Fermentation liquid extract of streptomyces M53 as well as preparation method and application of fermentation liquid extract

Abstract

The invention relates to the technical field of microorganisms, and discloses a fermentation liquor extract of streptomyces M53 as well as a preparation method and application of the fermentation liquor extract. The preparation method of the fermentation liquor extract comprises the following steps: inoculating streptomyces M53 into an SGTYP liquid culture medium, and performing shaking culture for 72-96 hours to obtain fermentation liquor; extracting the fermentation liquor with ethyl acetate, and removing a water phase to obtain a fermentation liquor extracting solution; ethyl acetate in the fermentation liquor extracting solution is removed, and a fermentation liquor extract of the streptomyces M53 is obtained; wherein the streptomycete M53 is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No: 65720. The fermentation liquor extract prepared by adopting the preparation method has a concentration gradient effect inhibiting effect on surging movement of pseudomonas aeruginosa PAO1, can reduce the drug resistance of the pseudomonas aeruginosa PAO1 to antibiotics, and inhibits the generation of biofilms and rhamnolipid of the pseudomonas aeruginosa PAO1, so that the fermentation liquor extract has a relatively good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to an extract of the fermentation broth of Streptomyces M53, a preparation method thereof, and an application thereof. Background Art

[0002] With the overuse and abuse of antibiotics in the fields of clinical medicine, agriculture, animal husbandry, aquaculture, etc., the problem of bacterial drug resistance has become increasingly severe. However, the research and development of new drugs lags far behind, and it is urgent to develop new antibacterial drugs with high efficiency and low cross-drug resistance.

[0003] Compared with traditional bactericidal strategies, the new drug development strategy targeting the pathogenicity of pathogenic bacteria exerts less selective pressure on bacteria, is less likely to cause drug resistance, and has strong specificity. It can be used in combination with antibiotics to reduce the use concentration of antibiotics and enhance the drug effect. Therefore, it is a new prevention and control strategy that conforms to sustainable development. The targets of pathogenic bacteria's pathogenicity are often related to their group behaviors. Common group behaviors include swarming motility (also known as swarming) and the formation of biofilms (BF).

[0004] Swarming motility is a collective migration behavior of bacteria driven by flagella on the surface of semi-solid media. Compared with individual behaviors such as swimming motility, cells in the swarming motility state enhance their environmental adaptability through the following characteristics: ① overexpression of flagellar genes to form super-flagellar structures; ② elongation of bacterial cells; ③ secretion of surfactants (such as rhamnolipids); some also inhibit the expression of outer membrane porins (such as OmpF). Therefore, swarming motility, as a special group behavior of bacteria, is closely related to drug resistance, pathogenicity, and biofilm formation. More importantly, there is no report on the swarming ability of probiotics. Compared with the action of antibiotics, swarming motility inhibitors can weaken the pathogenicity of pathogenic bacteria by selectively inhibiting their swarming motility, while having no impact on the probiotic community. Therefore, based on the strategy of targeting swarming motility rather than direct bactericidal action, the development of new swarming motility inhibitors has important research significance.

[0005] Currently, substances known to have swarming motility inhibitory activity mainly include plant active natural products and sub-inhibitory concentrations of antibiotics. However, plant active natural products often require concentrations of several hundred micromoles or even millimoles to exert their effects, and their activity efficiency needs to be further improved, and the molecular mechanisms of their swarming inhibition are mostly unclear; the working concentrations of antibiotics for exerting swarming inhibitory activity are similar to their bactericidal concentrations, and further increasing the working concentration may induce drug resistance in pathogenic bacteria.

[0006] Therefore, the prior art still needs to be improved and developed. Summary of the Invention

[0007] In view of the deficiencies of the above-mentioned prior art, the object of the present invention is to provide a fermentation broth extract of Streptomyces M53, a preparation method thereof and an application thereof. The fermentation broth extract can inhibit the swarming motility, drug resistance and biofilm formation of Pseudomonas aeruginosa, and can be used to solve the problems of low activity efficiency of existing swarming motility inhibitors and easy induction of drug resistance in pathogenic bacteria.

[0008] The technical solution of the present invention is as follows: In the first aspect, a preparation method of a fermentation broth extract of Streptomyces ( Streptomyces sp.) M53 is provided, including the steps of: (1) Inoculating Streptomyces M53 into SGTYP liquid medium and shaking culture for 72-96 h to obtain a fermentation broth; (2) Extracting the fermentation broth with ethyl acetate, removing the aqueous phase to obtain a fermentation broth extract; (3) Removing ethyl acetate from the fermentation broth extract to obtain a fermentation broth extract of Streptomyces M53; Among them, Streptomyces M53 is preserved in the Guangdong Provincial Microbial Culture Collection Center, and the preservation number is GDMCC No: 65720.

[0009] In a preferred technical solution, the conditions of the shaking culture are: temperature 25-28 °C, rotation speed 100-300 rpm.

[0010] In a preferred technical solution, step (1) specifically includes: After activating Streptomyces M53 on MA solid medium, taking a single colony and shaking culture in 5-15 mL of SGTYP liquid medium for 20-30 h to obtain a seed liquid; Inoculating the seed liquid into a new SGTYP liquid medium and shaking culture for 72-96 h to obtain a fermentation broth.

[0011] In a preferred technical solution, the volume ratio of the seed liquid to the new SGTYP liquid medium is 1: (50-100).

[0012] In the second aspect, a fermentation broth extract of Streptomyces M53 is provided, and the fermentation broth extract is prepared by the preparation method described in the first aspect.

[0013] In the third aspect, an application of the fermentation broth extract described in the second aspect in the preparation of a product for inhibiting the swarming motility of Pseudomonas aeruginosa is provided.

[0014] In the fourth aspect, an application of the fermentation broth extract described in the second aspect in the preparation of a product for inhibiting the antibiotic resistance of Pseudomonas aeruginosa is provided.

[0015] In a preferred technical solution, the antibiotic is gentamicin.

[0016] In a fifth aspect, there is provided an application of the fermentation broth extract as described in the second aspect in the preparation of a product for inhibiting the formation of Pseudomonas aeruginosa biofilm.

[0017] Beneficial effects: The present invention provides a fermentation broth extract of Streptomyces sp. M53. This fermentation broth extract has a concentration-gradient effect on the swarming motility of Pseudomonas aeruginosa PAO1, and has no effect on the swimming motility, twitching motility and growth of Pseudomonas aeruginosa PAO1, and is a specific inhibitor of swarming motility. In addition, this fermentation broth extract can reduce the antibiotic resistance of Pseudomonas aeruginosa PAO1 in the swarming motility state, and inhibit the production of Pseudomonas aeruginosa PAO1 biofilm and rhamnolipid, so it has good application prospects. Description of the drawings

[0018] Figure 1 It is the morphological observation result and phylogenetic tree of strain M53 in Example 1 and the solution of the fermentation broth extract of strain M53 prepared in Example 2.

[0019] Figure 2 It is the result diagram of the fermentation broth extract of strain M53 inhibiting the swarming motility of Pseudomonas aeruginosa PAO1 in Example 3.

[0020] Figure 3 It is the result diagram of the fermentation broth extract of strain M53 not affecting the growth of Pseudomonas aeruginosa PAO1 in Example 4.

[0021] Figure 4 It is the result diagram of the fermentation broth extract of strain M53 showing a gradient effect on the inhibitory activity of the swarming motility of Pseudomonas aeruginosa PAO1 in Example 5.

[0022] Figure 5 It is the result diagram of the fermentation broth extract of strain M53 not inhibiting the swimming motility and twitching motility of Pseudomonas aeruginosa PAO1 in Example 6.

[0023] Figure 6 It is the result diagram of the fermentation broth extract of strain M53 inhibiting the formation of Pseudomonas aeruginosa PAO1 biofilm in Example 7.

[0024] Figure 7 It is the result diagram of the fermentation broth extract of strain M53 inhibiting the antibiotic resistance of Pseudomonas aeruginosa PAO1 in Example 8.

[0025] Figure 8 It is the result diagram of adding rhamnolipid to partially restore the swarming motility of Pseudomonas aeruginosa PAO1 in Example 9.

[0026] Figure 9It is a result graph showing the inhibition of the rhamnolipid production of Pseudomonas aeruginosa PAO1 by the fermentation broth extract of strain M53 in Example 10. Detailed implementation manners

[0027] The present invention provides a fermentation broth extract of Streptomyces M53, a preparation method thereof, and an application thereof. To make the purpose, technical solution, and effects of the present invention clearer and more definite, the present invention is further described in detail below.

[0028] The ocean has the richest biological resources on the earth. Among them, due to their special living environments such as high pressure, high salt, oligotrophy, low temperature, limited light, and hypoxia, marine microorganisms produce a rich variety of secondary metabolites with unique structures, and many have unique activities such as anti-inflammatory, antibacterial, and anti-tumor. In recent years, searching for novel active compounds from marine microorganisms has become a hot topic. However, there are few studies on novel swarming motility inhibitors from marine sources.

[0029] Currently, novel swarming motility inhibitors from marine sources generally have the problems of low efficiency and unclear action mechanisms. For example, Document 1 discloses that saponins extracted by Payam B et al. from Holothuria leucospilota ) have a swarming motility inhibition rate of 77% against Aeromonas hydrophila ( Aeromonas hydrophila ) at a concentration of 1 / 2 MIC (15 μg / mL). Document 2 discloses that equisetin isolated by Zhang M et al. from the marine fungus Fusarium sp. Z10 has a swarming motility inhibition rate of only 50% against Pseudomonas aeruginosa at a concentration of 300 μM. Document 3 discloses that the crude extract extracted by the Naik D N team from the marine Streptomyces Streptomyces sp. NIO 10068 with methanol can achieve a swarming motility inhibition rate of 90% against Pseudomonas aeruginosa at a concentration of 0.1 mg / mL, but its active ingredients and action mechanisms are still unclear.

[0030] Document 1: Payam B, Soltani M, Mehrgan M S, et al. Saponins from seacucumber disrupt aeromonashydrophila quorum sensing to mitigate pathogenicity[J]. AMB Express, 2025, 15(1): 43. Document 2: Zhang M, Wang M, Zhu X, et al. Equisetin as potential quorumsensing inhibitor of pseudomonas aeruginosa[J]. Biotechnology Letters, 2018, 40(5): 865 - 870. Document 3: Naik D N, Wahidullah S, Meena R M. Attenuation of pseudomonas aeruginosa virulence by marine invertebrate–derived streptomyces sp [J].Letters in Applied Microbiology, 2013, 56(3): 197 - 207. Based on this, in October 2016, a strain of Streptomyces (< Streptomyces sp.) M53 was isolated from the root soil of

[0031] Acanthus ilicifolius in the Mai Po Streptomyces Mangrove Nature Reserve in Hong Kong, and relevant research on the swarming motility of its fermentation broth extract was carried out. (1) Inoculate Streptomyces M53 into SGTYP liquid medium and shake culture for 72 - 96 h to obtain a fermentation broth; (2) Extract the fermentation broth with ethyl acetate, remove the aqueous phase to obtain a fermentation broth extract; (3) Remove the ethyl acetate from the fermentation broth extract to obtain the fermentation broth extract of Streptomyces M53; Among them, Streptomyces M53 is preserved in the Guangdong Provincial Microbial Culture Collection Center, and the preservation number is GDMCC No: 65720.

[0032] Specifically, through research, it was found that the fermentation broth extract prepared in the embodiments of the present invention has no bactericidal effect on Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) PAO1, but can significantly inhibit the swarming motility of Pseudomonas aeruginosa PAO1 at a relatively low concentration (10 μg / mL), and has no inhibitory effect on other types of motility of Pseudomonas aeruginosa PAO1, such as swimming motility and twitching motility, indicating that the fermentation broth extract is a highly efficient and specific swarming motility inhibitor. In addition, the fermentation broth extract also has a significant inhibitory effect on other virulence factors of Pseudomonas aeruginosa PAO1, such as the production of rhamnolipids and the formation of biofilms, and can also reduce the antibiotic resistance of Pseudomonas aeruginosa PAO1. These results indicate that the fermentation broth extract of Streptomyces M53 has good application prospects and is expected to be developed into a new strategy for preventing and controlling pathogenic bacteria in the future.

[0033] In one embodiment, the conditions for shaking culture are as follows: temperature is 25-28 °C, and rotation speed is 100-300 rpm.

[0034] In one embodiment, step (1) specifically includes: After activating Streptomyces M53 on MA solid medium, pick a single colony and shake culture it in 5-15 mL of SGTYP liquid medium for 20-30 h to obtain a seed solution. Inoculate the seed solution into fresh SGTYP liquid medium and shake culture it for 72-96 h to obtain a fermentation broth.

[0035] In one embodiment, the volume ratio of the seed solution to the fresh SGTYP liquid medium is 1:(50-100).

[0036] The embodiment of the present invention provides an extract of the fermentation broth of Streptomyces M53, and the extract of the fermentation broth is prepared by the preparation method described above.

[0037] The embodiment of the present invention provides the application of the extract of the fermentation broth described above in the preparation of a product for inhibiting the swarming motility of Pseudomonas aeruginosa.

[0038] The embodiment of the present invention provides the application of the extract of the fermentation broth described above in the preparation of a product for inhibiting the antibiotic resistance of Pseudomonas aeruginosa.

[0039] In one embodiment, the antibiotic is gentamicin.

[0040] The embodiment of the present invention provides the application of the extract of the fermentation broth described above in the preparation of a product for inhibiting the biofilm formation of Pseudomonas aeruginosa.

[0041] In one embodiment, the Pseudomonas aeruginosa is Pseudomonas aeruginosa PAO1.

[0042] The present invention will be further described below through specific examples.

[0043] In the embodiment of the present invention, the involved culture medium is prepared with deionized water, and the formula is as follows: Marine Agar 2216 (MA) solid medium: 5 g / L of Protein peptone, 1 g / L of Yeast extract, 0.1 g / L of Iron citrate, 19.45 g / L of Sodium chloride (NaCl), 5.98 g / L of Magnesium chloride (MgCl2), 3.24 g / L of Sodium sulfate (Na2SO4), 1.8 g / L of Calcium chloride (CaCl2), 0.55 g / L of Potassium chloride (KCl), 0.16 g / L of Sodium carbonate (Na2CO3), 0.08 g / L of Potassium bromide (KBr), 0.034 g / L of Strontium chloride (SrCl2), 0.022 g / L of Boric acid (H3BO3), 0.004 g / L of Sodium silicate (Na2SiO3), 0.0024 g / L of Sodium fluoride (NaF), 0.0016 g / L of Ammonium nitrate (NH4NO3), 0.008 g / L of Disodium hydrogen phosphate (Na2HPO4), 15 g / L of Agar.

[0044] LA solid medium: 10 g / L of Tryptone, 1 g / L of Yeast extract, 5 g / L of Sodium chloride (NaCl), 15 g / L of Agar.

[0045] LB liquid medium: 10 g / L of Tryptone, 1 g / L of Yeast extract, 5 g / L of Sodium chloride (NaCl).

[0046] SGTYP liquid medium: 5 g / L of Starch, 5 g / L of Glucose, 1 g / L of Tryptone, 1 g / L of Bacteriological peptone, 1 g / L of Yeast extract, 17 g / L of Seasalt.

[0047] Example 1 Isolation and Identification of Strain M53 Strain M53 was isolated from the root soil of Acanthus ilicifolius in the Mai Po Mangrove Nature Reserve, Hong Kong in October 2016. Scrape strain M53 onto the MA solid medium for streaking, and culture it at 28 °C for 48 h. During this period, observe and record the colony morphology, pick the surface mycelium onto a glass slide, and stain it with a Gram staining kit. Observe the mycelium morphology under an optical microscope. It can be observed that the colony is dry with a neat edge, milky white in color, smooth and opaque on the surface; then it develops into a villous shape, the color changes from milky white to brownish red, and it is not easy to pick up, specifically as Figure 1As shown in a. Observation under a 100× oil immersion microscope showed that the hyphae were fine and filamentous, with abundant branches and intertwined with each other. The 16S rRNA sequence (SEQ ID NO.1) of strain M53 was uploaded to the sequence alignment website (https: / / www.ezbiocloud.net / ), and it was found that the strain with the highest similarity to it was Streptomyces cavourensis BBRC 13026, and the phylogenetic tree is as shown in Figure 1 c. Based on the comprehensive results of 16S rRNA sequence alignment, colony and hyphal morphology, and Gram staining, it was determined that strain M53 was Streptomyces ( Streptomyces sp.).

[0048] Strain M53 has been deposited in the Guangdong Microbial Culture Collection Center. The deposit address is the 5th floor, Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou. The deposit number is GDMCC No: 65720, and the deposit date is January 6, 2025. Its taxonomic name is Streptomyces sp.

[0049] Example 2 Preparation of the extract from the fermentation broth of strain M53 A single colony was taken from the MA solid medium containing strain M53 and inoculated into 10 mL of SGTYP liquid medium, and cultured with shaking (200 rpm) at 28 °C for 24 h to obtain a seed solution. 10 mL of the seed solution was added to 1 L of SGTYP liquid medium at a ratio of 1:100, and cultured with shaking (200 rpm) at 28 °C for 96 h to obtain a fermentation broth. The fermentation broth was extracted three times with 1 L of ethyl acetate and dried by rotary evaporation in a centrifugal concentrator. The temperature used for rotary evaporation was 30 - 40 °C. Finally, it was made up to 200 mg / mL with dimethyl sulfoxide (DMSO) to obtain a solution of the extract from the fermentation broth. The solution of the extract from the fermentation broth is as shown in Figure 1 b.

[0050] Example 3 Inhibition of the swarming motility of Pseudomonas aeruginosa PAO1 by the extract from the fermentation broth of strain M53 Pseudomonas aeruginosa PAO1 was activated on LA solid medium and cultured at 37 °C for 24 h. The activated Pseudomonas aeruginosa PAO1 was collected with a sterile inoculation loop and diluted to 1×10 9 CFU / mL with LB liquid medium. 1 μL was taken and dropped onto the swarming motility medium containing the extract from the fermentation broth (100 μg / mL), the swarming motility medium containing DMSO (0.2% (v / v)) (negative control group), and the swarming motility medium without addition (blank control group). After culturing upright at 37 °C for 14 h, photos were taken and recorded. The results are as shown in Figure 2 shown. Figure 2 In it, a is the colony morphology, and b is the statistical result of the swarming motility distance; each experiment was repeated three times, and **** representsp < 0.0001. From Figure 2 It can be seen that the Pseudomonas aeruginosa PAO1 in the negative control group and the blank control group had moved to the edge of the plate at 14 h, showing a typical colony morphology with "tentacle" shape; while under the action of the fermentation broth extract at 100 μg / mL, the swarming motility of Pseudomonas aeruginosa PAO1 was completely inhibited. Therefore, the fermentation broth extract of strain M53 has inhibitory activity against the swarming motility of Pseudomonas aeruginosa PAO1.

[0051] Example 4 The fermentation broth extract of strain M53 does not affect the growth of Pseudomonas aeruginosa PAO1 To rule out the possibility that the inhibitory effect on swarming motility is caused by the bactericidal effect of the fermentation broth extract of strain M53, in this example, the growth curve determination and the dilution plating colony counting method were used to detect the effect of the fermentation broth extract on the growth of Pseudomonas aeruginosa PAO1.

[0052] The seed solution of Pseudomonas aeruginosa PAO1 cultured overnight at 37 °C (200 rpm) was diluted with LB liquid medium, and the volume ratio of the seed solution of Pseudomonas aeruginosa PAO1 to LB liquid medium was 1:100. Then, fermentation broth extracts with final concentrations of 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL were added respectively. The negative control group was added with DMSO (0.1% (v / v) and 0.2% (v / v)), and the blank control group was not added. The mixed system was incubated at 37 °C and 200 rpm, and the OD was measured at 4, 7, 10, 12, 14, and 24 h respectively. 600 The experimental results are as Figure 3 shown. From Figure 3 It can be seen that under the action of the fermentation broth extract at a concentration of 25 - 400 μg / mL, the growth of Pseudomonas aeruginosa PAO1 was not inhibited. Therefore, the fermentation broth extract of strain M53 has no bactericidal activity against Pseudomonas aeruginosa PAO1, and it does not inhibit swarming motility by inhibiting the growth of PAO1. It is speculated that non-antibiotic swarming inhibitory active substances are produced.

[0053] Example 5 The inhibitory activity of the fermentation broth extract of strain M53 against the swarming motility of Pseudomonas aeruginosa PAO1 shows a gradient effect To further explore whether the inhibitory effect of the fermentation broth extract of strain M53 on the swarming motility of Pseudomonas aeruginosa PAO1 has a concentration gradient effect, a series of concentration gradients were designed in this example.

[0054] Pseudomonas aeruginosa PAO1 was activated in LA solid medium and cultured at 37 °C for 24 h. The activated Pseudomonas aeruginosa PAO1 was collected with a sterile inoculation loop and diluted with LB liquid medium to 1×109 CFU / mL, 1 μL was dripped into the surging medium containing fermentation broth extract (5 μg / mL, 7.5 μg / mL, 8.125 μg / mL, 8.75 μg / mL and 10 μg / mL), the surging medium containing DMSO (0.2% (v / v)) (negative control group) and the surging medium without addition (blank control group), and then cultured upright at 37°C for 14 h and photographed and recorded. The results are shown in Figure 4 shown. Figure 4 In the figure, a is the colony morphology, and b is the statistical result of inhibition rate. Figure 4 It can be seen that after 16 hours of culture, the colonies of Pseudomonas aeruginosa PAO1 in the blank control group and the negative control group moved to the edge of the plate and showed typical "tentacle" morphological characteristics; in sharp contrast, when the concentration of the fermentation broth extract increased from 5µg / mL to 10µg / mL, the surging movement of Pseudomonas aeruginosa PAO1 was significantly inhibited; among them, under the action of 7.5µg / mL and 8.125µg / mL of fermentation broth extract, the surging movement of Pseudomonas aeruginosa PAO1 was inhibited to a lesser extent; and under the action of 10µg / mL of fermentation broth extract, the surging movement of Pseudomonas aeruginosa PAO1 was almost completely inhibited, with an inhibition rate of nearly 100%. The inhibitory effect of the fermentation broth extract of strain M53 on the surging movement of Pseudomonas aeruginosa PAO1 was concentration-dependent.

[0055] Example 6 The extract of the fermentation broth of strain M53 does not inhibit the swimming and gliding motility of Pseudomonas aeruginosa PAO1 Swimming motility is driven by the rotation of monopolar flagella, mediating the rapid diffusion of bacteria in liquid environments, while gliding motility assists bacteria in short-distance movement on solid interfaces with the help of the periodic extension and contraction of type IV pili. In order to explore whether the fermentation broth extract of strain M53 affects swimming motility by inhibiting flagella function, or inhibits gliding motility by interfering with pili assembly, this example measured the swimming and gliding motility of Pseudomonas aeruginosa PAO1 under the action of fermentation broth extract.

[0056] Pseudomonas aeruginosa PAO1 was activated in LA solid medium and cultured at 37°C for 24 h. The activated Pseudomonas aeruginosa PAO1 was collected with a sterile inoculation loop and diluted to 1×10 9CFU / mL. 1 μL was respectively taken and dropped onto the swarming motility medium containing the fermentation broth extract (100 μg / mL), the twitching motility medium containing the fermentation broth extract (100 μg / mL), the swarming motility medium containing DMSO (0.1% (v / v)) (negative control group), the twitching motility medium containing DMSO (0.1% (v / v)) (negative control group), the swarming motility medium without addition (blank control group), and the twitching motility medium without addition (blank control group). The swarming motility experiment was cultured upright at 37°C, and the twitching motility experiment was cultured inverted at 37°C, with no more than 2 stacked together. After 14 h, photos were taken and recorded. The results are as Figure 5 shown. Figure 5 In it, a is the colony morphology in the swarming motility experiment, and c is the statistical result of the inhibition rate in the swarming motility experiment; b is the colony morphology in the twitching motility experiment, and d is the statistical result of the inhibition rate in the twitching motility experiment. It can be Figure 5 seen that the fermentation broth extract at 100 μg / mL has no inhibitory effect on the swarming motility and twitching motility of Pseudomonas aeruginosa PAO1. Therefore, it is speculated that the fermentation broth extract of strain M53 is a specific swarming motility inhibitor.

[0057] Example 7 Inhibition of biofilm formation of Pseudomonas aeruginosa PAO1 by the fermentation broth extract of strain M53 Biofilm is a macromolecular complex barrier formed by Pseudomonas aeruginosa PAO1 against external adverse conditions. Biofilm has a close relationship with drug resistance. To explore whether the fermentation broth extract of strain M53 reduces its drug resistance by inhibiting the biofilm formation of Pseudomonas aeruginosa PAO1, the biofilm formation status of Pseudomonas aeruginosa PAO1 after the action of the fermentation broth extract was detected in this example.

[0058] Pseudomonas aeruginosa PAO1 was cultured overnight at 37°C (200 rpm) in LB liquid medium. The bacterial solution was added to LB liquid medium supplemented with 5 g / L glucose at a volume ratio of 1:20, and different concentrations of the fermentation broth extract (50, 100, 200, 400 μg / mL) were added. The negative control group was added with DMSO (0.1% (v / v), 0.2% (v / v)), and the blank control group had no addition. After the above-mentioned mixed bacterial solution was statically cultured at 37°C in a 24-well plate (1 mL / well) for 16 h, the upper layer of the liquid was aspirated, heat-fixed at 70°C for 30 min, then stained with 0.1% (w / v) crystal violet for 7 min, washed 3 times with water after staining, air-dried naturally, and the bottom biofilm was washed away with 30% (v / v) acetic acid, air-dried and photographed. After recording, the biofilm on the wall was dissolved with 30% (v / v) acetic acid and placed in a 96-well plate to measure OD 550 , and the results are as Figure 6 shown. Figure 6In it, a is the morphology of Pseudomonas aeruginosa PAO1 biofilm, b is the quantitative determination result of Pseudomonas aeruginosa PAO1 biofilm, and c is the inhibition rate of the fermentation broth extract on Pseudomonas aeruginosa PAO1 biofilm. As can be seen from Figure 6 the figure, the fermentation broth extract of strain M53 significantly inhibits the formation of Pseudomonas aeruginosa PAO1 biofilm; the fermentation broth extract at 50 μg / mL has no obvious effect on the biofilm formed by Pseudomonas aeruginosa PAO1 on the side wall of the 24-well plate, but as the concentration increases, the formation of the biofilm gradually decreases; the inhibition rate of the fermentation broth extract at 100 μg / mL on Pseudomonas aeruginosa PAO1 biofilm is about 38%, and the inhibition rate of the fermentation broth extract at 200 μg / mL on Pseudomonas aeruginosa PAO1 biofilm is about 52%. As the concentration of the fermentation broth extract increases to 400 μg / mL, the inhibition rate of Pseudomonas aeruginosa PAO1 biofilm also rises to 60%. Therefore, the fermentation broth extract of strain M53 has a concentration-dependent inhibitory activity on the formation of Pseudomonas aeruginosa PAO1 biofilm.

[0059] Example 8 Inhibitory effect of the fermentation broth extract of strain M53 on the drug resistance of Pseudomonas aeruginosa PAO1 Enhanced drug resistance is a typical feature of bacteria entering the swarming motility state. When bacteria enter the swarming motility state, their drug resistance usually increases significantly. To verify whether the fermentation broth extract of strain M53 can reduce the drug resistance of Pseudomonas aeruginosa PAO1 by inhibiting its swarming motility, in this example, the fermentation broth extract of strain M53 was combined with gentamicin, and whether the minimum inhibitory concentration (MIC) of the antibiotic changed before and after the combination was analyzed.

[0060] Pseudomonas aeruginosa PAO1 was activated in LA solid medium and cultured at 37 °C for 24 h. The activated Pseudomonas aeruginosa PAO1 was collected with a sterile inoculation loop and diluted to 1×10 9 CFU / mL with LB liquid medium. 1 μL was taken and dropped onto the swarming motility medium containing gentamicin (0.5, 1, 2, 4, 8, 16 μg / mL), the swimming motility medium containing gentamicin (0.5, 1, 2, 4, 8, 16 μg / mL), the swarming motility medium without addition (blank control group), and the swimming motility medium (blank control group). After culturing upright at 37 °C for 14 h, photos were taken and recorded. The results are shown in Figure 7 a and b in the figure. Figure 7 In the figure, a is the colony morphology and b is the statistical result of the inhibition rate. As can be seen from Figure 7As can be seen from a and b, Pseudomonas aeruginosa PAO1 in the blank control group reached the edge of the plate at 14 h; when 0.5 μg / mL gentamicin was added, the swarming motility of Pseudomonas aeruginosa PAO1 was only slightly inhibited (inhibition rate about 30%), while the swimming motility of Pseudomonas aeruginosa PAO was significantly inhibited (inhibition rate about 90%) under the same concentration of gentamicin; when 1 μg / mL gentamicin was added, the inhibition rate of the swarming motility of Pseudomonas aeruginosa PAO1 was about 40%, while the swimming motility of Pseudomonas aeruginosa PAO1 was completely inhibited under the same concentration of gentamicin, and Pseudomonas aeruginosa PAO1 could not grow either. These results were as expected, indicating that the drug resistance of Pseudomonas aeruginosa PAO1 to gentamicin was significantly increased in the swarming motility state.

[0061] Pseudomonas aeruginosa PAO1 was activated in LA solid medium and cultured at 37 °C for 24 h. An aseptic inoculation loop was used to collect the activated Pseudomonas aeruginosa PAO1 and diluted it to 1×10 9 CFU / mL with LB liquid medium. 1 μL was taken and dropped onto the swarming motility medium, and phenotypic induction was carried out by culturing at 37 °C for 14 h. The motile cells at 2 - 3 mm from the edge of the motile area were picked with an aseptic toothpick and inoculated into the center of the swarming motility medium containing the fermentation broth extract (7.5 μg / mL), the swarming motility medium containing gentamicin (1, 2 μg / mL), the swarming motility medium containing gentamicin (1, 2 μg / mL) and the fermentation broth extract (7.5 μg / mL), the swarming motility medium containing gentamicin (1, 2 μg / mL) and DMSO (0.1% (v / v)), the swarming motility medium containing DMSO (0.1% (v / v)) (negative control group), and the swarming motility medium without addition (blank control group). After incubation at 37 °C for 14 h, photos were taken and the motility radius was measured, and the inhibition rate was calculated. The results are as Figure 7 shown in c and d of Figure 7 In Figure 7 As can be seen from c and d of p<0.01); when gentamicin at 2 μg / mL was used in combination with the fermentation broth extract at 7.5 μg / mL, the swarming motility of Pseudomonas aeruginosa PAO1 was completely inhibited, and the colonies could hardly grow. Compared with the use of gentamicin alone at the same concentration (the inhibition rate was about 40%), the inhibition rate increased significantly. Therefore, the fermentation broth extract of strain M53 can reduce the drug resistance of Pseudomonas aeruginosa PAO1 by inhibiting swarming motility, and the combination with low-concentration gentamicin can significantly enhance the drug efficacy.

[0062] Example 9 Adding rhamnolipid can partially restore the swarming motility of Pseudomonas aeruginosa PAO1 The initiation of swarming motility in Pseudomonas aeruginosa PAO1 requires the secretion of the surfactant rhamnolipid to reduce surface tension. To explore whether the inhibition of the swarming motility of Pseudomonas aeruginosa PAO1 is related to rhamnolipid, different concentrations of rhamnolipid were added in this example based on the swarming motility experiment to study the changes in the swarming motility of Pseudomonas aeruginosa PAO1.

[0063] Pseudomonas aeruginosa PAO1 was activated in LA solid medium and cultured at 37 °C for 24 h. An aseptic inoculation loop was used to collect the activated Pseudomonas aeruginosa PAO1 and diluted it with LB liquid medium to 1×10 9 CFU / mL. 1 μL was taken and dropped onto the swarming motility medium containing the fermentation broth extract (10, 20 μg / mL) and rhamnolipid (0.1, 1 mg / mL), the swarming motility medium containing the fermentation broth extract (10, 20 μg / mL), the swarming motility medium containing DMSO (0.1% (v / v)) and rhamnolipid (0.1, 1 mg / mL), the swarming motility medium containing DMSO (0.1% (v / v)), the swarming motility medium containing rhamnolipid (0.1, 1 mg / mL), and the swarming motility medium without addition. After culturing upright at 37 °C for 14 h, photos were taken and recorded. The results are as Figure 8 shown. As Figure 8 can be seen, the swarming motility of Pseudomonas aeruginosa PAO1 was completely inhibited by the fermentation broth extract at 10 μg / mL and 20 μg / mL; after supplementing 0.1 mg / mL rhamnolipid, the swarming motility of Pseudomonas aeruginosa PAO1 inhibited by the 10 μg / mL fermentation broth extract was partially restored, while the swarming motility of Pseudomonas aeruginosa PAO1 inhibited by the 20 μg / mL fermentation broth extract was not restored. After supplementing 1 mg / mL rhamnolipid, the swarming motility of Pseudomonas aeruginosa PAO1 inhibited by the fermentation broth extract was further restored, while the swarming motility of Pseudomonas aeruginosa PAO1 in the control group without adding the fermentation broth extract under this condition was partially inhibited. Therefore, adding rhamnolipid can partially restore the swarming motility of Pseudomonas aeruginosa PAO1 inhibited by the fermentation broth extract. However, high-concentration rhamnolipid can also inhibit the normal swarming motility of Pseudomonas aeruginosa PAO1.

[0064] Example 10 Inhibitory effect of the fermentation broth extract of strain M53 on the rhamnolipid production of Pseudomonas aeruginosa PAO1 The results of Example 9 showed that the inhibition of the swarming motility of Pseudomonas aeruginosa PAO1 might be related to the production of rhamnolipids. Therefore, in this example, the rhamnolipid production of Pseudomonas aeruginosa PAO1 under the action of the fermentation broth extract of strain M53 was measured.

[0065] Pseudomonas aeruginosa PAO1 was activated on LA solid medium and cultured at 37 °C for 24 h. A single colony of Pseudomonas aeruginosa PAO1 was scraped and inoculated into LB liquid medium. After culturing with shaking at 37 °C (200 rpm) for 16 h, it was transferred to M9 medium supplemented with 2% (w / v) glycerol, 0.05% (w / v) glutamate and 0.05% (w / v) Triton X-100 at a volume ratio of 1:100. Then, the fermentation broth extract (50, 100, 200 μg / mL) or 0.1% (v / v) DMSO (negative control group) was added respectively, and no addition was used as the blank control group. After culturing with shaking at 37 °C (200 rpm) for 24 h, OD was measured. 600 The supernatant was collected by centrifugation and extracted three times with 3 volumes of ethyl acetate. After the extract was dried by rotary evaporation using a centrifugal concentrator, it was dissolved in 1 mL of water. After dissolution, 100 μL was transferred to a new EP tube, and then 800 μL of 60% (w / v) dilute sulfuric acid and 100 μL of orcinol reagent were added. The reaction was carried out at 80 °C for 30 min. After the reaction was completed, it was allowed to stand at room temperature for 15 min, and OD was measured. 421 through OD 421 / OD 600 The ratio was used to evaluate the rhamnolipid production, and the results are shown in Figure 9 as follows. Figure 9 In, each experiment was repeated three times, and **** represents p <0.0001. As can be seen from Figure 9 , after treatment with 50 μg / mL of the fermentation broth extract, the rhamnolipid production of Pseudomonas aeruginosa PAO1 decreased by about 50%; after treatment with 100 μg / mL of the fermentation broth extract, the rhamnolipid production of Pseudomonas aeruginosa PAO1 decreased by about 75%; as the concentration of the fermentation broth extract increased to 200 μg / mL, the rhamnolipid production of Pseudomonas aeruginosa PAO1 decreased by 85%. Therefore, the fermentation broth extract of strain M53 can significantly reduce the rhamnolipid production of Pseudomonas aeruginosa PAO1, thereby inhibiting swarming motility.

[0066] It should be understood that the application of the present invention is not limited to the above examples. For those of ordinary skill in the art, it can be improved or transformed according to the above description, and all such improvements and transformations should fall within the protection scope of the appended claims of the present invention.

Claims

1. A method for preparing an extract from the fermentation broth of Streptomyces ( Streptomyces sp . ) M53, characterized in that comprises the steps of: (1) Inoculating Streptomyces M53 into SGTYP liquid medium and shaking culture for 72 - 96 h to obtain a fermentation broth; (2) Extracting the fermentation broth with ethyl acetate, removing the aqueous phase to obtain an extract of the fermentation broth; (3) Removing ethyl acetate from the extract of the fermentation broth to obtain an extract of the fermentation broth of Streptomyces M53; wherein, Streptomyces M53 is deposited in the Guangdong Provincial Culture Collection of Microorganisms, and the deposit number is GDMCC No: 65720.

2. The preparation method according to claim 1, characterized in that, The conditions for the shaking culture are: temperature 25 - 28 °C, rotation speed 100 - 300 rpm.

3. The preparation method according to claim 1, characterized in that, Step (1) specifically includes: After activating Streptomyces M53 on MA solid medium, taking a single colony and shaking culture in 5 - 15 mL of SGTYP liquid medium for 20 - 30 h to obtain a seed liquid; Inoculating the seed liquid into a new SGTYP liquid medium and shaking culture for 72 - 96 h to obtain a fermentation broth.

4. The preparation method according to claim 3, characterized in that, The volume ratio of the seed liquid to the new SGTYP liquid medium is 1:(50 - 100).

5. Fermentation broth extract of Streptomyces sp. M53, characterized in that, The extract of the fermentation broth is prepared by the preparation method according to any one of claims 1 - 4.

6. Use of the extract of the fermentation broth according to claim 5 in the preparation of a product for inhibiting the swarming motility of Pseudomonas aeruginosa.

7. Use of the extract of the fermentation broth according to claim 5 in the preparation of a product for inhibiting the antibiotic resistance of Pseudomonas aeruginosa.

8. The application according to claim 7, wherein The antibiotic is gentamicin.

9. Use of the extract of the fermentation broth according to claim 5 in the preparation of a product for inhibiting the biofilm formation of Pseudomonas aeruginosa.

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