A method for detecting phosphorus-containing natural products in fermentation broth

Abstract

This invention belongs to the field of nuclear magnetic resonance (NMR) detection technology, specifically relating to a method for detecting phosphorus-containing natural products in fermentation broth. The detection method includes: performing two-dimensional NMR spectroscopy on the fermentation broth to be tested, setting the J-coupling parameter to 20-30 Hz and 500-600 Hz respectively; when the J-coupling parameter is 20-30 Hz, the obtained... 1 H- 31 The signal in the p-NMR spectrum indicates a carbon-phosphorus natural product. 1 H- 31 P signal; when the J coupling parameter is 500~600Hz, the obtained 1 H- 31 The signal in the p-NMR spectrum indicates a hydrogen-phosphorus natural product. 1 H- 31 P signal; based on the obtained 1 H- 31 The p-NMR spectrum is used to determine whether the phosphorus-containing natural products in the fermentation broth are carbon-phosphorus or hydrogen-phosphorus natural products. This invention can directly detect carbon-phosphorus natural products in complex fermentation broths, providing a convenient method for rapid detection and identification of carbon-phosphorus natural products.

Description

Technical Field

[0001] This invention belongs to the field of nuclear magnetic resonance detection technology, specifically relating to a method for detecting phosphorus-containing natural products in fermentation broth. Background Technology

[0002] Carbon and phosphorus natural products are slow to discover, have complex biosynthetic pathways with numerous branches, and involve reversible multi-step reactions, making it difficult to guarantee the supply of precursors and ultimately resulting in insufficient yields of the final product. Furthermore, carbon and phosphorus natural products lack specific unsaturated bonds and characteristic absorption wavelengths, making them difficult to detect effectively using traditional chromatographic techniques.

[0003] Nuclear Magnetic Resonance (NMR), a powerful analytical technique, has been widely applied in the study of small molecule compounds. Its principle is as follows: In a strong magnetic field, the nuclei and electrons of certain elements, inherently magnetic, are split into two or more quantized energy levels. Absorption of electromagnetic radiation of appropriate frequencies can induce transitions between these magnetically induced energy levels. In a magnetic field, these nuclear magnetic molecules or nuclei absorb the energy difference between the two energy levels transitioning from a lower to a higher energy state, producing a resonance spectrum. This spectrum can be used to determine the number, type, and relative positions of certain atoms within a molecule. Compared to other structural analysis methods, NMR can also provide a variety of information about the functional groups, main chain, and chemical environment of the atomic nuclei in a target compound.

[0004] The main characteristics of nuclear magnetic resonance (NMR) include the following: Nuclear fingerprinting: Different atomic nuclei possess characteristic resonance frequencies under specific magnetic fields, directly corresponding to the chemical environment of the molecule. Chemical shift (δ): Reflects the electronic environment of the atomic nucleus (such as functional group type, adjacent groups, etc.), and is the basis for structural analysis. Coupling parameter (J): Reflects the interaction between adjacent atomic nuclei (transferred through chemical bonds), used to infer stereochemistry (such as cis-trans isomerism) and conformational information. Integral area: The signal peak area is proportional to the number of atomic nuclei and can be used to quantitatively analyze the proportion of hydrogen atoms in each functional group. NMR analysis is suitable for detecting small molecules, and samples can usually be completely recovered after testing, making it suitable for analyzing precious or small-volume samples (such as natural products and drug metabolites). It can detect dynamic processes such as conformational changes, chemical exchanges, and intermolecular interactions in solutions, with timescales covering milliseconds to seconds. Temperature-dependent experiments can be used to study energy barriers, reaction kinetics, and molecular stability.

[0005] The main detection methods for carbon and phosphorus natural products include GC-MS, LC-MS / MS, and NMR. Liquid NMR spectroscopy, with its unique advantage of providing atomic-resolution structural information and dynamic reaction details, has become an indispensable core tool in the study of carbon and phosphorus natural products. It can be used not only for the structural identification of the final products but also for in-depth research into reaction mechanisms, metabolic pathways, and interactions. For novel and complex carbon and phosphorus natural products, it is difficult to determine their precise structures using techniques such as mass spectrometry alone; liquid NMR plays a decisive role in this regard.

[0006] Currently, the detection of phosphorus-containing substances primarily relies on one-dimensional P-spectroscopy and one-dimensional H-spectroscopy combined with mass spectrometry to detect and identify the products. After scanning the one-dimensional P-spectrum to obtain a P signal, the product is separated. The separated substances, with simpler compositions, are then analyzed using NMR spectra to determine whether they are novel carbon-phosphorus compounds. However, carbon-phosphorus compounds are highly polar, making the separation process complex, with a loss rate as high as 70% during purification. This complexity hinders the discovery of novel carbon-phosphorus natural products. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide a method for detecting phosphorus-containing natural products in fermentation broth. The detection method provided by this invention can directly detect whether the phosphorus-containing natural products in the fermentation broth contain carbon-phosphorus natural products, and then determine whether novel phosphorus-containing substances have been generated by comparing with a database.

[0008] This invention provides a method for detecting phosphorus-containing natural products in fermentation broth, comprising the following steps: The fermentation broth was subjected to two-dimensional nuclear magnetic resonance spectroscopy, with J coupling parameters set to 20–30 Hz and 500–600 Hz, respectively. 1 H- 31 P NMR spectrum; When the J coupling parameter is 20~30Hz, the obtained 1 H- 31 The signal in the p-NMR spectrum indicates a carbon-phosphorus natural product. 1 H- 31 P signal; when the J coupling parameter is 500~600Hz, the obtained 1 H- 31 The signal in the p-NMR spectrum indicates a hydrogen-phosphorus natural product. 1 H- 31 P signal; According to the obtained 1 H- 31 The P-NMR spectrum was used to determine whether the phosphorus-containing natural products in the fermentation broth were carbon-phosphorus or hydrogen-phosphorus natural products.

[0009] Preferably, the parameters for the two-dimensional nuclear magnetic resonance spectroscopy detection also include: temperature: 298K, 1H pulse width 200ms, 15N pulse width 7ms, hydrogen dimension spectral width: 1.5~11ppm, phosphorus dimension spectral width: 1.5~36ppm, and number of scans: NS 8.

[0010] Preferably, the J coupling parameters are set to 25Hz and 550Hz respectively.

[0011] Preferably, if the phosphorus-containing natural product in the fermentation broth to be tested is a carbon-phosphorus natural product, the detection method further includes: comparing the chemical shift with that of known carbon-phosphorus natural products in the database to determine whether a novel compound is generated.

[0012] Preferably, the method for preparing the fermentation broth to be tested includes the following steps: Streptomyces was cultured, and the resulting culture broth was separated into solid and liquid components to obtain the supernatant. The water in the supernatant was removed, and the solid was reconstituted to obtain the fermentation broth to be tested.

[0013] Preferably, before culturing the Streptomyces, the method further includes feeding the Streptomyces with an intermediate substance of a carbon-phosphorus natural product, wherein the intermediate substance is 2-AEP.

[0014] Preferably, the culture time is 7 days, and the pH value of the culture medium system during the culture process is 7.2.

[0015] Preferably, the solvent for resolution is water, and the ratio of the solid to water is 0.25g:500μL.

[0016] Preferably, the process after reconstitution further includes centrifugation to remove insoluble impurities; the centrifugation speed is 12000 rpm and the time is 10 min.

[0017] Preferably, the process after resolution further includes: subjecting the resolution sample to alcohol precipitation, removing water from the resulting liquid, and then resolution.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for detecting phosphorus-containing natural products in fermentation broth, comprising the following steps: performing two-dimensional nuclear magnetic resonance spectroscopy on the fermentation broth to be tested, setting the J coupling parameters to 20~30Hz and 500~600Hz respectively, to obtain... 1 H- 31 P-NMR spectrum; obtained when J coupling parameter is 20~30Hz. 1 H- 31 The signal in the p-NMR spectrum indicates a carbon-phosphorus natural product. 1 H- 31P signal; when the J coupling parameter is 500~600Hz, the obtained 1 H- 31 The signal in the p-NMR spectrum indicates a hydrogen-phosphorus natural product. 1 H- 31 P signal; based on the obtained 1 H- 31 The P-NMR spectrum was used to determine whether the phosphorus-containing natural products in the fermentation broth were carbon-phosphorus or hydrogen-phosphorus natural products.

[0019] This invention utilizes two-dimensional spectra to directly scan complex fermentation broth components. The scanning is achieved by adjusting the J-coupling parameter, specifically setting it to 20-30 Hz. 1 H- 31 P HSQC (heteronuclear single quantum relation) was used to obtain relevant signal parameters of carbon and phosphorus natural products; the J coupling parameter was set to scan at 500~600Hz. 1 H- 31 P HSQC was used to obtain signals related to hydrogen and phosphorus natural products. After identifying the signals related to carbon and phosphorus natural products, the chemical shift information of known carbon and phosphorus natural products in the database can be compared to determine whether the signal appearing in the two-dimensional spectrum is a known product. If an unknown product is found, further separation can proceed; otherwise, the process can be abandoned. The detection method provided by this invention can directly detect whether phosphorus-containing natural products in fermentation broth contain carbon and phosphorus natural products. Furthermore, by comparing with a database, it can be determined whether novel phosphorus-containing substances have been generated. The method is simple and facilitates the discovery of novel carbon and phosphorus natural products.

[0020] In this embodiment of the invention, a Bruker nuclear magnetic resonance spectrometer with a room-temperature probe is used to detect carbon and phosphorus natural products in solution. By feeding the intermediate substance 2-AEP that synthesizes carbon and phosphorus natural products, the nuclear magnetic resonance spectrometer is used to detect and determine whether novel carbon and phosphorus natural products are generated. By modifying the operating parameters, the P atom signals under different coupling conditions are scanned and detected to determine the type of product.

[0021] Fermentation products are complex in composition, and most detection methods require pretreatment of the fermentation broth before detection. This invention, however, can directly detect carbon and phosphorus natural products within the complex components of fermentation broth, providing a convenient method for rapid detection and identification of these products. This invention utilizes in-situ NMR detection of fermentation products, improving the efficiency of determining the novelty of carbon and phosphorus compounds. 31 Phosphorus NMR is a "privileged" tool for studying phosphorus-containing natural products. Phosphorus nuclei (P0 NMR) 31 Phosphorus (P) has 100% natural abundance and high sensitivity, with a wide chemical shift range (up to hundreds of ppm) and is extremely sensitive to the local chemical environment of phosphorus atoms (such as phosphate esters, phosphonates, oxidation states, and coordination states). Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a pulse sequence diagram; Figure 2 The carbon and phosphorus natural product standard 2-AEP in Example 1 1 H- 31 P NMR spectrum; Figure 3 The carbon and phosphorus natural products in the fermentation broth screened in Example 1 1 H- 31 P NMR spectrum; Figure 4 The carbon and phosphorus natural products in the supernatant after alcohol precipitation of the fermentation sample in Example 1 1 H- 31 P NMR spectrum; Figure 5 The hydrogen and phosphorus substances in the fermentation broth screened in Example 1 1 H- 31 P NMR spectrum. Detailed Implementation

[0024] This invention provides a method for detecting phosphorus-containing natural products in fermentation broth, comprising the following steps: The fermentation broth was subjected to two-dimensional nuclear magnetic resonance spectroscopy, with J coupling parameters set to 20–30 Hz and 500–600 Hz, respectively. 1 H- 31 P NMR spectrum; When the J coupling parameter is 20~30Hz, the obtained 1 H- 31 The signal in the p-NMR spectrum indicates a carbon-phosphorus natural product. 1 H- 31 P signal; when the J coupling parameter is 500~600Hz, the obtained 1 H- 31 The signal in the p-NMR spectrum indicates a hydrogen-phosphorus natural product. 1 H- 31 P signal; According to the obtained 1 H- 31 The P-NMR spectrum was used to determine whether the phosphorus-containing natural products in the fermentation broth were carbon-phosphorus or hydrogen-phosphorus natural products.

[0025] Unless otherwise specified, all materials and equipment used in this invention are commercially available products in the field.

[0026] This invention involves performing two-dimensional nuclear magnetic resonance spectroscopy on the fermentation broth to be tested. Figure 1 The diagram shows a pulse sequence, where τ = 1 / (4J). Changing the J coupling parameter will produce different pulses.

[0027] In this invention, the fermentation broth is preferably a fermentation broth collected from Streptomyces fermentation culture, containing a complex mixture of components. The fermentation broth is preferably an enriched broth, specifically obtained by rotary evaporation to remove the water naturally present in the fermentation broth and then reconstituted with water, thereby enriching the water-soluble components in the fermentation broth.

[0028] In this invention, the method for preparing the fermentation broth to be tested preferably includes the following steps: Streptomyces were cultured, and the resulting mixture was separated into solid and liquid components to obtain the supernatant. The water in the supernatant was removed, and the solid was reconstituted to obtain the fermentation broth to be tested.

[0029] In this invention, the process of culturing Streptomyces preferably includes feeding Streptomyces with an intermediate substance of carbon and phosphorus natural products, wherein the intermediate substance is preferably 2-aminoethylphosphonic acid (2-AEP), and 2-AEP is an intermediate substance in the pathway of Streptomyces synthesis of carbon and phosphorus natural products. The addition of 2-AEP promotes the forward synthesis of carbon and phosphorus natural products by Streptomyces.

[0030] In this invention, the culture time is preferably 7 days, and the pH value of the culture medium during the culture process is preferably 7.2.

[0031] In this invention, the solvent for resolution is preferably water, and the ratio of the solid to water is preferably 0.25g:500μL.

[0032] In this invention, the process of reconstitution preferably includes centrifugation to remove insoluble impurities; the centrifugation speed is preferably 12000 rpm and the centrifugation time is preferably 10 min.

[0033] In this invention, the process of redissolving preferably further includes: subjecting the redissolved sample to alcohol precipitation, removing water from the resulting liquid, and then redissolving it again. The alcohol precipitation preferably uses ethanol, and the volume fraction of ethanol in the system during the precipitation is preferably 50%.

[0034] In this invention, the two-dimensional nuclear magnetic resonance spectroscopy detection is... 1 H- 31 P HSQC nuclear magnetic resonance spectroscopy detection, observed by setting different J coupling parameters 31 The situation of atoms connected to P.

[0035] In this invention, the preferred parameters for the two-dimensional nuclear magnetic resonance spectroscopy detection include: temperature: 298K, 1H pulse width 200ms, 15N pulse width 7ms, hydrogen dimension spectral width: 1.5~11ppm, phosphorus dimension spectral width: 1.5~36ppm, number of scans: NS 8, and scan time: 1 h 17 min 8 s.

[0036] In this invention, the J coupling parameters are preferably set to 25Hz and 550Hz respectively.

[0037] The present invention is based on the obtained 1 H- 31 The P-NMR spectrum was used to determine whether the phosphorus-containing natural products in the fermentation broth were carbon-phosphorus or hydrogen-phosphorus natural products.

[0038] In this invention, if the phosphorus-containing natural product in the fermentation broth to be tested is a carbon-phosphorus natural product, the detection method preferably further includes: comparing the chemical shift with that of known carbon-phosphorus natural products in the database to determine whether a novel compound is generated.

[0039] This invention utilizes liquid nuclear magnetic resonance spectroscopy to identify corresponding carbon-phosphorus or hydrogen-phosphorus natural products in complex fermentation broths. This overcomes the limitations of conventional detection methods that require complex separation of the fermentation broth before detection, enabling direct determination of the presence of novel phosphorus-containing substances from the fermentation broth. Furthermore, this invention scans and detects carbon-phosphorus natural products in complex compounds without isotopic labeling and provides a preliminary assessment of the atomic structure surrounding the phosphorus atom.

[0040] To further illustrate the present invention, the method for detecting phosphorus-containing natural products in fermentation broth provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0041] In this embodiment of the invention, after fermentation of an unknown Streptomyces, nuclear magnetic resonance (NMR) technology is used to detect whether it is a strain capable of producing carbon-phosphorus natural products, thereby screening for Streptomyces strains capable of producing carbon-phosphorus natural products. Specifically, wild-type Streptomyces strains 1 and 2 both showed phosphorus-containing natural products in their fermentation broth as detected by one-dimensional P-spectrum analysis; further determination is needed to determine whether these are carbon-phosphorus compounds or hydrogen-phosphorus compounds.

[0042] The methods for obtaining wild-type Streptomyces strain 1 and strain 2 are as follows: Sludge samples were collected from Fisherman's Wharf, Haiwan Town, Fengxian District, Shanghai. Sampling depth was 5–10 cm below the surface, with approximately 20 g of sample taken from each sampling point. The samples were placed in sterile 50 mL centrifuge tubes and transported at low temperature (4℃), with subsequent processing occurring within 24 hours. The samples were first air-dried at room temperature for 7 days. One g of the air-dried sample was then added to 9 mL of sterile physiological saline (0.85 wt% NaCl solution) and shaken at 200 rpm for 30 min to fully disperse the microbial community. The samples were then serially diluted 10-fold to 10... -6 spare.

[0043] The diluted solution (100 μL) was plated separately onto Starch-Casein agar (SCA) selective medium plates. Cycloheximide (50 μg / mL) was added to the medium to inhibit fungal growth, and nalidixic acid (20 μg / mL) was added to inhibit Gram-negative bacteria. The plates were incubated upside down in a constant temperature incubator at 2°C for 7–21 days, and colony growth was observed daily. Suspected Streptomyces colonies were initially screened based on colony morphology characteristics (aerial hyphae formation, powdery or fluffy surface, pigment production), and streak purification was performed at least three times to obtain pure cultures.

[0044] Example 1 1. Dissolve the 2-aminoethylphosphonic acid (2-AEP) standard sample directly in 500 μL of water with a D2O volume fraction of 10% (pH 7.0) to a final concentration of 0.5 mg / mL.

[0045] Two-dimensional nuclear magnetic resonance (NMR) detection was performed on the 2-AEP standard solution with the following parameters: temperature: 298 K, 1H pulse width: 200 ms, 15N pulse width: 7 ms, hydrogen spectrum width: 1.5~11 ppm, phosphorus spectrum width: 1.5~36 ppm, number of scans: NS 8, scan time: 1 h 17 min 8 s, and J coupling parameter set to 20 Hz.

[0046] Figure 2 2-AEP, a standard for carbon and phosphorus natural products 1 H- 31 The P NMR spectrum is compared with the chemical shifts of P in the database. The substance is identified by comparing the signals on the spectrum with the parameters (H, C, P chemical shifts) in the database.

[0047] 2. Preparation of Streptomyces fermentation samples: (1) Streaking the frozen wild-type Streptomyces strain 1 on MS solid medium plates, culturing at 30°C for 7 days, scraping the generated spores with a sterilized cotton swab and storing them in 25wt% glycerol, then freezing at -80°C. (2) Prepare a 2-AEP stock solution with a concentration of 3 g / 27 mL; (3) Take 50 μL of frozen spore solution and inoculate it into 167 mL of ISP2 medium (pH 7.2). Add 1.5 mL of 2-AEP stock solution to the medium to make the final concentration of 2-AEP 1 mg / mL, and culture in a shake flask at 30℃; (4) On the third day of cultivation, take out 1.67 mL of each bottle of fermentation broth, measure the pH value and add 1 M NaOH solution or HCl solution to keep the pH value at 7.2. Add the solution to the fermentation broth at a 100-fold scale and continue cultivation for a total of 7 days. (5) Centrifuge and collect the supernatant to obtain the Streptomyces fermentation broth; (6) The collected supernatant was evaporated by rotary evaporator to remove the water in the fermentation broth. The evaporated sample was transferred to an EP tube by a spatula and stored at 4°C. (7) Take about 0.25g of fermentation sample and dissolve it in 500 μL of water with a volume fraction of 10% D2O (pH 7.0). After dissolving completely, centrifuge at 12000 rpm for 10 min to remove impurities that are not easily dissolved. Take out the supernatant liquid sample and put it into an NMR tube.

[0048] The supernatant liquid sample (fermentation broth sample 1) was subjected to two-dimensional nuclear magnetic resonance detection with the same parameters as sample 1.

[0049] Figure 3 To screen the natural carbon and phosphorus products in the fermentation broth 1 H- 31 P NMR spectrum; the phosphorus-containing natural products in the fermentation broth are carbon-phosphorus natural products, and by comparing the chemical shifts with those of known carbon-phosphorus natural products in the database, many previously undiscovered substances were observed, proving that the fermentation broth contains a variety of novel carbon-phosphorus natural products worth exploring.

[0050] 3. Preparation of carbon and phosphorus natural product samples from the supernatant after alcohol precipitation of fermentation samples: (1) Streaking the frozen wild-type Streptomyces strain 1 on MS solid medium plates, culturing at 30°C for 7 days, scraping the generated spores with a sterilized cotton swab and storing them in 25wt% glycerol, then freezing at -80°C. (2) Prepare a 2-AEP stock solution with a concentration of 3 g / 27 mL; (3) Take 50 μL of frozen spore solution and inoculate it into 167 mL of ISP2 medium (pH 7.2). Add 1.5 mL of 2-AEP stock solution to the medium to make the final concentration of 2-AEP 1 mg / mL, and culture in a shake flask at 30℃; (4) On the third day of cultivation, take out 1.67 mL of each bottle of fermentation broth, measure the pH value and add 1 M NaOH solution or HCl solution to keep the pH value at 7.2. Add the solution to the fermentation broth at a 100-fold scale and continue cultivation for a total of 7 days. (5) Centrifuge and collect the supernatant to obtain the Streptomyces fermentation broth; (6) The collected supernatant was evaporated by rotary evaporator to remove the water in the fermentation broth. The evaporated sample was transferred to an EP tube by a spatula and stored at 4°C. (7) Take 3 g of the above fermentation sample, first redissolve the sample with 6 mL of ultrapure water, and after fully dissolving, add anhydrous ethanol to make the final volume concentration of ethanol 50%. After mixing thoroughly, place it in a -20℃ refrigerator and let it stand for 2 h. (8) Centrifuge at 12000 rpm for 30 min to remove the precipitate; (9) After the supernatant sample is dried by rotary evaporation, it is reconstituted with water containing 10% D2O by volume and then loaded into an NMR tube.

[0051] The reconstituted liquid sample was subjected to two-dimensional nuclear magnetic resonance detection with the same parameters as in step 1.

[0052] Figure 4 The carbon and phosphorus natural products in the supernatant after alcohol precipitation of fermentation samples 1 H- 31 Using P NMR spectra, some substances that are poorly soluble in ethanol at this concentration, as well as some more polar products, were removed by alcohol precipitation to attempt crude separation of substances and trace the whereabouts of carbon and phosphorus natural products.

[0053] 4. Preparation of hydrogen-phosphorus fermentation samples: (1) Take the bacterial culture of wild-type Streptomyces strain 2 stored at -80℃ and inoculate it into ISP2 liquid medium and culture it in a shake flask for 7 days; (2) Centrifuge and collect the supernatant to obtain the Streptomyces fermentation broth; (3) The collected supernatant was evaporated by rotary evaporator to remove the water in the fermentation liquid. The evaporated sample was transferred to an EP tube by a spatula and stored at 4°C. (4) Resuspend the fermentation sample in a small amount of water, mix thoroughly to dissolve, centrifuge, and take the supernatant; (5) Take 450 μL of the supernatant obtained in the previous step, add 50 μL of D2O (pH 7.2), mix thoroughly, and then load it into an NMR tube for scanning.

[0054] The mixed liquid sample (fermentation broth sample 2) was subjected to two-dimensional nuclear magnetic resonance detection with the same parameters as 1. No carbon and phosphorus signals were found. The J coupling parameter was changed to 500 Hz.

[0055] Figure 5To screen for hydrogen and phosphorus substances in the fermentation broth 1 H- 31 P NMR spectrum.

[0056] This invention utilizes the design 1 H- 31 P HSQC investigates phosphorus-containing natural products in fermentation broth and compares them with known natural products in a database. 31 The chemical shift of P was used to determine whether the product was a carbon-phosphorus or hydrogen-phosphorus natural product. The scan was performed under J coupling parameters of 20–30 Hz. 1 H- 31 The signal in the P HSQC spectrum is from carbon and phosphorus natural products. 1 H- 31 The P signal was obtained by scanning under J coupling parameters of 500~600 Hz. 1 H- 31 The signal shown in the P HSQC spectrum is a hydrogen-phosphorus natural product.

[0057] This invention first collected 2-AEP, a standard of carbon and phosphorus natural products. 1 H- 31 P HSQC NMR spectrum ( Figure 2 The chemical shift of P in the spectrum was compared with that in the database to confirm that the substance could be identified by comparing the signal on the spectrum with the parameters in the database. Next, the J coupling parameter was set to 500 Hz to scan fermentation broth sample 1, and the spectrum was obtained. Figure 3 The fermentation broth products contained phosphorus-containing natural products, which were carbon-phosphorus natural products. A comparison with the chemical shifts of known carbon-phosphorus natural products in the database revealed many previously undiscovered substances, indicating the presence of several novel carbon-phosphorus natural products worthy of further exploration in the fermentation broth. The spectrum of fermentation sample 2 was obtained by scanning with a J-coupling parameter of 20 Hz. Figure 5 The study observed numerous signal peaks characteristic of hydrogen-phosphorus natural substances, which, upon comparison with database information, can be defined as hydrogen-phosphorus natural products. The detection method of this invention provides data support for the necessity of subsequent separation and purification.

[0058] This invention utilizes liquid nuclear magnetic resonance (NMR) technology to develop a method for assisting in the discovery of novel carbon and phosphorus natural products, and explores the discovery process of these novel products. Example data illustrates that fermentation broth samples from certain *Streptomyces* strains (strain 1) contain a large number of previously undiscovered novel carbon and phosphorus natural products; while some *Streptomyces* strains (strain 2) can ferment to produce novel hydrogen-phosphorus natural products. The NMR method of this invention can be used to identify whether novel phosphorus-containing compounds are present in complex fermentation broths.

[0059] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for detecting phosphorus-containing natural products in fermentation broth, characterized in that, Includes the following steps: The fermentation broth was subjected to two-dimensional nuclear magnetic resonance spectroscopy, with J coupling parameters set to 20–30 Hz and 500–600 Hz, respectively. 1 H- 31 P NMR spectrum; When the J coupling parameter is 20~30Hz, the obtained 1 H- 31 The signal in the p-NMR spectrum indicates a carbon-phosphorus natural product. 1 H- 31 P signal; When the J coupling parameter is 500~600Hz, the obtained 1 H- 31 The signal in the p-NMR spectrum indicates a hydrogen-phosphorus natural product. 1 H- 31 P signal; According to the obtained 1 H- 31 The P-NMR spectrum was used to determine whether the phosphorus-containing natural products in the fermentation broth were carbon-phosphorus or hydrogen-phosphorus natural products.

2. The detection method according to claim 1, characterized in that, The parameters for the two-dimensional nuclear magnetic resonance spectroscopy detection also include: temperature: 298K, 1H pulse width 200ms, 15N pulse width 7ms, hydrogen dimension spectral width: 1.5~11ppm, phosphorus dimension spectral width: 1.5~36ppm, and number of scans: NS 8.

3. The detection method according to claim 1 or 2, characterized in that, Set the J coupling parameters to 25Hz and 550Hz respectively.

4. The detection method according to claim 1, characterized in that, If the phosphorus-containing natural product in the fermentation broth to be tested is a carbon-phosphorus natural product, the detection method further includes: comparing the chemical shift with that of known carbon-phosphorus natural products in the database to determine whether a novel compound is generated.

5. The detection method according to claim 1, characterized in that, The preparation method of the fermentation broth to be tested includes the following steps: Streptomyces was cultured, and the resulting culture broth was separated into solid and liquid components to obtain the supernatant. The water in the supernatant was removed, and the solid was reconstituted to obtain the fermentation broth to be tested.

6. The detection method according to claim 5, characterized in that, The process of culturing Streptomyces also includes feeding Streptomyces with an intermediate substance of carbon-phosphorus natural products, wherein the intermediate substance is 2-AEP.

7. The detection method according to claim 5, characterized in that, The culture time is 7 days, and the pH value of the culture medium system during the culture process is 7.

2.

8. The detection method according to claim 5, characterized in that, The solvent for resolution is water, and the ratio of the solid to water is 0.25g:500μL.

9. The detection method according to claim 8, characterized in that, The process of reconstitution also includes centrifugation to remove insoluble impurities; the centrifugation speed is 12000 rpm and the time is 10 min.

10. The detection method according to claim 8, characterized in that, The process of redissolution further includes: precipitating the redissoluted sample with alcohol, removing water from the resulting liquid, and then redissolutizing it again.

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