Streptomyces albus culture medium, fermentation method, biological bacteriostatic agent and application thereof

Abstract

The invention relates to the technical field of microbial fermentation engineering, in particular to a streptomyces albus culture medium, a fermentation method, a biological bacteriostatic agent and application of the biological bacteriostatic agent. The culture medium contains a carbon source, a nitrogen source and inorganic salt in a specific concentration range, and the pH is neutral. When the culture medium is used for fermenting specifically preserved streptomyces albus ZJU527, a biological bacteriostatic agent can be efficiently prepared. The effective components of the bacteriostatic agent comprise tetramycin A, tetramycin B, tetraene rhzomorph A and tetraene rhzomorph B, and the bacteriostatic agent has a remarkable inhibition effect on various microorganisms such as bacillus subtilis, escherichia coli, saccharomycetes and fusarium graminearum, especially on plant pathogenic fungi fusarium graminearum.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation engineering technology, and in particular to a Streptomyces white mold culture medium, fermentation method, biological antibacterial agent and its application. Background Technology

[0002] Plant pathogenic fungi are a significant factor limiting agricultural production, causing substantial losses globally each year. Wheat and maize, as the world's dominant food and feed crops, are crucial for ensuring global food security due to their stable yields. Among them, Fusarium graminearum (… Fusariumgraminearum Fusarium head blight (in wheat) and ear rot (in corn) caused by fungi are the leading fungal diseases affecting the yield and quality of these two crops. Besides directly causing ear rot and reduced grain yield, the pathogen also produces various mycotoxins, further threatening food safety. Currently, disease control still mainly relies on chemical fungicides. The long-term dependence on single-mechanism inhibitors has led to increasingly prominent problems of pathogen resistance, and pesticide residues pose a continuous threat to the environment and food safety. Therefore, the development of environmentally friendly, novel, and non-resistant bio-based inhibitors has become an urgent need in the field of sustainable agricultural development.

[0003] Streptomyces, as an important source of natural active products, can produce a rich variety of antibacterial secondary metabolites, making them an ideal resource for developing novel biopesticides. Current development strategies often focus on selectively enhancing the ability of Streptomyces to synthesize one or a few specific antibacterial substances (such as single antibiotics or specific antifungal compounds) through strain selection or process optimization. While such "targeted" methods have achieved some success on specific targets, they often overlook the overall potential of Streptomyces as a complex metabolic system, limiting its ability to produce compositions with multi-component synergistic antibacterial functions. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a Streptomyces white mold culture medium, fermentation method, biological antibacterial agent, and their applications. The aim is to optimize the fermentation system, fully stimulate the overall metabolic potential of the microorganisms, and efficiently prepare fermentation products with broad-spectrum antibacterial activity, providing a new solution for the green control of wheat scab and corn ear rot.

[0005] The technical solution adopted in this invention is as follows: In a first aspect, the present invention provides a white Streptomyces culture medium comprising the following components: It contains a carbon source, a nitrogen source, and inorganic salts, and the pH of the culture medium is neutral; The carbon source is selected from at least one of starch, dextrin, and glucose, and its total concentration in the culture medium is 5~45 g / L; The nitrogen source is selected from at least one of plant-derived protein powder, yeast extract, inorganic ammonium salt or nitrate, and its total concentration in the culture medium is 1~30g / L; The inorganic salts include magnesium sulfate and calcium carbonate, wherein the concentration of magnesium sulfate is 0.1~2.0 g / L and the concentration of calcium carbonate is 1.0~5.0 g / L.

[0006] Furthermore, the culture medium is selected from any of the following: (a) Glucose 5~15g / L, maltodextrin 15~25g / L, soluble cottonseed powder 15~25g / L, soluble starch 25~35g / L, yeast extract 3~8g / L, MgSO4·7H2O 0.5~2g / L, CaCO3 1~3g / L, pH 7.2~7.4; (b) Potato flour 5~15g / L, casein amino acids 1~3g / L, yeast extract 1.5~2.0g / L, KCl 0.1~0.3g / L, MgSO4·7H2O 0.1~0.3g / L, NaNO3 0.1~0.4g / L, FeSO4·7H2O 0.002~0.005g / L, pH 7.2~7.4.

[0007] Furthermore, the culture medium (a) contains the following components: Glucose 8~12g / L, maltodextrin 18~22g / L, soluble cottonseed flour 18~22g / L, soluble starch 28~32g / L, yeast extract 4~7g / L, MgSO4·7H2O 0.8~1.2g / L, CaCO3 1.5~2.5g / L, pH 7.2~7.4.

[0008] More preferably, the culture medium (a) is: glucose 10 g / L, maltodextrin 20 g / L, soluble cottonseed powder 20 g / L, soluble starch 30 g / L, yeast extract 5 g / L, MgSO4·7H2O 1 g / L, CaCO3 2 g / L, pH 7.2~7.4.

[0009] In a second aspect, the present invention provides a method for fermenting *Streptomyces albopictus*, comprising the following steps: (a) Inoculate Streptomyces whitei into the aforementioned culture medium for fermentation culture; (b) After fermentation, the fermentation broth is separated to obtain a supernatant containing antibacterial activity; The preferred Streptomyces whiteum strain is Streptomyces whiteum ZJU527, whose accession number is CCTCCNO:M20232443.

[0010] Furthermore, the inoculum size of the *Streptomyces albopictus* is 10. 5 ~10 7CFU / mL.

[0011] Further, in step (a), the fermentation conditions of the white Streptomyces are: 28~32°C, 150~200rpm, and shaking culture under dark conditions for 5~9 days.

[0012] Further, in step (b), the separation and purification includes: adsorbing the fermentation broth onto a macroporous resin, eluting with a gradient of 50% to 70% ethanol by volume, with an elution volume of 1 to 5 column volumes, and collecting the eluent.

[0013] More preferably, the macroporous resin is AB-8 type macroporous resin.

[0014] More preferably, the volume fraction of the ethanol solution is 60%.

[0015] More preferably, the elution volume is 3 column volumes.

[0016] In a third aspect, the present invention provides a biological antibacterial agent, which is prepared by the aforementioned method.

[0017] Furthermore, the effective antibacterial components of the biological antibacterial agent include tetramycin A, tetramycin B, tetravinyl A, and tetravinyl B, and the mass ratio of the four chemical components in the two culture media is 100:23:49:7 and 100:62:68:22.

[0018] The present invention provides, in a fourth aspect, the application of the aforementioned bio-antimicrobial agent in inhibiting microorganisms, wherein the microorganisms are selected from at least one of the following: A1) Bacillus subtilis; A2) Escherichia coli; A3) Yeast; A4) Fusarium graminearum; The preferred Bacillus subtilis strain is Bacillus subtilis 3610.

[0019] Compared with the prior art, the present invention has the following beneficial effects: Through extensive screening and optimization, this invention provides a culture medium formulation suitable for the fermentation of Streptomyces albopictus ZJU527, which significantly enhances the antibacterial potency of the fermentation broth.

[0020] The biological antibacterial agent prepared by the specific culture medium and method of the present invention has its core active ingredients identified as tetramycin A, tetramycin B, tetraencin A and tetraencin B, exhibiting broad-spectrum antibacterial properties. Attached Figure Description

[0021] Figure 1 The strain of this invention StreptomycesalbulusZJU527 is a phylogenetic tree constructed based on the whole genome.

[0022] Figure 2 Streptomyces albopictus ( Streptomycesalbulus The strain morphology is described, and the strain number is ZJU527.

[0023] Figure 3 The image shows the UV chromatogram of the fermentation broth from culture medium 1, with a detection wavelength of 320 nm.

[0024] Figure 4 The image shows the UV chromatogram of the fermentation broth from culture medium 2, with a detection wavelength of 320 nm.

[0025] Figure 5 The image shows the UV chromatogram of fermentation broth from culture medium 3, with a detection wavelength of 320 nm.

[0026] Figure 6 The image shows the UV chromatogram of fermentation broth from culture medium 4, with a detection wavelength of 320 nm.

[0027] Figure 7 A shows the UV-Vis image (254 nm) of the fermentation product obtained by liquid chromatography-mass spectrometry (LC-MS / MS). Figure 7 B represents the mass of the four compounds obtained during preparation.

[0028] Figure 8 Four compounds in the fermentation products were characterized using liquid chromatography-mass spectrometry (LC-MS).

[0029] Figure 9 Four compounds in the fermentation products were characterized using nuclear magnetic resonance.

[0030] Figure 10 To determine the antibacterial effect of fermentation products on Fusarium graminearum using the paper disc diffusion method.

[0031] Figure 11 To determine the antibacterial effect of monomeric compounds against Fusarium graminearum using the paper disc diffusion method. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments. The following are only specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto.

[0033] Example 1: Identification of Streptomyces whiteiformis ZJU527 Whole-genome sequencing was performed on the bacterial strains to assess the genomic DNA quality. Acceptable DNA samples were randomly fragmented to approximately 350 bp using a Covaris fragmenter. The fragmented DNA underwent sequential end repair, 5' phosphorylation, and 3' A-tailing. Illumina sequencing adapters were then ligated to both ends of the DNA fragments. The ligation products were screened using a two-step (Double Size Selection) method with SPRI magnetic beads. The selected libraries were enriched by high-fidelity PCR amplification. The concentration of the amplified products was determined using Qubit 3.0, the insert size was detected using an Agilent 5400 system, and the effective library concentration (target 1.5 nM) was accurately quantified using qPCR. Quality-tested libraries were sequenced on the Illumina Novaseq platform. The libraries were first amplified into DNA clusters using Bridge PCR on FlowCell, followed by PE150 (Pair-end 150 bp) paired-end sequencing. Sequencing yielded raw data in FASTQ format. The raw data was quality controlled using the FastP software, with the following filtering rules: 1) Remove read pairs containing adapters; 2) Read pairs with a nitrogen base ratio of 10% or higher were removed; 3) Remove low-quality read pairs (defined as reads in which more than 50% of the bases have a Q ≤ 5). The resulting clean data is used for subsequent genome assembly and bioinformatics analysis.

[0034] Gene analysis was performed using sequencing data, and the genome was assembled using SPAdesv 4.1.0. The "streptomycetales_odb10" database from BUSCOv 5.2.2 was used to identify conserved genes, thus assessing the quality of the genome assembly. Genome annotation was performed using the "light" database from baktav 1.9.4. The "streptomycetales_odb10" database from BUSCOv 5.2.2 (-mprot) was used to identify conserved genes, thus assessing the quality of the genome annotation.

[0035] Phylogenetic analysis was performed using genomes of 20 publicly available Streptomycetales species from the NCBI database, and their genome quality was assessed using BUSCO v5.2.2 software. A phylogenetic tree was constructed using 198 single-copy BUSCO genes, which are present in at least half of all Streptomycetales species. Each BUSCO gene was aligned using MAFFT v7.525 with the "-auto" option, and ambiguous alignment regions were removed using the "gappyout" function of trimAlv1.5. PhyKIT v2.0.1 software was then used to integrate the nucleotide sequence alignment results of these 198 BUSCO genes into a complete data matrix. Finally, IQ-TREE version 2.4.0 was used, and the optimal TVM+F+R10 model was found using -m MFP for maximum likelihood phylogenetic analysis. Figure 1 Analysis confirmed that the strain was *Streptomyces albopictus*. Streptomycesalbulus And it is deposited at the China Center for Type Culture Collection, with accession number CCTCCNO:M20232443 ( Figure 2 ).

[0036] Example 2: Screening and Fermentation Methods for Culture Media To screen for the optimal fermentation medium, the following four representative media were prepared for comparison: Culture medium 1 (per 1L): 10g glucose, 20g maltodextrin, 20g soluble cottonseed powder, 30g soluble starch, 5g yeast extract, 1g MgSO4·7H2O, 2g CaCO3, pH 7.2-7.4.

[0037] Culture medium 2 (per 1L): 30g corn starch, 7g soybean flour, 1.5g KNO3, 0.5g K2HPO4, pH=7.5.

[0038] Culture medium 3 (per 1L): 10g potato starch, 2g casein amino acids, 1.8g yeast extract, 0.2g KCl, 0.2g MgSO4·7H2O, 0.24g NaNO3, 0.004g FeSO4·7H2O, pH 7.2-7.4.

[0039] Culture medium 4 (per 1L): soluble starch 35g / L, NH4NO3 3.5g / L, K2HPO4 1g / L, MgSO4·7H2O 0.5g / L, CaCO3 3g / L, pH=7.0.

[0040] Fermentation method: Sterilus oryzae ZJU527 was fermented at 10 6Inoculate the culture medium with an inoculum of CFU / mL and ferment at 30°C, 180 rpm, and in the dark for 7 days with shaking. After fermentation, centrifuge the fermentation broth at 5000 rpm for 10 minutes, and the resulting supernatant is the composition with primary antibacterial activity.

[0041] Example 3: Chemical composition and antibacterial activity analysis of fermentation products The fermentation supernatants of the above four culture media were analyzed by ultraviolet chromatography (detection wavelength 320 nm), and the results are as follows: Figures 3-6 As shown, the main active chemical components are tetramycin A, tetramycin B, tetravinyl A, and tetravinyl B, but the relative contents of each component vary significantly depending on the culture medium. In culture medium 1, the mass ratio of the four chemical components is 100:23:49:7; in culture medium 2, the mass ratio is 100:139:163:188; in culture medium 3, the mass ratio is 100:62:68:22; and in culture medium 4, the mass ratio is 100:107:48:36. This difference in the content of chemical components directly leads to the difference in their antibacterial activity.

[0042] The inhibition rates of each fermentation supernatant against Bacillus subtilis, Escherichia coli, yeast, and Fusarium graminearum were determined using the microdilution method. The experimental method is as follows: Indicator bacterial suspensions were prepared by preparing Bacillus subtilis 3610 and Escherichia coli suspensions, respectively, and adjusting their final concentrations to 1×10⁻⁶. 5 CFU / mL. Spore suspensions of yeast and Fusarium graminearum were prepared separately, and their final concentrations were adjusted to 1×10⁻⁶. 4 Spores / mL; Add 100 µL of the fermentation product sample to be tested to each well of a sterile 48-well plate, followed by 900 µL of the corresponding indicator culture medium (MH medium for Bacillus subtilis and Escherichia coli, YPDA medium for yeast, and RPMI 1640 medium for Fusarium graminearum); A control group was also set up. (a) Positive control (growth control): 100 µL of sterile culture medium (in place of fermentation product) and 900 µL of indicator bacterial suspension were added to each well; (b) Blank control (background control): 100 µL of the fermentation product sample to be tested and 900 µL of sterile culture medium were added to each well to eliminate the background of the culture medium. (c) Negative control (sterile control): 1000 µL of sterile culture medium was added to each well.

[0043] 48-well plates inoculated with bacteria were fermented at 30°C for 24 hours, and 48-well plates inoculated with fungi were fermented at 25°C for 48 hours. After fermentation, the absorbance (OD) of each well was measured at 600 nm using a microplate reader.600 The inhibition rate is calculated using the following formula: Inhibition rate (%) = [1 - (OD)] / (OD) 样品 -OD 空白对照 ) / (OD 阳性对照 -OD 阴性对照 ×100%. The results are shown in Table 1.

[0044] Table 1 Antibacterial activity of fermentation broth from four different culture media

[0045] (Note: The influence of blank culture medium has been deducted in the antibacterial test.) Data show that the fermentation broth of culture medium 1, while maintaining a high inhibition rate (71.80%) against Fusarium graminearum, also exhibits comprehensive and excellent inhibitory activity against bacteria and yeast. Therefore, culture medium 1 is preferred as the optimal culture medium for preparing the broad-spectrum antibacterial composition of this invention.

[0046] Example 4: Purification, component verification, and application effects of fermentation products (1) Preparation of four monomeric compounds The fermentation supernatant from culture medium 1 in Example 2 was used for separation and purification using macroporous resin. Gradient elution was performed using 60% ethanol solution at 3 column volumes. The eluent fractions were collected to obtain the secondary purified fermentation product. Its UV chromatogram (detection wavelength 254 nm) is shown below. Figure 7 As shown in Figure A.

[0047] The secondary purified fermentation products were further separated by preparative liquid chromatography to prepare monomeric compounds, as follows: The antibacterial active composition was further separated and prepared by liquid preparation. Mobile phase A was ultrapure water, and mobile phase B was methanol. The mobile phase was 0-50 min, 50-100% methanol, with one fraction collected every 3 min, for a total of 16 secondary fractions. Tetramycin B was obtained by further purification of fraction 9 with 60% methanol and water; tetravinil A was obtained by further purification of fraction 11 with 65% methanol and water; tetravinil A was obtained by further purification of fraction 6 with 50% methanol and water; and tetravinil B was obtained by further purification of fraction 14 with 70% methanol and water. The final monomer yield was as follows: Figure 7 As shown in B, its structural information was characterized by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) analysis techniques, such as... Figure 8-9 As shown.

[0048] (2) Paper disc diffusion method was used to determine the antibacterial effect of fermentation products on Fusarium graminearum. Add Fusarium graminearum spores to the PDA medium that has been melted and is at a suitable temperature, to a final concentration of 0.5-1×10⁻⁶. 4CFU / mL, pour the culture medium into a 9cm diameter petri dish and let it stand to dry. Add equal volumes of the mixture dissolved in methanol (water, 20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol, and 95% ethanol) dropwise onto a 0.5mm diameter sterile filter paper disc, dry it in a laminar flow hood, transfer it to a spore plate, and incubate at 25℃. Observe, measure, and photograph the results after 24 hours. See attached table. Figure 10 The fermentation product dissolved in 60% ethanol showed the largest inhibition zone diameter and the best effect. Meanwhile, a control with 50% methanol was set up, and no inhibition zone was observed, thus eliminating the interference of the solvent.

[0049] (3) Paper disc diffusion method was used to determine the antibacterial effect of monomeric compounds on Fusarium graminearum. The secondary purified fermentation products were further separated by preparative liquid chromatography to obtain four monomeric compounds: tetramycin A, tetramycin B, tetraenzin A, and tetraenzin B. The inhibitory effects of each monomer on *Fusarium graminearum* were tested using the same disk diffusion method as described above. The results showed ( Figure 11 All four monomers exhibit antibacterial activity, but the inhibition zone diameter of the secondary purified fermentation product is larger than that of any single monomer, indicating that there may be a synergistic effect among the components, and the overall antibacterial effect of the composition is better than that of a single component.

[0050] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A fermentation medium for *Streptomyces albopictus*, characterized in that, It contains a carbon source, a nitrogen source, and inorganic salts, and the pH of the culture medium is neutral; The carbon source is selected from at least one of soluble starch, potato starch, corn starch, dextrin, and glucose, and its total concentration in the culture medium is 5~45 g / L; The nitrogen source is selected from at least one of plant-derived protein powder, yeast extract, inorganic ammonium salt or nitrate, and its total concentration in the culture medium is 1~30g / L; The inorganic salts include magnesium sulfate and calcium carbonate, wherein the concentration of magnesium sulfate is 0.1~2.0 g / L and the concentration of calcium carbonate is 1.0~5.0 g / L.

2. The culture medium according to claim 1, characterized in that, The culture medium is selected from any of the following: (a) Glucose 5~15g / L, maltodextrin 15~25g / L, soluble cottonseed powder 15~25g / L, soluble starch 25~35g / L, yeast extract 3~8g / L, MgSO4·7H2O 0.5~2g / L, CaCO3 1~3g / L, pH 7.2~7.4; (b) Potato flour 5~15g / L, casein amino acids 1~3g / L, yeast extract 1.5~2.0g / L, KCl 0.1~0.3g / L, MgSO4·7H2O 0.1~0.3g / L, NaNO3 0.1~0.4g / L, FeSO4·7H2O 0.002~0.005g / L, pH 7.2~7.

4.

3. The culture medium according to claim 2, characterized in that, Culture medium (a) contains the following components: Glucose 8~12g / L, maltodextrin 18~22g / L, soluble cottonseed flour 18~22g / L, soluble starch 28~32g / L, yeast extract 4~7g / L, MgSO4·7H2O 0.8~1.2g / L, CaCO3 1.5~2.5g / L, pH 7.2~7.

4.

4. A method for fermenting *Streptomyces albopictus*, characterized in that, Includes the following steps: (a) Inoculating Streptomyces whitei into the culture medium as described in any one of claims 1 to 3 for fermentation culture; (b) After fermentation, the fermentation broth is separated and purified to obtain a supernatant containing antibacterial activity; The preferred Streptomyces whiteum strain is Streptomyces whiteum ZJU527, whose accession number is CCTCCNO:M20232443.

5. The fermentation method as described in claim 4, characterized in that, In step (a), the inoculum size of *Streptomyces albopictus* is 10. 5 ~10 7 CFU / mL; And / or, the fermentation conditions of the white Streptomyces are: 28~32°C, 150~200rpm, and shaking culture in the dark for 5~9 days.

6. The fermentation method according to claim 4, characterized in that, In step (b), the separation and purification includes: adsorbing the fermentation broth onto a macroporous resin, eluting with a gradient of 50% to 70% ethanol (volume fraction) for a volume of 1 to 5 column volumes, and collecting the eluent.

7. The fermentation method according to claim 4, characterized in that, The macroporous resin is AB-8 type macroporous resin; The volume fraction of the ethanol solution is 60%. The elution volume is 3 column volumes.

8. A biological antibacterial agent, characterized in that, The bio-antibacterial agent is prepared by the method described in any one of claims 4-7.

9. The biological antibacterial agent according to claim 8, characterized in that, The effective antibacterial components of the biological antibacterial agent include tetramycin A, tetramycin B, tetravinyl A, and tetravinyl B, and the mass ratio of the four chemical components in the two culture media is 100:23:49:7 and 100:62:68:

22.

10. The application of the bio-antimicrobial agent as described in claim 8 or 9 in inhibiting microorganisms, characterized in that, The microorganism is selected from at least one of the following: A1) Bacillus subtilis; A2) Escherichia coli; A3) Yeast; A4) Fusarium graminearum; The preferred Bacillus subtilis strain is Bacillus subtilis 3610.

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