Method for detecting heparin precursor content and use thereof

Abstract

This invention provides a method for detecting heparin precursor content and its application. The method involves comparing the sample concentration with... 1 Characteristic hydrogen signal in H-NMR spectrum: peak area A and 360° pulse width θ 360 The product A×θ 360 Linear correlation is performed, and the method allows for rapid determination of heparin precursor concentration directly in the NMR sample without the need for an internal standard. Specifically, the method utilizes the heparin precursor 1H NMR spectrum (A×θ) established under different systems (e.g., D2O, D2O-H2O, or D2O-fermentation broth). 360 The linear relationship between the concentration of heparin precursor C and the concentration of heparin precursor in different systems was demonstrated, and the results showed high accuracy. This invention achieves efficient and rapid quantitative detection of heparin precursor, avoiding the complexity of sample processing and purification steps in traditional methods. This improves real-time monitoring capabilities during production, optimizes the efficiency and quality control of heparin production, and has significant application value in the industrial production of heparin.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for detecting heparosan content and application thereof, in particular to a method for detecting heparosan content in fermentation broth. BACKGROUND

[0002] Heparosan polysaccharide belongs to glycosaminoglycan, and is a disaccharide repeating unit alternately formed by glucuronic acid GlcUA and acetylglucosamine GlcNAc, and exists in large quantities in the capsules of various bacteria. At present, heparosan is mainly extracted from Escherichia coli K5. Heparosan has a polysaccharide skeleton structure similar to heparin / heparan sulfate, and can obtain non-animal source heparin or its analogs after appropriate modification. Therefore, heparosan becomes an ideal raw material for heparinase synthesis. The structural formula of heparosan is as follows:

[0003] .

[0004] On the one hand, heparosan has potential applications in ophthalmology, plastic surgery, dermatology and coating of medical devices; heparosan and heparosan derivatives are also high-quality biomaterials for gels and scaffolds used in tissue engineering and good candidates for drug delivery carriers; heparosan can also be made into a drug carrier to enhance the targeting of anticancer drugs, and has important application prospects in the field of pharmaceutical development. It has important application value and strategic significance to use microbial fermentation to obtain heparosan with controllable molecular weight efficiently and at low cost. However, in the process of heparosan biofermentation, the heparosan content in the fermentation broth is affected by a large amount of small molecules and substances such as acetic acid.

[0005] At present, there are few reports on the detection and quality control methods of heparosan content. The detection of heparosan content generally uses the sulfuric acid-carbazole method, which is suitable for determining the content of uronic acid in acidic polysaccharides. Acidic polysaccharides are hydrolyzed into glucuronic acid under the action of concentrated sulfuric acid, and react with carbazole in the presence of sulfuric acid to generate a purple red compound with carbonyl group, which has maximum absorption at a wavelength of 530 nm. Heparosan polysaccharide is composed of acetylglucosamine and glucuronic acid, and the percentage of glucuronic acid content is 45.56%. By establishing a standard curve, the content of heparosan in the sample can be detected according to the content of glucuronic acid.

[0006] However, the biggest problem of using the sulfuric acid-carbazole method to detect the content of heparosan is poor specificity, which is easily interfered by impurities (heparosan analogs, residual culture medium) in the reagent or sample, and has the disadvantages of complicated operation, harsh reaction reagents and reaction conditions, poor friendliness to personnel, poor reproducibility, many interference factors, poor specificity and low accuracy.

[0007] 1 H-NMR technology can effectively detect different chemical structures in samples. By distinguishing the chemical shifts of different structures, it can help identify heparin precursors and other impurities. Since the intensity of the NMR signal is proportional to the number of hydrogen atoms in the corresponding structure, this technology can be used not only for qualitative analysis but also for quantitative determination.

[0008] In quantitative nuclear magnetic resonance (NMR) experiments, if the analyte is an electrolyte, its conductivity increases with its concentration. However, this increased conductivity leads to a decrease in the signal-to-noise ratio, causing the relationship between the sample signal and concentration to become non-linear. To improve accuracy, an internal standard of known concentration (such as benzyl alcohol) is typically added. The concentration of the target substance is then calculated by integrating the signal and combining it with the concentration of the internal standard. However, heparin-like substances are biological macromolecules, and their... 1 The wide and complex distribution of H-NMR signals makes it difficult to find internal standards that do not overlap with the signals. Furthermore, internal standards may interact with heparin or impurities, affecting the quantitative results. Therefore, the quantification of heparin precursors using NMR technology remains challenging.

[0009] Controlling the content of heparin precursors has always been a key focus and challenge in their research, production, and utilization. Current methods for determining and detecting the content of heparin precursors have many shortcomings. Therefore, there is an urgent need to develop a method for controlling and detecting the content of heparin precursors to ensure the accuracy of detection results during fermentation preparation and application. Summary of the Invention

[0010] To address the shortcomings of existing technologies, the present invention aims to provide a method for detecting heparin precursor content and its application, particularly a method for detecting heparin precursor content in fermentation broth and its application. The method involves combining the sample concentration with... 1 The characteristic hydrogen signal in the H-NMR spectrum: peak area A and 360° pulse width θ 360 The product A×θ 360 Linear correlation is achieved, and the method allows for rapid determination of heparin precursor concentration directly in the NMR sample without the need for an internal standard. Specifically, the method utilizes the heparin precursor 1H NMR spectrum (A×θ) established under different systems (e.g., D2O, D2O-H2O, or D2O-fermentation broth). 360 The linear relationship between the concentration of heparin precursor C and the concentration of heparin precursor was successfully established, and the concentration of heparin precursor in different systems was determined with high accuracy. This invention enables efficient and rapid quantitative detection of heparin precursor, avoiding the complexity of sample processing and purification steps in traditional methods. This improves real-time monitoring capabilities during production, optimizes the efficiency and quality control of heparin production, and has significant application value in the industrial production of heparin.

[0011] To achieve the object of the present application, the present application adopts the following technical solutions:

[0012] In a first aspect, the present application provides a method for detecting the content of heparin precursors, the method comprising:

[0013] (S1) determining the 360° pulse width θ 360 and the peak area A of the characteristic hydrogen signal in the nuclear magnetic resonance hydrogen spectrum of the heparin precursor control solution and the heparin precursor test solution, respectively, and calculating the product A x θ 360 of the peak area A of the characteristic hydrogen signal in the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width θ 360 ;

[0014] (S2) applying the least square method to perform linear regression, and establishing the mathematical relationship between the product Y of the peak area A of the characteristic hydrogen signal in the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width θ 360 of the heparin precursor control solution and the concentration X of the heparin precursor control solution, Y = kX + b;

[0015] (S3) calculating the concentration of the heparin precursor in the heparin precursor sample solution to be measured according to the mathematical relationship of step (S2); wherein the calculation formula of the concentration of the heparin precursor in the heparin precursor sample solution to be measured is:

[0016]

[0017] In the above formula, A 供试品 is the peak area of the characteristic hydrogen signal in the nuclear magnetic resonance hydrogen spectrum of the heparin precursor test solution; θ 360 is the 360° pulse width of the heparin precursor test solution; k is the slope of the linear equation; b is the intercept of the linear equation; and N is the dilution multiple of the heparin precursor sample solution to be measured to the heparin precursor test solution.

[0018] According to the embodiments of the present application, the heparin precursor sample solution to be measured becomes the heparin precursor test solution after being diluted, and the dilution multiple is N, which is a number greater than or equal to 1; illustratively, N is the ratio of the volume of the heparin precursor test solution to the volume of the heparin precursor sample solution to be measured.

[0019] According to the embodiments of the present application, the method further comprises the following steps:

[0020] (S0) preparing the heparin precursor control into a heparin precursor control solution; and preparing the heparin precursor sample to be measured into a heparin precursor test solution.

[0021] According to the embodiment of the present application, the method is a simple and rapid NMR method for detecting the content of heparin precursor, in particular, a rapid and quantitative NMR method for detecting the content of heparin precursor in fermentation broth. The method linearly correlates the product A x θ of the peak area A of the characteristic hydrogen signal in the H-NMR spectrum and the 360° pulse width θ, and can directly and rapidly measure the content of heparin precursor in the NMR sample without adding an internal standard substance to the NMR sample. 1 H-NMR spectrum The product A x θ of the peak area A of the characteristic hydrogen signal in the H-NMR spectrum and the 360° pulse width θ 360 360 linearly correlates, and can directly and rapidly measure the content of heparin precursor in the NMR sample without adding an internal standard substance to the NMR sample.

[0022] According to the embodiment of the present application, in step (S1), the concentration of heparin precursor in the heparin precursor control solution is 0.25-20.0 mg / mL; preferably 0.75-15.0 mg / mL, for example 0.25 mg / mL, 0.625 mg / mL, 0.75 mg / mL, 1.5 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, 10 mg / mL, 15 mg / mL or 20.0 mg / mL.

[0023] According to the embodiment of the present application, in step (S1), the heparin precursor control solution comprises heparin precursor and heavy water D2O; or the heparin precursor control solution comprises heparin precursor, heavy water D2O and water H2O; or the heparin precursor control solution comprises heparin precursor, heavy water D2O and fermentation broth.

[0024] According to the embodiment of the present application, in step (S1), when the heparin precursor control solution comprises heparin precursor, heavy water D2O and water H2O, the volume ratio of heavy water D2O to water H2O is 5-20:95-80, for example 5:95, 10:90, 15:85 or 20:80.

[0025] According to the embodiment of the present application, in step (S1), when the heparin precursor control solution comprises heparin precursor, heavy water D2O and fermentation broth, the volume ratio of heavy water D2O to fermentation broth is 5-20:95-80, for example 5:95, 10:90, 15:85 or 20:80.

[0026] According to the embodiment of the present application, the fermentation broth comprises fermentation supernatant.

[0027] According to the embodiment of the present application, the fermentation broth does not comprise heparin precursor.

[0028] According to the embodiment of the present application, the fermentation broth can be prepared by a method known in the art or purchased by commercial means; for example, the fermentation broth can be prepared by the following method:

[0029] ​The E. coli glycerol bacteria not expressing heparin precursor is fermented in a culture medium, and the supernatant is collected.

[0030] According to the embodiments of the present application, the fermentation conditions are exemplarily known in the art, such as the fermentation conditions satisfying the following: after being cultured at 37℃, 200rpm for 8-12 hours, the culture is further cultured at 30℃, 200rpm for 48 hours. More specifically, 200mL sterilized culture medium is added with 3mL 50% glycerol (sterilized), and then 100μL E. coli glycerol bacteria not expressing heparin precursor is added; the mixture is transferred into a shaker, and cultured at 37℃, 200rpm overnight; the next day, the temperature is changed to 30℃, and the culture is continued for 2 days; after the fermentation is completed, the fermentation broth is transferred into a centrifuge tube, and centrifuged at 10000rpm for 10min to obtain the supernatant; the supernatant is placed in boiling water for 10min, and centrifuged at 10000rpm for 10min, and the supernatant is collected, thereby obtaining the fermentation broth.

[0031] According to the embodiments of the present application, the fermentation broth is known in the art to be capable of generating heparin precursor, and is exemplarily LB fermentation broth, SOC fermentation broth or TB fermentation broth.

[0032] According to the embodiments of the present application, the LB fermentation broth is selected from LB culture medium (Luria-Bertani Broth).

[0033] According to the embodiments of the present application, the LB culture medium comprises peptone, yeast, sodium chloride and water.

[0034] According to the embodiments of the present application, the LB culture medium comprises the following concentrations of each component: 5-30g / L peptone, 5-25g / L yeast and 1-10g / L sodium chloride, and the solvent is water. Exemplarily, the LB culture medium comprises the following concentrations of each component: 16g / L peptone, 10g / L yeast and 5g / L sodium chloride, and the solvent is water.

[0035] According to the embodiments of the present application, the SOC fermentation broth is selected from SOC culture medium (Super Optimal Broth with Catabolite repression).

[0036] According to the embodiments of the present application, the SOC culture medium comprises peptone, yeast, sodium chloride, glucose, magnesium chloride, magnesium sulfate and water.

[0037] According to an embodiment of the present application, the SOC culture medium comprises each component at the following concentration: 5-30 g / L of proteose peptone, 1-10 g / L of yeast, 5-25 g / L of sodium chloride, 10-30 mM of glucose, 5-20 mM of magnesium chloride, and 5-20 mM of magnesium sulfate, with water as the solvent. Exemplarily, the SOC culture medium comprises each component at the following concentration: 16 g / L of proteose peptone, 5 g / L of yeast, 10 g / L of sodium chloride, 20 mM of glucose, 10 mM of magnesium chloride, and 10 mM of magnesium sulfate, with water as the solvent.

[0038] According to an embodiment of the present application, the TB fermentation broth is selected from a TB culture medium (Terrific Broth).

[0039] According to an embodiment of the present application, the TB culture medium comprises proteose peptone, yeast, glycerol and / or glucose, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate.

[0040] According to an embodiment of the present application, the TB culture medium comprises each component at the following concentration: 10-20 g / L of proteose peptone, 20-30 g / L of yeast, 2-8 g / L of glycerol and / or glucose, 1.5-5 g / L of potassium dihydrogen phosphate, and 10-20 g / L of dipotassium hydrogen phosphate.

[0041] According to an embodiment of the present application, in step (S1), the concentration of heparin precursor in the heparin precursor test sample solution is 0.25-20.0 mg / mL; preferably, 0.75-15.0 mg / mL.

[0042] According to an embodiment of the present application, in step (S1), the heparin precursor control sample solution and the heparin precursor test sample solution are of the same composition, i.e., when the heparin precursor test sample solution comprises a fermentation broth, the heparin precursor control sample solution also comprises the fermentation broth, and the fermentation broth is of the same composition; when the heparin precursor test sample solution comprises water H2O, the heparin precursor control sample solution also comprises water H2O; when the heparin precursor test sample solution comprises heavy water D2O, the heparin precursor control sample solution also comprises heavy water D2O.

[0043] According to the embodiment of the present application, in step (S1), the heparosan test sample solution is prepared by fermenting the E. coli glycerol bacteria expressing heparosan in a culture medium, collecting the supernatant, and preparing the heparosan test sample solution; adding heavy water D2O and optionally adding or not adding a diluent to obtain a heparosan test sample solution with a heparosan concentration of 0.25-20.0 mg / mL. The diluent is heavy water D2O or a mixed solution of heavy water D2O and water H2O, or a fermentation broth containing heavy water D2O, wherein the volume ratio of heavy water D2O to water H2O is preferably 5-20:95-80, for example 5:95, 10:90, 15:85 or 20:80; the volume ratio of heavy water D2O to fermentation broth is 5-20:95-80, for example 5:95, 10:90, 15:85 or 20:80. If the concentration of the heparosan test sample solution is high (>20.0 mg / mL) after adding heavy water D2O, a diluent can be added to further dilute the heparosan test sample solution to obtain a heparosan test sample solution with a heparosan concentration of 0.25-20.0 mg / mL.

[0044] Illustratively, heavy water D2O is added to the heparosan test sample solution to obtain a heparosan test sample solution with a heparosan concentration of 0.25-20.0 mg / mL; or heavy water D2O and a mixed solution of heavy water D2O and water H2O are added to the heparosan test sample solution to obtain a heparosan test sample solution with a heparosan concentration of 0.25-20.0 mg / mL; or heavy water D2O and a fermentation broth containing heavy water D2O are added to the heparosan test sample solution to obtain a heparosan test sample solution with a heparosan concentration of 0.25-20.0 mg / mL.

[0045] According to the embodiment of the present application, the fermentation conditions of the E. coli glycerol bacteria expressing heparosan are the same as those of the E. coli glycerol bacteria not expressing heparosan. In the present application, the only difference between the E. coli glycerol bacteria expressing heparosan and the E. coli glycerol bacteria not expressing heparosan is that the E. coli glycerol bacteria expressing heparosan contains the relevant genes expressing heparosan, while the E. coli glycerol bacteria not expressing heparosan only contains empty plasmids.

[0046] According to the embodiments of the present application, factors affecting the nuclear magnetic resonance signal include magnetic field strength, temperature, concentration, instrument parameters, and chemical environment, etc., and the nuclear magnetic resonance signals in different solution environments can have great differences under the same magnetic field strength, temperature, concentration, and instrument parameters, etc. Therefore, in the present application, the composition of the heparin precursor control sample solution and the heparin precursor test sample solution is preferably the same to ensure the consistency of the solution conditions of the heparin precursor control sample solution and the heparin precursor test sample solution, thereby obtaining higher result accuracy.

[0047] As a preferred solution, when the heparin precursor test sample solution comprises heavy water D2O and fermentation broth, the heparin precursor control sample solution also comprises heavy water D2O and fermentation broth, and the composition of the fermentation broth in the heparin precursor control sample solution is the same as that of the fermentation broth in the heparin precursor test sample solution, and the volume ratio of the heavy water D2O and the fermentation broth in the heparin precursor control sample solution is the same as that of the heavy water D2O and the fermentation broth in the heparin precursor test sample solution. This can be obtained by preparing a series of heparin precursor control sample solutions with concentration gradients by using fermentation broth without heparin precursor.

[0048] As a preferred solution, when the heparin precursor test sample solution comprises heavy water D2O and water H2O, the heparin precursor control sample solution also comprises heavy water D2O and water H2O, and the volume ratio of the heavy water D2O and the water H2O in the heparin precursor control sample solution is the same as that of the heavy water D2O and the water H2O in the heparin precursor test sample solution. This can be obtained by dissolving heparin precursor in heavy water D2O and water H2O to prepare a series of heparin precursor control sample solutions with concentration gradients.

[0049] As a preferred solution, when the heparin precursor test sample solution comprises heavy water D2O, the heparin precursor control sample solution also comprises heavy water D2O. This can be obtained by dissolving heparin precursor in heavy water D2O to prepare a series of heparin precursor control sample solutions with concentration gradients.

[0050] According to the embodiments of the present application, in step (S1), the nuclear magnetic resonance spectrometer used in the nuclear magnetic resonance spectrum analysis is a pulse Fourier transform (PFT) spectrometer; and exemplarily, the nuclear magnetic resonance spectrometer is a Bruker nuclear magnetic resonance spectrometer.

[0051] According to the embodiments of the present application, for the sample, the 360° pulse width θ 360 Any pulse sequence can be used for detection (preferably a pulse sequence used for one-dimensional spectrum acquisition in nuclear magnetic resonance), because the 360° pulse width θ 360 of the sample detected by using any pulse sequence is equal. In order to facilitate detection, a relatively simple Zg pulse sequence can be selected for detection.

[0052] According to an embodiment of the present application, the detection condition of the nuclear magnetic resonance spectrum analysis method for measuring the 360° pulse width θ 360 The detection condition of the nuclear magnetic resonance spectrum analysis method is shown in Table a below.

[0053] Table a Detection condition of the nuclear magnetic resonance spectrum analysis method

[0054]

[0055] According to a preferred embodiment of the present application, the detection condition of the nuclear magnetic resonance spectrum analysis method for measuring the 360° pulse width θ 360 The detection condition of the nuclear magnetic resonance spectrum analysis method is shown in Table b below.

[0056] Table b Detection condition of the nuclear magnetic resonance spectrum analysis method

[0057]

[0058] According to an embodiment of the present application, the calculation step of the 360° pulse width θ 360 includes: collecting the hydrogen spectrum of the heparin precursor according to the above detection condition, performing Fourier transform (efp), correcting the phase and baseline, calculating the 90° pulse width, obtaining the 90° pulse width P1, multiplying the P1 value by 4 to obtain the 360° pulse width θ 360 . Wherein, the calculation of the 90° pulse width is realized by inputting the instruction “pulsecal”.

[0059] According to an embodiment of the present application, the peak area of the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum can be measured by using the ledbppg2s1d pulse sequence.

[0060] According to an embodiment of the present application, the detection condition of the nuclear magnetic resonance spectrum analysis method for measuring the peak area of the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum is shown in Table c below.

[0061] Table c Detection condition of the nuclear magnetic resonance spectrum analysis method

[0062]

[0063] According to a preferred embodiment of the present application, the detection condition of the nuclear magnetic resonance spectrum analysis method for measuring the peak area of the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum is shown in Table d below.

[0064] Table d Detection condition of the nuclear magnetic resonance spectrum analysis method

[0065]

[0066] According to an embodiment of the present application, the peak area of the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum can be measured by using the Zgcppr pulse sequence.

[0067] According to the embodiment of the present application, the detection condition of the nuclear magnetic resonance spectroscopy method for determining the peak area of the characteristic hydrogen signal in the hydrogen spectrum is shown in Table e below:

[0068] Table e Detection condition of the nuclear magnetic resonance spectroscopy method

[0069]

[0070] According to the preferred embodiment of the present application, the detection condition of the nuclear magnetic resonance spectroscopy method for determining the peak area of the characteristic hydrogen signal in the hydrogen spectrum is shown in Table f below:

[0071] Table f Detection condition of the nuclear magnetic resonance spectroscopy method

[0072]

[0073] According to the embodiment of the present application, the peak area of the characteristic hydrogen signal in the hydrogen spectrum can be determined by using the Zggpw5 pulse sequence.

[0074] According to the embodiment of the present application, the detection condition of the nuclear magnetic resonance spectroscopy method for determining the peak area of the characteristic hydrogen signal in the hydrogen spectrum is shown in Table g below:

[0075] Table g Detection condition of the nuclear magnetic resonance spectroscopy method

[0076]

[0077] According to the preferred embodiment of the present application, the detection condition of the nuclear magnetic resonance spectroscopy method for determining the peak area of the characteristic hydrogen signal in the hydrogen spectrum is shown in Table h below:

[0078] Table h Detection condition of the nuclear magnetic resonance spectroscopy method

[0079]

[0080] According to the embodiment of the present application, the peak area A of the characteristic hydrogen signal in the hydrogen spectrum is determined by using the ledbppg2s1d pulse sequence in the nuclear magnetic resonance spectroscopy method under the heavy water D2O and fermentation broth system, and the hydrogen spectrum A x θ of the heparin precursor is established 360 and the linear relationship with the heparin precursor concentration C, so that the concentration of the heparin precursor in the heavy water D2O and fermentation broth system can be determined. This method is suitable for detecting the heparin precursor content in the heparin precursor crude product in the preparation process (fermentation process).

[0081] According to the embodiment of the present application, the peak area A of the characteristic hydrogen signal in the hydrogen spectrum is determined by using the ledbppg2s1d pulse sequence in the nuclear magnetic resonance spectroscopy method under the heavy water D2O and water H2O system, and the hydrogen spectrum A x θ of the heparin precursor is established360 and the linear relationship between the characteristic hydrogen signal and the heparin precursor concentration C, so that the concentration of heparin precursor in the heavy water D2O system can be determined. This method is suitable for detecting the heparin precursor content in the prepared heparin precursor finished product.

[0082] According to the embodiment of the present application, in the heavy water D2O system, the peak area A of the characteristic hydrogen signal in the nuclear magnetic resonance hydrogen spectrum is determined by selecting the ledbppg2s1d pulse sequence in the nuclear magnetic resonance spectrum analysis method, and the hydrogen spectrum A x θ of the heparin precursor is established 360 and the linear relationship between the characteristic hydrogen signal and the heparin precursor concentration C, so that the concentration of heparin precursor in the heavy water D2O system can be determined. This method is suitable for detecting the heparin precursor content in the prepared heparin precursor finished product.

[0083] According to the embodiment of the present application, in the heavy water D2O and water H2O system, the peak area A of the characteristic hydrogen signal in the nuclear magnetic resonance hydrogen spectrum is determined by selecting the zggpw5 pulse sequence in the nuclear magnetic resonance spectrum analysis method, and the hydrogen spectrum A x θ of the heparin precursor is established 360 and the linear relationship between the characteristic hydrogen signal and the heparin precursor concentration C, so that the concentration of heparin precursor in the heavy water D2O and water H2O system can be determined. This method is suitable for detecting the heparin precursor content in the prepared heparin precursor finished product.

[0084] According to the embodiment of the present application, in the heavy water D2O system, the peak area A of the characteristic hydrogen signal in the nuclear magnetic resonance hydrogen spectrum is determined by selecting the zggpw5 pulse sequence in the nuclear magnetic resonance spectrum analysis method, and the hydrogen spectrum A x θ of the heparin precursor is established 360 and the linear relationship between the characteristic hydrogen signal and the heparin precursor concentration C, so that the concentration of heparin precursor in the heavy water D2O system can be determined. This method is suitable for detecting the heparin precursor content in the prepared heparin precursor finished product.

[0085] According to the embodiment of the present application, in the heavy water D2O system, the peak area A of the characteristic hydrogen signal in the nuclear magnetic resonance hydrogen spectrum is determined by selecting the zggpw5 pulse sequence in the nuclear magnetic resonance spectrum analysis method, and the hydrogen spectrum A x θ of the heparin precursor is established 360 and the linear relationship between the characteristic hydrogen signal and the heparin precursor concentration C, so that the concentration of heparin precursor in the heavy water D2O system can be determined. This method is suitable for detecting the heparin precursor content in the prepared heparin precursor finished product.

[0086] According to an embodiment of the present invention, by measuring the characteristic hydrogen signal NMR peak area A under the ledbppg2s1d pulse sequence, signal interference from culture medium components and small metabolite molecules in the fermentation broth can be effectively filtered out, greatly improving the spectral quality of the heparin precursor in the fermentation broth. Furthermore, the feasibility of using the ledbppg2s1d pulse sequence to determine the heparin precursor was verified in heavy water D2O, heavy water D2O and water H2O systems, and heavy water D2O and fermentation broth systems. Hydrogen spectra of heparin precursor reference solutions of different concentrations were collected, and the 360° pulse width θ was measured. 360 Its characteristic hydrogen signal NMR hydrogen spectrum peak area A and 360° pulse width θ 360 The product A×θ 360 It has a linear relationship with the concentration of heparin precursor.

[0087] According to an embodiment of the present invention, the feasibility of using the zggpw5 pulse sequence to determine heparin precursors was verified in heavy water D2O, heavy water D2O and water H2O systems by measuring the peak area A of the characteristic hydrogen signal NMR hydrogen spectrum under zggpw5 pulse sequence conditions. Hydrogen spectra of heparin precursor reference solutions of different concentrations were collected and the 360° pulse width θ was measured. 360 Its characteristic hydrogen signal NMR hydrogen spectrum peak area A and 360° pulse width θ 360 The product A×θ 360 It has a linear relationship with the concentration of heparin precursor.

[0088] According to an embodiment of the present invention, the feasibility of using the zgcppr pulse sequence to determine heparin precursors was verified in a heavy water D2O system by measuring the peak area A of the characteristic hydrogen signal NMR hydrogen spectrum under zgcppr pulse sequence conditions. Hydrogen spectra of heparin precursor reference solutions of different concentrations were collected, and the 360° pulse width θ was measured. 360 Its characteristic hydrogen signal NMR hydrogen spectrum peak area A and 360° pulse width θ 360 The product A×θ 360 It has a linear relationship with the concentration of heparin precursor.

[0089] According to an embodiment of the present invention, by comparing the detection effects of different pulse sequences zgcppr, zggpw5 and ledbppg2s1d, the results show that when the pulse sequence is set to ledbppg2s1d, it has a better detection effect on heparin precursors in fermentation broth; in contrast, the pulse sequences zgcpprr and zggpw5 are more suitable for the detection of high-purity heparin precursors, but not suitable for the detection of fermentation broth.

[0090] According to an embodiment of the present invention, the heparin precursor proton spectrum A×θ established under different systems (such as heavy water D2O, heavy water D2O-water H2O, or heavy water D2O-fermentation broth) is obtained. 360The linear relationship with the concentration of heparosan precursor in the fermentation liquor is successfully determined, the determination result has high accuracy, the recovery rate is greater than 99%, and the detection requirement is met.

[0091] According to the embodiment of the present application, the characteristic hydrogen signal of the heparosan precursor refers to the hydrogen atom signal of the methyl group on the N-acetyl (NAc) of the heparosan precursor.

[0092] In the second aspect, the present application provides an application of the detection method of the heparosan precursor content in the detection of the heparosan precursor.

[0093] According to the embodiment of the present application, the detection method of the heparosan precursor content is applied to the monitoring in the fermentation process of the heparosan precursor, so as to determine whether the fermentation is completed.

[0094] According to the embodiment of the present application, the detection method of the heparosan precursor content is applied to the screening of the E. coli with high heparosan precursor yield in the E. coli modification process, so as to obtain the E. coli with high heparosan precursor yield.

[0095] According to the embodiment of the present application, the detection method of the heparosan precursor content is applied to the monitoring in the preparation process and the purification process of the heparosan precursor, so as to determine whether the purity of the prepared heparosan precursor meets the quality standard requirement.

[0096] The numerical range of the present application includes not only the listed point values, but also any point values between the listed numerical ranges, and the specific point values included in the range are not listed due to the length and the consideration of simplicity.

[0097] Compared with the prior art, the present application has the following beneficial effects:

[0098] (1) The present application provides a detection method of the heparosan precursor content, in particular, a method for detecting the heparosan precursor content in the fermentation liquor by using nuclear magnetic resonance spectroscopy. The detection method can be used for detecting the heparosan precursor content in the heparosan crude product in the preparation process (fermentation process) and the prepared heparosan product. The detection method has the characteristics of mild operation condition and high sensitivity. The detection method can efficiently and quickly detect the heparosan precursor content in the heparosan crude product in the preparation process and the prepared heparosan product, and is convenient for application in industrial mass production.

[0099] (2) The detection method for the content of heparin precursor has the characteristics of high efficiency, rapidness and strong adaptability, the detection method can quickly and quantitatively detect the content of heparin precursor in the sample to be detected without pre-treating the sample to be detected or specially chemically modifying or marking the sample to be detected; the detection method is based on the hydrogen spectrum signal of the obtained sample to be detected, so as to analyze and judge the structure of the sample to be detected and deduce whether there is an impurity similar substance; the detection method can overcome the signal interference of other substances (such as culture medium components and metabolite small molecules in the fermentation liquid) in the reaction system on the reaction result. The detection method is suitable for high-throughput detection, and the detection method can obtain the nuclear magnetic spectrum of multiple samples in a short time. The detection method is more simple, rapid and efficient, the detection result is more objective, and is suitable for industrial mass production.

[0100] (3) In the experiment of determining the content of heparin precursor by the sulfuric acid-carbazole method, it can be known from the experimental results that the purified water group and the blank culture medium group in the control experiment group also have absorbance, which indicates that there is an error in determining the content of heparin precursor by the sulfuric acid-carbazole method. The nuclear magnetic resonance spectrum detection method of heparin precursor can effectively overcome the influence of the introduction of blank culture medium and carbazole reagent on the detection result in the experiment process, and the nuclear magnetic resonance spectrum detection method of heparin precursor has higher accuracy, better precision and can more directly provide the detection result. BRIEF DESCRIPTION OF DRAWINGS

[0101] Figure 1 The results of plotting the characteristic hydrogen signal of the H-NMR spectrum of the heparin precursor control solution (the concentration is 20 mg / mL, and the solvent is LB fermentation liquid containing 10 vol% heavy water D2O) collected by the ledbppg2s1d pulse sequence in Example 2. 1 H-NMR spectrum.

[0102] Figure 2 The results of plotting the characteristic hydrogen signal of the H-NMR spectrum of the heparin precursor control solution (the solvent is heavy water D2O) collected by the Zgcppr pulse sequence in Example 3. 1 The results of plotting the characteristic hydrogen signal of the H-NMR spectrum of the heparin precursor control solution (the solvent is heavy water D2O) collected by the Zgcppr pulse sequence in Example 3.

[0103] Figure 3 The results of plotting the characteristic hydrogen signal of the H-NMR spectrum of the heparin precursor control solution (the solvent is heavy water D2O) collected by the Zgcppr pulse sequence in Example 3. 1 The results of plotting the characteristic hydrogen signal of the H-NMR spectrum of the heparin precursor control solution (the solvent is heavy water D2O) collected by the Zgcppr pulse sequence in Example 3. 360 The results of plotting the characteristic hydrogen signal of the H-NMR spectrum of the heparin precursor control solution (the solvent is heavy water D2O) collected by the Zgcppr pulse sequence in Example 3.

[0104] Figure 4 The results of plotting the characteristic hydrogen signal of the H-NMR spectrum of the heparin precursor control solution (the solvent is heavy water D2O) collected by the Zgcppr pulse sequence in Example 3. 1Results of plotting the peak area A of the characteristic hydrogen signal of the H-NMR spectrum against the concentration C of heparosan.

[0105] Figure 5 Results of plotting the product A x theta of the peak area A of the characteristic hydrogen signal of the H-NMR spectrum and the 360° pulse width theta against the concentration C of heparosan for the heparosan reference solution (solvent: water solution containing 10 vol% heavy water D2O) acquired with the Zggpw5 pulse sequence in Example 3. 1 Results of plotting the peak area A of the characteristic hydrogen signal of the H-NMR spectrum against the concentration C of heparosan. 360 Results of plotting the product A x theta of the peak area A of the characteristic hydrogen signal of the H-NMR spectrum and the 360° pulse width theta against the concentration C of heparosan.

[0106] Figure 6 Results of plotting the peak area A of the characteristic hydrogen signal of the H-NMR spectrum against the concentration C of heparosan for the heparosan reference solution (solvent: water solution containing 10 vol% heavy water D2O) acquired with the ledbppg2s1d pulse sequence in Example 3. 1 Results of plotting the product A x theta of the peak area A of the characteristic hydrogen signal of the H-NMR spectrum and the 360° pulse width theta against the concentration C of heparosan.

[0107] Figure 7 Results of plotting the peak area A of the characteristic hydrogen signal of the H-NMR spectrum against the concentration C of heparosan for the heparosan reference solution (solvent: water solution containing 10 vol% heavy water D2O) acquired with the ledbppg2s1d pulse sequence in Example 3. 1 Results of plotting the product A x theta of the peak area A of the characteristic hydrogen signal of the H-NMR spectrum and the 360° pulse width theta against the concentration C of heparosan. 360 Results of plotting the product A x theta of the peak area A of the characteristic hydrogen signal of the H-NMR spectrum and the 360° pulse width theta against the concentration C of heparosan.

[0108] Figure 8 Results of plotting the peak area A of the characteristic hydrogen signal of the H-NMR spectrum against the concentration C of heparosan for the heparosan reference solution (solvent: water solution containing 10 vol% heavy water D2O) acquired with the Zggpw5 pulse sequence in Example 3. 1 Results of plotting the product A x theta of the peak area A of the characteristic hydrogen signal of the H-NMR spectrum and the 360° pulse width theta against the concentration C of heparosan.

[0109] Figure 9 Results of plotting the peak area A of the characteristic hydrogen signal of the H-NMR spectrum against the concentration C of heparosan for the heparosan reference solution (solvent: water solution containing 10 vol% heavy water D2O) acquired with the Zggpw5 pulse sequence in Example 3. 1 Results of plotting the product A x theta of the peak area A of the characteristic hydrogen signal of the H-NMR spectrum and the 360° pulse width theta against the concentration C of heparosan. 360 Results of plotting the product A x theta of the peak area A of the characteristic hydrogen signal of the H-NMR spectrum and the 360° pulse width theta against the concentration C of heparosan.

[0110] Figure 10 Results of plotting the peak area A of the characteristic hydrogen signal of the H-NMR spectrum against the concentration C of heparosan for the heparosan reference solution (solvent: water solution containing 10 vol% heavy water D2O) acquired with the ledbppg2s1d pulse sequence in Example 3. 1 Results of plotting the product A x theta of the peak area A of the characteristic hydrogen signal of the H-NMR spectrum and the 360° pulse width theta against the concentration C of heparosan.

[0111] Figure 11The heparin prodrug control solution (solvent being an aqueous solution containing 10 vol% heavy water D2O) obtained from the ledbppg2s1d pulse sequence acquisition in Example 3. 1 The characteristic hydrogen signal in the H-NMR spectrum is the product of the hydrogen peak area and the 360° pulse width, A×θ. 360 Results of plotting the concentration C of heparin precursor.

[0112] Figure 12 The heparin prodrug control solution (concentration 5 mg / mL, solvent is an aqueous solution containing 10 vol% heavy water D2O) obtained by ZG pulse sequence acquisition in Example 3. 1 H-NMR spectrum.

[0113] Figure 13 In Example 3, the heparin precursor control solution (concentration 5 mg / mL, solvent: LB fermentation broth containing 10 vol% heavy water D2O) was collected using the zggpw5 pulse sequence. 1 H-NMR spectrum.

[0114] Figure 14 The heparin precursor in Example 3 under different solvents and different pulse sequence conditions 1 An overlay of H-NMR spectra, in which... Figure 14 In Example 3, 'a' refers to the heparin precursor control solution (concentration 5 mg / mL, solvent: LB fermentation broth containing 10 vol% heavy water D2O) collected using the ledbppg2s1d pulse sequence. 1 H-NMR spectrum; Figure 14 In Example 3, 'b' refers to the heparin prodrug control solution (concentration 20 mg / mL, solvent: heavy water D2O) acquired using the zggpw5 pulse sequence. 1 H-NMR spectrum; Figure 14 In Example 3, 'c' refers to the heparin precursor control solution (concentration 5 mg / mL, solvent: LB fermentation broth containing 10 vol% heavy water D2O) acquired using the zggpw5 pulse sequence. 1 H-NMR spectrum.

[0115] Figure 15 The heparin precursor control solution (solvent: LB fermentation broth containing 10 vol% heavy water D2O) was obtained using the ledbppg2s1d pulse sequence in Example 3. 1 The result of plotting the characteristic hydrogen signal peak area A of the H-NMR spectrum against the heparin precursor concentration C.

[0116] Figure 16H-NMR spectra of heparosan precursor control solution (concentration 5 mg / mL, solvent LB broth containing 10 vol% heavy water D2O) acquired in Example 3 using zgcppr pulse sequence. 1 A x θ 360 Results plotted against heparosan precursor concentration C.

[0117] Figure 17 H-NMR spectra of heparosan precursor control solution (concentration 5 mg / mL, solvent LB broth containing 10 vol% heavy water D2O) acquired in Example 3 using zgcppr pulse sequence. 1 H-NMR spectra.

[0118] Figure 18 H-NMR spectra of heavy water D2O and 10 vol% D2O - 90 vol% LB broth acquired in Example 4 using ledbppg2s1d pulse sequence. 1 H-NMR spectra.

[0119] Figure 19 H-NMR spectra of heparosan precursor control solution (concentration 10.3 mg / mL, solvent LB broth containing 10 vol% heavy water D2O) acquired in Example 5 using ledbppg2s1d pulse sequence after storage at 4°C for 0 h, 24 h, 48 h. 1 H-NMR spectra.

[0120] Figure 20 H-NMR spectra of heparosan precursor control solution (concentration 10.3 mg / mL, solvent LB broth containing 10 vol% heavy water D2O) acquired in Example 5 using ledbppg2s1d pulse sequence after storage at room temperature for 0 h, 24 h, 48 h. 1 H-NMR spectra.

[0121] Figure 21 H-NMR spectra of test product 1 to test product 6 acquired in Example 6 using ledbppg2s1d pulse sequence. 1 H-NMR spectra. DETAILED DESCRIPTION

[0122] The technical solutions of the present application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.

[0123] Unless otherwise indicated, the following techniques and procedures were carried out in accordance with the techniques and procedures described in the literature or in accordance with the manufacturer's instructions. Unless otherwise indicated, the reagents or instruments used were conventional products that can be commercially obtained from regular channels.

[0124] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be construed as indefinite or unclear unless specifically defined, but should be understood according to the ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0125] All reagents and solvents used in the present application are commercially available and can be used without further purification. The E. coli Nissle 1917 used in the present application is obtained from Beijing Saibo Biotechnology Co., Ltd., which is a lyophilized powder.

[0126] In the present application, the term "heparin precursor content" refers to the concentration of heparin precursor, specifically the mass concentration of heparin precursor, unless otherwise specified.

[0127] In the present application, the term "360° pulse width" refers to "360° pulse width" unless otherwise specified.

[0128] In the present application, the "characteristic hydrogen signal" of heparin precursor refers to the methyl hydrogen atom signal in the NAc of heparin precursor. In the present application, A represents the peak area of the characteristic hydrogen signal of heparin precursor in nuclear magnetic resonance hydrogen spectrum. Unless otherwise specified, D2O contains 0.002% (w / v) of TSP.

[0129] The following abbreviations are used in the present application: mg / mL represents milligrams per milliliter; r represents the linear correlation coefficient, the larger the r, the better the quantitative ability; R 2 represents the square of the linear correlation coefficient (i.e. r x r), the larger the R 2 , the better the quantitative ability; RSD represents the relative standard deviation; M represents moles per liter (mol / L); mM represents millimoles per liter (mmol / L); TSP represents 3-(trimethylsilyl)propionic acid sodium salt (2,2,3,3-D4-3-(trimethylsilyl)propionic acid sodium salt); δ represents the chemical shift value of the atom, in ppm; δ H represents the chemical shift value of the H atom, in ppm; δ C represents the chemical shift value of the C atom, in ppm; 13 C-NMR (C-nuclear magnetic resonance) represents carbon nuclear magnetic resonance; 13 C-nuclear magnetic resonance) represents carbon nuclear magnetic resonance;1 H-NMR (1H nuclear magnetic resonance) represents the hydrogen spectrum of nuclear magnetic resonance; NAc represents the structure in which the N atom is connected with acetyl group (-(CO)-CH3). 1 H-nuclear magenatic resonance) represents the hydrogen spectrum of nuclear magnetic resonance; NAc represents the structure in which the N atom is connected with acetyl group (-(CO)-CH3).

[0130] The LB medium used in the following examples (by concentration) includes: 16 g / L proteose peptone, 5 g / L yeast, and 10 g / L sodium chloride, and the solvent is water. It is a culture medium for E. coli for preparing heparin precursors.

[0131] The preparation method of the LB medium used in the following examples is as follows: weigh 16 g of proteose peptone, 10 g of sodium chloride, and 5 g of yeast powder in a beaker, add 1 L of ultrapure water, completely dissolve, and then divide into shake flasks, each containing 50 mL, seal, and then sterilize in a sterilization pot, with sterilization conditions of 121℃ for 30 min.

[0132] Detection conditions of the nuclear magnetic resonance spectroscopy method

[0133] Table 1 Detection conditions of the nuclear magnetic resonance spectroscopy method for measuring the 360° pulse width θ of the characteristic hydrogen signal 360 Detection conditions of the nuclear magnetic resonance spectroscopy method

[0134]

[0135] Table 2 Detection conditions of the nuclear magnetic resonance spectroscopy method for measuring the peak area of the characteristic hydrogen signal in the hydrogen spectrum using the Zgcppr pulse sequence

[0136]

[0137] Table 3 Detection conditions of the nuclear magnetic resonance spectroscopy method for measuring the peak area of the characteristic hydrogen signal in the hydrogen spectrum using the Zggpw5 pulse sequence

[0138]

[0139] Table 4 Detection conditions of the nuclear magnetic resonance spectroscopy method for measuring the peak area of the characteristic hydrogen signal in the hydrogen spectrum using the ledbppg2s1d pulse sequence

[0140]

[0141] Example 1 Preparation of heparin precursor control

[0142] The heparin precursor was prepared by the method of Example 1 disclosed in Chinese patent document CN119595804A, and was used as a control in the research of the detection method of the heparin precursor content in the present application.

[0143] Example 2 Method for determination of heparosan content

[0144] 1. Instruments and analytical conditions

[0145] NMR spectrometer (Bruker AVIII 600 MHz) equipped with QCI cryoprobe, software version Topspin 3.0.

[0146] 2. Preparation of reference and test solutions

[0147] Diluent: LB broth containing 10 vol% heavy water D2O (10 vol% D2O - 90 vol% LB broth).

[0148] LB broth refers to a fermentation broth without heparosan precursor, which is prepared as follows: 200 mL of sterilized LB medium is added with 3 mL of 50% glycerol (sterilized), and then 100 μL of E. coli glycerol bacteria without expression of heparosan precursor is added. The mixture is transferred into a shaker, and incubated overnight at 37 °C, 200 rpm. The next day, the temperature is changed to 30 °C, and the incubation is continued for 2 days. After completion of the fermentation, the fermentation broth is transferred into a centrifuge tube, and centrifuged at 10,000 rpm for 10 min to obtain the supernatant. The supernatant is placed in boiling water for 10 min, and centrifuged at 10,000 rpm for 10 min. The supernatant is collected, and LB broth is obtained.

[0149] LB broth containing 10 vol% heavy water D2O: 1 mL of heavy water D2O (containing 0.002% (W / V) TSP) is placed in a 10 mL volumetric flask, and the above-prepared LB broth is added. The mixture is ultrasonicated and diluted to the mark, and shaken to obtain 10 vol% D2O - 90 vol% LB broth.

[0150] Preparation of heparosan reference solution (solvent: 10 vol% D2O - 90 vol% LB broth): 400 mg of heparosan prepared in Example 1 is accurately weighed and placed in a 10 mL volumetric flask. 10 vol% D2O - 90 vol% LB broth is added, the mixture is ultrasonicated and diluted to the mark, and shaken to obtain a heparosan stock solution with a concentration of 40.0 mg / mL. Based on the heparosan stock solution, heparosan reference solutions with concentrations of 20.0 mg / mL, 15.0 mg / mL, 10.0 mg / mL, 7.5 mg / mL, 5.0 mg / mL, 2.5 mg / mL, 1.5 mg / mL, 0.75 mg / mL, 0.625 mg / mL, and 0.25 mg / mL are prepared, respectively.

[0151] Preparation of heparin precursor test solution: Accurately measure 9 mL of the test liquid sample containing heparin precursor and 1 mL of heavy water D2O, mix well, and optionally add 10 vol% D2O-90 vol% LB fermentation broth to obtain the heparin precursor test solution.

[0152] The test liquid sample containing heparin precursor is the liquid sample obtained during the fermentation process of heparin precursor, specifically prepared as follows: Take 200 mL of sterile LB medium, add 3 mL of 50% glycerol (sterile), and then add 100 μL of Ecoli glycerol bacteria expressing heparin precursor. Transfer to a shaker and incubate overnight at 37°C and 200 rpm. The next day, the temperature is reduced to 30°C, and incubation continues for 2 days. Samples taken at any time during these 2 days of incubation are the test liquid samples containing heparin precursor.

[0153] 3. Experimental Procedure and Results

[0154] Take 0.6 mL of the heparin prodrug reference solution and the heparin prodrug test solution prepared above, respectively, transfer them into NMR tubes, sonicate for 5 min, and then perform 360° pulse width θ analysis on an NMR spectrometer. 360 The determination and 1 Acquisition of H-NMR spectra.

[0155] The heparin prodrug control solution was tested under the detection conditions (Zg pulse sequence) shown in Table 1 above. 1 The 1H-NMR spectrum is acquired, Fourier transform (EFP) is performed, and phase and baseline corrections are applied. Then, the command "pulsecal" is input to calculate the 90° pulse width. After the calculation is complete, the software will provide the calculated 90° pulse width P1 for this sample. Multiplying this P1 value by 4 gives the 360° pulse width θ. 360 .

[0156] The heparin prodrug reference solution was tested under the detection conditions (ledbppg2s1d pulse sequence) shown in Table 4 above. 1 H-NMR spectra were acquired. 1 After acquiring the H-NMR spectrum, Fourier transform (efp) and phase and baseline corrections were performed. The hydrogen signal of the methyl group on the N-acetyl group (NAc) of the heparin precursor was integrated in the range of 2.01-2.09 ppm, and the integrated result was derived as the peak area A of the characteristic hydrogen signal NMR hydrogen spectrum.

[0157] Take each heparin precursor control solution 1 The peak area A of the NAc methyl hydrogen signal (characteristic hydrogen signal) peak and the corresponding 360° pulse width θ in the H-NMR spectrum 360 Multiply to establish the peak area A of the characteristic hydrogen signal NMR hydrogen spectrum and the 360° pulse width θ.360 The mathematical relationship between the product and the concentration of the control sample, Y = kX + b, is shown in Table 5.

[0158] Table 5 Results of A value detected by ledbppg2s1d pulse sequence (LB fermentation broth)

[0159]

[0160] 0.6 mL of the test sample solution of heparin precursor was transferred into a NMR tube, and after ultrasonic treatment for 5 min, the 360° pulse width θ of the test sample solution was determined according to the above method. 360 and 1 H-NMR spectrum, detection 1 The peak area A of the NAc methyl hydrogen signal (characteristic hydrogen signal) peak of the H-NMR spectrum was calculated, and the product of the characteristic hydrogen signal NMR hydrogen spectrum peak area A and the 360° pulse width θ was calculated. 360

[0161] The mathematical relationship Y = 2.76 x 10 7 X + 1.16 x 10 7 in the foregoing steps was used for calculation, and the concentration C of heparin precursor in the heparin precursor sample solution to be tested was calculated according to the following formula: 肝素前体 .

[0162] The concentration calculation formula of heparin precursor in the heparin precursor sample to be tested is:

[0163]

[0164] In the above formula, A 供试品 is the peak area of the methyl hydrogen atom in the NAc hydrogen spectrum of the heparin precursor test sample solution; θ 360 is the 360° pulse width of the heparin precursor test sample solution; k is the slope of the linear equation; b is the intercept of the linear equation; and N is the dilution multiple of the heparin precursor test sample solution, i.e. the ratio of the volume of the heparin precursor test sample solution to the volume of the heparin precursor sample solution to be tested.

[0165] Figure 1 The H-NMR spectrum of the heparin precursor control sample solution (concentration of 20 mg / mL, solvent: LB fermentation broth containing 10 vol% heavy water D2O) collected by ledbppg2s1d pulse sequence in Example 2 is shown in 1 From Figure 1 it can be seen that the use of ledbppg2s1d pulse sequence for NMR spectroscopy analysis can effectively filter out the signal interference of medium components and small molecule metabolites in the fermentation broth, greatly improving the spectrum quality of the hydrogen spectrum of heparin precursor in the fermentation broth, and thus obtaining accurate test results of the heparin precursor content in the fermentation broth.

[0166] ​Example 3 Optimization of the conditions of nuclear magnetic resonance

[0167] The purpose of this example is to investigate the influence of different solvents and nuclear magnetic pulse sequences on the detection results, so as to screen the suitable detection conditions for the rapid and quantitative detection of heparin precursors in the fermentation broth containing heparin precursors, heparin precursor purification intermediates and other mixtures.

[0168] (1) Under the condition of D2O, the influence of each nuclear magnetic pulse sequence on the detection results was investigated

[0169] The heparin precursor test sample is mostly the fermentation broth of heparin precursor, the intermediate of heparin precursor process or the crude product of heparin precursor, which may contain a large amount of water solvent. This example mainly investigates the influence of each nuclear magnetic pulse sequence on the detection results under the condition of D2O, i.e. without the influence of other solvent impurities, and the linear correlation degree of the concentration C and the peak area A, A x θ 360

[0170] 1. Instruments and analysis conditions:

[0171] Nuclear magnetic resonance spectrometer (Bruker AVIII 600 MHz) equipped with QCI ultralow temperature probe, software version Topspin3.0.

[0172] 2. Preparation of reference solution

[0173] Preparation of heparin precursor reference solution (solvent is heavy water D2O): take 100 mg of heparin precursor prepared in Example 1, add 2 mL of D2O (containing 0.002% (W / V) TSP), completely dissolve, and prepare a heparin precursor stock solution with a concentration of 50 mg / mL. Based on the heparin precursor stock solution, heparin precursor reference solutions with concentrations of 20.0 mg / mL, 15.0 mg / mL, 10.0 mg / mL, 7.0 mg / mL, 5.0 mg / mL, 2.5 mg / mL, 1.5 mg / mL, 0.75 mg / mL, 0.5 mg / mL, 0.25 mg / mL are prepared respectively.

[0174] 3. Experimental process and results

[0175] The 360° pulse width θ of the heparin precursor reference solution was determined according to the method in Example 2 360 (using Zg pulse sequence for determination), and the H-NMR spectrum of the heparin precursor reference solution was collected using Zgcppr pulse sequence, Zggpw5 pulse sequence and ledbppg2s1d pulse sequence, and the peak area A of the characteristic hydrogen signal of the heparin precursor was determined. The product of the peak area of the characteristic hydrogen signal of the H-NMR spectrum and the 360° pulse width A x θ 1 360 ​​A linear regression equation was performed on the concentration (C, mg / ml) of the heparin prodrug reference solution, and a linear regression equation was also performed on the characteristic hydrogen signal NMR peak area A against the concentration (C, mg / ml). Under different pulse sequence conditions, the characteristic hydrogen signal NMR peak area A and the 360° pulse width θ for each sample were analyzed. 360 The linear regression equations are shown in Tables 6, 7, and 8, respectively.

[0176] Table 6. Results of A-value detection (D2O) using Zgcppr pulse sequences.

[0177]

[0178] Heparin prodrug control solution obtained by Zgcppr pulse sequence acquisition 1 The characteristic hydrogen signal of the H-NMR spectrum, plotted against the heparin precursor concentration C, is as follows: Figure 2 As shown, the heparin prodrug control solution obtained by Zgcppr pulse sequence acquisition. 1 The characteristic hydrogen signal in the H-NMR spectrum is the product of the hydrogen peak area and the 360° pulse width, A×θ. 360 The results of plotting the concentration C of heparin precursor are as follows: Figure 3 As shown. From Figure 2 and Figure 3 The comparison shows that the heparin precursors obtained from Zgcppr pulse sequences 1 The characteristic hydrogen signal in the H-NMR spectrum is the product of the hydrogen peak area and the 360° pulse width, A×θ. 360 There is a good linear relationship between A and the concentration C of heparin precursor, and the obtained A×θ 360 The linear regression equation (ten points) for concentration C is Y = 8.49 × 10⁻⁶. 7 X+1.31×10 7 R 2 =0.998; heparin precursor obtained from Zgcppr pulse sequence acquisition 1 The linear relationship between the characteristic hydrogen signal peak area A in the 1H-NMR spectrum and the heparin precursor concentration C is relatively poor. The obtained linear regression equation (ten points) of A on concentration C is Y = 1.63 × 10⁻⁶. 6 X + 1.83 × 10 6 R 2 =0.986.

[0179] Table 7 Results of A-value detection (D2O) using the Zggpw5 pulse sequence.

[0180]

[0181] Heparin prodrug control solution obtained by Zggpw5 pulse sequence acquisition 1 The characteristic hydrogen signal of the H-NMR spectrum, plotted against the heparin precursor concentration C, is as follows: Figure 4 As shown, the heparin prodrug control solution obtained by Zggpw5 pulse sequence acquisition. 1 The characteristic hydrogen signal in the H-NMR spectrum is the product of the hydrogen peak area and the 360° pulse width, A×θ. 360 The results of plotting the concentration C of heparin precursor are as follows: Figure 5 As shown. From Figure 4 and Figure 5 The comparison shows that the heparin precursors acquired by the Zggpw5 pulse sequence are... 1 The characteristic hydrogen signal in the H-NMR spectrum is the product of the hydrogen peak area and the 360° pulse width, A×θ. 360 There is a good linear relationship between A and the concentration C of heparin precursor, and the obtained A×θ 360 The linear regression equation (ten points) for concentration C is Y = 1.50 × 10⁻⁶. 9 X-1.06×10 8 R 2 =0.999; heparin precursor obtained from Zggpw5 pulse sequence acquisition 1 The linear relationship between the characteristic hydrogen signal peak area A in the 1H-NMR spectrum and the heparin precursor concentration C is relatively poor. The obtained linear regression equation (ten points) of A on concentration C is Y = 2.88 × 10⁻⁶. 7 X + 2.31 × 10 7 R 2 =0.991.

[0182] Table 8 Results of A-value detection (D2O) for the ledbppg2s1d pulse sequence.

[0183]

[0184] Heparin prodrug control solution obtained by ledbppg2s1d pulse sequence acquisition 1 The characteristic hydrogen signal of the H-NMR spectrum, plotted against the heparin precursor concentration C, is as follows: Figure 6 As shown, the heparin prodrug reference solution obtained by the ledbppg2s1d pulse sequence acquisition. 1 The characteristic hydrogen signal in the H-NMR spectrum is the product of the hydrogen peak area and the 360° pulse width, A×θ. 360 The results of plotting the concentration C of heparin precursor are as follows: Figure 7 As shown. From Figure 6 and Figure 7The comparison shows that the heparin precursors obtained from the ledbppg2s1d pulse sequence are... 1 The characteristic hydrogen signal in the H-NMR spectrum is the product of the hydrogen peak area and the 360° pulse width, A×θ. 360 There is a good linear relationship between A and the concentration C of heparin precursor, and the obtained A×θ 360 The linear regression equation (ten points) for concentration C is Y = 8.49 × 10⁻⁶. 7 X+1.31×10 7 R 2 =0.998; heparin precursor obtained from ledbppg2s1d pulse sequence acquisition 1 The linear relationship between the characteristic hydrogen signal peak area A in the 1H-NMR spectrum and the heparin precursor concentration C is relatively poor. The obtained linear regression equation (ten points) of A on concentration C is Y = 1.63 × 10⁻⁶. 6 X + 1.83 × 10 6 R 2 =0.986.

[0185] The test results above show that, by Figure 2 , Figure 4 and Figure 6 The comparison shows that the heparin prodrug reference solution obtained in D2O by pulse sequences of zgcppr, zggpw5, and ledbppg2s1d has... 1 The H-NMR spectrum shows a relatively poor linear relationship between the peak area A of the characteristic hydrogen signal and the concentration C of the heparin precursor. Figure 3 , Figure 5 and Figure 7 The comparison shows that the heparin prodrug reference solution obtained in D2O by pulse sequences of zgcppr, zggpw5, and ledbppg2s1d has... 1 The H-NMR spectrum shows the product of the peak area of ​​the characteristic hydrogen signal and the 360° pulse width, A×θ. 360 It shows a linear relationship with the concentration C of heparin precursor, and the linear relationship is good. 2 Greater than or equal to 0.998.

[0186] (2) The effects of different NMR pulse sequences on the detection results were investigated under the conditions of 10 vol% D2O-90 vol% H2O.

[0187] Heparin precursor test samples are often heparin precursor fermentation broth, heparin precursor process intermediates, or crude heparin precursors, and contain a large amount of water solvent. This example mainly investigates the effect of various NMR pulse sequences on the detection results in the presence of H2O, and the relationship between concentration C and peak area A, A×θ. 360 The degree of linear correlation.

[0188] The experiment was specifically selected under the condition of 10 vol% D2O-90 vol% H2O because the ultimate goal of the method developed in this study is to detect the concentration of heparin precursor in the fermentation broth. When performing nuclear magnetic experiments, the simplest sample preparation method is to add 10 vol% D2O to the fermentation broth containing heparin precursor, rather than freeze-drying the fermentation broth containing heparin precursor and then redissolving it with 100% D2O.

[0189] 1. Instruments and analysis conditions:

[0190] The nuclear magnetic resonance spectrometer (Bruker AVIII 600 MHz) is equipped with a QCI ultralow temperature probe, and the software version is Topspin3.0.

[0191] 2. Preparation of reference solution

[0192] Preparation of 10 vol% D2O-90 vol% H2O: Take 1 mL of D2O (containing 0.002% (W / V) TSP) in a 15 mL centrifuge tube, add 9 mL of H2O, mix well to obtain 10 vol% D2O-90 vol% H2O.

[0193] Preparation of heparin precursor reference solution (solvent is an aqueous solution containing 10 vol% heavy water D2O): Take 100 mg of heparin precursor prepared in Example 1, add 2 mL of 10 vol% D2O-90 vol% H2O, completely dissolve to prepare a heparin precursor stock solution with a concentration of 50 mg / mL. Based on the heparin precursor stock solution, heparin precursor reference solutions with concentrations of 20.0 mg / mL, 15.0 mg / mL, 10.0 mg / mL, 7.5 mg / mL, 5.0 mg / mL, 3.75 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL are prepared respectively.

[0194] 3. Experimental process and results

[0195] The 360° pulse width θ of the heparin precursor reference solution was determined according to the method in Example 2 360 .

[0196] Because under the condition of 10 vol% D2O-90 vol% H2O, the water peak signal is too strong, the zgcppr pulse sequence cannot effectively suppress the water peak signal, resulting in too poor signal collected, so only the Zggpw5 pulse sequence and the ledbppg2s1d pulse sequence are used to collect the 1 H-NMR spectrum of the heparin precursor reference solution, and the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A of the heparin precursor is determined. The product of the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area and the 360° pulse width A x θ 360A linear regression equation was performed on the concentration (C, mg / ml) of the heparin prodrug reference solution, and a linear regression equation was also performed on the peak area A against the concentration (C, mg / ml). The characteristic hydrogen signal NMR peak area A and 360° pulse width θ for each sample were determined under different pulse sequence conditions. 360 The linear regression equations are shown in Tables 9 and 10, respectively.

[0197] Table 9 Results of A-value detection using Zggpw5 pulse sequence (10 vol% D2O - 90 vol% H2O)

[0198]

[0199] Heparin prodrug control solution obtained by Zggpw5 pulse sequence acquisition 1 The characteristic hydrogen signal of the H-NMR spectrum, plotted against the heparin precursor concentration C, is as follows: Figure 8 As shown, the heparin prodrug control solution obtained by Zggpw5 pulse sequence acquisition. 1 The characteristic hydrogen signal in the H-NMR spectrum is the product of the hydrogen peak area and the 360° pulse width, A×θ. 360 The results of plotting the concentration C of heparin precursor are as follows: Figure 9 As shown. From Figure 8 and Figure 9 The comparison shows that the heparin precursors acquired by the Zggpw5 pulse sequence are... 1 The characteristic hydrogen signal in the H-NMR spectrum is the product of the hydrogen peak area and the 360° pulse width, A×θ. 360 There is a good linear relationship between A and the concentration C of heparin precursor, and the obtained A×θ 360 The linear regression equation (ten points) for concentration C is Y = 1.94 × 10⁻⁶. 9 X-7.39×10 8 R 2 =0.999; heparin precursor obtained from Zggpw5 pulse sequence acquisition 1 The linear relationship between the characteristic hydrogen signal peak area A in the 1H-NMR spectrum and the heparin precursor concentration C is relatively poor. The obtained linear regression equation (ten points) of A on concentration C is Y = 3.74 × 10⁻⁶. 7 X + 1.88 × 10 7 R 2 =0.992.

[0200] Table 10 Results of A-value detection for ledbppg2s1d pulse sequence (10 vol% D2O - 90 vol% H2O)

[0201]

[0202] Heparin prodrug control solution obtained by ledbppg2s1d pulse sequence acquisition 1 The characteristic hydrogen signal of the H-NMR spectrum, plotted against the heparin precursor concentration C, is as follows: Figure 10 As shown, the heparin prodrug reference solution obtained by the ledbppg2s1d pulse sequence acquisition. 1 The characteristic hydrogen signal in the H-NMR spectrum is the product of the hydrogen peak area and the 360° pulse width, A×θ. 360 The results of plotting the concentration C of heparin precursor are as follows: Figure 11 As shown. From Figure 10 and Figure 11 The comparison shows that the heparin precursors obtained from the ledbppg2s1d pulse sequence are... 1 The characteristic hydrogen signal in the H-NMR spectrum is the product of the hydrogen peak area and the 360° pulse width, A×θ. 360 There is a good linear relationship between A and the concentration C of heparin precursor, and the obtained A×θ 360 The linear regression equation (ten points) for concentration C is Y = 6.95 × 10⁻⁶. 7 X-4.29×10 7 R 2 =0.999; heparin precursor obtained from ledbppg2s1d pulse sequence acquisition 1 The linear relationship between the characteristic hydrogen signal peak area A in the 1H-NMR spectrum and the heparin precursor concentration C is relatively poor. The obtained linear regression equation (ten points) of peak area A on concentration C is Y = 1.34 × 10⁻⁶. 6 X + 2.32 × 10 5 R 2 =0.997.

[0203] The test results above show that, by Figure 8 and Figure 10 The comparison shows that the heparin precursors obtained by the zggpw5 pulse sequence and ledbppg2s1d pulse sequence under the conditions of 10 vol% D2O-90 vol% H2O are significantly different. 1 The H-NMR spectrum shows a relatively poor linear relationship between the peak area A of the characteristic hydrogen signal and the concentration C of the heparin precursor; the peak area A increases more and more slowly with increasing heparin precursor concentration C. Figure 9 and Figure 11 The comparison shows that the heparin precursors obtained by pulse sequences of zggpw5 and ledbppg2s1d under the conditions of 10 vol% D2O-90 vol% H2O are significantly better. 1 The H-NMR spectrum shows the product of the peak area of ​​the characteristic hydrogen signal and the 360° pulse width, A×θ. 360The concentration C of heparosan is linearly related to the peak area A, A x θ, and the linear relationship is good, R 2 is greater than or equal to 0.999.

[0204] The test also investigates the heparosan reference solution (solvent is 10vol% D2O-90vol% H2O) obtained by using zg pulse sequence 1 The test results are shown in Figure 12 From Figure 12 it can be seen that, due to the strong water peak signal under the condition of 10vol% D2O-90vol% H2O, the zg pulse sequence can only obtain the water signal, and cannot obtain the characteristic hydrogen signal of heparosan.

[0205] (3) Under the condition of 10vol% D2O-90vol% LB fermentation broth, the influence of various nuclear magnetic pulse sequences on the detection results is investigated

[0206] After fermentation of heparosan fermentation broth by various bacteria, a large amount of small molecule substances such as acetic acid will be produced. In this embodiment, 10vol% D2O-90vol% LB fermentation broth is introduced as a solvent, and the influence of various nuclear magnetic pulse sequences on the detection results in the presence of H2O and a large amount of impurities such as acetic acid, and the linear correlation degree of concentration C with peak area A, A x θ 360 are investigated.

[0207] 1. Instruments and analysis conditions:

[0208] Nuclear magnetic resonance spectrometer (Bruker AVIII 600 MHz) equipped with QCI ultralow temperature probe, software version Topspin3.0.

[0209] 2. Preparation of reference solution

[0210] LB fermentation broth: LB fermentation broth (fermentation broth without heparosan) is prepared by the method of Example 1.

[0211] 10vol% D2O-90vol% LB fermentation broth: 9mL of the above LB fermentation broth and 1mL of D2O (containing 0.002% (W / V) TSP) are added into a 15mL centrifuge tube, and mixed to obtain 10vol% D2O-90vol% LB fermentation broth.

[0212] Preparation of heparosan reference solution (solvent is 10vol% heavy water D2O containing LB fermentation broth): 4mg of heparosan prepared in Example 1 is taken, 1mL of 10vol% D2O-90vol% LB fermentation broth is added, and after complete dissolution, a heparosan reference solution with a concentration of 4mg / mL is prepared.

[0213] 3. Experimental process and results

[0214] The 360° pulse width θ of the heparin prodrug reference solution was determined according to the method described in Example 2. 360 .

[0215] Because the water peak signal was too strong under the fermentation broth conditions of 10 vol% D2O-90 vol% LB, the ZGCPPR pulse sequence could not effectively suppress the water peak signal, resulting in poor signal acquisition. Therefore, only the ZGGPW5 and LedBPPG2S1D pulse sequences were used to acquire the heparin precursor control solution. 1 H-NMR spectrum, measuring the characteristic hydrogen signal peak area A of heparin precursor.

[0216] The heparin prodrug reference solution was acquired using the zggpw5 pulse sequence. 1 H-NMR spectrum, test results as follows Figure 13 As shown. By analyzing... Figure 13 Observational analysis showed that the heparin precursor control solution (solvent: 10 vol% D2O - 90 vol% LB fermentation broth) 1 The H-NMR spectrum shows strong signal peaks near a chemical shift of 1.98 ppm. These peaks are relatively sharp and have narrow full width at half maximum (FWHM), suggesting that these excessively strong signals originate from small molecules such as glycerol and acetic acid in the LB fermentation broth. The large number of acetic acid methyl hydrogen signal peaks at 1.98 ppm obscures the characteristic hydrogen signal peak of the heparin precursor (around 2.04 ppm), making it impossible to accurately detect the peak area of ​​the characteristic hydrogen signal of the heparin precursor. Consequently, the zggpw5 pulse sequence cannot be used to detect the content of heparin precursor in mixtures such as fermentation broth.

[0217] Heparin prodrug control solution was acquired using the ledbppg2s1d pulse sequence. 1 H-NMR spectrum, test results as follows Figure 14 As shown in 'a'. Figure 14 The image also shows a heparin prodrug reference solution (concentration 20 mg / mL, solvent: heavy water D2O) acquired using the zggpw5 pulse sequence. 1 H-NMR spectrum (e.g.) Figure 14 (b) and heparin precursor control solution (concentration 5 mg / mL, solvent: LB fermentation broth containing 10 vol% heavy water D2O) acquired using the zggpw5 pulse sequence. 1 H-NMR spectrum (e.g.) Figure 14 c). Through the analysis of Figure 14Observational analysis revealed that, using the ledbppg2s1d pulse sequence to acquire the heparin precursor reference solution (solvent: 10 vol% D2O - 90 vol% LB fermentation broth) 1H NMR spectrum (green), most small molecule signals were filtered out, allowing clear observation of the characteristic hydrogen signals of the heparin precursor. Furthermore, the chemical shifts of these characteristic hydrogen signals were consistent with the chemical shifts of the characteristic hydrogen signals of the heparin precursor in D2O (see...). Figure 14 (a and b in the text) indicates Figure 14 The characteristic hydrogen signal (chemical shift value 2.04 ppm) left in 'a' is the characteristic hydrogen signal of heparin precursor in LB fermentation broth. Although some other signals are left, these signals do not overlap with the characteristic hydrogen signal of heparin precursor and do not affect the quantification of heparin precursor. Therefore, the ledbppg2s1d pulse sequence can be used to determine the concentration of heparin precursor in fermentation broth.

[0218] Through the Figure 14 Comparative analysis showed that the heparin precursor reference solution (LB fermentation broth containing 10 vol% heavy water D2O as solvent) could acquire characteristic hydrogen signal peaks (e.g., under ledbppg2s1d pulse sequence conditions) Figure 14 (a) but characteristic hydrogen signal peaks could not be acquired under zggpw5 pulse sequence conditions (e.g. Figure 14 (c) , while using the zggpw5 pulse sequence can collect characteristic hydrogen signal peaks (such as...) under conditions where the solvent is heavy water D2O. Figure 15 (b) in the middle.

[0219] Heparin prodrug control solution obtained by ledbppg2s1d pulse sequence acquisition 1 The characteristic hydrogen signal of the H-NMR spectrum, plotted against the heparin precursor concentration C, is as follows: Figure 16 As shown, the heparin prodrug reference solution obtained by the ledbppg2s1d pulse sequence acquisition. 1 The characteristic hydrogen signal in the H-NMR spectrum is the product of the hydrogen peak area and the 360° pulse width, A×θ. 360 The results of plotting the concentration C of heparin precursor are as follows: Figure 15 As shown. From Figure 16 and Figure 17 The comparison shows that the heparin precursors obtained from the ledbppg2s1d pulse sequence are... 1 The characteristic hydrogen signal in the H-NMR spectrum is the product of the hydrogen peak area and the 360° pulse width, A×θ. 360 There is a good linear relationship between A and the concentration C of heparin precursor, and the obtained A×θ 360 The linear regression equation (ten points) for concentration C is Y = 2.76 × 10⁻⁶. 7 X + 1.16 × 10 7R 2 =0.999; heparin precursor obtained from ledbppg2s1d pulse sequence acquisition 1 The linear relationship between the characteristic hydrogen signal peak area A in the 1H-NMR spectrum and the heparin precursor concentration C is relatively poor. The obtained linear regression equation (ten points) of A on concentration C is Y = 4.33 × 10⁻⁶. 5 X + 5.21 × 10 5 R 2 =0.998.

[0220] This test also examined the heparin precursor control solution (solvent: 10 vol% D2O - 90 vol% LB fermentation broth) obtained using zgcppr pulse sequence acquisition. 1 H-NMR spectrum, test results as follows Figure 17 As shown. From Figure 18 As can be seen, under the conditions of 10 vol% D2O-90 vol% LB fermentation broth, the water peak signal is too strong, and the zgcppr pulse sequence can only obtain the water signal, but cannot obtain the characteristic hydrogen signal of the heparin precursor.

[0221] In summary, under D2O-fermentation broth conditions, the heparin precursor obtained using the ledbppg2s1d pulse sequence was... 1 The characteristic hydrogen signal in the H-NMR spectrum is the product of the hydrogen peak area and the 360° pulse width, A×θ. 360 It is linearly related to the concentration C of heparin precursor; at the same time, since the ledbppg2s1d sequence has added a diffusion sequence, the small molecule signal in the heparin precursor sample can be filtered out. On the one hand, it can suppress the water peak signal, and on the other hand, it can filter out the signals of other small molecules, thereby increasing the quality of the sample spectrum.

[0222] Heparin precursors were acquired using the ledbppg2s1d pulse sequence under D2O and D2O-H2O conditions. 1 The H-NMR spectra were consistent with those of the zgcppr and zggpw5 pulse sequences. The linear relationship between the characteristic hydrogen signal peak area (A) and the concentration (C) of the heparin precursor was relatively poor, and the increase in the characteristic hydrogen signal peak area (A) became more gradual with increasing concentration. Meanwhile, the product of the characteristic hydrogen signal peak area and the 360° pulse width, A×θ, was... 360 It shows a linear relationship with the concentration C of heparin precursor, and the linear relationship is good. 2 A value greater than or equal to 0.998 can be used for the quantitative detection of heparin precursors in fermentation broth and preparation intermediates.

[0223] As demonstrated in Example 3, this invention successfully developed a direct and rapid quantitative detection method for heparin precursor content based on nuclear magnetic resonance (NMR) technology; the detection method involves establishing a relationship between sample concentration C and its... 1 The characteristic hydrogen signal in the H-NMR spectrum: peak area A and 360° pulse width θ 360 The product A×θ 360 The linear relationship between A×θ was established, thus enabling accurate determination of the concentration of heparin precursors. Comparison revealed that under different detection conditions, A×θ... 360 Ri of the linear regression equation for concentration C 2 The squared linear correlation coefficients are all higher than the R-squared value of the linear regression equation of the characteristic hydrogen signal NMR hydrogen spectrum peak area A on the concentration C. 2 This indicates that A×θ 360 The linear regression equation for concentration C has better quantitative ability.

[0224] This invention significantly reduces the interference of small molecule impurities (such as glycerol and acetic acid) in the fermentation broth on the heparin precursor signal by introducing the ledbppg2s1d pulse sequence into NMR experiments. This enhances the clarity and accuracy of characteristic hydrogen signal identification and successfully establishes the product A×θ of the peak area of ​​the characteristic hydrogen signal NMR hydrogen spectrum and the 360° pulse width. 360 The linear relationship with heparin precursor concentration demonstrates that this method is suitable for the quantitative analysis of heparin precursors.

[0225] The results from experiments (1), (2), and (3) show that in D2O, the characteristic hydrogen signal of heparin precursor can be observed using the zg pulse sequence, zgcppr pulse sequence, zggpw5 pulse sequence, and ledbppg2s1d pulse sequence. Under D2O-H2O conditions, the characteristic hydrogen signal of heparin precursor cannot be observed using the zg pulse sequence, while the zgcppr pulse sequence can observe the characteristic hydrogen signal of heparin precursor, but the signal is weak. The zggpw5 pulse sequence and ledbppg2s1d pulse sequence can observe the characteristic hydrogen signal of heparin precursor relatively well. Under D2O-fermentation broth conditions, due to the excessively strong signals from water molecules and small molecules such as acetic acid and glycerol, the characteristic hydrogen signal of heparin precursor cannot be observed using the zg pulse sequence, zgcppr pulse sequence, and zggpw5 pulse sequence. The ledbppg2s1d pulse sequence can observe the characteristic hydrogen signal of heparin precursor relatively well.

[0226] Example 4: Specificity assessment of the method for determining heparin precursor content

[0227] The purpose of this embodiment is to verify that the method for determining the content of heparin precursor of the present invention is not affected by blank solution or culture medium, so as to prove that the method for testing the content of heparin precursor has good specificity.

[0228] 1. Instruments and analytical conditions

[0229] The same as Example 2.

[0230] 2. Solution preparation

[0231] Blank solution 1: heavy water D2O (containing 0.002% (W / V) TSP).

[0232] Blank solution 2: 10 vol% D2O-90 vol% LB fermentation broth, prepared in the same manner as in Example 2.

[0233] 3. Experimental process

[0234] 0.6 mL of blank solution 1 and blank solution 2, respectively, were transferred into a nuclear magnetic tube, and after ultrasonic treatment for 5 min, the 1 H-NMR spectra of blank solution 1 and blank solution 2 were collected by referring to the pulse sequence in Table 4 of Example 2.

[0235] 4. Acceptable standard

[0236] The 1 H-NMR spectra of blank solution 1 and blank solution 2 did not show signal peaks at N-acetyl methyl 2.04 ppm.

[0237] 5. Experimental results

[0238] Figure 18 The 1 H-NMR spectra of blank solution 1 (heavy water D2O) and blank solution 2 (10 vol% D2O-90 vol% LB fermentation broth) collected in Example 4 using the ledbppg2s1d pulse sequence. From Figure 19 it can be seen that blank solution 1 and blank solution 2 do not show obvious signal peaks at a chemical shift value of 2.04 ppm, that is, blank solution 1 and blank solution 2 do not show peaks at 2.04 ppm (the characteristic hydrogen chemical shift of N-acetyl methyl), indicating that blank solution 1 and blank solution 2 do not interfere with the N-acetyl signal position of heparosan, and proving that the method has good specificity.

[0239] Example 5 Investigation of solution stability of heparosan precursor detection method

[0240] The purpose of this example is to determine the degree to which the test results are not affected after the heparosan precursor control solution used in the determination method of the application is placed under certain conditions for a period of time.

[0241] The storage temperature and storage time of the prepared heparosan precursor control solution were investigated.

[0242] Acceptable standard: the peak area RSD of all samples is ≤1.0%.

[0243] 1. Instruments and analytical conditions

[0244] The same as Example 2.

[0245] 2. Preparation of the control solution

[0246] The heparin precursor control solution (concentration: 10.3 mg / mL; solvent: 10 vol% D2O-90 vol% LB fermentation broth) was prepared by the method of Example 2.

[0247] 3. Experimental process and results

[0248] 0.6 mL of the prepared heparin precursor control solution was transferred into an NMR tube to prepare a test sample, and 6 test samples were prepared in parallel. The 6 test samples prepared above were divided into 2 groups (3 samples in each group) in average, and the 2 groups of samples were placed at room temperature and 4°C respectively, and 1 test sample was taken from each group at 0 h, 24 h and 48 h respectively for NMR detection. The same method as in Example 2 was used to perform 360° pulse width θ 360 measurement and 1 H-NMR spectrum acquisition in the NMR spectrometer, and the peak area A of the NAc methyl hydrogen signal (characteristic hydrogen signal) peak of each sample 1 H-NMR spectrum was recorded, and the change of the product of θ 360 and A was investigated, and the test results are shown in Table 11.

[0249] Table 11. Investigation results of the heparin precursor control solution under different storage temperatures and storage times

[0250]

[0251] Figure 20 The H-NMR spectrum of the heparin precursor control solution (concentration: 10.3 mg / mL; solvent: LB fermentation broth containing 10 vol% heavy water D2O) collected by using the ledbppg2s1d pulse sequence in Example 5 at 4°C after being placed for 0 h, 24 h and 48 h. 1 Figure 21 The H-NMR spectrum of the heparin precursor control solution (concentration: 10.3 mg / mL; solvent: LB fermentation broth containing 10 vol% heavy water D2O) collected by using the ledbppg2s1d pulse sequence in Example 5 at room temperature after being placed for 0 h, 24 h and 48 h. 1 Through the above tests and results, compared with the heparin precursor control solution placed at room temperature for 0 h, the RSD of the product of θ 360 and A of each heparin precursor control solution was ≤1.0%; compared with the heparin precursor control solution placed at 4°C for 0 h, the RSD of the product of θ​360 The product value RSD of A is ≤1.0%; it can be seen that the heparin precursor control solution is stable at room temperature and 4°C within 48 hours, which indicates that the heparin precursor control of the present application can be effectively and accurately used for testing the content of a sample within 48 hours.

[0252] Example 6 Reproducibility investigation experiment of the heparin precursor content detection method

[0253] This example is to determine the reproducibility of the same operator for the same batch of sample multiple measurement results.

[0254] 1. Instruments and analysis conditions

[0255] The same as Example 2.

[0256] 2. Preparation of control solution and sample solution

[0257] The heparin precursor control solution with a concentration of 9.6 mg / mL was prepared by the method of Example 2, and was divided into 6 parts in parallel as heparin precursor sample solutions.

[0258] 3. Experimental process and results

[0259] 0.6 mL of the above heparin precursor sample solution was taken into a NMR tube, and after ultrasonic treatment for 5 min, the θ 360 and the 1 H-NMR spectrum of the sample were measured and collected, respectively. 7 H-NMR spectrum of the sample were measured and collected, respectively. 7 The content of heparin precursor in each heparin precursor sample solution was calculated, and the RSD of the content determination results of the 6 heparin precursor sample solutions should be ≤1.0%. The reproducibility results are shown in Table 12.

[0260] Table 12 Reproducibility investigation experiment results of the heparin precursor content detection method

[0261]

[0262] ​ The 1 H-NMR spectrum of sample 1 to sample 6 collected by the ledbppg2s1d pulse sequence in Example 6. Through the experiment and the results, it can be seen that the RSD of the concentration test results of the heparin precursor sample solution is 0.35%, which is much smaller than the acceptable limit of 1.0%, and it can be seen that the analysis reproducibility of the method of the present application meets the acceptance standard, which proves that the method has good reproducibility.

[0263] Example 7 Recovery rate experiment of the heparin precursor content detection method

[0264] The method of Example 2 was used to prepare heparin precursor control solution with a concentration of 0.5 mg / mL, 3.0 mg / mL and 6.0 mg / mL (solvent: 10 vol% D2O-90 vol% LB fermentation broth) as heparin precursor sample solution; 0.6 mL of each heparin precursor sample solution was sequentially transferred into a NMR tube, and after ultrasonic treatment for 5 min, the 360° pulse width θ of the heparin precursor sample solution was determined by the same method as in Example 2 360 , the H-NMR spectrum of the heparin precursor sample solution was collected, and the peak area A of the characteristic hydrogen signal of the heparin precursor was determined. 1

[0265] Table 13 Results of recovery rate test of the content detection method of heparin precursor

[0266]

[0267] From the above test results, it can be seen that the heparin precursor content detection method of the present application can accurately determine the concentration of heparin precursor in the sample. It can be seen that the detection method has good sensitivity in the linear range, the detection result is reliable, the recovery rate is greater than 99%, the determination recovery rate is high, and meets the detection requirements.

[0268] When the present application is actually determined in the fermentation broth, the quantitative result of heparin precursor is accurate, the recovery rate is more than 99%, which proves the reliability of the detection method of the present application in complex samples.

[0269] Example 8 Detection of heparin precursor concentration by heparin precursor detection method

[0270] The heparin precursor sample to be tested 1 was prepared by the following method:

[0271] 1 mL of heavy water D2O (containing 0.002% (W / V) TSP) was taken in a 10 mL volumetric flask, fermentation broth was added, ultrasonic treatment and dilution to the mark, and shaking to obtain heparin precursor sample to be tested 1. The fermentation broth was prepared by the following method:

[0272] The colonies on the cultured plate were picked into a shake flask containing glucose synthesis medium, and cultured at 37°C, 200 rpm for 6-10 h in a shaker. The obtained primary seed liquid was inoculated into 100 mL of glucose synthesis medium at a 3% (V / V) inoculation amount. The secondary seed liquid was obtained by culturing at 37°C, 200 rpm for 6-10 h in a shaker. The secondary seed liquid was inoculated into a fermentation tank at a 4% (V / V) inoculation amount, and cultured at 37°C, 450 rpm and aeration amount of 3-4 L / min for 24 h, and the bacteria were collected and centrifuged to obtain the fermentation broth.

[0273] ​The heparin precursor sample 2 to be tested was prepared by the following method:

[0274] 1 mL of heavy water D2O (containing 0.002% (W / V) TSP) was taken into a 10 mL volumetric flask, the heparin precursor crude extract was added, ultrasonic was performed and diluted to the mark, and then shaken to obtain the heparin precursor sample 2 to be tested. The heparin precursor crude extract was prepared by the following method:

[0275] The fermentation broth prepared above was loaded into a 2.0 L centrifugal cup, the centrifugal speed was set to 3800 rpm, and the centrifugal time was 60 min. After centrifugation, if the supernatant was still turbid, centrifugation could be repeated for 1-2 times until the supernatant was relatively clear. The obtained fermentation broth was centrifuged at 3800 rpm for 60 min, and if the obtained supernatant was still turbid, centrifugation could be repeated for 1-2 times until the obtained supernatant was clear. A 0.45 μm filter membrane was used to filter and remove the insoluble particles in the supernatant to protect the next step of ultrafiltration membrane. The supernatant obtained in the previous step was subjected to ultrafiltration (the molecular weight cut-off of the ultrafiltration membrane was 1000 Da), 2.4 L of purified water was slowly added, and the ultrafiltration was performed while adding the purified water until the volume was concentrated to 800 mL, and the ultrafiltration was ended to obtain the heparin precursor crude extract.

[0276] The heparin precursor sample 1 and the heparin precursor sample 2 obtained above were used as the heparin precursor test sample solution; 0.6 mL of the heparin precursor test sample solution was taken and transferred into a nuclear magnetic tube, ultrasonic was performed for 5 min, and then the 360° pulse width θ of the heparin precursor test sample solution was determined by the same method as in Example 2. 360 The H-NMR spectrum of the heparin precursor test sample solution was collected. 1 The characteristic hydrogen signal peak area A of the heparin precursor was determined, and the mathematical relationship Y = 2.76 x 10 7 X + 1.16 x 10 7 The content of the heparin precursor in the heparin precursor test sample solution (i.e. the heparin precursor sample 1 and the heparin precursor sample 2) was calculated.

[0277] Table 14 Concentration test results of the heparin precursor detected by the heparin precursor detection method

[0278]

[0279] As shown in Table 14, the heparin precursor sample 1 and the heparin precursor sample 2 have a relatively high content of heparin precursor. The above detection method has good sensitivity in the linear range, and the detection results are reliable.

[0280] Example 9 Linear test of the heparin precursor detection method

[0281] The purpose of this embodiment is to determine, within a predetermined range, the linear relationship between the measurement result of the test solution of the detection method of the present invention and the concentration of heparin precursor.

[0282] The selected heparin prodrug reference solutions had concentrations of 0.25 mg / mL, 0.625 mg / mL, 0.75 mg / mL, 1.5 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, 10 mg / mL, 15 mg / mL, and 20.0 mg / mL. Each heparin prodrug reference solution was analyzed. The product A×θ of the characteristic hydrogen signal hydrogen NMR peak area and the 360° pulse width was plotted on the x-axis as the concentration of the heparin prodrug reference solution. 360 Plot a standard curve with the ordinate as the vertical axis and calculate the relationship between the concentration of the heparin precursor reference solution and A×θ. 360 The linear relationship.

[0283] 1. Instruments and analytical conditions

[0284] Same as Example 2.

[0285] 2. Solution preparation

[0286] Same as Example 2.

[0287] 3. Experimental Procedure and Results

[0288] 0.6 mL of each heparin precursor control solution (LB fermentation broth containing 10 vol% D2O) was sequentially transferred into an NMR tube, sonicated for 5 min, and the 360° pulse width θ of the heparin precursor control solution was measured using the same method as in Example 2. 360 Collect heparin precursor control solution 1 H-NMR spectrum, measuring the characteristic hydrogen signal peak area A of heparin precursor.

[0289] The product of the characteristic hydrogen signal NMR hydrogen spectrum peak area and the 360° pulse width, A×θ 360 A linear regression equation was constructed for the concentration (C, mg / mL) of the heparin prodrug reference solution: Ten concentration gradients were selected: 0.25 mg / mL, 0.625 mg / mL, 0.75 mg / mL, 1.5 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, 10 mg / mL, 15 mg / mL, and 20.0 mg / mL. The linear regression equation A was: Y = 2.76 × 10⁻⁶. 7 X + 1.16 × 10 7 R 2=0.999; 0.75 mg / mL, 1.5 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, 10 mg / mL, 15 mg / mL, and 20.0 mg / mL are selected to construct a linear regression equation B, specifically Y = 3 x 10 7 X + 1 x 10 7 , R 2 =0.999; 0.75 mg / mL, 1.5 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, 10 mg / mL, 15 mg / mL, and 20.0 mg / mL are selected to construct a linear regression equation B, specifically Y = 3 x 10 7 X + 1 x 10 7 , R 2 =0.999; 0.75 mg / mL, 1.5 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, 10 mg / mL, 15 mg / mL, and 20.0 mg / mL are selected to construct a linear regression equation B, specifically Y = 3 x 10 7 X + 1 x 10 6 , R 2 =0.999.

[0290] The above test and results show that the concentration of the heparin precursor control solution is in the range of 0.25 mg / mL-20.0 mg / mL, the concentration C of the heparin precursor is linearly related to A x θ 360 , and the linear relationship is good, R 2 is greater than or equal to 0.999, indicating that the quantitative ability of the detection method is high.

[0291] The linear regression equation of the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A to the concentration (C, mg / ml) of the heparin precursor control solution is: 0.25 mg / mL, 0.625 mg / mL, 0.75 mg / mL, 1.5 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, 10 mg / mL, 15 mg / mL, and 20.0 mg / mL are selected to construct a linear regression equation E, specifically Y = 4.33 x 10 5 X + 5.21 x 10 5 , R 2 =0.998; 0.75 mg / mL, 1.5 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, 10 mg / mL, 15 mg / mL, and 20.0 mg / mL are selected to construct a linear regression equation F, specifically Y = 4.45 x 10 5 X + 5.04 x 10 5 , R 2 =0.996.

[0292] From the above experiments and results, it can be seen that the linear relationship between the concentration C of heparin precursor and the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A is relatively poor when the concentration of heparin precursor control solution is in the range of 0.25 mg / mL-20.0 mg / mL, R 2 less than 0.999, indicating that the quantitative ability of the detection method is relatively poor.

[0293] Comparative Example 1: Determination of heparin precursor content in samples by sulfuric acid-carbazole method

[0294] The heparin precursor content in the sample was determined by the sulfuric acid-carbazole method of Comparative Example 1 disclosed in Chinese patent document CN119595804A. From the experimental results, it can be seen that the purified water group and the blank medium group in the control experiment group have absorbance, indicating that the determination of heparin precursor by the sulfuric acid-carbazole method will have natural errors.

[0295] In summary, the detection method of heparin precursor content provided by the present application can effectively overcome the influence of blank medium and carbazole reagent on the detection results during the experimental process. The detection method of heparin precursor content provided by the present application has higher accuracy, good precision, and can more intuitively provide detection results.

[0296] The above describes the embodiments of the present application. However, the present application is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for detecting the heparosan precursor content, characterized in that, The detection method comprises: (S1) The 360° pulse width θ of the heparin precursor control solution and the heparin precursor test solution is respectively determined by nuclear magnetic resonance spectrum analysis method 360 , and the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A is calculated 360 , and the product A x θ of the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A and the 360° pulse width θ 360 is calculated; (S2) Linear regression was performed using the least squares method to establish the characteristic hydrogen signal NMR peak area A and 360° pulse width θ of the heparin precursor reference solution. 360 The mathematical relationship between the product Y and the concentration X of the heparin precursor reference solution is Y = kX + b; (S3) calculating the concentration of heparin precursor in the heparin precursor sample solution to be detected according to the mathematical relationship of step (S2); wherein the calculation formula of the concentration of heparin precursor in the heparin precursor sample solution to be detected is: In the above formula, A 供试品 characteristic hydrogen signal of heparin precursor test sample solution; θ 360 is the 360° pulse width of heparin precursor test sample solution; k is the slope of linear equation; b is the intercept of linear equation; N is the dilution multiple of heparin precursor sample solution diluted into heparin precursor test sample solution; wherein the heparin precursor content and the heparin precursor concentration are both the mass concentration of heparin precursor; the characteristic hydrogen signal of heparin precursor refers to the signal of methyl hydrogen atom in NAc of heparin precursor.

2. The detection method according to claim 1, characterized in that, In step (S1), the concentration of heparin precursor in the heparin precursor control solution is 0.25-20.0 mg / mL; in step (S1), the concentration of heparin precursor in the heparin precursor sample solution is 0.25-20.0 mg / mL.

3. The method of claim 1, wherein In step (S1), the heparin precursor control solution comprises heparin precursor and heavy water D2O; or the heparin precursor control solution comprises heparin precursor, heavy water D2O and water H2O; or the heparin precursor control solution comprises heparin precursor, heavy water D2O and fermentation broth; when the heparin precursor control solution comprises heparin precursor, heavy water D2O and water H2O, the volume ratio of heavy water D2O to water H2O is 5-20:95-80; when the heparin precursor control solution comprises heparin precursor, heavy water D2O and fermentation broth, the volume ratio of heavy water D2O to fermentation broth is 5-20:95-80.

4. The detection method according to claim 3, characterized in that, The fermentation broth does not comprise heparin precursor; and / or, the fermentation broth is LB fermentation broth, SOC fermentation broth or TB fermentation broth.

5. The method of claim 1, wherein, In step (S1), the composition of the heparin precursor control solution and the heparin precursor sample solution is the same, that is, when the heparin precursor sample solution comprises fermentation broth, the heparin precursor control solution also comprises fermentation broth, and the composition of the fermentation broth is the same; when the heparin precursor sample solution comprises water H2O, the heparin precursor control solution also comprises water H2O; when the heparin precursor sample solution comprises heavy water D2O, the heparin precursor control solution also comprises heavy water D2O.

6. The method of claim 1, wherein, In step (S1), the heparin precursor sample solution is prepared by the following method: fermenting E. coli glycerol bacteria expressing heparin precursor in a culture medium, collecting the supernatant to prepare the heparin precursor sample solution to be detected; adding heavy water D2O and optionally adding or not adding a diluent to obtain a heparin precursor sample solution with a heparin precursor concentration of 0.25-20.0 mg / mL; wherein the diluent is heavy water D2O or a mixed solution of heavy water D2O and water H2O, or a fermentation broth containing heavy water D2O, wherein the volume ratio of heavy water D2O to water H2O is 5-20:95-80; and the volume ratio of heavy water D2O to fermentation broth is 5-20:95-80.

7. The method of claim 1, wherein, The nuclear magnetic resonance spectrometer used in the nuclear magnetic resonance spectrum analysis is a pulse Fourier transform spectrometer. measuring the 360° pulse width θ 360 The detection condition of the nuclear magnetic resonance spectrum analysis method is: the NMR frequency is 600 MHz; the probe is CPQCI; the accumulation is 1-8 times; the temperature is 298 K; the relaxation delay is 1-6 s; the spectrum width is 15 ppm; the window function is 0.3 Hz; the pulse sequence is Zg; the number of empty scans is 0; the sampling point number is 64k; the 90° pulse width is 1 μs; Or, the detection condition of the NMR spectrum analysis method for measuring the peak area of the characteristic hydrogen signal in the hydrogen spectrum is: pulse sequence is ledbppg2s1d; NMR frequency is 600 MHz; probe is CPQCI; accumulation is 8-32 times; temperature is 298 K; relaxation delay is 2-12 s; empty scanning times are 4; spectral width is 18 ppm; window function is 0.3 Hz; diffusion time is 0.03 s; gradient duration is 3 ms; gradient intensity is 100%; and acquisition requirement is non-rotation; Or, the detection condition of the NMR spectrum analysis method for measuring the peak area of the characteristic hydrogen signal in the hydrogen spectrum is: pulse sequence is Zgcppr; NMR frequency is 600 MHz; probe is CPQCI; accumulation is 8-24 times; temperature is 298 K; relaxation delay is 6-20 s; spectral width is 18 ppm; window function is 0.3 Hz; pre-saturation power is 71.8 dB; and acquisition requirement is non-rotation; Or, the detection condition of the NMR spectrum analysis method for measuring the peak area of the characteristic hydrogen signal in the hydrogen spectrum is: pulse sequence is Zggpw5; NMR frequency is 600 MHz; probe is CPQCI; accumulation is 32-128 times; temperature is 298 K; relaxation delay is 6-20 s; spectral width is 18 ppm; window function is 0.3 Hz; doublet water peak suppression delay is 0.0002 s; and acquisition requirement is non-rotation.

8. The detection method according to claim 7, characterized in that, In heavy water D2O and fermentation broth system, the peak area of characteristic hydrogen signal in 1H NMR spectrum is determined by using ledbppg2s1d pulse sequence to establish the hydrogen spectrum A x θ of heparosan 360 The concentration of heparosan in heavy water D2O and fermentation broth system is determined to establish the linear relationship with the concentration C of heparosan. Or, in heavy water D2O and water H2O system, select ledbppg2s1d pulse sequence to determine the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area, establish heparin precursor hydrogen spectrum A x θ 360 The concentration of heparin precursor in heavy water D2O and water H2O system is determined; Or, in heavy water D2O system, select ledbppg2s1d pulse sequence to determine the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area, establish heparin precursor hydrogen spectrum A x θ 360 The linear relationship with the concentration C of heparin precursor, the concentration of heparin precursor in heavy water D2O system is determined; Or, in heavy water D2O and water H2O system, the peak area of characteristic hydrogen signal in hydrogen spectrum is determined by zggpw5 pulse sequence to establish the hydrogen spectrum A x θ of heparin precursor 360 The concentration of heparin precursor in heavy water D2O and water H2O system is determined according to the linear relationship with the concentration C of heparin precursor. Or, in heavy water D2O system, select zggpw5 pulse sequence to determine the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area, establish heparin precursor hydrogen spectrum A x θ 360 The linear relationship with the concentration of heparin precursor C, the concentration of heparin precursor in heavy water D2O system is determined; Or, in heavy water D2O system, select zgcppr pulse sequence to determine the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area, establish heparin precursor hydrogen spectrum A x θ 360 The concentration of heparin precursor in heavy water D2O system is determined.

9. Use of the heparosan content detection method according to any one of claims 1-8 in the detection of heparosan.

10. Use according to claim 9, characterized in that, The heparosan content detection method is applied to the monitoring of the fermentation process of heparosan, so as to determine whether the fermentation is completed; or, The heparosan content detection method is applied to the process of Escherichia coli modification, and screening of Escherichia coli with high heparosan yield, so as to obtain Escherichia coli with high heparosan yield; Or, The heparosan content detection method is applied to the monitoring of the preparation process and purification process of heparosan, so as to determine whether the purity of the prepared heparosan meets the requirements of the quality standard.

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