Analysis method for differential metabolites in different fermentation periods of streptomyces fradiae

Abstract

The invention discloses a method for analyzing differential metabolites in different fermentation periods of streptomyces fradiae. The method comprises the following steps: collecting and quenching streptomyces fradiae cells, extracting intracellular metabolites, performing LC-MS (Liquid Chromatography-Mass Spectrometry) detection, and processing and analyzing data. According to the method, samples of streptomyces fradiae in different fermentation periods are studied through a non-targeted metabonomics method, the types, abundance and metabolic pathways of main differential metabolites in the cell fermentation process of streptomyces fradiae are analyzed, and the regulation mechanism of the differential metabolites influencing the cell fermentation of streptomyces fradiae is clarified; therefore, the metabonomics and metabolic engineering research of streptomyces fradiae is promoted, and the industrial production application value of streptomyces fradiae is further improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological analysis and detection, and specifically relates to an analysis method for differential metabolites of Streptomyces freundii at different fermentation periods. Background Art

[0002] Streptomyces freundii is an aerobic, gram-positive bacterium with well-developed mycelium, mainly found in the soil. It is widely used in agriculture to prepare safe and efficient microbial agents, which can effectively prevent and treat a variety of soil-borne diseases. In addition, Streptomyces freundii is also the main production strain of the aminoglycoside antibiotic neomycin, which has multiple biological activities such as antibacterial, anti-cancer and anti-HIV. The secondary metabolite of Streptomyces freundii, tylosin, belongs to the 16-membered macrolide antibiotics. It is recognized worldwide as the first choice for the treatment and prevention of mycoplasma infections in livestock and poultry, and has a very broad application space. Research on Streptomyces freundii has been ongoing at home and abroad, including strain selection, genetic level research, and industrial production optimization.

[0003] With the development of omics technology, the status of systems biology research has become increasingly prominent. Metabolites, as the terminal level of biological information transmission, have shown great potential in the interpretation of gene function and analysis of metabolic status. Metabolomics is a research method that qualitatively and quantitatively measures the types and concentration changes of metabolites of organisms, analyzes the comprehensive differences of a series of related metabolites, and thus discovers the response of biological systems to genetic and environmental changes. The application of metabolomics in the field of industrial microbiology is mainly reflected in the following aspects: rapid identification of the physiological and metabolic characteristics of microorganisms during cultivation; discovery of new metabolic pathways of strains; explanation of the microbial metabolic regulation mechanism from a system level; rational guidance of strain transformation, increase of target metabolite production and guidance of fermentation process optimization. With the continuous improvement of detection technology and data processing level, metabolomics plays an increasingly important role in the field of industrial microbiology. However, to date, no research has reported the metabolite process of Streptomyces freundii, so it is extremely important to study the changes in the relevant metabolites of Streptomyces freundii at different fermentation periods. Summary of the invention

[0004] In view of the above problems, the object of the present invention is to provide a method for analyzing differential metabolites of Streptomyces freundii at different fermentation stages.

[0005] The specific technical solutions are as follows:

[0006] A method for analyzing differential metabolites of Streptomyces freundii at different fermentation stages, characterized in that it comprises the following steps:

[0007] 1) Intracellular metabolite processing

[0008] ①Cell collection and quenching

[0009] The fermentation broth of Streptomyces freundii at different fermentation periods was centrifuged to remove most of the solid impurities in the culture medium, the cell pellet was washed and then added with a solution quencher, quickly mixed by vortexing and homogenizing, incubated under low temperature conditions, and quickly filtered by vacuum filtration to collect solid samples;

[0010] ② Metabolite extraction

[0011] The treated solid sample is placed in a centrifuge tube, and the metabolite extract and internal standard are added. After vortexing, the sample is placed in ice cubes for ultrasonic extraction of intracellular metabolites. After the ultrasonic extraction, the sample is centrifuged. After the centrifugation, the same volume of extract is added to the precipitate obtained after the centrifugation, the sample is placed on ice cubes for ultrasonic extraction again. After the ultrasonic extraction, the sample is centrifuged, and the two supernatants are combined. After centrifugation, the supernatant is taken out and passed through a membrane into a sample injection vial for determination by liquid chromatography-mass spectrometry.

[0012] 2) Liquid chromatography-mass spectrometry

[0013] 3) Data processing and analysis

[0014] The data obtained by the test in step 2) were imported into the metabolomics processing software for baseline filtering, peak identification, integration, retention time correction, peak alignment, and normalization to obtain a data matrix containing retention time, mass-to-charge ratio, and peak intensity information; then, a characteristic peak search library was performed to match the MS and MS / MS mass spectrum information with the Metlin, KEGG, and PubChem databases, and the MS mass error was set to less than 10 ppm. At the same time, metabolites were identified based on the secondary mass spectrum matching score; variance analysis (ANOVA) was used to perform statistical analysis on the metabolites of samples in different groups, including principal component analysis (PCA) in the unsupervised mode and PLS-DA analysis in the supervised mode, and compounds with VIP>1, significant difference p<0.05, and compound response value difference greater than 2 times (|FC|>2) were screened out, which were the differential metabolites between the groups; metabolic pathway enrichment and overall change analysis were performed on the screened differential metabolites through the KEGG database.

[0015] Further, the operating conditions of the liquid chromatography-mass spectrometry method in step 2) are as follows: liquid phase parameters: chromatographic column: ACQUITY BEH C18 column, 2.1mm×100mm, 1.7μm; column temperature: 35°C; flow rate: 0.2mL / min; injection volume: 2μL; mobile phase A: 0.1% formic acid water; mobile phase B: acetonitrile; the gradient elution program is as follows: 0-20min, the volume fraction of mobile phase A is reduced from 95% to 75%, and the volume fraction of mobile phase B is increased from 5% to 25%; 20-25min, the volume fraction of mobile phase A is reduced from 75% to 10%, and the volume fraction of mobile phase B is increased from 25% to 90%; 25-26min, the volume fraction of mobile phase A is increased from 10% to 95%, and the volume fraction of mobile phase B is reduced from 90% to 5%, 26-30min, the volume fraction of mobile phase A is 95%, and the volume fraction of mobile phase B is 5%;

[0016] Mass spectrometry parameters: The mass spectrometer is in full scan mode in the positive ion conversion mode, with a mass range of m / z 50 to 1500; capillary voltage: 3000 V; drying gas flow rate: 8 L / min; fragmentation voltage: 160 V; drying gas temperature: 325°C; sheath gas temperature: 325°C; sheath gas flow rate: 11 L / min; nebulizer gas pressure: 40 psi.

[0017] Furthermore, the specific method of cell collection and quenching in step 1) is as follows: take the fermentation broth of Streptomyces freundii at different fermentation periods, centrifuge at 10000-14000rpm at 4°C for 5min to remove most of the solid impurities in the culture medium; discard the supernatant, wash the cell pellet twice with PBS buffer, then wash it once with deionized ultrapure water, add a 60% methanol solution quencher pre-cooled at -80°C for 24h, vortex for 1min, and then incubate at 0-5°C for 5min, and then quickly filter the mixed solution by vacuum filtration to collect the solid sample.

[0018] Furthermore, each 1 mol / L of the PBS buffer contains 8.00 g of NaCl, 0.20 g of KCl, 1.42 g of Na2HPO4, and 0.27 g of KH2PO4.

[0019] Furthermore, the specific method for extracting metabolites in step 1) is to place the treated solid sample in a centrifuge tube, add a mixed extract of methanol and water in a volume ratio of 1:1 and pre-cooled at -80°C for 24 hours and 0.02 mg / mL internal standard L-2-chlorophenylalanine, vortex for 1 minute to mix evenly, and then place it in ice cubes for ultrasonication for 20 to 40 minutes to extract intracellular metabolites; after the end of the ultrasound, centrifuge at 8000-12000rpm and 0-5°C for 5 to 20 minutes, add the extract to the precipitate and place it in ice cubes for ultrasonication for 15 to 45 minutes for secondary extraction, and centrifuge at 8000-12000rpm and 0-5°C for 5 to 15 minutes after the end of the ultrasound, combine the two supernatants, vortex for 1 minute to mix evenly, and then centrifuge at 8000-12000rpm and 4°C for 1 to 10 minutes, take the supernatant, take the supernatant, filter it into an injection vial with a 0.22μm membrane, and perform liquid chromatography-mass spectrometry determination.

[0020] KEGG enrichment in step (3) revealed that, compared with the control group, a total of 84 differential metabolites common to the four different fermentation time periods were screened out.

[0021] In step (3), a KEGG metabolic pathway bubble diagram was drawn based on the screened differential metabolites. The differences in metabolites were mainly related to pathways such as glycerophospholipid metabolism, TCA cycle, pantothenic acid and coenzyme A biosynthesis, riboflavin metabolism, thiamine metabolism, biotin metabolism, sphingolipid metabolism, starch and sucrose metabolism, and various amino acid metabolism.

[0022] The beneficial effects of the present invention are:

[0023] The analytical method of the present invention involves the termination of metabolic reactions, the extraction of metabolites, and the analysis of metabolites. Samples of Streptomyces flexneri at different fermentation periods are studied by a non-targeted metabolomics method, thereby obtaining qualitative and quantitative results of major differential metabolites in different fermentation stages of Streptomyces flexneri, analyzing the types, abundances and metabolic pathways of major differential metabolites in the fermentation process of Streptomyces flexneri cells, and clarifying the regulatory mechanism of differential metabolites affecting the fermentation of Streptomyces flexneri cells, thereby promoting the metabolomics and metabolic engineering research of Streptomyces flexneri, and further improving its industrial production application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the two-dimensional PCA score diagram of the metabolic characteristics of Streptomyces freundii at different fermentation stages;

[0025] Figure 2 Heat map of cluster analysis of common differential metabolites of each sample in different fermentation periods of Streptomyces freundii;

[0026] Figure 3 Bubble diagram of metabolic pathways for the 120h group and the 48h group;

[0027] Figure 4 Bubble diagram of metabolic pathways for the 168h group and the 48h group;

[0028] Figure 5 Bubble diagrams of metabolic pathways for the 240h group and the 48th group. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but the protection scope of the present invention is not limited thereto.

[0030] Example 1

[0031] Select Materials:

[0032] Methanol (chromatographic grade, Merck, Germany), formic acid (mass spectrometry grade, Thermo Fisher Scientific, USA), L-2-chlorophenylalanine (Shanghai Yuanye Biotechnology Co., Ltd.).

[0033] Agilent 1290 / 6545 UHPLC-Q-TOF liquid spectrometer (Agilent, USA), Vortex QL-861 vortex mixer (Kylin-Bell, China), KQ-600DE digital ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., China), Heraeus Biofuge Stratos desktop high-speed centrifuge (Thermo Scientific, Switzerland).

[0034] Specific steps of the analysis method:

[0035] (1) Intracellular metabolite processing

[0036] ①Cell collection and quenching

[0037] At 48h, 120h, 168h, and 240h of the fermentation of Streptomyces freundii, 10ml of fermentation liquid was taken and centrifuged at 12000rpm and 4℃ for 5min to remove most of the solid impurities in the culture medium; the supernatant was discarded, the cell pellet was washed twice with PBS buffer, and then washed once with deionized ultrapure water, and 20mL of 60% methanol (0.9% sodium chloride) solution quencher precooled at -80℃ for 24h was added, vortexed for 1min, and then incubated at 4℃ for 5min, and then the mixture was quickly filtered by vacuum filtration to collect solid samples. There were 4 parallel samples in each period.

[0038] Each 1 mol / L of the PBS buffer contains 8 g of NaCl, 0.2 g of KCl, 1.42 g of Na2HPO4, and 0.27 g of KH2PO4.

[0039] ② Metabolite extraction

[0040] Accurately weigh 50±5 mg of cell solids into a 2 mL centrifuge tube, add 1 mL of a mixed extract of methanol and water with a volume ratio of 1:1 precooled at -80℃ for 24 hours and 50 μL of L-2-chlorophenylalanine with an internal standard of 0.02 mg / mL, vortex for 1 min to mix evenly, and then place it in ice cubes for ultrasonication for 30 min to extract intracellular metabolites; after the end of ultrasonication, centrifuge at 10000 rpm and 4℃ for 10 min, add 1 mL of the extract to the precipitate and place it in ice cubes for ultrasonication for 30 min for secondary extraction, and centrifuge at 10000 rpm and 4℃ for 10 min after the end of ultrasonication, combine the two supernatants, vortex for 1 min to mix evenly, and then centrifuge at 10000 rpm and 4℃ for 5 min, take the supernatant and pass it through a 0.22 μm membrane into an injection vial for liquid chromatography-mass spectrometry determination.

[0041] 50 μL of supernatant was transferred from each sample and mixed as quality control samples, which were processed and tested in the same way as the analytical samples. During the instrument analysis, one quality control sample was inserted into every four analytical samples to examine the stability of the entire detection process.

[0042] (2) Liquid chromatography-mass spectrometry conditions

[0043] Liquid phase parameters:

[0044] Chromatographic column: ACQUITY BEH C18 column (100 mm × 2.1 mm, 1.7 μm)

[0045] Column temperature: 35 °C;

[0046] Flow rate: 0.2 mL / min;

[0047] Injection volume: 2 μL;

[0048] Mobile phase A: 0.1% formic acid in water;

[0049] Mobile phase B: acetonitrile;

[0050] The gradient elution procedure is as follows:

[0051] 0min→20min, 95% mobile phase A, 5% mobile phase B, linear change to 75% mobile phase A, 25% mobile phase B;

[0052] 20min→25min, 75% mobile phase A, 25% mobile phase B, linear change to 10% mobile phase A, 90% mobile phase B;

[0053] 25 min → 26 min, 10% mobile phase A, 90% mobile phase B, linear change to 95% mobile phase A, 5% mobile phase B;

[0054] 26min→30min, 95% mobile phase A, 5% mobile phase B.

[0055] Mass spectrometry parameters:

[0056] The mass spectrometer was fully scanned in the positive ion conversion mode, with a mass range of m / z 50-1500; capillary voltage: 3000 V; drying gas flow rate: 8 L / min; fragmentation voltage: 160 V; drying gas temperature: 325°C; sheath gas temperature: 325°C; sheath gas flow rate: 11 L / min; and nebulizer gas pressure: 40 psi.

[0057] (3) Data processing and analysis

[0058] The raw data were imported into the metabolomics processing software for baseline filtering, peak identification, integration, retention time correction, peak alignment, and normalization to obtain a data matrix containing retention time, mass-to-charge ratio, and peak intensity information; then, a characteristic peak search library was performed to match the MS and MS / MS mass spectrum information with databases such as Metlin, KEGG, and PubChem. The MS mass error was set to less than 10 ppm, and metabolites were identified based on the secondary mass spectrum matching score; analysis of variance (ANOVA) was used to perform statistical analysis on the metabolites of samples from different groups, including principal component analysis (PCA) in the unsupervised mode and PLS-DA analysis in the supervised mode, and compounds with VIP>1, significant differences (p<0.05), and compound response value differences greater than 2 times (|FC|>2) were screened out, which were the differential metabolites between the groups; the differential compounds were imported, and the metabolic pathway enrichment and overall change analysis of the screened differential metabolites were performed through the KEGG database.

[0059] Result analysis:

[0060] Figure 1 The results of principal component analysis of Streptomyces freundii metabolites at different fermentation stages. The purpose is to analyze the overall metabolic characteristics of each sample. Samples at the same fermentation stage are relatively concentrated, and samples at different fermentation stages are relatively far away, with no outliers, indicating that the principal component analysis model can well reflect the data characteristics, and there are obvious differences in the metabolites of samples at different fermentation stages.

[0061] Taking fermentation for 48 h as the control group, there were significant changes in the contents of 251 differential metabolites after 120 h of fermentation compared with 48 h of fermentation, of which 200 were significantly up-regulated and 51 were significantly down-regulated; there were significant changes in the contents of 244 differential metabolites after 168 h of fermentation compared with 48 h of fermentation, of which 204 were significantly up-regulated and 40 were significantly down-regulated; there were significant changes in the contents of 245 differential metabolites after 240 h of fermentation compared with 48 h of fermentation, of which 110 were significantly up-regulated and 135 were significantly down-regulated.

[0062] A total of 84 differential metabolites common to the four different fermentation time periods were screened, and the results are listed in Table 1. Among them, 46 metabolites were directly involved in metabolic pathways such as TCA cycle, riboflavin metabolism, galactose metabolism, synthesis and degradation of fatty acids, and biosynthesis of various amino acids, and their contents changed significantly during the entire fermentation process.

[0063] Table 1 Summary of the common differential compounds in the four periods

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070] In order to more intuitively display the changing trends of differential metabolites in different groups, a cluster analysis heat map of common differential metabolites at different fermentation times was drawn, such as Figure 2 As shown in the figure. The color strips in the figure represent the abundance of metabolites in each sample, red represents high abundance, and blue represents low abundance. As can be seen from the figure, with the increase of fermentation time, the contents of 42 metabolites such as thymosin, sphingosine, spermidine, riboflavin and oxalosuccinic acid continued to increase, and the contents of 24 metabolites such as xanthate, thiamine monophosphate, thiamine acetate, stearamide and sn-glycero-3-phosphocholine continued to decrease. In addition, the contents of 10 metabolites including sulfoacetaldehyde, stearic acid, palmitamide, MG (0:0 / 20:1(11Z) / 0:0) and LysoPE (0:0 / 16:0) increased during the 48-168 h of fermentation and then decreased; the contents of 6 substances including 3-oxobutyryl-CoA, thiobenzamide S,S-dioxide, PS (18:0 / 20:0), LysoPE (0:0 / 20:1(11Z)), isovalerylglutamate and enterochelin increased during the 48-120 h of fermentation and then decreased; the content of 2-hydroxy-3-oxosuccinic acid increased during the 48-120 h of fermentation, decreased during the 120-168 h, and increased during the 168-240 h; the content of dimethylallyl pyrophosphate decreased during the 48-120 h of fermentation and then increased.

[0071] According to the KEGG database, pathway analysis of the differential metabolites revealed that the differential metabolites were mainly related to metabolic pathways such as glycerophospholipid metabolism, TCA cycle, pantothenic acid and coenzyme A biosynthesis, riboflavin metabolism, thiamine metabolism, biotin metabolism, sphingolipid metabolism, starch and sucrose metabolism, and various amino acid metabolism. Among them, compared with fermentation for 48 h, glycerophospholipid metabolism, valine, leucine and isoleucine biosynthesis, TCA cycle, pantothenic acid and coenzyme A biosynthesis, riboflavin metabolism, and thiamine metabolism were significantly changed at 120 h of fermentation; compared with fermentation for 48 h, metabolic pathways such as biotin metabolism, TCA cycle, pantothenic acid and coenzyme A biosynthesis, riboflavin metabolism, sphingolipid metabolism, and histidine metabolism were significantly changed at 168 h of fermentation; compared with fermentation for 48 h, metabolic pathways such as thiamine metabolism, TCA cycle, starch and sucrose metabolism, and D-amino acid metabolism were significantly changed at 240 h of fermentation ( Figure 3-Figure 5 ).

[0072] As the core link of cell energy metabolism, TCA cycle converts chemical energy in organic matter into ATP through a series of redox reactions, providing a large amount of energy for life activities such as growth, reproduction and synthesis of secondary metabolites of Streptomyces. Glycerophospholipids are one of the main components of cell membranes. Through glycerophospholipid metabolism, Streptomyces can synthesize and decompose phospholipids to maintain the integrity, fluidity and permeability of cell membranes, thereby ensuring the normal transport of substances inside and outside cells and the normal physiological functions such as cell signal transduction. Sphingolipids are another important lipid component of cell membranes, which participate in the construction and functional regulation of cell membranes, and work together with glycerophospholipids to maintain the structure and characteristics of cell membranes, and play a synergistic role in cell signal transduction, material transport and other processes. Some sphingolipid metabolites can act as cell signaling molecules, participate in the intracellular signal transduction process, regulate gene expression, cell proliferation, differentiation and other physiological activities, and thus affect the growth, development and metabolic regulation of Streptomyces. Riboflavin is the precursor of flavin coenzyme, thiamine is the precursor of thiamine pyrophosphate, and biotin is the precursor of biotin coenzyme. These coenzymes participate in a variety of metabolic reactions in cells and are essential for maintaining the normal growth and metabolism of Streptomyces. Amino acids play an important role in various physiological processes, especially branched-chain amino acids (BCAA) - leucine, isoleucine and valine, which provide energy through the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain α-ketoacid dehydrogenase (BCKDH) pathways. In addition, amino acid metabolism can also produce some special modification groups for structural modification of secondary metabolites, such as glycosyl modules.

[0073] As fermentation continues, the energy required increases, so the content of citric acid, pantothenic acid, sphingosine, L-leucine, L-isoleucine, L-valine, etc. is significantly increased, and a series of enzymatic reactions in the cycle provide a large amount of energy for cell activities. At the same time, as the fermentation time increases, at the end of the fermentation period (240h), the number of bacteria increases, and the carbon and nitrogen sources required for growth are exhausted, so the metabolic activity begins to weaken, and more and more metabolites such as N, N-diethylphenylacetamide, pyridoxamine, stearamide, D-glucosamine, dTMP, etc. begin to decrease.

[0074] The above describes the embodiments of the present invention in detail. However, the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A method for analyzing differential metabolites of Streptomyces freundii at different fermentation stages, characterized in that: The steps include: 1) Intracellular metabolite processing: ①Cell collection and quenching The fermentation broth of Streptomyces freundii at different fermentation periods was centrifuged to remove most of the solid impurities in the culture medium, the cell pellet was washed and then added with a solution quencher, quickly mixed by vortexing and homogenizing, incubated under low temperature conditions, and quickly filtered by vacuum filtration to collect solid samples; ② Metabolite extraction The treated solid sample is placed in a centrifuge tube, and the metabolite extract and internal standard are added. After vortexing, the sample is placed in ice cubes for ultrasonic extraction of intracellular metabolites. After the ultrasonic extraction, the sample is centrifuged. After the centrifugation, the same volume of extract is added to the precipitate obtained after the centrifugation, the sample is placed on ice cubes for ultrasonic extraction again. After the ultrasonic extraction, the sample is centrifuged, and the two supernatants are combined. After centrifugation, the supernatant is taken out and passed through a membrane into a sample injection vial for determination by liquid chromatography-mass spectrometry. 2) Determination by liquid chromatography-mass spectrometry; 3) Data processing and analysis: The data obtained by the test in step 2) were imported into the metabolomics processing software for baseline filtering, peak identification, integration, retention time correction, peak alignment, and normalization to obtain a data matrix containing retention time, mass-to-charge ratio, and peak intensity information; then, a characteristic peak search library was performed to match the MS and MS / MS mass spectrum information with the Metlin, KEGG, and PubChem databases, and the MS mass error was set to less than 10 ppm. At the same time, metabolites were identified based on the secondary mass spectrum matching score; variance analysis was used to perform statistical analysis on the metabolites of samples in different groups, including principal component analysis in an unsupervised mode and PLS-DA analysis in a supervised mode, and compounds with VIP>1, significant differences p<0.05, and compound response value differences greater than 2 times were screened out, which were the differential metabolites between groups; metabolic pathway enrichment and overall change analysis were performed on the screened differential metabolites through the KEGG database.

2. The method for analyzing differential metabolites of Streptomyces freundii at different fermentation periods according to claim 1, characterized in that: The operating conditions of the liquid chromatography-mass spectrometry method in step 2) are as follows: liquid phase parameters: chromatographic column: ACQUITY BEH C18 column, 2.1 mm × 100 mm, 1.7 μm; column temperature: 35 ° C; flow rate: 0.2 mL / min; injection volume: 2 μL; mobile phase A: 0.1% formic acid water; mobile phase B: acetonitrile; gradient elution program is as follows: 0-20 min, the volume fraction of mobile phase A is reduced from 95% to 75%, and the volume fraction of mobile phase B is increased from 5% to 25%; 20-25 min, the volume fraction of mobile phase A is reduced from 75% to 10%, and the volume fraction of mobile phase B is increased from 25% to 90%; At 25-26 min, the volume fraction of mobile phase A increased from 10% to 95%, and the volume fraction of mobile phase B decreased from 90% to 5%. At 26-30 min, the volume fraction of mobile phase A was 95%, and the volume fraction of mobile phase B was 5%. Mass spectrometry parameters: The mass spectrometer is in full scan mode in the positive ion conversion mode, with a mass range of m / z 50 to 1500; capillary voltage: 3000 V; drying gas flow rate: 8 L / min; fragmentation voltage: 160 V; drying gas temperature: 325°C; sheath gas temperature: 325°C; sheath gas flow rate: 11 L / min; nebulizer gas pressure: 40 psi.

3. The method for analyzing differential metabolites of Streptomyces freundii at different fermentation periods according to claim 1, characterized in that: The specific method of cell collection and quenching in step 1) is as follows: take the fermentation broth of Streptomyces freundii at different fermentation periods, centrifuge at 10000-14000rpm at 4°C for 5min to remove most of the solid impurities in the culture medium; discard the supernatant, wash the cell pellet twice with PBS buffer, then wash it once with deionized ultrapure water, add a 60% methanol solution quencher precooled at -80°C for 24h, vortex for 1min, and then incubate at 0-5°C for 5min, and then quickly filter the mixed solution by vacuum filtration to collect the solid sample.

4. The method for analyzing differential metabolites of Streptomyces freundii at different fermentation periods according to claim 3, characterized in that: Each 1 mol / L of the PBS buffer contains 8.00 g of NaCl, 0.20 g of KCl, 1.42 g of Na2HPO4, and 0.27 g of KH2PO4.

5. The method for analyzing differential metabolites of Streptomyces freundii at different fermentation periods according to claim 1, characterized in that: The specific method for extracting metabolites in step 1) is to place the treated solid sample in a centrifuge tube, add a mixed extract of methanol and water with a volume ratio of 1:1 and pre-cooled at -80°C for 24h and 0.02mg / mL internal standard L-2-chlorophenylalanine, vortex for 1min to mix evenly, and then place it in ice for ultrasonication for 20-40min to extract intracellular metabolites; after the end of ultrasonication, centrifuge at 8000-12000rpm0-5°C for 5-20min, add the extract to the precipitate and place it in ice for ultrasonication for 15-45min for secondary extraction, centrifuge at 8000-12000rpm0-5°C for 5-15min after the end of ultrasonication, combine the two supernatants, vortex for 1min to mix evenly, and then centrifuge at 8000-12000rpm4°C for 1-10min, take the supernatant, filter it into an injection vial with a 0.22μm membrane, and determine it by liquid chromatography-mass spectrometry.

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