Fermentation culture medium and culture method for improving yield and activity of polyketone compounds

Abstract

The invention belongs to the technical field of strain culture, and particularly relates to a fermentation culture medium and a culture method for improving the yield and activity of polyketone compounds. The fermentation culture medium is an improved LB liquid culture medium; according to the improved LB liquid culture medium, on the basis of the LB liquid culture medium, 6.15 g / L of NaNO3 is used for replacing an original nitrogen source in the LB liquid culture medium, and the pH value is 6.5. According to the fermentation culture medium, the culture rate of the bacillus velezensis DJ1 can be increased, the yield and activity of polyketone compounds produced by the bacillus velezensis DJ1 are improved, and an effective way is provided for increasing the yield of the polyketone compounds produced by the bacillus velezensis DJ1.

Description

Technical Field

[0001] The present invention belongs to the technical field of strain culture, and specifically relates to a fermentation medium and a culture method for improving the yield and activity of polyketide compounds. Background Art

[0002] Currently, agriculture is facing increasingly serious ecological security problems such as soil degradation, pesticide residues, and environmental pollution. The development of bio-agriculture is crucial for addressing many severe global challenges such as food security, environmental degradation, and plant diseases. Developing new biological control agents using natural metabolites of microorganisms meets the requirements of the development of green and efficient agriculture, can significantly reduce the use of chemical pesticides and fertilizers, improve the yield and quality of agricultural products, and is more competitive in the current agricultural production context.

[0003] Bacillus velezensis is an important biocontrol resource bacterium newly discovered in recent years. It can produce various antibacterial active metabolites such as lipopeptides, proteins, and polyketides. These substances have broad-spectrum antibacterial activity and have strong inhibitory effects on various vegetable crop pathogens such as soybean root rot pathogen, rice blast pathogen, and Chinese cabbage soft rot pathogen. It is an important source of substances for developing new biological pesticides and has received high attention from the academic and industrial circles.

[0004] Bacillus velezensis DJ1 is a strain of disease-preventing and growth-promoting bacterium isolated by the team of Heilongjiang Institute of Microbiology. It can produce polyketide compounds. However, when using a conventional medium to culture Bacillus velezensis, the yield and activity of the polyketide compounds produced by the strain are not stable and cannot be applied in commercial production. Therefore, how to provide a fermentation medium and a culture method that can improve the yield and activity of polyketide compounds produced by Bacillus velezensis has become an urgent problem to be solved in this field. Summary of the Invention

[0005] In order to solve the problems such as low yield and poor activity of polyketide compounds produced by Bacillus velezensis DJ1 in the prior art, the present invention provides a method for improving the yield and activity of polyketide compounds produced by Bacillus velezensis DJ1, which specifically includes the following technical solutions:

[0006] A fermentation medium suitable for Bacillus velezensis, wherein the fermentation medium is a modified LB liquid medium;

[0007] The modified LB liquid medium is: on the basis of the LB liquid medium, replacing the original nitrogen source in the LB liquid medium with 6.15 g / L of NaNO 3 and having a pH value of 6.5.

[0008] The present invention also provides a medium combination for culturing Bacillus velezensis, including a seed medium and a fermentation medium;

[0009] The seed culture medium is MLB liquid medium;

[0010] The fermentation culture medium is the fermentation culture medium as described above.

[0011] The present invention also provides the application of the fermentation culture medium or medium combination as described above in improving the yield and / or activity of polyketide compounds produced by Bacillus velezensis.

[0012] The present invention also provides a method for improving the yield and / or activity of polyketide compounds produced by Bacillus velezensis, comprising the following steps:

[0013] Inoculate Bacillus velezensis into the seed culture medium for seed culture to obtain a seed solution;

[0014] Inoculate the seed solution into the fermentation culture medium for fermentation culture to obtain a fermentation culture solution;

[0015] Isolate the polyketide compounds in the fermentation culture solution to obtain polyketide compounds;

[0016] The fermentation culture medium is the fermentation culture medium as described above.

[0017] Preferably, the seed culture medium is MLB liquid medium; the time for seed culture is 15 - 24 h.

[0018] Preferably, the inoculation amount of the seed solution is 4% - 6% of the volume of the fermentation culture medium.

[0019] Preferably, the temperature for fermentation culture is 28 - 32 °C, the duration is 48 - 60 h, and the rotation speed is 170 - 190 rpm.

[0020] Preferably, an organic solvent extraction method is used to isolate the polyketide compounds in the fermentation culture solution.

[0021] Preferably, the steps of the organic solvent extraction method are as follows:

[0022] Centrifuge the fermentation culture solution and collect the supernatant;

[0023] Mix the supernatant with n-butanol according to a volume ratio of 4:1, shake on a shaker for 1.5 - 2 h to obtain a mixed solution; let the mixed solution stand for 8 - 12 h to separate layers, take the upper organic phase, evaporate and dry to obtain polyketide compounds.

[0024] Preferably, the rotation speed for centrifugation is 4000 rpm and the centrifugation duration is 15 min.

[0025] The beneficial effects of the present invention are as follows:

[0026] The present invention provides a fermentation medium for Bacillus velezensis DJ1, and the fermentation medium is a modified LB liquid medium; the modified LB liquid medium is: on the basis of the LB liquid medium, 6.15 g / L of NaNO 3 replaces the original nitrogen source in the LB liquid medium, and the pH value is 6.5. The present invention adjusts the components of the fermentation medium according to the physiological characteristics of Bacillus velezensis DJ1, which can improve the culture rate of Bacillus velezensis DJ1, and further improve the yield and activity of polyketide compounds produced by Bacillus velezensis.

[0027] The present invention also provides a method for increasing the yield of polyketide compounds produced by Bacillus velezensis DJ1. Culturing with the fermentation medium as described above can increase the yield of polyketide compounds produced by Bacillus velezensis DJ1, and also improve the antibacterial activity of the polyketide compound product. The present invention provides a new idea for increasing the yield of polyketide compounds produced by Bacillus velezensis DJ1. Description of the Drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0029] Figure 1 It is the growth curve of the strain of Bacillus velezensis DJ1 during the seed culture measured in Experimental Example 1 of the present invention;

[0030] Figure 2 It is the yield of polyketide compounds of Bacillus velezensis DJ1 when inoculated with different seed ages in Experimental Example 1 of the present invention;

[0031] Figure 3 It is the fermentation growth curve of Bacillus velezensis DJ1 during the fermentation culture measured in Experimental Example 2 of the present invention;

[0032] Figure 4 It is the yield of polyketide compounds of Bacillus velezensis DJ1 at different fermentation durations in Experimental Example 2 of the present invention;

[0033] Figure 5 It is the yield of polyketide compounds of Bacillus velezensis DJ1 at different inoculation amounts in Experimental Example 3 of the present invention;

[0034] Figure 6 It is the yield of polyketide compounds of Bacillus velezensis DJ1 under different culture conditions of different media in Experimental Example 4 of the present invention;

[0035] Figure 7 It is the yield of polyketide compounds of Bacillus velezensis DJ1 in the culture medium with different component optimizations in Experimental Example 5 of the present invention;

[0036] Figure 8 Polyketide production of Bacillus velezensis DJ1 at different fermentation temperatures in Experimental Example 6 of the present invention;

[0037] Figure 9 Polyketide production of Bacillus velezensis DJ1 at different fermentation rotation speeds in Experimental Example 7 of the present invention;

[0038] Figure 10 Polyketide production of Bacillus velezensis DJ1 at different fermentation pH values in Experimental Example 8 of the present invention;

[0039] Figure 11 Response surface model diagram of the influence of various factors on polyketide production in Experimental Example 9 of the present invention;

[0040] Figure 12 Seed age - fermentation duration response surface and contour lines in Experimental Example 9 of the present invention;

[0041] Figure 13 Seed age - inoculum size response surface and contour lines in Experimental Example 9 of the present invention;

[0042] Figure 14 Seed age - fermentation temperature response surface and contour lines in Experimental Example 9 of the present invention;

[0043] Figure 15 Seed age - fermentation rotation speed response surface and contour lines in Experimental Example 9 of the present invention;

[0044] Figure 16 Seed age - pH response surface and contour lines in Experimental Example 9 of the present invention;

[0045] Figure 17 Fermentation duration - inoculum size response surface and contour lines in Experimental Example 9 of the present invention;

[0046] Figure 18 Fermentation duration - fermentation temperature response surface and contour lines in Experimental Example 9 of the present invention;

[0047] Figure 19 Fermentation duration - fermentation rotation speed response surface and contour lines in Experimental Example 9 of the present invention;

[0048] Figure 20 Fermentation duration - pH response surface and contour lines in Experimental Example 9 of the present invention;

[0049] Figure 21 Inoculum size - fermentation temperature response surface and contour lines in Experimental Example 9 of the present invention;

[0050] Figure 22 Inoculum size - fermentation rotation speed response surface and contour lines in Experimental Example 9 of the present invention;

[0051] Figure 23 This is the inoculation amount - pH response surface and contour lines in Experimental Example 9 of the present invention;

[0052] Figure 24 This is the fermentation temperature - fermentation rotation speed response surface and contour lines in Experimental Example 9 of the present invention;

[0053] Figure 25 This is the fermentation temperature - pH response surface and contour lines in Experimental Example 9 of the present invention;

[0054] Figure 26 This is the fermentation rotation speed - pH response surface and contour lines in Experimental Example 9 of the present invention;

[0055] Figure 12 、 Figures 17 - 20 In Figures 17 - 20 , the shown fermentation cycle is the fermentation duration;

[0056] Figure 27 This is the schematic diagram of the antibacterial effects of different groups in Experimental Example 10 of the present invention;

[0057] Figure 28 This is the schematic diagram of the polyketide synthesis mechanism catalyzed by type I PKS in Experimental Example 11 of the present invention;

[0058] Among them, Propionyl-CoA: propionyl coenzyme A; Methylmalonyl-CoA: methylmalonyl CoA; Module: the module responsible for the extension of two-carbon units; DEBS: PKS enzyme complex; EryF: C-6 hydroxylase; erythronolide B: erythromycin lactone; EryBV: C-3 glycosyltransferase; mycarose: L-mycarose; 3-0-α-mycarosylerythronolide B: 3-0-mycarosyl erythromycin lactone; EryCIII: C-5 glycosyltransferase; Desosamine: D-desosamine; Erythromycin-D: erythromycin D; EryK: C-12 hydroxylase; Erythromycin-C: erythromycin C; EryG: C-3 methyltransferase; Erythro mycin-B: erythromycin B; Erythromycin-A: erythromycin A.

[0059] Figure 29 This is the schematic diagram of the polyketide synthesis mechanism catalyzed by type II PKS in Experimental Example 11 of the present invention;

[0060] Among them, Propionyl-CoA: propionyl coenzyme A; Malonyl-CoA: malonyl coenzyme A; Dps, Dnr, Dox: a series of modification genes in the natural synthesis pathway of doxorubicin; 21-carbon decaketide: 21-carbon decaketide compound; I-daunosamine-TDP: TDP derivative of L-daunosamine thymidine diphosphate; E-rhodomycinone is ε-rhodomycinone; TDP: thiamine pyrophosphate; Rhodomycine D: erythromycin D; Doxorubicin: doxorubicin;

[0061] Figure 30 This is the schematic diagram of the polyketide synthesis mechanism catalyzed by type III PKS in Experimental Example 11 of the present invention;

[0062] Among them, medium chain acyl-CoA: medium chain acyl coenzyme A; ethylmalonyl-CoA: malonyl coenzyme A; Gc s: glycine cleavage system; β-Ketoacyl-X: β-ketoacyl coenzyme A; Methylmalonyl-CoA: methylmalonyl coenzyme A; Ethyl-Malonyl-CoA: ethylmalonyl coenzyme A; β,δ-diketothioester-CoA: β,δ-diketothioester of coenzyme a; triketide intermediate: triketide ester intermediate. Detailed implementation mode

[0063] The present invention provides a fermentation medium suitable for Bacillus velezensis DJ1, and the fermentation medium is a modified LB liquid medium; the modified LB liquid medium is: on the basis of the LB liquid medium, 6.15 g / L of NaNO 3 replaces the yeast powder in the LB liquid medium, and the pH value is 6.5.

[0064] The composition of the modified LB liquid medium is: using water as a solvent, and further including 10.0 g / L of peptone (Tryptone), NaNO 3 6.15 g / L and 10.0 g / L of NaCl.

[0065] The present invention also provides a medium combination for culturing Bacillus velezensis DJ1, including a seed medium and a fermentation medium; the seed medium is an MLB liquid medium; the fermentation medium is the fermentation medium as described above. As an implementation mode, the MLB liquid medium of the present invention: 2.0 g / L of anhydrous glucose, 7.0 g / L of peptone, 2.0 g / L of yeast powder, 6.0 g / L of NaCl, 0.06 g / L of KCl and MgCl 2 ·6H2 O 0.5 g / L.

[0066] The present invention also provides the use of the fermentation medium or medium combination as described above in increasing the yield and / or activity of polyketide compounds produced by Bacillus velezensis. As an embodiment, the Bacillus velezensis may be Bacillus velezensis DJ1. As an embodiment, the activity referred to in the present invention means antibacterial activity. As an embodiment, the antibacterial activity of the present invention includes any one or more of the activities of inhibiting Fusarium oxysporum f. sp. cucumerinum, inhibiting Fusarium graminearum, inhibiting Rhizoctonia solani of Acanthopanax senticosus, inhibiting Phytophthora sojae, inhibiting Colletotrichum lindemuthianum, inhibiting Botrytis cinerea of tomato, inhibiting Setosphaeria turcica of maize, and inhibiting Colletotrichum coccodes of tomato.

[0067] The present invention also provides a method for increasing the yield and / or activity of polyketide compounds produced by Bacillus velezensis DJ1, comprising the following steps:

[0068] Inoculating the Bacillus velezensis DJ1 strain into a seed medium for seed culture to obtain a seed solution;

[0069] Inoculating the seed solution into a fermentation medium for fermentation culture to obtain a fermentation broth;

[0070] Separating the polyketide compounds in the fermentation broth to obtain polyketide compounds;

[0071] The fermentation medium is the fermentation medium in the fermentation medium or medium combination as described above.

[0072] The present invention inoculates a Bacillus velezensis strain into a seed culture medium for seed culture to obtain a seed liquid. As an implementation manner, the seed culture medium of the present invention is an MLB liquid culture medium. As an implementation manner, the Bacillus velezensis includes Bacillus velezensis DJ1. As an implementation manner, the Bacillus velezensis DJ1 strain is the strain disclosed in the patent: CN115975875A. As an implementation manner, the temperature of the seed culture is 30-40°C, the rotation speed is 170-190 rpm, and the culture time is 15-24 h. As another implementation manner, the temperature of the seed culture can be any one of 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, and 40°C. As another implementation manner, the rotation speed of the seed culture can be any one of 170 rpm, 180 rpm, and 190 rpm. As an implementation manner, the culture time of the seed culture can be any one of 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, and 24 h. In a specific embodiment, the seed culture is carried out at 35°C and 180 rpm for 20 h.

[0073] After obtaining the seed culture solution, the present invention inoculates the seed liquid into a fermentation culture medium for fermentation culture to obtain a fermentation culture solution. As an implementation manner, the inoculation amount of the seed culture solution of the present invention is 4%-6% of the volume of the fermentation culture medium. As another implementation manner, the inoculation amount of the seed liquid of the present invention can be any one of 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, and 6.0% of the volume of the fermentation culture medium. As an implementation manner, the temperature of the fermentation culture of the present invention is 28-32°C. As another implementation manner, the temperature of the fermentation culture of the present invention can be any one of 28°C, 29°C, 30°C, 31°C, and 32°C. As an implementation manner, the duration of the fermentation culture of the present invention is 48-60 h. As another implementation manner, the duration of the fermentation culture of the present invention can be any one of 42 h, 43 h, 44 h, 45 h, 46 h, 47 h, 48 h, 49 h, 50 h, 51 h, 52 h, 53 h, 54 h, 55 h, 56 h, 57 h, 58 h, 59 h, and 60 h. As an implementation manner, the rotation speed of the fermentation culture of the present invention is 170-190 rpm. As another implementation manner, the rotation speed of the fermentation culture of the present invention can be any one of 170 rpm, 180 rpm, and 190 rpm.

[0074] After obtaining the fermentation broth, the present invention separates the polyketide compounds in the fermentation broth to obtain polyketide compounds. As an implementation manner, the present invention uses an organic solvent extraction method to separate the polyketide compounds in the fermentation broth. As an implementation manner, the steps of the organic solvent extraction method are as follows: centrifuge the fermentation broth and collect the supernatant; mix the supernatant with n-butanol according to a volume ratio of 4:1, shake on a shaker for 1.5 - 2 h to obtain a mixed solution; let the mixed solution stand for 8 - 12 h to separate layers, take the upper organic phase, evaporate and dry to obtain polyketide compounds. As another implementation manner, the shaking duration on the shaker can be any one of 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, and 2.0 h. As an implementation manner, the standing duration for layer separation of the mixed solution in the present invention can be any one of 8 h, 9 h, 10 h, 11 h, and 12 h. As an implementation manner, the present invention uses a separating funnel for standing layer separation. As an implementation manner, the rotation speed of the centrifugation in the present invention is 4000 rpm, and the centrifugation duration is 15 min. As another implementation manner, the evaporation and drying is to naturally dry the upper organic phase in a fume hood or perform rotary evaporation drying, and then place the upper organic phase in an oven at 30 °C and dry it to a constant weight.

[0075] To further illustrate the present invention, the following describes in detail a fermentation medium and a cultivation method for improving the yield and activity of polyketide compounds provided by the present invention with reference to the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0076] The present invention aims to evaluate the influence of important physical and chemical factors on the fermentation process, so as to optimize the fermentation process. In the present invention, the initial cultivation includes initial seed cultivation and initial fermentation cultivation, and the cultivation conditions of the initial seed cultivation and the initial fermentation cultivation are as follows:

[0077] Initial seed cultivation conditions: Fill 20 mL of MLB liquid medium into an Erlenmeyer flask with a specification of 250 mL, then inoculate the Bacillus velezensis DJ1 strain into the MLB medium according to a volume ratio of 4%, and perform seed cultivation at 180 rpm and 35 °C until the seed age reaches 24 h to obtain a seed solution.

[0078] Initial fermentation cultivation conditions: Inoculate the seed solution into the LB medium with a pH value of 6.7 according to a volume percentage of 2% for fermentation cultivation. The cultivation period of the fermentation cultivation is 48 h, the cultivation temperature is 30 °C, and the rotation speed is 180 rpm. The components of the MLB medium and the LB medium are shown in Table 4.

[0079] On this basis, an exploration experiment was carried out by the method of controlling variables. In Experimental Examples 1-8, only a single factor was adjusted, and other factors remained at the initial culture condition values. The same specification of culture medium was used in each experiment to evaluate the effects of different physical and chemical factors, namely seed age, fermentation duration, inoculum size, and physical and chemical conditions such as carbon source, nitrogen source, inorganic salts, temperature, rotation speed, and pH value on the fermentation process, and to compare the accumulation amounts of polyketides under different conditions.

[0080] Experimental Example 1 Seed Age Optimization Experiment

[0081] A suitable seed age can keep the microorganisms in a vigorous state and improve the product metabolism rate.

[0082] In this experimental example, the Bacillus velezensis DJ1 strain was inoculated into the MLB solid medium of the seed culture medium, and the Bacillus velezensis DJ1 strain was cultured under the initial seed culture conditions. During the seed culture process, samples were taken every 1 h to detect the cell concentration index, and the growth curve of the strain was plotted. The results are as Figure 1 shown.

[0083] According to Figure 1 the results shown, in the later stage of the logarithmic growth phase of Bacillus velezensis DJ1, that is, in the interval of 15-24 h of seed age, the optimal seed age was explored.

[0084] Bacillus velezensis was cultured according to the initial seed culture conditions, with the difference that different seed liquors were harvested at the seed ages of 15 h, 18 h, 21 h, and 24 h respectively. Then, the seed liquors with seed ages of 15 h, 18 h, 21 h, and 24 h were respectively fermented and cultured under the initial fermentation culture conditions. After the culture was completed, the polyketides in the fermentation culture broth obtained by fermenting the seed liquors of different seed ages were extracted, and the yields were calculated. The yields of polyketides in the fermentation cultures of the seed liquors of different seed ages are shown in Table 1 and Figure 2 shown.

[0085] In this example, the polyketides refer to the total content of the polyketides shown in Table 16.

[0086] Table 1 Effects of Different Seed Ages on the Yield of Polyketides

[0087] Seed age / h 15 18 21 24 Polyketide production / g 0.4 0.58 0.48 0.49

[0088] From the result tables 1 and Figure 2 it can be seen that when the seed age of the Bacillus velezensis DJ1 bacterial solution is 18 h, the yield of polyketides is the highest.

[0089] Experimental Example 2 Fermentation Duration Optimization Experiment

[0090] The fermentation duration can be adjusted specifically according to the fermentation growth curve. The stationary phase is the best harvest period, and the fermentation process is often terminated during this period in production. In this experimental example, the seed liquid of Bacillus velezensis strain DJ1 was inoculated into LB liquid medium and cultured under the initial culture conditions. During the fermentation process, samples were taken every 1 h to detect the cell concentration index, and the fermentation curve of the strain was plotted. The results are as Figure 3 shown.

[0091] As Figure 3 can be seen, the cell concentration of Bacillus velezensis DJ1 is relatively high at 37 - 68 h, and at this time, it is in the fermentation stationary phase of the strain. The optimal fermentation duration was evaluated within the range of 37 - 68 h of the fermentation of Bacillus velezensis DJ1. Starting from the 38th h of fermentation, samples were taken every 5 h to detect the yield of polyketide compounds in the fermentation products of Bacillus velezensis DJ1 until the 68th h ended. The results are as Figure 4 and Table 2 show.

[0092] Table 2 Effects of different fermentation durations on the yield of polyketide compounds

[0093] Fermentation duration / h 38 43 48 53 58 63 68 Polyketide production / g 0.32 0.44 0.62 0.72 0.6 0.5 0.29

[0094] As can be seen from Table 2 and Figure 4 the results, when the fermentation duration is 53 h, the yield of polyketide substances is the highest. Too long or too short fermentation duration is not conducive to the accumulation of polyketide yield.

[0095] Experimental Example 3 Inoculum size optimization experiment

[0096] The inoculum size is crucial for the production of metabolites. In this experimental example, the culture was carried out according to the initial culture conditions. The difference is that during the initial fermentation culture, the seed liquid of Bacillus velezensis strain DJ1 was inoculated into LB liquid medium for fermentation culture at inoculum sizes of 1%, 2% (i.e., under the initial conditions), 4%, 6%, 8%, and 10% respectively. After the fermentation culture ended, the yield of polyketide compounds in different inoculum size groups was measured. The results are as Figure 5 and Table 3 show.

[0097] Table 3 Effects of different inoculum sizes on the yield of polyketide compounds

[0098] Inoculum size 1％ 2％ 4％ 6％ 8％ 10％ Polyketide production / g 0.42 0.32 0.45 0.43 0.36 0.32

[0099] As shown in Table 3 and Figure 5 as shown, when the inoculum size of the seed liquid of Bacillus velezensis strain DJ1 is 4%, it is most conducive to the accumulation of polyketide substance yield. Whether the inoculum size is too high or too low, it will affect the yield of polyketide. When the inoculum size is 2%, whether the inoculum size is increased or decreased, the yield of polyketide compounds will instead increase. This may be due to the following reasons:

[0100] ①Cell competition and growth inhibition effect: At an inoculation amount of 1%, the initial cell density is low and nutrients are sufficient, so the microorganism can be in the logarithmic growth phase for a longer time, with higher metabolic efficiency, which is beneficial to the accumulation of polyketide products. At an inoculation amount of 4%, although the initial cell density is high, the nutrients in the culture medium are still sufficient to support rapid metabolism, and the metabolic products may have little impact on the synthesis of polyketides due to rapid accumulation in a short time. When the inoculation amount is 2%, at this density, the rate of nutrient consumption may be moderate, but it is not enough to enable the bacteria to fully enter the high-efficiency metabolism stage. At the same time, it may start to be inhibited by metabolic by-products (such as organic acids, ethanol, etc.), resulting in a decrease in metabolic efficiency.

[0101] ②Oxygen limitation: In liquid fermentation, the oxygen transfer efficiency fluctuates with the change of cell density. When the inoculation amount is 2%, the oxygen demand may have approached the limit, resulting in partial hypoxia of the microorganism, which inhibits the aerobic metabolic pathway related to the synthesis of polyketide products. The inoculation amounts of 1% and 4% are in the extreme states of low density (sufficient oxygen) and high density (rapid consumption but high yield in a short time) respectively, which are relatively more conducive to product accumulation.

[0102] ③Dynamic regulation of metabolic pathways: Different inoculation amounts may trigger different physiological states of the microorganism. When the inoculation amount is 1%, the cells maintain in the logarithmic growth phase for a longer time, with higher activity, and the accumulation of polyketide products is significant. When the inoculation amount is 4%, although the initial metabolic pressure is large, it may be partially compensated by metabolic adjustment (such as increasing the product synthesis rate). When the inoculation amount is 2%, it may be in a "critical state", and the nutrient distribution and metabolic regulation are difficult to support efficient product synthesis.

[0103] ④Changes in the local environment: At an inoculation amount of 2%, the cell distribution may cause non-uniformity in the local microenvironment (such as pH, oxygen concentration), which inhibits the metabolic pathway. Moreover, the shear force may affect the cell activity (especially in shaking or stirring culture).

[0104] ⑤Quorum Sensing: Microorganisms regulate the synthesis of secondary metabolites (such as polyketides) through quorum sensing. When the inoculation amount is 2%, it may trigger a certain signaling pathway to inhibit product accumulation, while 1% and 4% are in different physiological regulation ranges, thus showing better performance.

[0105] Experimental Example 4 Medium Optimization Experiment

[0106] In this experimental example, based on the 7 common applicable basic media shown in Table 4, the accumulation of target products of Bacillus velezensis strain DJ1 on these basic media was compared, and a relatively suitable basic medium was preliminarily screened out.

[0107] Table 4 Different Basic Media and Their Formulas

[0108]

[0109] The Bacillus velezensis strain DJ1 was cultured in LB medium and MLB medium respectively according to the initial culture conditions to obtain two kinds of seed solutions. Then, the above two kinds of seed solutions were inoculated into the basal medium except MLB medium as described above, and a total of twelve groups with different medium combinations were set up. The Bacillus velezensis DJ1 in the above different groups was cultured, and the culture conditions were as shown in the initial culture conditions. The yields of polyketide compounds produced by Bacillus velezensis in different groups were statistically analyzed, and the results are as Figure 6 and Table 5 show.

[0110] Table 5 Effects of different medium combinations on the yield of polyketide compounds

[0111]

[0112] As can be seen from Figure 6 and Table 5, when the seed medium is LB medium and the fermentation medium is W2 medium, the yield of polyketide compounds is the highest. Followed by the group with the seed medium being MLB medium and the fermentation medium being LB medium. The yields of polyketide compounds in the groups of the remaining cultures and combinations are significantly lower than those of the above two groups.

[0113] The polyketide compounds were extracted from the fermentation broth produced by the above different groups of medium combinations by the organic solvent extraction method. The steps of the organic solvent extraction method are as follows:

[0114] Centrifuge the fermentation broth and collect the supernatant; mix the supernatant with n-butanol according to a volume ratio of 4:1, shake in a shaker for 2 h to obtain a mixture; let the mixture stand for 10 h to separate layers, take the upper organic phase, evaporate and dry to obtain the polyketide compounds.

[0115] The antibacterial effects of the polyketide compounds extracted by different groups of medium combinations were verified. The experimental steps for verifying the antibacterial effects are as follows: Using the pathogenic fungi shown in Table 6 as targets respectively, the activities of the polyketide compounds produced by different groups of medium combinations were determined by the Oxford cup method. The plant pathogenic fungi used for the antibacterial experiment grow in PDA solid medium, and the final concentration of agar in the solid medium is 2.0%. The antibacterial effect was evaluated by the size of the inhibition zone. The results of the antibacterial experiment are shown in Table 6.

[0116] Table 6 Antibacterial activities (mm) of polyketide compounds extracted from different medium combinations

[0117]

[0118] Note: In Table 6, the pathogenic bacteria 1-11 are: Fusarium oxysporum f. sp. cucumerinum, Dickeya zeae, Rhizoctonia solani of Acanthopanax senticosus, Magnaporthe oryzae, Fusarium solani, Rhizoctonia solani of rice, Colletotrichum lindemuthianum, Botrytis cinerea of tomato, Setosphaeria turcica, Colletotrichum coccodes of tomato, and Pythium aphanidermatum of rice; "-" indicates that no inhibition zone appears.

[0119] As can be seen from the results in Table 6, although certain yields of polyketide compounds were obtained after culturing Bacillus velezensis DJ1 with different combinations of culture media, not all the polyketide compounds produced by each culture medium combination had antibacterial activity. Combining the results in Tables 5-6, it can be seen that the group with LB medium as the seed medium and W2 medium as the fermentation medium had the highest yield of polyketide compounds, but the polyketide compounds at this time had no antibacterial activity. The group with LB medium as the seed medium and W1 medium as the fermentation medium had the lowest yield of polyketide compounds, but the polyketide compounds it produced had good antibacterial activity against the pathogens of Rhizoctonia solani of Acanthopanax senticosus, Fusarium solani, Botrytis cinerea of tomato, and Setosphaeria turcica. Among them, when the seed medium was MLB medium and the fermentation medium was LB medium, it was most able to balance the yield and antibacterial activity of polyketide compounds. Therefore, MLB medium was used as the seed medium and LB medium-based fermentation medium was used as the culture medium for Bacillus velezensis DJ1.

[0120] Experimental Example 5 Optimization Experiment of Culture Medium Components

[0121] Different microorganisms have different abilities and preferences for using carbon sources, and their growth and metabolism are affected by the carbon source concentration. Based on relevant research, in this experimental example, the carbon source, nitrogen source, and inorganic salts in the original LB medium were replaced with the nitrogen source, carbon source, and inorganic salts in Table 7 respectively to conduct an optimization experiment on the components of the fermentation medium, and the accumulation amount of polyketide products was compared. For the convenience of description, the samples in this group of experiments were numbered as shown in Table 7:

[0122] Table 7 Compositions of Nitrogen Source, Carbon Source, and Inorganic Salts in Different Culture Media

[0123] Composition of nitrogen source, carbon source and inorganic salts Number Peptone (10 g / L) + Yeast extract (5 g / L) + NaCl (10 g / L) D1 Lactose (10 g / L) + Yeast extract (5 g / L) + NaCl (10 g / L) D2 <![CDATA[Peptone (10 g / L) + NaNO 3 (6.15 g / L) + NaCl (10 g / L)]]> D3 Peptone (10 g / L) + Casein (7 g / L) + NaCl (10 g / L) D4 <![CDATA[Peptone (10 g / L) + Yeast extract (5 g / L) + Na 2 HPO 4 (10 g / L)]]> D5 <![CDATA[Lactose (10 g / L) + NaNO 3 (6.15 g / L) + NaCl (10 g / L)]]> D6 Lactose (10 g / L) + Casein (7 g / L) + NaCl (10 g / L) D7 <![CDATA[Lactose (10 g / L) + Yeast extract (5 g / L) + Na 2 HPO 4 (10 g / L)]]> D8 <![CDATA[Peptone + NaNO 3 (6.15 g / L) + Na 2 HPO 4 (10 g / L)]]> D9 <![CDATA[Peptone + Casein (7 g / L) + Na 2 HPO 4 (10 g / L)]]> D10 <![CDATA[Lactose (10 g / L) + NaNO 3 (6.15 g / L) + Na 2 HPO 4 (10 g / L)]]> D11 <![CDATA[Lactose (10 g / L) + Casein (7 g / L) + Na 2 HPO 4 (10 g / L)]]> D12

[0124] After replacing the carbon source, nitrogen source, and inorganic salts in LB medium with the carbon source, nitrogen source, and inorganic salts of the above different groups, Bacillus velezensis DJ1 strain was fermented and cultured, and the culture conditions were as shown in the initial culture conditions. The yields of polyketide compounds produced by Bacillus velezensis DJ1 after fermentation in different groups were measured, and the results are as Figure 7 and Table 8 show.

[0125] Table 8 Effects of Different Nitrogen Sources, Carbon Sources, and Inorganic Salts on the Yield of Polyketide Compounds

[0126]

[0127] As can be seen from Table 8 and Figure 7 it can be seen that on the basis of the original LB medium, using 6.15 g / L of NaNO 3 to replace yeast powder as the nitrogen source is the best.

[0128] Experimental Example 6 Fermentation Temperature Optimization Experiment

[0129] In this experimental example, on the basis of the initial culture conditions, the fermentation temperature was modified to conduct a fermentation temperature optimization experiment. In this experimental example, the fermentation temperatures were set to 25 °C, 30 °C, 33 °C, 34 °C, and 37 °C respectively, and the accumulation amounts of polyketide compounds in the fermentation products of Bacillus velezensis DJ1 cultured under different fermentation temperature conditions were detected. The results are as Figure 8 and Table 9.

[0130] Table 9 Effects of Different Fermentation Temperatures on the Yield of Polyketide Compounds

[0131] Temperature / °C 25 30 33 34 37 Polyketide production / g 0.4 0.49 0.43 0.41 0.42

[0132] From Figure 8 and Table 9, it can be seen that when the fermentation temperature is 30 °C, it is most conducive to the production of polyketide substances, and too high or too low temperatures are not conducive to the accumulation of products.

[0133] Experimental Example 7 Fermentation Rotation Speed Optimization Experiment

[0134] During the fermentation process, the mixing of dissolved oxygen and the fermentation broth is mainly affected by the rotation speed. In this experimental example, on the basis of the initial culture conditions, while keeping other fermentation conditions unchanged, the rotation speeds were only set to 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, 200 rpm, 210 rpm, and 220 rpm respectively, and Bacillus velezensis DJ1 was fermented and cultured. The accumulation amounts of polyketones in the fermentation products of Bacillus velezensis DJ1 in different rotation speed groups were compared. The results are as Figure 9 shown in Table 10.

[0135] Table 10 Effects of Different Fermentation Rotation Speeds on the Yield of Polyketide Compounds

[0136] Rotational speed / rpm 150 160 180 200 220 Polyketide production / g 0.35 0.4 0.46 0.43 0.41

[0137] From Figure 9 and Table 10, it can be seen that when the fermentation rotation speed value is 180 rpm, it is most conducive to the production of polyketide substances, and too high or too low rotation speeds are not conducive to the accumulation of products.

[0138] Experimental Example 8 Medium pH Optimization Experiment

[0139] Based on the initial culture conditions, the pH value of the fermentation medium was adjusted in this experimental example. The specific operation was as follows: After preparing the LB liquid medium, the pH of the LB medium was adjusted to different gradients as shown below: 6.0, 6.5, 7.0, 7.5. After adjusting the pH of the medium, the LB medium was subjected to conventional sterilization operations.

[0140] The differences in the accumulation amounts of polyketide products of Bacillus velezensis DJ1 under the culture conditions of LB liquid medium with different pH values were compared, and the results are as Figure 10 shown in Table 11.

[0141] Table 11 Effects of different medium pH values on the yield of polyketides

[0142] pH 6.0 6.5 Original medium (6.7) 7.0 7.5 Polyketide production / g 0.50 0.55 0.52 0.53 0.43

[0143] From Figure 10 the results in Table 11, it can be seen that when the pH value is 6.5, it is most conducive to the accumulation of polyketides. Too high or too low pH values are not conducive to the accumulation of products, probably because the inappropriate pH value reduces the activity of polyketide-producing related enzymes, thus hindering the metabolism of the bacterial cells.

[0144] Results and analysis of response surface verification in Experimental Example 9

[0145] The present invention optimized the fermentation process of DJ1 for producing polyketides by the response surface method:

[0146] Referring to the central composite design method of Minitab17 statistical software and RSM. Appropriate ranges of seed age, fermentation duration, inoculum size, fermentation temperature, fermentation rotation speed, and fermentation broth pH value were respectively selected as variable factors, and the yield of polyketide compounds was used as the dependent variable, that is, the response value. 54 groups of experimental runs were performed at 3 defined levels (-1, 0, +1), 15 series of experiments. Series 1 optimized the fermentation cycle and seed age, Series 2 optimized the inoculum size and seed age, Series 3 optimized the fermentation temperature and seed age, Series 4 optimized the fermentation rotation speed and seed age, Series 5 optimized the pH value and seed age, Series 6 optimized the inoculum size and fermentation duration, Series 7 optimized the fermentation temperature and fermentation duration, Series 8 optimized the fermentation rotation speed and fermentation duration, Series 9 optimized the pH value and fermentation duration, Series 10 optimized the fermentation temperature and inoculum size, Series 11 optimized the fermentation rotation speed and inoculum size, Series 12 optimized the pH value and inoculum size, Series 13 optimized the fermentation rotation speed and fermentation temperature, Series 14 optimized the pH value and fermentation temperature, Series 15 optimized the pH value and fermentation rotation speed. In these 54 groups of experiments, except for the variable factors, other conditions were cultured according to the initial culture conditions.

[0147] The culture parameters for the optimal culture conditions are as follows: The Bacillus velezensis strain DJ1 is inoculated into the MLB medium at an inoculation amount of 4% and cultured. The fermentation temperature is 35°C, the rotation speed is 180 rpm, and the culture is carried out until the seed age reaches 18 h to obtain a seed solution; then the seed solution is inoculated into the LB medium at an inoculation amount of 4% for fermentation culture. The fermentation duration is 53 h, the fermentation temperature is 30°C, the fermentation rotation speed is 180 rpm, and the pH of the fermentation broth is 6.5.

[0148] The response surface experimental design consists of 6 factors (seed age, fermentation duration, inoculation amount, fermentation temperature, fermentation rotation speed, pH value of the fermentation broth) and 3 levels (-1, 0, +1), as shown in Table 12.

[0149] Table 12 Response surface experimental design

[0150]

[0151] The important terms and interactions of the model are described through an investigation of analysis of variance. The coefficient of determination R reveals the accuracy of the model, as shown in Table 13.

[0152] Table 13 Corresponding analysis of variance

[0153]

[0154] As can be seen from the results of the analysis of variance in Table 13, for the experimental model, R = 0.9430 and the P value < 0.0001, indicating that this model has statistical significance. There is a satisfactory correlation between the experimental data and the simulated prediction data, thus verifying the applicability of the design model. The results show that the significance of individual factors is less than that of interaction factors, indicating that the metabolism of polyketide production by Bacillus velezensis is more significantly affected by multiple factors. The insignificant lack of fit is determined, reflecting the verification of the design model for existing research and is analyzed to be of great significance for the impact on polyketide production.

[0155] Model diagrams of the effects of each factor on polyketide production are plotted, and the results are as Figures 11 - 26 shown.

[0156] From Figures 11 - 26 it can be seen that the optimal conditions are a seed age of 18 h, a fermentation duration of 53 h, an inoculation amount of 4%, a fermentation temperature of 30°C, a fermentation rotation speed of 180 rpm, a pH value of 6.5 for the fermentation broth, 10 g / L of tryptone, 6.15 g / L of sodium nitrate, and 10 g / L of NaCl. This is close to the expected response value, thus verifying the applicability of the design model. The analysis of variance shows that the significance of individual factors is less than that of interaction factors, indicating that the metabolism of polyketide production by Bacillus velezensis may be affected by multiple aspects.

[0157] Experimental Example 10 Verification experiment on the antibacterial activity of polyketide compounds

[0158] In this experimental example, the optimal culture conditions of Bacillus velezensis DJ1 screened in Experimental Example 9 were used as the standard: seed age of 18 h, fermentation duration of 53 h, inoculum size of 4%, fermentation temperature of 30 °C, fermentation rotation speed of 180 rpm, pH value of the fermentation broth of 6.5, 10 g / L of tryptone, and 6.15 g / L of sodium nitrate, 10 g / L of NaCl. The crude polyketide extracts obtained by the culture methods of Bacillus velezensis DJ1 with the optimal single factor screened in each experiment of Experimental Examples 1 to 3 and 5 to 8 were verified respectively. Except for the optimal conditions, other fermentation parameters were as shown in the initial culture conditions. For the convenience of description, the polyketide samples extracted from different groups were numbered, and the numbering results are shown in Table 14. The antibacterial activities of the crude polyketide extracts obtained from the above different groups against plant pathogenic bacteria from 5 fungi and 1 bacterium were measured, and the antibacterial experiments were carried out on the above groups by the Oxford cup method respectively. The procedure of the Oxford cup method is as follows:

[0159] In this experimental example, the following method was used to determine the antibacterial activity and component analysis of polyketide compounds:

[0160] Taking pathogenic bacteria from bacterial sources and fungal sources as targets respectively, the Oxford cup method was used to judge their inhibitory activities, that is, to judge their inhibitory activities by the size of the inhibition zone. The inhibition zone refers to a circular area without bacterial growth formed around the spotting groove, and its size reflects the strength of the inhibitory effect of the sample to be tested on the indicator bacteria.

[0161] The crude polyketide extract was subjected to component and structure identification of the sample by chromatography or mass spectrometry to preliminarily screen its possible antibacterial components. Taking the diameter (mm) of the inhibition zone as the reference standard, its antibacterial effect was evaluated. The results of the antibacterial experiment are as Figure 27 and Table 15 show.

[0162] Table 14 Numbers of polyketide samples extracted from different groups

[0163]

[0164] Table 15 Diameters (mm) of inhibition zones of different polyketide samples

[0165]

[0166] As can be seen from Figure 27 and Table 15, the polyketide compound samples cultured under the fermentation parameters of the best single factor screened in A4 - G2 of the present invention have good antibacterial activities against different pathogenic bacteria.

[0167] Experimental Example 11 Identification of types of polyketide compounds

[0168] The crude polyketide extract samples obtained by culturing the single-factor optimized Bacillus velezensis DJ1 using the culture methods in Experimental Examples 1 to 3 and 5 to 8 were analyzed by HPLC-MS. Referring to the molecular weights / mass-to-charge ratios of different polyketide compounds and using Masslynx software to compare with the PubCHEM database, the following 33 polyketides in the samples were preliminarily screened out:

[0169] Table 16 Polyketide Compounds in the Fermentation Products of Bacillus velezensis DJ1

[0170]

[0171]

[0172] Based on the content in Table 16, the polyketide components in the samples can be preliminarily determined. Subsequently, more advanced experimental methods can be designed based on this research to separate the above polyketide components and conduct qualitative and quantitative analyses on them.

[0173] The synthesis of polyketide compounds mainly depends on the catalytic action of polyketide synthase (PKS). Polyketide synthase can be divided into three categories according to the organization form of its domains: type I PKS, type II PKS, and type III PKS. Type I PKS, also known as modular polyketide synthase, contains multiple modular domains, and each module is responsible for the extension of an acyl group. Taking the biosynthesis process of erythromycin as an example, the PKS enzyme complex (DEBS) contains 3 protein subunits, each subunit consists of 2 modules, a total of 6 modules. These modules complete the biosynthesis of the macrolide ring (6-dEB) through multiple rounds of cycles such as condensation, ketone reduction, dehydration, and enoyl reduction, and then complete the synthesis of erythromycin through the post-modification (hydroxylation, glycosylation, methylation) process of 6-dEB. In Bacillus velezensis, a similar modular PKS system may exist for the synthesis of bioactive polyketide compounds.

[0174] Type II PKS, also known as aromatic polyketide synthase, taking the synthesis of anthracycline doxorubicin as an example. Type II PKS usually does not use ACP as the anchor point of the polyketide chain, but through the synergistic action of multiple enzymes, converts the poly-β-ketone chain into the core of aromatic compounds. In Bacillus velezensis, type II PKS may be involved in the synthesis of polyketide compounds with aromatic structures, and these compounds may have biological activities such as antibacterial and anti-tumor.

[0175] Type III PKS, also known as chalcone-type polyketide synthase, takes the synthesis of pyrrole-derived polyketide Germicidin as an example. Type III PKS does not use ACP as an anchor, but directly uses medium-chain acyl-CoA as the starting unit for iterative catalysis. In Bacillus velezensis, Type III PKS may be involved in the synthesis of structurally complex polyketide compounds, which possess unique biological activities. The synthesis mechanisms of different types of polyketide compounds are as Figures 28 - 30 shown.

[0176] Example 1 A method for improving the yield and activity of polyketide compounds produced by Bacillus velezensis DJ1

[0177] The steps are as follows:

[0178] Inoculate the Bacillus velezensis DJ1 strain into the seed medium: MLB liquid medium for seed culture to obtain a seed solution;

[0179] Inoculate the obtained seed solution into the fermentation medium at an inoculum size of 4% of the volume of the fermentation medium, and perform seed culture. Incubate at 180 rpm and 35 °C for 18 h to obtain a seed solution.

[0180] Inoculate the obtained seed solution into the fermentation medium for fermentation culture to obtain a fermentation broth. This fermentation medium is a modified LB liquid medium, and its composition is: 6.15 g / L of NaNO 3 , 10.0 g / L of peptone and 10 g / L of inorganic salts. The pH of the modified LB liquid medium is 6.5; among them, the conditions for fermentation culture are to incubate at 30 °C for 53 h, and the rotation speed for fermentation culture is 180 rpm.

[0181] Use the organic solvent extraction method to separate the polyketide compounds in the obtained fermentation broth to obtain polyketide compounds.

[0182] The steps of the organic solvent extraction method are as follows:

[0183] After obtaining the fermentation broth, take 300 ml of the fermentation broth, centrifuge the fermentation broth at 4000 rpm for 15 min, collect the supernatant, extract it with n-butanol at a ratio of 4:1, shake it on a shaker for 2 h to make it evenly mixed, and let it stand overnight in a 500 ml separating funnel for stratification. After n-butanol is mixed with the solution containing polyketide, a two-phase system will be formed. After waiting for the liquid to stratify, take the upper organic phase and evaporate it to dryness in a fume hood to obtain polyketide compounds.

[0184] After measurement, under the culture conditions of this example, the yield of polyketide compounds in 300 ml of the fermentation broth is 1770 mg / L.

[0185] In summary, through experimental verification, the present invention has screened out the optimal culture conditions for Bacillus velezensis DJ1. Under the optimal culture conditions screened out by the present invention, not only can the yield of polyketide compounds in the fermentation products of Bacillus velezensis DJ1 be increased, but also the activity of the polyketide compounds can be improved, making the polyketide compounds have good antibacterial activity, overcoming the problem that the polyketide compounds in the fermentation products of Bacillus velezensis DJ1 produced by the prior art cannot have both high yield and high activity, and thus solving the problem that Bacillus velezensis cannot be industrialized.

[0186] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments without creative efforts as in this embodiment, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A fermentation medium suitable for Bacillus Velezii, characterized in that: The fermentation medium is a modified LB liquid medium; The improved LB liquid culture medium is: based on the LB liquid culture medium, the original nitrogen source in the LB liquid culture medium is replaced with 6.15 g / L NaNO3, and the pH value is 6.

5.

2. A culture medium combination for culturing Bacillus Velezii, characterized in that: including seed culture medium and fermentation medium; The seed culture medium is MLB liquid culture medium; The fermentation medium is the fermentation medium according to claim 1.

3. Use of the fermentation medium according to claim 1 or the medium combination according to claim 2 in improving the yield and / or activity of polyketide compounds produced by Bacillus Velezii.

4. A method for increasing the yield and / or activity of polyketide compounds produced by Bacillus Velezii, characterized in that: The steps include: inoculating the Bacillus Velez strain into a seed culture medium for seed culture to obtain a seed solution; inoculating the seed liquid into a fermentation medium for fermentation culture to obtain a fermentation culture liquid; separating the polyketide compounds in the fermentation culture broth to obtain the polyketide compounds; The fermentation medium is the fermentation medium according to claim 1.

5. The method according to claim 4, characterized in that The seed culture medium is MLB liquid culture medium; the seed culture time is 15 to 24 hours.

6. The method according to claim 4, characterized in that The inoculation amount of the seed liquid is 4% to 6% of the volume of the fermentation medium.

7. The method according to claim 4, characterized in that The fermentation culture temperature is 28-32° C., the fermentation time is 48-60 hours, and the rotation speed is 170-190 rpm.

8. The method according to claim 4, characterized in that The polyketide compounds in the fermentation culture liquid are separated by an organic solvent extraction method.

9. The method according to claim 8, characterized in that The steps of the organic solvent extraction method are as follows: Centrifuging the fermentation broth and collecting the supernatant; The supernatant and n-butanol are mixed in a volume ratio of 4:1, and shaken on a shaker for 1.5 to 2 hours to obtain a mixed solution; the mixed solution is allowed to stand for 8 to 12 hours to separate layers, and the upper organic phase is taken and evaporated to dryness to obtain a polyketide compound.

10. The method according to claim 9, characterized in that The centrifugal speed is 4000 rpm and the centrifugal time is 15 min.

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