Cycloheptyl prodigiosin culture medium based on natural seawater and method
By using a culture medium based on natural seawater and optimized fermentation conditions, the problem of high fermentation cost of cycloheptamethrin erythrin was solved, enabling high-yield and low-cost industrial production.
Patent Information
- Application Number
- CN202511047507.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-29
- Publication Date
- 2025-11-14
AI Technical Summary
In existing technologies, the fermentation of cycloheptamethrin mainly uses commercially available Zobell Marine Broth 2216 medium, which is expensive and unsuitable for industrial production.
A culture medium based on natural seawater was used, consisting of peptone (5 g/L–11 g/L), yeast extract (1 g/L–1.5 g/L), carbon source (1.67 mL/L–13.33 mL/L), and magnesium chloride (2 g/L–3 g/L). This medium was used for the fermentation of *Spartinivicinus ruber* MCCC1K03745T. The fermentation conditions were 30℃–33℃ and 140 rpm for 30 h.
It increased the yield of cycloheptamethrin to 2.3 times the original yield, reduced production costs, and is suitable for industrial-scale fermentation.
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Figure CN120944749A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of biotechnology, and in particular to a cycloheptamethrin erythrocyte culture medium and method based on natural seawater. Background Technology
[0002] Prodiginines are a class of red pigments produced by various bacteria, such as Serratia marcescens, actinomycetes, and various marine bacteria. Structurally, various prodiginines share a core composed of a tripyrrole ring, but each member has unique substituents on the C-ring. To date, 33 prodiginines have been identified, exhibiting a variety of biological functions, such as antiparasitic, antimicrobial, and anticancer activities. Over the past two decades, the antitumor properties of prodiginines have attracted increasing attention due to their superior cytotoxicity against various cancer cell lines and relatively low cytotoxicity to normal tissues. Furthermore, studies have shown that prodiginines are not substrates for multidrug resistance proteins, which can protect cancer cells from the cytotoxic effects of drugs. These advantages make prodiginines ideal candidates for anticancer drugs.
[0003] Despite sharing the same parent nucleus, different members of the Spartinivicin family may have different mechanisms of action. (Based on Spartinivicinus ruber MCCC1K03745) T Cycloheptamethrin, obtained through fermentation culture (deposited on April 17, 2019, at the China Marine Microbial Culture Collection Center, registration number: MCCC1K03745), is a newly isolated spartinivicin from our laboratory. Studies have shown that cycloheptamethrin exhibits potent killing activity against various non-small cell lung cancer (NSCLC) cell lines with diverse genetic backgrounds; at low concentrations, it significantly inhibits the proliferation of NSCLC cell lines; while at high concentrations, it induces cell death in NSCLC cell lines through an unexplained pathway. Therefore, cycloheptamethrin possesses significant research value and potential for developing novel antitumor drugs. Currently, the fermentation of cycloheptamethrin mainly utilizes commercially available Zobell Marine Broth 2216 medium and its modified versions, which are relatively expensive and unsuitable for *Spartinivicinus* ruber MCCC1K03745. T Industrial production of bacteria. Summary of the Invention
[0004] This application provides a cycloheptamethrin-based culture medium and method for natural seawater to solve the technical problem.
[0005] To address the aforementioned technical problems, in a first aspect, this application provides a cycloheptamethrin erythromycin culture medium based on natural seawater, comprising the following components: peptone 5 g / L to 11 g / L, yeast extract 1 g / L to 1.5 g / L, carbon source 1.67 mL / L to 13.33 mL / L, magnesium chloride 2 g / L to 3 g / L, and natural seawater as the solvent.
[0006] In some of these embodiments, the pH of the culture medium is 7.6 ± 0.2.
[0007] In some of these embodiments, the carbon source includes one or more of soybean oil, peanut oil, rapeseed oil, and sesame oil.
[0008] In some of these embodiments, the following components are included:
[0009] The ingredients were peptone 11 g / L, yeast powder 1 g / L, soybean oil 5.833 mL / L, magnesium chloride 3 g / L, and natural seawater as the solvent.
[0010] Secondly, this application provides a method for producing cycloheptamethrin, comprising the following steps:
[0011] Preparation of seed culture: Marine bacterial strains were inoculated into MB2216 medium at 0.1% v / v and cultured at 35℃ and 140 rpm for 16 h to obtain seed culture;
[0012] Fermentation of cyclohexylpyridin: The seed culture was inoculated into the cyclohexylpyridin culture medium as described in any one of claims 1 to 4 at 1% v / v, and cultured at 30℃~33℃ and 140rpm for 30h to obtain the cyclohexylpyridin fermentation broth.
[0013] In some embodiments, the marine bacterium is *Spartinivicinus ruber* MCCC1K03745, a rhizosphere bacterium of *Spartina alterniflora*. T The bacteria were deposited at the China Marine Microbial Culture Collection Center on April 17, 2019, with the collection registration number MCCC1K03745.
[0014] Compared with the prior art, this application has at least the following beneficial effects:
[0015] This application uses natural seawater as the solvent for the culture medium, and employs peptone at 5 g / L to 11 g / L, yeast extract at 1 g / L to 1.5 g / L, carbon source at 1.67 mL / L to 13.33 mL / L, and magnesium chloride at 2 g / L to 3 g / L as the culture medium components. These components are inexpensive and increase the yield of cycloheptamethrin to 2.3 times the original yield, making it suitable for *Spartinivicinus ruber* MCCC1K03745.T Industrial-grade fermentation. Attached Figure Description
[0016] Figure 1 This is a schematic diagram showing the concentration results of cycloheptamethrin under different nitrogen source conditions in the embodiments of this application;
[0017] Figure 2 This is a schematic diagram showing the concentration results of cycloheptamethrin under different peptone concentration conditions in the embodiments of this application;
[0018] Figure 3 This is a schematic diagram showing the concentration results of cycloheptamethrin under different yeast powder concentration conditions in the embodiments of this application;
[0019] Figure 4 This is a schematic diagram showing the concentration results of cycloheptamethrin under different magnesium chloride concentration conditions in the embodiments of this application;
[0020] Figure 5 This is a schematic diagram showing the concentration results of cycloheptamethrin under different carbon source conditions in the embodiments of this application;
[0021] Figure 6 This is a schematic diagram showing the concentration results of cycloheptamethrin under different soybean oil concentration conditions in the embodiments of this application;
[0022] Figure 7 This is a diagram of the culture medium formulation for full factorial analysis shown in the embodiments of this application;
[0023] Figure 8 This is a schematic diagram illustrating the effect of different factors and conditions on the concentration of cycloheptamethrin in an embodiment of this application;
[0024] Figure 9 This is a schematic diagram of the variance analysis results of a full factorial analysis model shown in an embodiment of this application;
[0025] Figure 10 This is a diagram illustrating the formulation of a culture medium for a steepest ascent experiment, as shown in an embodiment of this application.
[0026] Figure 11 This is a diagram illustrating another steepest climbing experiment culture medium formulation, as shown in an embodiment of this application.
[0027] Figure 12 This is a schematic diagram illustrating the results of the steepest climb experiment shown in the embodiments of this application;
[0028] Figure 13 L shown in the embodiments of this application 27 (3 13 Orthogonal experimental variables and level plots;
[0029] Figure 14 L shown in the embodiments of this application 27 (3 13 Orthogonal experimental medium formulation and cycloheptamethrin fermentation yield;
[0030] Figure 15 L shown in the embodiments of this application 27 (3 13 ANOVA results of orthogonal experiments;
[0031] Figure 16 This is a schematic diagram showing the concentration results of cycloheptamethrin under different batches of seawater under different conditions, as illustrated in the embodiments of this application. Detailed Implementation
[0032] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.
[0033] As used herein, the terms “prepared from” and “comprising” are synonymous. The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.
[0034] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1 to 5” is disclosed, the described range should be interpreted as including the ranges “1 to 4”, “1 to 3”, “1 to 2”, “1 to 2 and 4 to 5”, “1 to 3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.
[0035] Furthermore, the indefinite articles “a” and “an” preceding the elements or components of this invention do not impose any limitation on the quantity requirement (i.e., the number of times) of the elements or components. Therefore, “an” or “a” should be interpreted as including one or at least one, and the singular form of an element or component also includes the plural form, unless the quantity clearly refers to the singular form.
[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.
[0037] This application uses commercially available MB2216 culture medium as a control. 37.4 g of commercially available MB2216 culture medium powder was weighed and dissolved in 950 mL of deionized water, adjusting the pH to 7.6 ± 0.2. At this point, its components and contents were: ammonium nitrate 0.0016 g / L, boric acid 0.022 g / L, calcium chloride 1.8 g / L, disodium hydrogen phosphate 0.008 g / L, ferric citrate 0.1 g / L, magnesium chloride 5.9 g / L, magnesium sulfate 3.24 g / L, peptone 5.0 g / L, potassium bromide 0.08 g / L, potassium chloride 0.55 g / L, sodium bicarbonate 0.16 g / L, sodium chloride 19.45 g / L, sodium fluoride 0.0024 g / L, sodium silicate 0.004 g / L, strontium chloride 0.034 g / L, yeast extract 1.0 g / L, pH... 7.6±0.2; sterilize at 121℃ for 20 min.
[0038] Example 1
[0039] Spartinivicinus ruber MCCC1K03745 T Preparation of seed culture: The bacterial strain stored in a 4°C refrigerator was inoculated into MB2216 medium at a ratio of 0.1% (v / v) and cultured at 35°C and 140 rpm for 16 hours.
[0040] (1) In the culture medium based on natural seawater, the nitrogen sources were selected as peptone, soybean peptone, and... Peptone was used at a fixed concentration of 5 g / L; yeast extract was used at a fixed concentration of 1 g / L; ferric phosphate was used at a concentration of 0.01 g / L; commercially available MB2216 medium was used as a control; seed culture was inoculated at a ratio of 1% v / v and cultured at 30°C and 140 rpm for 30 h. Figure 1 It is known that peptone is the optimal nitrogen source for the fermentation of cycloheptamethrin.
[0041] (2) In a natural seawater-based culture medium, the yeast extract concentration was fixed at 1 g / L; the ferric phosphate concentration was 0.01 g / L; peptone was selected as the nitrogen source, and its concentration was adjusted to 3 g / L, 5 g / L, 7 g / L, 9 g / L, and 11 g / L, respectively; commercially available MB2216 medium was used as a control. Seed culture was inoculated at a ratio of 1% v / v and cultured at 30℃ and 140 rpm for 30 h. Figure 2It can be seen that the concentration of peptone has a significant effect on the fermentation yield of cycloheptamethrin, and the yield of cycloheptamethrin reaches its highest level when the peptone concentration is 9 g / L.
[0042] (3) In a natural seawater-based culture medium, the peptone concentration was fixed at 5 g / L; the ferric phosphate concentration was 0.01 g / L; and the yeast extract concentration was adjusted to 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, and 2.5 g / L, respectively. Commercially available MB2216 medium was used as a control. Seed culture was inoculated at a ratio of 1% v / v and cultured at 30℃ and 140 rpm for 30 h. Figure 3 It can be seen that the concentration of yeast powder has a significant effect on the fermentation yield of cycloheptamethrin, and the yield of cycloheptamethrin reaches its highest level when the yeast powder concentration is 1.5 g / L.
[0043] (4) In a natural seawater-based culture medium, the peptone concentration was fixed at 5 g / L; the ferric phosphate concentration was 0.01 g / L; the yeast extract concentration was 1 g / L; and the amount of additional MgCl2·6H2O was controlled at 0 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, and 5 g / L. Seed culture was inoculated at a ratio of 1% v / v and cultured at 30℃ and 140 rpm for 30 h. Figure 4 It can be seen that the concentration of magnesium chloride has a significant effect on the fermentation yield of cycloheptamethrin. When the amount of MgCl2·6H2O added is 3 g / L, the yield of cycloheptamethrin reaches the highest level.
[0044] (5) In a natural seawater-based culture medium, the peptone concentration was fixed at 5 g / L; the ferric phosphate concentration was 0.01 g / L; the yeast extract concentration was 1 g / L; and additional glycerol (0.5% v / v), rapeseed oil (0.67% v / v), soybean oil (0.67% v / v), peanut oil (0.67% v / v), and sesame oil (0.67% v / v) were added as carbon sources. Seed culture was inoculated at a ratio of 1% v / v and cultured at 30℃ and 140 rpm for 30 h. Figure 5 It can be seen that the type of carbon source has a significant impact on the fermentation yield of cycloheptamethrin, and the yield of cycloheptamethrin reaches its highest level when soybean oil is selected as the carbon source.
[0045] (6) In a natural seawater-based culture medium, the peptone concentration was fixed at 5 g / L; the ferric phosphate concentration was 0.01 g / L; the yeast extract concentration was 1 g / L; and the additional soybean oil concentration was controlled at 0 mL / L, 1.67 mL / L, 3.33 mL / L, 6.67 mL / L, and 13.33 mL / L. Seed culture was inoculated at a ratio of 1% v / v and cultured at 30℃ and 140 rpm for 30 h. Figure 6It can be seen that the addition of soybean oil has a significant effect on the yield of cyclohexylpyridin, but the yield-increasing effect reaches saturation when the addition amount is 1.67 mL / L.
[0046] In summary, the following concentrations of peptone (5 g / L–11 g / L), yeast extract (1 g / L–1.5 g / L), carbon source (1.67 mL / L–13.33 mL / L), and magnesium chloride (2 g / L–3 g / L) significantly affected the yield of cyclohexylpyridin; while the effect of ferric phosphate on cyclohexylpyridin was low and negligible.
[0047] Example 2
[0048] Spartinivicinus ruber MCCC1K03745 T The seed solution was prepared as in Example 1.
[0049] according to Figure 7 (Culture medium formulation for full factorial analysis) Prepare natural seawater culture medium, inoculate with seed culture at a ratio of 1% v / v, and incubate at 30℃ and 140 rpm for 30 h. Figure 8 It can be seen that soybean oil, peptone, and yeast powder have a significant impact on the yield of cycloheptamethrin, while magnesium chloride has no significant effect on fermentation yield; Figure 9 (Analysis of variance results from full factorial analysis) show that there are interactions between soybean oil and peptone, as well as between soybean oil and yeast extract. Furthermore, the order of priority for factors significantly affecting cycloheptamethrin production is soybean oil > peptone > yeast extract.
[0050] The magnesium chloride concentration was fixed at 3 g / L, and according to... Figure 10 (Steepest Climb Experiment Medium Formulation 1 (Formulation per 30mL of medium)) and Figure 11 (Steepest Climb Experiment Medium Formula 2 (Formula per 30 mL)) Prepare the medium, inoculate with seed culture at a ratio of 1% (v / v), and incubate at 30℃ and 140 rpm for 30 h. Use the steepest climb experiment to explore the optimal fermentation conditions; such as... Figure 12 As shown, under the conditions of Step 2.5, the yield of cycloheptamethrin was the highest.
[0051] according to Figure 13 (L 27 (3 13 (orthogonal experimental variables and levels) and Figure 14 (L 27 (3 13 (Orthogonal experimental medium formulation and cycloheptamethrin fermentation yield) Prepare the medium, inoculate the seed culture at a ratio of 1% v / v, and incubate at 30℃ and 140rpm for 30h. Use L 27 (3 13Orthogonal experiments were conducted to optimize the composition of the culture medium for efficient fermentation. Figure 15 It can be seen that when the culture medium composition is 11 g / L peptone, 1 g / L yeast extract, 5.833 mL / L soybean oil, and 3 g / L magnesium chloride, the fermentation level of cycloheptamethrin is the highest, reaching 14.49 mg / L. Figure 15 The study also showed that peptone and yeast extract had a significant impact on yield, while soybean oil had no significant effect. In the validation experiment, three fermentations were carried out under the optimized formulation of the orthogonal experiment, and the yield of cycloheptamethrin was 14.22 mg / L, with a deviation from the predicted value of less than 2%.
[0052] Take three batches of seawater, according to Figure 14 Prepare fermentation medium, inoculate with seed culture at a ratio of 1% v / v, and incubate at 30℃ and 140 rpm for 30 h; results are as follows. Figure 16 As shown in the analysis of variance, different batches of seawater-based culture media had no significant effect on the fermentation yield of cycloheptamethrin.
[0053] Based on Examples 1 and 2, it can be seen that a method based on natural seawater and suitable for Spartinivicinus ruberMCCC1K03745 T The culture medium formula for high-yield fermentation of cycloheptamethrin is as follows: peptone 11 g / L, yeast extract 1 g / L, soybean oil 5.833 mL / L, magnesium chloride 3 g / L, solvent is natural seawater, and pH is 7.6. A suitable medium for *Spartinivicinus ruber* MCCC1K03745 T A high-yield fermentation method for producing spartinivicin using Spartinivicinus ruberMCCC1K03745 T Seed culture, inoculated at 1% v / v into the *Spartinivicinus* ruber MCCC1K03745 T The culture medium for high-yield cycloheptamethrin was fermented at 30℃ and 140 rpm for 30 hours. The concentration of cycloheptamethrin in the fermentation broth was measured to be 14.22 mg / L.
[0054] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0055] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of this application. It should be understood that the above descriptions are merely specific embodiments of this application and are not intended to limit the scope of protection of this application. In particular, it should be noted that any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application for those skilled in the art.
Claims
1. A cycloheptamethrin-based culture medium containing natural seawater, characterized in that, It includes the following components: peptone 5g / L~11g / L, yeast powder 1g / L~1.5g / L, carbon source 1.67mL / L~13.33mL / L, magnesium chloride 2g / L~3g / L, and natural seawater as solvent.
2. The cycloheptamethrin-based culture medium for natural seawater as described in claim 1, characterized in that, The pH value of the culture medium is 7.6 ± 0.
2.
3. The cycloheptamethrin-based culture medium for natural seawater as described in claim 1, characterized in that, The carbon source includes one or more of soybean oil, peanut oil, rapeseed oil, and sesame oil.
4. The cycloheptamethrin-based culture medium for natural seawater as described in claim 1, characterized in that, Includes the following components: The ingredients were peptone 11 g / L, yeast powder 1 g / L, soybean oil 5.833 mL / L, magnesium chloride 3 g / L, and natural seawater as the solvent.
5. A method for producing cycloheptamethrin, characterized in that, Includes the following steps: Preparation of seed culture: Marine bacterial strains were inoculated into MB2216 medium at 0.1% v / v and cultured at 35℃ and 140 rpm for 16 h to obtain seed culture; Fermentation of cyclohexylpyridin: The seed culture was inoculated into the cyclohexylpyridin culture medium as described in any one of claims 1 to 4 at 1% v / v, and cultured at 30℃~33℃ and 140rpm for 30h to obtain the cyclohexylpyridin fermentation broth.
6. The method for producing cycloheptamethrin as described in claim 5, characterized in that, The marine bacterium mentioned is *Spartinivicinus ruber* MCCC1K03745, a rhizosphere bacterium of *Spartina alterniflora*. T The bacteria were deposited at the China Marine Microbial Culture Collection Center on April 17, 2019, with the collection registration number MCCC1K03745.