃ refrigerator, a 40℃ water bath, and a 60℃ water bath for 10 min respectively. The results are shown in Table 8. As can be seen from Table 8, when the temperature is 4℃, squalene has a good antibacterial effect on Bacillus subtilis; temperature has no significant effect on the antibacterial activity of squalene against Staphylococcus aureus.

[0102] Table 8. Antibacterial activity of styraxin under different conditions.

[0103]

[0104]

[0105] Note: In Table 8, "-" indicates that the antibacterial effect is not obvious.

[0106] 3. Antifungal activity of styracil against fungi (antifungal activity was determined using the plate confrontation method).

[0107] 1) Prepare 6 PDA culture media, inoculate the 6 fungi shown in Table 9 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.

[0108] 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.

[0109] 3) Following step 1 of the preservation method for squalene, a concentrated pure squalene product was obtained. A squalene solution with a concentration of 0.4 mg / mL was prepared. 6 mm diameter filter paper was immersed in the squalene solution, and after draining off excess solution, two filter paper pieces were placed symmetrically into the glass culture dish from step 2), 3 cm from the center of each dish. The culture dish was then incubated at 28℃ for 4 days. The width of the inhibition zone of squalene against six fungi was measured using calipers. The width of the inhibition zone was the distance (mm) between the edge of the fungal colony and the edge of the fungal growth. The inhibition effect was expressed as the ratio of the diameter D of the inhibition zone to the diameter d of the filter paper. The results are shown in Table 9. Table 9 shows that squalene had a significant inhibitory effect on Rhizopus oryzae and Streptomyces chromis, but no inhibitory effect on Aspergillus niger, Stem blight pathogens, Morchella esculenta, and Morchella mycoides.

[0110] Table 9. Antifungal activity of styraxin against fungi

[0111]

[0112] Note: In Table 9, "-" indicates that the antibacterial effect is not obvious.

[0113] 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 method for high-yield production of erythromycin, characterized in that, The method includes obtaining a mutant strain of *Serratianematotophila*, screening *Serratianematotophila* strains with high and stable production of squalene from the mutant strains for fermentation culture, and isolating squalene from the fermentation culture product; the *Serratianematotophila* strain is *Serratianematotophila*, strain number S207, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M2025727.

2. The method according to claim 1, characterized in that, The method for obtaining mutant strains of Serratia nematodes includes one or more of the following: ultraviolet mutagenesis, microwave mutagenesis, low-energy ion implantation, laser mutagenesis, chemical reagent mutagenesis, and genetic engineering; preferably, the method for obtaining mutant strains of Serratia nematodes is to irradiate the Serratia nematodes under ultraviolet light for 1 to 7 minutes, preferably 1 to 6 minutes, and more preferably 1 to 5 minutes. And / or, the method for screening Serratia nematodes strains with high and stable squalene production from mutant strains is to select strains that meet 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 nematodes strain number S207; 2) Stable squalene production: The squalene production of the mutant strain is relatively similar between adjacent generations of strains after subculturing and fermentation to produce squalene.

3. The method according to claim 1 or 2, 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 MgCl2, CaCl2, KCl, ZnCl2, ZnSO4, and MgSO4.

4. The method according to claim 3, 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 further comprises 0.2 wt% inorganic salt, wherein the inorganic salt is one or more of KCl, ZnCl2, and ZnSO4; or, the culture medium used for fermentation 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 value of the culture medium used for fermentation is 5–8, preferably 6–7. And / or, the fermentation culture conditions include: a temperature of 26–30°C and a 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.

5. The styraxin produced by the method according to claims 1 to 4.

6. The method for protecting styraxin according to claim 5, characterized in that, The protection method includes storing styraxin in a protective agent with a pH value of 2-6, storing it at 4-60°C and avoiding exposure to ultraviolet light; preferably, the protective agent includes an organic solvent, a metal salt and / or an oxidizing agent, wherein the metal salt is selected from one or more of manganese salt, zinc salt, potassium salt, ferrous salt, sodium salt, magnesium salt, and calcium salt, and the oxidizing agent includes one or more of H2O2 and HNO3; more preferably, the organic solvent includes one or more of ethanol, methanol, petroleum ether, and ethyl acetate.

7. The protection method according to claim 6, characterized in that, The pH value of the protective agent is 3 to 5, preferably 3.5 to 4.5; And / or, the metal salt is selected from one or more of manganese salts, zinc salts, and potassium salts; preferably, the manganese salt includes potassium permanganate, manganese dioxide, manganese sulfate, manganese citrate, and manganese acetate, and / or, the zinc salt includes zinc chloride, zinc nitrate, and zinc sulfate, and / or, the potassium salt includes potassium chloride, potassium sulfate, potassium nitrate, and potassium acetate; And / or, the concentration of the metal salt in the protective agent is 0.00001–0.2 mol / L, preferably 0.0001–0.1 mol / L; And / or, the protective agent includes HNO3; preferably, the concentration of HNO3 in the protective agent is 0.0002 to 0.001 mol / L, more preferably 0.0004 to 0.0008 mol / L.

8. A method for inhibiting bacteria, characterized in that, The antibacterial method includes contacting the bacterial strain with an antibacterial agent containing the styraxin of claim 5, wherein the antibacterial conditions include pH ≤ 6 and temperature ≤ 40°C; preferably, the bacterial strain includes 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.

9. The antibacterial method according to claim 8, characterized in that, The concentration of styraxin in the antibacterial agent is 0.1–2 mg / mL; and / or The antibacterial conditions include a pH value of 4–6 and a temperature of 0–40°C; and / or The antibacterial agent further includes metal salts, wherein the metal salts include one or more of potassium salts, manganese salts, and zinc salts; preferably, the potassium salts include potassium chloride, potassium sulfate, potassium nitrate, potassium acetate, and / or, the manganese salts include potassium permanganate, manganese dioxide, manganese sulfate, manganese citrate, manganese acetate, and / or, the zinc salts include zinc chloride, zinc nitrate, and zinc sulfate.

10. The antibacterial method according to claim 9, characterized in that, The concentration of the metal salt in the antibacterial agent is 0.05–0.2 mol / L, preferably 0.08–0.15 mol / L; and / or The antibacterial agent includes potassium salt and / or manganese salt; preferably, the potassium salt is potassium chloride, and / or the manganese salt is manganese sulfate.

Images